TLR4 gene polymorphisms rs11536889 is associated with intracranial aneurysm susceptibility.

Intracranial aneurysm (IA) is a common lesion which often present asymptomatic until the time of rupture and result in subarachnoid hemorrhage (SAH). The pathogenesis of IA formation is complex and is influenced by both genetic and environmental risk factors. For exploring the detailed molecular and cellular mechanisms involved in the pathogenesis of IA, recent studies indicated inflammatory pathways and their genetic variants may as potential biomarkers. In this study, functionally relevant polymorphisms in the toll-like receptor 4 (TLR4) were screened in 330 IA patients and 313 controls from a Han Chinese population. Eight single nucleotide gene polymorphisms (SNPs) genotyped by the Improved Multiple Ligase Detection Reaction (iMLDR) method. Our results indicated that the presence of the minor allele (C) of the TLR4 SNP rs11536889 was associated with a decreased risk of IA (C vs. G, OR = 0.731; 95% CI 0.567-0.943; P = 0.017). This association was also present at the genotype level in a codominant model (GC vs. GG, OR = 0.447; 95% CI 0.226-0.884; P = 0.020) and a recessive model (CC vs. GG + GC, OR = 0.489; 95% CI 0.250-0.955; P = 0.035). In summary, we firstly found that the TLR4 SNP rs11536889 was significantly associated with the susceptibility of IA. Our results indicated TLR4 SNP rs11536889 may be a marker for IA risk, though the exact functional roles of TLR4 SNP rs11536889 in IA formation are still not very clear.

Association between polymorphism of SMAD3 gene and risk of sporadic intracranial arterial aneurysms in the Chinese Han population.

Intracranial arterial aneurysms (IAAs) are locally abnormal dilations of the cerebral arteries and often result in subarachnoid hemorrhages (SAH). Genetic, molecular and cellular mechanisms of sporadic IAAs forms are poorly understood. In this study, we investigate the association between mothers against decapentaplegic homolog 3 (SMAD3) genotypes and the risk of sporadic intracranial arterial aneurysms among the Chinese Han population. A case-control study was conducted examining 330 IAA patients and 313 controls. There were eight single nucleotide polymorphisms of SMAD3 selected and genotyped using the polymerase chain reaction-ligase detection reaction (PCR-LDR) method. Our results indicated that SMAD3 rs1065080 polymorphism was associated with a risk of IAAs in a codominant model (GA vs GG, OR=1.433; 95% CI 1.030-1.994; P=0.032). In summary, we observed that SMAD3 rs1065080 single nucleotide gene polymorphisms were significantly associated with patient susceptibility to IAAs.

CD36 Gene Polymorphisms Are Associated with Intracerebral Hemorrhage Susceptibility in a Han Chinese Population.

The CD36 gene encodes a membrane glycoprotein (type B scavenger receptor, SR-B2) that plays a crucial role in lipid sensing, innate immunity, atherogenesis, and glycolipid metabolism. In this study, we aimed to investigate the association between CD36 gene polymorphisms and intracerebral hemorrhage (ICH) in a Han Chinese population. We performed genotype and allele analyses for eleven single nucleotide polymorphisms (SNPs) of CD36 in a case-controlled study involving 292 ICH patients and 298 control participants. Eleven SNPs were genotyped by the Improved Multiple Ligase Detection Reaction (iMLDR) method. The results indicated that the SNP rs1194182 values were significantly different between ICH group and control group in a dominant model after adjusting for confounding factors. The subgroup analysis conducted for rs1194182 showed that the allele G frequencies were significantly different between ICH patients and controls in hypertension group via a dominant model. We then analyzed the rs1194182 genotype distributions among different groups of the serum lipid groups, including BMI, TC, TG, HDL, and LDL. However, no significant differences were found in the analysis of other subgroups. Taken together, these findings indicate that rs1194182 polymorphism in the CD36 gene was associated with ICH, and genotype GG could be an independent predictor.

Mfsd2a (Major Facilitator Superfamily Domain Containing 2a) Attenuates Intracerebral Hemorrhage-Induced Blood-Brain Barrier Disruption by Inhibiting Vesicular Transcytosis.

BACKGROUND: Blood-brain barrier (BBB) disruption aggravates brain injury induced by intracerebral hemorrhage (ICH); however, the mechanisms of BBB damage caused by ICH remain elusive. Mfsd2a (major facilitator superfamily domain containing 2a) has been known to play an essential role in BBB formation and function. In this study, we investigated the role and underlying mechanisms of Mfsd2a in BBB permeability regulation after ICH. METHODS AND RESULTS: Using ICH models, we found that Mfsd2a protein expression in perihematomal brain tissues was significantly decreased after ICH. Knockdown and knockout of Mfsd2a in mice markedly increased BBB permeability, neurological deficit score, and brain water contents after ICH, and these were rescued by overexpressing Mfsd2a in perihematomas. Moreover, we found that Mfsd2a regulation of BBB permeability after ICH correlated with changes in vesicle number. Expression profiling of tight junction proteins showed no differences in Mfsd2a knockdown, Mfsd2a knockout, and Mfsd2a overexpression mice. However, using electron microscopy following ICH, we observed a significant increase in pinocytotic vesicle number in Mfsd2a knockout mice and decreased the number of pinocytotic vesicles in mouse brains with Mfsd2a overexpression. Finally, using multiple reaction monitoring, we screened out 3 vesicle trafficking-related proteins (Srgap2, Stx7, and Sec22b) from 31 vesicle trafficking-related proteins that were markedly upregulated in Mfsd2a knockout mice compared with controls after ICH. CONCLUSIONS: In summary, our results suggest that Mfsd2a may protect against BBB injury by inhibiting vesicular transcytosis following ICH.

Regulatory T cells ameliorate intracerebral hemorrhage-induced inflammatory injury by modulating microglia/macrophage polarization through the IL-10/GSK3ß/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 (GSK3ß), which phosphorylates and inactivates Phosphatase and Tensin homologue (PTEN) in microglia, TGF-ß 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/GSK3ß/PTEN axis.

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.

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.