ISSN Print 2500–1094
ISSN Online 2542–1204
Bulletin of RSMU
BIOMEDICAL JOURNAL OF PIROGOV UNIVERSITY (MOSCOW, RUSSIA)
ORIGINAL RESEARCH
Adaptive immunity and genetic aspects of tuberculosis in children
About authors
Department of Pediatrics, Center of Continuing Professional Education and Retraining,Omsk State Medical University, Omsk, Russia
Correspondence should be addressed: Maria Plekhanova
ul. Lenina, d. 12, Omsk, Russia, 644099; ur.liam@sulp-anid
About paper
Funding: this work was supported by the Russian Foundation for Basic Research (Grant ID 16-16-55001).
Received: 2017-09-24
Accepted: 2017-10-25
Published online: 2018-01-13
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The research efforts of the recent years have proved the important role cellular immunity and interferon gamma (IFNγ) play in protecting human body from mycobacterium tuberculosis (MBT), but the mechanisms of development of immunological competence against tuberculosis (TB) remain poorly understood [1]. It is known that proteins secreted by MBT at early stages of infection support proliferation of lymphocytes producing IFNγ [2]. A number of studies states that TB patients with nonresistant IFNG genotype have the level of synthesis of this cytokine correlated to the severity of the disease [3, 4].
The search for candidate genes causing congenital and adaptive immunity disorders in the presence of TB is also underway [1, 5, 6, 7, 8, 9]. Previously, IFNG gene polymorphism in development of various diseases, including tuberculosis, received much attention [3, 10, 11, 12, 13].
Studying immunological and genetic factors affecting antituberculous immunity ensures better understanding of the underlying pathogenesis of the disease and, what is more, allows optimization of the prevention approaches. This research aimed to investigate the immune response to TB development and the relationship between polymorphic variant rs2069705 (T-1488C) of IFNG gene and the expression of specific immunological reactions in children.
METHODS
This prospective study was conducted in 2014–2016. 310 patients, all under 18 years of age, took part in it. They were divided into 3 groups depending on the TB status: TB group — 110 children, confirmed TB, mean age of 9.5 ± 0.5, every second child (46.6 %) under 8 years of age; LTB group — 156 children, latent TB confirmed (tuberculin diagnostics), mean age of 6.6 ± 0.3, 70.5 % of children under 8 years of age; NTB group — 44 children, no TB infection, mean age of 3.1 ± 0.4, 95.5 % of children under 8 years of age. Genderwise, the groups were not different (p > 0.05).
All children underwent specific immunoassays; 169 of them were also subjected to molecular-genetic tests. To make the comparison valid, only the immune status data describing children under 8 were compared.
TB diagnosing included various laboratory, bacteriological, molecular-genetic tests, radiological examinations. The results of tuberculin diagnostics were also taken into account: Mantoux tests with 2 tuberculin units (TU) of tuberculin (PPD-L) and recombinant tuberculosis allergen tests (RTA) Diaskintest (Generium, Russia), which contain 0.2 μg of CFP10-ESAT6 recombinant protein. A central medical-control commission checked and confirmed diagnoses.
In the TB group, 23 children were diagnosed with infiltrative pulmonary tuberculosis, 38 — with primary tuberculosis, 46 — with intrathoracic lymph nodes tuberculosis (one patient had it combined with the left proximal bronchus lesion and there were cases of focal pulmonary TB, disseminated pulmonary TB and pleuresia tuberculosa, one of each). In 4 cases, TB also affected other organs: peripheral lymph nodes, humerus, intestine. Also in 4 cases, TB caused complications (atelectasis, exudative pleurisy, pulmonary dissemination, hemoptysis). 60.9 % (n = 67) of patients had TB at the infiltration stage, 10.9 % (n = 12) showed disintegration and seeding phase, 0.9 % (n = 1) — sealing and resorption, 26.4 % (n = 29) — calcination phase. Bacterioexcretion was registered in 12 cases (10.9 %); 6 of them proved resistant to the basic antituberculosis drugs.
In the LTB group, 75 children were diagnosed with early primary tuberculosis (EPT), and 81 children had MTB for more than one year.
In the NTB group, 21 child had post-vaccination allergy and 23 showed positive tuberculin anergy.
TB group participants had the infiltrate measure the average of 12.6 ± 0.4 mm (95 % CI 11.8–13.5 mm) for the Mantoux test and 14.95 ± 0.5 mm (95 % CI 13.9–16.0 mm) for the RTA test. The mean values for the same tests in the LTB group were 10.3±0.3mm(95%CI9.7–10.9mm)and1.9±0.4mm (95 % CI 1.2–2.65 mm) respectively. As for the NTB group children that had postvaccinal allergy, the Mantoux test there brought the average results of 4.1 ± 0.4 mm (95 % CI 3.2– 5.0 mm); 9 (42.9 %) cases were confirmed positive reactions, the rest were questionable. All children in this group returned negative reaction to the RTA test.
We had the data on TB vaccination of children. BCG administration site scar and Mantoux test a year after vaccination helped determine its effectiveness: when the scar was 4–10 mm long and tuberculin response positive, the vaccination was regarded a success, in the absence of the scar and negative reaction to tuberculin it was considered ineffective, all other cases fell into minimally effective category. In the TB group, 108 children (98.2 %) were vaccinated, but only for 31 of them the vaccination was effective. In the LTB group, 155 children (99.4 %) received vaccination and for the most of them (n = 97) it was effective. 36 children (81.8 %) of the NTB group were vaccinated; it was effective in every fourth case.
Patients went through immunoassays and molecular- genetic tests when admitted to the special in-patient department of the TB dispensary. The tests were made at the Omsk R&D Institute for Natural Focal Infections (Russia).
Immune status was assessed with the standard level I immunological screening tests: measurement of immunoglobulin levels in the blood serum (IgG, IgA, IgM and IgE) through enzyme-linked immunoassay; evaluation of neutrophil function through measurement of their ability to absorb inert latex particles and two variations of nitroblue tetrazolium (NBT) test, spontaneous and stimulated; evaluation of subpopulation composition of T-cells (CD) using a panel of monoclonal antibodies (DAKO, Denmark). We have also measured the content of spontaneously synthesized interferon gamma and IFNγ the synthesis of which was induced by specific antigens (PPD-L, CFP32B, Rv2660c, ESAT6, 85a, ESAT6-CFP10) for 72 h. Enzyme immunoassay system by Vector-Best (Russia) was used for the purpose. Antigens were isolated by the translational biomedicine lab of the Department of genetics and molecular biology of bacteria of N. F. Gamaleya Scientific Research Institute of Epidemiology and Microbiology (Moscow, Russia) [14].
DNA-blood reagent kit (TestGen, Russia) helped isolate DNA from blood serum; iQ5 amplifier (BioRad, USA) and a set of polymerase chain reaction reagents (FLASH format, by TestGen) were used to identify polymorphic marker rs2069705 of IFNG gene, all following instructions provided by the manufacturers. Allelic Discrimination software supplied by the manufacturer of the amplifier enabled analysis of genotypes. The frequency distribution of genotypes at the examined loci was checked for compliance with the Hardy–Weinberg law.
OpenEpi v3.0 software calculated the minimal sample size ensuring conclusiveness of the empirical data obtained. The reliability of differences between groups was determined through nonparametric Kruskal–Wallis (H), Mann–Whitney (U) and χ2 tests. The differences were considered significant at p < 0.05. We have also calculated the odds ratio (OR): if the chance (risk) was above 1, development of the disease was considered statistically significant. OpenEpi v3.0 and Statistica v6.0 software were used to process the data obtained.
The study got the approval of Omsk State Medical Academy ethics committee (Minutes no. 51 of October 10, 2012). Parents or legal representatives have signed voluntarily informed consent forms and thus approved participation of their children in the study.
RESULTS
Tab. 1 shows the comparative analysis of results of clinical and laboratory examinations/tests of children from TB and LTB groups. Children from the LTB group suffered from intoxication syndrome less often (9 %) than the TB group patients (19.1 %) (p = 0.008), but they had more of nasal breathing disorders caused by adenoid vegetations (16 % vs. 1.8 %, p = 0.00008), chronic infection foci (18.6 % vs. 10 %, p = 0.027), respiratory allergies manifestations (15.4 % vs. 0 %, p = 0.000008) and excess weight (10.3 % vs. 4.5 %, p = 0.044). Such clinical symptoms as hepato- and splenomegaly (OR 2.583 and 3.800, respectively) and body mass deficiency (OR 1.898) were significant in the TB group. Lab tests have also shown significance of anemia (OR 1.872), accelerated ESR (OR 2.255), lymphocytosis (OR 1.634) and eosinophilia (OR 5.371). It should be noted that only 4 cases of eosinophilia (out of 40) resulted from parasitic invasions.
Tab. 2 shows the results of assessment of the immune status of all children that participated in the study. There were no significant differences in immunological parameters describing the status of children constituting LTB and NTB groups (p > 0.05). The exception here was the content of spontaneously synthesized IFNγ (p < 0.05). In all groups, the numbers of leukocytes have increased slightly (compared to the reference values). Leukocytes reflect the state of cellular immunity, so the increase can signal of its activation due to nonspecific processes. At the same time, the differences were significant for CD16, HLA DR, and spontaneously synthesized IFNγ. In the TB group, activation of humoral immunity was observed: within the reference values, the volumes of IgG, IgA have grown, and IgE have shown a significant increase to 306.6 ± 130.7 IU/ml. Phagocytic activity of cells has also increased significantly, while neutrophils' reserve capacity has decreased. The changes of all the aforementioned values were statistically significant compared to those seen in the NTB group.
Thus, the immune status of children from the LTB group was the same to that of children not hosting MTB; at that, cellular immunity has activated slightly. In the TB group, no immunodeficiency development signs were observed, but the changes that did manifest themselves were those peculiar to an inflammatory process associated with TB. The process had features of a chronic one, primarily due to insufficient functional activity of cells and insufficient production of interferon gamma.
To better understand the contribution IFNG gene makes to the immunological defense against MBT, we investigated its polymorphism rs2069705. 51.9 % of children from the TB group had a heterozygous genotype, so it was associated with the disease (OR 1.885, 95 % CI 1.019–3.487). Be it secondary (65 %) or primary (47.5 %) TB, the genotype was there, which suggests a higher risk of the disease development regardless of its genesis. Heterozygous genotype was also associated with the development of infiltrates (OR 1.737), signs of lung tissue destruction (OR 1.458), dissemination (OR 1.75) and pleural effusion (OR 1.9), bacterial excretion (OR 1.458) and such clinical manifestations of tuberculosis as paraspecific reactions (OR 2.059), peripheral lymphadenopathy (OR 2.4), bodyweight deficit (OR 1.429), hepato- and splenomegaly (OR 5.5), anemia (OR 2.059), accelerated ESR (OR 3.4).
Given the association of IFNG gene polymorphism rs2069705 with adaptive immunity to tuberculosis, we evaluated the genotype-wise effectiveness of BCG vaccination in children affected by primary TB at its early stage (n = 32). In 12 cases, the vaccination was minimally effective or ineffective; half of those patients had heterozygous genotype. The probability of minimal effectiveness of vaccination was 59.38 % (95 % CI 42.23–74.62 %). Homozygous genotype (T allele) was more common (40 %) among children for whom the vaccination was effective (n = 20).
Ozhegova et al. [7] have established that polymorphism T-1488C of IFNG gene defines its expression level. Therefore, we assumed there is a connection between the genotypes studied and the level of interferon gamma synthesis. Analysis of the content of spontaneously synthesized IFNγ did not reveal any significant differences dependent on genotype; this is true for both LTB (H = 1.663, p = 0.435) and TB (H = 4.810; p = 0.090) groups. Analysis of the content of IFNγ synthesized through induction by specific antigens [15] showed that in the development of TB, there is a relationship between genotype and level of cytokine synthesis: with heterozygous genotype, the content of IFNγ decreased significantly under the influence of CFP32B, Rv2660c, ESAT6, Ag85a antigens (tab. 3). With this in mind, we have also analyzed the frequency of negative results — no response to induction in children with TB (tab. 4) — and established the relationship with heterozygous genotype. Its association with decelerated IFNγ synthesis when induced with PPD-L, CFP32B, Rv2660c, ESAT6, 85a and ESAT6-CFP10 antigens was confirmed.
DISCUSSION
The vast majority of people carrying MTB show no symptoms of tuberculosis, and although they are not contagious, they run a risk of developing an active form of TB. According to experts, the risk of TB reactivation during the lifetime of an LTB patient is 5–10 %. Often, the switch from latent to active happens within 5 years from the day of infection [16, 17]. Nevertheless, a number of researchers believes the level of this risk depends on several factors, the most important of which is the body's immune status [18, 19, 20, 21]. The results of our study suggest presence of a specific inflammatory process and absence of secondary immunodeficiency.
We studied IFNγ, the main function of which is immunoregulation, including macrophages activation, enhancement of Th-1 mediated response, induction of expression of MHC class II antigens on antigen-presenting cells, etc. [22]. With cellular immunity activation in the background, IFNγ producers are activated Th1-lymphocytes (the main activation marker is HLA DR) and natural killer cells (CD16). Therefore, reduced numbers of those natural killer cells in the presence of TB could lead to lowered levels of cytokine synthesis, and IFNγ deficiency could cause a decrease in activity of cytotoxic cells. This could explain the growing volume of spontaneously synthesized IFNγ seen even when TB is latent (p < 0.05); at the same time, we can assume that the antigen load was not sufficient to trigger hyperactivation of cellular response. During the period of development of TB, this indicator remained at the level typical of LTB (p > 0.05). Given the increased volume of spontaneously synthesized IFNγ, one could expect a lower content of IgE, as IFNγ, being the product of Th1-lymphocyte, inhibits proliferation of Th2-lymphocytes and IL4-induced Ig to IdE synthesis switch and supports IgG2 synthesis instead [23]. However, we have witnessed high levels of IgE synthesis during TB development, which may indicate inadequate IFNγ production and onset of chronic inflammation. A number of researchers have also established a direct relationship between the appearance of chronic bacterial or fungal infection foci and hyperproduction of IgE [24, 25, 26].
IFNγ is also considered to be the most important factor in macrophage activation [22]. Macrophages lyse MTB and provide antimycobacterial protection; in particular, they regulate synthesis of pro- and anti-inflammatory cytokines [27, 28]. However, the balance between cells and mediators required to destroy MTB and prevent lung pathology is still unclear; the issue is subject of other research papers in progress [29]. In our study, we saw a decrease in phagocytic activity of cells (decrease in reserve capacity of neutrophils) against the backdrop of developing TB, which may also point to inadequate production of IFNγ.
Averbakh et al. proposed a hypothesis stating there are genes in lymphocytes that control activation of cells synthesizing mediators and that a depression of one part of the genome can mean a depression of its another part, which may lead to disruption of intercellular interaction in the presence of TB [30]. The researchers are trying to establish genetic risk factors affecting TB infection and development [8, 9]. In particular, they are actively studying the IFNG gene associated with production of IFNγ cytokine [3, 9] and T-1488C polymorphism that affects the synthesis of regulatory protein [7]. In the context of our study, the marker of high TB risk was its heterozygous genotype at the studied genetic locus (OR 4.667, 95 % CI 1.236–17.62, p = 0.008), regardless of the disease genesis (primary or secondary). Low effectiveness of BCG seen in the group with primary tuberculosis infection at its early stage is an indirect proof thereof. Some researchers also believe the low effectiveness of vaccination is a TB risk factor [18, 31]. We have also determined that the studied IFNG gene polymorphism (heterozygous genotype) influences the immune response to individual mycobacterial antigens when TB is developing: we have seen a significantly weaker response to early stage TB proteins — CFP32B, Rv2660c, ESAT6, 85a [15], and a slightly decreased level of IFNγ synthesis with PPD-L and ESAT6-CFP10 induction, which gives a yet another reason to look at these antigens in the context of TB. We have also established that homozygous genotype (T allele) means better protection against TB and found it affects development of antituberculous immunity in children.
The results of this study allow taking the IFNG gene polymorphism (T-1488C) as an additional genetic risk factor contributing to the development of tuberculosis infection in children and one of the reasons behind inadequate functional activity of cells regulating synthesis of IFNγ.
CONCLUSIONS
We studied the immune response to development of TB and seen activation of cellular immunity and insufficient functional activity of cells when TB turns from latent to active. The main adaptive immunity cytokine considered was IFNγ.
We have established that IFNG gene polymorphism T-1488C affects the severity of specific immunological reactions. Heterozygous genotype implies inadequate production of cytokine at the early stage of TB development. Homozygous genotype (T allele) means better protective immunity against the disease.
We have established that heterozygous genotype of the IFNG gene's polymorphic version (rs2069705) bears relation to TB switching from latent to active in children, which allows taking this genotype as an additional risk factor.