Pyroptosis and adaptive immunity mechanisms are promptly engendered in mesenteric lymph-nodes during pig infections with Salmonella enterica serovar Typhimurium

Abstract

In this study, we explored the transcriptional response and the morphological changes occurring in porcine mesenteric lymph-nodes (MLN) along a time course of 1, 2 and 6 days post infection (dpi) with Salmonella Typhimurium. Additionally, we analysed the expression of some Salmonella effectors in tissue to complete our view of the processes triggered in these organs upon infection. The results indicate that besides dampening apoptosis, swine take advantage of the flagellin and prgJ expression by Salmonella Typhimuriun to induce pyroptosis in MLN, preventing bacterial dissemination. Furthermore, cross-presentation of Salmonella antigens was inferred as a mechanism that results in a rapid clearance of pathogen by cytotoxic T cells. In summary, although the Salmonella Typhimurium strain employed in this study was able to express some of its major virulence effectors in porcine MLN, a combination of early innate and adaptive immunity mechanisms might overcome virulence strategies employed by the pathogen, enabling the host to protect itself against bacterial spread beyond gut-associated lymph-nodes. Interestingly, we deduced that clathrin-mediated endocytosis could contribute to mechanisms of pathogen virulence and/or host defence in MLN of Salmonella infected swine. Taken together, our results are useful for a better understanding of the critical protective mechanisms against Salmonella that occur in porcine MLN to prevent the spread of infection beyond the intestine.

Figure 1

Gene network analysis by Ingenuity Pathway Analysis (IPA). Visual representation of networks 1 and 4. Red and grey nodes are input and IPA-inserted molecules respectively. Colored lines highlight genes that take part in an enriched Canonical Pathway.

Figure 2

Top 10 enriched canonical pathways. Blue bars and yellow squares denote –log(p-value) and ratio respectively. Threshold line represents a p-value of 0.05.

Figure 3

Interaction between CTLA-4 signaling in cytotoxic T lymphocytes and Clathrin-mediated endocytosis signaling pathways. A: visual representation of CTLA-4 signaling in cytotoxic T lymphocyte pathway stressing the regulation of molecules taking part in Clathrin-mediated endocytosis signaling. Red and grey nodes are input and IPA-inserted molecules respectively. B: Western blot analysis of CTLA4 and CLTA in MLN at 1, 2 and 6 dpi. C: mRNA quantification of CTLA4 by qPCR. D: expression pattern of CLTA mRNA by microarray analysis.

Figure 4

Analysis of MHC molecules in porcine MLN after Salmonella Typhimurium infection. A: Western blot analysis of MHC class I and II at 1, 2 and 6 dpi. B-E: MHCII labeling in tissue at 0 (B), 1(C), 2 (D) and 6 (E) dpi by immunohistochemistry. Scale bar = 20 μm. F-G: mRNA quantification of SLA-DRB5 (MHCII) (F) and SLA-B (MHCI) (G) by qPCR. H-K: confocal analysis of Salmonella Typhimurium infected MLN demonstrate the presence of the pathogen in MHCII positive cells. Salmonella Typhimurium-FITC (H), MHCII-Alexa Fluor® 594 (I), DAPI (J) and merge (K).

Figure 5

Cell death and histopathologic analysis of MLN from Salmonella Typhimurium infected pigs. A-D: Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) analysis at 0 (A), 1(B), 2 (C) and 6 (D) dpi. Scale bar = 100 μm. E-H: H&E staining of at 0 (E), 1(F), 2 (G) and 6 (H) dpi. Scale bar = 50 μm. I: Quantification of TUNEL fluorescent labeling shows an increase of positive nuclei at 1 dpi and a decrease to levels inferior than the controls at 2 and 6 dpi. J-K: mRNA quantification of CASP1(J) and CASP3(K) by qPCR.

Figure 6

Salmonella Typhimurium labeling and gene expression in porcine MLN. A-B: Different labeling profiles found for Salmonella Typhimurium in porcine MLN. Scale bar = 10 μm. (A) Pathogen detection as spherical structures in the perinuclear zone of mononuclear cells. (B) Staining of bacilli shaped structures. C: Analysis of Salmonella Typhimurium gene expression by SCOTS in vivo and in vitro. Black dots and bars respectively represent individual and mean expression values from analysis of cDNA from pig infected MLN. Triangles (early logarithmic phase) and squares (late logarithmic phase) denote gene expression data from Salmonella Typhimurium cultures. Higher values mean higher expression levels and vice-versa.