CD169 Expressing Macrophage, a Key Subset in Mesenteric Lymph Nodes Promotes Mucosal Inflammation in Dextran Sulfate Sodium-Induced Colitis

Abstract

Inflammatory bowel disease (IBD) including Crohn's disease (CD) and ulcerative colitis is a relapsing-remitting illness. Patients with long-standing extensive colitis are easy to develop colorectal cancer (CRC). The increasing incidence of IBD and a substantial increase in the risk of CRC make the necessity to pay more attention on the regulation of inflammation especially by specific macrophages subset. The present study reported that a key subset of sinus macrophage expressing CD169 in mesenteric lymph nodes (mLNs) played an essential role in promoting mucosal inflammation. The results revealed that the subset expressing CD169 in mLNs increased significantly during the dextran sulfate sodium (DSS)-induced colitis. The colitic symptoms were alleviated in CD169-diphtheria toxin receptor (DTR) mice at least partially due to the deletion of CD169⁺ macrophages in mLNs. In addition, the levels of inflammatory cytokines as well as the percentage of Th17 cells in mLNs from CD169-DTR mice were much lower than those from WT mice with DSS-induced colitis. Further experiment in vitro demonstrated that the supernatant from whole cells of mLNs or colon tissues could promote the production of inflammatory factors by mLN cells or colon tissues from CD169-DTR mice. These results could be explained by the cell sorting result that CD11b⁺CD169⁺ macrophages expressed higher level of inflammatory factors directly. All these data indicated that CD169⁺ sinus macrophage in mLNs played an essential role on regulating mucosal inflammation.

Keywords: CD169+ macrophages; colitis; cytokines; dextran sulfate sodium; mesenteric lymph nodes.

Figure 1

Histopathological changes and cytokines, chemokines expression of mesenteric lymph nodes (mLNs) during the colitis course. C57BL/6 WT mice were treated with 3.5% dextran sulfate sodium (DSS), and mice were harvested after 3 or 7 days. (A) Histological analysis of mLN sections obtained from WT control mice and DSS-treated mice (WT-Day3 and WT-Day7). (B) Real-time PCR analysis of pro-inflammatory cytokines in WT mice and DSS-treated mice (WT-Day3 and WT-Day7). (C) Chemokines expression in mLNs obtained from representative mice from indicated groups. Statistical analysis was determined by Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. Data are representative of two independent experiments.

Figure 2

CD169⁺ macrophages are increased in mesenteric lymph nodes (mLNs) of colitis mice. C57BL/6 WT mice were treated with 3.5% dextran sulfate sodium (DSS), and mice were harvested after 3 or 7 days. (A) The percentage of CD11b⁺CD169⁺ macrophages in mLNs of WT control mice and DSS-treated mice were measured by FACS, and statistical analysis was shown, *P < 0.05, ***P < 0.001 compared to control. (B) The percentage of CD11b⁺CD169⁻ macrophages in mLNs of WT control mice and DSS-treated mice, *P < 0.05 compared to control. (C) CD169 mRNA expression in mLNs obtained from WT control mice and DSS-treated mice, **P < 0.01 compared to control. (D) Immunohistochemistry analysis of mLN sections obtained from representative mice of indicated groups.

Figure 3

CD11b⁺CD169⁺ macrophages in mesenteric lymph nodes (mLNs) from CD169-diphtheria toxin receptor (DTR) mice was deleted by diphtheria toxin (DT) administration. CD169-DTR mice were intraperitoneally injected with DT (day 0), Mice were harvested on day 3. (A) Flow cytometry analysis of CD11b⁺CD169⁺ macrophage in mLNs obtained from WT control mice and CD169-DTR mice with DT treatment, and statistical analysis of the result of FACS, *P < 0.05 compared to control. (B) CD169 mRNA expression in mLNs obtained from WT control mice and CD169-DTR mice with DT treatment, **P < 0.01 compared to control. (C) mLN sections obtained from representative mice from indicated groups were detected by Immunohistochemistry. Data are representative of two independent experiments.

Figure 4

Typical colitis in WT mice caused by dextran sulfate sodium (DSS) treatment were not observed in CD169-diphtheria toxin receptor (DTR) mice. WT and CD169-DTR mice with diphtheria toxin treatment were both orally treated with 3.5% DSS; 7 days later, mice were harvested. (A) Weight loss of WT mice (n = 5) and CD169-DTR mice (n = 5) treated with the indicated time points with DSS, *P < 0.05, ***P < 0.001compared to control. (B) Macroscopic observation of colons on day 7, such as the length of colons and bloody stools (left), and the statistical analysis of the length of WT colitis and CD169-DTR colitis mice colons (right) (n = 10), ***P < 0.001 compared to control. (C) Immunohistochemistry analysis of mesenteric lymph node sections obtained from WT colitis mice and CD169-DTR colitis mice. Data are representative of two independent experiments.

Figure 5

Th17-related cytokines and the percentage of Th17 cells could be affected by the presence or not of CD169⁺ subset in mesenteric lymph nodes (mLNs). WT and CD169-diphtheria toxin receptor (DTR) mice with diphtheria toxin treatment were both orally treated with 3.5% dextran sulfate sodium (DSS) for 7 days. (A) Th17-related cytokines expression levels in mLNs. (B) Real-time PCR analysis of chemokines in mLNs of DSS-treated WT mice and CD169-DTR mice. (C) Flow cytometry analysis of Th17 cells in mLNs obtained from WT control mice, WT colitis mice and CD169-DTR colitis mice and statistical analysis of the result of FACS. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. Data are representative of two independent experiments.

Figure 6

Supernatant of mesenteric lymph nodes (mLNs) and colons from colitic mice could induce inflammatory cytokines production by mLN cells and colons in vitro. The supernatant of mLN cells and colons of WT mice with dextran sulfate sodium (DSS) treatment was transferred to the mLN cells and colons of CD169-diphtheria toxin receptor (DTR) mice with DSS treatment, then the cultured cells and colon tissues were harvested. (A) Cytokines and chemokines mRNA expression levels of cultured mLNs. (B) Cytokines and chemokines mRNA expression levels of cultured colons. *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. Data are representative of two independent experiments.

Figure 7

CD169⁺ macrophages can secret pro-inflammatory cytokines and chemokines directly. Mice were orally treated with 3.5% dextran sulfate sodium (DSS) for 7 days, then mesenteric lymph node cells from WT control mice and DSS-treated WT mice were sorted into three subpopulations by flow cytometry. (A) Pro-inflammatory factors’ mRNA expression levels of three subpopulations sorted from WT mice. (B) Pro-inflammatory factors’ mRNA expression levels of three subpopulations sorted from WT colitis mice, *P < 0.05, **P < 0.01, ***P < 0.001 compared to control. Data are representative of two independent experiments.