Administration of a Recombinant Fusion Protein of IFN-γ and CD154 Inhibited the Infection of Chicks with Salmonella enterica

Abstract

The cytokines IFN-γ and CD154 have been well established, and they play pivotal roles in immune protection against Salmonella in mice, but their effects and specific mechanisms in Salmonella-infected chickens are less understood. In this study, we conducted animal experiments to screen the highly immunoprotective chIFN-γ-chCD154 fusion protein compared with single protein chIFN-γ or chCD154 in white Leghorn chickens. The results showed that compared with separate pretreatments with chIFN-γ and chCD154, the fusion protein, chIFN-γ-chCD154, synergistically increased survival of infected chickens, reduced bacterial load in feces and organs, and attenuated pathological damage to the liver and cecum. Pretreatment with chIFN-γ-chCD154 also increased humoral immune responses, expression of the tight junction proteins zo-1, occludin, and claudin-1, and the relative abundance of Enterococcus_cecorum, Lactobacillus_helveticus, and Lactobacillus_agilis, which protect against intestinal inflammation. Compared with single protein pretreatment, chIFN-γ-chCD154 significantly upregulated STAT1, IRF1, and GBP1 in infected chickens while decreasing mRNA expression of TLR4, MyD88, NF-κB, TNF-α, IL-6, and IL-1β. In summary, damage to the cecal epithelial barrier and the inflammation induced by S. typhimurium infection was alleviated by chIFN-γ-chCD154 pretreatment through a mechanism involving the TLR4/MyD88/NF-κB and IFN-γ/STAT/IRF1/GBP1 pathways.

Keywords: CD154; IFN-γ; Salmonella Typhimurium; cecum; inflammation.

Figure 1

ChIFN-γ-chCD154 pretreatment alleviated S. Typhimurium infection in chickens. (A) SDS-PAGE and (B) Western blot analysis of chIFN-γ, chCD154, and chIFN-γ-chCD154. Lane M: protein molecular weight standard (The original images are listed in the Supplementary Materials); Lane 1: 19 kDa chIFN-γ protein; Lane 2: 16 kDa chIFN-γ-chCD154 protein; Lane 3: 35 kDa chCD154 protein. (C) Schematic illustration of animal experiments. (D) Survival rates. (E) The weight gain rates (%). The data are expressed as the mean ± SD (n = 3 per group). ** p < 0.01, **** p < 0.0001, and ns = not significant applies to the S. Typhimurium-infected groups pretreated with chIFN-γ, chCD154, or chIFN-γ-chCD154 compared with the control group. (F) Bacterial load in liver, spleen, cecum, and feces of chickens infected with S. Typhimurium. The data are the mean ± SD (n = 3 per group). **** p < 0.0001 are the groups of S. Typhimurium pretreated by chIFN-γ, chCD154, or chIFN-γ-chCD154 versus the infected control group pretreated only with PBS.

Figure 2

ChIFN-γ-chCD154 pretreatment attenuated S. Typhimurium-induced cecal inflammation. (A) Histological examination of the liver (bar = 100 μm). (B) Histological examination of the cecum (bar = 100 μm). (C) Protein expression of zo-1, claudin-1, and occludin. (D) The mRNA expression of zo-1, claudin-1, and occludin (n = 3 per group). (E) Levels of anti-S. Typhimurium IgG antibody in chickens. (F) The sIgA concentration in the cecum (n = 3 per group). The data are expressed as the mean ± SD (n = 3 per group), and a one-way ANOVA test was performed, followed by Tukey’s test. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant.

Figure 3

ChIFN-γ-chCD154 pretreatment alleviated intestinal damage in S. Typhimurium-infected chickens. (A) Alpha diversity indicators. (B) NMDS analysis of beta diversity in different groups (stress = 0.0000999). Relative abundance of bacterial phyla in cecal contents. (C) Relative abundance of bacterial genera in cecal contents (D). (E) Significance analysis of four different genera. (F) LEfSe analysis in different groups. The data are expressed as the mean ± SD (n = 3 per group), and a one-way ANOVA test was performed, followed by Tukey’s test. * p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant.

Figure 4

ChIFN-γ-chCD154 pretreatment altered the transcriptome expression profile in cecal tissues of S. Typhimurium-infected chickens. The volcano plot depicts the up-, down-, and nonregulated genes between the two groups, UNPT (non-pretreated) vs. NC (A) and UNPT vs. T (B). (C) Gene Ontology (GO) enrichment analysis of the DEGs, UNPT vs. NC, and (D) UNPT vs. T. (E) KEGG enrichment analysis of the DEGs, UNPT vs. NC, and (F) UNPT vs. T. (G) Gene clustering tree (dendrogram) based on hierarchical clustering of genes with adjacency-based dissimilarity. The distinctive color band designates the results obtained from the automatic single-block analysis. n = 6 per group. (H) The integrated interaction network comprises significantly enriched signaling pathways and the genes they contain. The node size represents the number of genes enriched in the pathway. n = 6 per group.

Figure 5

ChIFN-γ-chCD154 pretreatment suppressed the TLR4/Myd88/NF-kB pathway in the cecum of S. Typhimurium-infected chickens. The mRNA expression of (A) TLR4, (B) MyD88, (C) NF-kB, (D) TNF-α, (E) IL-1β, and (F) IL-6 in cecal tissues from S. Typhimurium-infected chickens. n = 3 per group. Data are expressed as the mean ± SD, and a one-way ANOVA test was performed, followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns = not significant.

Figure 6

ChIFN-γ-chCD154 pretreatment promoted the IFN-induced JAK/STAT1/IRF1/GBP1 axis in the cecum of infected chickens. (A) Protein expression of STAT1, IRF-1, and GBP1. The mRNA expression of (B) STAT1, (C) IRF-1, and (D) GBP1 (n = 3 per group). Data are expressed as the mean ± SD, and a one-way ANOVA test was performed, followed by Tukey’s test. * p < 0.05, ** p < 0.01, *** p < 0.001, ns = not significant.