The International Journal of Mycobacteriology

ORIGINAL ARTICLE
Year
: 2019  |  Volume : 8  |  Issue : 2  |  Page : 190--195

Immunological host responses as surveillance and prognostic markers in tubercular infections


Pranita Jwalant Waghmare1, Trupti Lende1, Kalyan Goswami1, Anshu Gupta2, Anupama Gupta2, Nitin Gangane1, Satish Kumar1,  
1 Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Wardha, Maharashtra, India
2 Department of Pathology, Mahatma Gandhi Institute of Medical Sciences, Wardha, Maharashtra, India

Correspondence Address:
Satish Kumar
Department of Biochemistry, Mahatma Gandhi Institute of Medical Sciences, Sevagram, Wardha - 442 102, Maharashtra
India

Abstract

Background: Tuberculosis (TB) control is challenging due to failure of drug compliance and resistance. Mycobacterial antigen-induced cytokine secretions are helpful in detecting Mycobacterium tuberculosis infection and to determine prediction for the fate of TB infection and its cure. Considering immunological response to be a crucial factor in pathogenesis and cure of TB, it can be explored to determine clinical prospects in different categorical tubercular infections. This study was designed to compare serum levels of inflammatory (tumor necrosis factor [TNF]-alpha and interferon [IFN]-gamma) and anti-inflammatory cytokines/chemokines (interleukin [IL]-10 and IL-8) among different TB groups (freshly diagnosed, relapse, cases on antitubercular treatment [ATT], and healthy controls). Methods: This cross-sectional study included total 100 subjects. The study subjects were further divided into four study groups with 25 cases in each of freshly diagnosed TB, TB relapse cases, cases on ATT, and 25 healthy controls. Levels of serum cytokines/chemokines (TNF-alpha, IFN-gamma, IL-10, and IL-8) were measured by flow cytometry. Results: Data analysis observed statistically significant differences in serum levels of TNF-alpha and IFN-gamma among the studied groups with significantly low levels in subjects on ATT and markedly high levels in TB relapse subjects. No statistically significant difference was observed in IL-10 and IL-8 levels. However, subjects with relapse revealed low IL-8 and high IL-10 levels. Conclusion: TNF-alpha and IFN-gamma have important roles in immune response and might be considered as indicators for response to ATT. However, high levels of IL-10 with low IL-8 appear to be associated with poor outcome and possibility of relapse.



How to cite this article:
Waghmare PJ, Lende T, Goswami K, Gupta A, Gupta A, Gangane N, Kumar S. Immunological host responses as surveillance and prognostic markers in tubercular infections.Int J Mycobacteriol 2019;8:190-195


How to cite this URL:
Waghmare PJ, Lende T, Goswami K, Gupta A, Gupta A, Gangane N, Kumar S. Immunological host responses as surveillance and prognostic markers in tubercular infections. Int J Mycobacteriol [serial online] 2019 [cited 2019 Aug 25 ];8:190-195
Available from: http://www.ijmyco.org/text.asp?2019/8/2/190/260382


Full Text



 Introduction



The vicious cycle of poor compliance and nonadherence to long-term treatment schedules poses an alarming situation in tuberculosis (TB) control. This results in drug-resistant TB[1] and continues to put a huge burden on TB morbidity and mortality. This situation demands suitable and timely measures to be taken in this regard. While ongoing research focuses on discovery of novel drug candidates, presently available standard treatment needs to be given meticulously and also monitored for its adequacy and effectiveness. As per the current guidelines, besides monthly follow-up,[2] Ziehl–Neelsen staining is recommended at the end of ATTs.[2] Such long durations of treatment without any biochemical or microbiological tool to judge the response to treatment may put unnecessary burden on the patient as well as the health sector. The discovery of novel biomarkers which can assess the response to treatment as early as possible may play a key role in such situations.

The pathogenesis of TB involves cell-mediated immune responses against Mycobacterium tuberculosis (MTB). A multifaceted cellular interaction takes place within the host after infection with MTBbacilli. CD4+ T cells can be classified into two subsets: Th1 and Th2. Th1 cells give rise to cell-mediated immunity and are characterized by the production of inflammatory cytokines such as interleukin (IL-2), interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha. Th2 cells are more efficient in mediating antibody production and secrete IL-4, IL-5, IL-6, and IL-10 which are anti-inflammatory in action. There are considerable interactions between these two mutually reciprocal subsets to bring forth the resultant effectors' response.[3],[4] One of the major determinants for the establishment of infection depends on the net balance of proinflammatory and anti-inflammatory cytokines. TNF-alpha and IFN-gamma are particularly important in promoting the formation of granuloma. The production of anti-inflammatory cytokines such as IL-4, IL-10, and transforming growth factor (TGF)-β in response to MTB downregulates the immune response and limits tissue injury, but excessive production of these cytokines may result in failure to control the infection.[4]

As the immunological response corresponds to pathogenesis of TB, exploration of immunological parameters can be of significance in fresh TB subjects and to assess response to treatment. Further, it will be of interest to know the immunological status in patients with relapse of TB. Early detection of cases with inadequate immunological response may subsequently lower unnecessary drug burden on the patient. Taking all these factors in consideration, we designed a study to find the cytokine response profile in various TB categories such as patients with fresh new TB infection, cases after start of ATT, and in relapse cases. Here, we aimed to look for differential levels of cytokines and chemokines in different groups of study subjects with tubercular infection in reference to healthy controls and their possible clinical significance.

 Methods



Ethical consideration

This study was ethically approved of Institutional Ethical Committee, MGIMS, Sevagram (Chairperson Dr. Ashok Pawde, on September 6, 2014).

Study design

Selection and description of participants

All the study subjects were drawn from patients attending Kasturba Hospital, Sevagram, and Civil Hospital, Wardha, during the year 2014–2016. The diagnosis of TB was accomplished based on clinical and radiological evidence in extrapulmonary TB and confirmed with detection of presence of tubercle bacilli in the sputum of pulmonary TB (at least one positive sputum smear and culture result for acid-fast bacilli). Staining for acid-fast bacilli was performed using the standardized Ziehl–Neelsen method.

The study samples include a total of 75 subjects (pulmonary TB [68%] and extrapulmonary TB [32%]) and 25 healthy controls. Age of all study subjects was >18 years. Subjects who have history of autoimmune diseases (rheumatoid arthritis), immune comprehensive state, or taking any steroid treatment were excluded from the study. The study group was categorized into four different groups, each group containing 25 subjects. Group 1 included TB patients with relapse of disease who had completed anti-TB regimen recommended by the Revised National Tuberculosis Control Program, India, for fresh TB once before and then returned with the diagnosis of TB again within 2 years of the last treatment, whereas Group 2 comprised of freshly diagnosed TB patients. Group 3 included TB patients who were on anti-TB drugs (ATT). For those who were on ATT, the sample was collected after 1 month of start of ATT. Healthy controls formed Group 4.

Sample collection

Written informed consent for participation in the study was obtained from each subject. Venous blood was collected from the study subjects. Samples were centrifuged; serum was separated out and stored at −20°C until its use.

Cytokine and chemokine assay

Cytokines (TNF-alpha, IFN-gamma, and IL-10) and chemokines (IL-8) were estimated by Becton Dickinson (BD) Cytometric Bead Array (CBA) following standard protocol.[5] In brief, each BD CBA Flex Set comes with capture beads, detection reagent, and standards. Sufficient reagents were provided to run 100 tests including two standard curves. All assays were available off-the-shelf and ready for mixing. The assay was performed following the instructions in the Master Buffer Kit manual. Standards were prepared. Samples were run on a laser flow cytometer (Make: BD FACS Canto II). Flex Set reagents were verified for performance on a number of BD dual-laser flow cytometry platforms. The standard was run and standard curve was obtained for each cytokine, followed by the samples. Data were analyzed using FCAP Array multiplex analysis software (Make: BD FACS Canto II).[5]

Statistical analysis

Statistical analysis was performed using SPSS 20.0 software (version 20.0.0.0, Company IBM, country Unites State). The data were expressed as arithmetic means with standard error; for comparison of means, P < 0.05 was considered statistically significant. After testing data set for deviation from normality, differences in cytokine levels among groups were evaluated by the Kruskal–Wallis tests. Following the Kruskal–Wallis test, we have used Bonferroni test for within-group comparison.

 Results



The distribution of cases among the study group did not show any statistical difference. The Chi-square test revealed P = 0.19. The mean serum levels of cytokines and chemokines in each study group are tabulated in [Table 1]. Kruskal–Wallis tests was conducted to compare intergroup variation pairwise. The analysis revealed that in terms of TNF-alpha and IFN–gamma, each group was significantly different from others, with low means in ATT-treated groups, and was marked high in relapse TB subjects. A single subject in Group 2 with ATT showed high levels of TNF-alpha (102.36 pg/ml) compared to the mean value in subjects with freshly diagnosed TB [Table 1]. IL-10 showed a significant difference between all groups except between Group 1 and Group 2. IL-8 showed significant difference among all the groups except between Group 3 and Group 4.{Table 1}

To know differential presentation of cytokine and chemokine in pulmonary and extrapulmonary TB subjects, multiple regression analysis with Kruskal–Wallis tests was carried out in each group. Intergroup comparison and Bonferroni test were carried out in subjects with pulmonary TB and revealed that TNF-alpha was significantly different in all groups from others, with low mean levels in ATT-treated group and high in relapse TB subjects. IFN-gamma did show a significant difference. IL-10 showed a significant difference between Group 2 and Group 3. IL-8 showed significant difference among all the groups.

Intergroup comparison and Bonferroni test carried out in subjects with extrapulmonary TB revealed that TNF-alpha and IFN-gamma were significantly different in all groups from others. IL-10 showed a significant difference between Group 2 and Group 3. IL-8 showed significant difference among all the groups except between Group 1 and Group 3.

 Discussion



Immune responses on infection with the pathogen MTB are poorly understood, hampering the discovery of new treatment strategies and TB prognosis. An interaction of T-cells with macrophages in an infected host is a central immune defense against the MTB bacillary infection. T-lymphocytes, especially CD4+ T-lymphocytes, play an important role in the immune reaction against TB. Although a Th1 profile is necessary for a protective response, it causes immunopathologic damage. Therefore, Th2 responses are important in regulatory effects on protecting patients from collateral host tissue damage.[4],[6] Indeed, there are many inter- and intracellular processes involved; and these mechanisms can be explored, so that new strategies may be designed in support of TB control. Cytokines released during these interactions are crucial, resulting in either control of TB infection or its flare-up. It has been reported that IFN-gamma, TNF-alpha, and IL-12 stimulate and that IL-10, TGF-β, and IL-4 suppress the protective immune response against TB.[7] Stimulation of CD4+ T-cell can produce Th1-associated cytokines, which may increase Th1 response. TNF-alpha is important for walling off infection. TNF-alpha in synergy with IFN- gamma induces inducible nitric oxide synthases, which are critical components of an effective immune response. TNF-alpha gene and its receptors have significant suppressive effects on bacterial growth into macrophages. TNF receptor 1 (TNFR1) is more responsible for apoptosis of bacilli, whereas TNFR2 receptors are involved in cell survival. A study by Saif et al.[8] reported that single-nucleotide polymorphisms (SNPs) at TNF-857 and TNF-238 regions were associated with susceptibility to TB, indicating its importance in deciding the fate of MTB bacilli in causation of disease. The quantitative estimation of these inflammatory cytokines definitely plays a crucial role in conjecturing the outcome of ATT as early as possible. In this context, we measured a set of inflammatory (TNF-alpha and IFN- gamma) and anti-inflammatory cytokines (IL-8 and IL-10) in different TB groups, namely freshly diagnosed, cases on ATT, and relapse TB infection. Results reported that the levels of cytokines, namely TNF-alpha and IFN- gamma, were found to be the most consistent inflammatory cytokines with a significant difference across the disease status among all the three case groups compared (fresh diagnosed, cases on ATT, and relapse cases) to the control group. At physiological levels, TNF-alpha plays an important role in host response to MTB bacillary infection, not only in activating macrophages but also by playing a role in modulation of macrophage-mediated apoptosis and granuloma formation. TNF-alpha appears to be crucial for infection control with elimination of bacilli.[6] Deveci et al. evaluated changes in the serum levels of pro- and anti-inflammatory cytokines in active pulmonary TB and on treatment. They observed that TNF-alpha levels were decreased in subjects who were on ATT, and the difference was statistically significant.[7] Similarly, in our study, there were low levels of TNF-alpha, and the means were significant in treated patients, probably indicating the decreased level of inflammation suggestive of decreased bacillary load due to effective combat by the anti-tubercular drugs. A single subject in Group 2 high levels of TNF-alpha needs to be followed up as a slow responder for anti-TB treatment and should be cautiously considered for drug resistance or for multidrug-resistant MTB bacillary infection or other immunity-related disorders.

Th1 cells secrete IL-2, IFN- gamma, and other cytokines to participate in cellular immunity. IFN- gamma, the major cytokine, has a role in antigen presentation, leading to conscription of CD4+ T-lymphocytes and cytotoxic T–lymphocytes, which facilitate mycobacterial killing. It has been reported that individuals with defective IFN- gamma receptor gene are susceptible to mycobacterial infections with poor granuloma formation, limiting bacillary control locally resulting in progressive multibacillary lesion.[9] Humans gene defect in IFN- gamma or the IFN- gamma receptors are susceptible to serious tubercular infections.[4] A single base polymorphism (SNP) of IFNGR1 at position-56 was reported to be potential “biomarker” for identifying subjects at higher risk of tubercular infection among the among Iranian community[10] highlighting the role of IFN- gamma in deciding the fate of bacilli in host after their entry. Further, the authors reported the genotyping on three potential polymorphic alleles located across the promoter region of IFN- gamma R1 gene at positions −56 and +295 deletions, confirming their potential association in increasing risk of developing TB among Sudanese patients.[11] In this study, the levels of IFN- gamma were found to be statistically significant in all the studied groups (P < 0.001), validating its role in tubercular control. Levels of IFN- gamma were lowest in the ATT-treated group among three diseased groups. Tang et al.,[12] Kawaguchi et al.,[13] and Bekker et al.[14] have specified the decreased inflammation in terms of IFN- gamma levels in TB patients after therapy, highlighting the decreased bacillary load and hence the inflammation.

The increased plasma TNF-alpha level may be associated with the clinical deterioration observed early in treatment of severe TB.[15] Thus, early assessment of these cytokines can be considered as a tool to monitor the worsening of TB disease. Measuring serum levels of inflammatory cytokines may be useful for evaluating the clinical progress of tubercular infection and also for monitoring response to ATT. Here, we suggest that the differential levels of TNF-alpha and IFN- gamma have important roles in the immune response to MTB, and these parameters could be used in follow-up as indicators of the success of ATT. There was no difference in cytokine response presentation in pulmonary and extrapulmonary subjects [Table 2] and [Table 3].{Table 2}{Table 3}

In our study, relapse group showed the highest levels of TNF-alpha and IFN- gamma levels in comparison to all other groups and healthy control subjects [Table 1], [Table 2], [Table 3]. Relapse cases of TB are expected to harbor low bacillary load and are possibly burdened by continuous inflammation process. This might be further flared up as a disease and this might act as strong acute response, resulting in high levels of inflammatory cytokines. The study group from relapse category had the highest mean values among all studied cases for TNF-alpha and IFN- gamma (85.59 ± 10.68 pg/ml and 83.62 ± 23.23, pg/ml, respectively). Besides major cause of relapse of TB infection those who are nonadherent to the ATT treatment, the Th1/Th2 balance hypothesis might also be applicable for these subjects. It is suggested that “Th1” and “Th2” cells were important regulators of the class switch of immune response,[16] depending on the dominance of Th1 or Th2 helper cells. From a case–control study by Lienhardt et al.,[17] it was evident that clinically observed healing of TB is associated with a shift toward higher Th1 activity and lower Th2 activity, and further, they also suggested that full course of treatment was required to achieve this effect and that patients who had poor clinical outcomes did not achieve this shift.[17] From our data, it is evident that high values of inflammatory cytokines in relapse cases may reflect acute presentation of infection. Although a shift toward Th1 is probably necessary for combating the disease, in this particular setup of relapse cases, as explained before, the already sustained low-grade inflammation that failed to combat the disease actually gets upsurged due to positive alteration in immune status flaring the tubercular infection. Therefore, the observed Th1 response should not be considered as a favorable outcome but a mere reflection of inflammatory overactivity due to increased bacillary load as seen in fresh case. The same results were observed when we compared pulmonary TB and extrapulmonary TB subjects [Table 2],[Table 3] and [Figure 1].{Figure 1}

Serum levels of IL-10 did not show difference between active TB and relapse TB cases. However, these two groups have a significant difference in levels of IL-8. It has been reported that IL-10 secreted from MTB-infected monocytes downregulates IL-8 secretion. This downregulatory effect is partly because of reduced IL-8 mRNA expression.[18] At a cellular level, phagocytosis of MTB by monocytes and macrophages is an important stimulus of IL-8 secretion. IL-8 attracts the neutrophils and T-cells to the site of infection and help in clearance of bacilli. In relapse cases (Group 2), the mean values of IL-10 were high (1.32 ± 0.43 pg/ml) and may be the reason for low IL-8 levels (571.98 ± 315.58 pg/dl), when compared with freshly diagnosed cases. This may be the reason for poor clearance of MTB bacilli that has led to temporary bacillary dormancy, which may later get flared up to be described as relapsed disease. The production of anti-inflammatory cytokines such as IL-4, IL-10, and TGF-β in response to MTB may downregulate the immune response and limit tissue injury, but excessive production of these cytokines may result in failure to control the infection.[19]

Due to possible overactivity of Th2-type cytokines such as IL-10, imbalance between Th1 and Th2 with associated decrease in Th1 might be expected. This might explain poor compliance for ATT, and therefore, these subjects have relapse of TB infection.[14] Here, we conclude that high levels of IL-10 with low IL-8 might be the marker for poor clinical outcome and such subjects had chances for relapse of TB infection. Similar data were revealed in pulmonary and extrapulmonary tubercular cases [Table 2],[Table 3] and [Figure 1].

 Conclusion



In conclusion, it is evident that cytokine response is a vital marker of TB infection and also important in monitoring of its status either as remission with treatment or as a failure with relapses. However, basically being mediators of inflammatory response, cytokines actually reflect the immune status but not the actual disease status. Therefore, the use of cytokines as a pathological parameter can be considered as an important guide to corroborate with the actual clinical status for more effective understanding of the disease process in a given patient. This study may be stimuli to ignite further exploratory studies for immune responses upon infection with the pathogen MTB which is poorly understood, hampering the discovery of new treatments, the improvements in diagnosis, and the prognosis for TB.

Limitation of the study

It is a primary study with a small sample size of participants (we have included 25 study subjects in each so as to satisfy minimal statistical requisite due to financial constrain). All studied cytokines and chemokines are general immune markers (though we have tried to minimize confounding factors by excluding subjects with immune-related disorders) and not specific for tubercular infection; hence, we mentioned it as a limitation. We also suggest that a group of logistic groups of cytokines as discovered should be studied with larger study sample considering severity of TB, such as symptoms, course of disease, and sputum bacteriological factor etc., might influence the levels of some cytokines.

Financial support and sponsorship

This study was supported by Maharashtra University of Health Sciences, Nashik. Grant received was under the Long-Term Research Grant (LRTG) ref. MUHS/UDC.GFL/O4/2014-15/E-1/79.

Conflicts of interest

There are no conflicts of interest.

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