IL-17 receptor A functions to help maintain barrier integrity and limit activation of immunopathogenic response to H. pylori infection.

Activation of Th17 cell responses, including the production of IL-17A and IL-21, contributes to host defense and inflammatory responses by coordinating adaptive and innate immune responses. IL-17A and IL-17F signal through a multimeric receptor, which includes the IL-17 receptor A (IL-17RA) subunit and the IL-17RC subunit. IL-17RA is expressed by many cell types, and data from previous studies suggest that loss of IL-17 receptor is required to limit immunopathology in the Helicobacter pylori model of infection. Here, an Il17ra-/- mouse was generated on the FVB/n background, and the role of IL-17 signaling in the maintenance of barrier responses to H. pylori was investigated. Generating the Il17ra-/- on the FVB/n background allowed for the examination of responses in the paragastric lymph node and will allow for future investigation into carcinogenesis. While uninfected Il17ra-/- mice do not develop spontaneous gastritis following H. pylori infection, Il17ra-/- mice develop severe gastric inflammation accompanied by lymphoid follicle production and exacerbated production of Th17 cytokines. Increased inflammation in the tissue, increased IgA levels in the lumen, and reduced production of Muc5ac in the corpus correlate with increased H. pylori-induced paragastric lymph node activation. These data suggest that the cross talk between immune cells and epithelial cells regulates mucin production, IgA production, and translocation, impacting the integrity of the gastric mucosa and therefore activating of the adaptive immune response.

Identification of an Essential LolD-Like Protein in Helicobacter pylori.

The localization of lipoprotein (Lol) system is used by Gram-negative bacteria to export lipoproteins to the outer membrane. Lol proteins and models of how Lol transfers lipoproteins from the inner to the outer membrane have been extensively characterized in the model organism Escherichia coli, but in numerous bacterial species, lipoprotein synthesis and export pathways deviate from the E. coli paradigm. For example, in the human gastric bacterium Helicobacter pylori, a homolog of the E. coli outer membrane component LolB is not found, E. coli LolC and LolE correspond to a single inner membrane component (LolF), and a homolog of the E. coli cytoplasmic ATPase LolD has not been identified. In the present study, we sought to identify a LolD-like protein in H. pylori. We used affinity-purification mass spectrometry to identify interaction partners of the H. pylori ATP-binding cassette (ABC) family permease LolF and identified the ABC family ATP-binding protein HP0179 as its interaction partner. We engineered H. pylori to conditionally express HP0179 and showed that HP0179 and its conserved ATP binding and ATP hydrolysis motifs are essential for H. pylori growth. We then performed affinity purification-mass spectrometry using HP0179 as the bait and identified LolF as its interaction partner. These results indicate that H. pylori HP0179 is a LolD-like protein and provide a more complete understanding of lipoprotein localization processes in H. pylori, a bacterium in which the Lol system deviates from the E. coli paradigm. IMPORTANCE Lipoproteins are critical in Gram-negative-bacteria for cell surface assembly of LPS, insertion of outer membrane proteins, and sensing envelope stress. Lipoproteins also contribute to bacterial pathogenesis. For many of these functions, lipoproteins must localize to the Gram-negative outer membrane. Transporting lipoproteins to the outer membrane involves the Lol sorting pathway. Detailed analyses of the Lol pathway have been performed in the model organism Escherichia coli, but many bacteria utilize altered components or are missing essential components of the E. coli Lol pathway. Identifying a LolD-like protein in Helicobacter pylori is important to better understand the Lol pathway in diverse bacterial classes. This becomes particularly relevant as lipoprotein localization is targeted for antimicrobial development.

Essential role of Helicobacter pylori apolipoprotein N-acyltransferase (Lnt) in stomach colonization.

Bacterial lipoproteins are post-translationally modified with acyl chains, anchoring these proteins to bacterial membranes. In Gram-negative bacteria, three enzymes complete the modifications. Lgt (which adds two acyl chains) and LspA (which removes the signal peptide) are essential. Lnt (which adds a third acyl chain) is not essential in certain bacteria including Francisella tularensis, Neisseria gonorrhoeae, and Acinetobacter baumannii. Deleting lnt results in mild to severe physiologic changes. We previously showed lnt is not essential for Helicobacter pylori growth in vitro. Here, the physiologic consequences of deleting lnt in H. pylori and the role of Lnt in the host response to H. pylori were examined using in vitro and in vivo models. Comparing wild-type, Δlnt, and complemented mutant H. pylori, no changes in growth rates or sensitivity to acid or antibiotics were observed. Since deleting lnt changes the number of acyl chains on lipoproteins and the number of acyl chains on lipoproteins impacts the innate immune response through Toll-like receptor 2 (TLR2) signaling, primary human gastric epithelial cells were treated with a purified lipoprotein from wild-type or lnt mutant H. pylori. Differential gene expression analysis indicated that lipoprotein from the lnt mutant induced a more robust TLR2 response. In a complementary approach, we infected wild-type and Tlr2-/- mice and found that both the wild-type and complemented mutant strains successfully colonized the animals. However, the lnt mutant strain was unable to colonize either mouse strain. These results show that lnt is essential for H. pylori colonization and identifies lipoprotein synthesis as a target for therapeutic intervention.

CD8αα⁺ innate-type lymphocytes in the intestinal epithelium mediate mucosal immunity.

Innate immune responses are critical for mucosal immunity. Here we describe an innate lymphocyte population, iCD8α cells, characterized by expression of CD8α homodimers. iCD8α cells exhibit innate functional characteristics such as the capacity to engulf and kill bacteria. Development of iCD8α cells depends on expression of interleukin-2 receptor γ chain (IL-2Rγc), IL-15, and the major histocompatibility complex (MHC) class Ib protein H2-T3, also known as the thymus leukemia antigen or TL. While lineage tracking experiments indicated that iCD8α cells have a lymphoid origin, their development was independent of the transcriptional suppressor Id2, suggesting that these cells do not belong to the family of innate lymphoid cells. Finally, we identified cells with a similar phenotype in humans, which were profoundly depleted in newborns with necrotizing enterocolitis. These findings suggest a critical role of iCD8α cells in immune responses associated with the intestinal epithelium.

T Cell Cytokines Impact Epithelial Cell Responses during Helicobacter pylori Infection.

The goal of this Brief Review is to highlight literature that 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 H. pylori The focal point of this Brief 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. Furthermore, the modulation of epithelial-derived chemokines by H. pylori infection will be discussed.

Enhanced Fitness of a Helicobacter pylori babA Mutant in a Murine Model.

Helicobacter pylori genomes encode over 60 predicted outer membrane proteins (OMPs). Several OMPs in the Hop family act as adhesins, but the functions of most Hop proteins are unknown. To identify hop mutant strains exhibiting differential fitness in vivo compared to in vitro, we used a genetic barcoding method that allowed us to track changes in the proportional abundance of H. pylori strains within a mixed population. We generated a library of hop mutant strains, each containing a unique nucleotide barcode, as well as a library of control strains, each containing a nucleotide barcode in an intergenic region predicted to be a neutral locus unrelated to bacterial fitness. We orogastrically inoculated each of the libraries into mice and analyzed compositional changes in the populations over time in vivo compared to changes detected in the populations during library passage in vitro. The control library proliferated as a relatively stable community in vitro, but there was a reduction in the population diversity of this library in vivo and marked variation in the dominant strains recovered from individual animals, consistent with the existence of a nonselective bottleneck in vivo. We did not identify any OMP mutants exhibiting fitness defects exclusively in vivo without corresponding fitness defects in vitro. Conversely, a babA mutant exhibited a strong fitness advantage in vivo but not in vitro. These findings, when taken together with results of other studies, suggest that production of BabA may have differential effects on H. pylori fitness depending on the environmental conditions.

Resolution of Gastric Cancer-Promoting Inflammation: A Novel Strategy for Anti-cancer Therapy.

The connection between inflammation and cancer was initially recognized by Rudolf Virchow in the nineteenth century. During the last decades, a large body of evidence has provided support to his hypothesis, and now inflammation is recognized as one of the hallmarks of cancer, both in etiopathogenesis and ongoing tumor growth. Infection with the pathogen Helicobacter pylori is the primary causal factor in 90% of gastric cancer (GC) cases. As we increase our understanding of how chronic inflammation develops in the stomach and contributes to carcinogenesis, there is increasing interest in targeting cancer-promoting inflammation as a strategy to treat GC. Moreover, once cancer develops and anti-cancer immune responses are suppressed, there is evidence of a substantial shift in the microenvironment and new targets for immune therapy emerge. In this chapter, we provide insight into inflammation-related factors, including T lymphocytes, macrophages, pro-inflammatory chemokines, and cytokines, which promote H. pylori-associated GC initiation and growth. While intervening with chronic inflammation is not a new practice in rheumatology or gastroenterology, this approach has not been fully explored for its potential to prevent carcinogenesis or to contribute to the treatment of GC. This review highlights current and possible strategies for therapeutic intervention including (i) targeting pro-inflammatory mediators, (ii) targeting growth factors and pathways involved in angiogenesis in the gastric tumor microenvironment, and (iii) enhancing anti-tumor immunity. In addition, we highlight a significant number of clinical trials and discuss the importance of individual tumor characterization toward offering personalized immune-related therapy.

Interleukin-21 (IL-21) Downregulates Dendritic Cell Cytokine Responses to Helicobacter pylori and Modulates T Lymphocyte IL-17A Expression in Peyer's Patches during Infection.

Interleukin-21 (IL-21), a cytokine produced by many subsets of activated immune cells, is critical for driving inflammation in several models. Using Helicobacter pylori infection as a model for chronic mucosal infection, we previously published that IL-21 is required for the development of gastritis in response to infection. Concomitant with protection from chronic inflammation, H. pylori-infected IL-21-/- mice exhibited limited Th1 and Th17 responses in their gastric mucosa. Here we report that H. pylori-infected IL-21-/- mice express significantly higher levels of IL-17A than H. pylori-infected wild-type (WT) mice in the Peyer's patches and mesenteric lymph nodes. This led us to hypothesize that IL-21 may indirectly regulate H. pylori-specific T cell responses by controlling dendritic cell (DC) functions in mucosa-associated lymphoid tissue. It was found that IL-21 treatment reduced the ability of dendritic cells to produce proinflammatory cytokines in response to H. pylori While H. pylori increased the expression of costimulatory proteins on DCs, IL-21 reduced the expression of CD40 in the presence of H. pylori Also, Th17 recall responses were intact when DCs were used as antigen-presenting cells in the presence of IL-21, but IL-21 did impact the ability of DCs to induce antigen-specific proliferation. These data suggest that IL-21, while proinflammatory in most settings, downregulates the proinflammatory cytokine microenvironment through modulating the cytokine expression of DCs, indirectly modifying IL-17A expression. Understanding how these proinflammatory cytokines are regulated will advance our understanding of how and why H. pylori infection may be tolerated in some individuals while it causes gastritis, ulcers, or cancer in others.

Th17 Cells in Helicobacter pylori Infection: a Dichotomy of Help and Harm.

Helicobacter pylori is a Gram-negative bacterium that infects the gastric epithelia of its human host. Everyone who is colonized with these pathogenic bacteria can develop gastric inflammation, termed gastritis. Additionally, a small proportion of colonized people develop more adverse outcomes, including gastric ulcer disease, gastric adenocarcinoma, or gastric mucosa-associated lymphoid tissue lymphoma. The development of these adverse outcomes is dependent on the establishment of a chronic inflammatory response. The development and control of this chronic inflammatory response are significantly impacted by CD4+ T helper cell activity. Noteworthy, T helper 17 (Th17) cells, a proinflammatory subset of CD4+ T cells, produce several proinflammatory cytokines that activate innate immune cell antimicrobial activity, drive a pathogenic immune response, regulate B cell responses, and participate in wound healing. Therefore, this review was written to take an intricate look at the involvement of Th17 cells and their affiliated cytokines (interleukin-17A [IL-17A], IL-17F, IL-21, IL-22, and IL-26) in regulating the immune response to H. pylori colonization and carcinogenesis.

Purine Biosynthesis Metabolically Constrains Intracellular Survival of Uropathogenic Escherichia coli.

The ability to de novo synthesize purines has been associated with the intracellular survival of multiple bacterial pathogens. Uropathogenic Escherichia coli (UPEC), the predominant cause of urinary tract infections, undergoes a transient intracellular lifestyle during which bacteria clonally expand into multicellular bacterial communities within the cytoplasm of bladder epithelial cells. Here, we characterized the contribution of the conserved de novo purine biosynthesis-associated locus cvpA-purF to UPEC pathogenesis. Deletion of cvpA-purF, or of purF alone, abolished de novo purine biosynthesis but did not impact bacterial adherence properties in vitro or in the bladder lumen. However, upon internalization by bladder epithelial cells, UPEC deficient in de novo purine biosynthesis was unable to expand into intracytoplasmic bacterial communities over time, unless it was extrachromosomally complemented. These findings indicate that UPEC is deprived of purine nucleotides within the intracellular niche and relies on de novo purine synthesis to meet this metabolic requirement.

Dietary Composition Influences Incidence of Helicobacter pylori-Induced Iron Deficiency Anemia and Gastric Ulceration.

Epidemiologic studies have provided conflicting data regarding an association between Helicobacter pylori infection and iron deficiency anemia (IDA) in humans. Here, a Mongolian gerbil model was used to investigate a potential role of H. pylori infection, as well as a possible role of diet, in H. pylori-associated IDA. Mongolian gerbils (either H. pylori infected or uninfected) received a normal diet or one of three diets associated with increased H. pylori virulence: high-salt, low-iron, or a combination of a high-salt and low-iron diet. In an analysis of all infected animals compared to uninfected animals (independent of diet), H. pylori-infected gerbils had significantly lower hemoglobin values than their uninfected counterparts at 16 weeks postinfection (P < 0.0001). The mean corpuscular volume (MCV) and serum ferritin values were significantly lower in H. pylori-infected gerbils than in uninfected gerbils, consistent with IDA. Leukocytosis and thrombocytosis were also detected in infected gerbils, indicating the presence of a systemic inflammatory response. In comparison to uninfected gerbils, H. pylori-infected gerbils had a higher gastric pH, a higher incidence of gastric ulcers, and a higher incidence of fecal occult blood loss. Anemia was associated with the presence of gastric ulceration but not gastric cancer. Infected gerbils consuming diets with a high salt content developed gastric ulcers significantly more frequently than gerbils consuming a normal-salt diet, and the lowest hemoglobin levels were in infected gerbils consuming a high-salt/low-iron diet. These data indicate that H. pylori infection can cause IDA and that the composition of the diet influences the incidence and severity of H. pylori-induced IDA.

Helicobacter pylori adaptation in vivo in response to a high-salt diet.

Helicobacter pylori exhibits a high level of intraspecies genetic diversity. In this study, we investigated whether the diversification of H. pylori is influenced by the composition of the diet. Specifically, we investigated the effect of a high-salt diet (a known risk factor for gastric adenocarcinoma) on H. pylori diversification within a host. We analyzed H. pylori strains isolated from Mongolian gerbils fed either a high-salt diet or a regular diet for 4 months by proteomic and whole-genome sequencing methods. Compared to the input strain and output strains from animals fed a regular diet, the output strains from animals fed a high-salt diet produced higher levels of proteins involved in iron acquisition and oxidative-stress resistance. Several of these changes were attributable to a nonsynonymous mutation in fur (fur-R88H). Further experiments indicated that this mutation conferred increased resistance to high-salt conditions and oxidative stress. We propose a model in which a high-salt diet leads to high levels of gastric inflammation and associated oxidative stress in H. pylori-infected animals and that these conditions, along with the high intraluminal concentrations of sodium chloride, lead to selection of H. pylori strains that are most fit for growth in this environment.

Role of connexin 43 in Helicobacter pylori VacA-induced cell death.

Helicobacter pylori colonizes the human stomach and confers an increased risk for the development of peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. A secreted H. pylori toxin, VacA, can cause multiple alterations in gastric epithelial cells, including cell death. In this study, we sought to identify host cell factors that are required for VacA-induced cell death. To do this, we analyzed gene trap and short hairpin RNA (shRNA) libraries in AZ-521 human gastric epithelial cells and selected for VacA-resistant clones. Among the VacA-resistant clones, we identified multiple gene trap library clones and an shRNA library clone with disrupted expression of connexin 43 (Cx43) (also known as gap junction protein alpha 1 [GJA1]). Further experiments with Cx43-specific shRNAs confirmed that a reduction in Cx43 expression results in resistance to VacA-induced cell death. Immunofluorescence microscopy experiments indicated that VacA did not colocalize with Cx43. We detected production of the Cx43 protein in AZ-521 cells but not in AGS, HeLa, or RK-13 cells, and correspondingly, AZ-521 cells were the most susceptible to VacA-induced cell death. When Cx43 was expressed in HeLa cells, the cells became more susceptible to VacA. These results indicate that Cx43 is a host cell constituent that contributes to VacA-induced cell death and that variation among cell types in susceptibility to VacA-induced cell death is attributable at least in part to cell type-specific differences in Cx43 production.

Fatty acids of Helicobacter pylori lipoproteins CagT and Lpp20.

Bacterial lipoproteins are post-translationally modified by the addition of acyl chains that anchor the protein to bacterial membranes. This modification includes two ester-linked and one amide-linked acyl chain on lipoproteins from Gram-negative bacteria. Helicobacter pylori lipoproteins have important functions in pathogenesis (including delivering the CagA oncoprotein to mammalian cells) and are recognized by host innate and adaptive immune systems. The number and variety of acyl chains on lipoproteins impact the innate immune response through Toll-like receptor 2. The acyl chains added to lipoproteins are derived from membrane phospholipids. H. pylori membrane phospholipids have previously been shown to consist primarily of C14:0 and C19:0 cyclopropane-containing acyl chains. However, the acyl composition of H. pylori lipoproteins has not been determined. In this study, we characterized the acyl composition of two representative H. pylori lipoproteins, Lpp20 and CagT. Fatty acid methyl esters were prepared from both purified lipoproteins and analyzed by gas chromatography-mass spectrometry. For comparison, we also analyzed H. pylori phospholipids. Consistent with previous studies, we observed that the H. pylori phospholipids contain primarily C14:0 and C19:0 cyclopropane-containing fatty acids. In contrast, both the ester-linked and amide-linked fatty acids found in H. pylori lipoproteins were observed to be almost exclusively C16:0 and C18:0. A discrepancy between the acyl composition of membrane phospholipids and lipoproteins as reported here for H. pylori has been previously reported in other bacteria including Borrelia and Brucella. We discuss possible mechanisms.IMPORTANCEColonization of the stomach by Helicobacter pylori is an important risk factor in the development of gastric cancer, the third leading cause of cancer-related death worldwide. H. pylori persists in the stomach despite an immune response against the bacteria. Recognition of lipoproteins by TLR2 contributes to the innate immune response to H. pylori. However, the role of H. pylori lipoproteins in bacterial persistence is poorly understood. As the host response to lipoproteins depends on the acyl chain content, defining the acyl composition of H. pylori lipoproteins is an important step in characterizing how lipoproteins contribute to persistence.

IL-17RA-Mediated Epithelial Cell Activity Prevents Severe Inflammatory Response to Helicobacter pylori Infection.

Helicobacter pylori is a Gram-negative pathogen that colonizes the stomach, induces inflammation, and drives pathological changes in the stomach tissue, including gastric cancer. As the principal cytokine produced by Th17 cells, IL-17 mediates protective immunity against pathogens by inducing the activation and mobilization of neutrophils. Whereas IL-17A is largely produced by lymphocytes, the IL-17 receptor is expressed in epithelial cells, fibroblasts, and hematopoietic cells. Loss of the IL-17RA in mice results in impaired antimicrobial responses to extracellular bacteria. In the context of H. pylori infection, this is compounded by extensive inflammation in Il17ra-/- mice. In this study, Foxa3creIl17rafl/fl (Il17raΔGI-Epi) and Il17rafl/fl (control) mice were used to test the hypothesis that IL-17RA signaling, specifically in epithelial cells, protects against severe inflammation after H. pylori infection. The data indicate that Il17raΔGI-Epi mice develop increased inflammation compared with controls. Despite reduced Pigr expression, levels of IgA increased in the gastric wash, suggesting significant increase in Ag-specific activation of the T follicular helper/B cell axis. Gene expression analysis of stomach tissues indicate that both acute and chronic responses are significantly increased in Il17raΔGI-Epi mice compared with controls. These data suggest that a deficiency of IL-17RA in epithelial cells is sufficient to drive chronic inflammation and hyperactivation of the Th17/T follicular helper/B cell axis but is not required for recruitment of polymorphonuclear neutrophils. Furthermore, the data suggest that fibroblasts can produce chemokines in response to IL-17 and may contribute to H. pylori-induced inflammation through this pathway.

High dietary salt intake exacerbates Helicobacter pylori-induced gastric carcinogenesis.

Persistent colonization of the human stomach with Helicobacter pylori is a risk factor for gastric adenocarcinoma, and H. pylori-induced carcinogenesis is dependent on the actions of a bacterial oncoprotein known as CagA. Epidemiological studies have shown that high dietary salt intake is also a risk factor for gastric cancer. To investigate the effects of a high-salt diet, we infected Mongolian gerbils with a wild-type (WT) cagA(+) H. pylori strain or an isogenic cagA mutant strain and maintained the animals on a regular diet or a high-salt diet. At 4 months postinfection, gastric adenocarcinoma was detected in 100% of the WT-infected/high-salt-diet animals, 58% of WT-infected/regular-diet animals, and none of the animals infected with the cagA mutant strain (P < 0.0001). Among animals infected with the WT strain, those fed a high-salt diet had more severe gastric inflammation, higher gastric pH, increased parietal cell loss, increased gastric expression of interleukin 1ß (IL-1ß), and decreased gastric expression of hepcidin and hydrogen potassium ATPase (H,K-ATPase) compared to those on a regular diet. Previous studies have detected upregulation of CagA synthesis in response to increased salt concentrations in the bacterial culture medium, and, concordant with the in vitro results, we detected increased cagA transcription in vivo in animals fed a high-salt diet compared to those on a regular diet. Animals infected with the cagA mutant strain had low levels of gastric inflammation and did not develop hypochlorhydria. These results indicate that a high-salt diet potentiates the carcinogenic effects of cagA(+) H. pylori strains.

Intestinal epithelial cells modulate CD4 T cell responses via the thymus leukemia antigen.

The intestinal epithelium is comprised of a monolayer of intestinal epithelial cells (IEC), which provide, among other functions, a physical barrier between the high Ag content of the intestinal lumen and the sterile environment beyond the epithelium. IEC express a nonclassical MHC class I molecule known as the thymus leukemia (TL) Ag. TL is known to interact with CD8αα-expressing cells, which are abundant in the intestinal intraepithelial lymphocyte compartment. In this report, we provide evidence indicating that expression of TL by IEC modulates the cytokine profile of CD4(+) T cells favoring IL-17 production. We show in an adoptive transfer model of colitis that donor-derived cells become more pathogenic when TL is expressed on IEC in recipient animals. Moreover, TL(+)IEC promote development of IL-17-mediated responses capable of protecting mice from Citrobacter rodentium infection. We also show that modulation of IL-17-mediated responses by TL(+)IEC is controlled by the expression of CD8α on CD4(+) T cells. Overall, our results provide evidence for an important interaction between IEC and CD4(+) T cells via TL, which modulates mucosal immune responses.

Increased intestinal permeability and downregulation of absorptive ion transporters Nhe3, Dra, and Sglt1 contribute to diarrhea during Clostridioides difficile infection.

BACKGROUND & AIM: Clostridioides difficile infection (CDI) is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis. Two protein toxins, TcdA and TcdB, produced by C. difficile are the major determinants of disease. However, the pathophysiological causes of diarrhea during CDI are not well understood. Here, we investigated the effects of C. difficile toxins on paracellular permeability and apical ion transporters in the context of an acute physiological infection. METHODS: We studied intestinal permeability and apical membrane transporters in female C57BL/6J mice. Üssing chambers were used to measure paracellular permeability and ion transporter function across the intestinal tract. Infected intestinal tissues were analyzed by immunofluorescence microscopy and RNA-sequencing to uncover mechanisms of transporter dysregulation. RESULTS: Intestinal permeability was increased through the size-selective leak pathway in vivo during acute CDI in a 2-day-post infection model. Chloride secretory activity was reduced in the cecum and distal colon during infection by decreased CaCC and CFTR function, respectively. SGLT1 activity was significantly reduced in the cecum and colon, accompanied by ablated SGLT1 expression in colonocytes and increased luminal glucose concentrations. SGLT1 and DRA expression was ablated by either TcdA or TcdB during acute infection, but NHE3 was decreased in a TcdB-dependent manner. The localization of key proteins that link filamentous actin to the ion transporters in the apical plasma membrane was unchanged. However, Sglt1, Nhe3, and Dra were drastically reduced at the transcript level, implicating downregulation of ion transporters in the mechanism of diarrhea during CDI. CONCLUSIONS: CDI increases intestinal permeability and decreases apical abundance of NHE3, SGLT1, and DRA. This combination likely leads to dysfunctional water and solute absorption in the large bowel, causing osmotic diarrhea. These findings provide insights into the pathophysiological mechanisms underlying diarrhea and may open novel avenues for attenuating CDI-associated diarrhea.

The intermediate region of Helicobacter pylori VacA is a determinant of toxin potency in a Jurkat T cell assay.

Colonization of the human stomach with Helicobacter pylori is a risk factor for peptic ulceration, noncardia gastric adenocarcinoma, and gastric lymphoma. The secreted VacA toxin is an important H. pylori virulence factor that causes multiple alterations in gastric epithelial cells and T cells. Several families of vacA alleles have been described, and H. pylori strains containing certain vacA types (s1, i1, and m1) are associated with an increased risk of gastric disease, compared to strains containing other vacA types (s2, i2, and m2). Thus far, there has been relatively little study of the role of the VacA intermediate region (i-region) in toxin activity. In this study, we compared the ability of i1 and i2 forms of VacA to cause functional alterations in Jurkat cells. To do this, we manipulated the chromosomal vacA gene in two H. pylori strains to introduce alterations in the region encoding the VacA i-region. We did not detect any differences in the capacity of i1 and i2 forms of VacA to cause vacuolation of RK13 cells. In comparison to i1 forms of VacA, i2 forms of VacA had a diminished capacity to inhibit the activation of nuclear factor of activated T cells (NFAT) and suppress interleukin-2 (IL-2) production. Correspondingly, i2 forms of VacA bound to Jurkat cells less avidly than did i1 forms of VacA. These results indicate that the VacA i-region is an important determinant of VacA effects on human T cell function.

IL-17 Receptor Signaling through IL-17A or IL-17F Is Sufficient to Maintain Innate Response and Control of Helicobacter pylori Immunopathogenesis.

IL-17R signaling is required for control of extracellular pathogens and is also implicated in development of chronic inflammatory processes. The response to the human pathogen Helicobacter pylori results in Th1 and Th17 cell activation and a chronic inflammatory process that can lead to adverse outcomes, such as gastric cancer. Previously, we identified IL-17RA as a requirement for the recruitment of neutrophils and control of H. pylori colonization in the gastric mucosa. Unexpectedly, H. pylori-infected Il17ra -/- mice had significantly more chronic inflammation than H. pylori-infected wild-type mice. In this study, human epithelial cell lines and murine models were used to investigate differential roles for IL-17A, IL-17F, and IL-17A/F during H. pylori infection. Moreover, the hypothesis that IL-17RA signaling, specifically in lymphocytes, provides an autocrine feedback loop that downregulates Th17 cytokine production was investigated. The data indicate that epithelial cells exhibit a stronger response to IL-17A and IL-17A/F than IL-17F, and that IL-17A and IL-17A/F can synergize with TNF and IL-22 to induce antimicrobial genes of gastric epithelial cells. In vivo deficiencies of IL-17A or IL-17F alone did not significantly change the immunopathological response to H. pylori, but if both cytokines were absent, a hyperinflammatory lymphocytic response developed. Using a cre/flox targeting approach for IL-17RA combined with infection, our findings demonstrate that increased chronic inflammation in Il17ra -/- mice was not attributed to a T cell-intrinsic defect. These data imply that IL-17A and IL-17F may have overlapping roles in maintenance of the gastric mucosal response to infection.