Biology subjects

Nigrovic, P. A.

Publications and source records attributed to Nigrovic, P. A..

14 recordsLinked to original sources

bioRxiv2026

Megakaryocyte emperipolesis arms neutrophils via intracellular provisioning

Neutrophils are phenotypically heterogenous cells that mediate host defense and tissue homeostasis. Here, we identify emperipolesis - the evolutionarily conserved process by which neutrophils pass through megakaryocytes - as a phenotypically transformative route of egress from bone marrow. By intravital microscopy and 3-D histology, we show that the rapid form emperipolesis is markedly enhanced under inflammatory conditions. Neutrophils exit from megakaryocytes directly to the blood, acquiring exosomes enriched in proteins related to metabolism, migration, and immune function. This transfer induces a distinct neutrophil phenotype characterized by enhanced glycolysis, oxidative phosphorylation, cytokine release, and longevity. Correspondingly, emperipolesis-educated neutrophils display accelerated migration in vitro and in vivo. Disrupting emperipolesis does not alter circulating neutrophil abundance but impairs neutrophil infiltration into inflamed tissues, including Pseudomonas aeruginosa-infected lung. These findings establish emperipolesis as a mechanism by which megakaryocytes amplify neutrophil-mediated immunity.

immunology↗

bioRxiv2025

Precision Editing of Cyclophilin A Generates Cyclosporine and Voclosporin Resistant Cellular Therapies

Recipients of allogeneic transplants or patients with autoimmune disease require immune suppression, often with calcineurin inhibitors. There is an expanding repertoire of immune effector cell therapies, including CD19 CAR-T cells and viral-specific T cells, deployed in these patients; however, ongoing calcineurin inhibition may be detrimental to cell therapy function. We developed a CRISPR/Cas9-based approach to engineer dual cyclosporine/voclosporin resistant cell therapies by targeting PPIA (encoding cyclophilin A), a critical binding partner for both drugs. Because Cyclophilin A has homeostatic functions in T cells, a complete knock-out is detrimental to cell viability. We thus targeted its C-terminus, disrupting drug binding while leaving the majority of the protein intact. C-terminal editing was stable throughout expansion and preserved Cyclophilin A expression. Edited CD19 CAR-T cells retained effector function in the presence of cyclosporine and voclosporin, including proliferation, cytokine production, and target cell killing, resulting in improved survival in murine models of CD19+ leukemia. Edited CMV-specific T cells also demonstrated preserved antigen-specific proliferation and cytokine production in the presence of these drugs. C-terminal editing of Cyclophilin A offers a promising avenue for developing next-generation cell therapies for patients receiving calcineurin inhibitors.

immunology↗

bioRxiv2025

A GPVI-platelet-neutrophil-NET axis drives systemic sclerosis

Systemic sclerosis (SSc) is immune-mediate inflammatory disease characterized by progressive tissue fibrosis. We observed that circulating neutrophils from patients with diffuse SSc exhibit an activated phenotype, a finding echoed in blood and skin transcriptomes. Neutrophil depletion abrogated experimental SSc induced by cutaneous injection of hypochlorous acid (HOCl) or bleomycin (BLM), and adoptive transfer of HOCl and BLM neutrophils induced skin and lung fibrosis in healthy mice, establishing neutrophils as necessary and sufficient for fibrosis. We noted that SSc patients exhibited platelet activation, a phenotype that preceded neutrophil activation in mice, suggesting an upstream role. Indeed, platelet depletion abrogated neutrophil activation and tissue fibrosis, and exposure to HOCl or BLM platelets conferred upon wild-type neutrophils the capacity to induce skin and lung fibrosis via neutrophil extracellular traps (NETs). Genetic and therapeutic blockade of the platelet collagen receptor GPVI attenuated platelet and neutrophil activation, reduced circulating NETs, and protected animals from skin and lung fibrosis. These findings identify the GPVI-platelet-neutrophil-NET as a new source of therapeutic targets in SSc.

immunology↗

bioRxiv2023

High-throughput identification of functional regulatory SNPs in systemic lupus erythematosus

Genome-wide association studies implicate multiple loci in risk for systemic lupus erythematosus (SLE), but few contain exonic variants, rendering systematic identification of non-coding variants essential to decoding SLE genetics. We utilized SNP-seq and bioinformatic enrichment to interrogate 2180 single-nucleotide polymorphisms (SNPs) from 87 SLE risk loci for potential binding of transcription factors and related proteins from B cells. 52 SNPs that passed initial screening were tested by electrophoretic mobility shift and luciferase reporter assays. To validate the approach, we studied rs2297550 in detail, finding that the risk allele enhanced binding to the transcription factor Ikaros (IKZF1), thereby modulating expression of IKBKE. Correspondingly, primary cells from genotyped healthy donors bearing the risk allele expressed higher levels of the interferon / NF-{kappa}B regulator IKK{varepsilon}. Together, these findings define a set of likely functional non-coding lupus risk variants and identify a new regulatory pathway involving rs2297550, Ikaros, and IKK{varepsilon} implicated by human genetics in risk for SLE.

genetics↗

bioRxiv2022

Non-coding autoimmune risk variant accelerates T peripheral helper cell development via ICOS

Fine-mapping and functional studies implicate rs117701653, a common non-coding variant in the CD28/CTLA4/ICOS locus, as a contributor to risk for rheumatoid arthritis and type 1 diabetes. Using DNA pulldown, mass spectrometry, genome editing and eQTL analysis, we establish that the disease-associated allele reduces affinity for the inhibitory chromosomal regulator SMCHD1 to drive expression of inducible T-cell costimulator (ICOS), enhancing memory CD4+ T cell ICOS expression in individuals bearing the risk allele. Higher ICOS expression is paralleled by an increase in circulating T peripheral helper (Tph) cells, and in rheumatoid arthritis patients, of blood and joint fluid Tph cells and circulating plasmablasts, suggesting a causal link. Indeed, ICOS ligation accelerates T cell differentiation into CXCR5-PD-1high Tph cells producing IL-21 and CXCL13, as does carriage of the rs117701653 risk allele. Thus, mechanistic dissection of a causal non-coding variant in human autoimmunity discloses a new pathway through which ICOS regulates Tph abundance.

genetics↗

bioRxiv2022

Identification of a regulatory pathway governing TRAF1 via an arthritis-associated non-coding variant

TRAF1/C5 was among the first loci shown to confer risk for inflammatory arthritis in the absence of an associated coding variant, but its genetic mechanism remains undefined. Using ImmunoChip data from 3,939 juvenile idiopathic arthritis (JIA) patients and 14,412 controls, we identified 132 plausible common non-coding variants, reduced serially by SNP-seq, electrophoretic mobility shift, and luciferase studies to the single variant rs7034653 in the third intron of TRAF1. Genetically manipulated experimental cells and primary monocytes from genotyped donors establish that the risk G allele reduces binding of Fos-Related Antigen 2 (FRA2), resulting in reduced TRAF1 expression and enhanced TNF production. Conditioning on this variant eliminates attributable risk for rheumatoid arthritis, implicating a mechanism shared across the arthritis spectrum. These findings reveal that rs7034653, FRA2, and TRAF1 mediate a pathway through which a non-coding causal variant drives risk of inflammatory arthritis in children and adults.

genetics↗

bioRxiv2022

Human and murine neutrophils share core transcriptional programs in both homeostatic and inflamed contexts

Neutrophils are frequently studied in murine models, but the extent to which findings translate to humans remains poorly defined. Here, we performed an integrative transcriptomic analysis of 11 murine and 13 human datasets. In homeostasis, neutrophils exhibited the highest number of lineage-specific genes and the greatest degree of correlated expression among genes with one-to-one orthologs (r = 0.79, P < 2.2 x 10-16) compared to other leukocytes. In inflammation, neutrophils displayed considerable transcriptional diversity, but shared a core inflammation program across a broad range of conditions which was conserved between species. This core program included genes encoding IL-1 family members, CD14, IL-4R, CD69 and PD-L1. Chromatin accessibility of core inflammation genes increased significantly in blood compared to bone marrow and further with migration from blood to tissue. Transcription factor enrichment analysis nominated members of the NF-{kappa}B family and AP-1 complex as important drivers of the core inflammation program, and HoxB8 neutrophils with JUNB knockout showed a significantly reduced expression of core inflammation genes at baseline and upon stimulation. In vitro perturbations confirmed surface protein upregulation of core inflammation members in both species. Together, we demonstrate substantial transcriptional conservation in neutrophils in homeostasis and identify a core inflammation program conserved across species. This systems biology approach can be leveraged to improve transitions between the murine and human context. Key PointsO_LIThe transcriptome of resting neutrophils is substantially conserved between humans and mice C_LIO_LIA core inflammation program in neutrophils is shared across a broad range of conditions and conserved across humans and mice C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=52 SRC="FIGDIR/small/516246v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1f6b40borg.highwire.dtl.DTLVardef@1a67bb4org.highwire.dtl.DTLVardef@1039a8forg.highwire.dtl.DTLVardef@1982d10_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

Aging and interferon gamma response drive the phenotype of neutrophils in the inflamed joint

ObjectivesNeutrophils are typically the most abundant leukocyte in arthritic synovial fluid. We sought to understand changes that occur in neutrophils as they migrate from blood to joint. MethodsWe performed RNA sequencing of neutrophils from healthy human blood, arthritic blood, and arthritic synovial fluid, comparing transcriptional signatures with those from murine K/BxN serum transfer arthritis. We employed mass cytometry to quantify protein expression and sought to reproduce the synovial fluid phenotype ex vivo in cultured healthy blood neutrophils. ResultsBlood neutrophils from healthy donors and patients with active arthritis exhibited largely similar transcriptional signatures. By contrast, synovial fluid neutrophils exhibited more than 1,600 differentially expressed genes. Gene signatures identified a prominent response to interferon gamma (IFN{gamma}), as well as to tumor necrosis factor, interleukin 6, and hypoxia, in both humans and mice. Mass cytometry also found healthy and arthritic donor blood neutrophils largely indistinguishable but revealed a range of neutrophil phenotypes in synovial fluid defined by downregulation of CXCR1 and upregulation of Fc{gamma}RI, HLA-DR, PD-L1, ICAM-1 and CXCR4. Reproduction of key elements of this signature in cultured blood neutrophils required both IFN{gamma} and prolonged culture. ConclusionsCirculating neutrophils from arthritis patients resemble those from healthy controls, but joint fluid cells exhibit a network of changes, conserved across species, that implicate IFN{gamma} response and aging as complementary drivers of the synovial neutrophil phenotype. KEY MESSAGESO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LINeutrophils are central in the effector phase of inflammatory arthritis but their phenotypic heterogeneity in inflamed synovial fluid is poorly understood. C_LI What does this study add?O_LIRNA-seq and mass cytometry identify a hallmark phenotype of neutrophils in synovial fluid consisting of upregulated ICAM-1, HLA-DR, PD-L1, Fc receptors and CXCR4. C_LIO_LITranscriptomics highlight an IFN{gamma} response signature conserved across humans and mice. C_LIO_LIIn vitro experiments implicate aging and IFN{gamma} as complementary factors orchestrating the synovial fluid neutrophil phenotype. C_LI How might this impact on clinical practice or future developments?O_LIUnderstanding the specific features of neutrophils in the arthritic joint may disclose opportunities for safe therapeutic targeting of this lineage. C_LI

immunology↗

bioRxiv2021

Arthritis Flares Mediated by Tissue Resident Memory T Cells in the Joint

Although rheumatoid arthritis is a systemic disease, flares typically occur in a subset of joints that is distinctive for each patient. Pursuing this intriguing pattern, we show that arthritis recurrence is mediated by long-lived synovial resident memory T cells (TRM). In three murine models, CD8+ cells bearing TRM markers remain in previously inflamed joints during remission. These cells are bona fide TRM, exhibiting failure to migrate from joint to joint, preferential uptake of fatty acids, and long-term residency. Disease flares result from TRM activation by antigen, leading to CCL5-mediated recruitment of circulating effector cells. Correspondingly, TRM depletion ameliorates recurrence in a site-specific manner. Human rheumatoid arthritis joint tissues contain a comparable CD8+-predominant TRM population, most evident in late-stage non-inflamed synovium, exhibiting limited T cell receptor diversity and a pro-inflammatory transcriptomic signature. Together, these findings establish synovial TRM cells as a targetable mediator of disease chronicity in autoimmune arthritis.

immunology↗

bioRxiv2021

Neutrophil transit time and localization within the megakaryocyte define morphologically distinct forms of emperipolesis

In emperipolesis, neutrophils transit through megakaryocytes, but it is unknown whether this interaction represents a single type of cell-in-cell interaction or a set of distinct processes. Using an in vitro model of murine emperipolesis, we characterized neutrophils entering megakaryocytes using live-cell spinning disk microscopy and electron microscopy. Approximately half of neutrophils exited the megakaryocyte rapidly, typically in 10 minutes or less, displaying ameboid morphology as they passed through the host cell (fast emperipolesis). The remaining neutrophils assumed a sessile morphology, most remaining within the megakaryocyte for at least 60 minutes (slow emperipolesis). These neutrophils typically localized near the megakaryocyte nucleus. By ultrastructural assessment, all internalized neutrophils remained morphologically intact. Most neutrophils resided within emperisomes, but some could be visualized exiting the emperisome into the cell cytoplasm. Neutrophils in the cytoplasm assumed close contact with the platelet-forming demarcation membrane system or with the perinuclear endoplasmic reticulum, as confirmed by immunofluorescence microscopy. Together, these findings reveal that megakaryocyte emperipolesis reflects at least two processes, fast and slow emperipolesis, each with its own characteristic transit time, morphology, and intracellular localization, suggesting distinct functions. Key PointsO_LINeutrophil passage through megakaryocytes, termed emperipolesis, diverges into fast and slow forms that differ in transit time, morphology, and intracellular localization C_LIO_LIDuring emperipolesis, neutrophils can reside in vacuoles (emperisomes) or escape into the cell cytoplasm to assume positions near the megakaryocytes demarcation membrane system, endoplasmic reticulum, or nucleus. C_LI

cell biology↗

bioRxiv2019

A Systems Immunology Approach Identifies Cytokine-Induced STAT Signaling Pathways Critical to Rheumatoid Arthritis Disease Activity and Treatment Response

Rheumatoid arthritis (RA), a chronic autoimmune disease characterized by circulating autoantibodies, involves many cytokine-mediated signaling pathways in multiple immune cell subsets. Most studies of immune cells in RA have limitations, such as analysis of a small number of cell subsets or pathways, and limited longitudinal data on patient phenotypes. In this study, we used an innovative systems immunology approach to simultaneously quantify up to 882 signaling nodes (Jak/STAT signaling readouts modulated by cytokines and other stimuli) in 21 immune cell subsets. We studied 194 RA patients and 41 controls, including 146 well-characterized RA patients prior to, and 6 months after, initiation of methotrexate or biologic agents from the Treatment Efficacy and Toxicity in RA Database and Repository (TETRAD). There was strikingly attenuated signaling capacity in RA patients in IFN stimulation followed by measurement of phosphorylated STAT1 [IFN[->]p-STAT1] in six immune cell subsets. Multiple nodes showed negative association with disease activity, including IFN[->]STAT5 signaling in naive and memory B cells. In contrast, IL-6-induced STAT1 and STAT3 activation in central memory CD4-negative T cells showed a positive association with disease activity. Multiple nodes were associated with treatment response, including IFN[->]STAT1 in monocytes and IL-6[->]STAT3 in CD4+ naive T cells. Decision tree analysis identified a model combining these two nodes as a high-performing classifier of treatment response to TNF inhibitors. Our study provides novel information on RA disease mechanisms and serves as a framework for the discovery and validation of biomarkers of treatment response in RA.

immunology↗

bioRxiv2018

Megakaryocyte emperipolesis mediates membrane transfer from intracytoplasmic neutrophils to platelets

Bone marrow megakaryocytes engulf neutrophils in a phenomenon termed emperipolesis. We show here that emperipolesis is a dynamic process mediated actively by both lineages, in part through the {beta}2-integrin/ICAM-1/ezrin pathway. Tethered neutrophils enter in membrane-bound vesicles before penetrating into the megakaryocyte cytoplasm. Intracytoplasmic neutrophils develop membrane contiguity with the demarcation membrane system, thereby transferring membrane to the megakaryocyte and to daughter platelets. This phenomenon occurs in otherwise unmanipulated marrow in vivo, resulting in circulating platelets that bear membrane from non-megakaryocytic hematopoietic donors. Transit through megakaryocytes can be completed as rapidly as minutes, after which neutrophils egress intact. Emperipolesis is amplified in models of inflammation associated with platelet overproduction, contributing to platelet production in vitro and in vivo. These findings identify emperipolesis as a new cell-in-cell interaction that enables neutrophils and potentially other cells passing through the megakaryocyte cytoplasm to modulate the production and membrane content of platelets.

cell biology↗

bioRxiv2018

Progression of recent Mycobacterium tuberculosis exposure to active tuberculosis is a highly heritable complex trait driven by 3q23 in Peruvians

Among 1.8 billion people worldwide infected with Mycobacterium tuberculosis, 5-15% are expected to develop active tuberculosis (TB). Approximately half of these will progress to active TB within the first 18 months after infection, presumably because they fail to mount the initial immune response that contains the local bacterial spread. The other half will reactivate their latent infection later in life, likely triggered by a loss of immune competence due to factors such as HIV-associated immunosuppression or ageing. This natural history suggests that undiscovered host genetic factors may control early progression to active TB. Here, we report results from a large genome-wide genetic study of early TB progression. We genotyped a total of 4,002 active TB cases and their household contacts in Peru and quantified genetic heritability [Formula] of early TB progression to be 21.2% under the liability scale. Compared to the reported [Formula] of genome-wide TB susceptibility (15.5%), this result indicates early TB progression has a stronger genetic basis than population-wide TB susceptibility. We identified a novel association between early TB progression and variants located in an enhancer region on chromosome 3q23 (rs73226617, OR=1.19; P < 5x10-8). We used in silico and in vitro analyses to identify likely functional variants and target genes, highlighting new candidate mechanisms of host response in early TB progression.

genetics↗

bioRxiv2017

Single Cell Transcriptomics And Flow Cytometry Reveal Disease-Associated Fibroblast Subsets In Rheumatoid Arthritis

Fibroblasts mediate normal tissue matrix remodeling, but they can cause fibrosis or tissue destruction following chronic inflammation. In rheumatoid arthritis (RA), synovial fibroblasts expand, degrade cartilage, and drive joint inflammation. Little is known about fibroblast heterogeneity or if aberrations in fibroblast subsets relate to disease pathology. Here, we used an integrative strategy, including bulk transcriptomics on targeted subpopulations and unbiased single-cell transcriptomics, to analyze fibroblasts from synovial tissues. We identify 7 phenotypic fibroblast subsets with distinct surface protein phenotypes, and these collapsed into 3 subsets based on transcriptomics data. One subset expressing PDPN, THY1, but lacking CD34 was 3-fold expanded in RA relative to osteoarthritis (P=0.007); most of these cells expressed CDH11. The subsets were found to differ in expression of cytokines and matrix metalloproteinases, localization in synovial microanatomy, and in response to TNF. Our approach provides a template to identify pathogenic stromal cellular subsets in complex diseases.

immunology↗