NOD2 regulates CXCR3-dependent CD8+ T cell accumulation in intestinal tissues with acute injury

Abstract

Polymorphisms in NOD2 confer risk for Crohn's disease, characterized by intestinal inflammation. How NOD2 regulates both inflammatory and regulatory intestinal T cells, which are critical to intestinal immune homeostasis, is not well understood. Anti-CD3 mAb administration is used as therapy in human autoimmune diseases, as well as a model of transient intestinal injury. The stages of T cell activation, intestinal injury, and subsequent T tolerance are dependent on migration of T cells into the small intestinal (SI) lamina propria. Upon anti-CD3 mAb treatment of mice, we found that NOD2 was required for optimal small intestinal IL-10 production, in particular from CD8(+) T cells. This requirement was associated with a critical role for NOD2 in SI CD8(+) T cell accumulation and induction of the CXCR3 ligands CXCL9 and CXCL10, which regulate T cell migration. NOD2 was required in both the hematopoietic and nonhematopoietic compartments for optimal expression of CXCR3 ligands in intestinal tissues. NOD2 synergized with IFN-γ to induce CXCL9 and CXCL10 secretion in dendritic cells, macrophages, and intestinal stromal cells in vitro. Consistent with the in vitro studies, during anti-CD3 mAb treatment in vivo, CXCR3 blockade, CD8(+) T cell depletion, or IFN-γ neutralization each inhibited SI CD8(+) T cell recruitment, and reduced chemokine expression and IL-10 expression. Thus, NOD2 synergizes with IFN-γ to promote CXCL9 and CXCL10 expression, thereby amplifying CXCR3-dependent SI CD8(+) T cell migration during T cell activation, which, in turn, contributes to induction of both inflammatory and regulatory T cell outcomes in the intestinal environment.

Figures

Figure 1. NOD2 is required for optimal accumulation of IL-10-producing cells in the SI of mice during anti-CD3 mAb treatment

NOD2+/− IL-10-GFP and NOD2−/− IL-10-GFP mice were treated with 15 μg of anti-CD3 mAb or IgG isotype control at 0 and 48 h. Four hours after the last injection animals were sacrificed. (A) Representative flow cytometry plots of IL-10–producing cells in the SI LP (gated on live cells). (B) Percentage of IL-10-producing cells in the SI. (C) Number of IL-10-producing cells in the SI (mean+SEM; n=3 per group; representative of 5 independent experiments). (D) IL-10 mRNA expression in the SI. (E) Serum IL-10. D–E are shown as mean+SEM; n=6 per group from two independent experiments. Comparisons are between anti-CD3 mAb and IgG treatment in the same genotype or as indicated. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 2. CD8+ T cells are the major source of IL-10-producing cells in the SI of mice during anti-CD3 mAb treatment

Mice were treated with anti-CD3 mAb as in Fig 1. (A) Representative flow cytometry gated on IL-10–producing cells in the SI LP. Surface CD3 is internalized with anti-CD3 mAb treatment, whereas Thy1.2 expression remains intact. Cells were stained for Thy1.2, CD4 and CD8 expression. (B) Pie chart of the proportion of IL-10-producing cell types in the SI after anti-CD3 mAb injection. (mean+SEM; n=3). (C) Percentage and (D) number of IL-10-producing CD8+ T cells (CD4−Thy1.2+) in the SI of NOD2+/− and NOD2−/− mice (mean+SEM; n=3 per group). Data are representative of at least 5 independent experiments. **, p<0.01.

Figure 3. NOD2 is required for CD8+ T cell migration to and cytokine production in the SI upon anti-CD3 mAb treatment

Mice were treated with anti-CD3 mAb as in Fig 1. (A) Representative flow cytometry plots of cells from the SI gated on CD8+ T cells (CD4−Thy1.2+). (B) Percentage of IL-10-GFP+ cells in CD8+ T cells in the SI of NOD2+/− and NOD2−/− mice after anti-CD3 mAb treatment. (C) Percentage and (D) number of CD8+ T cells in the SI lamina propria. (mean+SEM; n=3 per group; representative of 5 independent experiments). (E) IFN-γ and IL-17A mRNA expression in the SI (mean+SEM; n=6–9 per group from 3 independent experiments). Comparisons are between anti-CD3 mAb and IgG treatment in the same genotype or as indicated. (F) Oral FITC-dextran was administered 4h before sacrifice (coincident with the 48h anti-CD3 mAb treatment time point) and measured in the serum at the time of sacrifice. (G–I) NOD2+/− IL-10-GFP mice were administered an antibiotic (ABX) regimen consisting of vancomycin, metronidazole, ampicillin, and neomycin for 4 weeks in the drinking water. Antibiotic-treated or –untreated (SPF, specific pathogen free) mice were then treated with anti-CD3 mAb as in Fig 1 and examined for: (G) percentage of IL-10-GFP+ cells (gated on live cells), (H) percentage of IL-10+CD8+ T cells, and (I) percentage of CD8+ T cells in the SI lamina propria. *, p<0.05; **, p<0.01; ***, p<0.001; NS, not significant.

Figure 4. NOD2 is required for optimal CXCR3 ligand induction in the SI upon anti-CD3 mAb treatment

(A–D) NOD2+/− and NOD2−/− mice were treated with anti-CD3 mAb as in Fig 1. CXCL9 and CXCL10 mRNA levels in (A) SI and (B) MLN (mean+SEM; n=6 per group; representative of two independent experiments). (C–D) Percentage of CD8+ T cells expressing CXCR3 in SI, MLN and spleen was assessed. (C) Representative flow cytometry plots for CXCR3 expression on CD8+ T cells (solid black line). Low CXCR3-expressing MHCII+ cells are shown for comparison (shaded grey histogram). (D) Summary graph for CXCR3-expressing cells within CD8+ T cells (mean+SEM; n=3 per group; representative of two independent experiments). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 5. CXCR3 is essential for the amplification of SI CD8+ T cell accumulation, chemokines and subsequent IL-10 induction upon anti-CD3 mAb treatment

To block CXCR3, mice were treated 100 μg anti-CXCR3 mAb (or Armenian hamster IgG isotype control) i.p. 2h before each anti-CD3 mAb injection (at 0 and 48h). Mice were harvested 4h after the 2nd anti-CD3 mAb injection. (A) Percentage and (B) number of CD8+ T cells in the SI LP (mean+SEM; n=3 per group; representative of 3 independent experiments). (C) CXCL9 and CXCL10 mRNA expression in the SI. (D) IL-10, IFN-γ, and IL-17A mRNA expression in SI. (E) Serum levels of IL-10, IFN-γ, and IL-17A (mean + SEM; n = 6/group from 2 independent experiments for C–E). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 6. NOD2 in hematopoietic and non-hematopoietic cells is required for optimal CD8+ T cell migration into the SI lamina propria upon anti-CD3 mAb treatment

Chimeric mice were generated and treated with anti-CD3 mAb as in Fig 1. (A–D) Transfer of NOD2+/− or NOD2−/− Thy1.1+ BM cells into irradiated Thy1.2+ WT mice. (E–H) Transfer of WT Thy1.2+ BM cells into NOD2+/− or NOD2−/− Thy1.1+ mice. (A&E) Percentage of CD8+ T cells donor cells in the SI LP. (B&F) Chemokine mRNA expression in SI. (C&G) Cytokine mRNA expression in SI. (D&H) Serum cytokine levels. Data are mean+SEM; n=3 per group; representative of 2 independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 7. NOD2 stimulation synergistically enhances IFN-γ-induced CXCL9 and CXCL10 expression in BMM, BMDC and intestinal stromal cells in vitro

(A) BMM, (B) BMDC or (C) intestinal stromal cells from NOD2+/− or NOD2−/− mice were stimulated for 24h with MDP (10 μg/ml for BMM & BMDC cells; 1μg/ml intestinal stromal cells), 1 ng/ml IFN-γ, or 0.1 μg/ml lipid A, alone or combination as indicated. Supernatants were assayed for CXCL9 and CXCL10. Data shown as mean+SEM, n=3 per group, and representative of 3 independent experiments. Significance is compared for conditions examining synergy as indicated (Bonferroni correction applied) and between NOD2+/− and NOD2−/− cells. **, p<0.01; ***, p<0.001.

Figure 8. Depletion of CD8+ T cells or neutralization of IFN-γ attenuates intestinal outcomes upon anti-CD3 mAb treatment

(A–D) CD8+ T cells were depleted by anti-CD8 mAb injection (or treated with rat IgG2b isotype control) as per Materials & Methods. (E–H) IFN-γ was neutralized by anti-IFN-γ mAb injection (or treated with rat IgG1 isotype control) as per Materials & Methods. (A&E) Percentage of CD8+ T cells in the SI LP (mean+SEM; n=3 per group; representative of 2 independent experiments). (B&F) CXCL9 and CXCL10 mRNA expression in the SI. (C&G) CXCL9 and CXCL10 protein expression per mg SI tissue. (D&H) IL-10, IFN-γ, and IL-17A mRNA expression in the SI (mean + SEM; n = 6/group from 2 independent experiments for B-D & F-H). *, p<0.05; **, p<0.01; ***, p<0.001.

Figure 9. Proposed model for mechanisms of NOD2-mediated CXCR3-dependent T cell migration into intestinal tissues

1. Acute activation of circulating and intestinal CD8+ T cells by anti-CD3 mAb; 2. IFN-γ secretion by activated T cells; 3. IFN-γ (T cell-derived) and MDP (microbiota-derived; stimulates intracellular NOD2) synergize to increase CXCL9 and CXCL10 secretion from myeloid-derived and intestinal stromal cells; 4. CXCR3-mediated chemotaxis of CD8+ T cells into the intestinal lamina propria secondary to increased CXCL9 and CXCL10 expression; 5. Newly recruited CD8+ T cells amplify recruitment of additional CD8+ T cell into intestinal tissues through the above sequence of events.