Blockade of IL-7Rα ameliorated Hb-induced colitis in Mdr1a−/− mice
Mdr1a−/− mice develop colitis when infected with certain enteric helicobacter species [11]. To determine the effectiveness of blocking IL-7Rα in colitis, we administered an anti-IL-7Rα antibody to Mdr1a−/− mice beginning one week prior to infection with Hb and continuing for the duration of the experiment. Previous studies with anti-IL-7Rα antibodies resulted in decreased lymphocyte numbers in naïve and diseased mice (Amgen Inc.; data not shown). Therefore, we used two doses of anti-IL-7Rα M595 to determine if a 10-fold lower dose would preserve lymphocyte numbers yet still afford protection from colitis. As shown in Figure 1A, there was significant suppression of IBD in all mice receiving either 500 μg or 50 μg of anti-IL-7Rα M595. The colons from isotype Hb-infected mice (positive controls) were irregularly thickened and opaque along the entire length with no formed fecal pellets in the distal colon and showed severe transmural lymphohistiocytic and proliferative colitis (Figure 1B; isotype Hb). Anti-IL-7Rα-treated Hb-infected mice were normal with well-formed fecal pellets in the distal colon and normal diameter of the proximal colon and were generally considered normal histologically (Figure 1B, anti-IL-7R M595/500 Hb) or had some mild focal colitis (anti-IL-7R M595/50 Hb). Colons from isotype-broth (negative controls) mice were normal grossly and histologically (Figure 1B, isotype broth). Associated with suppression of IBD, anti-IL-7Rα-treated Hb-infected mice had an overall trend of higher percent weight gain compared with Hb-infected isotype-treated mice, and the weight gain was similar to uninfected isotype-treated mice (Figure 1C). Mice were analyzed further to determine which immune cells and inflammatory mediators were altered with anti-IL-7Rα M595 treatment.
Anti-IL-7Rα M595 treatment reduced CD4+ and CD8+ T cells
We focused our analysis on T-cell subsets trafficking to the mesenteric lymph node (MLN) as T-cell expansion is associated with the development of colitis in Hb-infected Mdr1a−/− mice [11]. Flow cytometric analysis (FACS) revealed that both doses of anti-IL-7Rα M595 significantly prevented the expansion of total MLN cellularity and total CD4+ and CD8+ T cells compared to both the isotype-broth and isotype-Hb-infected mice. Treatment with both anti-IL-7Rα antibody doses further reduced MLN, CD4+, and CD8+ cellularity below that of Hb-infected mice treated with isotype control. However, there were significantly more cells in the anti-IL-7Rα 50 μg-treatment group compared to the 500 μg-treatment group (Figure 2A-C). The higher dose of anti-IL-7Rα M595 resulted in a larger decrease in CD4+ T-cell numbers (9.8-fold decrease vs. isotype-broth group) than in CD8+ T-cell numbers (4.5-fold decrease vs. isotype-broth group), suggesting total CD4+ T cells had a preferential requirement for IL-7R signaling compared to total CD8+ T cells. The lower dose of anti-IL-7Rα M595 preserved the ratio of CD4+ to CD8+ T cells similar to control groups (Figure 2D). We observed similar results for naïve to activated/memory CD4+ (Figure 2E) and CD8+ (Figure 2F) T-cell ratios with both doses of anti-IL-7Rα M595 treatment. The lower dose of anti-IL-7Rα M595 preserved the ratio of CD4+ naïve to memory T cells similar to control groups. There was a greater decrease in absolute numbers of naïve cells versus activated/memory cells (Additional file 1: Figure S1) with anti-IL-7Rα antibody treatment, suggesting that naïve T cells were more sensitive to reduced IL-7R signaling than activated/memory T cells. Our results indicate that a relatively low dose of anti-IL-7Rα M595 can protect mice from Hb-induced colitis without inducing lymphopenia to the extent seen with the high dose of anti-IL-7Rα M595. Associated with the alterations in T-cell numbers, we observed a reduction in Hb-specific IgG2a antibody titers with anti-IL-7Rα M595 treatment (Additional file 2: Figure S2), suggesting dysregulation of T-dependent-B-cell responses as well.
Blocking IL-7Rα reduced mediators of colonic inflammation and revealed a role for innate immunity
To investigate how anti-IL-7Rα antibody treatment reduced and/or prevented inflammation in Hb-infected Mdr1a-/- mice, we analyzed messenger (m) RNA and protein expression in colon tissues. Proximal colon mRNA expression is depicted in a heat map (Figure 3A) comparing expression changes occurring with infection (mean fold change of isotype Hb divided by mean fold change of isotype broth) and expression changes when Hb-infected mice were treated with anti-IL-7Rα M595 antibody (mean fold change of anti-IL-7R M595 divided by mean fold change of isotype Hb). Many cytokines, chemokines, and cell-surface markers associated with inflammatory responses were increased with Hb infection and were correspondingly decreased with anti-IL-7Rα M595 treatment (Figure 3A). Although both doses of anti-IL-7Rα M595 clearly prevented the inflammatory response to Hb infection, the 500 μg dose had an overall greater effect on regulating gene expression than the 50 μg dose. Specifically, inflammatory cytokines IFN-γ, IL-6, IL-1α, IL-1β, IL-12p35, and IL-17, as well as chemokines strongly induced by IFNγ, such as activated T-cell recruiter, CXCL11 and a more general inflammatory-cell recruiter, CXCL10, followed this pattern. Costimulatory molecules (CD80, CD86, CD40, GITR, and ICOS), adhesion molecules (VCAM-1 and P-selectin), and signaling molecules involved in cytokine regulation (Tbx21 and Socs1) were also increased with Hb infection and decreased with anti-IL-7Rα M595 treatment in a dose-dependent manner. Genes involved in apoptosis inhibition (Bcl2, Vegfa) and cytokine modulation (Socs2, Ski, Smad3) were downregulated with Hb infection and increased with anti-IL-7Rα M595 treatment. These data also indicate that colonic IL-7 and IL-15 gene expression was downregulated with Hb infection, and upregulated with anti-IL-7Rα M595 treatment. The decrease in these two cytokines with infection and increase with anti-IL-7Rα treatment may be due to homeostatic regulation.
RNA expression of immune-cell markers was also altered with anti-IL-7Rα treatment. Similar to changes of CD4+ T cell numbers in MLN, colonic CD4 mRNA expression was increased in Hb-infected mice and was decreased in Hb-infected mice treated with both doses of anti-IL-7Rα M595 (Figure 3B). The effect was specific to CD4 expression (presumably on CD4+ T cells) as there were no significant differences in CD3ε or CD8α mRNA expression in colonic tissue (data not shown). Although not significantly increased with Hb infection, colonic IL-7Rα mRNA expression was reduced following anti-IL-7Rα M595 treatment. The lower dose of anti-IL-7Rα M595 was not associated with a significant decrease in IL-7Rα mRNA expression compared to either control, whereas the high dose of anti-IL-7Rα M595 had 7-fold less IL-7Rα expression compared to isotype-broth and isotype-Hb groups (Figure 3C). Thus, not only did anti-IL-7Rα treatment reduce cellularity in draining lymph nodes, but blocking IL-7Rα reduced immune-cell markers and inflammatory mediators in the colon as well.
Mdr1a−/− mice were chosen to test the effects of anti-IL-7Rα in colitis because they are a T- and B-cell sufficient strain. Although many of the changes in gene expression in the colon were related to T cells, several of the regulated genes are involved in innate immunity. For example, genes involved in trafficking and activation of macrophages, natural killer (NK) cells, and dendritic cells (DC) (CCR2, CCL19/CCR7, CCL3, CCL2, Csf3), promotion of cellular responses (CD68, TNFα, IL-10, CCL3, GITR, IL-6, IL-1α, IL-1β, IL-12p35), and innate responses to bacterial infection (C3, CD40, Gusb, Nos2) were upregulated with Hb infection and downregulated with anti-IL-7Rα M595 treatment in a dose-dependent manner (Figure 3A).
To correlate gene expression changes in colonic tissue with changes in secreted proteins, we analyzed serum samples and proximal colon explant cultures by multi-analyte profiling. Serum protein for IL-17, IL-6, CXCL10 (IP-10), and CCL2 (MCP1) had similar expression-pattern changes to those seen in the gene array, showing increases with Hb infection and decreases with anti-IL-7Rα M595 treatment (Figure 4, Additional file 3: Table S1). Additional analytes detected in the serum (but not represented in the gene array panel) also revealed that blocking IL-7Rα regulated innate immune responses. These included MIP-1γ (CCL9), MDC (CCL22), MIP-3β (CCL19), and MPO. Analysis of local production of proteins in colon explants showed expression of CCL3, CXCL10, IFN-γ, IL-17, IL-1α and IL-1β all had similar expression patterns compared to gene expression in adjacent tissue (increased expression with Hb infection and a dose-dependent decreased expression with anti-IL-7Rα M595 treatment) (Additional file 3: Table S1). As an example, correlations between histopathology scores, serum and colon protein, and colon mRNA are shown for CXCL10 (IP-10) in Figure 5. Taken together, these results indicate that an innate-immune signature occurred both locally and systemically in Hb-infected mice and suggested that an anti-IL-7Rα antibody could regulate adaptive- as well as innate-immune responses in colitis.
Blocking IL-7Rα ameliorated colitis in the absence of T and B cells
Given our results successfully treating Hb-induced colitis in Mdr1a−/− mice by inhibiting IL-7R signaling, we determined whether non-lymphoid cells also respond to anti-IL-7Rα treatment as was suggested in the mRNA and protein analysis. Therefore, we used T- and B-cell deficient Rag2−/− mice to determine the effects of blocking IL-7Rα on non-lymphoid cells in Hb-induced colitis. We tested anti-IL-7Rα M595 at two doses (250 and 50 μg) in Hb-infected-Rag2−/− mice. Both doses suppressed inflammation in Hb-infected-Rag2−/− mice, and they were not statistically different from one another (Figure 6A). Percent weight change was examined throughout the duration of the study. All groups of Hb-infected mice had a slight amount of weight loss the first week after infection which did not occur in uninfected mice. Starting at 2 weeks after infection, all groups of Hb-infected mice started to gain weight which continued for the duration of the study and were not statistically different than uninfected mice. No differences in percent weight change were seen between Hb-infected mice and anti-IL-7Rα-treated Hb-infected mice (Figure 6B). Representative H&E-stained sections of proximal colon from uninfected control mice (isotype broth), and Hb-infected mice treated with isotype control or anti-IL-7Rα M595 are shown in Figure 6C. Hb-induced disease in Rag2−/− mice was less severe than that in Mdr1a−/− mice and was characterized by moderate hyperplastic and lymphohistiocytic colitis with elongation of colonic glands and expansion of lamina propria and submucosa with inflammatory cells (isotype Hb). In contrast, the colons from anti-IL-7Rα treated Hb-infected mice had very mild lesions.
Multiple cell subsets were altered with IL-7Rα blockade in Rag2−/− mice
Given that disruption of IL-7R signaling causes T cells to decline by blocking survival and proliferation signals, it was possible that non-lymphoid cell numbers in the Rag2−/− mice could also be affected by IL-7Rα blockade. To determine which cell types were involved in colitic disease in Hb-infected Rag2−/− mice and whether the anti-IL-7Rα antibodies had any effects on particular cell types, we used FACS to analyze splenic macrophages, DCs, and NK cells. Total myeloid cell numbers were significantly increased with Hb infection and significantly decreased with both doses of anti-IL-7Rα M595 (Figure 7A). Separation of splenocytes based on cell surface expression of CD11c and CD11b indicated 5 populations of DCs and macrophages: Plasmacytoid DCs (pDC; CD11b-CD11clo), myeloid DCs (mDCs; CD11bhiCD11chi), macrophage 1 (Mac1; CD11bloCD11c-), macrophage 2 (Mac2: CD11bhi CD11clo/-) and macrophage 3 (Mac3; CD11bloCD11clo) (Figure 7B). We enumerated NK cells using surface expression of DX5. The expression of IL-7Rα on each subtype was determined, and numbers of total or IL-7Rα+ pDC, mDC, NK, Mac2, and Mac3 cells were increased with Hb infection (Figure 7C). Mac1 cell numbers were not altered significantly with disease. Treatment of mice with anti-IL-7Rα antibodies either maintained cell numbers similar to uninfected mice or lowered cell numbers further from uninfected control levels (Figure 7C). Some IL-7Rα+ cell subsets appeared to be more sensitive to anti-IL-7Rα M595 antibody treatment than others. For example, the decrease in cell numbers compared to the uninfected isotype control group was greater for IL-7Rα+ pDC than for total pDC (13- vs. 2.2-, and 18.6- vs. 2.1-fold decrease in M595/250 and M595/50 groups, respectively). This was also true for NK cells where anti-IL-7Rα M595 treatment groups had 17- and 22-fold fewer numbers (M595/250 and M595/50 groups, respectively) of IL-7Rα+ NK cells compared to uninfected isotype controls. This was in contrast to total NK cells in which anti-IL-7Rα-M595-treated groups had similar NK-cell numbers to uninfected control mice. Unlike the results in Mdr1a−/− mice, there were no significant differences between anti-IL-7Rα M595 doses on IBD scores or cell numbers in these studies. In addition, we used F4/80 immunohistochemistry to identify macrophage/DCs in proximal colon (Figure 7D). Consistent with findings in spleen for macrophages and DCs, F4/80+ cells in the colon were increased with disease and treatment with anti-IL-7Rα M595 preserved F4/80+ cell numbers similar to uninfected control mice. These observations strongly suggest that innate immune cells, including those expressing IL-7Rα, are involved in Hb-induced colitis, and that anti-IL-7Rα can limit their expansion and protect from development of colitis.
Anti-IL-7Rα treatment of Hb-infected Rag2−/− mice reduced mediators of colonic inflammation
To assess the activity of innate cells, we measured serum proteins by multi-analyte profiling. In the absence of T and B cells, there were fewer analytes altered with infection or with anti-IL-7Rα M595 treatment compared with the number of changes seen in Hb-infected Mdr1a−/− mice. However, the majority of analytes altered in these mice were chemotactic proteins (Figure 8). Circulating concentrations of lymphotactin, MIP-1γ, MPO, MMP9, as well as VCAM-1, CRP, SAP, and haptoglobin were altered with Hb-infection and reduced in Hb-infected mice treated with anti-IL-7Rα M595. Some analytes were not significantly increased with infection but were significantly reduced by treatment with the anti-IL-7Rα M595: MCP-1, MCP-3, MDC, MIP-1α, MIP3β, GCP-2, IL-18, Tissue Factor, and TPO. Thus, in line with the reduction in myeloid cells, innate-immune mediators were also reduced with IL-7Rα blockade and strongly indicate that anti-IL-7Rα treatment controls colitis by multiple mechanisms.