Deletion of CD24 impairs development of heat shock protein gp96-driven autoimmune disease through expansion of myeloid-derived suppressor cells

Abstract

CD24 binds to and suppresses inflammation triggered by danger-associated molecular patterns such as heat shock proteins (HSPs) and high-mobility group box 1. Paradoxically, CD24 has been shown to enhance autoimmune disease. In this study, we attempt to reconcile this paradox by deletion of CD24 (24KO) in a lupus-like disease model driven by forced expression of HSP gp96 at the cell surface (transgenic mice [tm]). As expected, tm24KO mice showed increased CD11c(+) dendritic cell activation coupled to a significant increase in dendritic cell-specific IL-12 production compared with tm mice. However, tm24KO mice showed less CD4 T cell activation and peripheral inflammatory cytokine production in comparison with tm mice. We characterized an enhanced immune suppressive milieu in tm24KO mice distinguished by increased TGF-β and greater regulatory T cell-suppressive capacity. We found greater absolute numbers of myeloid-derived suppressor cells (MDSCs) in tm24KO mice and showed that the Ly6C(+) MDSC subset had greater suppressive capacity from tm24KO mice. Deletion of CD24 in tm mice led to diminished lupus-like pathology as evidenced by anti-nuclear Ab deposition and glomerulonephritis. Finally, we show that expanded MDSC populations were mediated by increased free high-mobility group box 1 in tm24KO mice. Thus, the deletion of CD24 in an HSP-driven model of autoimmunity led to the unexpected development of regulatory T cell and MDSC populations that augmented immune tolerance. Further study of these populations as possible negative regulators of inflammation in the context of autoimmunity is warranted.

Figure 1. DC activation and IL-12 production in tm and tm24KO mice

Tm and tm24KO mice were subjected to flow cytometric analysis. (A) Surface staining of peripheral blood CD11c⁺ single-positive populations gated against SSC. Data of one representative mouse from 3 pairs of mice is shown. Averages of 3 mice per group from PBMC (*=p<0.05) or splenic CD11c⁺ populations are shown. (B) Single-positive CD11c⁺ population is gated and surface staining for CD80, CD86, and CD40 populations are gated and represented as average of MFI for 3 pairs of mice, (*=p<0.05). (C) PBMC from tm or tm24KO mice CD11c⁺ single-positive populations were gated on and intracellular IL-12 production was assessed. Data are graphed as average MFI of n=3 mice per group, (*=p<0.05). (D) Serum level of IL-12p40 at 6 months averaged for 6 pairs of mice, (*=p<0.05).

Figure 2. Decreased T cell activation, proliferation, and cytokine production in tm24KO mice

Tm and tm24KO mice were subjected to flow cytometric analysis. CD4⁺ single-positive populations were gated on. (A) Splenocytes from 6 month tm or tm24KO mice were surface stained for CD69⁺ and intracellular stained for BrdU after 3 days of BrdU water treatment. Data of one representative pair of mice from 4 pairs of mice is shown. Graph depicts statistical significance for CD4/CD69⁺ and CD4/CD69/BrdU⁺ cells, (*=p<0.05) (B) Splenocytes or mesenteric lymph node cells were incubated with PMA/Ionomycin in the presence of brefeldin A for 4–6 hours. Intracellular stain of IFN-γ (shown) and TNF-α were performed. Top flow panels show CD4-specific IFN-γ production. Bottom panels are gated on CD4⁺ single-positive cells and assessed for CD44/IFN-γ double positive populations. Data shown are one pair of mice, representative of 4 pairs of mice. Graph depicts statistical significance for CD4/TNF-α⁺ or CD4/IFN-γ⁺ splenocytes and mesenteric lymph node cells. (C) Peripheral blood mononuclear cells were incubated with PMA/Ionomycin in the presence of brefeldin A for 4 -6 hours. Intracellular stain of IFN-γ (shown) and TNF-α were performed. Data of one representative pair of mice from 5 pairs of mice is shown. Graph depicts statistical significance for CD4/TNF-α⁺ or CD4/IFN-γ⁺ PBMCs, (*=p<0.05).

Figure 3. Measurement of Treg associated suppressive parameters in tm and tm24KO mice

(A) Averages of active TGF-β measured by ELISA from 24 hour PBMC cultured supernatants (3 pairs of mice) or serum (6 pairs of mice), (*=p<0.05). (B) Tregs from spleens of tm or tm24KO mice were isolated and counted. Graphs are the averages for 3 pairs of mice, (*=p<0.05). (C) Splenocyte or mesenteric lymph node single-cell suspensions were extracellularly stained for CD4/CD25 (top panel). Splenocyte single-cell suspensions were incubated with PMA/Ionomycin in the presence of brefeldin A for 4–6 hours. Cells were extracellularly stained for CD4/CD25 and intracellular staining for Foxp3/IL-10 was performed. Bottom panels are gated on Foxp3+/CD4+ cells (*=p<0.05). Splenic data of one representative pair of mice from 3 pairs of mice is shown D) CD8 T cells and Tregs were isolated from tm or tm24KO spleens. CD8 T cells were labeled with CFSE and plated at ratio of 10:1 with strain specific Tregs on CD3/28 coated plates. CFSE dilution was analyzed by flow cytometry 72 hours later. Representative histograms from 3 separate experiments are shown. Data are presented as average MFI of CFSE (*=p<0.05).

Figure 4. Characterization of MDSC populations in tm and tm24KO mice

Tm and tm24KO mice were subjected to flow cytometric analysis. Extracellular CD11b+/Gr1+ single-positive populations were gated, except for (D). (A) Spleen cells were counted and total number of Cd11b+/Gr1+ cells was calculated. Graph depicts averages from 3 pairs of mice, (*=p<0.05). (B) Splenic or (C) peripheral blood CD11b⁺/Gr1^high (red) CD11b⁺/Gr1^dim (blue) cells are gated and histogram represents Ly6C⁺ dim (red) or high (blue) staining of these populations. (D) CD8 T cells, Ly6G⁺ and Ly6C⁺ cell fractions were isolated from tm or tm24KO spleens. Effector cells were labeled with CFSE and plated at ratios of 1:1 and 10:1 with strain specific MDSC subsets. CFSE dilution was analyzed by flow cytometry 72 hours later. Representative histograms from 3 separate experiments are shown. Data are presented as average MFI of CFSE (*=p<0.05). (E) Ly6G⁺ and Ly6C⁺ cell fractions were isolated from tm or tm24KO spleens and 1×10⁵ cells were plated with or without 1μg/mL LPS for 24 hours. Greiss reaction was performed on cell supernatants (*=p<0.05).

Figure 5. Decreased autoimmune disease in tm24KO mice as compared to tm mice

Mean fluorescent staining intensity (MFI) of (A) ANA (B) GN in 6 month old tm or tm24KO mice categorized as clinical score 1–5 for disease severity (5 is sever disease). (C) Quantification of H&E number of nuclei per glomeruli (20X). Data are representative of one pair of 5 or more pairs of mice, (*=p<.05). (C) Serum level of IgM, IgA, IgG1, IgG3, IgG2b, IgG2c are averages of 5 pairs of mice, (*=p<.05).

Figure 6. HMGB1 recruits increased numbers of MDSC in tm24KO mice

(A) Western blot for HMGB1 on serum from tm (+) or tm24KO (−) mice aged 12 or 3 months. Data are representative of results from 4 pairs of mice. Albumin is used as a loading control from Comassie blue de-stained gel. Tm or tm24KO mice were treated with 100 ng/mouse of rHMGB1 and flow cytometryic analysis was performed on single-suspension PBMC pre (0 hr) and post (4 hr) injection for extracellular stain CD11b/Gr1⁺. (B) Data of one representative pair of mice from 3 pairs of mice is shown (C) Data are graphed as percentage increase of CD11b/Gr1⁺ cells from 0 hr to 4 hr time point (*=p<.01).