Morphine-induced microglial immunosuppression via activation of insufficient mitophagy regulated by NLRX1.

BACKGROUND: Chronic morphine exposure induces immunosuppression in the peripheral and central nervous system, resulting in susceptibility of patients to invading pathogens. Mitophagy is a crucial regulator of inflammation, and dysregulated mitophagy may cause immunosuppression, but whether mitophagy is linked with morphine-induced immunosuppression in the brain remains unknown. NLRX1 is the only mitochondrially localized NOD family receptor protein which serves as a critical regulator in immunity and mitophagy activation, but it remains an enigma how NLRX1 functions in the crosstalk between microglial inflammatory defense and mitophagy in the presence of morphine. METHODS: Primary microglia and astrocytes, BV2 and MA cell lines were utilized. Mice were stimulated with repeated morphine treatment to mimic chronic morphine exposure, and activation of mitophagy, lysosomal functions, and inflammation were assayed in specific brain regions and immune organs with or without NLRX1-silencing. RESULTS: Morphine induced microglial mitophagy in a LC3 (microtubule-associated proteins light chain 3)-dependent manner, which was mediated by NLRX1. Contrastingly, morphine impaired lysosomal functions, including generation, acidification and mitophagosome-lysosome fusion, thus leading to insufficient mitophagy activation in microglia. NLRX1-silencing inhibited mitophagy activity and rescued lysosomal functions including generation and acidification in microglia. The NLRX1-mediated incomplete mitophagy in microglial cells contributed to immunosuppression and vulnerability towards pathogenic challenge after morphine treatment. In vivo, NLRX1-mediated microglial mitophagy activation by morphine was mainly located in the murine brain cortex, striatum, and cerebellum, where NLRX1 functioned as a negative immune regulator and facilitated septic shock. Collectively, microglial immune responses to septic shock were amenable to NLRX1 silencing in the brain with morphine treatment. CONCLUSION: Morphine activated insufficient mitophagy in microglia which was regulated by NLRX1, ultimately leading to host immunosuppression and susceptible conditions in the brain.

Cerebral Hemorrhage and Alcohol Exposure: A Review.

AIMS: To investigate the dose-response relationships between alcohol and intracerebral hemorrhage (ICH), the impact of alcohol on the outcome of ICH and possible mechanisms underlying hypertensive ICH (HICH) caused by heavy drinking. METHODS: Literature search from 1985 to August 2019 in the PubMed database. RESULTS: The relationship between low-middle alcohol consumption and ICH remains controversial for various reasons, whereas chronic heavy drinking increases the incidence of ICH and exerts worse outcome. More attention is needed to clarify the characteristics of chronic alcohol intake and binge drinking. Chronic alcohol abuse tends to elevates blood pressure, resulting in increased occurrence of HICH and exaggerated HICH-contributed brain injury. CONCLUSION: It is important to develop strategies to promote reasonable intake categories, prevent alcoholism and thus reduce the risk of ICH.

Autophagy alleviates ethanol-induced memory impairment in association with anti-apoptotic and anti-inflammatory pathways.

Chronic excessive drinking leads to a wide spectrum of neurological disorders, including cognitive deficits, such as learning and memory impairment. However, the neurobiological mechanisms underlying these deleterious changes are still poorly understood. We conducted a comprehensive study to investigate the role and mechanism of autophagy in alcohol-induced memory impairment. To establish an ethanol-induced memory impairment mouse model, we allowed C57BL/6J mice intermittent access to 20% ethanol (four-bottle choice) to escalate ethanol drinking levels. Memory impairment was confirmed by a Morris water maze test. We found that mice exposed to EtOH (ethanol) and EtOH combined with the autophagy inhibitor 3-methyladenine (3-MA) showed high alcohol intake and blood alcohol concentration. We confirmed that the EtOH group exhibited notable memory impairment. Inhibition of autophagy by 3-MA worsened ethanol-induced memory impairment. Ethanol induced autophagy in the hippocampus of mice as indicated by western blotting, electron microscopy, RT-qPCR, and fluorescence confocal microscopy. We determined that the mTOR/BECN1 (S14) pathway is involved in ethanol-induced autophagy in vivo. Further, ethanol-induced autophagy suppressed the NLRP3 inflammatory and apoptosis pathways in the hippocampus in mice and in vitro. These findings suggest that autophagy activation in hippocampal cells alleviates ethanol-induced memory impairment in association with anti-apoptotic and anti-inflammatory pathways.

Up-regulated TLR2 and TLR4 expressions in liver and spleen during acute murine T. gondii infection.

Toll-like receptors (TLRs) play a central role in the pathogen clearance and pathological processes. The liver is an important innate immune organ, in which Kupffer cells and hepatocytes are important innate immune cells. However, the role of TLR2 and TLR4 in the liver caused by Toxoplasma gondii infection remains less clear. In this study, mice were infected with T. gondii RH strain and the grades of liver and spleen injuries were histopathologically evaluated. TLR2+ and TLR4+ cells in the livers and spleens were detected by immunohistochemistry, and their messenger RNA (mRNA) expressions were detected using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The pathological severities in the livers and spleens were increased with time in T. gondii-infected mice. Compared with uninfected controls, obvious TLR2+ and TLR4+ cells were observed in the livers and spleens infected with T. gondii at 8 days post-infection, accompanied with significantly over-expressed mRNA levels of TLR2 and TLR4 in the livers and spleens after infection. Our data indicated that increased levels of TLR2 and TLR4 in the liver and spleen may play an important role during acute T. gondii infection.

Mapping study of papillary thyroid carcinoma in China: Predicting EQ-5D-5L utility values from FACT-H&N.

Objective: To develop a mapping algorithm that can be used to predict EQ-5D-5L health utility scores from FACT-H&N and obtain health utility parameters for Chinese patients with papillary thyroid carcinoma (PTC), which can be used for cost-utility analysis in health economic. Methods: A total of 1,050 patients with PTC from a tertiary hospital in China were included, and they completed FACT-H&N and EQ-5D-5L. Four mapping algorithms of direct mapping functions were used to derive the models: Ordinary least squares (OLS), Tobit model (Tobit), Two-part model (TPM), and Beta mixture regression model (Beta). The goodness-of-fit of models was assessed by the mean absolute error (MAE), root mean square error (RMSE), Akaike information criteria (AIC), Bayesian information criteria (BIC), and absolute error (AE). A fivefold cross-validation method was used to test the stability of the models. Results: The mean utility value of the EQ-5D-5L was 0.870 ± 0.094. The mean EQ-VAS score was 76.5 ± 13.0. The Beta mixture regression model mapping FACT-H&N to EQ-5D-5L achieved the best performance [fivefold cross-validation MAE = 0.04612, RMSE = 0.06829, AIC = -2480.538, BIC = -2381.137, AE > 0.05 (%) = 32.48, AE > 0.1 (%) = 8.95]. The independent variables in this model were Physical Well-Being (PWB), Emotional Well-Being (EWB), Head & Neck Cancer Subscale (HNCS) scores and its square term and interaction term scores. Conclusions: This study calculated the health utility score of Chinese patients with PTC. The reported algorithms can be used to map the FACT-H&N into the EQ-5D-5L, which can be applied in the cost-utility related study of patients with PTC.

Transmission of NLRP3-IL-1ß Signals in Cerebral Ischemia and Reperfusion Injury: from Microglia to Adjacent Neuron and Endothelial Cells via IL-1ß/IL-1R1/TRAF6.

The pyrin domain-containing protein 3 (NLRP3) inflammasome drives the profound cerebral ischemia and reperfusion injury (I/R) and mediates the secretion of IL-1ß (interleukin-1ß), which exerts a subsequent cascade of inflammatory injury. The NLRP3-activated-microglial manipulation in adjacent neuronal and endothelial NLRP3 activation has been confirmed in our previous studies. In the present study, we extended the cognition of how microglia mediated neuronal and endothelial NLRP3-IL-1ß signaling during cerebral ischemia and reperfusion injury. In vitro, Neuro-2a and bEND3 cells were cultured alone or co-cultured with BV2 cells and oxygen-glucose deprivation/reoxygenation (OGD/R) was performed. In vivo, transient middle cerebral artery occlusion (tMCAO) rat models and lentiviral silencing targeting IL-1R1 were performed. The NLRP3 inflammasome activation was evaluated by enzyme-linked immunosorbent assay, western blotting, immunoprecipitation, immunohistochemistry, and immunofluorescence. In the co-culture system after OGD/R treatment, NLRP3 inflammasomes in neurons and endothelial cells were activated by microglial IL-1ß via IL-1ß/IL-1R1/TRAF6 signaling pathway, with the basal protein level of NLRP3. In addition, ruptured lysosomes engulfing ASC specks which were possibly secreted from microglia triggered the enhanced NLRP3 expression. In cortices of tMCAO rats at 24 h of reperfusion, silencing IL-1R1, mainly presented in neurons and endothelial cells, was efficient to block the subsequent inflammatory damage and leukocyte brain infiltration, leading to better neurological outcome. Neuronal and endothelial NLRP3 inflammasomes were activated by microglia in cerebral ischemia and reperfusion injury mainly via IL-1ß/IL-1R1/TRAF6 signaling, which might be therapeutically targetable.

PAG neuronal NMDARs activation mediated morphine-induced hyperalgesia by HMGB1-TLR4 dependent microglial inflammation.

Morphine is one of the most effective and widely used analgesic drugs. However, chronic morphine use caused opioid-induced hyperalgesia (OIH). The development of OIH limits the use of morphine. The mechanisms of OIH are not fully understood. Toll-like receptor4 (TLR4) and glutamate receptors in the periaqueductal gray (PAG) are critical in OIH, however, the association between TLR4 and N-methyl-D-aspartate Receptors (NMDARs) activation in PAG remains unclear. Microglia activation, increased TLR4/p65 nuclear factor-kappa B (p65 NF-κB) and proinflammatory cytokines in microglia, and phosphorylation of NMDAR1 subunit (NR1) and NMDAR2B subunit (NR2B) in neurons were observed in PAG of OIH mice. Up-regulations of TLR4/p65 NF-κB and proinflammatory cytokines (IL-1ß, IL-6, TNF-α) in BV2 cells were prevented by inhibiting and knocking down TLR4. By inhibiting myeloid differentiation factor 2 (MD2) and knocking down the High-mobility group box 1 (HMGB1), we found that morphine activated TLR4 by HMGB1 but not MD2. We co-cultured Neuro-2a (N2A) with BV2 microglial cell line and found that instead of directly phosphorylating NMDAR subunits, morphine increased the phosphorylation of NR1 and NR2B by inducing TLR4-mediated microglia inflammation. Knocking TLR4 out of PAG by Lentivirus-GFP-TLR4 shRNA reversed these changes and relieved OIH. Our findings suggested that the secretion of HMGB1 induced by morphine-activated TLR4 in microglia, and the proinflammatory factors released by activated microglia phosphorylated NR1 and NR2B of adjacent neurons, induced increased neuronal excitability. In conclusion, TLR4/NMDARs in PAG were involved in the development and maintenance of OIH and supported novel strategies for OIH treatment.

MPO/HOCl Facilitates Apoptosis and Ferroptosis in the SOD1G93A Motor Neuron of Amyotrophic Lateral Sclerosis.

BACKGROUND: Oxidative stress and reactive oxygen species (ROS) are important in the pathogenesis of amyotrophic lateral sclerosis (ALS). Hypochlorous acid (HOCl) is a powerful oxidant of the reactive oxygen species (ROS) family. HOCl's role in the progress of ALS remains unclear due to the lack of an effective HOCl detection method. Cumulative evidence supports oxidative damage incurred by mutant hSOD1 contributing to motor neuron death; however, whether HOCl as well as its catalytic enzyme myeloperoxidase (MPO) function in the cell death of SOD1G93A ALS remains elusive. METHODS: The hSOD1WT and hSOD1G93A NSC-34 cell and SOD1G93A ALS mouse models were employed. With a novel fluorescent HOCl probe, HKOCl-3, we detected the expressions of HOCl and its catalytic enzyme, MPO, in the above models in vitro and in vivo. The regulation of MPO/HOCl by hSOD1G93A mutation and cell deaths by MPO/HOCl were also assayed, including apoptosis, ferroptosis, and autophagy. RESULTS: Our results showed that hSOD1G93A mutation promoted the activation of the MPO/HOCl pathway in SOD1G93A ALS cell models. The activation of MPO/HOCl pathways facilitated apoptosis and ferroptosis through increasing the Bax/Bcl-2 ratio and expression of caspase-3 or inhibiting the expressions of GPX4 and NQO1 and thus leading to irreversible lipid peroxidation. Overexpressed FSP1, a glutathione-independent suppressor, could ameliorate ferroptosis. In vivo, we demonstrated that the activation of the MPO/HOCl pathway occurred differently in motor neurons of the motor cortices, brain stems, and spinal cords in male and female SOD1G93A transgenic mice. In addition, inhibiting MPO improved the motor performance of SOD1G93A transgenic mice, as demonstrated by the rotarod test. CONCLUSIONS: We concluded that aggregation of mutant hSOD1 proteins contributed to activation of the MPO/HOCl pathway, triggering apoptosis and ferroptosis in motor neuronal deaths and exerting impaired motor performance.

Chronic exposure of alcohol triggers microglia-mediated synaptic elimination inducing cognitive impairment.

BACKGROUND AND AIMS: Long-term alcohol intake leads to cognitive impairment and dementia. The impairment of the cerebral cortex and limbic structures in alcoholics is associated with the loss of synapses instead of neurons. Synapse loss is considered to be an early and key feature of many neurodegenerative diseases, in which microglia-mediated synapse elimination is vital. However, the underlying mechanisms of synapse loss and cognitive impairment caused by long-term alcohol intake are still largely unknown. METHODS: We investigated the relationship of synapse impairment, the microglial innate immune receptor-TREM2, and microglia-mediated synaptic elimination in long-term alcohol exposure. RESULTS: We found that long-term alcohol exposure increased expression of TREM2, decreased expression of synaptic proteins and glutamate receptor subunits, reduced dendrite spine density, and impaired long-term potentiation (LTP) in the hippocampus. Minocycline reduced the amount of the postsynaptic marker PSD95 in microglia, attenuated dendrite spine density loss, and slow down the forgetting process of already-formed memory. Furthermore, we found that TREM2 participated in microglia-mediated synapse elimination in chronic alcohol exposure in vivo. Significantly fewer PSD95 were detectable in microglial phagolysosomes in TREM2 knockdown mice. Besides, TREM2 gene silencing ameliorated synapse loss, LTP impairment, and forgetting of remote memories. CONCLUSIONS: Our data suggests that TREM2 is associated with synaptic plasticity impairment and memory deficits, indicating microglia-mediated synaptic pruning might be the underlying mechanism involved in synapse loss and memory impairment induced by long-term alcohol intake. These findings provide new evidence for the receptor's participation in neurodegeneration diseases.

Idebenone attenuates cerebral inflammatory injury in ischemia and reperfusion via dampening NLRP3 inflammasome activity.

BACKGROUND: Idebenone is a well-appreciated mitochondrial protectant while the mechanisms underlying the neuroprotection in cerebral ischemia and reperfusion (I/R) remain elusive. It has been manifested NLRP3 inflammasom activation contributed to I/R induced damage. It raises questions how exactly NLRP3 inflammasom was activated in microglia and neuron and whether idebenone reverses the process in I/R. METHODS: I/R rat model was utilized and BV2, primary microglia and PC12 cells were subjected to oxygen-glucose deprivation (OGD). Then, western-blotting, q-PCR, immunofluorescence staining, ELISA, flow cytometry and immunoprecipitation analysis were performed. RESULTS: We found ROS-NLRP3 singaling was activated in BV2 cells at OGD/R 24 h. Importantly, microglial NLRP3 activation was essential for NLRP3 activation in PC12 cells under microglial-neuronal co-culture circumstance, which has been confirmed to induced neuronal apoptosis. Further, we found mitochondrial dysfunction in OGD/R led to mt-DNA translocation as well as generation of mt-ROS, resulting cytosolic accumulation of oxidized mt-DNA. Ultimately, oxidized mt-DNA binding to NLRP3 contributed to further activation of NLRP3 and dramatically augmented inflammation in BV2 and PC12 cells. Furthermore, idebenone treatment inhibited the process, thus suppressing the NLRP3-mediated inflammatory injury after OGD/R. In vivo, NLRP3 was activated in microglia of I/R rats and inhibition of NLRP3 was observed in idebenone treatment group, which had less neurological deficit and less infarct volume. INTERPRETATION: Our data revealed the anti-inflammatory effects of idebenone via suppressing activation of NLRP3 and ameliorating NLRP3-mediating damage in I/R, which may provide new insight in therapeutic strategy for ischemic stroke.

Effect of alcohol use disorders and alcohol intake on the risk of subsequent depressive symptoms: a systematic review and meta-analysis of cohort studies.

BACKGROUND AND AIMS: Alcohol use disorders (AUD) are often comorbid with depressive symptoms. Cohort studies on the association between AUD and subsequent depressive symptoms have produced inconsistent results. Moreover, regarding alcohol intake, the risk of developing depressive symptoms might vary with alcohol intake level. We aimed to investigate the association between AUD, alcohol intake and subsequent depressive symptoms. DESIGN AND SETTING: We conducted a systematic search in PubMed, Embase and PsycINFO for cohort studies on the association between AUD or alcohol intake and subsequent depressive symptoms. PARTICIPANTS: We included 338 426 participants from 42 studies. Six and four studies analyzed only females and males, respectively. MEASUREMENTS: We combined risk estimates for developing depressive symptoms using a random-effects model. We divided alcohol intake into abstinence, light (0-84 g/week), moderate (85-168 g/week) and heavy drinking (> 168 g/week or > 48 g/day at least weekly). We conducted a categorical analysis to compare the risk of depressive symptoms between abstinence and different intake categories. Further, we conducted a dose-response analysis to investigate the alcohol-depression association. FINDINGS: We analyzed 42 studies (follow-up time: 1-40 years). AUD was associated with significantly increased risk of subsequent depressive symptoms [relative risk (RR) = 1.57, 95% confidence interval (CI) = 1.41-1.76]. Regarding alcohol intake, heavy drinking had an increased risk of depressive symptoms; however, the association was only significant when controls were limited to non-heavy drinkers (RR = 1.13, 95% CI = 1.05-1.22). Taking into consideration the possibility of publication bias and confounding factors made the association non-significant. We observed J-shaped associations in both categorical and dose-response analyses where light-moderate drinking had a significantly decreased risk of depression, while heavy drinking did not show a significant association with depressive symptoms compared with non-drinkers. CONCLUSION: Alcohol use disorders are associated with increased the risk of subsequent depressive symptoms. Heavy drinking does not significantly predict occurrence of depressive symptoms after adjusting for potential confounders.

An miR-340-5p-macrophage feedback loop modulates the progression and tumor microenvironment of glioblastoma multiforme.

MicroRNAs (miRNAs) have been shown to be involved in the progression and tumor microenvironment of glioblastoma multiforme (GBM). Our previous research has indicated that miR-340-5p has an antitumor effect in vitro. However, the role of miR-340-5p in GBM has not been fully elucidated. Here, we show that downregulation of miR-340-5p in GBM is correlated with tumor size, recurrence, and poor survival. Moreover, we found that miR-340-5p levels are correlated with the density of tumor-associated macrophages (TAMs) and M2-polarized TAMs in GBM. Biofunctional investigations revealed that downregulation of miR-340-5p promoted TAM recruitment and M2-TAMs polarization in vitro and in vivo. In addition, we found that upregulation of miR-340-5p inhibited tumor growth and was associated with good prognosis in vivo. Through gene expression profiles and bioinformatics analysis, we showed that miR-340-5p directly targets POSTN, which recruited TAMs through integrin αvß3. Downregulation of miR-340-5p in GBM did not induce the differentiation of TAMs into polarized M2 cells but was able to promote the M2 polarization of TAMs through directly targeting LTBP-1. Furthermore, we found that M2-TAMs promoted tumorigenesis and were associated with a poor prognosis in vivo. In an in vitro study, we demonstrated that M2-TAMs inhibited miR-340-5p expression in GBM cells by upregulation of TGFß-1, which increased HMGA-2 expression in GBM. A ChIP assay confirmed that HMGA-2 transcriptionally suppressed miR-340-5p expression. Patients with low-miR-340-5p expression, high CD163, high POSTN, high LIBP1 levels, and high HMGA-2 had a poor prognosis with shorter overall survival, confirming data from the TCGA database. These findings suggest that an miR-340-5p-macrophage feedback loop modulates the progression and tumor microenvironment of GBM and may represent a prognostic biomarker and therapeutic strategy for GBM.