|Year : 2016 | Volume
| Issue : 2 | Page : 63-71
Effect of vitamin A and zinc on circulating profile of IL-2, IL-12, and IFNγ cytokines in pulmonary tuberculosis patients
Irfan Ahmad1, Khalid Al-Ahmare2
1 Department of Clinical Laboratory Science, College of Applied Medical Sciences, King Khalid University, Abha, KSA
2 Department of Medical Rehabilitation Sciences, King Khalid University, KSA
|Date of Web Publication||11-Apr-2016|
Department of Clinical Laboratory Science, College of Applied Medical Sciences, King Khalid University, Abha
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Tuberculosis (TB) is a major public health problem throughout the world and one of the leading causes of mortality. Vitamin A and zinc deficiency is frequently observed in patients of pulmonary TB and thus, diminish the activity of their immune system. The aim of this study was to investigate the effect of vitamin A and zinc supplementation on sputum conversion time and immunological profile in pulmonary TB patients receiving antitubercular treatment. Materials and Methods: This was a double-blind, placebo-controlled study. The cases comprised those outpatients with active pulmonary TB (new sputum smear positive) who had attended the DOTS center in the Department of Pulmonary Medicine, King George's Medical University, Lucknow, Uttar Pradesh, India. Results: Total 260 patients were enrolled and randomly grouped into four categories of intervention. After completion of the follow-up, some patients dropped out from the study so that total 208 patients completed it. The level of interleukin-2 (IL-2), IL-12, and interferon gamma (IFNγ) was significantly (P = 0.001) changed and the sputum smear conversion was significantly early in the vitamin A and zinc supplemented group. Conclusion: We demonstrated that vitamin A and zinc may directly or indirectly influence the activation of cytokines. The circulating cytokines play an especially important role in the pathogenesis of active pulmonary TB.
Keywords: Cytokines, malnutrition, tuberculosis, vitamin A, zinc
|How to cite this article:|
Ahmad I, Al-Ahmare K. Effect of vitamin A and zinc on circulating profile of IL-2, IL-12, and IFNγ cytokines in pulmonary tuberculosis patients. Int J Nutr Pharmacol Neurol Dis 2016;6:63-71
|How to cite this URL:|
Ahmad I, Al-Ahmare K. Effect of vitamin A and zinc on circulating profile of IL-2, IL-12, and IFNγ cytokines in pulmonary tuberculosis patients. Int J Nutr Pharmacol Neurol Dis [serial online] 2016 [cited 2020 Aug 9];6:63-71. Available from: http://www.ijnpnd.com/text.asp?2016/6/2/63/179965
| Introduction|| |
Tuberculosis (TB) is as old as mankind. This remains a major public health problem. Nearly one-third of the world's population is infected with Mycobacterium tuberculosis, the causative organism of TB.  The Centers for Disease Control and Prevention (CDC) predicts that TB will claim 30 million lives in the present decade. It is also estimated that between 2002 and 2020, approximately one billion people will be newly diagnosed. Over 150 million will get sick and 36 million will die of TB if proper control measures are not instituted.  In 2006, nearly 9.2 million new cases and 1.7 million deaths were reported due to TB, and over 90% of these deaths occurred in the low and middle income countries.  In these regions, TB is the one of the leading cause of adult mortality, ranking third after human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and ischemic heart disease as a cause of death among those aged 15-59 years (and seventh globally among all age groups). , Each year, 1.9 million new cases of TB occur in India, of which about 0.8 million are infectious new smear positive pulmonary TB cases. 
Malnutrition is frequently observed in patients with pulmonary TB but their nutritional status, especially of micronutrients, is still poorly documented. Several studies reported that patients with active pulmonary TB are malnourished as indicated by reductions in visceral proteins, anthropometric indexes, and micronutrient status. , Among the micronutrients, vitamin A has an immunoprotective role against TB and its supplementation results in a modulation of the immune response in patients with TB  and zinc is essential for human growth, development, and immune function; deficiency of this micronutrient impairs the overall immune function and resistance to infection. 
Vitamin A, acting via all-trans retinoic acid, 9-cis retinoic acid, or other metabolites and nuclear retinoic acid receptors (RARs), plays an important role in the regulation of innate and cell-mediated immunity and humoral antibody response. , It also plays a central role in the growth and function of T and B cells, antibody responses, and maintenance of mucosal epithelia.  Vitamin A deficiency is associated with diminished phagocytic and oxidative burst activity of the macrophages activated during inflammation  and a reduced number and activity of natural killer (NK) cells.  The increased production of interleukin-12 (IL-12) (promoting T cell growth) and pro-inflammatory tumor necrosis factor alpha (TNFα) (activating microbicidal action of the macrophages) in a vitamin A-deficient state may promote an excessive inflammatory response; however, supplementation with vitamin A can reverse these effects.  Lymphocyte proliferation is caused by the activation of RARs and therefore, vitamin A plays an essential role in the development and differentiation of T-helper cell 1 (T h 1) and T h 2 lymphocyte subsets.  T h 1 and T h 2 cells are characterized by the production of IL-2, IFNγ, and IL-4, IL-5, IL-6, respectively. Vitamin A maintains the normal antibody-mediated T h 2 response by suppressing IL-12, TNFα, and IFNγ production of T h 1 lymphocytes. As a consequence, in vitamin A deficiency, there is an impaired ability to defend against the extracellular pathogens.  IL-12 is a cytokine produced by phagocytic and antigen-presenting cells; it is an important activator of IFNγ produced by T cells and NK cells and provides an early switch in the differentiation of cluster of differentiation 4 (CD4)+ T cells. Hence, IL-12 seems to be an important factor determining the outcome of TB. , Antibody-mediated immunity is strongly impaired in vitamin A deficiency.  Vitamin A supplementation by oral administration increases delayed type hypersensitivity (DTH) in infants that may reflect vitamin A-related upregulation of lymphocyte function.  In humans, vitamin A supplementation has been shown to improve antibody titer response to various vaccines. ,
Most of the immune effects of vitamin A are carried out by vitamin A derivatives, namely, the isomers of retinoic acid. The isomers of retinoic acid are steroid hormones that bind to retinoid receptors, which belong to the following two different classes: RARs and retinoid X receptors (RXRs). In the classical pathway, RAR must first heterodimerize with RXR and then bind to small sequences of DNA called retinoic acid response elements (RAREs) to initiate a cascade of molecular interactions that modulate the transcription of specific genes.  More than 500 genes are directly or indirectly regulated by retinoic acid.  Several of these genes control cellular proliferation and differentiation; thus, vitamin A has obvious importance in immunity.
Zinc is important for enzymes of all the six classes as well as for transcription and replication factors.  It is necessary for the normal functioning of the immune system.  Even mild zinc deficiency, which is widely spread in contrast to severe zinc deficiency, depresses the immunity of humans.  It is needed by NK cells for the recognition of major histocompatibility complex (MHC) class I molecules by the p58 killer cell inhibitory receptors on the NK cells to inhibit the killing activity.  It influences not only NK cell-mediated killing as mentioned above but also affects the activity of the cytolytic T cells.  The relative amount of CD8+ CD73+ T lymphocytes is found to decrease during zinc deficiency.  These cells are predominantly precursors of cytotoxic T lymphocytes (CD8+) and CD73+ is known to be needed on these cells for antigen recognition and proliferation as well as cytolytic process generation.  Besides cytotoxic CD4+ T cells are affected by zinc deficiency, which causes an imbalance between T h 1 and T h 2 functions. This was observed in altered secretion of the typical T h 1 and T h 2 cytokines during zinc depletion. The T h 2 products IL-4, -6, and -10 remain unchanged during zinc deficiency, whereas the T h 1 cell products IL-2 and IFNγ are decreased. Production of both IL-2 and IFNγ is corrected by zinc supplementation. 
It was earlier reported that small imbalances in zinc levels adversely affect the function of the immune system. International studies indicate that when malnourished children from countries such as South America (Argentina, Brazil, Bolivia, Chile, Colombia, Guyana, Ecuador, Paraguay, Netherlands Antilles, Peru), South Asia (Afghanistan, Bangladesh, Bhutan, Maldives, Nepal, Pakistan, Sri Lanka), India, and Africa (Algeria, Benin, Botswana, Uganda, Zimbabwe, Namibia, Niger, Mali, Senegal, Ghana, Ethiopia) are given zinc supplements ranging 4-40 mg/day, the duration of incidences of diarrhea infection is shortened (Black, 1998). Further studies shows that when zinc is given to individuals with low zinc levels, the number of T cell lymphocytes increased, along with their ability to fight infection. 
Zinc is critical for the normal development and functioning of cells that mediate both innate and adaptive immunities.  The cellular functions of zinc can be divided into the following three categories: (1) Catalytic, (2) structural, and (3) regulatory.  Because zinc is not stored in the body, regular dietary intake of this mineral is important in maintaining the integrity of the immune system. Thus, inadequate intake can lead to zinc deficiency and compromised immune responses.  With respect to innate immunity, zinc deficiency impairs the complement system, cytotoxicity of the NK cells, phagocytic activity of the neutrophils and macrophages, and ability of the cells of the immune system to generate oxidants that kill invading pathogens. ,, Zinc deficiency also compromises adaptive immune function, including the number of lymphocytes and their function.  Even marginal zinc deficiency, which is more common than severe zinc deficiency, can suppress the aspects of immunity.  Zinc-deficient individuals are known to experience an increased susceptibility to a variety of infectious agents.
The presence of micronutrient deficiency has raised a question whether micronutrient supplementation would give additional benefits to the patients of TB treatment program and to what extent the concentration of cytokine in active TB would be modulated by antitubercular treatment and by micronutrient supplementation. The cases of TB received antitubercular treatment at the Directly Observed Treatment, Short Course (DOTS) centers, established under the Revised National Tuberculosis Control Programme (RNTCP). Supplementation of vitamin A and zinc is not part of the DOTS regimen but it is known to affect the course of action of this disease. The aim of the present study was to investigate the effect of vitamin A and zinc supplementation on sputum conversion time and immunological profile in pulmonary TB patients receiving antitubercular treatment. The present study will certainly provide some new insights and lead to a breakthrough in the treatment and management of this disease.
| Materials And Methods|| |
Study design and subjects
This was a double-blind, placebo-controlled study. The cases comprised those outpatients with active pulmonary TB (new sputum smear positive) who had attended the DOTS center in the Department of Pulmonary Medicine, King George's Medical University, Lucknow Uttar Pradesh, India. Selection of the cases was based on the following criteria: Age ranging 18-55 years, at least two sputum specimens positive for acid-fast bacilli (AFB) by microscopy, and clinical and radiographic abnormalities consistent with pulmonary TB. Exclusion criteria for the cases and controls were as follows: Previous anti-TB treatment, pregnancy, lactation, use of corticosteroids or supplements containing vitamin A, zinc, or iron during the previous month, moderate to severe injury or surgery during the last month, and diseases such as abnormal liver function as measured by the elevated serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), diabetes mellitus as measured by elevated fasting blood glucose levels, neoplasm as determined by clinical examination, chronic renal failure as determined by elevated serum levels of urea and creatinine, and congestive heart failure.
Randomization and supplementation
Randomization was done using a computer program in which a treatment code was given to the subjects. The patients were randomly assigned into the following four groups: Vitamin A alone, zinc alone, combination of vitamin A and zinc, and placebo. All the study groups were given supplements or placebo, along with the anti-TB drugs on the DOTS day. Each micronutrient capsule contained 5,000 IU of vitamin A (as retinyl acetate) and 15 mg of zinc (as zinc sulfate). The placebo consisted of lactose only. The supplements and placebo capsules were similar in terms of shape, size, and color. The dosage was based on the recommendation of the World Health Organization and Recommended Dietary Allowances. ,
We assessed the patient's compliance by comparing the number of remaining capsules with the number recorded in a logbook provided to the patients. Those patients who did not take their medication regularly, missing even 1 day in the first 2 months, were dropped from the study. We as well as the patients were unaware of the treatment code until the study was completed. The patients who had severe adverse drug effects were excluded from the study and received further treatment under the guidance of clinicians. Those patients with strains of Mycobacterium tuberculosis resistance to one or more drugs after 2 months of antitubercular treatment were placed on a modified drug regimen and their data were excluded from the study.
A difference of 0.37 with a standard deviation of 0.6 increase in the mean was assumed between the treatment group in plasma zinc from 2 months to 6 months  using the formula 2C/δ2 + 1 was assumed, where, significance level = 5%, δ = 0.37 (mean difference between the treatment groups/standard deviation), and C = 10.5 for 90% power and 5% significance level. The calculated sample size per group was 55. After adjusting 20% loss to follow-up, total 65 cases were recruited in each group.
Collection of sputum samples and analyses
The patients were asked to come to deliver their sputum for direct AFB smear examination at weekly intervals till the end of the intensive phase. Three early-morning sputum specimens were taken from the patients and examined by direct microscopic examination after Ziehl-Neelsen staining. 
Collection of blood samples and analyses
Blood samples (5 mL) were collected from the subjects via vein puncture at 0 day, after 2 months, and thereafter 6 months of the treatment to determine the cytokine parameter. The serum was separated by centrifugation at 2,500 rpm for 10 min at room temperature (20-25°C) and was then stored at -80°C until analyzed for IL-2, IL-12, and IFNγ.
Cytokine examinations were carried out by the enzyme-linked immunosorbent assay (ELISA) method according to the instructions of the manufacturer (BioLegend, 9727 Pacific Heights Blvd., San Diego, CA) at the start of the treatment (0 day), after 2 months (end of the intensive phase), and thereafter 6 month of the treatment (end of the continuation phase).
The study has been approved by the Institutional Ethics Committee of King George's Medical University, Lucknow Uttar Pradesh, India. The data were collected after the subjects agreed to participate in the study and gave written informed consent voluntarily.
A one-sample Kolmogorov-Smirnov test was used to investigate whether the variables were normally distributed. Data on the patient's age and sex distributions at the time of enrolment were summarized and used to assess the comparability of the patients randomly assigned to the four treatment groups. Independent t-tests were used to compare sputum conversion of the two treatment groups ; it was compared by the normally distributed variables between the two groups. Differences in treatment effects within the groups and between the micronutrient and placebo groups were tested by a multivariate analysis of variance repeated measures analysis of variance design with the supplement type as a between-subject factor (two groups) and the treatment effect (baseline compared with that of 2 months and 6 months) as a within-subject factor. A significant P value for the treatment effect indicated a change over time in the combined values of the two groups and was further investigated using a paired t-test for each individual group. None of the three-way interactions (treatment effect × supplement type × baseline value) was significant. Statistical significance was based on a two-tailed P < 0.05. Analyses were performed using the SPSS software package (Windows version 7.5.2; SPSS Inc., Chicago, Il, USA).
| Results|| |
In this study, total 260 patients were enrolled and randomly grouped into four categories of intervention, i.e., 65 patients in each group. After completion of the follow-up, some patients dropped out from the study so that total 208 patients completed it. The age of the patients was almost similar across the groups at the time of enrolment. More than half of the patients of the vitamin A group (53.7%), zinc group (56.7%), vitamin A and zinc group (53.8%), and placebo (53.3%) group were males. There was no significant (P > 0.05) difference among the groups in terms of sex. Sputum grade 3+ was most prevalent. More than one-third of the patients of the vitamin A group (38.9%), zinc group (40%), vitamin A and zinc group (40.4%), and placebo (38.3%) group were in sputum grade 3+. However, 33.3% of those in vitamin A group, 29.1% in the zinc group, 36.5% in the vitamin A and zinc group, and 34% in the placebo group were in sputum grade 2+. An equal percentage of patients among all the groups were in sputum grade 1+ [Table 1].
The level of IL-2 was significantly (P = 0.001) increased in patients of vitamin A supplementation from 0 month (9.90 ± 0.94) to 2 months (12.05 ± 0.68) and 6 months (13.50 ± 0.89). A significant impact on IL-2 was also observed in zinc, and vitamin A and zinc supplemented patients. An insignificant change in the IL-2 level was found in the placebo supplemented patients. The level of IL-12 was significantly (P = 0.001) increased in patients of vitamin A supplementation from 0 month (10.05 ± 0.77) to 2 months (12.68 ± 0.86) and 6 months (14.64 ± 0.95). The level of IL-12 was also observed to have significantly (P = 0.001) increased in the zinc, and vitamin A and zinc supplemented patients. No significant change in IL-12 level was found in the placebo supplemented patients. The level of IFNγ was significantly (P = 0.01) decreased in patients of vitamin A supplementation from 0 month (19.68 ± 1.48) to 2 months (16.97 ± 1.56) and 6 months (14.90 ± 1.33). Similar observations were found in the zinc and vitamin A and zinc supplemented patients. No significant change in the IFNγ level was found in the patients of placebo supplementation [Table 2].
The higher percentage increase in IL-2 from 0 month to 2 months was observed in the vitamin A and zinc group [27.4%, 95%; confidence interval (CI) =25.6-29.1] than the zinc group (23.2%, 95%; CI = 21.2-25.2), vitamin A group (17.7%, 95%; CI = 15.5-19.9), and placebo group (14.9%, 95%; CI = 12.5-17.4). Almost similar observation was found from 0 month to 6 months for IL-2. The percent increase was also higher in IL-12 in the vitamin A and zinc group (22.2%, 95%CI = 20.0-24.3) than the zinc group (20.2%, 95%; CI = 18.7-21.7), vitamin A group (20.4%, 95%; CI = 18.6-22.3), and placebo group (14.9%, 95%; CI = 16.4-18.7). Almost similar observation was found from 0 month to 6 months for IL-12. The percent decrease in IFNγ was higher in the vitamin A and zinc group (21.1%, 95%; CI = 19.2-22.9) than the zinc group (17.1%, 95%; CI = 15.5-18.6), vitamin A group (16.5%, 95%; CI = 13.9-19.0), and placebo group (13.2, 95%; CI = 11.9-14.6). Similar observation was found from 0 month to 6 month [Figure 1] and [Table 3].
|Figure 1: Average percent change in cytokine levels from 0 month to 2 months and 6 months (pg/mL)|
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|Table - 3: Average percent change in cytokine levels from 0 to 2 and 6 month |
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The sputum conversion was significantly early among the patients of the vitamin A and zinc group (median = 7), zinc group (median = 8), and vitamin A group (median = 8) than the patients of the placebo group (median = 9) [Figure 2].
|Figure 2: Proportion of patients with sputum smears converting to negative during the first 12 weeks of intervention (pg/mL)|
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| Discussion|| |
The immune response upon active Mycobacterium tuberculosis infection is initiated by the recognition of the bacteria, mainly by macrophage and dendritic cells through toll-like receptors that stimulate the production of cytokines such as IL-12. IL-12 subsequently activates a T h 1 cell-dominated adaptive immune response that is largely responsible for containing the inflammation through the secretion of IFNγ. Increased concentrations of IL-12 and IFNγ are the hallmarks of active TB infection.
In this study, the sociodemographic characteristics of the patients were similar in all the groups. Sputum positivity grade 3 was most prevalent. More than one-third of patients of the vitamin A group (38.9%), zinc group (40%), vitamin A and zinc group (40.4%), and placebo group (38.3%) were in + 3 sputum smear grade. However, 33.3% of patients in the vitamin A group, 29.1% in zinc group, 36.5% in vitamin A and zinc group, and 34% in the placebo group were in +2 smear grade. An equal percentage of patients among all the groups were in +1 smear grade. However, differences among the groups were statistically insignificant (P > 0.05). Thus, all the four groups were statistically comparable.
In the present study, there was a significant increase in the IL-2 level in all the three intervention groups. Similar finding was reported by Christopher et al. in their study.  Sorayya et al. also assessed the effect of supplementation of high zinc or vitamin A on immune function. After 3 months of feeding with a zinc and vitamin A deficient diet, mice were assigned into four groups which, for an additional 2 months, received normal or high zinc, along with vitamin A deficient diet and normal or high vitamin A and a zincw deficient diet. Serum and intestinal mucosa immunoglobulin A (IgA) were determined and supernatants of splenocytes were used to assess IL-2. The mice, in that study, were maintained on a zinc deficient diet with normal or high vitamin A. It was also estimated that supplementation of high dose of vitamin A augmented production of the cytokine as compared to normal intake of the vitamin. High intake of zinc, along with vitamin A deficient diet significantly enhanced the secretion of IL-2. Moreover, animals fed with high doses of zinc showed increased IL-2 production than those that had normal intake of zinc. 
IL-12 is a cytokine produced by the phagocytic and antigen-presenting cells; it is an important activator of IFNγ production by the T cells and NK cells and provides an early switch in the differentiation of CD4+ T cells. Hence, IL-12 seems to be an important factor in determining the outcome of TB. , In the present study, the level of IL-12 was significantly increased from the baseline to 2 months and 6 months. The percentage increase was higher in IL-12 in the vitamin A and zinc group (22.2%, 95%; CI = 20.0-24.3) than the zinc group (20.2%, 95%; CI = 18.7-21.7), vitamin A group (20.4%, 95%; CI = 18.6-22.3), and placebo group (14.9%, 95%; CI = 16.4-18.7). An almost similar observation was found from 0 month to 6 months for IL-12. In a study, Verbon et al. reported that in patients with active TB, IL-12 concentrations were not different from those in contact and healthy controls.  Of considerable interest, however, was the fact that the patients with positive smears had significantly lower IL-12 serum levels than any of the other study groups.  Considering the essential role of IL-12 in a protective immune response to TB in mice  and the fact that positive smears are associated with a higher mycobacterial burden,  it is tempting to speculate that in humans too, IL-12 production plays a role in antimycobacterial host defence.
The study by Dlugovitzky et al. demonstrated that the serum levels of IFNγ were increased in patients with pulmonary TB, especially in mild and moderate TB cases compared to advanced TB.  There is in vitro evidence that TB patients with progressive disease fail to generate IFNγ in response to stimulation with mycobacterial antigens.  Other studies that evaluated serum IFNγ levels in TB patients showed increases relative to the control subjects. , According to our results, the level of IFNγ was significantly (P = 0.01) decreased in patients of vitamin A supplementation from 0 month (19.68 ± 1.48) to 2 months (16.97 ± 1.56) and 6 months (14.90 ± 1.33). Similar observations were found in zinc and vitamin A and zinc supplemented patients. No significant change in the IFNγ level was found in the placebo supplemented patients. In turn, IFNγ stimulates human macrophages to produce TNFα and 1,25-dihydroxyvitamin D3, both facilitate mycobacterial inhibition. 
| Conclusion|| |
Vitamin A and zinc deficiency affects the production of cytokines by diminishing the activity of T cells and B cells. The circulating cytokines play an especially important role in the pathogenesis of active pulmonary TB. We demonstrated that vitamin A and zinc may directly or indirectly influence the activation of cytokines. IL-2 helps in the mitosis of T cells that play an important role in cell mediated immunity. Under the influence of IL-12, these T cells produce IFNγ that make the macrophages super active against Mycobacterium tuberculosis. Vitamin A and zinc supplementation along with antitubercular treatment will certainly lead to a breakthrough and may reduce the duration of the therapy.
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Conflict of interest
There are no conflict of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]