T cell cytokines impact epithelial cell responses during Helicobacter pylori infection

Abstract

The goal of this review is to highlight literature which demonstrates how cytokines made by T lymphocytes impact the gastric epithelium, especially during Helicobacter pylori infection. These cytokines effect many of the diverse functions of the epithelium and the epithelium’s interactions with Helicobacter pylori. Focal point of this review will be on how T cell cytokines impact antimicrobial function and barrier function and how T cell cytokines influence the development and progression of cancer. Further, the modulation of epithelial-derived chemokines by H. pylori infection will be discussed.

Introduction

The health and function of the stomach and the intestines are essential for interacting with the external environment and with the internal microbiome through the epithelial cell layer. Specifically, the function of the stomach is to store food, provide early digestion of food by producing acid and enzymes and macerating that food with its muscular lining. There are several specialized epithelial cells which aid in the stomach performing its necessary functions. These include chief cells, enteroendocrine cells, parietal cells, stem cells and foveolar cells (both surface mucous cells and mucous neck cells)(1)(Figure 1). Chief cells are found in the fundic glands, and most common, deep in the glands closer to the muscularis mucosae. The digestive enzymes of the stomach, pepsins, are secreted by chief cells. Located deep in the gastric glands are the enteroendocrine cells including G cells, D cells, and enterochromaffin-like cells (ECL cells), which are responsible for production of gastrin, somatostatin (SOM), and histamine, respectively. Gastrin and histamine impact parietal cell and chief cell function. Gastrin stimulates ECL cells to release histamine and then synergistically these hormones activate parietal cells to produce hydrochloric acid and chief cells to release pepsin. Foveolar cells are simple columnar epithelial cells which line the stomach. They are also found in gastric pits and at the top of the gastric glands (necks) where they are referred to as surface mucous cells and mucous neck cells, respectively. Surface mucous cells contain large amounts of mucin at their apical surface. The mucus and bicarbonate ions produced by the foveolar cells play an important role in modulating the impact of acids on the gastric epithelium. Gastric mucous neck cells are most common in the upper region of the glands.

Figure 1.

Illustration of the Stomach. The architecture of the human stomach is similar to the mouse with the exception of the presence of a nonglandular forestomach in the mouse. In the stomach epithelium, gastric pits lead to gastric glands that secrete gastric juice. The cellular composition of the gastric glands and pits varies based on localization in the corpus verses the antrum. Enlarged gastric pits and glands are shown containing different localization specialized epithelial cells including foveolar cells (both surface mucous cells and mucous neck cells), cells that that secrete hydrochloride acid, gastrin, somatostatin and activate pepsin (parietal cells, enteroendocrine cells, and chief cells), and gastric stem cells. H. pylori colonization is denser in the antrum of the stomach than the corpus.

The maintenance of the epithelium functions is vital for a body’s health. The gastric epithelium can be disrupted under several conditions including use of pharmacological reagents, alcohol or infection (2–4). Helicobacter pylori is the most common bacterial infection of the gastric mucosa. H. pylori’s prevalence is greater than 50% of the population, but there is significant variation in the prevalence between regions of the world (5, 6). Long term colonization by H. pylori is the most consequential risk factor for the development of gastric cancer (GC)(7), which imposes a significant global burden with over 1 million new stomach cancers diagnosed in 2018 (8). Like many bacterial pathogens, H. pylori must overcome natural defenses and the immune response to colonize and cause disease. H. pylori overcomes natural defenses of the stomach through production of urease which neutralizes the local acidic environment and through its flagella which provides the ability to traverse the mucus layer and interact with epithelial cells. Other bacterial factors which aid survival of H. pylori include its adherence to the epithelium, its ability to produce of catalase to neutralize hydrogen peroxide and its ability to acquire nutrients. Virulence factors, including the vacuolating toxin (vacA), the Cytotoxin A Type 4 secretion system (CagA T4SS), adhesins, among others, target the epithelial cells disrupting cell-cell communication, tight junctions and cellular signaling of the epithelium (9–11). Both bacterial interactions with GECs and the ongoing chronic inflammatory response to H. pylori contribute to carcinogenesis. This review will focus on how the chronic inflammatory process, especially T cell activation, impacts the epithelial cell response.

The Importance of the T cell Response during Helicobacter pylori infection

The innate immune response to H. pylori is activated through direct interactions of H. pylori or its products with the GECs. Both humoral and cellular immunity become chronically active, but this response is not effective in bacterial clearance. The cellular immune infiltrate in response to H. pylori is dominated by innate immune cells and CD4⁺ T cells (Figure 2). The CD4⁺ T cells response is instrumental during infection in mice and humans for the development of gastritis and control of infection. T and B cell deficient mice (recombination-activating gene or severe combined-immunodeficient mice (SCID)) do not control H. pylori colonization, nor do they exhibit inflammation like wild-type (WT) mice (12, 13). Reconstituting these immune deficient mice with CD4⁺ T cells, results in severe gastritis suggesting that CD4⁺ T cells are both necessary and sufficient for disease. MHC Class II^−/− mice, which lack CD4⁺ T cells responses, are unable to control H. pylori colonization as well as WT mice and they do not develop a protective response to immunization (14). Utilizing antigen-specific adoptive transfer models has confirmed the importance of the CD4⁺ T cell on driving gastritis through epithelial cell damage, associated proliferative and metaplastic responses. Peterson et al. reconstituted SCID mice with congenic splenocytes from H. pylori infected or naïve WT mice, and found significant increases in GEC apoptosis and proliferation in infected recipient and donor, compared with non-recipient and uninfected mice at 3 months post transfer (15). These data suggest that splenocytes from H. pylori infected mice and the factors they produce contribute to GEC turnover by inducing apoptosis and proliferation.

Figure 2.

CD4⁺ T lymphocytes subsets migrate to the gastric mucosa in response to Helicobacter pylori infection and where they produce cytokines which impact epithelial cell function. In this figure, the immune cell filtrate in the lamina propria is represented. Green cells represent CD4⁺ T cells; blue cells represent B lymphocytes; purple cells represent dendritic cells and macrophages; while pink cells represent neutrophils. Th1 and Th17 cells dominate the response during gastritis producing IFNγ, IL-17, IL-21 and IL-22 (possibly produced by Th22 cells). Th2 lymphocytes are not commonly activated during H. pylori infection, but when present do impact epithelial cell responses. Finally, T regulatory cells are activated during H. pylori and their presence can influence not only other T cells but also epithelial cell biology through production of IL-10 and TFGβ.

It is important to recognize the double-edged sword of a T helper (Th) cell response. T cell derived cytokines are required for activating antimicrobial responses antibody responses against pathogens; but on the other hand, there is collateral damage chronic, exacerbated or unregulated pro-inflammatory responses. The balance between pro-inflammatory Th cells and Tregs cells effect H. pylori immune responses and gastric disease (16–18) whereby low Treg response leads to increased inflammation and gastric disease. CD4⁺ T cell cytokines are often categorized by the function of the T cell which produces them (19). For example, Th1 cells produce IFNγ; Th2 cells produce IL-4, IL-5 and IL-13; Th17 cells produce IL-17a, IL-17f, IL-21 and IL-22; Th22 cells produce IL-22; Tregs produce IL-10 and TGFβ. Many of these cytokines can play a role in activating antimicrobial responses, wound healing, proliferation and carcinogenesis through their signaling in epithelial cells.

The Epithelial Cell and Antigen Presenting Cell Response to H. pylori triggers Immune Activation

Gastritis is the culmination of innate and adaptive immune response which is initiated at the intersection of H. pylori contact with GECs. H. pylori activates the gastric epithelium to produce CXCL8 (IL-8, a neutrophil chemokine), IL-6, IL-1β, granulocytes-macrophage colony stimulating factor (GM-CSF), monocyte chemoattractant protein-1 (CCL2), macrophage migration inhibitory factor (MIF), tumor necrosis factor alpha (TNFα), and tumor growth factor beta (TGFβ) (20, 21). Many reviews have been written on the direct interaction between H. pylori and epithelial cells, so we will only briefly focus on the characteristics of H. pylori that lead to epithelial cell activation and strongly influence the T cell response.

One of the most important virulence factors of H. pylori is the the Cytotoxin A Type 4 Secretion system (Cag T4SS). It is encoded by the cag pathogenicity island (cagPAI), and this secretion system is responsible for translocating the effector protein CagA, peptidoglycan metabolites and DNA directly into epithelial cells. The functioning of this T4SS has been shown to be vital for carcinogenesis and has been referred to as an oncogenic factor. The biosynthesis of heptose-1,7-bisphosphate (HBP), an intermediate metabolite of LPS, contributes to cagPAI-dependent activation of epithelial cells (22). OipA is a phase-variable outer membrane protein which is postulated to bind to an integrin (23). Experimental analysis of phase on/off mutants of H. pylori demonstrate that OipA is necessary but not sufficient for CagA translocation into host epithelial cells (24). The significance of the T4SS dependent activation of epithelial cells is activation of proinflammatory signaling that induces the transcriptional activation and secretion of chemokines such as CXCL8 (IL-8) which recruits neutrophils to the site of infection. Polarization of the Th cell response is influenced in H. pylori infected humans both by whether the patient is infected with a CagA-positive strain and their stage of progression to gastric carcinogenesis (25). Patients colonized with CagA+ strains had Th1-mediated cellular immunity in earlier stages of gastric carcinogenesis. Patients colonized with CagA+ strains in advanced stages were dominated by Th2-mediated humoral immunity and negatively associated with a large number of Treg cells.

Several soluble or secreted proteins of H. pylori also directly impact the function of gastric epithelial cells and antigen presenting cells. VacA, a pore-forming toxin, induces mitochondrial damage, and contributes to epithelial cell death through apoptosis and programmed necrosis (26, 27). Large amounts of urease produced by H. pylori to accommodate its survival in the stomach’s acidic environment, induce production of IL-6 and TNFα in primary GECs and in the human GEC line MKN-45 (20). In addition, urease has been shown to contribute to apoptosis and inflammation through its interaction with host cells. The interaction of urease with CD74 on host cells contributes to CXCL8 production by GECs (28); while urease binding to MHC Class II contributes to apoptosis of GECs (29). These are relevant findings because they impact antigen uptake by recruited phagocytes and expression of T cell skewing cytokines.

The epithelial cell layer is intended to be a major defensive part of the innate immune response setting off alarms through toll-like receptors and other pattern recognition receptors (PRR). Disruption of the epithelial cell barrier by virulence factors leads to the innate activation of the immune response (reviewed in (23)) and trigger innate activation of antigen presenting cells (APCs) (30, 31). For example, high temperature regulator A (HtrA) disrupts epithelial barrier through cleavage of the ectodomain of E-cadherin (32, 33), permitting H. pylori, outer membrane vesicles, and H. pylori soluble factors to invade spaces between GECs (34).

As an extracellular bacterium that produces several secreted factors, H. pylori activates a strong CD4⁺ T cell response (31, 35). Activation, proliferation and differentiation of CD4⁺ T cells requires several signals (35–39). The first and second signals are mediated through the T cell’s receptor with its cognate peptide loaded MHC molecule on the APC (40); and through co-stimulatory molecule interactions between the T cell expressed CD28 molecule and B7.1 or B7.2 molecules on the APC (41). These two signals result in activation, clonal expansion and proliferation of the T cells. A third signal is required for T cell differentiation, and is provided by the cytokine microenvironment (42). Although it is widely accepted that H. pylori does not activate APCs as strongly as other GI pathogens, APC produce several T cell differentiating cytokines including (but not limited to) IL-1β, IL-10, IL-12, IL-18, IL-23, and TGFβ in response to H. pylori (43–45). These cytokines are induced through PRRs and inflammasome activation (46–48) triggering differentiation of Th1 and Th17 responses. Interestingly, it has been demonstrated that H. pylori factors including VacA and gamma-glutamyl transpeptidase (GGT) downregulate the magnitude of the proinflammatory T cell response. Specifically, GGT converts glutamine into glutamate and ammonia and converts glutathione into glutamate and cysteinylglycine (reviewed in (49)). H. pylori GGT induces immune tolerance through the inhibition of T cell-mediated immunity and antigen presenting cell cytokine production. VacA impacts several cell types as a pore-forming toxin. It can directly inhibit T cell proliferation and IL-2 production, but also indirectly impacts T cell differentiation to targeting myeloid cells especially in young mouse models to induce tolerance (reviewed in (50)) VacA and GGT similarly impact dendritic cell activity (promoting Treg differentiation) and inhibit T cell proliferation.

Th1-lymphocyte derived cytokines

IFNγ-producing CD4⁺ T cells, Th1 cells, are activated during H. pylori infection. Increased presence of Th1 cells (increased IFNγ) are associated with more detrimental outcomes of H. pylori infection (51, 52). IFNγ acts synergistically with TNFα to activate macrophages and NK cells, but most mucosal epithelial cells also express the IFNγ receptor at the basolateral membrane (53). IFNγ signals activation and nuclear translocation of signal transducer and activator of transcription 1 (STAT1). IFNγ induces several chemokines which regulate mucosal T cell trafficking. These CXC chemokines, CXCL9, CXCL10 and CXCL11, specifically attract CXCR3⁺CD4⁺ T cells. IFNγ and TNFα strongly induced secretion of these chemokines in GECs (Kato III, NCI, and AGS cells)(54). Interestingly, in some settings H. pylori may inhibit STAT1 activation by IFNγ (55); and soluble or membrane fractions of H. pylori have been shown to prevent the chemokine expression in the above mentioned GECs (54). Conversely, in another study cagPAI⁺ H. pylori strains augmented GEC responses to IFNγ and specifically increased CXCL9, CXCL10 and CXCL11 expression (56). Other chemokines are also induced by H. pylori and IFNγ. The murine GEC line, GSM06, responded synergistically to H. pylori and IFNγ to induce CXCL2 and iNOS expression supporting inflammation (57). Results from H. pylori infection of IFNγ^−/− mice suggest that IFNγ plays a primary role in driving inflammation with minimal effect on colonization levels (58).

The turnover of mucosal epithelial cells is vital for homeostasis when the epithelial cell layer is colonized with inflammation-inducing bacterial colonizers or virulent bacterium. Homeostasis is maintained through a process of apoptosis balanced with proliferation. Inflammation and cytokines can disrupt this balance. Specifically, IFNγ enhances apoptosis during H. pylori infection. This has been demonstrated in organoid cultures (53) and in cell culture assays with human GECs (AGS cells and Kato III cells)(59). Mechanistically, IFNγ directly enhances caspase 3 activation and indirectly induces increased expression of MHC Class II molecules on the surface of GECs to which H. pylori can bind and trigger apoptosis through Bax activation. IFNγ clearly has a pro-apoptotic role on GECs, but it is also vital for enhancing gastritis through activation of chemotactic gradients which enhance immune cell infiltration to the gastric mucosa.

There is also evidence that IFNγ can alter the lineage, and therefore, the function of GECs. The human GEC line, NCI-N87 increased it expression of mucus, mucin 6 (Muc6), trefoil factor 2 (Tff2) and pepsinogen II in response to IFNγ, suggesting NCI-N87 cells became more like a mucous neck cell with this treatment (60). Infusion of IFNγ (in mice) expanded the mucous neck cell compartment and led to the proliferation of a gastric progenitor cell in the antral gland which can give rise to all gastric lineages in those glands (61). These were key experiments for understanding connections between inflammation and carcinogenesis.

Th17-lymphocyte derived cytokines

The Th17 subset also contributes to the immunopathogenic response to H. pylori. Th17 cells are differentiated in the presence of IL-1β, IL-6 and TGFβ; the subset is transcriptionally stabilized and expanded in the presence of IL-23 and IL-21. Once differentiated, Th17 cells make several cytokines including IL-17a, IL-17f, IL-21 and IL-22.

IL-17a is the cytokine which has the greatest affinity for IL-17RA/RC on epithelial cells and its canonical activation of the IL-17R leads to activation of several transcription factors including NFκB, AP-1 and C/EBP (62). The major consequence of IL-17 signaling is induction of a chemotactic response recruiting PMNs (through activation of CXCL1, CXCL2, CCL2 and CCL5). This tight association between IL-17A expression and CXCL8 induction by epithelial cells has been investigated in H. pylori infected patients. Increased levels of IL-17A and CXCL8 are reported during H. pylori infection compared to uninfected patients (63, 64). Moreover, expression of IL-17A and CXCL8 correlates with PMN infiltration in the H. pylori infected patients. Further, it has been demonstrated that isolated lamina propria mononuclear cells from H. pylori infected patients can induce the production of CXCL8 in an IL-17A dependent fashion for inhibition of IL-17 using an anti-IL-17A antibody lead to decreased CXCL8 expression (63). Interestingly, IL-17 alone is not a strong inducer of NFκB, but it is its synergistic response with IFNγ, lymphotoxin and TNFα leads to a strong cellular response (65). In the case of co-stimulation with TNFα, IL-17 signaling leads to increases in mRNA stability of IL-17 target genes (66, 67).

Central to regulation of IL-17 signaling is the Act1 protein and several TNF associated factors (TRAFs). Act1, a U-box E3 ubiquitin ligase, is recruited to the IL-17R (and ubiquitinates TRAF6 (68)). These events are critical for NFκB activation; but not required for the mRNA stability processes mediated by IL-17 signaling (67). On the other hand, TRAF4 competes for the same binding site on Act1 as TRAF6. While TRAF4 restricts the level of NFκB activation as a result of IL-17 signaling (69), it can also activate signaling in a MEKK3 dependent pathway. TRAF4 mediated signaling has been studied extensively in keratinocytes (70) and while the importance of these signaling events in GECs is yet to be determined, they may have implications in ulcer-healing or carcinogenesis. In keratinocytes, TRAF4 can also mediate MEKK3-dependent extracellular signal-regulated kinase 5 (ERK5) activation inducing Steap4 which is critical for cellular metabolism and proliferation (70). Moreover, TRAF4 can also transactivate the epidermal growth factor receptor (EGFR). Chen et al. demonstrate that Act1–TRAF4–ERK5 signaling is a result of IL-17a induced EGFR transactivation and critical for stimulating epidermal hyperplasia and tumor growth in keratinocytes (71). Another regulator, TRAF3, interferes with Act1 binding to IL-17R providing a proximal regulation of IL-17 signaling (72). TRAF3 is also regulated by Nuclear Dbf2‐related kinase 1 (NDR1). NDR1 can bind to TRAF3 preventing it from binding to IL-17R, which allows for the formation of the IL-17R-Act1-TRAF6- signaling complex formation and canonical activation of IL-17 signaling.

IL-17a and IL-22 act synergistically to activate antimicrobial responses in epithelial cells (reviewed in (73)). It is difficult to separate the importance of Th17-mediated induction of neutrophil recruitment from the importance of activation of antimicrobial proteins (AMPs). Nevertheless, there is a clear trend toward the need for a Th17 response to control H. pylori; both PMNs and AMP production are likely contributing factors. IL-17a and IL-22 together enhanced the ability of GECs to upregulate expression of calprotectin, lipocalin and some β-defensins (74). Further, activating GECs with IL-17a and IL-22 increased their ability to kill H. pylori in vitro compared to treatment with each cytokine alone (74).

IL-17f also signals through the IL-17RA/RC complex, but research on mechanisms by which IL-17f acts on GECs or in the tissue during H. pylori infection is minimal. Since IL-17a appears to be the predominately produced cytokine compared to IL-17f during H. pylori infection (75), an effect of IL-17f may be minimal and/or some IL-17R signaling may be redundant. Several studies have investigated whether polymorphism in the IL-17f gene are linked to gastric cancer, but there is no consistency in their findings (76). Still, there is no profound understanding of expression of these receptors on GEC subsets and regulation of these receptors by inflammatory stimuli.

IL-22 is another T cell-derived cytokine which can activate antimicrobial and inflammatory epithelial cell functions, especially in the context of H. pylori infection(77). While we have placed the IL-22 under the Th17 cytokine umbrella for this review, this cytokine may also be produced by Th22 cells. In fact, during H. pylori infection, while IL-22 expression has been reported, it is not clear if the cells producing the cytokine are Th17 or Th22 cells. IL-22 signals through a heterodimeric receptor of IL-22RA1 and IL-10Rβ. Since IL-10Rβ is constitutively expressed, regulation of IL-22 signaling begins with regulated expression of IL-22RA1. IL-22 activates STAT3 signaling and enhances gene transcription of select genes involved in AMPs. Increased expression of IL-22 is reported in H. pylori infected mice and humans (74, 77), and its expression correlates with the level of H. pylori colonization and level of gastritis (77). Remarkably, GECs upregulated the IL-22RA1 in response to H. pylori in a CagA dependent manner (77). The influence of IL-22 on GEC is not clear cut. In one report, IL-22 downregulated CCL20 expression of H. pylori induced AGS cells, suggesting IL-22 may play a role protecting the gastric mucosa from damage induced by inflammation (78). On the other hand, IL-22^−/− mice have reduced expression of CXCL2 in response to H. pylori infection compared to WT Balb/c mice (77). This reduced CXCL2 expression correlates with reduced influx of myeloid derived suppressor cells, reduced expression of calprotectin, and increased Th1 responses (77) suggesting a proinflammatory role for IL-22 in this mouse model.

IL-22 has also been shown to induce expression of matrix metalloprotease-10 (MMP-10) in GECs activating host defense pathways (79). MMP-10 expression is increased in parietal cells and chief cells in the H. pylori infected gastric mucosa, and IL-22 (not IL-17a or IFNγ) enhanced H. pylori’s activation of MMP-10 expression in AGS cells in an ERK dependent pathway (79). MMP-10 activates GECs and H. pylori induced inflammation. MMP-10^−/− mice as well as MMP-10^−/−/IL-22^−/− mice have reduced gastritis associated with decreased expression of CXCL16 and reduced CD8+ T cell migration (79). Interestingly, the authors of this study also found that Reg3a, ZO-1 and occludin expression was inhibited by MMP-10, and they hypothesize that MMP-10 may facilitate persistence and damage to the epithelial cell layer through these pathways (79). Overall, these studies suggest that IL-22 dependent GEC activation contributes to regulating the gastritis response to H. pylori.

Th2-lymphocyte derived cytokines

Th2 cells are typically thought of as the CD4⁺ T cells which produce IL-4, IL-5 and IL-13 with the goal of helping to activate the humoral response and mast cells. Binding of IL-4 or of IL-13 initiates Jak-dependent tyrosine phosphorylation of the IL-4Rα-chain and STAT6. Activation of STAT6 in GECs is required for the gastrointestinal response to nematode infections or parasites (80). Th2 responses are not strongly activated during H. pylori infection, relative to Th1 or Th17 responses in humans, rhesus macaques, gerbils or mice (81–85); but the cytokines produced by Th2 cells may indirectly downregulate gastritis. In fact, even though C57Bl/6 WT mice do not demonstrate an increase in IL-4 expression in response to H. pylori infection, IL-4^−/− mice do exhibit an increase in IFNγ expression and increased gastritis (82). Therefore, understanding how IL-4 might regulate IFNγ responses may increase our understanding of how these cytokines inhibit or activate GEC responses. During chronic gastritis, atrophy of gastric glands has been reported resulting in a decrease in the number of D cells (SOM secreting cells) and an increase in the number of G cells (gastrin secreting cells) (86, 87). The balance of Th1 and Th2 cytokines, specifically IFNγ and IL-4, may influence the turnover and function of these neuroendocrine cell populations in the gastric mucosa of chronic Helicobacter infection; for IL-4 provides a stimulus for SOM release from D cells and therefore, provides protection from Th1 mediated inflammation in the H. felis model (88).

IL-13 is also produced by Th2 cells, but there is little data on the direct role of IL-13 in GEC function. The IL-13R has been detected on chief cells and in models of induced spasmolytic polypeptide-expressing metaplasia. IL-13 was shown to be necessary and sufficient for chief cell transdifferentiation into metaplasia following parietal cell loss (89). In a small study of 23 H. pylori positive subjects, IL-13 localized to the inflammatory infiltrate (90). More specifically, in cases where the patients had intestinal metaplasia or intestinal type gastric cancer, IL-13 also localized to the GECs (90). The impact of IL-13 on intestinal epithelial cells has been studied, but its impact on GECs is largely undescribed in the literature.

Treg-derived cytokines

The presence of Tregs reduces the pathological outcomes of H. pylori infection in mice and human (91–95). Generally, the presence of Tregs is correlated with reduced gastritis, but at the expense of greater levels of H. pylori colonization. Mouse models demonstrate that depletion of Tregs leads to increased levels of IFNγ and increased inflammation (96). Tregs may be recruited to the H. pylori colonized mucosa via CCL20/CCR6 mediated chemotaxis orchestrated by the GEC response. CCL20 is upregulated in gastric biopsies of H. pylori infected patients (97); and further, CCL20 induction in GECs is cagPAI dependent. CCL20 expression was shown to induce Treg migration; for instance, CD4⁺Foxp3⁺ T cells (Tregs) isolated from H. pylori infected patients migrated to recombinant CCL20 in vitro while CD4⁺Foxp3⁻ T cells did not (97).

Tregs are defined by their ability to regulate pro-inflammatory T cell responses through direct activation of apoptosis, through secretion of inhibitory cytokines, or by depleting the microenvironment of extracellular ATP and IL-2 (98, 99). Tregs not only target T cells, but also may target GECs through production of IL-10 and TGFβ. While IL-10 expression may be protective during inflammation, once cancer develops IL-10 be detrimental. In fact, increased IL-10 in the serum is an unfavorable prognostic marker for GC (100). IL-10 in the tumor microenvironment may be produced by a number of cell types. One potential mechanism by which IL-10 could stimulate GECs in the tumor is through activating c-Met-STAT3. GEC lines responded to culture supernatants cancer-associated macrophages (CAMs) in an IL-10 dependent way to induce proliferation and migration, while it also suppressed apoptosis (101).

TGF-β is also produced by Treg cells (and other immune cells), and may act as a tumor suppressor in early stages but a tumor promoter in later stages (102). TGFβ signals through many cell types including fibroblasts and mesothelial cells, inducing extracellular matrix protein synthesis and contributing to fibrogenesis (103). TGFβ1 and TGFβ2 bind to TGFβ receptor I through TGFβ receptor II (TGFβRII) to activate its serine/threonine kinase. The activated dimeric receptor then activates a complex of Smad proteins which facilitates transcription of target genes (104). To investigate the role of TGFβ signaling during H. pylori infection, mice which overexpress the dominant negative mutant of the TGFβ RII in their stomach were generated (pS2-dnRII) (105). When these mice and WT littermates were infected with H. pylori, the mice lacking TGFβ signaling developed gastric adenocarcinoma with high cell proliferation index in their epithelial cells compared to their WT littermates, suggesting TGFβ provides a protective effect suppressing carcinogenesis (105). In vitro studies performed on GEC lines (BGC823 and MKN-45) demonstrated that TGFβ treatment downregulated expression of E-cadherin significantly, while it upregulated expression of epithelial mesenchymal transition-related proteins, snail and vimentin (106); interestingly, upstream of TGFβ signaling, MFAP2 is activated and promotes this activity. Increased levels of TGFβ in the serum and gastric tissue is associated with advanced cancer (107–109) and expression appears to be higher in the tissues of intestinal type GC than diffuse type GC (110). Tumor growth may be promoted by TGFβ by inducing epithelial cells to undergo epithelial-mesenchymal transition through upregulating long noncoding RNA urothelial carcinoma associated 1 (LncRNA UCA1), which when silenced in GC cell lines reduced the levels of snail and vimentin and promoted expression of E-cadherin and ZO-1 (111). Moreover, erythrocyte membrane protein band 4.1-like 5 is also a target of TGFβ signaling and regulates GC cell metastasis (112). So, while Tregs may play a role downregulating the inflammatory response to H. pylori, Tregs may be detrimental during GC since Tregs and their cytokines activate pro-carcinogenic pathways in the tumor microenvironment.

Conclusions

The inflammatory response to H. pylori significantly influences the outcome of infection, and while this response involves the innate and the adaptive immune response, T lymphocytes play a major role in orchestrating the gastritis. CD4⁺ T cell-derived cytokines amplify the innate response by recruiting PMNs and macrophages to the site of infection, but they can also directly impact epithelial cell biology and carcinogenesis. Our understanding of how many of the T cell-derived cytokines impact GEC functions has increased in the last decade, but there are many gaps in knowledge to address. We lack understanding of the temporal activation of the epithelial cell response. Pro-inflammatory responses are necessary for control of the bacterial colonization, but chronically can lead to carcinogenesis; however, sustaining pro-inflammatory responses may allow for anti-tumor responses and a favorable prognosis. Secondly, many studies that have investigated the GEC response to H. pylori and/or T cell-derived cytokines have been performed in a reductionist manner when in fact, there appears to be significant cross-regulation of epithelial cell responses by cytokines. Further, there is evidence that increased numbers and mislocalized stem cell presence in gastric pits is associated with carcinogenesis. The links between inflammation and T cell- derived cytokines in the gastric tissue has largely been unexplored. Finally, the increased use of gastroids is vital for our increased understanding of the consequence of expression and signaling of these cytokines and H. pylori on the specialized cells of the gastric mucosa. Gastroids combined with single cell analyses will inform which cells respond to the stimuli and how. Research in this area will help identify appropriate promising therapeutic targets for persons with detrimental outcomes of H. pylori infection from chronic gastritis to the different types of GC.

Abbreviations and acronyms:

CCL or CXCL

Chemokine

Cag T4SS

Cytotoxin-associated Type IV secretion system

CagA

Cytotoxin A

ECL

enteroendocrine cells

GC

gastric cancer

GECs

gastric epithelial cells

H. pylori

Helicobacter pylori

IL-

Interleukin

IFNγ

Interferon gamma

NFκB

Nuclear Factor kappa-light-chain-enhancer of activated B cells

Treg

Regulatory CD4+ T cell

STAT

Signal Transducer And Activator Of Transcription

SOM

Somatostatin

Th

T helper cell

TNFα

Tumor Necrosis Factor alpha

VacA

Vacuolating toxin A

WT

Wild type

knockout

−/−