The Cxcr2+ subset of the S100a8+ gastric granylocytic myeloid-derived suppressor cell population (G-MDSC) regulates gastric pathology.

Introduction: Gastric myeloid-derived suppressor cells (MDSCs) are a prominent population that expands during gastric pre-neoplastic and neoplastic development in humans and mice. However, the heterogeneity of this population has circumvented the ability to study these cells or understand their functions. Aside from Schlafen-4+ (Slfn-4+) MDSCs in mouse studies, which constitute a subset of this population, limitations exist in characterizing the heterogeneity of the gastric CD11b+Ly6G+ population and targeting its different subsets. Here we identify S100a8 as a pan-specific marker for this population and utilize it to study the role of the S100a8+Cxcr2+ subset. Methods: We profiled gastric CD11b+Ly6G+ versus CD11b+Ly6G- myeloid cells by transcriptomic and single-cell RNA sequencing. We identified S100a8 as a pan-specific marker of the gastric granulocytic MDSC (G-MDSC) population, and generated S100a8CreCxcr2flox/flox to study the effects of Cxcr2 knockdown. Results: Following 6-months of Helicobacter felis infection, gastric CD11b+Ly6G+ G-MDSCs were highly enriched for the expression of S100a8, S100a9, Slfn4, Cxcr2, Irg1, Il1f9, Hcar2, Retnlg, Wfdc21, Trem1, Csf3R, Nlrp3, and Il1b. The expression of these distinct genes following 6mo H. felis infection marked heterogeneous subpopulations, but they all represented a subset of S100a8+ cells. S100a8 was identified as a pan-marker for CD11b+Ly6G+ cells arising in chronic inflammation, but not neutrophils recruited during acute gut infection. 6mo Helicobacter felis-infected S100a8CreCxcr2flox/flox mice exhibited worsened gastric metaplastic pathology than Cxcr2flox/flox mice, which was associated with dysregulated lipid metabolism and peroxidation. Conclusion: S100a8 is a pan-specific marker that can be used to target gastric G-MDSC subpopulations, of which the Cxcr2+ subset regulates gastric immunopathology and associates with the regulation of lipid peroxidation.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

Dual NADPH oxidases DUOX1 and DUOX2 synthesize NAADP and are necessary for Ca2+ signaling during T cell activation.

The formation of Ca2+ microdomains during T cell activation is initiated by the production of nicotinic acid adenine dinucleotide phosphate (NAADP) from its reduced form NAADPH. The reverse reaction­NAADP to NAADPH­is catalyzed by glucose 6-phosphate dehydrogenase (G6PD). Here, we identified NADPH oxidases NOX and DUOX as NAADP-forming enzymes that convert NAADPH to NAADP under physiological conditions in vitro. T cells express NOX1, NOX2, and, to a minor extent, DUOX1 and DUOX2. Local and global Ca2+ signaling were decreased in mouse T cells with double knockout of Duoxa1 and Duoxa2 but not with knockout of Nox1 or Nox2. Ca2+ microdomains in the first 15 s upon T cell activation were significantly decreased in Duox2−/− but not in Duox1−/− T cells, whereas both DUOX1 and DUOX2 were required for global Ca2+ signaling between 4 and 12 min after stimulation. Our findings suggest that a DUOX2- and G6PD-catalyzed redox cycle rapidly produces and degrades NAADP through NAADPH as an inactive intermediate.

DUOX2 variants associate with preclinical disturbances in microbiota-immune homeostasis and increased inflammatory bowel disease risk.

A primordial gut-epithelial innate defense response is the release of hydrogen peroxide by dual NADPH oxidase (DUOX). In inflammatory bowel disease (IBD), a condition characterized by an imbalanced gut microbiota-immune homeostasis, DUOX2 isoenzyme is the highest induced gene. Performing multiomic analyses using 2872 human participants of a wellness program, we detected a substantial burden of rare protein-altering DUOX2 gene variants of unknown physiologic significance. We identified a significant association between these rare loss-of-function variants and increased plasma levels of interleukin-17C, which is induced also in mucosal biopsies of patients with IBD. DUOX2-deficient mice replicated increased IL-17C induction in the intestine, with outlier high Il17c expression linked to the mucosal expansion of specific Proteobacteria pathobionts. Integrated microbiota/host gene expression analyses in patients with IBD corroborated IL-17C as a marker for epithelial activation by gram-negative bacteria. Finally, the impact of DUOX2 variants on IL-17C induction provided a rationale for variant stratification in case control studies that substantiated DUOX2 as an IBD risk gene. Thus, our study identifies an association of deleterious DUOX2 variants with a preclinical hallmark of disturbed microbiota-immune homeostasis that appears to precede the manifestation of IBD.

Dendritic cell-derived TGF-ß mediates the induction of mucosal regulatory T-cell response to Helicobacter infection essential for maintenance of immune tolerance in mice.

BACKGROUND: Helicobacter pylori infection leads to regulatory T-cell (Treg) induction in infected mice, which contributes to H. pylori immune escape. However, the mechanisms responsible for H. pylori induction of Treg and immune tolerance remain unclear. We hypothesized DC-produced TGF-ß may be responsible for Treg induction and immune tolerance. MATERIALS AND METHODS: To test this hypothesis, we generated TGF-ß∆DC mice (CD11c+ DC-specific TGF-ß deletion) and assessed the impact of DC-specific TGF-ß deletion on DC function during Helicobacter infection in vitro and in vivo. To examine the T cell-independent DC function, we crossed TGF-ß∆DC mice onto Rag1KO background to generate TGF-ß∆DC xRag1KO mice. RESULTS: When stimulated with H. pylori, TGF-ß∆DC BMDC/splenocyte cocultures showed increased levels of proinflammatory cytokines and decreased levels of anti-inflammatory cytokines compared to control, indicating a proinflammatory DC phenotype. Following 6 months of H. felis infection, TGF-ß∆DC mice developed more severe gastritis and a trend toward more metaplasia compared to TGF-ßfl/fl with increased levels of inflammatory Th1 cytokine mRNA and lower gastric H. felis colonization compared to infected TGF-ßfl/fl mice. In a T cell-deficient background using TGF-ß∆DC xRag1KO mice, H. felis colonization was significantly lower when DC-derived TGF-ß was absent, revealing a direct, innate function of DC in controlling H. felis infection independent of Treg induction. CONCLUSIONS: Our findings indicate that DC-derived TGF-ß mediates Helicobacter-induced Treg response and attenuates the inflammatory Th1 response. We also demonstrated a previously unrecognized innate role of DC controlling Helicobacter colonization via a Treg-independent mechanism. DC TGF-ß signaling may represent an important target in the management of H. pylori.

Aim2-mediated/IFN-ß-independent regulation of gastric metaplastic lesions via CD8+ T cells.

Development of gastric cancer is often preceded by chronic inflammation, but the immune cellular mechanisms underlying this process are unclear. Here we demonstrated that an inflammasome molecule, absent in melanoma 2 (Aim2), was upregulated in patients with gastric cancer and in spasmolytic polypeptide-expressing metaplasia of chronically Helicobacter felis-infected stomachs in mice. However, we found that Aim2 was not necessary for inflammasome function during gastritis. In contrast, Aim2 deficiency led to an increase in gastric CD8+ T cell frequency, which exacerbated metaplasia. These gastric CD8+ T cells from Aim2-/- mice were found to have lost their homing receptor expression (sphingosine-1-phosphate receptor 1 [S1PR1] and CD62L), a feature of tissue-resident memory T cells. The process was not mediated by Aim2-dependent regulation of IFN-ß or by dendritic cell-intrinsic Aim2. Rather, Aim2 deficiency contributed to an increased production of CXCL16 by B cells, which could suppress S1PR1 and CD62L in CD8+ T cells. This study describes a potentially novel function of Aim2 that regulates CD8+ T cell infiltration and retention within chronically inflamed solid organ tissue. This function operates independent of the inflammasome, IFN-ß, or dendritic cells. We provide evidence that B cells can contribute to this mechanism via CXCL16.

Dietary L-serine confers a competitive fitness advantage to Enterobacteriaceae in the inflamed gut.

Metabolic reprogramming is associated with the adaptation of host cells to the disease environment, such as inflammation and cancer. However, little is known about microbial metabolic reprogramming or the role it plays in regulating the fitness of commensal and pathogenic bacteria in the gut. Here, we report that intestinal inflammation reprograms the metabolic pathways of Enterobacteriaceae, such as Escherichia coli LF82, in the gut to adapt to the inflammatory environment. We found that E. coli LF82 shifts its metabolism to catabolize L-serine in the inflamed gut in order to maximize its growth potential. However, L-serine catabolism has a minimal effect on its fitness in the healthy gut. In fact, the absence of genes involved in L-serine utilization reduces the competitive fitness of E. coli LF82 and Citrobacter rodentium only during inflammation. The concentration of luminal L-serine is largely dependent on dietary intake. Accordingly, withholding amino acids from the diet markedly reduces their availability in the gut lumen. Hence, inflammation-induced blooms of E. coli LF82 are significantly blunted when amino acids-particularly L-serine-are removed from the diet. Thus, the ability to catabolize L-serine increases bacterial fitness and provides Enterobacteriaceae with a growth advantage against competitors in the inflamed gut.

Essential Role of NADPH Oxidase-Dependent Production of Reactive Oxygen Species in Maintenance of Sustained B Cell Receptor Signaling and B Cell Proliferation.

Reactive oxygen species (ROS) are not only toxic substances inducing oxidative stress but also play a role as a second messenger in signal transduction through various receptors. Previously, B cell activation was shown to involve prolonged ROS production induced by ligation of BCR. However, the mechanisms for ROS production and ROS-mediated activation in B cells are still poorly understood. In this study, we demonstrate that BCR ligation induces biphasic ROS production in both mouse spleen B cells and the mouse B cell line BAL17; transient and modest ROS production is followed by sustained and robust ROS production at 2-6 h after BCR ligation. ROS production in the late phase but not in the early phase augments activation of signaling pathways, such as the NF-κB and PI3K pathways, and is essential for B cell proliferation. ROS production in the late phase appears to be mediated by NADPH oxidases (NOXes) because prolonged ROS production is inhibited by various NOX inhibitors, including the specific inhibitor VAS2870. BCR ligation-induced ROS production is also inhibited by CRISPR/Cas9-mediated deletion of either the Cyba gene encoding p22phox, the regulator of NOX1-4 required for their activation, or NOX3, whereas ROS production is not affected by double deficiency of the DUOXA1 and DUOXA2 genes essential for the activation of the NOX isoforms DUOX1 and DUOX2. These results indicate that NOXes play a crucial role in sustained but not early BCR signaling and suggest an essential role of NOX-dependent sustained BCR signaling in B cell activation.

CD4+ Tissue-resident Memory T Cells Expand and Are a Major Source of Mucosal Tumour Necrosis Factor α in Active Crohn's Disease.

BACKGROUND AND AIMS: Tumour necrosis factor [TNF]α- and IL-17A-producing T cells are implicated in Crohn's disease [CD]. Tissue-resident memory T [TRM] cells are tissue-restricted T cells that are regulated by PR zinc finger domain 1 [PRDM1], which has been implicated in pathogenic Th17 cell responses. TRM cells provide host defence but their role in CD is unknown. We thus examined CD4+ TRM cells in CD. METHODS: Colon samples were prospectively collected at endoscopy or surgery in CD and control subjects. Flow cytometry and ex vivo assays were performed to characterise CD4+ TRM cells. RESULTS: CD4+ TRM cells are the most abundant memory T cell population and are the major T cell source of mucosal TNFα in CD. CD4+ TRM cells are expanded in CD and more avidly produce IL-17A and TNFα relative to control cells. There was a unique population of TNFα+IL-17A+ CD4+ TRM cells in CD which are largely absent in controls. PRDM1 was highly expressed by CD4+ TRM cells but not by other effector T cells. Suppression of PRDM1 was associated with impaired induction of IL17A and TNFA by CD4+ TRM cells. CONCLUSIONS: CD4+ TRM cells are expanded in CD and are a major source of TNFα, suggesting that they are important in CD. PRDM1 is expressed by TRM cells and may regulate their function. Collectively, this argues for prospective studies tracking CD4+ TRM cells over the disease course.

Effects of Anti-Helicobacter pylori Therapy on Incidence of Autoimmune Diseases, Including Inflammatory Bowel Diseases.

BACKGROUND & AIMS: Helicobacter pylori induces immune tolerance and is associated with a lower risk for immune-mediated disorders, such as autoimmune and inflammatory bowel diseases (IBD). We aimed to determine the effects of treatment for H pylori infection on the incidence of autoimmune disease and IBD. METHODS: We collected data from the National Health Insurance Research Database in Taiwan on patients younger than 18 years old without a prior diagnosis of autoimmune disease or IBD. Patients with peptic ulcer disease (PUD) with treatment of H pylori infection (PUD+HPRx), PUD without H pylori treatment (PUD-HPRx), a urinary tract infection (UTI) treated with cephalosporin, or without PUD (controls) were matched for age, sex, insurance, and Charlson's comorbidity index score. RESULTS: Of the 1 million patients we collected data from in 2005, we included 79,181 patients in the study. We compared the effects of treatment for H pylori infection on the risk of autoimmunity or IBD and found that PUD+HPRx has the highest adjusted hazard risk (aHR) for autoimmunity or IBD (aHR, 2.36), compared to PUD-HPRx (aHR, 1.91) or UTI (aHRs, 1.71) (P < .001). The increased risk of autoimmune disease was not completely accounted for by antibiotic therapy alone, because PUD+HPRx had a higher aHR than UTI (P < .001). A small but significant increase in mortality was observed in the PUD+HPRx cohort (aHR, 1.11; P = .001). CONCLUSION: In an analysis of data from the National Health Insurance Research Database in Taiwan, we found that treatment for H pylori infection is associated with a significant increase in the risk for autoimmune disease, including IBD.

Increased risk for inflammatory bowel disease in congenital hypothyroidism supports the existence of a shared susceptibility factor.

Loss-of-function mutations in dual oxidase (DUOX) 2 are the most common genetic variants found in congenital hypothyroidism (CH), and similar mutations have been recently reported in few very-early-onset inflammatory bowel disease (IBD) patients without CH. If DUOX2 variants indeed increase susceptibility for IBD, the enrichment of DUOX2 mutation carriers among CH patients should be reflected in higher risk for developing IBD. Using a database containing health insurance claims data for over 230 million patients in the United States, 42,922 subjects with CH were identified based on strict inclusion criteria using diagnostic codes. For subgroup analysis, CH patients with pharmacy records were stratified as transient or permanent CH based on the absence or presence of levothyroxine treatment, respectively. Patients were matched to an equal-sized, age- and gender-matched non-CH group. Compared to controls, CH patients had a 73% higher overall IBD prevalence (0.52% vs 0.30%; P < 0.0001). The CH-associated relative risk was higher for indeterminate or ulcerative colitis than Crohn's disease. Patients with transient CH had higher odds for IBD (OR 2.39 (95% CI 1.77-3.23) than those with permanent CH (1.69 (95% CI 1.31-2.18). We conclude that patients with CH are at an increased risk of developing IBD. The risk was highest for patients with transient CH, for which partial defects in the DUOX2 system are a particularly common finding.

Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases.

Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels.

Chemotaxis Allows Bacteria To Overcome Host-Generated Reactive Oxygen Species That Constrain Gland Colonization.

The epithelial layer of the gastrointestinal tract contains invaginations, called glands or crypts, which are colonized by symbiotic and pathogenic microorganisms and may function as designated niches for certain species. Factors that control gland colonization are poorly understood, but bacterial chemotaxis aids occupation of these sites. We report here that a Helicobacter pylori cytoplasmic chemoreceptor, TlpD, is required for gland colonization in the stomach. tlpD mutants demonstrate gland colonization defects characterized by a reduction in the percentage of glands colonized but not in the number of bacteria per gland. Consistent with TlpD's reported role in reactive oxygen species (ROS) avoidance, tlpD mutants showed hallmarks of exposure to high ROS. To assess the role of host-generated ROS in TlpD-dependent gland colonization, we utilized mice that lack either the ability to generate epithelial hydrogen peroxide or immune cell superoxide. tlpD gland colonization defects were rescued to wild-type H. pylori levels in both of these mutants. These results suggest that multiple types of innate immune-generated ROS production limit gland colonization and that bacteria have evolved specific mechanisms to sense and direct their motility in response to this signal and thus spread throughout tissue.

Helicobacter pylori Antimicrobial Susceptibility Testing-Guided Salvage Therapy in the USA: A Real Life Experience.

BACKGROUND: The current practice guidelines recommend that Helicobacter pylori (H. pylori) culture and antimicrobial susceptibility testing (AST) be considered after patients failed the second course of H. pylori eradication therapy. AIMS: Here we report the real life experience of following this recommendation in the USA. METHODS: We established an in-house H. pylori culture protocol for AST and identified retrospectively patients who previously failed ≥ 2 courses of anti-H. pylori therapy and underwent esophagogastroduodenoscopy with AST at University of Michigan from 2010 to 2017. We determined the rate of H. pylori antibiotic resistance, the success rates of AST-guided tailored therapy, and the risk factors associated with treatment failure. RESULTS: Forty-seven patients were identified and 34 (72.3%) had successful cultures and AST. The most common antibiotic resistance was to metronidazole (79.4%), followed by clarithromycin (70.6%) and ciprofloxacin (42.9%). None of the patients were resistant to amoxicillin or tetracycline. The overall success rate of AST-guided tailored therapy was low (44.4%, 12/27). In patients infected with metronidazole-resistant H. pylori, bismuth quadruple therapy appears to be superior compared to non-bismuth quadruple therapy (6/8 or 75.0% vs. 3/14 or 21.4%, P = 0.03). High body mass index was significantly associated with tailored therapy failure (OR 1.24, 95% CI 1.00-1.54, P = 0.049). CONCLUSIONS: The success rate of AST-guided salvage therapy in the USA is low particularly in those with high BMI. Bismuth-based therapy appears to be better than non-bismuth-based regimens.

Indoleamine 2,3-Dioxygenase 1, Increased in Human Gastric Pre-Neoplasia, Promotes Inflammation and Metaplasia in Mice and Is Associated With Type II Hypersensitivity/Autoimmunity.

BACKGROUND & AIMS: Chronic gastrointestinal inflammation increases the risk of cancer by mechanisms that are not well understood. Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-binding enzyme that regulates the immune response via catabolization and regulation of tryptophan availability for immune cell uptake. IDO1 expression is increased during the transition from chronic inflammation to gastric metaplasia. We investigated whether IDO1 contributes to the inflammatory response that mediates loss of parietal cells leading to metaplasia. METHODS: Chronic gastric inflammation was induced in Ido1-/- and CB57BL/6 (control) mice by gavage with Helicobacter felis or overexpression of interferon gamma in gastric parietal cells. We also performed studies in Jh-/- mice, which are devoid of B cells. Gastric tissues were collected and analyzed by flow cytometry, immunostaining, and real-time quantitative polymerase chain reaction. Plasma samples were analyzed by enzyme-linked immunosorbent assay. Gastric tissues were obtained from 20 patients with gastric metaplasia and 20 patients without gastric metaplasia (controls) and analyzed by real-time quantitative polymerase chain reaction; gastric tissue arrays were analyzed by immunohistochemistry. We collected genetic information on gastric cancers from The Cancer Genome Atlas database. RESULTS: H felis gavage induced significantly lower levels of pseudopyloric metaplasia in Ido1-/- mice, which had lower frequencies of gastric B cells, than in control mice. Blood plasma from H felis-infected control mice had increased levels of autoantibodies against parietal cells, compared to uninfected control mice, but this increase was lower in Ido1-/- mice. Chronically inflamed stomachs of Ido1-/- mice had significantly lower frequencies of natural killer cells in contact with parietal cells, compared with stomachs of control mice. Jh-/- mice had lower levels of pseudopyloric metaplasia than control mice in response to H felis infection. Human gastric pre-neoplasia and carcinoma specimens had increased levels of IDO1 messenger RNA compared with control gastric tissues, and IDO1 protein colocalized with B cells. Co-clustering of IDO1 messenger RNA with B-cell markers was corroborated by The Cancer Genome Atlas database. CONCLUSIONS: IDO1 mediates gastric metaplasia by regulating the B-cell compartment. This process appears to be associated with type II hypersensitivity/autoimmunity. The role of autoimmunity in the progression of pseudopyloric metaplasia warrants further investigation.

Novel Mutations in the NKX2.1 gene and the PAX8 gene in a Boy with Brain-Lung-Thyroid Syndrome.

OBJECTIVE: To elucidate the molecular mechanism which causes thyroid dysgenesis (TD) in a boy with brain-lung-thyroid syndrome. DESIGN, PATIENTS, MEASUREMENTS: We describe a patient with TD, respiratory disease and cerebral palsy who is heterozygous for mutations in two different genes, the PAX8 (p.E234K) and the NKX2.1 (p.A329GfsX108). In vitro studies were performed to functionally characterize these mutations. Congenital hypothyroidism (CH) was identified by neonatal screening associated with a hypoplastic thyroid gland. Postpartum he developed a brain-lung-thyroid syndrome with severe respiratory failure, symptomatic epilepsy and a considerable psychomotor retardation. The DNA-binding capability and the transcriptional activity of the two mutated transcription factors were investigated in vitro. RESULTS: The NKX2.1 mutation did not show any transcriptional activity and had almost no DNA-binding. The PAX8 mutation was normally located to the nucleus and showed a normal transactivation and a normal binding to the known downstream targets. CONCLUSIONS: The molecular defect explaining the phenotype of brain-lung-thyroid syndrome was identified. To what extent the PAX8 mutation contributes to the phenotype needs to be further investigated. We recommend to screen patients with CH and TD for mutations in all known TD candidate genes.

The effect of CT26 tumor-derived TGF-ß on the balance of tumor growth and immunity.

INTRODUCTION: TGF-ß is an important target for many cancer therapies under development. In addition to suppressing anti-tumor immunity, it has pleiotropic direct pro- and anti- tumor effects. The actions of increased endogenous TGF-ß production remain unclear, and may affect the outcomes of anti-TGF-ß cancer therapy. We hypothesize that tumor-derived TGF-ß (td-TGF-ß) plays an important role in maintaining tumor remission by controlling tumor proliferation in vivo, and that decreasing td-TGF-ß in the tumor microenvironment will result in tumor progression. The aim of this study was to examine the effect of TGF-ß in the tumor microenvironment on the balance between its anti-proliferative and immunosuppressive effects. METHODS: A murine BALB/c spontaneous colon adenocarcinoma cell line (CT26) was genetically engineered to produce increased active TGF-ß (CT26-TGF-ß), a dominant-negative soluble TGF-ß receptor (CT26-TGF-ß-R), or the empty neomycin cassette as control (CT26-neo). In vitro proliferation rates were measured. For in vivo studies, the three cell lines were injected into syngeneic BALB/c mice, and tumor growth was measured over time. Immunodeficient BALB/c nude mice were used to investigate the role of T and B cells. RESULTS: In vitro, CT26-TGF-ß-R and CT26-TGF-ß cells showed increased and suppressed proliferation, respectively, compared to control (CT26-neo), confirming TGF-ß has direct anti-tumor effects. In vivo, we found that CT26-TGF-ß-R cells displayed slower growth compared to control, likely secondary to reduced suppression of anti-tumor immunity, as this effect was ablated in immunodeficient BALB/c nude mice. However, CT26-TGF-ß cells (excess TGF-ß) exhibited rapid early growth compared to control, but later failed to progress. The same pattern was shown in immunodeficient BALB/c nude mice, suggesting the effect on tumor growth is direct, with minimal immune system involvement. There was minimal effect on systemic antitumor immunity as determined by peripheral antigen-specific splenocyte type 1 cytokine production and tumor growth rate of CT26-neo on the contralateral flank of the same mice. CONCLUSION: Although TGF-ß has opposing effects on tumor growth, this study showed that excessive td-TGF-ß in the tumor microenvironment renders the tumor non-proliferative. Depleting excess td-TGF-ß may release this endogenous tumor suppressive mechanism, thus triggering the progression of the tumor. Therefore, our findings support cautions against using anti-TGF-ß strategies in treating cancer, as this may tip the balance of anti-immunity vs. anti-tumor effects of TGF-ß, leading to tumor progression instead of remission.

Resistance to thyrotropin.

Resistance to thyrotropin (RTSH) is broadly defined as reduced sensitivity of thyroid follicle cells to stimulation by biologically active TSH due to genetic defects. Affected individuals have elevated serum TSH in the absence of goiter, with the severity ranging from nongoitrous isolated hyperthyrotropinemia to severe congenital hypothyroidism with thyroid hypoplasia. Conceptually, defects leading to RTSH impair both aspects of TSH-mediated action, namely thyroid hormone synthesis and gland growth. These include inactivating mutations in the genes encoding the TSH receptor and the PAX8 transcription factor. A common third cause has been genetically mapped to a locus on chromosome 15, but the underlying pathophysiology has not yet been elucidated. This review provides a succinct overview of currently defined causes of nonsyndromic RTSH, their differential diagnoses (autoimmune; partial iodine organification defects; syndromic forms of RTSH) and implications for the clinical approach to patients with RTSH.

Deficiency in Duox2 activity alleviates ileitis in GPx1- and GPx2-knockout mice without affecting apoptosis incidence in the crypt epithelium.

Mice deficient in glutathione peroxidase (GPx)-1 and -2 (GPx1-/-GPx2-/- double knockout or DKO mice) develop very-early-onset (VEO) ileocolitis, suggesting that lack of defense against reactive oxygen species (ROS) renders susceptibility to intestinal inflammation. Two members of ROS-generating NADPH oxidase family, NOX1 and DUOX2, are highly inducible in the intestinal epithelium. Previously, we reported that Nox1 deficiency ameliorated the pathology in DKO mice (Nox1-TKO). The role of Duox2 in ileocolitis of the DKO mice is evaluated here in Duoxa-TKO mice by breeding DKO mice with Duoxa-/- mice (Duoxa-TKO), which do not have Duox2 activity. Similar to Nox1-TKO mice, Duoxa-TKO mice no longer have growth retardation, shortened intestine, exfoliation of crypt epithelium, crypt abscesses and depletion of goblet cells manifested in DKO mice by 35 days of age. Unlike Nox1-TKO mice, Duoxa-TKO mice still have rampant crypt apoptosis, elevated proliferation, partial loss of Paneth cells and diminished crypt density. Treating DKO mice with NOX inhibitors (di-2-thienyliodonium/DTI and thioridazine/THZ) and an antioxidant (mitoquinone/MitoQ) significantly reduced gut pathology. Furthermore, in the inflamed human colon, DUOX protein expression is highly elevated in the apical, lateral and perinuclear membrane along the whole length of gland. Taken together, we conclude that exfoliation of crypt epithelium, but not crypt apoptosis, is a major contributor to inflammation. Both Nox1 and Duox2 induce exfoliation of crypt epithelium, but only Nox1 induces apoptosis. NOX1 and DUOX2 may be potential therapeutic targets for treating ileocolitis in human patients suffering inflammatory bowel disease (IBD).

Functional Characterization of Inflammatory Bowel Disease-Associated Gut Dysbiosis in Gnotobiotic Mice.

BACKGROUND & AIMS: Gut dysbiosis is closely involved in the pathogenesis of inflammatory bowel disease (IBD). However, it remains unclear whether IBD-associated gut dysbiosis contributes to disease pathogenesis or is merely secondary to intestinal inflammation. We established a humanized gnotobiotic (hGB) mouse system to assess the functional role of gut dysbiosis associated with 2 types of IBD: Crohn's disease (CD) and ulcerative colitis (UC). METHODS: Germ-free mice were colonized by the gut microbiota isolated from patients with CD and UC, and healthy controls. Microbiome analysis, bacterial functional gene analysis, luminal metabolome analysis, and host gene expression analysis were performed in hGB mice. Moreover, the colitogenic capacity of IBD-associated microbiota was evaluated by colonizing germ-free colitis-prone interleukin 10-deficient mice with dysbiotic patients' microbiota. RESULTS: Although the microbial composition seen in donor patients' microbiota was not completely reproduced in hGB mice, some dysbiotic features of the CD and UC microbiota (eg, decreased diversity, alteration of bacterial metabolic functions) were recapitulated in hGB mice, suggesting that microbial community alterations, characteristic for IBD, can be reproduced in hGB mice. In addition, colonization by the IBD-associated microbiota induced a proinflammatory gene expression profile in the gut that resembles the immunologic signatures found in CD patients. Furthermore, CD microbiota triggered more severe colitis than healthy control microbiota when colonized in germ-free interleukin 10-deficient mice. CONCLUSIONS: Dysbiosis potentially contributes to the pathogenesis of IBD by augmenting host proinflammatory immune responses. Transcript profiling: GSE73882.