Differential expression of Fas receptors (CD95) and Fas ligands (CD95L) in HIV infected and exposed uninfected children in Cameroon versus unexposed children

Introduction The number of HIV exposed uninfected (HEU) infants is increasing as vertical transmission is reducing. This subpopulation requires more investigations. This study aimed at comparing the expression level of soluble Fas receptors (FasR) and ligands (FasL) between HIV infected, HEU and unexposed children. Methods Eighty eight HIV-1infected, 86 HEU and 38 HIV unexposed children were recruited. Soluble FasR and FasL were measured in their plasma. Mann-Whitney U-Test was used to compare groups with 95% confidence. Spearman coefficient was used to test the correlation with CD4 and viral load (VL). Results Overall plasma levels of FasR were higher than that of FasL. The concentration of FasR and FasL were significantly higher in HIV-1 infected children in comparison to HEU and unexposed children. There was no difference in the plasma level of FasL in HIV infected compared to HEU children. A significant difference was observed between HIV infected children and HEU children (P=0.001) for the FasL. FasR were higher in both HIV infected and unexposed children compared to HEU children. There was a positive correlation (rs=+0.4; p=0.01) in ARV treated children between CD4 count and FasL concentration. Significant negative correlation (rs=-0.3; p=0.040) in ARV naïve children was observed between CD4 percentage and FasL. Significant and positive correlation (rs=+0.4; p=0.008) was observed between the VL and FasL in HIV infected, treated or not. Conclusion HEU children differ from HIV infected and unexposed children as the level of FasL/R expression is concerned. HEU should be considered different from HIV unexposed although exempt from virus as some immune dysfunctions have been reported among them.


Introduction
The AIDS epidemic has harshly affected child mortality all over the world but intervention to prevent mother to child transmission has successfully reduced the risk of HIV transmission to < 1% in resource rich settings [1]. In sub-Saharan African regions, people living with HIV/Aids account for 70% worldwide [2]. Most of children living in this part of the world are infected via their HIV-positive mothers during pregnancy, childbirth or breastfeeding [2]. Some of them remained asymptomatic for several years despite the absence of therapy [3], unfortunately 50% of them die before their second anniversary [4]. Despite the greatest success of ART in HIV infection history, prevention of mother to child transmission remains an important challenge especially in sub-Saharan African countries where HIV-exposed uninfected (HEU) infants are more susceptible to morbidity and mortality due to many infectious mechanisms than their unexposed peers [5]. HEU infants represent a growing population in pediatric, they can represent nearly 30% of the newborn population in some endemic nations [6]. Even if these infants are not infected, they are affected by the virus and by the treatment receive by their mother during pregnancy to prevent transmission [7]. Infection caused by the Human immunodeficiency virus type 1 (HIV-1) is characterized by a progressive and severe depletion of CD4+ T lymphocytes [8]. This phenomenon is observed in both adult and children. In adults, T cell immune activation appears to be higher in sub-Saharan Africa than in industrialized countries [9]. The understanding of the HIV-1 disease pathogenesis requires an appreciation of mechanisms involved in the loss of CD4+ T cells.
Apoptosis, one type of a programmed cell death, plays an important role in the mechanisms of HIV/AIDS infection. One apoptotic pathway is the Fas mediated mechanism during activation induced cell death (AICD) system, a key regulator of the process in normal and malignant T cells [10,11]. Previous studies carried out on adults in Cameroon, a country characterized by a high genetic variability of HIV virus have shown the increase in plasma levels of Fas receptors (FasR) and Fas ligands (FasL) in HIV-positive compared to HIV negative individuals [12]. To date, a number of studies have shown evidence of various immunological abnormalities among HEU children [13], but only few are documented on the expression level of Fas in Sub Saharan African pediatric context particularly in HIV-exposed uninfected children. This study aimed firstly, to evaluate the level of Blood collection: five milliliters of blood were collected from each child included in the study. Part of whole blood was used for CD4 count and the rest was centrifuged at 1,000 g for 10 minutes, plasma was aliquoted and stored at -20 o C for further analyses. Statistical analysis: data were analyzed using GraphPad prism version 6. Plasma levels of both Fas receptors and Fas ligands in HIV infected treated or naïve infants and in HIV negative, exposed uninfected and unexposed uninfected group, were compared using the Mann-Whitney U-Test. Correlation in each group of children between CD4+ cell counts and viral load was assessed using Spearman test, and a P-value of less than 0.05 was considered statistically significant.

Results
The overall population consists of 212 children aged from 9 months to 15 years with 112 girls (52.83%) and 100 boys (47.16). Our study population includes 88 HIV-1 infected children aged 1 to 15 years, mean ± SD (6.70 ± 3.23), 86 HIV exposed uninfected (HEU) children aged 9 months to 13 years, mean ± SD (5.53±2.01) and 38 healthy children as control group aged from 1 to 15 years, mean ± SD (7 ± 2.24). The description of the study population is summarized in Table   1. Plasma concentrations of Fas receptors were higher than that of  Table 2.

Discussion
The current study aimed at measuring and evaluating the impact of soluble Fas receptors and ligands in HIV/AIDS infected and exposed non-infected children and the correlation with CD4+ cell depletion and HIV-1 RNA load in the sub-group of those infected. Our results showed that the expression level of FasR was higher than that of FasL. Fas L or CD95L is an innate immune factor whose binding with  [13,14]. These two immune activation markers have been shown to correlate with HIV-1 disease progression in adults and children [15]. Massive CD4+ T cell depletion in acquired immunodeficiency syndrome (AIDS) patients may involve the death Fas receptor, but this role can also be played by other death receptors, such as the TNF and TRAIL receptors, using the same ligand ; explaining while the concentration of ligands was lower than that of receptors [16]. The activity of Fas L and Fas R can explain partly the depletion of CD4 but not fully. This result is contradictory to the findings of Sieg et al, 1997, where they showed deficiency of FasL activity on the PBMC surface of HIV infected patients [17].
There was no statistically significant difference in the concentration of FasL between the group of HIV infected children and that of HIV exposed and uninfected children (HEU). It has been shown that HIV upregulates the expression of FasL [18]. In utero, HEU children are exposed to the virus for average 9 month, so as their infected peers.
This implies that the immune system of the HEU children is as well activated like that of HIV infected children. In addition, HEU as well as infected infants during their early life, have been exposed to ARV treatment which impacts them negatively by altering their immune system, resulting in activation [19]. They are as well exposed to cotrimoxazole as recommended by WHO, given the increased burden of morbidity and mortality in this specific group of children, particularly from pneumonia [20,21]. The expression level of FasR in the exposed non-infected children was the lowest compared to control or HIV infected children. It has been shown that peripheral blood lymphocytes expressing FasR were more elevated in HIVinfected individuals than HIV-negative controls [22]. Low expression of FasR in HEU may be due to the elimination of the exposing agent or a bias due to low sample size or to the decrease of this FasR after exposition, transient, observed in early infancy, but no longer present later in life [23]. Further studies will be needed to elucidate on this value [25], comparable to what was found in the current study. These findings on adults versus infants, just agree with the fact that there is a difference between pediatric and adults immune system and that

Competing interests
The authors declare no competing interests.

Acknowledgments
We are grateful for the parents and guardians of children enrolled in the work.      rs= Spearman's correlation at 95% confidence interval (CI), VL= Viral load in ART-treated and ART-naives children