A pro-inflammatory gut mucosal cytokine response is associated with mild COVID-19 disease and superior induction of serum antibodies.

The relationship between gastrointestinal tract infection, the host immune response, and the clinical outcome of disease is not well understood in COVID-19. We sought to understand the effect of intestinal immune responses to SARS-CoV-2 on patient outcomes including the magnitude of systemic antibody induction. Combining two prospective cohort studies, International Severe Acute Respiratory and emerging Infections Consortium Comprehensive Clinical Characterisations Collaboration (ISARIC4C) and Integrated Network for Surveillance, Trials and Investigations into COVID-19 Transmission (INSTINCT), we acquired samples from 88 COVID-19 cases representing the full spectrum of disease severity and analysed viral RNA and host gut cytokine responses in the context of clinical and virological outcome measures. There was no correlation between the upper respiratory tract and faecal viral loads. Using hierarchical clustering, we identified a group of fecal cytokines including Interleukin-17A, Granulocyte macrophage colony-stimulating factor, Tumor necrosis factorα, Interleukin-23, and S100A8, that were transiently elevated in mild cases and also correlated with the magnitude of systemic anti-Spike-receptor-binding domain antibody induction. Receiver operating characteristic curve analysis showed that expression of these gut cytokines at study enrolment in hospitalised COVID-19 cases was associated negatively with overall clinical severity implicating a protective role in COVID-19. This suggests that a productive intestinal immune response may be beneficial in the response to a respiratory pathogen and a biomarker of a successful barrier response.

Alterations in the gut microbiome implicate key taxa and metabolic pathways across inflammatory arthritis phenotypes.

Musculoskeletal diseases affect up to 20% of adults worldwide. The gut microbiome has been implicated in inflammatory conditions, but large-scale metagenomic evaluations have not yet traced the routes by which immunity in the gut affects inflammatory arthritis. To characterize the community structure and associated functional processes driving gut microbial involvement in arthritis, the Inflammatory Arthritis Microbiome Consortium investigated 440 stool shotgun metagenomes comprising 221 adults diagnosed with rheumatoid arthritis, ankylosing spondylitis, or psoriatic arthritis and 219 healthy controls and individuals with joint pain without an underlying inflammatory cause. Diagnosis explained about 2% of gut taxonomic variability, which is comparable in magnitude to inflammatory bowel disease. We identified several candidate microbes with differential carriage patterns in patients with elevated blood markers for inflammation. Our results confirm and extend previous findings of increased carriage of typically oral and inflammatory taxa and decreased abundance and prevalence of typical gut clades, indicating that distal inflammatory conditions, as well as local conditions, correspond to alterations to the gut microbial composition. We identified several differentially encoded pathways in the gut microbiome of patients with inflammatory arthritis, including changes in vitamin B salvage and biosynthesis and enrichment of iron sequestration. Although several of these changes characteristic of inflammation could have causal roles, we hypothesize that they are mainly positive feedback responses to changes in host physiology and immune homeostasis. By connecting taxonomic alternations to functional alterations, this work expands our understanding of the shifts in the gut ecosystem that occur in response to systemic inflammation during arthritis.

A conserved population of MHC II-restricted, innate-like, commensal-reactive T cells in the gut of humans and mice.

Interactions with commensal microbes shape host immunity on multiple levels and play a pivotal role in human health and disease. Tissue-dwelling, antigen-specific T cells are poised to respond to local insults, making their phenotype important in the relationship between host and microbes. Here we show that MHC-II restricted, commensal-reactive T cells in the colon of both humans and mice acquire transcriptional and functional characteristics associated with innate-like T cells. This cell population is abundant and conserved in the human and murine colon and endowed with polyfunctional effector properties spanning classic Th1- and Th17-cytokines, cytotoxic molecules, and regulators of epithelial homeostasis. T cells with this phenotype are increased in ulcerative colitis patients, and their presence aggravates pathology in dextran sodium sulphate-treated mice, pointing towards a pathogenic role in colitis. Our findings add to the expanding spectrum of innate-like immune cells positioned at the frontline of intestinal immune surveillance, capable of acting as sentinels of microbes and the local cytokine milieu.

Defactinib inhibits PYK2 phosphorylation of IRF5 and reduces intestinal inflammation.

Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, and has a key pathogenic function in gut inflammation, but how IRF5 is modulated is still unclear. Having performed a kinase inhibitor library screening in macrophages, here we identify protein-tyrosine kinase 2-beta (PTK2B/PYK2) as a putative IRF5 kinase. PYK2-deficient macrophages display impaired endogenous IRF5 activation, leading to reduction of inflammatory gene expression. Meanwhile, a PYK2 inhibitor, defactinib, has a similar effect on IRF5 activation in vitro, and induces a transcriptomic signature in macrophages similar to that caused by IRF5 deficiency. Finally, defactinib reduces pro-inflammatory cytokines in human colon biopsies from patients with ulcerative colitis, as well as in a mouse colitis model. Our results thus implicate a function of PYK2 in regulating the inflammatory response in the gut via the IRF5 innate sensing pathway, thereby opening opportunities for related therapeutic interventions for inflammatory bowel diseases and other inflammatory conditions.

IL-1-driven stromal-neutrophil interactions define a subset of patients with inflammatory bowel disease that does not respond to therapies.

Current inflammatory bowel disease (IBD) therapies are ineffective in a high proportion of patients. Combining bulk and single-cell transcriptomics, quantitative histopathology and in situ localization across three cohorts of patients with IBD (total n = 376), we identify coexpressed gene modules within the heterogeneous tissular inflammatory response in IBD that map to distinct histopathological and cellular features (pathotypes). One of these pathotypes is defined by high neutrophil infiltration, activation of fibroblasts and vascular remodeling at sites of deep ulceration. Activated fibroblasts in the ulcer bed display neutrophil-chemoattractant properties that are IL-1R, but not TNF, dependent. Pathotype-associated neutrophil and fibroblast signatures are increased in nonresponders to several therapies across four independent cohorts (total n = 343). The identification of distinct, localized, tissular pathotypes will aid precision targeting of current therapeutics and provides a biological rationale for IL-1 signaling blockade in ulcerating disease.

Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice.

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.

Overexpression of Cancer-Associated Stem Cell Gene OLFM4 in the Colonic Epithelium of Patients With Primary Sclerosing Cholangitis.

BACKGROUND: To examine immune-epithelial interactions and their impact on epithelial transformation in primary sclerosing cholangitis-associated ulcerative colitis (PSC-UC) using patient-derived colonic epithelial organoid cultures (EpOCs). METHODS: The EpOCs were originated from colonic biopsies from patients with PSC-UC (n = 12), patients with UC (n = 14), and control patients (n = 10) and stimulated with cytokines previously associated with intestinal inflammation (interferon (IFN) γ and interleukin (IL)-22). Markers of cytokine downstream pathways, stemness, and pluripotency were analyzed by real-time quantitative polymerase chain reaction and immunofluorescence. The OLFM4 expression in situ was assessed by RNAscope and immunohistochemistry. RESULTS: A distinct expression of stem cell-associated genes was observed in EpOCs derived from patients with PSC-UC, with lower expression of the classical stem-cell marker LGR5 and overexpression of OLFM4, previously associated with pluripotency and early stages of neoplastic transformation in the gastrointestinal and biliary tracts. High levels of OLFM4 were also found ex vivo in colonic biopsies from patients with PSC-UC. In addition, IFNγ stimulation resulted in the downregulation of LGR5 in EpOCs, whereas higher expression of OLFM4 was observed after IL-22 stimulation. Interestingly, expression of the IL-22 receptor, IL22RA1, was induced by IFNγ, suggesting that a complex interplay between these cytokines may contribute to carcinogenesis in PSC-UC. CONCLUSIONS: Higher expression of OLFM4, a cancer stemness gene induced by IL-22, is present in PSC-UC, suggesting that IL-22 responses may result in alterations of the intestinal stem-cell niche in these patients.

The Interleukin 22 Pathway Interacts with Mutant KRAS to Promote Poor Prognosis in Colon Cancer.

PURPOSE: The cytokine IL22 promotes tumor progression in murine models of colorectal cancer. However, the clinical significance of IL22 in human colorectal cancer remains unclear. We sought to determine whether the IL22 pathway is associated with prognosis in human colorectal cancer, and to identify mechanisms by which IL22 can influence disease progression. EXPERIMENTAL DESIGN: Transcriptomic data from stage II/III colon cancers in independent discovery (GSE39582 population-based cohort, N = 566) and verification (PETACC3 clinical trial, N = 752) datasets were used to investigate the association between IL22 receptor expression (encoded by the genes IL22RA1 and IL10RB), tumor mutation status, and clinical outcome using Cox proportional hazard models. Functional interactions between IL22 and mutant KRAS were elucidated using human colorectal cancer cell lines and primary tumor organoids. RESULTS: Transcriptomic analysis revealed a poor-prognosis subset of tumors characterized by high expression of IL22RA1, the alpha subunit of the heterodimeric IL22 receptor, and KRAS mutation [relapse-free survival (RFS): HR = 2.93, P = 0.0006; overall survival (OS): HR = 2.45, P = 0.0023]. KRAS mutations showed a similar interaction with IL10RB and conferred the worst prognosis in tumors with high expression of both IL22RA1 and IL10RB (RFS: HR = 3.81, P = 0.0036; OS: HR = 3.90, P = 0.0050). Analysis of human colorectal cancer cell lines and primary tumor organoids, including an isogenic cell line pair that differed only in KRAS mutation status, showed that IL22 and mutant KRAS cooperatively enhance cancer cell proliferation, in part through augmentation of the Myc pathway. CONCLUSIONS: Interactions between KRAS and IL22 signaling may underlie a previously unrecognized subset of clinically aggressive colorectal cancer that could benefit from therapeutic modulation of the IL22 pathway.

Alpha kinase 1 controls intestinal inflammation by suppressing the IL-12/Th1 axis.

Inflammatory bowel disease (IBD) are heterogenous disorders of the gastrointestinal tract caused by a spectrum of genetic and environmental factors. In mice, overlapping regions of chromosome 3 have been associated with susceptibility to IBD-like pathology, including a locus called Hiccs. However, the specific gene that controls disease susceptibility remains unknown. Here we identify a Hiccs locus gene, Alpk1 (encoding alpha kinase 1), as a potent regulator of intestinal inflammation. In response to infection with the commensal pathobiont Helicobacter hepaticus (Hh), Alpk1-deficient mice display exacerbated interleukin (IL)-12/IL-23 dependent colitis characterized by an enhanced Th1/interferon(IFN)-γ response. Alpk1 controls intestinal immunity via the hematopoietic system and is highly expressed by mononuclear phagocytes. In response to Hh, Alpk1-/- macrophages produce abnormally high amounts of IL-12, but not IL-23. This study demonstrates that Alpk1 promotes intestinal homoeostasis by regulating the balance of type 1/type 17 immunity following microbial challenge.

Foxp3+ T reg cells control psoriasiform inflammation by restraining an IFN-I-driven CD8+ T cell response.

Psoriasis is a complex inflammatory skin disease affecting ∼3% of the population worldwide. Although type I interferons (IFN-I) are thought to be involved in its pathogenesis, the details of this relationship remain elusive. Here we show that in a murine model of imiquimod-driven psoriatic skin inflammation, Foxp3+ regulatory T cells (T reg cells) control inflammation severity by restraining IFN-I. Depletion of T reg cells induces IFN-I and IFN-stimulated gene expression, and leads to accumulation of CD8+ T cells in lesional skin. Mononuclear phagocytes (MNPs) were the source of IFN-I, and their depletion reversed the effect of T reg cell depletion. Blockade of IFN-I signaling abolished CD8+ T cell infiltration and excess inflammation in the skin of T reg cell-depleted mice. Depletion of CD8+ T cells attenuated pathology, confirming their role as critical effector cells downstream of IFN-I. Our results describe an unexpected role for T reg cells in restraint of an MNP-IFN-I-driven CD8+ T cell response during psoriasiform skin inflammation. These findings highlight a pathway with potential relevance for the treatment of early-stage disease.

A Large Polysaccharide Produced by Helicobacter hepaticus Induces an Anti-inflammatory Gene Signature in Macrophages.

Interactions between the host and its microbiota are of mutual benefit and promote health. Complex molecular pathways underlie this dialog, but the identity of microbe-derived molecules that mediate the mutualistic state remains elusive. Helicobacter hepaticus is a member of the mouse intestinal microbiota that is tolerated by the host. In the absence of an intact IL-10 signaling, H. hepaticus induces an IL-23-driven inflammatory response in the intestine. Here we investigate the interactions between H. hepaticus and host immune cells that may promote mutualism, and the microbe-derived molecule(s) involved. Our results show that H. hepaticus triggers early IL-10 induction in intestinal macrophages and produces a large soluble polysaccharide that activates a specific MSK/CREB-dependent anti-inflammatory and repair gene signature via the receptor TLR2. These data identify a host-bacterial interaction that promotes mutualistic mechanisms at the intestinal interface. Further understanding of this pathway may provide novel prevention and treatment strategies for inflammatory bowel disease.

Tumour-associated changes in intestinal epithelial cells cause local accumulation of KLRG1+ GATA3+ regulatory T cells in mice.

CD4+ Foxp3+ regulatory T (Treg) cells include differentiated populations of effector Treg cells characterized by the expression of specific transcription factors. Tumours, including intestinal malignancies, often present with local accumulation of Treg cells that can prevent tumour clearance, but how tumour progression leads to Treg cell accumulation is incompletely understood. Here using genetically modified mouse models we show that ablation of E-cadherin, a process associated with epithelial to mesenchymal transition and tumour progression, promotes the accumulation of intestinal Treg cells by the specific accumulation of the KLRG1+ GATA3+ Treg subset. Epithelial E-cadherin ablation activates the ß-catenin pathway, and we find that increasing ß-catenin signals in intestinal epithelial cells also boosts Treg cell frequencies through local accumulation of KLRG1+ GATA3+ Treg cells. Both E-cadherin ablation and increased ß-catenin signals resulted in epithelial cells with higher levels of interleukin-33, a cytokine that preferentially expands KLRG1+ GATA3+ Treg cells. Tumours often present reduced E-cadherin expression and increased ß-catenin signalling and interleukin-33 production. Accordingly, Treg cell accumulation in intestinal tumours from APCmin/+ mice was exclusively due to the increase in KLRG1+ GATA3+ Treg cells. Our data identify a novel axis through which epithelial cells control local Treg cell subsets, which may be activated during intestinal tumorigenesis.

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.

The human tissue-biomaterial interface: a role for PPARγ-dependent glucocorticoid receptor activation in regulating the CD163+ M2 macrophage phenotype.

In vivo studies of implanted acellular biological scaffolds in experimental animals have shown constructive remodeling mediated by anti-inflammatory macrophages. Little is known about the human macrophage response to such biomaterials, or the nature of the signaling mechanisms that govern the macrophage phenotype in this environment. The cellular events at the interface of a tissue and implanted decellularized biomaterial were examined by establishing a novel ex vivo tissue culture model in which surgically excised human urinary tract tissue was combined with porcine acellular bladder matrix (PABM). Evaluation of the tissue-biomaterial interface showed a time-dependent infiltration of the biomaterial by CD68(+) CD80(-) macrophages. The migration of CD68(+) cells from the tissue to the interface was accompanied by maturation to a CD163(hi) phenotype, suggesting that factor(s) associated with the biomaterial or the wound edge was/were responsible for the active recruitment and polarization of local macrophages. Glucocorticoid receptor (GR) and peroxisome proliferator activated receptor gamma (PPARγ) signaling was investigated as candidate pathways for integrating inflammatory responses; both showed intense nuclear labeling in interface macrophages. GR and PPARγ activation polarized peripheral blood-derived macrophages from a default M1 (CD80(+)) toward an M2 (CD163(+)) phenotype, but PPARγ signaling predominated, as its antagonism blocked any GR-mediated effect. Seeding on PABM was effective at polarizing peripheral blood-derived macrophages from a default CD80(+) phenotype on glass to a CD80(-) phenotype, with intense nuclear localization of PPARγ. These results endorse in vivo observations that the infiltration of decellularized biological scaffolds, exemplified here by PABM, is pioneered by macrophages. Thus, it appears that natural factors present in PABM are involved in the active recruitment and polarization of macrophages to a CD163(+) phenotype, with activation of PPARγ identified as the candidate pathway. The harnessing of these natural matrix-associated factors may be useful in enhancing the integration of synthetic and other natural biomaterials by polarizing macrophage activation toward an M2 regulatory phenotype.