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Schindler et al. Microparticles in neuroimmune signaling

to other endogenous molecules such as damage- DAMPs with immunomodulatory properties similar
associated molecular patterns (DAMPs) released by to HMGB1 or TFAM. [15,29,30] We demonstrated that
damaged or dying cells. DAMPs identified in the human mononuclear phagocytes release MPs when
[10]
CNS include the DNA-binding proteins high-mobility activated by pro-inflammatory molecules. We also
group box 1 (HMGB1) and mitochondrial transcription showed that THP-1 monocytic cell-derived MPs act in
factor A (TFAM). [11,12] an autocrine or paracrine manner to induce secretions
of pro-inflammatory cytokines and cytotoxins by these
Recently, microparticles (MPs) have emerged as novel monocytic cells.
intercellular signaling agents, which can be released
by all brain cell types. MP release from microglia is METHODS
upregulated following their activation and such microlia-
derived MPs appear to possess immunomodulatory Reagents
properties similar to HMGB1. [13-16] Originally considered Sodium dodecyl sulphate (SDS), N,N-dimethylformamide
as inert platelet by-products, MPs are now known to (DMF), 0.05% trypsin with ethylenediaminetetraacetic
be a heterogeneous population of membrane-derived acid (EDTA), fetal bovine serum (FBS), Dulbecco’s
vesicles ranging in diameter from 0.1 to 1 μm, which modified Eagle medium: nutrient mixture F-12 Ham
participate in intercellular signaling. [17,18] The structural (DMEM/F12), penicillin/streptomycin stock solutions,
composition and content of MPs differs based on a bovine serum albumin (BSA), diethanolamine, and
variety of factors, including the cell type of origin and the Pierce bicinchoninic acid (BCA) Protein Assay
the nature of the inducing stimulus. [18] Kit were purchased from ThermoFisher Scientific
(Ottawa, ON, Canada). Dimethylsulfoxide (DMSO),
As MPs can mediate intercellular communication, lipopolysaccharide (LPS) from Escherichia coli
they have been implicated in the regulation of various O55:B5 and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
physiological processes, including, cell proliferation, 2H-tetrazolium bromide (MTT) were purchased
coagulation, and inflammation. Recent studies have
[19]
also identified MP involvement in disease processes from Sigma-Aldrich (Oakville, ON, Canada).
and the contribution of MPs to the progression of Human interferon (IFN)-γ and enzyme-linked
some neurodegenerative disorders has become immunosorbent assay (ELISA) development kits for
increasingly evident. [20-24] Elevated levels of MPs have IL-6, tumor necrosis factor (TNF)-α, and monocyte
been detected in the plasma and cerebrospinal fluid of chemoattractant protein-1 (MCP-1) were purchased
individuals suffering from Alzheimer’s disease, multiple from Peprotech (Embrun, ON, Canada). The FITC
sclerosis, and cerebral malaria. [20,25,26] Annexin-V Apoptosis Detection Kit was purchased
from BD Biosciences (Mississauga, ON, Canada).
Upon activation by adenosine triphosphate, microglia Nanobead NIST traceable particle size standards
and astrocytes have been found to release MPs were purchased from Polysciences Inc. (Warrington,
carrying the pro-inflammatory cytokine interleukin PA, USA). Recombinant human TFAM was a gift
(IL)-1β. [15,27] Moreover, microglia-derived MPs have from Dr. K. Wolthers (University of British Columbia
been shown to transfer inflammatory stimuli to other Okanagan Campus, Kelowna, BC, Canada).
microglial cells, which then express pro-inflammatory
genes such as IL-1β and IL-6. These findings Cell culture models
[28]
suggest that inflammatory mediators may be liberated The human monocytic THP-1 cell line was purchased
from glia-derived MPs and subsequently interact with from the American Type Culture Collection (ATCC,
surrounding cells, thus contributing to the neurotoxic Manassas, VA, USA). The human neuroblastoma SH-
environment observed in neuroinflammatory diseases. SY5Y cell line was donated by Dr. R. Ross (Department
Therefore, elucidating MP involvement in glial cell- of Biological Sciences, Fordham University, Bronx,
mediated neuroinflammation may identify additional NY, USA). Cell cultures were grown in DMEM/F12
targets for the development of therapeutic strategies media containing 10% FBS, penicillin (100 U/mL) and
aimed at neuropathologies with neuroinflammatory streptomycin (100 μg/mL), and incubated at 37°C in
components. humidified 5% CO and 95% air atmosphere.
2
Although MP release by all major CNS cell types has MP isolation
been demonstrated, the role of MPs as mediators MPs were isolated using previously published
in glia-neuron communication remains to be fully protocols, which were modified as follows. [15,27,31]
characterized. [15,16,27] Since MP release is upregulated Human monocytic THP-1 cells were counted using a
upon activation and during apoptosis or necrosis; hemocytometer, centrifuged for 7 min at 450 g, and
we hypothesized that MPs may act as endogenous seeded into 12-well plates at 0.5 million cells/mL in
Neuroimmunology and Neuroinflammation ¦ Volume 3 ¦ October 28, 2016 233