COVID-19 is characterised by a broad spectrum of clinical and pathological features. Natural killer (NK) cells play an important role in innate immune responses to viral infections. Here, we analysed the phenotype and activity of NK cells in the blood of COVID-19 patients using flow cytometry, single-cell RNA-sequencing (scRNA-seq), and a cytotoxic killing assay. In the plasma of patients, we quantified the main cytokines and chemokines. Our cohort comprises COVID-19 patients hospitalised in a low-care ward unit (WARD), patients with severe COVID-19 disease symptoms hospitalised in intensive care units (ICU), and post-COVID-19 patients, who were discharged from hospital six weeks earlier. NK cells from hospitalised COVID-19 patients displayed an activated phenotype with substantial differences between WARD and ICU patients and the timing when samples were taken post-onset of symptoms. While NK cells from COVID-19 patients at an early stage of infection showed increased expression of the cytotoxic molecules perforin and granzyme A and B, NK cells from patients at later stages of COVID-19 presented enhanced levels of IFN-γ and TNF-α which were measured ex vivo in the absence of usual in vitro stimulation. These activated NK cells were phenotyped as CD49a+CD69a+CD107a+ cells, and their emergence in patients correlated to the number of neutrophils, and plasma IL-15, a key cytokine in NK cell activation. Despite lower amounts of cytotoxic molecules in NK cells of patients with severe symptoms, majority of COVID-19 patients displayed a normal cytotoxic killing of Raji tumour target cells. In vitro stimulation of patients blood cells by IL-12+IL-18 revealed a defective IFN-γ production in NK cells of ICU patients only, indicative of an exhausted phenotype. ScRNA-seq revealed, predominantly in patients with severe COVID-19 disease symptoms, the emergence of an NK cell subset with a platelet gene signature that we identified by flow and imaging cytometry as aggregates of NK cells with CD42a+CD62P+ activated platelets. Post-COVID-19 patients show slow recovery of NK cell frequencies and phenotype. Our study points to substantial changes in NK cell phenotype during COVID-19 disease and forms a basis to explore the contribution of platelet-NK cell aggregates to antiviral immunity against SARS-CoV-2 and disease pathology.
Macrophages undergo plasma membrane fusion and cell multinucleation to form multinucleated giant cells (MGCs) such as osteoclasts in bone, Langhans giant cells (LGCs) as part of granulomas or foreign-body giant cells (FBGCs) in reaction to exogenous material. While osteoclast multinucleation is a prerequisite for vertebrate bone homeostasis, the effector function resulting from LGC and FBGC multinucleation is less well-defined. More generally, how multinucleation per se contributes to functional specialization of mature mononuclear macrophages remains poorly understood in humans. Here, we integrated comparative transcriptomics with functional assays in purified mature mononuclear and multinucleated human osteoclasts, LGCs and FBGCs. Strikingly, in all three types of MGCs, multinucleation causes a pronounced down-regulation of mononuclear phagocyte identity. We show enhanced lysosome-mediated intracellular iron homeostasis promoting MGC formation. The transition from mononuclear to multinuclear state is accompanied by cell specialization specific to each polykaryon. Enhanced phagocytic and mitochondrial function associate with FBGCs and osteoclasts, respectively. Moreover, only B7-H3 (CD276)-expressing human LGCs can form granuloma-like clusters in vitro, suggesting that LGC multinucleation potentiates T cell activation. These findings demonstrate how cell-cell fusion and multinucleation reset human macrophage identity as part of an advanced maturation step that confers MGC-specific functionality.
Objective Systemic juvenile idiopathic arthritis (JIA) is a systemic inflammatory disease with childhood onset. Systemic JIA is associated with neutrophilia, including immature granulocytes, potentially driven by the growth factor granulocyte‐colony stimulating factor (G‐CSF). This study was undertaken to investigate the role of G‐CSF in the pathology of systemic JIA. Methods Injection of Freund's complete adjuvant (CFA) in BALB/c mice induces mild inflammation and neutrophilia in wild‐type (WT) mice and a more pronounced disease, reminiscent to that of JIA patients, in interferon‐γ–knockout (IFNγ‐KO) mice. Extramedullary myelopoiesis was studied in CFA‐immunized mice by single‐cell RNA sequencing, and the effect of G‐CSF receptor (G‐CSFR) blockage on neutrophil development and systemic JIA pathology was evaluated. Additionally, plasma G‐CSF levels were measured in patients. Results Both in systemic JIA patients and in a corresponding mouse model, plasma G‐CSF levels were increased. In the mouse model, we demonstrated that G‐CSF is responsible for the observed neutrophilia and extramedullary myelopoiesis and the induction of immature neutrophils and myeloid‐derived suppressor‐like cells. Administration of a G‐CSFR antagonizing antibody blocked the maturation and differentiation of neutrophils in CFA‐immunized mice. In IFNγ‐KO mice, treatment was associated with almost complete inhibition of arthritis due to reduced neutrophilia and osteoclast formation. Disease symptoms were ameliorated, but slight increases in interleukin‐6 (IL‐6), tumor necrosis factor, and IL‐17 were detected upon G‐CSFR inhibition in the IFNγ‐KO mice, and were associated with mild increases in weight loss, tail damage, and immature red blood cells. Conclusion We describe the role of G‐CSF in a mouse model of systemic JIA and suggest an important role for G‐CSF–induced myelopoiesis and neutrophilia in regulating the development of arthritis.
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