Inhibition of Microglial TGF? Signaling Increases Expression of Mrc1.
ABSTRACT: Microglia are constantly surveying their microenvironment and rapidly react to impairments by changing their morphology, migrating toward stimuli and adopting gene expression profiles characterizing their activated state. The increased expression of the M2-like marker Mannose receptor 1 (Mrc1), which is also referred to as CD206, in microglia has been reported after M2-like activation in vitro and in vivo. Mrc1 is a 175-kDa transmembrane pattern recognition receptor which binds a variety of carbohydrates and is involved in the pinocytosis and the phagocytosis of immune cells, including microglia, and thought to contribute to a neuroprotective microglial phenotype. Here we analyzed the effects of TGF? signaling on Mrc1 expression in microglia in vivo and in vitro. Using C57BL/6 wild type and Cx3cr1 CreERT2 :R26-YFP:Tgfbr2 fl/fl mice-derived microglia, we show that the silencing of TGF? signaling results in the upregulation of Mrc1, whereas recombinant TGF?1 induced the delayed downregulation of Mrc1. Furthermore, chromatin immunoprecipitation experiments provided evidence that Mrc1 is not a direct Smad2/Smad4 target gene in microglia. Altogether our data indicate that the changes in Mrc1 expression after the activation or the silencing of microglial TGF? signaling are likely to be mediated by modifications of the secondary intracellular signaling events influenced by TGF? signaling.
Project description:Brain disturbances, like injuries or aberrant protein deposits, evoke nucleotide release or leakage from cells, leading to microglial chemotaxis and ingestion. Recent studies have identified P2Y12 purinergic receptors as triggers for microglial chemotaxis and P2Y6 receptors as mediators for phagocytosis. However, pinocytosis, known as the internalization of fluid-phase materials, has received much less attention. We found that ATP efficiently triggered pinocytosis in microglia. Pharmacological analysis and knockdown experiments demonstrated the involvement of P2Y4 receptors and the phosphatidylinositol 3-kinase/Akt cascade in the nucleotide-induced pinocytosis. Further evidence indicated that soluble amyloid beta peptide 1-42 induced self-uptake in microglia through pinocytosis, a process involving activation of P2Y4 receptors by autocrine ATP signaling. Our results demonstrate a previously unknown function of ATP as a "drink me" signal for microglia and P2Y4 receptors as a potential therapeutic target for the treatment of Alzheimer's disease.
Project description:Microglia-mediated neuroinflammation plays a dual role in various brain diseases due to distinct microglial phenotypes, including deleterious M1 and neuroprotective M2. There is growing evidence that the peroxisome proliferator-activated receptor ? (PPAR?) agonist rosiglitazone prevents lipopolysaccharide (LPS)-induced microglial activation. Here, we observed that antagonizing PPAR? promoted LPS-stimulated changes in polarization from the M1 to the M2 phenotype in primary microglia. PPAR? antagonist T0070907 increased the expression of M2 markers, including CD206, IL-4, IGF-1, TGF-?1, TGF-?2, TGF-?3, G-CSF, and GM-CSF, and reduced the expression of M1 markers, such as CD86, Cox-2, iNOS, IL-1?, IL-6, TNF-?, IFN-?, and CCL2, thereby inhibiting NF?B-IKK? activation. Moreover, antagonizing PPAR? promoted microglial autophagy, as indicated by the downregulation of P62 and the upregulation of Beclin1, Atg5, and LC3-II/LC3-I, thereby enhancing the formation of autophagosomes and their degradation by lysosomes in microglia. Furthermore, we found that an increase in LKB1-STRAD-MO25 complex formation enhances autophagy. The LKB1 inhibitor radicicol or knocking down LKB1 prevented autophagy improvement and the M1-to-M2 phenotype shift by T0070907. Simultaneously, we found that knocking down PPAR? in BV2 microglial cells also activated LKB1-AMPK signaling and inhibited NF?B-IKK? activation, which are similar to the effects of antagonizing PPAR?. Taken together, our findings demonstrate that antagonizing PPAR? promotes the M1-to-M2 phenotypic shift in LPS-induced microglia, which might be due to improved autophagy via the activation of the LKB1-AMPK signaling pathway.
Project description:Peripheral nerve injury causes neuropathic pain accompanied by remarkable microgliosis in the spinal cord dorsal horn. However, it is still debated whether infiltrated monocytes contribute to injury-induced expansion of the microglial population. Here, we found that spinal microgliosis predominantly results from local proliferation of resident microglia but not from infiltrating monocytes after spinal nerve transection (SNT) by using two genetic mouse models (CCR2(RFP/+):CX3CR1(GFP/+) and CX3CR1(creER/+):R26(tdTomato/+) mice) as well as specific staining of microglia and macrophages. Pharmacological inhibition of SNT-induced microglial proliferation correlated with attenuated neuropathic pain hypersensitivities. Microglial proliferation is partially controlled by purinergic and fractalkine signaling, as CX3CR1(-/-) and P2Y12(-/-) mice show reduced spinal microglial proliferation and neuropathic pain. These results suggest that local microglial proliferation is the sole source of spinal microgliosis, which represents a potential therapeutic target for neuropathic pain management.
Project description:Tissue microarchitecture and mechanics are important in development and pathologies of the Central Nervous System (CNS); however, their coordinating mechanisms are unclear. Here, we report that during colonization of the retina, microglia contacts the deep layer of high stiffness, which coincides with microglial bipolarization, reduction in TGF?1 signaling and termination of vascular growth. Likewise, stiff substrates induce microglial bipolarization and diminish TGF?1 expression in hydrogels. Both microglial bipolarization in vivo and the responses to stiff substrates in vitro require intracellular adaptor Kindlin3 but not microglial integrins. Lack of Kindlin3 causes high microglial contractility, dysregulation of ERK signaling, excessive TGF?1 expression and abnormally-patterned vasculature with severe malformations in the area of photoreceptors. Both excessive TGF?1 signaling and vascular defects caused by Kindlin3-deficient microglia are rescued by either microglial depletion or microglial knockout of TGF?1 in vivo. This mechanism underlies an interplay between microglia, vascular patterning and tissue mechanics within the CNS.
Project description:Constitutive TGF? signaling is important in maintaining retinal neurons and blood vessels and is a factor contributing to the risk for age-related macular degeneration (AMD), a retinal disease involving neurodegeneration and microglial activation. How TGF? signaling to microglia influences pathological retinal neuroinflammation is unclear. We discovered that ablation of the TGF? receptor, TGFBR2, in retinal microglia of adult mice induced abnormal microglial numbers, distribution, morphology, and activation status, and promoted a pathological microglial gene expression profile. TGFBR2-deficient retinal microglia induced secondary gliotic changes in Müller cells, neuronal apoptosis, and decreased light-evoked retinal function reflecting abnormal synaptic transmission. While retinal vasculature was unaffected, TGFBR2-deficient microglia demonstrated exaggerated responses to laser-induced injury that was associated with increased choroidal neovascularization, a hallmark of advanced exudative AMD. These findings demonstrate that deficiencies in TGF?-mediated microglial regulation can drive neuroinflammatory contributions to AMD-related neurodegeneration and neovascularization, highlighting TGF? signaling as a potential therapeutic target.
Project description:Microglia are myeloid cells of the CNS that participate both in normal CNS function and in disease. We investigated the molecular signature of microglia and identified 239 genes and 8 microRNAs that were uniquely or highly expressed in microglia versus myeloid and other immune cells. Of the 239 genes, 106 were enriched in microglia as compared with astrocytes, oligodendrocytes and neurons. This microglia signature was not observed in microglial lines or in monocytes recruited to the CNS, and was also observed in human microglia. We found that TGF-? was required for the in vitro development of microglia that express the microglial molecular signature characteristic of adult microglia and that microglia were absent in the CNS of TGF-?1-deficient mice. Our results identify a unique microglial signature that is dependent on TGF-? signaling and provide insights into microglial biology and the possibility of targeting microglia for the treatment of CNS disease.
Project description: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-? expression was determined by Western blotting, immunohistochemistry, and ELISA. The TGF-? signaling pathway was blocked by the TGF-? antibody to assess the role of TGF-? 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-? 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-? promotes microglia to generate primitive branches, whose ramification is refined by glial fibers.
Project description:Neuroinflammation is considered to be an important and inevitable pathological process associated with all types of damages to, and disorders of, the central nervous system. The hallmark of neuroinflammation is the microglia activation. In response to different micro-environmental disturbances, microglia could polarize into either an M1 pro-inflammatory phenotype, exacerbating neurotoxicity, or an M2 anti-inflammatory phenotype, exerting neuroprotection. Therefore, shifting the polarization of microglia toward the M2 phenotype could possess a more viable strategy for the neuroinflammatory disorders treatment. Naringenin (NAR) is naturally a grapefruit flavonoid and possesses various kinds of pharmacological activities, such as anti-inflammatory and neuroprotective activities. In the present study, we aimed to investigate the potential effects of NAR on microglial M1/M2 polarization and further reveal the underlying mechanisms of actions. First, NAR inhibited lipopolysaccharide (LPS)-induced microglial activation. Then, NAR shifted the M1 pro-inflammatory microglia phenotype to the M2 anti-inflammatory M2 microglia state as demonstrated by the decreased expression of M1 markers (i.e., inducible TNF-? and IL-1?) and the elevated expression of M2 markers (i.e., arginase 1, IL-4, and IL-10). In addition, the effects of NAR on microglial polarization were dependent on MAPK signaling, particularly JNK inactivation, as evidenced by the fact that the selective activator of JNK abolished NAR-promoted M2 polarization and further NAR-inhibited microglial activation. Together, this study demonstrated that NAR promoted microglia M1/M2 polarization, thus conferring anti-neuroinflammatory effects via the inhibition of MAPK signaling activation. These findings might provide new alternative avenues for neuroinflammation-related disorders treatment.
Project description:Glioma tumors constitute a significant portion of microglial cells, which are known to support tumor progression. The present study demonstrates that transforming growth factor-? (TGF?) signaling pathway in microglia in a glioma environment is involved in tumor progression and pathogenesis. It has been shown that the TGF? level is elevated in higher grades of gliomas and its signaling pathway regulates tumor progression through phosphorylation of SMAD2 and SMAD3, which form a complex with SMAD4 to regulate target gene transcription. In an in vitro cell line-based model increased protein levels of pSMAD2/3, total SMAD2/3 and SMAD4 were observed in murine BV2 microglia cultured in glioma conditioned medium (GCM), indicative of the activated TGF? signaling pathway in microglia associated with glioma environment. Immunofluorescence labeling further revealed the expression of SMAD4 in microglial and non-microglial cells of human glioblastomas tissue in vivo. Functional analysis through shRNA-mediated stable knockdown of SMAD4 in microglia revealed the downregulation of the expression of matrix metalloproteinase 9 (MMP9), which has been shown to be involved in tumor progression and cell migration. Further, knockdown of SMAD4 in microglia decreased the migration of microglial cells towards GCM, indicating that SMAD4 promotes microglial migration in glioma environment. In addition, SMAD4 has been shown to be post-transcriptionally regulated by microRNA-146a, which was downregulated in microglia treated with GCM. Overexpression of miR-146a resulted in decreased expression of SMAD4 together with tumor supportive gene MMP9 in microglia, and subsequently suppressed microglial migration towards GCM, possibly through regulation of SMAD4. On the other hand, the cell viability assay revealed decreased viability of glioma cells when they were treated with conditioned medium derived from SMAD4 knockdown microglia or miR-146a overexpressed microglia as compared to glioma cells treated with the medium from control microglial cells. Taken together, the present study suggests that microglial SMAD4 which is epigenetically regulated by miR-146a promotes microglial migration in gliomas and glioma cell viability.
Project description:Microglia are the brain's immune cells and play an important role in regulating the microenvironment in the central nervous system. Activated microglia are capable of acquiring the pro-inflammatory (M1) phenotype and anti-inflammatory (M2) phenotype. Overactivation of microglia is neurotoxic and may lead to neuroinflammatory brain disorders. Neuroinflammation in the brain plays a crucial role part in the pathophysiology of many psychiatric and neurological diseases. The inhibition of M1 microglia and promotion of M2 microglia was demonstrated to treat and prevent these diseases through reduced neuroinflammation. Isovitexin (IVX) has anti-inflammatory properties and passes through the blood-brain barrier; however, the molecular mechanism that modulates IVX-mediated microglial polarization remains unclear. In BV-2 cells and mouse primary microglia, IVX suppressed the expression of M1 microglial markers, enhanced the expression of M2 microglial markers, and enhanced the release of interleukin 10 (IL-10). IVX promoted the expression of peroxisome proliferator-activated receptor-? (PPAR?) and PPAR? coactivator-1? (PGC-1?) in LPS-induced microglial activation. The inhibition of PPAR? and PGC-1? attenuated the regulatory effect of IVX in LPS-induced microglial polarization. IVX increased the expression of p-CaMKK?, p-AMPK, and PGC-1? in BV-2 cells. Inhibition of CaMKK? with STO-609 or knockdown of CaMKK? with CaMKK? siRNA attenuated IVX-mediated M2 microglial polarization in LPS-treated cells. In LPS-treated mice, the inhibition of CaMKK? and PGC-1? attenuated the IVX-mediated prevention of sickness behavior and enhanction of IVX-mediated M2 microglial polarization. IVX promoted M2 microglial polarization which exerted anti-inflammatory effects on LPS-induced neuroinflammation via the activation of the CaMKK?/AMPK-PGC-1? signaling axis.