Protective Mechanisms of Refined Functional Carbohydrates against Salmonella Typhimurium Involve Inhibition of Inflammatory Response and Activation of Apoptosis Related Regulators

Salmonella serovars can invade the host by inducing their own uptake into intestinal epithelial cells. This uptake is induced by virulence proteins delivered into cytoplasm of infected cells by a specialized mechanism known as type III protein secretion system (TTSS) (Bertelsen et al., 2003). These proteins activate signaling pathways involved in cytoskeleton rearrangements and cellular uptake processes (Galán and Zhou, 2000). Besides facilitating the invasion process, the interaction between invading pathogen and host epithelium also leads to activation of genes with pro-inflammatory functions (Hobbie et al., 1997). The ability of Salmonella to cause macrophage programmed cell death or apoptosis, may be important for the initiation of infection, bacterial survival, and escape of the host immune response (Monack et al., 1996). Enzymatic hydrolysis of yeasts produces Refined Functional Carbohydrates (RFCs) that show activity against gram negative bacteria. Earlier studies have shown that the RFCs can agglutinate as well as prevent adherence of several species of Salmonella to the intestinal epithelium preventing it from colonizing in the gastro-intestinal tract (Walker et al., 2017, 2018). Hens at sexual maturity are known to exhibit immunosuppression allowing for considerable surges in Salmonella prevalence in poultry houses (Johnston et al., 2012). The present study aims to investigate the effect of continuous feeding of RFCs on the host’s response to infection. Using the transcriptomics approach, gene expression profiles were generated to identify transcript regulators and pathways that control the response of 17-week old hens, fed diets with or without RFCs, four days post infection with Salmonella Typhimurium.

Ninety-six 1-day old commercial brown egg layer strain chickens were reared in separate floor pens (24 birds per pen, 2 pens per treatment group). Birds received the same basic ration of pullet starter and grower rations (produced at University of Sydney feed mill). Treatment groups were untreated Controls and continuous RFCs in feed (100g/MT) from day old. Oral challenge with 10⁶ CFU of Salmonella Typhimurium DT 135 was given to 16 birds per group at 17 weeks and then to 8 birds per group at 20, 25, 35 and 45 weeks of age and cloacal swabs used to detect Salmonella prevalence using specific PCR. Of the 16 birds per group challenged at 17 weeks, eight birds per treatment were euthanised at 4- and 10-days post infection respectively and their caecal tonsils were harvested and stored in Invitrogen™ RNAlater™ Stabilization Solution (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocols at –20°C until analysed. The caecal tonsils were chosen as it contains as much as 45.7% of the lymph nodules, and as such, are the main source of immune function in chicken guts. Based on PCR results at 4-days post infection, four birds were selected per group and RNA was isolated from their caecal tonsil tissue with a Qiagen RNeasy mini kit and quantity and quality were assessed with a NanoDrop ND-1000spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and an Agilent Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). Labelled cRNA for all samples was prepared using the Ambion (Paisley, UK) WT Expression Kit following the manufacturers’ instructions. Labelled cRNA was hybridised to GeneChip™ Chicken Gene 1.1 ST Array Plate for 16 h at 45°C, followed by washing, staining and finally scanning in an Affymetrix GeneAtlas® Imaging Station and Fluidics Station.

The Affymetrix CEL files were imported into Applied Biosystems™ Transcriptome Analysis Console (TAC) version 4.0 to perform quality analysis checks. Summary statistics were computed after the Robust Multi-Array (RMA) normalization and significance analysis was performed using the Limma (Linear Models for Microarray Data; Smyth, 2005) within the TAC 4.0. To identify the statistically significant changes in gene expression, microarray data were subjected to gene filtering based on a combination of a log 2 -fold change at ±1.3 as cut off and P value ≤0.05. Lists generated by pairwise comparisons were used as input into the bioinformatics tool Ingenuity Pathway Analysis (IPA, Ingenuity Systems, USA), which was used to interpret the gene expression data in the context of biological processes and networks.

Of the 18,214 gene transcripts that were included in the gene chip array, a total of 472 gene transcripts (163 up-regulated and 309 down-regulated) were found to be differentially expressed, at log 2 -fold change of ±1.3 and P < 0.05, in samples from sexually mature birds fed RFCs when compared to Control. The top functional annotations associated with this list of genes were: host response to infection (number of transcripts, n=22), inflammatory response (n=22), carbohydrate metabolism (n=25) and cell signalling (n=24). Inflammatory response network analysis identified molecules in the interferon signalling, pattern recognition receptors (PRRS) triggering innate immunity, Th1 and Th2 pathways indicating both pro-inflammatory and anti-inflammatory functions, role of receptors for recognition of bacteria, apoptosis signalling and JAK/Stat signalling all of which were inhibited in expression (Fig 1). Of the top upstream regulators, IFNA2, Interferon alpha and PRL were Inhibited while TRIM24 and MAPK1 were activated. The biological functions that were upregulated in birds fed control diets as compared to RFCs fed, were pro-inflammation, transmembrane helix/integral component of the membrane (n=11) and secretion (n=8) indicating increased gut modulation and bacterial translocation.

Microarray technology has the advantage of simultaneously investigating the expression of thousands of genes, to enable the study of gene interactions and signal pathways. Salmonella infection in control samples was shown to activate relevant intracellular signalling pathways associated with pro-inflammatory cytokines production, modulation of gut with proliferation through epithelial adhesion and secretory pathways all leading to consequent inflammation, ulceration and translocation of the bacteria. Treatment with RFCs, however, showed decreased activation of inflammatory and host response to infection, carbohydrate metabolism, gastrointestinal disease and apoptosis associated signalling pathways. This was associated with decreased bacterial translocation and decreased systemic inflammation.

The top regulators identified within these pathways were either responsible for the protective response seen by the addition of RFCs or represented the consequence of reduced infection with this treatment. Type I interferons (IFNs) activate intracellular host response cascades and influence the development of innate and adaptive immune responses. IFN signalling activates the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway, leading to transcription of IFN-stimulated genes (ISGs). Down regulation of IFNA2 indicates the host-pathogen interaction and the decreased regulation in response of cells to this signalling pathway, thus calibrating host defences while limiting tissue damage and preventing autoimmunity (Robinson et al., 2012). Tripartite motif-containing (TRIM) superfamily are expressed in response to IFNs and are involved in a broad range of biological processes that are associated with innate immunity. TRIM24 associated with precision autophagy, that leads to recognition and delivery of invasive cellular components to lysosomes for degradation was upregulated in response to RFCs treatment, indicating the clearing mechanisms of Salmonella (Allton et al., 2009). Inhibition of interferon alpha (IFN-α) is related to host survival while inhibition of PRL indicated lowered proliferation, suppression of apoptosis and decreased cytokine production by modulation of immune response to recognise reduction in infection (López-Meza et al., 2010). Activation of the Mitogen-Activated Protein Kinases MAPK1 involves a signalling cascade leading to early activated response to stimuli and the resultant clearance (Hobbie et al., 1997).

The modulatory effects of prebiotics on host response to infection can partly be attributed to altering of immune system related gene expression. RFCs are seen to have effects on cytokine gene expression, including the genes encoding for pro-inflammatory cytokines and T helper (Th1and Th2) cytokines thus boosting the immune function of sexually mature hens. Moreover, the increased apoptotic functions in the presence of prebiotic decreases the numbers of colonised bacteria in the caeca of chicks. The adhesion of pathogenic bacteria onto the surface of yeast derived RFCs instead of intestinal receptors could also be responsible for decreased activation of pro-inflammatory signalling pathways, and, consequently, translocation of bacteria.

Prebiotics like RFCs can benefit during sexual maturity in hen by boosting the immune response while also providing protection from infection. Unravelling the mechanisms involved in these positive outcomes helps to develop targeted feeding strategies. It will be useful to look at the immune modulatory effects at latter time points to look at the longer lasting effects of the prebiotic during period of lay and beyond in hens.

ACKNOWLEDGEMENTS: This research was funded by Australian Eggs. Help from the PRF and Birling team is also acknowledged.