David, Chella S.

Autoimmune hepatitis (AIH) in humans is a severe inflammatory liver disease characterized by interface hepatitis, the presence of circulating autoantibodies, and hyper‐gammaglobulinemia. There are two types of AIH, type 1 (AIH‐1) and type 2 (AIH‐2), characterized by distinct autoimmune serology. Patients with AIH‐1 are positive for anti–smooth muscle and/or antinuclear autoantibodies, whereas patients with AIH‐2 have anti–liver kidney microsomal type 1 and/or anti–liver cytosol type 1 autoantibodies. Cytochrome P4502D6 is the antigenic target of anti–liver kidney microsomal type 1, and formiminotransferase cyclodeaminase is the antigenic target of anti–liver cytosol type 1. It is known that AIH, both types 1 and 2, is strongly linked to the human leukocyte antigen (HLA) alleles ‐DR3, ‐DR4, and ‐DR7. However, direct evidence of the association of HLA with AIH is lacking. We developed a novel mouse model of AIH using the HLA‐DR3 transgenic mouse on the nonobese‐diabetic background by immunization of HLA‐DR3– and HLA‐DR3+ nonobese‐diabetic mice with a DNA plasmid, coding for human cytochrome P4502D6/formiminotransferase cyclodeaminase fusion protein. Immunization with cytochrome P4502D6/formiminotransferase cyclodeaminase leads to a sustained elevation of alanine aminotransferase, development of antinuclear autoantibodies and anti–liver kidney microsomal type 1/anti–liver cytosol type 1 autoantibodies, chronic immune cell infiltration, and parenchymal fibrosis on liver histology in HLA‐DR3+ mice. Immunized mice also showed an enhanced T helper 1 immune response and paucity of the frequency of regulatory T cells in the liver. Moreover, HLA‐DR3+ mice with exacerbated AIH showed reduced diversity and total load of gut bacteria. Conclusion: Our humanized animal model has provided a novel experimental tool to further elucidate the pathogenesis of AIH and to evaluate the efficacy and safety of immunoregulatory therapeutic interventions in vivo. (Hepatology 2015;62:1536–1550)

Abstract SAgs, produced by Staphylococcus aureus, play a major role in the pathogenesis of invasive staphylococcal diseases by inducing potent activation of the immune system. However, the role of SAgs, produced by S. aureus, associated with indwelling devices or tissues, are not known. Given the prevalence of device-associated infection with toxigenic S. aureus in clinical settings and the potency of SAgs, we hypothesized that continuous exposure to SAgs produced by catheter-associated S. aureus could have systemic consequences. To investigate these effects, we established a murine in vivo catheter colonization model. One centimeter long intravenous catheters were colonized with a clinical S. aureus isolate producing SAgs or isogenic S. aureus strains, capable or incapable of producing SAg. Catheters were subcutaneously implanted in age-matched HLA-DR3, B6, and AEo mice lacking MHC class II molecules and euthanized 7 d later. There was no evidence of systemic infection. However, in HLA-DR3 transgenic mice, which respond robustly to SSAgs, the SSAg-producing, but not the nonproducing strains, caused a transient increase in serum cytokine levels and a protracted expansion of splenic CD4+ T cells expressing SSAg-reactive TCR Vβ8. Lungs, livers, and kidneys from these mice showed infiltration with CD4+ and CD11b+ cells. These findings were absent in B6 and AEo mice, which are known to respond poorly to SSAgs. Overall, our novel findings suggest that systemic immune activation elicited by SAgs, produced by S. aureus colonizing foreign bodies, could have clinical consequences in humans.

Abstract Multiple sclerosis is an inflammatory, demyelinating disease of the CNS of presumed autoimmune origin. Of all the genetic factors linked with multiple sclerosis, MHC class II molecules have the strongest association. Generation of HLA class II transgenic (Tg) mice has helped to elucidate the role of HLA class II genes in chronic inflammatory and demyelinating diseases. We have shown that the human HLA-DRB1*0301 gene predisposes to proteolipid protein (PLP)–induced experimental autoimmune encephalomyelitis (EAE), whereas HLA-DQβ1*0601 (DQ6) was resistant. We also showed that the DQ6 molecule protects from EAE in DRB1*0301.DQ6 double-Tg mice by producing anti-inflammatory IFN-γ. HLA-DQβ1*0302 (DQ8) Tg mice were also resistant to PLP91–110–induced EAE, but production of proinflammatory IL-17 exacerbated disease in DRB1*0301.DQ8 mice. To further confirm the role of IFN-γ in protection, we generated DRB1*0301.DQ8 mice lacking IFN-γ (DRB1*0301.DQ8.IFN-γ−/−). Immunization with PLP91–110 peptide caused atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice characterized by ataxia, spasticity, and dystonia, hallmarks of brain-specific disease. Severe brain-specific inflammation and demyelination in DRB1*0301.DQ8.IFN-γ−/− mice with minimal spinal cord pathology further confirmed brain-specific pathology. Atypical EAE in DRB1*0301.DQ8.IFN-γ−/− mice was associated with increased encephalitogenicity of CD4 T cells and their ability to produce greater levels of IL-17 and GM-CSF compared with DRB1*0301.DQ8 mice. Further, areas with demyelination showed increased presence of CD68+ inflammatory cells, suggesting an important role for monocytes/microglia in causing brain pathology. Thus, our study supports a protective role for IFN-γ in the demyelination of brain through downregulation of IL-17/GM-CSF and induction of neuroprotective factors in the brain by monocytes/microglial cells.

HLA‐DRB1*0401 expression in humans has been associated with a predisposition to developing rheumatoid arthritis (RA) and collagen‐induced arthritis (CIA), while HLA‐DRB1*0402 is not associated with susceptibility. Here, we determined if mice transgenic (Tg) for human *0401 have a CD4+ T‐cell repertoire that predetermines proinflammatory cytokine production. The data show that both *0401 and *0402 Tg mice can produce TH1/TH17 cytokines, although the kinetics of response may be different. However, in the context of antigen‐specific responses in a CIA model, *0402 Tg mice generate a TH2 response that may explain their resistance to developing arthritis. In addition, a significant subset of naïve CD4+ T cells from *0402 Tg mice can be activated in polarizing conditions to differentiate into Treg cells that produce IFN‐γ. *0401 Tg mice harbor memory CD4+ T cells that differentiate into IL‐17+ cells in various polarizing conditions. Our data suggest that *0401 Tg mice generate a strong immune response to lipopolysaccharide and may be efficient in clearing infection, and may *0401 have been evolutionarily selected for this ability. Autoimmunity, such as RA, could likely be a bystander effect of the cytokine storm that, along with the presence of low Treg‐cell numbers in *0401 Tg mice, causes immune dysregulation.

Abstract Life-threatening infections caused by Staphylococcus aureus, particularly the community-acquired methicillin-resistant strains of S. aureus, continue to pose serious problems. Greater virulence and increased pathogenicity of certain S. aureus strains are attributed to higher prevalence of exotoxins. Of these exotoxins, the superantigens (SAg) are likely most pathogenic because of their ability to rapidly and robustly activate the T cells even in extremely small quantities. Therefore, countering SAg-mediated T cell activation using T regulatory cells (Tregs) might be beneficial in diseases such as toxic shock syndrome (TSS). As the normal numbers of endogenous Tregs in a typical host are insufficient, we hypothesized that increasing the Treg numbers by administration of IL-2/anti–IL-2 Ab immune complexes (IL2C) or by adoptive transfer of ex vivo expanded Tregs might be more effective in countering SAg-mediated immune activation. HLA-DR3 transgenic mice that closely recapitulate human TSS were treated with IL2C to increase endogenous Tregs or received ex vivo expanded Tregs. Subsequently, they were challenged with SAg to induce TSS. Analyses of various parameters reflective of TSS (serum cytokine/chemokine levels, multiple organ pathology, and SAg-induced peripheral T cell expansion) indicated that increasing the Tregs failed to mitigate TSS. On the contrary, serum IFN-γ levels were increased in IL2C-treated mice. Exploration into the reasons behind the lack of protective effect of Tregs revealed IL-17 and IFN-γ–dependent loss of Tregs during TSS. In addition, significant upregulation of glucocorticoid-induced TNFR family-related receptor on conventional T cells during TSS could render them resistant to Treg-mediated suppression, contributing to failure of Treg-mediated immune regulation.

Staphylococcus aureusis capable of causing a spectrum of human illnesses. During seriousS. aureusinfections, the staphylococcal pathogen-associated molecular patterns (PAMPs) such as peptidoglycan, lipoteichoic acid, and lipoproteins and even intactS. aureus, are believed to act in conjunction with the staphylococcal superantigens (SSAg) to activate the innate and adaptive immune system, respectively, and cause immunopathology. However, recent studies have shown that staphylococcal PAMPs could suppress inflammation by several mechanisms and protect from staphylococcal toxic shock syndrome, a life-threatening systemic disease caused by toxigenicS. aureus. Given the contradictory pro- and anti-inflammatory roles of staphylococcal PAMPs, we examined the effects ofS. aureus-derived molecular patterns on immune responses driven by SSAgin vivousing HLA-DR3 and HLA-DQ8 transgenic mice. Our study showed that neitherS. aureus-derived peptidoglycans (PGN), lipoteichoic acid (LTA), nor heat-killedStaphylococcus aureus(HKSA) inhibited SSAg-induced T cell proliferationin vitro. They failed to antagonize the immunostimulatory effects of SSAgin vivoas determined by their inability to attenuate systemic cytokine/chemokine response and reduce SSAg-induced T cell expansion. These staphylococcal PAMPs also failed to protect HLA-DR3 as well as HLA-DQ8 transgenic mice from either SSAg-induced toxic shock or pneumonia induced by a SSAg-producing strain ofS. aureus.

Abstract To evaluate the disease-modulating role of HLA-DR2 and DR3 molecules, which have been associated with systemic lupus erythematosus, a humanized mouse model was examined. HLA-DR2 (DRB1*1502)- and DR3 (DRB1*0301)-transgenic mice were backcrossed to the New Zealand Mixed 2410 (NZM 2410, H2z) strain. Seventh generation DR2 and DR3 transgene-positive animals along with their transgene-negative littermates and the parental strain NZM2410 were monitored for proteinuria, azotemia, autoantibody production, development of nephritis, and mortality. The results showed no significant differences in proteinuria, azotemia, or mortality between the backcrosses with and without HLA-DR2 or HLA-DR3. However, the genetic analysis of different backcrosses showed that heterozygosity at the endogenous H2-E locus (Ez/Eb) was strongly linked with acceleration of lupus nephritis in both HLA-DR2 and HLA-DR3 transgenics. More importantly, the presence of the HLA-DR2, but not the HLA-DR3, transgene significantly enhanced the production of anti-dsDNA, but not anti-ssDNA, anti-histone-dsDNA complex, or anti-histone, Abs. In contrast, neither HLA-DR2 nor HLA-DR3 influenced the development of glomerulonephritis or the degree of immune complex deposition. Moreover, nephritic kidneys from mice with and without HLA-DR2 or HLA-DR3 transgenes showed similar patterns of cytokine expression. Collectively, these findings provide molecular evidence that the association of HLA-DR2 or HLA-DR3 with lupus susceptibility is related to the type of autoantibody rather than to disease mortality. The use of a humanized mouse model provides a way of dissecting the roles of human MHC genes in systemic lupus erythematosus pathogenesis.

Abstract Abs to Ro/SSA Ags in the sera of patients with systemic lupus erythematosus and Sjögren’s syndrome are influenced by the HLA class II genes. To investigate the role of individual HLA class II genes in immune responses to human Ro60 (hRo60), mice lacking murine class II molecules and carrying either HLA genes DR2(DRB1*1502), DR3(DRB1*0301), DQ6(DQA1*0103/DQB1*0601), or DQ8(DQA1*0301/DQB1*0302), were immunized with rhRo60. The results show that hRo60 induces strong T and B cell responses in DR2, DR3, and DQ8 mice in comparison to weaker responses in DQ6 mice. In all mice, the majority of the dominant T cell epitopes were located in the amino portion (aa 61–185) and the carboxy portion (aa 381–525) of the hRo60 molecules. In contrast, the early dominant B cell epitopes were located in the middle and carboxy portion of the hRo60 molecule (aa 281–315 and 401–538). In DR2, DR3, and DQ8 mice, the B cell epitopes subsequently spread to the amino and carboxy portion of the hRo60 molecule but were limited to the middle and carboxy portion in DQ6 mice. The DR2 and DR3 mice produced the highest titers of immunoprecipitating Abs against hRo60 and native mouse Ro60. In addition, only DR2 mice exclusively produced immunoprecipitating Abs to native mouse Ro52 and Abs to mouse La by slot blot analysis, whereas in other strains of mice Abs to mouse La were cross-reactive with the immunogen. The results of the present study demonstrate the importance of HLA class II in controlling the immune responses to the Ro-ribonucleoprotein.

Abstract To investigate the role of CD4 and CD8 T cells in arthritis, we generated transgenic mice deficient in CD4 and CD8 molecules expressing RA-susceptible gene HLA-DQ8. DQ8·CD4−/− mice were resistant to developing collagen-induced arthritis (CIA). However, DQ8·CD8−/− mice developed CIA with increased incidence and more severity than DQ8 mice. Both DQ8·CD8−/− and DQ8 mice produced rheumatoid factor. In addition, DQ8·CD8−/− mice produced antinuclear Abs. The B cell compartment and expression of DQ8 were normal in all the strains, although frequency of cells expressing DQ8 was less in CD4−/− mice. An increased frequency of CD3+ double-negative (DN) T cells was found in DQ8·CD8−/− compared with DQ8·CD4−/− and DQ8 mice. These CD3+ DN T cells produced high amounts of IL-10 in CD8-deficient mice. Analysis of cell division using a cell cycle tracking dye showed a higher rate of division of CD3+ and CD3+ DN T cells in DQ8·CD8−/− mice compared with DQ8·CD4−/− and DQ8 mice. Decreased apoptosis was seen in CIA-susceptible DQ8 and CD8-deficient mice, indicating a defect in activation-induced cell death. These observations suggest that CD4 cells are necessary for initiation of CIA in DQ8 mice. We hypothesize that CD8+ T cells are not capable of initiating CIA in DQ8-transgenic mice but may have a regulatory/protective effect.

Abstract To investigate the role of HLA-DR4 in predisposition to arthritis, we generated transgenic mice carrying DRB1*0401 and DRB1*0402 genes. We have previously shown that DRB1*0401 molecule renders B10.RQB3 (H2Aq) mice susceptible to porcine and human type II collagen-induced arthritis. We report that the introduction of DRB1*0402 transgene does not lead to development of arthritis in mice when they are immunized with porcine and human type II collagen. In addition, DRB1*0402 protects B10.RQB3 mice against developing arthritis with bovine type II collagen. These data show that DRB1 can modulate the disease mediated by Aq. In vivo depletion of DRB1*0402 did not lead to induction of collagen-induced arthritis in transgenic mice. In vitro cytokine analysis shows that mice protected from collagen-induced arthritis produce lower amounts of Th1 and higher levels of Th2 type cytokines upon immunization with type II collagen. Protection of mice was also related to higher apoptosis in DW10 mice as indicated by higher amounts of BclII in response to type II collagen. On the basis of our observations in HLA transgenic mice, we hypothesize that DRB1 polymorphism can modulate disease by shaping the T cell repertoire in thymus and select autoreactive T cells.