Exosomes Derived From M2b Macrophages Attenuate DSS-Induced Colitis

Abstract

Macrophages are commonly classified as M1 macrophages or M2 macrophages. The M2 macrophages are further sub-categorized into M2a, M2b, M2c, and M2d subtypes. The M2a, M2b, and M2c subtypes play roles in anti-inflammatory activity, tissue remodeling, type 2 T helper cell (Th2) activation, and immunoregulation. Previous studies have shown that macrophage exosomes can affect some disease processes. Exosomes are 30-150-nm lipid bilayer membrane vesicles derived from most living cells, with important biological functions. The role of exosomes in preventing the development of autoimmune diseases, including inflammatory bowel disease (IBD), has evoked increasing interest. Here, we analyze the roles of exosomes derived from M2a, M2b, and M2c macrophage phenotypes in dextran sulfate sodium (DSS)-induced colitis. Exosomes were isolated from the supernatant of different types of macrophages and identified via transmission electron microscopy (TEM), western blotting, and NanoSight. The results showed that M2b macrophage exosomes significantly attenuated the severity of DSS-induced colitis in mice. The number of regulatory T (Treg) cells in the spleens of mice with colitis and levels of IL-4 both increased following treatment with M2b macrophage exosomes. In addition, key cytokines associated with colitis (IL-1β, IL-6, and IL-17A) were significantly suppressed, following treatment with M2b macrophage exosomes. The M2b macrophage exosomes exerted protective effects on DSS-induced colitis, mainly mediated by the CC chemokine 1 (CCL1)/CCR8 axis. These findings provide a novel approach for the treatment of IBD.

Keywords: CCL1/CCR8 axis; IL-1β; M2b macrophage; exosomes; inflammatory bowel disease.

Figure 1

Exosomes were purified from the supernatant of macrophages. (A) Exosomes were analyzed by negative-staining transmission electron microscopy (TEM). (B) Exosome specific markers CD63, CD9, and CD81 were detected by western blotting. (C) The size distribution profile of the exosomes was analyzed by NanoSight.

Figure 2

Body weight and disease activity index (DAI) of mice with acute dextran sulfate sodium (DSS)-induced colitis treated with macrophage exosomes. The following groups of mice were used in the study: water + phosphate-buffered saline (PBS); DSS + PBS; DSS + M0-cell; DSS + IL-13-cell; DSS + IL-10-cell; DSS + IL-1β-cell; DSS + M0-exo; DSS + IL-13-exo; DSS + IL-10-exo; and DSS + IL-1β-exo. (A) Daily changes in body weight of each group. (B) Changes in DAI scored from body weight loss, diarrhea, and bleeding.

Figure 3

Colon length and colon macroscopic scores of mice treated with macrophage exosomes. (A) Mice were sacrificed on day 8 and colon lengths (an indirect marker of inflammation) were measured. (B) Macroscopic appearance of colons. (C) Mean macroscopic scores of colons. Macroscopic scores were assessed by hyperemia, wall thickening, ulceration, and extent of inflammation and damage.

Figure 4

Histopathological changes in the colons of mice treated with macrophage exosomes. (A) Colon sections were examined with hematoxylin and eosin (H & E) (5× and 20×). (B) Histopathological scores were determined in a blinded fashion.

Figure 5

Splenic Treg subset levels in mice with experimental colitis. (A) Cells were isolated from the spleens of mice and stained with fluorochrome-conjugated antibodies against CD3e, CD4, CD25, and Foxp3. The number of Tregs in the spleens were expressed as percentages of the CD3e⁺CD4⁺CD25⁺Foxp3⁺ cell population. (B) Statistical analysis of Tregs.

Figure 6

Inflammatory cytokine expression in the colons of mice was determined by real-time PCR; the housekeeping gene, GAPDH, was used as an internal reference. (A) The IL-1β mRNA expression levels. (B) The IL-6 mRNA expression levels. (C) The IL-17A mRNA expression levels.

Figure 7

Ex vivo fluorescence imaging of exosomes in the colon; the CCL1 protein in exosomes was detected by western blotting. (A) Ex vivo fluorescence imaging of the distribution of DiR labeled exosomes in the colon. (B) Western blotting analysis of CCL1 protein expression in exosomes purified from various macrophage phenotypes, exosome marker CD63 used as internal reference.

Figure 8

CCL1, CCR8, and IL4 expression in the colons of mice treated with M2b macrophage exosomes. (A) The CCL1, CCR8, and IL4 mRNA expression levels in the colons of mice were determined by real-time PCR; the housekeeping gene, GAPDH, was used as an internal reference. (B) Western blotting analysis of CCL1, CCR8, and IL4 protein expression in the colons of mice. (C) CCL1, CCR8, and IL4 expression in colon tissue was detected by immunohistochemistry, and the sum of the integrated optical density (IOD) was analyzed.