Trichinellosis, a widespread zoonosis, is regarded as an emerging or reemerging disease. Effective treatment and prognosis of trichinellosis depends on early diagnosis of the infection. The objective of this study was to identify sensitive and specific antigens for early diagnosis or effective vaccine antigens for preventing infection. The somatic proteins of T. spiralis adult worms were separated by two-dimensional gel electrophoresis (2-DE). The separated proteins were probed with early infection sera from swine or mice infected with T. spiralis for 7 days. The primary immunoreactive spots were characterized by MALDI-TOF/TOF-MS analysis in combination with bioinformatics. The identified proteins were annotated using WEGO based on their functions. The immunodominant protein was chosen for expression as recombinant protein in E. coli and the purified recombinant protein was used to confirm its antigenicity by Western blot with the same infection sera. Approximately 300 spots were separated by 2-DE, with molecular weights ranging from 10 to 130 kDa, and pI values ranging from pH 4 to 10. The sera from swine and mice infected with T. spiralis for 7 days recognized 64 proteins. MALDI-TOF/TOF-MS analysis identified 55 proteins, some with different isoforms. Finally, 40 individual immunoreactive proteins were obtained with a wide range of biological functions. Several proteins, such as heat shock protein 70, 14-3-3 protein, and cysteine protease could be used as immunodiagnostic or vaccine antigens. Among these identified proteins, the highly immunodominant Ts14-3-3 was chosen for expression in E. coli and purified recombinant Ts14-3-3 was able to be strongly recognized by the same T. spiralis infected sera used for identifying these antigens, therefore the most promising antigen for early immunodiagnosis of Trichinella infection. A total of 64 proteins from the adult worm were recognized by early infection sera from swine and mice infected with T. spiralis for 7 days. Several proteins, are of particular interest as immunodiagnostic or vaccine antigens, especially with Ts14-3-3 as most promising due to its highly immunogenicity during early infection, expressed protein can be recognized by Trichinella early infection sera and the native Ts14-3-3 expression in both adult and larval stages.

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.

Dendritic cells (DCs) are antigen-presenting cells that regulate T cell responses for many infectious diseases. The tissue-dwelling nematode Trichinella spiralis expresses paramyosin (TsPmy) not only as a structural protein but also as an immunomodulator to alleviate complement attack by binding to some host complement components. Whether TsPmy is involved in other immunomodulatory pathway and how TsPmy interacts with host DCs is still unknown. Mouse bone marrow-derived DCs were incubated with recombinant TsPmy (rTsPmy) for activation. Maturation of DC was determined by the expression of surface markers CD40, CD80, CD86 and MHCII. The rTsPmy-pulsed DCs were co-incubated with T. spiralis-sensitized or naïve mouse CD4+ T cells to observe their activation on T cells and polarizing regulatory T cells using flow cytometry. Cytokines were measured by enzyme-linked immunosorbent assays (ELISA). TsPmy was able to activate mouse bone marrow-derived DCs to semi-mature status characterized by expressing surface CD40 and CD86, but not CD80 and MHCII. The semi-mature TsPmy-pulsed DCs were able to stimulate T. spiralis-sensitized CD4+ T cells to proliferate. Incubation of TsPmy-pulsed DCs with naïve CD4+ splenocytes polarized the latter to CD4+CD25+Foxp3+ regulatory T cells. However, mice immunized with rTsPmy only induce the CD4+CD25−Foxp3+ T cell population, associated with high level of IL-10, TGF-β and IL-17A. During T. spiralis infection, TsPmy plays an important role in modulating the host immune system by stimulating DCs to differentiate the CD4+ T cells to regulatory T cells, in addition to binding to components of the host complement cascade, as survival strategies to live in host.

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.

Background: Trichinella spiralis adult excretory-secretory antigen (TsAES) has been proposed as a potential immunomodulator capable of promoting naïve T cell differentiation into peripheral regulatory T cells (pTregs). This study aims to investigate the effects of TsAES on pTreg development and evaluate its role in alleviating dextran sulfate sodium (DSS)-induced acute colitis. Methods: Colonic lamina propria mononuclear cells (LPMC) from Foxp3^eGFP mice were cultured with interleukin-2 (IL-2) and TsAES for 3 days. The frequency of CD4⁺CD25⁺Foxp3⁺ Treg cells was quantified by flow cytometry. Foxp3^eGFP mice subsequently received intraperitoneal injections of TsAES (20 µg/mouse) biweekly for a total of three doses. On day 8 post-model induction, mice were euthanized and colonic tissues collected for immunofluorescence and flow cytometric analysis to assess pTreg populations. Splenic CD4⁺ T cells and dendritic cells (DCs), isolated from Foxp3^eGFP mice using magnetic beads, were cultured with IL-2 in either TsAES or phosphate buffered saline for 72 h. The content of IL-10 in culture supernatant was measured by an enzyme-linked immunosorbent assay (ELISA), and CD4⁺Foxp3⁺ Treg cells were sorted and adoptively transferred (40,000 cells/mouse) via tail vein injection into C57BL/6 J mice, followed by DSS administration to induce acute colitis. Body weight was monitored daily, and disease activity index (DAI) was calculated based on fecal characteristics. On day 8, mice were sacrificed, and colons were dissected and subjected to hematoxylin and eosin staining for histopathology study, and flow cytometry was performed to evaluate splenic Treg proportions. Results: Flow cytometry analysis demonstrated that TsAES stimulation promoted LPMC differentiation into Tregs, leading to a significant increase in pTregs within the colonic lamina propria of TsAES-immunized Foxp3^eGFP mice. Although no significant differences in body weight were observed between Treg-infused and non-infused groups, the Treg-infused cohort exhibited a significantly lower DAI. Histological examination showed reduced inflammatory cell infiltration and better-preserved crypt architecture in the colons of Treg-treated mice. The ELISA results showed an elevated IL-10 level in TsAES-treated splenocytes, and flow cytometric analysis confirmed the presence of Tregs in the spleens of recipient mice following adoptive transfer. Conclusions: TsAES demonstrates tumor growth factor-beta-like activity, driving pTreg differentiation and suppressing DSS-induced colitis with IL-10 upregulation. These findings highlight a beneficial clinical effect of helminth-derived antigens on the immunomodulation and management of inflammatory bowel disease. Graphical