Cytokine responses to various larval stages of equine strongyles and modulatory effects of the adjuvant G3 in vitro

Abstract

Aims: To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites.

Methods and results: EqPBMC were exposed to S. vulgaris larvae (L3, exsheated L3 and L4) and cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL-4, IL-5 and IL-13 was induced by both S. vulgaris and cyathostomin L3. Moulting of S. vulgaris from L3 to L4 stage was accompanied by a shift to high expression of IL-5 and IL-9 (exsheated L3 and L4) and IFN-γ (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL-4, IL-5 and IL-10 while concomitantly enhancing the expression of IFN-γ.

Conclusion: The L4 stage of S. vulgaris generated a cytokine profile different from that induced by the earlier L3 stage of S. vulgaris and cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.

Keywords: adjuvant; cytokine; gene expression; horse; parasite; peripheral blood mononuclear cells.

FIGURE 1

Various preparations of S. vulgaris larvae. (A) S. vulgaris L3 before UV irradiation, (B) UV‐irradiated S. vulgaris L3, (C) S. vulgaris L3 exsheated in 0.1% sodium hydrochlorite, (D) L2 cuticle from exsheated S. vulgaris L3, and (E) S. vulgaris moulting to L4 after five days of culture in cKW2 medium (Photo: Nikon Eclipse TS100/Nikon Coolpix 990)

FIGURE 2

Cultures of eqPBMC isolated from the same horse and exposed to UV‐irradiated cyathostomin L3 (A) or S. vulgaris L3 (B) for 4 hours. (Photo: Leitz Labvert/Canon EOS Rebel T3i)

FIGURE 3

Relative expression of cytokine genes in eqPBMC cultured in plain growth medium (medium control) or in the presence of UV‐irradiated S. vulgaris L3, cyathostomin L3, the G3 adjuvant, chitin or endotoxin. The cytokine gene expression was normalized to the geometric mean for the reference genes (SDHA and RPL32) and calibrated to that in the medium control. FC > 2 (indicated by dashed line) is considered as up‐regulated, and significant differences against the medium control are indicated in the figure by P < .05, P < .01, P < .001

FIGURE 4

Relative expression of cytokine genes in eqPBMC cultured in the presence of the G3 adjuvant, UV‐irradiated cyathostomin L3 or S. vulgaris L3, alone or combinations thereof. The effect of cyathostomin L3 or S. vulgaris L3 was evaluated in two separate experiments (closed and open circle, respectively). The cytokine gene expression was normalized to the geometric mean for the reference genes (SDHA and RPL32) and calibrated to that in the medium control. FC > 2 (indicated by dashed line) were considered as up‐regulated. Significant differences between stimulations are indicated by *<0.05, **<0.01, ***<0.001

FIGURE 5

Relative expression of cytokine genes in eqPBMC cultured in the presence of UV‐irradiated S. vulgaris at different stages of ecdysis; S. vulgaris L3, Exsheated S. vulgaris L3 or S. vulgaris L4 (closed circle). As controls, eqPBMC were exposed to plain cKW2 medium (cKW2 pre‐culture) or supernatants collected from the L4 culture (cKW2 post‐culture; open circle). The cytokine gene expression was normalized to the geometric mean for the reference genes (SDHA and RPL32) and calibrated to that in the medium control. FC > 2 (indicated by dashed line) was considered as up‐regulated, and significant differences between larval stages are indicated by *<0.05, **<0.01, ***<0.001