All ETDs from UAB

Advisory Committee Chair

Paul A Goepfert

Advisory Committee Members

Scott W Ballinger

Scott W Ballinger

Zdenek Hel

Edgar T Overton

Chandler Raman

Document Type

Dissertation

Date of Award

2023

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Neutrophils, the most abundant type of circulating leukocyte, represent a heterogeneous population with distinct roles in immune regulation and disease pathogenesis. A major obstacle to our understanding of neutrophil biology is that neutrophils are readily activated during preparation. The lack of standardized methodology for neutrophil characterization makes the comparison of results across studies challenging. Here we provide a careful comparison of eight characterization methods and present a novel, optimized protocol for the characterization of whole blood neutrophils minimizing activationinduced phenotypic alterations during processing. Despite successful virological control, HIV-1-infected individuals maintain an increased risk of life-threatening comorbidities including liver, cardiovascular, and other end-organ diseases. This excess risk is associated with continuous microbial translocation and resulting chronic inflammation; however, the specific mediators and mechanisms driving these pathologies remain unclear. Despite reports of reduced neutrophil functionality and phenotypic alterations in HIV-1-infected individuals, neutrophils and neutrophil subsets remain understudied. Here, we identify and characterize a circulating immature neutrophil population defined as CD16negCD10negCD64high that is expanded during HIV-1 infection and exhibits distinct phenotypic, transcriptional, and functional properties. CITE-Seq analysis demonstrates the presence of three distinct clusters (N1 – N3) within CD16neg neutrophils and two clusters (N4 and N5) within CD16pos neutrophils, representing sequential maturational stages and demonstrating previously unappreciated heterogeneity of circulating neutrophil populations. Detailed analyses demonstrate phenotypic alterations of CD16neg and CD16pos neutrophils in HIV-1-infected individuals, consistent with a shift toward earlier maturational states. Phenotypic changes correlate with key clinical parameters including current and nadir CD4+ T cell counts and FibroSCAN score. Transcriptional and metabolic analyses support a significant shift toward an immature neutrophil phenotype in HIV-1- infected individuals compared to healthy donors. Overall, the data presented here suggest underlying alternative granulopoiesis in inflammatory conditions, including HIV-1 infection, inducing shifts of specific neutrophil populations toward a less mature phenotype, increasing the frequency of circulating neutrophil progenitors, and resulting in phenotypic, functional, transcriptional, and metabolomic alterations during advanced disease. A comprehensive assessment of the specific neutrophil subsets described here will enhance our understanding of the role of innate immune mechanisms in chronic inflammation and provide novel therapeutic targets for disease management.

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