CCL25/CCR9 interactions regulate large intestinal inflammation in a murine model of acute colitis

Abstract

Background aims: CCL25/CCR9 is a non-promiscuous chemokine/receptor pair and a key regulator of leukocyte migration to the small intestine. We investigated here whether CCL25/CCR9 interactions also play a role in the regulation of inflammatory responses in the large intestine.

Methods: Acute inflammation and recovery in wild-type (WT) and CCR9(-/-) mice was studied in a model of dextran sulfate sodium (DSS)-induced colitis. Distribution studies and phenotypic characterization of dendritic cell subsets and macrophage were performed by flow cytometry. Inflammatory bowel disease (IBD) scores were assessed and expression of inflammatory cytokines was studied at the mRNA and the protein level.

Results: CCL25 and CCR9 are both expressed in the large intestine and are upregulated during DSS colitis. CCR9(-/-) mice are more susceptible to DSS colitis than WT littermate controls as shown by higher mortality, increased IBD score and delayed recovery. During recovery, the CCR9(-/-) colonic mucosa is characterized by the accumulation of activated macrophages and elevated levels of Th1/Th17 inflammatory cytokines. Activated plasmacytoid dendritic cells (DCs) accumulate in mesenteric lymph nodes (MLNs) of CCR9(-/-) animals, altering the local ratio of DC subsets. Upon re-stimulation, T cells isolated from these MLNs secrete significantly higher levels of TNFα, IFNγ, IL2, IL-6 and IL-17A while down modulating IL-10 production.

Conclusions: Our results demonstrate that CCL25/CCR9 interactions regulate inflammatory immune responses in the large intestinal mucosa by balancing different subsets of dendritic cells. These findings have important implications for the use of CCR9-inhibitors in therapy of human IBD as they indicate a potential risk for patients with large intestinal inflammation.

Figure 1. Expression of CCR9 and CCL25 in the large intestine.

(A) Quantification of CCR9 and CCL25 mRNA levels from intestinal tissues of WT, CCR9^−/− and CCL25^−/− mice. Y-axis shows expression levels normalized to β-actin mRNA (mean +/− SD, n = 3). PP: Peyer's Patches, SI: Small intestine, LI: Large intestine. Data represent the mean +/− SD of 3 independent experiments, performed in triplicate (B) Acute DSS-mediated colitis. Induction phase: 2% DSS in drinking water for 7 days. Recovery: 10 days water. Mice were analyzed at days 0 (d0), d7, d10, d13 and d17. (C) CCL25 mRNA expression increases during the recovery phase of DSS-mediated colitis, (mean +/− SD; d0, n = 3; d10, n = 3; d13, n = 5; d17, n = 5; where n corresponds to a single experiments of 3 to 5 pooled intestinal samples).

Figure 2. CCR9^−/− mice show increased mortality and delayed recovery during DSS-mediated colitis.

(A) 30.65% mortality was observed in CCR9^−/− mice (open circles, n = 78). WT mice are shown in gray circles (n = 66). Data are representative of 17 pooled experiments. (B) CCR9^−/− mice showed a more pronounced body weight loss than WT. Body weight was monitored daily and is expressed as percent of weight at d0 (data represent the mean +/− SD of 17 pooled experiments of 3–5 mice per group). Open circles show CCR9^−/− mice, gray circles WT.

Figure 3. Exacerbation of DSS-mediated colitis in CCR9^−/− mice.

(A) Higher IBD scores during the recovery phase in CCR9^−/− animals. WT mice are depicted in gray circles (n = 51), CCR9^−/− mice in open circles (n = 63). (B) Representative H&E sections from colons of WT and CCR9^−/− mice (d0, d13 and d17; 10×-magnification). CCR9^−/− intestinal mucosa displays exacerbated IBD symptoms. (C) Significant colon shortening in CCR9^−/− mice (open circles) at d17. Colon length was measured from d0 to d17. ***P<0.005, n.s. not significant.

Figure 4. The gastrointestinal tract of CCR9^−/− animals shows multiple signs of exacerbated tissue inflammation.

(A) Accumulation of inflammatory macrophages. Flow cytometry analysis of MLNs of WT and CCR9^−/− animals. Frequencies of inflammatory macrophages were determined in MLN suspensions with anti-CD11c, CD11b, F4/80, Ly6C and MHC-II (in black) overlaid on isotype control mAbs (in gray). Data represent the mean (+/− SD) of 3 independent experiments of 3–5 pooled mice per genotype. (B and C) mRNA profiling of large intestinal tissues. Total RNA was prepared from the colon of WT and CCR9^−/− mice at d0 and d17 and was analyzed with the nCounter™ system (mean +/− SD of 3–5 animals per group of 2 independent experiments). (B) Macrophage accumulation is reflected by increased mRNA levels of CD64 and Arginase1. (C) Comparison of tissue immunophenotype of the large intestinal mucosa before and after DSS-mediated colitis in WT and CCR9^−/− animals. (D and E) T cell re-stimulation assay for cytokine production. (D) Representative cytometry bead array assay for Th1/Th2/Th17 cytokines. (E) Production of TNFα, IFNγ, IL-2, IL-6 and IL-17A is upregulated while production of IL-10 and IL-4 is down-regulated in T cells from CCR9^−/− animals. Quantification of cytokine production from WT (gray bars) and CCR9^−/− cells (open bars). Data represent one experiment out of 3 independent experiments, performed in triplicate (mean +/− SD) of 3 pooled WT MLN and 3 pooled CCR9^−/− MLN cell suspensions reactivated in vitro with anti-CD3ε/anti-CD28 mAbs for 72 h.

Figure 5. CCR9^−/− mice show altered ratios of dendritic cell subpopulations at steady state and during DSS-mediated colitis.

(A) WT MLNs contain CCR9⁺ and CCR9⁻ pDCs gated as MHC II^int and PDCA-1^high (left dot plot). CCR9 expression in WT pDCs (black line, right histogram) is overlaid on CCR9^−/− pDCs (in gray, right histogram) (B) Steady state distribution of pDCs and cDCs is altered in SPL and MLN of CCR9^−/− mice. No significant difference between strains is observed in the colon. cDCs were defined as CD11c^high and MHC II^high cells by flow cytometry. Ratio of subpopulations is expressed on the y-axis as pDC/cDC cells. Data represent the mean +/− SD of 4 independent experiments of 3–5 pooled mice per group. (C) Accumulation of pDCs in MLNs of CCR9^−/− animals. Percentages of pDCs in WT and CCR9^−/− MLN are depicted in bold from d0 to d17 of DSS colitis. (D) Accumulation of pDCs correlates with the level of tissue damage. Increase of DSS concentration to 4% augments the number of CCR9^low/− B220⁺ PDCA-1⁺ MHC II^int/high cells in MLNs of WT animals. (E) DSS-mediated tissue inflammation alters pDC/cDC ratios in MLN of CCR9^−/− animals. Data points correspond to individual experiments corresponding to 17 DSS colitis experiments harvested at different time point using 3–5 pooled mice per genotype.

Figure 6. pDCs from CCR9^−/− MLNs are more activated than WT pDCs.

(A) pDCs of WT and CCR9^−/− mice express low levels of CD11b in contrast to inflammatory macrophages. pDCs were defined as CD11b^low MHC II^int/high PDCA-1⁺. (B) CCR9^−/− pDCs show signs of phenotypic maturation. Maturation levels were assessed by comparing expression levels of MHC II and costimulatory molecules CD80 and CD86. (C) Morphology of pDCs isolated from WT and CCR9^−/− animals.