Abstract
BACKGROUND: Trichinellosis is a globally significant zoonotic parasitic disease for which effective preventive strategies remain elusive. Our previous studies identified Ts-Hsp70 as a promising vaccine candidate antigen against Trichinella spiralis, although its precise mechanism of immune protection is not fully understood. Macrophages (Mφ) play pivotal roles in anti-helminth immunity and undergo dynamic M1/M2 polarization shifts during different stages of T. spiralis infection. OBJECTIVE: To investigate whether Ts-Hsp70 enhances host resistance against T. spiralis infection by reprogramming macrophage polarization to provide multi-stage protection. METHODS: The role of macrophages in worm expulsion was confirmed via macrophage depletion using clodronate liposomes (CloA). In vivo, cytokine levels (ELISA) and macrophage polarization (flow cytometry, immunofluorescence) were assessed in the spleen, mesenteric lymph nodes (MLN), intestine, and muscle of Ts-Hsp70-immunized mice at 1, 5, 15, and 30 days post-infection (dpi). In vitro studies evaluated the direct effects of Ts-Hsp70 on macrophage phenotype, phagocytic capacity, and T-cell activation. RESULTS: Macrophage depletion significantly abrogated Ts-Hsp70-mediated expulsion of both adult worms (early stage) and muscle larvae (mid-late stage). In vivo, Ts-Hsp70 immunization promoted a sustained M1-dominated response, increased pro-inflammatory cytokines (IFN-γ, TNF-α) and reduced anti-inflammatory cytokines (IL-4, IL-10) in serum. This shift significantly increased M1/M2 ratios across peripheral organs (spleen, MLN) and local infection sites (intestine, muscle) during mid-late infection. In vitro, Ts-Hsp70 directly induced M1 markers (iNOS, CD80, TNF-α), enhanced phagocytic activity, and promoted CD4(+)T-cell proliferation. CONCLUSION: Ts-Hsp70 confers dual-stage protection against T. spiralis by sustaining M1 macrophage polarization-promoting early adult worm expulsion and disrupting larval survival in later stages. These findings elucidate the mechanism underlying Ts-Hsp70-induced immune protection and reinforce its potential as an effective vaccine antigen against trichinellosis.