The pathways and mechanisms of muramyl dipeptide transcellular transport mediated by PepT1 in enterogenous infection

Abstract

Background: The transcellular transport of muramyl dipeptide (MDP) mediated by peptide transporter (PepT1) involves the translocation into intestinal epithelial cell (IEC) stage and the transport out of IEC stage. However, its mechanism has not been fully understood. This study aimed to investigate the pathways and mechanisms of MDP transcellular transport in enterogenous infection.

Methods: Firstly, experimental rats were randomly divided into three groups: sham-operation (sham group), MDP perfusion (MDP group), and PepT1 competitive inhibition (MDP + Gly-Gly group). Then, the overall survival (OS) and intestinal weight were measured in MDP and MDP + Gly-Gly group. HE staining was performed to observe the pathological changes of the small intestine. The levels of IL-6, IL-1b, IL-8, IL-10, TNF-α, and nitric oxide (NO) in rat serum and small intestine were determined by ELISA. To further verify the pathways and mechanisms of MDP transcellular transport from IEC in intestinal inflammatory damage, the NFκB inhibitor, PDTC, was used to treated lamina propria macrophages in small intestinal mucosa in sham, MDP, and MDP + Gly-Gly groups. Finally, the expression of CD80/86 and the antigen presentation of dendritic cells (DCs) were measured by flow cytometry.

Results: MDP infusion was able to induce death, weight loss, and intestinal pathological injury in rats. Competitive binding of Gly-Gly to PepT1 effectively inhibited these effects induced by MDP. As well, competitive of PepT1 by Gly-Gly inhibited inflammation-related cytokines induced by MDP in rat serum and small intestine. Furthermore, we also found that MDP transported by PepT1 contributes to activation of macrophages and antigen presentation of DCs.

Conclusions: PepT1-NFκB signal is pivotal for activation of intestinal inflammatory response and MDP transcellular transport.

Keywords: Muramyl dipeptide (MDP); PepT1; enterogenous infection; immune cells.

Figure 1

PepT1 mediated MDP transport induces small intestinal pathological injury. (A) The survival in each group was assessed in MDP and MDP + Gly-Gly groups. (B) The intestinal weight was measured at the 1st, 2nd, 3rd, 4th, 5th, 6th, and 7th day in MDP and MDP + Gly-Gly groups. (C) HE staining of small intestine in each group. 100× magnification. PepT1, peptide transporter; MDP, muramyl dipeptide; HE, hematoxylin and eosin.

Figure 2

The expression levels of serum IL-6, IL-1b, IL-8, IL-10, TNF-α, and NO were measured by ELISA. *P<0.05, **P<0.01 versus sham group; ^#P<0.05 versus MDP group. NO, nitric oxide; ELISA, enzyme-linked immunosorbent assay; MDP, muramyl dipeptide.

Figure 3

The expression levels of IL-6, IL-1b, IL-8, IL-10, TNF-α, and NO in rat small intestinal mucosa were measured by ELISA. *P<0.05, **P<0.01 versus sham group; ^#P<0.05 versus MDP group. NO, nitric oxide; ELISA, enzyme-linked immunosorbent assay; MDP, muramyl dipeptide.

Figure 4

MDP transported by PepT1 contributes to activation of macrophages and antigen presentation of DCs. (A) The expression levels of serum IL-6, TNF-α, and NO in rat macrophages were measured by ELISA. *P<0.05, **P<0.01 versus MDP-Blank; ^##P<0.01 versus MDP-Blank. (B) The expression levels of CD80 and CD86 in rat DCs were measured by flow cytometry. (C) The antigen presentation of rat DCs were measured by flow cytometry. MDP, muramyl dipeptide; PepT1, peptide transporter; DC, dendritic cell; NO, nitric oxide.