obtaining-human-microglia-from-adult-human-brain-tissue

Obtaining Human Microglia from Adult Human Brain Tissue

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Inflammation is a double-edged sword. While short-lived, acute inflammation is essential for the repair and resolution of infection and damage, uncontrolled and unresolved chronic inflammation is central to several diseases, including cancer, autoimmune diseases, allergy, metabolic disease, and cardiovascular disease. This report aims to review the literature regarding several members of the nucleotide-binding domain, leucine-rich repeat-containing receptor (NLR) family of pattern recognition sensors/receptors that serve as checkpoints for inflammation. Understanding the negative regulation of inflammation is highly relevant to the development of therapeutics for inflammatory as well as infectious diseases. 

Holding the inflammatory system in check: NLRs keep it cool.

NLRs (nucleotide-binding domain, leucine-rich repeat containing receptors) are pattern recognition receptors associated with immunity and inflammation in response to endogenous and exogenous pathogen and damage associated molecular patterns (PAMPs and DAMPs respectively). Dysregulated NLR function is associated with several diseases including cancers, metabolic diseases, autoimmune disorders and autoinflammatory syndromes. In the last decade, distinct cell and organ specific roles for NLRs have been identified however; their roles in cancer initiation, development and progression remain controversial. This review summarizes the emerging role of NLRs in cancer and their possible future as targets for cancer therapeutics.

NLR-regulated pathways in cancer: opportunities and obstacles for therapeutic interventions.

The inflammasome is a large multimeric protein complex comprising an effector protein that demonstrates specificity for a variety of activators or ligands; an adaptor molecule; and procaspase-1, which is converted to caspase-1 upon inflammasome activation. Inflammasomes are expressed primarily by myeloid cells and are located within the cell. The macromolecular inflammasome structure can be visualized by cryo-electron microscopy. This complex has been found to play a role in a variety of disease models in mice, and several have been genetically linked to human diseases. In most cases, the effector protein is a member of the NLR (nucleotide-binding domain leucine-rich repeat-containing) or NOD (nucleotide oligomerization domain)-like receptor protein family. However, other effectors have also been described, with the most notable being AIM-2 (absent in melanoma 2), which recognizes DNA to elicit inflammasome function. This review will focus on the role of the inflammasome in myeloid cells and its role in health and disease.

Inflammasomes in Myeloid Cells: Warriors Within.

Trigonella foenum-graecum (fenugreek) is an important medicinal plant, well known for its anti-inflammatory properties. However, the underlying cellular and molecular mechanisms of its action remain largely unknown. The apoptosis associated speck like protein containing a caspase recruitment domain (CARD) (ASC) is central to inflammatory and cell death pathways in innate and adaptive immunity. Here, we show that fenugreek seed extract provides cytoprotection to bacterial lipopolysaccharide (LPS) inflammed and nanosilica-treated fibroblasts via a reactive oxygen species independent pathway. All atom molecular dynamics simulations of ASC-ligand complex reveal that individual phytochemicals in fenugreek can bind to ASC via specific non-covalent interactions. These data show that a synergistic effect of fenugreek phytochemicals with the ASC protein alters its molecular properties resulting in altered cellular function. Such information is crucial to the development of targeted therapeutic interventions for inflammatory diseases.

Trigonella seed extract ameliorates inflammation via regulation of the inflammasome adaptor protein, ASC.

Gliomas are the most common solid tumors among central nervous system tumors. Most glioma patients succumb to their disease within two years of the initial diagnosis. The median survival of gliomas is only 14.6 months, even after aggressive therapy with surgery, radiation, and chemotherapy. Gliomas are heavily infiltrated with myeloid- derived cells and endothelial cells. Increasing evidence suggests that these myeloid- derived cells interact with tumor cells promoting their growth and migration. NLRs (nucleotide-binding oligomerization domain (NOD)-containing protein like receptors) are a class of pattern recognition receptors that are critical to sensing pathogen and danger associated molecular patterns. Mutations in some NLRs lead to autoinflammatory diseases in humans. Moreover, dysregulated NLR signaling is central to the pathogenesis of several cancers, autoimmune and neurodegenerative diseases. Our review explores the role of angiogenic factors that contribute to upstream or downstream signaling pathways leading to NLRs. Angiogenesis plays a significant role in the pathogenesis of variety of tumors including gliomas. Though NLRs have been detected in several cancers including gliomas and NLR signaling contributes to angiogenesis, the exact role and mechanism of involvement of NLRs in glioma angiogenesis remain largely unexplored. We discuss cellular, molecular and genetic studies of NLR signaling and convergence of NLR signaling pathways with angiogenesis signaling in gliomas. This may lead to re-appropriation of existing anti-angiogenic therapies or development of future strategies for targeted therapeutics in gliomas.

Role of NOD- like Receptors in Glioma Angiogenesis: Insights into future therapeutic interventions.

Inflammation in the brain accompanies several high-impact neurological diseases including multiple sclerosis (MS), stroke, and Alzheimer's disease. Neuroinflammation is sterile, as damage-associated molecular patterns rather than microbial pathogens elicit the response. The inflammasome, which leads to caspase-1 activation, is implicated in neuroinflammation. In this study, we reveal that lysophosphatidylcholine (LPC), a molecule associated with neurodegeneration and demyelination, elicits NLRP3 and NLRC4 inflammasome activation in microglia and astrocytes, which are central players in neuroinflammation. LPC-activated inflammasome also requires ASC (apoptotic speck containing protein with a CARD), caspase-1, cathepsin-mediated degradation, calcium mobilization, and potassium efflux but not caspase-11. To study the physiological relevance,  and  mice are studied in the cuprizone model of neuroinflammation and demyelination. Mice lacking both genes show the most pronounced reduction in astrogliosis and microglial accumulation accompanied by decreased expression of the LPC receptor G2A, whereas MS patient samples show increased G2A. These results reveal that NLRC4 and NLRP3, which normally form distinct inflammasomes, activate an LPC-induced inflammasome and are important in astrogliosis and microgliosis.

NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes.

Gliomas are the most prevalent primary brain tumors with immense clinical heterogeneity, poor prognosis and survival. The nucleotide-binding domain, and leucine-rich repeat containing receptors (NLRs) and absent-in-melanoma 2 (AIM2) are innate immune receptors crucial for initiation and progression of several cancers. There is a dearth of reports linking NLRs and AIM2 to glioma pathology. NLRs are expressed by cells of innate immunity, including monocytes, macrophages, dendritic cells, endothelial cells, and neutrophils, as well as cells of the adaptive immune system. NLRs are critical regulators of major inflammation, cell death, immune and cancer-associated pathways. We used a data-driven approach to identify NLRs, AIM2 and NLR-associated gene expression and methylation patterns in low grade glioma and glioblastoma, using The Cancer Genome Atlas (TCGA) patient datasets. Since TCGA data is obtained from tumor tissue, comprising of multiple cell populations including glioma cells, endothelial cells and tumor-associated microglia/macrophages we have used multiple cell lines and human brain tissues to identify cell-specific effects. TCGA data mining showed significant differential NLR regulation and strong correlation with survival in different grades of glioma. We report differential expression and methylation of NLRs in glioma, followed by NLRP12 identification as a candidate prognostic marker for glioma progression. We found that Nlrp12 deficient microglia show increased colony formation while Nlrp12 deficient glioma cells show decreased cellular proliferation. Immunohistochemistry of human glioma tissue shows increased NLRP12 expression. Interestingly, microglia show reduced migration towards Nlrp12 deficient glioma cells.

Differential Expression Profile of NLRs and AIM2 in Glioma and Implications for NLRP12 in Glioblastoma.

Apoptosis associated speck like protein containing CARD (ASC) is widely researched and recognized as an adaptor protein participating in inflammasome assembly and pyroptosis. It contains a bipartite structure comprising of a pyrin and a caspase recruitment domain (CARD) domain. These two domains help ASC function as an adaptor molecule. ASC is encoded by the gene PYCARD. ASC plays pivotal role in various diseases as well as different homeostatic processes. ASC plays a regulatory role in different cancers showing differential regulation with respect to tissue and stage of disease. Besides cancer, ASC also plays a central role in sensing, regulation, and/or disease progression in bacterial infections, viral infections and in varied inflammatory diseases. ASC is expressed in different types of immune and non-immune cells. Its localization pattern also varies with different kinds of stimuli encountered by cell. This review will summarize the literature on the structure cellular and tissue expression, localization and disease association of ASC.

Comprehensive review of ASC structure and function in immune homeostasis and disease.

Nanoparticles are promising bioengineering platforms facilitating various consumer product formulations, including packaged food, electrical, biosensor and biomedical tools. The unique surface and physicochemical properties of amorphous nanosilica supports advanced nano-biomolecular applications for various manufacturing, biotechnology, and healthcare industries including cosmetics, packaging, implants, drug delivery systems and cancer diagnostics. The increased technological and economic benefits of amorphous nanosilica, raises concerns regarding their adverse biological effects on humans. The cellular mechanisms underlying amorphous nanosilica internalization, evasion of biological barriers, inadvertent nano-bio interactions and unexpected long term exposure effects must be taken into consideration from the diverse ecosystems and human safety aspects. Recent research studies reveal cytotoxic, inflammatory and immunomodulatory effects of amorphous nanosilica particles. Our review focuses on studies demonstrating hazardous impact of amorphous nanosilica/bio-systems interface on the cellular and biochemical processes. The review further seeks to evaluate amorphous nanosilica-induced cytotoxicity, innate immune responses, inflammation and immune related dysfunctions, and discuss open research questions related to the use of amorphous nanosilica in biomedicine.

Article	Year
Holding the inflammatory system in check: NLRs keep it cool. F1000prime reports\| PubMed ID: 25750733	2015
NLR-regulated pathways in cancer: opportunities and obstacles for therapeutic interventions. Cellular and molecular life sciences : CMLS\| PubMed ID: 26708292	2016
Inflammasomes in Myeloid Cells: Warriors Within. Microbiology spectrum\| PubMed ID: 28102121	2017
Trigonella seed extract ameliorates inflammation via regulation of the inflammasome adaptor protein, ASC. Frontiers in bioscience (Elite edition)\| PubMed ID: 28199188	2017
Role of NOD- like Receptors in Glioma Angiogenesis: Insights into future therapeutic interventions. Cytokine & growth factor reviews\| PubMed ID: 28233643	2017
NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes. The Journal of experimental medicine\| PubMed ID: 28404595	2017
Differential Expression Profile of NLRs and AIM2 in Glioma and Implications for NLRP12 in Glioblastoma. Scientific reports\| PubMed ID: 31186453	2019
Comprehensive review of ASC structure and function in immune homeostasis and disease. Molecular biology reports\| PubMed ID: 32124174	2020
Amorphous nanosilica induced toxicity, inflammation and innate immune responses: A critical review. Toxicology\| PubMed ID: 32525085	2020

Sushmita Jha

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