Clostridium butyricum alleviates intestinal low-grade inflammation in TNBS-induced irritable bowel syndrome in mice by regulating functional status of lamina propria dendritic cells

Abstract

Background: Irritable bowel syndrome (IBS) is one of the most common functional gas-troenterological diseases characterized by abnormal visceral sensitivity and low-grade inflammation. The role of Clostridium butyricum (C. butyricum) in reducing intestinal low-grade inflammation via immune pathways has been well defined. However, the detailed mechanisms of the effects of C. butyricum on intestinal mucosal immunity, especially on immune cells of the lamina propria, remain unclear. Dendritic cells (DCs), which are important immune cells, secrete proinflammatory cytokines (IL-1β, IL-6, and others) and express T cell immuno-globulin and mucin domain-3 (TIM3), promoting proliferation and activation of DCs, and mediating Th1 and Th17 inflammatory responses.

Aim: To investigate the role of DCs in the development of IBS in a rat model and to understand the regulation of DCs after C. butyricum intervention.

Methods: An IBS animal model was established using C57BL/6 mice, and C. butyricum was continuously administered via the intragastric route to simulate different intestinal immune states. Intestinal visceral hypersensitivity and histopathology were assessed using the abdominal withdrawal reflex (AWR) test and hematoxylin & eosin (H&E) staining, respectively. The expression of proinflammatory cytokines (IL-1β and IL-6) and TIM3 was analyzed by Western blot analysis and real-time PCR. Flow cytometry was applied to analyze the quantity, function, and membrane molecule TIM3 of the lamina propria dendritic cells (LPDCs). The regulatory effect of C. butyricum was verified in bone marrow-derived dendritic cells by in vitro experiments.

Results: The secretion of proinflammatory cytokines (IL-1β and IL-6) in mice with IBS was significantly increased compared with that of the control group, which suggested that the intestinal mucosa in mice with IBS was in a low-grade inflammatory state. The expression of CD11C+CD80+ and CD11c+TIM3+ in intestinal LPDCs in mice with IBS increased significantly. Meanwhile, the cytokines (IL-1β and IL-6) were significantly reduced after the intervention with probiotic C. butyricum. The amount and function of LPDCs and the TIM3 on the surface of the LPDCs were decreased with the alleviation of the intestinal inflammatory response.

Conclusion: The results suggest that C. butyricum regulates the amount and functional status of LPDCs in the intestinal mucosa of mice with IBS, and therefore modulates the local immune response in the intestine.

Keywords: Clostridium butyricum; Irritable bowel syndrome; Lamina propria dendritic cells; Proinflammatory cytokines; T cell immunoglobulin and mucin domain-3.

Figure 1

Photomicrographs of hematoxylin and eosin staining. There was no difference with regard to morphology between control (A), IBS (B), IBS + C. butyricum (C), and IBS + NS (D) tissues in hematoxylin and eosin staining (original magnification, ×200) (n = 6 per group).

Figure 2

Effects of Clostridium butyricum on visceral hypersensitive in TNBS-induced irritable bowel syndrome mice. The abdominal withdrawal reflex (AWR) scores at 0.25 mL (A), 0.35 mL (B), and 0.50 mL (C) distension volumes were measured on day 28. Data are expressed as the mean ± SEM (n = 6). ^aP < 0.05, ^bP < 0.05, ^cP < 0.05 vs control group, IBS + normal saline group, and IBS group, respectively. IBS: Irritable bowel syndrome.

Figure 3

Effects of Clostridium butyricum on the production of proinflammatory cytokines in irritable bowel syndrome mice. Real-time PCR (A and B) and Western blot analysis(C-E) were used to measure the levels of proinflammatory cytokines IL-1β and IL-6. The results showed that IL-1β and IL-6 in the intestinal low-grade inflammatory state of IBS were significantly higher than those in the normal control group. The secretion of proinflammatory cytokines was significantly decreased after C. butyricum intervention. ^aP < 0.05, ^bP < 0.05, ^cP < 0.05 vs control group, IBS + normal saline group, and IBS group, respectively. IBS: Irritable bowel syndrome.

Figure 4

Effects of Clostridium butyricum on the quantity and functional status of the lamina propria dendritic cells. A: Flow cytometry of CD11c+ LPDCs expression; B: Flow cytometry of CD11C+CD80+ LPDCs expression; C: Flow cytometry of CD11c+Tim3+ LPDCs expression; D: Flow scatter plot of all test indicators; E: Tim-3 mRNA levels measured by real-time PCR; F: Tim3 protein expression measured by Western blot analysis. The results showed that the expression of CD11c+, CD11C+CD80+, and CD11c+Tim3+ LPDCs in the intestinal inflammatory state of IBS was significantly higher than in that in the normal control group, and the costimulatory molecule CD80 representing DC in the activated mature state was also significantly increased (P < 0.01). However, the above indicators were significantly decreased after C. butyricum intervention. Our experimental results suggest that the LPDCs participate in and promote the intestinal inflammatory immune response of IBS and C. butyricum suppresses intestinal inflammation by reducing the number, function, and immunoregulatory molecule Tim3 of LPDCs in IBS mice. ^aP < 0.05, ^bP < 0.05, ^cP < 0.05 vs control group, IBS + normal saline group, and IBS group, respectively. IBS: Irritable bowel syndrome; LPDCs: Lamina propria dendritic cells.

Figure 5

Effects of Clostridium butyricum on the expression of Tim3 and inflammatory cytokines in bone marrow-derived dendritic cells. LPS stimulated the proliferation and activation of bone marrow-derived dendritic cells (BMDCs). The expression of CD11c+CD80+ BMDCs and CD11c+Tim3+ BMDCs was significantly higher than that of the immature BMDCs group by flow cytometry (A-D). The inflammatory cytokines IL-1 and IL-6 detected by ELISA were also significantly elevated (E-F). Tim3 was highly expressed in mature activated BMDCs. Different forms of probiotics were co-cultured and stimulated with LPS. The expression of Tim3 was down-regulated with the decrease of inflammatory factors. It can be seen that Tim3 is the key point to regulate the activation of DCs. Clostridium butyricum can alter the activation status and function of DCs by regulating the expression of Tim3. ^aP < 0.05, ^bP < 0.05, ^cP < 0.05 vs ImBMDC group, BDMC group, and BDMC + LPS group, respectively. BMDCs: Bone marrow-derived dendritic cells.