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Year : 2020  |  Volume : 9  |  Issue : 2  |  Page : 185-189

Plasma levels of tumor necrosis factor-alpha, interferon-gamma, inducible nitric oxide synthase, and 3-nitrotyrosine in drug-resistant and drug-sensitive pulmonary tuberculosis patients, Ibadan, Nigeria

1 Department of Chemical Pathology, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria
2 Department of Immunology, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria
3 Department of Virology, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Oyo State, Nigeria
4 Centre for Innovation in Global Health Technologies, North-western University, Evanston, Illinois, USA

Date of Web Publication29-May-2020

Correspondence Address:
Elizabeth Bosede Bolajoko
Department of Chemical Pathology, Faculty of Basic Medical Sciences, College of Medicine, University of Ibadan, Ibadan, Oyo State
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_63_20

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Background: Nigeria is one of the countries with a high burden of tuberculosis (TB) in the world. TB associated inflammation is reported to be central to progression from latent TB to active TB or drug sensitive TB (DSTB) to drug resistant TB (DRTB). Inflammatory cytokines, especially interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), act synergistically in the control of TB infection. They activate macrophages to produce effector molecules such as inducible nitric oxide synthase (iNOS), nitric oxide, and ultimately 3-nitrotyrosines(3-NTs), which are involved in the control of TB. This study investigated the potential involvement of TNF-α, IFN-γ, iNOS, and 3-NT in differentiating DRTB and DSTB in Ibadan, Nigeria. Methods: One hundred participants above 18 years were recruited into this study and were grouped as follows: 32 DRTB, 34 DSTB, and 34 apparently healthy controls. Plasma from the patients was used for the analyses of inflammatory (TNF α and IFN-γ) and oxidative stress (iNOS and 3-NT) biomarkers using the ELISA. Mann–Whitney test was applied for the statistical test. Results: Mean levels of plasma TNF-α, IFN-γ, iNOS, and 3-NT were higher in DRTB (19.74 ± 3.62 pg/mL, 4.41 ± 0.96 pg/mL, 1791.07 ± 419.42 pg/mL, and 20.27 ± 1.80 ng/mL, respectively) and DSTB (17.02 ± 1.84 pg/mL, 5.59 ± 1.40 pg/mL, 2823.42 ± 685.32 pg/mL, and 25.06 ± 2.15 ng/mL, respectively) compared with controls (12.18 ± 0.92 pg/mL, 1.58 ± 0.21 pg/mL, 1275.86 ± 166.12 pg/mL, and 19.98 ± 1.23 ng/mL, respectively). In addition, higher plasma levels of IFN-γ (P > 0.05), iNOS (P > 0.05), and 3-NT (P < 0.05) were observed in DSTB compared with DRTB patients. Conclusion: The 3-NT may be used as differentiating markers of DSTB from DRTB.

Keywords: 3-nitrotyrosine, inducible nitric oxide synthase, inflammatory cytokines, pulmonary tuberculosis

How to cite this article:
Bolajoko EB, Arinola OG, Odaibo GN, Maiga M. Plasma levels of tumor necrosis factor-alpha, interferon-gamma, inducible nitric oxide synthase, and 3-nitrotyrosine in drug-resistant and drug-sensitive pulmonary tuberculosis patients, Ibadan, Nigeria. Int J Mycobacteriol 2020;9:185-9

How to cite this URL:
Bolajoko EB, Arinola OG, Odaibo GN, Maiga M. Plasma levels of tumor necrosis factor-alpha, interferon-gamma, inducible nitric oxide synthase, and 3-nitrotyrosine in drug-resistant and drug-sensitive pulmonary tuberculosis patients, Ibadan, Nigeria. Int J Mycobacteriol [serial online] 2020 [cited 2021 Dec 5];9:185-9. Available from: https://www.ijmyco.org/text.asp?2020/9/2/185/285236

  Introduction Top

Globally, tuberculosis (TB) is an infectious disease of significant public health importance, which is caused mainly by Mycobacterium tuberculosis (MTB).[1],[2],[3] In 2018, an estimate of 10 million people were affected with TB worldwide. The average incidence of new cases among the countries of the world was 130/100,000 population.[4] This burden of TB varies immensely geographically with the highest contributions found in the WHO regions of South-east Asia (44%), Africa (24%), and the Western Pacific (18%), whereas Eastern Mediterranean (8%), the Americas (3%), and Europe (3%) contribute smaller percentages.[4] Nigeria ranks the sixth among the 30 high-TB burden countries in the world,[4] with an average incidence of 429,000 cases of TB reported in 2018.[5]

The development of new TB or progression to active TB from latent TB is related to the host-immune status.[6],[7] Therefore, the quality of the host-defense system plays an important role in preventing the spread of MTB and also determines the severity of infection with MTB.[1] The host-protective immune response against the bacillus, during the early stage of infection, is mediated by the cellular immunity. During this process, certain cytokines such as tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukins-2,-6,-12,-15,-17,-18,-23, and-27, as well as T-helper-1 (Th1) cells play crucial roles.[6],[8],[9] Among these cytokines, IFN-γ and TNF-α have been reported to be at the cornerstone of the antimycobacterial cytokine cascade. They drive the formation and maintenance of inflammatory lesions during infection (granuloma and cavity) that are mediated by TNF-α acting in synergy with IFN-γ in the activation of macrophages. Consequently, producing effector molecules, including inducible nitric oxide synthase (iNOS) and nitric oxide (˙NO) among others.[9],[10],[11],[12],[13],[14] The ˙NO produced by iNOS in the activated macrophages reacts with superoxide radicals (O2˙¯) to form peroxynitrite (ONOO¯), which is an unstable metabolite. Protein tyrosine residues are highly susceptible to ONOO¯-dependent nitration reactions producing 3-nitrotyrosine (3-NT) that are more stable end-products.[15]

However, in Nigeria, there is a paucity of data on the potential role of TNF-α, IFN-γ, especially iNOS and 3-NT in drug-resistant (DR) and drug-sensitive (DS) pulmonary TB and their utility as biomarkers. Therefore, this study investigated the potentials of TNF-α, IFN-γ, iNOS, and 3-NT in differentiating DRTB from DSTB patients in Ibadan, Nigeria.

  Methods Top


The study protocol was carried out in accordance with the declaration of Helsinki and was approved by the joint University of Ibadan/University College Hospital Institutional Research Ethics Committee. Informed consent was sought and obtained from each participant before recruitment into this study.

Selection and description of participants

One hundred male and female participants were recruited into this study based on the following inclusion criteria: (i) >18 years of age, (ii) nonpregnant/lactating female, (iii) GeneXPert polymerase chain reaction positive pulmonary TB (DSTB or DRTB) participants, and (iv) apparently healthy participants without clinical symptoms of TB controls. Participants were excluded if they did not meet these criteria. Thirty-two DRTB and 34 DSTB were recruited from the Jericho Chest Clinic, Ibadan, and 34 controls were recruited from the University College Hospital, Ibadan, Nigeria.

Collection of blood

Four milliliters of whole blood samples were collected from each participant into ethylenediaminetetraacetic acid tubes. All blood samples were centrifuged at 4000 rpm for 10 min to extract plasma for the determination of TNF-α, IFN-γ, iNOS, and 3-NT. The plasma samples were stored at −20°C until analysis.

Determination tumor necrosis factor-alpha and interferon-gamma

The TNF-α and IFN-γ were measured in EDTA plasma using the ELISA kit (Abcam Inc. 1 Kendall Square, Suite B2304 Cambridge, MA 02139-1517). Samples were prepared according to the manufacturer's protocol and followed this principle. The wells of the microtiter strips have been coated with a monoclonal antibody specific for TNF-α or IFN-γ. Samples, including standards of known TNF-α or IFN-γ concentrations, control specimens or unknowns are pipetted into these wells. The standards or samples and a biotinylated monoclonal antibody specific for TNF-α or IFN-γ are simultaneously incubated for 3 h at room temperature and washed. After washing, the enzyme streptavidin-horseradish peroxidase (streptavidin-HRP), that binds the biotinylated antibody is added, incubated for 30 min at room temperature and then washed. Tetramethylbenzidine (TMB) substrate solution is added, which acts on the bound enzyme to induce a colored reaction product. This is read on spectrophotometer using 450 nm as the primary wavelength and 620 nm as the reference wavelength (ELISA reader: Spectra Max Plus 384 Molecular Devices LLC, USA). The intensity of these colored products is directly proportional to the concentration of TNF-α or IFN-γ present in the samples.

Determination of inducible nitric oxide synthase and 3-nitrotyrosine

The iNOS and 3-NT were measured in EDTA plasma using the ELISA kit (Elabscience Biotechnology Inc., Wuhan, Hubei, China). Samples were prepared according to the manufacturer's protocol. The method is based on the principle of competitive-ELISA. In this principle, iNOS or 3-NT has been precoated on the microtiter plate provided. During the reaction, iNOS or 3-NT in the sample or standard competes with fixed amount of iNOS or 3-NT on the solid-phase supporter for sites on the biotinylated detection antibody specific for iNOS or 3-NT. Excess conjugate and unbound sample or standard are washed from the plate and Avidin conjugated to HRP are added to each microplate well and incubated. Then, a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by the addition of stop solution, and the color change is measured spectrophotometrically at a wavelength of 450 nm with an ELISA reader (Spectra Max Plus 384 Molecular Devices LLC, USA). The concentration of iNOS or 3-NT in the samples is then determined by comparing the optical density of the samples to the standard curve.

Statistical analysis

All the data were presented as mean ± standard error of the mean. The data were subjected to the statistical analysis using the Statistical Package for the Social Sciences software version 20 (IBM SPSS, Armonk, NY, USA). The mean values of measured markers were compared between the groups using the Mann–Whitney tests. P < 0.05 was considered as being statistically significant.

  Results Top

The ages of participants were not significantly different and ranged from 18 to 70 years, with a mean age of 34.94 ± 1.72 for DRTB, 38.00 ± 2.45 for DSTB, and 34.59 ± 2.09 for control participants.

Plasma levels of IFN-γ were significantly higher, whereas the levels of TNF-α, 3-NT, and iNOS were higher but not statistically significant in DRTB compared with controls. Similarly, when DSTB were compared with controls, higher plasma levels of IFN-γ and 3-NT (P < 0.05), TNF-α and iNOS (P > 0.05) were found. However, when TNF-α, IFN-γ, 3-NT, and iNOS levels in DRTB were compared with DSTB, no significant differences were observed [Table 1].
Table 1: Comparison of mean tumor necrosis factor-alpha, interferon-gamma, 3-nitrotyrosine, and inducible nitric oxide synthase levels in plasma of participants

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  Discussion Top

The development of TB or reactivation of latent TB infection depends on the host-immune status. The protective immunity against the pathogen is mediated by certain cytokines, in which TNF-α and IFN-γ have been identified as the major cytokines involved in the control of bacilli infection.[14],[16] In this study, the data revealed the higher levels of plasma TNF-α in TB participants compared with apparently healthy controls. The involvement of TNF-α in the pathogenesis of both local and systemic inflammatory conditions has been reported.[17] The increase in levels of TNF-α in TB patients has been proposed to be one of the causes of systemic effects during active TB disease such as fever, weight loss, and immunosuppression among others in people with active TB.[18],[19] The finding in this study is consistent with other studies.[2],[19] In addition, high-plasma levels of TNF-α were seen in DRTB compared with DSTB. This agrees with the work of Onifade et al.[19] and Basingnaa et al.[20] The elevated levels of TNF-α, especially in the DRTB, may be due to significant tissue necrosis that occurs during the incident of the disease. Furthermore, the mutations on drug-resistance genes, which are essential genes for bacterial survival and interactions with the host, may lead to a stronger inflammatory response. The lack of TB drug-effectiveness on DRTB certainly contributes to an increased pathology in these patients.

IFN-γ, at the site of mycobacterial infection, is the main cytokine of Th1 cells that protect the host against MTB.[21] In synergy with TNF-α, it activates the macrophages to produce reactive nitrogen species that help in the killing of intracellular mycobacteria,[14] thereby proffering protection against the bacilli. This current study revealed a significantly higher plasma level of IFN-γ in TB participants compared with the controls. This may be because in mycobacterial infection, more IFN-γ is required to activate the macrophages, stimulating the anti-microbial activities that destroy the intracellular mycobacteria, therefore, proffering protection to the host. This finding agrees with other studies by Abhimanyu et al.[1] and Obeagu et al.[22] However, a higher level was observed in DSTB compared with DRTB, this is contrary to the study of Basingnaa et al.[20] where the higher levels of IFN-γ was reported in multidrug-resistant TB compared with DSTB. The higher values observed in DSTB may be a preventive mechanism against the intracellular mycobacteria from becoming resistant to anti-TB drugs.

The role of iNOS in mycobacterial infections has been emphasized in both clinical and especially experimental studies over the years.[23],[24] iNOS catalyzes the production of ˙NO by converting arginine into citrulline and ˙NO. This is a macrophage-derived radical that is highly toxic to intracellular mycobacteria.[23],[25] Therefore, evaluating the activities of iNOS can be used as a marker for ˙NO concentration. In Nigeria, there is a paucity of data on direct measurement of iNOS activities and 3-NT levels in TB; however, levels of ˙NO have been reported. In this study, plasma iNOS activities were evaluated and observed to be significantly higher in TB participants, with DSTB having higher activities than the DRTB, compared with controls. This finding is not surprising since the levels of IFN-γ, the main cytokine in mycobacterial infection, is also high. It is believed that the higher IFN-γ level in TB stimulates the catalytic activities of iNOS to produce more of the highly toxic radical ˙NO required for the killing of the pathogen. This study agrees with other studies that reported the presence of iNOS in human TB[26] and in bovine TB.[11] Since iNOS activities may be used as a marker for ˙NO concentration, the finding in this study is in contrast with the study of Edem and Arinola[27] where lower levels of ˙NO were reported in TB patients.

3-NT is an indicator of iNOS activity and also a marker of ˙NO.[11] It is formed when ONOŌ, generated from the reaction of ˙NO and O2˙̄ radical, react with tyrosinated proteins.[15] Indeed, NT has been described as a ”fingerprint” of ˙NO production, suggesting the functionally active iNOS.[15] A study has shown the possibility that nitration of mycobacteria with 3-NT production as well as damage to proteins and DNA may affect mycobacterial survival within the phagosome.[11] In this study, 3-NT was found to be higher, but not significant, in TB participants compared with the controls. This level was significantly higher in DSTB than DRTB. The higher level of 3-NT in TB participants of this study agrees with other studies, where 3-NT was found in TB patients.[26] The higher value, especially in DSTB, further collaborates the higher levels of IFN-γ, and higher activity of iNOS observed in this group of TB, and therefore, suggesting the prevention from progression to DRTB.

The result of this study proposed that the generation of reactive oxygen/nitrogen intermediates such as iNOS, ˙NO, and ultimately 3-NT by neutrophils and macrophages in TB patients could be modulated through micronutrient intervention for the benefit of the patients. In addition, the data of this study revealed higher IFN-γ and TNF-α in TB patients, which support previous findings of weight loss in TB patients.[18],[28],[29] The use of relatively small sample size confers the limitation on the discussion relating to inflammatory and oxidative stress biomarkers. Therefore, relating oxidative stress biomarkers with weight loss and large sample size in TB patients are suggested as further study.

  Conclusions Top

The levels of TNF-α, IFN-γ, iNOS, and 3-NT were higher in TB compared with apparently healthy control participants. DSTB patients demonstrated higher values than DRTB except for TNF-α, which was lower in DSTB patients. Therefore, 3-NT may be used as differentiating markers of DSTB from DRTB.


We appreciate Dr. El T. Osman, the Director of Damien Foundation Belgium, Ibadan, who facilitated our recruitment at the Jericho Chest Clinic. Dr. O. A. Adeyemo and Dr. F. A. Taleatu for patient recruitment at the Jericho Chest Clinic. Furthermore, the technical support of Dr. V. F. Edem of Department of Immunology, College of Medicine, University of Ibadan, Nigeria.

Financial support and sponsorship

This study was financially supported by the Award number D43TW010140 from the Fogarty International Center, National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Fogarty International Center of the National Institutes of Health.

Conflicts of interest

There are no conflicts of interest.

  References Top

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