# CD169+ and HLA-DR+ extracellular vesicles are highly represented in human plasma and dynamically expressed in SARS-CoV-2 infection and long COVID-associated sequelae

## TL;DR

This study explores how immune cells and extracellular vesicles change during and after SARS-CoV-2 infection, offering new ways to monitor long-term effects of COVID-19.

## Contribution

The study identifies dynamic expression of CD169+ and HLA-DR+ extracellular vesicles in SARS-CoV-2 infection and long COVID, linking them to immune dysregulation and persistent inflammation.

## Key findings

- HLA-DR+CD169+ extracellular vesicles are significantly elevated in both acute and post-acute SARS-CoV-2 infection.
- EVs correlate with inflammatory markers and coagulation parameters in patients with long COVID.
- EVs reflect immune perturbations and may serve as indicators of persistent inflammation in long COVID.

## Abstract

Elevated inflammation and immune dysregulation are the main consequences of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The dysregulated inflammatory state persists after coronavirus disease 2019 (COVID-19), establishing the post-acute sequelae of SARS-CoV-2 infection in individuals with long COVID (LC). The role of CD169+ monocytes in the early diagnosis of SARS-CoV-2 infection and their association with severe outcomes were demonstrated in COVID-19 patients (COV). We aimed to delineate specific myeloid activation that characterizes the acute and post-acute phases of SARS-CoV-2 infection, evaluating the correlation between cellular and extracellular vesicles (EVs).

Blood samples from COV, LC, and healthy donors (HD) were collected at Tor Vergata University Hospital in Rome. Plasmatic EVs were isolated by differential centrifugation and evaluated by flow cytometry and atomic force microscopy (AFM). Leukocyte subpopulations and different sizes of circulating EVs (100–200, 240–500, >500 nm) were characterized for HLA-DR and CD169 expression in COV, LC, and HD through flow cytometry. Serum inflammatory markers were assessed by the ELLA immunoassay system. The analyzed markers were associated with clinical and biochemical parameters in COV and LC.

The analysis of HLA-DR+, CD169+, and HLA-DR+CD169+ leukocytes confirmed our previous results in which the activated monocytes CD169+HLA-DR+ were found significantly high in COV, persisting in LC, and correlated differently with coagulation markers and inflammatory cytokines. Similar to cellular levels, the percentage and number of HLA-DR+CD169+ EVs were significantly elevated in COV and persisted in LC compared to HD. Different HLA-DR and CD169 expressions were found according to EV size in COV, LC, and HD, and correlations with biochemical parameters and circulating inflammatory markers were found. A positive correlation of HLA-DR and CD169 expression among monocytes and circulating EVs was found, supporting a possible connection between the two compartments and circulating inflammatory mediators. Moreover, the characterization by flow cytometry of EV cell derivation and cytokine cargo revealed EVs as sensitive indicators of both acute and persistent immune perturbations, bridging viral antigen persistence with inflammatory signaling in long COVID.

Myeloid activation markers and inflammatory cytokines are dynamically expressed between blood cells and circulating extracellular vesicles, underlining multilevel cell-to-cell communications, opening new possibilities to monitor COVID-19 and long COVID-associated sequelae.