Low-density granulocytes display immature cells with enhanced NET formation in people living with HIV

Abstract

While the protective role of neutrophil extracellular traps (NETs) in limiting human immunodeficiency virus (HIV) spread to susceptible cells has been documented, there is comparatively little insight into whether NET formation is harmful in people living with HIV (PLWH). To gain insight into neutrophil dysregulation and the pathological role of NETs in HIV, we examined expressions of NET-associated markers [cell-free DNA (cfDNA) and citrullinated histone H3 (CitH3)] in the plasmas from a cohort of the Hawaii Aging with HIV-cardiovascular and HIV-seronegative (HIV-) individuals. In a subset of participants, circulating low-density granulocyte (LDG) levels and their maturation and activation status were analyzed via flow cytometry. We demonstrated higher plasma levels of CitH3 in PLWH compared to HIV- individuals. LDGs from PLWH had heightened CD66b, but reduced CD16 expression. The percentages and counts of CD10⁺ LDGs were significantly decreased in PLWH. In addition, the CD16^Lo LDG subsets were enriched in PLWH, compared to HIV- group, indicating that immature LDGs are increased in PLWH. Moreover, LDGs from PLWH exhibited significantly higher NET forming capacity. In summary, our study presents evidence that LDGs from PLWH on ART display an immature and altered phenotype with increased NET formation. Among PLWH, plasma NET levels as well as LDG parameters correlated with blood markers for inflammation and coagulation, suggesting that neutrophil activation and NETs may exert proinflammatory and coagulation effects. Our data provide insights into the pathologic role of LDGs at least in part mediated through NET formation in PLWH.

Figure 1

PLWH had higher levels of NET-associated markers in plasma compared to HIV− individuals. (A,B) Plasma samples from PLWH, referred hereafter as HIV+ (n = 88) and HIV− individuals (n = 58) were assessed for cfDNA (A) and CitH3 (B). (C) Correlation between CitH3 and cfDNA in PLWH. (D) Plasma levels of CitH3 in PLWH with/without detectable viremia. UD undetectable, D detectable. Spearman’s rank correlation was calculated. (A,B,D) Mann–Whitney U test, N.S. non-significant.

Figure 2

Characterization of LDG population in HIV+ (n = 37) and HIV− (n = 48) individuals (A,B). Percentage (A) and numbers (B) of LDGs in HIV− and HIV+. (C–G) Comparison of expression of neutrophil markers on LDGs between groups. Representative histograms of expression of CD15, CD66b, CD11b, and CD16 in LDGs in HIV− (blue) and HIV+ (red) groups (C). CD15 MFI (D), CD66b MFI (E), CD11b MFI (F), and CD16 MFI (G) of in HIV− and HIV+ individuals. Mann Whitney-U test; ns = p > 0.05.

Figure 3

Decreased mature CD10⁺ LDGs were observed in PLWH. (A–D) The percentages and counts of CD10⁺ and CD10⁻ LDGs in HIV+ (n = 37) and HIV− (n = 48). CD10⁺ LDG percentage (A), CD10⁺ LDG count (B), CD10⁻ LDG percentage (C), CD10⁻ LDG count (D) in HIV− and HIV+ individuals. MPO expression in LDGs. MPO⁺ CD10⁺ LDG percentage (E), MPO⁺ CD10⁺ LDG count (F), and MPO MFI of LDG, CD10⁺ LDG, and CD10⁻ LDG in HIV− and HIV+ individuals (G). Mann Whitney-U test; ns = p > 0.05.

Figure 4

Comparison of CD16^Hi and CD16^Lo LDG subsets between groups. (A) Representative dot plot demonstrating CD16^Hi (2) and CD16^Lo (1) LDG populations in HIV+ (n = 37) and HIV− (n = 48) groups. (B) CD16^Hi and CD16^Lo LDG percentages in HIV− and HIV+ groups. (C) CD16^Hi and CD16^Lo LDG counts in HIV− and HIV+ groups. (D–I) CD10 MFI of CD16^Hi (D) and CD16^Lo LDG (E), CD11b MFI of CD16^Hi (F) and CD16 ^Lo LDG (G), CD66b MFI of CD16^Hi (H) and CD16^Lo LDG (I). Mann Whitney-U test; ns = p > 0.05.

Figure 5

NET-forming LDGs in HIV+ (n = 37) and HIV− (n = 48) individuals. (A,B) NET forming LDG percentage (A), NET-forming LDG count (B) in HIV− and HIV+ individuals. (C,D) CD10⁺ and CD10⁻ NET-forming LDG percentage (C), CD10⁺ and CD10⁻ NET forming LDG count (D) in HIV− and HIV+ individuals. Mann Whitney-U test; ns = p > 0.05.