Year : 2016 | Volume
: 9 | Issue : 5 | Page : 327--330
Alteration of serum inflammatory cytokines in active pulmonary tuberculosis, following antitubercular treatment
Nazish Fatima1, Mohammad Shameem2, Nabeela1, Haris M Khan1,
1 Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
2 Department of TB and Respiratory Diseases, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh
Introduction: Mycobacterium tuberculosis (Mtb) infects one third of the world population. Despite advance in therapy, tuberculosis (TB) remains responsible for 2-3 million deaths annually. During TB, cytokines play a role in host defense. Many cytokines are produced during TB with a predominance of Th1 cytokines during the early stage and Th2 cytokines in the later stages of the infection. The present study aims to screen tumor necrosis factor-alpha (TNF-α), interferon gamma (IFN-γ), interleukin (IL)-12p70, IL-4, IL-6, and IL-1β among TB patients and controls. Materials and Methods: The study was conducted at the Department Of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, India. Different cytokine levels were measured in 65 samples of TB patients of whom 30 (46.1%) were new TB cases and 35 (53.8%) were suspected multidrug-resistant (MDR)-TB cases along with 15 bacille Calmette-Guérin (BCG) vaccinated healthy controls by enzyme-linked immunosorbent assay (ELISA) (Diaclone, France). Complete clinical, radiological, and treatment data were collected. Informed consent was taken from all subjects. Study was approved by the Institutional Bioethical Committee, Jawahar Lal Nehru Medical College. Statistical analysis had been performed by using Sigma plot (10.0). Results: The serum concentration of TNF-α, IFN-γ, IL-12p 70 , IL-1β, and IL-6 in patients with active TB and MDR-TB was elevated than in patients after anti-TB treatment (P < 0.001), in contacts (P < 0.001) and in controls (P < 0.001). It was found that IL-6 seems critical in resistance to TB. IL-4 concentration increased only in MDR-TB patients (P < 0.001) as compared to controls. All these cytokines showed no significant variations according to the site of involvement in pulmonary versus extrapulmonary TB cases. Conclusions: Measuring the serum levels of several cytokines may be useful for evaluating the activity of TB disease and monitoring the clinical effects of anti-tubercular treatment (ATT). The serum TNF-α, IFN-γ, IL-12p 70 , IL-1β and IL-6 level can be a useful marker to diagnose the effectiveness of therapy in the patients.
|How to cite this article:|
Fatima N, Shameem M, Nabeela, Khan HM. Alteration of serum inflammatory cytokines in active pulmonary tuberculosis, following antitubercular treatment.Ann Trop Med Public Health 2016;9:327-330
|How to cite this URL:|
Fatima N, Shameem M, Nabeela, Khan HM. Alteration of serum inflammatory cytokines in active pulmonary tuberculosis, following antitubercular treatment. Ann Trop Med Public Health [serial online] 2016 [cited 2019 Nov 21 ];9:327-330
Available from: http://www.atmph.org/text.asp?2016/9/5/327/190179
Despite the availability of chemotherapies to combat tuberculosis (TB), the TB epidemic is not under control and caseloads increase every year. , It has been predicted that if TB control is not improved, one billion people will be newly infected with mycobacterium tuberculosis (Mtb) by 2020. 
Multidrug-resistant tuberculosis (MDR-TB), which is a worldwide clinical problem, is associated with high morbidity and mortality, in spite of long-term survival of infected immunocompetent patients. It is reported to be a frequent clinical infection and might be prevalent in as much as 70% of infected cases. 
When TB infection occurs, a variety of chemokines and cytokines are secreted from infected cells and tissue macrophages. Tumor necrosis factor-alpha (TNF-α) increase early in the disease and take part in the pathogenesis and prevention of mycobacterial infection. Additionally, TNF-α appears crucial for the formation of Mtb-constraining granulomas, infection control, and elimination of mycobacteria. 
Immunologic resistance and susceptibility to intracellular pathogens are mediated by cluster of differentiation 4 (CD4) + T cells with specific patterns of cytokine secretion. Th1 cells that produce gamma interferon (IFN-γ) confer resistance to infection with mycobacteria.  IFN-γ produced by a variety of cells is involved in the immune response against Mtb. IFN-γ is a soluble protein.
At the site of Mtb infection, it is the major cytokine of type 1 T helper (Th1) cells, therefore, as it protects against Mtb.  Interleukin (IL)-12p70 is known to play an important role in anti-TB cell mediated immunity  and is the major cytokine for directing primary Th1 differentiation in CD4+ T cells in vitro and in vivo. 
IL-1β is produced at the site of Mtb infection where IL-1β stimulates a protective proinflammatory response. The intense proinflammatory response is associated with tissue damage that is neutralized by the release of anti-inflammatory cytokines. 
IL-6 is produced by TH2 cells, but it may be produced by TH1 cells as well and it is associated with protection against Mtb. IL-6 seems critical in resistance to TB. 
IL-4 is produced by T-helper cells and acts as a cofactor in activation of humoral immunity by activation of B-cell and T-cell proliferation and differentiation. , The high expression of IL-4 has been implicated as a virulence factor, both for the anti-inflammatory ability and for its apparent capacity to promote tissue damage in association with TNF-α as well.  The present study aims to screen TNF-α, IFN-γ, IL-12p 70 , IL-4, IL-6, and IL-1β among TB patients and controls.
Materials and Methods
The present study was conducted at the Department Of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, India. Samples were collected from the Department of TB and Chest Diseases, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India. A complete clinical and radiological data was collected. Informed consent was taken from all subjects. Institutional Bioethical Committee, Jawahar Lal Nehru Medical College.
Microscopy and sputum culture
Morning sputum samples were collected from the TB patients on 3 consecutive days. TB diagnosis was based on clinical sign and symptoms and radiological confirmation of pulmonary and extrapulmonary TB, presence of acid-fast bacilli (AFB) in the sputum smear following Ziehl-Neelsen test. Sputum culture positivity for Mtb was confirmed by inoculation of samples on Löwenstein Jensen (LJ) media. The reference strain H37Rv was used as control.
A total number of 76 sera samples were obtained from patients with active TB before treatment. Patients of all age groups with both pulmonary and extrapulmonary TB were included. Extrapulmonary sites included were pleural, lymph nodes, soft tissues, meninges, gastrointestinal, bone and joints, and disseminated disease. Sera was additionally obtained from patients with TB who had been treated for at least 2 weeks, but had not yet completed therapy at the time of blood sampling and from patients who had completed anti-TB therapy. Records of all patients with active TB was reviewed for clinical data such as fever (rectal temperature >38 ° C), anorexia, skin test, chest X-Ray, bacille Calmette-Guιrin (BCG) vaccination, direct microscopy, and culture results. Furthermore, 15 sera samples were obtained from persons who had been in close contact with patients with smear-positive TB and from healthy (all skin tests - negative) controls.
Evaluation of chest radiograph
All TB patients had undergone plain posteroanterior and lateral chest radiography. Chest radiographs were evaluated for the presence and distribution of signs relating to the active pulmonary TB that include miliary patterns, cavity, fibro-cavity, segmental consolidation, lobar consolidation, infiltrate, and patchy opacity.
Measurements of all cytokines were performed by sandwich enzyme-linked immunosorbent assay (ELISA) method and using commercially available ELISA kits. ELISA test was performed following the supplier's instructions. The absorbance values were analyzed by the ELISA reader at 450 nm (Thermo Electron Corporation, Vantaa, Finland).
All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) Statistics (version-20). Receiver operating characteristic (ROC) curve and other performance measures were performed using the statistical software Med Calc (version 10.2.0.0). Pairwise analysis using Chi-square test was done to compare differences in cytokine levels between groups of patients. Analysis and results were considered significantly different at P < 0.05.
In this study, 76 patients were enrolled, in which 33 (43.42%) were new, 24 (31.57%) were under-treatment (UT), 19 (25%) were MDR-TB cases, one (1.31%) had pneumothorax, two (2.63%) had pyopneumothorax while one (1.31%) had diabetes mellitus. None of the patients were human immunodeficiency virus (HIV) positive. Fifteen BCG vaccinated healthy individuals were included as controls.
TNF-α levels was elevated in new and MDR-TB patients compared to healthy controls (P < 0.001, P < 0.001, respectively), but levels of TNF-α is less significant in undertreatment cases (P > 0.001).
The levels of IFN-γ were elevated in new and MDR-TB cases compared to controls (P < 0.001). There was less significant difference between undertreatment patients and controls.
IL-12p70 and IL-1β concentrations were increased in new TB cases and MDR TB cases compared to under-treatment and healthy controls (P < 0.001).
IL-6 levels were significantly elevated in patients with TB during and after treatment compared to controls (P < 0.001).
IL-4 serum levels were low in all groups, however, significantly increased levels were seen in MDR cases as compared to controls (P < 0.001).
All these cytokines (TNF-α, IFN-γ, IL-12p 70, IL-4, and IL-1β) showed no significant variations according to the site of involvement in pulmonary versus extrapulmonary TB cases (P > 0.001).
The serum levels of inflammatory cytokines TNF-α, IFN-γ, IL-12p 70, IL-4, and IL-1β were highly elevated in new and MDR-TB patients. Upon Mtb infection of host macrophages, a number of pro-inflammatory cytokines including TNF-α, IFN-γ, IL-12p 70, IL-4, and IL-1β, IL-6 are secreted in excess. 
In our study, we found raised levels of TNF-α in new and MDR-TB cases, on the other hand, levels of TNF-α decreased in undertreatment TB cases. TNF-α increases the capacity of phagocytes, kills mycobacteria, and stimulates apoptosis of macrophages depriving bacilli of host cells.  We found the increased levels of TNF-α in MDR-TB cases compared to the undertreatment in our previous study as well.  Periodic measurement of TNF-α through ELISA method could help to understand TB outcome. Elevated levels of TNF-α are related to an excessive inflammation with necrosis and chachexy. 
IFN-γ levels of confirmed TB patients were higher than that of clinically diagnosed TB patients. This may be due to the fact that most of clinically diagnosed TB patients were on ATT and, therefore, the healing effect on granuloma could reduce the number of local and circulating IFN-γ-producing activated T-cells.
There are some studies in which investigators have reported increased levels of IFN-γ in pleural effusion  or in the sputum  and observed a correlation between IFN-γ and disease activity. We did not perform this comparison because the majority of our community is randomly immunized with BCG vaccination that could produce variable and unpredictable results.
In Mtb infection, one of the earliest events of activation of cell-mediated immunity involves the production of IL-12p70.  In Mtb infection, IL-12p70 level is elevated due to the activation of macrophages upon successful interaction of the toll-like receptors (TLRs) present in the phagosome and cell surfaces with the Mtb-associated antigen/s.  We found an increased level of IL-12p70 in active TB cases compared to IL-6, which is usually thought of as a type 2 cytokine, but it may be produced by type 1 cells as well.  Although, it has been shown that IL-6 promotes the intracellular growth of mycobacteria in monocytes, , it was found that IL-6 seems critical in resistance to TB.  In our study, we showed that IL-6 serum levels were elevated in the vast majority of patients with active TB and MDR-TB. IL-6 levels do not increase in anti-TB treatment.
IL-1β is produced at the site of Mtb infection where IL-1β stimulates a protective proinflammatory response.  The intense proinflammatory response is associated with tissue damage that is neutralized by the release of anti-inflammatory cytokines.  Our study showed that the levels of IL-1β elevated in new and MDR-TB cases compared to controls.
IL-4 represents as one of the cytokines produced by Th 2 cells and acts as a cofactor in activation of humoral immunity by activation of B-cell and T-cell proliferation and differentiation. Our study revealed significantly raised IL-4 levels in MDR-TB patients. Shahemabadi et al. found elevated levels of IL-4 in TB patients.  We did not find elevated levels of IL-4 in new and undertreatment TB cases.
The present study identified that cytokines can serve as biomarkers of anti-TB treatment response. The above findings are encouraging, as they support the concept of host biomarkers for the prediction differential TB treatment responses. The levels of these cytokines is being used to diagnose active TB infection, therefore, where the prevalence of TB is low and clinical finding suggest TB, than a positive test may indicate active TB infection. Both " in vitro" and " in vivo" studies are required to establish the immunoregulatory and diagnostic roles of these cytokines in our populations.
Financial support and sponsorship
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.
Conflicts of interest
There are no conflicts of interest.
|1||Global Tuberculosis Control: Surveillance, Planning, Financing. WHO Report 2008. Geneva, Switzerland: Nonserial Publication; 2008. p. 17-37.|
|2||Dolin PJ, Raviglione MC, Kochi A. Global tubercolosis incidence and mortality during 1990-2000. Bull World Health Organ 1994;72:213-20.|
|3||World Health Organization. World Health Organization Fact Sheet. Geneva, Switzerland: World Health Organization; 2002. p. 12-56.|
|4||Goble M, Iseman MD, Madsen LA, Waite D, Ackerson L, Horsburgh CR Jr. et al. Treatment of 171 patients with pulmonary tuberculosis resistant to isoniazid and rifampin. N Engl J Med 1993;328:527-32.|
|5||Kindler V, Sappino AP, Grau GE, Piguet PF, Vassalli P. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 1989;56:731-40. |
|6||Orme IM, Roberts AD, Griffin JP, Abrams JS. Cytokine secretion by CD4 T lymphocytes acquired in response to Mycobacterium tuberculosis infection. J Immunol 1993;151:518-25.|
|7||Lewinsohn DM, Briden AL, Reed SG, Grabstein KH, Alderson MR. Mycobacterium tuberculosis-reactive CD8+ T lymphocytes: The relative contribution of classical versus nonclassical HLA restriction. J Immunol 2000;165:925-30. |
|8||Trinchieri G, Gerosa F. Immunoregulation by interleukin-12. J Leukoc Biol 1996;59:505-11.|
|9||Cooper AM, Roberts AD, Rhoades ER, Callahan JE, Getzy DM, Orme IM. et al. The role of interleukin-12 in acquired immunity to Mycobacterium tuberculosis infection. Immunology 1995; 84:423-32.|
|10||van Crevel R, Ottenhoff TH, van der Meer JW. Innate immunity to Mycobacterium tuberculosis. Clin Microbiol Rev 2002;15:294-309.|
|11||Verbon A, Juffermans N, Van Deventer SJ, Speelman P, Van Deutekom H, Van Der Poll T. et al. Serum concentrations of cytokines in patients with active tuberculosis (TB) and after treatment. Clin Exp Immunol 1999;115:110-3.|
|12||Surcel HM, Troye-Blomberg M, Paulie S, Andersson G, Moreno C, Pasvol G, et al. Th1/Th2 profiles in tuberculosis, based on the proliferation and cytokine response of blood lymphocytes to mycobacterial antigens. Immunology 1994;81:171-6.|
|13||Seah GT, Rook GA. IL-4 influences apoptosis of mycobacterium-reactive lymphocytes in the presence of TNF-alpha. J Immunol 2001;167:1230-7.|
|14||Lin Y, Zhang M, Hofman FM, Gong J, Barnes PF. Absence of a prominent Th2 cytokine response in human tuberculosis. Infect Immun 1996;64:1351-6.|
|15||Serbina 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.|
|16||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;8:258-61.|
|17||Dahir Ramos de Andrade Júnior, Sânia Alves dos Santos, Isac de Castro, Dahir Ramos de Andrade. Correlation between serum tumor necrosis factor alpha levels and clinical severity of tuberculosis. Braz J Infect Dis 2008;12:3.|
|18||Ribera E, Ocaña I, Martinez-Vazquez JM, Rossell M, Español T, Ruibal A. et al. High level of interferon gamma in tuberculous pleural effusion. Chest 1988;93:308-1.|
|19||Riberio-Rodrigues R, Resende Co T, Johnson JL, Ribeiro F, Palaci M, Sá RT, et al. Sputum cytokine levels in patients with pulmonary tuberculosis as early markers of mycobacterial clearance. Clin Diagn Lab Immunol 2002;9:818-23.|
|20||Flynn JL, Chan J. Immunology of tuberculosis. Annu Rev Immunol 2001;19:93-129.|
|21||Zuñiga J, Torres-García D, Santos-Mendoza T, Rodriguez-Reyna TS, Granados J, Yunis EJ. et al. Cellular and humoral mechanisms involved in the control of tuberculosis. Clin Dev Immunol 2012;2012:193923.|
|22||Van Snick J. Interleukin-6: An overview. Annu Rev Immunol 1990;8:253-78.|
|23||Wallis RS, Ellner JJ. Cytokines and tuberculosis. J Leukoc Biol 1994;55:676-81.|
|24||Shiratsuchi H, Johnson JL, Ellner JJ. Bidirectional effects of cytokines on the growth of Mycobacterium avium within human monocytes. J Immunol 1991;146:3165-70.|
|25||Ladel CH, Blum C, Dreher A, Reifenberg K, Kopf M, Kaufmann SH. et al. Lethal tuberculosis in interleukin-6-deficient mice. Infect Immun 1997;65:4843-9.|
|26||Tang S, Xiao H, Fan Y, Wu F, Zhang Z, Li H. et al. Changes of proinflammatory cytokines and their receptors in serum from patients with pulmonary tuberculosis. 2002;25:325-9.|
|27||Shahemabadi AS, Hosseini AZ, Shaghsempour S, Masjedi MR, Rayani M, Pouramiri M. et al. Evaluation of T cell immune responses in multi-drug resistant tuberculosis (MDR-TB) patients to mycobacterium tuberculosis total lipid antigens. Clin Exp Immunol 2007;149:285-94.|