Macrophages and dendritic cells (DC) play an essential role in the initiation and maintenance of immune response to pathogens. To analyze early interactions between Mycobacterium tuberculosis (Mtb) and immune cells, human peripheral blood monocyte-derived macrophages (MDM) and monocyte-derived dendritic cells (MDDC) were infected with Mtb. Both cells were found to internalize the mycobacteria, resulting in the activation of MDM and maturation of MDDC as reflected by enhanced expression of several surface Ags. After Mtb infection, the proinflammatory cytokines TNF-α, IL-1, and IL-6 were secreted mainly by MDM. As regards the production of IFN-γ-inducing cytokines, IL-12 and IFN-α, was seen almost exclusively from infected MDDC, while IL-18 was secreted preferentially by macrophages. Moreover, Mtb-infected MDM also produce the immunosuppressive cytokine IL-10. Because IL-10 is a potent inhibitor of IL-12 synthesis from activated human mononuclear cells, we assessed the inhibitory potential of this cytokine using soluble IL-10R. Neutralization of IL-10 restored IL-12 secretion from Mtb-infected MDM. In line with these findings, supernatants from Mtb-infected MDDC induced IFN-γ production by T cells and enhanced IL-18R expression, whereas supernatants from MDM failed to do that. Neutralization of IFN-α, IL-12, and IL-18 activity in Mtb-infected MDDC supernatants by specific Abs suggested that IL-12 and, to a lesser extent, IFN-α and IL-18 play a significant role in enhancing IFN-γ synthesis by T cells. During Mtb infection, macrophages and DC may have different roles: macrophages secrete proinflammatory cytokines and induce granulomatous inflammatory response, whereas DC are primarily involved in inducing antimycobacterial T cell immune response.

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-γ-inducing cytokines, IFN-αβ and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-αβ. Indeed, IFN-αβ neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4+, and CD8+ cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-αβ-induced expression of CXCL10, neutralization of IFN-αβ significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-αβ may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.

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The virulence of a Mycobacterium tuberculosis H37Rv sigE mutant was studied in immunodeficient and immunocompetent mice. The mutant was strongly attenuated in both animal models and induced formation of granulomas with different characteristics than those induced by the wild-type strain.During infection, bacteria often face different environments which result from the site in which the pathogen resides as well as activation of the host's immune response. To be successful, a pathogen must be able to adapt quickly to these differing milieus. Most bacterial adaptive mechanisms are based on the regulation of gene expression, which consequently plays a very important role in bacterial pathogenesis (13).Sigma factors play a major role in the regulation of bacterial gene expression. These proteins are interchangeable RNA polymerase subunits that are responsible for promoter recognition. Bacteria usually have a principal sigma factor, usually constitutively expressed, which is responsible for the transcription of essential housekeeping genes, and a number of alternative sigma factors that are transcriptionally and/or posttranslationally activated in response to specific environmental signals (14). The Mycobacterium tuberculosis genome encodes 13 putative sigma factors, 10 of which belong to the extra cytoplasmic function (ECF) family (4, 6). In previous work, our investigators studied the variation of factor gene expression in response to different environmental stresses and found that sigB and sigE were strongly induced after exposure to detergent-induced surface stress. The same two genes, together with sigH, were also induced after heat shock and after exposure to the thiol-specific oxidizing agent diamide (8, 9). Our investigators recently characterized two M. tuberculosis mutants lacking the ECF factors E and H . These mutants were sensitive to various environmental stresses; moreover, the sigE mutant showed a decreased ability to grow inside macrophages. Using DNA microarray technology, we have studied the E and H regulons and have identified several genes that are under direct or indirect control of these factors (9, 10). Interestingly, it has been recently reported that the M. tuberculosis sigE regulon is activated after phagocytosis (12). A , F , and the ECF factor H have a role in M. tuberculosis virulence, as shown in animal models of infection. A Mycobacterium bovis mutant with a mutated sigA was attenuated for virulence in guinea pigs (5), while an M. tuberculosis sigF strain was shown to be attenuated in immunocompetent mice (3). M. tuberculosis mutants lacking sigH produced a reduced immunopathology in infected animals (7), despite the observation that the growth kinetics in their organs were similar to that of the wild-type (wt) strain (7, 9).In the experiments described in this communication, we have studied the M. tuberculosis sigE mutant ST28 (10) in two different mouse models of infection: immunocompetent BALB/c mice and severe combined immunodeficient (SCID) mice, which lack functional B and T cells...

Resistance of Mycobacterium tuberculosis to fluoroquinolones (FQ) results mostly from mutations in the gyrA gene. We developed a reverse hybridization-based line probe assay in which oligonucleotide probes carrying the wild-type gyrA sequence, a serine-to-threonine (S95T) polymorphism, and gyrA mutations (A90V, A90V-S95T, S91P, S91P-S95T, D94A, D94N, D94G-S95T, D94H-S95T) were immobilized on nitrocellulose strips and hybridized with digoxigenin-labeled PCR products obtained from M. tuberculosis strains. When a mutated PCR product was used, hybridization occurred to the corresponding mutated probe but not to the wild-type probe. A panel of M. tuberculosis complex strains including 19 ofloxacin-resistant (OFL-R) and 9 ofloxacin-susceptible (OFL-S) M. tuberculosis strains was studied for detection and identification of gyrA mutations by the line probe assay and nucleotide sequencing, in comparison with testing of in vitro susceptibility to FQ. Results were 100% concordant with those of nucleotide sequencing. The S95T polymorphism, which is not related to FQ resistance, was found in 5 OFL-S and 2 OFL-R strains; the other 17 OFL-R strains harbored single mutations associated with serine or threonine at codon 95. No mutations were found in the other OFL-S strains. Overall, on the basis of the MICs on solid medium, the new line probe assay correctly identified all OFL-S and 17 out of 19 (89.5%) OFL-R strains. A nested-PCR protocol was also evaluated for the assay to amplify PCR products from M. tuberculosis-spiked sputa, with a good specificity and a sensitivity of 2 ؋ 10 3 M. tuberculosis CFU per ml of sputum. (5,(9)(10)(11)29). The use of these drugs as second-line antituberculosis agents is recommended for treating multidrug-resistant (MDR) tuberculosis (TB) (3,8). As with other antimicrobial agents, the use of FQ can generate resistant mutants (5, 6, 11-13). The principal target of the FQ in M. tuberculosis is DNA gyrase, a type II topoisomerase composed of two A and two B subunits encoded by the gyrA and gyrB genes, respectively (2,11,33). Mutations in the so-called quinolone resistance-determining region (QRDR) of gyrA are the primary mechanisms of FQ resistance in M. tuberculosis. Amino acids at positions 88, 90, 91, and 94 of gyrA are those most frequently substituted in the FQ-resistant M. tuberculosis clinical isolates (1,11,12,19,22,26,27,(31)(32)(33); mutations of gyrB have rarely been reported (15). Fluoroquinolones (FQ) are antimicrobial agents with good in vitro and in vivo activities against Mycobacterium tuberculosisDespite the increasing use of these drugs in TB therapy, standard 3-week culture-based FQ susceptibility testing in solid media is not always performed (11). PCR-based techniques provide new possibilities for the rapid diagnosis of FQ resistance; however, it can be difficult to put them into practice in the mycobacteriology laboratory (7,24,33,34). To this end, we developed a reverse hybridization-based line probe assay for rapid detection of gyrA mutations in M. tuberculosis. The test was ...