**6. Immunomodulatory functions of SAA in disease models**

Since most of the early studies were conducted using cell lines and isolated primary cells such as monocytes and neutrophils, these experimental findings are now examined in an *in vivo* setting. An early model created for the *in vivo* studies of SAA employed adenoviral expression of human SAA1, raising the circulatory levels of human SAA1 in the infected mice [106]. This approach was used in studies of the involvement of SAA1 in lipid metabolism [106] and fibril formation [107]. In a more recent study, the same group that created the adenoviral approach found a role for SAA3 in atherosclerosis [108].

Transgenic expression of human SAA1 in mice is another approach used in studies of the *in vivo* functions of SAA. Ji et al. reported transgenic expression of human SAA1 in mouse liver [89]. These mice exhibited more severe liver injury, increased hepatocyte apoptosis, and higher levels of hepatic enzymes than in their wildtype controls. After induction of hepatitis, liver infiltration of CD4+ T cells and macrophages was also increased more in the transgenic mice than in wildtype mice, along with elevated expression of several chemokines. The aggravated liver injury, increased hepatocyte apoptosis and elevated levels of hepatic enzymes in the transgenic mice were eased with the use of a TLR2 antagonist, suggesting that TLR2 mediates the effects of the transgenic SAA1. In a more recent study, Cheng et al. placed the human SAA1 under an inducible promoter of SR-A receptor, generating transgenic mice with elevated local production of SAA1 upon inflammatory stimulation [53]. The transgenic SAA1 was most abundant in mouse lungs and protected mice against acute lung injury caused by LPS administration and cecal ligation and puncture (CLP). Transgenic expression of SAA1 did not protect mice against acute lung injury induced by intratracheal instillation of TNFα. Binding studies showed that human SAA1, purified from either *E. coli* or transfected HEK293 cells, bound to LPS and formed a complex that promoted LPS clearance by macrophages. As a result, serum endotoxin concentration was significantly reduced in the transgenic mice than in their wildtype controls that went through the CLP procedure. Of note, injection of a SAA1-derived peptide that disrupted LPS-SAA1 interaction diminished the endotoxin-lowering effect in the SAA1 transgenic mice and increased serum endotoxin level in wildtype mice after CLP [53]. These findings suggest a mechanism by which acute-phase SAA protects host against bacterial infection-induced injury.

SAA gene knockout mice were generate to examine the physiological functions of the individual SAA proteins. After observing SAA1 and SAA2 expression in intestinal epithelial cells and conforming their cell-protecting effect in epithelial cell line co-cultured with *E. coli*, Eckhardt et al. examined the effect of Saa1/2 double knockout (DKO) in dextran sodium sulfate (DSS) induced colitis model [109]. They found that that epithelial expression of SAA1 and SAA2 protected colonic epithelium against bacterial infection. A more recent study using Saa3 gene knockout mice found that SAA3 is the predominant isoform of inducible SAA proteins in colonic epithelium following chemical injury [92]. Compared to wildtype mice, *Saa3*<sup>−</sup>/<sup>−</sup> mice exposed to DSS showed more severe damage to the colonic epithelial structure, significantly reduced expression of the anti-microbial peptides Reg3β and Reg3γ, and reduced lifespan of afflicted mice if not treated. Administration of exogenous SAA3 protein or adoptive transfer of SAA3-treated neutrophils partially ameliorated symptoms of DSS-induced colitis in part due to SAA3-induced

neutrophil expression of IL-22, a cytokine with epithelia-protection function [110]. Together, these results suggest that epithelial expression of SAA1 and SAA2 in healthy mice may be important for homeostasis of gut functions including host defense, whereas inducible expression of SAA3 serves to combat acute injury to the colonic epithelium.

A role for SAA as a mediator of local immune response has been reported recently. In a study of segmented filamentous bacteria (SFB) for its involvement in mucosal defenses and autoimmune diseases through RORγ<sup>+</sup> Th17 cells, Sano et al. found that direct contact of SFB with epithelium in the ileum could induce SAA1 and SAA2 expression and promote local IL-17A expression in RORγ(+) T cells. The mechanisms involved an IL-23R/IL-22 circuit and the participation of type 3 innate lymphoid cells (ILC3) that secretes IL-22 [111]. Likewise, Atarashi et al. investigated a group of intestinal microbes for their ability to induce Th17 response, and found that SFB could stimulate intestinal epithelial cells to generate SAA and ROS, creating an amplification loop for sustained production of SAA by both epithelial cells and myeloid cells that led to local Th17 response [112]. These findings provide direct evidence for the contribution of epithelial SAA to intestinal homeostasis in an environment where host interaction with gut microbiota influences the health states of individuals.

In addition to studies of the *in vivo* functions of SAA in innate immunity and inflammation, mice with genetically altered SAA genes were used in the investigation of these acute-phase proteins in animal models of atherosclerosis, osteoclast activation, adipogenesis, and neurodegenerative disorders such as Alzheimer's disease. Ahlin et al. generated transgenic mouse model expressing human SAA1 in the adipose tissue, and used the hSAA1+/<sup>−</sup> mice in studies of the effect of SAA1 on glucose metabolism and insulin resistance [114]. They found no evidence that adipose tissue-derived hSAA1 could influence the development of insulin resistance or obesity-related inflammation. The potential involvement of SAA in atherogenesis was investigated using the Saa1/2 DKO mice in the *Apoe*<sup>−</sup>/<sup>−</sup> background [115]. Surprisingly, the absence of *Saa1.1* and *Saa2.1* did not affect atherosclerotic lesion in the ApoE-deficient mice that were fed with Western diets. It was later reported that SAA3, instead of SAA1/2, is pro-atherogenic based on experiments using adeno-associated virus for overexpression of SAA3 and antisense oligonucleotidemediated suppression of Saa3 expression [108]. Using SAA3 KO mice, Liu et al. reported elevated Tau phosphorylation (hyperphosphorylation) compared to wildtype mice upon systemic LPS administration. Overexpression of SAA by intracerebral injection attenuated tau hyperphosphorylation in the brain, suggesting that SAA3 may be neuroprotective in the mouse AD model [116].

Several studies of the *in vivo* functions of SAA were conducted in wildtype mice. De Santo et al. reported that systemic SAA1 plays a role in the regulation of neutrophil plasticity through induction of the anti-inflammatory IL-10 and promotion of the interaction of invariant natural killer T cells (iNKT cells) with neutrophils. As a result, SAA1 indirectly limits the suppressive activity by diminishing IL-10 production and enhancing IL-12 production [113].

Collectively, results from the studies of SAA proteins in mice identified important functions of SAA that were previous unknown from *in vitro* studies. There are other functions revealed from the *in vivo* studies using genetically altered mice that are consistent with the *in vitro* findings. For example, the ability of SAA to interact with Gram-negative bacterial wall components [117] is consistent with the *in vivo* findings that SAA1 protects mice against LPS- and CLP-induced acute lung injury [53]. The *in vivo* findings strongly suggest that acute-phase SAA protects host against environmental insults such as chemical-induced intestinal epithelial injury and bacterial infection. Four of the animal models used in studies of SAA are

**121**

*Serum Amyloid A and Immunomodulation DOI: http://dx.doi.org/10.5772/intechopen.81617*

**7. Conclusion remarks**

**Figure 3.**

*immunity.*

**Acknowledgements**

**Conflict of interest**

summarized in **Figure 3**. Due to page limitation, *in vivo* studies on SAA functions other than those related to immunomodulation are not discussed in this chapter.

*Immunomodulatory functions of SAA in selected mouse models. Left: transgenic expression of human SAA1 in the lung tissue protects mice against LPS-induced acute lung injury [53]. The protection is conferred in part through SAA binding to LPS, forming a complex that promotes LPS clearance by macrophages. Middle: SAA1 and SAA2 expressed in epithelium of the ileum serves as a mediator of segmented filamentous bacteria-induced local Th17 response [111, 112], contributing to homeostasis of the microenvironment in the intestine [109]. In response to acute injury such as dextran sodium sulfate (DSS) treatment, SAA3 is induced in mouse colonic epithelium and serves as an inducer for neutrophil IL-22 expression [92]. Right: SAA1-producing melanomas induce neutrophil secretion of IL-10 for its suppressive effect. SAA1 also promotes neutrophil interaction with invariant natural killer T (iNKT) cells, thereby limiting IL-10 production but enhancing IL-12 production [113]. This mechanism may be explored to reduce the immunosuppressive neutrophils and restore tumor-specific* 

SAA has emerged from a precursor of AA to a modulator of immunity and inflammation. Several developments, including the ability to express recombinant SAA proteins, the generation of genetically altered mice expressing SAA transgenes or deletion of a specific SAA gene, and the availability of crystal structures of SAA proteins, have helped to advance our understanding of SAA for its functions in host defense, lipid metabolism, adipogenesis, and neuroprotection. In coming years, studies will likely focus on the comparison of SAA functions *in vitro* to those identified *in vivo,* and on the possible modifications and proteolytic processing of newly synthesized SAA in order to address several questions that remain unanswered today. A better understanding of SAA for its biological functions is expected to benefit human health through development of new diagnostic approaches and therapies.

This work was supported in part by grants from the Science and Technology

Development Fund of Macau (026/2016/A1) and the University of Macau

(MYRG2016-00152-ICMS-QRCM and CPG2015-00018-ICMS).

The authors declare that they have no conflict of interest.

*Serum Amyloid A and Immunomodulation DOI: http://dx.doi.org/10.5772/intechopen.81617*

#### **Figure 3.**

*Amyloid Diseases*

colonic epithelium.

states of individuals.

neutrophil expression of IL-22, a cytokine with epithelia-protection function [110]. Together, these results suggest that epithelial expression of SAA1 and SAA2 in healthy mice may be important for homeostasis of gut functions including host defense, whereas inducible expression of SAA3 serves to combat acute injury to the

A role for SAA as a mediator of local immune response has been reported recently. In a study of segmented filamentous bacteria (SFB) for its involvement in

found that direct contact of SFB with epithelium in the ileum could induce SAA1 and SAA2 expression and promote local IL-17A expression in RORγ(+) T cells. The mechanisms involved an IL-23R/IL-22 circuit and the participation of type 3 innate lymphoid cells (ILC3) that secretes IL-22 [111]. Likewise, Atarashi et al. investigated a group of intestinal microbes for their ability to induce Th17 response, and found that SFB could stimulate intestinal epithelial cells to generate SAA and ROS, creating an amplification loop for sustained production of SAA by both epithelial cells and myeloid cells that led to local Th17 response [112]. These findings provide direct evidence for the contribution of epithelial SAA to intestinal homeostasis in an environment where host interaction with gut microbiota influences the health

In addition to studies of the *in vivo* functions of SAA in innate immunity and inflammation, mice with genetically altered SAA genes were used in the investigation of these acute-phase proteins in animal models of atherosclerosis, osteoclast activation, adipogenesis, and neurodegenerative disorders such as Alzheimer's disease. Ahlin et al. generated transgenic mouse model expressing human SAA1 in the adipose tissue, and used the hSAA1+/<sup>−</sup> mice in studies of the effect of SAA1 on glucose metabolism and insulin resistance [114]. They found no evidence that adipose tissue-derived hSAA1 could influence the development of insulin resistance or obesity-related inflammation. The potential involvement of SAA in atherogenesis was investigated using the Saa1/2 DKO mice in the *Apoe*<sup>−</sup>/<sup>−</sup> background [115]. Surprisingly, the absence of *Saa1.1* and *Saa2.1* did not affect atherosclerotic lesion in the ApoE-deficient mice that were fed with Western diets. It was later reported that SAA3, instead of SAA1/2, is pro-atherogenic based on experiments using adeno-associated virus for overexpression of SAA3 and antisense oligonucleotidemediated suppression of Saa3 expression [108]. Using SAA3 KO mice, Liu et al. reported elevated Tau phosphorylation (hyperphosphorylation) compared to wildtype mice upon systemic LPS administration. Overexpression of SAA by intracerebral injection attenuated tau hyperphosphorylation in the brain, suggest-

ing that SAA3 may be neuroprotective in the mouse AD model [116].

tion and enhancing IL-12 production [113].

Several studies of the *in vivo* functions of SAA were conducted in wildtype mice. De Santo et al. reported that systemic SAA1 plays a role in the regulation of neutrophil plasticity through induction of the anti-inflammatory IL-10 and promotion of the interaction of invariant natural killer T cells (iNKT cells) with neutrophils. As a result, SAA1 indirectly limits the suppressive activity by diminishing IL-10 produc-

Collectively, results from the studies of SAA proteins in mice identified important functions of SAA that were previous unknown from *in vitro* studies. There are other functions revealed from the *in vivo* studies using genetically altered mice that are consistent with the *in vitro* findings. For example, the ability of SAA to interact with Gram-negative bacterial wall components [117] is consistent with the *in vivo* findings that SAA1 protects mice against LPS- and CLP-induced acute lung injury [53]. The *in vivo* findings strongly suggest that acute-phase SAA protects host against environmental insults such as chemical-induced intestinal epithelial injury and bacterial infection. Four of the animal models used in studies of SAA are

Th17 cells, Sano et al.

mucosal defenses and autoimmune diseases through RORγ<sup>+</sup>

**120**

*Immunomodulatory functions of SAA in selected mouse models. Left: transgenic expression of human SAA1 in the lung tissue protects mice against LPS-induced acute lung injury [53]. The protection is conferred in part through SAA binding to LPS, forming a complex that promotes LPS clearance by macrophages. Middle: SAA1 and SAA2 expressed in epithelium of the ileum serves as a mediator of segmented filamentous bacteria-induced local Th17 response [111, 112], contributing to homeostasis of the microenvironment in the intestine [109]. In response to acute injury such as dextran sodium sulfate (DSS) treatment, SAA3 is induced in mouse colonic epithelium and serves as an inducer for neutrophil IL-22 expression [92]. Right: SAA1-producing melanomas induce neutrophil secretion of IL-10 for its suppressive effect. SAA1 also promotes neutrophil interaction with invariant natural killer T (iNKT) cells, thereby limiting IL-10 production but enhancing IL-12 production [113]. This mechanism may be explored to reduce the immunosuppressive neutrophils and restore tumor-specific immunity.*

summarized in **Figure 3**. Due to page limitation, *in vivo* studies on SAA functions other than those related to immunomodulation are not discussed in this chapter.

#### **7. Conclusion remarks**

SAA has emerged from a precursor of AA to a modulator of immunity and inflammation. Several developments, including the ability to express recombinant SAA proteins, the generation of genetically altered mice expressing SAA transgenes or deletion of a specific SAA gene, and the availability of crystal structures of SAA proteins, have helped to advance our understanding of SAA for its functions in host defense, lipid metabolism, adipogenesis, and neuroprotection. In coming years, studies will likely focus on the comparison of SAA functions *in vitro* to those identified *in vivo,* and on the possible modifications and proteolytic processing of newly synthesized SAA in order to address several questions that remain unanswered today. A better understanding of SAA for its biological functions is expected to benefit human health through development of new diagnostic approaches and therapies.

### **Acknowledgements**

This work was supported in part by grants from the Science and Technology Development Fund of Macau (026/2016/A1) and the University of Macau (MYRG2016-00152-ICMS-QRCM and CPG2015-00018-ICMS).

#### **Conflict of interest**

The authors declare that they have no conflict of interest.

*Amyloid Diseases*
