Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation.
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ABSTRACT:
SUBMITTER: Lohman AW
PROVIDER: S-EPMC4824045 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The proinflammatory cytokine IL-1? is a significant risk factor in cardiovascular disease that can be targeted to reduce major cardiovascular events. IL-1? expression and release are tightly controlled by changes in intracellular Ca2+ ([Ca2+]i), which has been associated with ATP release and purinergic signaling. Despite this, the mechanisms that regulate these changes have not been identified. The pannexin 1 (Panx1) channels have canonically been implicated in ATP release, especially during inflammation. We examined Panx1 in human umbilical vein endothelial cells following treatment with the proinflammatory cytokine TNF-?. Analysis by whole transcriptome sequencing and immunoblot identified a dramatic increase in Panx1 mRNA and protein expression that is regulated in an NF-?B-dependent manner. Furthermore, genetic inhibition of Panx1 reduced the expression and release of IL-1?. We initially hypothesized that increased Panx1-mediated ATP release acted in a paracrine fashion to control cytokine expression. However, our data demonstrate that IL-1? expression was not altered after direct ATP stimulation in human umbilical vein endothelial cells. Because Panx1 forms a large pore channel, we hypothesized it may permit Ca2+ diffusion into the cell to regulate IL-1?. High-throughput flow cytometric analysis demonstrated that TNF-? treatments lead to elevated [Ca2+]i, corresponding with Panx1 membrane localization. Genetic or pharmacological inhibition of Panx1 reduced TNF-?-associated increases in [Ca2+]i, blocked phosphorylation of the NF-?B-p65 protein, and reduced IL-1? transcription. Taken together, the data in our study provide the first evidence, to our knowledge, that [Ca2+]i regulation via the Panx1 channel induces a feed-forward effect on NF-?B to regulate IL-1? synthesis and release in endothelium during inflammation.
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Project description:The exit of antigen-presenting cells and lymphocytes from inflamed skin to afferent lymph is vital for the initiation and maintenance of dermal immune responses. How such an exit is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels are unknown. We show that inflammatory cytokines trigger activation of dermal lymphatic endothelial cells (LECs), leading to expression of the key leukocyte adhesion receptors intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin, as well as a discrete panel of chemokines and other potential regulators of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity where they mediate lymph node trafficking of dendritic cells (DCs) via afferent lymphatics. Lastly, we show that tumor necrosis factor alpha stimulates both DC adhesion and transmigration of dermal LEC monolayers in vitro and that the process is efficiently inhibited by ICAM-1 and VCAM-1 adhesion-blocking monoclonal antibodies. These results reveal a CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in inflammation and highlight the process of lymphatic transmigration as a potential new target for antiinflammatory therapy.
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Project description:Sensing of extracellular metabolites controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin-1 (Panx1). Here, we report that T cell-specific Panx1 is needed for efficient CD8+ T cell responses to viral infection and cancer. Panx1 favors both the expansion of effector CD8+ T cells and the survival of memory CD8+ T cells. Our data suggests that, while memory CD8+ T cells require Panx1 for mitochondrial function, Panx1 promotes the glycolytic pathway in effector CD8+ T cells. Panx1 promotes memory CD8+ T cell survival together with the eATP sensor P2RX7. However, Panx1 induces effector CD8+ T cells independently of eATP. Rather, we found an unexpected link between Panx1, sodium lactate export and the activation of effector CD8+ T cells. Therefore, Panx1 regulates effector and memory CD8+ T cells through export of distinct metabolites and by engaging different intracellular pathways.
2023-08-02 | GSE218643 | GEO
Project description:Sensing of extracellular metabolites controls CD8+ T cell function. Their accumulation can occur through export by specialized molecules, such as the release channel Pannexin-1 (Panx1). Here, we report that T cell-specific Panx1 is needed for efficient CD8+ T cell responses to viral infection and cancer. Panx1 favors both the expansion of effector CD8+ T cells and the survival of memory CD8+ T cells. Our data suggests that, while memory CD8+ T cells require Panx1 for mitochondrial function, Panx1 promotes the glycolytic pathway in effector CD8+ T cells. Panx1 promotes memory CD8+ T cell survival together with the eATP sensor P2RX7. However, Panx1 induces effector CD8+ T cells independently of eATP. Rather, we found an unexpected link between Panx1, sodium lactate export and the activation of effector CD8+ T cells. Therefore, Panx1 regulates effector and memory CD8+ T cells through export of distinct metabolites and by engaging different intracellular pathways.
2023-08-02 | GSE217292 | GEO
Project description:Pannexin 1 (Panx1) is a membrane channel implicated in numerous physiological and pathophysiological processes via its ability to support release of ATP and other cellular metabolites for local intercellular signaling. However, to date, there has been no direct demonstration of large molecule permeation via the Panx1 channel itself, and thus the permselectivity of Panx1 for different molecules remains unknown. To address this, we expressed, purified, and reconstituted Panx1 into proteoliposomes and demonstrated that channel activation by caspase cleavage yields a dye-permeable pore that favors flux of anionic, large-molecule permeants (up to ~1 kDa). Large cationic molecules can also permeate the channel, albeit at a much lower rate. We further show that Panx1 channels provide a molecular pathway for flux of ATP and other anionic (glutamate) and cationic signaling metabolites (spermidine). These results verify large molecule permeation directly through caspase-activated Panx1 channels that can support their many physiological roles.
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Project description:BackgroundNeuroinflammation is a major component of secondary damage after traumatic brain injury (TBI). We recently reported that pharmacological inhibition of Pannexin-1 (Panx1) channels markedly reduced the inflammatory response after TBI. Panx1 channels have been shown to be important conduits for adenosine 5'-triphosphate (ATP) release and are associated with leukocyte infiltration and pyroptosis. Because Panx1 blockers significantly decrease ATP release and migration of activated microglia and other myeloid cells (such as monocyte-derived macrophages and dendritic cells) in vitro, we hypothesized that myeloid Panx1 channels play a specific role in immune cell infiltration promoting tissue damage following TBI.MethodsThe murine-controlled cortical impact (CCI) model was used on myeloid-specific Panx1 conditional knockout (Cx3cr1-Cre::Panx1fl/fl) mice to determine whether myeloid Panx1 mediates neuroinflammation and brain damage. Immune cell infiltration was measured using flow cytometry. Locomotor and memory functions were measured using the rotarod and Barnes maze test, respectively. The levels of biomarkers for tissue damage and blood-brain barrier leakage were measured using western blot and magnetic resonance imaging. Panx1 channel activity was measured with ex vivo dye uptake assays, using flow cytometry and confocal microscopy.ResultsCCI-injured Cx3cr1-Cre::Panx1fl/fl mice showed markedly reduced immune cell infiltration to the brain parenchyma compared with Panx1fl/fl mice. As expected, Panx1 dependent activity, assessed by dye uptake, was markedly reduced only in myeloid cells from Cx3cr1-Cre::Panx1fl/fl mice. The expression of biomarkers of tissue damage was significantly reduced in the CCI-injured Cx3cr1-Cre::Panx1fl/fl mice compared with Panx1fl/fl mice. In line with this, magnetic resonance imaging showed reduced blood-brain barrier leakage in CCI-injured Cx3cr1-Cre::Panx1fl/fl mice. There was also a significant improvement in motor and memory function in Cx3cr1-Cre::Panx1fl/fl mice when compared with Panx1fl/fl mice within a week post-CCI injury.ConclusionOur data demonstrate that CCI-related outcomes correlate with Panx1 channel function in myeloid cells, indicating that activation of Panx1 channels in myeloid cells is a major contributor to acute brain inflammation following TBI. Importantly, our data indicate myeloid Panx1 channels could serve as an effective therapeutic target to improve outcome after TBI.
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Project description:Allergic airway inflammation is driven by type-2 CD4+ T cell inflammatory responses. We uncover an immunoregulatory role for the nucleotide release channel, Panx1, in T cell crosstalk during airway disease. Inverse correlations between Panx1 and asthmatics and our mouse models revealed the necessity, specificity, and sufficiency of Panx1 in T cells to restrict inflammation. Global Panx1-/- mice experienced exacerbated airway inflammation, and T-cell-specific deletion phenocopied Panx1-/- mice. A transgenic designed to re-express Panx1 in T cells reversed disease severity in global Panx1-/- mice. Panx1 activation occurred in pro-inflammatory T effector (Teff) and inhibitory T regulatory (Treg) cells and mediated the extracellular-nucleotide-based Treg-Teff crosstalk required for suppression of Teff cell proliferation. Mechanistic studies identified a Salt-inducible kinase-dependent phosphorylation of Panx1 serine 205 important for channel activation. A genetically targeted mouse expressing non-phosphorylatable Panx1S205A phenocopied the exacerbated inflammation in Panx1-/- mice. These data identify Panx1-dependent Treg:Teff cell communication in restricting airway disease.
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Project description:The exit of antigen-presenting cells (APC) and lymphocytes from inflamed skin to afferent lymph is vital for the initiation and maintenance of dermal immune responses. How such exit is achieved and how cells transmigrate the distinct endothelium of lymphatic vessels is however unknown. Here we show that inflammatory cytokines trigger activation of dermal lymphatic endothelial cells (LEC) leading to expression of the key leukocyte adhesion receptors ICAM-1, VCAM-1 and E-selectin, as well as a discrete panel of chemokines and other potential regulators of leukocyte transmigration. Furthermore, we show that both ICAM-1 and VCAM-1 are induced in the dermal lymphatic vessels of mice exposed to skin contact hypersensitivity where they mediate lymph node trafficking of DC via afferent lymphatics. Lastly, we show that TNF_-stimulates both DC adhesion and transmigration of dermal LEC monolayers in vitro and that the process is efficiently inhibited by ICAM-1 and VCAM-1 adhesion-blocking mAbs. These results reveal a CAM-mediated mechanism for recruiting leukocytes to the lymph nodes in inflammation and highlight the process of lymphatic transmigration as a potential new target for anti-inflammatory therapy. Keywords: TNFalpha, Lymphatic endothelium, induction, Inflammation
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