DataSheet_1_Differential Functional Responses of Neutrophil Subsets in Severe COVID-19 Patients.pdf

Neutrophils play a significant role in determining disease severity following SARS-CoV-2 infection. Gene and protein expression defines several neutrophil clusters in COVID-19, including the emergence of low density neutrophils (LDN) that are associated with severe disease. The functional capabilities of these neutrophil clusters and correlation with gene and protein expression are unknown. To define host defense and immunosuppressive functions of normal density neutrophils (NDN) and LDN from COVID-19 patients, we recruited 64 patients with severe COVID-19 and 26 healthy donors (HD). Phagocytosis, respiratory burst activity, degranulation, neutrophil extracellular trap (NET) formation, and T-cell suppression in those neutrophil subsets were measured. NDN from severe/critical COVID-19 patients showed evidence of priming with enhanced phagocytosis, respiratory burst activity, and degranulation of secretory vesicles and gelatinase and specific granules, while NET formation was similar to HD NDN. COVID LDN response was impaired except for enhanced NET formation. A subset of COVID LDN with intermediate CD16 expression (CD16Int LDN) promoted T cell proliferation to a level similar to HD NDN, while COVID NDN and the CD16Hi LDN failed to stimulate T-cell activation. All 3 COVID-19 neutrophil populations suppressed stimulation of IFN-γ production, compared to HD NDN. We conclude that NDN and LDN from COVID-19 patients possess complementary functional capabilities that may act cooperatively to determine disease severity. We predict that global neutrophil responses that induce COVID-19 ARDS will vary depending on the proportion of neutrophil subsets.

中性粒细胞在SARS-CoV-2感染后疾病严重程度的确定中扮演着至关重要的角色。在COVID-19中，基因和蛋白表达定义了多个中性粒细胞簇，其中包括与重症疾病相关的低密度中性粒细胞（LDN）。这些中性粒细胞簇的功能特性及其与基因和蛋白表达的相关性尚不明确。为了界定COVID-19患者中正常密度中性粒细胞（NDN）和LDN的宿主防御和免疫抑制功能，我们招募了64名重症COVID-19患者和26名健康供体（HD）。我们对这些中性粒细胞亚群的内吞作用、呼吸爆发活性、脱颗粒、中性粒细胞外泌体（NET）形成以及T细胞抑制进行了测量。重症/危重症COVID-19患者的NDN表现出先导作用，其吞噬作用、呼吸爆发活性和分泌囊泡、明胶酶和特异性颗粒的脱颗粒能力增强，而NET形成与HD NDN相似。COVID LDN的反应受损，除了NET形成增强外。CD16表达介于中等（CD16Int LDN）的COVID LDN亚群促进了T细胞增殖至与HD NDN相似的水平，而COVID NDN和CD16Hi LDN未能刺激T细胞活化。与HD NDN相比，所有三种COVID-19中性粒细胞人群均抑制了IFN-γ的产生。我们得出结论，COVID-19患者的NDN和LDN具有互补的功能特性，可能协同作用以确定疾病严重程度。我们预测，诱导COVID-19急性呼吸窘迫综合征的全局中性粒细胞反应将因中性粒细胞亚群的比例而异。