Phenotypic modulation in Mycobacterium tuberculosis infected neutrophil during tuberculosis.

BACKGROUND & OBJECTIVE: Polymorphonuclear leucocytes (PMN) or neutrophils infiltrate to the inflammatory sites and phagocytose mycobacteria thereby inhibiting the bacillary spread initially until the accumulated macrophages get activated. The present study was carried out to highlight the interaction of neutrophils with the two clinical isolates (S7 and S10) of Mycobacterium tuberculosis and the subsequent morphological changes. METHODS: Dextran purified neutrophils from normal and TB patients infected with M. tuberculosis isolates were cultured for 3 and 18 h time points. At the end of termination, the cell surface expression of CD16, CD69, CXCR2 and induction of apoptosis were analyzed using flow cytometry. Cytokines and chemokines were estimated in supernatants by ELISA. RESULTS: All infected PMN showed decrease in CD16 at both time points in normals while at 18 h in TB group. Interestingly, CD69 expression was significantly high at early time point in TB-PMN compared to normals. The high expression of CXCR2 was sustained in infected TB-PMN at both the time points. S7 and S10 infected neutrophils showed high phagocytic indices compared to H37Rv in both the groups. A significant increase in apoptosis was observed at both the time points in infected TB-PMN but only at 18 h in normals. Increased pro-inflammatory cytokine (TNF-alpha) and chemokine (IL-8) response was observed in infected neutrophils at 3 h in both the groups. INTERPRETATION & CONCLUSION: This study demonstrates the varying degree of modulation of neutrophil functions in both the groups. TB-PMN was more competent in amplifying the innate immune response and conferring protection at the early phase of infection. However, the response was not strain specific in either of these groups.

Infection with prevalent clinical strains of Mycobacterium tuberculosis leads to differential maturation of monocyte derived dendritic cells.

The link between innate and adaptive host immune response to Mycobacterium tuberculosis (M.tb) is driven by dendritic cells (DC). In this study, we examined the ability of prevalent clinical strains from south India (S7, S10) and laboratory strain H37Rv (Rv) to induce maturation of monocyte derived dendritic cells (MoDC). The phenotypic and functional changes of DC upon infection with different strains of M.tb were evaluated. It was observed that S7 and Rv strains partially hampered the maturation of MoDC as reflected by the low expression of maturation markers and co-stimulatory markers when compared to LPS stimulated MoDC. In contrast, strain S10 infected DC showed a marked increase in the expression of these markers. The functional property was investigated by the ability of infected MoDC to induce T-cell proliferation and to stimulate secretion of IFN-gamma by CD4(+)T-cells. It was found that Rv and S7 infected MoDC were less efficient in inducing T-cell proliferation. The secretion of IL-12 by Rv and S7 infected MoDC was also found to be significantly lesser than LPS stimulated MoDC. On the other hand, S10 infected MoDC showed enhanced T-cell stimulation and cytokine secretion. Together these results indicate that there is a substantial variability in the capacity of M.tb clinical strains to induce maturation of DC which may be dependent upon their virulence.

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.

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-gamma-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.

Expression of co-stimulatory molecules B7.1 & B7.2 on macrophages infected with various strains of Mycobacterium tuberculosis & its influence on T-cell apoptosis.

BACKGROUND & OBJECTIVE: Activation of T cells is mediated through two critical signals provided by activated macrophages. The first signal is triggered when T cell receptor (TCR) binds to the major histocompatibility antigen (MHC/Ag) complex. The second signal is the interaction of co-stimulatory molecules with their respective ligands on T cells for their activation and proliferation. We undertook this study to observe the modulation in B7.1 (CD80) and B7.2 (CD86) co-stimulatory molecules on Mycobacterium tuberculosis infected monocyte derived macrophages (MDM) and their role in T helper (Th1) cell apoptosis. METHODS: M. tuberculosis clinical strains (S7 and S10) and laboratory strains (H37Ra and H37Rv) were used to infect the MDMs. The modulation of apoptosis was assessed by treating T cells with anti-CD3 and anti-CD28 antibodies. The infected MDMs were co-cultured with autologous PPD pulsed T cells to ascertain the role of co-stimulatory molecules during infection. RESULTS: In infected MDMs, all strains on day 1 but only S7 on day 2 showed significant decrease (P<0.05) in B7.1 expression compared to uninfected. The expression levels of B7.2 were also low on day 1 in S7, S10 and H37Ra infected MDMs. The anit-CD3 induced apoptosis in PPD pulsed Tcells showed further reduction with anti-CD28 antibodies. However, the modulation observed in B7.1 expression in infected MDMs was not reflected in T cell apoptosis in co-culture experiments. INTERPRETATION & CONCLUSION: Our results confirmed the role of B7.1 in rescuing the activated Tcells from undergoing apoptosis. During infection when the expression of B7.1 is downregulated, other co-stimulatory molecules may take over its crucial role to confer protective immune response against M. tuberculosis.

Leptin response in patients with tuberculous pleuritis.

BACKGROUND & OBJECTIVES: Tuberculous pleuritis is used as a model to understand the protective immune response in tuberculosis. It is predominated by Th1 response at the site of infection, where a possible role for the leptin, a known enhancer of Th1 response, could be speculated. Hence, we investigated leptin levels in pleural effusions in patients with both tuberculous (TP) and non-tuberculous (NTP) pleural effusion. METHODS: Leptin and cytokine levels were assessed in serum and pleural fluid of TP and NTP patients (N = 20 each) by ELISA. Multivariate regression analysis were performed to find the possible determinants of leptin taking leptin as the dependent and body mass index (BMI), gender, source of leptin [i.e., serum or pleural fluid (PF)], age and disease status as independent variables. RESULTS: PF leptin levels were significantly higher than serum leptin levels in both the groups however the PF leptin levels were significantly lower in TP subjects compared to NTP. The results showed that the leptin was found to be dependent on BMI but not on the other parameters. However, regression analysis based on the source of leptin showed males to be a better predictor of leptin. No correlation was observed between leptin and measured immune parameters. INTERPRETATION & CONCLUSION: Our findings demonstrated that the decreased leptin levels were associated with reduction in BMI but not with the disease status in tuberculous pleuritis.

TLR stimulation of human neutrophils lead to increased release of MCP-1, MIP-1α, IL-1ß, IL-8 and TNF during tuberculosis.

Neutrophils inform and shape immune responses. Toll-like receptors (TLRs) play an essential part in the perception of microbes and shape the complex host responses that occur during infection. The TLRs present on neutrophils play an indispensable role in neutrophil mediated pathogen recognition and elimination. This study was done to identify the role of significant TLRs in immune responses leading to differences in cytokine/chemokine release following stimulation. We evaluated the concentrations of various significant cytokines (IL-1ß, TNF, MIP-1α, MCP-1 and IL-8) secreted by neutrophils from healthy donors and pulmonary tuberculosis patients following TLR ligand stimulation. TLR stimulation increased the release of such cytokines in both the groups. Thus it is noted that TLR stimulation of neutrophils definitely lead to increased cytokine response. Also, the release of all the studied cytokines are found to be greatly increased in patient neutrophils, affirming that neutrophils undergo secretory level modifications during tuberculosis infection.

Proteomics Analysis of Three Different Strains of Mycobacterium tuberculosis under In vitro Hypoxia and Evaluation of Hypoxia Associated Antigen's Specific Memory T Cells in Healthy Household Contacts.

In vitro mimicking conditions are thought to reflect the environment experienced by Mycobacterium tuberculosis inside the host granuloma. The majority of in vitro dormancy experimental models use laboratory-adapted strains H37Rv or Erdman instead of prevalent clinical strains involved during disease outbreaks. Thus, we included the most prevalent clinical strains (S7 and S10) of M. tuberculosis from south India in addition to H37Rv for our in vitro oxygen depletion (hypoxia) experimental model. Cytosolic proteins were prepared from hypoxic cultures, resolved by two-dimensional electrophoresis and protein spots were characterized by mass spectrometry. In total, 49 spots were characterized as over-expressed or newly emergent between the three strains. Two antigens (ESAT-6, Lpd) out of the 49 characterized spots were readily available in recombinant form in our lab. Hence, these two genes were overexpressed, purified and used for in vitro stimulation of whole blood collected from healthy household contacts (HHC) and active pulmonary tuberculosis patients (PTB). Multicolor flow cytometry analysis showed high levels of antigen specific CD4(+) central memory T cells in the circulation of HHC compared to PTB (p < 0.005 for ESAT-6 and p < 0.0005 for Lpd). This shows proteins that are predicted to be up regulated during in vitro hypoxia in most prevalent clinical strains would indicate possible potential immunogens. In vitro hypoxia experiments with most prevalent clinical strains would also elucidate the probable true representative antigens involved in adaptive mechanisms.

Differentiation of highly prevalent IS6110 single-copy strains of Mycobacterium tuberculosis from a rural community in South India with an ongoing DOTS programme.

We have prospectively analysed the DNA fingerprinting of Mycobacterium tuberculosis strains in a rural community from high prevalence area in South India with an ongoing DOTS programme. Strains from 451 culture-positive cases, diagnosed during July 1999-December 2000, were fingerprinted initially by both IS6110 and DR probes followed by polymorphic GC-rich repeat sequences (PGRS) typing only on low-copy strains. The results were correlated with selected epidemiological and clinical data. Forty one percent of strains showed single copy of IS6110, which further got differentiated into 62 DR and 27 PGRS patterns. One predominant DR pattern (5B/2) was found in 20% of the low-copy strains and was also involved in clusters. In all, 183 patients out of 451 (40%) were clustered in total 44 clusters when analysed by IS6110 and DR probes. With additional PGRS typing, the number of patients clustered was further reduced to 106 (23%). More number of patients (131) were clustered in IS6110 single-copy group. The maximum number of clusters was found with two or three patients. Only a small percentage (16%) of the patients reported direct epidemiological links while remaining patients might have had indirect links or casual contacts. Thus, a combination of two to three genetic markers is able to differentiate the most endemic strains of M. tuberculosis in areas with a high incidence of tuberculosis. The epidemiological data do not suggest any major outbreaks or a hot-spot hypothesis of transmission in this region. Phylogenetic analysis using IS6110, DR and PGRS RFLP (restriction fragment length polymorphism, RFLP) fingerprints showed that isolates exhibited clonal evolutionary pattern. The predominance of certain genotypes and agreement between the phylogenetic trees indicated that these strains were closely related and might have evolved or propagated from the common ancestor.

Th2-type immune response observed in healthy individuals to sonicate antigen prepared from the most prevalent Mycobacterium tuberculosis strain with single copy of IS6110.

Different Mycobacterium tuberculosis strains operate different immune evasion strategies for their survival in the host. This mainly depends on the virulence of the strain and the host immune responses. The most virulent strains are actively involved in the transmission, widely spread in the community and induce differential immune responses. We evaluated the immune response of a sonicate antigen prepared from one predominant strain (S7) from M. tuberculosis harbouring a single copy of IS6110. Significant lymphoproliferative response against purified protein derivative from tubercle bacillus (PPD) and H37Rv antigens was observed in PPD positive normal individuals and tuberculosis patients. Interferon-gamma (IFN-gamma) levels against these antigens were significantly increased in normal individuals but not in tuberculosis patients. The antigen S7 showed marginal T-cell proliferation but did not induce IFN-gamma secretion in both groups. Conversely, it induced significantly high levels of cytokine interleukin 4 (IL-4) in normal individuals. The macrophage cytokines, IL-12 and tumour necrosis factor alpha (TNF-alpha), did not show S7 antigen specific stimulation. The intracellular cytokine further confirmed an increase in IL-4(+)/CD4+ T-cells and a decrease in IFN-gamma(+)/CD4+ T-cells upon stimulation. The antibody response showed an increase in IgG and IgA levels against this antigen in normal individuals. These observations suggest that antigen S7 modulates the immune response towards T helper cell type 2 by suppressing T helper cell type 1 protective immune response in PPD positive normal individuals. We speculate that some components of this sonicate antigen are associated with immunosuppressive response.

Mycobacterium tuberculosis strains modify granular enzyme secretion and apoptosis of human neutrophils.

Mycobacterium tuberculosis has evolved to employ multiple strategies to avoid an efficient host immune response. Accordingly, enzymes are important antimycobacterial elements and apoptosis decides the fate of any cell. Hence, we carried out this study to discern the amplitude of two clinical stains (S7 & S10) and a laboratory strain (H37Rv) of M. tuberculosis in modifying the release of lytic enzymes and apoptosis of neutrophils from healthy volunteers and pulmonary tuberculosis patients. We detected reduced levels of elastase in neutrophils from pulmonary tuberculosis patients. The laboratory strain H37Rv is found to increase the release of elastase and myeloperoxidase in neutrophils from both the groups. This strain is more efficient compared to clinical strains in inducing late apoptosis/necrosis in neutrophils. Our results proclaim the susceptibility of neutrophils in responding to infection with H37Rv. Also, at the functional level, neutrophils undergo changes related to release of enzymes after acquiring tuberculosis.

Cell proliferation and apoptosis: dual-signal hypothesis tested in tuberculous pleuritis using mycobacterial antigens.

Antigens and mitogens have the innate ability to trigger cell proliferation and apoptosis thus exhibiting a dual-signal phenomenon. This dual-signal hypothesis was tested with mycobacterial antigens (PPD and heat killed Mycobacterium tuberculosis - MTB) in tuberculous pleuritis patients where the immune response is protective and compartmentalized. We compared and correlated the cell-cycle analysis and antigen-induced apoptosis in normal and patients' peripheral blood mononuclear cells (PBMCs) and patients' pleural fluid mononuclear cells (PFMCs). In cell-cycle analysis, PFMCs showed good mitotic response with PPD and MTB antigens where 10% and 7% of resting cells entered the S and G2/M phases of cell cycle, respectively. This antigen-induced proliferation of PFMCs correlated well with the lymphocyte transformation test (LTT) results. On the other hand, PFMCs also showed 21% of spontaneous apoptosis, which further increased to 43%, by induction with known apoptotic agent like Dexamethasone (DEX) and the mycobacterial antigens PPD and MTB. Further we demonstrated by anti-CD3 induction experiments that prior activation of cells is prerequisite for them to undergo apoptosis. Our results showed that PPD and MTB antigens induced both cell proliferation and apoptosis in PFMCs, which were pre-sensitized to mycobacterial antigens in vivo. Thus the dual-signal phenomenon was operative against these antigens in tuberculous pleuritis. We also demonstrated that the activated cells are more predisposed to apoptosis.

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.

Neutrophils from pulmonary tuberculosis patients show augmented levels of chemokines MIP-1α, IL-8 and MCP-1 which further increase upon in vitro infection with mycobacterial strains.

Neutrophils being innate cells initiate the immune defence against mycobacteria by sending signals to other immune cells. Chemokines being the vital link in signaling processes, it is of interest to study their secretion by neutrophils as a response to tuberculosis infection. The levels of various chemokines (MIP-1α, MCP-1, IL-8 and IP-10) and chemokine receptors (CXCR1, CXCR2 and CCR1) in neutrophils from healthy individuals and pulmonary tuberculosis patients were studied following infection with Mycobacterium tuberculosis strains (clinical--S7 and S10 and laboratory--H37Rv). The release of MIP-1α, IL-8 and MCP-1 is found to be greatly increased in patient neutrophils. Mycobacterial strains differentially influenced neutrophils affecting the release of chemokines to different extent. H37Rv significantly increased the release of MIP-1α and IL-8 in both normals and tuberculosis patients, while S10 up regulated only the release of MIP-1α in patients. Thus, during tuberculosis, neutrophils undergo functional alteration to combat infection. While H37Rv is greatly recognized by neutrophils and triggers the release of chemokines, clinical strains by some means try to suppress immune activation of neutrophils in their favor.

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.

Mycobacterium tuberculosis H37Rv is more effective compared to vaccine strains in modulating neutrophil functions: an in vitro study.

Neutrophils are the primary cells contributing to initial defense against mycobacteria. Yet, little is known about the potential of various mycobacterial strains to stimulate neutrophils. This study was focused to compare the differential capacity of vaccine strains, Mycobacterium bovis bacillus Calmette-Guerin (BCG) and Mycobacterium indicus pranii (Mw), and laboratory strain H37Rv to activate and enhance neutrophil functions. The expression of phenotypic markers like Fcγ receptor, toll-like receptor (TLR), and chemokine receptor; secretion of pro-inflammatory cytokines; and the rate of apoptosis were studied in infected neutrophils. Increased expression of CD32, CD64, TLR4, and CXCR3; increased TNF-α secretion; and downregulation of early apoptosis were observed in H37Rv-infected neutrophils. Among the vaccine strains, BCG increased the expression of only CD32 on neutrophils, while Mw was comparatively ineffective. To understand the paracrine role of neutrophils, the supernatants from infected neutrophils were used to stimulate monocytes and T helper cells. The secretory molecules from all infected neutrophils increased the expression of CCR5 on monocytes, whereas only H37Rv-infected supernatant increased the expression of CCR7 on monocytes and CD69 on T cells. Thus, H37Rv was more effective in activating neutrophils and in turn stimulating monocytes and T cells. By comparison, vaccine strains were less effective in modulating neutrophil functions.

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.

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.

Impaired phenotype and function of monocyte derived dendritic cells in pulmonary tuberculosis.

Pulmonary tuberculosis (PTB) is often associated with impaired immunological functions. Blood monocytes, which can differentiate into dendritic cells upon cytokine stimulation, play a central role in adequate immune reactivity. Here, we investigated the morphologic, phenotypic and functional characteristics of in vitro-generated monocyte derived dendritic cells (MoDC) from PTB patients in comparison with healthy subjects. Phenotypic analysis revealed a defective differentiation of MoDC in PTB patients as assessed by a strong down regulation of CD1a, MHC II, CD80 and CD83 expression and impaired allostimulatory function under the influence of IL-4 and GM-CSF. In contrast, the expression of CD86 was not affected and remained same as in healthy subjects. Furthermore, the maturation status of lipopolysaccharide (LPS) stimulated MoDC was not optimal in PTB. However, the MoDC of PTB patients produced significantly higher levels of TNF-alpha and IL-6 but lower levels of IL-12 compared to healthy subjects. These findings suggest that there is a fundamental defect in the differentiation and maturation of dendritic cells during PTB that may compromise the antigen presentation and subsequent immune functions.