Intestinal CX3C chemokine receptor 1(high) (CX3CR1(high)) myeloid cells prevent T-cell-dependent colitis

Abstract

Adequate activation of CD4(+) T lymphocytes is essential for host defense against invading pathogens; however, exaggerated activity of effector CD4(+) T cells induces tissue damage, leading to inflammatory disorders such as inflammatory bowel diseases. Several unique subsets of intestinal innate immune cells have been identified. However, the direct involvement of innate immune cell subsets in the suppression of T-cell-dependent intestinal inflammation is poorly understood. Here, we report that intestinal CX(3)C chemokine receptor 1(high) (CX(3)CR1(high)) CD11b(+) CD11c(+) cells are responsible for prevention of intestinal inflammation through inhibition of T-cell responses. These cells inhibit CD4(+) T-cell proliferation in a cell contact-dependent manner and prevent T-cell-dependent colitis. The suppressive activity is abrogated in the absence of the IL-10/Stat3 pathway. These cells inhibit T-cell proliferation by two steps. Initially, CX(3)CR1(high) CD11b(+) CD11c(+) cells preferentially interact with T cells through highly expressed intercellular adhesion molecule-1/vascular cell adhesion molecule-1; then, they fail to activate T cells because of defective expression of CD80/CD86. The IL-10/Stat3 pathway mediates the reduction of CD80/CD86 expression. Transfer of wild-type CX(3)CR1(high) CD11b(+) CD11c(+) cells prevents development of colitis in myeloid-specific Stat3-deficient mice. Thus, these cells are regulatory myeloid cells that are responsible for maintaining intestinal homeostasis.

Fig. 1.

CX₃CR1^high CD11b⁺ CD11c⁺ cells in the intestinal lamina propria suppress T-cell proliferation. (A) Flow cytometric plots of IL-17-, IFN-γ-, or Foxp3-expressing CD4⁺ T cells cocultured with the indicated cells for 72 h. (B) [³H]thymidine uptake of CD4⁺ T cells cultured with the indicated cells. #P < 0.022. (C) The fluorescence intensity of CFSE-labeled CD4⁺ T cells cultured with the indicated cells at a ratio 1:1:1 for 72 h. (D) [³H]thymidine uptake by CD4⁺ T cells cocultured with CX₃CR1⁻ DCs in the presence of increasing ratios of splenic CD4⁺ CD25⁺ T_reg cells (closed rectangle) or colonic CX₃CR1^high cells (open circle). All data are representative of two independent experiments (means ± SD of duplicate well measurements).

Fig. 2.

CX₃CR1^high myeloid cells alleviate T-cell-dependent intestinal inflammation. (A) SCID mice were injected i.p. with 3 × 10⁵ CD45RB^high CD4⁺ T cells or PBS (closed circles). After 2 h, 3 × 10⁵ CX₃CR1^high cells were transferred (open triangles) or not (closed rectangles). Body weight change was monitored and is presented relative to initial body weight. *P < 0.005 (n = 8 per group). (B) Hematoxylin and eosin staining of colon sections at 4 wk after the transfer described in A (Left) and the colitis score (Right). *P < 0.0012. (Original magnification, 200×.) (C) Numbers of large intestinal lamina propria CD4⁺ T cells at 2 wk (n = 4 per group) (Left) and 4 wk (n = 5 per group) (Right) after transfer. *P < 0.02; **P < 0.015. (D) CD45RB^high T cells (3 × 10⁵) were labeled with CFSE and transferred into Rag2^−/− mice with or without 3 × 10⁵ CX₃CR1^high cells. After 12 d, CFSE dilution in colonic CD4⁺ T cells was analyzed. (E) Cryosection of the colon from a SCID mouse at 3 d after i.v. injection of CFSE-labeled CX₃CR1^high cells. (Original magnification, 200×.) Data are representative of three independent experiments (D and E).

Fig. 3.

Defective activity of Stat3^−/− M_reg cells. (A and B) Expression of Hpgd, Cd163, Hmox1, CD209f, CD209g, and Cebpb mRNA in CX₃CR1^high M_reg cells from wild-type, Il10^−/−, and LysM-cre; Stat3^fl/fl mice. Data are presentative of two independent experiments (means ± SD of at least triplicate PCRs on the identical sample). *, not detected; #P < 0.025. (C) [³H]thymidine uptake by CD4⁺ T cells cultured with CX₃CR1⁻ DCs in the presence of wild-type or Stat3^−/− CX₃CR1^high M_reg cells. Data are representative of four independent experiments (means ± SD of triplicate well measurements). *P < 0.047. (D) Hematoxylin and eosin staining of colon sections of Rag2^−/− mice given 3 × 10⁵ CD45RB^high CD4⁺ T cells with 3 × 10⁵ M_reg cells from wild-type or LysM-cre; Stat3^fl/fl mice (Left) and colitis score (Right). (Original magnification, 200×.) *P < 0.025 (n = 5 per group).

Fig. 4.

Two-step mechanism for suppression of T-cell growth by M_reg cells. (A) Green dye-labeled CD4⁺ T cells were cultured with nonlabeled CX₃CR1⁻ DCs and red dye-labeled M_reg cells from wild-type, LysM-cre; Stat3^fl/fl, or Il10^−/− mice (Left). The number of T-cell aggregation in twenty fields (Right). (Original magnification, 100×.) (B) Expression of adhesion molecules on the indicated cells from wild-type and LysM-cre; Stat3^fl/fl mice. (C) CD4⁺ T cells and wild-type CX₃CR1⁻ DCs were cocultured with M_reg cells pretreated with the indicated blocking Abs or control Ig for evaluation of T-cell proliferation. *P < 0.012. (D) Red dye-labeled CX₃CR1^high M_reg cells were treated with blocking Abs to ICAM-1 and VCAM-1 then added to the mixture of green dye-labeled CD4⁺ T cells and nonstained CX₃CR1⁻ DCs. (Original magnification, 100×.) (E) IL-10 (100 ng/mL) was added to the coculture of CD4⁺ T cells, wild-type DCs, and Il10^−/− M_reg cells. Then, T-cell proliferation was measured. *P < 0.02. (F) Wild-type and Il10^−/− M_reg cells were preincubated with or without 100 ng/mL IL-10 for 72 h. Then, the cells were analyzed for the suppressive activity of T-cell proliferation. *P < 0.016; **P < 0.034. (G) Surface expression of CD80, CD86, CD40, and MHC class II on CX₃CR1^high M_reg cells and CX₃CR1⁻ DCs. (H) Expression of CD80, CD86, and CD40 on M_reg cells from wild-type and Il10^−/− mice cultured for 48 h with or without 100 ng/mL IL-10. (I) Stat3^−/− and Il10^−/− M_reg cells were pretreated with the indicated blocking Abs. Then, M_reg cells were cultured with CD4⁺ T cells and wild-type CX₃CR1⁻ DCs, and T-cell proliferation was measured. *P < 0.025; #P < 0.045. All data are representative of at least two independent experiments (mean values ± SD of triplicate well measurements).

Fig. 5.

Defective M_reg cell function leads to development of colitis. (A, C, and D) LysM-cre; Stat3^fl/fl mice were transferred i.p. with 7 × 10⁴ wild-type CX₃CR1^high M_reg cells at 4 and 6 wk of age. At 2 wk after the last transfer, splenic (A) or colonic lamina propria (C) CD4⁺ T cells were analyzed for production of IFN-γ and IL-17A. *, not detected; ^#P < 0.008. Total number of CD4⁺ T cells in the colonic lamina propria was analyzed (D). ^#P < 0.0079. Data are from two independent experiments with four mice per group. (B) Hematoxylin and eosin staining of colon sections at 2 wk after the last transfer (Left) and the colitis score (Right). (Original magnification, 200×.) *P < 0.0002.