Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus.

Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions1,2. Expansion of T follicular helper (TFH) and T peripheral helper (TPH) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4. Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs. 5,6), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH/TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH/TFH cells on a polarization axis opposite from T helper 22 (TH22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states.

IL-33 drives influenza-induced asthma exacerbations by halting innate and adaptive antiviral immunity.

BACKGROUND: Influenza virus triggers severe asthma exacerbations for which no adequate treatment is available. It is known that IL-33 levels correlate with exacerbation severity, but its role in the immunopathogenesis of exacerbations has remained elusive. OBJECTIVE: We hypothesized that IL-33 is necessary to drive asthma exacerbations. We intervened with the IL-33 cascade and sought to dissect its role, also in synergy with thymic stromal lymphopoietin (TSLP), in airway inflammation, antiviral activity, and lung function. We aimed to unveil the major source of IL-33 in the airways and IL-33-dependent mechanisms that underlie severe asthma exacerbations. METHODS: Patients with mild asthma were experimentally infected with rhinovirus. Mice were chronically exposed to house dust mite extract and then infected with influenza to resemble key features of exacerbations in human subjects. Interventions included the anti-IL-33 receptor ST2, anti-TSLP, or both. RESULTS: We identified bronchial ciliated cells and type II alveolar cells as a major local source of IL-33 during virus-driven exacerbation in human subjects and mice, respectively. By blocking ST2, we demonstrated that IL-33 and not TSLP was necessary to drive exacerbations. IL-33 enhanced airway hyperresponsiveness and airway inflammation by suppressing innate and adaptive antiviral responses and by instructing epithelial cells and dendritic cells of house dust mite-sensitized mice to dampen IFN-ß expression and prevent the TH1-promoting dendritic cell phenotype. IL-33 also boosted luminal NETosis and halted cytolytic antiviral activities but did not affect the TH2 response. CONCLUSION: Interventions targeting the IL-33/ST2 axis could prove an effective acute short-term therapy for virus-induced asthma exacerbations.

Accelerating Lead Identification by High Throughput Virtual Screening: Prospective Case Studies from the Pharmaceutical Industry.

At the onset of a drug discovery program, the goal is to identify novel compounds with appropriate chemical features that can be taken forward as lead series. Here, we describe three prospective case studies, Bruton Tyrosine Kinase (BTK), RAR-Related Orphan Receptor γ t (RORγt), and Human Leukocyte Antigen DR isotype (HLA-DR) to illustrate the positive impact of high throughput virtual screening (HTVS) on the successful identification of novel chemical series. Each case represents a project with a varying degree of difficulty due to the amount of structural and ligand information available internally or in the public domain to utilize in the virtual screens. We show that HTVS can be effectively employed to identify a diverse set of potent hits for each protein system even when the gold standard, high resolution structural data or ligand binding data for benchmarking, is not available.

A Single Amino Acid Difference between Mouse and Human 5-Lipoxygenase Activating Protein (FLAP) Explains the Speciation and Differential Pharmacology of Novel FLAP Inhibitors.

5-Lipoxygenase activating protein (FLAP) plays a critical role in the metabolism of arachidonic acid to leukotriene A4, the precursor to the potent pro-inflammatory mediators leukotriene B4 and leukotriene C4 Studies with small molecule inhibitors of FLAP have led to the discovery of a drug binding pocket on the protein surface, and several pharmaceutical companies have developed compounds and performed clinical trials. Crystallographic studies and mutational analyses have contributed to a general understanding of compound binding modes. During our own efforts, we identified two unique chemical series. One series demonstrated strong inhibition of human FLAP but differential pharmacology across species and was completely inactive in assays with mouse or rat FLAP. The other series was active across rodent FLAP, as well as human and dog FLAP. Comparison of rodent and human FLAP amino acid sequences together with an analysis of a published crystal structure led to the identification of amino acid residue 24 in the floor of the putative binding pocket as a likely candidate for the observed speciation. On that basis, we tested compounds for binding to human G24A and mouse A24G FLAP mutant variants and compared the data to that generated for wild type human and mouse FLAP. These studies confirmed that a single amino acid mutation was sufficient to reverse the speciation observed in wild type FLAP. In addition, a PK/PD method was established in canines to enable preclinical profiling of mouse-inactive compounds.

Oxidative stress-induced mitochondrial dysfunction drives inflammation and airway smooth muscle remodeling in patients with chronic obstructive pulmonary disease.

BACKGROUND: Inflammation and oxidative stress play critical roles in patients with chronic obstructive pulmonary disease (COPD). Mitochondrial oxidative stress might be involved in driving the oxidative stress-induced pathology. OBJECTIVE: We sought to determine the effects of oxidative stress on mitochondrial function in the pathophysiology of airway inflammation in ozone-exposed mice and human airway smooth muscle (ASM) cells. METHODS: Mice were exposed to ozone, and lung inflammation, airway hyperresponsiveness (AHR), and mitochondrial function were determined. Human ASM cells were isolated from bronchial biopsy specimens from healthy subjects, smokers, and patients with COPD. Inflammation and mitochondrial function in mice and human ASM cells were measured with and without the presence of the mitochondria-targeted antioxidant MitoQ. RESULTS: Mice exposed to ozone, a source of oxidative stress, had lung inflammation and AHR associated with mitochondrial dysfunction and reflected by decreased mitochondrial membrane potential (ΔΨm), increased mitochondrial oxidative stress, and reduced mitochondrial complex I, III, and V expression. Reversal of mitochondrial dysfunction by the mitochondria-targeted antioxidant MitoQ reduced inflammation and AHR. ASM cells from patients with COPD have reduced ΔΨm, adenosine triphosphate content, complex expression, basal and maximum respiration levels, and respiratory reserve capacity compared with those from healthy control subjects, whereas mitochondrial reactive oxygen species (ROS) levels were increased. Healthy smokers were intermediate between healthy nonsmokers and patients with COPD. Hydrogen peroxide induced mitochondrial dysfunction in ASM cells from healthy subjects. MitoQ and Tiron inhibited TGF-ß-induced ASM cell proliferation and CXCL8 release. CONCLUSIONS: Mitochondrial dysfunction in patients with COPD is associated with excessive mitochondrial ROS levels, which contribute to enhanced inflammation and cell hyperproliferation. Targeting mitochondrial ROS represents a promising therapeutic approach in patients with COPD.

Mast cell mediators cause early allergic bronchoconstriction in guinea-pigs in vivo: a model of relevance to asthma.

One feature of allergic asthma, the EAR (early allergic reaction), is not present in the commonly used mouse models. We therefore investigated the mediators involved in EAR in a guinea-pig in vivo model of allergic airway inflammation. Animals were sensitized using a single OVA (ovalbumin)/alum injection and challenged with aerosolized OVA on day 14. On day 15, airway resistance was assessed after challenge with OVA or MCh (methacholine) using the forced oscillation technique, and lung tissue was prepared for histology. The contribution of mast cell mediators was investigated using inhibitors of the main mast cell mediators [histamine (pyrilamine) and CysLTs (cysteinyl-leukotrienes) (montelukast) and prostanoids (indomethacin)]. OVA-sensitized and challenged animals demonstrated AHR (airway hyper-responsiveness) to MCh, and lung tissue eosinophilic inflammation. Antigen challenge induced a strong EAR in the sensitized animals. Treatment with a single compound, or indomethacin together with pyrilamine or montelukast, did not reduce the antigen-induced airway resistance. In contrast, dual treatment with pyrilamine together with montelukast, or triple inhibitor treatment, attenuated approximately 70% of the EAR. We conclude that, as in humans, the guinea-pig allergic inflammation model exhibits both EAR and AHR, supporting its suitability for in vivo identification of mast cell mediators that contribute to the development of asthma. Moreover, the known mast cell mediators histamine and leukotrienes were major contributors of the EAR. The data also lend further support to the concept that combination therapy with selective inhibitors of key mediators could improve asthma management.

Identification of benzofuran central cores for the inhibition of leukotriene A(4) hydrolase.

Leukotrienes (LT's) are known to play a physiological role in inflammatory immune response. Leukotriene A(4) hydrolase (LTA(4)H) is a cystolic enzyme that stereospecifically catalyzes the transformation of LTA(4) to LTB(4). LTB(4) is a known pro-inflammatory mediator. This paper describes the identification and synthesis of substituted benzofurans as LTH(4)H inhibitors. The benzofuran series demonstrated reduced mouse and human whole blood LTB(4) levels in vitro and led to the identification one analog for advanced profiling. Benzofuran 28 showed dose responsive target engagement and provides a useful tool to explore a LTA(4)H inhibitor for the treatment of inflammatory diseases, such as asthma and inflammatory bowel disease (IBD).

Diversity and Clonality of T Cell Receptor Repertoire and Antigen Specificities in Small Joints of Early Rheumatoid Arthritis.

OBJECTIVE: CD4+ T cells are implicated in rheumatoid arthritis (RA) pathology from the strong association between RA and certain HLA class II gene variants. This study was undertaken to examine the synovial T cell receptor (TCR) repertoire, T cell phenotypes, and T cell specificities in small joints of RA patients at time of diagnosis before therapeutic intervention. METHODS: Sixteen patients, of whom 11 patients were anti-citrullinated protein antibody (ACPA)-positive and 5 patients were ACPA-, underwent ultrasound-guided synovial biopsy of a small joint (n = 13) or arthroscopic synovial biopsy of a large joint (n = 3), followed by direct sorting of single T cells for paired sequencing of the αß TCR together with flow cytometry analysis. TCRs from expanded CD4+ T cell clones of 4 patients carrying an HLA-DRB1*04:01 allele were artificially reexpressed to study antigen specificity. RESULTS: T cell analysis demonstrated CD4+ dominance and the presence of peripheral helper T-like cells in both patient groups. We identified >4,000 unique TCR sequences, as well as 225 clonal expansions. Additionally, T cells with double α-chains were a recurring feature. We identified a biased gene usage of the Vß chain segment TRBV20-1 in CD4+ cells from ACPA+ patients. In vitro stimulation of T cell lines expressing selected TCRs with an extensive panel of citrullinated and viral peptides identified several different virus-specific TCRs (e.g., human cytomegalovirus and human herpesvirus 2). Still, the majority of clones remained orphans with unknown specificity. CONCLUSION: Minimally invasive biopsies of the RA synovium allow for single-cell TCR sequencing and phenotyping. Clonally expanded, viral-reactive T cells account for part of the diverse CD4+ T cell repertoire. TRBV20-1 bias in ACPA+ patients suggests recognition of common antigens.

Cytotoxic CD8+ T cells target citrullinated antigens in rheumatoid arthritis.

The immune mechanisms that mediate synovitis and joint destruction in rheumatoid arthritis (RA) remain poorly defined. Although increased levels of CD8+ T cells have been described in RA, their function in pathogenesis remains unclear. Here we perform single cell transcriptome and T cell receptor (TCR) sequencing of CD8+ T cells derived from anti-citrullinated protein antibodies (ACPA)+ RA blood. We identify GZMB+CD8+ subpopulations containing large clonal lineage expansions that express cytotoxic and tissue homing transcriptional programs, while a GZMK+CD8+ memory subpopulation comprises smaller clonal expansions that express effector T cell transcriptional programs. We demonstrate RA citrullinated autoantigens presented by MHC class I activate RA blood-derived GZMB+CD8+ T cells to expand, express cytotoxic mediators, and mediate killing of target cells. We also demonstrate that these clonally expanded GZMB+CD8+ cells are present in RA synovium. These findings suggest that cytotoxic CD8+ T cells targeting citrullinated antigens contribute to synovitis and joint tissue destruction in ACPA+ RA.

Multifaceted immune dysregulation characterizes individuals at-risk for rheumatoid arthritis.

Molecular markers of autoimmunity, such as antibodies to citrullinated protein antigens (ACPA), are detectable prior to inflammatory arthritis (IA) in rheumatoid arthritis (RA) and may define a state that is 'at-risk' for future RA. Here we present a cross-sectional comparative analysis among three groups that include ACPA positive individuals without IA (At-Risk), ACPA negative individuals and individuals with early, ACPA positive clinical RA (Early RA). Differential methylation analysis among the groups identifies non-specific dysregulation in peripheral B, memory and naïve T cells in At-Risk participants, with more specific immunological pathway abnormalities in Early RA. Tetramer studies show increased abundance of T cells recognizing citrullinated (cit) epitopes in At-Risk participants, including expansion of T cells reactive to citrullinated cartilage intermediate layer protein I (cit-CILP); these T cells have Th1, Th17, and T stem cell memory-like phenotypes. Antibody-antigen array analyses show that antibodies targeting cit-clusterin, cit-fibrinogen and cit-histone H4 are elevated in At-Risk and Early RA participants, with the highest levels of antibodies detected in those with Early RA. These findings indicate that an ACPA positive at-risk state is associated with multifaceted immune dysregulation that may represent a potential opportunity for targeted intervention.

Serologic Biomarkers of Progression Toward Diagnosis of Rheumatoid Arthritis in Active Component Military Personnel.

OBJECTIVE: To identify a panel of serum biomarkers that could specifically identify imminent cases of rheumatoid arthritis (RA) before diagnosis. METHODS: Serum samples were collected at 4 time points from active component US military personnel, including 157 anti-citrullinated protein antibody (ACPA)-seropositive and 50 ACPA-seronegative RA subjects, 100 reactive arthritis (ReA) subjects, and 76 healthy controls. The cohorts were split into 2 phases, with samples tested on independent proteomic platforms for each phase. Classification models of RA diagnosis based on samples obtained within 6 months prior to diagnosis were developed both in univariate analyses and by multivariate random forest modeling of training sample sets and testing sample sets from each phase. RESULTS: Increases in serum analytes, including C-reactive protein levels, serum amyloid A, and soluble programmed cell death 1 (PD-1), were observed in seropositive RA subjects at the time point closest to diagnosis, up to several years before diagnosis. Only a small fraction of RA subjects had levels above the 95th percentile of healthy control levels until the time period within 6 months of diagnosis. For classification of RA diagnosis using samples obtained within 6 months prior to diagnosis, soluble PD-1 provided superior specificity compared to ReA cases (>89%), with a sensitivity of 48% for RA classification. An 8-analyte model provided superior sensitivity (69%), with comparable specificity relative to ReA (>82%). CONCLUSION: Our findings demonstrate that imminent RA diagnosis could be classified with high specificity, relative to healthy controls and ReA cases, using a panel of cytokines measured in serum samples collected within 6 months before actual diagnosis.

Differential expansion of T peripheral helper cells in early rheumatoid arthritis and osteoarthritis synovium.

OBJECTIVES: Programmed cell death protein 1 (PD-1)-expressing T cells are implicated in the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis. A subset of CXCR5- T cells, termed T peripheral helper (Tph) cells, which drive B cell differentiation, have been identified in ectopic lymphoid structures in established rheumatoid arthritis synovial tissue. Here, we aimed to characterise these in treatment-naïve, early rheumatoid arthritis to determine whether these cells accumulate prior to fully established disease. METHODS: Fresh dissociated tissue and peripheral blood mononuclear cell (PBMC) suspensions were stained with Zombie UV, followed by anti-CD45RO, PD-1, CD3, ICOS, CD8, CD4, CD20, CXCR5, TIGIT and CD38 antibodies prior to analysis. For histology, rheumatoid arthritis synovial sections were prepared for Opal multispectral immunofluorescence with anti-CD45RO, CD20, PD-1 and CXCR5 antibodies. Images were acquired on the Perkin Elmer Vectra V.3.0 imaging system and analysed using InForm Advanced Image Analysis software. RESULTS: Flow cytometry revealed T cell infiltration in the rheumatoid arthritis synovium with differential expression of PD-1, CD45RO, ICOS, TIGIT and CD38. We observed a higher frequency of PD1hiCXCR5- Tph in rheumatoid arthritis synovial tissue and PBMCs versus controls, and no significant difference in T follicular helper cell frequency. Microscopy identified a 10-fold increase of Tph cells in early rheumatoid arthritis synovial follicular and diffuse regions, and identified Tph adjacent to germinal centre B cells. CONCLUSIONS: These data demonstrate that PD-1hi Tph cells are present in early rheumatoid arthritis, but not osteoarthritis synovium, and therefore may provide a target for treatment of patients with early rheumatoid arthritis.

Discovery of JNJ-64264681: A Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase.

Bruton's tyrosine kinase (BTK) is a Tec family kinase that plays an essential role in B-cell receptor (BCR) signaling as well as Fcγ receptor signaling in leukocytes. Pharmacological inhibition of BTK has been shown to be effective in treating hematological malignancies and is hypothesized to provide an effective strategy for the treatment of autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. We report the discovery and preclinical properties of JNJ-64264681 (13), a covalent, irreversible BTK inhibitor with potent whole blood activity and exceptional kinome selectivity. JNJ-64264681 demonstrated excellent oral efficacy in both cancer and autoimmune models with sustained in vivo target coverage amenable to once daily dosing and has advanced into human clinical studies to investigate safety and pharmacokinetics.

CpG-induced tyrosine phosphorylation occurs via a TLR9-independent mechanism and is required for cytokine secretion.

Toll-like receptors (TLRs) recognize molecular patterns preferentially expressed by pathogens. In endosomes, TLR9 is activated by unmethylated bacterial DNA, resulting in proinflammatory cytokine secretion via the adaptor protein MyD88. We demonstrate that CpG oligonucleotides activate a TLR9-independent pathway initiated by two Src family kinases, Hck and Lyn, which trigger a tyrosine phosphorylation-mediated signaling cascade. This cascade induces actin cytoskeleton reorganization, resulting in cell spreading, adhesion, and motility. CpG-induced actin polymerization originates at the plasma membrane, rather than in endosomes. Chloroquine, an inhibitor of CpG-triggered cytokine secretion, blocked TLR9/MyD88-dependent cytokine secretion as expected but failed to inhibit CpG-induced Src family kinase activation and its dependent cellular responses. Knock down of Src family kinase expression or the use of specific kinase inhibitors blocked MyD88-dependent signaling and cytokine secretion, providing evidence that tyrosine phosphorylation is both CpG induced and an upstream requirement for the engagement of TLR9. The Src family pathway intersects the TLR9-MyD88 pathway by promoting the tyrosine phosphorylation of TLR9 and the recruitment of Syk to this receptor.

Leukotriene A(4) hydrolase inhibition attenuates allergic airway inflammation and hyperresponsiveness.

RATIONALE: Allergic asthma is characterized by reversible airway obstruction, lung inflammation, and airway hyperresponsiveness (AHR). Previous studies using leukotriene B(4) (LTB(4)) receptor 1-deficient mice and adoptive transfer experiments have suggested that LTB(4) plays a role in lung inflammation and AHR. OBJECTIVES: In this study, we used a leukotriene A(4) hydrolase (LTA(4)H) inhibitor as a pharmacological tool to directly examine the role of LTB(4) in a mast cell-dependent murine model of allergic airway inflammation. METHODS: We used the forced oscillation technique to test the effects of an LTA(4)H inhibitor dosed during the challenge phase on AHR. Lung tissue and lavage were collected for analysis. MEASUREMENTS AND MAIN RESULTS: Treatment with an LTA(4)H inhibitor improved multiple parameters encompassing AHR and lung function. Significant decreases in inflammatory leukocytes, cytokines, and mucin were observed in the lung lumen. Serum levels of antigen-specific IgE and IgG1 were also decreased. Labeled antigen uptake by lung dendritic cells and subsequent trafficking to draining lymph nodes and the lung were decreased on LTA(4)H inhibitor treatment. Provocatively, inhibition of LTA(4)H increased lipoxin A(4) levels in lung lavage fluid. CONCLUSIONS: These data suggest that LTB(4) plays a key role in driving lung inflammation and AHR. Mechanistically, we provide evidence that inhibition of LTA(4)H, affects recruitment of both CD4(+) and CD8(+) T cells, as well as trafficking of dendritic cells to draining lymph nodes, and may beneficially modulate other pro- and antiinflammatory eicosanoids in the lung. Inhibition of LTA(4)H is thus a potential therapeutic strategy that could modulate key aspects of asthma.

The role of posttranslational modifications in generating neo-epitopes that bind to rheumatoid arthritis-associated HLA-DR alleles and promote autoimmune T cell responses.

While antibodies to citrullinated proteins have become a diagnostic hallmark in rheumatoid arthritis (RA), we still do not understand how the autoimmune T cell response is influenced by these citrullinated proteins. To investigate the role of citrullinated antigens in HLA-DR1- and DR4-restricted T cell responses, we utilized mouse models that express these MHC-II alleles to determine the relationship between citrullinated peptide affinity for these DR molecules and the ability of these peptides to induce a T cell response. Using a set of peptides from proteins thought to be targeted by the autoimmune T cell responses in RA, aggrecan, vimentin, fibrinogen, and type II collagen, we found that while citrullination can enhance the binding affinity for these DR alleles, it does not always do so, even when in the critical P4 position. Moreover, if peptide citrullination does enhance HLA-DR binding affinity, it does not necessarily predict the generation of a T cell response. Conversely, citrullinated peptides can stimulate T cells without changing the peptide binding affinity for HLA-DR1 or DR4. Furthermore, citrullination of an autoantigen, type II collagen, which enhances binding affinity to HLA-DR1 did not enhance the severity of autoimmune arthritis in HLA-DR1 transgenic mice. Additional analysis of clonal T cell populations stimulated by these peptides indicated cross recognition of citrullinated and wild type peptides can occur in some instances, while in others cases the citrullination generates a novel T cell epitope. Finally, cytokine profiles of the wild type and citrullinated peptide stimulated T cells unveiled a significant disconnect between proliferation and cytokine production. Altogether, these data demonstrate the lack of support for a simplified model with universal correlation between affinity for HLA-DR alleles, immunogenicity and arthritogenicity of citrullinated peptides. Additionally they highlight the complexity of both T cell receptor recognition of citrulline as well as its potential conformational effects on the peptide:HLA-DR complex as recognized by a self-reactive cell receptor.

Discovery of a Potent and Selective Covalent Inhibitor of Bruton's Tyrosine Kinase with Oral Anti-Inflammatory Activity.

Bruton's tyrosine kinase (BTK) is a cytoplasmic tyrosine kinase that plays a critical role in the activation of B cells, macrophages, and osteoclasts. Given the key role of these cell types in the pathology of autoimmune disorders, BTK inhibitors have the potential to improve treatment outcomes in multiple diseases. Herein, we report the discovery and characterization of a novel potent and selective covalent 4-oxo-4,5-dihydro-3H-1-thia-3,5,8-triazaacenaphthylene-2-carboxamide BTK inhibitor chemotype. Compound 27 irreversibly inhibits BTK by targeting a noncatalytic cysteine residue (Cys481) for covalent bond formation. Compound 27 is characterized by selectivity for BTK, potent in vivo BTK occupancy that is sustained after it is cleared from systemic circulation, and dose-dependent efficacy at reducing joint inflammation in a rat collagen-induced arthritis model.

Pathogenic, glycolytic PD-1+ B cells accumulate in the hypoxic RA joint.

While autoantibodies are used in the diagnosis of rheumatoid arthritis (RA), the function of B cells in the inflamed joint remains elusive. Extensive flow cytometric characterization and SPICE algorithm analyses of single-cell synovial tissue from patients with RA revealed the accumulation of switched and double-negative memory programmed death-1 receptor-expressing (PD-1-expressing) B cells at the site of inflammation. Accumulation of memory B cells was mediated by CXCR3, evident by the observed increase in CXCR3-expressing synovial B cells compared with the periphery, differential regulation by key synovial cytokines, and restricted B cell invasion demonstrated in response to CXCR3 blockade. Notably, under 3% O2 hypoxic conditions that mimic the joint microenvironment, RA B cells maintained marked expression of MMP-9, TNF, and IL-6, with PD-1+ B cells demonstrating higher expression of CXCR3, CD80, CD86, IL-1ß, and GM-CSF than their PD-1- counterparts. Finally, following functional analysis and flow cell sorting of RA PD-1+ versus PD-1- B cells, we demonstrate, using RNA-Seq and emerging fluorescence lifetime imaging microscopy of cellular NAD, a significant shift in metabolism of RA PD-1+ B cells toward glycolysis, associated with an increased transcriptional signature of key cytokines and chemokines that are strongly implicated in RA pathogenesis. Our data support the targeting of pathogenic PD-1+ B cells in RA as a focused, novel therapeutic option.

Cohort profile of a US military population for evaluating pre-disease and disease serological biomarkers in rheumatoid and reactive arthritis: Rationale, organization, design, and baseline characteristics.

PURPOSE: The etiology of several autoimmune disorders, including rheumatoid arthritis, remains unknown. While there are clear phases of disease progression, the mechanisms of transition between these phases are poorly understood. Additionally, treatment focuses on an alteration of the biological processes to prevent joint damage and functional decline. A goal is to potentially treat the disease during the preclinical phase to mitigate the disease process. Reactive arthritis is another rheumatologic condition known to be secondary to a distal infection. While prevention of infection would mitigate risk, serologic profiling patients with the disease may assist in the elucidation of potential disease risk factors. This study was initiated to enable an assessment of pre-disease biomarkers in patients newly diagnosed with rheumatoid arthritis and reactive arthritis. PARTICIPANTS: A retrospective cohort of 500 rheumatoid and 500 reactive arthritis cases with 500 matched controls was drawn from a population of active component US military personnel. Appropriate inclusion criteria limited subject selection. Additionally, 4 serum samples (3 pre-disease and 1 disease-associated) were obtained for each case and control. FINDINGS TO DATE: The established cohort provides the framework for novel exploration of the host response through serum profiling and seroepidemiology prior to disease onset. FUTURE PLANS: This study establishes the framework for the evaluation of novel serum biomarkers enabling the identification of signals prior to clinical disease that may enable disease prediction, elucidate disease pathogenesis and identify novel exposures leading to increased disease risk and/or disease severity.

IRAK1: a critical signaling mediator of innate immunity.

The innate immune system is equipped with sensitive and efficient machineries to provide an immediate, first line defense against infections. Toll-like receptors (TLRs) detect pathogens and the IL-1 receptor (IL-1R) family enables cells to quickly respond to inflammatory cytokines by mounting an efficient protective response. Interleukin-1 receptor activated kinases (IRAKs) are key mediators in the signaling pathways of TLRs/IL-1Rs. By means of their kinase and adaptor functions, IRAKs initiate a cascade of signaling events eventually leading to induction of inflammatory target gene expression. Due to this pivotal role, IRAK function is also highly regulated via multiple mechanisms. In this review, we focus on IRAK1, the earliest known and yet the most interesting member of this family. An overview on its structure, function and biology is given, with emphasis on the different novel mechanisms that regulate IRAK1 function. We also highlight several unresolved questions in this field and evaluate the potential of IRAK1 as a target for therapeutic intervention.