P2x7 Receptor Signaling Blockade Reduces Lung Inflammation and Necrosis During Severe Experimental Tuberculosis.

The risk of developing severe forms of tuberculosis has increased by the acquired immunodeficiency syndrome (AIDS) epidemic, lack of effective drugs to eliminate latent infection and the emergence of drug-resistant mycobacterial strains. Excessive inflammatory response and tissue damage associated with severe tuberculosis contribute to poor outcome of the disease. Our previous studies using mice deficient in the ATP-gated ionotropic P2X7 receptor suggested this molecule as a promising target for host-directed therapy in severe pulmonary tuberculosis. In this study, we assessed the effects of P2X7 pharmacological blockade on disease severity. First, we observed an increase in P2RX7 gene expression in the peripheral blood of tuberculosis patients compared to healthy donors. Lung leukocytes of mice infected with hypervirulent mycobacteria also showed increased expression of the P2X7 receptor. P2X7 blockade in mice with advanced tuberculosis recapitulated in many aspects the disease in P2X7-deficient mice. P2X7-directed therapy reduced body weight loss and the development of inflammatory and necrotic lung lesions, as well as delayed mycobacterial growth. Lower TNF-α production by lung cells and a substantial reduction in the lung GR-1+ myeloid cell population were observed after P2X7 inhibition. The effector CD4+ T cell population also decreased, but IFN-γ production by lung cells increased. The presence of a large population with characteristics of myeloid dendritic cells, as well as the increase in IL-6 production by lung cells, also indicate a qualitative improvement in the pulmonary immune response due to P2X7 inhibition. These findings support the use of drugs that target the P2X7 receptor as a therapeutic strategy to improve the outcome of pulmonary tuberculosis.

P2X7 Receptor in Bone Marrow-Derived Cells Aggravates Tuberculosis Caused by Hypervirulent Mycobacterium bovis.

Tuberculosis (TB) remains a serious public health problem despite the great scientific advances in the recent decades. We have previously shown that aggressive forms of TB caused by hypervirulent strains of Mycobacterium tuberculosis and Mycobacterium bovis are attenuated in mice lacking the P2X7 receptor, an ion channel activated by extracellular ATP. Therefore, P2X7 receptor is a potential target for therapeutic intervention. In vitro, hypervirulent mycobacteria cause macrophage death by a P2X7-dependent mechanism that facilitates bacillus dissemination. However, as P2X7 receptor is expressed in both bone marrow (BM)-derived cells and lung structural cells, several cellular mechanisms can operate in vivo. To investigate whether the presence of P2X7 receptor in BM-derived cells contributes to TB severity, we generated chimeric mice by adoptive transfer of hematopoietic cells from C57BL/6 or P2X7-/- mice into CD45.1 irradiated mice. After infection with hypervirulent mycobacteria (MP287/03 strain of M. bovis), P2X7-/->CD45.1 mice recapitulated the TB resistance observed in P2X7-/- mice. These chimeric mice showed lower lung bacterial load and attenuated pneumonia compared to C57BL/6>CD45.1 mice. Lung necrosis and bacterial dissemination to the spleen and liver were also reduced in P2X7-/->CD45.1 mice compared to C57BL/6>CD45.1 mice. Furthermore, an immature-like myeloid cell population showing a Ly6Gint phenotype was observed in the lungs of infected C57BL/6 and C57BL/6>CD45.1 mice, whereas P2X7-/- and P2X7-/->CD45.1 mice showed a typical neutrophil (Ly6Ghi) population. This study clearly demonstrates that P2X7 receptor in BM-derived cells plays a critical role in the progression of severe TB.

P2X7 receptor in bone marrow-derived cells aggravates tuberculosis caused by hypervirulent mycobacterium bovi

Tuberculosis (TB) remains a serious public health problem despite the great scientific advances in the recent decades. We have previously shown that aggressive forms of TB caused by hypervirulent strains of Mycobacterium tuberculosis and Mycobacterium bovis are attenuated in mice lacking the P2X7 receptor, an ion channel activated by extracellular ATP. Therefore, P2X7 receptor is a potential target for therapeutic intervention. In vitro, hypervirulent mycobacteria cause macrophage death by a P2X7-dependent mechanism that facilitates bacillus dissemination. However, as P2X7 receptor is expressed in both bone marrow (BM)-derived cells and lung structural cells, several cellular mechanisms can operate in vivo. To investigate whether the presence of P2X7 receptor in BM-derived cells contributes to TB severity, we generated chimeric mice by adoptive transfer of hematopoietic cells from C57BL/6 or P2X7-/- mice into CD45.1 irradiated mice. After infection with hypervirulent mycobacteria (MP287/03 strain of M. bovis), P2X7-/->CD45.1 mice recapitulated the TB resistance observed in P2X7-/- mice. These chimeric mice showed lower lung bacterial load and attenuated pneumonia compared to C57BL/6>CD45.1 mice. Lung necrosis and bacterial dissemination to the spleen and liver were also reduced in P2X7-/->CD45.1 mice compared to C57BL/6>CD45.1 mice...

Effects of curine in HL-60 leukemic cells: cell cycle arrest and apoptosis induction.

Curine is a natural alkaloid isolated from Chondrodendron platyphyllum and it has been reported that this alkaloid has vasodilatory and anti-inflammatory effects. The aim of this study is to analyze the cytotoxic effects of curine in cancer cell lines HL-60, K562, and HT-29, and in primary cultures of peripheral blood mononuclear cells (PBMC). Cells were treated with curine (from 3 to 15 µM) for 24 and 48 h. Cell viability was analyzed by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry with propidium iodide (PI) assay. To assess the type of cell death induced in HL-60, the cell cycle, morphological, and biochemical alterations were analyzed, which were determined by differential staining with acridine orange/ethidium bromide, and annexin V/PI double-labeling and change in mitochondrial membrane potential assays. Curine demonstrated a potent cytotoxic effect on leukemic cell lines (HL-60 and K562). Its cytotoxic effects in HL-60 cells was related to plasma membrane damage and cell cycle arrest at the G1 phase from 43.4 ± 1.0 to 56.7 ± 1.4 % (p < 0.05). Curine (15 µM) also increased the apoptotic cells number by around 60 % in HL-60 cells and caused phosphatidylserine externalization, inducing about 57 % of apoptosis. Moreover, this alkaloid provoked 20 % of mitochondrial membrane depolarization. We conclude that curine presented a cytotoxic effect and induced apoptosis in HL-60 cells. Thus, it can be considered a promising pharmacological drug.

Pulmonary infection with hypervirulent Mycobacteria reveals a crucial role for the P2X7 receptor in aggressive forms of tuberculosis.

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.