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Myeloid Pannexin-1 mediates acute leukocyte infiltration and leads to worse outcomes after brain trauma.

ABSTRACT: BACKGROUND:Neuroinflammation 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. METHODS:The 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. RESULTS:CCI-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. CONCLUSION:Our 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.

SUBMITTER: Seo JH

PROVIDER: S-EPMC7441665 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Myeloid Pannexin-1 mediates acute leukocyte infiltration and leads to worse outcomes after brain trauma.

Seo Joon Ho JH Dalal Miloni S MS Calderon Frances F Contreras Jorge E JE

Journal of neuroinflammation 20200820 1

<h4>Background</h4>Neuroinflammation 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 ...[more]

PMID: 32819386

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Project description:BackgroundTraumatic brain injury (TBI) is a significant cause of death and disability worldwide. The TLR4-NFκB signaling cascade is the critical pro-inflammatory activation pathway of leukocytes after TBI, and modulating this signaling cascade may be an effective therapeutic target for treating TBI. Previous studies indicate that recombinant annexin A2 (rA2) might be an interactive molecule modulating the TLR4-NFκB signaling; however, the role of rA2 in regulating this signaling pathway in leukocytes after TBI and its subsequent effects have not been investigated.MethodsC57BL/6 mice were subjected to TBI and randomly divided into groups that received intraperitoneal rA2 or vehicle at 2 h after TBI. The peripheral leukocyte activation and infiltrating immune cells were examined by flow cytometry, RT-qPCR, and immunostaining. The neutrophilic TLR4 expression on the cell membrane was examined by flow cytometry and confocal microscope, and the interaction of annexin A2 with TLR4 was assessed by co-immunoprecipitation coupled with Western blotting. Neuroinflammation was measured via cytokine proteome profiler array and RT-qPCR. Neurodegeneration was determined by Western blotting and immunostaining. Neurobehavioral assessments were used to monitor motor and cognitive function. Brain tissue loss was assessed via MAP2 staining.ResultsrA2 administration given at 2 h after TBI significantly attenuates neutrophil activation and brain infiltration at 24 h of TBI. In vivo and in vitro data show that rA2 binds to and reduces TLR4 expression on the neutrophil surface and suppresses TLR4/NFκB signaling pathway in neutrophils at 12 h after TBI. Furthermore, rA2 administration also reduces pro-inflammation of brain tissues within 24 h and neurodegeneration at 48 h after TBI. Lastly, rA2 improves long-term sensorimotor ability and cognitive function, and reduces brain tissue loss at 28 days after TBI.ConclusionsSystematic rA2 administration at 2 h after TBI significantly inhibits activation and brain infiltration of peripheral leukocytes, especially neutrophils at the acute phase. Consequently, rA2 reduces the detrimental brain pro-inflammation-associated neurodegeneration and ultimately ameliorates neurological deficits after TBI. The underlying molecular mechanism might be at least in part attributed to rA2 bindings to pro-inflammatory receptor TLR4 in peripheral leukocytes, thereby blocking NFκB signaling activation pathways following TBI.

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Myeloid Pannexin-1 mediates acute leukocyte infiltration and leads to worse outcomes after brain trauma.

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Myeloid Pannexin-1 mediates acute leukocyte infiltration and leads to worse outcomes after brain trauma.

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