Regulation of granuloma fibrosis by nitric oxide during Mycobacterium avium experimental infection (original) (raw)
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Tubercle and Lung …, 1997
Setting: Although nitric oxide (NO) is a major proximate mediator of microbicidal activity in murine macrophages against intracellular pathogens including mycobacteria, its production by and effector role in human macrophages is not clear. Objective: To determine the capacity of Mycobacterium tuberculosis (MTB) to stimulate NO in human monocytes (MN) and alveolar macrophages (AM) and to assess the relationship between NO production and intracellular growth of MTB. Design: NO production (measured as nitrite) by MTB (H37Ra)-infected macrophages and intracellular growth of MTB were measured in cells from 17 healthy subjects. Results: MTB (5:1, MTB:cells) stimulated little to no NO by MN, but induced NO in AM at days 4 and 7 after infection. There was, however, variability in the response by AM to MTB: among seven subjects MTBqnduced NO was low (4 _+ 2 #M, mean + SE); six subjects were moderate (56 + 11); four subjects were high (502 _+ 167). NO synthase inhibitors inhibited the production of NO by AM but did not significantly affect the intracellular growth of MTB, although a trend towards increased intracellular growth was seen on day 4 of culture. Intracellular growth of MTB in AM from low NO producers was significantly higher than that in AM from moderate NO producers, P <_ 0.05. Inducible NO synthase (iNOS) mRNA by RT-PCR was constitutively expressed by both MN and AM, but was further stimulated by MTB in AM > MN; MTB-induced iNOS protein was present in both MN and AM by Western blot analysis. Conclusion: Thus, MTB-infected human AM are capable of producing NO and NO production correlates with intracellular growth inhibition of MTB in AM suggesting that NO may serve either directly or indirectly as a mycobactericidal mediator in human tissue macrophages. Mycobacterium tuberculosis (MTB) and the lungs are the major site of disease activity. Alveolar macrophages (AM) that reside along alveolar epithelial cells are believed to
2014
Background: In earlier studies, it was shown that ex vivo Mycobacterium tuberculosis-infected type II alveolar epithelial cells generate de novo nitric oxide (NO), but the mycobactericidal quantity of NO was released only by stimulation of these cells with proinflammatory cytokines, i.e. IFN-c, TNF-a and IL-1b. In the present communication, it was demonstrated that M. tuberculosis-infected/mycobacterial antigens stimulated cells utilize both, JAK-STAT and NF-jB pathways for the induction of inducible Nitric Oxide Synthase (iNOS) mRNA and NO production. Methods: Alveolar epithelial cell line A549 were either infected with M. tuberculosis or stimulated with M. tuberculosis components. Confocal microscopy, NO estimation and EMSA were performed on the infected/stimulated A549 cells. Results: Nuclear extracts prepared from M. tuberculosis infected A549 cells alone or stimulated with IFN-c or a combination of three cytokines (IFN-c, TNF-a and IL-1b) formed DNA protein complexes with probes from both À5.2 kb region (specific for binding of STAT-1 protein) and À5.8 kb region (specific for binding of both STAT-1 and NF-jB) of the iNOS promoter. However, TNF-a or IL-1b stimulated M. tuberculosis-infected A549 cells showed no protein DNA complexes with construct from À5.2 kb region. Conclusions: This differential response indicated that TNF-a/IL-1b does not allow STAT-1 production or its translocation to nucleus in M. tuberculosis-infected A549 cells in the absence of IFN-c. This differential signaling of iNOS induction in M. tuberculosis-infected alveolar epithelial cells by cytokines may be responsible for controlled production of NO intracellularly.
Immunology, 2004
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-c, tumour necrosis factor-a, interleukin-1b, 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 39AE29% % (from 10AE62 ± ± 0AE48-6AE392 ± ± 0AE54) following cytokine stimulation of the infected cells. Interestingly c-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.
Mycobacteria Inhibit Nitric Oxide Synthase Recruitment to Phagosomes during Macrophage Infection
Infection and Immunity, 2004
Inducible nitric oxide synthase (iNOS) is a cytoplasmic protein responsible for the generation of nitric oxide (NO ⅐ ) in macrophages. In this work, we hypothesized that the intracellular localization of iNOS is significant for effective delivery of NO ⅐ to phagosomes containing ingested microorganisms. Using immunofluorescence microscopy and Western blot analysis, iNOS was shown to localize in the vicinity of phagosomes containing latex beads in stimulated macrophages. iNOS also localized to phagosomes containing Escherichia coli. The colocalization of iNOS with ingested latex beads was an actin-dependent process, since treatment with the actin microfilament disrupter cytochalasin D prevented iNOS recruitment to latex bead phagosomes. In contrast to E. coli and inert particle phagosomes, mycobacterial phagosomes did not colocalize with iNOS. This study demonstrates that (i) iNOS can be recruited to phagosomes; (ii) this recruitment is dependent on a functional actin cytoskeleton; (iii) certain microorganisms have the ability to prevent or reduce colocalization with iNOS; and (iv) spatial exclusion of iNOS may play a role in Mycobacterium tuberculosis pathogenesis.
IFN-γ and NO in mycobacterial disease: new jobs for old hands
Trends in Microbiology, 2002
In several murine models of mycobacterial infection, the absence of IFN-γ γ and/or NO results in dysregulated granuloma formation and increased lymphocytic responses, which, in the case of M. avium infection, even leads to reduced bacterial growth.
Journal of immunology (Baltimore, Md. : 1950), 2013
Macrophages in granulomas are both antimycobacterial effector and host cell for Mycobacterium tuberculosis, yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial NO synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared with nongranulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, whereas epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68, and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1, and Arg2 proteins were identified in macrophages and localized similarly in granulomas across species, with...
IUBMB Life, 1997
Expression or possession of catalase gene may interfere with the iNOS/NO pathway in mycobacteria, hence altering their capacity to survive within macrophages. Therefore, strains of M. smegmatis with an inactive catalase-peroxidase gene (KatG), or into which the KatG gene of Mycobacterium tuberculosis had been transfected, were used to study the influence of catalase on nitric oxide (NO) production and mycobacterial survival within infected murine J774 macrophages. High levels of nitrite (40-70 nM) were detected in IFN-y and LPS activated, infected murine cell culture supernatants, however, NO2-titres produced by infected murine cells did not diffcr between various catalase phenotype strains. Similarly, no significant difference in mycobacterial killing was also observed among the five strains of M. smegmatis tested over a 3 day infection period.
2003
To explore the role of the 10-kDa Mycobacterium tuberculosis -specific secreted antigen (MTSA-10 or CFP-10) in modulation of macrophage function, J774 macrophages were transfected stably with DNA encoding MTSA-10. Compared to normal or mock-transfected controls, MTSA-10-expressing macrophages had markedly lower levels of co-stimulatory molecule B7·1 on their surface, while the expression of B7·2 and ICAM-1 was not affected. MTSA-transfected cells also produced significantly less microbicidal free radical nitric oxide (NO) upon stimulation with interferon (IFN)-g , lipopolysaccharide or M. tuberculosis cell lysate. Western blot analysis revealed the absence of tyrosine-phosphorylated protein slightly larger than 112 kDa in MTSA-transfected macrophages. Moreover, the treatment of control J774 cells with protein tyrosine kinase inhibitor genistein completely mimicked the effects of transfection with MTSA-10, selectively down-regulating NO and B7·1, but not B7·2 or ICAM-1 expression. The observed MTSA-10-mediated block of B7·1 expression and NO release might contribute to the suppression of antimycobacterial response in tuberculosis.