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A protein-truncating R179X variant in RNF186 confers protection against ulcerative colitis.
Protein-truncating variants protective against human disease provide in vivo validation of therapeutic targets. Here we used targeted sequencing to conduct a search for protein-truncating variants conferring protection against inflammatory bowel disease exploiting knowledge of common variants associated with the same disease. Through replication genotyping and imputation we found that a predicted protein-truncating variant (rs36095412, p.R179X, genotyped in 11,148 ulcerative colitis patients and 295,446 controls, MAF=up to 0.78%) in RNF186, a single-exon ring finger E3 ligase with strong colonic expression, protects against ulcerative colitis (overall P=6.89 × 10(-7), odds ratio=0.30). We further demonstrate that the truncated protein exhibits reduced expression and altered subcellular localization, suggesting the protective mechanism may reside in the loss of an interaction or function via mislocalization and/or loss of an essential transmembrane domain.
Exploring the genetic architecture of inflammatory bowel disease by whole-genome sequencing identifies association at ADCY7.
To further resolve the genetic architecture of the inflammatory bowel diseases ulcerative colitis and Crohn's disease, we sequenced the whole genomes of 4,280 patients at low coverage and compared them to 3,652 previously sequenced population controls across 73.5 million variants. We then imputed from these sequences into new and existing genome-wide association study cohorts and tested for association at ∼12 million variants in a total of 16,432 cases and 18,843 controls. We discovered a 0.6% frequency missense variant in ADCY7 that doubles the risk of ulcerative colitis. Despite good statistical power, we did not identify any other new low-frequency risk variants and found that such variants explained little heritability. We detected a burden of very rare, damaging missense variants in known Crohn's disease risk genes, suggesting that more comprehensive sequencing studies will continue to improve understanding of the biology of complex diseases.
Genome-wide association study implicates immune activation of multiple integrin genes in inflammatory bowel disease.
Genetic association studies have identified 215 risk loci for inflammatory bowel disease, thereby uncovering fundamental aspects of its molecular biology. We performed a genome-wide association study of 25,305 individuals and conducted a meta-analysis with published summary statistics, yielding a total sample size of 59,957 subjects. We identified 25 new susceptibility loci, 3 of which contain integrin genes that encode proteins in pathways that have been identified as important therapeutic targets in inflammatory bowel disease. The associated variants are correlated with expression changes in response to immune stimulus at two of these genes (ITGA4 and ITGB8) and at previously implicated loci (ITGAL and ICAM1). In all four cases, the expression-increasing allele also increases disease risk. We also identified likely causal missense variants in a gene implicated in primary immune deficiency, PLCG2, and a negative regulator of inflammation, SLAMF8. Our results demonstrate that new associations at common variants continue to identify genes relevant to therapeutic target identification and prioritization.
Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection.
The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.
ICG-001 affects DRP1 activity and ER stress correlative with its anti-proliferative effect.
Mitochondria form a highly dynamic network driven by opposing scission and fusion events. DRP1 is an essential modulator of mitochondrial fission and dynamics within mammalian cells. Its fission activity is regulated by posttranslational modifications such as activating phosphorylation at serine 616. DRP1 activity has recently been implicated as being dysregulated in numerous human disorders such as cancer and neurodegenerative diseases. Here we describe the development of a cell-based screening assay to detect DRP1 activation. We utilized this to undertake focused compound library screening and identified potent modulators that affected DRP1 activity including ICG-001, which is described as WNT/β-catenin signaling inhibitor. Our findings elucidate novel details about ICG-001's mechanism of action (MOA) in mediating anti-proliferative activity. We show ICG-001 both inhibits mitochondrial fission and activates an early endoplasmic reticulum (ER) stress response to induce cell death in susceptible colorectal cancer cell lines.
The Antiviral Efficacy of HIV-Specific CD8+ T-Cells to a Conserved Epitope is Heavilydependent on the Infecting HIV-1 Isolate
A major challenge to developing a successful HIV vaccine is the vast diversity of viral sequences, yet it is generally assumed that an epitope conserved between different strains will be recognised by responding T-cells. We examined whether an invariant HLA-B8 restricted Nef90-97 epitope FL8 shared between five high titre viruses and eight recombinant vaccinia viruses expressing Nef from different viral isolates (clades A-H) could activate antiviral activity in FL8-specific cytotoxic T-lymphocytes (CTL). Surprisingly, despite epitope conservation, we found that CTL antiviral efficacy is dependent on the infecting viral isolate. Only 23% of Nef proteins, expressed by HIV-1 isolates or as recombinant vaccinia-Nef, were optimally recognised by CTL. Recognition of the HIV-1 isolates by CTL was independent of clade-grouping but correlated with virus-specific polymorphisms in the epitope flanking region, which altered immunoproteasomal cleavage resulting in enhanced or impaired epitope generation. The finding that the majority of virus isolates failed to present this conserved epitope highlights the importance of viral variance in CTL epitope flanking regions on the efficiency of antigen processing, which has been considerably underestimated previously. This has important implications for future vaccine design strategies since efficient presentation of conserved viral epitopes is necessary to promote enhanced anti-viral immune responses. © 2011 Ranasinghe et al.
Elevation of intact and proteolytic fragments of acute phase proteins constitutes the earliest systemic antiviral response in HIV-1 infection
The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI) exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, b-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA) occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT), termed virus inhibitory peptide (VIRIP), was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies. © 2010 Kramer et al.
Human SNP links differential outcomes in inflammatory and infectious disease to a FOXO3-regulated pathway.
The clinical course and eventual outcome, or prognosis, of complex diseases varies enormously between affected individuals. This variability critically determines the impact a disease has on a patient's life but is very poorly understood. Here, we exploit existing genome-wide association study data to gain insight into the role of genetics in prognosis. We identify a noncoding polymorphism in FOXO3A (rs12212067: T > G) at which the minor (G) allele, despite not being associated with disease susceptibility, is associated with a milder course of Crohn's disease and rheumatoid arthritis and with increased risk of severe malaria. Minor allele carriage is shown to limit inflammatory responses in monocytes via a FOXO3-driven pathway, which through TGFβ1 reduces production of proinflammatory cytokines, including TNFα, and increases production of anti-inflammatory cytokines, including IL-10. Thus, we uncover a shared genetic contribution to prognosis in distinct diseases that operates via a FOXO3-driven pathway modulating inflammatory responses.
Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor-neutralizing therapy in patients with inflammatory bowel disease.
Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex chronic inflammatory conditions of the gastrointestinal tract that are driven by perturbed cytokine pathways. Anti-tumor necrosis factor-α (TNF) antibodies are mainstay therapies for IBD. However, up to 40% of patients are nonresponsive to anti-TNF agents, which makes the identification of alternative therapeutic targets a priority. Here we show that, relative to healthy controls, inflamed intestinal tissues from patients with IBD express high amounts of the cytokine oncostatin M (OSM) and its receptor (OSMR), which correlate closely with histopathological disease severity. The OSMR is expressed in nonhematopoietic, nonepithelial intestinal stromal cells, which respond to OSM by producing various proinflammatory molecules, including interleukin (IL)-6, the leukocyte adhesion factor ICAM1, and chemokines that attract neutrophils, monocytes, and T cells. In an animal model of anti-TNF-resistant intestinal inflammation, genetic deletion or pharmacological blockade of OSM significantly attenuates colitis. Furthermore, according to an analysis of more than 200 patients with IBD, including two cohorts from phase 3 clinical trials of infliximab and golimumab, high pretreatment expression of OSM is strongly associated with failure of anti-TNF therapy. OSM is thus a potential biomarker and therapeutic target for IBD, and has particular relevance for anti-TNF-resistant patients.
Innate immune receptors for cross-presentation: The expanding role of NLRs.
A critical role of pattern recognition receptors (PRRs) is to influence adaptive immune responses by regulating antigen presentation. Engagement of PRRs in dendritic cells (DCs) increases MHC class I antigen presentation and CD8+ T-cell activation by cross-presented peptides but the molecular mechanisms underlying these effects are not completely understood. Studies looking at the role of PRRs in cross-presentation have been largely limited to TLRs but the role of other PRRs such as cytosolic nucleotide-binding oligomerization domain-like (NOD-like) receptors remains particularly enigmatic. Here we discuss recent evidence of the role of PRRs on cross-presentation and consider how cytosolic NLR-associated pathways, such as NOD2, may integrate these signals resulting in effective adaptive CD8+ T cells responses.
HLA-dependent variation in SARS-CoV-2 CD8 + T cell cross-reactivity with human coronaviruses.
The conditions and extent of cross-protective immunity between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and common-cold human coronaviruses (HCoVs) remain open despite several reports of pre-existing T cell immunity to SARS-CoV-2 in individuals without prior exposure. Using a pool of functionally evaluated SARS-CoV-2 peptides, we report a map of 126 immunogenic peptides with high similarity to 285 MHC-presented peptides from at least one HCoV. Employing this map of SARS-CoV-2-non-homologous and homologous immunogenic peptides, we observe several immunogenic peptides with high similarity to human proteins, some of which have been reported to have elevated expression in severe COVID-19 patients. After combining our map with SARS-CoV-2-specific TCR repertoire data from COVID-19 patients and healthy controls, we show that public repertoires for the majority of convalescent patients are dominated by TCRs cognate to non-homologous SARS-CoV-2 peptides. We find that for a subset of patients, >50% of their public SARS-CoV-2-specific repertoires consist of TCRs cognate to homologous SARS-CoV-2-HCoV peptides. Further analysis suggests that this skewed distribution of TCRs cognate to homologous or non-homologous peptides in COVID-19 patients is likely to be HLA-dependent. Finally, we provide 10 SARS-CoV-2 peptides with known cognate TCRs that are conserved across multiple coronaviruses and are predicted to be recognized by a high proportion of the global population. These findings may have important implications for COVID-19 heterogeneity, vaccine-induced immune responses, and robustness of immunity to SARS-CoV-2 and its variants.
Invasive Salmonella exploits divergent immune evasion strategies in infected and bystander dendritic cell subsets.
Non-typhoidal Salmonella (NTS) are highly prevalent food-borne pathogens. Recently, a highly invasive, multi-drug resistant S. Typhimurium, ST313, emerged as a major cause of bacteraemia in children and immunosuppressed adults, however the pathogenic mechanisms remain unclear. Here, we utilize invasive and non-invasive Salmonella strains combined with single-cell RNA-sequencing to study the transcriptome of individual infected and bystander monocyte-derived dendritic cells (MoDCs) implicated in disseminating invasive ST313. Compared with non-invasive Salmonella, ST313 directs a highly heterogeneous innate immune response. Bystander MoDCs exhibit a hyper-activated profile potentially diverting adaptive immunity away from infected cells. MoDCs harbouring invasive Salmonella display higher expression of IL10 and MARCH1 concomitant with lower expression of CD83 to evade adaptive immune detection. Finally, we demonstrate how these mechanisms conjointly restrain MoDC-mediated activation of Salmonella-specific CD4+ T cell clones. Here, we show how invasive ST313 exploits discrete evasion strategies within infected and bystander MoDCs to mediate its dissemination in vivo.
Human Enteric Glia Diversity in Health and Disease: New Avenues for the Treatment of Hirschsprung Disease.
BACKGROUND & AIMS: The enteric nervous system (ENS), which is composed of neurons and glia, regulates intestinal motility. Hirschsprung disease (HSCR) results from defects in ENS formation; however, although neuronal aspects have been studied extensively, enteric glia remain disregarded. This study aimed to explore enteric glia diversity in health and disease. METHODS: Full-thickness intestinal resection material from pediatric controls and patients with HSCR was collected, dissociated, and enriched for the ENS population through fluorescence-activated cell sorting. Single-cell RNA sequencing was performed to uncover the transcriptomic diversity of the ENS in controls and HSCR patients, as well as in wild-type and ret mutant zebrafish. Immunofluorescence and fluorescence in situ hybridization confirmed the presence of distinct subtypes. RESULTS: Two major enteric glial classes emerged in the pediatric intestine: Schwann-like enteric glia, which are reminiscent of Schwann cells, and enteric glia expressing classical glial markers. Comparative analysis with previously published datasets confirmed our classification and revealed that although classical enteric glia are predominant prenatally, Schwann-like enteric glia become more abundant postnatally. In HSCR, ganglionic segments mirrored controls and aganglionic segments featured only Schwann-like enteric glia. Leveraging the regenerative potential of Schwann cells, we explored therapeutic options using a ret mutant zebrafish. Prucalopride, a serotonin-receptor (5-HT) agonist, induced neurogenesis partially rescuing the HSCR phenotype in ret+/- mutants. CONCLUSIONS: Two major enteric glial classes were identified in the pediatric intestine, highlighting the significant postnatal contribution of Schwann-like enteric glia to glial heterogeneity. Crucially, these glial subtypes persist in aganglionic segments of patients with HSCR, offering a new target for their treatment using 5-HT agonists.
Cannabis, Cannabinoids, and the Endocannabinoid System-Is there Therapeutic Potential for Inflammatory Bowel Disease?
Cannabis sativa and its extracts have been used for centuries, both medicinally and recreationally. There is accumulating evidence that exogenous cannabis and related cannabinoids improve symptoms associated with inflammatory bowel disease [IBD], such as pain, loss of appetite, and diarrhoea. In vivo, exocannabinoids have been demonstrated to improve colitis, mainly in chemical models. Exocannabinoids signal through the endocannabinoid system, an increasingly understood network of endogenous lipid ligands and their receptors, together with a number of synthetic and degradative enzymes and the resulting products. Modulating the endocannabinoid system using pharmacological receptor agonists, genetic knockout models, or inhibition of degradative enzymes have largely shown improvements in colitis in vivo. Despite these promising experimental results, this has not translated into meaningful benefits for human IBD in the few clinical trials which have been conducted to date, the largest study being limited by poor medication tolerance due to the Δ9-tetrahydrocannabinol component. This review article synthesises the current literature surrounding the modulation of the endocannabinoid system and administration of exocannabinoids in experimental and human IBD. Findings of clinical surveys and studies of cannabis use in IBD are summarised. Discrepancies in the literature are highlighted together with identifying novel areas of interest.
Goblet cell differentiation subgroups in colorectal cancer.
The poor prognosis of relatively undifferentiated cancers has long been recognized, suggesting that selection against differentiation and in favor of uncontrolled growth is one of the most powerful drivers of cancer progression. Goblet cells provide the mucous surface of the gut, and when present in colorectal cancers (CRC), the cancers are called mucinous. We have used the presence of MUC2, the main mucous product of goblet cells, and an associated gene product, TFF3, to classify a large panel of nearly 80 CRC-derived cell lines into five categories based on their levels of MUC2 and TFF3 expression. We have then shown that these five patterns of expression can be easily identified in the direct analysis of tumor specimens allowing a much finer characterization of CRCs with respect to the presence of goblet cell differentiation. In particular, about 30% of all CRCs fall into the category of expressing TFF3 but not MUC2, which has not previously been acknowledged. Using the cell line data, we suggest that there are up to 12 genes (MUC2, TFF3, ATOH1, SPDEF, CDX1, CDX2, GATA6, HES1, ETS2, OLFM4, TOX3, and LGR5) that may be involved in selection against goblet cell differentiation in CRC by changes in methylation rather than mutations. Of these, LGR5, which is particularly associated with lack of goblet cell features, may function in the control of differentiation rather than direct control of cell growth, as has so far mostly been assumed. These results emphasize the importance of methylation changes in driving cancer progression.
Bone Morphogenetic Protein Pathway Antagonism by Grem1 Regulates Epithelial Cell Fate in Intestinal Regeneration.
BACKGROUND & AIMS: In homeostasis, intestinal cell fate is controlled by balanced gradients of morphogen signaling. The bone morphogenetic protein (BMP) pathway has a physiological, prodifferentiation role, predominantly inferred through previous experimental pathway inactivation. Intestinal regeneration is underpinned by dedifferentiation and cell plasticity, but the signaling pathways that regulate this adaptive reprogramming are not well understood. We assessed the BMP signaling landscape and investigated the impact and therapeutic potential of pathway manipulation in homeostasis and regeneration. METHODS: A novel mouse model was generated to assess the effect of the autocrine Bmp4 ligand on individual secretory cell fate. We spatiotemporally mapped BMP signaling in mouse and human regenerating intestine. Transgenic models were used to explore the functional impact of pathway manipulation on stem cell fate and intestinal regeneration. RESULTS: In homeostasis, ligand exposure reduced proliferation, expedited terminal differentiation, abrogated secretory cell survival, and prevented dedifferentiation. After ulceration, physiological attenuation of BMP signaling arose through upregulation of the secreted antagonist Grem1 from topographically distinct populations of fibroblasts. Concomitant expression supported functional compensation after Grem1 deletion from tissue-resident cells. BMP pathway manipulation showed that antagonist-mediated BMP attenuation was obligatory but functionally submaximal, because regeneration was impaired or enhanced by epithelial overexpression of Bmp4 or Grem1, respectively. Mechanistically, Bmp4 abrogated regenerative stem cell reprogramming despite a convergent impact of YAP/TAZ on cell fate in remodeled wounds. CONCLUSIONS: BMP signaling prevents epithelial dedifferentiation, and pathway attenuation through stromal Grem1 upregulation was required for adaptive reprogramming in intestinal regeneration. This intercompartmental antagonism was functionally submaximal, raising the possibility of therapeutic pathway manipulation in inflammatory bowel disease.
Large-scale sequencing identifies multiple genes and rare variants associated with Crohn's disease susceptibility.
Genome-wide association studies (GWASs) have identified hundreds of loci associated with Crohn's disease (CD). However, as with all complex diseases, robust identification of the genes dysregulated by noncoding variants typically driving GWAS discoveries has been challenging. Here, to complement GWASs and better define actionable biological targets, we analyzed sequence data from more than 30,000 patients with CD and 80,000 population controls. We directly implicate ten genes in general onset CD for the first time to our knowledge via association to coding variation, four of which lie within established CD GWAS loci. In nine instances, a single coding variant is significantly associated, and in the tenth, ATG4C, we see additionally a significantly increased burden of very rare coding variants in CD cases. In addition to reiterating the central role of innate and adaptive immune cells as well as autophagy in CD pathogenesis, these newly associated genes highlight the emerging role of mesenchymal cells in the development and maintenance of intestinal inflammation.