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    Neutrophil-derived interleukin-17A participates in neuroinflammation induced by traumatic brain injury
  • Figure 2|TBI triggers morphological and cytoarchitectural abnormalities and widespread neuroinflammation at 7 days post-injury.

    To assess the extent of brain damage caused by CCI at 7 dpi, we first used non-invasive MRI. T2-weighted MRI images showed that the structural integrity of the cortex was destroyed at the impact site, although no prominent midline shift was identified. Moreover, extensive edematous tissues appeared in the ipsilateral hemisphere, expanding to involve the cortex, hippocampus, corpus callosum, and lateral ventricles (Figure 2A). Nissl staining indicated that the most abundant neurons underwent cell death other than cerebral cortical loss at the peri-injury site, and gliosis increased dramatically (Figure 2A). Consistent with this, Iba1 immunolabeling demonstrated that microglia were much more broadly and intensively activated after TBI, not only at the lesion but also in areas distal from the lesion or even in the contralateral hemisphere, indicating widespread neuroinflammation (Figure 2B). In addition, TBI induced significant levels of apoptosis, as evidenced by TUNEL-positivity of different cell types during the subacute phase (Figure 2B). Taken together, these results suggest that TBI evokes morphological and cytoarchitectural abnormalities and widespread neuroinflammation during the subacute phase.

    Figure 4|Neutrophils are the main cellular source of IL-17A at 7 dpi. 

    Besides well-known IL-17A–producing cells, such as T helper 17 cells and tissue-resident innate immune cells such as γδ T cells (Monin and Gaffen, 2018), emerging evidence shows that glial cells within the central nervous system, including microglial cells and astrocytes, can secrete IL-17A under the physiological and pathophysiological conditions (Kawanokuchi et al., 2008; Zhang et al., 2017; Luo et al., 2019; Di Filippo et al., 2021). To determine the cellular source of IL-17A in the ipsilateral peri-injury cortex during the subacute phase, double immunofluorescence was used to stain cells for IL-17A and one of three other markers: GFAP (astrocyte marker (Alawieh et al., 2021)), Iba-1 (microglia marker (Song et al., 2021)), or NeuN (neuronal marker (Liu et al., 2021)). The results demonstrated that IL-17A did not colocalize with GFAP or NeuN, and only infrequently colocalized with Iba-1 (Figure 4A–C). Given that TBI can facilitate parenchymal neutrophil infiltration (Jassam et al., 2017) and that neutrophils can produce IL-17A in specific disease states (Li et al., 2010; Hu et al., 2017), we speculated that neutrophils might express IL-17A during the subacute period of TBI. S100 proteins are a family of small calcium-binding cytosolic proteins, of which S100A8 and S100A9 are abundantly and constitutively expressed by neutrophils and account for approximately 45% of the cytosolic proteins in neutrophils (Xia et al., 2017; Wang et al., 2018). S100A8/A9 expression was recently used to determine neutrophil identity based on single-cell transcriptome analysis of the mouse brain (Ximerakis et al., 2019). Our immunofluorescence results showed that IL-17A colocalized with S100A8, which, coupled with the shape of the nuclei, implied that neutrophils were the main cellular source of IL-17A during the subacute phase of TBI (Figure 4D). 

    Figure 5|STATs and NF-κB may be involved in IL-17A-associated neuroinflammation. 

    In response to diverse physiological and pathological stimuli, transcription factors fine-tune spatiotemporal gene expression (Shaban and Seeber, 2020). We found that TBI induced differential expression of 118 transcription factors at the mRNA level, 103 of which were upregulated, including STATs (Figure 5A). Both qRT-PCR and immunofluorescence corroborated that mRNA and protein levels of STAT1 and STAT3 were significantly upregulated at 7 dpi (Figure 5A–C). On the one hand, STAT3 is essential for IL-17A production, and on the other hand, IL-17A can promote neuroinflammation through STATs, especially STAT3 (Zong et al., 2014; You et al., 2017). Generally, IL-17A induces a proinflammatory response through the transcription factor NF-κB, whose activity is controlled by phosphorylation (Christian et al., 2016; Chong et al., 2020). Therefore, the levels of phosphorylated NF-κB were determined by immunofluorescence although no change was observed at the mRNA level. The results showed that phosphorylated NF-κB was dramatically elevated at the peri-injury site (Figure 5D). Collectively, these findings suggest that STATs and NF-κB might be involved in IL-17A–mediated neuroinflammation. 

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  • 发布日期: 2022-11-01  浏览: 146
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