OBJECTIVES:Autoantibody internalisation has been implicated in autoimmune disease pathogenesis, yet its mechanisms and generalisability across diseases, cell types, and tissues remain poorly defined. We sought to address these gaps. METHODS:Bulk RNA sequencing was performed on 814 muscle biopsies from patients with autoimmune diseases and healthy controls, including an external validation cohort of 41 additional samples. Purified patient immunoglobulin (Ig) G was electroporated into primary human cells to assess functional effects in vitro. Immunofluorescence evaluated localisation of immunoglobulins across diseases and tissues. Spatial transcriptomics assessed disease-specific gene expression, cellular injury, and inflammatory responses. RESULTS:Using anti-Mi2 dermatomyositis and anti-PM/Scl scleromyositis as model diseases, we validated reproducible, autoantibody-specific transcriptomic signatures consistent with autoantigen dysfunction across cohorts and confirmed that electroporation of purified patient IgG into healthy primary cells recapitulates these disease-associated transcriptional programmes. Spatial transcriptomics linked these signatures to cellular injury and distinct inflammatory responses, including activation of type I interferon and TGFβ signalling in anti-Mi2 dermatomyositis and type II interferon signalling in anti-PM/Scl scleromyositis. These programmes were observed predominantly not only in muscle fibres but also in macrophages, endothelial cells, and fibroblasts/fibroadipogenic progenitors. Spatial transcriptomics further revealed transfer of immunoglobulin RNA from antibody-secreting cells to adjacent target cells expressing disease-specific transcriptional programmes. Antibody internalisation, previously described in myositis muscle, was also observed in skin from patients with anti-Mi2 and anti-PM/Scl and in tissues from anti-U1RNP mixed connective tissue disease, anti-Ku overlap syndrome, and anti-Scl70 systemic sclerosis. CONCLUSIONS:Together, these findings establish autoantibody internalisation as a mechanism of tissue injury in autoimmunity, suggesting its broader relevance across autoantibody-mediated diseases.

ABSTRACT Modeling neural-immune interactions in neurodegenerative and immune-mediated central nervous system (CNS) diseases requires human 3D models that capture cellular diversity and long-term tissue maturation. Here, we present an enhanced human induced pluripotent stem cell (hiPSC)-derived cerebral organoid (CO) platform optimized to mitigate core hypoxia for over 200 days. Timed pro-myelinating cues established organized neuronal layering and progressive axonal myelination through day 140, while vascular fusion yielded assembloids incorporating endothelial structures and microglia. Extended culture (>500–750 days) spontaneously reproduced hallmark features of human CNS aging, including cellular senescence signatures, neuroaxonal loss, hypomyelination, and the autonomous emergence of a neurotoxic astrocyte transcriptional profile in the complete absence of microglia or immune cells. Co-culture with autologous activated peripheral blood mononuclear cells (PBMC) resulted in transient immune infiltration and a pronounced type II interferon response across CNS lineages. High-plex spatial transcriptomics revealed that immune cell infiltration was associated with oligodendrocyte loss and in aged organoids also with downregulated oligodendrocyte myelin gene transcription. While not fully reproducing adult tissue stoichiometry, this platform enables longitudinal modeling of neural-immune crosstalk in age-related and neuroinflammatory CNS disorders.

RATIONALE: Recruitment of neutrophils to the lung is associated with acute lung inflammation (ALI). Characterization of pulmonary neutrophils during lung inflammation is limited, therefore, we sought to identify neutrophil subsets acutely transmigrating into an inflamed lung segment after lipopolysaccharide (LPS) instillation to enhance our understanding of ALI. METHODS: Five healthy subjects underwent a bronchoscopy with instillation of 4ng/kg of E. coli O:113 (CCRE) LPS into the right middle lobe (D0). Twenty-four hours later (day 1, D1), pulmonary neutrophils were collected from the lung segment via bronchoalveolar lavage (BAL). Blood was collected at baseline (D0) and D1. We performed flow cytometry with a 14-color panel and single cell-RNA-sequencing with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on the whole blood and BAL samples. Flow cytometry data was analyzed using OMIQ to characterize CD15+ neutrophils across samples. To eliminate bias, 100,000 CD15+ events from each sample were used to perform dimensional reduction visualized as tSNE plots and further characterized by FlowSOM. The number of neutrophil metaclusters for FlowSOM were determined using the elbow-criterion method (k=21). Additionally, CD15+ neutrophils were evaluated across sample types using traditional flow cytometry gating strategies. RESULTS: The elbow-criterion method identified 21 metaclusters (MC) across pulmonary and whole blood neutrophils (WBNs) (figure 1A-F). MC01, MC03, MC06 and MC14 consisted primarily of pulmonary neutrophils representing 17%, 36%, 16% and 5% of all pulmonary neutrophils respectively. Meta-clusters MC01, MC03, and MC06 expressed low amounts of the degranulation marker CD63, myeloperoxidase (MPO) and activation marker CD62L, and increased CD11b (figure 1E). MC03 and MC06 expressed high levels of CD10 compared to MC01 indicating a mature phenotype. MC14 expressed high levels of MPO and low levels of CD62L (figure 1E, G). Based on these characteristics, we identified a similar population equivalent to MC14 (MC14-EQ) using traditional flow cytometry gating that was MPOhi CD62Llo and specific to BAL neutrophils (figure 1H-J). Compared to all pulmonary neutrophils and WBNs at D0 and D1, the MC14-EQ population expressed lower CD10 and CD16, and higher CD63, suggesting an immature and activated state (figure 1K-M). CONCLUSION: Segmental LPS administration recruited a subset of mature (CD10hi) activated (CD11bhi CD62Llo) neutrophils that were potentially degranulating, evidenced by low MPO and CD63. Additionally, we identified a population of immature (CD10lo) and active (CD62Llo) neutrophils, with heightened MPO expression. Elucidating transcriptional differences between these neutrophil subsets warrants further investigation to determine their contribution to tissue inflammation and establish future therapeutic targets.

The mevalonate kinase (MVK) pathway is an essential metabolic pathway for sterol and isoprenoid synthesis. Non-sterol isoprenoids have critical biological functions in the post-translational modifications of numerous signaling molecules via prenylation, including inflammasomes. A defect in prenylation impacts protein trafficking and localization and increases proinflammatory cytokine production. Deficiency of the enzymes in the MVK pathway is known to cause systemic autoinflammatory diseases with a broad spectrum of clinical manifestations. Anticholesterol drug statins, which block the second enzyme in this pathway, HMG-CoA reductase, can lead to muscle inflammation. We report biallelic loss-of-function variants in the HMGCS1 gene, which encodes for the first enzyme in the MVK pathway, in four patients from three unrelated families who presented with recurrent fever, arthritis, abdominal pain, and progressive myositis. Three patients were homozygous for a rare variant, c.265C>T (p.Arg89Trp), predicted to be deleterious by multiple algorithms. One patient was homozygous for the novel c.572G>C (p.Arg191Pro) variant classified as VUS. The enzyme activity of both mutant HMGCS1 proteins was decreased, suggesting a deleterious effect on the protein function. Preliminary results showed that these missense variants do not affect protein expression or dimerization, which is critical for protein activity. The PBMCs from patients and CRISPR/cas9–edited HMGSC1-deficient cells showed a defect in prenylation, confirming the involvement of the MVK pathway. HMGCS1-deficient cells displayed an elevated level of IL-1β and IL-18 in response to stimulation with Pam3CSK4 and IFNγ, indicating that HMGCS1 deficiency led to inflammasome activation, which was independent of NLRP3 but was dependent on pyrin. UMAP analysis of peripheral blood single-cell RNA sequencing identified two major cell clusters: NK cells and monocytes. The most differentially upregulated pathways include oxygen/CO2 transport genes, cell death genes, and NF-κB signaling. Intracellular cytokine staining identified a significant increase of IL-1 β, IL-6, and IFNγ in all monocyte subsets of affected patients compared with unaffected relatives. RNA sequencing of frozen muscle tissues detected strong type I and II IFN signatures. Treatment with a JAK inhibitor ameliorated systemic and muscle inflammation in two patients. Further studies are in progress to understand the spectrum of immune dysregulation associated with the HMGCS1 deficiency.

Neutrophils, the most abundant immune cells in the human circulation, play a central role in the innate immune system. While neutrophil heterogeneity is a topic of increasing research interest, few efforts have been made to model the dynamics of neutrophil population subsets. We develop a mathematical model to describe the dynamics that characterizes the states and transitions involved in the maturation of human neutrophils. We use single-cell gene expression data to identify five clusters of healthy human neutrophils, and pseudo-time analysis to inform model structure. We find that precursor neutrophils transition into immature neutrophils, which then either transition to an interferon-responsive state or continue to mature through two further states. The key model parameters are the transition rates (the inverse of a transition rate is the mean waiting time in one state before transitioning to another). In this framework, the transition from the precursor to immature state (mean time less than an hour) is more rapid than subsequent transitions (mean times more than 12 hours). Approximately a quarter of neutrophils are estimated to follow the interferon-responsive path; the remainder continue along the standard maturation pathway. We use Bayesian inference to describe the variation, between individuals, in the fraction of cells within each cluster.

We describe a previously unappreciated role for Bruton’s tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, patients who were treated with BTKi, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in patients who are susceptible.

Introduction: Purinergic signaling through extracellular nucleotides such as adenosine is at the intersection of inflammation and coagulation pathways. CD73 is the key vascular enzyme involved generating AMP-derived extracellular adenosine to reduce inflammation, but its impact on thromboinflammation remains unexplored. Hypothesis: We hypothesized that CD73 suppresses innate immune activation and protects against thromboinflammation. Methods: To test this hypothesis, we recruited ten patients with ultrarare, loss of function mutations in the gene encoding CD73 which can result in an autosomal recessive, arterial calcification disease. Medical history and blood samples were obtained from patients for experiments. Preclinical experiments to study thromboinflammation were performed in mice with genetic and post-natal CD73 inhibition with appropriate controls. Results: We first confirmed loss of functional CD73 enzymatic activity in these patients using a novel, live cell phosphohydrolysis assay. Four of the 10 patients had a history of thrombosis (2 arterial, 2 venous). Neutrophils from patients lacking CD73 formed more spontaneous neutrophil extracellular traps (NETs) than healthy controls measured using multiple assays, which was recapitulated by bone marrow-derived neutrophils from mice genetically deficient for CD73 (CD73KO). Using single-cell RNA-seq of human neutrophils, we further characterized circulating neutrophil phenotypes in affected patients and healthy controls. We observed in these patients three distinct, overexpressed neutrophil subsets corresponding to mature and activated; immature; and IFN-responsive phenotypes. The differential gene expression of neutrophils from CD73-deficient patients was largely driven by genes involved in IFN response and granule genes. In contrast, CD14+ monocytes purified from patients lacking CD73 had similar cellular tissue factor expression as healthy controls, and similar tissue factor activity on extracellular vesicles released following monocyte stimulation with LPS. To mechanistically explore the role of CD73 in venous thrombosis, we used two murine models of deep vein thrombosis (electrolytic injury and stenosis). Genetic CD73 deletion and inhibition of CD73 in WT mice using a blocking mAb resulted in increased murine venous thrombogenesis, which was abrogated by exogenous apyrase administration. The increased thrombosis in CD73KO mice was attributed, in part to excess NETs formation characterized by increases in plasma cell-free DNA early after thrombosis induction, citrullinated histone H3 in the thrombosis, and attenuation of in vivo thrombosis by administration of DNase. Immunohistochemical analysis revealed increased myeloperoxidase in thrombi from CD73-deficient mice. Mechanistically, the severe thromboinflammation phenotype induced by genetic or post-natal CD73 inhibition in mice could be rescued by stimulation of the GPCR adenosine 2A receptor with CGS21680, suggesting CD73's enzymatic activity to generate adenosine and trigger downstream cAMP signaling as a key mechanism in mitigating venous thrombosis. Conclusions: These translational studies identify a previously unknown thromboinflammatory phenotype in patients with a rare, monogenic disease and establishes CD73 as a critical checkpoint in thromboinflammation using mechanistic investigations in human studies and animal models. CD73 inhibitors are also being tested in cancer clinical trials as an immune checkpoint inhibitor. Our ongoing studies will test whether a clinically used CD73 inhibitor is capable of triggering a thromboinflammatory phenotype in mice. These studies are especially relevant in the context of the large number of patients with prothrombotic cancers who may potentially receive a CD73 inhibitor for cancer treatment.

Spleen tyrosine kinase (SYK) is a previously unidentified therapeutic target that inhibits neutrophil and mac-rophage activation in coronavirus disease 2019 (COVID-19). Fostamatinib, a SYK inhibitor, was studied in a phase 2 placebo-controlled randomized clinical trial and was associated with improvements in many secondary end points related to efficacy. Here, we used a multiomic approach to evaluate cellular and soluble immune mediator responses of patients enrolled in this trial. We demonstrated that SYK inhibition was associated with reduced neutrophil activation, increased circulation of mature neutrophils (CD10+CD33???), and decreased circulation of low-density granulocytes and polymorphonuclear myeloid-derived suppressor cells (HLA-DR???CD33+CD11b???). SYK inhibition was also associated with normalization of transcriptional activity in circulating monocytes rela-tive to healthy controls, an increase in frequency of circulating nonclassical and HLA-DRhi classical monocyte populations, and restoration of interferon responses. Together, these data suggest that SYK inhibition may mit-igate proinflammatory myeloid cellular and soluble mediator responses thought to contribute to immunopa-thogenesis of severe COVID-19.

Abstract Background Coronavirus Disease 2019 (COVID-19) caused by the SARS-CoV-2 virus is associated with dysregulation in the innate immune response including NK cells. NK cells are integral in the innate immune response against viral infections. Canonical NK cells are classified as CD56dim CD16+ and CD56bright CD16-. An unconventional subset of CD56dim CD16- NK cells has previously been identified in COVID-19 that is not present in other viral infections. Here we characterize phenotypic changes in the NK cells of patients with severe COVID-19 as work towards determining the functional status of this unconventional subset. Methods Peripheral blood mononuclear cells (PBMCs) and plasma were isolated from healthy donors (n=5) and patients with severe COVID-19 on Extra Corporeal Membrane Oxygenation (ECMO) (n=15). Primary NK cells were stimulated in vitro with plasma from patients with severe COVID-19 or healthy donors. Flow cytometry was used to phenotype the NK cells. A separate cohort of PBMC samples (n=7) from patients requiring hospitalization for COVID-19 underwent Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) analysis. Results The CD56bright CD16- NK subset was expanded in PBMCs from patients with severe COVID-19 as compared to healthy controls. CITE-Seq demonstrated that NK cells without surface CD16 clustered separately based on transcriptional profiling and did express FCGR3A at the translational level. Stimulation with COVID-19 plasma recapitulated the loss of CD16 from primary human NK cells and led to increased activity of Caspase 3/7. Figure 1.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset in patients with severe COVID-19. a) Representative gating of NK cell subsets by Flow Cytometry in healthy and COVID-19 patient peripheral blood mononuclear cells (PBMCs). b) Percentage of total NK cells belonging to a particular cell subset compared between healthy donor samples (n=4) and COVID-19 patient samples (n=8). Data points represent an individual patient sample. Error bars represent the standard deviation of the mean. Differences between groups was analyzed using a two tailed t-test. *: p< 0.05, ns: not significant Figure 2.NK cells shift from the CD56dim CD16+ subset to the CD56dim CD16-subset after stimulation with COVID-19 plasma in vitro a) Representative gating of NK cell subsets by Flow Cytometry analysis in healthy donor NK cells stimulated by healthy plasma and COVID-19 patient plasma. b)Relative change in percentage of total NK cells belonging to a particular cell subset compared between healthy donor plasma (n=6)and COVID-19 patient plasma (n=15) stimulation conditions. Error bars represent the standard deviation of the mean and the difference between groups was analyzed using a two-tailed T-test. **: p< 0.01, ***: p< 0.001, ns: not significant. Conclusion We demonstrate and characterize a nonclassical population of CD56dim CD16- NK cells that are present in patients with severe COVID-19 and replicate this phenotype in vitro. Reproduction of this in vivo phenotype in an in vitro system will allow for additional studies on the functional state of NK cell subsets in COVID-19. The presence of this NK cell population may reflect a dysregulated innate immune response and immunopathogenesis of COVID-19. Disclosures All Authors: No reported disclosures.

Inflammatory and neuropathic-like components underlie rheumatoid arthritis (RA)-associated pain, and lysophosphatidic acid (LPA) is linked to both joint inflammation in RA patients and to neuropathic pain. Thus, we investigated a role for LPA signalling using the collagen antibody-induced arthritis (CAIA) model. Pain-like behavior during the inflammatory phase and the late, neuropathic-like phase of CAIA was reversed by a neutralizing antibody generated against LPA and by an LPA1/3 receptor inhibitor, but joint inflammation was not affected. Autotaxin, an LPA synthesizing enzyme was upregulated in dorsal root ganglia (DRG) neurons during both CAIA phases, but not in joints or spinal cord. Late-phase pronociceptive neurochemical changes in the DRG were blocked in Lpar1 receptor deficient mice and reversed by LPA neutralization. In vitro and in vivo studies indicated that LPA regulates pain-like behavior via the LPA1 receptor on satellite glia cells (SGCs), which is expressed by both human and mouse SGCs in the DRG. Furthermore, CAIA-induced SGC activity is reversed by phospholipid neutralization and blocked in Lpar1 deficient mice. Our findings suggest that the regulation of CAIA-induced pain-like behavior by LPA signalling is a peripheral event, associated with the DRGs and involving increased pronociceptive activity of SGCs, which in turn act on sensory neurons.

Neutrophils are the most abundant leukocytes in human blood and are essential components of innate immunity. Until recently, neutrophils were considered homogeneous and transcriptionally inactive cells, but both concepts are being challenged. Single-cell RNA sequencing (scRNA-seq) offers an unbiased view of cells along a continuum of transcriptional states. However, the use of scRNA-seq to characterize neutrophils has proven technically difficult, explaining in part the paucity of published single-cell data on neutrophils. We have found that modifications to the data analysis pipeline, rather than to the existing scRNA-seq chemistries, can significantly increase the detection of human neutrophils in scRNA-seq. We have then applied a modified pipeline to the study of human peripheral blood neutrophils. Our findings indicate that circulating human neutrophils are transcriptionally heterogeneous cells, which can be classified into one of four transcriptional clusters that are reproducible among healthy human subjects. We demonstrate that peripheral blood neutrophils shift from relatively immature (Nh0) cells, through a transitional phenotype (Nh1), into one of two end points defined by either relative transcriptional inactivity (Nh2) or high expression of type I IFN-inducible genes (Nh3). Transitions among states are characterized by the expression of specific transcription factors. By simultaneously measuring surface proteins and intracellular transcripts at the single-cell level, we show that these transcriptional subsets are independent of the canonical surface proteins that are commonly used to define and characterize human neutrophils. These findings provide a new view of human neutrophil heterogeneity, with potential implications for the characterization of neutrophils in health and disease.