Sulochana D. Das

Molecular Epidemiology of Tuberculosis in a Rural Area of High Prevalence in South India: Implications for Disease Control and Prevention

ABSTRACT Molecular and conventional epidemiologic techniques were used to study the mechanisms and risk factors for tuberculosis transmission in a rural area with high prevalence in south India. Restriction fragment length polymorphism analysis with IS6110 and direct repeat probes was performed with 378 Mycobacterium tuberculosis isolates from patients. Forty-one percent of M. tuberculosis isolates harbored a single copy of IS6110. Of 378 patients, 236 had distinct strains; 142 (38%) shared a strain with other patients, indicating recent infection. Older patients, those detected by a house-to-house community survey, and those hospitalized in a sanatorium were more likely to have had a recent infection. These findings suggest that the majority of the tuberculosis cases in south India were due to reactivation; therefore, efforts to control tuberculosis should be sustained.

Correlates of protective immune response in tuberculous pleuritis

Tuberculous pleuritis (TB) provides a good model to study the correlates of protective immune response at the site of infection. To study the in vivo correlates of immunity, cell subset profile and cytokine assay in plasma (BL) and pleural fluid (PF) of 82 patients were done. Lymphocyte proliferation and cytokine response to mycobacterial antigens were measured in 32 subjects to understand the in vitro correlates. Increase in CD4(+) cells and CD4(+)/CD8(+) ratio with selective concentration of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and interleukin (IL)-12 in PF suggests that the CD4(+) population may be of TH1 type. We observed an accelerated lymphoproliferative response to purified protein derivative (PPD) and heat killed Mycobacterium tuberculosis (MTB) in PF cells of both TB and non-TB (NTB) subjects. Interestingly, in in vitro studies, IL-4 levels together with IFN-gamma were significantly increased in the supernatants of PF mononuclear cells (PFMC) of TB patients and showed a shift in immune response towards TH0/TH2 type. PPD and MTB antigens induced an enhanced proliferation of PFMC and also increased in vitro IL-4 response together with apoptosis, thus eliciting a dual response.

Augmented chemokine levels and chemokine receptor expression on immune cells during pulmonary tuberculosis

The systemic changes in immune mediators such as cytokine and chemokines, and their synchronized interaction that regulates the cell trafficking during Mycobacterium tuberculosis (M. tuberculosis) infection, were studied. Cytokines and chemokines were evaluated by cytometric bead array (CBA) and enzyme-linked immunosorbent assay (ELISA) in 34 pulmonary tuberculosis (PTB) patients and 30 healthy subjects. The expression of chemokine receptors was assessed by flow cytometry. A significant increase in IP-10, MIG, interleukin-8, RANTES, and interleukin-6 levels was found, whereas a decrease in interferon-gamma, tumor necrosis factor-alpha, and transforming growth factor-beta was observed during PTB. Significant correlation within chemokines and between cytokines was observed in PTB. All immune cells except monocytes and B cells expressed significantly higher levels of CCR1, CCR2, and CXCR2 whereas CCR7 expression was upregulated only on monocytes and neutrophils in PTB. Both T and B cells expressed significantly high levels of CXCR3 which also correlated well with the chemokine levels in PTB. Thus, it was found that chemokines function coordinately and consistently during PTB. This balanced chemokine and cytokine relationship at the periphery may aid in amplified effector immune cell trafficking and retarded monocyte migration through differential chemokine receptor expression.

Expression of CXC and CC type of chemokines and its receptors in tuberculous and non-tuberculous effusions

Chemokines mediate their biological functions by transmigration of various immune cells to the site of infection. Tuberculous pleurisy provides an effective model to study the role of chemokines in the recruitment of immune cells to the pleura. Our aim was to understand the cumulative effect of chemokines (IP-10, MIG, IL-8, MCP-1, MIP-1alpha and RANTES) and its receptors (CXCR2, CXCR3, CCR1, CCR2, CCR5 and CCR7) in the recruitment of CD4(+) T cells obtained from blood (BL) and pleural fluid (PF) of tuberculous (TB) and non-tuberculous (NTB) patients. We observed significant increase in CD4(+) T cells in TB PF indicating lymphocytic rich effusion. All chemokines except RANTES were significantly high in PF compared to BL in TB group, whereas IL-8 and MCP-1 showed significant increase only in NTB PF. The significantly high levels of IFN-gamma and TauNuF-alpha in TB PF and their positive correlation with IP-10 and MIP-1alpha indicated their synergistic action to elicit a strong protective Th1 response. In spite of high levels of Th1 cytokines and chemokines in TB PF, significantly lower levels of RANTES indicated its limited role at the site. The CXC receptors in PF of both the groups and CC receptors except CCR5 in TB PF were significantly high compared to BL. Only CXCR2, CCR5 and CCR7 showed significant increase in TB compared to NTB. Thus a selective concentration of chemokines, cytokines and abundant expression of chemokine receptors confirm the accumulation of activated and memory T cells at the site of infection and help in polarizing Th1 immune response.

Diagnostic utility of interferon-γ–induced protein of 10 kDa (IP-10) in tuberculous pleurisy

Tuberculous pleuritis (TP) is characterized by predominant Th1 immune response. We observed significantly high levels of interferon gamma (IFN-gamma) and chemokines such as IP-10, monokine induced by IFN-gamma (MIG), interleukin 8 (IL-8), monocyte chemotactic protein (MCP)-1, and macrophage inflammatory protein (MIP)-1alpha in tuberculous pleural effusions. In the current study, we evaluated the diagnostic utility of IFN-gamma-dependent chemokine especially IP-10. The receiver operating characteristics (ROC) curve analyses based on cytometric bead array values depicted high sensitivity only for IP-10 (76.3%) followed by IFN-gamma (73.7%). The ELISA test further confirmed the significantly high levels of IFN-gamma and IP-10 in TP. The ROC curve analysis again demonstrated high area under the curve (AUC) for IP-10 (0.966) than the referred diagnostic marker IFN-gamma (0.930). The better sensitivity (84.2% for IFN-gamma and 89.2% for IP-10) and equal specificity (95.7%) of IP-10 assay compared with IFN-gamma suggest that IP-10 is a potential diagnostic marker for evaluating TP.

A Correlation between Phagocytosis and Apoptosis in THP-1 Cells Infected with Prevalent Strains of Mycobacterium tuberculosis

The innate ability of infected macrophages to undergo programmed cell death (apoptosis) and curtail the infection is crucial for the host defense. Although phagocytosis and intracellular killing mechanisms leading to apoptosis in macrophages are highly effective in eliminating the infecting tuberculous bacilli, some Mycobacterium tuberculosis (Mtb) strains have evolved strategies to inhibit this microbicidal function and make use of macrophage for its successful and prolonged survival. Two clinical strains of Mtb (S7 and S10) found to be prevalent and primitive, based on molecular epidemiological studies, were used to study the magnitude in induction of apoptosis in THP‐1 cells at various time points of infection and to correlate it with phagocytosis. The percentage of phagocytosis did not show any strain‐specific association with differentiated THP‐1 cells. But in the phagocytic index, the clinical strains showed a low dose of infection in the 1–10 bacilli category thereby exerting less burden on the cells. The induction of apoptosis was strain dependent The THP‐1 cells infected with H37Ra and S10 showed an increase in apoptosis at all time points while the S7 strain induced minimum apoptosis. A negative correlation between apoptosis and phagocytic index was observed in the 1–10 category and a positive correlation in the >20 category of the phagocytic index. This novel observation indicates that the magnitude of THP‐1 cell apoptosis is a function of the number of internalized mycobacteria. These results indicated a differential mode of infection by clinical strains and their adaptation to different survival strategies that may lead to immune suppression and pathogenesis of the disease.

Differential upregulation of chemokine receptors on CD56+ NK cells and their transmigration to the site of infection in tuberculous pleurisy

Chemokines and their receptors orchestrate leukocyte recruitment and confer immunity during Mycobacterium tuberculosis infection. The immunoregulatory and cytotoxic activities of natural killer (NK) cells are essential at the site of infection during tuberculous pleurisy. The frequency, subtypes, and expression of phenotype markers and chemokine receptors on NK cells were assessed by flow cytometry in tuberculous (TB) and nontuberculous (NTB) pleural fluid (PF). Chemotaxis was also shown in response to chemokines. A significant decrease in CD56(dim) with no change in CD56(bright) NK cells was observed, while a significant increase in activation markers and Toll-like receptors (TLRs) was observed on TB-PF CD56(bright) NK cells. Significantly increased expression of chemokine receptors CCR1, CCR2 and CCR7 on CD56(bright) and CCR5 on CD56(dim) NK cells was observed in the TB group. Transmigration of TB-PF NK cells was significantly high in response to IL-8, IP-10, MCP-1 and SLC. Transmigrated TB-NK cells showed a significant increase in CXCR2, CCR2 and CCR7 expression. The study suggests that CD56(bright) NK cells may recognize M. tuberculosis directly using TLRs, HLA-DR and express CD69 as an early activation marker. In addition, CC chemokines induce activation signals in chemokine receptors mediating differential NK cell migration to the site. Thus, NK cells act as first direct sensors and effectors in mycobacterial infection.

Comparison of localized versus systemic levels of Matrix metalloproteinases (MMPs), its tissue inhibitors (TIMPs) and cytokines in tuberculous and non-tuberculous pleuritis patients

The interaction of Matrix metalloproteinases (MMPs), its tissue inhibitors (TIMPs) and pro-inflammatory cytokines in response to Mycobacterium tuberculosis (MTB) infection is important to understand the immune response at the site of infection. We compared the levels of MMPs, TIMPs and cytokines in plasma (BL) and pleural fluid (PF) of tuberculosis (TB) and non tuberculosis (NTB) patients. Comparison between BL and PF showed significantly higher levels of MMP-1, TIMP-1 and -3 in TB PF; of MMP-7, -8, -9 in BL of both groups. Also, levels of MMP-1,-8,-9 and TIMP-3 were significantly higher in TB PF compared to NTB. Cytokines INF-γ, TNF-α, and IL-6 significantly increased in PF of both groups. A positive correlation of MMPs with TIMPs in TB, MMP-1 and -9 with IL-6 in TB PF and MMP-9 with IFN-γ in NTB PF was observed. This study implicates the possible usage of MMPs as bio-markers aiding diagnosis in TB pleuritis.

Differential migration of human monocyte-derived dendritic cells after infection with prevalent clinical strains of Mycobacterium tuberculosis

Dendritic cells (DCs) play a key role in the host immune response to infections. Mycobacterium tuberculosis (MTB) can inhibit the maturation of DCs and impair their ability to stimulate T cell proliferation. Here, we assessed in vitro migratory behavior of human monocyte-derived DCs (MoDC) when infected with various MTB strains (H37Rv and prevalent clinical strains S7 and S10 from South India). The migration of Rv and S7 infected MoDC towards secondary lymphoid chemokine (CCL21) was 50% lower after 1 day of infection compared to LPS stimulation. This reduced cell migration may be due to a block in the chemokine receptor switch from CCR5 to CCR7 expression on MoDC. Only clinical strain S10 infected MoDC showed an up-regulation of CCR7 and down-regulation of CCR5 expression, similar to LPS stimulated MoDC. While Rv and S7 infected MoDC did not display any alteration in expression of these receptors. Similarly, Rv and S7 infected MoDC did not induce IL-8, IP-10 and MCP-1 chemokine production. This reduction in chemokine levels was reflected in the reduced chemoattraction of CD4(+) T cells also. These findings suggest that there is variation in the stimulation of MoDC with different clinical strains of MTB and this variation may be dependent upon the virulence of the strain.

Cell-Mediated Immune Responses of Healthy Laboratory Volunteers to Sonicate Antigens Prepared from the Most Prevalent Strains of Mycobacterium tuberculosis from South India Harboring a Single Copy of IS6110

ABSTRACT Our restriction fragment length polymorphism (RFLP) studies have shown that the most prevalent (40%) strains of Mycobacterium tuberculosis from South India contain a single copy of the IS6110 insertion sequence and are of importance in studying virulence and immunity. Sonicate antigens from seven such strains were used to study in vitro T-cell proliferation and gamma interferon (IFN-γ) and interleukin-12 (IL-12) secretion as markers of protective immunity in 25 healthy subjects positive for purified protein derivative (PPD). The standard PPD and heat-killed H37Rv antigens induced the maximum levels of T-cell proliferation and IFN-γ secretion but low levels of IL-12. All sonicate antigens induced T-cell proliferation and IFN-γ secretion with strong positive correlation. Our results suggest that sonicate antigens from the most prevalent and recent strains of M. tuberculosis from clinical isolates have the potential to induce T-cell activation and may allow newer and specific antigens to be further characterized for diagnosis and vaccine development.