Gut microbiota regulate stress resistance by influencing microglia-neuron interactions in the hippocampus.

Communication among the brain, gut and microbiota in the gut is known to affect the susceptibility to stress, but the mechanisms involved are unclear. Here we demonstrated that stress resistance in mice was associated with more abundant Lactobacillus and Akkermansia in the gut, but less abundant Bacteroides, Alloprevotella, Helicobacter, Lachnoclostridium, Blautia, Roseburia, Colidextibacter and Lachnospiraceae NK4A136. Stress-sensitive animals showed higher permeability and stronger immune responses in their colon, as well as higher levels of pro-inflammatory cytokines in serum. Their hippocampus also showed more extensive microglial activation, abnormal interactions between microglia and neurons, and lower synaptic plasticity. Transplanting fecal microbiota from stress-sensitive mice into naïve ones perturbed microglia-neuron interactions and impaired synaptic plasticity in the hippocampus, translating to more depression-like behavior after stress exposure. Conversely, transplanting fecal microbiota from stress-resistant mice into naïve ones protected microglia from activation and preserved synaptic plasticity in the hippocampus, leading to less depression-like behavior after stress exposure. These results suggested that gut microbiota may influence resilience to chronic psychological stress by regulating microglia-neuron interactions in the hippocampus.

The secretome of microglia induced by IL-4 of IFN-γ differently regulate proliferation, differentiation and survival of adult neural stem/progenitor cell by targeting the PI3K-Akt pathway.

Microglia has been reported to be able to regulate the proliferation, differentiation and survival of adult neural stem/progenitor cells (NSPCs) by modulating the microenvironment, which results in different consequences of adult neurogenesis. However, whether the microglial activation is beneficial or harmful to NSPCs is still controversial because of the complexity and variability of microglial activation phenotypes. In this study, we systematically explored the activation phenotypes of IFN-γ- or IL-4-induced microglia at different time after stimulation, and investigated the effects of the secretome of different phenotype of microglia on the process of proliferation, differentiation and survival of NSPCs. Moreover, the possible molecular pathways of secretory influence on NSPCS were further explored using western blotting. The result showed that IFN-γ and IL-4 differently regulate microglial phenotypes, IL-4 induced a M2-like phenotype, while IFN-γ induced a M1-like phenotype. These phenotypes of microglia can only be maintained for 24 h after removal of IFN-γ or IL-4 intervention. The secretome from IFN-γ- or IL-4-induced microglia also had opposite effects on NSPCs proliferation, differentiation and survival. The secretome from the IL-4-treated microglia promoted NSPCs proliferation, survival and differentiation into neurons and oligodendrocytes, while factors secreted by the INF-γ-treated microglia stimulated the NSPCs differentiation into astrocyte, inhibited the neurogenesis and oligodendrogliogenesis, and induced NSPCs apoptosis. Furthermore, the PI3K-Akt pathway mediates the effects of the secretome from IFN-γ- or IL-4-induced microglia on NSPC proliferation, differentiation, and survival. In conclusion, our results suggested that the secretome of microglia induced by IL-4 of IFN-γ differently regulate proliferation, differentiation and survival of adult neural stem/progenitor cell by targeting the PI3K-Akt pathway. These findings will help further study the biological mechanism of microglia regulating neurogenesis, and provide a therapeutic strategy for neurological diseases by regulating microglial phenotypes to affect neurogenesis.

Asperosaponin VI ameliorates the CMS-induced depressive-like behaviors by inducing a neuroprotective microglial phenotype in hippocampus via PPAR-γ pathway.

BACKGROUND: The natural compound asperosaponin VI has shown potential as an antidepressant, but how it works is unclear. Here, we explored its effects on mice exposed to chronic mild stress (CMS) and the underlying molecular pathways. METHODS: Mice were exposed to CMS for 3 weeks followed by asperosaponin VI (40 mg/kg) or imipramine (20 mg/kg) for another 3 weeks. Depression-like behaviors were assessed in the forced swimming test (FST), sucrose preference test (SPT), tail suspension test (TST). Microglial phenotypes were evaluated using immunofluorescence staining, real-time quantitative PCR and enzyme-linked immunosorbent assays in hippocampus of mice. In some experiments, stressed animals were treated with the PPAR-γ antagonist GW9662 to examine its involvement in the effects of asperosaponin VI. Blockade of PPAR-γ in asperosaponin VI-treated primary microglia in the presence of lipopolysaccharide (LPS) was executed synchronously. The nuclear transfer of PPAR-γ in microglia was detected by immunofluorescence staining in vitro and in vivo. A co-cultured model of neuron and microglia was used for evaluating the regulation of ASA VI on the microglia-neuron crosstalk molecules. RESULTS: Asperosaponin VI ameliorated depression-like behaviors of CMS mice based on SPT, TST and FST, and this was associated with a switch of hippocampal microglia from a pro-inflammatory (iNOS+-Iba1+) to neuroprotective (Arg-1+-Iba1+) phenotype. CMS reduced the expression levels of PPAR-γ and phosphorylated PPAR-γ in hippocampus, which asperosaponin VI partially reversed. GW9662 treatment prevented the nuclear transfer of PPAR-γ in asperosaponin VI-treated microglia and inhibited the induction of Arg-1+ microglia. Blockade of PPAR-γ signaling also abolished the ability of asperosaponin VI to suppress pro-inflammatory cytokines while elevating anti-inflammatory cytokines in the hippocampus of CMS mice. The asperosaponin VI also promoted interactions between hippocampal microglia and neurons by enhancing CX3CL1/CX3CR1 and CD200/CD200R, and preserved synaptic function based on PSD95, CamKII ß and GluA levels, but not in the presence of GW9662. Blockade of PPAR-γ signaling also abolished the antidepressant effects of asperosaponin VI in the SPT, TST and FST. CONCLUSION: CMS in mice induces a pro-inflammatory microglial phenotype that causes reduced crosstalk between microglia and neuron, inflammation and synaptic dysfunction in the hippocampus, ultimately leading to depression-like behaviors. Asperosaponin VI may ameliorate the effects of CMS by inducing microglia to adopt a PPAR-γ-dependent neuroprotective phenotype.

Mapping the Plasticity of Morphology, Molecular Properties and Function in Mouse Primary Microglia.

Microglia exert diverse functions by responding in diverse ways to different stimuli, yet little is known about the plasticity of various phenotypes that microglia display. We used interferon (IFN)-γ, interleukin (IL)-4 and IL-10 to induce different phenotypes in mouse primary microglia. RNA sequencing was used to identify genes differentially expressed in response to stimulation, and the different stimulated populations were compared in terms of morphology, proliferative capacity, phagocytic ability and neurotoxicity. IFN-γ induced an "immunodefensive" phenotype characterizing both induction of filopodia and upregulation of inducible nitric oxide synthase (iNOS) and tumor necrosis factor α. Microglia with this phenotype mediated an acute inflammatory response accompanied by excellent proliferative capacity and neurotoxicity, and remained susceptible to remodeling for up to 48 h after initial stimulation. IL-4 induced an enduring "neuroimmunoregulatory" phenotype involving induction of lamellipodium and persistent upregulation of arginase (Arg)-1 and YM-1 expression. Microglia with this phenotype remained susceptible to remodeling for up to 24 h after initial stimulation. IL-10 induced an "immunosuppressive" phenotype involving induction of ameba-like morphology and upregulation of transforming growth factor ß and IL-10 as well as inhibition of inflammation. This phenotype was accompanied by inhibition of self-proliferation, while its morphology, molecular properties and function were the least susceptible to remodeling. IFN-γ, IL-4, or IL-10 appear to induce substantially different phenotypes in microglia. The immunodefensive microglia induced by IFN-γ showed remarkable plasticity, which may help repair CNS inflammation damage under pathological condition. Chronic activation with IL-10 decreases microglial plasticity, which may help protect the brain form the immune response. Our research justifies and guides further studies into the molecular pathways that operate in each phenotype to help multitasking microglia regulate homeostasis in the brain.

Asperosaponin VI inhibits LPS-induced inflammatory response by activating PPAR-γ pathway in primary microglia.

Microglia cells are the main mediators of neuroinflammation. Activation of microglia often aggravates the pathological process of various neurological diseases. Natural chemicals have unique advantages in inhibiting microglia-mediated neuroinflammation and improving neuronal function. Here, we examined the effects of asperosaponin VI (ASA VI) on LPS-activated primary microglia. Microglia were isolated from mice and pretreated with different doses of ASA VI, following lipopolysaccharide (LPS) administration. Activation and inflammatory response of microglia cells were evaluated by real-time fluorescence quantitative polymerase chain reaction (q-PCR), immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). Signaling pathways were detected by western blotting. We found that the ASA VI inhibited the morphological expansion of microglia cells, decreased the expression and release of proinflammatory cytokines, and promoted the expression of antiinflammatory cytokines in a dose-dependent manner. ASA VI also activated PPAR-γ signaling pathway in LPS-treated microglia. The anti-inflammatory effects of ASA VI in microglia were blocked by treating PPAR-γ antagonist (GW9662). These results showed that ASA VI promote the transition of microglia cells from proinflammatory to anti-inflammatory by regulating PPAR-γ pathway.

Mouse Astrocytes Promote Microglial Ramification by Releasing TGF-ß and Forming Glial Fibers.

The morphology of microglial cells is often closely related to their functions. The mechanisms that regulate microglial ramification are not well understood. Here we reveal the biological mechanisms by which astrocytes regulate microglial ramification. Morphological variation in mouse microglial cultures was measured in terms of cell area as well as branch number and length. Effects on microglial ramification were analyzed after microinjecting the toxin L-alpha-aminoadipic acid (L-AAA) in the mouse cortex or hippocampus to ablate astrocytes, and after culturing microglia on their own in an astrocyte-conditioned medium (ACM) or together with astrocytes in coculture. TGF-ß expression was determined by Western blotting, immunohistochemistry, and ELISA. The TGF-ß signaling pathway was blocked by the TGF-ß antibody to assess the role of TGF-ß on microglial ramification. The results showed that microglia had more and longer branches and smaller cell bodies in brain areas where astrocytes were abundant. In the mouse cortex and hippocampus, ablation of astrocytes by L-AAA decreased number and length of microglial branches and increased the size of cell bodies. Similar results were obtained with isolated microglia in culture. However, isolated microglia were able to maintain their multibranched structure for a long time when cultured on astrocyte monolayers. Ameboid microglia isolated from P0 to P3 mice showed increased ramification when cultured in ACM or on astrocyte monolayers. Microglia cultured on astrocyte monolayers showed more complex branching structures than those cultured in ACM. Blocking astrocyte-derived TGF-ß decreased microglial ramification. Astrocytes induced the formation of protuberances on branches of microglia by forming glial fibers that increased traction. These experiments in mice suggest that astrocytes promote microglial ramification by forming glial fibers to create traction and by secreting soluble factors into the surroundings. For example, astrocyte-secreted TGF-ß promotes microglia to generate primitive branches, whose ramification is refined by glial fibers.

IL-4 and IL-10 promotes phagocytic activity of microglia by up-regulation of TREM2.

Triggering receptor expressed on myeloid cells-2 (TREM2) is an innate immune receptor that promotes phagocytosis by microglia. However, whether TREM2 is related to the stimulus-dependent phagocytic activity of microglia is unclear. In this study, the primary cultured microglia were stimulated with interferon (IFN)-γ, interleukin (IL)-4, and interleukin (IL)-10, respectively, and their phagocytic activity against microbeads and apoptotic neural stem cells (NSCs) was measured. TREM2 of microglia was detected by qPCR and western blotting. The TREM2 signal was blocked in microglia using the siRNA technique. The results showed that IL-4 or IL-10 treatment significantly increased the number of microglia gathered around the apoptotic neurosphere. IL-4 and IL-10 treatment also promoted phagocytosis of microbeads and apoptotic NSCs by primary cultured microglia. The TREM2 expression was up-regulated in IL-4- or IL-10- treated microglia. TREM2 siRNA treatment blocked the phagocytic activity of IL-4- or IL-10-treated microglia. In conclusion, these results indicated that IL-4 and IL-10 promote the phagocytic activity of microglia by the up-regulation of TREM2, which suggested a new potential therapeutic target for neurodegenerative disease.

Priming of microglia with IFN-γ impairs adult hippocampal neurogenesis and leads to depression-like behaviors and cognitive defects.

Neuroinflammation driven by interferon-gamma (IFN-γ) and microglial activation has been linked to neurological disease. However, the effects of IFN-γ-activated microglia on hippocampal neurogenesis and behavior are unclear. In the present study, IFN-γ was administered to mice via intracerebroventricular injection. Mice received intraperitoneal injection of ruxolitinib to inhibit the JAK/STAT1 pathway or injection of minocycline to inhibit microglial activation. During a 7-day period, mice were assessed for depressive-like behaviors and cognitive impairment based on a series of behavioral analyses. Effects of the activated microglia on neural stem/precursor cells (NSPCs) were examined, as was pro-inflammatory cytokine expression by activated microglia. We showed that IFN-γ-injected animals showed long-term adult hippocampal neurogenesis reduction, behavior despair, anhedonia, and cognitive impairment. Chronic activation with IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, MHC II and CD68 up-regulation, and pro-inflammatory cytokine (IL-1ß, TNF-α, IL-6) and nitric oxide (NO) release. Microglia isolated from the hippocampus of IFN-γ-injected mice suppressed NSPCs proliferation and stimulated apoptosis of immature neurons. Inhibiting of the JAK/STAT1 pathway in IFN-γ-injected animals to block microglial activation suppressed microglia-mediated neuroinflammation and neurogenic injury, and alleviated depressive-like behaviors and cognitive impairment. Collectively, these findings suggested that priming of microglia with IFN-γ impairs adult hippocampal neurogenesis and leads to depression-like behaviors and cognitive defects. Targeting microglia by modulating levels of IFN-γ the brain may be a therapeutic strategy for neurodegenerative diseases and psychiatric disorders.

The antidepressant effects of asperosaponin VI are mediated by the suppression of microglial activation and reduction of TLR4/NF-κB-induced IDO expression.

AIM: Indoleamine 2,3-dioxygenase 1 (IDO) is responsible for the progression of the kynurenine pathway, which has been implicated in the pathophysiology of inflammation-induced depression. It has been reported that asperosaponin VI (ASA VI) could play a neuroprotective role through anti-inflammatory and antioxidant. In this study, we examined the antidepressant effect of ASA VI in lipopolysaccharide (LPS)-treated mice and further explored its molecular mechanism by looking into the microglial kynurenine pathway. METHODS: To generate the model, LPS (0.83 mg/kg) was administered intraperitoneally to mice. The mice received ASA VI (10 mg/kg, 20 mg/kg, 40 mg/kg, and 80 mg/kg, i.p.) 30 min before LPS injection. Depressive-like behaviors were evaluated based on the duration of immobility in the forced swim test. Microglial activation and inflammatory cytokines were detected by immunohistochemistry, real-time PCR, and ELISA. The TLR4/NF-κB signaling pathway and the expression of IDO, GluA2, and CamKIIß were also measured by western blotting. RESULTS: ASA VI exhibited significant antidepressant activity in the presence of LPS on immobility and latency times in the forced swim test. The LPS-induced activation of microglia and inflammatory response were inhibited by ASA VI, which showed a dose-dependent pattern. TLR4/NF-κB signaling pathway also was suppressed by ASA VI in the hippocampus and prefrontal cortex of LPS-treated mice. Furthermore, ASA VI inhibited the increase in IDO protein expression and normalized the aberrant glutamate transmission in the hippocampus and prefrontal cortex caused by LPS administration. CONCLUSION: Our results propose a promising antidepressant effect for ASA VI possibly through the downregulation of IDO expression and normalization of the aberrant glutamate transmission. This remedying effect of ASA VI could be attributed to suppress microglia-mediated neuroinflammatory response via inhibiting the TLR4/NF-κB signaling pathway.