A Cross-Disease Study Investigating the Phenotypic and Functional Heterogeneity in Neutrophil Subsets

Abstract

Neutrophils play a critical role in the innate immune response. They are the most abundant leukocytes in humans and are the first line of defence against invading microorganisms; growing evidence in the last decade has challenged the traditional view that neutrophils are functionally homogeneous cells and can contribute to the pathogenesis of various inflammatory diseases. Several studies have identified the presence of low-density neutrophils (LDN) that co-localise with the mononuclear cells during density separation of whole blood. Despite their ubiquitous presence in pathological conditions, there is little consistency in the literature regarding the isolation technique, source, function and origin of LDNs. Whether the differences reflect true intrinsic differences or are induced by isolation-induced activation is still debated. The major limitation in characterising these cells is due to the lack of consistency in the surface markers used to study these LDN populations across diseases. Thus precluding a direct comparison between these reported LDN subsets. In this study, we have systematically characterised and compared the phenotype and function of LDN populations across four diseases with distinct underlying pathology such as ANCA-Associated Vasculitis, lung cancer, sepsis and COVID-19 to understand whether LDN phenotype and functions vary under different inflammatory conditions, suggesting plasticity in these cells or are these cells merely a consequence of a generic response to inflammation. In this work, we have demonstrated that LDN frequency and immature neutrophil composition greatly vary depending on the severity of the underlying disease. Notably, we have shown that LDNs in sepsis patients are of primarily immature phenotype, making them distinct from other diseases. Immature LDNs displayed overlapping features between the four diseases, suggesting a common source, likely originating from emergency granulopoiesis. Lastly, we have identified the emergence of disease-specific LDN phenotypes in sepsis, AAV and lung cancer patients with unique phenotypic and functional features. These findings add to our understanding of the growing field of neutrophil heterogeneity and could potentially lead to the identification of pathogenic neutrophil subsets that can be exploited as putative prognostic and therapeutic targets.