Apoptosis characterization in mononuclear blood leukocytes of HIV patients during dengue acute disease

Dengue virus (DENV) co-circulation in Brazil represents a challenge for treatment and vaccine development. Despite public health impact, the occurrence of coinfections with other viruses is a common event. Increased T cell activation and altered inflammatory response are found during DENV coinfection with Human Immunodeficiency Virus (HIV) impacting HIV-pathogenesis. Even with Antiretroviral therapy (ART), HIV- treated patients had chronic immune activation and lymphocyte apoptosis. However, apoptotic mechanisms have not been investigated during coinfection with DENV. Our attention was attracted to apoptotic cell markers expressions in PBMCs from DENV and DENV/HIV coinfected patients. We found CD4/CD8 ratio inversion in most coinfected patients. CD4 T and CD8 T-cell subsets from DENV and DENV/HIV groups expressed low levels of anti-apoptotic protein Bcl-2. Furthermore, CD8 CD95 double positive cells frequency expressing low levels of Bcl-2 were significantly higher in these patients. Additionally, the density of Bcl-2 on classical monocytes (CD14++CD16−) was significantly lower during DENV infection. Upregulation of pro-apoptotic proteins and anti-apoptotic proteins were found in DENV and DENV/HIV, while catalase, an antioxidant protein, was upregulated mainly in DENV/HIV coinfection. These findings provide evidence of apoptosis triggering during DENV/HIV coinfection, which may contribute to knowledge of immunological response during DENV acute infection in HIV-patients treated with ART.

106) from patients and controls were thawed in water bath at 37 °C, centrifuged (350 g, 5 min) and washed once with 1 mL of PBS pH 7.4 supplemented with 2% FCS and 0,01% NaN3. Thawed cells were stained with panels comprising the antibodies listed above for flow cytometry analysis. Cells were labeled extracellularly and intracellularly as described previously 13 . Briefly, after extracellular stain with cocktail surface markers, cells were washed in Perm/Wash for 15 min and then were incubated with Cytofix/Cytoperm solution for 20 min at 4 °C (BD biosciences). Cells were washed again in Perm/Wash and stained for 30 min with PE conjugated anti apoptotic Bcl-2 protein mAb. About 1 × 10 5 events were acquired in the lymphocyte and monocyte gates. Data acquisition and analysis were performed using FACS Aria IIu and analyzed using FlowJo software version 10 respectively.
Expression profile assay of proteins with apoptotic and anti-apoptotic functions. Apoptosisrelated proteins expression profile was analyzed using Human Apoptosis Array Kit (R&D Systems). PBMCs lysates were obtained from DENV monoinfected, HIV monoinfected and DENV/HIV coinfected, as well as HD. The lysate volume was adjusted for 250 μL/array, according to the manufacturer protocol. Two kits, both containing 4 nitrocellulose membranes each with 35 different anti-apoptosis antibodies printed in duplicate, were performed. After washes and immunodetection antibodies procedures, each membrane was treated with chemiluminescent reagents, covered with plastic wrap and exposed to X-ray film for 5-10 minutes. The positive signals on developed film were identified by placing the transparency overlay on the array image and aligning it with the three pairs of positive control spots in the corners of each array. Apoptosis array data on developed X-ray film were quantified by scanning the film on a transmission-mode scanner and the array image file was evaluated using image analysis software quantity One (Bio-Rad Version 4.6.3). A template analyze pixel density in each spot of the array was created and subtraction of averaged background signal from each one performed. Data were export to Microsoft Excel and the average signal (pixel density) of the duplicate spots pairs, representing each apoptosis-related protein, calculated. Corresponding signals of each duplicate spot were determined and compared to analyses changes in apoptosis-related protein levels between controls and patient samples. Finally, normalization of the signal intensity based on positive and negative controls values were performed. Statistical analysis. Statistical analyses were performed by Anova analysis. Kruskal wallis and Dunn's multiple comparison tests were used for all pairs of groups using GraphPad P sofware (version 6). P values < 0.05 were considered statistically significant. A two-dimensional heat map with hierarchical clustering was built, with the expression of the apoptotic markers in the Y dimension and the HIV, DENV and DENV/HIV groups in the X dimension. The cluster for protein markers was constructed using the Euclidean distance between the medians of the scaled values, and Ward as a linkage algorithm. We ran 1000 bootstrap replications to verify the clustering support. We constructed a Principal Component Analysis (PCA) to verify the distribution pattern of the studied analytes among the individuals. This technique reduces the dimensionality space to reveal the analytes that contribute more to a group profile differentiation. Each Principal Component (PC1 and PC2) represents the total variation (%) explained the distribution of the individuals along with the multidimensional space. In the PCA plot, the individuals are represented as colored points, and the variables are drawn as arrows. The length of each arrow is proportional to the contribution of each variable to the overall variance. In this plot, the angles formed by two arrows (variables) represent the correlation between them and the closer the angle, the higher the correlation between them from orthogonal, independent variables, to more collinear, highly correlated pairs. Cluster and bootstrap analyses were performed using packages gplots 16 and pvclust 18 respectively, and PCA analysis was performed using vegan 19 , all of them in the statistical environment 17 .
There were no significant statistical differences in age, sex or other signs/symptoms between groups of patients analyzed. The Dengue infected and coinfected patients enrolled presented fever accompanied by one or more signs/symptoms such as myalgia, arthralgia, exanthema, headache, prostration, pruritus, conjunctival hyperemia, edema, nausea, vomiting, and retro orbital pain. Dengue monoinfected patients were classified according to the latest WHO classification 1 . Of these, 20 were classified as DwoWS, 17 as DwWS, and 6 as SD. According to groups mono or coinfected, we observed that 6 DENV monoinfected presented DwoWS, 9 patients DwWS and 5 were classified as SD. With respect to DENV/HIV coinfected patients, 14 had DwoWS, 8 DwWS and only one were classified as SD. Regardless mono DENV or DENV/HIV coinfected, the main warning signs presented by infected patients were abdominal pain, mucosal bleeding, liver enlargement and increased hematocrit concomitant with decreased platelets counts. Severe patients presented persistent abdominal pain, followed by uncontrollable vomiting, severe bleeding and severe plasma leakage. Most patients infected with dengue had a positive IgG reaction suggesting a previous heterologous DENV infection. According to laboratorial parameters, DENV monoinfected patients presented low platelet counts compared to HIV (p < 0.01) and high ALT levels compared to DENV/ Characterization of CD4 and CD8 T lymphocytes subsets from DENV and DENV/HIV coinfected patients. The frequencies of CD4 and CD8 T-cell subsets were analyzed by flow cytometric using specific monoclonal antibodies. Figure 1A shows gating strategy for analysis of T -cell subsets. The median of CD4 T absolute counts is lower in DENV/HIV coinfected patients as compared to HIV (p < 0.05). Additionally, DENV/ HIV coinfected patients had a higher CD8 T percentages and low CD4/CD8 ratio compared to DENV group (p < 0.05) ( Table 1).

DENV and DENV/HIV coinfected patients present a higher frequency of T lymphocytes expressing Fas/CD95 death receptor.
We have already shown that DENV infection induces up regulation of death receptor Fas/CD95 in both CD4 T and CD8 T-cell subsets 13 . In order to evaluate whether Fas/CD95 cell surface receptor plays a role during coinfection, we compared Fas/CD95 expression in T-cell subsets from different groups (Fig. 1B) (Fig. 1D). In addition, the median fluorescence intensity (MFI) of Fas/CD95 on CD4 and CD8 T-cell subsets in coinfected patients was significantly higher than in HD (Fig. 1E,F). CD4 T cells expressing Fas/CD95 are inversely associated with CD4 T cell count in DENV monoinfection (r = 0.5, p = 0.0403).

Ex vivo Bcl-2 levels are greatly reduced in DENV monoinfection and DENV/HIV coinfection. Studies from HIV infected patients demonstrated down-regulation of the anti-apoptotic protein Bcl-2
(Bcl-2 low ) on CD8 T cells making then susceptible to apoptosis in vitro. Most CD8 T cells from non-HIV infected individuals expressed homogeneous (intermediary or normal) levels of Bcl-2 20 . Besides, activated CD4 T and CD8 T cells expressed low levels of Bcl-2 during dengue infection as demonstrated previously by our group 7,13 . In this way, this prompted us toinvestigate whether coinfection could influence the levels of Bcl-2 protein expression in T-cell subsets. Figure     www.nature.com/scientificreports www.nature.com/scientificreports/ Up regulation of apoptotic and anti-apoptotic proteins suggests cell death modulation of pBMcs in HiV treated patients at acute Dengue infection. The analysis of apoptosis-related proteins expression profile is essential for understanding signaling molecules roles and their involvement in programmed cell death mechanisms during disease states. To investigate changes in apoptosis-related proteins, we evaluated the relative expression levels of 35 apoptosis-related proteins simultaneously detected in a single sample. We assessed the following representative samples: heathy donor -HD n = 2; DENV mono-infected patients n = 8; DENV/HIV coinfected patients n = 3 and HIV infected controls n = 2. The results allowed us to compare those different groups and estimate wich proteins would have changed their expression. Among the 35 apoptosis -related proteins analyses, 13 (37%) reached very lower density values and were not evaluated. However, the other 22 (62.8%) proteins had their expression increased or decreased.
Our study failed to demonstrated statistically significant differences in most proteins analyzed probable due small sample size. However, data analysis of pro-caspase 3 demonstrated an increased expression in DENV monoinfected compared with HD [HD: 39 (10-69) n = 2; DENV 125.9 (101-136) n = 8; median-minimum/ maximum; Kruskal-Wallis test with Dunn's multiple comparisons test p > 0,05] (Fig. 4A). Caspases are divided into two groups, the initiators and the effectors. They are expressed in inactive form (pro-caspases) and, when activated, (cleaved caspase) are involved in early and late regulatory apoptosis events. Effector caspase 3 is proteolytically activated in a cascade leading to cell disintegration 3 . No statistically significant differences were found in cleaved caspase 3 among groups, although the trend is toward increased medians in DENV monoinfected compared to coinfected [DENV 72.6 (39-105) n = 8; DENV/HIV: 51.2 (9.5-58.9) n = 3; median-minimum/ maximum; Kruskal-Wallis test with Dunn's multiple comparisons test p = 0,05].
The oxidative stress production caused by the reactive oxygen species (ROS) accumulation is another manner of apoptosis induction 23  As previously mentioned, no significant differences in the level of most apoptotic proteins were found between studied groups, probable due small sample size. However, we built a two-dimensional heat map with hierarchical clustering that grouped the expression of the apoptotic-cell markers in Y dimension and segregated the HIV, DENV, DENV/HIV and HD groups in the X dimension (Fig. 5). Additionally, to confirm the grouping and to determine the cluster robustness observed in Fig. 5, we ran a bootstrapped clustering (n = 1000 replicates). Here, we confirmed that separated clusters in the image had support ( p < 0.05). As observed in Fig. 5 A principal component analysis (PCA) was performed to evaluate the grouping of each individual from the studied groups in a multidimensional space. The PCA1 explained 69.77% of the variation. The variables with more contribution for the separation along PCA1 were ciAp-2, Bcl-2, XIAP, Bcl-x, FADD, ciAP-1, and TRAIL-2. Along the PCA2 the analytes that contributed more were Bad, Bax, and Pro caspase 3 (see Supplementary  Table S1 and Supplementary Fig. S1). Along the PCA1 dimension we observed a separation of all individuals from DENV/HIV and HIV groups. One of the HD presented low levels of Bad, Bax and pro caspase 3 (seen as the point opposed to the Bad, Bax and pro caspase 3 arrows along X axis). Two individuals of group monoinfected by DENV presented similar responses to DENV/HIV and HIV groups.

Discussion
Dengue and AIDS are considered public health emergency with higher incidence of morbidity and mortality. Brazil has been plagued by a triple epidemic caused by arboviruses (Dengue, Zika and Chikungunya) of worldwide impact 25 . In this scenario, DENV and HIV coinfection is a reality and it is unclear whether HIV infection could increase DENV severity. Literature data indicate that most coinfected patients had mild outcomes of dengue. Corroborating, the present data demonstrated that most coinfected patients had mild symptons confirming our previous data 14 . More recently, Hotzz et.al., reported that DENV infection in ART-treated HIV patients was associated with reduced vascular instability and liver damage 26 . However, coinfected patients could have severe dengue outcomes 27 .
In this study, quantitative and qualitative changes in T cells and monocytes subsets during DENV infection in HIV treated patients were evaluated. We found significantly decrease in CD4 and CD8 T-cell subsets confirming earlier results with infected Brazilian patients 28 . Interestingly, decreased CD4/CD8 T ratio and increased in the CD8 T percentages were observed in coinfected patients as compared to DENV infected. The ART era enable immune restoration and improved AIDS related and non-AIDS related morbidity as well as mortality turning HIV/AIDS into chronic disease. Despite ART effectiveness, late ART initiation is a Brazilian challenge [29][30][31] . A significant number of individuals start HIV/AIDS care at later stage of infection (later presenters) and usually present low T CD4 counts even with complete viral suppression. Several factors are associated with T CD4 recovery including age, specific ART regimens, comorbidities and coinfections 32  www.nature.com/scientificreports www.nature.com/scientificreports/ considered as a biomarker for treated HIV patients 33 . It is not clear whether altered CD4/CD8 T ratios could lead to dengue severity in ART treated HIV patients. However, besides decreased CD4/CD8 ratios, DENV/HIV coinfected patients evolved with a good prognosis.
Studies demonstrated the occurrence of massive apoptosis in lymphocytes during the acute phase of DENV infection 34,35 . In agreement with this, we found that frequencies of CD4 and CD8 T cells expressing Fas/CD95 were increased during DENV and DENV/HIV coinfection. Furthermore, Bcl-2 levels on T cells were greatly reduced in DENV and DENV/HIV coinfected compared to healthy individuals. More importantly, the Fas/CD95 upregulation on both CD4 and CD8 T cells expressing low levels of Bcl-2 were increased during DENV infection and also in CD8 T cells of co-infected patients suggesting apoptotic events on T lymphocytes. In fact, we found an inverse correlation between CD4 T absolute counts and CD4 T cells expressing Fas/CD95 in DENV monoinfection. During the extrinsic apoptotic pathway, the interaction between death receptor Fas/CD95 agonist and its natural ligand FasL promotes the recruitment of adapter proteins that in turn interact with caspase 8 to trigger the initial step of apoptosis 36 . We previously described phosphatidylserine exposure and DNA fragmentation on T lymphocytes besides increased Fas/CD95 death receptors expression and decreased Bcl-2 expression in naturally DENV infected patients 13 . Moreover, DENV-specific CD8 T cells were susceptible to apoptosis as demonstrated by Bcl-2 downregulation, Fas/CD95 upregulation and DNA fragmentation 37 . CD4 T cell depletion is more pronounced in coinfected patients suggesting that an apoptotic extrinsic pathway may be contributing to the T lymphocyte depletion. Persistent coinfection between GB virus C (GBV-C) and HIV lead to a slower disease progression and mortality in HIV infected patients 38 . Fas/CD95 expression was lower in GBV-C and non-treated HIV coinfected patients suggesting that reduced Fas/CD95-mediated apoptosis of T cells might be responsible for beneficial effect of GBV-C coinfection. Moenkemeyer and colleagues suggested that GBV-C inhibit host cell apoptosis and consequently maintain chronic GBV-C infection. In agreement with our study, Fas/CD95 expression on T cells were not significantly different in patients receiving ART and degree of Fas/CD95-expressing lymphocytes was comparable between GBV-C co-infected and GBV-C infected 39 . A recent study demonstrated that apoptosis of platelets were similar between DENV infected and coinfected with HIV as evidenced by phosphatidylserine exposure, mitochondrial membrane potential and caspase -9 activation 26 .
Blood monocytes have been divided into 3 subsets based on relative expression of CD14 and FCγIII receptor CD16 on classical monocytes (CD14 ++ CD16 − ), intermediate (CD14 ++ CD16 + ) and non-classical monocytes (CD14 + CD16 ++ ). CD14 ++ CD16 − classical monocytes presented high phagocytic activity and they are critical for initial inflammatory responses while CD14 ++ CD16 + intermediate monocytes are associated with inflammatory response, and CD14 + CD16 ++ non-classical are considered as patrolling monocytes 21 . Monocyte activation has been reported during DENV and HIV infections and they are associated with disease pathogenesis 22,40 . Our results demonstrated significant changes of monocyte subsets during DENV and DENV/HIV coinfection. As demonstrated previously 41,42 , we found that in the acute DENV infection, CD14 ++ CD16 − classical monocytes www.nature.com/scientificreports www.nature.com/scientificreports/ were significantly decreased while intermediate were increased. Similarly, an expansion of intermediate monocytes and a reduction of classical monocytes during coinfection were found. Monocytes play important roles in the innate and adaptive response and are key players in mediating anti-DENV immune responses. They are the main target cells of DENV infection and after activation produce pro-inflammatory mediators involved in dengue severity 11 . Perturbations of monocyte subsets were also found in treated HIV patients 43, 44 . In agreement we observed that some HIV treated individuals presented increased percentages of intermediate and non-classical monocytes suggesting chronic immune monocyte activation despite virus suppression after ART. More importantly, we report, for the first time (to our knowledge) that DENV/HIV coinfected patients showed increased frequencies of CD14 ++ CD16 + intermediate monocytes. Interestingly we found that, in the context of DENV infection, the MFI of Bcl-2 on CD14 ++ CD16 − classical monocytes were significantly lower as compared to DENV/HIV coinfection. Activation of apoptosis pathway by DENV infected target cells have been described previously 11 . Our group and others have previously shown that DENV is capable of apoptosis induction in infected monocytes by extrinsic and intrinsic pathways 12,45 . Is unknown whether apoptosis could occur as directed mechanism of viral dissemination and evasion or simply represents an appropriate host response to limit virus replication. More recently, it was demonstrated that a pro-survival protein, Bcl-xl, is involved in the survival of DENV, Zika virus (ZIKV) and Japanese encephalitis virus (JEV) HuH7 infected cells. Suzuki and colleagues proposed that Flavivirus infection induces delayed cell apoptosis and consequently virus spread to neighboring cells leading to higher viral loads and disease severity. Bcl-xL inhibition accelerates apoptosis leading to enhanced viral clearance through macrophage phagocytosis 46 .
Screening of 35 apoptosis-related proteins expression on PBMCs of study groups showed upregulation of pro caspase 3 and cleaved caspase 3 in DENV monoinfected group. Additionally, pro-apoptotic proteins involved in extrinsic apoptosis (Fas/CD95, TRAIL1, TRAIL2 and FADD) were down regulated in DENV/HIV and HIV, whereas these molecules were not downregulated in DENV infected. Our study confirmed extrinsic apoptotic death receptor Fas/CD95 and Bcl-2 family involvement in apoptosis regulation during DENV infection. However, other regulatory molecules might be involved in apoptosis of activated cells during infection. TRAIL appears to be capable of apoptosis inducement on T lymphocytes in DENV infected patients 35 . Matsuda et al. (2005) show that hepatocytes express TRAIL-RII and produce soluble TRAIL during DENV infection, undergoing apoptosis 47 . Endothelial cells infected by DENV-2 showed decreased TRAIL expression suggesting evasion of TRAIL induced apoptosis 48 . Taken together, these results indicate that differential expression of apoptotic proteins observed in dengue monoinfection may be involved in apoptosis susceptibility during infection as reported previously 11 .
Our results indicate that pro-apoptotic proteins (Fas/CD95, TRAIL1, TRAIL2 and FAD), anti-apoptotic Bcl-2, Bcl-x) and IAPs (ciAP1 ciAP2, XIAP) were downregulated in DENV/HIV and HIV. It has been reported that HIV encoded proteins induce pro-and anti-apoptotic activities 49,50 . On the other hand, ART reduce direct cytopathic effect of viral proteins, immune responses to viral antigens and decreases spontaneous T cell apoptosis in successfully treated individuals 51 . PI as well as NRTI regimens induce intrinsic anti-apoptotic activity and consequently reduce lymphocyte apoptosis 52,53 . Indeed, most HIV patients of our study received PI and NRTI therapies. Although a reduction in immune activation and apoptosis can be observed after initiation of ART therapy 54 , viral latent reservoirs could support immune cell activation and CD4 T cell apoptosis 55 . In this context, we observed that Bad and Bax were upregulated not only in DENV monoinfection but also in DENV/HIV coinfection.
Smac/DIABLO and michocondrial serine protease HtrA2/Omi are reported to promote apoptosis by inhibiting IAP activity 56,57 . The IAP family of proteins are important regulators of both intrinsic and extrinsic apoptosis pathways 58 . The XIAP and survivin remain the better-known members of this family 59 . Our data also suggested an opposite balance between an increase of those apoptotic proteins and downregulation of IAPs proteins expressions during coinfection.
Of particular interest were catalase antioxidant protein, found to be significantly upregulated in DENV/HIV coinfection. Reactive oxygen species (ROS) accumulation generate oxidative stress and induce apoptosis by increasing the collapse of mitochondrial membrane electric potential, an important step in early apoptosis. Also, ROS react with many biological molecules, including lipids, proteins and carbohydrates, causing loss of cellular integrity and cellular dysfunctions as well. We already show that catalase, an antioxidant protein, is up-regulated in severe dengue patients, indicating a possible scenario of oxidative stress in response to infection 13 . Here, for the first time, we observed data that suggests a similar scenario during Dengue/HIV coinfection.
There may be some possible limitations in this study. Quantification of DENV viral load in the serum of coinfected patients was not possible in our study. Although, we observed that levels of circulating NS1 protein were not different between DENV monoinfected and DENV/HIV coinfected (data not shown). In fact, Hottz and colleagues demonstrated no difference in the DENV viremia between DENV infection or coinfection with HIV 26 . Unfortunately, due limited sample size we could not evaluate spontaneous apoptosis in vitro.
Our findings have to be interpreted carefully specially concerning to limited sample size. However, we demonstrated here several evidences supporting the concomitant up regulation of apoptosis in DENV/HIV coinfected patients corroborating previous data observed during DENV infection 13 . Importantly, pathogenesis of ART treated HIV patients with acutedengue disease remains largely unknown. In the present study, we report, for the first time that DENV/HIV coinfected patients show increased frequencies of apoptotic molecules on PBMCs. Further in vivo and in vitro studies are needed to evaluate the outcomes of coinfection in risk areas of Brazil, especially due co-circulation of other arboviruses of medical importance such as ZIKV and chikungunya virus (CHIKV). Further understanding about regulation of extrinsic and intrinsic apoptotic pathways during HIV coinfection is necessary to develop new therapies for both infections.