The main goal of this study was detection of “first-response” genes that have a major impact on development of Salmonella-induced inflammation latter on. To avoid repetition with studies in which the transcriptional response to Salmonella in the intestine of rats [23, 24], mouse , chicken [26, 27], and pigs [9, 28, 29] was recorded after longer infection periods than 2 and 4 hours, the pathways/genes called significant after 8 hours were discussed briefly in the results section (section isogenic comparisons). In this discussion we focus on 2 and 4 hours genes/processes which may play a crucial role in the regulation of inflammation in the intestine in general.
One of the most important observations in this study was the failure of pig 3 to produce an ongoing IL1B response even though this pig produced a faint IL1B and high IL8 response at 2 hours. Already after 2 hours of perfusion we detected invasion of Salmonella in all 3 test pigs . It is known that crossing of Salmonella over the epithelial barrier is also supported by Microfold (M) cells. Research in humans and mouse revealed that M cells are enterocyte-like cells formed in the Peyers’s patches of the jejunums and ileum. These cells lack microvilli and are able to phagocytize pathogenic organisms/particles and transport them over the epithelial barrier into the lamina propia . After these cells become injected with Salmonella effector proteins  or invaded with whole Salmonella bacteria, M cells undergo cytoskeletal rearrangements to support forming of Salmonella containing vacuoles  and produce an array of cytokines, among them IL8, IL1B and macrophage inflammatory proteins (MIP’s) . IL8 and MIP’s attract and activate neutrophils, basophils, monocytes (macrophages and DC’s), and T-cells. If M cells in pigs are also capable to produce a similar cytokine response to Salmonella, attraction of these cells may have occurred in pig 2 and 4, and stayed behind in pig 3, and with this, also inflammation induced by these cells. In analogy, we observed a response of IL1B and MIP’s mRNA (CXCL2, 5), and CXCL6 (alias; granulocyte chemotactic protein 2) at 4 and 8 hours in pig 2 and 4, but not in pig 3. However, it had to be noted that “normal” enterocytes and residing immune cells can also account for this “first” IL8 and IL1B response, respectively.
Using the “tissue expression” module of DAVID equal proportions of IL8-high and -low genes (17 and 15%, respectively) were mapped to a BDCA4 + DCs expression library (result not shown) indicating that activation of DC’s also occurred in the absence of an IL8 response. Whether this are residing or infiltrating DC’s is not clear. We detected a significant number of genes part of the TSLP Signaling pathway in IL8-high pigs (CISH, GAB1, IL8, MAP2K1, NFKB2, NFKBIA) at 2 hours, a pathway responsible for cross-talk between enterocyte-conditioned DC’s (EC-DC’s) and enterocytes . TSLP mediated cross-talk directs T cell polarization towards a non-inflammatory T helper type 2 (Th2) response , and over production of TSLP results in an exaggerated basophil responses, believed to be responsible for induction of Th2 cytokine-associated inflammatory diseases like asthma and food allergy . However, all TSLP-pathway genes we detected are also essential factors in many other immunological signaling systems. Therefore, we cannot conclude whether TSLP-crosstalk, and with this T cell polarization important for development of inflammation, was differently regulated in the IL8-low pig and two high pigs.
Neutrophils are the first and predominant cells that invade the intestinal site of Salmonella infection [32, 33]. Besides neutrophils engulf pathogenic bacteria, degrade them in fused phago-lysosomes, and express degraded material as MHC class II on their surface, they also produce chemokine’s like the above mentions IL8, CXCL5, and CXCL6. Moreover, neutrophils express cytokines that activate (TNF-α), or suppress (IL10 and TGFβ) T cell responses, regulate Th1/Th2 polarization (IL4 and IL12, respectively), and supports Th2-Th17 differentiation (IL6) . These functions make neutrophils important orchestrators of the first (innate) response in the intestine to bacterial pathogens. In relation to this, IL8-high genes mapped to the “NRF2-mediated Oxidative Stress Response” pathway may support the destruction of Salmonella in phago-lysosomes of neutrophils. The gene SCARB1, mapped to this pathway, was higher expressed in the IL8-low pig 2. SCARB1 is a scavenger receptor located in lipid rafts that facilitates the flux of free and esterified cholesterol between the cell surface and extracellular donors such as high-density lipoproteins (HDL’s). Through its phosphatidylserine-binding activity SCARB1 also plays a role in phagocytosis. Recently it was shown that HDL’s were potent attenuators of neutrophil activity and that free cholesterol alters neutrophil lipid raft structure, and consequently, Ca2+ entry and respiratory burst in these cells . Therefore, SCARB1-mediated cholesterol efflux may influence the activity of neutrophils. In addition, the internal cholesterol load in these phagocytes may be regulated by SCARB1, affecting cortisol metabolism and with this the inflammatory status of the intestine. Besides SCARB1 several other genes, directly or indirectly involved in conversion, transport, or regulation of steroids, were differently expressed in the IL8-low and high pig(s) (e.g. HSD17B6, HMGCS1, SC4MOL, CYP2C8, 9 and 18). The most interesting question to be answered now is which type of phagocytic cell plays a dominant role in early regulation of steroid metabolism.
Two pigs (3,4) that produced an early IL8 and IL1B response at 2 hours activated TLR, RIG1, cytosolic DNA-sensing, and/or NOD signaling pathways responsible for sensing of bacterial “danger signals”. In contrast, in pig 2, in which the IL8 and IL1B response was delayed, no pattern-recognition pathways were called significant. Remarkably, regulation of genes coding for T-cell receptor delta (δ; TRD) and gamma (γ; CD3G) chains was observed in this pig, suggesting that γδ T cells were active at the site of exposure to Salmonella. γδ T cells are the predominant T cells within the population of intraepithelial lymphocytes (IELs) in the intestinal mucosa. They play an early and essential role in sensing ‘danger’ by invading pathogens. In case of an acute Salmonella infection their concentration expands dramatically . γδ T cells do not require presentation of antigens by the MHC-complex and are believed to have a prominent role in recognition of lipid antigens . We observed a different expression of mRNA’s coding for enzymes supporting the oxidation of fatty acids in the peroxisome in IL8-low pig 2 than in the two other pigs. Among these enzymes ACSL3, a key enzyme in the β-oxidation of unsaturated fatty acid, which transcription is regulated by the PPARG - retinoid X receptor (RXR) signaling. It was reported that IL1B-mediated inflammation targets the RXR-alpha receptor (RXRA) for nuclear export and degradation . In addition, our analysis showed that a complete different set of genes belonging to the “LPS/IL-1 mediated inhibition of RXR function” pathway was regulated in the IL8-low pig than in the two high pigs. This suggests that inhibition of PPARG-RXRA signaling influences gene expression of enzymes responsible for β-oxidation of unsaturated fatty acid, and with this peroxisome-mediated processing of lipid antigens recognized by γδ T cells. It would be interesting to investigate by which cells peroxisome processing and presentation of such bacterial lipid antigens is facilitated, M cells or other antigen presenting cells (APC’s), and whether γδ T cells play a major role in recognition of these lipids antigens.
Differences in the level of expression of specific genes between IL8-high and -low pig(s), which may be important for T cell proliferation, survival and activation, were observed. In the IL8-low pig, transducer of ERBB2-1 (TOB1) and MEF2C were expressed higher, and in the high pig NR4A1, FOS, CISH, and NFKBIA. TOB1 associates with SMAD4 and exerts an inhibitory effect on IL2 transcription in T cells [39–41]. IL2 expression by antigen-activated T cells plays a critical role for orchestrating the immune response. It stimulates the proliferation of T and B cells, NK cells, and cells of the monocyte lineage (macrophages, DC’s). In normal T cells, triggering of the TCR-CD3 complex leads to the activation of transcription factors involved in immune processes, including the here detected genes NFKB2, EGR1, and FOS. MEF2C is necessary for the transcriptional activation of IL2 and plays a crucial role in T-cell apoptosis by regulating expression of NR4A1 . As shown in Figure 3, EGR1, FOS, and especially NR4A1, a potent regulator of transcription of steroid enzymes , all play a role in tempering inflammation in pig 3. Transcriptional regulation by TOB1 and MEF2C in the IL8-low pig may therefore be crucial for the function of IELs and with this the production of cytokines that orchestrate communication between the various immune cells in the intestine early after exposure to Salmonella.
Glucocorticoid Receptor (NR3C1) and ERK signaling were the most prominent pathways called in the IL8-high pigs. The GCR-GC complex associates with CEBPA, resulting in CDKN1A (alias p21) production and induction of cell-cycle arrest. CDKN1A was higher expressed in ILB-high pigs 2 and 4 at 4 hours. In addition to Salmonella effector protein AvrA , GC’s represses NFKB-driven production of interleukins and other inflammatory cytokines mediated by transcription factor NFKB in APC’s (macrophages, DC’s) and granulocytes. GC’s specifically stimulate the expression of NFKB inhibitor NFKBIA . Interestingly, ANGPT2, part of both these pathways, was expressed to a higher level in the IL8 low pig 2. ANGPT2 (angiopoietin 2) binds to the same receptor (angiotensin II receptor type 1) as angiotensin II. Activation of this receptor leads to nuclear translocation of ERK1/2 resulting in activation of transcription factors we detected in this study, like FOS, CITED2, NFIL3, PER1 (NFIL3 and PER1 are discussed below), and member of the ETS family of transcription factors (see IL8-low list: ETS2, ELF3). Angiotensin II is a potent activator of cortisol by stimulating NR4A1 binding to its transcriptional response element .
The level of Bactericidal/permeability-increasing protein (BPI) mRNA in IL1B-low pig 3 was higher than in pig 2, the pig with a delayed IL8 and IL1B response and that responded most vigorous to Salmonella. BPI possesses antibacterial, endotoxin-neutralizing and opsonic activity against Gram-negative bacteria, among them Salmonella. Expression of BPI was detected in mucosal epithelia and neutrophils. Recently it was shown that all-trans retinoic acid promotes binding of CEBPB (up-regulated in case an elevated IL8 and/or IL1B expression was observed; see Additional file 1: Table S1) or CEBPE to the BPI promoter and stimulates BPI expression in human myeloid cells . After normalization of IL1B levels in pig 3 at 4 hours degradation of RXRA may stop (see above) and signaling through this receptor may be restored, and with this CEBPB-driven BPI expression. With respect to BPI, it would be interesting to investigate whether the expression level of this bactericidal correlates with colonization and survival of Salmonella bacteria in the GI tract of pigs under natural conditions.
In the IL1B low pig three transcription factors/regulators, PER1, NFIL3 (nuclear factor regulated by IL-3) and TEF, were mapped to the circadian rhythm pathway. TEF and NFIL3 compete for the same “PAR DNA”-transcriptional binding site and both play an important role in transcriptional regulation from the interleukin-3 promoter [49–51] and with this, in IL3-mediated production and differentiation of granulocytes and monocytes (macrophages and DC’s). In addition, NFIL3 represses PER1 transcription , a factor that affects transcription from the “Hypoxia-Responsive Element” . Also, transcription of four other transcription factors/regulators from our gene lists (ALAS1, EGR1, IRF1 and DBP) is regulated by the circadian rhythm system. NFIL3 is highly expressed in CD14+ monocytes (BioGPS), suggesting that transcriptional regulation by TEF, NFIL3 and PER1 could affect the respiratory burst-mediated destruction of Salmonella in the phagozomes of macrophages.
We observed an effect on mRNA expression of Salmonella-response genes IL8 and NFKBIA in IPEC-J2 cells for 9 out of the 20 chemicals. It has to be noted that we tested pure chemicals in a “clean, in vitro environment and in one type of intestinal cells. In vivo, these substances are part of a complex matrix (food-feed) and are subjected to modifications induced by other chemicals, host enzymes, and micro biota during their route from intake to the intestine. Therefore, further in vivo studies, in which all types of functional intestinal cells and immune cells are exposed to pre-digested food or feed preparations, have to prove if these chemicals of natural origin indeed influence inflammatory processes in the intestine of pigs. Moreover, in such experiments the influence of these chemicals on Salmonella colonization and invasion of the intestinal mucosa of the pig may be studied under natural conditions.