Brain Macrophages Research Articles

Neural responses to peripheral infection in Alzheimer’s disease model mice

AbstractBackgroundClinical and animal studies have suggested that peripheral inflammation affects the central nervous system (CNS) and may be a major factor in the pathophysiology of neurodegenerative diseases like AD. Furthermore, a variety of signaling factors originating in the periphery, such as inflammatory mediators, have been associated with the regulation of brain barrier functions. Here, we hypothesized that the presence of peripheral inflammation dysregulates brain barriers’ integrity and induces neuroinflammation with consequences on amyloid pathology.MethodIn this study, we employed an AD mouse model nasally infected with Staphylococcus aureus to assess the impact of chronic or acute peripheral inflammation on brain transcriptome and amyloid pathology. We utilized both single cell and Spatial Transcriptomics to better determine transcriptional response to peripheral inflammation.ResultThe chronic exposure increased the diffuse and compact amyloid plaques and blood cytokine levels. Following a short‐term exposure, single‐cell and spatial transcriptomics uncovered cell type‐ and spatial‐specific transcriptional changes indicating a dysregulation of the brain barriers, including the blood‐brain and the blood‐cerebrospinal fluid barriers. Brain macrophages exhibited increased Apoe expression and macrophage‐specific genes were upregulated at ventricular areas of infected mice. In addition, we report an increase of disease associated microglia genes around the Ab plaques, together with disbalances in neuronal expression in response to peripheral inflammation.ConclusionWe observed that low‐grade peripheral infection triggers transcriptional responses linked to brain barriers and brain infiltrated/resident macrophages indicate a fundamental importance of them in the mechanisms linking peripheral inflammation and AD pathogenesis. Finally, we showed that bacterial infection caused molecular changes restricted to specific cell types and/or brain spatial microenvironments, which highlights the relevance of studying brain molecular responses with high resolution in order to have new insights on AD etiology.

Modulation of Heme-Induced Inflammation Using MicroRNA-Loaded Liposomes: Implications for Hemolytic Disorders Such as Malaria and Sickle Cell Disease.

Hemolytic disorders, like malaria and sickle cell disease (SCD), are responsible for significant mortality and morbidity rates globally, specifically in the Americas and Africa. In both malaria and SCD, red blood cell hemolysis leads to the release of a cytotoxic heme that triggers the expression of unique inflammatory profiles, which mediate the tissue damage and pathogenesis of both diseases. MicroRNAs (miRNAs), such as miR-451a and let-7i-5p, contribute to a reduction in the pro-inflammatory responses induced by circulating free hemes. MiR-451a targets both IL-6R (pro-inflammatory) and 14-3-3ζ (anti-inflammatory), and when this miRNA is present, IL-6R is reduced and 14-3-3ζ is increased. Let-7i-5p targets and reduces TLR4, which results in anti-inflammatory signaling. These gene targets regulate inflammation via NFκB regulation and increase anti-inflammatory signaling. Additionally, they indirectly regulate the expression of key heme scavengers, such as heme-oxygenase 1 (HO-1) (coded by the HMOX1 gene) and hemopexin, to decrease circulating cytotoxic heme concentration. MiRNAs can be transported within extracellular vesicles (EVs), such as exosomes, offering insights into the mechanisms of mitigating heme-induced inflammation. We tested the hypothesis that miR-451a- or let-7i-5p-loaded artificial EVs (liposomes) will reduce heme-induced inflammation in brain vascular endothelial cells (HBEC-5i, ATCC: CRL-3245) and macrophages (THP-1, ATCC: TIB-202) in vitro. We completed arginase and nitric oxide assays to determine anti- and pro-inflammatory macrophage presence, respectively. We also assessed the gene expression of IL-6R, TLR4, 14-3-3ζ, and NFκB by RT-qPCR for both cell lines. Our findings revealed that the exposure of HBEC-5i and THP-1 to liposomes loaded with miR-451a or let-7i-5p led to a reduced mRNA expression of IL-6R, TLR4, 14-3-3ζ, and NFκB when treated with a heme. It also resulted in the increased expression of HMOX1 and hemopexin. Finally, macrophages exhibited a tendency toward adopting an anti-inflammatory differentiation phenotype. These findings suggest that miRNA-loaded liposomes can modulate heme-induced inflammation and can be used to target specific cellular pathways, mediating inflammation common to hematological conditions, like malaria and SCD.

CSF1R Inhibition Promotes Neuroinflammation and Behavioural Deficits during Graft-Versus-Host Disease in Mice

Chronic graft-versus-host disease (cGVHD) remains a significant complication of allogeneic hematopoietic stem cell transplantation. Central nervous system (CNS) involvement is becoming increasingly recognised, where brain-infiltrating donor MHC class II + bone marrow-derived macrophages (BMDM) are implicated mediators of pathology. Colony-stimulating factor 1 receptor (CSF1R)-dependent BMDM are established mediators of cutaneous and pulmonary cGVHD, and clinical trials assessing the efficacy of CSF1R antibody blockade to deplete macrophages are promising. We hypothesized that CSF1R antibody blockade may also be a useful strategy to prevent/treat CNS cGVHD. We leveraged studies in acute GVHD (aGVHD) to determine the feasibility of an antibody-based approach for depleting brain microglia and macrophages. Moreover, we queried whether CSF1R blockade may be a viable therapy for CNS aGVHD, which has been shown to be mediated by host microglia. Increased blood-brain barrier permeability during aGVHD facilitated CNS antibody access. However, CSF1R blockade early post-transplant induced region-specific microglia depletion and was associated with unexpected exacerbation of aGVHD neuroinflammation, including increased T cell and inflammatory monocyte infiltration, and whole brain cytokine levels. In established cGVHD, vascular changes and anti-CSF1R efficacy was more limited. Anti-CSF1R-treated mice retained donor BMDM, activated microglia, CD8 + and CD4 + T cells, and local cytokine expression in the brain. These findings were recapitulated in GVHD recipients where CSF1R was conditionally depleted in donor CX3CR1 + BMDM. Notably, inhibition of CSF1R signalling post-transplant failed to reverse either acute or chronic GVHD-induced behavioral changes. Taken together, these data suggest that ongoing clinical trials of CSF1R inhibition should assess neurological adverse events in patients. In contrast to the lack of efficacy with CSF1R inhibition, transfer of Ifngr-/- grafts reduced MHC class II + BMDM infiltration, resulting in improved behaviour. Furthermore, single nuclei RNA sequencing of brains from mice receiving wild-type or Ifngr-/- allografts revealed a disease-associated interferon-inducible mature oligodendrocyte population in the cGVHD brain. This was markedly reduced in recipients of Ifngr-/- grafts, indicating that targeting this axis can promote brain repair beyond the myeloid compartment during cGVHD. In summary, our findings reveal unexpected neurological immune toxicity during CSF1R blockade and highlight the IFNGR pathway as an alternative target for the treatment of cGVHD within the CNS.

Characterization of the responses of brain macrophages to focused ultrasound-mediated blood-brain barrier opening.

The opening of the blood-brain barrier (BBB) by focused ultrasound (FUS) coupled with intravenously injected microbubbles can be leveraged as a form of immunotherapy for the treatment of neurodegenerative disorders. However, how FUS BBB opening affects brain macrophages is not well understood. Here by using single-cell sequencing to characterize the distinct responses of microglia and central nervous system-associated macrophages (CAMs) to FUS-mediated BBB opening in mice, we show that the treatment remodels the immune landscape via the recruitment of CAMs and the proliferation of microglia and via population size increases in disease-associated microglia. Both microglia and CAMs showed early and late increases in population sizes, yet only the proliferation of microglia increased at both timepoints. The population of disease-associated microglia also increased, accompanied by the upregulation of genes associated with gliogenesis and phagocytosis, with the depletion of brain macrophages significantly decreasing the duration of BBB opening.

Clearing and replacing tissue-resident myeloid cells with an anti-CD45 antibody–drug conjugate

AbstractTissue-resident myeloid (TRM) cells in adults have highly variable lifespans, and may be derived from early embryonic yolk sac, fetal liver, or bone marrow. Some of these TRM cells are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplantation can replace long-lived brain TRM cells, resulting in clinical improvements in metabolic storage diseases. With the advent of antibody–drug conjugate (ADC)-targeted cell killing as a cell-selective means of transplant conditioning, we assessed the impact of anti-CD45–ADC on TRM cells in multiple tissues. Replacement of TRM cells ranged from 40% to 95% efficiencies in liver, lung, and skin tissues, after a single anti-CD45–ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM cells in tissues returned to pretreatment levels, suggesting a regulated control of TRM cell abundance. As expected, brain microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45–ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque-resident myeloid cells were efficiently replaced with anti-CD45–ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that the anti-CD45–ADC clears both hematopoietic stem and TRM cells from their niches, enabling cell replacement to achieve disease modification in a resident myeloid cell–driven disease.

The role of tumor-associated macrophages in glioma cohort: through both traditional RNA sequencing and single cell RNA sequencing.

Gliomas are the leading cause in more than 50% of malignant brain tumor cases. Prognoses, recurrences, and mortality are usually poor for gliomas that have malignant features. In gliomas, there are four grades, with grade IV gliomas known as glioblastomas (GBM). Currently, the primary methods employed for glioma treatment include surgical removal, followed by chemotherapy after the operation, and targeted therapy. However, the outcomes of these treatments are unsatisfactory. Gliomas have a high number of tumor-associated macrophages (TAM), which consist of brain microglia and macrophages, making them the predominant cell group in the tumor microenvironment (TME). The glioma cohort was analyzed using single-cell RNA sequencing to quantify the genes related to TAMs in this study. Furthermore, the ssGSEA analysis was utilized to assess the TAM-associated score in the glioma group. In the glioma cohort, we have successfully developed a prognostic model consisting of 12 genes, which is derived from the TAM-associated genes. The glioma cohort demonstrated the predictive significance of the TAM-based risk model through survival analysis and time-dependent ROC curve. Furthermore, the correlation analysis revealed the significance of the TAM-based risk model in the application of immunotherapy for individuals diagnosed with GBM. Ultimately, the additional examination unveiled the prognostic significance of PTX3 in the glioma group, establishing it as the utmost valuable prognostic indicator in patients with GBM. The PCR assay revealed the PTX3 is significantly up-regulated in GBM cohort. Additionally, the assessment of cell growth further confirms the involvement of PTX3 in the GBM group. The analysis of cell proliferation showed that the increased expression of PTX3 enhanced the ability of glioma cells to proliferate. The prognosis of glioblastomas and glioma is influenced by the proliferation of tumor-associated macrophages.

Changes in the number of macrophages, T-lymphocytes, activity of antioxidant enzymes in the brain, behavior and structure of the central nervous system neurons in adult and aging mice of different strains with the MPTP-induced model of parkinsonism

Background. Oxidative stress and immune cell (T-lymphocytes, macrophages) products are important for the development of morpho-functional disorders of the nervous system in Parkinson’s disease. Connection of Parkinson’s disease with age and functioning of the major histocompatibility complex genes are also shown. The purpose was to assess changes in the brain of T-lymphocytes, macrophages, malondialdehyde (MDA) contents, the activity of antioxidant enzymes, the structure of brain and spinal cord neurons, as well as behavior in adult and aging mice with different H-2 haplotypes and toxic model of parkinsonism. Materials and methods. Adult and aging female mice of FVB/N (genotype H-2q) and 129/Sv (genotype H-2b) strains were once injected with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at the dose of 30 mg/kg. Contents of CD3+, CD3–CD11b+, CD3+CD11b+ cells, MDA and the activity of antioxidant enzymes in the brain were evaluated. The structure of neurons of the substantia nigra, lumbar spine, behavior in the open field test, as well as in rigidity and rotarod performance tests were studied. Results. In adult mice of both strains, motor and non-motor (spatial-exploratory, emotional) activity is impaired under the influence of MPTP. At the same time, linear differences in behavior changes were revealed in their directions and expressiveness. In aging FVB/N experimental mice, motor behavior disorders prevailed and were combined with non-motor changes in 129/Sv mice. Violations in the structure of substantia nigra neurons after MPTP administration were more severe in FVB/N mice while those in the lumbar spinal cord neurons were more pronounced in the 129/Sv mice. In aging experimental mice of both strains, the percentage of damaged neurons in the brain and spinal cord was significantly lower than in adult animals. After MPTP administration to adult mice, the direction of changes in the brain T-lymphocytes and macrophages (increase or decrease) depended on their strains. In aging experimental mice, the linear heterogeneity of changes was preserved for the T-lymphocyte content, while the macrophage level was increased regardless of the animal strains. Under MPTP influence, the MDA content increased in the brain of mice of all experimental groups. The direction of changes in superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities (decrease or increase) depended on the strains of animals and their age. Conclusions. MPTP-induced changes (directions, expressiveness) in T-lymphocyte, macrophage contents, antioxidant enzymes activity, the structure of neurons of the substantia nigra and lumbar spine, as well as behavioral reactions largely depended on the adult mice H-2 haplotype. There were age-related effects of the neurotoxin on changes in the studied indicators in mice of different strains. At the same time, dependence of changes in most above indicators on the H-2 haplotype preserved.

SCH 644343 alleviates ischemic stroke-induced neuroinflammation by promoting microglial polarization via the IL-4/SREBP-1 signaling pathway

Ischemic stroke (IS), a kind of acute cerebrovascular disease, is one of the most common diseases, and it endangers the lives and health of elderly individuals. Inflammation is a key factor leading to stroke, making it a potential therapeutic target. Previous studies have found that neuroinflammation is closely associated with microglial polarization. Due to the various side effects of current drugs used to treat neuroinflammation, it is important to explore alternative drugs with anti-inflammatory activity for neuroinflammation treatment. In the present study, we investigated the effect of SCH 644343 (SCH), a natural compound, on neuroinflammation induced by IS and explored the mechanism. We found that SCH meliorated the phenotypes of IS in vivo, which was correlated with the increased percentage of infiltrated M2 macrophages in brain after stroke. SCH exerted a significant effect against oxygen–glucose deprivation/reoxygenation (OGD/R) in BV2 cells in vitro by inhibiting M1 microglial polarization and promoting M2 microglial polarization. Furthermore, suppression of SREBP-1 expression by pretreatment with the SREBP-1 inhibitor 25-HC attenuated the effect of SCH on IS in vitro. Taken together, SCH exerts anti-IS effect by promoting microglial polarization via the IL-4/SREBP-1 signaling pathway.

Translatome Profiling of Tissue-Resident Macrophages Using the RiboTag Approach.

Global gene expression profiling has provided valuable insights into the specific contributions of different cell types to various physiological processes. Notably though, both bulk and single-cell transcriptomics require the prior retrieval of the cells from their tissue context to be analyzed. Isolation protocols for tissue macrophages are, however, notoriously inefficient and, moreover, prone to introduce considerable bias and artifacts. Here, we will discuss a valuable alternative, originally introduced by Amieux and colleagues. This so-called RiboTag approach allows, in combination with respective macrophage-specific Cre transgenic lines, to retrieve macrophage translatomes from crude tissue extracts. We will review our experience with this ingenious method, focusing on the study of brain macrophages, including microglia and border-associated cells. We will elaborate on the advantages of the RiboTag approach that render it a valuable complement to standard cell sorting-based profiling strategies, especially for the investigation of tissue macrophages.

Aggravation of lipopolysaccharide-induced depressive-like behavior in CCR4-deficient mice

Increasing evidence indicates that immune abnormalities are associated with the pathogenesis of depression. CCR4 is a chemokine receptor that regulates regulatory T cell (Treg) and Th17 cell migration. Here, using a lipopolysaccharide (LPS)-induced depression mouse model, we demonstrated that CCR4 deficiency exacerbated depressive-like behavior. Tregs and M2 macrophages, but not Th17 cells, were decreased in the brain of CCR4-deficient mice. Consistently, treatment with a CCR4 inhibitor reduced Tregs and M2 macrophages in the brain and exacerbated depressive-like behavior. Thus, CCR4 may contribute to the reduction of depressive symptoms by promoting Treg recruitment to the brain and subsequent M2 macrophage polarization.

SYST-25 MICROGLIA-SECRETED LIPID SPECIES DRIVE GLIOMA INVASION

Abstract The capacity of gliomas, including GBMs, to invade and infiltrate normal brain tissues makes their complete surgical removal impossible. The most crucial survival predictor is still the extent of surgical resection, urging the need to identify treatments that block the invasive growth of glioma to improve surgical resection and overall survival. Using a combination of single-cell transcriptomics, lipidomics, and high-resolution spatial metabolomics in genetically engineered GBM mouse models, reminiscent of human disease, and validation in human GBM samples, we found that 1) tumor cells infiltrating the normal brain parenchyma associate with and activate specific pathways in microglial cells (MGs), the residential brain macrophages. In turn, MGs undergo metabolic rewiring and secrete lipid species that promote glioma invasion of the different GBM subtypes by activating distinct signaling cues. Thus, blocking the MG-tumor cell axis or metabolic MG rewiring targets several GBM types and, in combination with standard therapy, results in small circumscribed tumors that should be easier excised and lead to a substantial increase in progression-free survival in glioma patients.

The effects of high plasma levels of Aβ1-42 on mononuclear macrophage in mouse models of Alzheimer’s disease

More and more evidences are proving that microglia play a crucial role in the pathogenesis of Alzheimer’s disease (AD) and the plasma Aβ1-42 levels significantly increased 15 years before the onset of dominantly inherited AD. However, the effects of high plasma levels of Aβ1-42 on mononuclear macrophage, the peripheral counterparts of microglia, remain unclear. In the present study, we used APP/PS1 transgenic (Tg) mice and a parabiotic model of wild type (Wt) mice and Tg mice (Parabiotic Wt-Tg, Pa (Wt-Tg)) to investigate the effects of high plasma levels of Aβ1-42 on peripheral mononuclear macrophage. Our results showed that in the early stage of Tg mice (7 months) and Pa (Wt-Tg) mice (4 months), the proportions of pro-inflammatory macrophages in peritoneal cavity, myeloid derived suppressor cells (MDSCs) in spleen, granulocyte-monocyte progenitors (GMPs) in bone marrow, and the plasma levels of interleukin-6 (IL-6) were significantly decreased. While the proportions of pro-inflammatory macrophages, MDSCs, GMPs, and the plasma levels of IL-6 and tumor necrosis factor (TNF)-α, as well as the numbers of bone marrow-derived macrophages (BMDMs) in mice brain were increased in the late stage of Tg mice (11 months) and Pa (Wt-Tg) mice (8 months). In addition, the proportions of monocytes in spleen and the proliferation of bone marrow cells (BMCs) were enhanced consistently, and the phagocytic function of macrophages kept stably after high plasma levels of Aβ1-42 sustaining stimulation. These results demonstrated that high plasma levels of Aβ1-42 play a biphasic regulating role at different stages of the disease, namely inhibiting effects on peripheral pro-inflammatory macrophages in the early stage of AD model, while promoting effects in the late stage of AD model. The mechanism behind this may be associated with their effects on MDSCs in spleen and myeloid progenitor cells in bone marrow. Therefore, intervening the effects of plasma Aβ1-42 on pro-inflammatory macrophages might offer a new therapeutic approach to AD.

Not just Glia-Dissecting brain macrophages in the mouse.

Macrophages have emerged as critical cellular components of the central nervous system (CNS), promoting development, maintenance, and immune defense of the CNS. Here we will review recent advances in our understanding of brain macrophage heterogeneity, including microglia and border-associated macrophages, focusing on the mouse. Emphasis will be given to the discussion of strengths and limitations of the experimental approaches that have led to the recent insights and hold promise to further deepen our mechanistic understanding of brain macrophages that might eventually allow to harness their activities for the management of CNS pathologies.

IMMU-01. EVALUATING THE IMPACT OF CAR DESIGN ON THE TUMOR IMMUNE MICROENVIRONMENT AND ANTI-TUMOR RESPONSE IN SYNGENEIC GLIOMA MODELS

Abstract B7-H3 CAR T-cells have limited efficacy in immunocompetent mouse models indicating a suppressive role of the tumor immune microenvironment (TIME). In this study, we set to optimize B7-H3 CAR structure and examine the effects of CAR design on brain TIME. We generated a panel of second-generation B7-H3-specific CARs with different transmembrane (CD8, CD28), costimulatory (CD28, 4-1BB), and activation (ζ, mutζ) domains. We then compared the cytolytic activity, expansion, and anti-tumor activity of T-cells expressing these CARs. Results showed that T-cells expressing B7-H3 CARs with CD28 transmembrane/costimulatory domains have superior efficacy in vitro. Yet, providing optimized costimulation and signaling did not induce superior anti-glioma efficacy of B7-H3 CAR T-cells in vivo. Additionally, incorporating 4-1BB signaling into CD28-based CAR T-cells by expressing 4-1BBL on their cell surface enhances the therapeutic efficacy and persistence of B7-H3 CAR T-cells in vitro. However, transgenic expression of 4-1BBL in CD28-based CARs was not associated with further enhanced anti-glioma efficacy of B7-H3 CAR T-cells in vivo. Next, using high-dimensional flow cytometry and spatial transcriptomic analyses, we show that suppressive tumor-associated macrophages (TAMs) infiltrate tumors in non-responder mice and they predominantly surround the tumor edges. Additionally, minimal T-cell infiltrates were observed in these tumors with predominantly exhausted and dysfunctional phenotypes. Analysis of gene-expression signatures early after CAR T-cell treatment revealed that tumors treated with CD28-based CARs differentially upregulate genes associated with inflammatory myeloid responses. While treatment with other signaling domains’ containing CARs upregulates pathways predictive of suppressive TAM and regulatory/dysfunctional T-cell responses. We show that global depletion of brain macrophages using CSF1R inhibitor exacerbates CAR T-cell anti-tumor activity. In summary, our results show that specific CAR domains induce an early inflammatory immune hub predictive of potent anti-tumor responses. Additionally, inflammatory macrophages are needed for successful CAR T-cell activation and anti-glioma responses.

Modeling brain macrophage biology and neurodegenerative diseases using human iPSC-derived neuroimmune organoids.

Modeling brain macrophage biology and neurodegenerative diseases using human iPSC-derived neuroimmune organoids.

Focused Ultrasound-Mediated Blood–Brain Barrier Opening Best Promotes Neuroimmunomodulation through Brain Macrophage Redistribution

Neuroimmunomodulation is a promising form of drug-free treatment for neurological diseases ranging from Alzheimer’s disease to depression. The evidence supporting the efficacy of focused ultrasound (FUS) neuroimmunomodulation is encouraging; however, the method has yet to be standardized, and its mechanism remains poorly understood. Methods of FUS neuroimmunomodulation can be categorized into three paradigms based on the parameters used. In the first paradigm, focused ultrasound blood–brain barrier opening (FUS-BBBO) combines FUS with microbubbles (MB) to transiently and safely induce BBB opening. In the second paradigm, focused ultrasound neuromodulation (FUS-N) harnesses the acoustic effects of FUS alone (without MB). In the third paradigm, focused ultrasound with microbubbles without BBBO (FUS + MB) combines MB with FUS below the BBBO pressure threshold—harnessing the mechanical effects of FUS without opening the barrier. Due to the recent evidence of brain macrophage modulation in response to FUS-BBBO, we provide the first direct comparison of brain macrophage modulation between all three paradigms both in the presence and absence of Alzheimer’s disease (AD) pathology. Flow cytometry and single-cell sequencing are employed to identify FUS-BBBO as the FUS paradigm, which maximizes brain macrophage modulation, including an increase in the population of neuroprotective, disease-associated microglia and direct correlation between treatment cavitation dose and brain macrophage phagocytosis. Next, we combine spatial and single-cell transcriptomics with immunohistochemical validation to provide the first characterization of brain macrophage distribution in response to FUS-BBBO. Given their relevance within neurodegeneration and perturbation response, we emphasize the analysis of three brain macrophage populations—disease- and interferon-associated microglia and central-nervous-system-associated macrophages. We find and validate the redistribution of each population with an overall trend toward increased interaction with the brain–cerebrospinal fluid barrier (BCSFB) after FUS-BBBO, an effect that is found to be more pronounced in the presence of disease pathology. This study addresses the prior lack of FUS neuroimmunomodulation paradigm optimization and mechanism characterization, identifying that FUS-BBBO best modulates brain macrophage response via complex redistribution.

Microglial inflammation modulates opioid analgesic tolerance.

As we all know, opioids are the drugs of choice for treating severe pain. However, very often, opioid use leads to tolerance, dependence, and hyperalgesia. Therefore, understanding the mechanisms underlying opioid tolerance and designing strategies for increasing the efficacy of opioids in chronic pain are important areas of research. Microglia are brain macrophages that remove debris and dead cells from the brain and participate in immune defense of the central nervous system during an insult or injury. However, recent studies indicate that microglial activation and generation of proinflammatory molecules (e.g., cytokines, nitric oxide, eicosanoids, etc.) in the brain may contribute to opioid tolerance and other side effects of opioid use. In this review, we will summarize the evidence and possible mechanisms by which proinflammatory molecules produced by activated microglia may antagonize the analgesic effect induced by opioids, and thus, lead to opioid tolerance. We will also delineate specific examples of studies that suggest therapeutic targets to counteract the development of tolerance clinically using suppressors of microglial inflammation.

Reaction of different cell types of the brain on neurotoxin cuprizone and hormone melatonin treatment in young and aging mice.

The brain myelin and neurons destruction in multiple sclerosis may be associated with the production of neuroinflammatory cells (macrophages, astrocytes, T-lymphocytes) of pro-inflammatory cytokines and free radicals. The age-associated changes of the above cells can influence on the response of nervous system cells to toxic damaging and regulatory factors of humoral/endocrine nature, in particular pineal hormone melatonin. The study aim was (1) to evaluate changes of the brain macrophages, astrocytes, T-cells, neural stem cells, neurons, and central nervous system (CNS) functioning in the neurotoxin cuprizone-treated mice of different age; and (2) to assess in such mice the effects of exogenous melatonin and possible courses of its action. A toxic demyelination and neurodegeneration model was induced in 129/Sv mice aged 3-5 and 13-15 months by adding cuprizone neurotoxin to their food for 3 weeks. From the 8th day of the cuprizone treatment, melatonin was injected intraperitoneally at 6 p.m. daily, at a dose of 1 mg/kg. The brain GFPA + -cells were evaluated by immunohistochemical method, the proportion of CD11b+, CD3+CD11b+, CD3+, CD3+CD4+, CD3+CD8+, Nestin+-cells was determined via flow cytometry. Macrophage activity was evaluated by their ability to phagocytose latex beads Morphometric analysis of the brain neurons and the behavioral reactions ("open field" and rotarod tests) were performed. To assess the involvement of the bone marrow and thymus in the action of melatonin, the amount of granulocyte/macrophage colony-forming cells (GM-CFC), and blood monocytes and thymic hormone thymulin were evaluated. The numbers of the GFAP+-, CD3+-, CD3+CD4+, CD3+CD8+, CD11b+, CD3+CD11b+, Nestin+-cells and macrophages phagocytic latex beads and malondialdehyde (MDA) content were increased in the brain of young and aging mice under cuprizone influence. The proportion of undamaged neurons within the brain, motor, affective, and exploratory activities, and muscle tone decreased in mice of both ages. Introducing melatonin to mice of any age reduced the number of GFAP+-, CD3+- cells and their subpopulations, macrophage activation, and MDA content. At the same time, the percentage of brain neurons that were unchanged increased as the number of Nestin+ cells decreased. The behavioral responses were also improved. Besides, the number of bone marrow GM-CFC and the blood level of monocytes and thymulin increased. The effects of both neurotoxin and melatonin on the brain astrocytes, macrophages T-cells, and immune system organs as well as the structure and functioning of neurons were more pronounced in the young mice. We have observed the involvement of the astrocytes, macrophages, T-cells, neural stem cells, and neurons in the brain reaction of mice different age after administration of neurotoxin cuprizone and melatonin. The brain cell composition reaction has the age features. The neuroprotective effects of melatonin in cuprizone-treated mice have been realized through an improvement of the brain cell composition and oxidative stress factors and functioning of bone marrow and thymus.

Syk inhibitors protect against microglia-mediated neuronal loss in culture.

Microglia are brain macrophages and play beneficial and/or detrimental roles in many brain pathologies because of their inflammatory and phagocytic activity. Microglial inflammation and phagocytosis are thought to be regulated by spleen tyrosine kinase (Syk), which is activated by multiple microglial receptors, including TREM2 (Triggering Receptor Expressed on Myeloid Cells 2), implicated in neurodegeneration. Here, we have tested whether Syk inhibitors can prevent microglia-dependent neurodegeneration induced by lipopolysaccharide (LPS) in primary neuron-glia cultures. We found that the Syk inhibitors BAY61-3606 and P505-15 (at 1 and 10 μM, respectively) completely prevented the neuronal loss induced by LPS, which was microglia-dependent. Syk inhibition also prevented the spontaneous loss of neurons from older neuron-glia cultures. In the absence of LPS, Syk inhibition depleted microglia from the cultures and induced some microglial death. However, in the presence of LPS, Syk inhibition had relatively little effect on microglial density (reduced by 0-30%) and opposing effects on the release of two pro-inflammatory cytokines (IL-6 decreased by about 45%, TNFα increased by 80%). Syk inhibition also had no effect on the morphological transition of microglia exposed to LPS. On the other hand, inhibition of Syk reduced microglial phagocytosis of beads, synapses and neurons. Thus, Syk inhibition in this model is most likely neuroprotective by reducing microglial phagocytosis, however, the reduced microglial density and IL-6 release may also contribute. This work adds to increasing evidence that Syk is a key regulator of the microglial contribution to neurodegenerative disease and suggests that Syk inhibitors may be used to prevent excessive microglial phagocytosis of synapses and neurons.

A Cre-deleter specific for embryo-derived brain macrophages reveals distinct features of microglia and border macrophages

Genetic tools to target microglia specifically and efficiently from the early stages of embryonic development are lacking. We generated a constitutive Cre line controlled by the microglia signature gene Crybb1 that produced nearly complete recombination in embryonic brain macrophages (microglia and border-associated macrophages [BAMs]) by the perinatal period, with limited recombination in peripheral myeloid cells. Using this tool in combination with Flt3-Cre lineage tracer, single-cell RNA-sequencing analysis, and confocal imaging, we resolved embryonic-derived versus monocyte-derived BAMs in the mouse cortex. Deletion of the transcription factor SMAD4 in microglia and embryonic-derived BAMs using Crybb1-Cre caused a developmental arrest of microglia, which instead acquired a BAM specification signature. By contrast, the development of genuine BAMs remained unaffected. Our results reveal that SMAD4 drives a transcriptional and epigenetic program that is indispensable for the commitment of brain macrophages to the microglia fate and highlight Crybb1-Cre as a tool for targeting embryonic brain macrophages.