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CD8+ T cells are recognized as key players in exacerbation of ischemic stroke; however, the underlying mechanism in modulating the function of CD8+ T cells has not been completely elucidated. Here, we uncovered that FasL enhanced the cytotoxicity of CD8+ T cells to neurons after ischemic stroke. Inactivation of FasL specific on CD8+ T cells protected against brain damage and neuron loss. Proteomic analysis identified that PDPK1 functioned downstream of FasL signaling and inhibition of PDPK1 effectively reduced cytotoxicity of CD8+ T cells and improved ischemic neurological deficits. Taken together, these results highlight an intrinsic FasL-PDPK1 pathway regulating the cytotoxicity of CD8+ T cells in ischemic stroke.

Birt–Hogg–Dubé syndrome (BHDS, MIM #135150), caused by germline mutations of FLCN gene, is a rare autosomal dominant inherited disorder characterized by skin fibrofolliculomas, renal cancer, pulmonary cysts and spontaneous pneumothorax. The syndrome is considered to be under-diagnosed due to variable and atypical manifestations. Herein we present a BHDS family. Targeted next generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA) revealed a novel FLCN intragenic deletion spanning exons 10-14 in four members including the proband with pulmonary cysts and spontaneous pneumothorax, one member with suspicious skin lesions and a few pulmonary cysts, as well as two asymptomatic family members. In addition, a linkage analysis further demonstrated one member with pulmonary bullae to be a BHDS-ruled-out case, whose bullae presented more likely as an aspect of paraseptal emphysema. Furthermore, the targeted NGS and MLPA data including our previous and present findings were reviewed and analyzed to compare the advantages and disadvantages of the two methods, and a brief review of the relevant literature is included. Considering the capability of the targeted NGS method to detect large intragenic deletions as well as determining deletion junctions, and the occasional false positives of MLPA, we highly recommend targeted NGS to be used for clinical molecular diagnosis in suspected BHDS patients.

Objectives: Cerebral autoregulation and blood brain barrier (BBB) are often impaired in hypertensive and aging individuals, contributing to the development of stroke. Vascular smooth muscle cells (VSMC) play a vital role in cerebral blood flow control and maintenance of BBB. Ischemic injury triggers VSMC to switch into a detrimental phenotype in peripheral blood vessels, however, the pathological phenotype switching in cerebral VSMC and the impact of VSMC on BBB are not elucidated thoroughly in stroke with hypertension. Methods: Hypertensive mice with smooth muscle–specific deletion of MYPT1 (MYPT1 SMKO mice) were generated. Middle cerebral artery occlusion (MCAO) was conducted in adult MYPT1 SMKO mice and their wild-type (WT) mice for 60 min. Ischemic infarct volumes and neurological function were evaluated. Evens blue, immunofluorescence (IF), western blot (WB) and so on were performed to evaluate the permeability of BBB after stroke. The phenotype switching of VSMC was measured by WB, IF and quantitative real-time polymerase chain reaction (q-PCR) in oxygen glucose deprivation (OGD) and co-cultured in vitro models. Proteomic analysis was performed in cerebral capillaries. The q-PCR and WB were used for further validation. Results: It was found that the infarct volumes and neurological deficits were aggravated in MYPT1 SMKO mice and the permeability of BBB was increased. The loss of pericyte cells and degradation of basement membrane of BBB were more severe in MYPT1 SMKO mice. Furthermore, the protein expression of α-SMA in MYPT1 SMKO mice after MCAO was reduced and the protein expression of Calponin and SM-22α was reduced. The ability of migration and proliferation in VSMC of MYPT1-knockout was altered after OGD. Proteomic analysis showed that the expression of protein associated with inflammation in VSMC of MYPT1 SMKO mice was increased than that of WT mice, but the specific protein underlying the mechanism needs further verification. Conclusions: The damage of BBB was increased in MYPT1 SMKO hypertensive mice. And there is a phenotype switching in cerebral VSMC after stroke in MYPT1 SMKO hypertensive mice. The potential mechanism may involve the increasing expression of protein associated with inflammation in VSMC that needs further verification.

Summary Vascular smooth muscle cells (VSMCs) have been widely recognized as key players in regulating blood-brain barrier (BBB) function, and their roles are unclear in ischemic stroke. Myosin phosphatase target subunit 1 (MYPT1) is essential for VSMC contraction and maintaining healthy vasculature. We generated VSMC-specific MYPT1 knockout (MYPT1SMKO) mice and cultured VSMCs infected with Lv-shMYPT1 to explore phenotypic switching of VSMCs and the accompanied impacts on BBB integrity. We found that MYPT1 deficiency induced phenotypic switching of synthetic VSMCs, which aggravated BBB disruption. Proteomic analysis identified evolutionarily conserved signaling intermediates in Toll pathways (ECSIT) as a downstream molecule that promotes activation of synthetic VSMCs and contributed to IL-6 expression. Knocking down ECSIT rescued phenotypic switching of VSMCs and BBB disruption. Additionally, inhibition of IL-6 decreased BBB permeability. These findings reveal that MYPT1 deficiency activated phenotypic switching of synthetic VSMCs and induced BBB disruption through ECSIT-IL-6 signaling after ischemic stroke., Graphical abstract, Highlights • MYPT1 deficiency induces activation of synthetic VSMCs and aggravates BBB disruption • Synthetic VSMCs release more IL-6 to destroy BBB in a contact-independent way • MYPT1-ECSIT-IL-6 signaling pathway regulates synthetic VSMC-mediated BBB disruption, Immunology; Molecular physiology; Cell biology; Proteomics

IL-37 is a newly identified immune-suppressive factor; however, the function, cellular sources, and mechanism of IL-37 in humoral immunity and Myasthenia gravis (MG) are still unclear. In this study, we found IL-37 were substantially downregulated in the serum and PBMCs of MG patients compared with healthy controls. The lower IL-37 was associated with severer disease (quantitative MG score) and higher follicular Th (Tfh)/Tfh17 and B cell numbers. Flow cytometry analysis revealed that IL-37 was mainly produced by CD4+ T cells without overlapping with Th1, Th17, and Tfh subsets in MG patients. Regulatory IL-37+ T cell rarely expressed Foxp3 and CD25 but produced numerous IL-4. Tfh and B cell expressed high levels of SIGIRR, the receptor of IL-37, in MG patients. Mechanically, IL-37 directly bond to SIGIRR, repressed the proliferation, cytokine production of Tfh and B cells, and the secretion of autoantibody via inhibition of STAT3 signaling in Tfh and B cells.

CD8

Significance In the present study, we found a significantly up-regulated CD3+CD4−CD8− T cell (double-negative T cell; DNT) population in the peripheral blood of patients with acute stroke compared with age-matched healthy subjects. Interestingly, infiltrating DNTs were mainly located close to microglia in the ischemic brain tissues of both stroke patients and middle cerebral artery occlusion mice. DNTs amplified the proinflammatory microglia and subsequently enhanced the neuroinflammation and exacerbated the brain injury after ischemic stroke. Mechanistically, DNT-intrinsic FasL and PTPN2 collaboratively regulate the level of CD86+ microglia via production of TNF-α. Taken together, these data reveal that infiltrating DNTs are the critical driving force in promoting microglia-mediated neuroinflammation and ischemic brain injury., CD3+CD4−CD8− T cells (double-negative T cells; DNTs) have diverse functions in peripheral immune-related diseases by regulating immunological and inflammatory homeostasis. However, the functions of DNTs in the central nervous system remain unknown. Here, we found that the levels of DNTs were dramatically increased in both the brain and peripheral blood of stroke patients and in a mouse model in a time-dependent manner. The infiltrating DNTs enhanced cerebral immune and inflammatory responses and exacerbated ischemic brain injury by modulating the FasL/PTPN2/TNF-α signaling pathway. Blockade of this pathway limited DNT-mediated neuroinflammation and improved the outcomes of stroke. Our results identified a critical function of DNTs in the ischemic brain, suggesting that this unique population serves as an attractive target for the treatment of ischemic stroke.

Enhancer of zeste homolog-2 (EZH2), a histone methyltransferase, has been recognized to play a pivotal role in regulating the immune response in various diseases. However, its role in the inflammatory response induced by ischaemic stroke remains to be further investigated. The aim of this study was to determine the role of EZH2 in microglia-associated inflammation in ischaemic stroke and to further detect the effects of the EZH2 inhibitor, 3-deazaadenosine A (DZNep), in ischaemic brain injury. Here, we found that both in vivo ischemic/reperfusion (I/R) injury and in vitro oxygen-glucose deprivation (OGD) treatment induced a marked upregulation of EZH2 in microglia. The administration of the EZH2 inhibitor DZNep improved behavioural performance and reduced the infarct volume in mice after experimental stroke. Furthermore, we showed that DZNep blocked pro-inflammatory (CD86+) microglial activation and triggered anti-inflammatory (CD206+) microglial polarization in experimental stroke. Pro-inflammatory cytokines such as IL-1β, IL-6, TNF-α and CXCL10 were also significantly downregulated by DZNep. In addition, it was found that DZNep blocked the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in microglia, which was increased by I/R injury and OGD. Collectively, we demonstrated that EZH2 is implicated in regulating microglial activation and exacerbates neurological deficits after ischaemic stroke, probably via activating STAT3, and that the EZH2 inhibitor DZNep can exert neuroprotective effects after ischaemic stroke.

We report a new kind of polyethlene oxide (PEO)-Li based composite polymer electrolyte containing active nanocomposite particles with ethylene carbonate (EC)/propylene carbonate (PC) plasticizers embedded in mesoporous SiO2 (EC/PC-SiO2). A very large enhancement of the ionic conductivity was observed for the PEO-Li/(EC/PC-SiO2) electrolytes. This could be attributed mainly to conducting EC/PC nanochannels in the active nanocomposite particles. This unique mechanism is absent in currently known nanocomposite polymer electrolytes with inert oxide nanoparticles. Both high ionic conductivity and excellent stability of such new nanocomposite polymer electrolytes potentially makes it one of the most promising electrolyte materials.