The Multifaceted Role of Neutrophil Heterogeneity in COVID-19 Pathogenesis

COVID-19 is a respiratory illness caused by the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The disease is characterized by an excessive inflammatory response and dysfunction of the immune system triggered by immunopathological mechanisms. Neutrophils are the most abundant circulating leukocytes, playing a critical role in the pathogenesis of multiple inflammatory conditions. Several studies have demonstrated that neutrophils are key contributors to COVID-19 disease pathogenesis; however, their role in disease progression remains unclear.

Here, we demonstrate immune dysregulation in COVID-19 patients characterized by a significant expansion of neutrophil numbers and reduced frequency of T-cell populations. We identify an immature CD16^neg CD64^high neutrophil population that is significantly expanded in the circulation of COVID-19 patients and strongly correlates with increased neutrophil-to-lymphocyte ratio (NLR).

Using CITE-Seq analysis, a method allowing concurrent analysis of surface protein levels and transcripts on a single cell, we demonstrated the presence of two distinct immature clusters (16N1 and 16N2) within the CD16^neg population that exhibit distinct phenotypic and transcriptional profiles. The 16N1 subset is consistent with progenitor neutrophils (proNeu) and represents the earliest stage of neutrophil lineage in the circulation of COVID-19 patients. The 16N2 subset displays characteristics of precursor neutrophils (preNeu). Both populations are significantly expanded in COVID-19 patients. 

CD16^pos neutrophils, in COVID-19 patients, exhibit increased levels of neutrophil activation and degranulation and a shift to a less mature phenotype. Detailed CITE-Seq analysis reveals six CD16^pos neutrophil clusters in COVID-19 patients along the neutrophil maturation trajectory, including an expanded population consistent with previously described immunosuppressive CD62L^low CXCR4^high neutrophils. Importantly, CD62L^low CXCR4^high neutrophils that are expanded in severe COVID-19 patients display immunosuppressive characteristics, including increased production of ROS and high surface levels of PD-L1 and CD25. Moreover, this potential immunosuppressive population expresses higher levels of CD73, a novel immune checkpoint effector that mediates immunosuppression by converting AMP to adenosine (ADO) Overall, the findings presented here demonstrate altered myelopoiesis in COVID-19 disease indicated by increased frequencies of neutrophil progenitor and precursor subsets in the circulation, a shift of CD16^pos neutrophils towards an immature phenotype, and an expansion of neutrophils with an immunosuppressive phenotype. Detailed characterization of the role of specific neutrophil subsets in COVID-19 patients significantly enhances our understanding of the role of these subsets in disease pathogenesis and opens new avenues for the identification of novel therapeutic strategies for the management of infectious and inflammatory diseases.