Study of the roles of caspase-3 and nuclear factor kappa B in myenteric neurons in a P2X7 receptor knockout mouse model of ulcerative colitis.

BACKGROUND: The literature indicates that the enteric nervous system is affected in inflammatory bowel diseases (IBDs) and that the P2X7 receptor triggers neuronal death. However, the mechanism by which enteric neurons are lost in IBDs is unknown. AIM: To study the role of the caspase-3 and nuclear factor kappa B (NF-κB) pathways in myenteric neurons in a P2X7 receptor knockout (KO) mouse model of IBDs. METHODS: Forty male wild-type (WT) C57BL/6 and P2X7 receptor KO mice were euthanized 24 h or 4 d after colitis induction by 2,4,6-trinitrobenzene sulfonic acid (colitis group). Mice in the sham groups were injected with vehicle. The mice were divided into eight groups (n = 5): The WT sham 24 h and 4 d groups, the WT colitis 24 h and 4 d groups, the KO sham 24 h and 4 d groups, and the KO colitis 24 h and 4 d groups. The disease activity index (DAI) was analyzed, the distal colon was collected for immunohistochemistry analyses, and immunofluorescence was performed to identify neurons immunoreactive (ir) for calretinin, P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, and total NF-κB. We analyzed the number of calretinin-ir and P2X7 receptor-ir neurons per ganglion, the neuronal profile area (µm²), and corrected total cell fluorescence (CTCF). RESULTS: Cells double labeled for calretinin and P2X7 receptor, cleaved caspase-3, total caspase-3, phospho-NF-κB, or total NF-κB were observed in the WT colitis 24 h and 4 d groups. The number of calretinin-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (2.10 ± 0.13 vs 3.33 ± 0.17, P < 0.001; 2.92 ± 0.12 vs 3.70 ± 0.11, P < 0.05), but was not significantly different between the KO groups. The calretinin-ir neuronal profile area was increased in the WT colitis 24 h group compared to the WT sham 24 h group (312.60 ± 7.85 vs 278.41 ± 6.65, P < 0.05), and the nuclear profile area was decreased in the WT colitis 4 d group compared to the WT sham 4 d group (104.63 ± 2.49 vs 117.41 ± 1.14, P < 0.01). The number of P2X7 receptor-ir neurons per ganglion was decreased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (19.49 ± 0.35 vs 22.21 ± 0.18, P < 0.001; 20.35 ± 0.14 vs 22.75 ± 0.51, P < 0.001), and no P2X7 receptor-ir neurons were observed in the KO groups. Myenteric neurons showed ultrastructural changes in the WT colitis 24 h and 4 d groups and in the KO colitis 24 h group. The cleaved caspase-3 CTCF was increased in the WT colitis 24 h and 4 d groups compared to the WT sham 24 h and 4 d groups, respectively (485949 ± 14140 vs 371371 ± 16426, P < 0.001; 480381 ± 11336 vs 378365 ± 4053, P < 0.001), but was not significantly different between the KO groups. The total caspase-3 CTCF, phospho-NF-κB CTCF, and total NF-κB CTCF were not significantly different among the groups. The DAI was recovered in the KO groups. Furthermore, we demonstrated that the absence of the P2X7 receptor attenuated inflammatory infiltration, tissue damage, collagen deposition, and the decrease in the number of goblet cells in the distal colon. CONCLUSION: Ulcerative colitis affects myenteric neurons in WT mice but has a weaker effect in P2X7 receptor KO mice, and neuronal death may be associated with P2X7 receptor-mediated caspase-3 activation. The P2X7 receptor can be a therapeutic target for IBDs.

Effects of experimental ulcerative colitis on myenteric neurons in P2X7-knockout mice.

This study aimed to investigate the distal colon myenteric plexus and enteric glial cells (EGCs) in P2X7 receptor-deficient (P2X7-/-) animals after the induction of experimental ulcerative colitis. 2,4,6-Trinitrobenzene sulfonic acid (TNBS) was injected into the distal colon of C57BL/6 (WT) and P2X7 receptor gene-deficient (P2X7-/-, KO) animals. Distal colon tissues in the WT and KO groups were analyzed 24 h and 4 days after administration. The tissues were analyzed by double immunofluorescence of the P2X7 receptor with neuronal nitric oxide synthase (nNOS)-immunoreactive (ir), choline acetyltransferase (ChAT)-ir, and PGP9.5 (pan neuronal)-ir, and their morphology was assessed by histology. The quantitative analysis revealed 13.9% and 7.1% decreases in the number of P2X7 receptor-immunoreactive (ir) per ganglion in the 24 h-WT/colitis and 4 day-WT/colitis groups, respectively. No reduction in the number of nNOS-ir, choline ChAT-ir, and PGP9.5-ir neurons per ganglion was observed in the 4 day-KO/colitis group. In addition, a reduction of 19.3% in the number of GFAP (glial fibrillary acidic protein)-expressing cells per ganglion was found in the 24 h-WT/colitis group, and a 19% increase in the number of these cells was detected in the 4 day-WT/colitis group. No profile area changes in neurons were observed in the 24 h-WT and 24 h-KO groups. The 4 day-WT/colitis and 4 day-KO/colitis groups showed increases in the profile neuronal areas of nNOS, ChAT, and PGP9.5. The histological analysis showed hyperemia, edema, or cellular infiltration in the 24 h-WT/colitis and 4 day-WT/colitis groups. Edema was observed in the 4 day-KO/colitis group, which showed no histological changes compared with the 24 h-KO/colitis group. We concluded that ulcerative colitis differentially affected the neuronal classes in the WT and KO animals, demonstrating the potential participation and neuroprotective effect of the P2X7 receptor in enteric neurons in inflammatory bowel disease.

Disruption of Purinergic Receptor P2X7 Signaling Increases Susceptibility to Cerebral Toxoplasmosis.

Toxoplasmosis is a neglected disease that affects millions of individuals worldwide. Toxoplasma gondii infection is an asymptomatic disease, with lethal cases occurring mostly in HIV patients and organ transplant recipients. Nevertheless, atypical strains of T. gondii in endemic locations cause severe pathology in healthy individuals. Toxoplasmosis has no cure but it can be controlled by the proinflammatory immune response. The purinergic receptor P2X7 (P2X7) is involved in many inflammatory events and has been associated with genes that confer resistance against toxoplasmosis in humans. In vitro studies have reported parasite death after P2X7-receptor activation in various cell types. To understand the contribution of P2X7 during cerebral toxoplasmosis, wild-type and P2rx7 knockout mice were infected orally with T. gondii and their pathologic profiles were analyzed. We found that all P2rx7-/- mice died 8 weeks after infection with an increased number of cysts and fewer inflammatory infiltrates in their brains. The cytokines interleukin-1ß, interleukin-12, tumor necrosis factor-α, and reactive oxygen species were absent or reduced in P2rx7-/- mice. Taken together, these data suggest that the P2X7 receptor promotes inflammatory infiltrates, proinflammatory cytokines, and reactive oxygen species production in the brain, and that P2X7 signaling mediates major events that confer resistance to cerebral toxoplasmosis.

Sulfate-reducing bacteria stimulate gut immune responses and contribute to inflammation in experimental colitis.

The intestinal microbiota is critical for mammalian immune system development and homeostasis. Sulfate-reducing bacteria (SRB) are part of the normal gut microbiota, but their increased levels may contribute to colitis development, likely in association with hydrogen sulfide (H2S) production. Here, we investigated the effects of SRB in the gut immune response in germ-free mice, and in experimental colitis. After 7days of colonization with Desulfovibrio indonesiensis or with a human SRB consortium (from patients with colitis), germ-free mice exhibited alterations in the colonic architecture, with increased cell infiltration in the lamina propria. SRB colonization upregulated the Th17 and Treg profiles of cytokine production/cell activation, in T cells from mesenteric lymph nodes. These alterations were more pronounced in mice colonized with the human SRB consortium, although D. indonesiensis colonization produced higher levels of H2S. Importantly, the colon of C57BL/6 mice with colitis induced by TNBS or oxazolone had increased SRB colonization, and the administration of D. indonesiensis to mice with TNBS-induced colitis clearly exacerbated the alterations in colonic architecture observed in the established disease, and also increased mouse weight loss. We conclude that SRB contribute to immune response activation in the gut and play an important role in colitis development.

The role of the P2X7 receptor in murine cutaneous leishmaniasis: aspects of inflammation and parasite control.

Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis. The immunopathology of leishmaniasis caused by L. amazonensis infection is dependent on the pathogenic role of effector CD4+ T cells. Purinergic signalling has been implicated in resistance to infection by different intracellular parasites. In this study, we evaluated the role of the P2X7 receptor in modulating the immune response and susceptibility to infection by L. amazonensis. We found that P2X7-deficient mice are more susceptible to L. amazonensis infection than wild-type (WT) mice. P2X7 deletion resulted in increased lesion size and parasite load. Our histological analysis showed an increase in cell infiltration in infected footpads of P2X7-deficient mice. Analysis of the cytokine profile in footpad homogenates showed increased levels of IFN-γ and decreased TGF-ß production in P2X7-deficient mice, suggesting an exaggerated pro-inflammatory response. In addition, we observed that CD4+ and CD8+ T cells from infected P2X7-deficient mice exhibit a higher proliferative capacity than infected WT mice. These data suggest that P2X7 receptor plays a key role in parasite control by regulating T effector cells and inflammation during L. amazonensis infection.

Infection with Leishmania amazonensis upregulates purinergic receptor expression and induces host-cell susceptibility to UTP-mediated apoptosis.

Nucleotides are released into the extracellular milieu from infected cells and cells at inflammatory sites. The extracellular nucleotides bind to specific purinergic (P2) receptors and thereby induce a variety of cellular responses including anti-parasitic effects. Here we investigated whether extracellular nucleotides affect leishmanial infection in macrophages, and found that UTP reduces strongly the parasite load in peritoneal macrophages. Ultrastructural analysis of infected cells revealed that UTP induced morphological damage in the intracellular parasites. Uridine nucleotides also induced dose-dependent apoptosis of macrophages and production of ROI and RNI only in infected macrophages. The intracellular calcium measurements of infected cells showed that the response to UTP, but not UDP, increased the sensitivity and amplitude of cytosolic Ca(2+) changes. Infection of macrophages with Leishmania upregulated the expression of P2Y(2) and P2Y(4) receptor mRNA. The data suggest indirectly that Leishmania amazonensis infection induces modulation and heteromerization of P2Y receptors on macrophages. Thus UTP modulates the host response against L. amazonensis infection. UTP and UTP homologues should therefore be considered as novel components of therapeutic strategies against cutaneous leishmaniasis.