Over the past two decades, it has become increasingly apparent that Alzheimer’s disease neuropathology is characterized by activated microglia (brain resident macrophages) as well as the classic features of amyloid plaques and neurofibrillary tangles. The intricacy of microglial biology has also become apparent, leading to a heightened research interest in this particular cell type. Over the years a number of different microglial cell culturing techniques have been developed to study either primary mammalian microglia, or immortalized cell lines. Each microglial system has advantages and disadvantages and should be selected for its appropriateness in a particular research context. This review summarizes several of the most common microglial cell culture systems currently being employed in Alzheimer’s research including primary microglia; BV2 and N9 retroviral immortalized microglia; human immortalized microglia (HMO6); and spontaneously immortalized rodent microglial lines (EOC lines and HAPI cells). Particularities of cell culture requirements and characteristics of microglial behavior, especially in response to applied inflammogen stimuli, are compared and discussed across these cell types.
Neuron-microglia co-cultures treated with pro-inflammatory agents are a useful tool to study neuroinflammation in vitro, where to test the potential neuroprotective effect of anti-inflammatory compounds. However, a great diversity of experimental conditions can be found in the literature, making difficult to select the working conditions when considering this approach for the first time. We compared the use of neuron-primary microglia and neuron-BV2 cells (a microglial cell line) co-cultures, using different neuron:microglia ratios, treatments and time post-treatment to induce glial activation and derived neurotoxicity. We show that each model requires different experimental conditions, but that both neuron-BV2 and neuron-primary microglia LPS/IFN-γ-treated co-cultures are good to study the potential neuroprotective effect of anti-inflammatory agents. The contribution of different pro-inflammatory parameters in the neurotoxicity induced by reactive microglial cells was determined. IL-10 pre-treatment completely inhibited LPS/IFN-γ-induced TNF-α and IL-6 release, and COX-2 expression both in BV2 and primary microglial cultures, but not NO production and iNOS expression. However, LPS/IFN-γ induced neurotoxicity was not inhibited in IL-10 pre-treated co-cultures. The inhibition of NO production using the specific iNOS inhibitor 1400 W totally abolished the neurotoxic effect of LPS/IFN-γ, suggesting a major role for NO in the neurotoxic effect of activated microglia. Consequently, among the anti-inflammatory agents, special attention should be paid to compounds that inhibit NO production.
Methamphetamine is a drug of abuse that can cause neurotoxic damage in humans and animals. Modafinil, a wake-promoting compound approved for the treatment of sleeping disorders, is being prescribed off label for the treatment of methamphetamine dependence. The aim of the present study was to investigate if modafinil could counteract methamphetamine-induced neuroinflammatory processes, which occur in conjunction with degeneration of dopaminergic terminals in the mouse striatum. We evaluated the effect of a toxic methamphetamine binge in female C57BL/6 mice (4×5 mg/kg, i.p., 2 h apart) and modafinil co-administration (2×90 mg/kg, i.p., 1 h before the first and fourth methamphetamine injections) on glial cells (microglia and astroglia). We also evaluated the striatal expression of the pro-apoptotic BAX and anti-apoptotic Bcl-2 proteins, which are known to mediate methamphetamine-induced apoptotic effects. Modafinil by itself did not cause reactive gliosis and counteracted methamphetamine-induced microglial and astroglial activation. Modafinil also counteracted the decrease in tyrosine hydroxylase and dopamine transporter levels and prevented methamphetamine-induced increases in the pro-apoptotic BAX and decreases in the anti-apoptotic Bcl-2 protein expression. Our results indicate that modafinil can interfere with methamphetamine actions and provide protection against dopamine toxicity, cell death, and neuroinflammation in the mouse striatum.
The chronic effects of repetitive head impacts (RHI) on the development of neuroinflammation and its relationship to chronic traumatic encephalopathy (CTE) are unknown. Here we set out to determine the relationship between RHI exposure, neuroinflammation, and the development of hyperphosphorylated tau (ptau) pathology and dementia risk in CTE. We studied a cohort of 66 deceased American football athletes from the Boston University-Veteran’s Affairs-Concussion Legacy Foundation Brain Bank as well as 16 non-athlete controls. Subjects with a neurodegenerative disease other than CTE were excluded. Counts of total and activated microglia, astrocytes, and ptau pathology were performed in the dorsolateral frontal cortex (DLF). Binary logistic and simultaneous equation regression models were used to test associations between RHI exposure, microglia, ptau pathology, and dementia. Duration of RHI exposure and the development and severity of CTE were associated with reactive microglial morphology and increased numbers of CD68 immunoreactive microglia in the DLF. A simultaneous equation regression model demonstrated that RHI exposure had a significant direct effect on CD68 cell density (p < 0.0001) and ptau pathology (p < 0.0001) independent of age at death. The effect of RHI on ptau pathology was partially mediated through increased CD68 positive cell density. A binary logistic regression demonstrated that a diagnosis of dementia was significantly predicted by CD68 cell density (OR = 1.010, p = 0.011) independent of age (OR = 1.055, p = 0.007), but this effect disappeared when ptau pathology was included in the model. In conclusion, RHI is associated with chronic activation of microglia, which may partially mediate the effect of RHI on the development of ptau pathology and dementia in CTE. Inflammatory molecules may be important diagnostic or predictive biomarkers as well as promising therapeutic targets in CTE.
Rett syndrome is an X-linked autism spectrum disorder. The disease is characterized in most cases by mutation of the MECP2 gene, which encodes a methyl-CpG-binding protein. Although MECP2 is expressed in many tissues, the disease is generally attributed to a primary neuronal dysfunction. However, as shown recently, glia, specifically astrocytes, also contribute to Rett pathophysiology. Here we examine the role of another form of glia, microglia, in a murine model of Rett syndrome. Transplantation of wild-type bone marrow into irradiation-conditioned Mecp2-null hosts resulted in engraftment of brain parenchyma by bone-marrow-derived myeloid cells of microglial phenotype, and arrest of disease development. However, when cranial irradiation was blocked by lead shield, and microglial engraftment was prevented, disease was not arrested. Similarly, targeted expression of MECP2 in myeloid cells, driven by Lysm(cre) on an Mecp2-null background, markedly attenuated disease symptoms. Thus, through multiple approaches, wild-type Mecp2-expressing microglia within the context of an Mecp2-null male mouse arrested numerous facets of disease pathology: lifespan was increased, breathing patterns were normalized, apnoeas were reduced, body weight was increased to near that of wild type, and locomotor activity was improved. Mecp2(+/-) females also showed significant improvements as a result of wild-type microglial engraftment. These benefits mediated by wild-type microglia, however, were diminished when phagocytic activity was inhibited pharmacologically by using annexin V to block phosphatydilserine residues on apoptotic targets, thus preventing recognition and engulfment by tissue-resident phagocytes. These results suggest the importance of microglial phagocytic activity in Rett syndrome. Our data implicate microglia as major players in the pathophysiology of this devastating disorder, and suggest that bone marrow transplantation might offer a feasible therapeutic approach for it.
ZIKA virus (ZIKV) is an emerging pathogen responsible for neurological disorders and congenital microcephaly. However, the molecular basis for ZIKV neurotropism remains poorly understood. Here, we show that Axl is expressed in human microglia and astrocytes in the developing brain and that it mediates ZIKV infection of glial cells. Axl-mediated ZIKV entry requires the Axl ligand Gas6, which bridges ZIKV particles to glial cells. Following binding, ZIKV is internalized through clathrin-mediated endocytosis and traffics to Rab5+ endosomes to establish productive infection. During entry, the ZIKV/Gas6 complex activates Axl kinase activity, which downmodulates interferon signaling and facilitates infection. ZIKV infection of human glial cells is inhibited by MYD1, an engineered Axl decoy receptor, and by the Axl kinase inhibitor R428. Our results highlight the dual role of Axl during ZIKV infection of glial cells: promoting viral entry and modulating innate immune responses. Therefore, inhibiting Axl function may represent a potential target for future antiviral therapies.
Activated microglia and infiltrating lymphocytes are neuropathological hallmarks of amyotrophic lateral sclerosis (ALS), a fatal motoneuron disease. Although both cell types play pivotal roles in the ALS pathogenic process, the interactions between microglia and lymphocytes, specifically regulatory CD4+CD25High T lymphocytes (Tregs) and cytotoxic CD4+CD25- T lymphocytes (Teffs), have not been addressed. When co-cultured with mSOD1 adult microglia, mSOD1 Tregs suppressed the cytotoxic microglial factors NOX2 and iNOS through an IL-4-mediated mechanism, whereas Teffs were only minimally effective; IL-4 inhibitory antibodies blocked the suppressive function of mSOD1 Tregs, and conditioned media from mSOD1 Tregs or the addition of IL-4 reduced microglial NOX2 expression. During the stable disease phase, the total number of Tregs, specifically the numbers of CD4+CD25HighIL-4+, CD4+CD25HighIL-10+ and CD4+CD25HighTGF-β+ Tregs, were increased in ALS mice compared with WT mice; Tregs isolated during this phase reduced Teff proliferation. In contrast, during the rapidly progressing phase, the number of mSOD1 Tregs decreased while the proliferation of mSOD1 Teffs increased. The combination of IL-4, IL-10, and TGF-β was required to inhibit the proliferation of mSOD1 Teffs by mSOD1 Tregs that were isolated during the slow phase, while inhibition of mSOD1 Teffs by mSOD1 Tregs during the rapid phase, as well as WT Teffs, was not dependent on these factors. Thus, mSOD1 Tregs at the slow phase suppressed microglial toxicity and SOD1 Teff proliferation through different mechanisms; microglial activation was suppressed through IL-4 whereas mSOD1 Teffs were suppressed by IL-4, IL-10 and TGF-β. These data suggest that mSOD1 Tregs contribute to the slowly progressing phase in ALS mice and may offer a novel therapeutic option for ALS patients.
Hypoxia has been shown to promote inflammation, including the release of proinflammatory cytokines, but it is poorly investigated how hypoxia directly affects inflammasome signaling pathways. To explore whether hypoxic stress modulates inflammasome activity, we examined the effect of cobalt chloride (CoCl2)-induced hypoxia on caspase-1 activation in primary mixed glial cultures of the neonatal mouse brain. Unexpectedly, hypoxia induced by oxygen-glucose deprivation or CoCl2 treatment failed to activate caspase-1 in microglial BV-2 cells and primary mixed glial cultures. Of particular interest, CoCl2-induced hypoxic condition considerably inhibited NLRP3-dependent caspase-1 activation in mixed glial cells, but not in bone marrow-derived macrophages. CoCl2-mediated inhibition of NLRP3 inflammasome activity was also observed in the isolated brain microglial cells, but CoCl2 did not affect poly dA:dT-triggered AIM2 inflammasome activity in mixed glial cells. Our results collectively demonstrate that CoCl2-induced hypoxia may negatively regulate NLRP3 inflammasome signaling in brain glial cells, but its physiological significance remains to be determined.
Minocycline strongly inhibits microglial activation, which contributes to central sensitization, a major mechanism underlying chronic pain development. We hypothesized that the perioperative administration of minocycline might decrease persistent pain after lumbar discectomy. We randomly assigned 100 patients undergoing scheduled lumbar discectomy to placebo and minocycline groups. The minocycline group received 100mg minocycline orally, twice daily, beginning the evening before surgery and continuing for 8days. The primary outcome was the change in lower limb pain intensity at rest between baseline and 3months. Secondary outcomes were pain intensity on movement, the incidence of persistent pain and chronic neuropathic pain, back pain intensity at rest and on movement, and changes in Neuropathic Pain Symptom Inventory, Brief Pain Inventory, and Roland-Morris scores at 3months. An intention-to-treat analysis was performed for patients assessed from the day before surgery to 3months. The decrease in lower limb pain intensity was similar in the placebo and minocycline groups, both at rest -1.7±1.6 vs -2.3±2.4 and on movement -2.5±2.1 vs -3.4±2.9. The incidence and intensity of neuropathic pain and functional scores did not differ between the minocycline and placebo groups. Exploratory analysis suggested that minocycline might be effective in a subgroup of patients with predominantly deep spontaneous pain at baseline. Perioperative minocycline administration for 8days does not improve persistent pain after lumbar discectomy.
The branched-chain amino acids (BCAAs) valine, leucine and isoleucine are essential amino acids involved in several important brain functions. Although commonly used as nutritional supplements, excessive intake of BCAAs might favour the establishment of neurotoxic conditions as indicated by the severe neurological symptoms characterising inherited disorders of BCAA catabolism such as maple syrup urine disease (MSUD). Recent evidence indicates that BCAAs induce excitotoxicity through mechanisms that require the presence of astrocytes. In the present study, we evaluated the effects of BCAAs on microglia, the main immune cells of the brain. As an experimental model we used primary microglial cells harvested from mixed glial cultures that had been kept in normal or high BCAA medium (H-BCAA). We show that H-BCAA microglial cells exhibit a peculiar phenotype characterized by a partial skewing toward the M2 state, with enhanced IL-10 expression and phagocytic activity but also increased free radical generation and decreased neuroprotective functions. We suggest that such an intermediate M1/M2 phenotype might result in a less efficient microglial response, which would promote the establishment of a low grade chronic inflammation and increase the likelihood of neurodegeneration. Although based on in vitro evidence, our study adds on to an increasing literature indicating that the increasing use of dietary integrators might deserve consideration for the possible drawbacks. In addition to excitotoxicity, the altered immune profile of microglia might represent a further mechanism by which BCAAs might turn into toxicants and facilitate neurodegeneration.