Aflatoxin-Related Immune Dysfunction in Health and in Human Immunodeficiency Virus Disease

Both aflatoxin and the human immunodeficiency virus (HIV) cause immune suppression and millions of HIV-infected people in developing countries are chronically exposed to aflatoxin in their diets. We investigated the possible interaction of aflatoxin and HIV on immune suppression by comparing immune parameters in 116 HIV positive and 80 aged-matched HIV negative Ghanaians with high (≥0.91 pmol/mg albumin) and low (<0.91 pmol/mg albumin) aflatoxin B1 albumin adduct (AF-ALB) levels. AF-ALB levels and HIV viral load were measured in plasma and the percentages of leukocyte immunophenotypes and cytokine expression were determined using flow cytometry. The cross-sectional comparisons found that (1) among both HIV positive and negative participants, high AF-ALB was associated with lower perforin expression on CD8+ T-cells (P = .012); (2) HIV positive participants with high AF-ALB had significantly lower percentages of CD4+ T regulatory cells (Tregs; P = .009) and naive CD4+ T cells (P = .029) compared to HIV positive participants with low AF-ALB; and (3) HIV positive participants with high AF-ALB had a significantly reduced percentage of B-cells (P = .03) compared to those with low AF-ALB. High AF-ALB appeared to accentuate some HIV associated changes in T-cell phenotypes and in B-cells in HIV positive participants.

However, only two studies have been conducted on the immune effects of aflatoxin in humans exposed to low levels of aflatoxin in contaminated foods. One study conducted in Gambian children reported that secretory immunoglobulin A in saliva may be reduced by dietary levels of aflatoxin [21]. We previously reported that the percentages of CD8+ T-cells that expressed perforin, or both perforin and granzyme A were significantly lower in participants with high AFB1-albumin adduct (AF-ALB) levels in plasma compared to those with low AF-ALB [22]. We also found that low levels of CD3+CD69+ and CD19+CD69+ cells were significantly associated with high AF-ALB levels. These alterations in immunological parameters in participants with high AF-ALB levels could result in impairments in cellular immunity in these 2 Clinical and Developmental Immunology individuals that could decrease their resistance to infections.
Hendrickse et al. [23] investigated the reasons for the rapid progression of human immunodeficiency virus (HIV) and acquired immune deficiency syndrome (AIDS) in heroin addicts in the Netherlands and Scotland. They found that street heroin was often contaminated with aflatoxin, and that aflatoxin derivatives were commonly found in the body fluids of the addicts. They speculated that the accelerated rate of HIV progression was due to aflatoxinrelated immune suppression, but did not undertake studies to examine this. This suggestion of synergy between aflatoxin and HIV progression is also supported by the broad correlation between estimated aflatoxin exposure and the commonly perceived faster rate of HIV progression in Africa than in developed countries in Europe or the United States of America [24,25]. The HIV pandemic is critical enough for this possibility to be investigated as a matter of urgency.
HIV infection results in impaired immune function that can be measured by changes in immunphenotypically defined lymphocyte subsets and other in vitro functional assays. The altered expression of lymphocyte surface antigens reflects the dynamic interaction between the immune system and HIV. Investigation of the effect of aflatoxin on the immune system in HIV positive individuals is urgently needed since both aflatoxin and HIV are immune suppressive and millions of people who are chronically exposed to aflatoxin in their diet in developing countries are also HIV positive. In this study, we examined the potential immune suppressive interaction of aflatoxin and HIV by measuring a broad array of immune indices in HIV-infected individuals with high and low AF-ALB levels. HIV negative participants were included as a control group.

Study design and study participants
This was a cross-sectional analysis of 116 HIV positive individuals and 80 HIV negative control participants. The HIV positive patients and HIV negative controls of comparable age were recruited from the St. Markus Hospital and surrounding communities in Kumasi, Ghana. The protocol for the study was approved by the Institutional Review Board of the University of Alabama at Birmingham (UAB) and the Medical School Ethics Committee of the Kwame Nkrumah University of Science and Technology (KNUST) and participants gave informed consent. The participants were asked to complete a survey on socio-demographic characteristics, and a 20 mL blood sample was collected from each in EDTA vacutainer tubes. Plasma was separated and peripheral blood mononuclear cells (PBMCs) were prepared using ficoll-hypaque density gradients as previously done [26]. PBMCs were stored frozen in liquid nitrogen and shipped to UAB for analysis.

Determination of AF-ALB levels in plasma by radioimmunoassay
AF-ALB levels in plasma of study participants were determined by radioimmunoassay (RIA) as published previously [27]. Briefly, human plasma samples were concentrated by high-speed centrifugal filtration using Microcon-50 microconcentrator with a 50 000 mol. wt. filter cutoff. The concentrated protein was resuspended in 100-150 µL PBS and the amount of human plasma albumin determined in each sample using a bromocresol purple dye binding method. In addition, the amount of total protein was determined by the procedure of Bradford (Pierce Biotechnology Inc., Rockford, IL). Total protein was then digested with Pronase (Calbiochem, La Jolla, Calif, USA; 70 000 proteolytic units/g dry weight was dissolved in PBS at 10 mg/mL) at a ratio of 4 : 1 (Protein : pronase) in a shaking water bath (50 strokes/min) at 37 • C for 16-18 hours. Digestion was stopped by cooling on ice. Two volumes ice-cold acetone were added and the sample mixed and allowed to remain at 4 • C for 1 hour. The suspension was then centrifuged at 11 000 rpm (9800 g) for 15 minutes. The resulting supernatant containing the bound aflatoxin was decanted and dried in vacuo using a savant speed-vac concentrator. The RIA procedure was used to quantify aflatoxin B1-albumin adducts in duplicate human plasma protein digests each containing 2 mg protein.
Nonspecific inhibition in the assay was determined by processing pooled normal human plasma standards obtained from Sigma Aldrich (St. Louis, MO, USA) and the average value of the background was subtracted from the values of test samples in calculating AFB1-albumin adduct levels. The standard curve for the RIA was determined using a nonlinear regression method [28] and values were expressed as the amount of AFB1 per mg albumin [27]. The detection limit of the assay was 0.01 pmol/mg albumin.

Determination of cytokine expressing CD8+ and CD3-CD56+ cells
CD8+ T-cell cytokine expression (perforin and granzyme A) was measured by intracellular cytokine staining and multiparameter flow cytometry. Also, the cytotoxicity potential of NK-cells was examined by detecting perforin expression in phenotypically defined NK-cells (CD3-CD56+).
For intracellular cytokine staining, PBMCs (1 × 10 6 ) were collected in dPBS and washed once with cold dPBS containing 1% BSA. Cells were then resuspended in 100 µL of staining buffer (PBS supplemented with 0.1% sodium azide and 1% FBS pH 7.4) and the phenotypic MAb (CD3, CD8, and CD56) and incubated at 4 • C for 30 minutes. After staining, the cells were washed with PBS and resuspended in 1 mL of fix/perm buffer (BD PharMingen, San Diego, Calif). The cells were then fixed for 30 minutes at 4 • C, washed, resuspended in 3 mL perm staining buffer and incubated with cytokine antibodies (antiperforin, antigranzyme A) (BD PharMingen, San Diego, Calif) in the presence of 50 uL of permeabilization buffer for 30 minutes at 4 • C. The cells were then washed with perm buffer and resuspended in 300 µL of fixative buffer (BD PharMingen) for flow cytometric analysis on a Becton Dicknson FACS using CELLQuest software.

Quantitative viral load assay
HIV RNA was measured using a quantitative reverse transcriptase polymerase chain reaction assay (Amplicor Monitor, Roche Diagnostic System, Brandersburg, NJ, USA). Virus from 0.2 mL of plasma was lysed using the kit lysis buffer and the HIV RNA was precipitated using isopropanol and pelleted by centrifugation. After washing with ethanol, the RNA was resuspended using the kit dilution buffer. Extracted RNA was amplified and detected according to the manufacturer's instructions, and results were reported as HIV RNA copies/mL. All undetectable values (below 400 copies) were assigned a value of 399.

Statistical analysis
Data were entered and analyzed using Windows SPSS version 11.5 (SPSS Inc., Clay, NC, USA). Data are expressed as the means ± SD and the median. For analysis, HIV negative and HIV positive participants were divided into high and low AF-ALB subgroups based on the median AF-ALB level for the group. Groups were compared by intragroup comparisons (high versus low, among either HIV positive or HIV negative participants) and by intergroup comparisons (HIV positive versus HIV negative participants, among either high or low AF-ALB), using Mann-Whitney U-tests. Possible correlates of AF-ALB-associated accelerated HIV disease progression included those indices that were similarly and significantly associated with AF-ALB, as determined by intragroup comparisons among both HIV positive and HIV negative participants, and with HIV infection, as determined by at least 1 intergroup comparison among high and low AF-ALB participants. A probability value of P < .05 was considered statistically significant. Correlations using nonparametric methods were also conducted to examine the association between AF-ALB and the immune parameters for the entire group and for HIV positive and HIV negative groups separately.

T, B, and NK cell phenotypes of intergroup and intragroup comparisons
Nominally, significant inter-and intragroup differences are summarized in Figure 1 and Table 2. When intergroup comparisons were made, HIV associated differences were mostly similar among both high AF-ALB and low AF-ALB groups. Relative and absolute CD4+ T-cells were significantly decreased in HIV positive participants compared to HIV negative controls (Figure 1(a)) and the proportions of CD8+ T-cells were higher in HIV positive participants than in the negative controls (P = .000, Figure 1(b)). Differences in lymphocyte antigen expression that were evident in the HIV positive group were the CD28+ and HLA-DR+CD38+ percentages within both the CD4+ and CD8+ lymphocyte populations (all P ≤ .001, Table 2, Figures 1(c), 1(d), 1(i), and 1(j)). There were fewer CD4+CD25+ and CD4+CD25+CD45RO+ regulatory T-cells in HIV positive participants than HIV negative controls among both high and low AF-ALB groups (Table 2, Figure 1(k). The difference was statistically significant among the high AF-ALB group (P = .000) and was not statistically significant among the low AF-ALB group (P = .061).
There was lower CD69 expression on CD19+ B-cells in HIV positive participants among both high and low AF-ALB group (Table 2, Figure 1(p)), but this difference was only statistically significant in the low AF-ALB group (P = .000).
No significant HIV associated difference in CD3-CD56+ and perforin expressing NK-cells were apparent by intergroup comparison among either the high AF-ALB or low AB-ALB groups. With the exception of CD19+CD69+, CD8+CD27-CD45RA+, and CD8+CD27+CD45RA+ cells, many of the significant T-cell perturbations that were associated with HIV infection in high AF-ALB HIV positive participants were present in their low AF-ALB counterparts.
When intragroup comparisons were made, aflatoxin associated differences depended in part on the HIV serostatus of the participants. Among both the HIV positive and HIV negative participants, higher AF-ALB was associated with lower expression of perforin on CD8+ T-cells (Table 2, Figure 1(m)). Lower perforin and granzyme A expressing CD8+ T-cells also were seen in both groups (Table 2, Figure 1(n)), but the difference was only statistically significant for the HIV negative groups (P = .01). Additional aflatoxin associated differences among HIV positive participants included lower percentage of CD4+CD25+CD45RO+ regulatory T-cells (P = .009), which was associated with HIV infection in both high and low AF-ALB HIV positive participants (Table 2, Figure 1(k)), and lower percentage of naive CD4+ (CD4+CD45RA+CD62L+) T-cells (Table 2, Figure 1(e)) in the high AF-ALB group.
AF-ALB associated reduction in the B-cells was apparent in HIV positive participants (P = .03) but not in HIV negative participants (Table 2, Figure 1(o)). High AF-ALB associated differences among HIV negative controls included less CD69 expression on both CD3+ T-cells (P = .024) and CD19+ B-cells (P = .027) (Table 2, Figure 1(p)).
Yi Jiang et al. When we conducted correlation analyses between AF-ALB and the immune parameters among HIV positive participants, we found significant correlations between AF-ALB and perforin-expressing CD8+ T-cells (r = −0.170; P = .045), T-regulatory cells (r = −0.395; P = .002), and Bcells (r = −0.212; P = .012). These findings are similar to our findings above. Also our results were consistent with those presented above for the entire study group and for HIV negative participants.

DISCUSSION
To identify possible correlates that may underlie the interaction of AF-ALB with HIV disease progression, we sought to identify immune perturbations that are common to both conditions. This study demonstrated, for the first time, associations of aflatoxin with immune parameters in HIVinfected people.
The changes observed in CD3+, CD4+, and CD8+ Tcell phenotypes, CD19+ B-cells and CD3-CD56+ NK-cells in HIV positive compared to HIV negative participants (intergroup comparison) in this study are consistent with previously well-characterized, HIV-associated changes in these cells. HIV-associated immune perturbations were largely similar in participants with high and low AF-ALB levels. HIV infection induced a decrease in CD4+ T-cell numbers and concomitantly activated the immune system. HIV infection was associated with greater expression of HLA-DR/CD38 and lower expression of CD28 on CD4+ and CD8+ T-cells. We found that the surface expression of HLA-DR/CD38 in both CD4+ and CD8+ T-cells was significantly increased in HIV positive participants. The means of CD4+ and CD8+ cells expressing HLA-DR/CD38 progressively increased with advancing clinical disease as determined by CDC stage (data not shown). Also there was a strong negative correlation between both CD4+ T-cell percentage and CD4+ T-cell count with HLA-DR+CD38+ expressing CD4+ T-cells (data is not shown). This activated immune phenotype has been extensively validated in prior studies and demonstrates T-cell activation to be a strong prognostic indicator for progression to AIDS [31][32][33]. T-cell activation is believed to be the major cause of CD4+ T-cell depletion in HIV infection, through a progression of activationinduced cell death (AICD) [34][35][36][37]. The increased immune activation together with increased viral replication causes severe depletion of CD4+ T-cells, eventually leading to the development of AIDS. Therefore, our data suggest that HLA-DR/CD38 could be used as a progression marker in HIVinfected Ghanaians as in HIV-infected North Americans [38].
In the present study, the principal costimulatory molecule CD28 has been uniformly downmodulated in CD4+ and CD8+ T-cells in HIV infected participants. Engagement of the CD28 molecule on CD8+ T lymphocytes in HIV positive individuals during activation has been reported to increase CD8+ T-cell proliferation and differentiation and to prevent apoptosis [39][40][41]. T-cell receptor stimulation in the absence of CD28 often leads to anergy and to cell death via apoptosis in HIV-infected patients [42].
Many studies have suggested that HIV induces dysfunction of CD4+ and CD8+ cells by CD28 downregulation [43,44]. The loss of CD28 expression on CD4+ and CD8+ cells in HIV infected participants may be associated with the functional defect of the T-cells and progression to AIDS.
CD8+ T-cells are very important lymphocyte subsets in the immune response against HIV infection. Naive CD8+ T-cells can differentiate into effector-type CD8+ Tcells after they have recognized MHC-matched antigens and then express cytolytic molecules, such as perforin and granzymes which are stored in intracellular granules. Therefore, perforin is expressed in antigen-primed CD8+ T-cells with a cytolytic activity potential that contribute to the inhibition of pathogen spread through immediate lysis of infected cells [45]. In our study, the percentage of naive CD8+ T-cells in HIV infected participants was lower than in HIV negative controls, and the percentage of perforin expressing CD8+ T-cells was significantly increased in the HIV-infected participants than in HIV negative controls. Our observation is consistent with other reports [33,34]. Overexpression of perforin in HIV-infected participants may be the consequence of CD8+ T-cell hyperactivation and expansion as a part of feedback regulation of anti-HIV cytotoxic T-lymphocyte (CTL) activity [46,47]. However, among both the HIV infected and HIV negative control groups, those with high AF-ALB showed a lower percentage of perforin expressing CD8+ T-cells compared to those with low AF-ALB levels. This may indicate that CD8+ Tcells synthesizing perforin to enhance the CTL response are impaired in individuals with high AF-ALB [22]. Thus cellular immune function against infectious diseases, such HIV infection will be affected.
T-regulatory cells (Tregs) represent 5-10% of peripheral CD4+ T-cells in healthy individuals and are characterized by constitutive expression of CD25+ and CD45RO+. Tregs (CD4+CD25+CD45RO+) have been implicated in controlling responses to chronic pathogens [48][49][50] and are known to profoundly inhibit both CD4+ and CD8+ T-cell activation, proliferation, and effector function, although the mechanism of this inhibition remains unclear. Thus Tregs may play a critical role in limiting immunopathology that results from persistent high level immune stimulation from chronic viral infections [51]. We evaluated this population of cells in HIV positive and HIV negative participants for both high and low AF-ALB groups. We found that CD4+CD25+CD45RA+ Tregs showed a tendency to decrease in HIV infected participants with both high and low AF-ALB level compared to HIV negative controls. In addition, we found that the HIV positive group with high AF-ALB had the lowest percentage of Tregs of all the groups suggesting that there is a loss of Tregs in HIV infected participants with high AF-ALB. This loss may facilitate the immune hyperactivation associated with HIV and lead to more severe disease in those with high aflatoxin levels.
The activation molecule CD69 is a costimulatory molecule for lymphocyte proliferation. It is expressed early on the membranes of T-and B-lymphocytes through the stimulated antigen receptor/CD3 complex or cross-linking of surface immunoglobulins, respectively. T-lymphocyte High aflatoxin appeared to accentuate some HIVassociated changes in T-cell phenotypes and B-cells in HIVinfected participants. Because of the cross-sectional design of this study, however, we cannot exclude the possibility that these intragroup differences may simply reflect an imbalance in the severity of HIV disease, with more advanced disease (not evident by our CDC classification) in the high AF-ALB group. However, despite these limitations, we believe that this study has a potentially significant impact on understanding the effects of aflatoxin in HIV disease progression.
A better understanding of the interaction of high aflatoxin with HIV disease might provide insight into key mechanisms that underlie the immunopathogenesis of both processes. Potential immune correlates of this interaction may include reduced Tregs, impaired CD8+ T-cell function, plus impaired B-cell CD69-expressing ability. In HIVinfected patients with higher AF-ALB, loss of Tregs, and persistent immune activation might lead to exhaustion of the naive CD8+ T-cell pool, uniform downmodulated of the principal costimulatory molecule CD28 on CD8+ and CD4+ cells, impaired function of CD8+ T-cells synthesizing perforin (which results in failure of cytolysis by CD8+ Tcells), plus impaired ability of B-cell to express CD69. This will result in increased viral replication and increase in the occurrence of viral escape mutants. Because of viral escape, viral load will increase resulting in further loss of functional HIV specific CD4+ T-cell and a progressive deterioration of the immune system.
Given these observations, high aflatoxin may promote more rapid HIV disease progression in HIV-infected Ghanaians. Our findings should be considered exploratory, given the cross-sectional design of this study and because there may be other variables not considered that may affect immune cell distribution and function. Further clarification of these and other possible immune correlates of the interaction of high aflatoxin with HIV disease progression might be afforded by additional investigation.

CONCLUSION
High AF-ALB appeared to accentuate some HIV-associated changes in T-cell phenotypes and B-cells in HIV positive participants. These results may indicate that CD8+ T-cells synthesizing perforin to enhance the CTL response are impaired in individuals with high AF-ALB. The loss of Tregs in HIV positive participants with high AF-ALB may facilitate HIV associated immune hyperactivation and lead to more severe disease.