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.

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.

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.

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.

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.

Current perspectives on the interleukin-1 family as targets for inflammatory disease.

Since the first description of interleukin-1 (IL-1) and the genesis of the field of cytokine biology, the understanding of how IL-1 and related cytokines play central orchestrating roles in the inflammatory response has been an area of intense investigation. As a consequence of these endeavours, specific strategies have been developed to target the function of the IL-1 family in human disease realizing significant impacts for patients. While the most significant advances to date have been associated with inhibition of the prototypical family members IL-1α/ß, approaches to target more recently identified family members such as IL-18, IL-33 and the IL-36 subfamily are now beginning to come to fruition. This review summarizes current knowledge surrounding the roles of the IL-1 family in human disease and describes the rationale and strategies which have been developed to target these cytokines to inhibit the pathogenesis of a wide range of diseases in which inflammation plays a centrally important role.

Transcriptional Profiling of Immune-Related Genes in Leishmania infantum-Infected Mice: Identification of Potential Biomarkers of Infection and Progression of Disease.

Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4) or remain activated throughout the experiment (Il18bp). The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi), suggesting the generation of a classical "protective response" against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that counteracts the Th1/M1 response. This large pool of data was also used to identify potential biomarkers of infection and parasitic burden in spleen, on the bases of two different regression models. Given the results, gene expression signature analysis appears as a useful tool to identify mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development.

Deletion of the BH3-only protein Noxa alters electrographic seizures but does not protect against hippocampal damage after status epilepticus in mice.

Several members of the Bcl-2 gene family are dysregulated in human temporal lobe epilepsy and animal studies show that genetic deletion of some of these proteins influence electrographic seizure responses to chemoconvulsants and associated brain damage. The BH3-only proteins form a subgroup comprising direct activators of Bax-Bak that are potently proapoptotic and a number of weaker proapoptotic BH3-only proteins that act as sensitizers by neutralization of antiapoptotic Bcl-2 family members. Noxa was originally characterized as a weaker proapoptotic, 'sensitizer' BH3-only protein, although recent evidence suggests it too may be potently proapoptotic. Expression of Noxa is under p53 control, a known seizure-activated pathway, although Noxa has been linked to energetic stress and autophagy. Here we characterized the response of Noxa to prolonged seizures and the phenotype of mice lacking Noxa. Status epilepticus induced by intra-amygdala kainic acid caused a rapid increase in expression of noxa in the damaged CA3 subfield of the hippocampus but not undamaged CA1 region. In vivo upregulation of noxa was reduced by pifithrin-α, suggesting transcription may be partly p53-dependent. Mice lacking noxa developed less severe electrographic seizures during status epilepticus in the model but, surprisingly, displayed equivalent hippocampal damage to wild-type animals. The present findings indicate Noxa does not serve as a proapoptotic BH3-only protein during seizure-induced neuronal death in vivo. This study extends the comprehensive phenotyping of seizure and damage responses in mice lacking specific Bcl-2 gene family members and provides further evidence that these proteins may serve roles beyond control of cell death in the brain.

The Challenge of Stability in High-Throughput Gene Expression Analysis: Comprehensive Selection and Evaluation of Reference Genes for BALB/c Mice Spleen Samples in the Leishmania infantum Infection Model.

The interaction of Leishmania with BALB/c mice induces dramatic changes in transcriptome patterns in the parasite, but also in the target organs (spleen, liver…) due to its response against infection. Real-time quantitative PCR (qPCR) is an interesting approach to analyze these changes and understand the immunological pathways that lead to protection or progression of disease. However, qPCR results need to be normalized against one or more reference genes (RG) to correct for non-specific experimental variation. The development of technical platforms for high-throughput qPCR analysis, and powerful software for analysis of qPCR data, have acknowledged the problem that some reference genes widely used due to their known or suspected "housekeeping" roles, should be avoided due to high expression variability across different tissues or experimental conditions. In this paper we evaluated the stability of 112 genes using three different algorithms: geNorm, NormFinder and RefFinder in spleen samples from BALB/c mice under different experimental conditions (control and Leishmania infantum-infected mice). Despite minor discrepancies in the stability ranking shown by the three methods, most genes show very similar performance as RG (either good or poor) across this massive data set. Our results show that some of the genes traditionally used as RG in this model (i.e. B2m, Polr2a and Tbp) are clearly outperformed by others. In particular, the combination of Il2rg + Itgb2 was identified among the best scoring candidate RG for every group of mice and every algorithm used in this experimental model. Finally, we have demonstrated that using "traditional" vs rationally-selected RG for normalization of gene expression data may lead to loss of statistical significance of gene expression changes when using large-scale platforms, and therefore misinterpretation of results. Taken together, our results highlight the need for a comprehensive, high-throughput search for the most stable reference genes in each particular experimental model.