FasL incapacitation alleviates CD4+ T cells-induced brain injury through remodeling of microglia polarization in mouse ischemic stroke
Graphical abstract
Introduction
Ischemic stroke is one of the leading causes of death and mortality worldwide (Tsai et al., 2015). Inflammation, which occurs throughout the whole process of cerebral ischemia reperfusion, is considered to be the most important pathological mechanism of ischemic stroke (Chamorro et al., 2012). After brain ischemia, neuronal death triggers local immune responses, leading to microglia activation and peripheral inflammatory cells migration into the lesion area (Waisman et al., 2015). Activated microglia act synergistically with infiltrating inflammatory cells and release large amounts of pro-inflammatory mediators such as interleukin-1 (IL-1), IL-6, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and other deleterious substances (Iadecola and Anrather, 2011), resulting in additional neuronal death, disruption of blood brain barrier (BBB), brain edema and aggravation of ischemic brain damage (Brait et al., 2010; Ceulemans et al., 2010).
Microglia are resident innate immune cells in the central nervous system which act as a sensor (Czeh et al., 2011). In recent years, large amounts of studies indicate that microglia are highly plastic cells and can perform different phenotypes and functions in response to different environment stimulus (Perry et al., 2010). Activated microglia can polarize to pro-inflammatory (classically activated, or M1) or anti-inflammatory (alternatively activated, or M2) phenotypes (David and Kroner, 2011; Mosser and Edwards, 2008). M1 microglia produce high levels of pro-inflammatory cytokines which are essential for the clearance of invading pathogens and injured tissue products, but resulting in collateral tissue damage at the same time (Benarroch, 2013). Inversely, M2 microglia secret anti-inflammatory cytokines, such as transforming growth factor-β (TGF-β) and neurotrophins, play crucial roles in promoting wound healing and suppressing destructive immune responses (Martinez et al., 2009). These dual and opposing activation phenotypes of microglia have been found in ischemic stroke and determine the regulation of inflammatory responses after brain infarction (Hu et al., 2012; Yenari et al., 2010). However, the mechanisms of how microglia change phenotypes after ischemic stroke are still unknown.
As the major effector cells of lymphocytes, CD4+ T cells (helper T cells, Th) are involved in the evolution of ischemic stroke and accompanying neurological deficits. CD4+ T cells invade into the ischemic brain within 24 h and peak on day 3 after focal infarction (Gelderblom et al., 2009; Jander et al., 1995). Infiltrated CD4+ T cells regulate brain inflammation though their cytokines production. Typically, CD4+ T cells can be divided into Th1, Th2, Th17, and regulatory T (Treg) cells based on their cytokine secretion profiles (Zhu et al., 2010). Recent studies suggest that different CD4+ T cell subsets may have distinct effects on the injured brain. Th1 and Th17 cells produce specific cytokines such as IL-1, IFN-γ, IL-17, IL-22 and so on, so they are characterized by high pro-inflammatory function and aggravating brain damage (Arumugam et al., 2005; Stockinger et al., 2007). On the contrary, Th2 and Treg cells are characterized by suppressing excessive inflammation, achieving self-tolerance and brain repair via producing anti-inflammatory cytokines such as IL-4, IL-10 and TGF-β (Liesz et al., 2009; Xie et al., 2015; Yilmaz and Granger, 2010). The balance between Th1/Th17 and Th2/Treg cells predominantly affect the function of CD4+ T cells and determine the outcome of brain inflammation (Afzali et al., 2007; Gu et al., 2015). Substantial evidence proves that CD4+ T cells can interact with microglia after brain injury, resulting in injurious or neurotrophic outcomes in vicinity (Strachan-Whaley et al., 2014). However, the molecular mechanisms of their crosstalk are still debated.
Fas ligand (FasL), a member of TNF protein family, has been shown to induce apoptosis in Fas-expressing cells and serves as a key death factor in the immune system (Lettau et al., 2008). Emerging evidence suggests that FasL plays important roles in inflammation since its capacity of inducing production of pro-inflammatory cytokines and leukocyte infiltration (Kunes et al., 2009). In previous studies, we proved that FasL mutation profoundly reduced infarct volume, neutrophil recruitment and microglia activation after ischemic stroke (Meng et al., 2016; Niu et al., 2012). However, little is known about the effect of FasL regulating CD4+ T cell subsets and crosstalk between CD4+ T cells and microglia.
In the present study, we intended to investigate the influence of FasL depletion on brain inflammation by virtue of ischemic stroke using middle cerebral artery occlusion (MCAO) models and explore the underlying mechanism of microglia polarization using co-culturing system involving CD4+ T cells from FasL-mutant (gld) mice and primary microglia. As a result, we found that FasL mutant CD4+ T cells could remodel microglia polarization. More importantly, we showed that proportion of CD4+ T cells were reduced both in the brain and periphery of FasL mutant mice after middle cerebral artery occlusion (MCAO), and that Th17/Treg cells balance was skewed into Treg cells.
Section snippets
Middle cerebral artery occlusion model in mice
Fourteen-week-old male recombination activating gene 1–deficient (Rag1−/−) mice, FasL mutant (gld) mice (22-24 g) and their wild-type control C57BL/6 J (B6) mice were purchased from the Animal Model Research Center of Nanjing University. All animal experiments were approved by the Animal Care Committee in China and performed according to institutional guidelines. Mice were subjected to middle cerebral artery occlusion (MCAO) by intraluminal filament technique for 60 min as previously described (
FasL incapacitation remodels CD4+ T cells reversed M1 microglia polarization
Initially,the crosstalk between CD4+ T cells and microglia were explored using co-culturing models involving CD4+ T cells from mice and primary microglia induced by LPS into an inflammatory state before co-culture. And gene expression of M1 makers (iNOS, TNF-α and CD86) and M2 makers (CD206 and Ym1/2) of microglia were measured by q-PCR. We showed that CD4+ T cells from wild type mice of MCAO models could significantly up-regulate mRNA expression of M1 markers and down-regulate M2 markers of
Discussion
In this study, we reported some novel findings including: (1) FasL deficiency changes the subsets of CD4+ T cells and alleviates inflammatory reaction after MCAO. (2) CD4+ T cells facilitated microglial pro-inflammation through NF-κB signaling pathway and FasL of CD4+ T cells played an important role in M1 microglia polarization. (3) FasL-mediated crosstalk between CD4+ T cells and microglia aggravated neuronal death and apoptosis.
In central nervous system, FasL has been observed to elevate in
Acknowledgments
This research was supported by the National Natural Science Foundation of China (81230026, 81630028, 81402065), the Science and Technology Department of Jiangsu Province (BE2016610) and Jiangsu Province Key Medical Discipline (ZDXKA2016020) and Nanjing Medical Science and technique Development Foundation (QRX17118).
Conflicts of interest
The authors declare no conflicts of interest.
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These authors contributed equally to this study.