Qi Zhong

Heme activates TLR4-mediated inflammatory injury via MyD88/TRIF signaling pathway in intracerebral hemorrhage

BackgroundInflammatory injury plays a critical role in intracerebral hemorrhage (ICH)-induced neurological deficits; however, the signaling pathways are not apparent by which the upstream cellular events trigger innate immune and inflammatory responses that contribute to neurological impairments. Toll-like receptor 4 (TLR4) plays a role in inflammatory damage caused by brain disorders.MethodsIn this study, we investigate the role of TLR4 signaling in ICH-induced inflammation. In the ICH model, a significant upregulation of TLR4 expression in reactive microglia has been demonstrated using real-time RT-PCR. Activation of microglia was detected by immunohistochemistry, cytokines were measured by ELISA, MyD88, TRIF and NF-κB were measured by Western blot and EMSA, animal behavior was evaluated by animal behavioristics.ResultsCompared to WT mice, TLR4−/− mice had restrained ICH-induced brain damage showing in reduced cerebral edema and lower neurological deficit scores. Quantification of cytokines including IL-6, TNF-α and IL-1β and assessment of macrophage infiltration in perihematoma tissues from TLR4−/−, MyD88−/− and TRIF−/− mice showed attenuated inflammatory damage after ICH. TLR4−/− mice also exhibited reduced MyD88 and TRIF expression which was accompanied by decreased NF-κB activity. This suggests that after ICH both MyD88 and TRIF pathways might be involved in TLR4-mediated inflammatory injury possibly via NF-κB activation. Exogenous hemin administration significantly increased TLR4 expression and microglial activation in cultures and also exacerbated brain injury in WT mice but not in TLR4−/− mice. Anti-TLR4 antibody administration suppressed hemin-induced microglial activation in cultures and in the mice model of ICH.ConclusionsOur findings suggest that heme potentiates microglial activation via TLR4, in turn inducing NF-κB activation via the MyD88/TRIF signaling pathway, and ultimately increasing cytokine expression and inflammatory injury in ICH. Targeting TLR4 signaling may be a promising therapeutic strategy for ICH.

HMBG1 mediates ischemia–reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling

High-mobility group protein box-1 (HMGB1) has recently been recognized as a novel candidate in a specific upstream pathway promoting inflammation after brain ischemia. However, its downstream pathway and underlying mechanism have yet to be elucidated. The HMGB1 level in the acute cerebral infarct (ACI) group was significantly increased compared with that of control group, and correlated with the severity of neurologic impairment of ACI patients. Further, recombinant human HMGB1 (rhHMGB1) had no effect on microglia derived from mice lacking the Toll-like receptor 4 (TLR4−/–). Intracerebroventricular injection of rhHMGB1 in TLR4+/+ mice cause significantly more injury after cerebral ischemia–reperfusion than control group. But, TLR4−/– mice administered with rhHMGB1 showed moderate impairment after ischemia–reperfusion than TLR4+/+ mice. To determine the potential downstream signaling of HMGB1/TLR4 in cerebral ischemic injury, we used the ischemic–reperfusion model with Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β knockout mice (TRIF−/–) and evaluated the activity and expression of TRIF pathway-related kinases. The results suggest that the TRIF pathway is not likely to be involved in TLR4-mediated ischemia brain injury. Finally, we found that TLR4 expressed by immigrant macrophages was involved in the development of ischemic brain damage. These results suggest that HMBG1 mediates ischemia–reperfusion injury by TRIF-adaptor independent Toll-like receptor 4 signaling. The TLR4 expressed by immigrant macrophages may be involved in the development of ischemic brain damage.

High-mobility group protein box-1 and its relevance to cerebral ischemia

High-mobility group box-1 (HMGB1) was originally identified as a ubiquitously expressed, abundant, nonhistone DNA-binding protein. It has well-established functions in the maintenance of nuclear homeostasis. The HMGB1 can either be passively released into the extracellular milieu in response to necrotic signals or actively secreted in response to inflammatory signals. Extracellular HMGB1 interacts with receptors, including those for advanced glycation endproducts (RAGEs) as well as Toll-like receptor 2 (TLR2) and TLR4. The HMGB1 functions in a synergistic manner with other proinflammatory mediators and acts as a potent proinflammatory cytokine-like factor that contributes to the pathogenesis of diverse inflammatory and infectious disorders. Numerous reports point to HMGB1 as a novel player in the ischemic brain. This review provides an appraisal of the emerging roles of HMGB1 in cerebral ischemia injury, highlighting the relevance of HMGB1-blocking agents as potent therapeutic tools for neuroprotection.

Elevation of High-Mobility Group Protein Box-1 in Serum Correlates with Severity of Acute Intracerebral Hemorrhage

High-mobility group protein box-1 (HMGB1) is a proinflammatory involved in many inflammatory diseases. However, its roles in intracerebral hemorrhage (ICH) remain unknown. The purpose of this study was to examine the correlation between changes in serum levels of HMGB1 following acute ICH and the severity of stroke as well as the underlying mechanism. Changes in serum levels of HMGB1 in 60 consecutive patients with primary hemispheric ICH within 12 hours of onset of symptoms were determined. The correlation of HMGB1 with disease severity, IL-6, and TNF-α was analyzed. Changes in HMGB1 levels were detected with ELISA and Western blot. Compared with normal controls, patients with ICH had markedly elevated levels of HMGB1, which was significantly correlated with the levels of IL-6 and TNF-α, NIHSS score at the 10th day, and mRS score at 3 months. In comparison with the control group, the levels of HMGB1 in the perihematomal tissue in mice with ICH increased dramatically, peaked at 72 hours, and decreased at 5 days. Meanwhile, heme could stimulate cultured microglia to release large amounts of HMGB1 whereas Fe2+/3+ ions failed to stimulate HMGB1 production from microglia. Our findings suggest that HMGB1 may play an essential role in the ICH-caused inflammatory injury.

Regulatory T cells ameliorate intracerebral hemorrhage-induced inflammatory injury by modulating microglia/macrophage polarization through the IL-10/GSK3β/PTEN axis

Inflammation mediated by the peripheral infiltration of inflammatory cells plays an important role in intracerebral hemorrhage (ICH) induced secondary injury. Previous studies have indicated that regulatory T lymphocytes (Tregs) might reduce ICH-induced inflammation, but the precise mechanisms that contribute to ICH-induced inflammatory injury remain unclear. Our results show that the number of Tregs in the brain increases after ICH. Inducing Tregs deletion using a CD25 antibody or Foxp3DTR-mice increased neurological deficient scores (NDS), the level of inflammatory factors, hematoma volumes, and neuronal degeneration. Meanwhile, boosting Tregs using a CD28 super-agonist antibody reduced the inflammatory injury. Furthermore, Tregs depletion shifted microglia/macrophage polarization toward the M1 phenotype while boosting Tregs shifted this transition toward the M2 phenotype. In vitro, a transwell co-culture model of microglia and Tregs indicated that Tregs changed the polarization of microglia, decreased the expression of MHC-II, IL-6, and TNF-α and increased CD206 expression. IL-10 originating from Tregs mediated the microglia polarization by increasing the expression of Glycogen Synthase Kinase 3 beta (GSK3β), which phosphorylates and inactivates Phosphatase and Tensin homologue (PTEN) in microglia, TGF-β did not participate in this conversion. Thus, Tregs ameliorated ICH-induced inflammatory injury by modulating microglia/macrophage polarization toward the M2 phenotype through the IL-10/GSK3β/PTEN axis.

Toll-Like Receptor 4/MyD88-Mediated Signaling of Hepcidin Expression Causing Brain Iron Accumulation, Oxidative Injury, and Cognitive Impairment After Intracerebral Hemorrhage.

Background: Disturbance of brain iron metabolism after intracerebral hemorrhage (ICH) results in oxidative brain injury and cognition impairment. Hepcidin plays an important role in regulating iron metabolism, and we have reported that serum hepcidin is positively correlated with poor outcomes in patients with ICH. However, the roles of hepcidin in brain iron metabolism after ICH remain largely unknown. Methods: Parabiosis and ICH models combined with in vivo and in vitro experiments were used to investigate the roles of hepcidin in brain iron metabolism after ICH. Results: Increased hepcidin-25 was found in serum and primarily in astrocytes after ICH. The brain iron efflux, oxidative brain injury, and cognition impairment were improved in Hepc−/− ICH mice but aggravated by the human hepcidin-25 peptide in C57BL/6 ICH mice. Data obtained in in vitro studies showed that increased hepcidin inhibited the intracellular iron efflux of brain microvascular endothelial cells but was rescued by a hepcidin antagonist, fursultiamine. Using parabiosis ICH models also shows that increased serum hepcidin prevents brain iron efflux. In addition, Toll-like receptor 4 (TLR4)/MyD88 signaling pathway increased hepcidin expression by promoting interleukin-6 expression and signal transducer and activator of transcription 3 phosphorylation. TLR4−/− and MyD88−/− mice exhibited improvement in brain iron efflux at 7, 14, and 28 days after ICH, and the TLR4 antagonist (6R)-6-[N-(2-chloro-4-fluorophenyl) sulfamoyl] cyclohex-1-ene-1–carboxylate significantly decreased brain iron levels at days 14 and 28 after ICH and improved cognition impairment at day 28. Conclusions: The results presented here show that increased hepcidin expression caused by inflammation prevents brain iron efflux via inhibition of the intracellular iron efflux of brain microvascular endothelial cells entering into circulation and aggravating oxidative brain injury and cognition impairment, which identifies a mechanistic target for muting inflammation to promote brain iron efflux and to attenuate oxidative brain injury after ICH.

Interleukin-23 Secreted by Activated Macrophages Drives γδT Cell Production of Interleukin-17 to Aggravate Secondary Injury After Intracerebral Hemorrhage.

Background Neuroinflammation plays a key role in intracerebral hemorrhage (ICH)–induced secondary brain injury, but the specific roles of peripheral inflammatory cells such as macrophages and lymphocytes remain unknown. The purpose of this study was to explore the roles of macrophages, T lymphocytes, and the cytokines they secrete as potential targets for treating secondary brain injury after ICH. Methods and Results Our results showed that peripheral macrophages and T lymphocytes successively infiltrated the brain, with macrophage counts peaking 1 day after ICH and T‐lymphocyte counts peaking after 4 days. These peaks in cellular infiltration corresponded to increases in interleukin (IL)‐23 and IL‐17 expression, respectively. We found that hemoglobin from the hematoma activated IL‐23 secretion by infiltrating macrophages by inducing the formation of toll‐like receptor (TLR) 2/4 heterodimer. This increased IL‐23 expression stimulated γδT‐cell production of IL‐17, which increased brain edema and neurologic deficits in the model mice as a proinflammatory factor. Finally, we found that sparstolonin B (SsnB) could ameliorate brain edema and neurologic deficits in ICH model mice via inhibition of TLR2/TLR4 heterodimer formation, and notably, SsnB interacted with myeloid differentiation factor 88 Arg196. Conclusions Together, our results reveal the importance of the IL‐23/IL‐17 inflammatory axis in secondary brain injury after ICH and thus provide a new therapeutic target for ICH treatment.

A20 Ameliorates Intracerebral Hemorrhage–Induced Inflammatory Injury by Regulating TRAF6 Polyubiquitination

Reducing excessive inflammation is beneficial for the recovery from intracerebral hemorrhage (ICH). Here, the roles and mechanisms of A20 (TNFAIP3), an important endogenous anti-inflammatory factor, are examined in ICH. A20 expression in the PBMCs of ICH patients and an ICH mouse model was detected, and the correlation between A20 expression and neurologic deficits was analyzed. A20 expression was increased in PBMCs and was negatively related to the modified Rankin Scale score. A20 expression was also increased in mouse perihematomal tissues. A20−/− and A20-overexpressing mice were generated to further analyze A20 function. Compared with wild-type (WT) mice, A20−/− and A20-overexpressing mice showed significant increases and decreases, respectively, in hematoma volume, neurologic deficit score, mortality, neuronal degeneration, and proinflammatory factors. Moreover, WT-A20−/− parabiosis was established to explore the role of A20 in peripheral blood in ICH injury. ICH-induced damage, including brain edema, neurologic deficit score, proinflammatory factors, and neuronal apoptosis, was reduced in A20−/− parabionts compared with A20−/− mice. Finally, the interactions between TRAF6 and Ubc13 and UbcH5c were increased in A20−/− mice compared with WT mice; the opposite occurred in A20-overexpressing mice. Enhanced IκBα degradation and NF-κB activation were observed in A20−/− mice, but the results were reversed in A20-overexpressing mice. These results suggested that A20 is involved in regulating ICH-induced inflammatory injury in both the central and peripheral system and that A20 reduces ICH-induced inflammation by regulating TRAF6 polyubiquitination. Targeting A20 may thus be a promising therapeutic strategy for ICH.

A novel lipopolysaccharide-antagonizing aptamer protects mice against endotoxemia.

A growing number of researchers have recognized the importance of using lipopolysaccharide (LPS) as target for the prevention and treatment of sepsis. However, no drugs targeting LPS have been applied clinically. In this study, LPS-inhibiting aptamers were screened by Systematic Evolution of Ligands by Exponential Enrichment (SELEX), and their therapeutic effects for experimental sepsis were observed. After 12 rounds of screening, 46 sequences were obtained. Primary structure analysis indicated that they had identical sequences, partly conserved sequences, or non-conserved sequences. Secondary structure analysis showed these sequences usually contained hairpin or stem-loop structures. Aptamer 19 significantly decreased NF-kappaB activation of monocytes challenged by LPS and reduced the IL-1 and TNF-alpha concentration in the media of LPS-challenged monocytes. Furthermore, aptamer 19 significantly increased the survival rate of mice with endotoxemia. The results suggest that a novel LPS antagonizing aptamer was obtained by SELEX, which successfully treated experimental sepsis.

Foxp3 exhibits antiepileptic effects in ictogenesis involved in TLR4 signaling

Inflammatory processes play critical roles in epileptogenesis, but the exact mechanisms that underlie these processes are still not completely understood. In this study, we investigated the role of forkhead transcription factor 3 (Foxp3), a transcription factor that is involved in T‐cell differentiation, in epileptogenesis. In both human epileptic tissues and experimental seizure models, we found significant up‐regulation of Foxp3 in neurons and glial cells. Of importance, Foxp3‐/‐ mice were susceptible to kainic acid–induced seizures, whereas overexpression of Foxp3 reduced acute seizure occurrence and decreased chronic seizure recurrence. In addition, in vitro experiments revealed that Foxp3 inhibited neuronal excitability via glial cells and not neurons. The protective effects of Foxp3 were manifested as a reduction in glial cell activation and proinflammatory cytokine production and increased neuronal survival. Moreover, we showed that beneficial effects of Foxp3 involved the attenuation of TLR4 signaling and inflammation, which led to the inactivation of NR2B‐containing NMDA receptors. These results suggest that Foxp3 in glial cells may play an antiepileptic role in epileptogenesis and may act as a modulator of TLR4. Taken together, our results indicate that Foxp3 may represent a novel therapeutic target for achieving anticonvulsant effects in patients with epilepsy that is currently resistant to drugs.—Wang, F.‐X., Xiong, X.‐Y., Zhong, Q., Meng, Z.‐Y., Yang, H., Yang, Q.‐W. Foxp3 exhibits antiepileptic effects in ictogenesis involved in TLR4 signaling. FASEB J. 31, 2948–2962 (2017). www.fasebj.org