Essential Role Of Interleukin-17A From Reactive Astrocytes In Post-stroke Subventricular Zone Neurogenesis And Behavioral Recovery

Part Ⅰ Temporal and spatial expression profile of IL-17A and effects of IL-17A on the neurogenesis and functional recovery after ischemic stroke in miceObjective:To establish the expression profile of IL-17A following stroke and to determine whether IL-17A can modulate neurogenesis and functional recovery after stroke.Methods:Adult male IL-17A knock-out (il-17a-/-) or wide-type (WT) mice were subjected to60minutes of middle cerebral artery occlusion (MCAO) and subsequent reperfusion. Some WT mice were treated intranasally with recombinant mouse-IL-17A (rIL-17A) or vehicle, and/or intracerebroventricularly with neutralizing anti-IL-17A monoclonal antibody or mouse IgGl isotype control mAb for7days beginning at14days post-ischemia (dpi). We first assessed cell proliferation by treating mice twice with BrdU with an8-hour interval between injections at27dpi; on the following day, the animals were humanely killed to analyze BrdU labeling of dividing cells. In the second experiment, BrdU was given i.p.2times daily for1week starting at7dpi. BrdU labeling was analyzed at35dpi to assess the survival rate of SVZ NSCs. Mice were sacrificed at1,3,7,14,21,28,42and70dpi to measure IL-17A expression in the ipsilateral ischemic hemisphere by real-time PCR and western blotting. IL-17A levels in the hippocampal or subventricular zone (SVZ) were analyzed by immunofluorescence and western blotting at7,14,21and28dpi. Immuo-histochemistry staining with antibodies against bromodeoxyuridine (BrdU), Ki67, phospho-histone H3(PH3) or doublecortin (DCX) was performed at28or35dpi. Western blotting analysis of synaptosomal-associated protein-25kDa (SNAP-25), immuo-histochemistry staining with synaptophysin or β3-tubulin, and functional testing was performed at35dpi.Results:IL-17A gene and protein expression increased significantly at7,14,21and28dpi in the ipsilateral ischemic hemisphere, and peaked at28dpi compared to mice in sham-operated group. IL-17A positive cells were significantly increased in the ipsilateral SVZ and striatum at7,14,21and28dpi. Blocking IL-17A significantly reduced the number of BrdU+/DCX+cells, whereas rIL-17A treatment significantly increased the number of BrdU+/DCX+cells in the ipsilateral striatum at28dpi. However, IL-17A had no effects on the number of BrdU+, Ki67+or PH3+cells in the ipsilateral SVZ at28dpi. Furthermore, blocking IL-17A significantly reduced the number of BrdU+、BrdU+/DCX+cells, and significantly reduced the number of BrdU+/DCX+cells in the ipsilateral SVZ at35dpi. Blocking IL-17A also significantly decreased the number of BrdU+/βⅢ-tubulin+cells and the expression of synaptophysin and SNAP-25in the ipsilateral striatum, and ultimately inhibited functional recovery at35dpi. However, rIL-17A treatment had the opposite effects.Conclusion:The delayed upregulation of IL-17A in ipsilateral ischemic SVZ and striatum promotes the survival of SVZ NSCs, promotes neuronal differentiation and synaptogenesis, and untimately ptomotes the functional recovery in the later phases of stroke recovery. Part Ⅱ Effects of p38mitogen-activated protein kinase (MAPK)/calpain1signaling pathway on the IL-17A-induced neurogenesis and functional recovery after ischemic stroke in miceObjective:To explore the potential molecular mechanisms of IL-17A-induced neurogenesis after stroke.Methods:Additional WT mice were also subjected to60min MCAO using the same protocol. Some WT mice were treated intranasally with recombinant mouse-IL-17A (rIL-17A) or vehicle, and/or intracerebroventricularly with neutralizing anti-IL-17A monoclonal antibody or mouse IgGl isotype control mAb for7days beginning at14days post-ischemia (dpi). Cell proliferation was assessed by treating mice twice with BrdU with an8-hour interval between injections at27dpi; on the following day, the animals were humanely killed to analyze BrdU labeling of dividing cells. In addition to the IL-17A and/or anti-IL-17A mAb treatment, some mice were also received intraperitoneally with p38MAPK inhibitor SB203580(or1%DMSO), or intracerebroventricularly with calpain1inhibitor PD151746(or1%DMSO). The protein levels of IL-17A, phospho-ERK, phospho-JNK, phospho-AKT, phospho-p38(p-p38), all-spectrin, calpain1and calpain2were analyzed by western blotting at28dpi. Western blotting analysis of synaptosomal-associated protein-25kDa (SNAP-25) and immuo-histochemistry staining with BrdU, DCX, β3-tubulin or synaptophysin were performed at35dpi.Results:IL-17A deficiency, anti-IL-17A mAb or rIL-17A treatment did not alter the levels of phospho-ERK, phospho-JNK, phospho-AKT and calpain2. Blocking IL-17A significantly reduced, whereas rIL-17A treatment significantly increased or reduced p-p38levels. In addition, the SBDP145levels and calpain1activity was significantly increased in il-17a-/-mice or anti-IL-17A mAb-treated WT mice at28dpi, whereas rIL-17A treatment markedly reduced the SBDP145levels and calpain1activity. SB203580treatment significantly increased calpain1activity in the ipsilateral ischemic hemisphere at28dpi. We also found that the inhibitory effect of IL-17A on SBDP145expression and calpain1activity was significantly blocked by SB203580. Furthermore, PD151746had no effect on the protein levels of IL-17A and p-p38. In addition, calpain1inhibitor PD151746significantly increased the number of BrdU+/DCX+and BrdU+/β3-tubulin+cells and the synaptophysin expression in the ipsilateral striatum.Conclusion:IL-17A promotes neurogenesis through the p38MAPK/calpain1signaling pathway after ischemic stroke. Part Ⅲ Cellular source of IL-17A in the recovery phase of ischemic stroke and memechanisms that control IL-17A secretionObjective:To determine the main cellular source of IL-17A in the recovery phase of stroke and to elucidate the mechanisms underlying the secretion of IL-17A.Methods:Adult male wide-type (WT) mice were subjected to60minutes of middle cerebral artery occlusion (MCAO) and subsequent reperfusion. Double immunofluorescence staining for IL-17A and astrocyte marker GFAP, neuronal marker NeuN or microglia marker Iba-1was performed to identify which cells were responsible for the elevation of IL-17A at28dpi in mice. In primary astrocytes cultures from the SVZ of ischemic mice at14dpi, IL-17A levels in the cell lysate and culture supernatant of astrocytes treated with lipopolysaccharides (LPS) or PBS were analyzed by western blotting and immuno fluorescence, respectively. The p38MAPK specific inhibitor SB203580or1%DMSO was added to the culture medium30minutes prior to LPS treatment.Results:Our in vivo data showed that IL-17A immunoreactivity was colocalized with the astrocyte marker GFAP but not with the microglia marker Iba-1or the neuronal marker NeuN. Our in vitro data showed that LPS treatment significantly increased the production and secretion of IL-17A as detected by the expression of IL-17A in both the cell lysate and culture supernatant. Inhibiting p38MAPK by administration of SB203580significantly blocked the promoting effect of LPS on the production and secretion of IL-17A.Conclusion:Astrocytes are the major cellular source of IL-17A and the p38MAPK signaling pathway is essential for the production and secretion of IL-17A from astrocytes. Part Ⅳ Effects of IL-17A on the proliferation and differentiation of neural stem/progenitor cells (NSCs) in vitroObjective:To investigate the direct effects of IL-17A on NSCs in vitro and to elucidate the underlying mechanisms.Methods:Adult male IL-17A knock-out (il-17a-/-) or wide-type (WT) mice were subjected to60minutes of middle cerebral artery occlusion (MCAO) and subsequent reperfusion. We prepared neurosphere cultures from the SVZ of ischemic mice at14dpi. The passage2neurosphere nubmer and the percentage of cells that incorporated BrdU was measured24hours after the treatment of rIL-17A at different concentrations (0,10,50,100ng/ml). Double immunofluorescence staining with antibodies against β3-tubulin and GFAP was performed on NSCs dissociated from il-17a-/-or WT mice, or on NSCs dissociated from WT mice treated with rIL-17A (0,10,50,100ng/ml), or with culture supernatant of activated astrocytes with or without the presence of anti-IL-17A mAb. The neutralizing anti-IL-17A monoclonal antibody or mouse IgG1isotype control mAb was added to the differentiation medium simultaneously with IL-17A2h after differentiation induction daily for7days. The p38MAPK specific inhibitor SB203580was added to the differentiation medium30minutes prior to rIL-17A treatment. The calpain1specific inhibitor PD151746was added to the differentiation medium2h after differentiation induction daily for7days. The protein levels of p-p38, calpain1and calpain2were analyzed by western blotting.Results:NSCs from il-17a-/-mice differentiated into fewer β3-tubulin+neurons. NSCs treated with10or50ng/ml rIL-17A or the culture supernatant of activated astrocytes significantly increased the percentage of β3-tubulin+neurons and reduced the percentage of GFAP+astrocytes. When anti-IL-17A mAb was added, the effects of the supernatant on NSCs differentiation into neurons were significantly blocked. Treatment with rIL-17A significantly increased p-p38levels and calpain1activity. When anti-IL-17A mAb was added, the promoting or inhibitory effects of rIL-17A on the p-p38MAPK expression or calpain1activity were significantly blocked. Pre-treatment with SB203580significantly blocked the inhibitory effect of rIL-17A on calpain1activity. Furthermore, PD151746treatment had no effect on the expression of p-p38MAPK. PD151746significantly increased the percentage of β3-tubulin+neurons and reduced the percentage of GFAP+astrocytes.Conclusion:IL-17A promotes the neuronal differentiation of NSCs through the p38MAPK/calpain1in vitro.