Stable L-forms are cell wall-deficient bacteria which are able to multiply and propagate indefinitely, despite the absence of a rigid peptidoglycan cell wall. We investigated whether L-forms of the intracellular pathogen L. monocytogenes possibly retain pathogenicity, and if they could trigger an innate immune response. While phagocytosis of L. monocytogenes L-forms by non-activated macrophages sometimes resulted in an unexpected persistence of the bacteria in the phagocytes, they were effectively eliminated by IFN-γ preactivated or bone marrow-derived macrophages (BMM). These findings were in line with the observed down-regulation of virulence factors in the cell-wall deficient L. monocytogenes. Absence of Interferon-β (IFN-β) triggering indicated inability of L-forms to escape from the phagosome into the cytosol. Moreover, abrogated cytokine response in MyD88-deficient dendritic cells (DC) challenged with L. monocytogenes L-forms suggested an exclusive TLR-dependent host response. Taken together, our data demonstrate a strong attenuation of Listeria monocytogenes L-form pathogenicity, due to diminished expression of virulence factors and innate immunity recognition, eventually resulting in elimination of L-form bacteria from phagocytes.

Humoral autoimmunity paralleled by the accumulation of follicular helper T cells (T(FH) cells) is linked to mutation of the gene encoding the RNA-binding protein roquin-1. Here we found that T cells lacking roquin caused pathology in the lung and accumulated as cells of the T(H)17 subset of helper T cells in the lungs. Roquin inhibited T(H)17 cell differentiation and acted together with the endoribonuclease regnase-1 to repress target mRNA encoding the T(H)17 cell-promoting factors IL-6, ICOS, c-Rel, IRF4, IκBNS and IκBζ. This cooperation required binding of RNA by roquin and the nuclease activity of regnase-1. Upon recognition of antigen by the T cell antigen receptor (TCR), roquin and regnase-1 proteins were cleaved by the paracaspase MALT1. Thus, this pathway acts as a 'rheostat' by translating TCR signal strength via graded inactivation of post-transcriptional repressors and differential derepression of targets to enhance T(H)17 differentiation.

Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.

Inflammatory bowel disease (IBD) is a chronic intestinal disorder, often leading to an impaired quality of life in affected patients. The importance of environmental factors in the pathogenesis of IBD, including their disease-modifying potential, is increasingly recognised. Hypoxia seems to be an important driver of inflammation, as has been reported by our group and others. The aim of the study is to evaluate if hypoxia can alter disease activity of IBD measured by Harvey-Bradshaw Activity Index in Crohn's disease (increase to ≥5 points) and the partial Mayo Score for ulcerative colitis (increase to ≥2 points). To test the effects of hypoxia under standardised conditions, we designed a prospective and controlled investigation in healthy controls and patients with IBD in stable remission.

Spermidine is a dietary polyamine that is able to activate protein tyrosine phosphatase non-receptor type 2 (PTPN2). As PTPN2 is known to be a negative regulator of interferon-gamma (IFN-γ)-induced responses, and IFN-γ stimulation of immune cells is a critical process in the immunopathology of inflammatory bowel disease (IBD), we wished to explore the potential of spermidine for reducing pro-inflammatory effects in vitro and in vivo.

Fecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infections (RCDIs). However, long-term effects on the patients' gut microbiota and the role of viruses remain to be elucidated. Here, we characterized bacterial and viral microbiota in the feces of a cured RCDI patient at various time points until 4.5 yr post-FMT compared with the stool donor. Feces were subjected to DNA sequencing to characterize bacteria and double-stranded DNA (dsDNA) viruses including phages. The patient's microbial communities varied over time and showed little overall similarity to the donor until 7 mo post-FMT, indicating ongoing gut microbiota adaption in this time period. After 4.5 yr, the patient's bacteria attained donor-like compositions at phylum, class, and order levels with similar bacterial diversity. Differences in the bacterial communities between donor and patient after 4.5 yr were seen at lower taxonomic levels. C. difficile remained undetectable throughout the entire timespan. This demonstrated sustainable donor feces engraftment and verified long-term therapeutic success of FMT on the molecular level. Full engraftment apparently required longer than previously acknowledged, suggesting the implementation of year-long patient follow-up periods into clinical practice. The identified dsDNA viruses were mainly Caudovirales phages. Unexpectedly, sequences related to giant algae-infecting Chlorella viruses were also detected. Our findings indicate that intestinal viruses may be implicated in the establishment of gut microbiota. Therefore, virome analyses should be included in gut microbiota studies to determine the roles of phages and other viruses-such as Chlorella viruses-in human health and disease, particularly during RCDI.

Pathogenesis of inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), involves interaction between environmental factors and inappropriate immune responses in the intestine of genetically predisposed individuals. Bile acids and their nuclear receptor, FXR, regulate inflammatory responses and barrier function in the intestinal tract.

In Crohn's disease (CD) the stress-shield of intestinal subepithelial myofibroblasts (ISEMF) provided by intact tissue is disturbed due to inflammation and thus, cells start with remodelling activities. This is characterized by increased numbers of collagen-producing ISEMF causing an uncontrolled, irreversible wound-healing response to the chronic inflammation of the gastrointestinal tract. Reconstitution of the original ECM leads ISEMF to exit this cycle. In contrast, during fibrosis, ISEMF persist. It is known that ISEMF produce and deposit collagen types I, III, IV and V; however synthesis and the role of fibrillar peripheral molecules like collagen type XVI have not been addressed yet. Here, we have analyzed the distribution of collagen XVI in the normal and inflamed bowel wall, its gene and protein expression by ISEMF of different inflammation stages, the cell-matrix interactions in different phases of the inflammatory process and their effect on cell spreading, proliferation and migration. Collagen XVI is deposited in the submucosa of the intestinal wall where it co-localizes with fibrillin-1 and integrin alpha1. ISEMF reveal increasing gene and protein expression of collagen XVI concurrent to increasing inflammation. ISEMF reveal more mature focal adhesion contacts when seeded on collagen XVI resulting in an extensive cell spreading. This involves recruitment of alpha1beta1 integrin, which shows increased cell surface expression on ISEMF in late stages of inflammation. We assume that collagen XVI promotes persistence of ISEMF in the normal and, even stronger in the inflamed bowel wall by stabilizing focal adhesion contacts via cell-matrix interaction preferentially through recruitment of alpha1ss1 integrin into the tips of the focal adhesion contacts.

Genetic risk factors, intestinal microbiota and a dysregulated immune system contribute to the pathogenesis of inflammatory bowel disease (IBD). We have previously demonstrated that dysfunction of protein tyrosine phosphatase non-receptor type 2 (PTPN2) and PTPN22 contributes to alterations of intestinal microbiota and the onset of chronic intestinal inflammation in vivo. Here, we investigated the influence of PTPN2 and PTPN22 gene variants on intestinal microbiota composition in IBD patients.

Nonalcoholic steatohepatitis (NASH), a primary cause of liver disease, leads to complications such as fibrosis, cirrhosis, and carcinoma, but the pathophysiology of NASH is incompletely understood. Epstein-Barr virus-induced G protein-coupled receptor 2 (EBI2) and its oxysterol ligand 7α,25-dihydroxycholesterol (7α,25-diHC) are recently discovered immune regulators. Several lines of evidence suggest a role of oxysterols in NASH pathogenesis, but rigorous testing has not been performed. We measured oxysterol levels in the livers of NASH patients by LC-MS and tested the role of the EBI2-7α,25-diHC system in a murine feeding model of NASH. Free oxysterol profiling in livers from NASH patients revealed a pronounced increase in 24- and 7-hydroxylated oxysterols in NASH compared with controls. Levels of 24- and 7-hydroxylated oxysterols correlated with histological NASH activity. Histological analysis of murine liver samples demonstrated ballooning and liver inflammation. No significant genotype-related differences were observed in Ebi2-/- mice and mice with defects in the 7α,25-diHC synthesizing enzymes CH25H and CYP7B1 compared with wild-type littermate controls, arguing against an essential role of these genes in NASH pathogenesis. Elevated 24- and 7-hydroxylated oxysterol levels were confirmed in murine NASH liver samples. Our results suggest increased bile acid synthesis in NASH samples, as judged by the enhanced level of 7α-hydroxycholest-4-en-3-one and impaired 24S-hydroxycholesterol metabolism as characteristic biochemical changes in livers affected by NASH.

The human hepatitis B virus (HBV) is a major cause of chronic hepatitis and hepatocellular carcinoma, but molecular mechanisms driving liver disease and carcinogenesis are largely unknown. We therefore studied cellular pathways altered by HBV infection.

Plasma levels of high-density lipoprotein (HDL) cholesterol are frequently found decreased in patients with inflammatory bowel disease (IBD). Therefore, and because HDL exerts anti-inflammatory activities, we investigated whether HDL and its major protein component apolipoprotein A-I (apoA-I) modulate mucosal inflammatory responses in vitro and in vivo.

Forward genetic screens with genome-wide CRISPR libraries are powerful tools for resolving cellular circuits and signaling pathways. Applying this technology to organoids, however, has been hampered by technical limitations. Here we report improved accuracy and robustness for pooled-library CRISPR screens by capturing sgRNA integrations in single organoids, substantially reducing required cell numbers for genome-scale screening. We applied our approach to wild-type and APC mutant human intestinal organoids to identify genes involved in resistance to TGF-β-mediated growth restriction, a key process during colorectal cancer progression, and validated hits including multiple subunits of the tumor-suppressive SWI/SNF chromatin remodeling complex. Mutations within these genes require concurrent inactivation of APC to promote TGF-β resistance and attenuate TGF-β target gene transcription. Our approach can be applied to a variety of assays and organoid types to facilitate biological discovery in primary 3D tissue models.

Hepatic innate immune control of viral infections has largely been attributed to Kupffer cells, the liver-resident macrophages. However, hepatocytes, the parenchymal cells of the liver, also possess potent immunological functions in addition to their known metabolic functions. Owing to their abundance in the liver and known immunological functions, we aimed to investigate the direct antiviral mechanisms employed by hepatocytes.

Proton-sensing ovarian cancer G-protein coupled receptor (OGR1) plays an important role in pH homeostasis. Acidosis occurs at sites of intestinal inflammation and can induce endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), an evolutionary mechanism that enables cells to cope with stressful conditions. ER stress activates autophagy, and both play important roles in gut homeostasis and contribute to the pathogenesis of inflammatory bowel disease (IBD). Using a human intestinal epithelial cell model, we investigated whether our previously observed protective effects of OGR1 deficiency in experimental colitis are associated with a differential regulation of ER stress, the UPR and autophagy. Caco-2 cells stably overexpressing OGR1 were subjected to an acidic pH shift. pH-dependent OGR1-mediated signalling led to a significant upregulation in the ER stress markers, binding immunoglobulin protein (BiP) and phospho-inositol required 1α (IRE1α), which was reversed by a novel OGR1 inhibitor and a c-Jun N-terminal kinase (JNK) inhibitor. Proton-activated OGR1-mediated signalling failed to induce apoptosis, but triggered accumulation of total microtubule-associated protein 1 A/1B-light chain 3, suggesting blockage of late stage autophagy. Our results show novel functions for OGR1 in the regulation of ER stress through the IRE1α-JNK signalling pathway, as well as blockage of autophagosomal degradation. OGR1 inhibition might represent a novel therapeutic approach in IBD.

The use of molecular markers for inflammation in the gastrointestinal tract could empower optical imaging modalities for early diagnosis and eventually personalized timely treatments. A major hurdle to the widespread use of functional fluorescence imaging is the absence of suitable contrast agents, in particular to be administered via the oral route due to the usual proteolytic susceptibility of the biomarkers. By designing a retro-inverso peptide, starting from a previously described sequence specific for N-cadherin, we achieved resistance to gastrointestinal degradation and even slightly improved specificity towards the target, both in ex vivo and in vivo experimental colitis. Simulations at fundamental molecular level suggested that the introduced retro-inverso modifications did not affect the folding of the peptide, leaving its ability to interact with the binding pocket of the monomeric N-cadherin unaltered, even when fluorescently labeled. Possible further derivatization of this sequence could be envisaged to further extend the potential of the designed retro-inverso peptide as diagnostic or theranostic agent for the oral route.

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.

Nonalcoholic steatohepatitis (NASH)-induced hepatocellular carcinoma (HCC) and its precursor, nonalcoholic fatty liver disease (NAFLD) are an unmet health issue due to widespread obesity. We assessed copy number changes of genes associated with hepatocarcinogenesis and oxidative pathways at a single-cell level. Eleven patients with NASH-HCC and 11 patients with NAFLD were included. Eight probes were analyzed using multiplex interphase fluorescence in situ hybridization (miFISH), single-cell imaging and phylogenetic tree modelling: Telomerase reverse transcriptase (TERT), C-Myc (MYC), hepatocyte growth factor receptor tyrosine kinase (MET), tumor protein 53 (TP53), cyclin D1 (CCND1), human epidermal growth factor receptor 2 (HER2), the fragile histidine triad gene (FHIT) and FRA16D oxidoreductase (WWOX). Each NASH-HCC tumor had up to 14 distinct clonal signal patterns indicating multiclonality, which correlated with high tumor grade. Changes frequently observed were TP53 losses, 45%; MYC gains, 36%; WWOX losses, 36%; and HER2 gains, 18%. Whole-genome duplications were frequent (82%) with aberrant tetraploid cells evolving from diploid ancestors. Non-tumorous NAFLD/NASH biopsies did not harbor clonal copy number changes. Fine mapping of NASH-HCC using single-cell multiplex FISH shows that branched tumor evolution involves genome duplication and that multiclonality increases with tumor grade. The loss of oxidoreductase WWOX and HER2 gains could be potentially associated with NASH-induced hepatocellular carcinoma.

Integrin αvβ6 is a heterodimeric cell surface protein whose cellular expression is determined by the availability of the integrin β6 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 αvβ6 is associated with aggressive disease and poor prognosis in numerous carcinoma entities. Integrin αvβ6 is one of the major physiological activators of transforming growth factor-β (TGF-β), 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 αvβ6 inhibition on the tumor immune response in colorectal cancer.

Alveolar (AE) and cystic echinococcosis (CE) are severe parasitic zoonoses caused by the larval stages of Echinococcus multilocularis and E. granulosus sensu lato, respectively. A panel of 7 monoclonal antibodies (mAbs) was selected against major diagnostic epitopes of both species. The binding capacity of the mAbs to Echinococcus spp. excretory/secretory products (ESP) was analyzed by sandwich-ELISA, where mAb Em2G11 and mAb EmG3 detected in vitro extravesicular ESP of both E. multilocularis and E. granulosus s.s. These findings were subsequently confirmed by the detection of circulating ESP in a subset of serum samples from infected hosts including humans. Extracellular vesicles (EVs) were purified, and the binding to mAbs was analyzed by sandwich-ELISA. Transmission electron microscopy (TEM) was used to confirm the binding of mAb EmG3 to EVs from intravesicular fluid of Echinococcus spp. vesicles. The specificity of the mAbs in ELISA corresponded to the immunohistochemical staining (IHC-S) patterns performed on human AE and CE liver sections. Antigenic small particles designated as ''spems'' for E. multilocularis and ''spegs'' for E. granulosus s.l. were stained by the mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2, while mAb Em2G11 reacted with spems and mAb Eg2 with spegs only. The laminated layer (LL) of both species was strongly visualized by using mAb EmG3IgM, mAb EmG3IgG1, mAb AgB, and mAb 2B2. The LL was specifically stained by mAb Em2G11 in E. multilocularis and by mAb Eg2 in E. granulosus s.l. In the germinal layer (GL), including the protoscoleces, a wide staining pattern with all structures of both species was observed with mAb EmG3IgG1, mAb EmG3IgM, mAb AgB, mAb 2B2, and mAb Em18. In the GL and protoscoleces, the mAb Eg2 displayed a strong E. granulosus s.l. specific binding, while mAb Em2G11 exhibited a weak granular E. multilocularis specific reaction. The most notable staining pattern in IHC-S was found with mAb Em18, which solely bound to the GL and protoscoleces of Echinococcus species and potentially to primary cells. To conclude, mAbs represent valuable tools for the visualization of major antigens in the most important Echinococcus species, as well as providing insights into parasite-host interactions and pathogenesis.