OBJECTIVE:Genetic associations and blockade of the interleukin (IL)-23/IL-17 axis with monoclonal antibodies support a role for this pathway in patients with psoriatic arthritis (PsA). This study examines the requirement of IL-23 for IL-17 production and the role of the metabolic microenvironment in the expansion of Th17-derived cells in patients with PsA. METHODS:Th17 cell frequencies in synovial fluid or peripheral blood from patients with PsA were evaluated by flow cytometry using chemokine receptor 6, CD161, and T-bet as phenotypic markers, and the cytokines interferon γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-17 were assessed by flow cytometry and enzyme-linked immunosorbent assay. The impact of IL-23 and metabolic stress on T cell differentiation was investigated. RESULTS:Polyfunctional positive IL-17 (IL-17pos) CD4 (P < 0.0001) and CD8 (P < 0.0001), and GM-CSFpos Th17-derived cells (P < 0.0001) were increased in the inflamed joints of patients with PsA, with a proportional decrease in the peripheral blood of patients. We demonstrate IL-23-independent IL-17 release by CD4 T cells in patients with PsA, in which the absence of IL-23 during Th17 differentiation reduced IL-17 by mean ± SEM 31% ± 5.8%. Exogenous IL-23 increased IL-17, negatively regulated GM-CSF, and cooperated with transforming growth factor β to augment IL-17. Polyfunctional Th17 and Th17-derived cells, but not Th1 cells, were expanded by metabolic stress in patients with PsA. CONCLUSION:We confirmed the abundance of polyfunctional type 17 CD4 and CD8 cells in the inflamed joints of patients with PsA. We demonstrate IL-23-independent expansion of Th17 cells, for which IL-23 negatively regulates GM-CSF. This may account for therapeutic differences in IL-17 and IL-23 inhibition in patients with PsA or other spondyloarthritides. Polyfunctional IL-17pos Th17 and Th17-derived but not Th1 cells were expanded by metabolic stress, and metabolic stress may itself represent a unique therapeutic target.

Heart failure and associated cachexia is an unresolved and important problem. We report a new model of severe heart failure that consistently results in cachexia. Mice lacking the integrated stress response (ISR) induced eIF2α phosphatase, PPP1R15A, exhibit a dilated cardiomyopathy and severe weight loss following irradiation, whilst wildtype mice are unaffected. This is associated with increased expression of Gdf15 in the heart and increased levels of GDF15 in the circulation. We provide evidence that blockade of GDF15 activity prevents cachexia and slows the progression of heart failure. Our data suggests that cardiac stress mediates a GDF15 dependent pathway that drives weight loss and worsens cardiac function. We show relevance of GDF15 to lean mass and protein intake with patients with heart failure. Blockade of GDF15 could constitute a novel therapeutic option to limit cardiac cachexia and improve clinical outcomes in patients with severe systolic heart failure.

Interleukin-1 alpha (IL-1α) is a powerful cytokine that modulates immunity, and requires canonical cleavage by calpain for full activity. Mature IL-1α is produced after inflammasome activation and during cell senescence, but the protease cleaving IL-1α in these contexts is unknown. We show IL-1α is activated by caspase-5 or caspase-11 cleavage at a conserved site. Caspase-5 drives cleaved IL-1α release after human macrophage inflammasome activation, while IL-1α secretion from murine macrophages only requires caspase-11, with IL-1β release needing caspase-11 and caspase-1. Importantly, senescent human cells require caspase-5 for the IL-1α-dependent senescence-associated secretory phenotype (SASP) in vitro, while senescent mouse hepatocytes need caspase-11 for the SASP-driven immune surveillance of senescent cells in vivo. Together, we identify IL-1α as a novel substrate of noncanonical inflammatory caspases and finally provide a mechanism for how IL-1α is activated during senescence. Thus, targeting caspase-5 may reduce inflammation and limit the deleterious effects of accumulated senescent cells during disease and Aging.

Thymic stromal lymphopoietin (TSLP) is a functionally pleotropic cytokine important in immune regulation, and TSLP dysregulation is associated with numerous diseases. TSLP is produced by many cell types, but has predominantly been characterized as a secreted factor from epithelial cells which activates dendritic cells (DC) that subsequently prime T helper (TH) 2 immunity. However, DC themselves make significant amounts of TSLP in response to microbial products, but the functional role of DC-derived TSLP remains unclear. We show that TSLPR signaling negatively regulates IL-1β production during dectin-1 stimulation of human DC. This regulatory mechanism functions by dampening Syk phosphorylation and is mediated via NADPH oxidase-derived ROS, HIF-1α and pro-IL-1β expression. Considering the profound effect TSLPR signaling has on the metabolic status and the secretome of dectin-1 stimulated DC, these data suggest that autocrine TSLPR signaling could have a fundamental role in modulating immunological effector responses at sites removed from epithelial cell production of TSLP.

Since the concept of the inflammasome was introduced by Martinon, Burns and Tschopp in 2002, there has been an exponential increase in our understanding of how inflammasomes (caspase activating molecular platforms) regulate innate inflammatory responses to infectious microorganisms. Advances in understanding inflammasome biology have been developed using a range of bacterial pathogens. Recent studies investigating inflammasome responses during Chlamydia infection have provided interesting mechanistic insights in to inflammasome activation during intracellular bacterial infection. This review highlights new concepts regulating inflammasome activation to bacterial infections including: interferon-regulated loss of compartmentalisation, mechanisms of canonical and non-canonical inflammasome activation and their relevance to Chlamydia infections are discussed.

Aim: Tuberculosis granulomas share similar features with arterial atherosclerotic plaques. The presence of a necrotic core and the accumulation of lipid promote progression towards an unstable atherosclerotic lesion. Clec4e (Mincle) receptor mediates macrophage activation in response to mycobacterial glycolipid, but also senses damaged cells and drives inflammatory activation in response to necrotic cell death. Our aims were to examine the role of Clec4e in atherosclerosis development and identify the signaling pathways that mediate macrophage responses to Clec4e stimulation

Dectin-1/CLEC7A is a pattern recognition receptor that recognizes β-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. β-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate β-glucans are thought to be poor activators of innate immunity. We show that β-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large β-glucan-stimulated DC generate significantly more IL-1β, IL-6, and IL-23 compared to those stimulated with the smaller β-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to β-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1β production determined by β-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that β-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.

The innate immune system is a critical component of host defence against microbial pathogens, but effective responses require an ability to distinguish between infectious and non-infectious insult to prevent inappropriate inflammation. Using the important obligate intracellular human pathogen Chlamydia trachomatis; an organism that causes significant immunopathology, we sought to determine critical host and pathogen factors that contribute to the induction of inflammasome activation. We assayed inflammasome activation by immunoblotting and ELISA to detect IL-1β processing and LDH release to determine pyroptosis. Using primary murine bone marrow derived macrophages or human monocyte derived dendritic cells, infected with live or attenuated Chlamydia trachomatis we report that the live organism activates both canonical and non-canonical inflammasomes, but only canonical inflammasomes controlled IL-1β processing which preceded pyroptosis. NADPH oxidase deficient macrophages were permissive to Chlamydia trachomatis replication and displayed elevated type-1 interferon and inflammasome activation. Conversely, attenuated, non-replicating Chlamydia trachomatis, primed but did not activate inflammasomes and stimulated reduced type-1 interferon responses. This suggested bacterial replication or metabolism as important factors that determine interferon responses and inflammasome activation. We identified STING but not cGAS as a central mediator of interferon regulated inflammasome activation. Interestingly, exogenous delivery of a Chlamydia trachomatis metabolite and STING ligand-cyclic di-AMP, recovered inflammasome activation to attenuated bacteria in a STING dependent manner thus indicating that a bacterial metabolite is a key factor initiating inflammasome activation through STING, independent of cGAS. These data suggest a potential mechanism of how the innate immune system can distinguish between infectious and non-infectious insult and instigate appropriate immune responses that could be therapeutically targeted.

Background Psoriasis [Ps] is a common chronic inflammatory skin condition affecting about 1–2% of the world population; approximately 20–30% of patients have psoriatic arthritis [PsA]. PsA can affect peripheral and axial joints, eyes, ileum, colon and skin. Genetic studies reveal common candidate genes including IL23R, IL12B, STAT3, and CARD9, all associated with IL-23 signalling. We demonstrated increased frequencies of Th17 cells in patients with Ps and PsA and the clinical importance of IL-23/Th17 is revealed by the efficacy of biologics targeting this pathway. Whilst IL-17 was thought to be pathogenic in inflammatory conditions such as experimental autoimmune encephalomyelitis, the redundancy of IL-17 but essential requirement for GM-CSF release by Th17 cells was shown. GM-CSF+ T cells were identified in cerebrospinal fluid from patients with multiple sclerosis but a role in the aetio-pathogenesis of PsA has not been established. Objectives 1) Are GM-CSF+ T cells enriched in peripheral blood and synovial fluid obtained from patients with PsA? 2) Is GM-CSF co-produced with IFN-g? 3) Do CD4+GM-CSF+ T cells express surface markers characteristic of Th17? 4) Is GM-CSF release augmented by exogenous IL-23 and/or IL-12? Methods PBMC or synovial fluid derived from patients with PsA, or healthy donor (HD) PBMC, were stimulated with anti-CD3/anti-CD28 +/− recombinant IL-23 or IL-12. Supernatants were harvested and analysed by ELISA for IL-17, IFN-γ or GM-CSF. Alternatively, cells were activated with phorbol myristate acetate (PMA) and ionomycin, and evaluated for the expression of the Th17-associated cell surface markers CCR6, CD161 & IL-23R plus IL-17A, IFN-g and GM-CSF by flow cytometry. Results GM-CSF release from PsA PBMC was higher than that of HD, whereas IFN-g levels were higher in HD. This was most significant on comparing the ratio of IFN-g to GM-CSF. On examining the co-expression of GM-CSF and IFN-g in CD4+ T cells, PsA patients had higher proportions of GM-CSF+ single positive and fewer GM-CSF+IFN-g+ relative to HD. There was also marked enrichment of CD4+GM-CSF+ cells in synovial fluid. On evaluating the expression of the Th17-associated markers CCR6, CD161 and IL-23R, we demonstrated that whereas almost all CD4+IL-17+ cells expressed CCR6, approximately 50% of CD4+GM-CSF+ cells expressed this chemokine receptor. Surprisingly, there were fewer PsA patient CD4+GM-CSF+ peripheral blood T cells co-expressing CCR6, CD161 and IL-23R relative to HD. Finally, IL-23 has been shown to render Th17 cells more pathogenic, hence we examined the effect of this cytokine on GM-CSF release. We showed that whilst IL-17 release was enhanced by exogenous IL-23, GM-CSF release was significantly downregulated with little effect on IFN-g. In contrast, IL-12 increased IFN-g and GM-CSF, and reduced IL-17. Conclusions IL-23 and Th17 cells are therapeutic targets in PsA, and mouse models of inflammation suggest that pathogenic Th17 cells release GM-CSF. We reveal that GM-CSF release is elevated in patients with PsA, and GM-CSF+ cells are enriched in diseased joints. However, only a proportion of GM-CSF+ cells exhibited features of Th17. Furthermore, IL-23 downmodulated GM-CSF whilst enhancing IL-17. Given that GM-CSF was mainly produced by cells not co-expressing IFN-g in PsA patients [in contrast to healthy donors], this subset may be pathogenic in PsA and warrants further investigation. Disclosure of Interest None declared

FOXP3+ regulatory T (Treg) cells are indispensable for immune homeostasis, but their study in humans is complicated by heterogeneity within Treg, the difficulty in purifying Tregs using surface marker expression (e.g. CD25) and the transient expression of FOXP3 by activated effector cells. Here, we report that expression of CD39 and CD45RO distinguishes three sub-populations within human CD4 + CD25 hi T cells. Initial phenotypic and functional analysis demonstrated that CD4 + CD25 hi CD39 + CD45RO + cells had properties consistent with effector Treg, CD4 + CD25 hi CD39 − CD45RO − cells were naïve Treg and CD4 + CD25 hi CD39 − CD45RO + cells were predominantly non-Treg with effector T-cell function. Differences in these two newly identified Treg subsets were corroborated by studies of gene expression and TCR analysis. To apply this approach, we studied these two newly identified Treg subsets in ankylosing spondylitis, and showed impairment in both effector and naïve Treg. This work highlights the importance of discriminating Treg subsets to enable proper comparisons of immune regulatory capacity in healthy individuals and those with inflammatory disease.

Background: Atherosclerotic lesion expansion is characterized by the development of a lipid-rich necrotic core known to be associated with the occurrence of complications. Abnormal lipid handling, inflammation, and alteration of cell survival or proliferation contribute to necrotic core formation, but the molecular mechanisms involved in this process are not properly understood. C-type lectin receptor 4e (Clec4e) recognizes the cord factor of Mycobacterium tuberculosis but also senses molecular patterns released by necrotic cells and drives inflammation. Methods: We hypothesized that activation of Clec4e signaling by necrosis is causally involved in atherogenesis. We addressed the impact of Clec4e activation on macrophage functions in vitro and on the development of atherosclerosis using low-density lipoprotein receptor–deficient ( Ldlr −/− ) mice in vivo. Results: We show that Clec4e is expressed within human and mouse atherosclerotic lesions and is activated by necrotic lesion extracts. Clec4e signaling in macrophages inhibits cholesterol efflux and induces a Syk-mediated endoplasmic reticulum stress response, leading to the induction of proinflammatory mediators and growth factors. Chop and Ire1a deficiencies significantly limit Clec4e-dependent effects, whereas Atf3 deficiency aggravates Clec4e-mediated inflammation and alteration of cholesterol efflux. Repopulation of Ldlr −/− mice with Clec4e −/− bone marrow reduces lipid accumulation, endoplasmic reticulum stress, and macrophage inflammation and proliferation within the developing arterial lesions and significantly limits atherosclerosis. Conclusions: Our results identify a nonredundant role for Clec4e in coordinating major biological pathways involved in atherosclerosis and suggest that it may play similar roles in other chronic inflammatory diseases.

FOXP3+ regulatory T (Treg) cells are indispensable for immune homeostasis, but their study in humans is complicated by heterogeneity within Treg, the difficulty in purifying Tregs using surface marker expression (e.g. CD25) and the transient expression of FOXP3 by activated effector cells. Here, we report that expression of CD39 and CD45RO distinguishes three sub‐populations within human CD4 + CD25 hi T cells. Initial phenotypic and functional analysis demonstrated that CD4 + CD25 hi CD39 + CD45RO + cells had properties consistent with effector Treg, CD4 + CD25 hi CD39 − CD45RO − cells were naïve Treg and CD4 + CD25 hi CD39 − CD45RO + cells were predominantly non‐Treg with effector T‐cell function. Differences in these two newly identified Treg subsets were corroborated by studies of gene expression and TCR analysis. To apply this approach, we studied these two newly identified Treg subsets in ankylosing spondylitis, and showed impairment in both effector and naïve Treg. This work highlights the importance of discriminating Treg subsets to enable proper comparisons of immune regulatory capacity in healthy individuals and those with inflammatory disease.

Protein kinase RNA activated (PKR) is a crucial mediator of anti-viral responses but is reported to be activated by multiple non-viral stimuli. However, mechanisms underlying PKR activation, particularly in response to bacterial infection, remain poorly understood. We have investigated mechanisms of PKR activation in human primary monocyte-derived dendritic cells in response to infection by Chlamydia trachomatis. Infection resulted in potent activation of PKR that was dependent on TLR4 and MyD88 signalling. NADPH oxidase was dispensable for activation of PKR as cells from chronic granulomatous disease (CGD) patients, or mice that lack NADPH oxidase activity, had equivalent or elevated PKR activation. Significantly, stimulation of cells with endoplasmic reticulum (ER) stress-inducing agents resulted in potent activation of PKR that was blocked by an inhibitor of IRE1α RNAse activity. Crucially, infection resulted in robust IRE1α RNAse activity that was dependent on TLR4 signalling and inhibition of IRE1α RNAse activity prevented PKR activation. Finally, we demonstrate that TLR4/IRE1α mediated PKR activation is required for the enhancement of interferon-β production following C. trachomatis infection. Thus, we provide evidence of a novel mechanism of PKR activation requiring ER stress signalling that occurs as a consequence of TLR4 stimulation during bacterial infection and contributes to inflammatory responses.

SummaryExpression of the adhesion molecule, CD146/MCAM/MelCAM, on T cells has been associated with recent activation, memory subsets and T helper type 17 (Th17) effector function, and is elevated in inflammatory arthritis. Th17 cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) and spondyloarthritides (SpA). Here, we compared the expression of CD146 on CD4+ T cells between healthy donors (HD) and patients with RA and SpA [ankylosing spondylitis (AS) or psoriatic arthritis (PsA)] and examined correlations with surface markers and cytokine secretion. Peripheral blood mononuclear cells (PBMC) were obtained from patients and controls, and synovial fluid mononuclear cells (SFMC) from patients. Cytokine production [elicited by phorbol myristate acetate (PMA)/ionomycin] and surface phenotypes were evaluated by flow cytometry. CD146+ CD4+ and interleukin (IL)-17+ CD4+ T cell frequencies were increased in PBMC of PsA patients, compared with HD, and in SFMC compared with PBMC. CD146+ CD4+ T cells were enriched for secretion of IL-17 [alone or with IL-22 or interferon (IFN)-γ] and for some putative Th17-associated surface markers (CD161 and CCR6), but not others (CD26 and IL-23 receptor). CD4+ T cells producing IL-22 or IFN-γ without IL-17 were also present in the CD146+ subset, although their enrichment was less marked. Moreover, a majority of cells secreting these cytokines lacked CD146. Thus, CD146 is not a sensitive or specific marker of Th17 cells, but rather correlates with heterogeneous cytokine secretion by subsets of CD4+ helper T cells.

Background: The spondylarthritides, AS, PsA and enteropathic arthritis, cause chronic inflammation of peripheral and axial joints, eyes, skin, ileum and colon.Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3 and CARD9, all associated with IL-23 signalling.We have recently shown elevated frequencies of CD4 þ IL-17 þ cells in PsA in blood, joints and skin.Recent mouse studies identified a central role for GM-CSF released by T helper 17 (Th17) cells in experimental autoimmune encephalomyelitis.Here we investigate GM-CSF release by Th17 cells in PsA.Methods: Peripheral blood mononuclear cells (PBMCs) or SF derived from patients with PsA, or healthy donor (HD) PBMCs, were stimulated with anti-CD3/anti-CD28 human T activator beads in the presence or absence of recombinant IL-23.Supernatants were harvested and analysed by ELISA for IL-17, IFNg or GM-CSF.Alternatively, PBMC or SF were activated with phorbol myristate acetate (PMA) and ionomycin, and CD4 T cells evaluated for the expression of Th17associated cell surface markers including CCR6, CD161 and IL-23R.Cells were fixed, permeabilized and stained for IL-17A, IFNg and GM-CSF.Results: As previously reported, GM-CSF was released by SF MCs derived from patients with PsA.GM-CSF release from PsA PBMCs was higher than that of HD PBMCs, whereas IFNg responses were higher in HDs.This reached significance when comparing the ratio of IFNg to GM-CSF, in that the ratio was 10.5 AE 2.6 in PsA patients and 35.1 AE 8.2 in HD (P < 0.01).GM-CSF release was significantly downregulated (P < 0.0001) in pre-incubating PBMCs with IL-23, in contrast to an increased production of IL-17 (P < 0.01).These data led to further evaluation of GM-CSF and IL-17 release by immunophenotyping.50-expressed this Th17-associated marker in addition to CD161 and IL-23R.This suggested that a proportion of GM-CSF was indeed being released by a CD4 þ T cell subset other than Th17; this will be further evaluated by cell sorting.Given that chemokine receptors are down-modulated by PMA and ionomycin, CD4 þ T cells will be sorted according to the differential expression of CCR4, CCR6, CCR10 and CXCR3 prior to activation, to further interrogate the subset(s) responsible for GM-CSF release.Conclusion: IL-23 is a therapeutic target in PsA.We demonstrate that GM-CSF release is elevated in patients with PsA, with a significant proportion of GM-CSF-producing cells exhibiting characteristics of Th17 cells.However, given that many GM-CSF þ cells also co-express IFNg, and that GM-CSF release is down-modulated by IL-23, further characterization of this putative therapeutic target is required.