Chemokine receptor CCR7 regulates the intestinal TH1/TH17/Treg balance during Crohn's-like murine ileitis

Abstract

The regulation of T cell and DC retention and lymphatic egress within and from the intestine is critical for intestinal immunosurveillance; however, the cellular processes that orchestrate this balance during IBD remain poorly defined. With the use of a mouse model of TNF-driven Crohn's-like ileitis (TNF(Δ) (ARE)), we examined the role of CCR7 in the control of intestinal T cell and DC retention/egress during experimental CD. We observed that the frequency of CCR7-expressing TH1/TH17 effector lymphocytes increased during active disease in TNF(Δ) (ARE) mice and that ΔARE/CCR7(-/-) mice developed exacerbated ileitis and multiorgan inflammation, with a marked polarization and ileal retention of TH1 effector CD4(+) T cells. Furthermore, adoptive transfer of ΔARE/CCR7(-/-) effector CD4(+) into lymphopenic hosts resulted in ileo-colitis, whereas those transferred with ΔARE/CCR7(+/+) CD4(+) T cells developed ileitis. ΔARE/CCR7(-/-) mice had an acellular draining MLN, decreased CD103(+) DC, and decreased expression of RALDH enzymes and of CD4(+)CD25(+)FoxP3(+) Tregs. Lastly, a mAb against CCR7 exacerbated ileitis in TNF(Δ) (ARE) mice, phenocopying the effects of congenital CCR7 deficiency. Our data underscore a critical role for the lymphoid chemokine receptor CCR7 in orchestrating immune cell traffic and TH1 versus TH17 bias during chronic murine ileitis.

Keywords: CD103+ dendritic cells; Crohn’s disease; effector memory T cells; inflammatory bowel disease; lymphatics.

Figure 1.. The onset of ileitis in TNF^∆ARE mice is characterized by increased T_H1/T_H17 CD4⁺ T cells expressing CCR7.

(A and D) Cellular composition of WT and TNF^∆ARE (∆ARE) ileal infiltrate and MLN, with correlation of CD4⁺ and CD4⁺ CD44⁺ (T_EM; CD44^high CD62L^neg) lymphocytes by flow cytometry. (B and E) Flow cytometry analysis of CD4⁺ T cells from ilea and MLN of WT and TNF^∆ARE mice assessing the relative expression of IFN-γ and IL-17a. Gating was performed on live, CD45⁺MHCII^neg CD4⁺ T cells from indicated compartments at 8–10 wk of age. (C and F) Relative mRNA expression of CCR7 from sorted CD4⁺CD44⁺ from MLN and ileum of WT and TNF^∆ARE mice. Data expressed as mean ± sem; *P < 0.05; **P < 0.01 versus age-matched WT mice from 2 independent experiments (n = 5 mice/strain; A and D, ANOVA; C and F, t-test).

Figure 2.. CCR7 deficiency exacerbates ileitis in TNF^∆ARE mice.

(A and B) Cellular composition of WT, CCR7^−/−, TNF^∆ARE/CCR7^+/+ (∆ARE) versus TNF^∆ARE/CCR7^−/− (∆ARE/CCR7^−/−) ileal infiltrate and MLN. (C) Histologic assessment of TNF^∆ARE/CCR7^+/+ and TNF^∆ARE/CCR7^−/− mice ilea at indicated ages, according to acute and chronic leukocytic infiltrates, villus distortion, and a combinatorial total inflammatory index. (D) Representative micrographs of ileum H&E from TNF^∆ARE/CCR7^+/+ and TNF^∆ARE/CCR7^−/− mice between 4 and 20 wk of age. Data expressed as mean ± sem; *P < 0.05; **P < 0.01 versus age-matched WT mice (n = 10–18 mice/strain, ANOVA). Original scale bars, 100 μm.

Figure 3.. CCR7 deficiency increased ileal retention of CD4⁺ T_EM and inhibits egress to draining MLN.

(A) Flow cytometric analyses characterizing the absolute cell numbers of T_EM, T_CM, and T_Naive CD4⁺ T cells in the ilea and MLN of WT, CCR7^−/−, ΔARE^+/+ and ΔARE^−/− mice. (B) Representative flow cytometry contour plots depicting frequency of T_EM, T_CM, and T_Naive CD4⁺ T cells in the ilea and MLN of ΔARE/CCR7^+/+ or ΔARE/CCR7^−/− and WT mice. Data expressed as mean ± sem; **P < 0.01 versus its indicated counterpart from 3 independent experiments (n = 6 mice/strain, ANOVA).

Figure 4.. Altered T_H1/T_H17 CD4⁺ ratio in ∆ARE/CCR7^−/− mice.

(A–D) Absolute numbers and representative flow cytometry plots of IFN-γ⁺ versus IL-17a⁺ from ileum and MLN CD4⁺ T cells. Gating was performed on live, CD45⁺ MHCII^neg CD4⁺ T cells at 8–10 wk of age. Data expressed as mean ± sem; **P < 0.01 versus age-matched indicated counterparts from 2 independent experiments (t-test). (E, I–XII) Cytokine mRNA analysis was performed by real-Time PCR on ileal tissues from CCR7-sufficient or -deficient WT and ∆ARE/CCR7^+/+ or ∆ARE/CCR7^−/− mice at 10 wk of age. Data expressed as mean ± sem; *P < 0.05; **P < 0.01 versus indicated counterparts from 2 independent experiments (n = 6 mice/strain, ANOVA).

Figure 5.. Changes in CD103⁺ versus CD11b⁺ DC subsets, loss of RALDH enzymes, and altered T_reg profile in ∆ARE/CCR7^−/− mice.

(A) Frequency of ileal CD103⁺ and CD11b⁺ DC population and representative flow cytometry plots of ileal CD103⁺ and CD11b⁺ DC. SSC-A, Side-scatter-area. (B) Relative mRNA expression analysis of ileal RALDHs (RALDH1, RALDH2, RALDH3) from indicated genotypes. (C) Absolute numbers of CD4⁺CD25⁺FoxP3⁺ T_regs from the ileum of CCR7-sufficient or -deficient WT and ∆ARE/CCR7^+/+ or ∆ARE/CCR7^−/− mice. (D) Frequency of MLN CD103⁺ and CD11b⁺ DC population. (E) Relative mRNA expression analysis of RALDHs (RALDH1, RALDH2, RALDH3) in the MLN from indicated genotypes. (F) Absolute numbers of CD4⁺CD25⁺FoxP3⁺ T_regs from the MLN of CCR7-sufficient or -deficient WT and ∆ARE/CCR7^+/+ or ∆ARE/CCR7^−/− mice. Experiments were performed from indicated compartments and genotypes at 8–10 wk of age. Data expressed as mean ± sem from 3 independent experiments; *P < 0.05; **P < 0.01 (n = 4–6 mice/strain, ANOVA).

Figure 6.. Antibody blockade of CCR7 exacerbates ileitis in TNF^ΔARE mice by inhibiting lymphatic egress and promoting retention of effector/memory CD4⁺ T cells.

Eight-wk-old TNF^∆ARE mice received 4 injections (i.p.) of anti-CCR7 (4B12; 500 μg) or IgG2a vehicle every 4 days for 2 wk. (A) Histology indices were assessed for ileal tissue post-treatment with representative micrographs. (B) Cytokine mRNA analysis was performed by real-time PCR on ileal tissues of isotype (IgG2a)- and anti-CCR7-treated mice. (C) Frequency of CD103⁺ and CD11b⁺ DC subsets from ilea post-treatment. Data expressed as mean ± sem; *P < 0.05; **P < 0.01 versus IgG2a from 2 independent experiments (n = 6–7 mice/treatment). (A and B) t-test versus IgG2a; (C) ANOVA). Original scale bars, 100 μm.

Figure 7.. CCR7-deficient CD4⁺ effector T cells from TNF^ΔARE mice drive a dysregulated ileo-colitis following adoptive transfer into RAG1^−/− mice.

CD4⁺ T_EM (CD44^high CD62L^neg) from the spleen of 4- to 6-wk-old ΔARE/CCR7^+/+ and ΔARE/CCR7^−/− mice were isolated by FACS and adoptively transferred (1 × 10⁶; i.p.) into RAG1^−/− recipients. (A) Representative assessment of CCR7 on adoptively transferred CD4⁺ T cells by flow cytometry. (B) Cell counts were assessed 8 wk post-transfer in ilea, colon, and MLN. (C and D) Percentage of Ki67⁺ in proliferating CD4⁺ T cells in the ilea and colon and representative flow cytometry plots from the ileum. (E) Representative flow cytometry histograms of ileal Tbet⁺ and RORγt⁺ CD4⁺. MFI, Mean fluorescence intensity. (F and G) Percentage of ileal IFN-γ⁺ CD4⁺ T cells and representative flow cytometry plots. (H and I) Percentage of colonic IL-17a⁺ CD4⁺ and representative flow cytometry plots. (J and K) Total inflammatory indices from adoptively transferred RAG1^−/− recipient ilea and colons. Data expressed as mean ± sem; *P < 0.05; **P < 0.01 versus ΔARE/CCR7^+/+ from 2 independent experiments (n = 6–7 mice/treatment). (B, J, and K, t-test; F and H, ANOVA). Original scale bars, 100 μm.

Figure 8.. ∆ARE/CCR7^−/− mice induce multiorgan inflammation and associated pathology.

(A) Histologic assessment and pathologic evaluation of ∆ARE/CCR7^+/+ and ∆ARE/CCR7^−/− mice at 10 wk of age. (B) Representative micrographs of tissue H&E from ∆ARE/CCR7^+/+ and ∆ARE/CCR7^−/− mice. (C) Representative macroscopic assessment at necropsy of ∆ARE/CCR7^−/− mice displays a clear inability to thrive by 8 wk of age. Data expressed as mean ± sem; *P < 0.05; ***P < 0.001 versus age-matched ∆ARE/CCR7^+/+ counterparts (n = 3–5 mice/strain, t-test). Original scale bars, 100 μm.