TiO2 nanoparticles abrogate the protective effect of the Crohn's disease-associated variation within the PTPN22 gene locus.

OBJECTIVE: Inflammatory bowel disease (IBD) is a multifactorial condition driven by genetic and environmental risk factors. A genetic variation in the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene has been associated with autoimmune disorders while protecting from the IBD subtype Crohn's disease. Mice expressing the murine orthologous PTPN22-R619W variant are protected from intestinal inflammation in the model of acute dextran sodium sulfate (DSS)-induced colitis. We previously identified food-grade titanium dioxide (TiO2, E171) as a neglected IBD risk factor. Here, we investigate the interplay of the PTPN22 variant and TiO2-mediated effects during IBD pathogenesis. DESIGN: Acute DSS colitis was induced in wild-type and PTPN22 variant mice (PTPN22-R619W) and animals were treated with TiO2 nanoparticles during colitis induction. Disease-triggering mechanisms were investigated using bulk and single-cell RNA sequencing. RESULTS: In mice, administration of TiO2 nanoparticles abrogated the protective effect of the variant, rendering PTPN22-R619W mice susceptible to DSS colitis. In early disease, cytotoxic CD8+ T-cells were found to be reduced in the lamina propria of PTPN22-R619W mice, an effect reversed by TiO2 administration. Normalisation of T-cell populations correlated with increased Ifng expression and, at a later stage of disease, the promoted prevalence of proinflammatory macrophages that triggered severe intestinal inflammation. CONCLUSION: Our findings indicate that the consumption of TiO2 nanoparticles might have adverse effects on the gastrointestinal health of individuals carrying the PTPN22 variant. This demonstrates that environmental factors interact with genetic risk variants and can reverse a protective mechanism into a disease-promoting effect.

pH-Sensing G Protein-Coupled Receptor OGR1 (GPR68) Expression and Activation Increases in Intestinal Inflammation and Fibrosis.

Local extracellular acidification occurs at sites of inflammation. Proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1, also known as GPR68) responds to decreases in extracellular pH. Our previous studies show a role for OGR1 in the pathogenesis of mucosal inflammation, suggesting a link between tissue pH and immune responses. Additionally, pH-dependent signalling is associated with the progression of intestinal fibrosis. In this study, we aimed to investigate OGR1 expression and OGR1-mediated signalling in patients with inflammatory bowel disease (IBD). Our results show that OGR1 expression significantly increased in patients with IBD compared to non-IBD patients, as demonstrated by qPCR and immunohistochemistry (IHC). Paired samples from non-inflamed and inflamed intestinal areas of IBD patients showed stronger OGR1 IHC staining in inflamed mucosal segments compared to non-inflamed mucosa. IHC of human surgical samples revealed OGR1 expression in macrophages, granulocytes, endothelial cells, and fibroblasts. OGR1-dependent inositol phosphate (IP) production was significantly increased in CD14+ monocytes from IBD patients compared to healthy subjects. Primary human and murine fibroblasts exhibited OGR1-dependent IP formation, RhoA activation, F-actin, and stress fibre formation upon an acidic pH shift. OGR1 expression and signalling increases with IBD disease activity, suggesting an active role of OGR1 in the pathogenesis of IBD.

Protein Tyrosine Phosphatase Nonreceptor Type 2 Expression Does Not Correlate with Viral Load or Response to Direct-Acting Antiviral Therapy in Hepatitis C Virus Infections-Infected Patients.

BACKGROUND/AIMS: The hepatitis C virus nonstructural 3/4A protease has been shown to cleave protein tyrosine phosphatase nonreceptor type 2 (PTPN2, also known as T cell protein tyrosine phosphatase), thereby inducing a shift from a Th1 toward a nonantiviral Th2 immunity. Ribavirin therapy reverses these effects and supports direct-acting antiviral (DAA) therapy as an immunomodulatory compound and ultimately improves the response to DAA therapy. Here we aimed to assess whether intrahepatic levels of PTPN2 might be used as a clinical prognostic marker for the response to DAA therapy. METHODS: Liver biopsies from hepatitis C virus-infected patients with and without cirrhosis were immunohistochemically stained for PTPN2 and scored for staining intensity as well as percentage of hepatocytes positive for nuclear PTPN2 localization. PTPN2 scores were correlated to sustained virologic response after DAA therapy, viral load, serum levels of alanine aminotransferase, aspartate aminotransferase, gamma-glutamyl transferase (GGT), and the Model for End-Stage Liver Disease (MELD) score at the time of liver biopsy. RESULTS: We did not detect a difference in intrahepatic PTPN2 levels between responders with cirrhosis, responders without cirrhosis, and nonresponders to DAA therapy. There was no correlation between intrahepatic PTPN2 levels and viral load or clinical markers such as liver transaminases, GGT, or the MELD score. CONCLUSION: Intrahepatic PTPN2 levels assessed via IHC staining do not represent a clinical prognostic marker for the response to DAA therapy.

Titanium Dioxide Presents a Different Profile in Dextran Sodium Sulphate-Induced Experimental Colitis in Mice Lacking the IBD Risk Gene Ptpn2 in Myeloid Cells.

Environmental and genetic factors have been demonstrated to contribute to the development of inflammatory bowel disease (IBD). Recent studies suggested that the food additive; titanium dioxide (TiO2) might play a causative role in the disease. Therefore, in the present study we aimed to explore the interaction between the food additive TiO2 and the well-characterized IBD risk gene protein tyrosine phosphatase non-receptor type 2 (Ptpn2) and their role in the development of intestinal inflammation. Dextran sodium sulphate (DSS)-induced acute colitis was performed in mice lacking the expression of Ptpn2 in myeloid cells (Ptpn2LysMCre) or their wild type littermates (Ptpn2fl/fl) and exposed to the microparticle TiO2. The impact of Ptpn2 on TiO2 signalling pathways and TiO2-induced IL-1ß and IL-10 levels were studied using bone marrow-derived macrophages (BMDMs). Ptpn2LysMCre exposed to TiO2 exhibited more severe intestinal inflammation than their wild type counterparts. This effect was likely due to the impact of TiO2 on the differentiation of intestinal macrophages, suppressing the number of anti-inflammatory macrophages in Ptpn2 deficient mice. Moreover, we also found that TiO2 was able to induce the secretion of IL-1ß via mitogen-activated proteins kinases (MAPKs) and to repress the expression of IL-10 in bone marrow-derived macrophages via MAPK-independent pathways. This is the first evidence of the cooperation between the genetic risk factor Ptpn2 and the environmental factor TiO2 in the regulation of intestinal inflammation. The results presented here suggest that the ingestion of certain industrial compounds should be taken into account, especially in individuals with increased genetic risk.

Macrophages Compensate for Loss of Protein Tyrosine Phosphatase N2 in Dendritic Cells to Protect from Elevated Colitis.

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) plays a critical role in the pathogenesis of inflammatory bowel diseases (IBD). Mice lacking PTPN2 in dendritic cells (DCs) develop skin and liver inflammation by the age of 22 weeks due to a generalized loss of tolerance leading to uncontrolled immune responses. The effect of DC-specific PTPN2 loss on intestinal health, however, is unknown. The aim of this study was to investigate the DC-specific role of PTPN2 in the intestine during colitis development. PTPN2fl/flxCD11cCre mice were subjected to acute and chronic DSS colitis as well as T cell transfer colitis. Lamina propria immune cell populations were analyzed using flow cytometry. DC-specific PTPN2 deletion promoted infiltration of B and T lymphocytes, macrophages, and DCs into the lamina propria of unchallenged mice and elevated Th1 abundance during acute DSS colitis, suggesting an important role for PTPN2 in DCs in maintaining intestinal immune cell homeostasis. Surprisingly, those immune cell alterations did not translate into increased colitis susceptibility in acute and chronic DSS-induced colitis or T cell transfer colitis models. However, macrophage depletion by clodronate caused enhanced colitis severity in mice with a DC-specific loss of PTPN2. Loss of PTPN2 in DCs affects the composition of lamina propria lymphocytes, resulting in increased infiltration of innate and adaptive immune cells. However, this did not result in an elevated colitis phenotype, likely because increased infiltration of macrophages in the intestine upon loss of PTPN2 loss in DCs can compensate for the inflammatory effect of PTPN2-deficient DCs.

ß6 -integrin serves as a novel serum tumor marker for colorectal carcinoma.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify ß6 -integrin (ITGB6) as a novel serum tumor marker for diagnosis, prognosis, and surveillance of CRC. ITGB6 serum levels were validated in retro- and prospective CRC patient cohorts. ITGB6 serum levels were analyzed by ELISA. Using an initial cohort of 60 CRC patients, we found that ITGB6 is present in the serum of CRC, but not in non-CRC control patients. A cut-off of ≥2 ng/mL ITGB6 reveals 100% specificity for the presence of metastatic CRC. In an enlarged study cohort of 269 CRC patients, ITGB6 predicted the onset of metastatic disease and was associated with poor prognosis. Those data were confirmed in an independent, prospective cohort consisting of 40 CRC patients. To investigate whether ITGB6 can also be used for tumor surveillance, serum ITGB6-levels were assessed in 26 CRC patients, pre- and post-surgery, as well as during follow-up visits. After complete tumor resection, ITGB6 serum levels declined completely. During follow-up, a new rise in ITGB6 serum levels indicated tumor recurrence or the onset of new metastasis as confirmed by CT scan. ITGB6 was more accurate for prognosis of advanced CRC and for tumor surveillance as the established marker carcinoembryonic antigen (CEA). Our findings identify ITGB6 as a novel serum marker for diagnosis, prognosis, and surveillance of advanced CRC. This might essentially contribute to an optimized patient care.

The impact of the rs8005161 polymorphism on G protein-coupled receptor GPR65 (TDAG8) pH-associated activation in intestinal inflammation.

BACKGROUND: Tissue inflammation in inflammatory bowel diseases (IBD) is associated with a decrease in local pH. The gene encoding G-protein-coupled receptor 65 (GPR65) has recently been reported to be a genetic risk factor for IBD. In response to extracellular acidification, proton activation of GPR65 stimulates cAMP and Rho signalling pathways. We aimed to analyse the clinical and functional relevance of the GPR65 associated single nucleotide polymorphism (SNP) rs8005161. METHODS: 1138 individuals from a mixed cohort of IBD patients and healthy volunteers were genotyped for SNPs associated with GPR65 (rs8005161, rs3742704) and galactosylceramidase (rs1805078) by Taqman SNP assays. 2300 patients from the Swiss IBD Cohort Study (SIBDC) were genotyped for rs8005161 by mass spectrometry based SNP genotyping. IBD patients from the SIBDC carrying rs8005161 TT, CT, CC and non-IBD controls (CC) were recruited for functional studies. Human CD14+ cells were isolated from blood samples and subjected to an extracellular acidic pH shift, cAMP accumulation and RhoA activation were measured. RESULTS: In our mixed cohort, but not in SIBDC patients, the minor variant rs8005161 was significantly associated with UC. In SIBDC patients, we observed a consistent trend in increased disease severity in patients carrying the rs8005161-TT and rs8005161-CT alleles. No significant differences were observed in the pH associated activation of cAMP production between IBD (TT, CT, WT/CC) and non-IBD (WT/CC) genotype carriers upon an acidic extracellular pH shift. However, we observed significantly impaired RhoA activation after an extracellular acidic pH shift in IBD patients, irrespective of the rs8005161 allele. CONCLUSIONS: The T allele of rs8005161 might confer a more severe disease course in IBD patients. Human monocytes from IBD patients showed impaired pH associated RhoA activation upon an acidic pH shift.

Titanium dioxide nanoparticles exacerbate DSS-induced colitis: role of the NLRP3 inflammasome.

OBJECTIVE: Western lifestyle and diet are major environmental factors playing a role in the development of IBD. Titanium dioxide (TiO2) nanoparticles are widely used as food additives or in pharmaceutical formulations and are consumed by millions of people on a daily basis. We investigated the effects of TiO2 in the development of colitis and the role of the nucleotide-binding oligomerisation domain receptor, pyrin domain containing (NLRP)3 inflammasome. DESIGN: Wild-type and NLRP3-deficient mice with dextran sodium sulfate-induced colitis were orally administered with TiO2 nanoparticles. The proinflammatory effects of TiO2 particles in cultured human intestinal epithelial cells (IECs) and macrophages were also studied, as well as the ability of TiO2 crystals to traverse IEC monolayers and accumulate in the blood of patients with IBD using inductively coupled plasma mass spectrometry. RESULTS: Oral administration of TiO2 nanoparticles worsened acute colitis through a mechanism involving the NLRP3 inflammasome. Importantly, crystals were found to accumulate in spleen of TiO2-administered mice. In vitro, TiO2 particles were taken up by IECs and macrophages and triggered NLRP3-ASC-caspase-1 assembly, caspase-1 cleavage and the release of NLRP3-associated interleukin (IL)-1ß and IL-18. TiO2 also induced reactive oxygen species generation and increased epithelial permeability in IEC monolayers. Increased levels of titanium were found in blood of patients with UC having active disease. CONCLUSION: These findings indicate that individuals with a defective intestinal barrier function and pre-existing inflammatory condition, such as IBD, might be negatively impacted by the use of TiO2 nanoparticles.

The pH-sensing receptor OGR1 improves barrier function of epithelial cells and inhibits migration in an acidic environment.

The pH-sensing receptor ovarian cancer G protein-coupled receptor 1 (OGR1; GPR68) is expressed in the gut. Inflammatory bowel disease is typically associated with a decrease in local pH, which may lead to altered epithelial barrier function and subsequent gastrointestinal repair involving epithelial cell adhesion and migration. As the mechanisms underlying the response to pH changes are not well understood, we have investigated OGR1-mediated, pH-dependent signaling pathways in intestinal epithelial cells. Caco-2 cells stably overexpressing OGR1 were created and validated as tools to study OGR1 signaling. Barrier function, migration, and proliferation were measured using electric cell-substrate impedance-sensing technology. Localization of the tight junction proteins zonula occludens protein 1 and occludin and the rearrangement of cytoskeletal actin were examined by confocal microscopy. Paracellular permeability and protein and gene expression analysis using DNA microarrays were performed on filter-grown Caco-2 monolayers. We report that an acidic pH shift from pH 7.8 to 6.6 improved barrier function and stimulated reorganization of filamentous actin with prominent basal stress fiber formation. Cell migration and proliferation during in vitro wound healing were inhibited. Gene expression analysis revealed significant upregulation of genes related to cytoskeleton remodeling, cell adhesion, and growth factor signaling. We conclude that acidic extracellular pH can have a signaling function and impact the physiology of intestinal epithelial cells. The deconstruction of OGR1-dependent signaling may aid our understanding of mucosal inflammation mechanisms.

Serum REG3alpha and C-Reactive Protein Levels in Crohn's Disease Patients Undergoing Immunoablation and Autologous Hemopoetic Stem Cell Transplantation in the ASTIC Trial.

BACKGROUND: REG3α has been recently shown to be a highly accurate biomarker for graft-versus-host-disease (GvHD). Given the unmet need of such biomarkers in Crohn's disease (CD) and the similarities between CD and GvHD, we aimed at investigating the role of serum REG3α as a diagnostic and prognostic biomarker in CD patients undergoing autologous hemopoetic stem cell transplantation (HSCT) in the multicenter Autologous Stem Cell Transplantation International Crohn's Disease (ASTIC) trial and to compare it to C-reactive protein (CRP). METHODS: Stored serum samples from the ASTIC trial were analyzed using a commercially available human PAP1 ELISA-kit to measure REG3α levels. CRP was available from prior analysis in the ASTIC trial. These levels were correlated with clinical and endoscopic disease activity as well as overall clinical and endoscopic outcome 1 year after autologous HSCT. RESULTS: One hundred thirty two serum samples were available for analysis. The mean concentration of REG3α was 101.8 ng/ml (95% CI 22.6-258.3). No significant elevation of REG3α was found among patients with active disease compared to those in remission (106.3 vs. 91.4). Patients with moderate to severe endoscopic disease activity showed substantially, although not significantly elevated REG3α levels compared to those in remission (95.4 vs. 52.4, p = 0.052). Baseline serum REG3α levels of patients without clinical or endoscopic remission 1 year after HSCT were not elevated compared to those in remission (63.1 vs. 66.9, and 68.4 vs. 59.2, respectively). In contrast, CRP was significantly elevated in patients with active disease compared to patients in remission (14.1 vs. 6.0 mg/dl, p = 0.032). In addition, CRP was elevated, although not significantly, in patients with severe endoscopic disease compared to those in endoscopic remission (18.7 vs. 4.1, p = 0.062). Furthermore, baseline CRP was reduced in patients with clinical and endoscopic remission after HSCT compared to those without remission, although not significantly (8.8 vs. 21.4, n.s. and 8.1 vs. 12.4, n.s.). CONCLUSION: Given the divergent findings compared to GvHD, we conclude that serum REG3α is not an accurate diagnostic and predictive biomarker in CD patients undergoing HSCT. In contrast, CRP is a valuable biomarker in order to differentiate active disease from remission. However, CRP does not seem to be of prognostic value for HSCT outcome.

Common requirement for the relaxosome of plasmid R1 in multiple activities of the conjugative type IV secretion system.

Macromolecular transport by bacterial type IV secretion systems involves regulated uptake of (nucleo)protein complexes by the cell envelope-spanning transport channel. A coupling protein receptor is believed to recognize the specific proteins destined for transfer, but the steps initiating their translocation remain unknown. Here, we investigate the contribution of a complex of transfer initiation proteins, the relaxosome, of plasmid R1 to translocation of competing transferable substrates from mobilizable plasmids ColE1 and CloDF13 or the bacteriophage R17. We found that not only does the R1 translocation machinery engage the R1 relaxosome during conjugative self-transfer and during infection by R17 phage but it is also activated by its cognate relaxosome to mediate the export of an alternative plasmid. Transporter activity was optimized by the R1 relaxosome even when this complex itself could not be transferred, i.e., when the N-terminal activation domain (amino acids 1 to 992 [N1-992]) of TraI was present without the C-terminal conjugative helicase domain. We propose that the functional dependence of the transfer machinery on the R1 relaxosome for initiating translocation ensures that dissemination of heterologous plasmids does not occur at the expense of self-transfer.

Structure of a translocation signal domain mediating conjugative transfer by type IV secretion systems.

Relaxases are proteins responsible for the transfer of plasmid and chromosomal DNA from one bacterium to another during conjugation. They covalently react with a specific phosphodiester bond within DNA origin of transfer sequences, forming a nucleo-protein complex which is subsequently recruited for transport by a plasmid-encoded type IV secretion system. In previous work we identified the targeting translocation signals presented by the conjugative relaxase TraI of plasmid R1. Here we report the structure of TraI translocation signal TSA. In contrast to known translocation signals we show that TSA is an independent folding unit and thus forms a bona fide structural domain. This domain can be further divided into three subdomains with striking structural homology with helicase subdomains of the SF1B family. We also show that TSA is part of a larger vestigial helicase domain which has lost its helicase activity but not its single-stranded DNA binding capability. Finally, we further delineate the binding site responsible for translocation activity of TSA by targeting single residues for mutations. Overall, this study provides the first evidence that translocation signals can be part of larger structural scaffolds, overlapping with translocation-independent activities.

Downregulation of duodenal SLC transporters and activation of proinflammatory signaling constitute the early response to high altitude in humans.

Solute carrier (SLC) transporters mediate the uptake of biologically active compounds in the intestine. Reduced oxygenation (hypoxia) is an important factor influencing intestinal homeostasis. The aim of this study was to investigate the pathophysiological consequences of hypoxia on the expression and function of SLCs in human intestine. Hypoxia was induced in human intestinal epithelial cells (IECs) in vitro (0.2; 1% O2 or CoCl2). For human in vivo studies, duodenal biopsies and serum samples were obtained from individuals (n = 16) acutely exposed to 4,554 meters above sea levels. Expression of relevant targets was analyzed by quantitative PCR, Western blotting, or immunofluorescence. Serum levels of inflammatory mediators and nucleosides were determined by ELISA and LC/MS-MS, respectively. In the duodenum of volunteers exposed to high altitude we observed decreased mRNA levels of apical sodium-dependent bile acid transporter (ASBT), concentrative nucleoside transporters 1/2 (CNT1/2), organic anion transporting polypeptide 2B1 (OATP2B1), organic cation transporter 2 (OCTN2), peptide transporter 1 (PEPT1), serotonin transporter (SERT), and higher levels of IFN-γ, IL-6, and IL-17A. Serum levels of IL-10, IFN-γ, matrix metalloproteinase-2 (MMP-2), and serotonin were elevated, whereas the levels of uridine decreased upon exposure to hypoxia. Hypoxic IECs showed reduced levels of equilibrative nucleoside transporter 2 (ENT2), OCTN2, and SERT mRNAs in vitro, which was confirmed on the protein level and was accompanied by activation of ERK1/2, increase of hypoxia-inducible factor (HIF) proteins, and production of IL-8 mRNA. Costimulation with IFN-γ and IL-6 during hypoxia further decreased the expression of SERT, ENT2, and CNT2 in vitro. Reduced oxygen supply affects the expression pattern of duodenal SLCs that is accompanied by changes in serum levels of proinflammatory cytokines and biologically active compounds demonstrating that intestinal transport is affected during systemic exposure to hypoxia in humans.

Loss of protein tyrosine phosphatase nonreceptor type 22 regulates interferon-γ-induced signaling in human monocytes.

BACKGROUND & AIMS: A gain-of-function variation within the locus that encodes protein tyrosine phosphatase nonreceptor type (PTPN)22 is associated with a reduced risk for Crohn's disease (CD), whereas a loss-of-function variant seems to promote autoimmune disorders. We investigated how loss of PTPN22 could contribute to chronic inflammation of the intestine. METHODS: Intestinal tissue samples from patients with or without inflammatory bowel disease (controls) were analyzed for levels of PTPN22 messenger RNA (mRNA) and protein. In human THP-1 monocytes, protein levels were analyzed by immunoblotting, mRNA levels by real-time polymerase chain reaction, and cytokine release by enzyme-linked immunosorbent assay. RESULTS: Intestinal tissue samples from patients with CD had reduced levels of PTPN22 mRNA and protein, compared with samples from controls. In human THP-1 monocytes, interferon-γ (IFN-γ) induced expression and activity of PTPN22. Loss of PTPN22 increased levels of p38-mitogen-activated protein kinase, but reduced phosphorylation of nuclear factor-κB subunits. Increased activity of suppressor of cytokine signaling-1 was accompanied by reduced phosphorylation of signal-transducer and activator of transcription protein 1 and signal-transducer and activator of transcription protein 3 in PTPN22-deficient cells incubated with cytokines. PTPN22 knockdown increased secretion of the inflammatory cytokines interleukin (IL)-6 and IL-17, but reduced expression or secretion of T-bet, intercellular adhesion molecule-1, nucleotide-binding oligomerization domain containing-2, monocyte chemoattractant protein-1, IL-2, and IL-12p40 in response to IFN-γ. CONCLUSIONS: PTPN22 expression is reduced in intestinal tissues of patients with active CD. PTPN22 regulates intracellular signaling events and is induced by IFN-γ in human monocytes. Knockdown of PTPN22 alters activation of inflammatory signal transducers, increasing secretion of T-helper 17-related inflammatory mediators. Genetic variants that reduce PTPN22 activity might contribute to the pathogenesis of CD by these mechanisms.

Bilberry-derived anthocyanins prevent IFN-γ-induced pro-inflammatory signalling and cytokine secretion in human THP-1 monocytic cells.

BACKGROUND/AIMS: Anthocyanins are plant-derived dietary components that are highly abundant, for example, in bilberries. We have previously demonstrated that anthocyanins exert anti-inflammatory properties in mouse colitis models and ameliorate disease activity in ulcerative colitis patients. Here, we studied the molecular mechanisms through which anthocyanin-containing bilberry extract (BE) exerts anti-inflammatory effects in human monocytic THP-1 cells. METHODS: THP-1 cells were pre-incubated with BE 20 min prior to TNF-α or IFN-γ (100 ng/ml each) stimulation. Signalling protein activation was studied by Western blotting, mRNA expression by quantitative PCR and cytokine secretion by ELISA. RESULTS: IFN-γ-induced phosphorylation of STAT1 and STAT3 was significantly reduced by BE co-treatment. Consequently, levels of mRNA expression and/or cytokine secretion of MCP-1, IL-6, TNF-α, ICAM-1, and T-bet were lower with BE co-treatment. In contrast, BE enhanced TNF-α-mediated p65-NF-κB phosphorylation but reduced ERK1/2 phosphorylation. BE co-treatment further increased TNF-α-induced mRNA expression and secretion of NF-κB target genes, such as IL-6, IL-8, and MCP-1, while mRNA levels of ICAM-1 were reduced. CONCLUSIONS: BE co-treatment reduced IFN-γ-induced signal protein activation, pro-inflammatory gene expression, and cytokine secretion, whereas it enhanced TNF-α-induced responses. These findings suggest a distinct role for anthocyanins in modulating inflammatory responses that need to be further studied to fully understand anthocyanin-mediated effects.

Glycoprotein (GP)96 Is Essential for Maintaining Intestinal Epithelial Architecture by Supporting Its Self-Renewal Capacity.

BACKGROUND & AIMS: Glycoprotein (GP)96 is an endoplasmic reticulum-resident master chaperone for cell surface receptors including the Wnt co-receptors low-density lipoprotein-receptor-related protein 5/6. Intestinal epithelial cell (IEC)-specific deletion of Gp96 is embryonically lethal. However, the role of GP96 in adult intestinal tissue and especially within the intestinal stem cell (ISC) niche is unknown. Here, we investigated how GP96 loss interferes with intestinal homeostasis by compromising viability, proliferation, and differentiation of IECs. METHODS: Tamoxifen was used to induce Cre-mediated deletion of Gp96 in GP96-VillincreERT2 (Cre recombinase-Estrogen-Receptor Transgene 2) mice and intestinal organoids. With H&E and immunofluorescence staining we assessed alterations in intestinal morphology and the presence and localization of IEC types. Real-time polymerase chain reaction and Western blot analysis were performed to explore the molecular mechanisms underlying the severe phenotype of Gp96 KO mice and organoids. RESULTS: IEC-specific deletion of Gp96 in adult mice resulted in a rapid degeneration of the stem cell niche, followed by complete eradication of the epithelial layer and death within a few days. These effects were owing to severe defects in ISC renewal and premature ISC differentiation, which resulted from defective Wnt and Notch signaling. Furthermore, depletion of GP96 led to massive induction of endoplasmic reticulum stress. Although effects on ISC renewal and adequate differentiation were partly reversed upon activation of Wnt/Notch signaling, viability could not be restored, indicating that reduced viability was mediated by other mechanisms. CONCLUSIONS: Our work shows that GP96 plays a fundamental role in regulating ISC fate and epithelial regeneration and therefore is indispensable for maintaining intestinal epithelial homeostasis.

An activation domain of plasmid R1 TraI protein delineates stages of gene transfer initiation.

Bacterial conjugation is a form of type IV secretion that transports protein and DNA to recipient cells. Specific bacteriophage exploit the conjugative pili and cell envelope spanning protein machinery of these systems to invade bacterial cells. Infection by phage R17 requires F-like pili and coupling protein TraD, which gates the cytoplasmic entrance of the secretion channel. Here we investigate the role of TraD in R17 nucleoprotein uptake and find parallels to secretion mechanisms. The relaxosome of IncFII plasmid R1 is required. A ternary complex of plasmid oriT, TraD and a novel activation domain within the N-terminal 992 residues of TraI contributes a key mechanism involving relaxase-associated properties of TraI, protein interaction and the TraD ATPase. Helicase-associated activities of TraI are dispensable. These findings distinguish for the first time specific protein domains and complexes that process extracellular signals into distinct activation stages in the type IV initiation pathway. The study also provided insights into the evolutionary interplay of phage and the plasmids they exploit. Related plasmid F adapted to R17 independently of TraI. It follows that selection for phage resistance drives not only variation in TraA pilins but diversifies TraD and its binding partners in a plasmid-specific manner.

General requirements for protein secretion by the F-like conjugation system R1.

Bacterial conjugation disseminates genes among bacteria via a process requiring direct cell contact. The cell envelope spanning secretion apparatus involved belongs to the type IV family of bacterial secretion systems, which transport protein as well as nucleoprotein substrates. This study aims to understand mechanisms leading to the initiation of type IV secretion using conjugative plasmid paradigm R1. We analyze the general requirements for plasmid encoded conjugation proteins and DNA sequence within the origin of transfer (oriT) for protein secretion activity using a Cre recombinase reporter system. We find that similar to conjugative plasmid DNA strand transfer, activation of the R1 system for protein secretion depends on binding interactions between the multimeric, ATP-binding coupling protein and the R1 relaxosome including an intact oriT. Evidence for DNA independent protein secretion was not found.

MMP9 expression in intestinal fistula from patients with fistulizing CD and from human xenograft mouse model.

Fistula treatment represents a major unmet medical need in the therapy of Crohn's disease (CD). Current medical therapies, such as anti-TNF antibody treatments, are often insufficient and do not achieve permanent fistula closure. Previously published data point toward a critical role for metalloproteinase-9 (MMP-9)/gelatinase B in fistula pathogenesis. The aim of this project was to investigate in detail MMP-9 expression in different fistula types and to confirm that MMP-9 is a potential target for fistula therapy in CD patients.Immunohistochemistry for total and active MMP-9, Cytokeratin 8 (CK-8) and co-staining of active MMP-9/CK-8 was performed in specimen derived from perianal fistulas, entero-enteric fistulas and fistulas from patients not responding to anti-TNF therapy. In addition, fistulas from the xenograft mouse model (anti-TNF treated or untreated) were analyzed.Total and active MMP-9 protein was detectable in cells lining the tracts of perianal and entero-enteric fistulas. Of note, total and active MMP-9 was also expressed in fistulas of CD patients non-responding to anti-TNF treatment. Interestingly, we detected considerable co-staining of active MMP-9 and CK-8 in particular in cells lining the fistula tract and in transitional cells around the fistulas. Furthermore, total and active MMP-9 are detectable in both anti-TNF treated and untreated xenograft fistulas.Taken together, our data suggest that MMP-9 is involved in fistula pathogenesis in CD patients, in fistulas of different origins and particularly in patients non-responding to anti-TNF therapy. Our xenograft fistula model is suitable for in vivo studies investigating a possible therapeutic role for MMP-9 targeting as fistula therapy.

Inhibition of integrin αvß6 sparks T-cell antitumor response and enhances immune checkpoint blockade therapy in colorectal cancer.

BACKGROUND: Integrin αvß6 is a heterodimeric cell surface protein whose cellular expression is determined by the availability of the integrin ß6 subunit (ITGB6). It is expressed at very low levels in most organs during tissue homeostasis but shows highly upregulated expression during the process of tumorigenesis in many cancers of epithelial origin. Notably, enhanced expression of integrin αvß6 is associated with aggressive disease and poor prognosis in numerous carcinoma entities. Integrin αvß6 is one of the major physiological activators of transforming growth factor-ß (TGF-ß), which has been shown to inhibit the antitumor T-cell response and cause resistance to immunotherapy in mouse models of colorectal and mammary cancer. In this study, we investigated the effect of ITGB6 expression and antibody-mediated integrin αvß6 inhibition on the tumor immune response in colorectal cancer. METHODS: Using orthotopic and heterotopic tumor cell injection, we assessed the effect of ITGB6 on tumor growth and tumor immune response in wild type mice, mice with defective TGF-ß signaling, and mice treated with anti-integrin αvß6 antibodies. To examine the effect of ITGB6 in human colorectal cancer, we analyzed RNAseq data from the colon adenocarcinoma dataset of The Cancer Genome Atlas (TCGA-COAD). RESULTS: We demonstrate that expression of ITGB6 is an immune evasion strategy in colorectal cancer, causing inhibition of the antitumor immune response and resistance to immune checkpoint blockade therapy by activating latent TGF-ß. Antibody-mediated inhibition of integrin αvß6 sparked a potent cytotoxic T-cell response and overcame resistance to programmed cell death protein 1 (PD-1) blockade therapy in ITGB6 expressing tumors, provoking a drastic increase in anti-PD-1 treatment efficacy. Further, we show that the majority of tumors in patients with colorectal cancer express sufficient ITGB6 to provoke inhibition of the cytotoxic T-cell response, indicating that most patients could benefit from integrin αvß6 blockade therapy. CONCLUSIONS: These findings propose inhibition of integrin αvß6 as a promising new therapy for colorectal cancer, which blocks tumor-promoting TGF-ß activation, prevents tumor exclusion of cytotoxic T-cells and enhances the efficacy of immune checkpoint blockade therapy.