Salmonella invasion of the chicken cecum induces a short-lived inflammation mediated by the increased expression of pro-inflammatory cytokine and chemokine genes in the intestinal tissue (Withanage et al., 2005; Setta et al., 2012; Matulova et al., 2013; Rychlik et al., 2014). The activation of the innate immune response induces an influx of heterophils (granulocytes) to the intestine that limits bacterial invasion (Kogut et al., 1994; Kogut et al., 2012) but does not lead to a pathological inflammation that is seen in mammals (Foster et al., 2003; Patel and McCormick, 2014). However, this heterophil infiltration of the intestine does not have a significant protective response against the salmonellae bacteria that remain in the lumenal side of the ceca. Interestingly, this inflammatory response is largely resolved by 2-3 days post-infection (Kogut et al., 2012; Kogut et al., 2016; Kogut et al., 2017) and is characterized by the reduction of pro-inflammatory cytokines mRNA transcription in the cecum to non-infected control levels yet Salmonella can persist in the intestine and be shed in the feces for several weeks (Withanage et al., 2005).

We have previously used a chicken-specific kinome peptide array analysis to define changes in cecal tissue phenotype during the establishment of a Salmonella persistent infection in chickens. Using the kinome array, we described a Salmonella-mediated reprogramming of host signaling pathways around 3 days post-infection to be the principal mechanism developed by the bacteria to establish a successful persistent long-term colonization in chicken cecum (Kogut et al., 2017). A wide range of host immune and metabolic pathways acted as Salmonella targets, including the T cell receptor signaling pathway, JAK-STAT signaling pathway, the Wnt-Ca²⁺ signaling pathway, NF-κB signaling, the mTOR and AMPK metabolic signaling pathways (Kogut and Arsenault, 2015; Kogut et al., 2017; Kogut and Arsenault, 2017). These pathways control cellular functions such as immunity and metabolism and are an indication of the complexity and scale with which Salmonella infection interferes and thwarts host defense mechanisms to establish and maintain long-term cecal colonization.

However, until now, there has been no characterization of the acute host signaling interactome in the chicken intestine during the initial (first 24 h) infection with Salmonella. In the murine Salmonella infection model, Salmonella enterica serovars use self-induced pathologic inflammation which provides a growth advantage in the host gut that favors intestinal colonization (Winter et al., 2010; Winter and Baumler, 2011; Thiennimitr et al., 2012; Patel and McCormick, 2014). An inflammatory response is generated in chickens, but the signaling pathways that are activated or altered are unknown. In this study, we used a chicken-specific global immunologic peptide array to study the changes in cecal immune signaling pathways during the first 24 h following Salmonella Enteritidis infection.

Understanding both sides of the host –Salmonella interaction is essential in discovering alternative strategies to manage infections in poultry. The host’s antimicrobial immunity and the bacteria’s evasion of that immunity both rely on appropriate regulation of transcriptional and signaling networks. To date, the majority of the studies in the chicken-Salmonella interaction has been microbe-centric, concentrating on the pathogen’s infection of the bird intestine by using the Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that encode a specialized type III secretion system (T3SS) to secrete multiple protein effectors into the intestinal epithelium which manipulate the host cell biology to aid in bacteria invasion, intracellular survival, and to modulate host immune responses (Barrow et al., 2012; Figueira and Holden, 2012; Wigley, 2014; Eng et al., 2015). On the host side of the interaction, the transcriptional and signaling descriptions of the interaction has concentrated on the post-infection period starting 2 days after the initial infection by the pathogen. This breach results in a directed innate immune response to purge the bacterium and includes the release of antimicrobial peptides (Crhanova et al., 2011; Garcia et al., 2020), activation of pattern-recognition receptors (Keestra et al., 2013, Kogut, 2022a; Kogut, 2022b), release of chemokines and cytokines (Withanage et al., 2005; Bernd et al., 2007; Rychlik et al., 2014; Meijerink et al., 2021), and recruitment of a variety of immune cells (Van Immerseel et al., 2003; Bernd et al., 2007; Pieper et al., 2011; Meijerink et al., 2021).

Therefore, what is needed is more insight into the immediate response of the chicken to the Salmonella infection process (first 24 hours). For example, chickens infected with Salmonella do not develop clinical disease and the acute inflammatory response is short-lived (Wigley, 2014). Thus, it is vital to understand how the chicken responds to a Salmonella breach of the mucosal barrier system and what is involved in the regulation of the intestinal response that prevents excessive intestinal inflammation and damage yet does not eliminate pathogen colonization. Our recent development of chicken-specific peptide arrays for kinome analysis of host phosphorylation-based cellular signaling responses provided us with the opportunity to develop a more detailed understanding of the chicken host-pathogen interactions with Salmonella (Arsenault and Kogut, 2015; Perry et al., 2020). We have used this technology to describe the immunometabolic phenotypic changes in the avian cecum of Salmonella-infected chickens that decreased the host responsiveness resulting in the establishment of persistent colonization starting around 4 days after the initial infection (Kogut et al., 2016).

Overall, in all the pathways described here, the majority of the differential phosphorylation events between the infected cecal tissue compared to the non-infected control tissue occurred with the first 4-8 hours post-infection. In general, from 8-24 hours post-infection, there was a decrease in phosphorylation alterations in the infected tissue with a return to control levels of phosphorylation. Based on the signaling pathways that were differentially activated (TLR, NOD, chemokine, apoptosis, JAK-STAT), the chicken responds very rapidly with an orchestrated innate response: recognition (TLR and NOD signaling pathway), attraction of activated immune cells (chemokine and JAK-STAT signaling pathways), and the prevention/reduction in pathogen numbers (apoptosis).

Based on the findings described here, the chicken pattern recognition receptors, Toll-like receptors (TLR) and nucleotide-binding oligomerization domains receptors (NOD), recognized molecular associated microbial patterns (MAMPs) associated with S. Enteritidis ( Tables 4 , 5 ; Supplemental Figures 1 and 2 ). The activity of both pathways was activated (phosphorylated) 4-8 hours post-infection and their activity decreased by 24 post-infection. Pathways analysis suggests that NOD1 and NOD2 are specifically involved in the immediate recognition of a Salmonella in chickens. NOD1 and NOD2, two prototypic NLRs, sense the cytosolic presence of bacterial peptidoglycan fragments that escaped from endosomal compartments, driving the activation of NF-κB and MAPK, cytokine production and apoptosis (Li et al., 2017; Tao et al., 2017). However, NOD2 appears to be absent in birds (Boyle et al., 2013). There is no evidence, based on the pathway analysis, that inflammasome-mediated pathways were involved the chicken recognition of S. Enteritidis.

Although kinome analysis of the S. Enteritidis-infected tissue does not provide the specific TLR that initiated the TLR signaling pathway in the chicken, previous studies have convincingly shown that recognition of flagellin by TLR5 is the primary recognition receptor that stimulates the bird’s innate response (Iqbal et al., 2003). Is also possible that TLR4 that recognizes LPS on the Salmonella cell wall and TLR21 which recognizes CpG (bacterial unmethylated DNA) could be involved in initiating the TLR signaling pathway as both have been found in chickens (Higgs et al., 2006). However, chickens are very refractory to LPS except at high levels not found in bacterial cell walls (Adler and DaMassa, 1979; Keestra and Van Putten, 2008).

Chemokines are small chemoattractant peptides that provide directional cues for the cell trafficking and thus are vital for protective host response. In addition, chemokines regulate several biological processes of hematopoietic cells that lead to cellular activation, differentiation and survival. The chemokine signal is transduced by chemokine receptors expressed on the hematopoietic immune cells. After receptor activation, several diverse downstream pathways are activated which encompass a number of biological activities of hematopoietic cells that lead to cellular activation, differentiation and survival. Based on the findings here, the kinomic analysis of the chemokine signaling pathway demonstrates the activation of three downstream pathways: (1) the MAPK/JAK-STAT cascades involved in cell migration, cytokine production, and cell survival, (2) the PI3K → AKT→ Itk cascade that regulates leukocyte transmembrane migration and actin reorganization, and (3) the CCL5 → PLC → PKC cascade that causes the induction of nitric oxide, production of reactive oxygen species, and degranulation (Hughes et al., 2001; Wang et al., 2005; Sick et al., 2006; Hughes et al., 2007). All three cascades were highly phosphorylated in the 4 h post-infection cecal tissue when compared to the non-infected control tissue. From 8-24 h post-infection, there is a dramatic increase in either dephosphorylation events or no differences from the control inn the S. Enteritidis-infected tissues. The kinome analysis of the timing of the chemokine signaling pathway coincides with our previous studies where we found that paratyphoid Salmonella serovars induced a rapid (within 4 h) inflammatory response characterized by an influx of heterophils (the primary granulocytic cell the gastrointestinal tract of chickens (Kogut et al., 1993; Kogut et al., 1994; Genovese et al., 1999; Swaggerty et al., 2006).

Apoptosis is a genetically regulated process of caspase-dependent form of cell death of damaged or infected cells in response to intrinsic or extrinsic signaling cascades (Galluzzi et al., 2018; Wemyss and Pearson, 2019). Typically, in the intestine apoptosis of epithelial cells from the mucosa occurs with little, if any, inflammation and disruption of the epithelial barrier integrity (Schwerk and Schultze-Osthoff, 2005). However, Salmonella-induced cell death (apoptosis, necroptosis, pyroptosis) are crucial components of bacterial-mediated gastroenteritis in mammals (Kim et al., 1998; Fink and Cookson, 2007; Hefele et al., 2018; Wemyss and Pearson, 2019). Alternatively, the major paratyphoid Salmonella serovar infections of poultry leads to little or no clinical signs of disease (Barrow et al., 2012) which may coincide with the fact that there have been no reports of Salmonella-induced cell death in the chicken intestine during colonization and infection. Based on the findings here, S. Enteritidis infection of the cecum induces the apoptosis signaling pathway when compared to the non-infected control cecal tissue. More specifically, in fact, based on the alterations in phosphorylation, S Enteritidis infection stimulated both intrinsic and extrinsic apoptotic signaling cascades ( Table 7 ; Supplemental Figure 4 ). The lack of clinical signs of disease in chickens during paratyphoid Salmonella infection, the importance of maintaining both intestinal homeostasis and the intestinal barrier during the initial phase of infection, strongly suggests that the apoptotic pathway outlined here is a host defense strategy to limit bacterial replication and survival (Sellin et al., 2014). Limiting the initial infection and replication of the pathogen may be an important component of the ‘disease tolerance’ mechanism of host defense that is unique to the chicken-Salmonella interaction that we have described previously (Kogut and Arsenault, 2017; Lee et al., 2020). Finally, we found no evidence, based on phosphorylation events, of either of the pro-inflammatory forms of cell death, necroptosis nor pyroptosis in the Salmonella-infected cecal tissues. Specifically, necroptosis is an inflammatory-mediated, caspase-independent cell death mechanism. We have shown clearly ( Table 7 ; Supplemental Figure 4 ) that caspases 3, 6, and 8 are all phosphorylated; thus activated as part of the extrinsic apoptosis pathway. Further, pyroptosis is a highly inflammatory cell death pathway that requires the development of an inflammasome which mediates the event. We found no evident of an inflammasome development in our studies.

Further kinome pathway analysis of the S. Enteritidis-infected cecal tissue revealed the differential phosphorylation of individual peptides in the JAK-STAT pathway ( Table 8 ). The JAK-STAT pathway is a signaling cascade that provides a direct mechanism to translate an extracellular signal into a transcriptional response, in S. Enteritidis-infected cecal tissue. The JAK-STAT system consists of three main components: (1) a receptor; (2) Janus kinase (JAK); and (3) Signal Transducers and Activator of Transcription (STAT) (Hu et al., 2021). Based on the results from the present experiment ( Table 8 ), the IFN-α, IFN-γ, IL-2, and IL-6 cytokine family (gp130) receptors were phosphorylated; whereas, IL-4, IL-10 were dephosphorylated, and IL-7 receptor showed no change from the non-infected control cecal tissue, at 4 and 8 hours after infection, but was dephosphorylated at 24 h post-infection. IFN-γ is characteristic of a Th1 response whereas IL-2 is normally produced by T cells during an immune response and involved in growth, proliferation, and differentiation of T cells to become “effector” T cells (Cantrell and Smith, 1984; Smith, 1988). Traditionally, IL-6 is a pro-inflammatory cytokine involved in stimulating an immune response during infection which may very well be the case with these findings. However, we also consider strong evidence that IL-6 is involved in metabolic function, rather than just immune response. IL-6 induction has been found to stimulate a metabolic reprogramming [Pavlov and Tracey (2012); Flint et al., 2016 and Ghanemi and St Amand (2018)] which could be happening here with the initiation of an innate response to Salmonella infection. Salmonella infection activates the innate immune system as described above but activating the innate immune system places considerable energy and resource demands on the chicken that requires amplified metabolic requirements and nutrient consumption (Klasing, 2007; Klasing, 2017). The increased phosphorylation of the IL6ST (gp130) receptor throughout the initial 24 hours of infection with Salmonella may be an immunometabolic signature of the instigation of the innate immune response.

Neither the IL-4R nor the IL7R were activated (phosphorylated) during the first 24 h after S. Enteritidis infection in the cecum when compared to the non-infected control tissues. Since IL-4 normally induces differentiation of naïve T helper cells to Th2 cells and IL-7 is involved in early T cell development, it is undoubtedly too early in the immune process for responses, the results are suggestive of premature involvement of T cell-mediated immune responsiveness so early in the infection process. Interestingly, the IL2RB was phosphorylated throughout the 24 hr initial infection period. Classically, IL-2 was described as a T cell growth factor and to also stimulate growth promoting activity in B cells and NK cells (Henney et al., 1981; Robb et al., 1981; Waldmann et al., 1984). However, we showed 20 years ago that IL-2 can directly activate chicken heterophils to exert effector functions and induce heterophil activation (Kogut et al., 2002). It has subsequently been proven that IL-2 has functional activity that connects innate and acquired immunity (reviewed Bendickova and Fric, 2020).

Both the type I interferon (IFNAR1, IFN-α) and type II interferon receptors (IFNGR1, IFN-γ) were phosphorylated 4-8 hours after cecal infection with S. Enteritidis. Traditionally, IFN-α is an antiviral cytokine, but recent evidence suggests that it is involved further in the development of innate immunity by playing a role in restricting bacterial infections, including Salmonella (Alphonse et al., 2021). In fact, further evidence is that IFN-α signaling can be regulated by the NOD-1 receptor (Kienes et al., 2021). We can speculate that the sensing of the S. Enteritidis infection by NOD-1, as described above, can lead to the production of IFN-α that controls the level of colonization and infection by the pathogenic bacteria in the chicken cecum. We have further shown that IFN-γ can activate in vitro heterophil functional activity against Salmonella (Kogut et al., 2001; Kogut et al., 2005) and the presence of IFN-γ and IL-2 in a S. Enteritidis immune cytokine cocktail protects day-old chicks against Salmonella infections mediated by activated heterophils (Kogut et al., 1996; Verduzzo et al., 2009).

The dephosphorylation of SOCS3 suggests that Salmonella virulence factors are not directly involved in the inhibition of cytokine signaling during the early infection period in the cecum. Alternatively, the host is initiating the innate response necessary to fight infection through the release of cytokines and that the activation of the negative regulator of cytokine signaling is not required at this time.

Cytokine receptor proteins lack enzymatic activity, thus are dependent upon JAKs to initiate signaling upon binding of their ligands. The JAK family has four members: JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) (Hu et al., 2021). The STAT family consist of seven members: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b, and STAT6 (Hu et al., 2021; Nobel et al., 2021). We observed a selective activation of the JAK proteins with JAK2 and JAK3 the only JAK family members that were phosphorylated in the ceca from the S. Enteritidis-infected chickens ( Table 8 ). JAK2 signaling is activated by interferon receptors and the gp130 receptor family (IL-6R) and is an essential tyrosine kinase for initiating the innate immune response (Pena et al., 2010). JAK3 is predominantly expressed in hematopoietic lineage and intestinal epithelial cells, but its role in cytokine signaling is more restricted than other JAKs with IL-2R, IL-4R, and IL-7R being the most common receptors (Kumar et al., 2007; Mishra et al., 2012). In the intestinal epithelium, IL-2 induces activation of Jak3 to facilitate/maintain mucosal homeostasis including mucin secretion and tight junction proteins (Kumar et al., 2021).

STAT5b is the sole member of the STAT family that is phosphorylated in the cecum during the initial infection process (4-24 hours) by S. Enteritidis. JAK-STAT5B signaling in the intestine is involved in the host response to inflammation and infection (Nobel et al., 2021). In the intestine, STAT5B activation is regulated by specific members of IL-2 family of cytokines (Awasthi et al., 2021) and IL-6 (Jiang et al., 2009; Rani and Murphy, 2016) is involved in multiple intestinal physiological responses including mucosal barrier function through tight junction permeability, mucin production, and regeneration and proliferation of intestinal epithelial stem cells (Han et al., 2009; Gilbert et al., 2015; Karin and Clevers, 2016; Degirmenci et al., 2018). However, activated STAT5 is short-lived and undergo rapid deactivation (Grimley et al., 1999). Combined with the cytokine receptor phosphorylation data discusses previously (IL2R and IL6R), it is reasonable to assume that STAT5B phosphorylation found here is involved in maintaining the cecal function during the initial Salmonella infection stage.

Stimulation of the T cell receptor results in the activation of the TCR signal transduction pathway. This pathway can activate the transcription factors nuclear factor κB (NF-κB), nuclear factor of activated T-cells (NFAT), and activator protein 1 (AP-1), that induce expression of cytokine genes (Smith-Garvin et al., 2009). However, based on up-stream regulators, neither NFAT nor NF-κB are activated through the T cell receptor pathway. First, the NFAT transcription factor family are regulated by calcium signaling which is critical to activation of NFAT because calmodulin activates the serine/threonine phosphatase calcineurin. Nuclear import of NFAT is dependent on the calcium level inside of a cell. If the calcium level drops, the exporting kinases in a nucleus rephosphorylate NFAT causing the transcription factor to revert into its inactive state and is exported back to the cytosol where maintenance kinases finish the rephosphorylation to keep it in the inactivated state (Baba and Kuroski, 2016; Park et al., 2020). Here we found a significant dephosphorylation in calmodulin, meaning that there is a dramatic reduction in calcium levels in the S. Enteritidis-infected tissue thus inhibiting NFAT activity. Second, NFAT requires the activity of PLC-1, which generates the second messengers, diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 induces an increase in the concentration of cytoplasmic calcium (Ca²⁺) and activation of the Ca²⁺-dependent phosphatase calcineurin, which results in the rapid activation of NFAT, which is followed by its translocation to the nucleus (Baine et al., 2009). Thus, the dephosphorylation of PLCG1 prevents NFAT activation due to the decrease in intracellular calcium transport into the immune cells. Likewise, the dephosphorylation of PLCG1 inhibits the generation of DAG which is a vital second messenger in NF-κB activation via the T cell receptor. However, activation of NF-κB by PRRs in the innate response does not require PLCG1 generation of the DAG molecule to activate downstream proteins. PRRs use MyD88-dependent pathways to activate NF-κB activity (Akira et al., 2006, Kumar et al., 2011).

One intriguing finding from the current studies that links with our previous report on Salmonella persistence is the consistent dephosphorylation of phospholipase cγ1 (PLCG1) enzyme in the S. Enteritidis -infect cecal tissue (Kogut et al., 2017). This ‘targeting’ of PLCG1 by Salmonella seems to be a feature of infection between 24-96 hours post-infection. Even though we are not aware of what bacterial factors may be involved in dephosphorylating PLCG1, it is reasonable to assume that this action is part of the asymptomatic inflammatory response of chickens that leads to the development of a persistent Salmonella infection in the chicken. Further it appears possible that Salmonella inhibiting PLCG1 phosphorylation is part of the unique survival strategy of paratyphoid Salmonella in poultry that minimizes host defenses mechanisms (disease resistance) during the initial infection and then further uses the inhibition of PLCG1 activity as part of the immunometabolic reprogramming that converts host defense strategy to disease tolerance. Disease tolerance is the ability of the host to limit the damage caused by both the pathogen and the host immune response, i.e., immunopathology (Ayres and Schneider, 2012). PLCG1 would be a logical focal point of the initial Salmonella colonization/infection repertoire since it is involved in regulating innate immune functions including phagocytosis, the oxidative burst, cell migration and TLR-mediated signaling (Bae et al., 2017; Zhu et al., 2018; Jing et al., 2021).

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

These studies were approved by the Animal Care and Use Committee (ACUC) at the Southern Plains Agricultural Research Center, Agricultural Research Service, United States Department of Agriculture (ACUC #2012007), which meets all federal requirements as defined in the Animal Welfare Act, the Public Health Service Policy, and the Humane Care and Use of Laboratory Animals.

All authors contributed to the conception and design of the study. MK, KG, JB, HH, and CS conducted the infection studies and samples collections. RA conducted the kinome array and MK and RA conducted the bioinformatic analysis of the kinome data. YF and RA conducted the statistical analyses of all the data. MK wrote the first draft of the manuscript. All of the authors contributed to the manuscript revision and read and approved the submitted version.

The research was conducted as part of the ARS CRIS Project: ‘Immunological and Practical Approaches to Manipulate the Ecological Niches and Reduce Foodborne Pathogens in Poultry’ Project #3091-32000-037-00D).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.