Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice

Abstract

In inflammatory bowel diseases (IBDs), particularly ulcerative colitis, intestinal macrophages (MΦs), eosinophils, and the eosinophil-selective chemokine CCL11, have been associated with disease pathogenesis. MΦs, a source of CCL11, have been reported to be of a mixed classical (NF-κB-mediated) and alternatively activated (STAT-6-mediated) phenotype. The importance of NF-κB and STAT-6 pathways to the intestinal MΦ/CCL11 response and eosinophilic inflammation in the histopathology of experimental colitis is not yet understood. Our gene array analyses demonstrated elevated STAT-6- and NF-κB-dependent genes in pediatric ulcerative colitis colonic biopsies. Dextran sodium sulfate (DSS) exposure induced STAT-6 and NF-κB activation in mouse intestinal F4/80(+)CD11b(+)Ly6C(hi) (inflammatory) MΦs. DSS-induced CCL11 expression, eosinophilic inflammation, and histopathology were attenuated in RelA/p65(Δmye) mice, but not in the absence of STAT-6. Deletion of p65 in myeloid cells did not affect inflammatory MΦ recruitment or alter apoptosis, but did attenuate LPS-induced cytokine production (IL-6) and Ccl11 expression in purified F4/80(+)CD11b(+)Ly6C(hi) inflammatory MΦs. Molecular and cellular analyses revealed a link between expression of calprotectin (S100a8/S100a9), Ccl11 expression, and eosinophil numbers in the DSS-treated colon. In vitro studies of bone marrow-derived MΦs showed calprotectin-induced CCL11 production via a p65-dependent mechanism. Our results indicate that myeloid cell-specific NF-κB-dependent pathways play an unexpected role in CCL11 expression and maintenance of eosinophilic inflammation in experimental colitis. These data indicate that targeting myeloid cells and NF-κB-dependent pathways may be of therapeutic benefit for the treatment of eosinophilic inflammation and histopathology in IBD.

Figure 1. Eosinophil recruitment and CCL11 expression are STAT-6-independent in DSS-induced colitis

A, Representative flow cytometry plots of F4/80⁺CD11b⁺Ly6C^hi colonic MΦs and representative histograms of phospho-STAT-6 expression in DSS-treated WT mice (open histogram) compared to STAT-6^−/− mice (filled histogram) and mean fluorescence intensity of phospho-STAT-6. B, Histological score. C, Representative photomicrographs of H&E-stained colonic sections from baseline and DSS-treated mice (day 7, 2.5% DSS). D, Eosinophil levels in the colon and E, representative photomicrographs of anti-MBP-stained colonic sections from baseline and DSS-treated mice (day 7, 2.5% DSS). F, CCL11 levels in punch biopsy supernatants from WT and STAT-6^−/− mice at baseline and following 5 days of DSS. (A). Data represents the mean ± SEM of n = 3–7 mice per group from duplicate experiments. (B-F) Data represents the mean ± SEM of n = 5–6 mice per group. Data is representative of triplicate experiments. Significant differences (*p<0.05; **p<0.01; ***p<0.001) between groups.

Figure 2. Specificity of RelA/p65 deletion in RelA/p65^Δmye mice

A, Representative flow cytometry plots and histograms of phospho-RelA/p65 expression in F4/80⁺CD11b⁺Ly6C^hi colonic MΦs from DSS-treated WT (open histogram) and RelA/p65^Δmye mice (filled histogram). B. Quantification of Nfkbia expression from Ly6C^hi MΦs/monocytes sorted from the peripheral blood or colon of DSS-treated mice. Data represent the mean ± SEM of n = 5 mice per group. Representative western blot analysis of WT (+) and RelA/p65^Δmye (−) (C) BMDMs stimulated with 1 ug/mL LPS for 1 hour, (D), colonic epithelium and spleen of total RelA/p65 and/or phospho-RelA/p65. E, representative western blot analysis of IKKα, IκBα, IκBβ, c-Rel, p105 and actin expression in BMDMs stimulated with LPS for indicated times (minutes). F, WT and RelA/p65^Δmye BMDMs were stimulated for 24 hours with vehicle or 1 ug/mL LPS and levels of IL-6, TNF-α, IL-1β, NO₂ and IL-12p40 were measured in the supernatants. G, Representative flow cytometry plots and graph of BMDMs stimulated for 24 hours with control, IL-4 or IFN-γ + LPS and assessed for active caspase-3. (F and G) Data represent the mean ± SEM of n = 3 individual samples per group. (A, C, E–G) Data is representative of duplicate experiments. Significant differences (*p<0.05**, p < 0.01; ***p < 0.001) between groups.

Figure 3. DSS-induced colitis is attenuated in RelA/p65^Δmye mice

A, Percent weight change and (B) Disease activity index (DAI) of WT and p65^Δmye mice following DSS exposure. C, colon lengths D, representative photomicrographs and (E) quantitative histological score of H&E-stained colonic sections from of WT and p65^Δmye mice following 7 days of DSS exposure. F, Cytokine Profile of colonic punch biopsy samples from WT and p65^Δmye mice following DSS-exposure. Data represent the mean ± SEM of n = 3–5 mice per group. Data is representative of triplicate (A–E) and duplicate (F) experiments. Significant differences (*p < 0.05; **p < 0.01; ***p < 0.001) compared with WT vehicle or as indicated. Magnification of photomicrographs is ×100. n.s. – not significant.

Figure 4. Colonic recruitment of myeloid cells is not impaired in p65^Δmye mice

A, Representative flow cytometry plots and B, quantification of the F4/80⁺CD11b⁺Ly6C^hi MΦ populations from the colon of WT and RelA/p65^Δmye mice following 5 days of vehicle (baseline) or DSS exposure. MΦs were initially gated by SSC vs. FSC followed by F4/80⁺, CD11b⁺ and Ly6C^hi. C, Representative flow cytometry plots and D, quantification of the F4/80⁻CD11b^hi neutrophil population from the colon of WT and RelA/p65^Δmye mice following 5 days of vehicle (baseline) or DSS exposure. MΦs were initially gated by SSC vs. FSC followed by F4/80⁻ and CD11b^hi. B and D, data represents the mean ± SEM of n = 5–6 mice per group and is representative of duplicate experiments. Significant differences (*p < 0.05; ***p < 0.001) between groups. n.s. – not significant.

Figure 5. Lack of induction of CCL11 and eosinophils in RelA/p65^Δmye mice during DSS-induced colitis

A, Representative photomicrographs of anti-MBP-stained colonic sections from baseline and DSS-treated mice (day 6, 2.5%) and colonic eosinophil levels in the colon. B, CCL11 levels in punch biopsy supernatants from WT and RelA/p65^Δmye mice in vehicle- (baseline) and DSS-treated mice (day 5, 2.5%) Data indicates the mean ± SEM of n = 6–8 mice per group from triplicate experiments. Significant differences (*p < 0.05; ***p < 0.001) between groups. Magnification of photomicrographs is ×100.

Figure 6. Phenotype Ly6C^hi colonic MΦs in WT vs. RelA/p65^Δmye mice during DSS-induced colonic injury

A, Representative flow cytometry plot of CD11b⁺Ly6C^hi colonic MΦs flow sorted on day 5 of DSS treatment. B, gene expression was analyzed by qRT-PCR. 3–4 mice were pooled per sample from DSS-treated WT and RelA/p65^Δmye mice (3–4 samples per group). Data represents the mean ± SEM. Data is representative of duplicate experiments. Significant differences (*p < 0.05; **p < 0.01) between groups. n.s. – not significant.

Figure 7. Relationship between eosinophil-specific gene (Ear5), Ccl11 and Calprotectin (S100a8 and S100a9) expression in DSS-induced colitis

A. Heat map showing the clustering of the top 25 differentially-expressed genes following DSS-induced colitis (day 6 vs. day 0, p < 0.05). B. Top 10 differentially-expressed genes following DSS-induced colitis (day 6 vs. day 0, p < 0.05, fold change > 10) that correlated with Ccl11 expression (r > 0.75). C. Pearson product-moment correlation between normalized microarray expression values of Ear5, S100a8 and S100a9 are plotted against Ccl11 from individual animals. (D) Pearson product-moment correlation between S100a8 and S100a9/Hprt ratio and eosinophils/hpf in the colon of WT C57BL6 mice on day 3, 5 and 7 after DSS exposure. E. Representative flow plots of RAGE expression on (E.) colonic F4/80⁺ CD11b⁺ Ly6C^hi monocytes/MΦ from DSS-treated mice and (F.) BMMΦ. G. CCL11 levels in supernatants from WT and RelA/p65^Δmye BMMΦ following 24 hour stimulation with S100a8/S100a9 complex. G. Data represents the mean ± SEM. Data is representative of duplicate experiments. Significant differences (**p < 0.01) between groups.