Effect of IFN-γ, IL-12 and IL-10 cytokine production and mRNA expression in tuberculosis patients with diabetes mellitus and their household contacts

Cytokine 81 (2016) 127–136

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Effect of IFN-c, IL-12 and IL-10 cytokine production and mRNA expression in tuberculosis patients with diabetes mellitus and their household contacts Ponnana Meenakshi a, Sivangala Ramya a, Joshi Lavanya a, Valluri Vijayalakshmi a,b, Gaddam Sumanlatha a,c,⇑ a

Department of Immunology, Bhagwan Mahavir Medical Research Center, Hyderabad, India Department of Immunology and Molecular Biology, LEPRA Society, Hyderabad, India c Department of Genetics, Osmania University, Hyderabad, India b

a r t i c l e

i n f o

Article history: Received 22 November 2015 Received in revised form 3 March 2016 Accepted 9 March 2016

Keywords: Tuberculosis with diabetes mellitus Household contacts Cytokine production Fold change Antigen85A

a b s t r a c t Objective: The study was carried out to understand the influence of IFN-c, IL-12 and IL-10 cytokine production and expression in tuberculosis patients with diabetes mellitus (TBDM) and their household contacts (HHC). Methodology: The study involved a total of 300 subjects, 50 in each category of TBDM, TBDM HHC, pulmonary tuberculosis patients (PTB), PTB HHC, DM and healthy controls (HC). TBDM, PTB and their HHC, 25 each were followed at different intervals to determine their immune responses in Ag85A stimulated culture supernatants by Enzyme Linked Immunosorbent Assay (ELISA). mRNA expression by TRIZOL method in 5 cases of each category and follow-up studies were performed. Results: IFN-c and IL-12 cytokine production markedly decreased and that of IL-10 increased after Ag85A M.tb stimulation, however anti TB treatment reconstituted the response in TBDM and PTB patients. The household contacts revealed cytokine gene expression similar to that of patients and two of them developed the disease during follow-up. Conclusion: Cytokine responses of the patients retained after treatment highlighting the antigen importance, hence further studies with recombinant cytokines may help in coming up with a biomarker. Analogous immune responses of household contacts with the TBDM and PTB patients may assist in recognizing the high risk individuals. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Many clinical and epidemiological studies have discovered that type 2 diabetes mellitus is one of the major risk factors for the development of infection [1]. This heightened severity of infection increases the risk of Mycobacterium tuberculosis (M.tb) by 8-fold and thereby development of pulmonary tuberculosis [2]. Tuberculosis patients are the main source of transmission of infection to the individuals living in the same household or who are in frequent

Abbreviations: TBDM, tuberculosis patients with diabetes mellitus; PTB, pulmonary tuberculosis; DM, diabetes mellitus; HHC, household contacts; PPM, public private mix; ANOVA, analysis of variance; ROC, receiver operating characteristic; PBMC, peripheral blood mononuclear cells. ⇑ Corresponding author at: Immunology Department, Bhagwan Mahavir Medical Research Centre, 10-1-1, A.C. Guards, Hyderabad, Andhra Pradesh 500 004, India. E-mail address: [email protected] (G. Sumanlatha). http://dx.doi.org/10.1016/j.cyto.2016.03.009 1043-4666/Ó 2016 Elsevier Ltd. All rights reserved.

contact with these patients [3–6]. However, for reasons unexplained, most of these infected contacts may or may not develop full-blown TB and it is currently impossible to predict who will or will not develop the disease. Diabetes mellitus (DM) not only aggravates the latent tuberculosis infection, but also affects the tuberculosis (TB) disease presentation and response to its management that could be due to the compromised immune response in the diabetic individuals [7]. This compromised adaptive and innate immune response in these diabetics is due to chronic hyperglycemia; these immune impairments are restricted by glycemic control [8,9]. The induction of type 1 cellular immunity vital for protection is mediated by the cytokines of innate and adaptive immune system that synchronize the response to M.tb [10,11]. The proinflammatory cytokines like IFN-c and IL-12 were implicated in protection from TB; however the anti-inflammatory cytokines,

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mostly IL-10 are associated with increased susceptibility to TB disease [12]. Recently, a mouse model was developed for TB and diabetes that had elevated bacterial load and altered IFN-c expression in mice with chronic diabetes [13]. Certain studies reported on the reduced pro-inflammatory cytokine production in TB patients with DM [14,15]. The mycobacterial cell contains various secreted proteins, a major portion of which is formed by the Ag85 complex, a 30–32 kDa family of three proteins (Ag85A, Ag85B, Ag85C) that induces strong T cell proliferation and IFN-c production in peripheral blood mononuclear cell (PBMC) cultures of most healthy individuals infected with M.tb [16,17]. Blastogenic responses are not stimulated in the lymphocytes of patients with TB. Cytokine expression profiles that correlate with either active or latent TB are limited in comparing responses in different clinical groups. The central hypothesis lies in the fact that TB patients at the time of recruitment had advanced disease due to their failed immune response that might improve with treatment, on the other hand healthy individuals who are infected i.e., the household contacts revealed a protective immune response. In this perspective, we assessed differences in IFN-c, IL-12 and IL-10 cytokine secretion and expression in response to M. tuberculosis antigen in tuberculosis patients with diabetes and without diabetes and their household contacts at different intervals of follow-up. 2. Materials and methods 2.1. Subjects A total of 300 subjects including tuberculosis patients with diabetes mellitus (TBDM), Pulmonary Tuberculosis patients (PTB), their household contacts (TBDM HHC and PTB HHC), diabetes mellitus patients (DM) and Healthy Controls (HCs), 50 in each category during the period 2010–2014 were recruited in the study. TBDM, PTB patients and their HHC were those who attended the PPMDOTS (Public Private Mix-Directly Observed Treatment Shortcourse) free chest clinic, Tuberculosis Unit (TU) under the Revised National Tuberculosis Control Programme (RNTCP) implemented at Bhagwan Mahavir Medical Research Center (BMMRC) and DM patients were from Mahavir Hospital. HCs without any family history of TB and DM were taken in the study. TBDM, PTB patients and their HHC were from a lower socioeconomic background. The sputum microscopy for AFB using the Ziehl-Neelsen stain was performed as per the RNTCP with a definite diagnosis of sputum, culture and chest X-ray in patients. Diabetes was confirmed based on blood sugar levels. All the subjects were HIV (Human Immunodeficiency Virus) negative. Body mass index (BMI) was calculated in all the subjects. Tuberculin skin test (TST) was performed in all the subjects except in DM and HC by administering 5Tuberculin Units (TU) purified protein derivative (PPD) on the volar surface of the left forearm and an induration >10 mm within 48–72 h was considered positive (TST+). The study was approved by the institutional ethical committee. Informed consent, clinical details and personal history were obtained from all the subjects included in the study. 2.2. Sampling A total of 10 ml blood was drawn from each subject and collected in Heparin tubes for PBMC assay, mRNA expression studies and cytokine production profile.

2.4. T-cell proliferation is assessed by lymphocyte proliferation assay PBMCs were cultured from the heparinised blood, wherein the assay described by Mosmann [18], has been employed based on the cellular conversion of a tetrazolium salt. The PBMCs were stimulated with concanavalin A (Con-A at a concentration of 1  10 used as positive control for cell reactivity) and h37rvAg 85A M.tb antigen, incubated for 3 days and 5 days respectively at 37 °C in an atmosphere of 5% CO2 for further studies. After addition of MTT (3-(4-5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) the optical density (O.D) was recorded at a dual wavelength of 570 nm and 620 nm reference filter. Data were expressed as a stimulation index (S.I) i.e. ratio of the mean O.D of experimental cultures to the mean O.D of control cultures. 2.5. Enzyme Linked Immunosorbent Assay (ELISA) ELISA was performed to measure the cytokine levels of IFN-c, IL-10 and IL-12 (pg/ml) in the culture supernatants (stimulated with h37rvAg 85A antigen) of TBDM, PTB patients, their HHC, DM patients and healthy controls using the BD biosciences kit as per the manufacturer’s instructions. 2.6. RNA isolation from stimulated cells, cDNA synthesis and Reverse Transcription Polymerase Chain Reaction (RT-PCR) The stimulated PBMCs were stored in 500 ll trizol at 80 °C for further mRNA isolation and cDNA synthesis. The synthesized cDNA as per the manufacturer’s instructions was amplified using the mRNA primers (shown in Table 1). The relative quantitative method was used to detect gene expression in cells. The IFN-c, IL-12, IL-10 and the housekeeping gene glyceraldehyde-3phosphate dehydrogenase (GAPDH) expression was carried out using SYBR green in iCycler IQ multicolor real-time detection system (Bio-Rad). Amplifications and the thermal-cycle parameters were followed according to the manufacturer’s directions. The delta-delta method (i.e., comparative ct [Dct] method) for quantitative analysis of gene expression was used. The results were normalized to those for GAPDH in the same sample and were expressed as fold increase. Gene expressions were determined based on results of stimulated cells as a fold change compared with basal levels in unstimulated cells. 2.7. Statistical analysis The mean and Standard deviation (SD) of cytokine levels and fold-change of the cytokine expression was calculated for each group using the non-parametric Mann-Whitney T-test and unpaired t test wherein the difference between the groups was checked. Shapiro-Wilk normality test was run to check the normal distribution for each group of the independent variable using graph pad prism software. Using IBM SPSS Statistics (version 20), a oneway analysis of variance (ANOVA) with a Tukey posthoc test was

Table 1 mRNA primer sequences of IFN-c, IL-12, IL-10 and GAPDH for expression analysis. GENE

Primer sequence

Amplicon size

IFN-c

F-GACCAGAGCATCCAAAAGAGT R-ATTGCTTTGCGTTGGACATTC F-GACATTCTGCGTTCAGGTCCAG R-CATTTTTGCGGCAGATGACCGTG F-GAGAACAGCTGCACCCACTT R-CTCAGACAAGGCTTGGCAAC F-GCCAATCAATGACCCCTTCATT R-TTGACGGTGCCATGGAATTT

143 bp

IL-12

2.3. Antigen

IL-10

Purified recombinant h37rvAg 85A antigen of M. tuberculosis has been procured from bei resource, USA.

GAPDH

203 bp 177 bp 320 bp

P. Meenakshi et al. / Cytokine 81 (2016) 127–136

used for comparison of 3 groups; Receiver-operating-characteristic (ROC) analysis was performed and the areas under the curve (AUC) were obtained for each cytokine marker by comparing the patient and control groups. The optimal cutoff value of each marker was obtained to determine the high and low risk groups. The cutoff derived for each marker was used to determine the likelihood of disease susceptibility in a group of household contacts. A p-value at confidence level 0.05 (two tailed) was considered significant for all the statistical analysis.

3. Results Lympho-proliferative responses and IFN-c, IL-12 and IL-10 cytokine production were measured in 50 cases each of TBDM, PTB, their HHC, DM and HCs at 0 M (0 month i.e., start of treatment). Some of the subjects have discontinued at different intervals of follow-up and out of them 25 cases each of TBDM, TBDM HHC, PTB and PTB HHC were followed for a period of one year (intervals of 4 M, 6 M and 12 M). The mRNA expression was carried out in 30 cases (5 cases in each category of TBDM, TBDM HHC, PTB, PTB HHC, DM and HC at 0 M (0 month i.e., start of treatment). 5 cases in each category of TBDM, TBDM HHC, PTB, PTB HHC were followed for one year at different intervals of 4 M, 6 M and 12 M. The lympho-proliferative responses were measured to determine the T-cell reactivity of Ag85A M.tb antigen. The mean stimulation index was significantly lower in TBDM (2.27 ± 0.729), TBDM HHC (2.775 ± 0.870), PTB (2.547 ± 0.836), PTB HHC (2.964 ± 0.876) and DM (3.281 ± 0.616) when compared to HCs (3.882 ± 1.278) at p < 0.0001, 0.0001, 0.0001, 0.0001 and 0.006 respectively. However, significant difference was not observed within the groups. The mean stimulation index at 0 M, 4 M, 6 M and 12 M respectively was 2.298 ± 0.726, 1.944 ± 0.855, 2.801 ± 0.882 and 2.886 ± 0.888 in TBDM, 2.369 ± 1.093, 2.347 ± 1.047, 2.723 ± 0.865 and 2.711 ± 0.711 in TBDM HHC, 2.569 ± 0.811, 2.379 ± 0.87, 2.764 ± 0.823 and 3.127 ± 0.998 in PTB, 3.335 ± 0.525, 3.11 ± 0.478, 3.361 ± 0.654 and 3.376 ± 0.592 in PTB HHC. It was significantly lower in TBDM at 0 M when compared to that at 6 M and 12 M at p < 0.045 and p < 0.039 respectively, and was statistically high at 12 M when compared to 0 M at p < 0.019 in the PTB. Statistically significant difference was not observed in the HHC at different stages of follow up (Fig. 1).

3.1. Cytokine production overtime in Ag85A stimulated culture supernatants of TBDM, PTB patients, their HHC, DM and HC The PBMC’s produced significantly low IFN-c in TBDM, TBDM HHC, PTB, PTB HHC (28.41 ± 12.82, 36.46 ± 18.71, 35.76 ± 25.8, 33.19 ± 26.94 pg/ml, p < 0.0001) and DM (49.06 ± 27.18, p < 0.0034) compared to HC (70.05 ± 42.88). A significant difference was observed in TBDM vs. TBDM HHC (p < 0.023), TBDM vs. DM (p < 0.0001) and PTB vs. DM (p < 0.0002). IFN-c levels increased with treatment at 6 M (p < 0.039, p < 0.008) and at 12 M (p < 0.0006, p < 0.001) in TBDM and TBDM HHC respectively; at 6 M (p < 0.02) in the PTB. In PTB HHC, the mean levels were almost similar at different phases of follow up (Fig. 2). Low IL-12 was produced in TBDM, TBDM HHC, PTB, PTB HHC and DM compared to HC and were found to be significant (24.68 ± 10.64, 58.68 ± 29.6, 25.56 ± 13.01, 44.16 ± 33.96, 59.10 ± 33.94, 122.0 ± 70.53 pg/ml, p < 0.0001). Statistically significant difference was observed in TBDM vs. TBDM HHC; TBDM vs. DM (p < 0.0001), PTB vs. PTB HHC (p < 0.003) and PTB vs. DM (p < 0.0001). IL-12 levels significantly increased with treatment in TBDM (p < 0.0002, p < 0.0001 and p < 0.0001), PTB (p < 0.0001) at 4 M, 6 M and 12 M respectively, when compared to 0 M; in TBDM at 6 M (p < 0.005) and

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12 M (p < 0.0001) when compared to 4 M; in TBDM 12 M vs. 6 M (p < 0.012) and in TBDM HHC at 4 M vs. 0 M (p < 0.02) (Fig. 3). Significantly high IL-10 was produced in TBDM, TBDM HHC, PTB, PTB HHC and DM compared to HC (72.64 ± 42.12, 60.15 ± 36.35, 94.53 ± 31.27, 30.60 ± 33.05, 79.36 ± 50.82, 16.46 ± 8.474 pg/ml, p < 0.0001). Statistically significant difference was observed in TBDM vs. PTB (p < 0.0002), TBDM HHC vs. PTB HHC, PTB vs. PTB HHC (p < 0.0001) and PTB vs. DM (p < 0.038). IL-10 levels significantly increased with treatment at 6 M and 12 M (p < 0.0008 and p < 0.0001) in TBDM and at 4 M, 6 M and 12 M (p < 0.018, p < 0.0003, p < 0.0001) in PTB when compared to 0 M; at 6 M (p < 0.008) and 12 M (p < 0.0002) in TBDM when compared to 4 M; in TBDM HHC at 12 M vs. 0 M (p < 0.029) (Fig. 4). The mean cytokine levels at different intervals of follow-up were shown in Table 2. 3.2. One-way Analysis of Variance (ANOVA) One way ANOVA analysis was carried out illustrating a significant difference in gender (p < 0.035), age, BMI, BCG, blood sugar levels and IFN-c, IL-12 and IL-10 cytokine levels at p < 0.0005. Post hoc tests for Turkey HSD for gender show significant differences between TBDM vs. PTB HHC at p < 0.017. Turkey HSD for age revealed significant differences between TBDM vs. TBDM HHC (<0.002), TBDM vs. PTB and TBDM vs. HC (<0.0005) & PTB vs. DM (<0.001). However Dunnett T3 for BMI showed significant difference between TBDM vs. HC at p < 0.004, PTB vs. PTB HHC (<0.001), PTB vs. DM (<0.003), PTB vs. HC (<0.0005). Turkey HSD for BCG showed significant difference between TBDM vs. HC and TBDM HHC vs. HC (<0.001), PTB vs. HC, PTB HHC vs. HC and DM vs. HC (<0.0005). Turkey HSD for IFN-c levels were significantly different between TBDM vs. DM (p < 0.003), TBDM vs. HC, TBDM HHC vs. HC, PTB vs. HC, PTB HHC vs. HC (<0.0005) and DM vs. HC (<0.002). Turkey HSD for IL-12 and IL-10 levels were significantly different between TBDM vs. TBDM HHC, TBDM vs. DM, TBDM vs. HC, TBDM HHC vs. HC, PTB vs. DM, PTB vs. HC and PTB HHC vs. HC (<0.0005) (Table 3). 3.3. Receiver Operating Characteristic (ROC) analysis The ROC curves were constructed by computing the sensitivity and specificity of varying IFN-c, IL-12 and IL-10 cytokine markers in TBDM, TBDM HHC, PTB, PTB HHC, DM vs. HC where the area under curve (AUC) with 95% confidence interval was obtained with statistical significance. The cutoff value is derived and sensitivity, specificity determined based on the cutoff value to identify the individual’s risk with respect to the marker. The AUC for IFN-c (0.824, 0.748, 0.773, 0.788, 0.632) and IL-10 (0.017, 0.038, 0.002, 0.406, 0.045) was less and that for IL-12 (0.992, 0.843, 0.986, 0.896 and 0.825) was more in TBDM, TBDM HHC, PTB, PTB HHC, DM respectively when compared to HC indicating that the test was performed more accurately in IL-12 with statistical significance (p < 0.0005). The cutoff value, sensitivity and specificity were the best for IL-12 which was 26.16 pg/ml, 98 and 38 in TBDM, 51.1 pg/ml, 98 and 44 in TBDM HHC, 49.26 pg/ml, 98 and 6 in PTB, 52.03 pg/ml, 98 and 28 in PTB HHC and 51.93 pg/ml, 98 and 50 in DM when compared to HC. Thus ROC analysis highlights the importance of IL-12 cytokine marker in the risk analysis (Table 4). 3.4. Expression of cytokine mRNA at different intervals of follow-up in TBDM, PTB patients, their HHC, DM and HC The IFN-c mRNA expression was significantly low by 3.6-fold in TBDM (p < 0.016), 3.8-fold in TBDM HHC (p < 0.008), 3.0-fold in PTB (p < 0.012), 4.0-fold in PTB HHC (p < 0.003) when compared to the HCs. At 12 M a statistically significant increase in expression was observed by 3.7-fold in TBDM (p < 0.008), 2.9-fold in TBDM

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<0.0001

Stimulation index (S.I)

<0.0001 <0.006

6

6

Stimulation index (S.I)

<0.0001 <0.0001

8

4 2

<0.039 <0.045

4

2

12

M

6M

C

C

H

M

H

H M

D

D TB

M

TB

D

H

C H H

H M D

4M

0M

M

C

12

H

M D

TB

TB

CATEGORY (N=50)

TB

4M

M TB

D

M D

TB

TB

D

C H

M D

H H

M

0M

D

PT

M

B

H

C

PT B

H

C

M D TB TB

6M

0 0

CATEGORY (N=25)

stimulatin index (S.I)

6 <0.019

4

2

P

H P

TB

TB P

P

TB HC H 6M H C 12 M

4M

H

H

C

C

0M

M H H

TB

P

TB

12

6M TB

4M P

TB P

P

TB

0M

0

CATEGORY (N=25) Fig. 1. Lympho-proliferative responses in TBDM, PTB patients, their HHC, DM and HC after stimulation with M.tb Ag85A and during follow up of TBDM, PTB patients and their HHC. Bars indicate the mean & SD for each group. Horizontal lines indicate a statistically significant difference between groups. Differences between the groups were analyzed by Mann-Whitney U test. p < 0.05 was considered statistically significant.

(a)

(b)

<0.0001 <0.0001 <0.0001 <0.034

<0.0001

150

200 175

IFN-γ (pg/ml)

IFN-γ (pg/ml)

200

<0.0002 <0.023

100 50

150 125

<0.0006 <0.039

<0.001 <0.008

<0.02

100 75 50 25

C

M

H

H PT

B

D

C H

B PT

C H H

M D TB

CATEGORY (N=50)

TB TBDM TBDM 0M TB T D 4M B M TB DM D 6 M M D T MH 1 TBBD H HC 2M D M HHC 0M M H 4 H C M H 6 C M P 12 TB M P 0 T M P B4 T P PT B M T 6 P B HB 1 M T 2 P B HHC M P TB H 0M TB H C H HC 4M H 6 C M 12 M

0

0

CATEGORY (N=25) Fig. 2. IFN-c levels in M.tb Ag85A stimulated culture supernatants of (a) TBDM, PTB patients, their HHC, DM and HC (b) TBDM, PTB patients and their HHC during their follow up. Bars indicate the mean & SD for each group. Horizontal lines indicate a statistically significant difference between groups. Differences between the groups were analyzed by Mann-Whitney U test. ⁄p < 0.05 was considered statistically significant.

HHC (p < 0.03), by 3.0-fold in PTB (p < 0.012) and at 6 M in PTB HHC (p < 0.016) when compared to its expression at 0 M. Significant difference was not observed at 4 M and 6 M in TBDM and TBDM HHC and at 4 M and 12 M in PTB HHC when compared to 0 M (Fig. 5).

IL-12 mRNA expression was significantly low by 2.9-fold in TBDM (p < 0.03), 3.0-fold in TBDM HHC (p < 0.008), 4.5-fold in PTB (p < 0.012) and 5.0-fold in PTB HHC (p < 0.008) when compared to HCs. In TBDM it increased significantly by 2.7-fold at 6 M and by 2.3-fold at 12 M (p < 0.0078) with treatment, while

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(b)

(a) 350

<0.0001

300

<0.0001

100

IL-12 (pg/ml)

IL-12 (pg/ml)

<0.0001

<0.0001

250 <0.0012

<0.0001

200 <0.0001

150 100

75

<0.0001 <0.0001 <0.0002 <0.0001

<0.02

50 25

50 0

C

P

TB

CATEGORY (N=50)

TB TBDM TBDM 0M T B TB D 4M M TB DM DM 6 M D T B M H H 12 TB DM H C M H D H C 0M M H 4 H C M H 6 C M P 12 T P B M T 0 P B4M TB M P P T T 6 P B HB 1 M TB H 2 P H CM P TB H 0M TB H C H HC 4M H 6 C M 12 M

D

H TB

H

M

C H

TB P

D

M

TB

H

D

H

C

M

0

CATEGORY (N=25) Fig. 3. IL-12p40 levels in M.tb Ag85A stimulated culture supernatants of (a) TBDM, PTB patients, their HHC, DM and HC (b) TBDM, PTB patients and their HHC during their follow up. Bars indicate the fold change for each group. Horizontal lines indicate a statistically significant difference between groups. Differences between the groups were analyzed by Mann-Whitney U test. ⁄p < 0.05 was considered statistically significant.

(b)

(a) <0.0001

250

<0.0001

100

IL-10 (pg/ml)

150

80

C

<0.029

TB TB DM TB DM 0M TB T D 4 B M TB DM D 6 M M M D TB M H 12 H M TB DM H C H D H C 0M M H 4 H C M H 6 C M P 1 TB 2 P 0M T M P B4 T P PT B M TB B 6 P H 1M T 2 P B H HC M P TB H 0M TB H C H HC 4M H 6 C M 12 M

TB

P

D

M

TB

H

M

H

P

H

D

H

TB

C H

C

0

M

0

TB

<0.0001

40 20

-50

<0.0008

60

50

D

IL-10 (pg/ml)

<0.0001

<0.0001

200

<0.0003

100

<0.0001

CATEGORY (N=50)

CATEGORY (N=25)

Fig. 4. IL-10 levels in M.tb Ag85A stimulated culture supernatants of (a) TBDM, PTB patients, their HHC, DM and HC (b) TBDM, PTB patients and their HHC during their follow up. Bars indicate the fold change for each group. Horizontal lines indicate a statistically significant difference between groups. Differences between the groups were analyzed by Mann-Whitney U test. ⁄p < 0.05 was considered statistically significant.

Table 2 Mean IFN-c, IL-12 and IL-10 cytokine levels in Ag85A stimulated culture supernatants of TBDM, PTB patients, their HHC during follow-up. Cytokine gene

Follow-up

TBDM Mean ± SD

TBDM HHC Mean ± SD

PTB Mean ± SD

PTB HHC Mean ± SD

IFN-c

0M 4M 6M 12 M

28.46 ± 9.85 36.11 ± 15.82 40.43 ± 20.22 51.01 ± 25.31

31.37 ± 17.12 37.13 ± 21.80 42.75 ± 19.14 53.30 ± 26.70

33.34 ± 21.00 30.15 ± 25.36 49.45 ± 29.40 46.37 ± 27.96

26.64 ± 12.01 38.40 ± 35.58 26.55 ± 26.94 50.05 ± 45.14

IL-12

0M 4M 6M 12 M

25.63 ± 10.47 37.75 ± 11.42 63.65 ± 27.38 82.11 ± 34.05

42.84 ± 12.70 36.80 ± 19.80 41.75 ± 20.12 43.98 ± 22.07

18.94 ± 6.35 43.82 ± 14.10 49.95 ± 24.77 57.65 ± 25.97

41.56 ± 35.37 36.10 ± 32.85 28.88 ± 24.54 42.25 ± 22.58

IL-10

0M 4M 6M 12 M

57.61 ± 27.64 56.19 ± 27.37 39.51 ± 21.13 32.65 ± 19.47

52.95 ± 36.17 55.07 ± 36.03 37.62 ± 17.62 35.39 ± 18.76

88.04 ± 23.13 63.25 ± 47.89 54.64 ± 34.94 44.28 ± 17.71

29.61 ± 29.80 36.20 ± 22.08 36.15 ± 54.90 33.98 ± 31.48

TBDM – tuberculosis patients with diabetes mellitus; TBDM HHC – household contacts of TBDM; PTB – pulmonary tuberculosis patients; PTB HHC – household contacts of PTB

PTB patients have shown low expression at 0 M and increased significantly with treatment at 6 M by 2.5-fold and at 12 M by 2.4-fold (p < 0.021) when compared to its expression at 0 M. Significant difference was not observed in DM vs. HC and with treatment in TBDM HHC and PTB HHC (Fig. 6).

IL-10 mRNA expression was significantly low by 2.6-fold in TBDM (p < 0.01), 2-fold in TBDM HHC (p < 0.03) when compared to HCs. It was low at 0 M and significantly increased with treatment at 6 M by 2.4-fold in TBDM (p < 0.015), by 3.2-fold in TBDM HHC (p < 0.02) and by 1.9-fold at 12 M in PTB HHC (p < 0.04) when

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Table 3 One-way ANOVA analysis for clinical parameters and IFN-c, IL-10 and IL-12 levels in TBDM, PTB patients, their HHC, DM and HC. Variable

Mean square

F

p value

Gender

Between Groups Within groups

0.597 0.245

2.44

0.035

Age

Between Groups Within groups

1816.92 157.39

11.54

0.0005

BMI

Between Groups Within groups

245.28 27.41

8.95

0.0005

Skin test

Between Groups Within groups

0.18 0.141

1.275

0.284

BCG

Between Groups Within groups

1.69 0.216

7.81

0.0005

Blood sugar levels

Between Groups Within groups

37.82 0.34

111.17

0.0005

IFN-c levels

Between Groups Within groups

11680.68 747.23

9.79

0.0005

IL-10 levels

Between Groups Within groups

41325.64 1491.39

27.71

0.0005

IL-12 levels

Between Groups Within groups

64240.03 1406.38

45.68

0.0005

⁄ p < 0.05 was considered statistically significant. Groups-TBDM, PTB patients, their HHC, DM and HC.

compared to its expression at 0 M. Significant difference was not observed in DM vs. HC and with respect to fold change at 6 M and 12 M in TBDM HHC and PTB HHC with treatment (Fig. 7). 4. Discussion The Antigen85 complex composed of three closely related proteins 85A(32 kDa), 85B(30 kDa) and 85C (32.5 kDa) of mycobacteria is a major secretory product inducing T cell proliferation and antibody synthesis against TB [19–21]. When the PBMCs were stimulated with Ag85A, stimulation was low in patients which enhanced after treatment completion. This suggests an improvement in cell mediated immunity upon treatment. However, statistical significance was not observed in the HHCs. A depressed immune response and a low proliferative response to mycobacterial antigens was further reported in the PBMC of TB patients [22,23] which may be due to the secretion of monocytes and lymphocytes that act as suppressive factors and may also be due to a shift from Th1 to Th2 cytokine response. Immunosuppression may also be due to preferential abolition of antigen specific T cells

in the infected areas leading to their absence in the PBMCs. A study by Chang Hwa song et al., reported a difference in the lympho proliferative response among TB patients and their healthy tuberculin reactors using 30 kDa antigen unlike our study [24]. Contradictory results were shown in a Brazilian population study wherein the mean stimulation index in the PBMC upon treatment withAg85A was found to be higher in PTB patients and improved with treatment [25]. There were no reports in TBDM, their HHC and in DM. IFN-c is the most important cytokine marker in T cell stimulation assays involved with protective immunity in the host towards mycobacterial antigens and it mediates immunopathology. It activates macrophages in conjunction with TNF-a to facilitate the killing of intracellular mycobacteria. TBDM patients showed low IFN-c levels at the time of diagnosis and increased gradually with treatment. A similar result was observed in Chennai, India, where IFN-c production was low in TBDM when compared to PTB and DM patients [26] and increased after 6 months of treatment in PTB patients similar to a Pakistan study [27,28] and Indonesia [29]. Nevertheless IFN-c production in response to ESAT-6 but not Ag85B and PPD were significantly higher in PTB patients when compared to the controls [30]. In contrast the average IFN-c production was significantly higher in 38 kDa/CFP10 stimulated cells of PTB patients [31]. One study in the United Kingdom had shown decreased IFN–c production upon treatment. T cells produced higher IFN–c one month after the initiation of treatment in LTBI but towards the end decreased. PBMCs infected with M.tb and treated invitro led to an increase in IFN-c producing cells [32]. Decreased IFN-c production may be due to the lack of IL-12 production which induces IFN-c production. A study by Hussain et al., has reported significant differences in the IFN-c levels of PTB patients and healthy controls [33]. Furthermore the concentration of IFN-c in PTB patients negatively correlated with the duration of anti-tuberculosis therapy. IFN-c levels were high in the ESAT-6, CFP-10 and TB7.7 stimulated cells of LTBI individuals when compared to HCs [34]. Our findings indicate that Ag85A may be strongly implicated in the protective immune responses in humans as it brings out a lympho-proliferative response and IFN-c production in PTB patients after an appropriate anti tuberculosis therapy [35]. TBDM patients were found to have lower IFN-c mRNA expression similar to a Japanese study. Similar to our study, Martens et al., has not reported any significant difference between TBDM and DM patients [36]. PTB patients elicited slight IFN-c expression when compared to the Healthy controls similar to a Korean study where they expressed limited mRNA with response to Ag85B in PTB patients [37]. Different reports indicate that the PBMC of PTB patients produce low IFN-c mRNA than PTB HHC [38] which was

Table 4 Area under the curve, sensitivity, specificity and cut-off values of IFN-c, IL-12 and IL-10 cytokines in TBDM, PTB patients, their HHC and DM vs. HC.

⁄

Category

Variable

Area

Std. Error

Sig

95% CI

Sensitivity

Specificity

Cut-off

TBDM vs. HC

IFN-c IL-12 IL-10

0.824 0.992 0.017

0.058 0.008 0.009

0.956 0.0005 0.0005

0.389–0.617 0.977–1.00 0.0005–0.034

92 98 6

58 38 86

23.11 26.16 35.4

TBDM HHC vs. HC

IFN-c IL-12 IL-10

0.748 0.843 0.038

0.049 0.04 0.016

0.0005 0.0005 0.0005

0.651–0.844 0.765–0.922 0.006–0.069

64 98 4

28 44 62

43.99 51.1 39.16

PTB vs. HC

IFN-c IL-12 IL-10

0.773 0.986 0.002

0.047 0.009 0.002

0.0005 0.0005 0.0005

0.681–0.865 0.968–1.00 0.0005–0.006

94 98 4

58 6 96

21.75 49.26 39.47

PTB HHC vs. HC

IFN-c IL-12 IL-10

0.788 0.896 0.406

0.045 0.033 0.058

0.0005 0.0005 0.106

0.700–0.876 0.832–0.961 0.292–0.521

96 98 6

68 28 24

18.36 52.03 37.56

DM vs. HC

IFN-c IL-12 IL-10

0.632 0.825 0.045

0.056 0.042 0.018

0.022 0.0005 0.0005

0.522–0.742 0.743–0.907 0.10–0.08

64 98 16

42 50 88

43.99 51.93 24.36

p < 0.05 was considered statistically significant.

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Fig. 5. IFN-c mRNA expression in response to M.tb Ag85A stimulation in (a) TBDM, PTB patients, their HHC, DM and HC (b) TBDM patients and TBDM HHC during their follow up and (c) PTB patients and PTB HHC during their follow up. Bars indicate the fold change for each group. Horizontal lines indicate a statistically significant difference between groups. Differences between the groups were analyzed by Mann-Whitney U test. ⁄p < 0.05 was considered statistically significant.

distinct from a study in Brazil where it was high in PTB patients compared to HCs [39]. This impairment may be due to the inability in bacterial clearance and the consequent progress of the disease. There were no studies reported till date on mRNA expression in TBDM and TBDM HHC during their follow-up. IFN-c mRNA expression levels increased with treatment in PTB patients in contrast to a study in Brazil where they decrease after anti tuberculosis therapy [39]. Similar to the Brazilian population, no difference was observed at 4 M. There was no significant difference between PTB and PTB HHC analogous to an Ethiopian study where no statistical significance was reported. Even post treatment PTB patients have shown their IFN-c mRNA unchanged and PTB HHC have shown a slight increase [40]. In the present study, low IFN-c production was observed at the time of diagnosis in all the patients and household contacts. IL-12 composed of p35 and p40 chains is a heterodimeric cytokine has potent immunological effects in vitro. Defective IL-12 production was seen during M.tb infection, wherein diabetes is likely to be a strong risk factor contributing to anti-mycobacterial defenses through several mechanisms. It enhances cytotoxicity by CD4+ T cells against the macrophages pulsed with M.tb [41]. IL-12 production was low in TBDM, PTB patients and their HHC that may be due to a deficiency in intracellular reduced Glutathione (GSH) concentrations in diabetic patients. IL-12, the strong inducer of IFN-c was similar in M.tb H37Rv stimulated culture supernatants of both TBDM and PTB groups similar to an Indonesian population study [15]. Divergent results were reported in ESAT6/CFP10 stimulated culture supernatants of PTB patients where it was high when compared to the PPD + and PPD- household contacts [42]. DM patients were found to have significantly less IL-12 production, which was not in agreement to a study by NP Kumar et al., where higher production was reported [26].

IL-12 production was low before treatment in PTB but increased after anti-TB therapy similar to studies from Japan [43], Korea [24] and Hyderabad [44]. This increase in the IL-12 production with treatment may be due to an augmentation in the immune responses and variation in antigen load. IL-12 mRNA expression increased with treatment in PTB patients similar to a Brazilian population study [41]. Significant increase was reported at 4 M by Oliveira et al., unlike in our study where a significant increase was observed at the end of treatment, but not at 4 M [45]. A southern blot analysis indicated that PBMC from healthy tuberculin reactors exhibited much higher IL-12 expression than those from TB patients [24]. In TB pleuritis, IL-12 mRNA and protein are concentrated at the site of disease produced in response to M.tb [38] was in contrast to our study where decreased IL-12 was seen in PTB patients. IL-12 mRNA was low in DM patients compared to HCs similar to LPS stimulated PBMCs of DM patients [46]. Increased IL-12 mRNA was witnessed in M.tb infected mice, which decreased dramatically later in the micro-dissected granulamatous lesions and in guinea pigs [47]. Formation of granulomas by the Th1 cytokines helps in the containment of mycobacteria. Interleukin-10 is a potent immunomodulatory cytokine that directly or indirectly affects multiple cell types including macrophages, monocytes, dendritic cells, CD4 T cells and CD8 T cells in vitro [48]. The dominant function of IL-10 is to deactivate macrophages, resulting in diminished Th1 cytokine production [49], decreased production of reactive nitrogen or oxygen species [50] and reduced antigen presentation which may have far-reaching consequences on both innate and acquired immunity in vivo. The production of anti-inflammatory cytokine IL-10 in M.tb H37Rv antigen stimulated culture supernatants was significantly higher in PTB patients at 0 M and decreased to normal after treatment which was in agreement to a study by Hari Sai Priya et al. [44]. This

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Fig. 6. IL-12 mRNA expression in response to M.tb Ag85A stimulation in (a) TBDM, PTB patients, their HHC and DM vs. HC (b) TBDM patients and TBDM HHC during their follow up (c) PTB patients and PTB HHC during their follow up. Bars indicate the fold change for each group. Horizontal lines indicate a statistically significant difference between groups. Differences between the groups were analyzed by Mann-Whitney U test. ⁄p < 0.05 was considered statistically significant.

Fig. 7. IL-10 mRNA expression in response to M.tb Ag85A stimulation in (a) TBDM, PTB patients, their HHC and DM vs. HC (b) TBDM patients and TBDM HHC during their follow up (c) PTB patients and PTB HHC during their follow up. Bars indicate the fold change for each group. Horizontal lines indicate a statistically significant difference between groups. Differences between the groups were analyzed by Mann-Whitney U test. ⁄p < 0.05 was considered statistically significant.

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decrease may be due to the modification in cytokine expression in the infected individuals with treatment. Indonesian population [15] did not report any difference in IL-10 between PTB and HC and was significantly lower in TBDM when compared to PTB patients dissimilar to our study. IL-10 production was significantly higher in ESAT-6, CFP 10 and TB 7.7 stimulated PBMCs of PTB and LTBI individuals compared to the HCs [51]. The proliferative response towards M.tb H37Rv stimulation enhanced in all the patients with therapy that may be due to cell mediated immune progress and similar results were observed in a Brazilian population. Significant difference was not observed in PTB patients before and after treatment in a Brazilian population [52]. Increased IL-10 production may be due to the decrease in blastogenic response and IFN-c production in active PTB patients. IL-10 production was significantly higher in TBDM when compared to PTB patients similar to a study in Chennai [26]. IL-10 mRNA expression was low in PTB patients similar to a Norwegian population study wherein it was low in latently infected mice when compared to the HCs [53]. Similar results were shown in the M.tb stimulated PBMCs of TB and LTBI individuals [54]. Divergent results were reported where the IL-10 expression was found to be high at 0 M and 6 M but significant difference was not shown during treatment analogous to our study [45]. Among the 50 household contacts (TBDM HHC and PTB HHC 25 each) that were followed 2 of them have developed the disease during their sixth month of follow up. The mean IFN-c, IL-12 and IL-10 cytokine levels remained almost similar at different intervals of follow-up indicating the suppressed pro-inflammatory response by the anti-inflammatory cytokine response that could be due to an imbalance between pro and anti inflammatory responses. The antigen 85A plays a pivotal role in treatment monitoring, thereby protecting the high risk individuals by their prior identification, and thereby highlighting the importance of this antigen. 5. Conclusion The mean PBMC proliferative response to Ag85A M.tb leading to the cytokine release was positive and reveals the clinical status of the patients and household contacts. After treatment completion, a significant increase in the stimulation indices (SI) was observed and was almost equal to that of healthy controls. This antigen may thus be used in immunodiagnostics and immunotherapeutics. The IFN-c and IL-12 cytokine levels were low at the time of diagnosis and increased to normal levels at the end of treatment with least immune response in TBDM and PTB patients. IL-10 levels were high and decreased with treatment in all the groups of patients and household contacts. The mean cytokine levels were almost similar both in the patients and household contacts and even not much difference was observed during their follow up. This might be due to the latent infection developed by household contacts due to the similar environment shared by them with the patients, but due to their anti inflammatory response they may have not developed the disease. Thereby follow up of these patients and household contacts may help in identifying the patients who are likely to return to be categorized as retreatment cases, and for early detection of the high risk household contacts. The IL-12 cytokine gene was considered as a candidate gene and the most potential biomarker based on the ROC curve analysis with the highest sensitivity and specificity, hence may be used in the diagnostics. The pro-inflammatory cytokine gene expression correlates with protective immunity against TB, however, higher level post infection signifies a risk factor for developing active TB. Thus, our data suggest that the mechanisms that control the excessive inflammatory responses during M.tb infection may have a critical role in the immunopathology of TB.

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