Imbalance of essential trace elements viz. Zinc, Selenium, Iron, Copper and Magnesium has been reported to influence disease course in HIV-1, HBV and HCV infections by altering immune status. A study was taken up to examine plasma levels of Th1 (IFN- γ and IL-2) and Th2 (IL-4 and IL-10) categories of cytokines and immune activation markers (TNF- α, TNFR I and TNFR II) in an asymptomatic group of HIV-1, HBV and HCV infected blood donors in relation to trace elements. Plasma levels of Zn, Se and Mg were depressed in all the three groups of blood donors (P < 0.001 for all). Levels of Cu and Fe were depressed in HIV-1 infection (P < 0.001 for both), but elevated in HBV and HCV infections (P < 0.015 and < 0.001 for Cu and < 0.001 for Fe in case of HBV and HCV infections respectively). IL-2 and IFN- γ were depressed in all the three groups of blood donors (P < 0.001). IL-4 and IL-10 levels were elevated in HBV and HCV infections (P < 0.001 for both). Immune activation markers were elevated in all the three groups of blood donors (P < 0.001 for all). HIV-1 infection showed positive correlations between Cu and IL-2, Zn and IFN- γ, and in HBV infection while positive correlations were found between Mg and TNFR I and TNFR II and Se with TNFR II. HCV infection showed a positive correlation between Se and IFN- γ (P < 0.001), Mg and IL-4 (P = 0.02), Fe and IL-10 (P < 0.01). The present study reveals possible relationship between trace element level alterations and alterations in cytokine and immune activation levels in HIV-1, HBV and HCV infection.
High prevalence of Human immune deficiency virus type 1 (HIV-1), Hepatitis B virus (HBV) and Hepatitis C virus (HCV) among blood donors has been recorded globally [
The project was approved by institutional review board including ethical clearance. The donors were explained the purpose of the study. Written consent for the study was obtained from the donors using a predesigned consent form in local language. During the period from July to December 2016, all the major blood banks including the private ones in the national capital territory of Delhi were requested to refer the blood donors found seropositive for HIV, HBV and HCV at their centres to the Centre for AIDS and Related Diseases, National Centre for Disease Control (NCDC). A total of 120 blood donors (all males), 40 each confirmed to be seropositive for HIV-1, HBV and HCV at NCDC were selected for the study. The period between detection of seropositivity for HIV-1, HBV and HCV in the blood banks referring the donors and confirmation of the same at Centre for AIDS and Related Diseases department, NCDC was considered duration of infection since diagnosis. In addition, since the donors were repeat donors donating blood at intervals of 4 - 8 months, whenever a seropositive blood donor was referred by a blood bank to our centre, test results for HIV, HBV and HCV at preceding blood bank attended by the seropositive donors were checked from the records available with blood banks to confirm seronegativity at the time of preceding donation. Forty, age and socioeconomically matched male blood donors seronegative for HIV-1, HBV and HCV infections and donating at the same blood banks were included as controls.
A confidential interview was taken from the donors regarding age, marital status, frequency of blood donation (for preceding one year), HIV related risk behaviours in terms of frequency (per week) of unprotected sex with heterosexual and/or homosexual partners as well as number of sex partners (for preceding one year). History of other risk factors viz. parenteral drug abuse as well as receipt of blood/ blood products were also obtained from the donors.
A quantitative assessment of dietary intake was conducted by recall procedure for immediate past three typical days employing a food frequency questionnaire. Average daily intakes of total energy and protein were calculated as per Indian Council of Medical Research (ICMR) guidelines based on the quantities of various categories of raw food materials consumed as described earlier [
Height (±0.1 cm) and weight (±0.1 Kg) were measured using standard techniques and body mass index (BMI) calculated by the formula weight (kg)/height (m2) [
Fasting blood was collected and distributed as about 8 ml in plain sterile tube to yield serum for estimation of enzyme markers, serological assays for HIV-1, HBV and HCV infections, while about 4 ml of blood was distributed ethylene-diamine-tetra-acetic acid (EDTA) vial for routine haemoglobin, peripheral CD4 T lymphocyte count, cytokine, immune markers and trace element analysis.
Diagnosis of HIV-1 infection was based on commercial Enzyme- Linked Immunosorbent Assay (ELISA) test carried out at various blood banks in the city of Delhi and confirmatory western blot test carried out at AIDS Reference Centre, National Centre for Disease control (formerly National Institute of Communicable Diseases), Delhi [
Estimation of three enzyme markers as indices of liver function including alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin in serum were carried out using auto-analyzer (Hitachi 912, Hitachi Ltd., Japan). Estimation of serum levels of albumin were carried out by techniques as described earlier [
Enumeration of peripheral CD4 + T lymphocytes was done by flow cytometry. In brief, a standard flow cytometry method with lysed whole blood and a panel of two color combinations of fluorescein isothyocyanate and phycoerythrinconjuagated monoclonal antibody regents obtained from a single manufacturer (Becton-Dickinson, San Jose, California) were used to determine the expression of each antigen or antigen combination as described earlier [
Estimation of trace elements viz., Zn, Se, Fe, Cu and Mg in plasma was carried out using Flame Atomic Absorption Spectrophotometer (FAAS) (Thermo Electron Corporation, UK M6 Spectro with integrated software SOLAAR AA) as described earlier [
Levels of T helper type1 (Th1) cytokines viz. interleukin-2 (IL-2) and gamma interferon (IFN-γ) and T helper type 2 (Th2) cytokines viz., Interleukin-4 (IL-4) and Interleukin-10 (IL-10) were measured in plasma using commercial ELISA kits (R&D Systems, Minneapolis, MI, USA) with sensitivity limits of 0.06 pg/ml, 8 pg/ml, 0.22 pg/ml and 0.17 pg/ml for IL-2, IFN-γ, IL-4 and IL-10 respectively. Plasma levels of immune activation markers viz., tumour necrosis factor alpha (TNF-α), tumour necrosis factor alpha receptor type l (TNFR p 55) and type ll (TNFR p 75) were measured using commercial ELISA kits from source as above with sensitivity levels of 0.49 pg/ml, 1.2 pg/ml and 2.3 pg/ml respectively. Coefficient variation for all of these assays was found to be less than 10%. Readings below detection range were assigned zero values.
Statistical analysis was carried out employing SPSS version 20. Characteristics of blood donors as assessed at enrolment were compared using χ2 test for categorical variables and one way ANOVA for continuous variables with normal distribution while continuous variables not following normal distribution were compared by Kruskal Wallis test followed by multiple comparison using Mann Whitney test. P value < 0.05 was considered as statistically significant and it was adjusted for multiple comparisons. Fisher’s exact test was used in which any of the expected cell frequency was less than five. Correlation of cytokine levels with trace element levels was evaluated by Spearman rank correlation test (due to lack of normal distribution in certain parameters).
All the seropositive donors were males and unmarried belonging to the age group 24 - 33 years without any significant difference compared to seronegative blood donor control (mean ± SD for HIV-1, HBV, HCV and seronegative control as 29.2 ± 3.3, 30.5 ± 2.6, 29.2 ± 2.9, 30.0 ± 2.5 respectively). The mean duration between the preceding seronegative donation and detection of seropositivity for HIV-1, HBV and HCV in infected blood donors and seronegative donors ranged between 4 - 6 months with mean ± SD for HIV-1, HBV and HCV as 5.1 ± 0.8, 4.57 ± 0.5, 5.0 ± 0.8 and 4.8 ± 0.6 respectively (data not shown in table). Magnitude of exposure to unprotected sex with female CSWs could be elicited most in HIV-1 infected donors followed by HBV and HCV (
There was no alteration in CD4 T + lymphocyte count in all the subgroups of seropositive donors. On the other hand liver enzymes showed marginal elevation in all the subgroups of seropositive donors although the levels were comparable to seronegative controls. Serum bilirubin levels were unaltered in all the subgroups of seropositive donors. Energy intake and protein intake in all the three subgroups of seropositive donors were comparable to seronegative blood donor controls. There was no difference in the status of BMI and serum levels of albumin among the various groups of donors and controls (Supplementary
HIV-1 (n = 40) | HBV (n = 40) | HCV (n = 40) | Control (n = 40) | Statistical Comparisons | |||
---|---|---|---|---|---|---|---|
Group I | Group II | Group III | Group IV | ||||
H/O unprotected sex with female CSW* | Positive history | No (%) | 31 (77.5%) | 16 (40%) | 9 (22.5%) | NA | χ2 = 26.86 P < 0.000 |
Frequency of Exposure/week | Range** | 1 - 3 | 0 - 1 | 0 - 1 | NA | f = 23.94 P < 0.000 | |
Mean ± SD** | 2.1 ± 0.8 | 1.0 ± 0 | 1.0 ± 0 | NA | |||
No. of partners | Range** | 4 - 6 | 2 - 3 | 2 - 3 | NA | f = 77.56 P < 0.000 | |
Mean ± SD** | 4.5 ± 0.8 | 2.4 ± 0.5 | 2.1 ± 0.3 | ||||
Consumption of alcoholic drink* | Positive history | No (%) | 15 (37.5) | 14 (35) | 12 (30) | 10 (25) | |
Sessions/week | Range** | 2 - 4 | 2 - 3 | 2 - 3 | 1 - 3 | f = 0.63 P = NS (0.55) | |
Mean ± SD** | 2.1 ± 0.38 | 2.2 ± 0.46 | 2.2 ± 0.45 | 2.1 ± 0.8 |
*During one year of pre-enrolment period; **Calculated for the donors with positive history only; None of the donors gave history of parenteral drug abuse or receipt of blood transfusion.
There was significant depression in plasma levels of Zn, Se, Fe, Cu and Mg in the group of blood donors infected with HIV-1 (P < 0.001 for all comparisons). In case of HBV and in HCV infected blood donors, plasma levels of Cu and Fe were significantly elevated as compared to controls. However, Zn, Se and Mg, levels in plasma were significantly depressed in both HBV and HCV infected blood donors compared to seronegative controls (P < 0.001 for all comparisons) (
The plasma concentrations of Th1 type of cytokines i.e., IL-2 and IFN-γ, were significantly decreased in all the three asymptomatic infected groups i.e., HIV-1, HBV and HCV (P < 0.001). On the other hand, levels of Th2 type cytokines viz. IL-4 and IL-10 remained unaltered compared to control groups in case of HIV-1 (P = 0.39, P = 0.42 respectively). But in case of HBV and HCV, Th2 type of cytokines i.e., IL-4 and IL-10 were found to be significantly elevated (P < 0.001 for all comparisons). Levels of three immune activation markers studied viz. TNF α, TNFR l and TNFR II were significantly increased in all the three groups of donors compared to seronegative controls (P < 0.001) (
Correlation of trace element status with plasma cytokine levels in HIV-1, HBV and HCV infected blood donors showed that in case of HIV-1 infection a
Blood donor groups | Control | |||
---|---|---|---|---|
Trace element level | HIV-1 (n = 40) Group I | HBV (n = 40) Group II | HCV (n = 40) Group III | (n = 40) Group IV |
Zn(µg /dl) | 50.78 ± 4.53* | 54.28 ± 12.25* | 54.40 ± 3.12* | 83.08 ± 12.48 |
Se(µg /dl) | 49.75 ± 14.60* | 63.28 ± 12.70* | 51.43 ± 7.94* | 93.55 ± 9.08 |
Fe(µg /dl) | 63.52 ± 8.94* | 181.76 ± 16.92** | 183.63 ± 13.55** | 105.58 ± 25.50 |
Cu (µg /dl) | 67.23 ± 12.98* | 95.25 ± 10.60** | 128.85 ± 16.89** | 89.63 ± 12.46 |
Mg (mg/dl) | 1.16 ± 0.27* | 1.57 ± 0.50* | 1.35 ± 0.21* | 2.03 ± 0.62 |
*Significant depression (P < 0.05) compared to controls; **Significant elevation (P < 0.05) compared to controls; Multiple comparisons between the groups(by ANOVA): Cu: Overall P < 0.001; II vs. IV P < 0.03; rest all comparisons <0.001; Fe: Overall P < 0.001: II vs. III P = NS (0.63); rest all comparisons <0.001; Mg: Overall <0.001; I vs. III P = NS (0.12); rest all comparisons <0.001; Se: Overall P < 0.001; I vs. III P = NS (0.53): rest all comparisons <0.001; Zn: Overall P < 0.001; I vs. II P = NS (0.10); II vs. III < 0.95; rest all comparisons <0.001.
Immunological markers and their plasma levels (pg/ml) | Blood donor groups | Control | ||
---|---|---|---|---|
HIV-1 (n = 40) Group I | HBV (n = 40) Group II | HCV (n = 40) Group III | (n = 40) Group IV | |
IL-2 | 4.19 ± 1.89* | 5.53 ± 1.69* | 0.30 ± 0.24* | 16.37 ± 4.08 |
IFN-γ | 23.1 ± 2.22* | 23.93 ± 3.68* | 27.53 ± 2.41* | 50.53 ± 3.87 |
IL-4 | 2.5± 1.18 | 3.83 ± 1.97** | 27.15 ± 6.37** | 2.60 ± 1.27 |
IL-10 | 1.2 ± 0.27 | 25.03 ± 4.87** | 29.98 ± 8.12** | 1.23 ± 0.72 |
TNF-α | 12.3 ± 5.21** | 11.03 ± 2.37** | 61.86 ± 21.67** | 3.11 ± 0.48 |
TNFR I | 1.6 ± 0.36** | 1.31 ± 0.76** | 1.02 ± 0.65** | 0.31 ± 0.13 |
TNFR II | 5.8 ± 1.38** | 7.36 ± 2.76** | 4.15 ± 2.01** | 1.23 ± 0.36 |
*Significant depression (P < 0.05) compared to controls; **Significant elevation (P < 0.05) compared to controls; Multiple comparisons between the groups(by ANOVA): IL2: Overall P < 0.001; all comparisons <0.001; IFN-γ: Overall P < 0.001; I vs. II P = NS (0.23); rest all comparisons P < 0.001; IL-4: Overall P < 0.001; I vs. IV P = NS (0.80); rest all comparisons P < 0.001; IL-10: Overall P < 0.001; I vs. IV P = NS (0.85); rest all comparisons P < 0.001; TNF α: Overall P < 0.001; I vs. II P = NS (0.14); rest all comparisons P < 0.001; TNFR I: Overall P < 0.001; I vs. II P = NS (0.12); rest all comparisons <0.001; TNFR II: Overall P < 0.001; all comparisons P < 0.001.
marginal but significant positive correlation was found between Cu level and IL-2, Zn and IFN-γ (r = 0.33, P = 0.03 for both Cu and Zn) (
In case of HBV infected blood donors significant positive correlations were found between Mg and TNFR l (r = 0.33, P = 0.04) and both Mg and Se with immune marker TNFR II (r = 0.33, P = 0.04; r = 0.42, P = 0.006 respectively) (Figures 2(a)-(c)). No correlation could be demonstrated in HBV infected blood donors between the levels of various trace elements and any of the Th1 and Th2 types of cytokines (Supplementary
In HCV infected blood donors samples, there was a positive correlation between Se andIFN-γ (r = 0.60 , P < 0.001), Mg with IL-4 ( r = 0.36 , P = 0.02), Fe with IL-10 (r = 0.37, P = 0.01) (Figures 3(a)-(c)). However, a negative correlation was demonstrated between Cu level with Th2 type of cytokines IL-4 and IL-10 (r = −0.39, P = 0.01, r = −0.32, P = 0.03 respectively) (
The HIV-1 seropositive donors in the present study showed higher magnitude of exposure to unsafe sex compared to HBV and HCV positive donors. However,
absence of such risk factor in perceptible proportion (22.5%) of the HIV-1 positive donors suggests acquisition of HIV-1 by other means like blood donation in unhygienic setup as pointed out in our earlier study [
Trace elements play important roles in various components of both innate and acquired immune function [
activity of numerous immune cells, such as B and T lymphocytes and natural killer cells [
Present study showed significantly depressed plasma levels of Zn, Se, Fe, Mg and Cu in blood donors infected with HIV-1. Zinc is a component of HIV-1 nucleocapsid proteins and is used for gene expression, multimerization and integration in HIV-1 and this may explain the low plasma zinc levels observed in our study [
The plasma trace element status in both HBV and HCV infected blood donors in the present study showed significantly low level of Zn, Se and Mg concentrations and elevated level of Cu and Fe as compared to seronegative controls which is in agreement with the study by Hatano et al., [
The present study demonstrated a significantly low level of Th1 type of cytokines (IL-2 and IFN-γ) in blood among HIV-1 infected group when compared to seronegative controls, while the Th2 type of cytokines i.e. (IL-4 and IL-10) remained unaltered. Many reports describe a shift from Th1 to Th2 cytokine profile during the progression of HIV-1 disease [
Both HBV and HCV infections in the present study showed dominance of Th2 category of cytokines and elevation of immune activation markers. It could be hypothesized that these donors were likely to be future potential persistent carriers for both these infections [
The present study showed a significant positive correlation between depression of Zn and Cu levels and Th1 type of cytokines in HIV-1 infection which is in agreement with studies [
It has been shown that in Zn deficiency generation of IL-2 and IFN-γ are decreased where as the production of Th2 type of cytokines IL-4 and IL-10 are not affected [
Positive correlation between Se with IFN-γ in HCV infection observed in the present study has been demonstrated in mouse model experiments [
Information on the inter-relationship between trace elements, cytokines and immune activation is limited to HIV-1 infection only [
Nevertheless, the present report provides information on the association between trace element alteration and cytokine imbalance at early asymptomatic stage of HIV, HBV and HCV infections in absence of obvious protein calorie malnutrition that tends to point out that trace element alterations can exist at early asymptomatic stage since the donors in our study had unaltered parameters like BMI, protein intake, calorie intake, serum albumin levels comparable to control population.
We declare that we have no conflict of interest.
This study was carried out by institutional support.
Verghese, A., Singh, S.N. and Chattopadhya, D. (2017) Trace Element Levels, Cytokine Profile and Immune Activation Status in Plasma among Repeat Blood Donors with Asymptomatic HIV-1, HBV and HCV Infection. Journal of Biosciences and Medicines, 5, 75-94. https://doi.org/10.4236/jbm.2017.59008
Supplementary
Note: There was no statistical difference in the above parameters between HIV-1, HBV, HCV; infected donors and that of seronegative controls in multiple comparisons as well as in individual comparisons between the above subgroups ( by ANOVA).
Supplementary
(a)
(b)
*indicates significant correlation.
(c)
*indicates significant correlation.