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

Introduction: Tuberculosis (TB) remains a significant public health problem, with an estimated one-third of the world’s 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 “in vitro“-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.

Keywords: Cytokine, multidrug-resistant tuberculosis (MDR-TB), tuberculosis (TB), tumor necrosis factor-α (TNF-α)

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.

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.

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].

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].

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].

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.

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.

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.

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/1755-6783.162627

[Figure 1], [Figure 2], [Figure 3], [Figure 4]