on DSS-induced colitis were found to associate with PD-1 upregulation and M2 macrophage polarization. Thus, PD-1-mediated M2 macrophage polarization is a key mechanism of helminth-induced modulation of the host immune system.

Trichinella spiralis maintains chronic infections within its host, involving a variety of immunomodulatory properties, the mechanisms of which have not been completely elucidated. In this study, we found that T. spiralis infection induced strong regulatory T cell responses through parasite excretory–secretory (ES) products, characterized by increase of CD4+CD25+Foxp3+ and CD4+CD25−Foxp3+ Treg cells accompanied by high levels of IL-10 and TGF-β. T. spiralis adult worm excretory–secretory products (AES) and muscle larvae excretory–secretory products (MES) were both able to activate BMDCs in vitro to facilitate their maturation and to create regulatory cytokines IL-10 and TGF-β. The T. spiralis AES- and MES-pulsed dendritic cells (DCs) possessed abilities not only to present antigens to sensitized CD4+ T cell to stimulate their proliferation but also to induce naive CD4+ T cells to differentiate to Treg cells secreting IL-10 and TGF-β. The passive transfer of T. spiralis AES- and MES-pulsed bone marrow-derived dendritic cells (BMDCs) conferred the naive mice to acquire the differentiation of Treg cells. T. spiralis AES possesses a better ability to induce Treg cells than did MES, although the latter has the ability to induce CD4+CD25−Foxp3+ Treg cells. The results obtained in this study suggested that T. spiralis ES products stimulate the differentiation of host Treg cells possibly through activating dendritic cells to create a regulatory environment that benefits the survival of the parasite in the host.

Helminths develop strategies to escape host immune responses that facilitate their survival in the hostile host immune environment. Trichinella spiralis, a tissue-dwelling nematode, has developed a sophisticated strategy to escape complement attack. Our previous study demonstrated that T. spiralis secretes calreticulin (TsCRT) to inhibit host classical complement activation through binding to C1q; however, the C1q binding site in TsCRT and the specific mechanism involved with complement-related immune evasion remains unknown. Using molecular docking modeling and fragment expression, we determined that TsCRT-S, a 153-aa domain of TsCRT, is responsible for C1q binding. Recombinant TsCRT-S protein expressed in Escherichia coli had the same capacity to bind and inhibit human C1q-induced complement and neutrophil activation, as full-length TsCRT. TsCRT-S inhibited neutrophil reactive oxygen species and elastase release by binding to C1q and reduced neutrophil killing of newborn T. spiralis larvae. Binding of TsCRT-S to C1q also inhibited formation of neutrophil extracellular traps (NETs), which are involved in autoimmune pathologies and have yet to be therapeutically targeted. These findings provide evidence that the TsCRT-S fragment, rather than the full-length TsCRT, is a potential target for vaccine or therapeutic development for trichinellosis, as well as for complement-related autoimmune disease therapies.

Helminth infection modulates host regulatory immune responses to maintain immune homeostasis. Our previous study identified Trichinella spiralis paramyosin (TsPmy) as a major immunomodulatory protein with the ability to induce regulatory T cells (Tregs). However, whether TsPmy regulates gut Tregs and contributes to intestinal immune homeostasis remains unclear. Here we investigated the therapeutic effect of recombinant TsPmy protein (rTsPmy) on experimental colitis in mice, and elucidated the roles and mechanisms of colonic Tregs induced by rTsPmy in ameliorating colitis. Acute colitis was induced by dextran sodium sulfate (DSS) in C57BL/6J mice, and chronic colitis was induced by naïve T cells in Rag1 KO mice. Mice with colitis were pre-treated with rTsPmy intraperitoneally, and clinical manifestations and colonic inflammation were evaluated. Colonic lamina propria (cLP) Tregs phenotypes and functions in DSS-induced colitis were analyzed by flow cytometry. Adoptive transfer of cLP Tregs treated by rTsPmy into Rag1 KO chronic colitis was utilized to verify Tregs suppressive function. rTsPmy ameliorated the disease progress of DSS-induced colitis, reduced pro-inflammatory responses but enhanced regulatory cytokines production in DSS-induced colitis. Moreover, rTsPmy specifically stimulated the expansion of thymic-derived Tregs (tTregs) rather than the peripherally derived Tregs (pTregs) in the inflamed colon, enhanced the differentiation of effector Tregs (eTregs) with higher suppressive function and stability in colitis. This study describes the mechanisms of colonic Tregs induced by the Trichinella-derived protein rTsPmy in maintaining gut immune homeostasis during inflammation. These findings provide further insight into the immunological mechanisms involved in the therapeutic effect of helminth-derived proteins in inflammatory bowel diseases.

BackgroundTrichinella spiralis is a tissue-dwelling parasite has developed the ability to evade the host immune attack to establish parasitism in a host. One of the strategies evolved by the nematode is to produce proteins that immunomodulate the host immune system. TsPmy is a paramyosin secreted by T. spiralis on the surface of larvae and adult worms that can interact with complement components C1q and C8/C9 to compromise their activation and functions. To better understand the mechanism of TsPmy involved in the C1q inactivation and immune evasion, the C1q-binding site on TsPmy was investigated.MethodsThe TsPmy C1q-binding site was investigated by sequential narrow-down fragment expression in bacteria and peptide binding screening. C1q binding activity was identified by Far-Western blotting and ELISA assays.ResultsAfter several runs of sequential fragment expression, the C1q binding site was narrowed down to fragments of N-terminal TsPmy226-280aa and TsPmy231-315aa, suggesting the final C1q binding site is probably located to TsPmy231-280aa. A total of nine peptides covering different amino acid sequences within TsPmy231-280aa were synthesized. The binding assay to C1q determined that only P2 peptide covering TsPmy241-280aa binds to C1q, indicating that the C1q binding domain may need both the linearized sequence and conformational structure required for binding to C1q. The binding of peptide P2 to C1q significantly inhibited both C1q-initiated complement classical activation and C1q-induced macrophage chemotaxis.ConclusionsThis study identifies the C1q binding site within TsPmy which provides helpful information for developing a vaccine against trichinellosis by targeting the C1q-binding activity of TsPmy.