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Gut Microbes 1:5, 316-324; September/October 2010; © 2010 Landes Bioscience
Enteric pathogens and gut function: Role of cytokines and STATs
Terez Shea-Donohue,1,3* Alessio Fasano,2,3 Allen Smith4 and Aiping Zhao1,3 Department of Medicine; 2 Department of Pediatrics; 3 Mucosal Biology Research Center; University of Maryland School of Medicine, Baltimore, MD USA; 4Beltsville Human Nutrition Research Center; USDA; Beltsville, MD USA
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Key words: cytokine, STAT4, STAT6, epithelial cell, smooth muscle, mucosal permeability. A/E pathogens, enteric nematodes The gut harbors the largest immune system in the body. The mucosa is considered to be the initial site of interaction with commensal and pathogenic organisms; therefore, it is the first line of defense against the pathogens. In response to the invasion of various pathogens, naïve CD4+ cells differentiate into subsets of T helper (Th) cells that are characterized by different cytokine profiles. Cytokines bind to cell surface receptors on both immune and non-immune cells leading to activation of JAK-STAT signaling pathway and influence gut function by upregulating the expression of specific target genes. This review considers the roles of cytokines and receptor-mediated activation of STATs on pathogen-induced changes in gut function. The focus on STAT4 and STAT6 is because of their requirement for the full development of Th1 and Th2 cytokine profiles.
for humoral-mediated immunity against extracellular pathogens such as enteric helminths. The discovery of another subset of cytokines derived from Th17 cells expanded this paradigm. The Th17 lineage is thought to coordinate the early response to bacterial pathogens and is also important in autoimmune-mediated inflammatory pathologies of the gut. The cytokine profiles elaborated by each of these different lineages regulate a variety of biological processes that are designed to promote the elimination of pathogens. These cytokines use transcriptional regulatory networks that program the expression of genes necessary for the specific functioning of both immune and non-immune cells. In this manner, enteric pathogens upregulate specific cytokine profiles that act to influence gut function directly or indirectly by binding to receptors on hematopoietic and non-hematopoietic cells. JAK-STAT Signaling
The success of human pathogens depends on their ability to adapt to the host environment. The gut harbors the largest immune system in the body and the mucosa is considered to be the initial site of interaction with commensal and pathogenic organisms. The innate immune system operates to limit the passage of microbiota across the mucosal barrier and, thus, epithelial cells acting in concert with antigen-presenting cells (APC) form the first line of defense. There is a growing recognition of the importance of T cells to mucosal barrier function and differentiation of naïve CD4 + T cells into subsets of T helper (Th) is a critical process. Cytokine binding to the T cell receptor promotes T cell expansion of distinct Th subsets or to regulatory T cells (Tregs). The divergent Th1/Th2 lineages, with their polarized cytokine profiles and counter-regulatory abilities, represent the classical paradigm to explain orchestration of the host response to pathogens and establishment of memory responses. The Th1 cells elaborate pro-inflammatory cytokines including IFN-g and TNF-a that are important for cell mediated immunity directed against most bacteria.1,2 In contrast, Th2 cells produce antiinflammatory cytokines including IL-4 and IL-13 that are critical *Correspondence to: Terez Shea-Donahue; Email:
[email protected] Submitted: 11/09/10; Revised: 01/18/10 and 04/03/10; Accepted: 05/12/10 Previously published online: www.landesbioscience.com/journals/gutmicrobes/article/13329 DOI: 10.4161/gmic.1.5.13329
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The seminal work on the role of the janus kinase (JAK) and signal transducers and activators of transcription (STAT) in cytokine signaling was performed by Darnell and Stark in an effort to determine the downstream events of receptor binding of type-1 and type-2 IFN on the transcriptional activation of genes involved in the immune response.3,4 This resulted in the identification of STAT1 and STAT2 more than a decade ago and since then, five other mammalian STATs have been identified (STAT3, STAT4, STAT5A, STAT5B and STAT6), all of which respond to discrete stimuli (reviewed in references 1 and 2). STATs play a role in cell function, but are especially important for differentiation of Th cells. STAT activation is transient and thus, the inhibition or activation of STATs is equally important in their ability to regulate biological functions.1,2 Cytokines also upregulate the expression of suppressors of cytokine signaling (SOCS) that inhibit the activity of JAKs and STATs.4,5 In this way cytokines act in a feedback inhibition loop to limit the amplitude and duration of their actions. Cytokines bind to cell surface receptors leading to receptor oligomerization and activation by one or more members of the JAK family of tyrosine kinases. Once activated, JAKs phosphorylate the cytoplasmic receptor domain as well as STATs. To date, STATs are the only transcription factors reported to be activated by tyrosine phosphorylation. STATs also can be activated through G-protein-coupled receptors and by receptor tyrosine kinases such as epidermal growth factor receptor and platelet derived growth factor. STATs have three separate functional domains:
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Figure 1. The STAT gene contains a coiled coil (cc) domain, a DNA-binding domain (DBD), a SRC-homology (SH2) domain and a single tyrosine at the carboxy terminus. Removal of the SH2 ablates the STAT gene and STAT protein as it is needed for receptor binding and dimerization with the SH2 domain of another STAT to allow reciprocal SH2-phosphotyrosine interaction. STATs can be activated by a number of different mechanisms. Cytokines can bind to cell surface receptors leading to receptor oligomerization and activation by one or more members of the JAK family of tyrosine kinases. Once activated, JAKs or other protein kinases (PTK) phosphorylate the cytoplasmic receptor domain as well as STATs. Phosphorylated STATs dimerize, translocate to the nucleus and bind specific DNA targets. STATs can be activated also though G-protein-coupled receptors and by receptor tyrosine kinases.
a conserved N-terminus that is required for dimer formation, a DNA-binding domain that is needed for binding of the promoters of target genes, and a C-terminus that works in transcriptional activation. The conformation of the tyrosine phosphorylated dimers allows STATs to bind to the promoter region of the target genes.2 In the classical signaling model, STATs are localized to the cytoplasm until they are phosphorylated, followed by dimerization and translocation to the nucleus, where they increase the transcription rate of a select panel of genes (Figure 1). Inactivation of STATs is accomplished by dephosphorylation by tyrosine phosphatases in the nucleus and transport back to the cytoplasm.1 This classic signaling paradigm was revised in the last several years to include the concepts of the existence of dimers composed of nonphosphorylated STATs in the cytoplasm that may target genes distinct from those of the phosphorylated STATs.6 A number of STATs exist as alternatively spliced isoforms a and b that differ at the C-terminal domain, including STAT1, 3, 4 and 5. For most STATs, the truncated b isoforms function as dominant negative factors that suppress the gene expression mediated by the full-length a forms. With regard to STAT4, the a and b isoforms have shared as well as distinct roles in mediating IL-12
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responses. Studies in STAT4-deficient mice specifically expressing either STAT4a or STAT4b in T cells showed that IL-12 induced 98 genes that were linked to both isoforms, with 32 of these genes induced only by STAT4a and 29 of the genes only by STAT4b.7 Although both isoforms were involved in Th1 differentiation, STAT4a was required for maximal IFN-g production, while STAT4b was important in mediating IL-12-stimulated cell proliferation. Modifications in STAT4 signaling are linked to a number of autoimmune diseases8,9 including diabetes10,11 and inflammatory bowel disease (IBD).12 Polymorphisms in STAT4 are associated with an increased risk of lupus, systemic sclerosis and rheumatoid arthritis.8 STAT6 is a therapeutic target for asthma and chromic obstructive pulmonary disease (COPD) because of the role of the Th2 cytokines in airway hypersensitivity and remodeling. Indeed, there is correlation between IL-13 single nucleotide polymorphisms (SNPs) and COPD susceptibility.13 STAT6 is also a target in a number of cancers including prostate cancer and Hodgkins lymphoma.14,15 Of interest is that both STAT4 and STAT6 SNPs are associated with increased cardiovascular complications in dialysis patients.16 The contribution of STAT6 to gut pathologies, however, has not been investigated.
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Figure 2. Protective immunity is dependent on the ability to elicit a pathogen appropriate immune response. Naïve CD4+ T cells can differentiate into a number of effector T helper (Th) and regulatory cells. Protection against microorganisms requires cell-mediated immunity, which is promoted by differentiation of native T cells into the Th1 subset of CD4+ T cells. IL-12 generated by antigen-presenting dendritic cells binds to receptors linked to STAT4 with the subsequent upregulation of T-bet and differentiation into Th1 cells that release IFN-γ. A number of factors, including retinoic-acidreceptor-related orphan receptor-γ (RORγ), transforming growth factor-β (TGFβ), and IL-23, promote Th17-cell differentiation, and these effector cells are involved in resistance to extracellular bacteria and to autoimmune inflammatory pathologies. In contrast, protection against helminths is dependent on Th2 cells that induce humorally-mediated immune responses. IL-4/IL-13 binding to receptors leads to activation of STAT6 and upregulation of GATA-3. Expression of the transcription factor forkhead box P3 (FOXP3) along with TGFβ, leads to differentiation of regulatory T cells (Treg cells) that can inhibit the immune response. In addition, development of the Th1 response suppresses development of Th1 and Th17 cytokine production.
STATs and Adaptive Immunity (Fig. 2) Although there are a number of STATs expressed in the gut, the focus of this review is on STAT4 and STAT6 because of their importance in the control of adaptive immunity against enteric pathogens. The full development of the Th1 response requires IL-12, STAT4, and T-bet, while the Th2 response requires IL-4, STAT6, and GATA-3. Thus, STAT4 and STAT6 are likely to be more important for regulating genes involved in infectioninduced changes in gut function. STAT4 was discovered in a search for homologs of STAT1.17,18 It was originally thought to be limited to lymphoid cells, but was found later to be expressed in the myeloid compartment, particularly in activated monocytes, macrophages, and dendritic cells.19 Microbial antigens promote the migration of dendritic cells and macrophages to the gut where they secrete IL-12. In the presence of antigen, IL-12 acts through STAT4 to promote the development of Th1 cells that generate the pro-inflammatory cytokine IFN-g. STAT4 directly binds the Ifng gene promoter to provide a transient increase in IFN-g that then upregulates expression of the T-box transcription factor, T-bet, which in turn augments Ifng expression. While both IL-12 and IFN-g activate STAT4, IFN-g also acts through STAT1. It is now thought that binding of IFN-g to the receptor, IFNGR,
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and subsequent activation of STAT1 are required for the initiation of Th1-based immunity, while IL-12-induced activation of STAT4 is essential for maintenance of the Th1-based immune response. Mice deficient in IL-12, STAT4, or T-bet all have impaired Th1 cell differentiation. Although Th17 cells produce a distinct profile of cytokines and develop along a separate pathway from Th1 cells, both STAT4 and T-bet are needed for the differentiation of Th17 cells in response to IL-23.20-22 TGFb is required for Th17 development, but suppresses Th1 development by inhibiting STAT4 and T-bet signaling23 as well as Th2 development by reducing GATA-3 expression.24 In addition, IL-12, working through STAT4, also inhibits the development of Tregs that are critical to the mucosal immune cell tolerance needed for homeostasis. STAT6 is activated by IL-4 and IL-13 leading to activation of GATA-3. In the classical paradigm, IL-4 binds IL-4Ra, which together with the common gamma chain (gc) forms the type-I IL-4R. IL-4 can also act through the type-II IL-4R that contains two chains: IL-4Ra, which is the component of receptor complex that is needed for STAT6 signaling, and IL-13Ra1.25 The formation of IL-13/IL-13Ra1 is slow because of the low efficiency of IL-13 binding. In contrast, formation of IL-4/IL-4Ra is relatively fast because of the high efficiency of IL-4 binding.
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As Th2 cytokines must bind to their receptors to induce biological responses, the distribution and location of these receptors are of interest. The type-I IL-4R is expressed exclusively on hematopoietic cells, while the type-II IL-4R is not expressed on T or B cells, but is present on other immune cells as well as structural cells including epithelial cells, smooth muscle cells, and enteric neurons. There are a few cells, such as macrophages, which express both type-I and type-II IL-4R. There are three tyrosine residues on the IL-4Ra that are critical for STAT6 activation. This process is rapid with phosphorylation of STAT6 occurring within several minutes of IL-4 binding.26 Receptor-mediated activation of STAT6 by IL-4/ IL-13 and downstream activation of GATA-3 are required for the full development of Th2-based immune response in that mice deficient in STAT6 exhibit impaired immune responses.27 Constitutively, structural cells have a similar expression of both IL-4Ra and IL-13Ra1; however, in response to enteric nematode infection, expression of IL-13Ra1 is actually reduced.28,29 Thus, the strong upregulation of IL-13 during nematode infection is needed to achieve the high concentrations of IL-13 to form the IL-13/IL-13Ra1 complex that then efficiently binds IL-4Ra. This renders IL-13 more important than IL-4 for the biological actions that facilitate nematode expulsion. While responses to pathogens often require polarized cytokine profiles acting through STAT4 or STAT6 for protective immunity, the ability to survive bacterial sepsis requires both STAT4 and STAT6.30 Role of the Immune System in the Response to Pathogens The major role of the gut is the absorption of nutrients or ions, secretion of fluid, maintenance of mucosal barrier function, and movement of the luminal contents. Most enteric pathogens alter one or more of these functions. The gastrointestinal mucosa has adapted to potential invading pathogens by limiting access to the surface epithelium, maintaining an effective barrier to their invasion, and activating appropriate local immune responses when the barrier is breached. These interactions form the basis for the innate immune response, which plays a key role in initiating the adaptive immune response. The area of the gut preferentially colonized by each pathogen is also a critical factor, not only with regard to repertoire of stratagems used to circumvent host defense, but also for the mechanisms available to the host to promote removal of the pathogen. Pathogens have a greater ability to adapt rapidly to changes in the host response, and one aspect of the “Red Queen hypothesis” is that pathogen mutations are part of a constant race just to maintain their advantage against the host response.31 The host must rely on more flexible, but less mutable mechanisms, which are already in place and can be amplified by recruitment and activation of immune cells. A critical step in host immunity, therefore, is the elaboration of polarized cytokine profiles to avoid developing a new or separate immune response for each pathogen and to allow exploitation of existing physiological mechanisms by the immune response. Of interest therefore, are the immune-mediated changes in gut function that promote pathogen clearance, particularly in the context
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of vast differences between Th1/Th17 and Th2 cytokines. These polarized profiles provide a means for maintaining these functional alterations over time and may be linked to STAT4 and STAT6-dependent upregulation of target genes. Infection and epithelial cell function. Control of intestinal permeability is arguably one of the more critical aspects of host defense as enhanced permeability facilitates passage of large numbers of intraluminal bacteria, antigens, or other pathogengenerated molecules across the mucosal barrier that may trigger immune responses. Tight junctions are an important component of epithelial barrier function, and there is an emerging interest in pathogen-induced changes in the functionality of tight junctions in addition to alterations in the expression of these proteins. Much of the information on regulation of epithelial barrier function, however, is derived from studies in cultured epithelial cell lines and more in vivo studies are needed. Discussion of the different host immune response to all noncommensal bacteria is beyond the scope of this review. One group of pathogens is characterized by destruction of epithelial cells as a result of their intimate attachment and effacing (A/E) and includes enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC).32 There is a wealth of information indicating that A/E pathogens are associated with an upregulation of Th1 cytokines such as TNF-a and IFN-g. These bacteria have evolved a number of strategies that allow them to directly augment, disrupt, or inhibit epithelial cell function including invasion of epithelial cells, molecular mimicry, activation of intracellular signaling pathways, formation of pores, and alteration of cytoskeleton-dependent functions.33-36 There is a general consensus that A/E pathogens increase epithelial permeability37 and induce changes in epithelial secretion and absorption that result in diarrhea. This topic was the focus of several recent excellent reviews.37,38 Although co-option of epithelial cell function to promote colonization is the initial action of A/E pathogens, the resulting upregulation of Th1 cytokines is important in sustaining initial bacterial effects on permeability. Of interest is that loss of absorptive surface is a consequence of infection, but the effects on epithelial permeability do not appear to be due to epithelial apoptosis.39 It should be noted, however, that phagocytosis of infected apoptosis epithelial cells is a prerequisite for the development of the Th17 response in A/E infections40 and is critical for pathogen clearance. In addition, recent evidence suggests that a number of A/E pathogens, in turn, generate factors that down-regulate the inflammatory response thereby preserving the integrity of epithelial cells that is critical for colonization.41 These data emphasize the importance of immune-mediated regulation of bacterial and host cell interactions. A major drawback in studying the effects of A/E bacteria is the lack of suitable animal models as EPEC and EHEC, which infect humans, do not naturally infect mice. As a result, in vivo studies on A/E pathogens have lagged significantly behind in vitro studies using epithelial cell lines. Citrobacter rodentium (C. rodentium) is a non-invasive A/E bacterium that colonizes the colon inducing mucosal hyperplasia and accumulation of actin beneath the site of attachment. This infection shares a number of features with human EPEC infection42 and currently is
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considered the best model available for the investigation of A/E pathogen interactions at the applical surface in vivo. Both the pathology and effective clearance of C. rodentium are dependent on upregulation of both Th1 and Th17 cytokines.43 IL-17 and IL-22 increase the expression of a number of antimicrobial peptides and chemokines that are key to host defense.44 Recent studies showed that C. rodentium infection did not alter colonic epithelial permeability in vivo,45 consistent with the observation that bacterial colonization was limited to the colon with little evidence of bacteria in the spleen or mesenteric lymph nodes.45 In addition, secretion was reduced during active infection45 and recent data indicate that this hypo-responsiveness to secretagogs was dependent, in part, on STAT4 (Shea-Donohue, unpublished results). There are additional reports in which the antisecretory effects of C. rodentium infection in vivo were attributed to reduced expression of the anion exchanger DRA (SLC26A3) on the apical membrane in the colon46 and are similar to the observed effects of EPEC in vitro.47 The antisecretory effect of infection, however, may be offset in part by the inhibition of fluid absorption through reduced expression of aquaporins.46 Missing from in vitro models, therefore, is the highly integrated environment that pathogens encounter in vivo. It is likely that the interplay among bacterial factors, enteroendocrine factors such as 5-HT48 and galanin,49 and immune mediators all contribute to A/E bacterial-mediated changes in the intraluminal fluid content. This may contribute to differences in susceptibility to infection among mouse strains as well as to differences in patient symptoms during infection. The consequences of nematode infection on epithelial cell function are not as well characterized. There is also a concern that much of the information on Th2 cytokine-induced alterations in mucosal permeability is derived from studies in cell cultures and is not always consistent with results obtained in vivo. The upregulation of Th2 cytokines is critical to the increased mucosal permeability and impaired glucose absorption in nematode infection. These effects are not observed in STAT6-, IL-4-, or IL-13-deficient mice resulting in impaired clearance of nematodes.50-53 It is important to note that receptors for IL-4/IL-13 as well as STAT6 are expressed both on immune cells and epithelial cells, allowing for direct effects of these cytokines.28 Thus, changes in receptor expression are important in controlling epithelial cell function in nematode infection.29 Nematode infection is described as eliciting a “weep and sweep” (increased secretion and motility) response in the small intestine; however, data do not entirely support this concept. Upregulation of Th2 cytokines induces a STAT6-dependent stereotypic inhibition of epithelial secretion in response to secretagogues such as acetylcholine and 5-HT and an inhibition of sodium-linked glucose absorption.51-53 The increase in intraluminal fluid, therefore, is due to decreased absorption rather than increased secretion, similar to the effects of A/E pathogens. These changes constitute a number of shared effects among pathogens that may promote movement of contents into the colon and/or to limit access of the pathogen to the mucosal surface of the small intestine. Nematodes generate a number of factors such as serine proteases that are important in tissue penetration, a retained feature
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that may contribute to their ability to migrate, adapt, and survive host defenses. Nematodes that preferentially infect the small intestine induce a stereotypic and STAT6-dependent increase in permeability52,53 that is mimicked by exogenous IL-13 administration.51 The increased permeability facilitates exposure of worm products to resident immune cells. One mechanism for this change in permeability may be that infection results in a mastocytosis associated with an increased release of tryptase (mMCP-1 is the mouse homolog), which activates proteaseacti vated receptor-2 (PAR2). Activation of PAR2 has been linked to mast cell-mediated changes in permeability.54 Exposure to IL-13 for prolonged periods (days) increased permeability in intestinal epithelial cell lines, an effect attributed to increased expression of claudin-2 by a mechanism involving PI3-kinase.55 The pore-forming claudin-2 is linked to cation-selective channels in the tight junctions that govern paracellular permeability to sodium ions.56 Infection-induced upregulation of IL-13 does not alter occludin expression but increases expression of claudin 1 (Shea-Donohue, unpublished data). Thus, the mechanism of the prolonged changes in epithelial permeability in response to nematode infection in vivo remains elusive but may be mediated, in part, by STAT6-mediated elevation in PAR2 expression.57 It should be noted that neither Trichuris muris (T. muris), a nematode that preferentially infects the colon, nor exogenous administration of IL-13, alters colonic permeability. Moreover, mice that have chronically elevated levels of IL-13 have increased permeability in the small intestine, but not in the colon.61 This may contribute to the lack of diarrhea in nematode infection. There are reports of a STAT6-dependent increase in both enterochrommaffin cells and 5-HT levels in response to T. muris infection of the colon.58 This may be due, in part, to the unique host response that accelerates epithelial cell turnover in the colon to enhance worm expulsion by STAT6-, IL-13-, and CXCL10- dependent mechanisms.59 Thus, there may be unappreciated regional differences in the host response to enteric infection. Smooth muscle function. Contractions of gut smooth muscle are necessary for mixing contents with digestive secretions, increasing exposure to the absorptive surface to promote epithelial absorption, and propelling contents along the GI tract. Alterations of gut smooth muscle function, in concert with changes in epithelial cell function, constitute a major part of the host immunity against the invasion of enteric pathogens. Upregulation of various cytokines as part of the adaptive immune responses is implicated in the regulation of gut smooth muscle function. These cytokines can act either directly on smooth muscle cells, which express functional receptors for cytokines such as TNF-a, IL-1b, TGF-b, IL-4, and IL-13, or indirectly through release of mediators from either immune or non-immune cells. Members of the STAT family, especially STAT4 and STAT6, are involved in this regulation by orchestrating the effects of Th1/ Th17 and Th2 cytokines. Luminal pathogens have little direct access to smooth muscle; therefore, responses are likely mediated by changes in the cytokine content and types of cells present in the immune environment. The upregulation of Th1/Th17 cytokines in response to A/E infection is similar to that observed in inflammatory bowel
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Figure 3. Divergent effects of Th1 versus Th2 cytokine on gut smooth muscle function: Th1 cytokines, primarily IL-12 and IFN-γ, bind to cell surface receptors that lead to activation of STAT1 or STAT4 pathways resulting in smooth muscle hypo-contractility. IL-1β and NOS-2 are among the downstream effector molecules mediating this event. Th2 cytokines, mainly IL-4 and -13, acting through STAT6 pathway, cause smooth muscle hypercontractility via upregulating the effector molecules PAR1, PAR2 and 5-HT2A.
disease (IBD). Much of the information on the effects of these cytokines on smooth muscle function, therefore, is derived from animal models of colitis. A characteristic feature of inflammation in vivo60 and in vitro61,62 is a smooth muscle hypo-responsiveness, which may impair the colonic absorption of ions and fluid. There are also increases in the number and frequency of abnormal motility patterns that contribute to diarrhea.63,64 Smooth muscle expresses receptors for IL-1b and TNFa and receptor-mediated activation leads to downstream changes in the expression of signaling molecules RGS4 and CPI1765,66 or NFkB-mediated upregulation of ICAM-1 expression,67 resulting in reduced contractility. Despite the presence of receptors for a variety of immune mediators, smooth muscle is devoid of expression of STAT4 even in response to upregulation of Th1 or Th17 cytokines. There are receptors for IL-17 on smooth muscle (Zhao et al., submitted), but their functional importance is unknown. In response to C. rodentium infection, there is a STAT4-dependent decrease in smooth muscle response to acetylcholine that is associated with an increase in the expression of both nitric oxide synthase (NOS)-2 and IL-1b. Increased production of macrophage-generated nitric oxide, a potent inhibitor of smooth muscle contractility, may also contribute to suppression of smooth muscle function. It is known that a dominant Th2 response is necessary for expulsion of enteric nematodes. Nematode infection induces a hyper-contractility of smooth muscle68-70 that promotes expulsion. Although IL-4 and IL-13 have similar effects on epithelial function, nematode infection-induced changes in smooth muscle function are mediated primarily by IL-13 and can be mimicked by exogenous administration of IL-13. The effects of IL-13 are characteristic of a number of infections including N. brasiliensis, Trichinella spiralis (T. spiralis), Heligmosomoides polygyrus (H. polygyrus) and T. muris and are STAT6-dependent.51,52 There
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are a number of factors that contribute to the hyper-contractility including enteric nerves, mast cells, and macrophages. Nematode infection induces a mastocytosis that is dependent on IL-4, IL-3, and IL-9.71,72 Mast cells located near sensory afferents release leukotrienes that enhance sensitivity to nerve stimulation.73,74 Upregulation of Th2 cytokines also induces the development of alternatively activated macrophages (AAMf) that secrete insulinlike growth factor-1, transforming growth factor-b1 (TGF-b1), and arginase 1 that may contribute to smooth muscle hyperplasia and hypertrophy.75 Finally, smooth muscle cells express receptors for IL-4/IL-13 and STAT6 and nematode infection upregulates an array of effector molecules that are involved in the regulation of smooth muscle function, such as muscarinic receptors, PAR1, PAR2, 5-HT2A that are important in hyper-contractilty.68,76 The interaction between immune and smooth muscle cells, therefore, plays a key role in infection-induced changes in smooth muscle function. IL-25, also known as IL-17E, is a recently described member of the IL-17 cytokine family78,79 that is upregulated during enteric nematode infection. Unlike other members of this family that have biological activities typical of type 1 inflammatory responses,80 IL-25 promotes Th2 and inhibits Th1/Th17 cytokine responses.81-83 Exogenous administration of IL-25 in mice induces the characteristic smooth muscle hyper-contractility of intestine observed in response to nematode infection or exogenous IL-13, while nematode-induced changes in smooth muscle function are absent in IL-25-deficient mice (Zhao et al., submitted). The effects of IL-25 may be mediated by STAT6 as IL-25 increased production of IL-4 and IL-13 and the effects of IL-25 on smooth muscle function were abolished in IL-13-deficient mice (Zhao et al., submitted). Thus, IL-13 is the major downstream molecule responsible for the hyper-contractile activity of IL-25.
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Target Genes for STAT4 and STAT6 (Figure 3) Th1/Th2 dominant responses result in the acquisition and persistence of expression and silencing of genes in specific cell types. The major function of cytokine activation of STATs is to increase the rate of transcription of target genes. Recent studies have implicated the importance of epigenetic modification of histones, a component of chromatin, in the commitment to T cell-specific silencing of genes.84 STAT4, in concert with T-bet and STAT6, along with GATA-3, regulates histone acetylation and controls the recruitment of enzymes that produce changes in chromatin that are associated with either gene activation or gene silencing.85 The importance of STAT4 and STAT6 for adaptive immunity has generated considerable interest in characterization of the panel of genes that are activated. There is little information, however, on how STATs perform this function, how selectivity of activation is achieved, and what controls the scope and identity of target genes. The development of new technologies, such as chip-on-chip analysis, has facilitated the investigation of the STAT4- and STAT6-regulated genes.86 The lack of expression of STAT4 in structural cells suggests that targeted genes that affect gut function are most likely linked to cytokine mediated STAT4-activation in recruited immune and inflammatory cells. It is the mediators derived from upregulation of genes in these cells that impact smooth muscle function. It is known that STAT4 deficiency protects against the development of autoimmune diseases and attenuates the severity of pathology in animal models of colitis. In contrast, lack of STAT4 is detrimental in bacterial infection, with exaggerated inflammation and impaired clearance. Mice that lack STAT4 exhibit a constitutive hypo-contractility of colonic smooth muscle that may be due to the increased expression of IL-17 and IL-1b and/or to the low level of inflammation in the local environment (Shea-Donohue, unpublished data). This suggests that STAT4 has constitutive anti-inflammatory activity. Recent studies identified five classes of STAT4 target genes, Th1 (e.g Myd88, IFN-g), TCR signaling, cytokines (IFN-a), receptors (IFN-g, TNF, IL-17) and IFN-inducible.86 Another target of STAT4 is furin, which plays a role in tolerance86 and may be linked to the constitutive antiinflammatory role of STAT4. Although T-bet is considered to be the master regulator of the Th1 response, recent studies indicate that gene subsets require either T-bet or STAT4 while others require both factors. In addition, STAT4 is needed for T-bet to activate STAT-4-dependent genes.87 IL-12 is the major activator of STAT4 signaling yet there are also genes activated by IL-12 that are STAT4-independent.88
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Activation of the STAT6 signaling pathways leads to transcription of a number of genes controlling the expression of cytokines (IL-4), immune receptors (IL-4Ra, CD40), and chemokines (e.g eotaxin).89 Members of the CCAAT/enhancer binding proteins (C/EBP) family of transcription factors play a key role in STAT6-mediated promoter regulation. These proteins contribute to a number of cellular processes including proliferation, differentiation, and immune functions.90 Mice that lack STAT6 do not have any significant alterations in gut function51,52,64 indicating that the major role of this factor is the development of protective immunity. The genes involved in Th2-mediated changes in epithelial function have not been characterized. In contrast, there are a number of genes upregulated specifically by IL-13 and STAT6 that contribute to the smooth muscle hyper-contractility by increasing the number of receptors on smooth muscle for agonists (e.g.PAR-1. 5-HT2A) or that are critical for the recruitment and activation of AAMf. Although IL-4/IL-13 are major inducers of STAT6, there are other putative activators including the hormone leptin,91 which is involved in obesity. Equally important is the ability of STAT6 activation in suppressing the development of the Th1/Th17 response (Figure 2). Summary The nature of the interaction between enteric pathogens and the mammalian gut is a continually evolving process. The ability of the gastrointestinal tract to discriminate between commensal and pathogenic organisms is a critical function for host survival. Considering the myriad of functions the gut must perform, it is unlikely that the gut will develop pathogen-specific changes in gut function. Rather, the host makes use of the available repertoire of physiological actions, such as changes in permeability that promote antigen presentation, which may also be exploited by the pathogens in an effort to circumnavigate host defenses. In addition, this may explain why a common feature of many enteric infections is increased intestinal permeability in the small intestine. The activation of innate immune responses leads to the development of polarized Th1/Th17 and Th2 cytokines. These differing cytokine profiles coordinate the infiltration of immune/ inflammatory cells that modulate or amplify gut function to promote clearance of the pathogen. STATs are the downstream transcription factors of cytokine binding to membrane receptors. STAT4 and STAT6 are particularly important in the adaptive immune response to enteric pathogens. The major action of STATs is to increase the rate of transcription of specific target genes, many of which are involved in pathogen-induced changes in gut function.
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Volume 1 Issue 5
