Regulation Of Macrophages And Myoblast Function By Excretory/secretory Products From Trichinella Spiralis In Vitro

Trichinella spiralis is an intracellular parasite of skeletal muscle, which can infect a wide variety of mammalian species and some carnivorous birds. Trichinellosis is a widespread food-borne zoonosis. Human trichinellosis outbreaks occur in many parts of the world, and it has been estimated that as many as 11 million people are infected with Trichinella. There is still a high prevalence in China. Trichinella differs from other helminthes because its life cycle which is reproduced in the same host involves two distinct intracellular habitats, intestianal epithelium and skeletal cell and can be divided into three main stages: adult worms, newborn larvae, and muscle larvae. During the infection, T. spiralis release a variety of biologically active proteins to regulate host immune and invasive the muscle cell to construct their home, named nurse cell, which are very important to parasitize successfully.Macrophages play crucial roles in the immune response agaist infections, as they can initiate, modulate and also be final effector cells during immune responses. During helminth infections, macrophages, the important targets for immunomodulation, have been shown to play a central role in helminth survival. Moreover, macrophages play an important role in repairing injured muscle, which do not only play important role as scavengers to clear cell debris, but also can regulate satellite cell proliferation and differentiation by releasing some active factors. Now, how ES products modulate macrophage and muscle cell functions as well as signal transduction pathways is largely unknown. Research on the impact of different stages of ES products on these host cell function will be benefical for us to clarify the mechanism of immune evasion and nurse cell formation.First of all, we investigated the effect of ES products from different stages of T. spiralis on modulating J774A.1 macrophage activities. The ELISA and real-time PCR results indicated that ES products from different stages of T. spiralis reduced the capacity of macrophages to express pro-inflammatory cytokines (tumor necrosis factor (TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6), and interleukin-12 (IL-12)) in response to lipopolysaccharide (LPS) challenge. However, only ES products from 3-day-old adult worms (AD3) and 5-day-old adult worms/newborn larvae (AD5-NBL) significantly inhibited inducible nitric oxide synthase (iNOS) gene expression in LPS-induced macrophages. In addition, ES products from different stages of T. spiralis alone boosted the expression of anti-inflammatory cytokines interleukin-10 (IL-10) and transforming growth factor-β(TGF-β) and effector molecule arginase 1 (Arg1) in J774A.1 macrophages. In contrast, ES products alone reduced iNOS expression. Signal transduction studies showed that ES products significantly inhibited nuclear factor-κB (NF-κB) translocation into the nucleus and the phosphorylation of both extracellular signal-regulated protein kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) in LPS-stimulated J774A.1 macrophages. Therefore, we hypothesize that during T. spiralis infection, ES products least partly depend on inhibiting these pathway to suppress pro-inflammatory products. These results suggest that ES products regulate host immune response at the macrophage level, which may be important for worm survival and host health. Secondly, utilizing a C2C12 myoblast cell line, the ability of ES from muscle larvae of T. spiralis to influence proliferation and differentiation of murine myoblasts was evaluated in vitro by CCK-8, real-time PCR, immunofluorescence and western-blot technique. CCK-8 results showed that ML ES products significantly enhance cell proliferation under low-serum conditions and dose-dependent manner. Moreover, immunofluorescence and western-blot analysis demonstrated that in differentiation condition, ML ES increase the PCNA and cyclin D1 expression, compared with control group. In addition, our biochemical analysis showed that ML ES reduce the expression of MHC in transcription and post transcription, a marker of sarcomere assembley during terminal differentiation. This result suggests that ML ES reversibly inhibited their differentiation. Furthermore, we exam the mechamism by which ML ES inhibit C2C12 myoblast differentiation, the results showed that ML ES inhibitd the expression of MRFs (MyoD and Myogenin), as well as p21 expression. ML ES also suppressed the phophoryation of p38 MAPK in C2C12 myoblast. Take together, these results imply that some important mediators in ML ES are able to promote proliferation but inhibit differentiation in murine skeletal myoblast, which provide an important insight for full understanding of the mechanisms of T. spiralis to induce infected muscle cell phenotype change and nurse cell formation.Finally, we investigated the effect of ML ES-treated macrophage on C2C12 myoblasts differentiation program in vitro using the co-culture technique. In the treated group, increase expression of PCNA and cyclin D1 indicated that cocultured ML ES-treated macrophages enhance the C2C12 myoblast proliferation. In contrast, the immunostaining showed that the expression of MRFs (MyoD and Myogenin), p21 and MHC were reduced as compared with control group, indicating that cocultured ML ES-treated macrophages inhibit myoblasts differentiation. The inhibitory effects of ML ES on the expression MRFs (MyoD and Myogenin) and MHC were confirmed by western-blot analysis. Our study showed that ML ES not only directly regulate myoblast differentiation, but also can indirectly influence myoblasts differentiation by regulating macrophages activity, which provide new opinion for elucidate complex mechanism of cell-parasite and cell-cell interaction during T. spiralis infection.