Increased alpha-defensins 1-3 production by dendritic cells in HIV-infected individuals is associated with slower disease progression

Abstract

Background: Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce alpha-defensins1-3 and that alpha-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of alpha-defensins1-3 production by DC on disease progression.

Methodology/principal findings: Monocyte-derived DC (MDDC) were analyzed comparatively in healthy controls (HC) and HIV-infected patients, including untreated "elite" and "viremic" controllers, untreated viremic non-controllers and antiretroviral-treated patients. We found that production of alpha-defensins1-3 was significantly increased in MDDC from HIV-infected patients versus HC, and this increase was mainly due to that observed in controllers, while in non-controllers the increase was not statistically significant (controllers vs. HC, p<0.005; controllers vs. non-controllers p<0.05). Secreted alpha-defensins1-3 by immature MDDC positively correlated with CD4 T cell counts in controllers, but not in non-controllers. Moreover, independently of their clinical classification, HIV-infected patients with higher alpha-defensins1-3 secretion by immature MDDC showed slower disease progression, measured as no decrease in the number of CD4+ T-cells below 350 cell/mm(3), lower increase of plasma viral load and no initiation of treatment over time. Plasma alpha-defensins1-3 levels lacked any relationship with immunologic and virologic parameters.

Conclusions/significance: High production of alpha-defensins1-3 by immature DCs appears as a host protective factor against progression of HIV-1 infection, suggesting potential diagnostic, therapeutic and preventive implications. This protective effect may arise from the activity of alpha-defensins1-3 to damage the virions prior and/or after their internalization by immature DC, and hence favoring a more efficient viral processing and presentation to HIV-specific CD4+ T cells, without or with a minor rate of transmission of infectious HIV-1 virions.

Figure 1. Production of α-defensins 1-3 by immature (im) and mature (m) MDDCs.

(A) Immature and mature MDDC immunophenotype from one representative healthy control individual and one HIV-infected subject. X-axis represents HLA-DR expression and Y-axis represents the surface markers indicated. The percentage of positive cells are shown in each quadrant. (B) Relative expression of α-defensins 1-3 mRNA by real time RT-PCR in healthy control individuals (n = 15; open circles) and HIV-infected subjects (n = 39; open rhombus). Dots represent each patient and horizontal lines represent median ± interquartile ranges. Undetectable samples from mMMDC in HC and HIV-infected patients are not represented in the figure. (C) Levels of α-defensins 1-3 detected in MDDC culture supernatants from healthy non-infected controls (n = 15; white boxes) and HIV-infected subjects (n = 39; grey boxes). Boxes represent interquartile ranges, horizontal lines inside each box indicate the median and whiskers indicate maximum and minimum values. (D) Intracellular staining and flow cytometry analysis of α-defensins 1-3 in immature MDDCs. MDDCs were treated with monensin for 8h and double-stained for CD40 and α-defensins 1-3. CD40 positive cells were selected and the histogram corresponds to positive cells for intracellular α-defensins 1-3. Histogram represents the isotype control (dots line), healthy control (open line) and HIV-infected subject (filled histogram). Numbers indicate the percent of positive cells for healthy control (normal number) or HIV-infected patient (bold numbers). Representative result of four different healthy controls and four HIV-infected patients. (E) Geometric mean fluorescence intensity for intracellular α-defensins 1-3 in non-infected individuals (n = 4; white box) and HIV-infected subjects (n = 4; grey box). Boxes represent interquartile ranges, horizontal lines inside each box indicate the median and whiskers indicate maximum and minimum values.

Figure 2. Levels of α-defensins 1-3 produced by imMDDC from HIV-infected individuals.

(A) Comparison between the secreted levels of α-defensins 1-3 by imMDDC from HIV-controllers (elite controllers and viremic controllers; n = 19) and HIV non-controllers (viremic non-controllers and patients with HAART; n = 20). (B) Secreted levels of α-defensins 1-3 by imMDDC from healthy non-infected (NI; n = 15), elite controllers (ELITE; n = 4), viremic controllers (VC; n = 15), viremic non-controllers (VNC; n = 11) and patients receiving HAART (HAART; n = 9). Dots indicate each patient and lines represent median ± interquartile ranges.

Figure 3. Positive correlation between secreted levels of α-defensins 1-3 by imMDDC and CD4 T cell counts.

(A) HIV-infected patients (n = 39), (B) patients not receiving HAART (n = 30), (C) controllers (elite and viremic controllers; n = 19), and (D) non-controllers (viremic non-controllers and patients with HAART; n = 20). Spearman correlation test was applied.

Figure 4. No correlation between secreted levels of α-defensins 1-3 by imMDDC and plasmatic viral load (PVL).

(A) HIV-infected patients (n = 39), (B) patients not receiving HAART (n = 30), (C) controllers (elite and viremic controllers; n = 19), and (D) non-controllers (viremic non-controllers and patients with HAART; n = 20). Spearman correlation test was applied.

Figure 5. Analysis of plasma levels of α-defensins 1-3.

(A) Plasma levels of α-defensins 1-3 detected in the different groups of HIV-infected patients, elite controllers (ELITE; n = 3), viremic controllers (VC; n = 10), viremic non-controllers (VNC; n = 5) and patients receiving HAART (HAART; n = 9). Dots indicate each patient and lines represent median ± interquartile ranges. (B) Correlation between CD4 T cell counts and plasma levels of α-defensins 1-3. (C) Correlation between PVL and plasma levels of α-defensins 1-3. (D) Plasma levels of α-defensins 1-3 in two different determinations over time in patients that remained controllers (VC-VC; white boxes; n = 13), patients that were controllers in the first determination but lost viremic control in the second one (VC-VNC; grey pointed boxes; n = 5), controller patients in the first determination with disease progression and HAART in the second one (VC-ART; grey boxes; n = 8) and patients receiving HAART during both determinations (ART-ART; grey lined boxes; n = 9). Boxes represent interquartile ranges, horizontal lines inside each box indicate the median and whiskers indicate maximum and minimum values. Mann-Whitney test was applied for the statistical analysis of every pair and the differences were not significant (NS).

Figure 6. Levels of α-defensins 1-3 secreted by imMDDC were associated with indicators of disease progression.

Patients without treatment at the moment of blood extraction for the differentiation of MDDC were divided in two groups according to their secreted levels of α-defensins 1-3 by imMDDC. Levels higher than the median (650 pg/ml) of the whole group of patients (black squares; n = 18) and secreted levels lower than the median (grey circles; n = 12). (A) Percentage of patients with a decrease in the number of CD4 T cells below 350 cel/mm³ during follow up time (months). (B) Percentage of patients with an increase in PVL greater than 0.5 logarithms and (C) percentage of patients without HAART at the moment of determination of α-defensins 1-3 levels but started treatment later during the follow up. P values and hazard ratio (HR) with 95% confidence interval values are represented in the figure. Log-Rank test was used to determine statistical differences between curves.