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. 2023 Nov 15:14:1256182.
doi: 10.3389/fimmu.2023.1256182. eCollection 2023.

Aging dysregulates neutrophil extracellular trap formation in response to HIV in blood and genital tissues

Laura Moreno de Lara  1   2 , Alexandra Werner #  1 , Anna Borchers #  1 , Francisco J Carrillo-Salinas  1 , Wendelin Marmol  3 , Siddharth Parthasarathy  1 , Vidya Iyer  4 , Alison Vogell  4 , Diego Illanes  4 , Ana C Abadía-Molina  2   5 , Christina Ochsenbauer  6 , Charles R Wira  7 , Marta Rodriguez-Garcia  1
Affiliations

Aging dysregulates neutrophil extracellular trap formation in response to HIV in blood and genital tissues

Laura Moreno de Lara et al. Front Immunol. .

Abstract

Women acquire HIV through sexual transmission, with increasing incidence in women >50 years old. Identifying protective mechanisms in the female genital tract (FGT) is important to prevent HIV-acquisition in women as they age. Human genital and blood neutrophils inactivate HIV by releasing neutrophil extracellular traps (NETs), an innate protective mechanism against HIV-infection. However, how NET formation is triggered by HIV in different tissues and whether this mechanism is affected by aging remain unknown. We demonstrate that the mechanisms that trigger NET release in response to HIV are different in blood and genital tissues, and that NET release decreases with aging. In blood neutrophils, HIV stimulation independently activated calcium pathways and endosomal TLR8, but aging reduced calcium responses, resulting in delayed NET release. In contrast, calcium responses were absent in genital neutrophils and NET release was triggered preferentially through TLR8 activation, but aging impaired this pathway. HIV induced NET formation through non-lytic pathways in blood and FGT neutrophils, except for a small subset of NETs that incorporated annexin V and lactoferrin predominantly in blood, suggesting proinflammatory and lytic NET release. Our findings demonstrate that blood neutrophils cannot model genital neutrophil responses which has important implications to understanding protection against HIV acquisition.

Keywords: HIV prevention; NETs; aging; female genital tract; menopause; neutrophil extracellular traps; neutrophils; women.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Aging delays NET release in response to HIV. (A) Representative NET release images by blood neutrophils after HIV stimulation (2h). Arrows indicate NETs. Scale bar: 50μm. (B) Representative confocal images of HIV-NETs after 1h stimulation. Scale bar: 10 μm. (C) Representative quantification of NET release over time by premenopausal (black) and postmenopausal women (white) after HIV stimulation (circles). Unstimulated neutrophils (triangles) overlap at the bottom. (D) Comparison of NET release by blood neutrophils from premenopausal (n=18) and postmenopausal women (n=19) at total (0-2h), early (0-15min), late (15min-2h) times. Mann-Whitney test. Data represent median with interquartile range. (E) Correlation of early NET release (15min) and women’s age (premenopausal=18; postmenopausal=19) in blood. Spearman r correlation. Vertical dotted line indicates menopause. (F) Representative NET release images by genital neutrophils after HIV stimulation (2h). Scale bar: 50μm. (G) Comparison of NET release by genital neutrophils from premenopausal (n=34) and postmenopausal women (n=17) at total (0-2h), early (0-15min) and late (15min-2h) times. Mann-Whitney test. Data represent median with interquartile range. (H) Correlation of early NET release (15min) and women’s age (premenopausal=34; postmenopausal=17) in genital tissues and (I) separated by anatomical compartment: endometrium and uterine cervix (endocervix + ectocervix). Spearman r correlation. Vertical dotted line indicates menopause. (J) Correlation between women’s age and number of neutrophils (left graph) or neutrophil percentage within immune cells (CD45+) (right graph) in endometrium (n=11), endocervix (n=7) and ectocervix (n=7). *p<0.05; **p<0.01.
Figure 2
Figure 2
TLR8 and TLR7 mediate HIV-induced NET-release in a temporal sequential manner. (A) Representative quantification of HIV-induced NET release over time in HIV only condition (black), dual iODN (dark gray), or CU-CPT9a (light gray) treatment in blood neutrophils (CU-CUPT9a: n=25; Dual iODN: n=17). (B, C) NET release in HIV alone condition (black) or after TLR or RLR inhibition (red) in blood neutrophils. Wilcoxon test. 1 outlier removed from ODN 20958 condition by Grubbs’s test (Alpha = 0.05). (D) NET release by blood neutrophils from premenopausal and postmenopausal women after TLR8 (left graph) and TLR7/9 (right) inhibition. Wilcoxon test. (E) Normalized reduction in NET release after TLR8 (left graph) or TLR7/9 (right graph) inhibition with respect to HIV only control condition in blood neutrophils from pre and postmenopausal women. One sample t test (F) NET release by genital neutrophils in response to HIV stimulation alone (black) and following treatment with TLR inhibitors (red) (CU-CUPT9a: n=13; Dual iODN: n=14; ODN 20958: n=11). Premenopausal women’s age (average): 44; postmenopausal women´s age (average): 60. Wilcoxon test. 1 outlier removed from ODN 20958 condition by Grubbs’s test (Alpha = 0.05). (G) NET release in response to HIV by genital neutrophils from premenopausal and postmenopausal women following treatment with TLR inhibitors. Wilcoxon test. (H) Normalized reduction in NET release after TLR8 (left graph) or TLR7/9 (right graph) inhibition with respect to HIV only condition in genital neutrophils from pre and postmenopausal women. One sample t test; horizontal lines represent median with interquartile range. *p<0.05, **p<0.01.
Figure 3
Figure 3
Induction of cytosolic calcium by HIV stimulation in blood neutrophils from younger women. (A) Representative images of intracellular calcium increase (Rhod-3 AM) in blood neutrophils after HIV stimulation (HIV-GFP, green). (B) Representative quantification of intracellular calcium in blood neutrophils after HIV stimulation. (C) Comparison of intracellular calcium levels in the absence (black) or presence (gray) of BAPTA in resting blood neutrophils and after HIV stimulation (5 min). Wilcoxon test. (D) Comparison of intracellular calcium accumulation in blood neutrophils from pre (n=11; black) and postmenopausal women (n=6; white) after 5 and 15 min of HIV stimulation. Wilcoxon test. (E) Correlation between age and intracellular calcium increase after HIV stimulation at 5 min. Spearman r correlation (one-tailed). (F) Correlation between early NET release by blood neutrophils and intracellular calcium increase after HIV stimulation at 5 min. Spearman r correlation (two-tailed). (G) Percentage of HIV-induced NETs reduction in the presence of BAPTA in blood neutrophils from premenopausal (black) and postmenopausal (white) women. One sample t test. (H) Lack of effect of TLR inhibition in intracellular calcium levels in blood neutrophils stimulated with HIV. (I) Intracellular calcium increase in blood neutrophils stimulated with HIV with envelope (black) or HIV without envelope (white). One sample t test. (J) Representative images of calcium staining (Rhod-3 AM) in genital neutrophils after HIV stimulation (HIV-GFP, green). (K) Representative quantification of intracellular calcium in genital neutrophils after HIV stimulation. (L) Intracellular calcium accumulation in genital neutrophils after HIV stimulation (circles) or control condition (triangles). (M) Lack of correlation between early NET release by genital neutrophils and intracellular calcium after HIV stimulation. (N) NET release by genital neutrophils after HIV stimulation (black) or HIV stimulation plus BAPTA (gray). Horizontal lines represent median with interquartile range. *p<0.05, ***p<0.001. Scale bar: 100µm.
Figure 4
Figure 4
HIV stimulation induces ROS production through differential mechanisms in blood and genital neutrophils. (A) Representative images of blood neutrophils stained with CellROX dye (red) prior to stimulation with HIV-VLPs (HIV-GFP, green). Scale bar: 100µm. (B) Representative quantification of intracellular ROS production by blood neutrophils in control condition (white) or after stimulation with HIV (black), or HIV lacking envelope proteins (HIV-Δenv) (gray). (C) Intracellular ROS production by blood neutrophils from premenopausal (black) and postmenopausal (white) women after HIV or HIV-Δenv stimulation. Wilcoxon test. (D) Intracellular ROS production by blood neutrophils stimulated with HIV alone (black) or in the presence of NADPH oxidase inhibitors (DPI) or calcium chelators (BAPTA). Wilcoxon test. (E) Fold-change of intracellular ROS production after HIV stimulation in the presence of TLR inhibitors ((HIV+TLR inhibitor)/HIV) by blood neutrophils from premenopausal (black) and postmenopausal (white) women. Mann–Whitney U test. (F) Representative images of genital neutrophils stained with CellROX dye (red) after stimulation with HIV-VLPs (HIV-GFP, green). Scale bar: 50µm. (G) Representative quantification of intracellular ROS production by genital neutrophils in control condition (white) or after stimulation with HIV (black), or HIV lacking envelope proteins (HIV-Δenv) (gray). *p<0.05.
Figure 5
Figure 5
HIV is internalized by neutrophils. (A) Representative flow cytometry plots for CD4, CCR5 and CXCR4 expression on blood and genital neutrophils. (B) Percentage of expression of CD4, CXCR4, and CCR5 in blood neutrophils (top graph) and in genital neutrophils (bottom graph). (C) Representative images of confocal microscopy of Rab5 expression (red) in blood neutrophils before (control) or after 5 min of HIV stimulation. Scale bar: 25 µm. (D) Representative confocal image (zoom) of Rab5+ blood neutrophils after 5 min of HIV stimulation. Scale bar: 50 µm. (E) Representative images of Rab5+ genital neutrophils after 15 min of HIV stimulation using confocal microscopy. Scale bar: 25 µm. (F) Quantification of the percentage of Rab5+ cells in resting versus HIV-stimulated blood neutrophils (left graph) and genital mixed cell suspensions (right graph). Wilcoxon test. (G) NET release in response to HIV stimulation (black) and HIV stimulation in the presence of dynamin inhibitor (white) in blood neutrophils (left graph) and genital neutrophils (right graph). Wilcoxon test. Horizontal lines represent median with interquartile range. *p<0.05.
Figure 6
Figure 6
Annexin V identifies proinflammatory NETs. (A) Representative images of blood neutrophils stained with red cytotox in resting and HIV-stimulated conditions (30 minutes). White circles show the mask applied to quantify cytotox+ cells. Scale bar: 50 μm. (B) Quantification of cytotox+ blood neutrophils in control and HIV-stimulated conditions at 30 minutes. (C) Representative images of HIV-stimulated blood neutrophils stained with annexin V or cytotox (30 minutes). Scale bar: 50 μm. (D) Representative images of annexin V+ and annexin V- NETs after HIV stimulation of blood neutrophils. scale bar: 100 μm. (E) Representative curve and (F) quantification of annexin V+ (yellow) and annexin V- (green) NET production by blood neutrophils after HIV stimulation. Wilcoxon test. (G) Representative images of blood neutrophils stimulated with calcium ionophore in the presence of green cytotox and red annexin V (3 h). Scale bar: 50 μm. (H, I) Annexin V+ and annexin V- NET production after HIV stimulation of blood neutrophils preincubated with TLR8 inhibitor (H) or TLR7 inhibitor (I). Wilcoxon test. (J) Representative images of the lack of annexin V+ NET production by genital neutrophils. Scale bar: 150 μm (K) Comparison of annexin V+ NET production by blood and genital neutrophils. Mann-Whitney test; (L, M) Representative confocal microscopy images of annexin V and lactoferrin expression in NETs from blood neutrophils (L) (Scale bar: 5 μm), and genital neutrophils (M) following 30min-2h stimulation with HIV (HIV-GFP, green). Scale bar: 10 μm. *p<0.05; ns, not significant..
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