Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rβ1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.

SUMMARY Developing strategies that promote the resolution of vascular inflammation and atherosclerosis remains a major therapeutic challenge. Here, we show that exosomes produced by naive bone marrow-derived macrophages (BMDM-exo) contain anti-inflammatory microRNA-99a/146b/378a that are further increased in exosomes produced by BMDM polarized with IL-4 (BMDM-IL-4-exo). These exosomal microRNAs suppress inflammation by targeting NF-κB and TNF-α signaling and foster M2 polarization in recipient macrophages. Repeated infusions of BMDM-IL-4-exo into Apoe −/− mice fed a Western diet reduce excessive hematopoiesis in the bone marrow and thereby the number of myeloid cells in the circulation and macrophages in aortic root lesions. This also leads to a reduction in necrotic lesion areas that collectively stabilize atheroma. Thus, BMDM-IL-4-exo may represent a useful therapeutic approach for atherosclerosis and other inflammatory disorders by targeting NF-κB and TNF-α via microRNA cargo delivery.

BackgroundData from next generation sequencing technologies uncovered the existence of many classes of small RNAs. Recent studies reported that small RNAs are released by cells and can be detected in the blood. In this report, we aimed to discover the occurrence of novel circulating small RNAs in coronary artery disease (CAD).MethodsWe used high-throughput sequencing of small RNAs from human and mouse apoptotic primary macrophages, and analyzed the data by empirical Bayes moderated t-statistics to assess differential expression and the Benjamini and Hochberg method to control the false discovery rate. Results were then confirmed by Northern blot and RT-qPCR in foam cells and in two animal models for atherosclerosis, namely ApoE−/− and Ldlr−/− mouse lines. Quantitative RT-PCR to detect identified small RNAs, the RNY-derived small RNAs, was performed using sera of 263 patients with CAD compared to 514 matched healthy controls; the Student t-test was applied to statistically assess differences. Associations of small RNAs with clinical characteristics and biological markers were tested using Spearman’s rank correlations, while multivariate logistic regressions were performed to test the statistical association of small RNA levels with CAD.ResultsHere, we report that, in macrophages stimulated with pro-apoptotic or pro-atherogenic stimuli, the Ro-associated non-coding RNAs, called RNYs or Y-RNAs, are processed into small RNAs (~24–34 nt) referred to as small-RNYs (s-RNYs), including s-RNY1-5p processed from RNY1. A significant upregulation of s-RNY expression was found in aortic arches and blood plasma from ApoE−/− and Ldlr−/− mice and in serum from CAD patients (P <0.001). Biostatistical analysis revealed a positive association of s-RNY1-5p with hs-CRP and ApoB levels; however, no statistical interaction was found between either of these two markers and s-RNY1-5p in relation to the CAD status. Levels of s-RNY1-5p were also independent from statin and fibrate therapies.ConclusionOur results position the s-RNY1-5p as a relevant novel independent diagnostic biomarker for atherosclerosis-related diseases. Measurement of circulating s-RNY expression would be a valuable companion diagnostic to monitor foam cell apoptosis during atherosclerosis pathogenesis and to evaluate patient’s responsiveness to future therapeutic strategies aiming to attenuate apoptosis in foam cells in advanced atherosclerotic lesions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-015-0489-y) contains supplementary material, which is available to authorized users.

Ultracentrifugation (UC) is recognized as a robust approach for the isolation of extracellular vesicles (EVs). However, recent studies have highlighted limitations of UC including low recovery efficiencies and aggregation of EVs that could impact downstream functional analyses. We tested the benefit of using a liquid cushion of iodixanol during UC to address such shortcomings. In this study, we compared the yield and purity of EVs isolated from J774A.1 macrophage conditioned media by conventional UC and cushioned-UC (C-UC). We extended our study to include two other common EV isolation approaches: ultrafiltration (UF) and polyethylene glycol (PEG) sedimentation. After concentrating EVs using these four methods, the concentrates underwent further purification by using OptiPrep density gradient ultracentrifugation (DGUC). Our data show that C-DGUC provides a two-fold improvement in EV recovery over conventional UC-DGUC. We also found that UF-DGUC retained ten-fold more protein while PEG-DGUC achieved similar performance in nanoparticle and protein recovery compared to C-DGUC. Regarding purity as assessed by nanoparticle to protein ratio, our data show that EVs isolated by UC-DGUC achieved the highest purity while C-DGUC and PEG-DGUC led to similarly pure preparations. Collectively, we demonstrate that the use of a high-density iodixanol cushion during the initial concentration step improves the yield of EVs derived from cell culture media compared to conventional UC. This enhanced yield without substantial retention of protein contaminants and without exposure to forces causing aggregation offers new opportunities for the isolation of EVs that can subsequently be used for functional studies.

Objective— The physiological function of the ATP–binding cassette G1 (ABCG1) transporter in humans is not yet elucidated, as no genetic disease caused by ABCG1 mutations has been documented. The goal of our study was, therefore, to investigate the potential role(s) of ABCG1 in lipid metabolism in humans. Methods and Results— Here we report that among the 104 polymorphisms present in the ABCG1 gene, the analysis of the frequent functional rs1893590 and rs1378577 single nucleotide polymorphisms located in the regulatory region of ABCG1 in the Regression Growth Evaluation Statin Study population revealed that both ABCG1 single nucleotide polymorphisms were significantly associated with plasma lipoprotein lipase (LPL) activity. Moreover, we observed that plasma LPL activity was modestly reduced in Abcg1 −/− mice as compared with control mice. Adipose tissue and skeletal muscle are the major tissues accounting for levels and activity of plasma LPL in the body. However, beyond its lipolytic action in the plasma compartment, LPL was also described to act locally at the cellular level. Thus, macrophage LPL was reported to promote foam cell formation and atherosclerosis in vivo. Analysis of the relationship between ABCG1 and LPL in macrophages revealed that the knockdown of ABCG1 expression (ABCG1 knockdown) in primary cultures of human monocyte–derived macrophages using small interfering RNAs led to a marked reduction of both the secretion and activity of LPL. Indeed, LPL was trapped at the cell surface of ABCG1 knockdown human monocyte–derived macrophages, likely in cholesterol-rich domains, thereby reducing the bioavailability and activity of LPL. As a consequence, LPL–mediated lipid accumulation in human macrophage foam cells in the presence of triglyceride-rich lipoproteins was abolished when ABCG1 expression was repressed. Conclusion— We presently report that ABCG1 controls LPL activity and promotes lipid accumulation in human macrophages in the presence of triglyceride-rich lipoproteins, thereby suggesting a potential deleterious role of macrophage ABCG1 in metabolic situations associated with high levels of circulating triglyceride-rich lipoproteins together with the presence of macrophages in the arterial wall.

IL-23 activates the synthesis and production of Leukotriene B4 (LTB4) in myeloid cells, which modulate inflammatory arthritis. In this manuscript we investigate the role of LTB4 and its receptor LTB4R1 (BLT1) in synovial inflammation and osteoclast differentiation. Specifically, we utilized IL-23 in vivo gene transfer to induce arthritis in mice and showed that elevated serum LTB4 and synovial expression of 5-LO correlated with increased disease severity by histological evaluation and paw swelling compared to GFP-gene transfer controls. To further investigate the effect of LTB4 pathway in bone loss we performed osteoclast differentiation assays by stimulating with M-CSF and RANKL bone marrow cells derived from BLT1+/+ and/or BLT1−/− mice and utilized qPCR for gene expression analysis in terminally differentiated osteoclasts. Deficiency in BLT1 resulted in the up-regulation of osteoclast related genes and an increase in the formation of giant, multi-nucleated TRAP+ cells capable of F-actin ring formation. In addition, BLT1 deficiency showed an increase of phosphorylated NF-κB and phosphorylated IκB levels in osteoclasts. We also performed real time calcium imaging to study the effect of BLT1 deficiency in RANKL-induced activation of intracellular calcium flux in vitro. Our data show that LTB4 and its receptor BLT1 exacerbate synovial inflammation in vivo and bone resorption in vitro suggesting LTB4 and BLT1 could be effectively targeted for the treatment of musculoskeletal diseases.

In inflammatory arthritis, the dysregulation of osteoclast activity by proinflammatory cytokines, including TNF, interferes with bone remodeling during inflammation through Ca-dependent mechanisms causing pathological bone loss. Ca-dependent CREB/c-fos activation via Ca-calmodulin kinase IV (CaMKIV) induces transcriptional regulation of osteoclast-specific genes via NFATc1, which facilitate bone resorption. In leukocytes, Ca regulation of NFAT-dependent gene expression oftentimes involves the activity of the Ca-activated K channel KCa3.1. In this study, we evaluate KCa3.1 as a modulator of Ca-induced NFAT-dependent osteoclast differentiation in inflammatory bone loss. Microarray analysis of receptor activator of NF-κB ligand (RANKL)-activated murine bone marrow macrophage (BMM) cultures revealed unique upregulation of KCa3.1 during osteoclastogenesis. The expression of KCa3.1 in vivo was confirmed by immunofluorescence staining on multinucleated cells at the bone surface of inflamed mouse joints. Experiments on in vitro BMM cultures revealed that KCa3.1 and TRAM-34 treatment significantly reduced the expression of osteoclast-specific genes ( < 0.05) alongside decreased osteoclast formation ( < 0.0001) in inflammatory (RANKL+TNF) and noninflammatory (RANKL) conditions. In particular, live cell Ca imaging and Western blot analysis showed that TRAM-34 pretreatment decreased transient RANKL-induced Ca amplitudes in BMMs by ∼50% ( < 0.0001) and prevented phosphorylation of CaMKIV. KCa3.1 reduced RANKL+/-TNF-stimulated phosphorylation of CREB and expression of in BMMs ( < 0.01), culminating in decreased NFATc1 protein expression and transcriptional activity ( < 0.01). These data indicate that KCa3.1 regulates Ca-dependent NFATc1 expression via CaMKIV/CREB during inflammatory osteoclastogenesis in the presence of TNF, corroborating its role as a target candidate for the treatment of bone erosion in inflammatory arthritis.

Interleukin 17A (IL-17A) has been strongly associated with epidermal hyperplasia in many cutaneous disorders. However, as IL-17A is mainly produced by αβ and γδT cells in response to IL-23, the role of T cells and IL-23 have overshadowed any IL-17A independent actions. Herein, we report that IL-17A gene transfer induces epidermal hyperplasia in Il23r−/−Rag1−/− and Tcrδ deficient mice, which can be prevented by neutrophil depletion. Moreover adoptive transfer of CD11b+Gr-1hi cells, following IL-17A gene transfer, was sufficient to phenocopy the disease. We further show that the IL-17A-induced pathology was prevented in transgenic mice with impaired NETosis and/or neutrophils with conditional deletion of the master regulator of selective autophagy, Wdfy3. Our data demonstrate a novel T-cell independent mechanism that is associated with NETosis and selective autophagy in IL-17A-mediated epidermal hyperplasia.