Crohn's disease susceptibility variants in Colombian tuberculosis patients.

BACKGROUND: The molecular basis of genetic predisposition to pulmonary tuberculosis (PTB) in adults remains largely elusive. A chronic granulomatous inflammatory reaction is one of the main characteristics of the immune response to TB; however, a similar reaction is observed in other diseases, such as Crohn's disease. OBJECTIVE: To assess the association of genetic polymorphisms previously associated with Crohn's disease and PTB in a Colombian population of PTB patients and controls. DESIGN: A case-control study was performed among 500 newly diagnosed PTB patients and 320 healthy control subjects. Thirty-one single nucleotide polymorphisms (SNPs) identified in a previous meta-analysis of genome-wide association studies of Crohn's disease were used for genotyping using MassARRAY technology. RESULTS: In this study, we identified an association with borderline significance (P = 0.0009433 and P = 0.029 after multiple testing by Bonferroni's correction) of SNP rs10995271 with PTB. SNP rs10995271 is in linkage disequilibrium with SNPs belonging to the zinc finger protein (ZNF365) gene. CONCLUSIONS: Our results suggest that human PTB shares a genetic basis with Crohn's disease, and that SNPs in the ZNF365 gene would have a role in the occurrence of chronic granulomatous inflammatory reaction in TB as well as Crohn's disease.

Host genomics and control of tuberculosis infection.

Tuberculosis (TB), caused by the human pathogenic bacterium Mycobacterium tuberculosis, poses a major global health problem. The tubercle bacillus is transmitted from person to person by aerosol, but only a proportion of those in contact with infectious aerosol particles will become infected. If infection occurs, less than 10% of those infected will develop clinical signs of TB, while the majority will develop latent TB infection (LTBI). The identification and treatment of LTBI persons is a major aspect of TB control, especially in low-incidence, highly developed nations. In the absence of a gold standard test for latent TB, infection is inferred with the help of either the in vivo tuberculin skin test or in vitro interferon gamma release assays of anti-mycobacterial immunity. Recent work has observed high heritability of these immune assays indicating the critical role of the host genetic background on the establishment of infection and latency. Additional genetic studies have identified the host genetic background as an important covariate for the proper interpretation of the results obtained from LTBI assays. Taken together, these data suggest TB surveillance and control can likely be improved by including host genetic information into the interpretation of these widely used assays.

Evaluation of Toll-like receptor and adaptor molecule polymorphisms for susceptibility to tuberculosis in a Colombian population.

Immunological studies have supported the idea that innate immunity is critical for the control of Mycobacterium tuberculosis (Mtb) infection in humans. Despite the overwhelming evidence showing the critical role of Toll-like receptors (TLRs) in the in vitro recognition of Mtb, the in vivo significance of individual TLRs has been more difficult to demonstrate consistently. We were interested in examining the role of genes of TLRs and molecules involved in their signalling cascades, and a case-control study was designed to test the association of polymorphisms of these innate immune genes with pulmonary tuberculosis (TB) in a Colombian population. In this study, we did not find an association with TLR2, TLR4, TLR9, MyD88 or MAL/TIRAP polymorphic variants. These findings suggest that those genes are not involved as risk factors for pulmonary TB in our population.

TNF-alpha and IL-10 modulate the induction of apoptosis by virulent Mycobacterium tuberculosis in murine macrophages.

The Bcg/Nramp1 gene controls early resistance and susceptibility of macrophages to mycobacterial infections. We previously reported that Mycobacterium tuberculosis-infected (Mtb) B10R (Bcgr) and B10S (Bcgs) macrophages differentially produce nitric oxide (NO-), leading to macrophage apoptosis. Since TNF-alpha and IL-10 have opposite effects on many macrophage functions, we determined the number of cells producing TNF-alpha and IL-10 in Mtb-infected or purified protein derivative-stimulated B10R and B10S macrophages lines, and Nramp1+/+ and Nramp1-/- peritoneal macrophages and correlated them with Mtb-mediated apoptosis. Mtb infection and purified protein derivative treatment induced more TNF-alpha+Nramp1+/+ and B10R, and more IL-10+Nramp1-/- and B10S cells. Treatment with mannosylated lipoarabinomannan, which rescues macrophages from Mtb-induced apoptosis, augmented the number of IL-10 B10R+ cells. Anti-TNF-alpha inhibited apoptosis, diminished NO- production, p53, and caspase 1 activation and increased Bcl-2 expression. In contrast, anti-IL-10 increased caspase 1 activation, p53 expression, and apoptosis, although there was no increment in NO- production. Murine rTNF-alpha induced apoptosis in noninfected B10R and B10S macrophages that was reversed by murine rIL-10 in a dose-dependent manner with concomitant inhibition of NO- production and caspase 1 activation. NO- and caspase 1 seem to be independently activated in that aminoguanidine did not affect caspase 1 activation and the inhibitor of caspase 1, Tyr-Val-Ala-Asp-acylooxymethylketone, did not block NO- production; however, both treatments inhibited apoptosis. These results show that Mtb activates TNF-alpha- and IL-10-dependent opposite signals in the induction of macrophage apoptosis and suggest that the TNF-alpha-IL-10 ratio is controlled by the Nramp1 background of resistance/susceptibility and may account for the balance between apoptosis and macrophage survival.

Induction of apoptosis in murine macrophages by Mycobacterium tuberculosis is reactive oxygen intermediates-independent.

Infection with Mycobacterium tuberculosis induces apoptosis in murine macrophage lines. Resistant macrophages B10R (Bcgr) are more prone to undergo apoptosis than susceptible B10S (Bcgs) macrophages. Apoptosis and inhibition of intracellular growth of the mycobacteria seem to be dependent on the production of nitric oxide, since both can be reverted by aminoguanidine (AMG). Although B10R macrophages produce more superoxide anion than B10S macrophages after infection with M. tuberculosis, reactive oxygen intermediate (ROIs) scavengers did not affect uptake of 3H-uracil incorporation by the mycobacteria nor the induction of apoptosis. These results further suggest that both phenomena are dependent on the production of nitric oxide by the infected macrophages.

Inhibition of virulent Mycobacterium tuberculosis by Bcg(r) and Bcg(s) macrophages correlates with nitric oxide production.

The Nramp1 gene controls macrophage resistance or susceptibility to several intracellular microorganisms; however, there is conflicting evidence regarding its role during infection with virulent Mycobacterium tuberculosis. Nitric oxide (NO) is a potent antimycobacterial agent produced by macrophages, which is also regulated by Nramp1. The in vitro ability of B10R (resistant) and B10S (susceptible) murine macrophages to inhibit M. tuberculosis H37Rv and to produce NO in response to infection and interferon-gamma (IFN-gamma) was compared. Infected B10R macrophages inhibited [3H]uracil incorporation by M. tuberculosis and produced higher amounts of NO than did B10S macrophages. IFN-gamma increased the inhibitory activity of both cells. Inhibition of M. tuberculosis by IFN-gamma-activated B10R macrophages was reversed by N(G)-monomethyl-L-arginine (N(G)MMA). L-arginine restored NO production and increased the antimycobacterial activity by IFN-gamma-stimulated N(G)MMA-treated macrophages. The Bcg/Nramp1 gene may regulate macrophage resistance or susceptibility to virulent M. tuberculosis by a differential capability of these cells to produce NO.

Differential induction of apoptosis by virulent Mycobacterium tuberculosis in resistant and susceptible murine macrophages: role of nitric oxide and mycobacterial products.

Resistance and susceptibility of macrophages to mycobacteria are under the control of the Bcg/Nramp1 gene, which also controls the NO- production in response to macrophage activators. There is recent evidence indicating that mycobacteria induces apoptosis in infected macrophages. Using murine macrophage lines, congenic at the Bcg/Nramp1 gene, this report shows that B10R are more prone than B10S macrophages to undergo apoptosis after exposure to live virulent Mycobacterium tuberculosis H37Rv (Mtb) or PPD, as determined by cell viability, DNA fragmentation, hypoploidy, and the terminal deoxynucleotide transferase dUTP-biotin nick-end labeling assay. Induction of apoptosis correlated with NO- production. Aminoguanidine and anti-TNF-alpha inhibited NO- production and apoptosis. B10R and B10S macrophages were equally affected by sodium nitroprusside, a donor of NO-, but its effect, mainly in B10R cells, was enhanced by the presence of Mtb. Nonvirulent mycobacteria induced lower levels of NO- and did not cause cell death. Killed Mtb, mannose-capped lipoarabinomannan (ManLAM), and LPS rescued macrophages from apoptosis albeit induce NO-. These findings suggest the existence of opposite pathways: metabolically active mycobacteria promotes apoptosis whereas their structural components inhibit it. Apoptosis may be a critical mechanism by which Nramp1 gene controls the macrophage infection with virulent mycobacteria.

I-A beta gene expression regulation in macrophages derived from mice susceptible or resistant to infection with M. bovis BCG.

The innate capacity of mice to control mycobacterial multiplication early after infection is controlled by the resistant allele of the Nramp-1/Bcg gene. The Bcg gene seems to be involved in a pathway leading to macrophage activation. It differentially affects the ability of BCG-resistant and -susceptible strains of mice to express important macrophage genes including Major Histocompatibility Complex (MHC) class II genes. An inhibition of Nramp1 gene by Nramp1-ribozyme transfection in macrophages resulted in the impairment of MHC class II gene induction by IFN gamma. In this study, we have investigated the molecular mechanisms involved in IFN-gamma-induced MHC class II expression using macrophages derived from mice resistant or susceptible to mycobacterial infections (B10R and B10S, respectively). We have found that the difference in the IFN gamma-induced Ia surface protein expression between B10R and B10S macrophages correlate with a higher rate of I-A beta gene transcription. We have also studied the binding of proteins prepared from nuclear extracts of non-stimulated and IFN-gamma-stimulated B10R and B10S macrophages to the S, X and Y cis-acting elements of the I-A beta promoter. Differences observed in protein binding to the X box may explain the difference in transcription activation of the I-A beta gene. We have also found that I-A alpha and I-A beta mRNA half-lives measured in IFN gamma-stimulated cells are significantly longer in B10R, compared to B10S macrophages. Overall, our data suggest that both transcriptional and posttranscriptional regulatory mechanisms are responsible for the more efficient expression of I-A beta gene in macrophages carrying a resistant allele of Nramp1 gene.

Nitrite production by macrophages derived from BCG-resistant and -susceptible congenic mouse strains in response to IFN-gamma and infection with BCG.

Reactive nitrogen intermediates (RNI) have been implicated in the interferon-gamma (IFN-gamma)-induced anti-microbial action of macrophages against a wide variety of pathogens. We have been studying the production of NO2- by macrophage lines derived from the bone marrow of either B10.A (Bcgs) strain mice (B10S cell lines), or their congenic BCG-resistant partners of the B10A.Bcgr (Bcgr) strain (B10R cell lines). We have discovered that there is a significant difference in the production of NO2- of B10S compared with B10R macrophages in response to IFN-gamma. By 48 hr following treatment with 10 U/ml IFN-gamma, B10R macrophages had produced an approximately threefold higher level of NO2- than B10S macrophages. Similar results were obtained when experiments were performed with total splenic cells harvested from the spleens of B10.A.Bcgr and B10.A strain mice. The bacteriostatic activity, as assessed by the [3H]uracil incorporation by Mycobacterium bovis BCG, was higher in B10R macrophages compared to B10S macrophages. The bacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide produced by the macrophages. The anti-mycobacterial activity was inhibited by NgMMLA, a specific inhibitor of nitrite and nitrate synthesis from L-arginine. Addition of L-arginine to IFN-gamma-stimulated macrophages in the presence of NgMMLA restored nitrite production and bacteriostatic activity of macrophages. Northern blot analysis of macrophage nitric oxide synthase (iNOS) revealed that the difference in NO2- production by IFN-gamma-treated B10S and B10R lines was reflective of the difference in iNOS mRNA expression.

Assessment of mycobacterial infection and multiplication in macrophages by polymerase chain reaction.

The amplification of mycobacterium-specific DNA sequences from samples obtained from infected patients by the polymerase chain reaction (PCR) has been useful in the clinical diagnosis of mycobacterial diseases. Using 20 bp oligonucleotide primers that recognize a 123 bp repeated sequence present in M. bovis and M. tuberculosis DNA, we describe in detail the conditions of the PCR reaction that allow an assessment of the mycobacterial content of infected macrophages. The results of the highly reproducible, time-efficient PCR technique show good correlation with the widely used colony forming unit (CFU) and [3H]uracil incorporation methods for the detection of Mycobacterium. Our method allows an assessment of the level of M. bovis BCG infection from a variety of sources, including peritoneal macrophages and macrophage lines, within a few hours, making it the assay of choice for rapid determination of the level of mycobacterial growth in infected cells, in experimental models of mycobacterial infection.

Natural killer cell activity in patients with pulmonary tuberculosis and in healthy controls.

Natural killer (NK) cell activity of freshly isolated peripheral blood mononuclear cells (MNC) or cells stimulated for 72 h with 10 micrograms/ml of a sonicate antigen of Mycobacterium tuberculosis H37Rv were studied in healthy responder and non-responder controls, as detected by lymphocyte proliferation with specific antigen, and in patients with pulmonary tuberculosis. K-652 cells were used as targets in a 4 h 51Cr release assay. MNC from patients exhibited a significant decrease in NK function as compared with responder controls (p less than 0.02). NK activity in responder individuals was highest 72 h after incubation with antigen. Non-stimulated cells were not cytotoxic. MNC from healthy responder and non-responder subjects incubated for 72 h with antigen yielded a significant increase in the percentage NK cytotoxicity at all effector/target ratios studied (p less than 0.01) as well as in the number of lytic units per culture (p less than 0.004). However, this increase was higher in responder individuals as compared to non-responder subjects (p = 0.02). The response to antigen was not significant in the group of patients although a net increase was also observed in the whole group. Only 4 of 9 patients exhibited significant increased responses after antigenic stimulation, 3 showed moderate responses, 1 did not respond and in a further patient a decrease was observed. The decreased NK activity could be secondary to abnormalities in the production of lymphokines by tuberculous patients. Although the role of non-specific cytotoxic cells in tuberculosis is unknown, their alterations could contribute to the pathogenesis of the disease.