Synovial fibroblast gene expression is associated with sensory nerve growth and pain in rheumatoid arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We developed a machine-learning approach (graph-based gene expression module identification or GbGMI) to identify an 815-gene expression module associated with pain in synovial biopsy samples from patients with established RA who had limited synovial inflammation at arthroplasty. We then validated this finding in an independent cohort of synovial biopsy samples from patients who had early untreated RA with little inflammation. Single-cell RNA sequencing analyses indicated that most of these 815 genes were most robustly expressed by lining layer synovial fibroblasts. Receptor-ligand interaction analysis predicted cross-talk between human lining layer fibroblasts and human dorsal root ganglion neurons expressing calcitonin gene-related peptide (CGRP+). Both RA synovial fibroblast culture supernatant and netrin-4, which is abundantly expressed by lining fibroblasts and was within the GbGMI-identified pain-associated gene module, increased the branching of pain-sensitive murine CGRP+ dorsal root ganglion neurons in vitro. Imaging of solvent-cleared synovial tissue with little inflammation from humans with RA revealed CGRP+ pain-sensing neurons encasing blood vessels growing into synovial hypertrophic papilla. Together, these findings support a model whereby synovial lining fibroblasts express genes associated with pain that enhance the growth of pain-sensing neurons into regions of synovial hypertrophy in RA.

Machine Learning Reveals Synovial Fibroblast Genes Associated with Pain Affect Sensory Nerve Growth in Rheumatoid Arthritis.

It has been presumed that rheumatoid arthritis (RA) joint pain is related to inflammation in the synovium; however, recent studies reveal that pain scores in patients do not correlate with synovial inflammation. We identified a module of 815 genes associated with pain, using a novel machine learning approach, Graph-based Gene expression Module Identification (GbGMI), in samples from patients with longstanding RA, but limited synovial inflammation at arthroplasty, and validated this finding in an independent cohort of synovial biopsy samples from early, untreated RA patients. Single-cell RNA-seq analyses indicated these genes were most robustly expressed by lining layer fibroblasts and receptor-ligand interaction analysis predicted robust lining layer fibroblast crosstalk with pain sensitive CGRP+ dorsal root ganglion sensory neurons. Netrin-4, which is abundantly expressed by lining fibroblasts and associated with pain, significantly increased the branching of pain-sensitive CGRP+ neurons in vitro . We conclude GbGMI is a useful method for identifying a module of genes that associate with a clinical feature of interest. Using this approach, we find that Netrin-4 is produced by synovial fibroblasts in the absence of inflammation and can enhance the outgrowth of CGRP+ pain sensitive nerve fibers. One Sentence Summary: Machine Learning reveals synovial fibroblast genes related to pain affect sensory nerve growth in Rheumatoid Arthritis addresses unmet clinical need.

Oral mucosal breaks trigger anti-citrullinated bacterial and human protein antibody responses in rheumatoid arthritis.

Periodontal disease is more common in individuals with rheumatoid arthritis (RA) who have detectable anti-citrullinated protein antibodies (ACPAs), implicating oral mucosal inflammation in RA pathogenesis. Here, we performed paired analysis of human and bacterial transcriptomics in longitudinal blood samples from RA patients. We found that patients with RA and periodontal disease experienced repeated oral bacteremias associated with transcriptional signatures of ISG15+HLADRhi and CD48highS100A2pos monocytes, recently identified in inflamed RA synovia and blood of those with RA flares. The oral bacteria observed transiently in blood were broadly citrullinated in the mouth, and their in situ citrullinated epitopes were targeted by extensively somatically hypermutated ACPAs encoded by RA blood plasmablasts. Together, these results suggest that (i) periodontal disease results in repeated breaches of the oral mucosa that release citrullinated oral bacteria into circulation, which (ii) activate inflammatory monocyte subsets that are observed in inflamed RA synovia and blood of RA patients with flares and (iii) activate ACPA B cells, thereby promoting affinity maturation and epitope spreading to citrullinated human antigens.

DRUL for school: Opening Pre-K with safe, simple, sensitive saliva testing for SARS-CoV-2.

To address the need for simple, safe, sensitive, and scalable SARS-CoV-2 tests, we validated and implemented a PCR test that uses a saliva collection kit use at home. Individuals self-collected 300 µl saliva in vials containing Darnell Rockefeller University Laboratory (DRUL) buffer and extracted RNA was assayed by RT-PCR (the DRUL saliva assay). The limit of detection was confirmed to be 1 viral copy/µl in 20 of 20 replicate extractions. Viral RNA was stable in DRUL buffer at room temperature up to seven days after sample collection, and safety studies demonstrated that DRUL buffer immediately inactivated virus at concentrations up to 2.75x106 PFU/ml. Results from SARS-CoV-2 positive nasopharyngeal (NP) swab samples collected in viral transport media and assayed with a standard FDA Emergency Use Authorization (EUA) test were highly correlated with samples placed in DRUL buffer. Direct comparison of results from 162 individuals tested by FDA EUA oropharyngeal (OP) or NP swabs with co-collected saliva samples identified four otherwise unidentified positive cases in DRUL buffer. Over six months, we collected 3,724 samples from individuals ranging from 3 months to 92 years of age. This included collecting weekly samples over 10 weeks from teachers, children, and parents from a pre-school program, which allowed its safe reopening while at-risk pods were quarantined. In sum, we validated a simple, sensitive, stable, and safe PCR-based test using a self-collected saliva sample as a valuable tool for clinical diagnosis and screening at workplaces and schools.

Vaccine Breakthrough Infections with SARS-CoV-2 Variants.

Emerging variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are of clinical concern. In a cohort of 417 persons who had received the second dose of BNT162b2 (Pfizer-BioNTech) or mRNA-1273 (Moderna) vaccine at least 2 weeks previously, we identified 2 women with vaccine breakthrough infection. Despite evidence of vaccine efficacy in both women, symptoms of coronavirus disease 2019 developed, and they tested positive for SARS-CoV-2 by polymerase-chain-reaction testing. Viral sequencing revealed variants of likely clinical importance, including E484K in 1 woman and three mutations (T95I, del142-144, and D614G) in both. These observations indicate a potential risk of illness after successful vaccination and subsequent infection with variant virus, and they provide support for continued efforts to prevent and diagnose infection and to characterize variants in vaccinated persons. (Funded by the National Institutes of Health and others.).

RNA Identification of PRIME Cells Predicting Rheumatoid Arthritis Flares.

BACKGROUND: Rheumatoid arthritis, like many inflammatory diseases, is characterized by episodes of quiescence and exacerbation (flares). The molecular events leading to flares are unknown. METHODS: We established a clinical and technical protocol for repeated home collection of blood in patients with rheumatoid arthritis to allow for longitudinal RNA sequencing (RNA-seq). Specimens were obtained from 364 time points during eight flares over a period of 4 years in our index patient, as well as from 235 time points during flares in three additional patients. We identified transcripts that were differentially expressed before flares and compared these with data from synovial single-cell RNA-seq. Flow cytometry and sorted-blood-cell RNA-seq in additional patients were used to validate the findings. RESULTS: Consistent changes were observed in blood transcriptional profiles 1 to 2 weeks before a rheumatoid arthritis flare. B-cell activation was followed by expansion of circulating CD45-CD31-PDPN+ preinflammatory mesenchymal, or PRIME, cells in the blood from patients with rheumatoid arthritis; these cells shared features of inflammatory synovial fibroblasts. Levels of circulating PRIME cells decreased during flares in all 4 patients, and flow cytometry and sorted-cell RNA-seq confirmed the presence of PRIME cells in 19 additional patients with rheumatoid arthritis. CONCLUSIONS: Longitudinal genomic analysis of rheumatoid arthritis flares revealed PRIME cells in the blood during the period before a flare and suggested a model in which these cells become activated by B cells in the weeks before a flare and subsequently migrate out of the blood into the synovium. (Funded by the National Institutes of Health and others.).

Rheumatoid Arthritis Morning Stiffness Is Associated With Synovial Fibrin and Neutrophils.

OBJECTIVE: Morning stiffness is a hallmark symptom of rheumatoid arthritis (RA), but its etiology is poorly understood. This study was undertaken to determine whether any histologic features of synovium are associated with this symptom. METHODS: Data on patient-reported morning stiffness duration and severity, and Disease Activity Score in 28 joints (DAS28) were collected from 176 patients with RA undergoing arthroplasty. Synovium was scored for 10 histopathologic features: synovial lining hyperplasia, lymphocytes, plasma cells, Russell bodies, binucleate plasma cells, fibrin, synovial giant cells, detritus, neutrophils, and mucin. Fibrinolysis of clots seeded with various cell types was measured in turbidimetric lysis assays. RESULTS: Stiffness severity and morning stiffness duration were both significantly associated with DAS28 (P = 0.0001 and P = 0.001, respectively). None of the synovial features examined were associated with patient-reported stiffness severity. The presence of neutrophils and fibrin in RA synovial tissue were significantly associated (P < 0.0001) with patient-reported morning stiffness of ≥1 hour, such that 73% of patients with both synovial fibrin and neutrophils reported morning stiffness of ≥1 hour. Further, neutrophils and fibrin deposits colocalized along the synovial lining. In in vitro analyses, fibrin clots seeded with necrotic neutrophils were more resistant to fibrinolysis than those seeded with living neutrophils or no cells (P = 0.008). DNase I treatment of necrotic neutrophils abrogated the delay in fibrinolysis. CONCLUSION: In RA, prolonged morning stiffness may be related to impaired fibrinolysis of neutrophil-enmeshed fibrin deposits along the synovial membrane. Our findings also suggest that morning stiffness severity and duration may reflect distinct pathophysiologic phenomena.

ZFP36 RNA-binding proteins restrain T cell activation and anti-viral immunity.

Dynamic post-transcriptional control of RNA expression by RNA-binding proteins (RBPs) is critical during immune response. ZFP36 RBPs are prominent inflammatory regulators linked to autoimmunity and cancer, but functions in adaptive immunity are less clear. We used HITS-CLIP to define ZFP36 targets in mouse T cells, revealing unanticipated actions in regulating T-cell activation, proliferation, and effector functions. Transcriptome and ribosome profiling showed that ZFP36 represses mRNA target abundance and translation, notably through novel AU-rich sites in coding sequence. Functional studies revealed that ZFP36 regulates early T-cell activation kinetics cell autonomously, by attenuating activation marker expression, limiting T cell expansion, and promoting apoptosis. Strikingly, loss of ZFP36 in vivo accelerated T cell responses to acute viral infection and enhanced anti-viral immunity. These findings uncover a critical role for ZFP36 RBPs in restraining T cell expansion and effector functions, and suggest ZFP36 inhibition as a strategy to enhance immune-based therapies.

T cells presenting viral antigens or autoantigens induce cytotoxic T cell anergy.

In the course of modeling the naturally occurring tumor immunity seen in patients with paraneoplastic cerebellar degeneration (PCD), we discovered an unexpectedly high threshold for breaking CD8+ cytotoxic T cell (CTL) tolerance to the PCD autoantigen, CDR2. While CDR2 expression was previously found to be strictly restricted to immune-privileged cells (cerebellum, testes, and tumors), unexpectedly we have found that T cells also express CDR2. This expression underlies inhibition of CTL activation; CTLs that respond to epithelial cells expressing CDR2 fail to respond to T cells expressing CDR2. This was a general phenomenon, as T cells presenting influenza (flu) antigen also fail to activate otherwise potent flu-specific CTLs either in vitro or in vivo. Moreover, transfer of flu peptide-pulsed T cells into flu-infected mice inhibits endogenous flu-specific CTLs. Our finding that T cells serve as a site of immune privilege, inhibiting effector CTL function, uncovers an autorepressive loop with general biologic and clinical relevance.

cTag-PAPERCLIP Reveals Alternative Polyadenylation Promotes Cell-Type Specific Protein Diversity and Shifts Araf Isoforms with Microglia Activation.

Alternative polyadenylation (APA) is increasingly recognized to regulate gene expression across different cell types, but obtaining APA maps from individual cell types typically requires prior purification, a stressful procedure that can itself alter cellular states. Here, we describe a new platform, cTag-PAPERCLIP, that generates APA profiles from single cell populations in intact tissues; cTag-PAPERCLIP requires no tissue dissociation and preserves transcripts in native states. Applying cTag-PAPERCLIP to profile four major cell types in the mouse brain revealed common APA preferences between excitatory and inhibitory neurons distinct from astrocytes and microglia, regulated in part by neuron-specific RNA-binding proteins NOVA2 and PTBP2. We further identified a role of APA in switching Araf protein isoforms during microglia activation, impacting production of downstream inflammatory cytokines. Our results demonstrate the broad applicability of cTag-PAPERCLIP and a previously undiscovered role of APA in contributing to protein diversity between different cell types and cellular states within the brain.

High-Titer Rheumatoid Arthritis Antibodies Preferentially Bind Fibrinogen Citrullinated by Peptidylarginine Deiminase 4.

OBJECTIVE: Most patients with rheumatoid arthritis (RA) harbor antibodies to citrullinated autoantigens such as citrullinated fibrinogen. Two isoforms of peptidylarginine deiminase (PAD), PAD type 2 (PAD2) and PAD4, which catalyze citrullination with different substrate specificities, can be detected in the synovium of RA patients. This study was undertaken to determine whether RA antibodies preferentially bind PAD2- or PAD4-citrullinated fibrinogen. METHODS: RA patient and normal donor plasma specimens were tested for binding to PAD2- or PAD4-citrullinated fibrinogen, native fibrinogen, or citrullinated fibrinogen peptides in various dilutions by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Bands corresponding to masses demonstrating RA antibody reactivity by Western blotting were excised and analyzed by mass spectrometry. RESULTS: At low antibody titers (1:40 and 1:100), there was no significant difference between RA antibody reactivity to PAD2- and PAD4-citrullinated fibrinogen. When plasma was further diluted to 1:250 and 1:1,000, RA patient plasma bound PAD4-citrullinated fibrinogen significantly more than PAD2-citrullinated fibrinogen, as measured by ELISA and Western blotting. An increased antibody titer was associated with increased avidity for both PAD2- and PAD4-citrullinated fibrinogen. Both enzymes hypercitrullinated fibrinogen, but PAD4 citrullinated arginines more intermittently, generating a mix of citrullinated and noncitrullinated arginines. Peptide ELISA and preadsorption assays confirmed that the region of intermittent citrullination accounts for the majority of RA antibody binding to the ß-chain of citrullinated fibrinogen. CONCLUSION: At high titers, RA antibodies preferentially bind fibrinogen modified by PAD4, because intermittent citrullination offers a more diverse assortment of citrullinated epitopes.

Inflammatory but not apoptotic death of granulocytes citrullinates fibrinogen.

BACKGROUND: Neutrophil activation induces citrullination of intracellular targets of anticitrullinated peptide antibodies (ACPA), which are specific for rheumatoid arthritis (RA). Citrullinated fibrinogen is bound by ACPA but it is less well understood how extracellular proteins are citrullinated. The cells that produce fibrinogen, hepatocytes, do not express peptidyl arginine deiminase (PAD) enzymes nor do PAD enzymes include N-terminal signal peptides to direct them into the secretory pathway. We hypothesized that dying neutrophils release PAD in the extracellular space, and that this could cause citrullination of target extracellular antigens relevant to RA such as fibrinogen. METHODS: HL60 cells were differentiated into neutrophil-like cells by treatment with all-trans retinoic acid (ATRA). Differentiation was confirmed by CD11b staining, PAD4, PAD2 and myeloperoxidase expression, cell division, and nuclear morphology. Death was induced with various stimuli, including freeze-thaw to induce necrosis, Ionomycin and PMA to induce NETosis, and UV-B to induce apoptosis. Death markers were assessed by immunohistochemistry and flow cytometry. To quantify extracellular citrullination, dying ATRA-differentiated HL60 cells were cultured with fibrinogen for 24 hours and supernatants were probed for fibrinogen citrullination, PAD2 and PAD4 by western blot. RESULTS: While both NETotic and necrotic ATRA differentiated HL60 cells citrullinated fibrinogen, apoptotic cells did not citrullinate fibrinogen, even when allowed to undergo secondary necrosis. Incubation of necrotic neutrophil lysates with fibrinogen also causes fibrinogen citrullination. PAD2 and PAD4 were detected by western blot of supernatants of ATRA-differentiated HL60 cells undergoing necrotic and NETotic death, but not apoptotic or secondarily necrotic cell death. CONCLUSION: We implicate granulocytes undergoing inflammatory cell death as a mechanism for altering extracellular self-proteins that may be targets of autoimmunity linked to inflammatory diseases such as rheumatoid arthritis.

Neuroinflammation: Ways in Which the Immune System Affects the Brain.

Neuroinflammation is the response of the central nervous system (CNS) to disturbed homeostasis and typifies all neurological diseases. The main reactive components of the CNS include microglial cells and infiltrating myeloid cells, astrocytes, oligodendrocytes, and the blood-brain barrier, cytokines, and cytokine signaling. Neuroinflammatory responses may be helpful or harmful, as mechanisms associated with neuroinflammation are involved in normal brain development, as well as in neuropathological processes. This review examines the roles of various cell types that contribute to the immune dysregulation associated with neuroinflammation. Microglia enter the CNS very early in embryonic development and, as such, play an essential role in both the healthy and diseased brain. B-cell diversity contributes to CNS disease through both antibody-dependent and antibody-independent mechanisms. The influences of these B-cell mechanisms on other cell types, including myeloid cells and T cells, are reviewed in relationship to antibody-mediated CNS disorders, paraneoplastic neurological diseases, and multiple sclerosis. New insights into neuroinflammation offer exciting opportunities to investigate potential therapeutic targets for debilitating CNS diseases.

A destructive feedback loop mediated by CXCL10 in central nervous system inflammatory disease.

OBJECTIVE: Paraneoplastic neurologic disorders (PND) are autoimmune diseases associated with cancer and ectopic expression of a neuronal antigen in a peripheral tumor. Patients with PND harbor high-titer antibodies and T cells in their serum and cerebrospinal fluid (CSF) that are specific to the tumor antigen, and treatment with the immunosuppressant FK506 (tacrolimus) decreases CSF white blood cell counts. The objective of this study was to determine the effect of FK506 on CSF chemokine levels in PND patients. METHODS: CSF samples before and after FK506 treatment were tested by multiplex assay for the presence of 27 cytokines. Follow-up in vitro experiments aimed to determine whether T cells secrete CXCL10 in response to cognate antigen. RESULTS: Here we report that PND patients harbor high levels of the chemokine CXCL10 in their CSF. CXCL10 is a cytokine that recruits CXCR3(+) cells such as activated T cells, and we found that FK506 treatment specifically decreased CSF CXCL10 from among 27 cytokines tested. In vitro, CXCL10 was only produced during antigen-specific cognate interactions between T cells and antigen-presenting cells (APCs) when interferon-γ (IFNγ) receptors were present on the T cell. INTERPRETATION: These results support a model in which antigen-specific T cell stimulation by PND APCs triggers IFNγ, followed by CXCL10 production and further lymphocyte recruitment, suggesting that treatments targeting T cells or CXCL10 in the central nervous system (CNS) may interrupt a destructive positive feedback loop present in CNS inflammation.

Dendritic cell vaccines containing lymphocytes produce improved immunogenicity in patients with cancer.

BACKGROUND: Dendritic cells are currently under investigation for their ability to generate anti-cancer immune responses. No consensus has been reached as to the optimal method of dendritic cell vaccine preparation and is a barrier to success in the field. METHODS: Over a course of three separate dendritic cell vaccine studies to treat cancer, we tested two different methods for preparing dendritic cells from peripheral blood mononuclear cells: adherence and antibody-selected CD14+ cells. RESULTS: Surprisingly, we found that patients who received dendritic cell vaccines generated by the adherence method mounted increased T cell proliferation in response to vaccination. This difference could not be accounted for by dendritic cell vaccine dose, cell surface phenotype or dendritic cell function in vitro. One notable difference between the two vaccine preparation methods was that the dendritic cell vaccine cultures generated by the adherence method contained up to 10% lymphocytes, and these lymphocytes were proliferating and producing IFNγ in response to antigen in vitro at the time of administration. CONCLUSIONS: Enhanced immunogenicity of adherence dendritic cell vaccinations may be due to the presence of lymphocytes during dendritic cell culture. TRIAL REGISTRATION: Clinicaltrials.gov identifiers: NCT00289341, NCT00345293, and NCT00893945.

T cells targeting a neuronal paraneoplastic antigen mediate tumor rejection and trigger CNS autoimmunity with humoral activation.

Paraneoplastic neurologic diseases (PND) involving immune responses directed toward intracellular antigens are poorly understood. Here, we examine immunity to the PND antigen Nova2, which is expressed exclusively in central nervous system (CNS) neurons. We hypothesized that ectopic expression of neuronal antigen in the periphery could incite PND. In our C57BL/6 mouse model, CNS antigen expression limits antigen-specific CD4+ and CD8+ T-cell expansion. Chimera experiments demonstrate that this tolerance is mediated by antigen expression in nonhematopoietic cells. CNS antigen expression does not limit tumor rejection by adoptively transferred transgenic T cells but does limit the generation of a memory population that can be expanded upon secondary challenge in vivo. Despite mediating cancer rejection, adoptively transferred transgenic T cells do not lead to paraneoplastic neuronal targeting. Preliminary experiments suggest an additional requirement for humoral activation to induce CNS autoimmunity. This work provides evidence that the requirements for cancer immunity and neuronal autoimmunity are uncoupled. Since humoral immunity was not required for tumor rejection, B-cell targeting therapy, such as rituximab, may be a rational treatment option for PND that does not hamper tumor immunity.