Annals of Tropical Medicine and Public Health

ORIGINAL ARTICLE
Year
: 2015  |  Volume : 8  |  Issue : 6  |  Page : 258--261

Association of TNF-α serum levels with response to antitubercular treatment in MDR tuberculosis patients


Mohammad Shameem1, Nazish Fatima2, Nabeela2, Haris Manzoor Khan2,  
1 Department of TB and Respiratory Diseases, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
2 Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India

Correspondence Address:
Nazish Fatima
Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh - 202 002, Uttar Pradesh
India

Abstract

Introduction: Tuberculosis (TB) remains a significant public health problem, with an estimated one-third of the worldSQs population being infected. Cytokines play a major role in protection against Mycobacterium tuberculosis (M. tuberculosis) infection and regulate the immune responses at a cellular level. Most studies on cytokines during TB are from DQin vitroDQ-stimulated lymphoid cells with few reports on in vivo plasma levels. The aim of this study was to evaluate the levels of tumor necrosis factor-α (TNF-α) in new, undertreatment (UT), and multidrug-resistant (MDR) pulmonary and extrapulmonary cases. Materials and Methods: The study was conducted in the Department of Microbiology, Jawaharlal Nehru Medical College (J.N.M.C.), Aligarh Muslim University (A.M.U.), Aligarh, Uttar Pradesh, India. Results: The levels of TNF-α were measured in 76 serum samples from TB patients by an enzyme linked immunosorbent assay (ELISA) kit (Diaclone Sas, 1 BD, A Flemming Besancon Cedax, France), along with 10 healthy controls. Complete clinical, radiological and treatment data were collected. The TNF-α levels were elevated in new cases (P < 0.05) and MDR cases (P < 0.05) but not significantly for UT cases (P > 0.05). Conclusions: An understanding of this response may lead to an insight into the pathogenesis and novel therapies for TB.



How to cite this article:
Shameem M, Fatima N, Nabeela, Khan HM. Association of TNF-α serum levels with response to antitubercular treatment in MDR tuberculosis patients.Ann Trop Med Public Health 2015;8:258-261


How to cite this URL:
Shameem M, Fatima N, Nabeela, Khan HM. Association of TNF-α serum levels with response to antitubercular treatment in MDR tuberculosis patients. Ann Trop Med Public Health [serial online] 2015 [cited 2019 Nov 19 ];8:258-261
Available from: http://www.atmph.org/text.asp?2015/8/6/258/162627


Full Text

 Introduction



Tuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), remains the single largest infectious disease causing two to three million deaths annually. [1] In India, there are 500,000 deaths occurring annually due to TB. [2]

TB progression is associated with the immune status. It is known that the host protective immune response against this pathogen is mediated by cellular immunity, in which certain cytokines and T helper cell type 1 (T h 1) cells have a critical role. [3] Cytokines are molecules that mediate mainly the intercellular communication in the immune system, being produced by different cell types. Cytokines have pleiotropic and regulatory effects and participate in the host's defense and in inflammatory and tissue reparation processes [4] and also play a major role in the protection against M. tuberculosis infection and regulate the immune response at a cellular level.

TNF-α is one of the principal immune that plays a role in the control of infection and elimination of mycobacteria. [5],[6]

The main TNF-α-producing cells are activated macrophages, T-lymphocytes, and dendritic cells. [7],[8] There is a parity of data from in vivo studies as most studies on cytokines during TB are in vitro stimulated lymphoid cells; [9] understanding the mechanism involved in cell-mediated immune response against M. tuberculosis is of significant relevance in the development of effective control. With this background, we undertook this study to determine the variations in serum TNF-α levels in new TB-cured cases and multidrug-resistant (MDR)-TB cases and to correlate these variations with the usefulness of TNF-α as an early marker of MDR-TB cases.

 Materials and Methods



The present study was conducted at the Department of Microbiology, Jawaharlal Nehru Medical College (J.N.M.C.), Aligarh Muslim University (A.M.U.), Aligarh, Uttar Pradesh, India. Complete clinical, radiological, and treatment data were collected. Informed consent was obtained from all the subjects.

Sputum culture and drug susceptibility test

Sputum culture was positive for M. tuberculosis and was confirmed by the inoculation of samples on the Lφwenstein-Jensen (LJ) medium. Briefly, the sputum specimen were decontaminated with 4% NaOH and inoculated into the LJ medium. When the growth was detected as positive, drug susceptibility test was carried out on the LJ medium containing antiTB drugs. The "absolute concentration method" was used as previously described. The critical concentrations for isoniazid and rifampin were 0.2 μg/mL and 40 μg/mL, respectively.

Blood collection

Seventy six blood samples (3-5 mL) were collected in plain vacutainers from the patients before starting antiTB treatment (ATT) and after 3 months of ATT. The blood samples were centrifuged at 5,000 rpm for 10 min and then the serum samples were collected and stored at -20΀C until they were assayed.

Cytokines assay

For cytokine analysis, sandwich enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody sets (Diaclone SAS, Besanηon Cedex, France), Streptavidin-horseradish peroxidase (HRP) conjugate, and recombinant cytokines as the standard were used.

Briefly, 96 well plates were coated with cytokines (TNF-α) according to the manufacturers protocols. The samples were added to all the wells. Diluted biotinylated antiTNF-α samples were added and incubated at room temperature for 3 h. After washing twice, streptavidin-HRP was added and incubated for 30 min. 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well and incubated at room temperature for 12-15 min and the reaction was stopped by adding stop solution. The plate was read at 450 nm in an ELISA reader (Thermo Electron Corporation, Vantaa, Uusimaa, Greater Helsinki, Finland). The detection range of the assay was less than 8 pg/mL for TNF-α.

Statistical analysis

Receiver operating characteristic (ROC) curve and the other performance measures were performed using the statistical software MedCalc version 10.2.0.0 (Medcalc Bvba, Acacialan, Belgium). The graphs were all created using Microsoft Excel. The conventional 5% level of significance was used for all statistical tests.

 Results



Of the 76 patients who were enrolled, 33 were new TB cases, 22 new MDR-TB cases and 21 undertreatment (UT) MDR-TB cases, one was a case of pneumothorax, two cases were of pyopneumothorax, and one had diabetes mellitus. None of the patients were human immunodeficiency virus (HIV)-positive. Ten healthy individuals were included as healthy controls. The level of TNF-α was the highest in the new cases of TB but significantly decreased in the UT MDR-TB cases in comparison to the healthy controls (P < 0.05) [Figure 1] and [Figure 2], respectively].{Figure 1}{Figure 2}

The TNF-α level was significantly increased in the new MDR-TB cases compared to the UT MDR-TB cases (P < 0.05) [Figure 3]. {Figure 3}

The discriminatory power of assessing TNF-α as a marker for active TB is good. The TNF-α levels show no significant variations according to the site of involvement in the pulmonary versus the extra pulmonary cases [Figure 4].{Figure 4}

 Discussion



In the past, immunological studies conducted on cytokine production have focused on the "ex vivo0" cytokine production capacity of isolated peripheral blood mononuclear cells (PBMCs) or CD4 + T cells with inconsistent results. [10],[11],[12] Additionally "ex vivo" stimulated production of cytokines will not provide an insight into the exact interplay of various cytokines "in vivo."

The main TNF-α producing cells are activated macrophages, T-lymphocytes, and dendritic cells. [8] TNF-α increases the capacity of macrophages to phagocytose and kill the mycobacteria and stimulates apoptosis of the macrophages, depriving bacilli of the host cells and leading to death and presentation by the dendritic cells of the mycobacterial antigens. [13] In vivo TNF-α is required for the formation and maintenance of granulomas. Neutralization of TNF-α produced by mice chronically infected with M. tuberculosis-specific monoclonal antibodies disrupts the integrity of granulomas, exacerbates infection, and increases mortality. [14] TNF-α is the head mediator of the destruction of the pulmonary tissue. [15] Elevated levels of TNF-α are related to an excessive inflammation with necrosis and cachexy. [16]

TNF-α is produced at the site of disease in TB patients. [17] Early clinical deterioration is associated with a selective increase of TNF-α in plasma. In particular, patients with pulmonary TB accompanied by systemic manifestations (persistent fever, weight loss) showed increased TNF-α compared to the controls. [18]

Previous studies have shown higher serum levels of TNF-α in active TB patients than the controls. [6],[19],[20],[21] We found significantly raised serum TNF-α levels in the new TB cases than the healthy controls. Also, we found that serum TNF-α levels declined significantly in the UT cases (P < 0.05) but were elevated again in the MDR cases.

Similarly, Tang et al., [22] Portales-Pérez et al., [17] and Kawaguchi et al. [23] found decreased TNF-α levels in TB patients after therapy. However, Moura et al. [24] did not observe significant differences in TNF-α levels after treatment. These studies reinforce the believe that TNF-α has a role in both the physiopathology and in protective immunity against TB.

MDR-TB patients were found to have a higher TNF-α level as compared to the newly diagnosed TB patients (P < 0.005). We can, thus, hypothesize that TNF-α could be used as an early marker of drug sensitivity in patients undergoing TB treatment. We found a decrease in the TNF-α levels in MDR-TB patients following initiation of the treatment. It is worth pointing out that TNF-α could be used as a marker of response to second line antitubercular drugs in MDR-TB patients. TNF-α would prove to be an early indicator for discontinuation of therapy, especially among cases who are treated for more than 2 years. TNF-α levels showed no significant variations according to the site of involvement in the pulmonary or extrapulmonary TB cases.

 Conclusion



TNF-α is a dynamic cytokine and plays an important role in the pathogenesis of TB. Our study suggests that TNF-α serum levels could be used as surrogate markers of response to treatment in the new and the MDR-TB cases, along with the clinical parameters of disease activity such as x-ray of the chest and culture results.

 Acknowledgment



We are indebted to the Council of Science & Technology, Lucknow, Uttar Pradesh, India for providing financial assistance to carry out this research work. We thank Mr. Sanjay Sharma for his excellent technical expertise.

Conflict of interest

There are no conflicts of interest.

References

1World Health Organization. Anti-Tuberculosis Drug Resistance in the World: The WHO/IUTLD Global Project on Anti-Tuberculosis Drug Resistance Surveillance. Geneva, Switzerland: World Health Organization; 2001.
2Raja A. Immunology of tuberculosis. Indian J Med Res 2004;120:213-32.
3Kinjo Y, Kawakami K, Uezu K, Yara S, Miyagi K, Koguchi Y, et al. Contribution of IL-18 to Th1 response and host defense against infection by Mycobacterium tuberculosis: A comparative study with IL-12p40. J Immunol 2002;169:323-9.
4Gupta A, Kaul A, Tsolaki AG, Kishore U, Bhakta S. Mycobacterium tuberculosis: Immune evasion, latency and reactivation. Immunobiology 2012; 217:363-74.
5Andrade Júnior DR, Santos SA, Castro Id, Andrade DR. Correlation between serum tumor necrosis factor alpha levels and clinical severity of tuberculosis. Braz J Infect Dis 2008;12:226-33.
6Kart L, Buyukoglan H, Tekin IO, Altin R, Senturk Z, Gulmez I, et al. Correlation of serum tumor necrosis factor-alpha, interleukin-4 and soluble interleukin-2 receptor levels with radiologic and clinical manifestations in active pulmonary tuberculosis. Mediators Inflamm 2003;12:9-14.
7Barnes PF, Lu S, Abrams JS, Wang E, Yamamura M, Modlin RL. Cytokine production at the site of disease in human tuberculosis. Infect Immun 1993;61:3482-9.
8Serbina NV, Flynn JL. Early emergence of CD8(+) T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect Immun 1999;67:3980-8.
9Geffner L, Yokobori N, Basile J, Schierloh P, Balboa L, Romero MM, et al. Patients with multidrug-resistant tuberculosis display impaired Th1 responses and enhanced regulatory T-cell levels in response to an outbreak of multidrug-resistant Mycobacterium tuberculosis M and Ra strains. Infect Immun 2009;77:5025-34.
10Surcell HM, Troye-Blomberg M, Paulie S, Anderson G, Moreno C, Pasvol G, et al. Th1/Th2 profiles in tuberculosis based on the proliferation and cytokine responses of blood lymphocytes to mycobacterial antigens. Immunology 1994;81:171-6.
11Zhang M, Lin Y, Iyer DV, Gong J, Abrams JS, Barnes PF. T-cell cytokine response in human infection with Mycobacterium tuberculosis. Infect Immun 1995;63:3231-4.
12Lai CK, Ho S, Chan CH, Chan J, Choy D, Leung R, et al. Cytokine gene expression profile of circulating CD4+ T cells in active pulmonary tuberculosis. Chest 1997;111:606-11.
13Keane J, Remold HG, Kornfeld H. Virulent Mycobacterium tuberculosis strains evade apoptosis of infected alveolar macrophages. J Immunol 2000;164:2016-20.
14Robinson CM, Jung JY, Nau GJ. Interferon-g, tumor necrosis factor, and interleukin-18 cooperate to control growth of Mycobacterium tuberculosis in human macrophages. Cytokine 2012;60:233-41.
15Flynn JL, Chan J, Lin PL. Macrophages and control of granulomatous inflammation in tuberculosis. Mucosal Immunol. 2011;4:271-8.
16Ehlers S, Benini J, Held HD, Roeck C, Alber G, Uhlig S. Alphabeta T cell receptor-positive cells and interferon-gamma, but not inducible nitric oxide synthase, are critical for granuloma necrosis in a mouse model of mycobacteria-induced pulmonary immunopathology. J Exp Med 2001;194:1847-59.
17Portales-Pérez DP, Baranda L, Layseca E, Fierro NA, de la Fuente H, Rosenstein Y, et al. Comparative and prospective study of different immune parameters in healthy subjects at risk for tuberculosis and in tuberculosis patients. Clin Diagn Lab Immunol 2002;9:299-307.
18Bekker LG, Maartens G, Steyn L, Kaplan G. Selective increase in plasma tumor necrosis factor-alpha and concomitant clinical deterioration after initiating therapy in patients with severe tuberculosis. J Infect Dis 1998;178:580-4.
19Pereira CB, Palaci M, Leite OH, Duarte AJ, Benard G. Monocyte cytokine secretion in patients with pulmonary tuberculosis differs from that of healthy infected subjects and correlates with clinical manifestations. Microbes Infect 2004;6:25-33.
20Ameglio F, Casarini M, Capoluongo E, Mattia P, Puglisi G, Giosuè S. Post-treatment changes of six cytokines in active pulmonary tuberculosis: Differences between patients with stable or increased fibrosis. Int J Tuberc Lung Dis 2005;9:98-104.
21Nakaya M, Yoneda T, Yoshikawa M, Tsukaguchi K, Tokuyama T, Fu A, et al. The evaluation of interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) level in peripheral blood of patients with active pulmonary tuberculosis. Kekkaku 1995;70:461-6.
22Tang S, Xiao H, Fan Y, Wu F, Zhang Z, Li H, Yang Y. Changes of proinflammatory cytokines and their receptors in serum from patients with pulmonary tuberculosis. Zhonghua Jie He He Hu Xi Za Zhi 2002;25:325-9.
23Kawaguchi H, Ina Y, Ito S, Sato S, Sugiura Y, Tomita H, et al. Serum levels of solubule tumor necrosis factor (TNF) receptors in patients with pulmonary tuberculosis. Kekkaku 1996;71:259-65.
24Moura EP. Answer rating immune cellular and humoral before and after the treatment of tuberculoses pulmonary. Tese 2002. p. 90.