S100a9 Protects Male Lupus-Prone NZBWF1 Mice From Disease Development

Abstract

Systemic lupus erythematosus (SLE) is an autoimmune disorder disproportionally affecting women. A similar sex difference exists in the murine New Zealand Black/White hybrid model (NZBWF1) of SLE with all females, but only 30-40% of males, developing disease within the first year of life. Myeloid-derived suppressor cells (MDSCs) are prominent in NZBWF1 males and while depletion of these cells in males, but not females, promotes disease development, the mechanism of suppression remains unknown. S100a9, expressed by neutrophils and MDSCs, has previously been shown to exert immunosuppressive functions in cancer and inflammation. Here we investigated if S100a9 exerts immunosuppressive functions in NZBWF1 male and female mice. S100a9^+/+, S100a9^+/- and S100a9^-/- NZBWF1 mice were followed for disease development for up to 8 months of age. Serum autoantibody levels, splenomegaly, lymphocyte activation, glomerulonephritis and proteinuria were measured longitudinally or at the time of harvest. In accordance with an immunosuppressive function of MDSCs in male mice, S100a9-deficient male NZBWF1 mice developed accelerated autoimmunity as indicated by increased numbers of differentiated effector B and T cells, elevated serum autoantibody levels, increased immune-complex deposition and renal inflammation, and accelerated development of proteinuria. In contrast, female mice showed either no response to S100a9-deficiency or even a slight reduction in disease symptoms. Furthermore, male, but not female, S100a9^-/- NZBWF1 mice displayed an elevated type I interferon-induced gene signature, suggesting that S100a9 may dampen a pathogenic type I interferon signal in male mice. Taken together, S100a9 exerts an immunosuppressive function in male NZBWF1 mice effectively moderating lupus-like disease development via inhibition of type I interferon production, lymphocyte activation, autoantibody production and the development of renal disease.

Keywords: MDSC; S100A9; autoantibody; lupus; mouse model; proteinuria; sex specific effects; type I interferon.

Figure 1

S100a9 expression is elevated within Gr1^highCD11b⁺ cells of male lupus-prone mice and regulates B cell differentiation in vitro and antibody production in vivo. Gr1^highCD11b⁺ and Gr1^lowCD11b⁺ cells were isolated from 9 week old male and female NZBWF1 lupus-prone mice by flow cytometry and levels of S100a9 (A), S100a8 (B), and S100a4 (C) mRNA were determined by RT-PCR analysis (n = 4). Graphs show mean +/- SEM. (D) Supernatants from overnight cultured Gr1^highCD11b⁺ cells from 4 wk old NZBWF1 male mice suppress IFNα/CD40L-driven B cell differentiation. Shown is the average (± SEM) of 4 independent experiments. (E) S100A8/A9 heterodimer levels in serum, spleen and bone marrow from 9 wk old NZBWF1 mice. Each symbol represents one mouse. Male vs. female: p = 0.07; two-way ANOVA. (F) Gr1^highCD11b⁺ cells from 9 wk old S100a9-deficienct male NZBWF1 mice fail to suppress cytokine-driven B cell differentiation in vitro. IgM/IgG-secreting cells were enumerated by ELISPOT and are presented as number of cells per 10⁵ plated B cells. Data shown represent the mean ± SEM of 4 independent assays. *P < 0.05; **P < 0.01, Student’s unpaired t test.

Figure 2

Enhanced response to T-dependent Ag immunization in male S100a9^-/- NZBWF1 mice. Male (A) S100a9^+/+ (black squares), S100a9 ^+/- (grey diamonds), S100a9^-/- (symbol X), and female (B) S100a9^+/+ (black triangles, up), S100a9 ^+/- (grey triangles, down), S100a9^-/- (open circles) NZBWF1 mice (n = 3-4) were immunized on day 0 with NP₂₇-CGG in CFA. Levels of NP-specific IgG1 antibodies were determined on days -1, 7, 14, 21 and 28. *p < 0.05, two-way ANOVA. (C) GC B cells were identified by flow cytometry four weeks post immunization. *p < 0.05, Student’s unpaired t test with Welch’s correction. One-way ANOVA: p < 0.05.

Figure 3

S100a9^-/- male BWF1 mice develop splenomegaly and elevated anti-nuclear autoantibodies. Spleens were harvested from eight month old BWF1 lupus-prone mice. Spleen weight (A) and splenocyte count (B) were measured (n = 5-11). Each symbol represents one individual mouse, horizontal lines represent mean values. C-G) Serum was obtained from five months old BWF1 lupus-prone mice and serum autoantibody levels were determined by ELISA: serum IgM (C), serum IgG (D), anti-histone IgG (E), anti-chromatin IgG (F), anti-dsDNA (G). Each symbol represents one individual mouse (n = 5-11). (H) Hep2 Assay depicting nuclear and perinuclear antibody staining. Representative pictures shown from 5-11 mice/group. *p < 0.05; **p < 0.01; ***p < 0.001, Student’s unpaired t test with Welch’s correction. One-way ANOVA: p = 0.21 (A), p < 0.05 (B), p < 0.01 (C), p < 0.01 (D), p < 0.01 (E), p < 0.0001 (F), p = 0.87 (G).

Figure 4

S100a9-deficiency results in increased spontaneous germinal reactions in male BWF1 mice. Spleens were harvested from eight month old BWF1 lupus-prone mice and percentages of B cell subsets were determined by flow cytometry: (A) total B220+ cells, (B) Follicular mature (B220⁺CD23^highCD21^lowIgM^low) B cells, (C) Marginal Zone B cells (B220⁺CD21^highCD23^low IgM^high), (D) Germinal center B cells (B220⁺GL7⁺CD38^lowIgM^low), (E) memory B cells (B220⁺ CD38^highGL7^-IgM^-) and (F) Plasma cells (B220^-/lowCD138⁺IgM^-IgD^-). G) Frozen spleen sections were stained for B cells (B220-FITC) and germinal centers (GL-7-TexasRed). Representative pictures are shown. H) Germinal center areas from (G) were determined using the Keyence BZ-X analysis software. (A–F, H) Each symbol represents one individual mouse (n = 5-11). *p < 0.05; **p < 0.01, Student’s unpaired t test with Welch’s correction. One-way ANOVA: p = 0.13 (A), p = 0.14 (B), p = 0.33 (C), p < 0.01 (D), p = 0.36 (E), p < 0.01 (F), p < 0.01 (H).

Figure 5

S100a9-deficiency promotes the activation and differentiation of CD4+ T naïve cells to effector memory cells in S100a9^{-/ -} male BWF1 mice. Spleens were harvested from eight month old BWF1 lupus-prone mice and percentages of T lymphocytes and their subsets were determined by flow cytometry: total CD4⁺ cells (A), CD25⁺ CD4⁺ cells (B), CD69⁺ CD4⁺ cells (C), naïve (CD44^lowCD62L^high) CD4⁺ cells (D), effector memory (CD44^highCD62L^low) CD4⁺ cells (E), and the ratio between naïve and effector memory cells (F). Each symbol represents one individual mouse. *p < 0.05; **p < 0.01, Student’s unpaired t test with Welch’s correction. One-way ANOVA: p < 0.05 (A), p < 0.05 (B), p < 0.0001 (C), < 0.0001 (D), p < 0.05 (E), p < 0.01 (F).

Figure 6

S100a9-deficiency promotes proteinuria and renal damage in male BWF1 mice. Kidneys were harvested from eight month old BWF1 lupus-prone mice (n = 5-11) and stained. (A) H&E (a-f) and Masson’s Trichrome (g-m) were performed to assess glomerulonephritis, mesangial proliferation (black open-head arrows), interstitial inflammation, tubular atrophy, collagen deposits (blue closed-head arrows), and cast formation (red asterisk). All images were taken at similar settings and magnifications. (B, C) Deposition of IgG-immune complexes (red) and fixation of complement factor 3 (green) were determined by immunofluorescent staining of kidneys (B, n-s). Colocalization of IgG and C’3 can be seen as yellow. IgG deposition was quantified (C). (D) Proteinuria readings at the time of harvest. (E) Renal histology scores. Each symbol represents one individual mouse. *p < 0.05; **p < 0.01, Student’s unpaired t test with Welch’s correction (C, D), Student’s paired t- test (E). One-way ANOVA: p < 0.01 (C), p < 0.01 (D) and p < 0.01 (E).

Figure 7

Male S100a9-deficient BWF1 mice express elevated levels of mature neutrophils, but unaltered levels of BAFF. (A) Levels of Gr1^highCC11b⁺SSC^high mature neutrophils was measured in spleens of male and female S100a9 ^+/+, S100a9 ^+/- and S100a9 ^-/- mice by flow cytometry. (B) Serum BAFF levels were measured by ELISA on serum from 4 month old mice. Each dot represent one mouse. Shown is Mean ± SEM. *p < 0.05; **p < 0.01. One-way ANOVA: p < 0.05 (A), p = 0.14 (B).

Figure 8

Male S100a9-deficient BWF1 mice express elevated expression of interferon-stimulated genes and increased levels of low-density granulocytes. (A–C) Real-time RT-PCR was performed on total splenocytes obtained from a subset of S100a9 ^+/+ and S100a9 ^-/- male and female BWF1 mice at the time of harvest (≥ 7 months of age) and levels of Ifi202 (A), Irf7 (B), and Isg15 (C) were determined. Levels of total pDCs (D), SigH⁺ pDCs (E), and Low-density granulocytes (LDGs) (F), were determined by flow cytometry. Each dot represent one mouse. Shown is Mean ± SEM. *p < 0.05; **p < 0.01, ns, not statistically significant. One-way ANOVA: p < 0.05 (A), p = 0.08 (B), p = 0.07 (C), p = 0.09 (D), p = 0.67 (E), p = 0.0001 (F).