Introduction Tuberculosis

Introduction
Tuberculosis (TBC), a highly infectious chronic respiratory disease caused by Mycobacterium tuberculosis (Mtb) infection, remains one of the world’s major causes of illness and death despite of various anti-mycobacterial therapies. According to WHO global Tuberculosis Report from 2014, infection developed into active tuberculosis in 9.0 million cases, resulting in 1.5 million deaths worldwide (1). It is known that host protective immune response against Mtb is mediated by cellular immunity, in which certain cytokines and Th1 cells have a critical role (2). Understanding the mechanisms involved in this response, and in particular the function of the cytokine network involved in this disease, is of significant relevance to reach advances in the development of effective control and prevention (3).
Tumor necrosis factor (TNF-?) plays a major role in the initial and long-term control of TBC. It was described that mycobacteria decreases the production of TNF-? in human peripheral blood mononucleated cells (PBMCs), skill which probably contributes to its ability to establish chronic infections (4). Produced by macrophages, lymphocytes, neutrophils, and some endothelial cells, TNF-? coordinates the inflammatory response via induction of other cytokines (IL-1 and IL-6), and the recruitment of immune and inflammatory cells through the induction of chemokine and supraregulation of adhesion molecules (5). TNF-? increases the capacity of macrophages to phagocytose and kill mycobacteria and stimulates apoptosis of macrophages, depriving bacilli of host cells and leading to death and presentation by dendritic cells of mycobacterial antigens (6).
In vivo TNF-? is required for the formation and maintenance of granulomas. Experimental models have shown that TNF-? plays an important role not only in host response against Mtb but also in the immunopathology of TBC like the head mediator of the destruction of the pulmonary tissue (7,8). Elevated levels of TNF-? are related to an excessive inflammation with necrosis and cachexy (9, 10).
Interleukin 6 (IL-6) is a multifunctional cytokine and its increased production is a hallmark of many human chronic inflammatory diseases. First, IL-6, together with TNF-? and IL-1?, initiates early pro-inflammatory responses and is a known inducer of acute-phase proteins. Second, IL-6 is involved in the promotion of T-cell and B-cell responses. In active TBC, the role of IL-6 may be predominantly negative. This is supported by several facts: (a) IL-6 promotes the growth of mycobacteria in peripheral blood monocytes, (b) IL-6 inhibits the production of TNF-? and IL-1?, which may enhance intracellular killing of microorganisms and development of granulomata (11), favoring dissemination of the disease. In IL-6 has also been shown to play a role in the priming of a TBC subunit vaccine. (12).
Interleukin-1 receptor-associated kinase 1 (IRAK1) is an enzyme that is encoded by the IRAK1gene. IRAK 1 is one of two putative serine/threonine kinases that become associated with the interleukin-1 receptor (IL1R) upon stimulation. This gene is partially responsible for IL1-induced upregulation of the transcription factor NF-kappa B (NF-?B). Orchestrating action of specific factors, such as miR-146a, inhibits the expression of IRAK1 and impairs NF-?B activity, which in turn suppresses expression of IL-6, IL-8, IL-1?, and TNF-? genes (13).
In this study, we investigated expression of IRAK1, TNF-? and IL-6 genes and their correlation with clinical severity of TBC. Analyses of changes in gene expression during Mtb infection as well during treatment, could help better understanding of mechanisms of host response to Mtb, and point specific molecular markers of disease state and progression.
Materials and methods
Patients and controls
Patients were recruited at the Clinic for Pulmonary Diseases, Clinical Center of Serbia, between February 2013 and February 2014. Patient’s group consisted of 33 patients with active pulmonary TBC, with a mean age of 52.34±17.61 years, 13 females and 20 males. The control group consisted of 10 healthy volunteers, matched to patient’s group by ethnicity, gender and age. None of the patients with active TBC or the controls had a history of severe pathologies, including HIV infection, cardiovascular diseases, diabetes, asthma, atopy or autoimmune diseases, and cancer. The study was performed in conformance with the Declaration of Helsinki ethical guidelines. Informed consent was obtained from each patient and the protocol for the research project was approved by the Ethics Committee of the Clinical Centre of Serbia.
Patients were all HIV negative and they were receiving anti-tuberculous therapy at the time of blood sampling. All TBC patients were drug responders to the first line treatment (isoniazid, rifampin, ethambutol, pyrazinamide). Medical records of all patients were analyzed for clinical data such as anemia, weight loss, sputum, as well as diagnostic tests such as thoracic X-Ray (XR), computed tomographic (CT) scan of the chest, and blood counts. The TBC diagnosis was made if acid-fast bacilli had been positive in search and/or culture in each case. We considered anemia as hemoglobin ; 12 g/dL (woman) or 2 months) (median 14.79 (13.47-34.59) compared to those with shorter therapy duration ( 2 months) compared to ones treated for less than two months points the signifficance ot TNF-? in the defense from mycobacterium. Previous studies have showed controversial results concerning the levels of TNF-? upon antitubercular therapy. Study of Tang et al., (24) and Portales-Pérez et al. (25) found decreased TNF-? levels in TBC patients after therapy. However, Moura et al. (26) did not observe significant differences in TNF-? levels after treatment. These studies reinforce the believe that TNF-? has a role in both the physiopathology and in protective immunity against TBC.
Both antiinflammatory and proinflammatory role of IL-6 could contribute to the pathogenesis of TBC. Our results showed increased levels of IL-6 mRNA in patients group compared to healthy subjects. Levels of IL-6 were found to be increased in women compared to man, as well as in patients with complication and in patients without anemia, but the result did not reach statistical significance.
Mycobacterial components firstly activate TLR signaling pathways and cause the miRNA expression via NF-?B pathway. The inductive miRNA in turn modulates TLR signaling pathway by targeting its downstream molecules, such as IRAK1 and then limits inflammation (15). This, on the one hand, could avoid the additional damage caused by excessive immune responses; on the other hand it could also be utilized by mycobacteria for better replication (21). Lower levels of IRAK1 shown in our study supports the strategy of Mtb to downregulate inflammatory processes in the granuloma, which is in concordance with other studies, in which IRAK1 down-regulation robustly reduced the inflammatory responses (22,23).
Our study indicates that TNF-? is the important parameter in evaluating the severity of disease and monitoring of the clinical effect of the antituberculosis drugs (DOTS) over a period of prescribed drug therapy.