Jorge Castagnino

Mycobacterium tuberculosis-Induced Gamma Interferon Production by Natural Killer Cells Requires Cross Talk with Antigen-Presenting Cells Involving Toll-Like Receptors 2 and 4 and the Mannose Receptor in Tuberculous Pleurisy

ABSTRACT Tuberculous pleurisy allows the study of human cells at the site of active Mycobacterium tuberculosis infection. In this study, we found that among pleural fluid (PF) lymphocytes, natural killer (NK) cells are a major source of early gamma interferon (IFN-γ) upon M. tuberculosis stimulation, leading us to investigate the mechanisms and molecules involved in this process. We show that the whole bacterium is the best inducer of IFN-γ, although a high-molecular-weight fraction of culture filtrate proteins from M. tuberculosis H37Rv and the whole-cell lysate also induce its expression. The mannose receptor seems to mediate the inhibitory effect of mannosylated lipoarabinomannan, and Toll-like receptor 2 and 4 agonists activate NK cells but do not induce IFN-γ like M. tuberculosis does. Antigen-presenting cells (APC) and NK cells bind M. tuberculosis, and although interleukin-12 is required, it is not sufficient to induce IFN-γ expression, indicating that NK cell-APC contact takes place. Indeed, major histocompatibility complex class I, adhesion, and costimulatory molecules as well as NK receptors regulate IFN-γ induction. The signaling pathway is partially inhibited by dexamethasone and sensitive to Ca2+ flux and cyclosporine. Inhibition of p38 and extracellular-regulated kinase mitogen-activated protein kinase pathways reduces the number of IFN-γ+ NK cells. Phosphorylated p38 (p-p38) is detected in ex vivo PF-NK cells, and M. tuberculosis triggers p-p38 in PF-NK cells at the same time that binding between NK and M. tuberculosis reaches its maximum value. Thus, interplay between M. tuberculosis and NK cells/APC triggering IFN-γ would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a type 1 profile.

Sex, ethnicity, and slow acetylator profile are the major causes of hepatotoxicity induced by antituberculosis drugs.

Treatment with antituberculosis (TB) drugs produces liver damage in a large proportion of patients. Isoniazid, an antibacterial drug, is primarily responsible for this hepatotoxicity. Several polymorphisms of the N‐acetyltransferase 2 (NAT‐2) and cytochrome P450 2E1 enzymes, which are involved in the metabolism of isoniazid, may be directly associated with the development of hepatotoxicity. This study was designed to analyze the association between the NAT‐2 and CYP2E1 polymorphisms with the development of anti‐TB drug‐induced hepatotoxicity (ATDH).

Humoral response to Mycobacterium tuberculosis in patients with human immunodeficiency virus infection.

The effect of the human immunodeficiency virus (HIV) on mycobacterial antibody production was investigated. Using an enzyme-linked immunosorbent assay (ELISA) for detecting IgG against Mycobacterium tuberculosis PPD, it was observed that individuals at risk of HIV infection show a pattern of humoral response to the tubercle bacillus similar to that previously found in the immunocompetent population not exposed to risk factors: 6 of 12 (50.0%) tuberculosis cases had elevated levels of antibodies to PPD and 27 of 30 (90.0%) asymptomatic individuals had antibody levels within the normal range. In an HIV-seropositive group without AIDS indicator diseases, 8 of 22 (36.4%) tuberculous patients had detectable mycobacterial antibodies whereas 156 of 164 (95.1%) non-tuberculous subjects did not. Among AIDS cases, only 1 of 20 (5.0%) patients with tuberculosis and none of 53 non-tuberculous subjects showed a positive result. The study evidenced an increasing humoral unresponsiveness to PPD in the progression of HIV infection to AIDS. Thus, a serodiagnostic method for detecting tuberculosis such as the ELISA here employed noticeably decreases its utility in the latency stage of the HIV infection, and it is practically useless in clinical AIDS.

Paradoxical role of CD16+CCR2+CCR5+ monocytes in tuberculosis: efficient APC in pleural effusion but also mark disease severity in blood

The role of CD16– and CD16+ Mo subsets in human TB remains unknown. Our aim was to characterize Mo subsets from TB patients and to assess whether the inflammatory milieu from TB pleurisy modulate their phenotype and recruitment. We found an expansion of peripheral CD16+ Mo that correlated with disease severity and with TNF‐α plasma levels. Circulating Mo from TB patients are activated, showing a higher CD14, CD16, and CD11b expression and Mtb binding than HS. Both subsets coexpressed CCR2/CCR5, showing a potential ability to migrate to the inflammatory site. In tuberculous PF, the CD16+ subset was the main Mo/MΦ population, accumulation that can be favored by the induction of CD16 expression in CD16– Mo triggered by soluble factors found in this inflammatory milieu. CD16+ Mo in PF were characterized by a high density of receptors for Mtb recognition (DC‐SIGN, MR, CD11b) and for lipid‐antigens presentation (CD1b), allowing them to induce a successful, specific T cell proliferation response. Hence, in tuberculous PF, CD16+ Mo constitute the main APC population; whereas in PB, their predominance is associated with the severity of pulmonary TB, suggesting a paradoxical role of the CD16+ Mo subset that depends on the cellular localization.

Impaired dendritic cell differentiation of CD16-positive monocytes in tuberculosis: Role of p38 MAPK

Tuberculosis (TB) is one of the worlds most pernicious diseases mainly due to immune evasion strategies displayed by its causative agent Mycobacterium tuberculosis (Mtb). Blood monocytes (Mos) represent an important source of DCs during chronic infections; consequently, the alteration of their differentiation constitutes an escape mechanism leading to mycobacterial persistence. We evaluated whether the CD16+/CD16− Mo ratio could be associated with the impaired Mo differentiation into DCs found in TB patients. The phenotype and ability to stimulate Mtb‐specific memory clones DCs from isolated Mo subsets were assessed. We found that CD16− Mos differentiated into CD1a+DC‐SIGNhigh cells achieving an efficient recall response, while CD16+ Mos differentiated into a CD1a−DC‐SIGNlow population characterized by a poor mycobacterial Ag‐presenting capacity. The high and sustained phosphorylated p38 expression observed in CD16+ Mos was involved in the altered DC profile given that its blockage restored DC phenotype and its activation impaired CD16− Mo differentiation. Furthermore, depletion of CD16+ Mos indeed improved the differentiation of Mos from TB patients toward CD1a+DC‐SIGNhigh DCs. Therefore, Mos from TB patients are less prone to differentiate into DCs due to their increased proportion of CD16+ Mos, suggesting that during Mtb infection Mo subsets may have different fates after entering the lungs.

CD4+CD25highforkhead box protein 3+ regulatory T lymphocytes suppress interferon‐γ and CD107 expression in CD4+ and CD8+ T cells from tuberculous pleural effusions

Tuberculous pleural effusion is characterized by a T helper type 1 (Th1) profile, but an excessive Th1 response may also cause tissue damage that might be controlled by regulatory mechanisms. In the current study we investigated the role of regulatory T cells (Treg) in the modulation of Th1 responses in patients with tuberculous (TB) pleurisy. Using flow cytometry we evaluated the proportion of Treg (CD4+CD25highforkhead box protein 3+), interferon (IFN)‐γ and interleukin (IL)‐10 expression and CD107 degranulation in peripheral blood (PB) and pleural fluid (PF) from patients with TB pleurisy. We demonstrated that the proportion of CD4+CD25+, CD4+CD25highFoxP3+ and CD8+CD25+ cells were increased in PF compared to PB samples. Mycobacterium tuberculosis stimulation increased the proportion of CD4+CD25low/negIL‐10+ in PB and CD4+CD25low/negIFN‐γ+ in PF; meanwhile, CD25high mainly expressed IL‐10 in both compartments. A high proportion of CD4+CD107+ and CD8+CD107+ cells was observed in PF. Treg depletion enhanced the in‐vitro M. tuberculosis‐induced IFN‐γ and CD4+ and CD8+ degranulation responses and decreased CD4+IL‐10+ cells in PF. Our results demonstrated that in TB pleurisy Treg cells effectively inhibit not only IFN‐γ expression but also the ability of CD4+ and CD8+ cells to degranulate in response to M. tuberculosis.

NK cells from tuberculous pleurisy express high ICAM-1 levels and exert stimulatory effect on local T cells

Tuberculous pleurisy, one of the most common manifestations of extrapulmonary tuberculosis, is characterized by a T‐cell‐mediated hypersensitivity reaction along with a Th1 immune profile. In this study, we investigated functional cross‐talk among T and NK cells in human tuberculous pleurisy. We found that endogenously activated pleural fluid‐derived NK cells express high ICAM‐1 levels and induce T‐cell activation ex vivo through ICAM‐1. Besides, upon in vitro stimulation with monokines and PAMP, resting peripheral blood NK cells increased ICAM‐1 expression leading to cellular activation and Th1 polarization of autologous T cells. Furthermore, these effects were abolished by anti‐ICAM‐1 Ab. Hence, NK cells may contribute to the adaptive immune response by a direct cell‐contact‐dependent mechanism in the context of Mycobacterium tuberculosis infection.

CD3 expression distinguishes two γδT cell receptor subsets with different phenotype and effector function in tuberculous pleurisy

Tuberculous pleurisy is a naturally occurring site of Mycobacterium tuberculosis (Mtb) infection. Herein, we describe the expression of activation, natural killer (NK) and cell migration markers, as well as effector functions from γδT cells in peripheral blood (PB) and pleural effusion (PE) from tuberculosis patients (TB). We observed a decreased percentage of circulating γδT from TB patients and differential expression of NK as well as of chemokine receptors on PB and PE. Two subsets of γδT cells were differentiated by the CD3/γδT cell receptor (γδTCR) complex. The γδTCRlow subset had a higher CD3 to TCR ratio and was enriched in Vδ2+ cells, whereas most Vδ1+ cells belonged to the γδTCRhigh subset. In PB from TB, most γδTCRhigh were CD45RA+CCR7‐ and γδTCRlow were CD45RA+/−CCR7+CXCR3+. In the pleural space the proportion of CD45RA‐CCR7+CXCR3+ cells was higher. Neither spontaneous nor Mtb‐induced interferon (IFN)‐γ production was observed in PB‐γδT cells from TB; however, PE‐γδT cells showed a strong response. Both PB‐ and PE‐γδ T cells expressed surface CD107a upon stimulation with Mtb. Notably, PE‐γδTCRlow cells were the most potent effector cells. Thus, γδT cells from PB would acquire a further activated phenotype within the site of Mtb infection and exert full effector functions. As γδT cells produce IFN‐γ within the pleural space, they would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a T helper type 1 profile.

Human pleural B-cells regulate IFN-γ production by local T-cells and NK cells in a Mycobacterium tuberculosis-induced delayed hypersensitivity reaction

DTH (delayed type hypersensitivity) reactions are secondary cellular immune responses that appear 24-72 h after antigen exposure. Tuberculous pleurisy is a common manifestation of extrapulmonary TB (tuberculosis) and is considered a human model of Th1-mediated DTH. In order to identify functional cross-talk among cellular populations sited in this inflammatory microenvironment, we analysed phenotypic and functional features of human B-cells isolated from the PF (pleural fluid) of TB patients. Freshly isolated PF-B-cells displayed a lower expression of CD20, CD1d and HLA-DR, and a higher expression of CD95, CD38, CD25, CXCR3 (CXC chemokine receptor 3) and CXCR4 (CXC chemokine receptor 4) than their PB (peripheral blood) counterparts, suggesting a non-classical in situ activation. Although memory PF-T-cell frequencies were increased, the frequencies of memory PF-B-cells were not. We demonstrated that, upon stimulation with γ-irradiated M. tuberculosis, mycobacterially secreted proteins or a lectin mitogen, PF-B-cells had a strong activation and produced IL-10 by a mechanism that was dependent on bystander activation of CD19(-) PF cells. Besides, within PF cells, B-cells diminished in vitro M. tuberculosis-induced IFN (interferon)-γ production by T-cells and NK (natural killer) cells in an IL-10-dependent manner. Finally, we found that the lower the frequency of B-cells, the higher the ratio of IFN-γ/IL-10 within PF. Thus our results suggest that B-cells can regulate a human DTH reaction induced by M. tuberculosis.

The distribution of allelic and genotypic frequencies of N-Acetyltransferase-2 variants in an Argentine population

INTRODUCTION Arylamine N-acetyltransferase-2 (NAT-2) is a key human enzyme in drug detoxification and elimination. Mutations in NAT-2 affect the activity of anti-tuberculosis drugs and result in three different phenotypes: rapid (RA), intermediate (IA) and slow acetylators (SA). METHODOLOGY The allelic, genotypic and phenotypic frequencies of NAT-2 were studied in 185 patients from Buenos Aires by restriction fragment length polymorphism. RESULTS The following allele frequencies were obtained: *4 = 29.9%, *5 = 37.0, *6 = 25.6%, *7 = 8% and *14 = 1.3%. With regard to the phenotype, we observed that 53.6% of the population was SA, 35.7% was IA and 10.7% was RA. CONCLUSION A high prevalence of SA might have an impact on anti-TB drug-induced hepatotoxicity.