Development of a 3D Brain Model to Study Sex-Specific Neuroinflammation After Hemorrhagic Stroke.

Subarachnoid hemorrhage (SAH) accounts for 5% of stroke, with women having a decreased inflammatory response compared to men; however, this mechanism has yet to be identified. One hurdle in SAH research is the lack of human brain models. Studies in murine models are helpful, but human models should be used in conjunction for improved translatability. These observations lead us to develop a 3D system to study the sex-specific microglial and neuroglial function in a novel in vitro human SAH model and compare it to our validated in vivo SAH model. Our lab has developed a 3D, membrane-based in vitro cell culture system with human astrocytes, microglia, and neurons from both sexes. The 3D cultures were incubated with male and female cerebrospinal fluid from SAH patients in the Neuro-ICU. Furthermore, microglial morphology, erythrophagocytosis, microglial inflammatory cytokine production, and neuronal apoptosis were studied and compared with our murine SAH models. The human 3D system demonstrated intercellular interactions and proportions of the three cell types similar to the adult human brain. In vitro and in vivo models of SAH showed concordance in male microglia being more inflammatory than females via morphology and flow cytometry. On the contrary, both in vitro and in vivo models revealed that female microglia were more phagocytic and less prone to damaging neurons than males. One possible explanation for the increased phagocytic ability of female microglia was the increased expression of CD206 and MerTK. Our in vitro, human, 3D cell culture SAH model showed similar results to our in vivo murine SAH model with respect to microglial morphology, inflammation, and phagocytosis when comparing the sexes. A human 3D brain model of SAH may be a useful adjunct to murine models to improve translation to SAH patients.

In Vitro and In Vivo Drug-Response Profiling Using Patient-Derived High-Grade Glioma.

BACKGROUND: Genomic profiling cannot solely predict the complexity of how tumor cells behave in their in vivo microenvironment and their susceptibility to therapies. The aim of the study was to establish a functional drug prediction model utilizing patient-derived GBM tumor samples for in vitro testing of drug efficacy followed by in vivo validation to overcome the disadvantages of a strict pharmacogenomics approach. METHODS: High-throughput in vitro pharmacologic testing of patient-derived GBM tumors cultured as 3D organoids offered a cost-effective, clinically and phenotypically relevant model, inclusive of tumor plasticity and stroma. RNAseq analysis supplemented this 128-compound screening to predict more efficacious and patient-specific drug combinations with additional tumor stemness evaluated using flow cytometry. In vivo PDX mouse models rapidly validated (50 days) and determined mutational influence alongside of drug efficacy. We present a representative GBM case of three tumors resected at initial presentation, at first recurrence without any treatment, and at a second recurrence following radiation and chemotherapy, all from the same patient. RESULTS: Molecular and in vitro screening helped identify effective drug targets against several pathways as well as synergistic drug combinations of cobimetinib and vemurafenib for this patient, supported in part by in vivo tumor growth assessment. Each tumor iteration showed significantly varying stemness and drug resistance. CONCLUSIONS: Our integrative model utilizing molecular, in vitro, and in vivo approaches provides direct evidence of a patient's tumor response drifting with treatment and time, as demonstrated by dynamic changes in their tumor profile, which may affect how one would address that drift pharmacologically.

Gender differences in Alzheimer's may be associated with TLR4-LYN expression in damage associated microglia and neuronal phagocytosis.

The role of Aß plaques and neurofibrillary tangles in Alzheimer's disease (AD) pathogenesis have recently come into question due to failure of many pharmaceutical agents targeting these deposits and detection of these misfolded proteins in normal human brains. Therefore, we investigated correlations between microglial activation and toll like receptor 4 (TLR4) and Lck/Yes novel tyrosine (LYN) kinase signaling in an AD mouse model. In this study, we used 5-6-month-old 5XFAD and wild type (WT) male and female mice. Immunohistochemistry (IHC) and flow cytometry (FC) were performed on their brains. Cognitive performance was assessed with the Barnes-Maze. IHC showed more Ab aggregation in microglia of female 5XFAD mice compared to their male counterparts. Increased co-localization of microglial TLR4 and LYN was also observed in AD more than WT and females more than males. IHC also suggests microglial phagocytosis of neurons in AD mice, which is supported by FC data. Our FC data also support the involvement of disease associated microglia (DAMs) in this process based on cytokine secretion. Cognitive assessment by the Barnes maze showed 5XFAD females performed worse than males. In this study, we investigated the relationship between microglial TLR4 and LYN kinase in 5XFAD male and females. Our data reveals a correlation between microglial TLR4 and LYN co-localization and AD pathogenesis, more in females than males. Targeting microglial TLR4 and Lyn in DAMs may offer new therapeutic opportunities in the treatment of AD.

Betuletol, a Propolis Component, Suppresses IL-33 Gene Expression and Effective against Eosinophilia.

Propolis, a resinous substance produced by honeybees, has been used in folk medicine since ancient times due to its many biological benefits such as antitumor, antioxidant, antimicrobial, anti-inflammatory, and immunomodulatory effects. Propolis contains flavonoids, terpenoids, aromatic aldehydes, and alcohols, which vary with different climate and environmental conditions. In our study, we examined the antiallergic activity of Brazilian green propolis (BGP) and isolated the active compound that can suppress an allergy-sensitive gene, IL-33, expression and eosinophilia. Ethanolic extract of BGP freeze-dried powder was fractionated with several solvent systems, and the active fractions were collected based on activity measurement. The single active compound was found by thin-layer chromatography. Using column chromatography and NMR, the active compound was isolated and identified as 3,5,7-trihydroxy-6,4'-dimethoxyflavone, also known as betuletol. Further, the antiallergic activity of that has been examined in PMA-induced up-regulation of IL-33 gene expression in Swiss 3T3 cells. Our data showed the IL-33 gene suppression both by BGP and the isolated active compound, betuletol. We also found that betuletol suppressed ERK phosphorylation, suggesting it could be effective in suppressing IL-33 mediated eosinophilic chronic inflammation and will provide new insights to develop potent therapeutics against allergic inflammations.

PD-1 and ICOS counter-regulate tissue resident regulatory T cell development and IL-10 production during flu.

Regulatory T cells that express the transcription factor Foxp3 (Treg cells) are a highly heterogenous population of immunoregulatory cells critical for maintaining immune homeostasis and preventing immunopathology during infections. Tissue resident Treg (TR-Treg) cells are maintained within nonlymphoid tissues and have been shown to suppress proinflammatory tissue resident T cell responses and promote tissue repair. Human populations are repetitively exposed to influenza infections and lung tissue resident effector T cell responses are associated with flu-induced long-term pulmonary sequelae. The kinetics of TR-Treg cell development and molecular features of TR-Treg cells during repeated and/or long-term flu infections are unclear. Utilizing a Foxp3RFP/IL-10GFP dual reporter mouse model along with intravascular fluorescent in vivo labeling, we characterized the TR-Treg cell responses to repetitive heterosubtypic influenza infections. We found lung tissue resident Treg cells accumulated and expressed high levels of co-inhibitory and co-stimulatory receptors post primary and secondary infections. Blockade of PD-1 or ICOS signaling reveals that PD-1 and ICOS signaling pathways counter-regulate TR-Treg cell expansion and IL-10 production, during secondary influenza infection. Furthermore, the virus-specific TR-Treg cell response displayed distinct kinetics, when compared to conventional CD4+ tissue resident memory T cells, during secondary flu infection. Our results provide insight into the tissue resident Foxp3+ regulatory T cell response during repetitive flu infections, which may be applicable to other respiratory infectious diseases such as tuberculosis and COVID.

Microglial TLR4 is Critical for Neuronal Injury and Cognitive Dysfunction in Subarachnoid Hemorrhage.

BACKGROUND: Toll-like receptor 4 (TLR4) activation causes excessive production of proinflammatory mediators and an increased expression of costimulatory molecules that leads to neuroinflammation after subarachnoid hemorrhage (SAH). Although TLR4-mediated inflammatory pathways have long been studied in neuroinflammation, the specific glia implicated in initiation and propagation of neuroinflammation in SAH have not been well elucidated. In this study, we investigated the involvement of glial TLR4 including microglia and astrocytes in brain damage and poor neurological outcome. METHODS: In this study, global TLR4 knockout, cell-specific TLR4 knockout, and floxxed control male and female mice were used. The mice were injected with 60 µl autologous blood near the mesencephalon to induce SAH; animals were euthanized on postoperative day 7 for immunohistochemistry of glia and apoptotic cells. Microglial morphology was evaluated by using immunofluorescence density quantification to determine correlations between morphology and neuroinflammation. Microglial depletion was accomplished with the intracerebroventricular administration of clodronate liposomes. Cognitive function was assessed with Barnes maze. RESULTS: On postoperative day 7 after SAH induction, neuronal apoptosis was markedly reduced in the clodronate liposome group compared with phosphate-buffered saline control liposomes, and cognitive performance in the clodronate group was improved, as well. Differences in microglial activation, assessed by morphometric analysis, and neuronal apoptosis were significantly greater in wildtype knockouts compared with cell-specific and global TLR4 knockouts. The mice lacking TLR4 on astrocytes and neurons showed no differences compared with wildtype mice on any end points. CONCLUSIONS: Our data suggest that microglial depletion with the intracerebroventricular administration of clodronate can improve the cognitive function in an SAH mouse model, and TLR4 is critical for microglial activation and neuronal injury. Only microglial TLR4 is necessary for brain damage and poor cognitive outcome rather than astrocyte or neuronal TLR4. Thus, microglial TLR4 could be a potent therapeutic target to treat SAH-associated neuronal injury and protect against cognitive dysfunction.

An overview on microglial origin, distribution, and phenotype in Alzheimer's disease.

Alzheimer's disease (AD) is the most common neurodegenerative disease that is responsible for about one-third of dementia cases worldwide. It is believed that AD is initiated with the deposition of Ab plaques in the brain. Genetic studies have shown that a high number of AD risk genes are expressed by microglia, the resident macrophages of brain. Common mode of action by microglia cells is neuroinflammation and phagocytosis. Moreover, it has been discovered that inflammatory marker levels are increased in AD patients. Recent studies advocate that neuroinflammation plays a major role in AD progression. Microglia have different activation profiles depending on the region of brain and stimuli. In different activation, profile microglia can generate either pro-inflammatory or anti-inflammatory responses. Microglia defend brain cells from pathogens and respond to injuries; also, microglia can lead to neuronal death along the way. In this review, we will bring the different roles played by microglia and microglia-related genes in the progression of AD.

Effect of wild grape on the signaling of histamine H1 receptor gene expression responsible for the pathogenesis of allergic rhinitis.

As expression level of allergic disease-sensitive genes are correlated with allergic symptom severity, suppression of these gene expressions could be good therapeutics. We have demonstrated that PKCδ signaling and NFAT signaling, involve in histamine H1 receptor (H1R) and IL-9 gene expressions, respectively, are responsible for the pathogenesis of allergic rhinitis. We explore anti-allergic compounds that suppress these signaling pathways and found that wild grape (WG) contains such compounds. Here, we investigated the effect of WG hot water extract (WGE) on the signaling pathways for PKCδ-mediated H1R and NFAT-mediated IL-9 gene expressions. WGE suppressed histamine/PMA-induced H1R gene up-regulation in HeLa cells. Toluene-2,4-diisocyanate (TDI)-induced H1R mRNA elevation in TDI-sensitized rats was also suppressed by WGE treatment. Treatment with WGE in combination with Awa-tea, suppresses NFAT signaling-mediated IL-9 gene, markedly alleviated nasal symptoms. Furthermore, WGE suppressed PMA-induced IL-33 gene up-regulation in Swiss 3T3 cells. Data suggest that combination of WGE, suppresses PKCδ signaling with Awa-tea, suppresses NFAT signaling would have distinct clinical and therapeutic advantages as a substitute for anti-allergic drugs. In addition, as the expression level of IL-33 mRNA was correlated with the blood eosinophils number in patients with pollinosis, WG could alleviate eosinophilic inflammation through the suppression of IL-33 gene expression. J. Med. Invest. 65:242-250, August, 2018.