Cytokine Gene Polymorphisms across Tuberculosis Clinical Spectrum in Pakistani Patients

BACKGROUND: Pakistan ranks 7(th) globally in terms of tuberculosis (TB) disease burden (incidence 181/100000 pop./yr; prevalence of 329/pop./yr). Reports from different populations show variable associations of TB susceptibility and severity with cytokine gene polymorphisms. Tuberculosis clinical severity is multi-factorial and cytokines play a pivotal role in the modulation of disease severity. We have recently reported that the ratio of two key cytokines (IFNgamma and IL10) show significant correlation with the severity spectrum of tuberculosis. The objective of the current study was to analyze the frequency of cytokine gene polymorphisms linked to high and low responder phenotypes (IFNgamma +874 T(hi)-->A(lo) and IL10 -1082 G(lo)-->A(hi)) in tuberculosis patients. METHODS AND FINDINGS: STUDY GROUPS WERE STRATIFIED ACCORDING TO DISEASE SITE AS WELL AS DISEASE SEVERITY: Pulmonary N = 111 (Minimal, PMN = 19; Moderate, PMD = 63; Advance, PAD = 29); Extra-pulmonary N = 67 (Disseminated DTB = 20, Localized LTB = 47) and compared with healthy controls (TBNA = 188). Genotype analyses were carried out using amplification refractory mutation system-PCR (ARMS-PCR) and stimulated whole blood (WB) culture assay was used for assessing cytokine profiles. Our results suggest that the IFNgamma +874 TT genotype and T allele was overrepresented in PMN (p = 0.01) and PMD (p = 0.02). IFNgamma +874 TT in combination with IL10 GG(lo) genotypes showed the highest association (chi(2) = 6.66, OR = 6.06, 95% CI = 1.31-28.07, p = 0.01). IFNgamma AA(lo) on the other hand in combination with IL10 GG(lo) increased the risk of PAD (OR = 5.26; p = 0.005) and DTB (OR = 3.59; p = 0.045). CONCLUSION: These findings are consistent with the role of IL10 in reducing collateral tissue damage and the protective role of IFNgamma in limiting disease in the lung

Interferonγ /IL10 ratio defines the disease severity in pulmonary and extra pulmonary tuberculosis

Several cytokines (IFNγ, TNFα, IL10 and IL6) show an association with either disease localization or dissemination in tuberculosis. There are also reports of involvement of extra-pulmonary sites in tuberculosis with differential clinical severity. However, no comparative study of biomarkers across the disease severity spectrum is available. This was the purpose of the current study. Cytokines (IFNγ, TNFα, IL10 and IL6) secreted in response to a panel of stimulants (PHA, LPS or mycobacterial antigens) in whole blood were determined in eighty-two tuberculosis patients. WHO criteria was applied for stratification of patients according to disease severity: disseminated and or severe disease (EPTB1; N=29); disease localized to lung parenchyma (PTB; N=32) and disease localized to peripheral sites without lung involvement (EPTB2; N=21). Mycobacterial antigens induced IFNγ/IL10 ratio showed a direct relationship with disease severity ranking (median ratios: EPTB1=0.21; PTB=0.85; EPTB2=7.7) and the highest correlation (Spearman Rank; rho=0.673, pγ/IL10 ratio also rank ordered clinical severity as it relates to anatomic sites. IFNγ/IL10 ratio may therefore provide a useful objective marker of disease severity in both pulmonary and extra-pulmonary tuberculosis

Diagnostic modality used for confirmation of tuberculosis.

<p>Note: Primary diagnostic modality used diagnosis of tuberculosis.</p>*<p>Criteria for disease category given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004778#s4" target="_blank">material and methods</a>.</p>¶<p>Imaging tests included chest×rays for pulmonary patients, CT scan and or MRI for disseminated disease.</p

Relationship of IFNγ and IL10 SNPs with mycobacterial antigen induced cytokine secretion.

<p>Whole blood from TB not affected (TBNA) Tuberculin skin test positive donors was stimulated with <i>M. tuberculosis</i> culture filtrate (CF) proteins [5 µg/ml] and supernatants tested at 2 days for IL10 secretion and day 5 for IFNγ secretion using ELISA method as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004778#s4" target="_blank">material and methods</a>. Results are expressed as pg/ml after deducting secretion in un-stimulated whole blood.</p

Demographic characteristics of TB Patients and Controls.

<p><b>Note:</b> Patient stratification is as given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004778#s4" target="_blank">Materials and Methods</a>. Abbreviations for groups are given in brackets. TBA included 10 previously treated patients.</p

Genotype combination in relation to disease severity.

<p>Genotype combination in relation to disease severity.</p

Differences in allele frequencies in healthy controls and tuberculosis patients

<p>Note: Patient stratification is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004778#s4" target="_blank">material and methods</a>. N for each group is given in brackets. Abbreviations used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004778#pone-0004778-t001" target="_blank">Table 1</a>.</p

Genotype frequencies in healthy controls and different clinical forms of tuberculosis (TB).

<p>Note: Patient stratification is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004778#s4" target="_blank">Materials and Methods</a>. N for each group given in brackets. Abbreviations used as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004778#pone-0004778-t001" target="_blank">Table 1</a>. Number (frequency) of genotypes is indicated. Pearson chi analysis was carried to determine the significance of differences.</p><p>All significant p values are indicated in bold. P approaching significance is given in italics. <i>p</i><0.05 is considered significant.</p