Mucosal Host Defense Research Articles

08 IL-17A REGULATES INTESTINAL EPITHELIAL CELLS REGENERATION VIA ATONAL HOMOLOG (ATOH)1

IL-17A, derived from Th17 cells, plays an important role in intestinal host defense. However, the interaction and potential molecular synergies between IL-17A and epithelial cell lineages, intestinal stem cells (ISCs), and progenitors of the intestine remain unclear. Entire gut epithelium (Il17rafl/fl;Villin-cre)and secretory progenitor and epithelium(Il17rafl/fl;Atoh1-cre)-specific IL-17RA knockoutmice were generated. Dextran Sulfate Sodium (DSS) and g-irradiation-mediated epithelial injury and regeneration models were used. Cohoused littermate mice were used in all experiments. RT-PCR, immunofluorescence, and primary enteroid culture were used to study impact of IL-17A signaling on specific cell types. We found that IL-17A/F receptor (IL-17RA/IL-17RC) is expressed on functionally distinct absorptive cells (enterocytes), secretory cells (Paneth), Lgr5+ intestinal stem cells (ISCs) and transit amplifying (TA) progenitor cells of the intestine. Our data show that knockout of IL-17RA from the entire intestinal epithelia in Il17rafl/fl;Villin-cre+miceare more susceptible to DSS- and g-irradiation-mediated epithelial injury. We found a defect in secretory goblet number in DSS-administered Il17rafl/fl;villin-cremice. Atonal homolog 1 (Atoh1) is a transcription factor required for intestinal secretory cell differentiation including goblet and Paneth cells. We found that recombinant IL-17A induced Atoh1 expression in ISCs. Our preliminary data show a more severe DSS and g-irradiation-mediated intestinal injury in Il17rafl/fl;Atoh1-cre+ mice. Next, we found reduced Sox9 expression as well as reduced Ki67+stained cells in the colon of DSS administered Il17rafl/fl;Atoh1-cre+mice. Furthermore, we show that IL-17A does not regulate mature goblet cell-specific functions which indicates a stem/progenitor cell-specific defect. Our data suggest that IL-17A via Atoh1 regulates intestinal mucosal host defense. Our data show a novel role of IL-17A in regulating Atoh1-dependent epithelial cell regeneration.

08 IL-17A REGULATES INTESTINAL EPITHELIAL CELLS REGENERATION VIA ATONAL HOMOLOG (ATOH)1

IL-17A, derived from Th17 cells, plays an important role in intestinal host defense. However, the interaction and potential molecular synergies between IL-17A and epithelial cell lineages, intestinal stem cells (ISCs), and progenitors of the intestine remain unclear. Entire gut epithelium (Il17rafl/fl;Villin-cre)and secretory progenitor and epithelium(Il17rafl/fl;Atoh1-cre)-specific IL-17RA knockoutmice were generated. Dextran Sulfate Sodium (DSS) and g-irradiation-mediated epithelial injury and regeneration models were used. Cohoused littermate mice were used in all experiments. RT-PCR, immunofluorescence, and primary enteroid culture were used to study impact of IL-17A signaling on specific cell types. We found that IL-17A/F receptor (IL-17RA/IL-17RC) is expressed on functionally distinct absorptive cells (enterocytes), secretory cells (Paneth), Lgr5+ intestinal stem cells (ISCs) and transit amplifying (TA) progenitor cells of the intestine. Our data show that knockout of IL-17RA from the entire intestinal epithelia in Il17rafl/fl;Villin-cre+miceare more susceptible to DSS- and g-irradiation-mediated epithelial injury. We found a defect in secretory goblet number in DSS-administered Il17rafl/fl;villin-cremice. Atonal homolog 1 (Atoh1) is a transcription factor required for intestinal secretory cell differentiation including goblet and Paneth cells. We found that recombinant IL-17A induced Atoh1 expression in ISCs. Our preliminary data show a more severe DSS and g-irradiation-mediated intestinal injury in Il17rafl/fl;Atoh1-cre+ mice. Next, we found reduced Sox9 expression as well as reduced Ki67+stained cells in the colon of DSS administered Il17rafl/fl;Atoh1-cre+mice. Furthermore, we show that IL-17A does not regulate mature goblet cell-specific functions which indicates a stem/progenitor cell-specific defect. Our data suggest that IL-17A via Atoh1 regulates intestinal mucosal host defense. Our data show a novel role of IL-17A in regulating Atoh1-dependent epithelial cell regeneration.

Thymopentin ameliorates dextran sulfate sodium-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes.

Background: Ulcerative colitis (UC) is a chronic inflammatory gastrointestinal disease, notoriously challenging to treat. Previous studies have found a positive correlation between thymic atrophy and colitis severity. It was, therefore, worthwhile to investigate the effect of thymopentin (TP5), a synthetic pentapeptide corresponding to the active domain of the thymopoietin, on colitis.Methods: Dextran sulfate sodium (DSS)-induced colitis mice were treated with TP5 by subcutaneous injection. Body weight, colon length, colon weight, immune organ index, disease activity index (DAI) score, and the peripheral blood profile were examined. The immune cells of the spleen and colon were analyzed by flow cytometry. Histology was performed on isolated colon tissues for cytokine analysis. Bacterial DNA was extracted from mouse colonic feces to assess the intestinal microbiota. Intestinal lamina propria mononuclear cells (LPMCs), HCT116, CT26, and splenocytes were cultured and treated with TP5.Results: TP5 treatment increased the body weight and colon length, decreased the DAI score, and restored colon architecture of colitic mice. TP5 also decreased the infiltration of immune cells and expression levels of pro-inflammatory cytokines such as IL-6. Importantly, the damaged thymus and compromised lymphocytes in peripheral blood were significantly restored by TP5. Also, the production of IL-22, both in innate and adaptive lymphoid cells, was triggered by TP5. Given the critical role of IL-22 in mucosal host defense, we tested the effect of TP5 on mucus barrier and gut microbiota and found that the number of goblet cells and the level of Mucin-2 expression were restored, and the composition of the gut microbiome was normalized after TP5 treatment. The critical role of IL-22 in the protective effect of TP5 on colitis was further confirmed by administering the anti-IL-22 antibody (αIL-22), which completely abolished the effect of TP5. Furthermore, TP5 significantly increased the expression level of retinoic acid receptor-related orphan receptor γ (RORγt), a transcription factor for IL-22. Consistent with this, RORγt inhibitor abrogated the upregulation of IL-22 induced by TP5.Conclusion: TP5 exerts a protective effect on DSS-induced colitis by triggering the production of IL-22 in both innate and adaptive lymphocytes. This study delineates TP5 as an immunomodulator that may be a potential drug for the treatment of UC.

Dietary Factors in Prevention of Pediatric Escherichia coli Infection: A Model Using Domestic Piglets.

Enterotoxigenic Escherichia coli (ETEC) is the major etiological agent causing acute watery diarrhea that is most frequently seen in young children in lower-income countries. The duration of diarrheal symptom may be shortened by antibiotic treatment, but ETEC is relative refractory to common antibiotics. Burgeoning evidence suggests bioactive components that naturally occur in human milk (e.g., lysozyme and oligosaccharides) and plants (e.g., nondigestible carbohydrates and phytochemicals) contain antimicrobial functions are promising preventive measures to control ETEC infection. Although the exact protective mechanisms may vary for each compound and are still not completely understood, they generally act to (1) competitively inhibit the binding of pathogenic bacteria and toxins to gut epithelium; (2) directly kill pathogens; and (3) stimulate and/or enhance host mucosal and systemic immune defense against pathogenic microorganisms. An appropriate ETEC-challenge animal model is critical to evaluate the effect and unveil the mechanism of bioactive compounds in prevention of enteric infection. Despite wide application in biomedical research, rodents do not usually manifest typical clinical signs of enteric infections. The remarkable differences in digestive physiology, immune response, and gut microbiota between rodents and human beings necessitate the use of alternative animal models. Pigs are closely related to humans in terms of genomes, physiology, anatomy of gastrointestinal tracts, digestive enzymes, components of immune system, and gut microbiota. Like human infants and young children, nursing and nursery piglets are more susceptible to ETEC infection and reproduce the clinical signs as observed in humans. Hence, the ETEC-challenge piglet represents a valuable translational model to study pathogenesis and evaluate dietary factors (e.g., milk bioactive compounds, nondigestible carbohydrates, and phytochemicals) as preventive measures for ETEC infection in pediatrics.

Opioid Use Potentiates the Virulence of Hospital Acquired Infection, Increases Systemic Bacterial Dissemination and Exacerbates Gut Dysbiosis in a Murine Model of Citrobacter Rodentium Infection

Background: Opioid analgesics are frequently prescribed in the United States and worldwide. However, serious side effects such as addiction, immunosuppression and gastrointestinal symptoms limit their use. It was recently demonstrated that morphine treatment results in a significant disruption in gut barrier function, leading to an increased translocation of gut commensal bacteria. Further studies have indicated distinct alterations in the gut microbiome and metabolome following morphine treatment, contributing to the negative consequences that are associated with opioid use. However, it is unclear how opioids modulate gut homeostasis in the context of a hospital acquired bacterial infection. Methods: In the current study, a mouse model of C. rodentium infection was used to investigate the role of morphine in the modulation of gut homeostasis in the context of a hospital acquired bacterial infection. Citrobacter rodentium is a natural mouse pathogen that models intestinal infection by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and causes attaching and effacing lesions and colonic hyperplasia. Findings: Morphine treatment resulted in 1) the promotion of C. rodentium systemic dissemination, 2) an increase in the expression of the virulence factors of C. rodentium colonization in intestinal contents, 3) altered gut microbiome, 4) damaged integrity of gut epithelial barrier function, 5) inhibition of the C. rodentium-induced increase in goblet cells, and 6) dysregulated IL-17A immune response. Interpretation: This is the first study to demonstrate that morphine promotes pathogen dissemination in the context of an intestinal C. rodentium infection, indicating that morphine modulates the virulence factor-mediated adhesion of pathogenic bacteria and induces disruption of mucosal host defense during C. rodentium intestinal infection in mice. This study demonstrates and further validates a positive correlation between opioid drug use/abuse and an increased risk of infections, suggesting that the overprescription of opioids may increase the risk of the emergence of pathogenic strains and should therefore be prescribed cautiously. Therapeutics that are directed at maintaining gut homeostasis during opioid use may reduce the comorbidities associated with opioid use for pain management. Funding Statement: This work was supported by the NIH grants R01 DA043252, R01 DA037843, R01 DA044582, and R01 DA047089 to SR. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animals were maintained in specific-pathogen-free facilities, and all procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). The IACUC protocol number was 1203A11091. All procedures were conducted in line with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Neuronal regulation of intestinal immune functions in health and disease.

Nerve-mucosa interactions control various elements of gastrointestinal functions, including mucosal host defense, gut barrier function, and epithelial cell growth and differentiation. In both intestinal and extra-intestinal diseases, alterations of autonomic nerve activity have been observed to be concurrent with the disease course, such as in inflammatory and functional bowel diseases, and neurodegenerative diseases. This is relevant as the extrinsic autonomic nervous system is increasingly recognized to modulate gut inflammatory responses. The molecular and cellular mechanisms through which the extrinsic and intrinsic nerve pathways may regulate digestive mucosal functions have been investigated in several pre-clinical and clinical studies. The present review focuses on the involvement of neural pathways in gastrointestinal disease, and addresses the current strategies to intervene with neuronal pathway as a means of treatment.

Nucleotide-binding oligomerization domain 1 and Helicobacter pylori infection: A review.

Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular innate immune sensor for small molecules derived from bacterial cell components. NOD1 activation by its ligands leads to robust production of pro-inflammatory cytokines and chemokines by innate immune cells, thereby mediating mucosal host defense systems against microbes. Chronic gastric infection due to Helicobacter pylori (H. pylori) causes various upper gastrointestinal diseases, including atrophic gastritis, peptic ulcers, and gastric cancer. It is now generally accepted that detection of H. pylori by NOD1 expressed in gastric epithelial cells plays an indispensable role in mucosal host defense systems against this organism. Recent studies have revealed the molecular mechanism by which NOD1 activation caused by H. pylori infection is involved in the development of chronic gastritis and gastric cancer. In this review, we have discussed and summarized how sensing of H. pylori by NOD1 mediates the prevention of chronic gastritis and gastric cancer.

Salmonella Typhi Colonization Provokes Extensive Transcriptional Changes Aimed at Evading Host Mucosal Immune Defense During Early Infection of Human Intestinal Tissue

Commensal microorganisms influence a variety of host functions in the gut, including immune response, glucose homeostasis, metabolic pathways and oxidative stress, among others. This study describes how Salmonella Typhi, the pathogen responsible for typhoid fever, uses similar strategies to escape immune defense responses and survive within its human host. To elucidate the early mechanisms of typhoid fever, we performed studies using healthy human intestinal tissue samples and “mini-guts,” organoids grown from intestinal tissue taken from biopsy specimens. We analyzed gene expression changes in human intestinal specimens and bacterial cells both separately and after colonization. Our results showed mechanistic strategies that S. Typhi uses to rearrange the cellular machinery of the host cytoskeleton to successfully invade the intestinal epithelium, promote polarized cytokine release and evade immune system activation by downregulating genes involved in antigen sampling and presentation during infection. This work adds novel information regarding S. Typhi infection pathogenesis in humans, by replicating work shown in traditional cell models, and providing new data that can be applied to future vaccine development strategies.

Airway Mucosal Host Defense Is Key to Genomic Regulation of Cystic Fibrosis Lung Disease Severity.

The severity of cystic fibrosis (CF) lung disease varies widely, even for Phe508del homozygotes. Heritability studies show that more than 50% of the variability reflects non-cystic fibrosis transmembrane conductance regulator (CFTR) genetic variation; however, the full extent of the pertinent genetic variation is not known. We sought to identify novel CF disease-modifying mechanisms using an integrated approach based on analyzing "in vivo" CF airway epithelial gene expression complemented with genome-wide association study (GWAS) data. Nasal mucosal RNA from 134 patients with CF was used for RNA sequencing. We tested for associations of transcriptomic (gene expression) data with a quantitative phenotype of CF lung disease severity. Pathway analysis of CF GWAS data (n = 5,659 patients) was performed to identify novel pathways and assess the concordance of genomic and transcriptomic data. Association of gene expression with previously identified CF GWAS risk alleles was also tested. Significant evidence of heritable gene expression was identified. Gene expression pathways relevant to airway mucosal host defense were significantly associated with CF lung disease severity, including viral infection, inflammation/inflammatory signaling, lipid metabolism, apoptosis, ion transport, Phe508del CFTR processing, and innate immune responses, including HLA (human leukocyte antigen) genes. Ion transport and CFTR processing pathways, as well as HLA genes, were identified across differential gene expression and GWAS signals. Transcriptomic analyses of CF airway epithelia, coupled to genomic (GWAS) analyses, highlight the role of heritable host defense variation in determining the pathophysiology of CF lung disease. The identification of these pathways provides opportunities to pursue targeted interventions to improve CF lung health.

Innate Immunity to Mucosal Candida Infections.

Mucosal epithelial tissues are exposed to high numbers of microbes, including commensal fungi, and are able to distinguish between those that are avirulent and those that cause disease. Epithelial cells have evolved multiple mechanisms to defend against colonization and invasion by Candida species. The interplay between mucosal epithelial tissues and immune cells is key for control and clearance of fungal infections. Our understanding of the mucosal innate host defense system has expanded recently with new studies bringing to light the importance of epithelial cell responses, innate T cells, neutrophils, and other phagocytes during Candida infections. Epithelial tissues release cytokines, host defense peptides, and alarmins during Candida invasion that act in concert to limit fungal proliferation and recruit immune effector cells. The innate T cell/IL-17 axis and recruitment of neutrophils are of central importance in controlling mucosal fungal infections. Here, we review current knowledge of the innate immunity at sites of mucosal Candida infection, with a focus on infections caused by C. albicans.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

Entamoeba histolytica-Induced Mucin Exocytosis Is Mediated by VAMP8 and Is Critical in Mucosal Innate Host Defense.

ABSTRACTIntestinal mucus secretion is critical in maintaining mucosal host defense against a myriad of pathogens by preventing direct association with the epithelium. Entamoeba histolytica specifically binds colonic MUC2 mucin and also induces potent hypersecretion from goblet cells; however, characterization of the nature of the mechanisms controlling mucus release remains elusive. In this report, we identify vesicle SNARE vesicle-associated membrane protein 8 (VAMP8) present on mucin granules as orchestrating regulated exocytosis in human goblet cells in response to the presence of E. histolytica. VAMP8 was specifically activated during E. histolytica infection, and ablation of VAMP8 led to impaired mucin secretion. As a consequence, loss of VAMP8 increased E. histolytica adherence to epithelial cells associated with enhanced cell death through apoptosis characterized by caspase 3 and 9 cleavages and DNA fragmentation. With the mucosal barrier compromised in Vamp8−/− animals, E. histolytica induced an aggressive proinflammatory response with elevated levels of interleukin-1 alpha (IL-1α), IL-1β, and tumor necrosis factor alpha (TNF-α) secretion. This report is the first to characterize regulated mucin exocytosis in intestinal goblet cells in response to a pathogen and the downstream consequences of improper mucin secretion in mucosal barrier defense.

Intranasal delivery of Duox2 DNA using cationic polymer can prevent acute influenza A viral infection in vivo lung.

We studied the contribution of Duox2 in mucosal host defense against influenza A virus (IAV) infection in in vivo lung. We found that Duox2 was required for the induction of type I and III interferon (IFN)s and transient Duox2 overexpression using cationic polymer polyethyleneimine (PEI) leads to suppression of IAV infection in in vivo lung. Twenty mice (C57BL/6J) were anesthetized and challenged by intranasal administration of 213pfu/30μl of IAV (WS/33/H1N1), and IAV-infected mice were euthanized at 1, 3, 5, 7, 10, 14days post infection (dpi). Duox2 small hairpin RNA (shRNA) and pCMV-Duox2 formulated with PEI were inoculated to mice to assess the regulatory mechanism between Duox2 and IFN secretion. Following intranasal IAV inoculation, viral infection was significantly aggravated from 3dpi in in vivo lung and viral titer was highest at 7dpi. Consistent with this, Duox2 messenger RNA (mRNA) and protein expressions were significantly induced from 3dpi in the lung tissue of IAV-infected mice. Viral titer was much higher in IAV-infected mice that were inoculated with Duox2 shRNA accompanied with lower survival rate and extensive lung pathologies. Interestingly, severe lung pathologies in IAV-infected mice were not observed and viral titer was significantly reduced in mice with pulmonary administration of pCMV-Duox2 formulated with PEI before IAV inoculation. Both mRNA and secreted protein levels of IFN-β and IFN-λ2/3 were highly elevated in IAV-infected mice with pCMV-Duox2 formulated with PEI. Duox2 is necessary for the regulation of IFN secretion in in vivo lung, and pulmonary administration of Duox2 DNA using cationic polymer triggers the induction of type I and III IFNs resulting in more complete suppression of IAV infection.

Abstract 1343: DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization

Abstract Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is closely associated with epithelial-to-mesenchymal transition (EMT) and enhanced tumor invasiveness and metastasis. However, the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. In fact, recent studies have indicated that nuclear localization of EGFR in cancer cells is associated with metastatic cell behavior and poor clinical outcome, and the nuclear EGFR localization depends on Src-dependent phosphorylation of EGFR at Y1101. We observed that overexpression of DUOX1 in alveolar lung cancer A549 cells, which possess EMT-like features and in which DUOX1 is normally silenced, results in redistribution Src to the plasma membrane and decreased nuclear accumulation. DUOX1 overexpression in A549 cells also suppressed EGF-stimulated nuclear translocation of EGFR, which was associated with reduced EGFR phosphorylation at Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292, which normally expresses DUOX1 expression, was found to promote EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Since nuclear EGFR is thought to enhance the transcription of target genes related to cell cycle progression and proliferation (e.g. CDK1, Myc, others), we evaluated gene expression of these target genes in our cell models. Indeed, in cells lacking DUOX1, EGF stimulation significantly enhanced mRNA levels of CDK1, Myc, and other target genes for nuclear EGFR, whereas no such induction was seen in cells that express DUOX1. Our findings indicate that DUOX1 silencing in lung cancer may be associated with worse prognosis, partly due to altered spatiotemporal regulation of EGFR and Src and increased nuclear targeting. Since both EGFR and Src are subject to redox regulation by cysteine oxidation, we are currently aiming to elucidate the molecular mechanisms by which these mechanism are affected by altered DUOX1 status. Citation Format: Andrew C. Little, Karamathullah Danyal, David Heppner, Milena Hristova, Albert van der Vliet. DUOX1 silencing in lung cancer is associated with enhanced nuclear EGFR localization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1343. doi:10.1158/1538-7445.AM2017-1343

Nucleotide-binding oligomerization domain 1 and gastrointestinal disorders.

Nucleotide-binding oligomerization domain 1 (NOD1) is an intracellular sensor that detects small peptides derived from the cell wall component of intestinal microflora. NOD1 is expressed in both non-hematopoietic cells such as epithelial cells and hematopoietic cells such as antigen-presenting cells. Detection of its ligand by NOD1 leads to innate immune responses through activation of nuclear factor kappa B and type I interferon as well as induction of autophagy. Innate immune responses through NOD1 activation play an indispensable role both in host defense against microbial infection and in the development of gastrointestinal disorders. Of particular importance, NOD1-mediated innate immune responses are associated with mucosal host defenses against Helicobacter pylori (H. pylori) infection of the stomach and with the development of pancreatitis. In this review, we discuss the molecular mechanisms by which NOD1 activation leads to the development of H. pylori-related gastric diseases and pancreatitis.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients.

In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients.

Novel applications for a noninvasive sampling method of the nasal mucosa

Reliable methods for sampling the nasal mucosa provide clinical researchers with key information regarding respiratory biomarkers of exposure and disease. For quick and noninvasive sampling of the nasal mucosa, nasal lavage (NL) collection has been widely used as a clinical tool; however, limitations including volume variability, sample dilution, and storage prevent NL collection from being used in nonlaboratory settings and analysis of low abundance biomarkers. In this study, we optimize and validate a novel methodology using absorbent Leukosorb paper cut to fit the nasal passage to extract epithelial lining fluid (ELF) from the nasal mucosa. The ELF sampling method limits the dilution of soluble mediators, allowing quantification of both high- and low-abundance soluble biomarkers such as IL-1β, IL-8, IL-6, interferon gamma-induced protein 10 (IP-10), and neutrophil elastase. Additionally, we demonstrate that this method can successfully detect the presence of respiratory pathogens such as influenza virus and markers of antibiotic-resistant bacteria in the nasal mucosa. Efficacy of ELF collection by this method is not diminished in consecutive-day sampling, and percent recovery of both recombinant IL-8 and soluble mediators are not changed despite freezing or room temperature storage for 24 h. Our results indicate that ELF collection using Leukosorb paper sampling of ELF provides a sensitive, easy-to-use, and reproducible methodology to collect concentrated amounts of soluble biomarkers from the nasal mucosa. Moreover, the methodology described herein improves upon the standard NL collection method and provides researchers with a novel tool to assess changes in nasal mucosal host defense status.

Infection Dynamics of Foot-and-Mouth Disease Virus in Cattle Following Intranasopharyngeal Inoculation or Contact Exposure

For the purpose of developing an improved experimental model for studies of foot-and-mouth disease virus (FMDV) infection in cattle, three different experimental systems based on natural or simulated natural virus exposure were compared under standardized experimental conditions. Ante-mortem infection dynamics were characterized in cattle exposed to FMDV through a novel, simulated natural intranasopharyngeal (INP) inoculation system or through standardized and controlled systems of within- or between-species direct contact exposure (cattle-to-cattle or pig-to-cattle). All three systems were efficient in causing synchronous, generalized foot-and-mouth disease in cattle exposed to one of three different strains of FMDV representing serotypes O, A and Asia1. There was more within-group variation in the timing of clinical infection following natural and simulated natural virus exposure systems when compared with the conventionally used system of needle inoculation (intraepithelial lingual inoculation). However, the three optimized exposure systems described herein have the advantage of closely simulating field conditions by utilizing natural routes of primary infection, thereby facilitating engagement of mucosal host defence mechanisms. Overall, it is concluded that INP inoculation and standardized systems of direct contact exposure provide effective alternatives to conventional (needle) inoculation systems for studies in which it is desirable to simulate the natural biology of FMDV infection.

Abstract 1681: DUOX1 expression in lung cancer disrupts pro-oncogenic activation mechanisms and localization of Src and EGFR

Abstract Non-small cell lung cancer (NSCLC) remains to be one of the leading causes of cancer-related mortalities worldwide. The NADPH oxidase homolog, Dual Oxidase 1 (DUOX1), is an H2O2 producing enzyme located in the airway epithelium with key roles in mucosal host defense and wound repair mechanisms. Recent studies indicate that DUOX1 is epigenetically silenced in many forms of NSCLC via hypermethylation of its promoter. We previously demonstrated that DUOX1 silencing in lung cancer cells is associated with epithelial-to-mesenchymal transition (EMT), a key feature of tumor invasiveness and metastasis, and that RNAi-mediated DUOX1 suppression can promote EMT, but the mechanism(s) by which DUOX1 silencing promotes these outcomes are not understood. Previous findings indicate that DUOX1-dependent epithelial host defense pathways are mediated by redox-dependent activation of epithelial signaling via the non-receptor tyrosine kinase, Src, and the receptor tyrosine kinase, EGFR. We therefore hypothesized that loss of DUOX1 in lung cancer may be associated with aberrant regulation of Src and/or EGFR, tyrosine kinases that are frequently overexpressed and activated in lung cancer and strongly contribute to tumor growth and survival. Furthermore, nuclear Src/EGFR localization and phosphorylation of EGFR-Y1101 in lung cancers was recently associated with metastatic cell behavior and poor clinical outcome. Preliminary findings in alveolar lung cancer A549 cells, which possesses some EMT-like features, indicate that DUOX1 overexpression redistributes Src localization to the plasma membrane and decreases its nuclear accumulation. Moreover, DUOX1 overexpression in A549 cells also suppressed EGF-stimulated EGFR internalization and nuclear translocation, in association with reduced EGFR phosphorylation on its Src target, Y1101. Conversely, RNAi-mediated silencing of DUOX1 in the epithelial cancer cell line H292 (which has retained DUOX1 expression) promoted EGF-mediated EGFR nuclear translocation and Y1101 phosphorylation. Further mechanistic studies will be performed to elucidate the molecular mechanisms by which DUOX1 is able to alter these events. Collectively, our findings indicate that DUOX1 silencing in lung cancer may contribute to EMT and/or tumor invasiveness by altering Src/EGFR localization and activation mechanisms. Citation Format: Andrew C. Little, Karamatullah Danyal, Robert A. Bauer, David E. Heppner, Milena Hristova, Christopher Dustin, Aida Habibovic, Albert van der Vliet. DUOX1 expression in lung cancer disrupts pro-oncogenic activation mechanisms and localization of Src and EGFR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1681.

Beneficial Effects of Sodium Phenylbutyrate Administration during Infection with Salmonella enterica Serovar Typhimurium.

Sodium phenylbutyrate (PBA) is a derivative of the short-chain fatty acid butyrate and is approved for treatment of urea cycle disorders and progressive familial intrahepatic cholestasis type 2. Previously known functions include histone deacetylase inhibitor, endoplasmic reticulum stress inhibitor, ammonia sink, and chemical chaperone. Here, we show that PBA has a previously undiscovered protective role in host mucosal defense during infection. Administration of PBA to Taconic mice resulted in the increase of intestinal Lactobacillales and segmented filamentous bacteria (SFB), as well as an increase of interleukin 17 (IL-17) production by intestinal cells. This effect was not observed in Jackson Laboratory mice, which are not colonized with SFB. Because previous studies showed that IL-17 plays a protective role during infection with mucosal pathogens, we hypothesized that Taconic mice treated with PBA would be more resistant to infection with Salmonella enterica serovar Typhimurium (S Typhimurium). By using the streptomycin-treated mouse model, we found that Taconic mice treated with PBA exhibited significantly lower S Typhimurium intestinal colonization and dissemination to the reticuloendothelial system, as well as lower levels of inflammation. The lower levels of S Typhimurium gut colonization and intestinal inflammation were not observed in Jackson Laboratory mice. Although PBA had no direct effect on bacterial replication, its administration reduced S Typhimurium epithelial cell invasion and lowered the induction of the proinflammatory cytokine IL-23 in macrophage-like cells. These effects likely contributed to the better outcome of infection in PBA-treated mice. Overall, our results suggest that PBA induces changes in the microbiota and in the mucosal immune response that can be beneficial to the host during infection with S Typhimurium and possibly other enteric pathogens.