In the current study, the Salmonella -induced pathway and network changes were mainly observed to show inflammatory inhibition and oxidative stress in mitochondria at the early stage of infection, while at the late stage of infection, the dramatic changes in thousands of gene expression are characterized.
Two networks for up-regulated genes around IFN-γ and TNF-α were identified and cross-talked with some identified signaling pathways. Furthermore, a series of pathways associated with inflammatory/immune response, cell proliferation, cell apoptosis, and developmental disorder were appraised. The biochemical and pathologic data were consistent with the microarray analysis and confirmed the biological role of Salmonella in inducing inflammation and epithelium cell proliferation through the regulation of multiple signaling pathways.
Salmonella infection and apoptosis
Intestinal epithelial apoptosis is a response to bacterial infection . Salmonella effector AvrA dampened the proapoptotic innate immune response to Salmonella at the mouse intestinal mucosa . Our microarray data also showed that a number of genes involved in apoptosis presented Salmonella -induced expression changes, including up-regulated Caspase family members (CASP3, CASP7, CASP8 and CASP12), Poly (ADP-ribose) polymerase family members (PARP3, PARP9, PARP12 and PARP14) and some down-regulated genes (Famim3, Stk4, Stk17 and Nalp1). Accordingly, as shown in Table 2 strong induction of apoptosis-related pathways were involved in response to Salmonella infection at 4 days, such as IL-9 (anti-apoptosis), retinoic acid mediated apoptosis (pro-apoptosis), caspase family mediated apoptosis(pro-apoptosis), and LPS-stimulated MAPK pathway (pro-apoptosis). These apparently contradictory pathways may reflect the complexity of the apoptosis process in mouse colon mucosa responded to Salmonella infection.
Salmonella effector protein SigD/SopB protects epithelial cells from apoptosis by sustained activation of Akt [75, 76]. Our microarray analysis along with the Western blots and immunostaining in vivo confirmed these previous researches. Overall, these results suggest that Salmonella infection in vivo increased Akt protein levels and induced Akt activation, thus regulating multiple signaling pathways.
Epidermal growth factor receptor (EGFR) is involved in Salmonella infection in vivo
EGFR is a transmembrane glycoprotein with an intrinsic tyrosine kinase. Ligand binding to the EGFR activates cell signaling. Galan et al. (1992)  reported that stimulation of the EGF receptor is involved in the invasion of cultured Henle-407 cells by Salmonella infection. EGFR downstream signaling proteins initiate several signal transduction cascades, principally the Stat3/Stat1, MAPK, Akt and JNK pathways, leading to DNA synthesis and cell proliferation . Bertelson et a l. further reported Salmonella effector SigD can activate the EGFR signaling in T84 epithelial monolayers cells through downstream signaling PI3K, and pointed out that this activation may induce different actions than what is observed in the EGFR pathway . We observed the mRNA level of EGFR to be up-regulated (Additional file 22 Table S22 and Figure S1), and downstream signaling protein, such as STAT3, STAT1, AKT3 and MKK4 also showed up-regulation at 4 days post infection. Hence, our microarray data confirms previous research and extends the down-stream signaling of EGFR response to Salmonella infection and provides more comprehensive information about the EGFR pathway involved in Salmonella infection.
Oxidative stress response signaling and metabolism
NRF2-mediated oxidative stress response signaling was the most significant pathway at 4 days post infection (Table 2). This pathway involved 55 up-regulated genes and 24 down-expressed genes. Oxidative stress is caused by an imbalance between the production of reactive oxygen and the detoxification of reactive intermediates. Severe oxidative stress can trigger apoptosis and necrosis. The cellular defense response to oxidative stress includes induction of detoxifying enzymes, heat shock proteins, and antioxidant enzymes (microarray data suggested). Roland Nilsson et al found that LPS stimulation is a pivotal role for NRF-2 in orchestrating the LPS response in macrophages . NRF2-mediated oxidative stress response signaling in the mouse colon intestine may be activated by Salmonella LPS. Interestingly, heat shock protein 40 (DNJ family) showed significant change in this pathway (Additional file 23 Table S23). Of the DNJ members, DNJ5 showed the most significant up-regulation. Takaya A et al (2004) reported that DnaK/DnaJ chaperone machinery is involved in the bacterial invasion of intestine epithelial cells. Recently, ERdj3, an endoplasmic reticulum luminal chaperone of the Hsp40/DnaJ family, is further indentified as a target for Salmonella effector protein SlrP in HeLa cells . Taken together, Salmonella effector Slrp may play a role in transmitting NRF2-mediated oxidative stress response signaling in colon mucosa.
As shown in Additional file 10, Figure S6 and Additional file 24 Table S24, all genes involved in antigen presentation pathway were up-regulated. These results are consistent with the gene expression patterns observed in the porcine lung during Salmonella infection . These data illustrate that the antigen processing pathway was activated by pathogenic Salmonella infection in colon mucosa (Table 2 Figure S6).
Most genes, such as CD80, FAS, PLA2G12A, PLA2G12B, PTGS, TNFRSF1A, TNFSF1B, IIGP1, TRAF1, TIRAP, AKT3, TLR2, TLR3, TLR4, STAT1, STAT2, SLC11A1, SLC16A3, IRF9 IFIT2, and IL1B, which are known to be involved in innate/inflammatory pathway, increase their RNA expression levels significantly at 4 days post-infection. Accordingly, p38 MAPK signaling, MIF regulation of innate immunity, and LPS-stimulated MAPK signaling pathways were all activated. Most of interferon-induced protein, such as IFI35, IFI73, IFNAR2 and IFNG, were up-regulated by Salmonella. At 4 days post-infection, interferon signaling pathways were strongly affected (Table 2 Additional file 10 Figure S7 and Additional file 15 Table S15). Top functions of these genes were associated with antigen presentation, cell morphology and cell to cell signaling.
As shown Additional file 25 Table S25, 43 enzymes in the valine, leucine, and isoleucine degradation pathway were down-regulated at 4 days post-infection, including acetyl-Coenzyme A acyltransferase family member (ACAA1, ACAA2 and ACAA1B), acyl-Coenzyme A dehydrogenase family member (ACAD8, ACAD10, ACADL, ACADM and ACADS), and aldehyde dehydrogenase family member (ALDH2, ALDH1A1, ALDH1A3, ALDH1A7, ALDH1B1, ALDH1B1, ALDH3A2 and ALDH6A1, ALDH7A1). Interestingly, we observed that these enzymes are also involved in other metabolic pathways including valine, leucine, and isoleucine degradation, propanoate metabolism, fatty acid metabolism, and fatty acid elongation in mitochondria. Thus, down-regulation of these important genes may play key role in disordering embolism activities of colon mucosa.
NF-κB is a key transcriptional regulator of innate and adaptive immunity. We found that S100A1, MUC1, and TRIP6 around NF-κB increases NF-κB activity, but rather, BEX2, GLRX3, GPX1 and PXCARD decreases NF-κB activity. Interestingly, our microarray data showed that the expression level of BEX2, GLRX3, GPX1 and PXCARD were down-regulated at 4 days post-infection, which is different from the up-regulated mRNA level at 8 hours post infection. However, S100A1, MUC1, and TRIP6 showed a continued up-regulated status at 4 days post-infection.
I κ Bα and I κ Bz as inhibitory genes are activated by NF-κB in a negative feed back loop, which provides an effective mechanism for controlling the NF-κB activity [83, 84]. However, we found both genes were not indentified in this network. Further microarray data also showed mRNA level of I κ Bα and IκBz remained unchanged at 8 hours post infection, but showed prominent change at 4 days post infection.
Based on the above microarray information, we speculate that NF-κB activity undergoes early stimulation without demonstrable feedback regulation, but at with demonstrable feedback regulation at the late stage of infection. Porcine MLN during Salmonella infection also showed the similar regulation process .
IFN-γ and TNF-α
IFN-γ is a remarkable cytokine that orchestrates many distinct cellular programs through transcriptional controlling over large numbers of genes . The role of IFN-γ is related to host defense against Salmonella infection . Actually, the network analysis supports that interferon signaling was activated by Salmonella infection. We further pointed out the central role that IFN-γ plays in mice colonic against bacterial infection (Figure S7 and Table S15).
GTPase family is clearly regulated by IFN-γ-induced genes , which regulate the survival of pathogens residing in phagosomes vacuoles. We observed that GTPase family members, such as GViN1, Gbp8, Gbp5, IIGP1 and IRGM, are directly targeted by IFN-γ (Figure 6A). The data correlate with the observation in rat colonic cells infected with Salmonella [58, 88]. In particular, IIGP1 was found to be highly up-regulated in our microarray data (90 fold times). Uthaiah RC et al (2003)  also reported that recombinant IIGP1 showed cooperative enzymatic activity and GTP-dependent multimerization.
TNF-α encodes a multifunctional proinflammatory cytokine that belongs to the tumor necrosis factor (TNF) superfamily. This cytokine is involved in the regulation of a wide spectrum of biological processes including cell proliferation, differentiation, apoptosis and lipid metabolism [90, 91]. As expected, the genes in this network are associated with TNF function. Interestingly, we observed GBP4 and GBP6 as IFN-γ induced genes (Figure 6A) that are also involved in TNF-α network. GBP4 showed highly up-regulated in microarray data. Degrandi et al. (2007) reported mouse TNF-α protein increases expression of mouse GBP4 mRNA in ANA-1 cells, but we did not find other reports showing that GBP6 were TNF-α-induced genes. Therefore, further experiment is needed to establish whether this gene is up-regulated by TNF-α in mouse colonic mucosa after Salmonella infection.
Clare et al. (2003) used ICAM (-/-) knockout mice to demonstrate that ICAM-1 plays a critical role during the rechallenge of immunized mice with virulent Salmonella . Our network and microarray data also confirmed that the intracellular adhesion molecule ICAM was induced by TNF-α. We further observed CTSZ as an antigen presentation molecule is also up-regulated. Thus, the network analysis is consistent with the previous experiment results: production of TNF-α in the intestinal tract following S. typhimurium infection and the observation that early pathology induced by Salmonella infection of the gastrointestinal tract is mediated by immune mechanisms .
Overall, the number of connections among the molecules other than TNF-α or IFN-γ is quite limited (Figure 6A and Figure 7A). Most of genes are targeted directly by TNF-α or IFN-γ, which are very different from that of NF-κB network shown in Figure 4. Hence, these TNF-α or IFN-γ networks further reflect the pleiotropic action of proinflammatory cytokine involved in host defense against Salmonella infection by regulating extensive biological process.
T helper 2 (Th2) immune response
Both Interleukin-4 (IL-4) and Interleukin-9 (IL-9) are multifunctional cytokine secreted by T helper 2 (Th2) lymphocytes. IL-9 stimulates the growth and proliferation of T cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors [95, 96]. IL-4 plays a critical role in the regulation of immune responses  and the pathogenesis of inflammatory bowel disease [98, 99].
Previous research study reveled that IL-9 receptor and IL-4 receptor ligation results in auto and/or trans-phosphorylation of Janus kinases 1 and 3 (JAK1 and JAK3) phosphorylation of the receptor, and activation of the pathways involved in IL-9 signaling and IL-4 signaling [100, 101]. These pathways include signal transducer and activator of transcription 1, 3, 5 and 6 (STAT1, STAT3, and STAT5 and STAT6), Insulin receptor substrate 1 and 2 (IRS-1 and IRS-2)/Phosphoinositide-3-kinase (PI3K regulatory subunit) and Extracellular signal regulated kinases 1 and 2 (ERK1/2) [102–104].
We observed the mRNA level of IL-9 receptor (IL-2 R) and IL-4 receptor (IL-2R, IL-4R IL-13 R) are up-regulated and that downstream signaling protein, such as JAK2 JAK3, STAT1, STAT2, STAT3, IRS1, SOCS1 and SOCS3 showed up-regulation at 4 days post infection (Figure S2, Figure S8, Additional file 26 Table S26, and Additional file 27 Table S27). Dumoutier et al. reported that STAT1 and STAT3, activated by IL-9, then up-regulate the transcription of IL-3 and IL-22, which are involve in the generation of inflammatory and allergic responses . Accordingly, we also observed that Interleukin-3 and 22 were up-regulated in mouse colon mucosa with Salmonella infection at four days (Additional file 2 Table S2). IL-4 is produced in response to IL-18 or IL-33 stimulation from mouse basophils . We also found IL-18b and IL-33 to be up-regulated (Additional file 2 Table S2). Overall, these data illustrate that the IL-4 and IL-9 signaling pathway associated with TH2 immune response was activated by pathogenic Salmonella infection in colon mucosa.
Recent advances have called attention to the the involvement of allergen- and parasite product-mediated activation of epithelial cells, basophils and dendritic cells and the functions of the cytokines IL-4, IL-25, IL-33 in the initiation and amplification of TH2-type immune responses in vivo [106, 107].
Cytokines play a key role in IBD that determine T cell differentiation of Th1, Th2, T regulatory and newly described Th17 cells . Hence, IL-4 and IL-9 signaling pathway activated in mouse mucosa with Salmonella infection provides more comprehensive information about how the Th2 immune system interplays with signaling transducers in colon mucosal inflammation.
In Drosophila, the Janus kinases-signal transducers and activators of transcription (Jak-Stat) pathway plays an important role in hematopoiesis, stress response, stem cell proliferation, and antiviral immunity in intestine [87, 108–110]. Interestingly, mouse microarray data showed Jak2, Stat1 and Stat3 as vital proteins in this pathway and were up-regulated at the 4 days post-infection. The mouse colon mucosal complex system is different from Drosophila gut, stat proteins are intracellular effector molecules of cytokine-modulated signaling in mammalian immune system . Further research is needed to validate our analysis and how JAK-Stat signaling regulates the host response during Salmonella infection.
However, even if we confirmed the coherence of our microarray data by other molecular biology approaches, this study has limitations: transcriptional changes not representing the changes at the post-transcriptional level, posttransductional behavior of the differentially expressed genes, and statistical error. For example, our published data showed that Salmonella effector AvrA can activate the beta-catenin pathway through deubquitination . However, this activated pathway was not revealed in this analysis. Further studies combining genomic and proteomic are necessary to find out more details of host cell interplay with Salmonella. Moreover, the colon mucosa tissue samples used in this experiment contained several cell types. Diverse pathways may be activated in different cell types, not necessarily within one kind of cells. Future research on structured information and pathways occurring in individual kind of cells is required.