In vitro cytokine response of circulating mononuclear cells from healthy dogs to stage-specific antigens of Angiostrongylus vasorum.

Abstract

Background: Circulating lymphocytes are essential for the immune response to helminth infections, as they secrete cytokines and thus coordinate subsequent immunological processes. In canine angiostrongylosis, a potentially fatal disease caused by the metastrongylid nematode Angiostrongylus vasorum, the underlying immune mechanisms driving disease progression remain poorly understood despite the severe clinical consequences. Canine peripheral blood mononuclear cells (PBMCs) were isolated from healthy dogs and stimulated with three different antigens of A vasorum, including adult excretory-secretory products (ESP), adult full-worm somatic antigen, and first-stage larval (L1) somatic antigen. Temporal dynamics and magnitude of relative cytokine expression (IFNγ, TNF, IL-4, IL-6, IL-10, IL-13) was evaluated after 4 h, 24 h, and 5 days of antigen exposure via quantitative reverse transcription PCR. To determine whether the cytokine expression changes translated into shifts in circulating mononuclear cell subsets or proliferative activity, phenotypic characterisation by flow cytometry was conducted after a 72 h stimulation with L1 antigen or ESP, and compared to unstimulated controls.

Results: Early responses varied across antigen types, with ESP promoting a regulatory cytokine profile with modest upregulation of IL-4, IL-6, and IL-10, and downregulation of IFNγ, TNF, and IL-13. Adult antigen induced increased expression of all examined cytokines, while L1 antigen triggered the strongest inflammatory response compared to the other antigens. At 24 h, all responses were amplified, particularly those to L1 and adult antigen, and showed a shift towards a Th2 cytokine profile with increased IL-4 and IL-13 expression. By five days, IL-4 and IL-13 remained predominant. No change in the relative abundance of major immune cell populations (CD4⁺ T cells, CD8⁺ T cells, B cells, neutrophils, monocytes, and CD14⁻ CD22⁻ antigen-presenting cells) was observed in flow cytometry following stimulation. However, a notable increase in Ki67 expression, a marker of cell proliferation, was detected in CD8⁺ T cells after L1 antigen stimulation.

Conclusions: Distinct cytokine profiles elicited by different A. vasorum antigens suggest that the parasite's modulation of host immunity and induction of stage-specific responses are key to persistence and clinical presentation of canine angiostrongylosis. Further investigation into the antigenic components and immune pathways may lead to tailored therapies, improved clinical management, and to a deeper understanding of stage-specific aspects of helminth infections.