**3. MiRNAs at the cross-roads between innate and adaptive immune responses**

#### **3.1. Innate immune responses**

208 Immunodeficiency

cell development.

T cell differentiation and leukemogenesis.

**2. The role of MiRNAs in hematopoietic cell development** 

The development of hematopoietic and immune system requires an integrated network of survival, proliferation and apoptotic signals that are finely tuned along differentiation. miRNAs represent efficient modulators of such a system as they can affect the expression of multiple genes at different stages. The identification of putative miRNA involved in hematopoietic ontogeny has been one of primary topic of miRNA studies since their discovery. To clarify the role of specific miRNAs, Chen et al. [9] first cloned about 100 previously identified miRNAs and analyzed only those expressed in hematopoietic cells (miR181, 142 and 223). miR181 was found in lineage negative (Lin -) mouse bone marrow undifferentiated cells and strongly upregulated in mature B cells and within the thymus; miR142 was more ubiquitously expressed, while 223 was mostly confined to myeloid lineage. miR181 was then cloned into a GFP gene carrying retroviral vector and ectopically expressed in Lin- bone marrow cells. Infected cells were then followed in vitro to check their lineage commitment. A preferential development of B cells was observed. When Lin- miR181+ cells were transplanted in irradiated mice, lymphoid repopulation showed a prevalence of B cell population as compared to control (80% vs 32%, respectively). This study was of primary relevance as it has shown the effects of a single miRNA in lymphopoiesis and addressed a method to study next candidate miRNAs. De Yebenes and colleagues [10] observed that miR181b is involved also in immunoglobulin class switch at activated B cell level. Hence, miR 181 family is involved in early (switch from pro B cell to pre B cell) and late (from centroblasts to activated B cells) stages of B

The expression of miR181 in mouse thymus prompted investigators to evaluate their role in T cell selection. Interestingly, miR181 is expressed at higher levels in early T cell differentiation as its expression drops from double negative/double positive cells to single positive CD4/CD8 cells [11] MiR17-92 cluster (miR17, 18a, 19a, 20a, 19b and 92) has been implicated in B cell lymphopoiesis (transition pro B to pre B cells) by Ventura et al. [12]. Interestingly, in mice this cluster is homologous to the miR-106a-63 (except miR18 and 19), although only mice lacking miR17-92 show a relevant phenotype, including B cell differentation arrest. It is likely that miR17-92 cluster controls apoptotic signals through suppression of Bim and PTEN [13] miR-150 has been involved in the transition pro B to pre B cells through suppression of c-Myb [14-16], a transcription factor that leads this phase. miR-150 is strongly upregulated along T cell development beginning from double positive stage and modulates expression of NOTCH3 [17]. NOTCH3 gene is known to be involved in

Overall, these data indicate that miR181/miR17-92/miR150 are among the main regulators of

An analogue role is played by miR 223 in myeloid lineage. Chen et al [9] observed that miR223 is highly expressed in mouse bone marrow. Indeed, miR223 tunes granulocytic differentation both at an early and late phase [18]. MiR223 knock out mice show expansion

early T and B lymphopoiesis from the common lymphoid precursor.

Innate responses imply the final differentiation and interaction of intervening cells to the site where inflammatory stimuli were generated. Several miRNAs have been described as implicated in a complex network, that controls the on and off phases of the response.

miR146a is upregulated upon LPS stimulation in monocytes and is likely to be responsible of the phenomenon known as hyporesponsiveness to prolonged LPS exposure. Indeed, miR146a acts as a negative regulator of LPS induced responses. LPS-induced NF Kb promotes miR146a upregulation, which in turns suppresses TRAF6 (TNF receptor associated factor 6), IRAK1/2 (Interleukin 1 receptor associated kinase 1). These genes encode key adaptor molecules along TLR related pathways and are involved in innate responses through TNF activation and production of IL-1 dependent molecules such as IL-8 and RANTES [20-22]. Therefore, monocytes become hyporesponsive to further stimulation with LPS and relevant pro inflammatory molecules are reduced in the microenvironment.

miR155 seems to act on the same pathways but with opposite effects to miR146a. Indeed, engagement of several TLRs (3,4 and 9) promotes miR155 transcription through AP1 and NFkb. miR155 main targets are SOCS1 (suppressor of cytokine signaling 1) and SHIP1(Src Homology-2 domain-containing inositol-5'-phosphatase 1) that lead to release of proinflammatory cytokines in the microenvironment such as TNF alpha and IFN gamma [23-26].

miR223 can regulate also monocyte-macrophage differentiation by targeting IKK-alpha (IKb kinase) and leaving NFKb to promote inflammatory genes transcription. The final result is the transition of monocyte to macrophage [27].

The emerging data suggest that miRNA regulation of inflammatory and innate responses is timely tuned according to microenviromental stimuli. Indeed, TLR stimulation evokes miR155 upregulation within 2hr , while other miRNAs, such as miR21 are produced according to a delayed time frame. These observations are likely related to a differential role

of miRNAs in turning on and off the inflammatory/innate responses. In this setting, miR21 should turn off the response, by increasing IL-10 levels [28].

Emerging Role of MicroRNAs in the Pathophysiology of Immune System 211

epidermal DC population is heterogeneous and not pure, it is possible that high levels of miR155 promote attenuation of T cell responses through APCs different from differentiated

The plasticity of miRNAs in controlling overall immune responses is further demonstrated by the miR29 activity. Interestingly, recent findings of Ma et al. [32], who have shown that miR29 suppresses IFN-γ secretion in NK and T cells, thereby linking together innate and adaptive responses. Indeed, responses to pathogen are mainly regulated through this

The emerging picture of a central role played by miRNAs in the onset, development and turning off of immune responses is strictly related to the findings of their involvement in autoimmune diseases. In some cases, the functions of specific miRNAs have been first elucidated in the disease and then in immune system physiology. The possibility to use murine models of autoimmunity allows investigators to study the selected miRNAs *in vivo* in order to understand how they facilitate or attenuate the disease. However, the identification of a specific miRNA in the mouse model does not mean a direct translation into human disease. Overall, disregulation of miRNAs observed in autoimmunity promote either activation of immune effectors and/or suppression of immune regulatory cells, thus contributing to disease development. In the following section, the contribution of miRNAs

SLE is a chronic autoimmune disease with a complex pathogenesis, involving different organs [33]. Since systemic inflammation is the hallmark of the disease, deregulation of critical pro inflammatory pathways have been described [34]. Indeed, miRNAs deregulated

Type I IFN pathway is widely recognized as a primary deregulation of inflammatory responses in SLE pathogenesis [34]. IFN I pathway is elicited by TLR engagement. Among TLRs, TLR-7 contributes the most to this phenomenon. In 2009, Tang et al. [35] have shown that underexpression of miR146a is tightly related to the upregulation of type I IFN pathway. They analyzed 52 patients with SLE, 6 with Behcet'disease and 29 normal subjects and evaluated miR146a levels from PBMCs. Interestingly, miR146a was proportionally decreased according to disease state (no disease, inactive SLE and active SLE, with active SLE having the lowest levels). miR146a levels were inversely related to IFN score, which was calculated considering the expression of three representative

mechanism as for the case of L. Monocytogenes and M. Tuberculosis.

will be discussed according to the specific autoimmune disorder.

in SLE target genes involved in the inflammatory responses

**5. Systemic Lupus Erythematosus (SLE)** 

**5.1. miR146a** 

**4. MiRNAs and autoimmune diseases** 

DCs.

#### **3.2. Adaptive responses**

Immune responses require an integration between the innate/inflammatory and adaptive arm. According to their specific functions, miRNAs represent a perfect set of molecules to finely regulate and coordinate also adaptive responses. miRNAs that are involved in developmental stages of hematopoiesis can show additional functions in differentiated immune cells. Indeed, miR181a, which is implicated in thymic selection, is able to strengthen TCR signaling and reinforce T cell activation upon antigen engagement [11]. This effect likely relies on phosphatase suppression and increase in ERK phosphorylation. A member of the same family, miR181b has been proposed as regulating CSR (class switch recombination) of B cells. CSR is induced by activation induced cytidine deaminase (AID) and is likely targeted by miR181b. Indeed, IgG switch promoted by LPS and IL-4 stimulation is impaired when levels of endogenous miR181b are increased [10].

The role of miRNAs in T cell responses can be also variable according to its endogenous levels and/or contemporary expression in antigen presenting cells (APCs). This might be the case of miR155. miR155 is encoded within the BIC region (B cell integration cluster), which is often involved in lymphomas. BIC deficient mice, which lacked miR155 production, did not show significant impairment in hematopoiesis. When immunization with different bacterial strains and subsequent challenge with the same pathogens were administered to BIC deficient mice, the animals died of infection. Indeed, immunizations did not translate into protective immunity as compared with wild type mice. The authors have shown that T cell activity was compromised because there was a shift towards Th2 phenotype due to downregulation of c-Maf, which is a transcription factor that drives Th2 cytokine secretion [29]. Furthermore, BIC deficient DCs failed to adequately activate T cell responses. In this model, B cells were not able to differentiate to plasmablasts and showed alterations in CSR (class switch recombination). This phenomenon may be due to miR155, that targets AID [30].

Overall, these data indicate that miR155 has a pivotal role in sustaining adaptive immune responses.

However, these data are partly in contrast with the study from Mao et al. [31], who showed that miR155 is upregulated upon TLR stimulation in murine bone marrow derived dendritic cells. Furthermore, transfection of murine epidermal DCs with miR155 coding plasmid increased its endogenous levels and attenuated T cells responses driven by DCs. These effects were reverted when a miR155 antisense sequence was co-transfected into epidermal DCs. The authors try to reconcile these conflicting data, explaining that endogenous levels may induce different effects of the same miRNA in different cell types. However, since epidermal DC population is heterogeneous and not pure, it is possible that high levels of miR155 promote attenuation of T cell responses through APCs different from differentiated DCs.

The plasticity of miRNAs in controlling overall immune responses is further demonstrated by the miR29 activity. Interestingly, recent findings of Ma et al. [32], who have shown that miR29 suppresses IFN-γ secretion in NK and T cells, thereby linking together innate and adaptive responses. Indeed, responses to pathogen are mainly regulated through this mechanism as for the case of L. Monocytogenes and M. Tuberculosis.
