Biology subjects

Grieshaber-Bouyer, R.

Publications and source records attributed to Grieshaber-Bouyer, R..

6 recordsLinked to original sources

bioRxiv2023

Distinct Functional States of Neutrophils by Actin Disassembly and NF-kB/STAT3 Signaling

Polymorphonuclear neutrophils (PMNs) can differentiate into distinct states, which can either exacerbate or resolve inflammation. Our study shows that mice challenged with TLR agonists exhibited PMN differentiation along two major paths characterized by the expression of CCR5 or PD-L1. Similar differentiation was observed in the blood of severe COVID-19 patients and the synovial fluid of osteoarthritis patients. Prolonged in vitro priming of human PMNs modeled the differentiation paths. Actin disassembly favored CCR5 upregulation, while NF-kB activation stabilized the actin cytoskeleton and suppressed the development of CCR5+ PMNs. Additionally, PD-L1 upregulation was triggered by STAT3 signaling and NF-kB activation. Functionally, CCR5 expressing PMNs were pro-NETotic, while PD-L1+ PMNs showed immunosuppressive functions by inhibiting T cell proliferation via PD1. Together, PMN differentiation depended on the priming conditions, and the balance between actin disassembly and NF-kB/STAT3 activation translated the present micro-milieu into phenotypic and functional diversification of PMNs. SynopsisNeutrophils underwent phenotypical and functional diversification both in vivo and in vitro. Actin disassembly led to the generation of CCR5high neutrophils with increased spontaneous NETosis, whereas NF-kB and STAT3 induced PD-L1 expression with T-cell suppressive properties as a deviation from the default pathway. O_LIPMN of mice challenged with TLR agonists develop two distinct phenotypes, CCR5high and PD-L1high. C_LIO_LICCR5 and PD-L1-defined neutrophil phenotypes were found in blood of patients with severe COVID-19 and in the synovial fluid of osteoarthritis patients. C_LIO_LIIn vitro priming induced a similar bifurcation of PMN phenotypes marked by either CCR5 or PD-L1. C_LIO_LIActin disassembly preceded canonical development of CCR5+ PMN. C_LIO_LINF-kB halted actin disassembly by LPL regulation. C_LIO_LIDuring neutrophil priming, STAT3 aided NF-kB in the expression of PD-L1. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=159 HEIGHT=200 SRC="FIGDIR/small/548975v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@10305f3org.highwire.dtl.DTLVardef@17bada8org.highwire.dtl.DTLVardef@c61021org.highwire.dtl.DTLVardef@14ce7f3_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

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

Inflammation induces pro-NETotic neutrophils via TNFR2 signaling

Cytokines released during chronic inflammatory diseases induce pro-inflammatory properties in polymorphonuclear neutrophils (PMN). Here we show that in vitro cytokine treatment leads to the development of a subgroup of human PMN expressing CCR5, termed CCR5+ cytokine-induced PMN (CCR5+ cPMN). Auto/paracrine TNF signaling increases intracellular neutrophil elastase (ELANE) abundance and induces NETosis in CCR5+ cPMN. Triggering of CCR5 amplifies NETosis. Membranous TNF (mTNF) outside-in signaling induces the formation of reactive oxygen species, a known activator of NETosis. In vivo, we find an increased number of CCR5+ cPMN in the peripheral blood and inflamed lamina propria of patients with ulcerative colitis (UC) but not Crohns disease (CD). Notably, failure of anti-TNF therapy is associated with higher frequencies of CCR5+ cPMN. In conclusion, we identify a phenotype of pro-NETotic, CCR5 positive PMN present in inflamed tissue in vivo and inducible in vitro. These cells may reflect an important component of tissue damage during chronic inflammation and could be of diagnostic value.

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↗