[pt] ESCRITA: CELEBRANDO UMA DÉCADA

The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R-/- mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R-/- mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis

Lung gross pathology in C57BL/6 and P2X7R^−/− mice on day 28 p.i. with hypervirulent mycobacteria.

<p>C57BL/6 and P2X7R^−/− mice were infected i.t. with approx. 100 bacilli of H37Rv <i>Mtb</i>, Beijing 1471 <i>Mtb</i> and MP287/03 <i>Mbv</i>. Non-infected mice were used as controls. (A) Representative images of the right lungs are shown. (B) Right lung weights and (C) relative masses (circles) were evaluated (<i>n</i> = 9). The mean values are represented by horizontal lines. The relative lung masses were calculated by the ratios of the mean values of the lung weights in the indicated groups and the control group. Significant differences were observed for the indicated groups (**<i>p</i><0.01 and ***<i>p</i><0.001). The data are representative of three separate experiments.</p

Lung infiltrating cells and cytokine production in C57BL/6 and P2X7R^−/− mice on day 28 p.i. with hypervirulent mycobacteria.

<p>C57BL/6 and P2X7R^−/− mice were infected i.t. with approx. 100 bacilli of H37Rv <i>Mtb</i>, Beijing 1471 <i>Mtb</i> and MP287/03 <i>Mbv</i>. Non-infected mice were used as controls. (A) The numbers of total, CD11b⁺, CD19⁺, CD4⁺ and CD8⁺ cells in the lungs are shown (means ± SD, <i>n</i> = 3). (B) IL-1β, IFNγ and IL-10 were quantified in the supernatants of lung cells after 48 h of culture (means ± SD, <i>n</i> = 3). Significant differences were observed for the indicated groups (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001). The data are representative of three separate experiments.</p

Schematic illustration of a hypothetical model to explain the high and low involvement of the P2X7R during severe and mild TB.

<p>In severe TB, the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The eATP released by damaged cells may engage the P2X7R on their surfaces or on neighboring cells. The autocrine or paracrine P2X7R signaling cooperates with mycobacterial components exhibiting membrane-lysing activity and accelerates the necrotic death of infected macrophages and the spread of bacilli. The resistance of hypervirulent mycobacteria to the protective mechanisms elicited in macrophages by eATP contributes to disease dissemination. The release of large amounts of eATP triggers a vicious cycle that exacerbates the pulmonary recruitment of pathogen-permissive monocytes and macrophages and thereby leads to further intracellular bacillus growth. The suppressive environment that results from an excess of adenosine, a byproduct of eATP degradation, may also facilitate the survival of hypervirulent mycobacteria. According to this model, lung necrosis derives from programmed cell death that is triggered by P2X7R signaling. The modest levels of tissue damage induced by less virulent strains and the susceptibility of these mycobacteria to eATP-induced intracellular killing explain, respectively, the minor role and the protective effect of the P2X7R in the mild forms of TB.</p

Induction of necrotic cell death and the release of bacteria and IL-1β in P2X7R^−/− and C57BL/6 BMDMs infected with hypervirulent mycobacteria.

<p>C57BL/6 and P2X7R^−/− BMDMs were infected with H37Rv <i>Mtb</i>, Beijing 1471 <i>Mtb</i> and MP287/03 <i>Mbv</i> at an MOI of 10. (A) On day 4 p.i., necrotic cells were identified by AO/EthBr incorporation. (B) Images show C57BL/6 and P2X7R^−/− BMDMs on day 4 p.i. with Beijing 1471 <i>Mtb</i> (200× magnification; bar scales correspond to 100 µm). (C) The release of mycobacteria was examined by CFU quantification in the supernatants of the infected cultures described in A. (D) On day 3 p.i., IL-1β was quantified in the culture supernatants. Significant differences were observed for the indicated experimental conditions (***<i>p</i><0.001). The data represent the means ± SD of samples in triplicate. The data are representative of three separate experiments.</p

Survival curves and bacillary burdens in the lungs, liver and spleen of C57BL/6 and P2X7R^−/− mice infected with hypervirulent mycobacteria.

<p>C57BL/6 and P2X7R^−/− mice were infected i.t. with approx. 100 bacilli of H37Rv <i>Mtb</i>, Beijing 1471 <i>Mtb</i> and MP287/03 <i>Mbv</i>. Non-infected mice were used as controls. (A) The infected mice were examined daily to determine the survival curves (<i>n</i> = 13–19). (B) The number of CFU/g of tissue was evaluated in the left lung, liver and spleen on day 28 p.i. (means ± SD, <i>n</i> = 9). Significant differences were observed for the indicated groups (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001). The data are representative of three separate experiments.</p