Lactate-driven macrophage polarization in the inflammatory microenvironment alleviates intestinal inflammation

Abstract

Background: Lactate has long been considered an intermediate by-product of glucose metabolism. However, in recent years, accumulating evidence reveals that lactate has unique biological activities. In previous studies, lactate signaling was shown to inhibit inflammation. Furthermore, in vitro experiments have shown that lactate can promote the transformation of pro-inflammatory macrophages into anti-inflammatory macrophages. However, no in vivo studies have shown whether lactate can alleviate inflammation.

Methods: RAW 264.7 macrophages were stimulated by LPS to induce an M1 phenotype, and cultured with low and high concentrations of lactate. The cells were then observed for phenotypic transformations and expression of inflammatory mediators and surface markers. The expression of inflammatory factors was also analyzed in the cell-free supernatant fraction. Further, a mouse model of DSS-induced colitis was established and treated with lactate. Colonic tissue injury was monitored by histopathological examinations.

Results: The in vitro experiments showed that lactate promoted the transformation of activated macrophages to M2 phenotype and decreased the expression of TLR4-mediated NF-κB signaling proteins and inflammatory factors. In the DSS-induced colitis mouse model, lactate promoted the phenotypic transformation of macrophages in colonic tissue, reduced inflammation and organ damage, inhibited the activation of TLR4/NF-κB signaling pathway, decreased the serum levels of pro-inflammatory factors, increased the expression of anti-inflammatory factors, promoted the repair of the intestinal mucosal barrier and reduced the severity of colitis.

Conclusions: Lactate inhibits the TLR/NF-κB signaling pathway and the production of pro-inflammatory factors by promoting polarization of macrophages. In addition, lactate promotesthe repair of the intestinal mucosal barrier and protects intestinal tissue in inflammation. Furthermore, lactate is relatively safe. Therefore, lactate is a promising and effective drug for treating inflammation through immunometabolism regulation.

Keywords: colitis; inflammation; inflammatory microenvironment; lactic acid; macrophage polarization.

Figure 1

Lactate promoted M2 polarization of macrophages in vitro. (A) Representative images of macrophage phenotypes after lactate treatment. Polarization of macrophages toward the M2 phenotype was associated with elongated cell shape (n = 3). scale bar, 5μm. (B) Morphological changes following the phenotypic transformation of macrophages at different time points (n = 3). scale bar, 5μm. (C) Representative fluorescence images showing the macrophage phenotypes after lactate treatment (n = 3). scale bar, 5μm. (D) Representative fluorescence images of macrophage phenotypes transformation after lactate treatment at different time points (n = 3). scale bar, 5μm. (E) Western blotting assay showing the expression of phenotype-related markers in LPS-stimulated macrophages after lactate treatment at different time points (n = 3). (F) Expression level of M1 macrophages-related markers after a 24-h treatment with different concentrations of lactate as determined by western blotting (n = 3).

Figure 2

Lactate inhibited inflammation in vitro. (A) Intracellular ROS generation was observed using a ROS fluorescence probe DCFH-DA after lactate treatment (n = 3). scale bar, 5μm. (B) Intracellular ROS generation by macrophages at different time points (n = 3). scale bar, 5μm. (C) Western blotting results showing expression of phenotypic markers of LPS-stimulated macrophages after lactate treatment (n = 3). (D) Inflammatory cytokines released by macrophages in different treatment groups determined by ELISA (n=6). Pro-inflammatory cytokines, TNF-α and IL-1β; Anti-inflammatory factors, TGF-β and IL-10.

Figure 3

Lactate improved intestinal injury and intestinal mucosal barrier in mice with colitis. (A) Therapeutic design of drug-induced acute colitis model mice (n = 6). (B) Body weight changes in mice after lactate treatment (n = 6). (C) Changes of disease activity index in mice after lactate treatment (n = 6). (D) Colon length of mice after different treatments (n = 6). (E) Representative images of HE staining of colon tissues and histological scores in different groups (n = 6). Scale bar, 100μm. The length of intestinal villi of mice after different treatments (n = 6). (F) Immunohistochemistry images showing the level of tight junction proteins in colon tissues in different groups (n = 6). Scale bar, 100μm.

Figure 4

Lactic acid alleviated intestinal inflammation in mice with colitis. (A) Serum levels of inflammatory factors in different groups (n = 6). (B) Western blotting analysis of inflammatory signals TLR4 and P65 in colon tissues of different groups.

Figure 5

Lactic acid promoted M2 polarization of macrophages in colon tissues. (A) Representative fluorescent images of the macrophage phenotypes in colon tissues in different groups. Scale bar, 100μm. (B) Western blotting analysis showing expression level of macrophage phenotypic markers in colon tissues for different groups.

Figure 6

Safety evaluation of lactate. (A) The cytotoxicity results determined by the CCK8 assay (n=3). (B) Lactate was added to the cell culture media. pH was measured over 2 hours. (C) Effects of lactate on body weight in normal mice (N=6). (D) Effect of exogenous lactic acid on serum lactic acid in normal mice (n=3). (E) Effect of lactate on organs of normal mice (n=3).