Abstract: We evaluated the role of regulatory T cells (CD4(+) CD25(+) Foxp3(+) cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin reactors, but not in tuberculin-negative individuals. The M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19-kDa protein and heat shock protein 65 had no effect. Anti-IL-10 and anti-TGF-beta alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme-2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat-killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amounts of TGF-beta and IL-10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis-responsive CD4(+) IFN-gamma cells. Culturing M. tuberculosis-expanded Tregs with autologous CD8(+) cells decreased the frequency of IFN-gamma(+)cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.

Abstract: We evaluated the capacity of NK cells to influence expansion of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) in response to microbial Ags, using Mycobacterium tuberculosis as a model. We previously found that Tregs expand when CD4(+) cells and monocytes are exposed to M. tuberculosis. Addition of NK cells that were activated by monokines (IL-12, IL-15, and IL-18) or by exposure to M. tuberculosis-stimulated monocytes reduced Treg expansion in response to M. tuberculosis. NK cell inhibition of Treg expansion was not mediated through IFN-gamma. Activated NK cells lysed expanded, but not freshly isolated Tregs. Although monokines increased NK cell expression of the activating receptors NKp46, NKG2D, 2B4, CD16, and DNAM-1, only anti-NKG2D and anti-NKp46 inhibited NK cell lysis of expanded Tregs. Of five NKG2D ligands, only UL16-binding protein 1 (ULBP1) was up-regulated on M. tuberculosis-expanded Tregs, and anti-ULBP1 inhibited NK cell lysis of expanded Tregs. M. tuberculosis-stimulated monocytes activated NK cells to lyse expanded Tregs, and this was also inhibited by anti-NKG2D and anti-ULBP1, confirming the physiological relevance of this effect. Our study identifies a potential new role for NK cells in maintaining the delicate balance between the regulatory and effector arms of the immune response.

Abstract: Recent report from our laboratory showed that A549 cells representing alveolar epithelial cells produce chemokine interleukin-8 and nitric oxide (NO) when challenged with Mycobacterium tuberculosis. Interferon-gamma (IFN-gamma) played a critical role in priming these cells to generate NO in vitro. In the present study, we report that M. tuberculosis-infected A549 cells are capable of elaborating IFN-gamma as shown by enzyme-linked immunosorbent assay and intracellular staining for IFN-gamma. Secretion profile indicated that M. tuberculosis-infected A549 released significantly high concentration of IFN-gamma at 48 and 72 h post-infection. Low level of IFN-gamma release was also seen to be induced by gamma-irradiated M. tuberculosis and subcellular components of M. tuberculosis. Cell surface receptor analysis showed that the M. tuberculosis-infected A549 cells expressed enhanced levels of IFN-gamma receptors. This observation suggests that the endogenously produced IFN-gamma in response to M. tuberculosis infection plays a role in intracellular regulation of innate immunity against intracellular pathogen such as M. tuberculosis. This observation is further strengthened by the fact that infected A549 cells expressed signal transducer and activator of transcription 1 (STAT1), an important mediator for IFN-gamma signaling pathway, leading to expression of inducible NO synthase and subsequent release of NO in sufficient concentration to be mycobactericidal. Our results show that production of IFN-gamma and enhanced expression of IFN-gamma receptors by infected A549 cells is a local phenomenon occurring as de novo intracellular activity, in response to M. tuberculosis infection. To the best of our knowledge, this is the first report to show that A549 cells interact actively with M. tuberculosis to produce IFN-gamma that might play an important role in innate immunity against tuberculosis.

Abstract: We previously showed that human NK cells used the NKp46 receptor to lyse Mycobacterium tuberculosis H37Ra-infected monocytes. To identify ligands on H37Ra-infected human mononuclear phagocytes, we used anti-NKp46 to immunoprecipitate NKp46 from NK cells bound to its ligand(s) on H37Ra-infected monocytes. Mass spectrometry analysis identified a 57-kDa molecule, vimentin, as a putative ligand for NKp46. Vimentin expression was significantly up-regulated on the surface of infected monocytes, compared with uninfected cells, and this was confirmed by fluorescence microscopy. Anti-vimentin antiserum inhibited NK cell lysis of infected monocytes, whereas antiserum to actin, another filamentous protein, did not. CHO-K1 cells transfected with a vimentin construct were lysed much more efficiently by NK cells than cells transfected with a control plasmid. This lysis was inhibited by mAb-mediated masking of NKp46 (on NK cells) or vimentin (on infected monocytes). ELISA and Far Western blotting showed that recombinant vimentin bound to a NKp46 fusion protein. These results indicate that vimentin is involved in binding of NKp46 to M. tuberculosis H37Ra-infected mononuclear phagocytes.

Abstract: In view of the presence of a large number of epithelial cells in the alveoli of the lung and their ability to produce various cytokines and chemokines, the possible role of alveolar epithelial cells in the innate immune response to tuberculosis was examined. The human alveolar epithelial cell line A549 was used as a model. The ability of A549 cells to induce nitric oxide (NO) in response to Mycobacterium tuberculosis infection was taken as an in vitro correlate of innate immunity. M. tuberculosis infection induced A549 cells to produce significant levels of NO and to express inducible nitric oxide synthase mRNA at 48 hr of infection. However, the amount of NO released at this point was not mycobactericidal. Cytokine stimulation (interferon-gamma, tumour necrosis factor-alpha, interleukin-1beta, alone or in combination) of the infected A549 cells induced a higher concentration of NO. The study of colony-forming units (CFU) as a measure of the mycobactericidal capacity of A549 cells revealed a reduction in CFU of M. tuberculosis by 39.29% (from 10.62 +/- 0.48 - 6.392 +/- 0.54) following cytokine stimulation of the infected cells. Interestingly gamma-irradiated M. tuberculosis H37Rv could also induce higher than basal level of NO. Therefore we examined mycobacterial antigenic components for their possible role in NO production. We observed that A549 cells produced significantly higher amounts of NO at 48 hr when treated with mycobacterial whole cell lysates, cell wall or cell membrane preparations. The release of NO and the resultant mycobactericidal activity could be further enhanced by simultaneously conditioning the M. tuberculosis infected A549 cells with cytokine and mycobacterial components. These results suggest that alveolar epithelial cells respond to their microenvironment, which is constituted of various cytokines and macrophage-processed antigens and may contribute to the innate immune response to tuberculosis.

Abstract: Mycobacterium tuberculosis is an intracellular pathogen that readily survives and replicates in human macrophages. Host cells have developed various mycobactericidal and immunoregulatory mechanisms, such as the production of nitric oxide and inflammatory cytokines to control intracellular replication of M. tuberculosis. Inducible nitric oxide synthase (iNOS) is transcriptionally under the control of IFN-gamma and TNF-alpha. IL-12 provides a crucial link between activated mononuclear phagocytes and T cells by regulating the production of IFN-gamma. In this study, we investigated the production of nitric oxide (NO), TNF-alpha and IL-12 by the peripheral blood monocytes (PB Mn) of patients suffering from multidrug-resistant tuberculosis (MDR-TB). The cells were infected with M. tuberculosis and stimulated with IFN-gamma or activated with mycobacterial subcellular components. The results were compared with those from cases of newly diagnosed TB and healthy controls. Nitric oxide production was significantly depressed in PB Mn from MDR-TB patients. Infected monocytes from newly diagnosed TB patients produced significantly higher levels of NO as compared to those from MDR-TB patients or normal controls. The subcellular fraction of M. tuberculosis-like whole cell lysate (WCL), culture filtrate protein (CFP) and lipoarabinomannan (LAM) induced higher concentrations of NO release in PB Mn from newly diagnosed TB patients as compared to those from MDR-TB patients. Cell culture supernatant from PB Mn assayed at 48 h after infection or stimulation demonstrated significantly depressed release of TNF-alpha and IL-12 from MDR-TB cases as compared to the fresh cases. We observed a definite correlation between nitric oxide release and TNF-alpha production, irrespective of low or high production in MDR-TB or fresh cases, respectively. The present data suggest that peripheral blood monocytes of MDR-TB patients typically show signs of immunosuppression. Whether such immunodepression is the cause or the effect of MDR-TB merits further investigation.