Immunoregulatory molecule expression on extracellular microvesicles in people living with HIV.

Introduction: People living with HIV (PLWH) now benefit from combined antiviral treatments that durably control viral replication. These antiretroviral treatments decrease mortality and improve quality of life in PLWH, but do not completely control the excessive non-specific activation of the immune system in PLWH. This chronic immune activation is a key element of HIV immunopathology that contributes to the pathophysiology of inflammatory comorbid conditions, such as cardiovascular disorders, cancer and autoimmune diseases. Circulating non-exosomal extracellular vesicles, also known as microparticles (MPs) are detected in these diseases and have been linked to immune activation. The objective of this study was to characterize the MPs present in PLWH and to assess their association with chronic immune activation. Methods: We performed flow cytometry for the complete phenotypic characterization of MPs from fresh plasma from PLWH and from people without HIV as the control group. The absolute number, size and cellular origin of MPs were evaluated. The immunoregulatory profile was determined by cell origin, for MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs). Results: PLWH had significantly more circulating MPs than controls, for MPs of all sizes originating from T lymphocytes, red blood cells, neutrophils, dendritic cells, B lymphocytes and endothelial cells. PMPs and MMPs were not more numerous in PLWH, but the immunoregulatory phenotypes of these MPs differed between PLWH and controls. These differences in immunoregulatory molecule expression profile were also observed for LMPs. PDL1, ICOSL, CCR5, TGFß1, MHC classes I and II, TRAIL, CXCR4, OX40, DC-SIGN, CTLA4 and PDL2 were more strongly expressed on the surface of MPs from PLWH than on those from controls. Conclusion: MPs are an important element in intercellular communication, making it possible to transfer phenotypes and functions to immune cells. The significantly higher numbers of MPs expressing diverse immunomodulatory molecules in PLWH may make a major contribution to the maintenance and/or the development of immune-cell activation in these individuals.

G-CSF reshapes the cytosolic PCNA scaffold and modulates glycolysis in neutrophils.

Cytosolic proliferating cell nuclear antigen (PCNA) is involved in neutrophil survival and function, in which it acts as a scaffold and associates with proteins involved in apoptosis, NADPH oxidase activation, cytoskeletal dynamics, and metabolism. While the PCNA interactome has been characterized in neutrophils under homeostatic conditions, less is known about neutrophil PCNA in pathophysiological contexts. Granulocyte colony-stimulating factor (G-CSF) is a cytokine produced in response to inflammatory stimuli that regulates many aspects of neutrophil biology. Here, we used isolated normal-density neutrophils from G-CSF-treated haemopoietic stem cell donors (GDs) as a model to understand the role of PCNA during inflammation. Proteomic analysis of the neutrophil cytosol revealed significant differences between GDs and healthy donors (HDs). PCNA was one of the most upregulated proteins in GDs, and the PCNA interactome was significantly different in GDs compared with HDs. Importantly, while PCNA associated with almost all enzymes involved in glycolysis in HDs, these associations were decreased in GDs. Functionally, neutrophils from GDs had a significant increase in glycolysis compared with HDs. Using p21 competitor peptides, we showed that PCNA negatively regulates neutrophil glycolysis in HDs but had no effect on GD neutrophils. These data demonstrate that G-CSF alters the PCNA scaffold, affecting interactions with key glycolytic enzymes, and thus regulates glycolysis, the main energy pathway utilized by neutrophils. By this selective control of glycolysis, PCNA can organize neutrophils functionality in parallel with other PCNA mechanisms of prolonged survival. PCNA may therefore be instrumental in the reprogramming that neutrophils undergo in inflammatory or tumoral settings.

Specific circulating neutrophils subsets are present in clinically stable adults with cystic fibrosis and are further modulated by pulmonary exacerbations.

The progressive lung destruction in cystic fibrosis (CF) is tightly associated with chronic bacterial infection and neutrophil-dominated airway inflammation. CF pulmonary disease is complicated by episodes of acute exacerbations, contributing to irreversible lung damage. We hypothesized that circulating subsets of neutrophils from clinically stable adults with CF present some phenotypic specificities that could amplify their activation during an infectious episode. The aim of the present study was to examine the different neutrophil subsets in whole blood and in the low density neutrophils (LDN) that co-purify with peripheral blood mononuclear cells (PBMC) in clinically stable adults with CF and in CF adults during pulmonary exacerbations compared to healthy donors. Blood samples were obtained from 22 adults with CF (16 in stable state and 6 during pulmonary exacerbations) and from 20 healthy donors. Flow cytometry analysis of 13 different markers related to lineage (CD45, CD15), maturity (CD16, CD10, and CD33), activation (CD62L, CD11b, CD66b, and CD114), metabolism (GLUT-1, LOX1) and immunosuppression (PD1, PD-L1) was carried out within whole blood and within the LDN fraction. Unsupervised analysis of flow cytometry data was performed using visual t-distributed stochastic neighbor embedding (vi-tSNE). A significant increase in the CD11b expression in neutrophils from CF patients during exacerbations was observed compared to neutrophils from stable CF patients or to healthy donors, indicative of a circulating activation state due to an infectious status. The percentage of LDN was not increased in stable CF patients but increased during exacerbations. Analysis of neutrophil subsets using the double CD16/CD62L labeling revealed a significant increase in the CD16high/CD62Llow subset in all CF patients compared to healthy donors. In contrast, an increase in the CD16low/CD62Lhigh subset was observed only in CF patients during exacerbations. Unsupervised analysis identified a PD-L1high/CD114high population that was present in stable CF patients and as well as in CF patients during exacerbations.