Extracellular vesicles in systemic juvenile idiopathic arthritis.

Systemic juvenile idiopathic arthritis is a chronic pediatric inflammatory disease of unknown etiology, characterized by fever, rash, hepatosplenomegaly, serositis, and arthritis. We hypothesized that intercellular communication, mediated by extracellular vesicles, contributes to systemic juvenile idiopathic arthritis pathogenesis and that the number and cellular sources of extracellular vesicles would differ between inactive and active states of systemic juvenile idiopathic arthritis and healthy controls. We evaluated plasma from healthy pediatric controls and patients with systemic juvenile idiopathic arthritis with active systemic flare or inactive disease. We isolated extracellular vesicles by size exclusion chromatography and determined total extracellular vesicle abundance and size distribution using microfluidic resistive pulse sensing. Cell-specific extracellular vesicle subpopulations were measured by nanoscale flow cytometry. Isolated extracellular vesicles were validated using a variety of ways, including nanotracking and cryo-electron microscopy. Extracellular vesicle protein content was analyzed in pooled samples using mass spectrometry. Total extracellular vesicle concentration did not significantly differ between controls and patients with systemic juvenile idiopathic arthritis. Extracellular vesicles with diameters <200 nm were the most abundant, including the majority of cell-specific extracellular vesicle subpopulations. Patients with systemic juvenile idiopathic arthritis had significantly higher levels of extracellular vesicles from activated platelets, intermediate monocytes, and chronically activated endothelial cells, with the latter significantly more elevated in active systemic juvenile idiopathic arthritis relative to inactive disease and controls. Protein analysis of isolated extracellular vesicles from active patients showed a proinflammatory profile, uniquely expressing heat shock protein 47, a stress-inducible protein. Our findings indicate that multiple cell types contribute to altered extracellular vesicle profiles in systemic juvenile idiopathic arthritis. The extracellular vesicle differences between systemic juvenile idiopathic arthritis disease states and healthy controls implicate extracellular vesicle-mediated cellular crosstalk as a potential driver of systemic juvenile idiopathic arthritis disease activity.

Identification of Distinct Inflammatory Programs and Biomarkers in Systemic Juvenile Idiopathic Arthritis and Related Lung Disease by Serum Proteome Analysis.

OBJECTIVE: Recent observations in systemic juvenile idiopathic arthritis (JIA) suggest an increasing incidence of high-mortality interstitial lung disease often characterized by a variant of pulmonary alveolar proteinosis (PAP). Co-occurrence of macrophage activation syndrome (MAS) and PAP in systemic JIA suggests a shared pathology, but patients with lung disease associated with systemic JIA (designated SJIA-LD) also commonly experience features of drug reaction such as atypical rashes and eosinophilia. This study was undertaken to investigate immunopathology and identify biomarkers in systemic JIA, MAS, and SJIA-LD. METHODS: We used SOMAscan to measure ~1,300 analytes in sera from healthy controls and patients with systemic JIA, MAS, SJIA-LD, or other related diseases. We verified selected findings by enzyme-linked immunosorbent assay and lung immunostaining. Because the proteome of a sample may reflect multiple states (systemic JIA, MAS, or SJIA-LD), we used regression modeling to identify subsets of altered proteins associated with each state. We tested key findings in a validation cohort. RESULTS: Proteome alterations in active systemic JIA and MAS overlapped substantially, including known systemic JIA biomarkers such as serum amyloid A and S100A9, and novel elevations in the levels of heat-shock proteins and glycolytic enzymes. Interleukin-18 levels were elevated in all systemic JIA groups, particularly MAS and SJIA-LD. We also identified an MAS-independent SJIA-LD signature notable for elevated levels of intercellular adhesion molecule 5 (ICAM-5), matrix metalloproteinase 7 (MMP-7), and allergic/eosinophilic chemokines, which have been previously associated with lung damage. Immunohistochemistry localized ICAM-5 and MMP-7 in the lungs of patients with SJIA-LD. The ability of ICAM-5 to distinguish SJIA-LD from systemic JIA/MAS was independently validated. CONCLUSION: Serum proteins support a systemic JIA-to-MAS continuum; help distinguish systemic JIA, systemic JIA/MAS, and SJIA-LD; and suggest etiologic hypotheses. Select biomarkers, such as ICAM-5, could aid in early detection and management of SJIA-LD.

Repression of CTSG, ELANE and PRTN3-mediated histone H3 proteolytic cleavage promotes monocyte-to-macrophage differentiation.

Chromatin undergoes extensive reprogramming during immune cell differentiation. Here we report the repression of controlled histone H3 amino terminus proteolytic cleavage (H3ΔN) during monocyte-to-macrophage development. This abundant histone mark in human peripheral blood monocytes is catalyzed by neutrophil serine proteases (NSPs) cathepsin G, neutrophil elastase and proteinase 3. NSPs are repressed as monocytes mature into macrophages. Integrative epigenomic analysis reveals widespread H3ΔN distribution across the genome in a monocytic cell line and primary monocytes, which becomes largely undetectable in fully differentiated macrophages. H3ΔN is enriched at permissive chromatin and actively transcribed genes. Simultaneous NSP depletion in monocytic cells results in H3ΔN loss and further increase in chromatin accessibility, which likely primes the chromatin for gene expression reprogramming. Importantly, H3ΔN is reduced in monocytes from patients with systemic juvenile idiopathic arthritis, an autoinflammatory disease with prominent macrophage involvement. Overall, we uncover an epigenetic mechanism that primes the chromatin to facilitate macrophage development.

A positive feedback loop reinforces the allergic immune response in human peanut allergy.

Food allergies are a leading cause of anaphylaxis, and cellular mechanisms involving antigen presentation likely play key roles in their pathogenesis. However, little is known about the response of specific antigen-presenting cell (APC) subsets to food allergens in the setting of food allergies. Here, we show that in peanut-allergic humans, peanut allergen drives the differentiation of CD209+ monocyte-derived dendritic cells (DCs) and CD23+ (FcєRII) myeloid dendritic cells through the action of allergen-specific CD4+ T cells. CD209+ DCs act reciprocally on the same peanut-specific CD4+ T cell population to reinforce Th2 cytokine expression in a positive feedback loop, which may explain the persistence of established food allergy. In support of this novel model, we show clinically that the initiation of oral immunotherapy (OIT) in peanut-allergic patients is associated with a decrease in CD209+ DCs, suggesting that breaking the cycle of positive feedback is associated with therapeutic effect.

Multicohort Analysis Identifies Monocyte Gene Signatures to Accurately Monitor Subset-Specific Changes in Human Diseases.

Monocytes are crucial regulators of inflammation, and are characterized by three distinct subsets in humans, of which classical and non-classical are the most abundant. Different subsets carry out different functions and have been previously associated with multiple inflammatory conditions. Dissecting the contribution of different monocyte subsets to disease is currently limited by samples and cohorts, often resulting in underpowered studies and poor reproducibility. Publicly available transcriptome profiles provide an alternative source of data characterized by high statistical power and real-world heterogeneity. However, most transcriptome datasets profile bulk blood or tissue samples, requiring the use of in silico approaches to quantify changes in cell levels. Here, we integrated 853 publicly available microarray expression profiles of sorted human monocyte subsets from 45 independent studies to identify robust and parsimonious gene expression signatures, consisting of 10 genes specific to each subset. These signatures maintain their accuracy regardless of disease state in an independent cohort profiled by RNA-sequencing and are specific to their respective subset when compared to other immune cells from both myeloid and lymphoid lineages profiled across 6160 transcriptome profiles. Consequently, we show that these signatures can be used to quantify changes in monocyte subsets levels in expression profiles from patients in clinical trials. Finally, we show that proteins encoded by our signature genes can be used in cytometry-based assays to specifically sort monocyte subsets. Our results demonstrate the robustness, versatility, and utility of our computational approach and provide a framework for the discovery of new cellular markers.

High Dimensional Analyses of Circulating Immune Cells in Psoriatic Arthritis Detects Elevated Phosphorylated STAT3.

Psoriatic arthritis (PsA) is a chronic inflammatory arthritis, affecting up to 40% of patients with psoriasis. Constitutive expression by CD4+ T cells of an active form of STAT3, a signal transducer and transcription factor, has been shown to induce many of the major features of PsA in an animal model. We used high dimensional mass cytometry (CyTOF) to probe ex-vivo levels of phosphorylated STAT3 (pSTAT3) in circulating immune cell subpopulations from PsA patients during active and inactive states. We evaluated the frequency of 16 immune cell populations and the levels of the activated forms of STAT3 (pSTAT3) and, for comparison, STAT1 (pSTAT1) and Src (pSrc) in whole blood fixed shortly after collection. In addition to PsA patients, we studied active rheumatoid arthritis (RA) patients. Increased levels of pSTAT3 were found in all the CD4+ T cell subsets analyzed, specifically, Th1, Th2, Th17, T follicular helper (Tfh) and T regulatory (Treg) as well as in CD14+CD16- (classical) monocytes from active PsA patients compared to inactive patients. After correcting for body mass index (BMI), smoking and conventional disease modifying antirheumatic drugs (c-DMARDs), levels of pSTAT3 levels remained increased in Th1 and Tfh CD4+ T cells, and in CD14+CD16- monocytes from active patients compared to inactive patients. No differences between the patient groups were observed for pSTAT1 or pSrc. No differences were found between the active PsA and active RA groups after correction for multiple testing. During active PsA, circulating Th1 and Tfh CD4+ T cells, and CD14+CD16- monocytes expressing high levels of pSTAT3 may play a role in PsA pathophysiology, perhaps by migration to inflamed sites.

Author Correction: In vivo clonal expansion and phenotypes of hypocretin-specific CD4+ T cells in narcolepsy patients and controls.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

In vivo clonal expansion and phenotypes of hypocretin-specific CD4+ T cells in narcolepsy patients and controls.

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer+/TRAJ24+/CD4+ T cells in DQ6+ individuals with and without narcolepsy. We identify related TRAJ24+ TCRαß clonotypes encoded by identical α/ß gene regions from two patients and two controls. TRAJ24-G allele+ clonotypes only expand in the two patients, whereas a TRAJ24-C allele+ clonotype expands in a control. A representative tetramer+/G-allele+ TCR shows signaling reactivity to the epitope HCRT87-97. Clonally expanded G-allele+ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24+ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development.

Tmem178 negatively regulates store-operated calcium entry in myeloid cells via association with STIM1.

Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER)-localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.

Epithelial MHC Class II Expression and Its Role in Antigen Presentation in the Gastrointestinal and Respiratory Tracts.

As the primary barrier between an organism and its environment, epithelial cells are well-positioned to regulate tolerance while preserving immunity against pathogens. Class II major histocompatibility complex molecules (MHC class II) are highly expressed on the surface of epithelial cells (ECs) in both the lung and intestine, although the functional consequences of this expression are not fully understood. Here, we summarize current information regarding the interactions that regulate the expression of EC MHC class II in health and disease. We then evaluate the potential role of EC as non-professional antigen presenting cells. Finally, we explore future areas of study and the potential contribution of epithelial surfaces to gut-lung crosstalk.

Interleukin-1 in monocyte activation phenotypes in systemic juvenile idiopathic arthritis: Observations from a clinical trial of rilonacept, an interleukin-1 inhibitor.

Systemic juvenile idiopathic arthritis (sJIA) is a childhood rheumatic disease of unknown origin. Dysregulated innate immunity is implicated in disease pathology. We investigated if IL-1 inhibition affects circulating cytokines and monocyte gene expression. CD14+ monocytes from patients in the RAPPORT trial were analyzed by RT-PCR for expression of IL1B and transcription factors associated with monocyte activation. Serum IL-1ra decreased with treatment, and IL-18BP transiently increased. Serum levels of IL-1ß, IL-6, IL-10 and IL-18 were unchanged. IRF5 and STAT6 were decreased, and PPARG was increased, independent of clinical response, and may represent a skew toward a PPARG-driven M2-like phenotype. IL1B expression was decreased in early clinical responders. A transient increase in STAT1, and a decrease in SOCS1 preceded the reduction in IL1B in early clinical responders. Changes in IL1B/STAT1/SOCS1 could be associated with crosstalk between IL-1 and IFN pathways in sJIA. These transcriptional changes might be useful as drug response biomarkers.

Autoantibody Profiling in Lupus Patients using Synthetic Nucleic Acids.

Autoantibodies to nuclear components of cells (antinuclear antibodies, ANA), including DNA (a-DNA), are widely used in the diagnosis and subtyping of certain autoimmune diseases, including systemic lupus erythematosus (SLE). Despite clinical use over decades, precise, reproducible measurement of a-DNA titers remains difficult, likely due to the substantial sequence and length heterogeneity of DNA purified from natural sources. We designed and tested a panel of synthetic nucleic acid molecules composed of native deoxyribonucleotide units to measure a-DNA. ELISA assays using these antigens show specificity and reproducibility. Applying the ELISA tests to serological studies of pediatric and adult SLE, we identified novel clinical correlations. We also observed preferential recognition of a specific synthetic antigen by antibodies in SLE sera. We determined the probable basis for this finding using computational analyses, providing valuable structural information for future development of DNA antigens. Synthetic nucleic acid molecules offer the opportunity to standardize assays and to dissect antibody-antigen interactions.

The MHC class II antigen presentation pathway in human monocytes differs by subset and is regulated by cytokines.

Monocytes play a critical role in the innate and adaptive immune systems, performing phagocytosis, presenting antigen, and producing cytokines. They are a heterogeneous population that has been divided in humans into classical, intermediate, and non-classical subsets, but the roles of these subsets are incompletely understood. In this study, we investigated the expression patterns of MHC class II (MHCII) and associated molecules and find that the intermediate monocytes express the highest levels of the MHC molecules, HLA-DR (tested in n = 30 samples), HLA-DP (n = 30), and HLA-DQ (n = 10). HLA-DM (n = 30), which catalyzes the peptide exchange on the MHC molecules, is also expressed at the highest levels in intermediate monocytes. To measure HLA-DM function, we measured levels of MHCII-bound CLIP (class II invariant chain peptide, n = 23), which is exchanged for other peptides by HLA-DM. We calculated CLIP:MHCII ratios to normalize CLIP levels to MHCII levels, and found that intermediate monocytes have the lowest CLIP:MHCII ratio. We isolated the different monocyte subsets (in a total of 7 samples) and analyzed their responses to selected cytokines as model of monocyte activation: two M1-polarizing cytokines (IFNγ, GM-CSF), an M2-polarizing cytokine (IL-4) and IL-10. Classical monocytes exhibit the largest increases in class II pathway expression in response to stimulatory cytokines (IFNγ, GM-CSF, IL-4). All three subsets decrease HLA-DR levels after IL-10 exposure. Our findings argue that intermediate monocytes are the most efficient constitutive antigen presenting subset, that classical monocytes are recruited into an antigen presentation role during inflammatory responses and that IL-10 negatively regulates this function across all subsets.

The Other Function: Class II-Restricted Antigen Presentation by B Cells.

Mature B lymphocytes (B cells) recognize antigens using their B cell receptor (BCR) and are activated to become antibody-producing cells. In addition, and integral to the development of a high-affinity antibodies, B cells utilize the specialized major histocompatibility complex class II (MHCII) antigen presentation pathway to process BCR-bound and internalized protein antigens and present selected peptides in complex with MHCII to CD4+ T cells. This interaction influences the fate of both types of lymphocytes and shapes immune outcomes. Specific, effective, and optimally timed antigen presentation by B cells requires well-controlled intracellular machinery, often regulated by the combined effects of several molecular events. Here, we delineate and summarize these events in four steps along the antigen presentation pathway: (1) antigen capture and uptake by B cells; (2) intersection of internalized antigen/BCRs complexes with MHCII in peptide-loading compartments; (3) generation and regulation of MHCII/peptide complexes; and (4) exocytic transport for presentation of MHCII/peptide complexes at the surface of B cells. Finally, we discuss modulation of the MHCII presentation pathway across B cell development and maturation to effector cells, with an emphasis on the shaping of the MHCII/peptide repertoire by two key antigen presentation regulators in B cells: HLA-DM and HLA-DO.

Novel Phospholipid-Protein Conjugates Allow Improved Detection of Antibodies in Patients with Autoimmune Diseases.

Reliable measurement of clinically relevant autoimmune antibodies toward phospholipid-protein conjugates is highly desirable in research and clinical assays. To date, the development in this field has been limited to the use of natural heterogeneous antigens. However, this approach does not take structural features of biologically active antigens into account and leads to low reliability and poor scientific test value. Here we describe novel phospholipid-protein conjugates for specific detection of human autoimmune antibodies. Our synthetic approach includes mild oxidation of synthetic phospholipid cardiolipin, and as the last step, coupling of the product with azide-containing linker and copper-catalyzed click chemistry with ß2-glycoprotein I and prothrombin. To prove utility of the product antigens, we used enzyme-linked immunosorbent assay and three cohorts of samples obtained from patients in Denmark (n = 34) and the USA (n = 27 and n = 14). Afterwards we analyzed correlation of the obtained autoantibody titers with clinical parameters for each patient. Our results prove that using novel antigens clinically relevant autoantibodies can be detected with high repeatability, sensitivity and specificity. Unlike previously used antigens the obtained autoantibody titers strongly correlate with high disease activity and in particular, with arthritis, renal involvement, anti-Smith antibodies and high lymphocyte count. Importantly, chemical composition of antigens has a strong influence on the correlation of detected autoantibodies with disease activity and manifestations. This confirms the crucial importance of antigens' composition on research and diagnostic assays, and opens up exciting perspectives for synthetic antigens in future studies of autoimmunity.

Altered signaling in systemic juvenile idiopathic arthritis monocytes.

Systemic juvenile idiopathic arthritis (sJIA) is characterized by systemic inflammation and arthritis. Monocytes are implicated in sJIA pathogenesis, but their role in disease is unclear. The response of sJIA monocytes to IFN may be dysregulated. We examined intracellular signaling in response to IFN type I (IFNα) and type II (IFNγ) in monocytes during sJIA activity and quiescence, in 2 patient groups. Independent of disease activity, monocytes from Group 1 (collected between 2002 and 2009) showed defective STAT1 phosphorylation downstream of IFNs, and expressed higher transcript levels of SOCS1, an inhibitor of IFN signaling. In the Group 2 (collected between 2011 and 2014), monocytes of patients with recent disease onset were IFNγ hyporesponsive, but in treated, quiescent subjects, monocytes were hyperresponsive to IFNγ. Recent changes in medication in sJIA may alter the IFN hyporesponsiveness. Impaired IFN/pSTAT1 signaling is consistent with skewing of sJIA monocytes away from an M1 phenotype and may contribute to disease pathology.

Tmem178 acts in a novel negative feedback loop targeting NFATc1 to regulate bone mass.

Phospholipase C gamma-2 (PLCγ2)-dependent calcium (Ca(2+)) oscillations are indispensable for nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) activation and downstream gene transcription driving osteoclastogenesis during skeletal remodeling and pathological bone loss. Here we describe, to our knowledge, the first known function of transmembrane protein 178 (Tmem178), a PLCγ2 downstream target gene, as a critical modulator of the NFATc1 axis. In surprising contrast to the osteopetrotic phenotype of PLCγ2(-/-) mice, Tmem178(-/-) mice are osteopenic in basal conditions and are more susceptible to inflammatory bone loss, owing to enhanced osteoclast formation. Mechanistically, Tmem178 localizes to the ER membrane and regulates RANKL-induced Ca(2+) fluxes, thus controlling NFATc1 induction. Importantly, down-regulation of Tmem178 is observed in human CD14(+) monocytes exposed to plasma from systemic juvenile idiopathic arthritis patients. Similar to the mouse model, reduced Tmem178 expression in human cells correlates with excessive osteoclastogenesis. In sum, these findings identify an essential role for Tmem178 to maintain skeletal mass and limit pathological bone loss.

Comparison of biomarkers for systemic juvenile idiopathic arthritis.

BACKGROUND: Differentiation of systemic juvenile idiopathic arthritis (SJIA) fever from other childhood fevers is often delayed due to the lack of reliable, specific biomarkers. We hypothesized that PD-L1 expression is dysregulated in SJIA monocytes and compared it to other candidate SJIA biomarkers. METHODS: This pilot study enrolled children with fever without source and compared PD-L1 expression on myeloid cells to C-reactive protein, erythrocyte sedimentation rate, leukocyte counts, S100A12, S100A8, S100A9, calprotectin, and procalcitonin. Logistic regression models were fit to test SJIA diagnosis with each marker used as an independent predictor. Receiver operating characteristic curves and area under curve were calculated. Gene expression profiling on a subset of samples was performed. RESULTS: Twenty subjects (10 active SJIA, 10 febrile non-SJIA) were enrolled. S100 proteins were significantly elevated in SJIA with >80% sensitivity and >90% specificity. PD-L1 expression was significantly lower in SJIA. Other markers were not specific for SJIA. On exploratory gene analysis, 106 genes were significant for SJIA association, and several of these are associated with immune response pathways. CONCLUSION: In this small cohort, S100 proteins were specific diagnostic biomarkers for SJIA in children with fever. Decreased PD-L1 surface expression on circulating myeloid cells in SJIA suggests possible mechanism for loss of peripheral immune regulation.

Synthesis of Phospholipid-Protein Conjugates as New Antigens for Autoimmune Antibodies.

Copper(I)-catalyzed azide-alkyne cycloaddition, or CuAAC click chemistry, is an efficient method for bioconjugation aiming at chemical and biological applications. Herein, we demonstrate how the CuAAC method can provide novel phospholipid-protein conjugates with a high potential for the diagnostics and therapy of autoimmune conditions. In doing this, we, for the first time, covalently bind via 1,2,3-triazole linker biologically complementary molecules, namely phosphoethanol amine with human ß2-glycoprotein I and prothrombin. The resulting phospholipid-protein conjugates show high binding affinity and specificity for the autoimmune antibodies against autoimmune complexes. Thus, the development of this work might become a milestone in further diagnostics and therapy of autoimmune diseases that involve the production of autoantibodies against the aforementioned phospholipids and proteins, such as antiphospholipid syndrome and systemic lupus erythematosus.