Immunogenic Eimeria tenella glycosylphosphatidylinositol-anchored surface antigens (SAGs) induce inflammatory responses in avian macrophages

Abstract

Background: At least 19 glycosylphosphatidylinositol (GPI)-anchored surface antigens (SAGs) are expressed specifically by second-generation merozoites of Eimeria tenella, but the ability of these proteins to stimulate immune responses in the chicken is unknown.

Methodology/principal findings: Ten SAGs, belonging to two previously defined multigene families (A and B), were expressed as soluble recombinant (r) fusion proteins in E. coli. Chicken macrophages were treated with purified rSAGs and changes in macrophage nitrite production, and in mRNA expression profiles of inducible nitric oxide synthase (iNOS) and of a panel of cytokines were measured. Treatment with rSAGs 4, 5, and 12 induced high levels of macrophage nitric oxide production and IL-1β mRNA transcription that may contribute to the inflammatory response observed during E. tenella infection. Concomitantly, treatment with rSAGs 4, 5 and 12 suppressed the expression of IL-12 and IFN-γ and elevated that of IL-10, suggesting that during infection these molecules may specifically impair the development of cellular mediated immunity.

Conclusions/significance: In summary, some E. tenella SAGs appear to differentially modulate chicken innate and humoral immune responses and those derived from multigene family A (especially rSAG 12) may be more strongly linked with E. tenella pathogenicity associated with the endogenous second generation stages.

Figure 1. Effect of rSAGs on the nitrite production by chicken macrophage HTC cells.

Chicken macrophage HTC cells were exposed to 100 µg/mL of each rSAG for 24 h and the levels of nitrite was measured by Griess assay. The results are expressed as mean±SD of three independent experiments. *, P<0.05 was considered significant compared to untreated cells as analysed by unpaired Student's t-test.

Figure 2. Effects of various concentration of rSAGs 4, 5, 12 and Trx on the viability and nitrite production of chicken macrophage HTC cells.

Chicken macrophage HTC cells were exposed to different concentrations (5, 10 and 20 µg/mL) of rSAGs 4, 5 and 12 and Trx for 24 h. (A) Macrophages viability was determined by trypan blue exclusion assay. The viability of macrophages became affected at 20 µg/mL of each rSAG. The results are expressed as mean±SD of three independent experiments. (B) The nitrite production by macrophages was determined by Griess assay. Nitrite production increased from treatment with 5 µg/mL to 20 µg/mL rSAG. *, P<0.05 was considered significant as analysed by unpaired Student's t-test.

Figure 3. Effects of Eimeria tenella merozoite crude lysate, rSAGs 4, 5 and 12 and Trx on chicken macrophage cytokines and iNOS expression.

Chicken HTC macrophages were treated with 10 µg/mL of Eimeria tenella merozoite crude lysate, rSAGs 4, 5 and 12 and Trx for 2, 6, 12 and 24 h. The mRNA transcriptional levels of (A) iNOS; (B) IL-1β; (C) IL-10; (D) IL-12 and (E) IFN-γ were determined by qPCR. Bars represent fold change relative to untreated cells from three independent experiments. *, P<0.05 was considered significant compared to cells treated with Trx as analysed by unpaired Student's t-test.

Figure 4. Reactivity of day 8 and day 14 post-infected chickens sera with crude sporulated oocyst lysate and rSAGs in ELISA.

Crude sporulated oocyst lysate and rSAGs were screened against sera collected from individual Eimeria tenella infected chickens on (A) day 8 (n = 6) and (B) day 14 (n = 6) post-infection. Cut off values are indicated by the horizontal line, mean values are indicated by asterisk symbol.