TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy

*Xuehui He and Xinhui Wang*

## **Abstract**

TNF has both proinflammatory and antiinflammatory effects. It binds to two structurally related but functionally distinct receptors TNFR1 and TNFR2. Unlike TNFR1 that is ubiquitously expressed, TNFR2 expression is more limited to myeloid and lymphoid cell lineages including a fraction of regulatory T cells (Treg). In general, TNFR1 is responsible for TNF-mediated cell apoptosis and death, and mostly induces proinflammatory reactions. However, TNFR2 mainly leads to functions related to cell survival and immune suppression. Treg play an indispensable role in maintaining immunological self-tolerance and restraining excessive immune reactions deleterious to the host. Impaired Treg-mediated immune regulation has been observed in various autoimmune diseases as well as in cancers. Therefore, Treg might provide an ideal therapeutic target for diseases where the immune balance is impaired and could benefit from the regulation of Treg properties. TNFR2 is highly expressed on Treg in mice and in humans, and TNFR2+ Treg reveal the most potent suppressive capacity. TNF-TNFR2 ligation benefits Treg proliferation, although the effect on Treg suppressive function remains controversial. Here, we will describe in detail the TNF-mediated regulation of Treg and the potential clinical applications in cancer immunotherapy as well as in autoimmune diseases, with the focus on human Treg subsets.

**Keywords:** TNF, TNF receptor 2, regulatory T cells, immunotherapy, autoimmune disease, cancer immunotherapy

## **1. Introduction**

CD4+FOXP3+ regulatory T cells (Treg) have an indispensable role in maintaining immune homeostasis and immune tolerance. They control unwanted immune responses that are involved in the regulation of immune tolerance to self as well as to foreign antigens. Loss-of-function mutation in *FOXP3* locus, a gene encoding Treg lineage transcription factor FOXP3, leads to multiorgan associated autoimmunity. Abnormal numbers of Treg and/or impaired suppressive function of Treg are often found in various autoimmune diseases like type 1 diabetes (T1D) [1], multiple sclerosis (MS) [2], rheumatoid arthritis (RA) [3], psoriasis [4–6], and systemic lupus erythematosus (SLE) [7–9]. On the other hand, tumor-infiltrating Treg generally show potent suppressive functions, indicating that they regulate tumorspecific immune responses and might help tumor immune escape [10]. It seems

logical to use Treg as a therapeutic target for diseases where the immune balance is impaired and could benefit from the regulation of Treg properties. Nevertheless, due to the intrinsic properties of Treg, i.e. heterogeneity and plasticity, several key questions need to be clarified before making Treg an ideal candidate for clinical applications.

Tumor necrosis factor (TNF) is initially expressed on cell surface as a membrane bound cytokine (mTNF), which can be cleaved by a metalloprotease TNF converting enzyme (TACE) to generate soluble form of TNF (sTNF) [11]. TNF binds to receptors, TNF receptor 1 (TNFR1) and 2 (TNFR2). In contrast to TNFR1, TNFR2 expression is restricted in certain cell types including lymphocytes [12]. TNF-TNFR1 interaction mostly induces proinflammatory reactions, whereas TNFR2 generally leads to the suppressive function of TNF [13]. It is known that TNFR2 is constitutively expressed on both murine and human Treg, and TNFR2+ Treg are the most suppressive Treg subpopulation [14–17]. The effect of TNF on Treg suppressor function remains controversial. In this chapter, we will describe in detail the TNFmediated signal transduction pathways, its effect on Treg cells, and the potential clinical applications in various immunopathologies.

## **2. Regulatory T cells and its plasticity**

Treg exert their function in primary and secondary lymphoid organs and nonlymphoid tissues. FOXP3, as the lineage transcription factor of Treg, facilitates Treg thymic development by stabilizing its own expression and inhibiting transcription factors needed for the development of other helper T-cell (Th) lineages like T-bet for Th1, GATA3 for Th2, and RORγt for Th17 cells [18]. Next to FOXP3, Treg constitutively express a high level of the IL-2 receptor α chain (CD25) and a low level of the IL-7 receptor α chain (CD127) compared to human activated non-Treg. The combination of CD4+, CD25high, and CD127low has been used to isolate Treg for functional studies and for adoptive immunotherapy [19]. However, no unique Treg marker has been identified so far, although many molecules are proposed. These Treg-related cell markers include CD27 [20], CD62L [21], CTLA4 (cytotoxic T-lymphocyte-associated protein) [22], CD39 and CD73 ectoenzymes [23], Helios [24], Neuropilin-1 [25], HLA-DR [26], and the most recently identified combination of TIGIT and FcRL3, which results in the identification of human Helios+ memory Treg [27].

Compelling evidence indicates that both mouse and human Treg consist of various subpopulations and have a more or less plastic phenotype depending on the microenvironment they are in [28]. Based on the site of Treg generation, two major Treg subsets are classified, namely, thymus-derived Treg (tTreg) that develop in the thymus from CD4 single positive thymocytes which in general display high-affinity self-reactive T-cell receptors (TCRs), and peripherally induced Treg (pTreg) which emerge in the periphery from conventional CD4+ T lymphocytes (Tconv) in response to environmental antigens and tolerogenic stimuli. Studies in mice have shown that pTreg and tTreg are both required for full protection against colitis and lymphoproliferative disease [29, 30], indicating that these two Treg subsets play distinct roles in protecting against immunopathology. However, the relative contribution of tTreg and pTreg in human immune tolerance remains a major unresolved issue, partially due to the lack of specific markers to definitively distinguish them. In fact, the transcription factor Helios was the first marker proposed to distinguish both mice and human tTreg from pTreg [31]. However, this has been disputed by studies showing that Helios can also be expressed by activated Tconv [32] and by pTreg upon in vitro and in vivo stimulation [33], precluding its

**43**

of PI3K/Akt pathway and cell survival.

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy*

use as tTreg-specific marker. Another cell surface marker that has been proposed to harbor the specificity necessary to distinguish between murine tTreg and pTreg is the coreceptor Neuropilin-1 [25]. Unfortunately, human Treg do not uniquely

TNF is firstly discovered as an inflammatory cytokine that is induced by the endotoxin [35]. Various immune cells produce TNF including macrophages, monocytes, dendritic cells, B cells, activated natural killer cells, and activated T cells. TNF is initially expressed on the cell surface as a trimeric type II transmembrane protein mTNF, which is then cleaved by the metalloproteinase TACE (also known as ADAM17) and released as soluble extracellular sTNF [36]. Both forms of TNF are present as bioactive homotrimers. There exist two structurally related but functionally distinct receptors, TNFR1 (p55) and TNFR2 (p75). TNFR1 is ubiquitously expressed on most mammalian cell types, and it binds to mTNF as well as sTNF, whereas TNFR2 expression is restricted to immune cells, neurons, and endothelial cells. TNFR2 binds with higher affinity to mTNF than sTNF compared

TNFR1 and TNFR2 share the similar extracellular TNF-binding motifs but differ in their intracellular domains. Both receptors lack intrinsic enzyme activity; thus, upon the ligand binding, they need to recruit the cytosolic proteins to initiate the intracellular signal transduction. Specifically, TNFR1 contains a homologous intracellular region called "death domain", which preferentially interacts with the adaptor protein named TNFR1-associated death-domain (TRADD) protein [37]. TRADD further recruits another two adaptor proteins, receptor interacting protein kinase 1 (RIPK1) and TNFR-associated factor (TRAF) 2, thus forming an enzymatic complex signalosome, which is also known as signaling complex 1. One of the main targets of the complex 1 is the enzyme complex called IkB kinase (IKK). Phosphorylation of IKK in turn leads to the canonical activation of the transcription factor NFkB as well as members of the family of MAPKs such as c-jun kinase (JNK) and p38 MAPK. The TRADD containing signaling complex 1 may further be converted to a death-inducing signaling complex, so-called complex 2, by adaptor protein Fas-associated protein with death domain (FADD). The complex 2 is able to further initiate downstream

caspase cascades, thus inducing cell apoptosis and cell death [37].

The pathways induced by TNFR2 are slightly different from TNFR1. Due to the lack of death domain, TNFR2 is unable to recruit TRADD protein, but it can directly interact with TRAF2 [38]. In contrast to TNFR1 that drives apoptosis and cell death, TNFR2 induces the noncanonical activation of NFκB via the activation of the NFκB-inducing kinase (NIK), which further leads to the phosphorylation of IKKα and the processing of p100, a crucial step in the nuclear translocation of p52/RelB [38, 39]. Interestingly, TRAF2 binding to TNFR2 is considerably weaker than its binding to TRADD protein. Upon binding to TRAF2, TNFR2 could also recruit cIAP1/2 proteins [39] that are involved in the TNFR1-mediated NFκB activation, indicating that there exists a crosstalk between TNFR1 and TNFR2 pathways. Another interesting adaptor protein called endothelial/epithelial protein tyrosine kinase (Etk) interacts with the C-terminal domain of TNFR2 in a ligandindependent manner [40]. TNFR2-mediated Etk phosphorylation is able to partially activate the growth factor receptor VEGFR2, which in turn results in the activation

A number of proteins are essential for the negative regulation of the TNF-TNFR

pathways. A20, also named as TNF alpha-induced protein 3, is one of the most

*DOI: http://dx.doi.org/10.5772/intechopen.85632*

**3. TNF/TNFR signaling pathways**

express Neuropilin-1 [34].

to TNFR1.

use as tTreg-specific marker. Another cell surface marker that has been proposed to harbor the specificity necessary to distinguish between murine tTreg and pTreg is the coreceptor Neuropilin-1 [25]. Unfortunately, human Treg do not uniquely express Neuropilin-1 [34].

## **3. TNF/TNFR signaling pathways**

*Cytokines*

applications.

logical to use Treg as a therapeutic target for diseases where the immune balance is impaired and could benefit from the regulation of Treg properties. Nevertheless, due to the intrinsic properties of Treg, i.e. heterogeneity and plasticity, several key questions need to be clarified before making Treg an ideal candidate for clinical

Tumor necrosis factor (TNF) is initially expressed on cell surface as a membrane bound cytokine (mTNF), which can be cleaved by a metalloprotease TNF converting enzyme (TACE) to generate soluble form of TNF (sTNF) [11]. TNF binds to receptors, TNF receptor 1 (TNFR1) and 2 (TNFR2). In contrast to TNFR1, TNFR2 expression is restricted in certain cell types including lymphocytes [12]. TNF-TNFR1 interaction mostly induces proinflammatory reactions, whereas TNFR2 generally leads to the suppressive function of TNF [13]. It is known that TNFR2 is constitutively expressed on both murine and human Treg, and TNFR2+ Treg are the most suppressive Treg subpopulation [14–17]. The effect of TNF on Treg suppressor function remains controversial. In this chapter, we will describe in detail the TNFmediated signal transduction pathways, its effect on Treg cells, and the potential

Treg exert their function in primary and secondary lymphoid organs and nonlymphoid tissues. FOXP3, as the lineage transcription factor of Treg, facilitates Treg thymic development by stabilizing its own expression and inhibiting transcription factors needed for the development of other helper T-cell (Th) lineages like T-bet for Th1, GATA3 for Th2, and RORγt for Th17 cells [18]. Next to FOXP3, Treg constitutively express a high level of the IL-2 receptor α chain (CD25) and a low level of the IL-7 receptor α chain (CD127) compared to human activated non-Treg. The combination of CD4+, CD25high, and CD127low has been used to isolate Treg for functional studies and for adoptive immunotherapy [19]. However, no unique Treg marker has been identified so far, although many molecules are proposed. These Treg-related cell markers include CD27 [20], CD62L [21], CTLA4 (cytotoxic T-lymphocyte-associated protein) [22], CD39 and CD73 ectoenzymes [23], Helios [24], Neuropilin-1 [25], HLA-DR [26], and the most recently identified combination of TIGIT and FcRL3, which results in the identification of human Helios+

Compelling evidence indicates that both mouse and human Treg consist of various subpopulations and have a more or less plastic phenotype depending on the microenvironment they are in [28]. Based on the site of Treg generation, two major Treg subsets are classified, namely, thymus-derived Treg (tTreg) that develop in the thymus from CD4 single positive thymocytes which in general display high-affinity self-reactive T-cell receptors (TCRs), and peripherally induced Treg (pTreg) which emerge in the periphery from conventional CD4+ T lymphocytes (Tconv) in response to environmental antigens and tolerogenic stimuli. Studies in mice have shown that pTreg and tTreg are both required for full protection against colitis and lymphoproliferative disease [29, 30], indicating that these two Treg subsets play distinct roles in protecting against immunopathology. However, the relative contribution of tTreg and pTreg in human immune tolerance remains a major unresolved issue, partially due to the lack of specific markers to definitively distinguish them. In fact, the transcription factor Helios was the first marker proposed to distinguish both mice and human tTreg from pTreg [31]. However, this has been disputed by studies showing that Helios can also be expressed by activated Tconv [32] and by pTreg upon in vitro and in vivo stimulation [33], precluding its

clinical applications in various immunopathologies.

**2. Regulatory T cells and its plasticity**

**42**

memory Treg [27].

TNF is firstly discovered as an inflammatory cytokine that is induced by the endotoxin [35]. Various immune cells produce TNF including macrophages, monocytes, dendritic cells, B cells, activated natural killer cells, and activated T cells. TNF is initially expressed on the cell surface as a trimeric type II transmembrane protein mTNF, which is then cleaved by the metalloproteinase TACE (also known as ADAM17) and released as soluble extracellular sTNF [36]. Both forms of TNF are present as bioactive homotrimers. There exist two structurally related but functionally distinct receptors, TNFR1 (p55) and TNFR2 (p75). TNFR1 is ubiquitously expressed on most mammalian cell types, and it binds to mTNF as well as sTNF, whereas TNFR2 expression is restricted to immune cells, neurons, and endothelial cells. TNFR2 binds with higher affinity to mTNF than sTNF compared to TNFR1.

TNFR1 and TNFR2 share the similar extracellular TNF-binding motifs but differ in their intracellular domains. Both receptors lack intrinsic enzyme activity; thus, upon the ligand binding, they need to recruit the cytosolic proteins to initiate the intracellular signal transduction. Specifically, TNFR1 contains a homologous intracellular region called "death domain", which preferentially interacts with the adaptor protein named TNFR1-associated death-domain (TRADD) protein [37]. TRADD further recruits another two adaptor proteins, receptor interacting protein kinase 1 (RIPK1) and TNFR-associated factor (TRAF) 2, thus forming an enzymatic complex signalosome, which is also known as signaling complex 1. One of the main targets of the complex 1 is the enzyme complex called IkB kinase (IKK). Phosphorylation of IKK in turn leads to the canonical activation of the transcription factor NFkB as well as members of the family of MAPKs such as c-jun kinase (JNK) and p38 MAPK. The TRADD containing signaling complex 1 may further be converted to a death-inducing signaling complex, so-called complex 2, by adaptor protein Fas-associated protein with death domain (FADD). The complex 2 is able to further initiate downstream caspase cascades, thus inducing cell apoptosis and cell death [37].

The pathways induced by TNFR2 are slightly different from TNFR1. Due to the lack of death domain, TNFR2 is unable to recruit TRADD protein, but it can directly interact with TRAF2 [38]. In contrast to TNFR1 that drives apoptosis and cell death, TNFR2 induces the noncanonical activation of NFκB via the activation of the NFκB-inducing kinase (NIK), which further leads to the phosphorylation of IKKα and the processing of p100, a crucial step in the nuclear translocation of p52/RelB [38, 39]. Interestingly, TRAF2 binding to TNFR2 is considerably weaker than its binding to TRADD protein. Upon binding to TRAF2, TNFR2 could also recruit cIAP1/2 proteins [39] that are involved in the TNFR1-mediated NFκB activation, indicating that there exists a crosstalk between TNFR1 and TNFR2 pathways. Another interesting adaptor protein called endothelial/epithelial protein tyrosine kinase (Etk) interacts with the C-terminal domain of TNFR2 in a ligandindependent manner [40]. TNFR2-mediated Etk phosphorylation is able to partially activate the growth factor receptor VEGFR2, which in turn results in the activation of PI3K/Akt pathway and cell survival.

A number of proteins are essential for the negative regulation of the TNF-TNFR pathways. A20, also named as TNF alpha-induced protein 3, is one of the most

studied negative regulatory proteins. A20 is an ubiquitin editing enzyme. It limits NFκB signaling after activation by TNF [41]. Consistent with this, A20-deficient mice are hypersensitive to TNF exposure and die perinatally because of severe inflammation and multiorgan failure [42]. Intriguingly, A20 is recently shown to regulate the de novo generation of tTreg in a cell-intrinsic manner, while the suppressor function of A20-deficient Treg is unchanged in vitro [43].

## **4. Effect of TNFR2 on Treg**

Although TNFR1 expression is not different between Treg and non-Treg cells, human Treg constitutively express high levels of TNFR2 compared to CD25- Tconv. Moreover, TNFR2+ Treg reveal the most potent suppressive capacity [14, 44]. The effect of TNF on Treg suppressor function remains controversial. Several groups including ours demonstrated that sTNF preserved or even increased FOXP3 expression as well as Treg suppressive capacity in both mice and humans [15, 45–47]. The TNF-TNFR2 is crucial for sustaining FOXP3 expression and maintaining the stability of murine Treg in an inflammatory environment [44]. A similar phenomenon is also observed for human Treg in vitro [48]. There is also evidence for the negative effects of TNF on Treg function. Studies show that TNF impairs Treg function by reducing FOXP3 expression or enhancing its dephosphorylation [47, 49]. In clinical practices, RA patients responding to anti-TNF antibody adalimumab showed an increased percentage of FOXP3 + cells as well as the restored regulatory function [50]. It should be noted that the nature of the TNFR2 antibodies used in these studies was likely different (agonistic versus antagonistic) [46]. Recent studies highlight that TNFR2 agonisms and antagonisms might regulate the phenotype and the suppressor function of Treg in a complete different way [46].

TNF priming induces the proliferation and activation of Treg in vitro [15, 51] as well as in vivo via TNFR2 in an acute mouse GvHD model [52]. Our group have found that stimulation of human Treg with a TNFR2-agonist antibody preserved a stable Treg phenotype and function after ex vivo expansion [48]. Using TNFR2 agonist only was enough to prevent the loss of FOXP3 expression, whereas the sustained hypomethylation of TSDR (Treg-specific demethylated region) of *FOXP3* gene locus required both rapamycin and TNFR2 agonist, suggesting that stabilization of FOXP3 expression requires both mTOR and NFκB signal pathways. In vitro restimulation of TNFR2 agonist plus rapamycin-expanded Treg led neither to the loss of FOXP3 protein nor the enhancement of IL-17A production, especially under proinflammatory conditions, indicating a well-preserved Treg stability. TNFR2 knockout CD4+ T cells have increased expression of RORγt and IL-17 production, which is dependent on the impairment of TNFR2-mediated activation of NFκB [53]. We speculate that a similar process of regulation may exist in human Treg where TNFR2/NFκB signaling might act as a double-edged sword to enhance FOXP3 but also to inhibit RORγt expression, thus contributing to Treg stability. Another possible explanation is that TNFR2 engagement results in an autocrine TNF-TNFR2 loop, which further regulates the expression of histone methyltransferase EZH2 [51], a subunit of the polycomb repressor complex 2 (PRC2). EZH2 is known to bind to FOXP3 thus helping FOXP3 to regulate the gene transcriptional repression [54].

## **5. TNFR2 agonists and autoimmune diseases**

Defect in the function of Treg as well as the low numbers are the main properties of various autoimmune diseases. Therefore, restoring the proper functional Treg

**45**

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy*

thus favoring the immune tolerance induction has become a final goal of treatment for patients with autoimmune diseases. As discussed above, ample studies show that either TNF and/or TNFR2 agonism has capacity to enhance Treg proliferation and activation. Furthermore, TNF-TNFR2 is essential to maintain the Treg function and stability in the inflammatory environment [44, 48]. Impaired TNF-TNFR signaling pathways occur in several human diseases including T1D, SLE, IBD, and MS. For instance, a single-nucleotide polymorphism (SNP) in the first intron is linked to a decreased level of TNFR2 in carriers of the SNP and a high risk of disease suscep-

The rationale for using TNFR2 agonists as a therapeutic option for autoimmune diseases was first shown in T1D. Using blood from patients with T1D, a dose-response relationship between TNFR2 agonism and the destroying of pathogenic autoreactive CD8 T cells was observed [56], suggesting inducing of TNF-TNFR2 pathway is

Currently used biologics targeting TNF include the anti-TNF antibodies infliximab, adalimumab, certolizumab, and the decoy receptor etanercept that binds to sTNF. Although they have a good safety profile, with increasing use of these drugs, paradoxical adverse events involving the skin, joints, and lungs have been described [57]. Skin manifestations are the most common adverse event and occur in about 25% of patients receiving anti-TNFs. The underlying mechanism is recently attributed to the TNFR2/A20 signal axis which is specifically responsible for TNF-mediated IL-17A inhibition [58]. Termination of NFκB activation is critical to prevent aberrant inflammatory responses. In memory CD4 T cells, A20 is identified as one of the strongest TNF-responsive genes with a strong inverse correlation to IL-17A expression.

Tumor microenvironment preferably recruits TNFR2+ Treg cells which possess a highly immunosuppressive capacity, thus facilitating tumor immune escape. That TNFR2 knockout mice show improved immune responses to tumors might be caused by the lack of TNFR2 expressing Treg or have failed to develop systemic autoimmunity [59] or the decreased numbers and the impaired function of MDSCs [60]. In humans, the high level of TNFR2+ Treg is found in the peripheral blood of lung cancer patients [10] and in the tumor-associated ascites in ovarian cancer patients [61]. Moreover, the increased TNFR2 gene expression on Treg cells has been shown to be associated with exhaustion of CD8 cytotoxic T lymphocytes in metastatic melanoma patients.

In addition to being an inducer of Treg expansion, TNFR2 also acts as an oncogene which has been identified on at least 25 tumor types. Enhanced expression of TNFR2 on tumor itself has been also reported but not limited in human renal cell carcinoma, multiple myeloma, colon cancer, ovarian cancer, and cutaneous T-cell lymphomas (CTCL) [62]. In general, the overexpression of TNFR2 exploits this cytokine receptor for increased tumor cell proliferation and tumor growth. Genetic mutation/genomic gains of *TNFRSF1B*, a gene encoding TNFR2 protein, occur in patients with Sézary syndrome (SS), a rare form of CTCL often refractory to treatment. SS is characterized with high expression of TNFR2 on the tumor cells and Treg. Such gain-of-function mutation in TNFR2 leads to the enhanced noncanonical NKκB activation [63], a pathway primarily involved in cell expansion and growth. It seems being desirable to apply one approach that could successfully inhibit potent suppressive Treg and also directly prevent tumor growth by using the antagonistic molecules against TNFR2. Such TNFR2-specific blocking molecules would ideally inhibit Treg and permit Tconv proliferation and function, thus enabling to restore

the antitumor immune responses and to induce tumor regression.

Treg compared to healthy controls.

*DOI: http://dx.doi.org/10.5772/intechopen.85632*

tibility [55]. T1D patients have higher TNFR2+

an effective approach of selectively killing autoreactive T cells.

**6. TNFR2 antagonists and cancer immunotherapy**

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.85632*

thus favoring the immune tolerance induction has become a final goal of treatment for patients with autoimmune diseases. As discussed above, ample studies show that either TNF and/or TNFR2 agonism has capacity to enhance Treg proliferation and activation. Furthermore, TNF-TNFR2 is essential to maintain the Treg function and stability in the inflammatory environment [44, 48]. Impaired TNF-TNFR signaling pathways occur in several human diseases including T1D, SLE, IBD, and MS. For instance, a single-nucleotide polymorphism (SNP) in the first intron is linked to a decreased level of TNFR2 in carriers of the SNP and a high risk of disease susceptibility [55]. T1D patients have higher TNFR2+ Treg compared to healthy controls. The rationale for using TNFR2 agonists as a therapeutic option for autoimmune diseases was first shown in T1D. Using blood from patients with T1D, a dose-response relationship between TNFR2 agonism and the destroying of pathogenic autoreactive CD8 T cells was observed [56], suggesting inducing of TNF-TNFR2 pathway is an effective approach of selectively killing autoreactive T cells.

Currently used biologics targeting TNF include the anti-TNF antibodies infliximab, adalimumab, certolizumab, and the decoy receptor etanercept that binds to sTNF. Although they have a good safety profile, with increasing use of these drugs, paradoxical adverse events involving the skin, joints, and lungs have been described [57]. Skin manifestations are the most common adverse event and occur in about 25% of patients receiving anti-TNFs. The underlying mechanism is recently attributed to the TNFR2/A20 signal axis which is specifically responsible for TNF-mediated IL-17A inhibition [58]. Termination of NFκB activation is critical to prevent aberrant inflammatory responses. In memory CD4 T cells, A20 is identified as one of the strongest TNF-responsive genes with a strong inverse correlation to IL-17A expression.

#### **6. TNFR2 antagonists and cancer immunotherapy**

Tumor microenvironment preferably recruits TNFR2+ Treg cells which possess a highly immunosuppressive capacity, thus facilitating tumor immune escape. That TNFR2 knockout mice show improved immune responses to tumors might be caused by the lack of TNFR2 expressing Treg or have failed to develop systemic autoimmunity [59] or the decreased numbers and the impaired function of MDSCs [60]. In humans, the high level of TNFR2+ Treg is found in the peripheral blood of lung cancer patients [10] and in the tumor-associated ascites in ovarian cancer patients [61]. Moreover, the increased TNFR2 gene expression on Treg cells has been shown to be associated with exhaustion of CD8 cytotoxic T lymphocytes in metastatic melanoma patients.

In addition to being an inducer of Treg expansion, TNFR2 also acts as an oncogene which has been identified on at least 25 tumor types. Enhanced expression of TNFR2 on tumor itself has been also reported but not limited in human renal cell carcinoma, multiple myeloma, colon cancer, ovarian cancer, and cutaneous T-cell lymphomas (CTCL) [62]. In general, the overexpression of TNFR2 exploits this cytokine receptor for increased tumor cell proliferation and tumor growth. Genetic mutation/genomic gains of *TNFRSF1B*, a gene encoding TNFR2 protein, occur in patients with Sézary syndrome (SS), a rare form of CTCL often refractory to treatment. SS is characterized with high expression of TNFR2 on the tumor cells and Treg. Such gain-of-function mutation in TNFR2 leads to the enhanced noncanonical NKκB activation [63], a pathway primarily involved in cell expansion and growth. It seems being desirable to apply one approach that could successfully inhibit potent suppressive Treg and also directly prevent tumor growth by using the antagonistic molecules against TNFR2. Such TNFR2-specific blocking molecules would ideally inhibit Treg and permit Tconv proliferation and function, thus enabling to restore the antitumor immune responses and to induce tumor regression.

*Cytokines*

**4. Effect of TNFR2 on Treg**

studied negative regulatory proteins. A20 is an ubiquitin editing enzyme. It limits NFκB signaling after activation by TNF [41]. Consistent with this, A20-deficient mice are hypersensitive to TNF exposure and die perinatally because of severe inflammation and multiorgan failure [42]. Intriguingly, A20 is recently shown to regulate the de novo generation of tTreg in a cell-intrinsic manner, while the sup-

Although TNFR1 expression is not different between Treg and non-Treg cells, human Treg constitutively express high levels of TNFR2 compared to CD25- Tconv. Moreover, TNFR2+ Treg reveal the most potent suppressive capacity [14, 44]. The effect of TNF on Treg suppressor function remains controversial. Several groups including ours demonstrated that sTNF preserved or even increased FOXP3 expression as well as Treg suppressive capacity in both mice and humans [15, 45–47]. The TNF-TNFR2 is crucial for sustaining FOXP3 expression and maintaining the stability of murine Treg in an inflammatory environment [44]. A similar phenomenon is also observed for human Treg in vitro [48]. There is also evidence for the negative effects of TNF on Treg function. Studies show that TNF impairs Treg function by reducing FOXP3 expression or enhancing its dephosphorylation [47, 49]. In clinical practices, RA patients responding to anti-TNF antibody adalimumab showed an increased percentage of FOXP3 + cells as well as the restored regulatory function [50]. It should be noted that the nature of the TNFR2 antibodies used in these studies was likely different (agonistic versus antagonistic) [46]. Recent studies highlight that TNFR2 agonisms and antagonisms might regulate the phenotype and

TNF priming induces the proliferation and activation of Treg in vitro [15, 51] as well as in vivo via TNFR2 in an acute mouse GvHD model [52]. Our group have found that stimulation of human Treg with a TNFR2-agonist antibody preserved a stable Treg phenotype and function after ex vivo expansion [48]. Using TNFR2 agonist only was enough to prevent the loss of FOXP3 expression, whereas the sustained hypomethylation of TSDR (Treg-specific demethylated region) of *FOXP3* gene locus required both rapamycin and TNFR2 agonist, suggesting that stabilization of FOXP3 expression requires both mTOR and NFκB signal pathways. In vitro restimulation of TNFR2 agonist plus rapamycin-expanded Treg led neither to the loss of FOXP3 protein nor the enhancement of IL-17A production, especially under proinflammatory conditions, indicating a well-preserved Treg stability. TNFR2 knockout CD4+ T cells have increased expression of RORγt and IL-17 production, which is dependent on the impairment of TNFR2-mediated activation of NFκB [53]. We speculate that a similar process of regulation may exist in human Treg where TNFR2/NFκB signaling might act as a double-edged sword to enhance FOXP3 but also to inhibit RORγt expression, thus contributing to Treg stability. Another possible explanation is that TNFR2 engagement results in an autocrine TNF-TNFR2 loop, which further regulates the expression of histone methyltransferase EZH2 [51], a subunit of the polycomb repressor complex 2 (PRC2). EZH2 is known to bind to FOXP3 thus helping FOXP3 to regulate the gene transcriptional repression [54].

Defect in the function of Treg as well as the low numbers are the main properties of various autoimmune diseases. Therefore, restoring the proper functional Treg

pressor function of A20-deficient Treg is unchanged in vitro [43].

the suppressor function of Treg in a complete different way [46].

**5. TNFR2 agonists and autoimmune diseases**

**44**

## **7. Strategies for blocking of TNF/TNFR2 signaling**

A number of agonistic or antagonistic biological agents targeting to TNF and/or TNFR2 have been developed. Two potent dominant TNFR2 antagonist antibodies are developed by Faustman et al. group [64]. They report that these TNFR2 antagonists lock the TNFR2 receptor in the form of antiparallel dimmers, which further prevents the TNF binding as well as the intracellular scaffolding. Consequently, these dominant TNFR2 antagonists, even in the presence of TNF, could kill Treg isolated from ovarian cancer ascites more potently than it kills Treg from healthy donors. Interestingly, TNFR2 antagonistic mAbs are also able to directly kill TNFR2-expression ovarian cancer cell lines in vitro [64]. Similar effect is observed in another in vitro study where the cancer cells and lymphocytes were isolated from the end-stage SS patients [65]. In mouse model of colon and breast cancers, combining a blocking TNFR2 antibody with a kind of immune stimulant markedly enhances the antitumor efficacy of immunotherapy through reducing the number of tumor-infiltrating TNFR2+ Treg and increasing the number of IFNγ-producing CD8 cells [66].

Some pharmacological agents are found to regulate TNF and/or its receptors expression. Thalidomide and its analogues prevent the surface expression of TNFR2 on activated T cells, which is associated with the inhibition of TNFR2 protein trafficking to the cell membrane [67]. Treating acute myeloid leukemia patients with azacitidine and lenalidomide, a thalidomide derivative can reduce TNFR2 expression on T cells as well as TNFR2+ Treg in vivo, leading to enhanced effector immune function [68]. Cyclophosphamide is a DNA alkylating agent. It is commonly used as a cytotoxic chemotherapy in cancer treatment. In a mouse model, it is shown that cyclophosphamide treatment depletes TNFR2+ Treg via inducing the death of replicating Treg that co-express TNFR2 and KI-67 [69]. A re-expansion of Treg from lymphodepletion suppresses the effective antitumor immunity developed after cyclophosphamide treatment. Intriguingly, blockade of TNF signaling using etanercept inhibits TNFR2+ Treg cell expansion during recovery from cyclophosphamide-induced lymphodepletion and markedly inhibits the growth of established CT26 tumors in mice [70]. Altogether, it suggests that a TNFR2-targeted approach to inactive host Treg, especially in only tumor microenvironment, may offer optimal options for antitumor immune reactions.

### **8. Conclusions**

Many surface receptors of Treg are also expressed on other immune cells, with TNFR2 being a prominent exception with highest density in the tumor microenvironment. TNFR2 is a functional receptor on Treg. Cell surface expression of TNFR2 not only identifies the potent Treg subsets but also is the property of tumor-infiltrating Treg. TNFR2 expression on some cancer-infiltrating Treg is about 100 times higher than on circulating Treg in control subjects. In other types of cancer, the abundance of TNFR2+ Treg in peripheral blood is higher than healthy ones. Targeting TNFR2 using small molecule agonists or antagonists is a promising but also a challenging task. Considering the suppressive property of Treg and its impaired functions in various immunopathologies, there is no doubt that novel (tumor-specific) antagonists against TNFR2 are promising for cancer immunotherapy. From the clinical utilities point of view, combination of TNFR2 inhibition with immune checkpoint inhibitors seems to be an attractive approach in reshaping modern cancer immunotherapy.

**47**

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy*

The authors would like to thank the A FACTT network (Cost Action BM1305: http: //www.afactt.eu) for supporting this work by positive discussion. XH is also supported by NSFC 61263039 and NSFC 11101321. XW is supported by NSFC

The funders had no role in study design, data collection and analysis, decision to

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of

*DOI: http://dx.doi.org/10.5772/intechopen.85632*

61263039, NSFC 11101321, and 2018-ZJ-776.

publish, or preparation of the manuscript**.**

IBD Inflammatory bowel disease CTCL Cutaneous T-cell lymphomas

MAPK Mitogen-activated protein kinase

SNP Single-nucleotide polymorphism

TSDR Treg-specific demethylated region

MS Multiple sclerosis

SS Sézary syndrome T1D Type 1 diabetes

TCR T-cell receptor TNFR TNF receptor

mTNF Membrane-bound TNF NFκB Nuclear factor κB RA Rheumatoid arthritis

TACE TNF-converting enzyme

TRAF TNFR-associated factor Treg Regulatory T cells

**Acknowledgements**

**Conflict of interest**

interest.

**Nomenclature**

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.85632*

## **Acknowledgements**

*Cytokines*

CD8 cells [66].

antitumor immune reactions.

modern cancer immunotherapy.

**8. Conclusions**

**7. Strategies for blocking of TNF/TNFR2 signaling**

A number of agonistic or antagonistic biological agents targeting to TNF and/or TNFR2 have been developed. Two potent dominant TNFR2 antagonist antibodies are developed by Faustman et al. group [64]. They report that these TNFR2 antagonists lock the TNFR2 receptor in the form of antiparallel dimmers, which further prevents the TNF binding as well as the intracellular scaffolding. Consequently, these dominant TNFR2 antagonists, even in the presence of TNF, could kill Treg isolated from ovarian cancer ascites more potently than it kills Treg from healthy donors. Interestingly, TNFR2 antagonistic mAbs are also able to directly kill TNFR2-expression ovarian cancer cell lines in vitro [64]. Similar effect is observed in another in vitro study where the cancer cells and lymphocytes were isolated from the end-stage SS patients [65]. In mouse model of colon and breast cancers, combining a blocking TNFR2 antibody with a kind of immune stimulant markedly enhances the antitumor efficacy of immunotherapy through reducing the number of tumor-infiltrating TNFR2+ Treg and increasing the number of IFNγ-producing

Some pharmacological agents are found to regulate TNF and/or its receptors expression. Thalidomide and its analogues prevent the surface expression of TNFR2 on activated T cells, which is associated with the inhibition of TNFR2 protein trafficking to the cell membrane [67]. Treating acute myeloid leukemia patients with azacitidine and lenalidomide, a thalidomide derivative can reduce TNFR2 expression on T cells as well as TNFR2+ Treg in vivo, leading to enhanced effector immune function [68]. Cyclophosphamide is a DNA alkylating agent. It is commonly used as a cytotoxic chemotherapy in cancer treatment. In a mouse model, it is shown that cyclophosphamide treatment depletes TNFR2+ Treg via inducing the death of replicating Treg that co-express TNFR2 and KI-67 [69]. A re-expansion of Treg from lymphodepletion suppresses the effective antitumor immunity developed after cyclophosphamide treatment. Intriguingly, blockade of TNF signaling using etanercept inhibits TNFR2+ Treg cell expansion during recovery from cyclophosphamide-induced lymphodepletion and markedly inhibits the growth of established CT26 tumors in mice [70]. Altogether, it suggests that a TNFR2-targeted approach to inactive host Treg, especially in only tumor microenvironment, may offer optimal options for

Many surface receptors of Treg are also expressed on other immune cells, with TNFR2 being a prominent exception with highest density in the tumor microenvironment. TNFR2 is a functional receptor on Treg. Cell surface expression of TNFR2 not only identifies the potent Treg subsets but also is the property of tumor-infiltrating Treg. TNFR2 expression on some cancer-infiltrating Treg is about 100 times higher than on circulating Treg in control subjects. In other types of cancer, the abundance of TNFR2+ Treg in peripheral blood is higher than healthy ones. Targeting TNFR2 using small molecule agonists or antagonists is a promising but also a challenging task. Considering the suppressive property of Treg and its impaired functions in various immunopathologies, there is no doubt that novel (tumor-specific) antagonists against TNFR2 are promising for cancer immunotherapy. From the clinical utilities point of view, combination of TNFR2 inhibition with immune checkpoint inhibitors seems to be an attractive approach in reshaping

**46**

The authors would like to thank the A FACTT network (Cost Action BM1305: http: //www.afactt.eu) for supporting this work by positive discussion. XH is also supported by NSFC 61263039 and NSFC 11101321. XW is supported by NSFC 61263039, NSFC 11101321, and 2018-ZJ-776.

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript**.**

## **Conflict of interest**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## **Nomenclature**


*Cytokines*

## **Author details**

Xuehui He1,2\* and Xinhui Wang1,3

1 College of Computer Science, Qinghai Normal University, Xining, Qinghai, China

2 Department of Laboratory Medicine, Laboratory Medical Immunology, Radboud University Medical Center, Nijmegen, The Netherlands

3 Department of Public and Occupational Health, Amsterdam Public Health Research Institute, Amsterdam University Medical Center, Amsterdam, The Netherlands

\*Address all correspondence to: xuehui.he@radboudumc.nl

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**49**

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy*

European Journal of Immunology.

[9] Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nature Genetics.

[10] Yan F, Du R, Wei F, Zhao H, Yu J, Wang C, et al. Expression of TNFR2 by regulatory T cells in peripheral blood is correlated with clinical pathology of lung cancer patients. Cancer Immunology, Immunotherapy: CII.

[8] Lyssuk EY, Torgashina AV, Soloviev SK, Nassonov EL, Bykovskaia SN. Reduced number and function of CD4+CD25 high FoxP3+ regulatory T cells in patients with systemic lupus erythematosus. Advances in Experimental Medicine and Biology.

2015;**45**(2):344-355

2007;**601**:113-119

2001;**27**(1):18-20

2015;**64**(11):1475-1485

[11] Moss ML, Jin SL, Milla ME, Bickett DM, Burkhart W, Carter HL, et al. Cloning of a disintegrin metalloproteinase that processes

Nature. 1997;**385**(6618):733-736

[12] Grell M, Douni E, Wajant H, Lohden M, Clauss M, Maxeiner B, et al. The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor. Cell. 1995;**83**(5):793-802

[13] Kim EY, Priatel JJ, Teh SJ, Teh HSTNF. Receptor type 2 (p75) functions as a costimulator for antigen-driven T cell responses in vivo. Journal of Immunology (Baltimore Md: 1950).

[14] Chen X, Subleski JJ, Hamano R, Howard OM, Wiltrout RH, Oppenheim

2006;**176**(2):1026-1035

precursor tumour-necrosis factor-alpha.

*DOI: http://dx.doi.org/10.5772/intechopen.85632*

[1] Bluestone JA, Buckner JH, Fitch M, Gitelman SE, Gupta S, Hellerstein MK, et al. Type 1 diabetes immunotherapy using polyclonal regulatory T cells. Science Translational Medicine.

[2] Haas J, Fritzsching B, Trubswetter P, Korporal M, Milkova L, Fritz B, et al. Prevalence of newly generated naive regulatory T cells (Treg) is critical for Treg suppressive function and determines Treg dysfunction in multiple sclerosis. Journal of Immunology.

[3] Komatsu N, Okamoto K, Sawa S, Nakashima T, Oh-hora M, Kodama T, et al. Pathogenic conversion of Foxp3+ T cells into TH17 cells in

autoimmune arthritis. Nature Medicine.

[4] Bovenschen HJ, van de Kerkhof PC, van Erp PE, Woestenenk R, Joosten I, Koenen HJ. Foxp3+ regulatory T cells of psoriasis patients easily differentiate into IL-17A-producing cells and are found in lesional skin. Joural of Investigative Dermatology.

[5] Sugiyama H, Gyulai R, Toichi E, Garaczi E, Shimada S, Stevens SR, et al. Dysfunctional blood and target tissue CD4+CD25 high regulatory T cells in psoriasis: Mechanism underlying unrestrained pathogenic effector T cell proliferation. Journal of Immunology.

[6] Keijsers RR, van der Velden HM, van Erp PE, de Boer-van Huizen RT, Joosten I, Koenen HJ, et al. Balance of Treg vs. T-helper cells in the transition from symptomless to lesional psoriatic skin. The British Journal of Dermatology.

[7] Ohl K, Tenbrock K. Regulatory T cells in systemic lupus erythematosus.

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2015;**7**(315):315ra189

2007;**179**(2):1322-1330

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2013;**168**(6):1294-1302

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.85632*

## **References**

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**48**

**Author details**

The Netherlands

Xuehui He1,2\* and Xinhui Wang1,3

University Medical Center, Nijmegen, The Netherlands

\*Address all correspondence to: xuehui.he@radboudumc.nl

provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

1 College of Computer Science, Qinghai Normal University, Xining, Qinghai, China

2 Department of Laboratory Medicine, Laboratory Medical Immunology, Radboud

3 Department of Public and Occupational Health, Amsterdam Public Health Research Institute, Amsterdam University Medical Center, Amsterdam,

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*Cytokines*

JJ. Co-expression of TNFR2 and CD25 identifies more of the

2010;**40**(4):1099-1106

1950). 2007;**179**(1):154-161

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[21] Ermann J, Hoffmann P, Edinger M, Dutt S, Blankenberg FG, Higgins JP,

2002;**196**(3):379-387

2012;**30**:531-564

2007;**319**(1-2):41-52

2006;**67**(9):665-675

functional CD4+FOXP3+ regulatory T cells in human peripheral blood. European Journal of Immunology.

et al. Only the CD62L+ subpopulation of CD4+CD25+ regulatory T cells protects from lethal acute GVHD. Blood.

[22] Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science.

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T cells. Journal of Immunology.

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2015;**36**(6):344-353

maintenance. Trends in Immunology.

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2006;**176**(8):4622-4631

2004;**34**(3):623-630

2005;**105**(5):2220-2226

2008;**322**(5899):271-275

Immunology. 2014;**5**:304

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**50**

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[48] He X, Landman S, Bauland SC, van den Dolder J, Koenen HJ, Joosten I. A TNFR2-agonist facilitates high purity expansion of human low purity Treg cells. PLoS One. 2016;**11**(5):e0156311

[49] Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood. 2006;**108**(1):253-261

[50] McGovern JL, Nguyen DX, Notley CA, Mauri C, Isenberg DA, Ehrenstein MR. Th17 cells are restrained by Treg cells via the inhibition of interleukin-6 in patients with rheumatoid arthritis responding to anti-tumor necrosis factor antibody therapy. Arthritis and Rheumatism. 2012;**64**(10):3129-3138

[51] Urbano PCM, Koenen H, Joosten I, He X. An autocrine TNFalpha-tumor necrosis factor receptor 2 loop promotes epigenetic effects inducing human Treg stability in vitro. Frontiers in Immunology. 2018;**9**:573

[52] Leclerc M, Naserian S, Pilon C, Thiolat A, Martin GH, Pouchy C, et al. Control of GVHD by regulatory T cells depends on TNF produced by T cells and TNFR2 expressed by regulatory T cells. Blood. 2016;**128**(12):1651-1659

[53] Miller PG, Bonn MB, McKarns SC. Transmembrane TNF-TNFR2 impairs Th17 differentiation by promoting Il2 expression. Journal of Immunology (Baltimore Md: 1950). 2015;**195**(6):2633-2647

[54] Arvey A, van der Veeken J, Samstein RM, Feng Y, Stamatoyannopoulos JA, Rudensky AY. Inflammation-induced repression of chromatin bound by the transcription factor Foxp3 in regulatory T cells. Nature Immunology. 2014;**15**(6):580-587

[55] Li D, Silverberg MS, Haritunians T, Dubinsky MC, Landers C, Stempak JM, et al. TNFRSF1B is associated with ANCA in IBD. Inflammatory Bowel Diseases. 2016;**22**(6):1346-1352

[56] Ban L, Zhang J, Wang L, Kuhtreiber W, Burger D, Faustman DL. Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(36):13644-13649

[57] Cleynen I, Vermeire S. Paradoxical inflammation induced by anti-TNF agents in patients with IBD. Nature Reviews Gastroenterology and Hepatology. 2012;**9**(9):496-503

[58] Urbano PCM, Aguirre-Gamboa R, Ashikov A, van Heeswijk B, Krippner-Heidenreich A, Tijssen H, et al. TNF-alpha-induced protein 3 (TNFAIP3)/A20 acts as a master switch in TNF-alpha blockade-driven IL-17A expression. The Journal of Allergy and Clinical Immunology. 2018;**142**(2):517-529

[59] Chen X, Hamano R, Subleski JJ, Hurwitz AA, Howard OM, Oppenheim JJ. Expression of costimulatory TNFR2

**53**

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy*

immunotherapeutic effect of CpG ODN in a mouse model of colon cancer. Science Signaling. 2018;**11**(511). pii:

[67] Marriott JB, Clarke IA, Dredge K, Muller G, Stirling D, Dalgleish AG. Thalidomide and its analogues have distinct and opposing effects on TNFalpha and TNFR2 during co-stimulation of both CD4(+) and CD8(+) T cells. Clinical and Experimental Immunology.

[68] Govindaraj C, Madondo M, Kong YY, Tan P, Wei A, Plebanski M. Lenalidomide-based maintenance therapy reduces TNF receptor 2 on CD4 T cells and enhances immune effector function in acute myeloid leukemia patients. American Journal of Hematology. 2014;**89**(8):795-802

[69] van der Most RG, Currie AJ, Mahendran S, Prosser A, Darabi A, Robinson BW, et al. Tumor eradication after cyclophosphamide depends on concurrent depletion of regulatory T cells: A role for cycling TNFR2 expressing effector-suppressor T cells in limiting effective chemotherapy. Cancer Immunology, Immunotherapy: CII.

[70] Chang LY, Lin YC, Chiang JM, Mahalingam J, Su SH, Huang CT, et al. Blockade of TNF-alpha signaling benefits cancer therapy by suppressing effector regulatory T cell expansion. Oncoimmunology. 2015;**4**(10):e1040215

2009;**58**(8):1219-1228

11/511/eaan0790

2002;**130**(1):75-84

*DOI: http://dx.doi.org/10.5772/intechopen.85632*

[60] Polz J, Remke A, Weber S, Schmidt

[61] Govindaraj C, Scalzo-Inguanti K, Madondo M, Hallo J, Flanagan K, Quinn M, et al. Impaired Th1 immunity in ovarian cancer patients is mediated by TNFR2+ Tregs within the tumor microenvironment. Clinical Immunology. 2013;**149**(1):97-110

[62] Vanamee ES, Faustman DL. TNFR2: A novel target for cancer immunotherapy. Trends in Molecular Medicine. 2017;**23**(11):1037-1046

2015;**47**(9):1056-1060

2017;**10**(462). pii: eaaf8608

[63] Ungewickell A, Bhaduri A, Rios E, Reuter J, Lee CS, Mah A, et al. Genomic analysis of mycosis fungoides and Sezary syndrome identifies recurrent alterations in TNFR2. Nature Genetics.

[64] Torrey H, Butterworth J, Mera T, Okubo Y, Wang L, Baum D, et al. Targeting TNFR2 with antagonistic antibodies inhibits proliferation of ovarian cancer cells and tumorassociated Tregs. Science Signaling.

[65] Torrey H, Khodadoust M, Tran L, Baum D, Defusco A, Kim YH, et al. Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic antibodies in advanced Sezary syndrome. Leukemia. 2018 Oct 24. pii: 10.1038/s41375-018-0292-9

[66] Nie Y, He J, Shirota H, Trivett AL, Yang KDM, et al. Blockade of TNFR2 signaling enhances the

D, Weber-Steffens D, Pietryga-Krieger A, et al. Myeloid suppressor cells require membrane TNFR2 expression for suppressive activity. Immunity, Inflammation and Disease.

induces resistance of CD4+FoxP3 conventional T cells to suppression by CD4+FoxP3+ regulatory T cells. Journal of Immunology (Baltimore Md: 1950).

2010;**185**(1):174-182

2014;**2**(2):121-130

*TNFR2 and Regulatory T Cells: Potential Immune Checkpoint Target in Cancer Immunotherapy DOI: http://dx.doi.org/10.5772/intechopen.85632*

induces resistance of CD4+FoxP3 conventional T cells to suppression by CD4+FoxP3+ regulatory T cells. Journal of Immunology (Baltimore Md: 1950). 2010;**185**(1):174-182

*Cytokines*

2016;**22**(1):16-17

Reports. 2013;**3**:3153

2013;**19**(3):322-328

[45] Zaragoza B, Chen X, Oppenheim JJ, Baeyens A, Gregoire S, Chader D, et al. Suppressive activity of human regulatory T cells is maintained in the presence of TNF. Nature Medicine.

depends on TNF produced by T cells and TNFR2 expressed by regulatory T cells. Blood. 2016;**128**(12):1651-1659

[53] Miller PG, Bonn MB, McKarns SC. Transmembrane TNF-TNFR2 impairs Th17 differentiation by promoting Il2 expression. Journal of Immunology (Baltimore Md: 1950).

[54] Arvey A, van der Veeken J, Samstein RM, Feng Y, Stamatoyannopoulos JA, Rudensky AY. Inflammation-induced repression of chromatin bound by the transcription factor Foxp3 in regulatory T cells. Nature Immunology.

[55] Li D, Silverberg MS, Haritunians T, Dubinsky MC, Landers C, Stempak JM, et al. TNFRSF1B is associated with ANCA in IBD. Inflammatory Bowel Diseases. 2016;**22**(6):1346-1352

[57] Cleynen I, Vermeire S. Paradoxical inflammation induced by anti-TNF agents in patients with IBD. Nature Reviews Gastroenterology and Hepatology. 2012;**9**(9):496-503

[58] Urbano PCM, Aguirre-Gamboa R, Ashikov A, van Heeswijk B, Krippner-Heidenreich A, Tijssen H, et al. TNF-alpha-induced protein 3 (TNFAIP3)/A20 acts as a master switch in TNF-alpha blockade-driven IL-17A expression. The Journal of Allergy and Clinical Immunology.

[59] Chen X, Hamano R, Subleski JJ, Hurwitz AA, Howard OM, Oppenheim JJ. Expression of costimulatory TNFR2

2018;**142**(2):517-529

[56] Ban L, Zhang J, Wang L, Kuhtreiber W, Burger D, Faustman DL. Selective death of autoreactive T cells in human diabetes by TNF or TNF receptor 2 agonism. Proceedings of the National Academy of Sciences of the United States of America. 2008;**105**(36):13644-13649

2015;**195**(6):2633-2647

2014;**15**(6):580-587

[46] Okubo Y, Mera T, Wang L,

Faustman DL. Homogeneous expansion of human T-regulatory cells via tumor necrosis factor receptor 2. Scientific

[47] Nie H, Zheng Y, Li R, Guo TB, He D, Fang L, et al. Phosphorylation of FOXP3 controls regulatory T cell function and is inhibited by TNF-alpha in rheumatoid arthritis. Nature Medicine.

[48] He X, Landman S, Bauland SC, van den Dolder J, Koenen HJ, Joosten I. A TNFR2-agonist facilitates high purity expansion of human low purity Treg cells. PLoS One. 2016;**11**(5):e0156311

downmodulates the function of human CD4+CD25hi T-regulatory cells. Blood.

[51] Urbano PCM, Koenen H, Joosten I, He X. An autocrine TNFalpha-tumor necrosis factor receptor 2 loop promotes epigenetic effects inducing human Treg stability in vitro. Frontiers in

[52] Leclerc M, Naserian S, Pilon C, Thiolat A, Martin GH, Pouchy C, et al. Control of GVHD by regulatory T cells

[49] Valencia X, Stephens G, Goldbach-Mansky R, Wilson M, Shevach EM, Lipsky PE. TNF

[50] McGovern JL, Nguyen DX, Notley CA, Mauri C, Isenberg DA, Ehrenstein MR. Th17 cells are restrained

by Treg cells via the inhibition of interleukin-6 in patients with rheumatoid arthritis responding to anti-tumor necrosis factor antibody therapy. Arthritis and Rheumatism.

2012;**64**(10):3129-3138

Immunology. 2018;**9**:573

2006;**108**(1):253-261

**52**

[60] Polz J, Remke A, Weber S, Schmidt D, Weber-Steffens D, Pietryga-Krieger A, et al. Myeloid suppressor cells require membrane TNFR2 expression for suppressive activity. Immunity, Inflammation and Disease. 2014;**2**(2):121-130

[61] Govindaraj C, Scalzo-Inguanti K, Madondo M, Hallo J, Flanagan K, Quinn M, et al. Impaired Th1 immunity in ovarian cancer patients is mediated by TNFR2+ Tregs within the tumor microenvironment. Clinical Immunology. 2013;**149**(1):97-110

[62] Vanamee ES, Faustman DL. TNFR2: A novel target for cancer immunotherapy. Trends in Molecular Medicine. 2017;**23**(11):1037-1046

[63] Ungewickell A, Bhaduri A, Rios E, Reuter J, Lee CS, Mah A, et al. Genomic analysis of mycosis fungoides and Sezary syndrome identifies recurrent alterations in TNFR2. Nature Genetics. 2015;**47**(9):1056-1060

[64] Torrey H, Butterworth J, Mera T, Okubo Y, Wang L, Baum D, et al. Targeting TNFR2 with antagonistic antibodies inhibits proliferation of ovarian cancer cells and tumorassociated Tregs. Science Signaling. 2017;**10**(462). pii: eaaf8608

[65] Torrey H, Khodadoust M, Tran L, Baum D, Defusco A, Kim YH, et al. Targeted killing of TNFR2-expressing tumor cells and Tregs by TNFR2 antagonistic antibodies in advanced Sezary syndrome. Leukemia. 2018 Oct 24. pii: 10.1038/s41375-018-0292-9

[66] Nie Y, He J, Shirota H, Trivett AL, Yang KDM, et al. Blockade of TNFR2 signaling enhances the

immunotherapeutic effect of CpG ODN in a mouse model of colon cancer. Science Signaling. 2018;**11**(511). pii: 11/511/eaan0790

[67] Marriott JB, Clarke IA, Dredge K, Muller G, Stirling D, Dalgleish AG. Thalidomide and its analogues have distinct and opposing effects on TNFalpha and TNFR2 during co-stimulation of both CD4(+) and CD8(+) T cells. Clinical and Experimental Immunology. 2002;**130**(1):75-84

[68] Govindaraj C, Madondo M, Kong YY, Tan P, Wei A, Plebanski M. Lenalidomide-based maintenance therapy reduces TNF receptor 2 on CD4 T cells and enhances immune effector function in acute myeloid leukemia patients. American Journal of Hematology. 2014;**89**(8):795-802

[69] van der Most RG, Currie AJ, Mahendran S, Prosser A, Darabi A, Robinson BW, et al. Tumor eradication after cyclophosphamide depends on concurrent depletion of regulatory T cells: A role for cycling TNFR2 expressing effector-suppressor T cells in limiting effective chemotherapy. Cancer Immunology, Immunotherapy: CII. 2009;**58**(8):1219-1228

[70] Chang LY, Lin YC, Chiang JM, Mahalingam J, Su SH, Huang CT, et al. Blockade of TNF-alpha signaling benefits cancer therapy by suppressing effector regulatory T cell expansion. Oncoimmunology. 2015;**4**(10):e1040215

**55**

**Chapter 5**

Memory

**Abstract**

ders (ASD).

**1. Introduction**

immune metabolic processes

*Harumi Jyonouchi*

Innate Immunity and

Neuroinflammation in

Neuropsychiatric Conditions

Disorders: Role of Innate Immune

The neuroimmune network represents a dense network of multiple signals mediated by neurotransmitters, hormones, growth factors, and cytokines produced by multiple lineage cells and is crucial for maintaining neuroimmune homeostasis. Endogenous and exogenous stimuli, which are dangerous to the body, are detected by sensor cells, and they rapidly inform the brain through this network. Innate immunity is thought to play a major role in the neuroimmune network, through cytokines and other mediators released from secretary innate immune cells. Recent research has revealed that innate immunity has its own memory. This is accomplished by metabolic and epigenetic changes. Such changes may result in augmenting immune protection with a risk of excessive inflammatory responses to subsequent stimuli (trained immunity). Alternatively, innate immune memory can induce suppressive effects (tolerance), which may impose a risk of impaired immune defense. Innate immune memory affects the neuroimmune network for a prolonged period, and dysregulated innate immune memory has been implicated with pathogenesis of neuropsychiatric conditions. This chapter summarizes a role of innate immune memory (trained immunity vs. tolerance) in neuroinflammation in association with neuropsychiatric conditions including autism spectrum disor-

**Keywords:** innate immunity, cytokines, neuroinflammation, neuroimmune network,

It is well accepted that inflammation in the peripheral organs can influence homeostasis and immune responses in the central nervous system (CNS) [1]. In common neuropsychiatric conditions such as schizophrenia and depression, evidence indicates that neuroinflammation plays a role in the disease pathogenesis [2]. Long-lasting effects of neuroinflammation in such neuropsychiatric conditions are

Including Autism Spectrum

## **Chapter 5**

Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism Spectrum Disorders: Role of Innate Immune Memory

*Harumi Jyonouchi*

## **Abstract**

The neuroimmune network represents a dense network of multiple signals mediated by neurotransmitters, hormones, growth factors, and cytokines produced by multiple lineage cells and is crucial for maintaining neuroimmune homeostasis. Endogenous and exogenous stimuli, which are dangerous to the body, are detected by sensor cells, and they rapidly inform the brain through this network. Innate immunity is thought to play a major role in the neuroimmune network, through cytokines and other mediators released from secretary innate immune cells. Recent research has revealed that innate immunity has its own memory. This is accomplished by metabolic and epigenetic changes. Such changes may result in augmenting immune protection with a risk of excessive inflammatory responses to subsequent stimuli (trained immunity). Alternatively, innate immune memory can induce suppressive effects (tolerance), which may impose a risk of impaired immune defense. Innate immune memory affects the neuroimmune network for a prolonged period, and dysregulated innate immune memory has been implicated with pathogenesis of neuropsychiatric conditions. This chapter summarizes a role of innate immune memory (trained immunity vs. tolerance) in neuroinflammation in association with neuropsychiatric conditions including autism spectrum disorders (ASD).

**Keywords:** innate immunity, cytokines, neuroinflammation, neuroimmune network, immune metabolic processes

## **1. Introduction**

It is well accepted that inflammation in the peripheral organs can influence homeostasis and immune responses in the central nervous system (CNS) [1]. In common neuropsychiatric conditions such as schizophrenia and depression, evidence indicates that neuroinflammation plays a role in the disease pathogenesis [2]. Long-lasting effects of neuroinflammation in such neuropsychiatric conditions are

implicated with altered innate immune responses in the absence of specific pathogens [2]. However, until recently, it is not well understood how innate immunity, which was thought to have no lasting memory unlike adaptive immunity, can exert prolonged actions on the CNS. The recent discovery of innate immune memory (trained immunity vs. tolerance) shed a light in a long postulated role of innate immunity in neuropsychiatric diseases [3, 4].

Since the existence of the immune system was recognized more than 50 years ago, the immune system has been thought to be comprised of two components, innate immunity and adaptive immunity. Innate immunity is the arm that mounts nonspecific, acute immune responses, by sensing microbial by-products called pathogen-associated molecular patterns (PAMPs) or by-products derived from tissue injuries called damage-associated molecular patterns (DAMPs) [5]. Signaling through PAMPs and DAMPs are thought to play a major role in plant immunity [6]. In animals, adaptive immunity is the arm that develops antigen (Ag)-specific responses. The development of Ag-specific responses requires lengthy processes including antigen (Ag) processing by Ag-presenting cells (APCs), Ag presentation to T and B cells, and TCR or immunoglobulin gene arrangements of T and B lymphocytes, respectively, which lead to the development of Ag-specific T and B cells and finally antibodies (Abs) [7]. Adaptive immunity effectively eliminates hazards from the body through Ag-specific cellular and humoral immune responses [7]. Adaptive immunity results in the development of long-lasting Ag-specific memory T/B cells [8]. In this way, the body retains immune memory against specific pathogens for a prolonged time. It is well known that individuals who have survived measles will retain measles-specific immune defense for life.

In contrast, immunology textbooks have long taught us that innate immunity does not have any lasting effects or memory, and it is mainly effective in containing infection until adaptive immunity takes over. Innate immunity has also been known to shape adaptive immunity through multiple mechanisms such as affecting actions of APCs, thereby indirectly modifying adaptive immune responses [7]. However, recent exciting research revealed that innate immunity can have its own memory, following an immune stimulus, and this depends on time, amount, and the kinds of stimuli through metabolic and epigenetic changes [3, 9]. More importantly, the stimuli that evoke innate immune memory are not restricted to microbes; nonpathogenic challenges such as stress and obesity are also found to cause innate immune memory [3, 10].

As described previously, despite the accumulating evidence, it was difficult to understand how innate immunity exerts lasting effects, in the absence of specific pathogens or other persistent environmental stimuli, in neuropsychiatric conditions. The recognition of innate immune memory (trained immunity vs. tolerance) has provided us new insights with regard to the role of innate immunity in physiological as well as pathogenic consequences in the brain. In this chapter, research efforts shaping a concept of innate immune memory (trained immunity vs. innate immune tolerance) will be discussed first. In the latter part of the chapter, a potential role of innate immune memory in neuropsychiatric conditions, especially in ASD, will be discussed.

#### **2. Innate immune memory**

#### **2.1 Trained immunity**

The presence of innate immune memory was first suspected because of unexpected, nonspecific effects of vaccinations. This is best known for a Bacillus

**57**

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism…*

Calmette-Guérin (BCG) vaccine. Epidemiological studies and subsequent randomized trials showed that the BCG vaccination not only provided protection for tuberculosis but also protection against other pathogens, especially those causing respiratory infection, which resulted in a reduction in infant mortality greater than expected for reducing tuberculosis-associated mortality [11, 12]. Likewise, the measles vaccination resulted in a striking reduction in children's mortality, which was again not to be explained by the reduction in mortality caused by measles [11]. These epidemiological observations were further explored by researchers in the Netherlands. They first demonstrated that innate immune memory does exist in animal models [13]. Namely, these researchers showed that BCG provided enhanced protection against *Candida albicans* through nonspecific adaptation of innate immunity, independent of lymphocytes [13]. They proposed to name this process of innate immune memory "trained immunity." The following studies by the same group also revealed that such adaptive changes in innate immunity are present not only in monocyte-macrophage lineage cells but also in other innate immune cells such as natural killer (NK) cells [14] and progenitor cells of innate immune cells in the bone marrow [15, 16]. Further studies revealed the presence of trained immunity in humans [17–19]. It became clear that trained immunity is similar to plant immunity which does not develop Ag-specific immunity, but develops prolonged immune defense by metabolic and epigenetic modulation [20]. Mounting evidence has now repeatedly shown that trained immunity is Ag nonspecific; the second stimulus (DAMP or PAMP) causing innate immune activation can be different from

Adaptive changes observed in "in vitro" models of trained immunity with β-glucan, a representative PAMP from *Candida albicans*, have been extensively studied. It was revealed that ß-glucan treatment induces activation of the dectin-1/ Akt/PTEN/mTOR/HIF-1α signaling pathway in innate immune cells [21]. That is, β-glucan activates dectin-1 which recruits Akt, leading to activation of mammalian target of rapamycin (mTOR) with suppression of PTEN expression and phosphorylation of the tuberous sclerosis complex (TSC) [22]. Activation of this pathway switches cellular metabolism from oxidative phosphorylation (ATP synthesis) to glycolysis, thereby reducing basal cellular respiration and increasing in glucose consumption, resulting in higher production of lactate [21]. Such metabolic changes lead to the exportation of citrate to the cytoplasm for cholesterol synthesis and

This metabolic shift described above results in the replenishment of the Krebs cycle by metabolization of glutamine into glutamate and α-keto-glutamate, leading to an accumulation of fumarate [23, 24]. Higher concentration of fumarate inhibits the KDM5 family of H3K4 demethylase that eventually leads to epigenetic reprogramming [23]. It has been reported that in the initial phase of trained immunity, lysine 27 of histone 3 (H3K27) is acetylated and lysine 4 of histone 3 (H3K4) is methylated rapidly [25]. Although H3K27Ac gradually returns to the baseline over time, H3K4me3 was found to remain elevated in the trained immunity [25]. Such epigenetic histone modification (accumulation of H3K4me3) is known to lead to the remodeling of the local chromatin into an open and accessible state, resulting in the facilitation of the loading of transcriptional machinery. The remaining accumulation of H3K4me3 on chromatin has been implicated in the establishment of the epigenetic memory in the trained immunity [25, 26]. It was hypothesized that H3K4me3 increases the local hydrophobicity of the chromatin, allowing for liquidliquid phase separated transcription factors to engage with the DNA in the aqueous

*DOI: http://dx.doi.org/10.5772/intechopen.87167*

the first stimulus [3].

**2.2 Mechanisms of trained immunity**

phospholipid synthesis [23, 24].

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism… DOI: http://dx.doi.org/10.5772/intechopen.87167*

Calmette-Guérin (BCG) vaccine. Epidemiological studies and subsequent randomized trials showed that the BCG vaccination not only provided protection for tuberculosis but also protection against other pathogens, especially those causing respiratory infection, which resulted in a reduction in infant mortality greater than expected for reducing tuberculosis-associated mortality [11, 12]. Likewise, the measles vaccination resulted in a striking reduction in children's mortality, which was again not to be explained by the reduction in mortality caused by measles [11]. These epidemiological observations were further explored by researchers in the Netherlands. They first demonstrated that innate immune memory does exist in animal models [13]. Namely, these researchers showed that BCG provided enhanced protection against *Candida albicans* through nonspecific adaptation of innate immunity, independent of lymphocytes [13]. They proposed to name this process of innate immune memory "trained immunity." The following studies by the same group also revealed that such adaptive changes in innate immunity are present not only in monocyte-macrophage lineage cells but also in other innate immune cells such as natural killer (NK) cells [14] and progenitor cells of innate immune cells in the bone marrow [15, 16]. Further studies revealed the presence of trained immunity in humans [17–19]. It became clear that trained immunity is similar to plant immunity which does not develop Ag-specific immunity, but develops prolonged immune defense by metabolic and epigenetic modulation [20]. Mounting evidence has now repeatedly shown that trained immunity is Ag nonspecific; the second stimulus (DAMP or PAMP) causing innate immune activation can be different from the first stimulus [3].

#### **2.2 Mechanisms of trained immunity**

Adaptive changes observed in "in vitro" models of trained immunity with β-glucan, a representative PAMP from *Candida albicans*, have been extensively studied. It was revealed that ß-glucan treatment induces activation of the dectin-1/ Akt/PTEN/mTOR/HIF-1α signaling pathway in innate immune cells [21]. That is, β-glucan activates dectin-1 which recruits Akt, leading to activation of mammalian target of rapamycin (mTOR) with suppression of PTEN expression and phosphorylation of the tuberous sclerosis complex (TSC) [22]. Activation of this pathway switches cellular metabolism from oxidative phosphorylation (ATP synthesis) to glycolysis, thereby reducing basal cellular respiration and increasing in glucose consumption, resulting in higher production of lactate [21]. Such metabolic changes lead to the exportation of citrate to the cytoplasm for cholesterol synthesis and phospholipid synthesis [23, 24].

This metabolic shift described above results in the replenishment of the Krebs cycle by metabolization of glutamine into glutamate and α-keto-glutamate, leading to an accumulation of fumarate [23, 24]. Higher concentration of fumarate inhibits the KDM5 family of H3K4 demethylase that eventually leads to epigenetic reprogramming [23]. It has been reported that in the initial phase of trained immunity, lysine 27 of histone 3 (H3K27) is acetylated and lysine 4 of histone 3 (H3K4) is methylated rapidly [25]. Although H3K27Ac gradually returns to the baseline over time, H3K4me3 was found to remain elevated in the trained immunity [25]. Such epigenetic histone modification (accumulation of H3K4me3) is known to lead to the remodeling of the local chromatin into an open and accessible state, resulting in the facilitation of the loading of transcriptional machinery. The remaining accumulation of H3K4me3 on chromatin has been implicated in the establishment of the epigenetic memory in the trained immunity [25, 26]. It was hypothesized that H3K4me3 increases the local hydrophobicity of the chromatin, allowing for liquidliquid phase separated transcription factors to engage with the DNA in the aqueous

*Cytokines*

implicated with altered innate immune responses in the absence of specific pathogens [2]. However, until recently, it is not well understood how innate immunity, which was thought to have no lasting memory unlike adaptive immunity, can exert prolonged actions on the CNS. The recent discovery of innate immune memory (trained immunity vs. tolerance) shed a light in a long postulated role of innate

Since the existence of the immune system was recognized more than 50 years ago, the immune system has been thought to be comprised of two components, innate immunity and adaptive immunity. Innate immunity is the arm that mounts nonspecific, acute immune responses, by sensing microbial by-products called pathogen-associated molecular patterns (PAMPs) or by-products derived from tissue injuries called damage-associated molecular patterns (DAMPs) [5]. Signaling through PAMPs and DAMPs are thought to play a major role in plant immunity [6]. In animals, adaptive immunity is the arm that develops antigen (Ag)-specific responses. The development of Ag-specific responses requires lengthy processes including antigen (Ag) processing by Ag-presenting cells (APCs), Ag presentation to T and B cells, and TCR or immunoglobulin gene arrangements of T and B lymphocytes, respectively, which lead to the development of Ag-specific T and B cells and finally antibodies (Abs) [7]. Adaptive immunity effectively eliminates hazards from the body through Ag-specific cellular and humoral immune responses [7]. Adaptive immunity results in the development of long-lasting Ag-specific memory T/B cells [8]. In this way, the body retains immune memory against specific pathogens for a prolonged time. It is well known that individuals who have survived

In contrast, immunology textbooks have long taught us that innate immunity does not have any lasting effects or memory, and it is mainly effective in containing infection until adaptive immunity takes over. Innate immunity has also been known to shape adaptive immunity through multiple mechanisms such as affecting actions of APCs, thereby indirectly modifying adaptive immune responses [7]. However, recent exciting research revealed that innate immunity can have its own memory, following an immune stimulus, and this depends on time, amount, and the kinds of stimuli through metabolic and epigenetic changes [3, 9]. More importantly, the stimuli that evoke innate immune memory are not restricted to microbes; nonpathogenic challenges such as stress and obesity are also found to cause innate

As described previously, despite the accumulating evidence, it was difficult to understand how innate immunity exerts lasting effects, in the absence of specific pathogens or other persistent environmental stimuli, in neuropsychiatric conditions. The recognition of innate immune memory (trained immunity vs. tolerance) has provided us new insights with regard to the role of innate immunity in physiological as well as pathogenic consequences in the brain. In this chapter, research efforts shaping a concept of innate immune memory (trained immunity vs. innate immune tolerance) will be discussed first. In the latter part of the chapter, a potential role of innate immune memory in neuropsychiatric conditions, especially in

The presence of innate immune memory was first suspected because of unexpected, nonspecific effects of vaccinations. This is best known for a Bacillus

immunity in neuropsychiatric diseases [3, 4].

measles will retain measles-specific immune defense for life.

**56**

immune memory [3, 10].

ASD, will be discussed.

**2.1 Trained immunity**

**2. Innate immune memory**

environment of the nucleus, subsequently rendering loading of transcriptional machinery onto promoters [27–29]. This will allow cells to start rapid transcription of the genes necessary for immune responses, thereby causing a much stronger Ag nonspecific pro-inflammatory response.

Long noncoding RNAs (lncRNAs) can function as a molecular scaffold where multiple protein complexes can assemble, and they also guide these complexes to specific gene loci [30]. Recent research disclosed a new class of lncRNAs named immune gene-priming lncRNAs (IPLs), and IPLs were found to have a crucial role in the accumulation of H3K4me3 on chromatin [31]. A candidate IPL, termed upstream master lncRNA of the inflammatory chemokine locus (UMLILO), was found to be crucial for trained immunity; ablation of the UMLILO transcript abolished β-glucan-induced trained immunity in both human and murine monocytes [30].

As shown in epidemiological studies of vaccinations, trained immunity, caused by metabolic and epigenetic changes, will be beneficial in providing broader immune defense and promoting tissue repair [32]. On the other hand, maladapted trained immunity can be detrimental to human health. Chronic inflammatory conditions including neuropsychiatric conditions have been implicated with maladapted changes in trained immunity [2, 9]. It should also be noted that induction of trained immunity appears to be associated with doses of PAMP, perhaps DAMP in humans; depending on the dose and the kinds of PAMP/DAMP, tolerance can be induced, instead of trained immunity [2]. It has been shown that low to moderate doses of β-glucan, tri-DAP, and muramyl dipeptides are reported to induce trained immunity [33]. It also needs to be cautioned that the effects of trained immunity are likely associated with individual's genetic and epigenetic background. For example, nonspecific effects of infant BCG vaccination are reported to be heterogeneous, affected by multiple genetic and environmental factors including age, gender, interactions with other vaccines, and exposure to infectious pathogens at the time of BCG vaccination [34].

#### **2.3 Mediators of trained immunity**

It has been reported that pre-administration of pro-inflammatory innate cytokines [interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and IL-6] provided protection against a variety of microbes [35]. Among the cytokines administered, IL-1 showed superior effects over TNF-α or IL-6 [35]. In BCG-vaccinated individuals, increase in production of these innate cytokines by monocytes in response to other microbes, other than BCG, was also found; this effect was again the most dependent on IL-1β [32]. IL-1β has also been reported to be crucial in the induction of trained immunity in NK cells [36]. On the other hand, in individuals with chronic mucocutaneous candidiasis, STAT-1-mediated type II interferon (IFN) induction was found to be crucial for induction of trained immunity [37]. The role of type II IFN (IFN-γ) in animal models was also reported by Kaufmann et al. [16]. However, in humans, innate immunity-associated protection (trained immunity) has been mainly implicated with IL-1β and other IL-1 families [38].

As detailed in the previous section, a metabolic shift from oxidative phosphorylation to aerobic glycolysis through the Hypoxia inducible factor-1α (HIF-1α) pathway downstream to mTOR is crucial for the development of trained immunity, since inhibition of this pathway is abolished induction of trained immunity [21]. Namely, in HIF-1α knockout mice, trained immunity was not induced [21]. IL-1β is known to be a direct target of HIF-1α [39], having a HIF-1α binding site in the promoter region of IL-1β gene [40]. It is now thus proposed that HIF-1α-induced

**59**

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism…*

IL-1β also plays a role in epigenetic changes, through histone modifications [35].

Given the role of IL-1β in trained immunity, excessive, dysregulated production of IL-1β is likely to cause maladapted trained immunity and resultant pathogenic consequences. This may be observed in patients with autoinflammatory syndromes associated with gene mutation that lead to overproduction of IL-1β, including cryopyrin associated periodic syndrome (CAPS) [38, 42]. On the other hand, impaired induction of trained immunity can also cause detrimental effects. It was reported that patients with chronic mucocutaneous candidiasis exhibit impaired induction of STAT-1-dependent, trained immunity in response to β-glucan [37]. The above-described metabolic shift is not limited to glucose metabolism. Changes in glutamine and cholesterol metabolism have also shown to be crucial in trained immunity [24]. Consequently, it is thought that increased cholesterol content also plays a role in the development of trained immunity. Interestingly, increased levels of oxidized low-density lipoprotein (OxLDL) caused by dysregulated cholesterol metabolism are found to induce trained immunity in human monocytes [10]. Such a finding indicates a pathogenic role for maladapted trained immunity in atherosclerosis, since monocyte and macrophage cells are known to play a major role in plaque formation in vascular endothelium, a major histologic

As detailed in the previous section, trained immunity causes a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis, rendering macrophage and monocyte lineage cells to classically activated cells or M1 phenotype; these cells exhibit impaired OXPHOS and anabolic repurposing of the tricarboxylic acid (TCA) cycle [43, 44]. In contrast, alternatively activated or the M2 phenotype of macrophages and monocytes has balanced processes of OXPHOS and TCA cycle activation; enhanced glycolytic generation of pyruvate fuels the TCA cycle, paralleling the induction of OXPHOS [44]. Trained macrophages via ß-glucan exposure are shown to reveal M1 phenotype [21]. Generation of M1 vs. M2 phenotypes of macrophages indicates the importance of regulating innate immune responses for prevention of excessive, potentially harmful inflammatory responses. In addition to generation of M2 phenotype, hypo-responsiveness of innate immunity has been described as endotoxin tolerance and compensatory anti-inflammatory response syndrome (CARS) [45]. Such regulatory mechanisms also have lasting effects, as

Endotoxin tolerance in innate immunity was first shown in rodent models of sepsis. Namely, survival from sepsis is associated with diminished or absence of responses to LPS, an endotoxin [46]. Subsequently, it was shown that previous exposure to a sublethal dose of LPS led to resistance to a lethal dose of LPS in rodents [46]. Endotoxin tolerance is thought to be a result of innate immune memory with lasting immune hypo-responsiveness, even to non-LPS stimulants [47]. Phenotypic changes of tolerant innate immune cells are characterized with less production of inflammatory cytokines (TNF-α, IL-12, IL-6) and increase in production of counterregulatory cytokines (IL-10 and TGF-β) upon stimulation [48, 49]. CARS was recognized as a clinical syndrome which is thought to represent a phase of immune "exhaustion," following initial potent immune activation, known as systemic inflammatory response syndrome (SIRS) [50]. Peripheral blood monocytes and neutrophils from CARS patients are reported to reveal similar phenotype to endotoxin-tolerant cells observed in rodent models [45, 49]. Recent research revealed that

Alternatively, IL-1β has been shown to upregulate HIF-1α [41].

*DOI: http://dx.doi.org/10.5772/intechopen.87167*

change in atherosclerosis [10].

observed in trained immunity.

**2.4 Tolerance in innate immunity**

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism… DOI: http://dx.doi.org/10.5772/intechopen.87167*

IL-1β also plays a role in epigenetic changes, through histone modifications [35]. Alternatively, IL-1β has been shown to upregulate HIF-1α [41].

Given the role of IL-1β in trained immunity, excessive, dysregulated production of IL-1β is likely to cause maladapted trained immunity and resultant pathogenic consequences. This may be observed in patients with autoinflammatory syndromes associated with gene mutation that lead to overproduction of IL-1β, including cryopyrin associated periodic syndrome (CAPS) [38, 42]. On the other hand, impaired induction of trained immunity can also cause detrimental effects. It was reported that patients with chronic mucocutaneous candidiasis exhibit impaired induction of STAT-1-dependent, trained immunity in response to β-glucan [37].

The above-described metabolic shift is not limited to glucose metabolism. Changes in glutamine and cholesterol metabolism have also shown to be crucial in trained immunity [24]. Consequently, it is thought that increased cholesterol content also plays a role in the development of trained immunity. Interestingly, increased levels of oxidized low-density lipoprotein (OxLDL) caused by dysregulated cholesterol metabolism are found to induce trained immunity in human monocytes [10]. Such a finding indicates a pathogenic role for maladapted trained immunity in atherosclerosis, since monocyte and macrophage cells are known to play a major role in plaque formation in vascular endothelium, a major histologic change in atherosclerosis [10].

#### **2.4 Tolerance in innate immunity**

*Cytokines*

cytes [30].

nonspecific pro-inflammatory response.

environment of the nucleus, subsequently rendering loading of transcriptional machinery onto promoters [27–29]. This will allow cells to start rapid transcription of the genes necessary for immune responses, thereby causing a much stronger Ag

As shown in epidemiological studies of vaccinations, trained immunity, caused by metabolic and epigenetic changes, will be beneficial in providing broader immune defense and promoting tissue repair [32]. On the other hand, maladapted trained immunity can be detrimental to human health. Chronic inflammatory conditions including neuropsychiatric conditions have been implicated with maladapted changes in trained immunity [2, 9]. It should also be noted that induction of trained immunity appears to be associated with doses of PAMP, perhaps DAMP in humans; depending on the dose and the kinds of PAMP/DAMP, tolerance can be induced, instead of trained immunity [2]. It has been shown that low to moderate doses of β-glucan, tri-DAP, and muramyl dipeptides are reported to induce trained immunity [33]. It also needs to be cautioned that the effects of trained immunity are likely associated with individual's genetic and epigenetic background. For example, nonspecific effects of infant BCG vaccination are reported to be heterogeneous, affected by multiple genetic and environmental factors including age, gender, interactions with other vaccines, and exposure to

It has been reported that pre-administration of pro-inflammatory innate cytokines [interleukin-1 (IL-1), tumor necrosis factor-α (TNF-α), and IL-6] provided protection against a variety of microbes [35]. Among the cytokines administered, IL-1 showed superior effects over TNF-α or IL-6 [35]. In BCG-vaccinated individuals, increase in production of these innate cytokines by monocytes in response to other microbes, other than BCG, was also found; this effect was again the most dependent on IL-1β [32]. IL-1β has also been reported to be crucial in the induction of trained immunity in NK cells [36]. On the other hand, in individuals with chronic mucocutaneous candidiasis, STAT-1-mediated type II interferon (IFN) induction was found to be crucial for induction of trained immunity [37]. The role of type II IFN (IFN-γ) in animal models was also reported by Kaufmann et al. [16]. However, in humans, innate immunity-associated protection (trained immunity)

As detailed in the previous section, a metabolic shift from oxidative phosphory-

lation to aerobic glycolysis through the Hypoxia inducible factor-1α (HIF-1α) pathway downstream to mTOR is crucial for the development of trained immunity, since inhibition of this pathway is abolished induction of trained immunity [21]. Namely, in HIF-1α knockout mice, trained immunity was not induced [21]. IL-1β is known to be a direct target of HIF-1α [39], having a HIF-1α binding site in the promoter region of IL-1β gene [40]. It is now thus proposed that HIF-1α-induced

infectious pathogens at the time of BCG vaccination [34].

has been mainly implicated with IL-1β and other IL-1 families [38].

**2.3 Mediators of trained immunity**

Long noncoding RNAs (lncRNAs) can function as a molecular scaffold where multiple protein complexes can assemble, and they also guide these complexes to specific gene loci [30]. Recent research disclosed a new class of lncRNAs named immune gene-priming lncRNAs (IPLs), and IPLs were found to have a crucial role in the accumulation of H3K4me3 on chromatin [31]. A candidate IPL, termed upstream master lncRNA of the inflammatory chemokine locus (UMLILO), was found to be crucial for trained immunity; ablation of the UMLILO transcript abolished β-glucan-induced trained immunity in both human and murine mono-

**58**

As detailed in the previous section, trained immunity causes a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis, rendering macrophage and monocyte lineage cells to classically activated cells or M1 phenotype; these cells exhibit impaired OXPHOS and anabolic repurposing of the tricarboxylic acid (TCA) cycle [43, 44]. In contrast, alternatively activated or the M2 phenotype of macrophages and monocytes has balanced processes of OXPHOS and TCA cycle activation; enhanced glycolytic generation of pyruvate fuels the TCA cycle, paralleling the induction of OXPHOS [44]. Trained macrophages via ß-glucan exposure are shown to reveal M1 phenotype [21]. Generation of M1 vs. M2 phenotypes of macrophages indicates the importance of regulating innate immune responses for prevention of excessive, potentially harmful inflammatory responses. In addition to generation of M2 phenotype, hypo-responsiveness of innate immunity has been described as endotoxin tolerance and compensatory anti-inflammatory response syndrome (CARS) [45]. Such regulatory mechanisms also have lasting effects, as observed in trained immunity.

Endotoxin tolerance in innate immunity was first shown in rodent models of sepsis. Namely, survival from sepsis is associated with diminished or absence of responses to LPS, an endotoxin [46]. Subsequently, it was shown that previous exposure to a sublethal dose of LPS led to resistance to a lethal dose of LPS in rodents [46]. Endotoxin tolerance is thought to be a result of innate immune memory with lasting immune hypo-responsiveness, even to non-LPS stimulants [47]. Phenotypic changes of tolerant innate immune cells are characterized with less production of inflammatory cytokines (TNF-α, IL-12, IL-6) and increase in production of counterregulatory cytokines (IL-10 and TGF-β) upon stimulation [48, 49]. CARS was recognized as a clinical syndrome which is thought to represent a phase of immune "exhaustion," following initial potent immune activation, known as systemic inflammatory response syndrome (SIRS) [50]. Peripheral blood monocytes and neutrophils from CARS patients are reported to reveal similar phenotype to endotoxin-tolerant cells observed in rodent models [45, 49]. Recent research revealed that

persistent effects of endotoxin tolerance and CARS are mediated by lncRNAs as well as microRNAs (miRNAs).

LPS activates TLR4 which leads to the activation of the myeloid differentiation factor 88 (MyD88)-mediated pathway and the TIR-domain-containing adaptorinducing interferon-β (TRIF) pathway [45]. The molecular signature of endotoxin tolerance involves downregulation of TLR4, decreased recruitment of MyD88 or TRIF to TLR4, decreased activation of IL-1 receptor-associated kinase (IRAK)1 and IRAK4, diminished nuclear factor κ chain of B-cell (NF-κB) signaling, as well as upregulation of negative regulatory molecules including SH2 domain-containing inositol phosphatase 1 (SHIP1) [51].

#### **2.5 Regulators of innate immune tolerance**

Recent research revealed a role of miRNAs in the regulation of endotoxin tolerance. Specifically, miR-155 and miR-146α have been shown to regulate endotoxin tolerance [52]. MiR-146α reduces TLR signaling, by targeting IRAK1 and TRAF6, key components of TLR signaling pathway [53]. In contrast, miR-155 is reported to inhibit expression of SHIP1 and SOCS1, negative regulators of TLR signaling, prohibiting or attenuating tolerance induction by endotoxin [54, 55]. Several other miRNAs are also implicated with regulation of endotoxin intolerance [45]. It was shown recently that miR-221/miR-222 regulates functional reprogramming of macrophages during LPS-induced tolerization [47]. miR-221/miR-222 targets brahma-regulated gene 1 (Brg1), rendering transcriptional silencing of a subset of inflammatory genes that depend on SWI/SNF and STAT-mediated chromatin remodeling [47].

Recent research also revealed a role of lncRNAs in endotoxin tolerance; lncRNAs exert transcriptional, posttranscriptional, and translational regulation of gene expression [56–58]. Multiple lncRNAs are reported to regulate target molecules of TLR4 signaling pathways. LPS-responsive lncRNAs Mirt2, THRIl, MALTAT1, NKILA, lincRNA-21, and SeT have been reported to suppress expression of proinflammatory mediators including TNF-α [45]. For example, Mirt2 is reported to inhibit TRAF6 ubiquitination, leading to a decrease in TNF-α production [59]. However, at this time, relationships between actions of miRNAs and lncRNA in innate immune tolerance are not well understood. Other soluble mediators such as cytokines (IL-1β, IL-10, TGF-β, and TNF-α) are also reported to induce crosstolerance or cytokine-mediated tolerance, causing a signaling cascade similar to that observed in TLR signaling [60]. In contrast, interferons (IFN-γ, α2-IFN, etc.) are known to abrogate endotoxin tolerance [61, 62]. Again these soluble mediators exert their actions on endotoxin tolerance via modulation of intracellular lncRNAs [45].

This type of innate immune memory (tolerance) is thought to be important in maintaining brain homeostasis, and impaired tolerance of innate immunity has been suspected in chronic neurodegenerative conditions such as Alzheimer's disease [9]. Aging is associated with an increased load of gram-negative bacteria in the GI tract and mouth mucosa, resulting in an increase in endotoxin levels in the blood and the brain [62]. However, aging individuals tolerate higher LPS levels in the brain through developing endotoxin tolerance [63].

#### **3. Role of innate immunity in the nervous system**

It is known that innate immunity does exist in the brain, playing a crucial role in brain morphogenesis and homeostasis. The major innate immune cells in the central nervous system (CNS) are microglial cells which are endogenously generated in the

**61**

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism…*

brain, but they can also be developed from bone marrow-derived monocytes, which are called BM-derived microglial cells (BMDM) [64, 65]. BMDM-induced inflammation has been implicated in neuropsychiatric conditions [64, 65]. It has also been reported that peripherally derived macrophages modulate microglial function after CNS injury; in this case, they are reported to exert anti-inflammatory effects [66]. Other innate immune cells in the CNS such as astrocytes are also known to exert

Inflammation in the periphery can prompt immune responses in the brain [1, 4]. Given the effects of trained immunity (activation vs. tolerance) in rodent models and humans, the development of maladapted innate immune memory in the CNS is expected to result in undesired, hazardous effects to the brain. However, reports concerning the effects of trained immunity and/or innate immune tolerance in the brain have been limited. Nevertheless, it was shown that microglial cells isolated from adult rats that were exposed to *E. coli* during the newborn period had increased expression of IL-1β mRNA [68]. The rats exposed to *E. coli* as newborns were also found to have impaired memory when they were challenged with a low dose of LPS, which was blocked by minocycline [2]. In experiments employing microglial cells obtained from sheep fetuses whose mother was given LPS intravenously, these fetal microglial cells were shown to have metabolic and epigenetic

Independent of the studies concerning trained immunity in the brain, persistent

effects of maternal immune activation (MIA) on fetuses have been extensively studied, as one of the best studied rodent models of ASD [70]. In this model, sterile inflammation in pregnant rodents was induced with the use of PAMPs such as LPS, poly I:C, resulting in impaired neuropsychiatric symptoms in offspring in their adult years [70]. That is, offspring of MIA mothers have been shown to suffer from persistent behavioral symptoms and cognitive deficits frequently seen in ASD subjects later in life [70]. In addition, MIA also causes persistent alteration of adaptive immunity [71]. However, in this model, it is not yet well understood how innate immune memory (most likely trained immunity in this model) plays a role in a MIA model, causing persistent behavioral changes and impaired cognitive development. Children exposed to stressful events during the fetal and newborn period have also been reported to have higher levels of pro-inflammatory cytokines and neurodevelopmental impairment than control children [2]. Given the research findings in molecular mechanisms of trained immunity described in the previous section, there is a possibility that maladapted trained immunity contributes to the

Tolerized innate immunity in the brain is thought to be crucial for limiting excessive inflammatory responses during brain tissue repair that involves phagocytosis of apoptotic cells and damaged tissue debris by tolerant phagocytes [72]. In rodent models, disruption of this pathway leads to neuroinflammation and subsequent neuronal damage [73]. An important regulator of this pathway is the triggering receptor expressed on myeloid cells 2 (TREM-2), which is expressed on microglial cells [74]. Blockade of TREM-2 was shown to exacerbate experimental autoimmune encephalitis (EAE), a rodent model of multiple sclerosis (MS) [75]. Apolipoprotein E (ApoE) which is a TREM-2 ligand was shown to have a role in maintaining tolerized phenotype of phagocytic cells [74]. This interaction was

modulation, as has been reported in trained immunity [69].

onset and progress of some neuropsychiatric disorders.

**3.2 Innate tolerance in the brain**

*DOI: http://dx.doi.org/10.5772/intechopen.87167*

important physiological roles [9, 67].

**3.1 Trained immunity in the CNS**

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism… DOI: http://dx.doi.org/10.5772/intechopen.87167*

brain, but they can also be developed from bone marrow-derived monocytes, which are called BM-derived microglial cells (BMDM) [64, 65]. BMDM-induced inflammation has been implicated in neuropsychiatric conditions [64, 65]. It has also been reported that peripherally derived macrophages modulate microglial function after CNS injury; in this case, they are reported to exert anti-inflammatory effects [66]. Other innate immune cells in the CNS such as astrocytes are also known to exert important physiological roles [9, 67].

#### **3.1 Trained immunity in the CNS**

*Cytokines*

as microRNAs (miRNAs).

remodeling [47].

inositol phosphatase 1 (SHIP1) [51].

**2.5 Regulators of innate immune tolerance**

brain through developing endotoxin tolerance [63].

**3. Role of innate immunity in the nervous system**

persistent effects of endotoxin tolerance and CARS are mediated by lncRNAs as well

LPS activates TLR4 which leads to the activation of the myeloid differentiation factor 88 (MyD88)-mediated pathway and the TIR-domain-containing adaptorinducing interferon-β (TRIF) pathway [45]. The molecular signature of endotoxin tolerance involves downregulation of TLR4, decreased recruitment of MyD88 or TRIF to TLR4, decreased activation of IL-1 receptor-associated kinase (IRAK)1 and IRAK4, diminished nuclear factor κ chain of B-cell (NF-κB) signaling, as well as upregulation of negative regulatory molecules including SH2 domain-containing

Recent research revealed a role of miRNAs in the regulation of endotoxin tolerance. Specifically, miR-155 and miR-146α have been shown to regulate endotoxin tolerance [52]. MiR-146α reduces TLR signaling, by targeting IRAK1 and TRAF6, key components of TLR signaling pathway [53]. In contrast, miR-155 is reported to inhibit expression of SHIP1 and SOCS1, negative regulators of TLR signaling, prohibiting or attenuating tolerance induction by endotoxin [54, 55]. Several other miRNAs are also implicated with regulation of endotoxin intolerance [45]. It was shown recently that miR-221/miR-222 regulates functional reprogramming of macrophages during LPS-induced tolerization [47]. miR-221/miR-222 targets brahma-regulated gene 1 (Brg1), rendering transcriptional silencing of a subset of inflammatory genes that depend on SWI/SNF and STAT-mediated chromatin

Recent research also revealed a role of lncRNAs in endotoxin tolerance; lncRNAs

It is known that innate immunity does exist in the brain, playing a crucial role in brain morphogenesis and homeostasis. The major innate immune cells in the central nervous system (CNS) are microglial cells which are endogenously generated in the

exert transcriptional, posttranscriptional, and translational regulation of gene expression [56–58]. Multiple lncRNAs are reported to regulate target molecules of TLR4 signaling pathways. LPS-responsive lncRNAs Mirt2, THRIl, MALTAT1, NKILA, lincRNA-21, and SeT have been reported to suppress expression of proinflammatory mediators including TNF-α [45]. For example, Mirt2 is reported to inhibit TRAF6 ubiquitination, leading to a decrease in TNF-α production [59]. However, at this time, relationships between actions of miRNAs and lncRNA in innate immune tolerance are not well understood. Other soluble mediators such as cytokines (IL-1β, IL-10, TGF-β, and TNF-α) are also reported to induce crosstolerance or cytokine-mediated tolerance, causing a signaling cascade similar to that observed in TLR signaling [60]. In contrast, interferons (IFN-γ, α2-IFN, etc.) are known to abrogate endotoxin tolerance [61, 62]. Again these soluble mediators exert their actions on endotoxin tolerance via modulation of intracellular lncRNAs [45]. This type of innate immune memory (tolerance) is thought to be important in maintaining brain homeostasis, and impaired tolerance of innate immunity has been suspected in chronic neurodegenerative conditions such as Alzheimer's disease [9]. Aging is associated with an increased load of gram-negative bacteria in the GI tract and mouth mucosa, resulting in an increase in endotoxin levels in the blood and the brain [62]. However, aging individuals tolerate higher LPS levels in the

**60**

Inflammation in the periphery can prompt immune responses in the brain [1, 4]. Given the effects of trained immunity (activation vs. tolerance) in rodent models and humans, the development of maladapted innate immune memory in the CNS is expected to result in undesired, hazardous effects to the brain. However, reports concerning the effects of trained immunity and/or innate immune tolerance in the brain have been limited. Nevertheless, it was shown that microglial cells isolated from adult rats that were exposed to *E. coli* during the newborn period had increased expression of IL-1β mRNA [68]. The rats exposed to *E. coli* as newborns were also found to have impaired memory when they were challenged with a low dose of LPS, which was blocked by minocycline [2]. In experiments employing microglial cells obtained from sheep fetuses whose mother was given LPS intravenously, these fetal microglial cells were shown to have metabolic and epigenetic modulation, as has been reported in trained immunity [69].

Independent of the studies concerning trained immunity in the brain, persistent effects of maternal immune activation (MIA) on fetuses have been extensively studied, as one of the best studied rodent models of ASD [70]. In this model, sterile inflammation in pregnant rodents was induced with the use of PAMPs such as LPS, poly I:C, resulting in impaired neuropsychiatric symptoms in offspring in their adult years [70]. That is, offspring of MIA mothers have been shown to suffer from persistent behavioral symptoms and cognitive deficits frequently seen in ASD subjects later in life [70]. In addition, MIA also causes persistent alteration of adaptive immunity [71]. However, in this model, it is not yet well understood how innate immune memory (most likely trained immunity in this model) plays a role in a MIA model, causing persistent behavioral changes and impaired cognitive development. Children exposed to stressful events during the fetal and newborn period have also been reported to have higher levels of pro-inflammatory cytokines and neurodevelopmental impairment than control children [2]. Given the research findings in molecular mechanisms of trained immunity described in the previous section, there is a possibility that maladapted trained immunity contributes to the onset and progress of some neuropsychiatric disorders.

#### **3.2 Innate tolerance in the brain**

Tolerized innate immunity in the brain is thought to be crucial for limiting excessive inflammatory responses during brain tissue repair that involves phagocytosis of apoptotic cells and damaged tissue debris by tolerant phagocytes [72]. In rodent models, disruption of this pathway leads to neuroinflammation and subsequent neuronal damage [73]. An important regulator of this pathway is the triggering receptor expressed on myeloid cells 2 (TREM-2), which is expressed on microglial cells [74]. Blockade of TREM-2 was shown to exacerbate experimental autoimmune encephalitis (EAE), a rodent model of multiple sclerosis (MS) [75]. Apolipoprotein E (ApoE) which is a TREM-2 ligand was shown to have a role in maintaining tolerized phenotype of phagocytic cells [74]. This interaction was

found to be impaired in patients with Alzheimer's disease [9]. In animal models of Alzheimer's disease treated with trained immunity vs. tolerance inducing stimuli, it was reported that long-term modulation of brain immune responses were observed, and the authors attributed this prolonged effects on innate immune memory to reprogramming of microglial cells [4].

#### **3.3 mTOR-related pathology in neuropsychiatric disorders**

In the previous section describing molecular pathways associated with trained immunity, the importance of mTOR signaling has been repeatedly shown. One thing we learned from the research on trained immunity is that multiple lineage cells reveal metabolic and epigenetic reprogramming in the process of innate immune memory, which, in animal models, can also be applied to microglial cells [4]. Interestingly, brain dysfunction caused by dysregulated mTOR signaling has been implicated in several neuropsychiatric disorders. In the next paragraph, we summarize mTOR-related brain dysfunctions and proposed mechanisms.

One of the expected consequences of excessive mTOR signaling caused by trained immunity is the impairment of lysosomal degradation of intracellular components, since mTOR activation inhibits autophagy via inhibition of the early steps of autophagosome biogenesis [76, 77]. Autophagy is a key physiological cellular function that clears intracellular molecules and thought to be developed to adjust the state of nutrient depletion [76, 77]. However, this is also an important mechanism to remove misfolded proteins that naturally occur in living cells [22]. In addition to degradation of misfolded proteins, autophagy also degrades altered subcellular organelles, such as the mitochondria [22]. Prolonged dysfunction in autophagy can lead to detrimental effects and is implicated in the pathogenesis of multiple neuropsychiatric conditions including dementia, movement disorders, seizures, brain ischemia, ASD, affective disorder, and schizophrenia [78–82]. In rodent models of depression, tuberous sclerosis, and ASD, rapamycin (sirolimus), a representative mTOR inhibitor, has been shown to attenuate social interactions and reverse behavioral effects on their neuropsychiatric symptoms [83–86]. Thus metabolic and epigenetic changes caused by trained immunity may have profound effects through altered levels of autophagy, as a result of metabolic and epigenetic reprograming, as detailed in the previous section.

#### **3.4 ASD and a possible role of trained immunity**

In this section, we discuss a possible role for trained immunity in the onset and progress of ASD. As a clinician, the author observed that an apparent strong immune stimulus altered the responses to subsequent immune stimuli in some, but not all ASD children and these ASD children also exhibit fluctuating neuropsychiatric symptoms, following microbial infection [87, 88]. As discussed in the previous section, in the MIA model of ASD, prolonged effects of MIA on the offspring brain can be explained through a concept of trained immunity occurring to the fetus at the time of sterile immune activation in the mother. This may have also happened in ASD subjects as described above. However, it should be noted that ASD is a behaviorally defined syndrome, diagnosed on the basis of behavioral symptoms, except for a minority of ASD cases that have well-defined gene mutations [89]. Therefore, based on the author's clinical experience, it is likely that trained immunity plays a role in a subset of ASD subjects for whom neuroinflammation is associated in their ASD pathogenesis.

In ASD patients, just like in other neuropsychiatric conditions, a role of inflammation has been long suspected, and more and more evidence has been accumulating [90–92]. In the research of innate immune abnormalities in ASD children, we have

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*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism…*

also found evidence of dysregulated innate immune responses, shifting to proinflammatory responses in a subset of ASD subjects [88, 93, 94]. We also experienced that these ASD subjects suffer from various comorbid medical conditions involving the gastrointestinal (GI) tract and other organs [87]. Retrospectively, our findings may be reflecting maladapted innate immunity as a form of trained immunity in such ASD subjects; these ASD subjects may fall into an ASD subset which we have called inflammatory autism, mimicking the rodent ASD model of MIA [93]. Our previous findings that may indicate altered innate immune memory

• In some but not all the ASD subjects, we found significant changes in innate immune abnormalities which are best reflected in changes in IL-1β/IL-10 ratios produced by purified peripheral blood monocytes (PBMo) [88, 93]. Namely, some patients reveal high ratios of IL-1β/IL-10, while others showed low ratios, and these rations can change from time to time, depending on their exposure to

• ASD subjects who revealed high and/or low IL-1β/IL-10 ratios also revealed fluctuating behavioral symptoms following immune insults [94]. Parents of these subjects often describe more severe, prolonged illnesses and frequent respiratory infection following microbial infection [87]. They also seem to reveal significant changes in their behavioral symptoms and cognitive activity with immune stimuli not associated with microbial infection; these ASD children may exhibit worsening neuropsychiatric symptoms, following flareups of aeroallergen allergy, delayed-type food allergy, and adverse reactions to

• ASD subjects who revealed high and/or low IL-1β/IL-10 ratios also revealed changes in production of inflammatory monocyte cytokines including TNF-α

• PBMo from ASD subjects who revealed altered IL-1β/IL-10 ratios also revealed changes in miRNA expression by PBMo, as compared to cells obtained from

• In recent studies, we also found changes in miRNA in sera of ASD subjects, when tested by high-throughput deep sequencing. Again, changes in serum miRNA levels are closely associated with changes in IL-1β/IL-10 ratios by PBMo, production of monocyte cytokines (TNF-β, IL-6, IL-10, CCL2 mostly), along with parameters of mitochondrial respiration (manuscript submitted for publication). Interestingly, in ASD subjects, miRNA levels are mostly decreased, as compared to non-ASD controls (submitted for publication). Targeted genes by miRNAs that are altered in serum levels in ASD subjects with high or low IL-1β/ IL-10 ratios are associated with pathways involved in innate immune responses,

• We also studied changes in mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) obtained from ASD subjects and non-ASD controls. Our results revealed evidence of altered mitochondrial respiration in association with changes in IL-1β/IL-10 ratios by PBMo in ASD subjects [95].

including the mTOR signaling pathway (unpublished observation).

The above-described findings may be best explained by altered innate immune responses associated with innate immune memory (trained immunity

*DOI: http://dx.doi.org/10.5772/intechopen.87167*

in such ASD patients are as follows:

immune insults [93].

and IL-6 [93, 95].

medications including vaccinations [87, 94].

neurotypical, non-ASD controls [93].

### *Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism… DOI: http://dx.doi.org/10.5772/intechopen.87167*

also found evidence of dysregulated innate immune responses, shifting to proinflammatory responses in a subset of ASD subjects [88, 93, 94]. We also experienced that these ASD subjects suffer from various comorbid medical conditions involving the gastrointestinal (GI) tract and other organs [87]. Retrospectively, our findings may be reflecting maladapted innate immunity as a form of trained immunity in such ASD subjects; these ASD subjects may fall into an ASD subset which we have called inflammatory autism, mimicking the rodent ASD model of MIA [93]. Our previous findings that may indicate altered innate immune memory in such ASD patients are as follows:


The above-described findings may be best explained by altered innate immune responses associated with innate immune memory (trained immunity

*Cytokines*

reprogramming of microglial cells [4].

**3.3 mTOR-related pathology in neuropsychiatric disorders**

reprograming, as detailed in the previous section.

**3.4 ASD and a possible role of trained immunity**

found to be impaired in patients with Alzheimer's disease [9]. In animal models of Alzheimer's disease treated with trained immunity vs. tolerance inducing stimuli, it was reported that long-term modulation of brain immune responses were observed, and the authors attributed this prolonged effects on innate immune memory to

In the previous section describing molecular pathways associated with trained immunity, the importance of mTOR signaling has been repeatedly shown. One thing we learned from the research on trained immunity is that multiple lineage cells reveal metabolic and epigenetic reprogramming in the process of innate immune memory, which, in animal models, can also be applied to microglial cells [4]. Interestingly, brain dysfunction caused by dysregulated mTOR signaling has been implicated in several neuropsychiatric disorders. In the next paragraph, we summarize mTOR-related brain dysfunctions and proposed mechanisms.

One of the expected consequences of excessive mTOR signaling caused by trained immunity is the impairment of lysosomal degradation of intracellular components, since mTOR activation inhibits autophagy via inhibition of the early steps of autophagosome biogenesis [76, 77]. Autophagy is a key physiological cellular function that clears intracellular molecules and thought to be developed to adjust the state of nutrient depletion [76, 77]. However, this is also an important mechanism to remove misfolded proteins that naturally occur in living cells [22]. In addition to degradation of misfolded proteins, autophagy also degrades altered subcellular organelles, such as the mitochondria [22]. Prolonged dysfunction in autophagy can lead to detrimental effects and is implicated in the pathogenesis of multiple neuropsychiatric conditions including dementia, movement disorders, seizures, brain ischemia, ASD, affective disorder, and schizophrenia [78–82]. In rodent models of depression, tuberous sclerosis, and ASD, rapamycin (sirolimus), a representative mTOR inhibitor, has been shown to attenuate social interactions and reverse behavioral effects on their neuropsychiatric symptoms [83–86]. Thus metabolic and epigenetic changes caused by trained immunity may have profound effects through altered levels of autophagy, as a result of metabolic and epigenetic

In this section, we discuss a possible role for trained immunity in the onset and progress of ASD. As a clinician, the author observed that an apparent strong immune stimulus altered the responses to subsequent immune stimuli in some, but not all ASD children and these ASD children also exhibit fluctuating neuropsychiatric symptoms, following microbial infection [87, 88]. As discussed in the previous section, in the MIA model of ASD, prolonged effects of MIA on the offspring brain can be explained through a concept of trained immunity occurring to the fetus at the time of sterile immune activation in the mother. This may have also happened in ASD subjects as described above. However, it should be noted that ASD is a behaviorally defined syndrome, diagnosed on the basis of behavioral symptoms, except for a minority of ASD cases that have well-defined gene mutations [89]. Therefore, based on the author's clinical experience, it is likely that trained immunity plays a role in a subset of ASD subjects for whom neuroinflammation is associated in their ASD pathogenesis.

In ASD patients, just like in other neuropsychiatric conditions, a role of inflammation has been long suspected, and more and more evidence has been accumulating [90–92]. In the research of innate immune abnormalities in ASD children, we have

**62**

vs. tolerance). So, if this is the case, for these ASD subjects, can clinical features that indicate an alternation of innate immune memory be detected? The author is a pediatric immunologist and, as indicated before, as stated previously, observes exacerbation of neuropsychiatric symptoms, following immune insults. Herein, a representative ASD case, in which trained immunity may be associated with the onset and progression of ASD, is presented.

#### **3.5 Case presentation**

A 10-year-old female child presented to the pediatric allergy/immunology clinic at our institution secondary to fluctuating behavioral symptoms. Fluctuation of behavioral symptoms often occurred, following microbial infection.

The patient was born at 41 weeks of gestation via cesarean section due to breech presentation, following an uneventful pregnancy. The patient was developing typically until 24 months of age and then suffered from significant developmental regression. Prior to the onset of the developmental regression, parents took the patient to South Asia to visit other family members and friends. During this visit, the patient suffered an insect bite which was complicated by a secondary bacterial skin infection. When treated with oral antibiotics abroad, the patient developed generalized hives and severe GI symptoms (nausea, vomiting, diarrhea, and bloating): the patient then became intolerant to multiple foods. After returning to the United States, the patient was given multiple vaccinations including live vaccines to catch up the vaccination schedule. All these vaccines were given while the patient was still suffering from GI symptoms and an active skin infection. Within several days after vaccinations (multiple vaccines given all together), noticeable loss of cognitive and motor skills became apparent in the patient. The patient was eventually diagnosed with ASD around 2.5 years of age.

Eventually, the patient's GI symptoms subsided, but this subject never regained the cognitive skills that this patient had once acquired prior to the onset of developmental regression. Prior to advancing to pre-kindergarten, the patient was given booster doses of vaccines which were well tolerated. However, after starting pre-kindergarten, the patient started getting sick frequently with upper respiratory infections, which often evolved into ear infection. The patient missed many days of school, since the patient suffered a prolonged course of illness and more severe symptoms, as compared to peers. While the patient presented with symptoms of upper respiratory infection, this patient's behavioral symptoms continue to fluctuate, most evident in worsening of obsessive compulsive behaviors and frequency of "rage" episodes. Worsening behavioral symptoms would always follow immune insults, worse in a convalescence stage. Avoidance of sick contacts by placing the patient in home schooling attenuated the fluctuating behavioral symptoms. At 7–8 years of age, the fluctuating behavioral symptoms seen were mainly associated with teething. After the completion of teething, behavioral symptoms became more stable. However, the patient stopped growing, falling under the first percentile of the growth curve in height and weight. An exhausting workup for primary mitochondrial diseases, endocrine diseases, primary immunodeficiency with known gene mutations, and congenital metabolic and genetic diseases was unrevealing. However, video electric encephalogram revealed a focal epileptic activity. Family history is negative for neuropsychiatric, genetic, autoimmune, immune, and metabolic diseases.

In the case presented above, did neuroinflammation caused by maladapted trained immunity have a role in her clinical features? It is hard to prove, but it may be speculated that the initial stressful events that occurred abroad shaped trained immunity in this patient, and the subsequent multiple unrelated immune stimuli

**65**

**4. Conclusions**

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism…*

may have caused prolonged maladapted trained immunity, leading to persistent neuroinflammation and impairment of cognitive activity, as observed in the MIA models of ASD. Interestingly, changes in GI conditions, such as changes in microbiome, have been implicated with neuropsychiatric diseases, triggering maladapted trained immunity [96]. It is also reported that trained innate immunity can be induced in human monocytes by cow's milk [97]. Thus her severe GI symptoms and subsequent intolerance to multiple foods may be associated with excessive trained

*IL-1β/IL-10 ratios produced by purified peripheral blood monocytes in response to medium only (no stimulus), LPS (TLR4 agonist), zymosan (TLR2/TLR6 agonist), CL097 (TLR7/TLR8 agonist), and β-glucan in the presented case (patient) and control cells from a non-ASD neurotypical subject. IL-1β/IL-10 ratios are shown* 

As summarized in the previous section, we have found that IL-1β/IL-10 ratios produced by PBMo are altered in some ASD subjects in association with fluctuating behavioral symptoms [94]. Thus if innate immune memory (trained immunity) is associated with her above-described remarkable clinical symptoms, we may also find altered IL-1β/IL-10 ratios, as an indicator of altered innate immune responses. Thus we assessed IL-1β/IL-10 ratios produced by PBMo in response to a panel of innate immune stimuli, including β-glucan, as reported previously [95]. As shown in **Figure 1**, the presented case revealed increase in IL-1β/IL-10 ratios in response to zymosan, CL097, and β-glucan. High IL-1β/IL-10 ratio in response to CL097, an agonist of TLR7/TLR8, was especially striking. We also observed increase in production of TNF-α and IL-6 and decrease in the production of IL-10, as well. Given these findings, it is possible that maladapted trained immunity may have caused excessive inflammatory responses to various innate immune stimuli, which then led to developmental regression and fluctuating behavioral symptoms in this presented case.

Our deepening knowledge of innate immune memory (trained immunity vs. tolerance) has shed light on the understanding of nonspecific effects of microbial infection and other immune stimuli, which have been implicated in the onset and

*DOI: http://dx.doi.org/10.5772/intechopen.87167*

immunity in the gut of this patient.

**Figure 1.**

*in a log scale.*

**3.6 Evidence of impaired trained immunity**

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism… DOI: http://dx.doi.org/10.5772/intechopen.87167*

#### **Figure 1.**

*Cytokines*

vs. tolerance). So, if this is the case, for these ASD subjects, can clinical features that indicate an alternation of innate immune memory be detected? The author is a pediatric immunologist and, as indicated before, as stated previously, observes exacerbation of neuropsychiatric symptoms, following immune insults. Herein, a representative ASD case, in which trained immunity may be associated with the

A 10-year-old female child presented to the pediatric allergy/immunology clinic at our institution secondary to fluctuating behavioral symptoms. Fluctuation of

The patient was born at 41 weeks of gestation via cesarean section due to breech

Eventually, the patient's GI symptoms subsided, but this subject never regained the cognitive skills that this patient had once acquired prior to the onset of developmental regression. Prior to advancing to pre-kindergarten, the patient was given booster doses of vaccines which were well tolerated. However, after starting pre-kindergarten, the patient started getting sick frequently with upper respiratory infections, which often evolved into ear infection. The patient missed many days of school, since the patient suffered a prolonged course of illness and more severe symptoms, as compared to peers. While the patient presented with symptoms of upper respiratory infection, this patient's behavioral symptoms continue to fluctuate, most evident in worsening of obsessive compulsive behaviors and frequency of "rage" episodes. Worsening behavioral symptoms would always follow immune insults, worse in a convalescence stage. Avoidance of sick contacts by placing the patient in home schooling attenuated the fluctuating behavioral symptoms. At 7–8 years of age, the fluctuating behavioral symptoms seen were mainly associated with teething. After the completion of teething, behavioral symptoms became more stable. However, the patient stopped growing, falling under the first percentile of the growth curve in height and weight. An exhausting workup for primary mitochondrial diseases, endocrine diseases, primary immunodeficiency with known gene mutations, and congenital metabolic and genetic diseases was unrevealing. However, video electric encephalogram revealed a focal epileptic activity. Family history is negative for neuropsychiatric, genetic, autoimmune, immune, and

In the case presented above, did neuroinflammation caused by maladapted trained immunity have a role in her clinical features? It is hard to prove, but it may be speculated that the initial stressful events that occurred abroad shaped trained immunity in this patient, and the subsequent multiple unrelated immune stimuli

presentation, following an uneventful pregnancy. The patient was developing typically until 24 months of age and then suffered from significant developmental regression. Prior to the onset of the developmental regression, parents took the patient to South Asia to visit other family members and friends. During this visit, the patient suffered an insect bite which was complicated by a secondary bacterial skin infection. When treated with oral antibiotics abroad, the patient developed generalized hives and severe GI symptoms (nausea, vomiting, diarrhea, and bloating): the patient then became intolerant to multiple foods. After returning to the United States, the patient was given multiple vaccinations including live vaccines to catch up the vaccination schedule. All these vaccines were given while the patient was still suffering from GI symptoms and an active skin infection. Within several days after vaccinations (multiple vaccines given all together), noticeable loss of cognitive and motor skills became apparent in the patient. The patient was eventu-

behavioral symptoms often occurred, following microbial infection.

onset and progression of ASD, is presented.

ally diagnosed with ASD around 2.5 years of age.

**3.5 Case presentation**

**64**

metabolic diseases.

*IL-1β/IL-10 ratios produced by purified peripheral blood monocytes in response to medium only (no stimulus), LPS (TLR4 agonist), zymosan (TLR2/TLR6 agonist), CL097 (TLR7/TLR8 agonist), and β-glucan in the presented case (patient) and control cells from a non-ASD neurotypical subject. IL-1β/IL-10 ratios are shown in a log scale.*

may have caused prolonged maladapted trained immunity, leading to persistent neuroinflammation and impairment of cognitive activity, as observed in the MIA models of ASD. Interestingly, changes in GI conditions, such as changes in microbiome, have been implicated with neuropsychiatric diseases, triggering maladapted trained immunity [96]. It is also reported that trained innate immunity can be induced in human monocytes by cow's milk [97]. Thus her severe GI symptoms and subsequent intolerance to multiple foods may be associated with excessive trained immunity in the gut of this patient.

#### **3.6 Evidence of impaired trained immunity**

As summarized in the previous section, we have found that IL-1β/IL-10 ratios produced by PBMo are altered in some ASD subjects in association with fluctuating behavioral symptoms [94]. Thus if innate immune memory (trained immunity) is associated with her above-described remarkable clinical symptoms, we may also find altered IL-1β/IL-10 ratios, as an indicator of altered innate immune responses.

Thus we assessed IL-1β/IL-10 ratios produced by PBMo in response to a panel of innate immune stimuli, including β-glucan, as reported previously [95]. As shown in **Figure 1**, the presented case revealed increase in IL-1β/IL-10 ratios in response to zymosan, CL097, and β-glucan. High IL-1β/IL-10 ratio in response to CL097, an agonist of TLR7/TLR8, was especially striking. We also observed increase in production of TNF-α and IL-6 and decrease in the production of IL-10, as well. Given these findings, it is possible that maladapted trained immunity may have caused excessive inflammatory responses to various innate immune stimuli, which then led to developmental regression and fluctuating behavioral symptoms in this presented case.

#### **4. Conclusions**

Our deepening knowledge of innate immune memory (trained immunity vs. tolerance) has shed light on the understanding of nonspecific effects of microbial infection and other immune stimuli, which have been implicated in the onset and progress of various neuropsychiatric diseases. Recent research indicates a possibility for a role of maladapted innate immune memory in various neuropsychiatric conditions. The finding of innate immune memory is especially exciting in the field of neuroimmunology, since we now likely have better tools for addressing the longsuspected role of immune-mediated inflammation that is not associated with specific pathogens or environmental factors, in various neuropsychiatric conditions. The concept of innate immune memory will be especially important in addressing insults to the brain during the early years of CNS development, and the resultant lasting intellectual disabilities, as seen in MIA models [70]. More importantly, an improved understanding of the role of innate immune memory (trained immunity vs. tolerance) in pathogenic neuroinflammation can lead to novel therapeutic measures that are desperately needed for the treatment of neuropsychiatric diseases.

## **Acknowledgements**

The part of the study presented in this manuscript was funded from the Jonty Foundation, St. Paul, MN. The author is thankful for the critical review of this manuscript by Dr. L. Huguenin.

## **Conflict of interest**

The author has nothing to declare.

## **Abbreviations**


**67**

**Author details**

Harumi Jyonouchi1,2

New Brunswick, NJ, United States

provided the original work is properly cited.

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism…*

*DOI: http://dx.doi.org/10.5772/intechopen.87167*

NK natural killer

TCA tricarboxylic acid TLR Toll-like receptor TNF tumor necrosis factor

NF-κB nuclear factor of κ chain of B cells

OxLDL oxidized low-density lipoprotein OXPHOS oxidative phosphorylation

SPUH Saint Peter's University Hospital

TSC tuberous sclerosis complex

PAMPs pathogen-associated molecular patterns PBMCs peripheral blood mononuclear cells PBMo peripheral blood monocytes

SHIP1 SH2 domain-containing inositol phosphatase 1

TREM-2 triggering receptor expressed on myeloid cells 2

TRIF TIR-domain-containing adaptor-inducing interferon-ß

UMLILO upstream master lncRNAs of the inflammatory chemokine locus

1 Department of Pediatrics, Rutgers-RWJ, Saint Peter's University Hospital (SPUH),

2 Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, United States

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: hjyonouchi@saintpetersuh.com

*Innate Immunity and Neuroinflammation in Neuropsychiatric Conditions Including Autism… DOI: http://dx.doi.org/10.5772/intechopen.87167*


## **Author details**

*Cytokines*

**Acknowledgements**

**Conflict of interest**

**Abbreviations**

Ab antibody Ag antigen

APC Ag-presenting cells ApoE apolipoprotein E

ASD autism spectrum disorders BCG Bacillus Calmette-Guérin Brg1 brahma-regulated gene 1

CNS central nervous system

lncRNAs long noncoding RNAs

LPS lipopolysaccharide

MS multiple sclerosis

HIF-1α hypoxia inducible factor-1α

IPLs immune gene-priming lncRNAs

MIA maternal immune activation

MyD88 myeloid differentiation factor 88 mTOR mammalian target of rapamycin

IRAK interleukin-1 receptor-associated kinase

GI gastrointestinal

IFN interferon IL interleukin

BMDM cell, bone marrow-derived microglial cell CAPS cryopyrin-associated periodic syndrome

DAMPs damage-associated molecular patterns EAE experimental autoimmune encephalitis

CARS compensatory anti-inflammatory response syndrome

manuscript by Dr. L. Huguenin.

The author has nothing to declare.

progress of various neuropsychiatric diseases. Recent research indicates a possibility for a role of maladapted innate immune memory in various neuropsychiatric conditions. The finding of innate immune memory is especially exciting in the field of neuroimmunology, since we now likely have better tools for addressing the longsuspected role of immune-mediated inflammation that is not associated with specific pathogens or environmental factors, in various neuropsychiatric conditions. The concept of innate immune memory will be especially important in addressing insults to the brain during the early years of CNS development, and the resultant lasting intellectual disabilities, as seen in MIA models [70]. More importantly, an improved understanding of the role of innate immune memory (trained immunity vs. tolerance) in pathogenic neuroinflammation can lead to novel therapeutic measures that are desperately needed for the treatment of neuropsychiatric diseases.

The part of the study presented in this manuscript was funded from the Jonty Foundation, St. Paul, MN. The author is thankful for the critical review of this

**66**

Harumi Jyonouchi1,2

1 Department of Pediatrics, Rutgers-RWJ, Saint Peter's University Hospital (SPUH), New Brunswick, NJ, United States

2 Rutgers-Robert Wood Johnson Medical School, New Brunswick, NJ, United States

\*Address all correspondence to: hjyonouchi@saintpetersuh.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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[18] Jensen KJ, Larsen N, Biering-Sorensen S, Andersen A, Eriksen HB, Monteiro I, et al. Heterologous immunological effects of early BCG vaccination in low-birthweight infants in Guinea-Bissau: A randomized-controlled trial. The Journal of Infectious Diseases. 2015;**211**(6):956-967

[19] Netea MG, Joosten LA, Latz E, Mills KH, Natoli G, Stunnenberg HG, et al. Trained immunity: A program of innate immune memory in health and disease. Science. 2016;**352**(6284):aaf1098

[20] Reimer-Michalski EM, Conrath U. Innate immune memory in plants. Seminars in Immunology. 2016;**28**(4):319-327

[21] Cheng SC, Quintin J, Cramer RA, Shepardson KM, Saeed S, Kumar V, et al. mTOR- and HIF-1alpha-mediated aerobic glycolysis as metabolic basis for trained immunity. Science. 2014;**345**(6204):1250684

[22] Ryskalin L, Limanaqi F, Frati A, Busceti CL, Fornai F. mTOR-related brain dysfunctions in neuropsychiatric disorders. International Journal of Molecular Sciences. 2018;**19**(8):1-29

[23] Arts RJ, Joosten LA, Netea MG. Immunometabolic circuits in trained immunity. Seminars in Immunology. 2016;**28**(5):425-430

[24] Arts RJ, Novakovic B, Ter Horst R, Carvalho A, Bekkering S, Lachmandas E, et al. Glutaminolysis and fumarate accumulation integrate immunometabolic and epigenetic programs in trained immunity. Cell Metabolism. 2016;**24**(6):807-819

[25] Saeed S, Quintin J, Kerstens HH, Rao NA, Aghajanirefah A, Matarese F, et al. Epigenetic programming of monocyte-tomacrophage differentiation and trained innate immunity. Science. 2014;**345**(6204):1251086

[26] Novakovic B, Habibi E, Wang SY, Arts RJW, Davar R, Megchelenbrink W, et al. Beta-glucan reverses the epigenetic state of LPS-induced immunological tolerance. Cell. 2016;**167**(5):1354-1368 e14

[27] Su X, Wellen KE, Rabinowitz JD. Metabolic control of methylation and acetylation. Current Opinion in Chemical Biology. 2016;**30**:52-60

[28] Shin Y, Chang YC, Lee DSW, Berry J, Sanders DW, Ronceray P, et al. Liquid nuclear condensates mechanically sense and restructure the genome. Cell. 2018;**175**(6):1481-1491 e13

[29] Cho WK, Spille JH, Hecht M, Lee C, Li C, Grube V, et al. Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science. 2018;**361**(6400):412-415

[30] Fok ET, Davignon L, Fanucchi S, Mhlanga MM. The lncRNA connection between cellular metabolism and epigenetics in trained immunity. Frontiers in Immunology. 2018;**9**:3184

[31] Fanucchi S, Fok ET, Dalla E, Shibayama Y, Borner K, Chang EY, et al. Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments. Nature Genetics. 2019;**51**(1):138-150

**68**

*Cytokines*

**References**

[1] Perry VH, Cunningham C, Holmes C. Systemic infections and inflammation affect chronic neurodegeneration. Nature Reviews. Immunology. 2007;**7**(2):161-167

Trained innate immunity: A salient factor in the pathogenesis of neuroimmune psychiatric disorders. Molecular Psychiatry.

2018;**23**(2):170-176

2018;**29**(11):1023-1040

2018;**556**(7701):332-338

2019;**2019**:2471215

2018;**31**(4):403-409

2016;**16**(2):124-128

[5] Krakauer T. Inflammasomes,

[6] Peng Y, van Wersch R, Zhang Y. Convergent and divergent signaling

[7] McCoy KD, Ronchi F, Geuking MB. Host-microbiota interactions and adaptive immunity. Immunological

[8] Farber DL, Netea MG, Radbruch

RM. Immunological memory: Lessons

Reviews. 2017;**279**(1):63-69

A, Rajewsky K, Zinkernagel

from the past and a look to the future. Nature Reviews. Immunology.

[9] Sfera A, Gradini R, Cummings M, Diaz E, Price AI, Osorio C. Rusty

in PAMP-triggered immunity and effector-triggered immunity. Molecular Plant-Microbe Interactions.

[2] Salam AP, Borsini A, Zunszain PA.

microglia: Trainers of innate immunity in Alzheimer's disease. Frontiers in

[10] Sohrabi Y, Lagache SMM, Schnack L, Godfrey R, Kahles F, Bruemmer D, et al. mTOR-dependent oxidative stress regulates oxLDL-induced trained innate immunity in human monocytes. Frontiers in Immunology. 2018;**9**:3155

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in astrocyte cultures. Journal of

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2014;**21**(4):534-545

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2017;**21**(3):297-300

2014;**155**(2):213-219

2018;**281**(1):28-39

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[52] Doxaki C, Kampranis SC, Eliopoulos AG, Spilianakis C, Tsatsanis C. Coordinated regulation of miR-155 and miR-146a genes during induction of endotoxin tolerance in macrophages. Journal of Immunology. 2015;**195**(12):5750-5761

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[70] Careaga M, Murai T, Bauman MD. Maternal immune activation and autism Spectrum disorder: From rodents to nonhuman and human primates. Biological Psychiatry.

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[73] Neher JJ, Neniskyte U, Brown GC. Primary phagocytosis of neurons by inflamed microglia: Potential roles in neurodegeneration. Frontiers in

[74] Painter MM, Atagi Y, Liu CC, Rademakers R, Xu H, Fryer JD, et al. TREM2 in CNS homeostasis and neurodegenerative disease. Molecular Neurodegeneration. 2015;**10**:43

C, Novakova L, Heslegrave A, Blennow K, et al. Soluble TREM-2 in cerebrospinal fluid from patients with multiple sclerosis treated with natalizumab or mitoxantrone. Multiple Sclerosis. 2016;**22**(12):1587-1595

[76] Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, et al. Nutrient-dependent mTORC1 association with the ULK1-Atg13- FIP200 complex required for autophagy.

Molecular Biology of the Cell.

2009;**20**(7):1981-1991

[75] Ohrfelt A, Axelsson M, Malmestrom

2014;**49**(3):1422-1434

Pharmacology. 2012;**3**:27

Neuroscience. 2015;**9**:294

2017;**81**(5):391-401

by alpha2-interferon. Journal of Leukocyte Biology. 2015;**98**(4):651-659

[63] Zhan X, Stamova B, Sharp FR. Lipopolysaccharide associates with amyloid plaques, Neurons and Oligodendrocytes in Alzheimer's Disease Brain: A Review. Frontiers in Aging Neuroscience. 2018;**10**:42

[64] Wlodarczyk A, Cedile O, Jensen KN, Jasson A, Mony JT, Khorooshi R, et al. Pathologic and protective roles for microglial subsets and bone marrowand blood-derived myeloid cells in central nervous system inflammation. Frontiers in Immunology. 2015;**6**:463

[65] Ataka K, Asakawa A, Nagaishi K, Kaimoto K, Sawada A, Hayakawa Y, et al. Bone marrow-derived microglia infiltrate into the paraventricular nucleus of chronic psychological stress-loaded mice. PLoS One.

[66] Greenhalgh AD, Zarruk JG, Healy LM, Baskar Jesudasan SJ, Jhelum P, Salmon CK, et al. Peripherally derived macrophages modulate microglial function to reduce inflammation after CNS injury. PLoS Biology.

2013;**8**(11):e81744

2018;**16**(10):e2005264

2011;**31**(43):15511-15521

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[80] Giorgi FS, Biagioni F, Lenzi P, Frati A, Fornai F. The role of autophagy in epileptogenesis and in epilepsyinduced neuronal alterations. Journal of Neural Transmission (Vienna). 2015;**122**(6):849-862

[81] Tramutola A, Triplett JC, Di Domenico F, Niedowicz DM, Murphy MP, Coccia R, et al. Alteration of mTOR signaling occurs early in the progression of Alzheimer disease (AD): Analysis of brain from subjects with pre-clinical AD, amnestic mild cognitive impairment and late-stage AD. Journal of Neurochemistry. 2015;**133**(5):739-749

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[87] Jyonouchi H, Geng L, Streck DL, Toruner GA. Children with autism spectrum disorders (ASD) who exhibit chronic gastrointestinal (GI) symptoms and marked fluctuation of behavioral symptoms exhibit distinct innate immune abnormalities and transcriptional profiles of peripheral blood (PB) monocytes. Journal of Neuroimmunology. 2011;**238**(1-2):73-80

[88] Jyonouchi H, Geng L, Davidow AL. Cytokine profiles by peripheral blood monocytes are associated with changes in behavioral symptoms following immune insults in a subset of ASD subjects: An inflammatory subtype? Journal of Neuroinflammation. 2014;**11**:187

[89] Turner TN, Eichler EE. The role of De novo noncoding regulatory mutations in neurodevelopmental disorders. Trends in Neurosciences. 2019;**42**(2):115-127

[90] Cristiano C, Lama A, Lembo F, Mollica MP, Calignano A, Mattace RG. Interplay between peripheral and central inflammation in autism Spectrum disorders: Possible nutritional and therapeutic strategies. Frontiers in Physiology. 2018;**9**:184

[91] Jiang NM, Cowan M, Moonah SN, Petri WA Jr. The impact of systemic inflammation on neurodevelopment. Trends in Molecular Medicine. 2018;**24**(9):794-804

[92] Siniscalco D, Schultz S, Brigida AL, Antonucci N. Inflammation and neuro-immune dysregulations in autism spectrum disorders. Pharmaceuticals (Basel). 4 June 2018;**11**(2):56

[93] Jyonouchi H, Geng L, Streck DL, Dermody JJ, Toruner GA. MicroRNA expression changes in association with changes in interleukin-1ss/interleukin10 ratios produced by monocytes in autism spectrum disorders: Their association with neuropsychiatric symptoms and comorbid conditions (observational study). Journal of Neuroinflammation. 2017;**14**(1):229

[94] Jyonouchi H, Geng L, Buyske S. Interleukin-1β/Interleukin10 ratio produced by monocytes as a biomarker of neuroinflammation in autism. Journal of Clinical & Cellular Immunology. 2017;**8**(3):1000503

[95] Jyonouchi H, Geng L, Rose S, Bennuri SC, Frye RE. Variations in mitochondrial respiration differ in IL-1ß/IL-10 ratio based subgroups in autism spectrum disorders. Frontiers in Psychiatry. 2019;**10**(Feburary):1-15

[96] Rudzki L, Szulc A. "Immune gate" of psychopathology-the role of gut derived immune activation in major psychiatric disorders. Frontiers in Psychiatry. 2018;**9**:205

[97] van Splunter M, van Osch TLJ, Brugman S, Savelkoul HFJ, Joosten LAB, Netea MG, et al. Induction of trained innate immunity in human monocytes by bovine milk and milk-derived immunoglobulin G. Nutrients. 27 Sept 2018;**10**(10)

**75**

**Chapter 6**

**Abstract**

**1. Introduction**

Cord Injury

Cytokines in Scar Glial Formation

after an Acute and Chronic Spinal

*Roxana Rodrígez-Barrera, Adrián Flores-Romero,* 

*Marcela Garibay-López and Elisa García-Vences*

phases, as well as the modulating treatments of glial scar.

determined by the level and completeness of the injury [1, 3].

*Julián García-Sánchez, Lisset Karina Navarro-Torres,* 

The inflammatory response after a spinal cord injury (SCI) is a secondary mechanism of damage, this involves alterations at the local and systemic level, and it is mediated by cytokine participation that takes part actively. The excessive inflammatory response causes an autoreactive response that targets against components of the nervous tissue; this response lengthens the inflammatory process initiated during the acute phase. The participation of immune cells in acute phases is characterized by the arrival of neutrophils, macrophages, and microglia, as well as T lymphocytes, which express their peaks on different days post-injury (1st, 3rd, and 11th respectively). The chronic phase of the injury begins 14 days after it occurred, reaching its highest point at 60 days, and can still be detected the following 180 days. One of the outcomes of the inflammatory process and cytokine synthesis is the generation of glial scar. In this chapter, we will review the different cytokine mechanisms involved in the formation of glial scar in acute and chronic

**Keywords:** spinal cord injury, immune cells, scar glial, modulating treatments

Spinal cord injury (SCI) causes catastrophic damaged to patients, and the incidence is getting higher each year. Most of them are occasioned by physical trauma from sports injuries, car accidents, falls, and more [1, 2]. This life-changing neurological condition also comes with socioeconomic implications for patients and their caregivers, besides the functional and sensitive consequences that are largely

After SCI, the acute and focal inflammation triggers a multicellular and multifunctional complex response which induces resident and infiltrating cells to form the glial scar (GS) at the site of the lesion [3]. The GS is a complicated phenomenon which has been considered as one of the main causes of limited regenerative capacity by inhibiting axonal regeneration and preventing functional recovery [4]. It has been proven that the GS creates both a physical barrier for neural repair as well as a chemical inhibition by the secretion of inhibitory extracellular matrix

## **Chapter 6**

*Cytokines*

and therapeutic strategies. Frontiers in

[91] Jiang NM, Cowan M, Moonah SN, Petri WA Jr. The impact of systemic inflammation on neurodevelopment. Trends in Molecular Medicine.

[92] Siniscalco D, Schultz S, Brigida AL, Antonucci N. Inflammation and neuro-immune dysregulations in autism spectrum disorders. Pharmaceuticals

[93] Jyonouchi H, Geng L, Streck DL, Dermody JJ, Toruner GA. MicroRNA expression changes in association with changes in interleukin-1ss/interleukin10 ratios produced by monocytes in autism spectrum disorders: Their association with neuropsychiatric symptoms and comorbid conditions (observational study). Journal of Neuroinflammation.

[94] Jyonouchi H, Geng L, Buyske S. Interleukin-1β/Interleukin10 ratio produced by monocytes as a biomarker of neuroinflammation in autism. Journal of Clinical & Cellular Immunology. 2017;**8**(3):1000503

[95] Jyonouchi H, Geng L, Rose S, Bennuri SC, Frye RE. Variations in mitochondrial respiration differ in IL-1ß/IL-10 ratio based subgroups in autism spectrum disorders. Frontiers in Psychiatry. 2019;**10**(Feburary):1-15

[96] Rudzki L, Szulc A. "Immune gate" of psychopathology-the role of gut derived immune activation in major psychiatric disorders. Frontiers in

[97] van Splunter M, van Osch TLJ, Brugman S, Savelkoul HFJ, Joosten LAB, Netea MG, et al. Induction of trained innate immunity in human monocytes by bovine milk and milk-derived immunoglobulin G. Nutrients. 27 Sept

Psychiatry. 2018;**9**:205

(Basel). 4 June 2018;**11**(2):56

Physiology. 2018;**9**:184

2018;**24**(9):794-804

2017;**14**(1):229

**74**

2018;**10**(10)
