Increased neutrophil apoptosis in chronically SIV-infected macaques

Polymorphonuclear neutrophils (PMN) from chronically HIV-infected individuals have been reported to be more prone to die. However, although non-human primates models have been extensively used for improving our knowledge on T cell immunity, the impact of SIV-infection on PMN, in relationships with disease severity, has never been assessed. In our study, we demonstrate that PMN from Rhesus macaques (RMs) of Chinese origin chronically infected with the virulent strain SIVmac251 display increased susceptibility to undergo apoptosis as compared to PMN from RMs infected with the non-pathogenic SIVΔnef strain. PMN apoptosis was significantly increased in RMs progressing faster to AIDS as compared to non-progressors RMs. Furthermore, the percentage of apoptotic cells correlated with PMN activation state reflected by increased CD11b expression and reactive oxygen species production. Interestingly, whereas inflammatory cytokines IL-8 and IL-1β prevent in vitro PMN death, the levels of those cytokines were low in RMs progressing towards AIDS. Altogether, increased PMN death during SIV infection is a new pathogenic effect associated with AIDS progression, adding to the long list of markers associated with disruption of defense against infection.


Findings
Polymorphonuclear neutrophils (PMN) are key components of the first line of defense against pathogens [1]. PMN are terminally differentiated cells with a short life span; they die spontaneously by apoptosis and are then recognized and phagocytosed by macrophages [2]. Shortened PMN survival due to apoptosis may explain susceptibility to severe and recurrent infections in some pathological situations [3,4].
It has been reported that PMN functions are impaired in the latter stages of HIV infection [5]; increased PMN apoptosis has also been observed in HIV-infected patients having less than 200 CD4 + cells/mm 3 [6][7][8][9][10][11]; however, the introduction of HAART has reduced spontaneous PMN apoptosis. Several lines of evidence suggest a key role of PMN, at least through defensin expression, in controlling viruses other than HIV or SIV [12][13][14]. In addition, human neutrophil α-defensins 1-4 have been reported to inhibit HIV-1 replication in vitro [15][16][17], and activated neutrophils have been demonstrated to exert cytotoxic activity against HIV-infected cells [18].
The use of non-human primate models, particularly SIVinfected Rhesus macaques (RMs), has allowed the detailed and sequential investigation of the events of SIV infection in terms of virus dynamics, immune response, and changes in the pool of CD4 + cells [19]. Once the setpoint phase is reached, the level of viral load predicts the rate of progression to AIDS [20][21][22]. Thus, SIVmac infection of RMs has proved to be an invaluable animal model for studies of AIDS pathogenesis, therapeutics, and vaccines. In particular, we and others have demonstrated that RMs of Chinese origin is a particularly relevant model to study human diseases [23][24][25][26][27][28]. Paradoxically, no studies have investigated, in SIV-infected RMs, possible PMN dysregulation, especially the propensity of PMNs to die, in relationships with disease severity.
All the animals were challenged with the same batch of virus, titrated in vivo in rhesus macaques, and were followed post-infection and studied 8 months later. RNA was extracted from plasma of SIV-infected monkeys, using the TRI REAGENT BD Kit (Molecular Research Center Inc., Cincinnati, Ohio). Real-time quantitative reverse tran-scriptase-polymerase chain reaction (RT-PCR) was used to determine viral loads as previously described [22]. Among SIV + macaques, slow progressors (n = 6) and moderate progressors (n = 5) were defined according to plasma viral load (<10 3 copies/ml and 10 3 -10 5 copies/ml, respectively), that predicts further disease progression in RMs of Chinese origin [20,21]. In contrast, in SIVΔnef-infected monkeys, plasma viral load was always lower than 1.5 × 10 2 copies/ml. Moreover, as shown in Table 1, CD4 + T cell counts were significantly decreased in moderate progressors as compared to SIV + slow progressors, SIVΔnef or healthy (SIV -) macaques consistent with previous reports [20,21].
To quantify PMN apoptosis, samples were analyzed directly on the whole blood cooled to 4°C to avoid nonspecific activation due to isolation procedure [29,30]. PMN apoptosis was measured after 4 hours of incubation in 24-well tissue culture-plates at 37°C with 5% CO 2 . Apoptosis was quantified by allophycocyanin (APC)-conjugated annexin V and 7-amino-actinomycin D (7-AAD) staining as previously described [29,30]. Whole blood samples (100 μl) were washed twice in PBS, incubated with FITC-anti-CD14 and PE-anti-CD11b monoclonal antibodies (mAbs) for 15 minutes, and then incubated with APC-annexin V for 15 minutes. After dilution in PBS (500 μl) the samples were incubated with 7-AAD at room temperature for 15 minutes and analyzed immediately by flow cytometry. PMN were identified as CD11b + CD14 low cells ( Figure 1A and 1B).
After 4 hours of incubation at 37°C, PMN apoptosis was significantly increased in SIV + macaques relative to SIVΔnef and healthy controls ( Figure 1D). The fact that SIVΔnef macaques showed reduced susceptibility to apoptosis as compared to SIV + animals is in keeping with a previous report that the apathogenic strain is associated with milder immune dysfunction and has a lower plasma viral Both CD11b expression ( Figure 2A) and ROS production ( Figure 2B) of resting PMN (maintained at 4°C) were significantly increased in both slow and moderate progressors relative to healthy and SIVΔnef controls. Furthermore, PMN from moderate progressors exhibited increased CD11b expression and ROS production as compared to slow progressors. Interestingly, the percentage of apoptotic cells correlated with basal PMN activation status (ρ = 0.69, p = 0.01 and ρ = 0.71, p = 0.01, for CD11b expression and ROS production, respectively  bone marrow lymphopoiesis results in the specific and reciprocal expansion of the granulocytic compartment in bone marrow [39]. Our results showed that, at 8 months post-inoculation (p.i.), the extent of PMN apoptosis is higher than that observed at 2 months p.i., while the levels of viral replication remain quite similar [30]. In addition, during the acute phase, the levels of 10 7 copies/ml in RMs infected with the pathogenic strain is associated with PMN death; interestingly in RMs infected with the live attenuated Δnef strain, despite a viral load of 10 5 copies/ml at the peak (day 14 p.i.), no PMN death was observed [30]. This level of viral replication corresponds to that observed during the chronic phase. Altogether, these data suggest that, while a certain threshold of viral particles is required for a direct effect on cell death, extracellular factors could participate in PMN dysregulation. Because it has been shown that inflammatory cytokines inhibit PMN apoptosis [40,41], we then determined in the plasma the amount of IL-8, TNF-α, and IL-1β. Blood was collected in sterile EDTA-treated vacuum tubes and immediately centrifuged at 400 g for 15 minutes at 4°C. IL-8, TNF-α, and IL-1β were detected simultaneously by using the human inflam-matory cytokine cytometric bead array (CBA) kit (BD Pharmingen), which has been validated for cytokine measurements in RMs after Toll-like receptor (TLR) stimulation (data not shown).
Firstly, we found that IL-8 and IL-1β plasma levels were significantly lower in SIV + moderate progressors as compared to SIV + slow progressors and SIVΔnef macaques ( Figure 3A). Secondly, pre-incubation of whole blood samples from moderate progressors with IL-8 (100 ng/ml) or IL-1β (100 ng/ml) for 4 hours significantly reduced PMN apoptosis as compared to samples incubated with PBS [percentage of annexin V + cells: 8. macaques. The absence of detection of TNF-α in chronically SIV-infected rhesus macaques is consistent with a recent report [42]. Indeed, the authors did not observe increased levels of pro-inflammatory cytokines despite increased levels of plasma LPS. Thus, the absence of inflammatory cytokines might lead to an abnormal tendency of PMN to die.
Finally, one consequence of such abnormal PMN apoptosis could be to facilitate the dissemination of SIV/HIV in vivo by modulating immune responses. Apoptotic cells are sources of biologically active oxidized phospholipids which serve as recognition signals on apoptotic cells, facilitating phagocytosis by macrophages [43]. Engulfment of apoptotic PMN has been shown to inhibit the production of pro-inflammatory mediators by macrophages, through the secretion of anti-inflammatory cytokines such as TGFβ [44]. In this context, we recently demonstrated that TGFβ is increased in the tissues of SIV-infected RMs [25]. Such anti-inflammatory events can inhibit antigen presentation and promote microbial growth within macrophages [45], HIV replication [46], as well as the expansion of IL-17producing cells [47].
In conclusion, our data demonstrate for the first time, to our knowledge, in SIV-infected macaques abnormal PMN deaths that increased in monkeys progressing faster to AIDS. This abnormality might therefore participate in the general immune defect leading to clinical outcomes in SIV infection.