**4. Discussion**

A unique feature of *M.tb* is the ability to persist in the host for a long time, despite functioning mechanisms of acquired immunity. In turn, approximately 2.3 billion individuals have latent tuberculosis infection without evidence of the clinical manifestation of active TB. It is hypothesized that active TB is usually caused by the reactivation of endogenous infection and untreated LTB is a

major source of new active TB infections and transmission. The risk for severe active tuberculosis reactivation is increased several times among the immunocompromised individuals, diabetics, organ transplantation recipients, patients with hematologic malignancies, or HIV-infected subjects. Several treatment regimens of LTB are recommended, between isoniazid monotherapy for six months, rifampicin plus isoniazid for three months or rifapentine plus isoniazid for three months [27]. Currently, interferon-gamma release assays (IGRA) are used to test LTB based on the production of IFN-γ by Th1 cells responding to specific *M.tb* antigens, although the IGRA's accuracy in immunocompromised individuals is still limited. Moreover, the definition of active tuberculosis infection might not be accurate using IFN-γ responses to *M.tb* antigens even in combination with tuberculin skin test [28]. At the same time, neither the IGRA test nor the tuberculin skin test can distinguish between latent and active TB infection. Strategies for rapid di fferentiation of patients with active TB and people with LTB and prevention of tuberculosis reactivation in LTB individuals are urgently needed.

To meet such needs, we compared the expression of genes of the IL-18 pathway, functional receptor of this cytokine IL-18R, and IFN-γ, as well as the expression of IL-18BP and IL-37 genes in groups of patients with active TB, healthy individuals with LTB and healthy controls without *M.tb* infection.

Our results for the first time showed a significant increase in the relative expression of IL-18 and IL-18BP mRNA in the group of patients with active TB and LTB individuals when compared to healthy controls, according to available literature data. It might sugges<sup>t</sup> a permanent activation of the immune cell signaling pathways in the course of *M.tb* infection either in the control or progression to active TB disease. Moreover, no significant di fferences in relative IL-18 and IL-18BP mRNA expression were observed between active TB patients and LTB individuals. Pechkovsky et al. demonstrated an increased expression of IL-18 mRNA in type II lung epithelial cells obtained from patients with pulmonary TB. Pneumocytes that were cultured in the presence of *M.tb* cell lysate showed an increased IL-18 mRNA expression in comparison with unstimulated cells and pneumocytes stimulated with PPD or LPS [29]. Higher levels of IL-18 mRNA expression were observed in monocytes responding to *M. leprae* antigens [30]. Corbaz et al. showed significantly higher levels of IL-18 and IL-18BP mRNA expression in the intestinal mucosa of patients with Crohn's disease as compared to healthy controls. We detected a similar increase in IL-18 and IL-18BP mRNA expression in the group of patients with active TB and healthy LTB individuals. These results are somewhat out of line with our earlier definition of a simultaneous increase in serum IL-18 and IL-18BP protein expression, which might be treated as a discriminatory biomarker of active tuberculosis and LTB. Yet, it is worth noting that only the complex co-expression of serum IL-18BP and IL-37, IP-10, and IFN-γ were identified as the most accurate discriminative biomarker set for diagnosis of active TB [21].

Our study allowed for the discovery of a novel relation—the significantly lower expression of functional IL-18R receptor mRNA in the LTBI group as compared to the active TB and healthy controls. This low expression of IL-18R mRNA in LTB individuals was accompanied by IFN-γ mRNA expression at the 'baseline' level, characterizing healthy individuals without *M.tb* infection. Similarly, Taha et al. pointed out that the expression of IFN-γ genes was significantly higher in the group of patients with active TB as compared to those who were infected but did not develop active TB [31].

Among many possible mediators of host response to *M.tb* is the activity of the indoleamine 2,3-dioxygenase-1 (IDO1), the enzyme of tryptophan metabolism, which leads to the formation of tryptophan metabolites, including quinolinic and picolinic acids [32]. In animal models, increased IDO-1 expression and the activation of the tryptophan-kynurenine pathway were indicated to play a crucial role in *M.tb* pathogenesis [29,30,33,34]. The depletion of tryptophan, which is required for microbial growth, as well as the accumulation of biologically active tryptophan metabolites, impaired e ffective anti-mycobacterial immune response and, thus, favoured survival and persistence of the pathogen [32]. In *M.tb*-infected mice, IFN-γ receptor-deficiency in nonhaematopoietic cells led to a lack of IDO-1 expression and it was associated with exuberant neutrophil recruitment and increased mortality [33]. In macaques, the suppression of IDO activity led to the reduction of the bacterial burden and clinical symptoms of active TB that was accompanied by increased lung T cell proliferation, the induction of inducible bronchus-associated lymphoid tissue, and the relocation of e ffector T cells to the center of the granuloma [34]. Mehra et al. demonstrated that IDO induction in the periphery of the granuloma correlated with active TB disease [30]. A few studies have investigated IDO-mediated tryptophan metabolism and its metabolites in humans in the context TB. Li et al. demonstrated increased IDO expression and activity in the pleural fluid from TB patients [35]. Almeida et al. found significantly higher expression levels of immune-suppressive mediators, including IDO-1, in patients with active pulmonary TB as compared to patients with other infectious lung diseases and healthy volunteers [36]. The authors suggested that the increased levels of immunosuppressive mediators may render the immune activation and counteract the development of Th1-type immune response against *M.tb*. The IDO levels were elevated at the time of TB diagnosis and declined after TB treatment, which serves as evidence that IDO expression might be both: a useful diagnostic marker of active TB as well as prognostic factor in TB treatment of HIV-negative patients [36]. Additionally, Adu-Gyamfi et al. showed that plasma IDO expression is a potential biomarker of active TB in HIV-positive patients [37], while Shi et al. confirmed that IDO activity might have an auxiliary diagnosis value for the early discrimination of multi-drug resistant TB patients [38]. The increase in IDO activity was noticed in both HIV-infected and uninfected active TB patients as compared with individuals with latent TB infection [36,37,39]. In relation to this statement, the simultaneous reduction of IL-18R mRNA expression together with significant overexpression of IL-18 mRNA observed by us in the LTB group is of particular interest.

The presented results entitle us to hypothesize that: the increase in IL-18 gene expression, the lack of increase in IFN-γ gene expression, and the remarkably reduced expression of IL-18R gene may be a novel set of conditions that partially describe the homeostasis between *M.tb* and host-immunity in latent tuberculosis infection. As an obligate intracellular pathogen, *M.tb* has numerous adaptive mechanisms of modifying cellular processes in the fight against the host immune response. In latent TB infection, *M.tb* bacilli benefit from epigenetic changes that occurred in the host immune system under mycobacterial infection [40]. These changes make the *M.tb* favorable environment in the host cells and promote mycobacterial survival, growth, and latency. In a study that was conducted among Chinese patients with pulmonary TB and healthy controls, single nucleotide polymorphisms in the IL-18R promoter were associated with genotype-specific methylation status and genotype-specific IL-18R expression [41]. In the author's opinion, the relationship between decreased mRNA expression of IL-18R that is caused by an SNP and increased DNA methylation can partially mediate the susceptibility to TB risk. No statistically significant di fferences were observed in the relative mRNA IL-37 expression among the groups in our study. IL-37 is a new member of the IL-1 family, which reduces systemic and local inflammation. IL-37 is expressed in various cells and tissues and it is regulated by numerous inflammatory stimuli and cytokines via di fferent signal transduction pathways [42]. Mannose-capped lipoarabinomannan purified from *M.tb* induces IL-37 production via enhancing TLR2 expression in human type II alveolar epithelial cells; this process might contribute to the persistence of *M.tb* infection [43]. Zhao et al. indicated that IL-37 is also a negative regulator of immune responses in *Listeria monocytogenes* infection due to reduced production of colony-stimulating factors and increased macrophage apoptosis [44]. In our earlier studies, the serum concentration of the IL-37 protein was similar in the group of patients with active pulmonary TB and healthy individuals with or without latent *M.tb* infection [21]. However, the complex co-expression between the two IL-18 inhibitors, IL-18BP and IL-37, was identified as the strongest discriminative biomarker of active TB disease.
