Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic, life-threatening disorder of the gastrointestinal tract that often arises during childhood. Pathogenesis is driven by dysregulated immune responses and inflammatory cytokine signaling, including interleukin-36 (IL-36)/IL-36 receptor (IL-36R) pathways. Work from our laboratory and others has demonstrated the key role of IL-36 cytokines and their signaling in the pathogenies of ulcerative colitis both in humans and preclinical mouse models. IL-36R regulates innate and T cell immunity, and IL-36 ligands are known to modulate dendritic cell (DC) functions, highlighting the importance of investigating their role in UC. Here, we aimed to investigate the specific role of IL-36R in mediating DC responses in IBD pathogenesis. We first analyzed single-cell RNA sequencing (scRNA-seq) datasets and colon biopsies from UC patients to assess IL-36R expression on gut-resident immune cells. Transcriptomic analysis revealed high IL36R expression in classical DC (cDC) subsets in UC, while digital spatial profiling of colon biopsies showed increased IL-36R expression in the lamina propria and barrier-adjacent regions, correlating positively with gut-resident DC markers (CD11c, CD103). Based on these findings, we generated cDC-specific Il36r knockout mice (Il36rΔcDC) to determine the role of IL-36R signaling in cDCs during experimental colitis. Age-matched, littermate controls were administered 2% dextran sulfate sodium (DSS) for 5 days and sacrificed on day 7. Il36rΔcDC mice were markedly protected from DSS-induced acute colitis, exhibiting reduced disease activity index, less body weight loss, reduced colon shortening, decreased inflammatory gene expression, and improved histology compared to wild-type counterparts. Under homeostasis, no baseline microbiome alterations were observed in Il36rΔcDC mice. However, RNA sequencing of colon tissue revealed upregulation of pathways linked to immune tolerance and barrier protection, including antigen processing and presentation, retinol metabolism, and mitogen-activated protein kinase signaling. These findings suggest that IL-36R deficiency in cDCs shapes the colonic microenvironment critical for barrier function and tolerance. Collectively, our data indicate that IL-36R signaling is altered in colonic cDCs of UC patients and that IL-36R deletion in cDCs protects mice against acute colitis, supporting further exploration of IL-36R-targeted therapies in UC.

Abstract Deficiency of the Interleukin-36 Receptor antagonist (DITRA) is a rare autoinflammatory condition which commonly manifests as severe, recurrent episodes of Generalized Pustular Psoriasis (GPP). Loss-of-function mutations in the IL36RN gene result in unopposed IL-36 cytokine signalling leading to severe psoriatic inflammation, which can be successfully treated with Anti-IL-36 receptor (IL-36R) monoclonal antibodies. Despite such advances, there remain some key questions concerning how loss of a functional IL-36R antagonist predisposes to GPP, including identifying the potential impacts of IL36RN mutations on skin homeostasis. To address this question, we investigated the consequences of IL-36Ra deficiency using Il36rn -/- mice, which recapitulate the severe psoriatic inflammation observed in DITRA patients. Here, we demonstrate, that in overtly healthy Il36rn -/- mice, prior to disease onset, there is disrupted dermal immune homeostasis, characterised by decreased expression of the chemokine CCL27. Altered skin homeostasis occurred in association with dysbiosis of the skin microbiome, characterised by a significant outgrowth of the commensal bacteria, Cutibacterium acnes . Importantly, intradermal administration of recombinant CCL27, prior to disease induction, significantly reduced the enhanced severity of psoriasiform inflammation, demonstrating a central role for this chemokine in regulating predisposition to increased severity. Transcriptomic analysis of GPP patient’s skin also revealed decreased CCL27 expression in non-lesional, as well as lesional, compared to healthy skin, indicating that this chemokine may also play a key instructive role among DITRA patients. Together, these data identify a novel mechanism through which IL-36Ra deficiency alters dermal homeostasis and predisposes to increased severity of psoriatic disease observed in DITRA patients.

Coeliac Disease (CeD) is a chronic gastrointestinal inflammatory disease initiated by dietary gluten in genetically predisposed individuals. While the inflammatory processes which drive tissue destruction in the coeliac duodenum have been extensively characterized, an increased oxidative stress (OS) response has also been suggested to contribute to CeD pathogenesis. However, the precise mechanisms which regulate OS in the coeliac mucosa and whether they impact inflammation remain ill defined. The master anti-oxidant transcriptional regulator Nuclear factor erythroid 2-related factor 2 (Nrf2), and its inhibitor, Kelch like ECH-associated protein 1 (Keap1) have been implicated in chronic gastrointestinal inflammatory diseases, such as ulcerative colitis but have been largely unexplored in the context of CeD. To investigate redox balance in the CeD duodenum, we utilized single cell transcriptomics to assess overall OS and cytoprotective Nrf2 activation across cell subsets in duodenal biopsies from CeD patients. OS induced gene expression was broadly increased across multiple cell subsets in the CeD mucosa. Simultaneously, specific markers of Nrf2 activation were decreased in cell subtypes central to pathogenesis of CeD, including activated CD4+ T cells and intraepithelial T lymphocytes, indicating a distinct redox imbalance in these cells. Furthermore, pharmacological activation of Nrf2 significantly decreased gliadin induced IFNG expression in CeD duodenal biopsies. Taken together, our findings demonstrate that redox imbalance represents a therapeutic opportunity for the modulation of proinflammatory responses that drive the pathogenesis of CeD.

OBJECTIVES:There is a paucity of validated predictors of response to anti-tumor necrosis factor (TNF) in pediatric Crohn's disease (CD). We aimed to evaluate the predictive utility of intestinal gene expression to predict response to anti-TNF in children with CD. METHODS:We enrolled children with CD before initiating anti-TNF as part of the prospective biobank of the pediatric inflammatory bowel disease Porto group of ESPGHAN. Genes potentially associated with therapeutic response were first preselected from a systematic literature review. Ribonucleic acid was extracted and sequenced from inflamed ileal biopsies of 20 children before initiating anti-TNF (13 with steroid-free remission [SFR] at 12 months, and seven with primary nonresponse [PNR]). An external validation cohort including 22 children (21 SFR, 1 PNR) was enrolled from Germany and Canada. Using maximum relevance-minimum redundancy (mRMR) methods, we constructed a support vector machine-learning model evaluated via leave-one-out cross-validation and permutation testing. RESULTS:Of 1799 studies identified in the systematic review, 24 met the inclusion criteria, reporting on 150 genes possibly associated with anti-TNF response in children or adults. In the Porto group cohort, 30 genes were associated with treatment response, of which five (TREM1, IL23R, CCL7, IL17F, and YES1) were most frequently selected. A multivariable model of these genes achieved high predictive utility (area under receiver operating characteristic curve: 0.88 [95% confidence interval: 0.69-1.0], sensitivity/specificity/positive predictive value/negative predictive value: 92%/71%/86%/83%). The same genomic signature in external validation achieved accuracy of 82% (i.e., 18/22 samples were classified correctly, including the single PNR patient). CONCLUSION:Increased expression of five genes is associated with higher rate of anti-TNF response in pediatric CD. Prospective studies are now warranted to validate these genes as biomarkers for treatment selection.

Patients with inflammatory bowel disease (IBD) have an increased risk of venous thromboembolism (VTE), but the underlying mechanistic basis remains poorly defined. Here, we find that colitogenic CD4+ T cells express tissue factor (TF) and promote rapid TF-dependent plasma thrombin generation. TF+CD3+CD4+ T cells are present in both the colons of mice with experimental colitis and blood and colonic tissue from patients with IBD. Expression of genes involved in regulating coagulation, including Protein C (PC; encoded by PROC) and its receptor (PROCR), are dysregulated in IBD patient gut biopsy tissues. Moreover, activated PC signalling reduces the procoagulant activity mediated by TF+CD4+ T cells. Our data thus identify TF-induced, colitogenic T cell-mediated thrombogenicity, and also demonstrate a new function for activated PC signalling in regulating T cell thrombo-inflammatory activity.

Abstract Background IBD patients exhibit up to 6-fold increased risk of venous thromboembolism (VTE) compared to the general population, although the mechanistic basis for this increased risk is unknown. Pre-clinical studies suggest an important role for diminished protein C (PC) pathway activity in IBD pathophysiology. Aims To evaluate the procoagulant activity of colitogenic T cells and assess the impact of activated protein C (APC) signalling in regulating T cell immunothrombotic activity. Methods The pro-thrombotic potential of T cells was determined using bespoke T-cell-dependent calibrated automated thrombinography assays, flow cytometry and microscopy. Meta-transcriptomic and gene expression analysis were used to assess the dysregulated expression of coagulation-associated genes in IBD patient gut biopsies. To elucidate the potential anti-thromboinflammatory effect of APC on T cells, ex vivo functional assays were established using paediatric IBD patients or donor PBMC-derived T cells treated with APC variants. Results We identified the presence of tissue factor (TF)+CD4+CD3+ T cells in the colons of both colitogenic mice and paediatric IBD patients during active disease (Fig 1.A-E). TF is best known as the predominant cellular initiator of blood coagulation, and subsequent ex vivo functional studies revealed that pro-inflammatory CD4+ T cells upregulate TF expression in response to T cell receptor ligation, resulting in rapid TF-dependent plasma thrombin generation and clot formation. TF is typically expressed in an ‘encrypted’ state and requires decryption for optimal procoagulant activity. Notably, activated and inflammatory CD4+ T cells were found to express significantly increased levels of acid sphingomyelinase and protein disulfide isomerase, critical mediators for TF decryption, on their cell membrane compared to naïve T cells. To understand how this process might be regulated, we performed meta-transcriptomic and gene expression analysis of IBD patient gut biopsy tissue, identifying dysregulated expression of genes involved in the regulation of coagulation, including PC (PROC) and its receptor (EPCR; PROCR) in IBD patients (Fig 1.F), revealing an environment permissive to, but deficient in, PC pathway anti-inflammatory activity. Subsequent functional studies revealed that activated protein C (APC) anti-inflammatory signalling reduced colitogenic T cell generation and activity, potently impaired TF decryption and significantly reduced T cell-mediated thrombin generation and clot formation (Fig 1.G-K). Conclusion These data implicate activated T cell thrombogenicity in IBD and demonstrate a prominent role for APC signalling in regulating colitogenic T cell thrombo-inflammatory activity.

Pathological increases in vascular permeability cause edema and swelling, driving retinal and neurological disorders. Few factors are known to specifically enhance barrier integrity and prevent fluid leakage. Here, we examine the effects of IL-36 receptor (IL-36R) activation on vascular permeability in vivo in mice, ex vivo in tissue explants, and in vitro in primary mouse and human microvascular endothelial cells. Using a soluble, biologically active DEVD-modified recombinant IL-36β cytokine, we find that the processed DEVDIL-36β strengthens endothelial barrier function, reduces vascular leakage, and limits pathology. Cell-specific knockdown of IL-36R confirms that endothelial IL-36R signaling mediates these barrier-promoting effects. IL-36R localizes to the plasma membrane in response to the loss of endothelial cell-cell contact, consistent with its role in tissue stress responses. Mechanistically, IL-36R signaling enhances adherens and tight junctions, induces vasculoprotective processes, and drives vessel remodeling and stabilization. RNA sequencing supports these findings, establishing IL-36R as a regulator of vascular integrity.

Although the orchestrating role of Interleukin-36 cytokines in regulating inflammation at barrier tissue sites, is well established, whether they play a significant role in the settings of metabolic health and disease, has yet to be fully established. Several recent studies have demonstrated that IL-36 cytokine expression is elevated among adult patients with obesity, and can play roles in regulating both insulin sensitivity and driving inflammation. In this report, we have extended these analyses to paediatric patients and identified an association between elevated serum levels of expression of the specific Interleukin-36 subfamily member, IL-36β, among children with obesity displaying insulin sensitivity, compared to children with obesity who are insulin resistant. While these data further indicate a possible protective role for IL-36 in metabolic health, they also differ with previous findings from an adult patient cohort, where elevated levels of the related cytokine, IL-36γ, were found to occur in association with improved metabolic health. While highlighting important differences between paediatric and adult patient cohorts in the context of metabolic disease associated with obesity, these data underscore the need for a deeper mechanistic analysis of the role of IL-36 cytokines in disease.

Recently, we reported that, in the naked mole-rat (Heterocephalus glaber) ovary, there is mitotic expansion of the primordial germ cells (PGCs), and the initiation of the meiotic program occurs postnatally. This is opposite to almost all other mammals, including humans and mice, whose reproductive cycle begins very early in development. In both mouse and human, the ovaries become populated with PGCs in utero; these PGCs will later generate the oogonia. After mitotic proliferation, these cells will trigger the meiotic program and initiate meiotic prophase I. Given that all these processes happen in utero, their analysis has been very challenging; so the ability to study them postnatally and to manipulate them with inhibitors or other substances, in the naked mole-rat, opens new possibilities in the field. In this chapter, we present a comprehensive collection of protocols that permit the culture of whole naked mole-rat ovaries, followed by analysis of germ cells, from PGCs to oocytes, in meiotic prophase I, as well the obtention of single-cell suspension or single-nuclei suspension for RNASeq.

ABSTRACT Background Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease, however, its role in inflammation-induced hypercoagulability is poorly understood. Objective/Methods Using novel myeloid cell-based global haemostasis assays and murine models of immunometabolic disease, we evaluated the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. Results Glycolysis was essential for enhanced activated myeloid cell tissue factor expression and decryption, driving increased cell-dependent thrombin generation in response to inflammatory challenge. Similarly, inhibition of glycolysis enhanced activated macrophage fibrinolytic activity via reduced plasminogen activator inhibitor 1 (PAI-1)-activity. Macrophage polarisation or activation markedly increased endothelial protein C receptor (EPCR) expression on monocytes and macrophages, leading to increased myeloid cell-dependent protein C activation. Importantly, inflammation-dependent EPCR expression on tissue-resident macrophages was also observed in vivo . Adipose tissue macrophages from obese mice fed a high-fat diet exhibited significantly enhanced EPCR expression and APC generation compared to macrophages isolated from the adipose tissue of healthy mice. Similarly, the induction of colitis in mice prompted infiltration of EPCR + innate myeloid cells within inflamed colonic tissue that were absent from the intestinal tissue of healthy mice. Conclusion Collectively, this study identifies immunometabolic regulation of myeloid cell hypercoagulability, opening new therapeutic possibilities for targeted mitigation of thrombo-inflammatory disease. ESSENTIALS Inflammation-mediated glycolytic reprogramming enables myeloid cell-induced hypercoagulability and antifibrinolytic activity. 2-Deoxy-D-glucose (2-DG) inhibits the expression of transcription factors necessary for inflammation-induced procoagulant gene expression. Myeloid cell membrane regulation of tissue factor procoagulant activity is glycolysis-dependent. Activation of myeloid innate immunity dysregulates activated protein C anticoagulant pathway activity.

Recent advances in understanding how the microbiome can influence both the physiology and the pathogenesis of disease in humans have highlighted the importance of gaining a deeper insight into the complexities of the host-microbial dialogue. In tandem with this progress, has been a greater understanding of the biological pathways which regulate both homeostasis and inflammation at barrier tissue sites, such as the skin and the gut. In this regard, the Interleukin-1 family of cytokines, which can be segregated into IL-1, IL-18 and IL-36 subfamilies, have emerged as important custodians of barrier health and immunity. With established roles as orchestrators of various inflammatory diseases in both the skin and intestine, it is now becoming clear that IL-1 family cytokine activity is not only directly influenced by external microbes, but can also play important roles in shaping the composition of the microbiome at barrier sites. This review explores the current knowledge surrounding the evidence that places these cytokines as key mediators at the interface between the microbiome and human health and disease at the skin and intestinal barrier tissues.

In the long-lived naked mole-rat (NMR), the entire process of oogenesis occurs postnatally. Germ cell numbers increase significantly in NMRs between postnatal days 5 (P5) and P8, and germs cells positive for proliferation markers (Ki-67, pHH3) are present at least until P90. Using pluripotency markers (SOX2 and OCT4) and the primordial germ cell (PGC) marker BLIMP1, we show that PGCs persist up to P90 alongside germ cells in all stages of female differentiation and undergo mitosis both in vivo and in vitro. We identified VASA+ SOX2+ cells at 6 months and at 3-years in subordinate and reproductively activated females. Reproductive activation was associated with proliferation of VASA+ SOX2+ cells. Collectively, our results suggest that highly desynchronized germ cell development and the maintenance of a small population of PGCs that can expand upon reproductive activation are unique strategies that could help to maintain the NMR's ovarian reserve for its 30-year reproductive lifespan.

Pattern recognition receptors (PRRs) of the innate immune system represent the critical front-line defense against pathogens, and new vaccine formulations target these PRR pathways to boost vaccine responses, through activation of cellular/Th1 immunity. The majority of pediatric vaccines contain aluminum (ALUM) or monophosphoryl lipid A (MPLA) as adjuvants to encourage immune activation. Evidence suggests that elements of the innate immune system, currently being targeted for vaccine adjuvanticity do not fully develop until puberty and it is likely that effective adjuvants for the neonatal and pediatric populations are being overlooked due to modeling of responses in adult systems. We recently reported that the activity of the cytosolic nucleic acid (CNA) sensing family of PRRs is strong in cord blood and peripheral blood of young children. This study investigates the function of CNA sensors in subsets of neonatal innate immune cells and shows that myeloid cells from cord blood can be activated to express T cell costimulatory markers, and also to produce Th1 promoting cytokines. CD80 and CD86 were consistently up-regulated in response to cytosolic Poly(I:C) stimulation in all cell types examined and CNA activation also induced robust Type I IFN and low levels of TNFα in monocytes, monocyte-derived macrophages, and monocyte-derived dendritic cells. We have compared CNA activation to adjuvants currently in use (MPLA or ALUM), either alone or in combination and found that cytosolic Poly(I:C) in combination with MPLA or ALUM can improve expression of activation marker levels above those observed with either adjuvant alone. This may prove particularly promising in the context of improving the efficacy of existing ALUM- or MPLA-containing vaccines, through activation of T cell-mediated immunity.

Obesity rates are continually increasing both in the USA and around the world. As rates of obesity continue to grow, public health concerns remain a top priority in order to curtail mortality and morbidity. An increase in visceral adipose tissue is correlated with dysfunction in multiple organs, including the kidneys. Increases in basal metabolic index, or BMI, has been linked to increases in triglyceride accumulation in the kidneys. Obesity accounts for ~25% of kidney diseases observed in women; however, its effects on kidney function have not been defined in post-menopausal women. In an ovarian hormone deficient mouse model (ovariectomized animals), we measured a significant weight gain compared with sham-operated control, associated with perirenal adipose tissue accumulation, and an increase in 1. glomerular filtration rate (GFR) used to determine renal function, 2. protein level of the NGAL, an early biomarker of kidney injury and 3. intrarenal lipid accumulation found in the renal tubules. In this study, we aim to determine whether obesity affects the levels of renal injury and lipid accumulation measured in a model of ovarian hormone deficiency. We studied four groups of mice: the first and second groups underwent ovariectomy (OVX); group one was fed a 60% high-fat (HF) diet, and group two was on a 10% low-fat (LH) diet. The third and fourth groups (controls), underwent surgery without ovariectomy (SHAM) and were fed a HF or LF diet. All animals had ad libitum access to their respective diet for fourteen weeks (i.e., ten weeks prior to surgery and four weeks after). Significant increase in body weight was measured in mice on HF compared to LF diet. The size of triglyceride droplets in the renal tubules was significantly higher (20-25%) in OVX compared to SHAM-operated control mice on both HF and LF diets. The Neutrophil Gelatinase Associated Lipocalin (NGAL) protein levels were measured as a biomarker of kidney injury. Levels of NGAL protein expression increased in OVX mice when compared to SHAM control in the renal tubules of mice on both HF (211.3 ± 3.7%, p<0.0001) and LF diet (111.33 ± 1.7%, p=0.02). In the glomeruli, NGAL protein expression levels increased in OVX mice compared to SHAM control in HF (188.8 ± 9.9%, p<0.0001) and LF diet fed groups (113.1 ± 4.9%, p=0.64). Furthermore, mice on HF diet had significant higher NGAL levels in OVX and sham-control conditions than mice in the same conditions on LF diet. These findings indicate that weight gain exacerbates renal injury induced by OVX, and that estrogen may be protective against excessive accumulation of triglycerides in kidney structures. Our study supports the understanding of the mechanisms implicated in the development of kidney diseases in obese post-menopausal women.