Aim2 deficiency in mice suppresses the expression of the inhibitory Fcgamma receptor (FcgammaRIIB) through the induction of the IFN-inducible p202, a lupus susceptibility protein

Murine Aim2 and Ifi202 genes (encoding for the Aim2 and p202 proteins) are members of the IFN-inducible Ifi200 gene family. The Aim2 deficiency in mice activates IFN signaling and stimulates the expression of the lupus susceptibility gene, the Ifi202, located within the NZB autoimmunity 2 (Nba2) interval. Given that the deficiency in the expression of the Fcgr2b gene (encoding for the inhibitory FcγRIIB receptor) is associated with increased lupus susceptibility in mice, we investigated whether the Aim2 protein could regulate the expression of Fcgr2b gene. In this article, we report that Aim2 deficiency in mice suppresses the expression of the FcγRIIB receptor. Interestingly, the Fcgr2b-deficient cells expressed increased levels of the IFN-β, activated IFN signaling, and expressed reduced levels of the Aim2 protein. Treatment of splenic cells with IFN-α or -γ reduced levels of the FcγRIIB mRNA and protein and also decreased the activity of the FcγRIIB p(-729/+585) Luc reporter. Moreover, levels of the FcγRIIB receptor were significantly higher in the Stat1-deficient splenic cells than in the wild-type cells. Accordingly, increased expression of IFN-β in lupus-prone B6.Nba2-ABC mice, as compared with non-lupus-prone C57BL/6 (B6) or B6.Nba2-C mice, was associated with reduced expression of the FcγRIIB receptor. Notably, overexpression of the p202 protein in cells decreased the expression of the Aim2 gene, activated the IFN response, and suppressed the expression of the Fcgr2b gene. These observations demonstrate that the expression of Aim2 protein is required to maintain the expression of the Fcgr2b gene and also predict epistatic interactions between the Ifi200 genes and the Fcgr2b gene within the Nba2 interval.     

Aim2 deficiency stimulates the expression of IFN-inducible Ifi202, a lupus susceptibility murine gene within the Nba2 autoimmune susceptibility locus

Murine Aim2 and p202 proteins (encoded by the Aim2 and Ifi202 genes) are members of the IFN-inducible p200 protein family. Both proteins can sense dsDNA in the cytoplasm. However, upon sensing dsDNA, only the Aim2 protein through its pyrin domain can form an inflammasome to activate caspase-1 and induce cell death. Given that the p202 protein has been predicted to inhibit the activation of caspase-1 by the Aim2 protein and that increased levels of the p202 protein in female mice of certain strains are associated with lupus susceptibility, we compared the expression of Aim2 and Ifi202 genes between Aim2-deficient and age-matched wild-type mice. We found that the Aim2 deficiency in immune cells stimulated the expression of Ifi202 gene. The increased levels of the p202 protein in cells were associated with increases in the expression of IFN-β, STAT1, and IFN-inducible genes. Moreover, after knockdown of Aim2 expression in the murine macrophage cell line J774.A1, IFN-β treatment of cells robustly increased STAT1 protein levels (compared with those of control cells), increased the activating phosphorylation of STAT1 on Tyr-701, and stimulated the activity of an IFN-responsive reporter. Notably, the expression of Aim2 in non-lupus-prone (C57BL/6 and B6.Nba2-C) and lupus-prone (B6.Nba2-ABC) splenic cells and in a murine macrophage cell line that overexpressed p202 protein was found to be inversely correlated with Ifi202. Collectively, our observations demonstrate an inverse correlation between Aim2 and p202 expressions. We predict that defects in Aim2 expression within immune cells contribute to increased susceptibility to lupus.     

Contribution of NZB autoimmunity 2 to Y-linked autoimmune acceleration-induced monocytosis in association with murine systemic lupus

The accelerated development of systemic lupus erythematosus (SLE) in BXSB male mice is associated with the presence of the Y-linked autoimmune acceleration (Yaa) mutation, which induces an age-dependent monocytosis. Using a cohort of C57BL/6 (B6) x (NZB x B6)F1 backcross male mice bearing the Yaa mutation, we defined the pathogenic role and genetic basis for Yaa-associated monocytosis. We observed a remarkable correlation of monocytosis with autoantibody production and subsequent development of lethal lupus nephritis, indicating that monocytosis is an additional useful indicator for severe SLE. In addition, we identified an NZB-derived locus on chromosome 1 predisposing to the development of monocytosis, which peaked at Fcgr2b encoding FcgammaRIIB and directly overlapped with the previously identified NZB autoimmunity 2 (Nba2) locus. The contribution of Nba2 to monocytosis was confirmed by the analysis of Yaa-bearing B6 mice congenic for the NZB-Nba2 locus. Finally, we observed a very low-level expression of FcgammaRIIB on macrophages bearing the NZB-type Fcgr2b allele, compared with those bearing the B6-type allele, and the development of monocytosis in FcgammaRIIB haploinsufficient B6 mice carrying the Yaa mutation. These data suggest that the Nba2 locus may play a supplementary role in the pathogenesis of SLE by promoting the development of monocytosis and the activation of effector cells bearing stimulatory FcgammaR, in addition to its implication in the dysregulated activation of autoreactive B cells.     

Effects of MHC and gender on lupus-like autoimmunity in Nba2 congenic mice

The lupus-like disease that develops in hybrids of NZB and NZW mice is genetically complex, involving both MHC- and non-MHC-encoded genes. Studies in this model have indicated that the H2d/z MHC type, compared with H2d/d or H2z/z, is critical for disease development. C57BL/6 (B6) mice (H2b/b) congenic for NZB autoimmunity 2 (Nba2), a NZB-derived susceptibility locus on distal chromosome 1, produce autoantibodies to nuclear Ags, but do not develop kidney disease. Crossing B6.Nba2 to NZW results in H2b/z F1 offspring that develop severe lupus nephritis. Despite the importance of H2z in past studies, we found no enhancement of autoantibody production or nephritis in H2b/z vs H2b/b B6.Nba2 mice, and inheritance of H2z/z markedly suppressed autoantibody production. (B6.Nba2 x NZW)F1 mice, compared with MHC-matched B6.Nba2 mice, produced higher levels of IgG autoantibodies to chromatin, but not to dsDNA. Although progressive renal damage with proteinuria only occurred in F1 mice, kidneys of some B6.Nba2 mice showed similar extensive IgG and C3 deposition. We also studied male and female B6.Nba2 and F1 mice with different MHC combinations to determine whether increased susceptibility to lupus among females was also expressed within the context of the Nba2 locus. Regardless of MHC or the presence of NZW genes, females produced higher levels of antinuclear autoantibodies, and female F1 mice developed severe proteinuria with higher frequencies. Together, these studies help to clarify particular genetic and sex-specific influences on the pathogenesis of lupus nephritis.     

Autoimmune-prone mice share a promoter haplotype associated with reduced expression and function of the Fc receptor FcgammaRII

Human autoimmune diseases thought to arise from the combined effects of multiple susceptibility genes include systemic lupus erythematosus (SLE) and autoimmune diabetes. Well-characterised polygenic mouse models closely resembling each of these diseases exist, and genetic evidence links receptors for the Fc portion of immunoglobulin G (FcR) with their pathogenesis in mice and humans [1] [2] [3]. FcRs may be activatory or inhibitory and regulate a variety of immune and inflammatory processes [4] [5]. FcgammaRII (CD32) negatively regulates activation of cells including B cells and macrophages [6]. FcgammaRII-deficient mice are prone to immune-mediated disease [7] [8] [9]. The gene encoding FcgammaRII, Fcgr2, is contained in genetic susceptibility intervals in mouse models of SLE such as the New Zealand Black (NZB) contribution to the (NZB x New Zealand White (NZW)) F1 strain [1] [10] [11] and the BXSB strain [12], and in human SLE [1] [2] [3]. We therefore sequenced Fcgr2 and identified a haplotype defined by deletions in the Fcgr2 promoter region that is present in major SLE-prone mouse strains (NZB, BXSB, SB/Le, MRL, 129 [13]) and non-obese diabetic (NOD) mice but absent in control strains (BALB/c, C57BL/6, DBA/2, C57BL/10) and NZW mice. The autoimmune haplotype was associated with reduced cell-surface expression of FcgammaRII on macrophages and activated B cells and with hyperactive macrophages resembling those of FcgammaRII-deficient mice, and is therefore likely to play an important role in the pathogenesis of SLE and possibly diabetes.     

Separation of the New Zealand Black genetic contribution to lupus from New Zealand Black determined expansions of marginal zone B and B1a cells

The F(1) hybrid of New Zealand Black (NZB) and New Zealand White (NZW) mice develop an autoimmune disease similar to human systemic lupus erythematosus. Because NZB and (NZB x NZW)F(1) mice manifest expansions of marginal zone (MZ) B and B1a cells, it has been postulated that these B cell abnormalities are central to the NZB genetic contribution to lupus. Our previous studies have shown that a major NZB contribution comes from the Nba2 locus on chromosome 1. C57BL/6 (B6) mice congenic for Nba2 produce antinuclear Abs, and (B6.Nba2 x NZW)F(1) mice develop elevated autoantibodies and nephritis similar to (NZB x NZW)F(1) mice. We studied B cell populations of B6.Nba2 mice to better understand the mechanism by which Nba2 leads to disease. The results showed evidence of B cell activation early in life, including increased levels of serum IgM, CD69(+) B cells, and spontaneous IgM production in culture. However, B6.Nba2 compared with B6 mice had a decreased percentage of MZ B cells in spleen, and no increase of B1a cells in the spleen or peritoneum. Expansions of these B cell subsets were also absent in (B6.Nba2 x NZW)F(1) mice. Among the strains studied, B cell expression of beta(1) integrin correlated with differences in MZ B cell development. These results show that expansions of MZ B and B1a cells are not necessary for the NZB contribution to lupus and argue against a major role for these subsets in disease pathogenesis. The data also provide additional insight into how Nba2 contributes to lupus.     

Differential role of three major New Zealand Black-derived loci linked with Yaa-induced murine lupus nephritis

By assessing the development of Y-linked autoimmune acceleration (Yaa) gene-induced systemic lupus erythematosus in C57BL/6 (B6) x (New Zealand Black (NZB) x B6.Yaa)F(1) backcross male mice, we mapped three major susceptibility loci derived from the NZB strain. These three quantitative trait loci (QTL) on NZB chromosomes 1, 7, and 13 differentially regulated three different autoimmune traits: anti-nuclear autoantibody production, gp70-anti-gp70 immune complex (gp70 IC) formation, and glomerulonephritis. Contributions to the disease traits were further confirmed by generating and analyzing three different B6.Yaa congenic mice, each carrying one individual NZB QTL. The chromosome 1 locus that overlapped with the previously identified Nba2 (NZB autoimmunity 2) locus regulated all three traits. A newly identified chromosome 7 locus, designated Nba5, selectively promoted anti-gp70 autoantibody production, hence the formation of gp70 IC and glomerulonephritis. B6.Yaa mice bearing the NZB chromosome 13 locus displayed increased serum gp70 production, but not gp70 IC formation and glomerulonephritis. This locus, called Sgp3 (serum gp70 production 3), selectively regulated the production of serum gp70, thereby contributing to the formation of nephritogenic gp70 IC and glomerulonephritis, in combination with Nba2 and Nba5 in NZB mice. Among these three loci, a major role of Nba2 was demonstrated, because B6.Yaa Nba2 congenic male mice developed the most severe disease. Finally, our analysis revealed the presence in B6 mice of an H2-linked QTL, which regulated autoantibody production. This locus had no apparent individual effect, but most likely modulated disease severity through interaction with NZB-derived susceptibility loci.     

Differences in Mucosal Gene Expression in the Colon of Two Inbred Mouse Strains after Colonization with Commensal Gut Bacteria

The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the animals with denaturing gradient gel electrophoresis revealed the development of a mouse strain-specific microbiota. Microarray-based gene expression analysis in the colonic mucosa identified 202 genes whose expression differed significantly by a factor of more than 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of more than 4 and observed a higher expression in C57BL/10 mice of the genes coding for Tlr1 and Ang4 which are involved in the recognition and response to gut bacteria. A higher expression of Pla2g2a was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1, Mal, Oasl2, Ifi202b, Rtp4, Ly6g6c, Ifi27l2a, Usp18, Ifit1, Ifi44, and Ly6g indicating that interferons may play an essential role in microbiota regulation. However, genes coding for interferons, their receptors, factors involved in interferon expression regulation or signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon.     

Autoimmune glomerulonephritis induced in congenic mouse strain carrying telomeric region of chromosome 1 derived from MRL/MpJ

In lupus erythematosus-prone mice, including the BXSB, NZW and NZB strains, telomeric regions of chromosome 1 (Chr.1) contain major glomerulonephritis susceptibility loci such as Bxs3, Sle1, and Nba2. To assess whether strain MRL, a model for lupus erythematosus, had glomerulonephritis susceptibility loci on Chr.1, we created B6.MRLc1(82-100) congenic mice carrying MRL/MpJ Chr.1 (82-100 cM) based on the C57BL/6 background and investigated renal pathology. From 6 months of age, B6.MRLc1 (82-100) showed the onset of diseases such as splenomegaly due to proliferation of CD3- or B220-positive cells, glomerular damage, and an increased serum anti-dsDNA antibody concentration, and these were earlier and severer in females. The score for glomerular damage was higher in B6.MRLc1(82-100) mice over 12 months old than in C57BL/6 or even in wild-type MRL/MpJ. Immune-complex depositions were demonstrated on glomerular basement membrane in B6.MRLc1(82-100) by immunohistochemistry and electron microscopy. For the percentage of IgG1-positive glomeruli, B6.MRLc1 (82-100) had significantly higher values than C57BL/6. In evaluations of clinical parameters, serum levels of blood urea nitrogen and the anti-dsDNA antibody in B6.MRLc1(82-100) were significantly higher than those in C57BL/6. In conclusion, B6.MRLc1(82-100) clearly developed autoimmune-mediated glomerulonephritis, and we demonstrated that MRL Chr.1 contained a novel glomerulonephritis susceptibility locus. We named this locus Mag (MRL autoimmune glomerulonephritis) and it provided new insights into the genetic basis and pathogenesis of lupus nephritis.