Serum IL-36ß levels are associated with Insulin sensitivity in paediatric patients with obesity.

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-36ß, 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.

Glycolytic reprogramming fuels myeloid cell-driven hypercoagulability.

BACKGROUND: Myeloid cell metabolic reprogramming is a hallmark of inflammatory disease; however, its role in inflammation-induced hypercoagulability is poorly understood. OBJECTIVES: We aimed to evaluate the role of inflammation-associated metabolic reprogramming in regulating blood coagulation. METHODS: We used novel myeloid cell-based global hemostasis assays and murine models of immunometabolic disease. 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 through reduced plasminogen activator inhibitor 1 activity. Macrophage polarization 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 activated protein C generation compared with 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 thromboinflammatory disease.

Interleukin-1 family cytokines at the crossroads of microbiome regulation in barrier health and disease.

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.

Sex-specific trajectories of molecular cardiometabolic traits from childhood to young adulthood.

BACKGROUND: The changes which typically occur in molecular causal risk factors and predictive biomarkers for cardiometabolic diseases across early life are not well characterised. METHODS: We quantified sex-specific trajectories of 148 metabolic trait concentrations including various lipoprotein subclasses from age 7 years to 25 years. Data were from 7065 to 7626 offspring (11 702 to14 797 repeated measures) of the Avon Longitudinal Study of Parents and Children birth cohort study. Outcomes were quantified using nuclear magnetic resonance spectroscopy at 7, 15, 18 and 25 years. Sex-specific trajectories of each trait were modelled using linear spline multilevel models. RESULTS: Females had higher very-low-density lipoprotein (VLDL) particle concentrations at 7 years. VLDL particle concentrations decreased from 7 years to 25 years with larger decreases in females, leading to lower VLDL particle concentrations at 25 years in females. For example, females had a 0.25 SD (95% CI 0.20 to 0.31) higher small VLDL particle concentration at 7 years; mean levels decreased by 0.06 SDs (95% CI -0.01 to 0.13) in males and 0.85 SDs (95% CI 0.79 to 0.90) in females from 7 years to 25 years, leading to 0.42 SDs (95% CI 0.35 to 0.48) lower small VLDL particle concentrations in females at 25 years. Females had lower high-density lipoprotein (HDL) particle concentrations at 7 years. HDL particle concentrations increased from 7 years to 25 years with larger increases among females leading to higher HDL particle concentrations in females at 25 years. CONCLUSION: Childhood and adolescence are important periods for the emergence of sex differences in atherogenic lipids and predictive biomarkers for cardiometabolic disease, mostly to the detriment of males.

Postnatal oogenesis leads to an exceptionally large ovarian reserve in naked mole-rats.

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.

Myeloid cell-derived proteases produce a proinflammatory form of IL-37 that signals via IL-36 receptor engagement.

Interleukin-1 (IL-1) family cytokines are key barrier cytokines that are typically expressed as inactive, or partially active, precursors that require proteolysis within their amino termini for activation. IL-37 is an enigmatic member of the IL-1 family that has been proposed to be activated by caspase-1 and to exert anti-inflammatory activity through engagement of the IL-18R and SIGIRR. However, here we show that the longest IL-37 isoform, IL-37b, exhibits robust proinflammatory activity upon amino-terminal proteolysis by neutrophil elastase or cathepsin S. In sharp contrast, caspase-1 failed to process or activate IL-37 at concentrations that robustly activated its canonical substrate, IL-1ß. IL-37 and IL-36 exhibit high structural homology, and, consistent with this, a K53-truncated form of IL-37, mimicking the cathepsin S-processed form of this cytokine, was found to exert its proinflammatory effects via IL-36 receptor engagement and produced an inflammatory signature practically identical to IL-36. Administration of K53-truncated IL-37b intraperitoneally into wild-type mice also elicited an inflammatory response that was attenuated in IL-36R-/- animals. These data demonstrate that, in common with other IL-1 family members, mature IL-37 can also elicit proinflammatory effects upon processing by specific proteases.

Discovery and multi-parametric optimization of a high-affinity antibody against interleukin-25 with neutralizing activity in a mouse model of skin inflammation.

Background: Interleukin (IL)25 has been implicated in tissue homeostasis at barrier surfaces and the initiation of type two inflammatory signaling in response to infection and cell injury across multiple organs. We sought to discover and engineer a high affinity neutralizing antibody and evaluate the antibody functional activity in vitro and in vivo. Methods: In this study, we generated a novel anti-IL25 antibody (22C7) and investigated the antibody's therapeutic potential for targeting IL25 in inflammation. Results: A novel anti-IL25 antibody (22C7) was generated with equivalent in vitro affinity and potency against the human and mouse orthologs of the cytokine. This translated into in vivo potency in an IL25-induced air pouch model where 22C7 inhibited the recruitment of monocytes, macrophages, neutrophils and eosinophils. Furthermore, 22C7 significantly reduced ear swelling, acanthosis and disease severity in the Aldara mouse model of psoriasiform skin inflammation. Given the therapeutic potential of IL25 targeting in inflammatory conditions, 22C7 was further engineered to generate a highly developable, fully human antibody while maintaining the affinity and potency of the parental molecule. Conclusions: The generation of 22C7, an anti-IL25 antibody with efficacy in a preclinical model of skin inflammation, raises the therapeutic potential for 22C7 use in the spectrum of IL25-mediated diseases.

The IL-1 cytokine family as custodians of barrier immunity.

The interleukin-1 (IL-1) family of cytokines and receptors are implicated in the functioning of innate and adaptive immunity and the genesis of inflammation. They are widely expressed in structural and immune cells with marked expression within barrier mucosal surfaces. In the lung, gut and skin, which are common entry sites for pathogens, they play essential functions in maintaining the functional integrity of the barrier and manage innate and adaptive immunity in response to insult and infections. In tissue sites, the IL-1 cytokines are tightly regulated by mechanisms involving decoy receptors and protease degradation. Dysregulation of these processes are associated with aberrant tissue inflammation leading to a number of inflammatory diseases. This review will address the roles of the different IL-1 cytokines at the lung, gut and skin barrier surfaces at homeostasis, and their roles as inflammatory mediators in diseases such as asthma, chronic obstructive pulmonary disease, inflammatory bowel diseases, atopic dermatitis and psoriasis.

Senolytic treatment reverses obesity-mediated senescent cell accumulation in the ovary.

Senescent cells are in a cell cycle arrest state and accumulate with aging and obesity, contributing to a chronic inflammatory state. Treatment with senolytic drugs dasatinib and quercetin (D + Q) can reduce senescent cell burden in several tissues, increasing lifespan. Despite this, there are few reports about senescent cells accumulating in female reproductive tissues. Therefore, the aim of the study was to characterize the ovarian reserve and its relationship with cellular senescence in genetically obese mice (ob/ob). In experiment 1, ob/ob (n = 5) and wild-type (WT) mice (n = 5) at 12 months of age were evaluated. In experiment 2, 2-month-old female ob/ob mice were treated with senolytics (D + Q, n = 6) or placebo (n = 6) during the 4 months. Obese mice had more senescent cells in ovaries, indicated by increased p21 and p16 and lipofuscin staining and macrophage infiltration. Treatment with D + Q significantly reduced senescent cell burden in ovaries of obese mice. Neither obesity nor treatment with D + Q affected the number of ovarian follicles. In conclusion, our data indicate that obesity due to leptin deficiency increases the load of senescent cells in the ovary, which is reduced by treatment by senolytics. However, neither obesity nor D + Q treatment affected the ovarian reserve.

IL-36 cytokines imprint a colitogenic phenotype on CD4+ T helper cells.

IL-36 cytokines are emerging as potent orchestrators of intestinal inflammation and are being implicated in the pathogenesis of inflammatory bowel diseases (IBD). However, the mechanisms through which these cytokines mediate these effects remain to be fully uncovered. Here, we report specifically elevated expression of IL-36α, and not IL-36ß or IL-36γ in the serum of newly diagnosed, treatment naïve, paediatric IBD patients and identify T cells as primary cellular mediators of IL-36 responses in the inflamed gut. IL-36R expression on CD4+ T cells was found to promote intestinal pathology in a murine model of colitis. Consistent with these effects, IL-36R can act as a potent instructor of CD4+ T cell differentiation in vivo, enhancing Th1 responses, while inhibiting the generation of Tregs. In addition, loss of IL-36 responsiveness significantly reduced the migration of pathogenic CD4+ T cells towards intestinal tissues and IL-36 was found to act, uniquely among IL-1 family members, to induce the expression of gut homing receptors in proinflammatory murine and human CD4+ T cells. These data reveal an important role for IL-36 cytokines in driving the colitogenic potential of CD4+ T cells and identify a new mechanism through which they may contribute to disease pathogenesis.

Cytosolic dsRNA improves neonatal innate immune responses to adjuvants in use in pediatric vaccines.

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.

Regulation of CEACAM Family Members by IBD-Associated Triggers in Intestinal Epithelial Cells, Their Correlation to Inflammation and Relevance to IBD Pathogenesis.

Carcinoembryogenic antigen cellular adhesion molecules (CEACAMs) are intercellular adhesion molecules highly expressed in intestinal epithelial cells. CEACAM1, -3, -5, -6, -7 are altered in patients suffering from colon cancer and inflammatory bowel diseases (IBD), but their role in the onset and pathogenesis of IBD is not well known. Herein, we aim to correlate CEACAM1, -3, -5, -6, -7 expression to the degree of inflammation in pediatric and adult IBD colon biopsies and to examine the regulation of CEACAMs on human intestinal epithelial cell lines (C2BBe1/HT29) by different IBD-associated triggers (cytokines, bacteria/metabolites, emulsifiers) and IBD-drugs (6-Mercaptopurine, Prednisolone, Tofacitinib). Biopsies from patients with pediatric Crohn's disease (CD) and adult ulcerative colitis (UC, active/inactive disease) showed a significant increase in CEACAM3, -5, -6 expression, while CEACAM5 expression was reduced in adult CD patients (active/inactive disease). Intestinal epithelial cells cultured with a pro-inflammatory cytokine cocktail and Adherent-invasive Escherichia coli (AIEC) showed a rapid induction of CEACAM1, -5, -7 followed by a reduced RNA and protein expression overtime and a constant expression of CEACAM3, correlating with IL-8 expression. Cells cultured with the emulsifier polysorbate-80 resulted in a significant induction of CEACAM3, -5, -6, -7 at a late time point, while SCFA treatment reduced CEACAM1, -5, -7 expression. No major alterations in expression of CEACAMs were noted on cells cultured with the commensal Escherichia coli K12 or the pathogen Salmonella typhimurium. IBD drugs, particularly Tofacitinib, significantly reduced cytokine-induced CEACAM1, -3, -5, -6, -7 expression associated with a reduced IL-8 secretion. In conclusion, we provide new evidence on the regulation of CEACAMs by different IBD-associated triggers, identifying a role of CEACAMs in IBD pathogenesis.

SIGIRR Negatively Regulates IL-36-Driven Psoriasiform Inflammation and Neutrophil Infiltration in the Skin.

SIGIRR has been described as a negative regulator of several IL-1R/TLR family members and has been implicated in several inflammatory disease conditions. However, it is unknown whether it can suppress IL-36 family cytokines, which are members of the broader IL-1 superfamily that have emerged as critical orchestrators of psoriatic inflammation in both humans and mice. In this study, we demonstrate that SIGIRR is downregulated in psoriatic lesions in humans and mice, and this correlates with increased expression of IL-36 family cytokines. Using Sigirr -/- mice, we identify, for the first time (to our knowledge), SIGIRR as a negative regulator of IL-36 responses in the skin. Mechanistically, we identify dendritic cells and keratinocytes as the primary cell subsets in which IL-36 proinflammatory responses are regulated by SIGIRR. Both cell types displayed elevated IL-36 responsiveness in absence of SIGIRR activity, characterized by enhanced expression of neutrophil chemoattractants, leading to increased neutrophil infiltration to the inflamed skin. Blockade of IL-36R signaling ameliorated exacerbated psoriasiform inflammation in Sigirr -/- mice and inhibited neutrophil infiltration. These data identify SIGIRR activity as an important regulatory node in suppressing IL-36-dependent psoriatic inflammation in humans and mice.

Opportunities to Target T Cell Trafficking in Pediatric Inflammatory Bowel Disease.

T cell subsets are considered central orchestrators of inflammation and homeostasis in the intestine and are established targets for the treatment of inflammatory bowel disease. While approaches aimed at the neutralization of T cell effector cytokines have provided significant benefits for pediatric and adult patients, more recent strategies aimed at inhibiting the infiltration of pathogenic T cell subsets have also emerged. In this review, we describe current knowledge surrounding the function of T cell subsets in pediatric inflammatory bowel disease and outline approaches aimed at targeting T cell trafficking to the intestine which may represent a new treatment option for pediatric inflammatory bowel disease.

The Diverse Roles of the IL-36 Family in Gastrointestinal Inflammation and Resolution.

The interleukin (IL)-36 family is a member of the IL-1 superfamily of cytokines and, in common with other IL-1 family members, has been shown to exhibit pleiotropic effects in homeostasis and inflammation. Although the important role these cytokines play in the skin has been widely reported, recent evidence suggests that IL-36 family members are expressed and can also exert significant influence at the intestinal mucosa. In this review, we summarize current knowledge surrounding the role of the IL-36 in the intestines. In particular, we examine its likely dichotomous role as a mediator of both inflammation and resolution, highlighting its overlapping roles in innate and adaptive inflammation at the mucosa and its contribution to pathophysiology of inflammatory bowel disease. We also summarize the complexities of targeting this cytokine family in a clinical setting.

UCP3 reciprocally controls CD4+ Th17 and Treg cell differentiation.

Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier superfamily that can mediate the transfer of protons into the mitochondrial matrix from the intermembrane space. We have previously reported UCP3 expression in thymocytes, mitochondria of total splenocytes and splenic lymphocytes. Here, we demonstrate that Ucp3 is expressed in peripheral naive CD4+ T cells at the mRNA level before being markedly downregulated following activation. Non-polarized, activated T cells (Th0 cells) from Ucp3-/- mice produced significantly more IL-2, had increased expression of CD25 and CD69 and were more proliferative than Ucp3+/+ Th0 cells. The altered IL-2 expression observed between T cells from Ucp3+/+ and Ucp3-/- mice may be a factor in determining differentiation into Th17 or induced regulatory (iTreg) cells. When compared to Ucp3+/+, CD4+ T cells from Ucp3-/- mice had increased FoxP3 expression under iTreg conditions. Conversely, Ucp3-/- CD4+ T cells produced a significantly lower concentration of IL-17A under Th17 cell-inducing conditions in vitro. These effects were mirrored in antigen-specific T cells from mice immunized with KLH and CT. Interestingly, the altered responses of Ucp3-/- T cells were partially reversed upon neutralisation of IL-2. Together, these data indicate that UCP3 acts to restrict the activation of naive T cells, acting as a rheostat to dampen signals following TCR and CD28 co-receptor ligation, thereby limiting early activation responses. The observation that Ucp3 ablation alters the Th17:Treg cell balance in vivo as well as in vitro suggests that UCP3 is a potential target for the treatment of Th17 cell-mediated autoimmune diseases.

Keratinocyte interleukin-36 receptor expression orchestrates psoriasiform inflammation in mice.

The IL-36 family cytokines have emerged as important mediators of dermal inflammation in psoriasis and have been reported to provide a proinflammatory stimulus to a variety of immune and stromal cell subsets in the inflamed skin. However, it remains to be determined which cell type, if any, in the skin plays a predominant role in mediating IL-36 cytokines instructive role in disease. Here, we demonstrate that targeted deletion of Il36r in keratinocytes results in similar levels of protection from psoriasiform inflammation observed in "global" Il36r-deficient mice. Mice with deficiency in IL-36 receptor expression on keratinocytes had significantly decreased expression, comparable with Il36r-deficient mice, of established mediators of psoriatic inflammation, including, IL-17a, IL-23, IL-22, and a loss of chemokine-induced neutrophil and IL-17A-expressing γδ T-cell subset infiltration to the inflamed skin. These data demonstrate that keratinocytes are the primary orchestrating cell in mediating the effects of IL-36-driven dermal inflammation in the imiquimod model of psoriasiform inflammation and shed new light on the cell-specific roles of IL-36 cytokines during psoriatic disease.

Interleukin-36 cytokines alter the intestinal microbiome and can protect against obesity and metabolic dysfunction.

Members of the interleukin-1 (IL-1) family are important mediators of obesity and metabolic disease and have been described to often play opposing roles. Here we report that the interleukin-36 (IL-36) subfamily can play a protective role against the development of disease. Elevated IL-36 cytokine expression is found in the serum of obese patients and negatively correlates with blood glucose levels among those presenting with type 2 diabetes. Mice lacking IL-36Ra, an IL-36 family signalling antagonist, develop less diet-induced weight gain, hyperglycemia and insulin resistance. These protective effects correlate with increased abundance of the metabolically protective bacteria Akkermansia muciniphila in the intestinal microbiome. IL-36 cytokines promote its outgrowth as well as increased colonic mucus secretion. These findings identify a protective role for IL-36 cytokines in obesity and metabolic disease, adding to the current understanding of the role the broader IL-1 family plays in regulating disease pathogenesis.