Proinflammatory Response In Microglia Research Articles

The Alteration of Microglial Calcium Homeostasis in Central Nervous System Disorders: A Comprehensive Review

Microglia, the unique and motile immune cells of the central nervous system (CNS), function as a security guard in maintaining CNS homeostasis, primarily through calcium signaling. The calcium dynamics in microglia control important functions such as phagocytosis, cytokine release, and migration. Calcium dysregulation in microglia has been linked to several CNS disorders, like Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), and ischemic stroke (IS). Calcium entering through channels such as voltage-gated calcium channels (VGCCs), store-operated calcium entry (SOCE), and transient receptor potential (TRP) channels is essential for microglial activation and pro-inflammatory responses. Under pathological conditions, like the formation of amyloid-β plaques in AD, aggregation of α-synuclein in PD, and oxidative stress in MS, calcium dysregulation exacerbates neuroinflammation, mitochondrial dysfunction, and neurodegeneration. Therapeutic strategies targeting calcium signaling pathways, using calcium channel blockers and antioxidant interventions, show promise for alleviating microglial activation and slowing down disease progression. This review summarizes the underlying mechanisms of microglial calcium dysregulation and potential therapeutic benefits for restoring microglial calcium balance in CNS disorders.

Acute postnatal inflammation alters adult microglial responses to LPS that are sex-, region- and timing of postnatal inflammation-dependent

BackgroundAdverse events in early life can have impact lasting into adulthood. We investigated the long-term effects of systemic inflammation during postnatal development on adult microglial responses to lipopolysaccharide (LPS) in two CNS regions (cortex, cervical spinal cord) in male and female rats.MethodsInflammation was induced in Sprague-Dawley rats by LPS (1 mg/kg) administered intraperitoneally during postnatal development at P7, P12 or P18. As adults (12 weeks of age), the rats received a second LPS dose (1 mg/kg). Control rats received saline. Microglia were isolated 3 h post-LPS followed by gene expression analysis via qRT-PCR for pro-inflammatory (IL-6, iNOS, Ptgs2, C/EBPb, CD14, CXCL10), anti-inflammatory (CD68, Arg-1), and homeostatic genes (P2Y12, Tmemm119). CSF-1 and CX3CL1 mRNAs were analyzed in microglia-free homogenates.ResultsBasal gene expression in adult microglia was largely unaffected by postnatal inflammation. Adult cortical microglial pro-inflammatory gene responses to LPS were either unchanged or attenuated in rats exposed to LPS during postnatal development. Ptgs2, C/EBPb, CXCL10 and Arg-1 were the most affected genes, with expression significantly downregulated vs. rats without postnatal LPS. Spinal microglia were affected most by LPS at P18, with mixed and sometimes opposing effects on proinflammatory genes in males vs. females. Overall, male cortical vs. spinal microglia were more affected by postnatal LPS. Females were affected in both cortex and spinal cord, but the effect was dependent on timing of postnatal LPS. Overall, inflammatory challenge at P18 had greater effect on adult microglia vs. challenge at P12 or P7.ConclusionsLong-lasting effects of postnatal inflammation on adult microglia depend on postnatal timing, CNS region and sex.

Extracellular vesicles in sepsis plasma mediate neuronal inflammation in the brain through miRNAs and innate immune signaling

BackgroundNeuroinflammation reportedly plays a critical role in the pathogenesis of sepsis-associated encephalopathy (SAE). We previously reported that circulating plasma extracellular vesicles (EVs) from septic mice are proinflammatory. In the current study, we tested the role of sepsis plasma EVs in neuroinflammation.MethodsTo track EVs in cells and tissues, HEK293T cell-derived EVs were labeled with the fluorescent dye PKH26. Cecal ligation and puncture (CLP) was conducted to model polymicrobial sepsis in mice. Plasma EVs were isolated by ultracentrifugation and their role in promoting neuronal inflammation was tested following intracerebroventricular (ICV) injection. miRNA inhibitors (anti-miR-146a, -122, -34a, and -145a) were applied to determine the effects of EV cargo miRNAs in the brain. A cytokine array was performed to profile microglia-released protein mediators. TLR7- or MyD88-knockout (KO) mice were utilized to determine the underlying mechanism of EVs-mediated neuroinflammation.ResultsWe observed the uptake of fluorescent PKH26-EVs inside the cell bodies of both microglia and neurons. Sepsis plasma EVs led to a dose-dependent cytokine release in cultured microglia, which was partially attenuated by miRNA inhibitors against the target miRNAs and in TLR7-KO cells. When administered via the ICV, sepsis plasma EVs resulted in a marked increase in the accumulation of innate immune cells, including monocyte and neutrophil and cytokine gene expression, in the brain. Although sepsis plasma EVs had no direct effect on cytokine production or neuronal injury in vitro, the conditioned media (CM) of microglia treated with sepsis plasma EVs induced neuronal cell death as evidenced by increased caspase-3 cleavage and Annexin-V staining. Cytokine arrays and bioinformatics analysis of the microglial CM revealed multiple cytokines/chemokines and other factors functionally linked to leukocyte chemotaxis and migration, TLR signaling, and neuronal death. Moreover, sepsis plasma EV-induced brain inflammation in vivo was significantly dependent on MyD88.ConclusionsCirculating plasma EVs in septic mice cause a microglial proinflammatory response in vitro and a brain innate immune response in vivo, some of which are in part mediated by TLR7 in vitro and MyD88 signaling in vivo. These findings highlight the importance of circulating EVs in brain inflammation during sepsis.

Escape of Kdm6a from X chromosome is detrimental to ischemic brains via IRF5 signaling.

The role of chromatin biology and epigenetics in disease progression is gaining increasing recognition. Genes that escape X chromosome inactivation (XCI) can impact neuroinflammation through epigenetic mechanisms. Our prior research has suggested that the X escapee genes Kdm6a and Kdm5c are involved in microglial activation after stroke in aged mice. However, the underlying mechanisms remain unclear. We hypothesized that Kdm6a/5c demethylate H3K27Me3/H3K4Me3 in microglia respectively, and mediate the transcription of interferon regulatory factor 5 (IRF5) and IRF4, leading to microglial pro-inflammatory responses and exacerbated stroke injury. Aged (17-20 months) Kdm6a/5c microglial conditional knockout (CKO) female mice (one allele of the gene) were subjected to a 60-min middle cerebral artery occlusion (MCAO). Gene floxed females (two alleles) and males (one allele) were included as controls. Infarct volume and behavioral deficits were quantified 3 days after stroke. Immune responses including microglial activation and infiltration of peripheral leukocytes in the ischemic brain were assessed by flow cytometry. Epigenetic modification of IRF5/4 by Kdm6a/5c were analyzed by CUT&RUN assay. The demethylation of H3K27Me3 by kdm6a increased IRF5 transcription; meanwhile Kdm5c demethylated H3K4Me3 to repress IRF5. Both Kdm6a fl/fl and Kdm5c fl/fl mice had worse stroke outcomes compared to fl/y and CKO mice. Gene floxed females showed more robust expression of CD68 in microglia, elevated brain and plasma levels of IL-1β or TNF-α, after stroke. We concluded that IRF5 signaling plays a critical role in mediating the deleterious effect of Kdm6a; whereas Kdm5c's effect is independent of IRF5.

ROS exhaustion reverses the effects of hyperbaric oxygen on hemorrhagic transformation through reactivating microglia in post-stroke hyperglycemic mice

Acute ischemic stroke (AIS) is a major global health concern due to its high mortality and disability rates. Hemorrhagic transformation, a common complication of AIS, leads to poor prognosis yet lacks effective treatments. Preclinical studies indicate that hyperbaric oxygen (HBO) treatment within 12 h of AIS onset alleviates ischemia/reperfusion injuries, including hemorrhagic transformation. However, clinical trials have yielded conflicting results, suggesting some underlying mechanisms remain unclear. In this study, we confirmed that HBO treatments beginning within 1 h post reperfusion significantly alleviated the haemorrhage and neurological deficits in hyperglycemic transient middle cerebral arterial occlusion (tMCAO) mice, partly due to the inhibition of the NLRP3 inflammasome-mediated pro-inflammatory response in microglia. Notably, reactive oxygen species (ROS) mediate the anti-inflammatory and protective effect of early HBO treatment, as edaravone and N-Acetyl-l-Cysteine (NAC), two commonly used antioxidants, reversed the suppressive effect of HBO treatment on NLRP3 inflammasome-mediated inflammation in microglia. Furthermore, NAC countered the protective effect of early HBO treatment in tMCAO mice with hyperglycemia. These findings support that early HBO treatment is a promising intervention for AIS, however, caution is warranted when combining antioxidants with HBO treatment. Further assessments are needed to clarify the role of antioxidants in HBO therapy for AIS.

Geraniol (GER) attenuated chronic sleep restriction (CSR)-induced neuroinflammation in adolescent mice

Sleep insufficiency is a significant health problem worldwide, and adolescent sleep restriction (SR) could induce multiple neurodevelopmental disorders in the central nervous system (CNS). Microglial-mediated neuroinflammation plays a vital role in multiple neurological diseases, and recent research showed the regulation effect of immunoproteasome on microglia functions. Geraniol (GER), an important ingredient in many essential oils, possesses diverse pharmacological properties like anti-inflammatory and antioxidant. The present study was designed to evaluate the neuroprotective effect of GER on SR in adolescent mice and further investigate the underlying mechanisms. Our results displayed that 14 days of chronic sleep restriction (CSR) induced cognitive decline, and anxiety-like and attention-deficit behaviors, which were mitigated by GER pretreatment. GER administration also reversed microglial pro-inflammatory response under CSR stimulation in the anterior cingulate cortex (ACC) regions by reducing the expression and secretion of cytokines like IL-1β and TNF-α. Mechanism research showed that LMP7 mRNA was selectively up-regulated under CSR treatment but down-regulated by GER administration. Proteasome activity and protein expression of LMP7 were consistent with mRNA data. ONX-0914 was applied to inhibit LMP7 selectively, and data validated that GER might alleviate CSR-induced neuroinflammation by regulating LMP7. Our study provides evidence that LMP7 is a critical regulator of CSR-induced proinflammation, and geraniol might be a promising therapy against CSR-induced neurodevelopmental disorders.

Reduced SH3RF3 may protect against Alzheimer's disease by lowering microglial pro-inflammatory responses via modulation of JNK and NFkB signaling.

Understanding how high-risk individuals are protected from Alzheimer's disease (AD) may illuminate potential therapeutic targets. A previously identified non-coding SNP in SH3RF3/POSH2 significantly delayed disease onset in a Caribbean Hispanic cohort carrying the PSEN1 G206A mutation sufficient to cause early-onset AD and microglial expression of SH3RF3 has been reported to be a key driver of late-onset AD. SH3RF3 acts as a JNK pathway scaffold and can activate NFκB signaling. While effects of SH3RF3 knockdown in human neurons were subtle, including decreased phospho-tau S422, knockdown in human microglia significantly reduced inflammatory cytokines in response to either a viral mimic or oligomeric Aβ42. This was associated with reduced activation of JNK and NFκB pathways in response to these stimuli. Pharmacological inhibition of JNK or NFκB signaling phenocopied SH3RF3 knockdown. We also found PSEN1 G206A microglia have reduced inflammatory responses to oAβ42. Thus, further reduction of microglial inflammatory responses in PSEN1 mutant carriers by protective SNPs in SH3RF3 might reduce the link between amyloid and neuroinflammation to subsequently delay the onset of AD.

Usp14 deficiency removes α-synuclein by regulating S100A8/A9 in Parkinson’s disease

Ubiquitin-proteasome system dysfunction triggers α-synuclein aggregation, a hallmark of neurodegenerative diseases, such as Parkinson’s disease (PD). However, the crosstalk between deubiquitinating enzyme (DUBs) and α-synuclein pathology remains unclear. In this study, we observed a decrease in the level of ubiquitin-specific protease 14 (USP14), a DUB, in the cerebrospinal fluid (CSF) of PD patients, particularly females. Moreover, CSF USP14 exhibited a dual correlation with α-synuclein in male and female PD patients. To investigate the impact of USP14 deficiency, we crossed USP14 heterozygous mouse (USP14+/−) with transgenic A53T PD mouse (A53T-Tg) or injected adeno-associated virus (AAV) carrying human α-synuclein (AAV-hα-Syn) in USP14+/− mice. We found that Usp14 deficiency improved the behavioral abnormities and pathological α-synuclein deposition in female A53T-Tg or AAV-hα-Syn mice. Additionally, Usp14 inactivation attenuates the pro-inflammatory response in female AAV-hα-Syn mice, whereas Usp14 inactivation demonstrated opposite effects in male AAV-hα-Syn mice. Mechanistically, the heterodimeric protein S100A8/A9 may be the downstream target of Usp14 deficiency in female mouse models of α-synucleinopathies. Furthermore, upregulated S100A8/A9 was responsible for α-synuclein degradation by autophagy and the suppression of the pro-inflammatory response in microglia after Usp14 knockdown. Consequently, our study suggests that USP14 could serve as a novel therapeutic target in PD.

Obligatory role of microglia-mobilized hippocampal CREB-BDNF signaling in the prophylactic effect of β-glucan on chronic stress-induced depression-like behaviors in mice

Our previous studies have reported that pre-stimulation of microglia before stress stimulation is a possible strategy to prevent depression-like phenotypes; however, the molecular mechanisms underlying this effect are still unclear. Here, we used β-glucan, a polysaccharide from Saccharomyces cerevisiae with immunomodulatory activities that cannot elicit pro-inflammatory responses in microglia, to address this issue. Our results showed that a single injection of β-glucan one day before stress exposure dose-dependently prevented the depression-like behaviors triggered by chronic unpredictable stress (CUS), which peaked at 20 mg/kg and prevented the impairment of hippocampal brain-derived neurotrophic factor (BDNF) signaling, a pathological process critical for the progression of depression-like phenotypes. Inhibition of BDNF signaling by infusion of an anti-BDNF antibody into the hippocampus, knock-in of the mutant BDNF Val68Met allele, or blockade of the BDNF receptor in the hippocampus abolished the preventive effect of β-glucan on CUS-induced depression-like behaviors. Further analysis showed that cAMP-response element binding protein (CREB)-mediated increase of BDNF expression in the hippocampus was essential for the prevention of depression-like phenotypes by β-glucan. Pretreatment with minocycline or PLX3397 before β-glucan injection to suppress microglia abolished the preventive effect of β-glucan on impaired CREB-BDNF signaling in the hippocampus and depression-like behaviors in CUS mice. These results suggest that an increase in hippocampal BDNF following CREB activation triggered by β-glucan-induced microglia stimulation and subsequent TrkB signaling mediates the preventive effect of β-glucan on depression. β-Glucan may be a more suitable immunostimulant for the prevention of depression due to its inability to promote pro-inflammatory responses in microglia.

Abstract WP312: Microglial Tmem119 Inhibits IRAK4 Activation and Confers Neuroprotection After Ischemic Stroke

Introduction: Ischemic microglia trigger post-stroke inflammation leading to secondary neuronal death. Microglial responses are regulated through membrane protein signals that in turn activate inflammatory transcription factors. Transmembrane 119 (Tmem119) is a type 1A membrane protein that is exclusively expressed by microglia. It has been suggested that Tmem119 interferes with myeloid differentiation primary response 88 (MyD88)-interleukin-1 receptor-associated kinase 4 (IRAK4) axis that is involved in microglial activation. However, the exact role of Tmem119 in microglial activation has been elusive. Hypothesis: Tmem119 inhibits IRAK4 activation to suppress microglial pro-inflammatory responses and is protective against ischemic injury. Methods: Young (6-8-week-old) C57BL/6 WT mice were injected stereotaxically Lenti-Tmem119 to induce overexpression in microglia. Six weeks later, the mice were subjected to a 60-min middle cerebral artery occlusion (MCAO) model. Oxygen-glucose deprivation (OGD) was also performed in primary microglia cultures treated with Lenti-Tmem119 or Tmem119 siRNA. Western blotting was performed on brain homogenates or microglia cells respectively for expression of p-IRAK4. ELISA was conducted to measure cytokine levels in the plasma and cell culture supernatant. Results: p-IRAK4 protein levels were significantly lower in ischemic brains from Lenti-Tmem119 vs. Lenti-GFP injected mice (p=0.0035) at 3d after MCAO (n=4-6), accompanied by smaller infarct size in Lenti-Tmem119 injected mice. Cultured microglia treated with Lenti-Tmem119 or Tmem119 siRNA had decreased and increased expression of p-IRAK4 respectively than controls after OGD (p=0.0090 and p=0.0152 respectively, n=3-4). Pro-inflammatory cytokine (TNF-α, IL-1β, IL-6) levels were significantly lower in the plasma of Lenti-Tmem119 treated mice compared to controls. Levels of these cytokines were decreased or increased in microglia culture medium treated with Lenti-Tmem119 or Tmem119 siRNA respectively than in control treated medium. Conclusion: Tmem119 signaling regulates microglial inflammatory responses through interaction with IRAK4 and is beneficial to stroke. Keywords: Stroke; microglia; Tmem119; IRAK4; inflammation

The S1 subunits of SARS-CoV-2 variants differentially trigger the IL-6 signaling pathway in human brain endothelial cells and downstream impact on microglia activation.

Cerebrovascular complications are prevalent in COVID-19 infection and post-COVID conditions; therefore, interactions of SARS-CoV-2 with cerebral microvascular cells became an emerging concern. We examined the inflammatory responses of human brain microvascular endothelial cells (HBMEC), the main structural element of the blood-brain barrier (BBB), following exposure to the S1 subunit of the spike protein of different SARS-CoV-2 variants. Specifically, we used the S1 subunit derived from the D614 variant of SARS-CoV-2, which started widely circulating in March of 2020, and from the Delta variant, which started widely circulating in early 2021. We then further examined the impact of the HBMEC secretome, produced in response to the S1 exposure, on microglial proinflammatory responses. Treatment with S1 derived from the D614 variant and from the Delta variant resulted in differential alterations of the IL-6 signaling pathway. Moreover, the HBMEC secretome obtained after exposure to the S1 subunit of the D614 variant activated STAT3 in microglial cells, indicating that proinflammatory signals from endothelial cells can propagate to other cells of the neurovascular unit. Overall, these results indicate the potential for different SARS-CoV-2 variants to induce unique cellular signatures and warrant individualized treatment strategies. The findings from this study also bring further awareness to proinflammatory responses involving brain microvasculature in COVID-19 and demonstrate how the surrounding microglia react to each unique variant derived response.

Nanotechnology for microglial targeting and inhibition of neuroinflammation underlying Alzheimer’s pathology

BackgroundAlzheimer's disease (AD) is considered to have a multifactorial etiology. The hallmark of AD is progressive neurodegeneration, which is characterized by the deepening loss of memory and a high mortality rate in the elderly. The neurodegeneration in AD is believed to be exacerbated following the intercoupled cascades of extracellular amyloid beta (Aβ) plaques, uncontrolled microglial activation, and neuroinflammation. Current therapies for AD are mostly designed to target the symptoms, with limited ability to address the mechanistic triggers for the disease. In this study, we report a novel nanotechnology based on microglial scavenger receptor (SR)-targeting amphiphilic nanoparticles (NPs) for the convergent alleviation of fibril Aβ (fAβ) burden, microglial modulation, and neuroprotection.MethodsWe designed a nanotechnology approach to regulate the SR-mediated intracellular fAβ trafficking within microglia. We synthesized SR-targeting sugar-based amphiphilic macromolecules (AM) and used them as a bioactive shell to fabricate serum-stable AM–NPs via flash nanoprecipitation. Using electron microscopy, in vitro approaches, ELISA, and confocal microscopy, we investigated the effect of AM–NPs on Aβ fibrilization, fAβ-mediated microglial inflammation, and neurotoxicity in BV2 microglia and SH-SY5Y neuroblastoma cell lines.ResultsAM–NPs interrupted Aβ fibrilization, attenuated fAβ microglial internalization via targeting the fAβ-specific SRs, arrested the fAβ-mediated microglial activation and pro-inflammatory response, and accelerated lysosomal degradation of intracellular fAβ. Moreover, AM–NPs counteracted the microglial-mediated neurotoxicity after exposure to fAβ.ConclusionsThe AM–NP nanotechnology presents a multifactorial strategy to target pathological Aβ aggregation and arrest the fAβ-mediated pathological progression in microglia and neurons.Graphical

Regulation of microglia-mediated inflammation by host lncRNA Gm20559 upon flaviviral infection

BackgroundJapanese Encephalitis Virus (JEV) and West Nile Viruses (WNV) are neurotropic flaviviruses which cause neuronal death and exaggerated glial activation in the central nervous system. Role of host long non coding RNAs in shaping microglial inflammation upon flavivirus infections has been unexplored. This study attempted to decipher the role of lncRNA Gm20559 in regulating microglial inflammatory response in context of flaviviruses. MethodsAntisense oligonucleotide LNA Gapmers designed against lncRNA Gm20559 and non-specific site (negative control) were used for Gm20559 knockdown in JEV and WNV-infected N9 microglial cells. Upon establishing successful Gm20559 knockdown, expression of various proinflammatory cytokines, chemokines, interferon-stimulated genes (ISGs) and RIG-I were checked by qRT-PCR and cytometric bead array. Western Blotting was done to analyse the phosphorylation level of various inflammatory markers and viral non-structural protein expression. Plaque Assays were employed to quantify viral titres in microglial supernatant upon knocking down Gm20559. Effect of microglial supernatant on HT22 neuronal cells was assessed by checking expression of apoptotic protein and viral non-structural protein by Western Blotting. ResultsUpregulation in Gm20559 expression was observed in BALB/c pup brains, primary microglia as well as N9 microglia cell line upon both JEV and WNV infection. Knockdown of Gm20559 in JEV and WNV-infected N9 cell led to the reduction of major proinflammatory cytokines – IL-1β, IL-6, IP-10 and IFN-β. Inhibition of Gm20559 upon JEV infection in N9 microglia also led to downregulation of RIG-I and OAS-2, which was not the case in WNV-infected N9 microglia. Phosphorylation level of P38 MAPK was reduced in case of JEV-infected N9 microglia and not WNV-infected N9 microglia. Whereas phosphorylation of NF-κB pathway was unchanged upon Gm20559 knockdown in both JEV and WNV-infected N9 microglia. However, treating HT22 cells with JEV and WNV-infected microglial supernatant with and without Gm20559 could not trigger cell death or influence viral replication. ConclusionKnockdown studies on lncRNA Gm20559 suggests its pivotal role in maintaining the inflammatory milieu of microglia in flaviviral infection by modulating the expression of various pro-inflammatory cytokines. However, Gm20559-induced increased microglial proinflammatory response upon flavivirus infection fails to trigger neuronal death.

Central IRF4/5 Signaling Are Critical for Microglial Activation and Impact on Stroke Outcomes.

Microglia and monocytes play a critical role in immune responses to cerebral ischemia. Previous studies have demonstrated that interferon regulatory factor 4 (IRF4) and IRF5 direct microglial polarization after stroke and impact outcomes. However, IRF4/5 are expressed by both microglia and monocytes, and it is not clear if it is the microglial (central) or monocytic (peripheral) IRF4-IRF5 regulatory axis that functions in stroke. In this work, young (8-12weeks) male pep boy (PB), IRF4 or IRF5 flox, and IRF4 or IRF5 conditional knockout (CKO) mice were used to generate 8 types of bone marrow chimeras, to differentiate the role of central (PB-to-IRF CKO) vs. peripheral (IRF CKO-to-PB) phagocytic IRF4-IRF5 axis in stroke. Chimeras generated from PB and flox mice were used as controls. All chimeras were subjected to 60-min middle cerebral artery occlusion (MCAO) model. Three days after the stroke, outcomes and inflammatory responses were analyzed. We found that PB-to-IRF4 CKO chimeras had more robust microglial pro-inflammatory responses than IRF4 CKO-to-PB chimeras, while ameliorated microglial response was seen in PB-to-IRF5 CKO vs. IRF5 CKO-to-PB chimeras. PB-to-IRF4 or IRF5 CKO chimeras had worse or better stroke outcomes respectively than their controls, whereas IRF4 or 5 CKO-to-PB chimeras had similar outcomes compared to controls. We conclude that the central IRF4/5 signaling is responsible for microglial activation and mediates stroke outcomes.

Stimulation of Angiotensin II Type 2 Receptor Modulates Pro-Inflammatory Response in Microglia and Macrophages: Therapeutic Implications for the Treatment of Stroke.

Sustained microglial activation contributes to the development of post-stroke cognitive impairment (PSCI). Compound 21 (C21), an angiotensin II type 2 receptor agonist, has shown some neurovascular protection after stroke. This study aimed to investigate the direct anti-inflammatory effects of C21 on macrophages, as well as brain innate immune cells. Murine microglial cell line (C8-B4) and RAW 264.7 macrophages were exposed to lipopolysaccharide (LPS) and co-treated with C21. Pro-inflammatory mediators were assessed via RT-qPCR and ELISA. Cellular reactive oxygen species (ROS) were evaluated via CellROXGreen staining, and nitrate production was assessed using Griess assay. C21 suppressed LPS-induced inflammation and ROS generation in both cells. In microglia, C21 blunted LPS-induced mRNA expression of IL-1β, IL-12b, COX-1, iNOS, and IL-6. A similar pattern was observed in macrophages, where C21 suppressed LPS-induced IL-1β, TNF-α, and CXCL1 expression. These anti-inflammatory effects in microglia and macrophages were associated with increased neuroprotective gene expression, including GDNF and BDNF, in a dose-dependent manner. Our findings suggest a protective effect of C21 against the inflammatory response, in both macrophages and microglia, via suppression of the release of pro-inflammatory cytokines/chemokines and the generation of ROS while stimulating the production of neurotrophic factors.

Xiaoyao San ameliorates high-fat diet-induced anxiety and depression via regulating gut microbiota in mice

Obesity, a growing health problem in the world, is related to a series of mental disorders, including anxiety and depression. XiaoYao San (XYS), a prescription of traditional Chinese medicine (TCM), has been widely used in the clinical treatment of anxiety and depression in China. However, the efficacy of XYS on obesity-related neuropsychiatric dysfunction and the underlying neural mechanisms remain unclear. Here, using a high-fat diet (HFD)-induced obese model, we found that XYS treatment significantly improves obesity-related anxiety- and depression-like behaviors and alters the gut microbiome, particularly by increasing the relative abundance of Faecalibaculum rodentium (F. rodentium), in mice. Interestingly, selective supplementation with F. rodentium or its metabolic products, short-chain fatty acids (SCFAs), is sufficient to rescue anxiety- and depression-like behaviors in HFD-fed mice. Next, we determined that the transcriptional level of dopamine D2 receptor (DRD2), which activation usually inhibits inflammation in the central nervous system (CNS), is significantly increased in the medial prefrontal cortex (mPFC) of XYS-treated mice when compared with that of vehicle-treated controls. Moreover, enriched pathways analysis with the differential expression genes (DEGs) showed that some of these DEGs are enriched in neuroinflammatory pathways. We further noticed that treatment with XYS contributes to controlling microglial activation and proinflammatory responses in the mPFC and hippocampus of HFD-fed mice. Overall, this study reveals that XYS rescues HFD-induced anxiety and depression via modulating gut microbiota-derived metabolites and that XYS is a potential therapeutic strategy for treating obesity-associated mental disorders.

P2Y6 receptor-mediated signaling amplifies TLR-induced pro-inflammatory responses in microglia.

TLR-induced signaling initiates inflammatory responses in cells of the innate immune system. These responses are amongst others characterized by the secretion of high levels of pro-inflammatory cytokines, which are tightly regulated and adapted to the microenvironment. Purinergic receptors are powerful modulators of TLR-induced responses, and we here characterized the effects of P2Y6 receptor (P2RY6)-mediated signaling on TLR responses of rhesus macaque primary bone marrow-derived macrophages (BMDM) and microglia, using the selective P2RY6 antagonist MRS2578. We demonstrate that P2RY6-mediated signaling enhances the levels of TLR-induced pro-inflammatory cytokines in microglia in particular. TLR1, 2, 4, 5 and 8-induced responses were all enhanced in microglia, whereas such effects were much less pronounced in BMDM from the same donors. Transcriptome analysis revealed that the overall contribution of P2RY6-mediated signaling to TLR-induced responses in microglia leads to an amplification of pro-inflammatory responses. Detailed target gene analysis predicts that P2RY6-mediated signaling regulates the expression of these genes via modulation of the activity of transcription factors NFAT, IRF and NF-κB. Interestingly, we found that the expression levels of heat shock proteins were strongly induced by inhibition of P2RY6-mediated signaling, both under homeostatic conditions as well as after TLR engagement. Together, our results shed new lights on the specific pro-inflammatory contribution of P2RY6-mediated signaling in neuroinflammation, which might open novel avenues to control brain inflammatory responses.

Abstract 84: The Role of IRF5-IRF4 Regulatory Axis in Stroke in Aged Mice: A Translational Study

Introduction: Inflammatory responses are a fundamental pathological consequence of stroke, in which microglial activation plays a central role. Our previous study has found interferon regulatory factor 5 (IRF5) and 4 (IRF4) regulate microglial activation in young stroke mice. However, whether the IRF5-IRF4 regulatory axis has the same effect in aged brains is not known. Numerous neurochemical changes occur with aging, and stroke mainly affects the elderly. Therefore, to study the IRF5-IRF4 axis with aged animals is clinically more relevant and translational. In this study, we exclusively used aged mice (18-20 months) to examine the role of IRF5-IRF4 axis in stroke. Methods: Aged, microglial IRF5 or IRF4 conditional knockout (CKO) male mice (with LysM Cre ) were subjected to a 60-min middle cerebral artery occlusion (MCAO) (n=6/gp for stroke and =4/gp for sham). Infarct volumes and neurological deficits were quantified at 3d after MCAO. Flow cytometry was performed to evaluate microglial activation and peripheral leukocyte infiltration in the ischemic brain. ELISA was conducted to determine the plasma cytokine levels. Results: IRF5 CKO mice had significantly smaller cortical infarct size than the flox controls (48.07±5.51% vs. 66.9±5.51%, p=0.0206); larger infarct volumes were seen in IRF4 CKO vs. flox controls in the cortex and hemisphere (Cortex: 58.93±5.16% vs. 38.38±5.16%, p= 0.0049; Hemisphere: 68.73±5.16% vs. 44.63±5.16%, p=0.0008). Significantly improvement on the corner test and neurological deficit score was seen in IRF5 CKO vs. flox mice, and an opposite pattern showed in IRF4 CKO vs. flox controls. Flow cytometry showed IRF5 CKO microglia had significantly lower levels of IL-1β and CD68 than controls; whereas significantly higher levels of IL-1β and TNF-α were seen in IRF4 CKO vs. control microglia. Plasma levels of IL-6 and IL-12 p 40 were decreased in IRF5 CKO vs. flox mice, and the anti-inflammatory cytokines (IL-4/IL-10) levels were higher in IRF5 CKO, and lower in IRF4 CKO mice vs. their flox controls respectively. Conclusion: IRF5 signaling drives microglial pro-inflammatory responses and is detrimental to stroke; whereas IRF4 signaling leads to a microglial anti-inflammatory response and protects against ischemia in aged mice.

Dopamine D3 receptor in the nucleus accumbens alleviates neuroinflammation in a mouse model of depressive-like behavior

We recently reported that dopamine D3 receptor (D3R) was involved in inflammation-related depression. Nucleus accumbens (NAc) inflammation is implicated in the development and progression of depression, but its regulatory mechanism remains largely unknown. In a mouse model of NAc neuroinflammation induced by bilateral NAc injection of lipopolysaccharide (LPS), we observed that NAc neuroinflammation triggered depressive-like behaviors, and D3R expression decline and microglial activation in the NAc. A selective knockdown of D3R in the NAc elicited depressive-like behaviors, while re-expression of D3R in the NAc of global D3RKO mice alleviated depressive-like behaviors induced by D3R deficiency. D3R downregulation in the NAc shifted microglia toward a proinflammatory state, which was validated with cultured mouse microglial cultures. Further in vitro results demonstrated that D3R inhibition induced microglia to enter a proinflammatory state primarily through the Akt signaling pathway. In conclusion, our results suggest that D3R expression in the NAc may inhibit microglial proinflammatory responses in the NAc, thus alleviating NAc neuroinflammation and subsequent depressive-like behaviors through the Akt signaling pathway.

MiR-124 Prevents the Microglial Proinflammatory Response by Inhibiting the Activities of TLR4 and Downstream NLRP3 in Palmitic Acid-Treated BV2 Cells.

Neuroinflammation is a mechanism by which obesity or a high-fat diet leads to cognitive impairment. MiR-124, a highly expressed microRNA in the brain, can alleviate neuroinflammation by regulating microglial activation, but its mechanism is unclear. The aim of the study was to explore whether miR-124 exerted this effect through TLR4/MyD88/NF-κB p65/NLRP3 signaling in palmitic acid-treated BV2 cells. Prepared BV2 cells were treated with palmitic acid to establish an in vitro model of a high-fat diet. An miR-124 mimic and inhibitor were adopted to upregulate and downregulate the expression of miR-124, respectively. TAK-242 and NLRP3 siRNA were used to downregulate the expression of TLR4 and NLRP3. The expression levels of miR-124, signaling proteins (TLR4, MyD88, and NF-κB p65), inflammasome markers (NLRP3 and IL-1β), and microglial activated markers (CD206, Arg-1, CD86, and iNOS) were measured by qPCR and western blotting. The pyroptosis rate was assessed using flow cytometry. First, palmitic acid upregulated TLR4/MyD88/NF-κB p65 signaling, increased NLRP3 expression, elevated the pyroptosis rate, and promoted the microglial proinflammatory response in BV2 cells. Second, the miR-124 mimic and inhibitor separately alleviated and aggravated the effect of palmitic acid on microglial activation and NLRP3 expression. The miR-124 mimic also downregulated TLR4/MyD88/NF-κB p65 signaling. Third, TAK-242 did not affect the expression of miR-124 but simulated the protective effect of the miR-124 mimic on microglial activation and NLRP3 expression. Fourth, NLRP3 siRNA also inhibited the microglial proinflammatory response in BV2 cells. MiR-124 prevented the microglial proinflammatory response through TLR4/MyD88/NF-κB p65/NLRP3 signaling in palmitic acid-treated BV2 cells.