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Figueroa-Hall et al. TLR4-mediated signaling in CHME-5 cells

TLR4 gene expression and immunofluorescence were significantly increased after LPS treatment. Conclusion: These data demonstrate
that CHME-5 cells are not human, but are indeed a beneficial tool for studying microglial inflammatory signaling.

INTRODUCTION TLR4 is the most widely studied TLR, and its
expression is more abundant in microglia than in any
The innate immune response is instrumental in other resident cell in the CNS [17,18] . TLR4 signaling
combatting infection and in response to stress and is well defined in peripheral immune cells, but less
physical injury. One family of receptors expressed clear in the CNS. Here, we characterized LPS-
on innate immune cells is toll-like receptors (TLRs). induced TLR4 inflammatory signal transduction in
TLRs can recognize a host of patterns produced by the CNS using a fetal-derived microglial cell line,
bacteria, viruses, and fungi, known as pathogen- CHME-5. To our knowledge, this is the first report
associated molecular patterns, or self-products to validate the finding that CHME-5 is not a human cell
released from apoptotic cells, called damaged- line, and show that microglial responses in CHME-5
[1]
associated molecular patterns . To date, 10 TLRs cells are comparative to human primary microglia.
have been identified in humans, 12 in rodents [2,3] . Therefore, we demonstrate that CHME-5 can be used
as an experimental tool for microglial research and
TLR4 was discovered as the receptor responsible neuroinflammatory signaling.
f o r d ete c t i o n of G r a m - n e g at i ve b ac te r i a l
lipopolysaccharide （LPS） , which leads to production METHODS
of pro-inflammatory cytokines and up-regulation of co-
stimulatory molecules necessary for initiation of the Cell culture
adaptive immune response [4-6] . TLR4 recognizes LPS CHME-5 cells were originally developed by Janabi et al.
[19]
with the aid of several accessory proteins, including and were gifted to our lab by Dr. Pierre Talbot, Quebec,
LPS-binding protein (LBP), cluster of differentiation Canada. CHME-5 growth media consisted of Dulbecco’s
14, and myeloid differentiation 2. Activation of TLR4 modified eagle medium (DMEM) with 4.5 g/L glucose
leads to initiation of 2 distinct signaling pathways, and sodium pyruvate without L-glutamine, 10% fetal
MyD88-dependent and TRIF-dependent pathways [3,7] . bovine serum, 200 mmol/L L-glutamine, penicillin-
streptomycin (100 U/mL potassium penicillin, 100 μg/mL
Until the past decade the central nervous system streptomycin sulfate), and 250 μg/mL amphotericin B.
(CNS) has been generally considered as an “immune CHME-5 cells were maintained in growth media at 37 °C,
privileged” site because of the extensive defense with 5% CO 2 . For experimental assays, growth medium
and regulatory mechanisms available to protect this was aspirated from cells and replaced with serum-free
[8]
organ from foreign cells and pathogens . But we now media (SFM) for no less than 16 h at 37 °C.
know there are cells expressed in the CNS that detect
infection or injury and respond accordingly. LPS treatment
Lipopolysaccharide from Escherichia coli O55:B5
Microglia, the primary immune cells, are also known (#L2880, Sigma-Aldrich, St. Louis, MO, USA) was
as “macrophages of the CNS”. Microglia interact with purified by phenol extraction. The lyophilized powder
other glia, namely astrocytes, to support neuronal was reconstituted in HyPure cell culture grade water and
function, survey/patrol for and clear foreign or sterile filtered to a stock concentration of 1.2 mg/mL.
harmful particles, and regulate neuroinflammation Cells were stimulated with 1.0 μg/mL LPS unless
through pro-inflammatory mediators [9,10] . Microglial otherwise noted. For dose-response studies, 0.001-
activation is characterized by morphological changes, 10 μg/mL was used for stimulation.
proliferation, and up-regulation of receptors including
scavenger, complement, cytokine/chemokine STR genotyping
and pattern recognition receptors. Recognition of PowerPlex 21 System (Promega-#DC8902) was
pathogens or insult leads to activation and release used to validate Short tandem repeats (STR)
of pro-inflammatory and neurotoxic factors including regions in CHME-5 cells. Reactions were set up
tumor necrosis factor-α (TNFα), interleukin-1β, using PowerPlex 21 5× Master Mix [5.0 µL/reaction
interferon gamma inducible protein-10 (CXCL10), nitric (rxn)], PowerPlex 21 5× Primer Pair Mix (5.0 µL/
oxide (NO) and reactive oxygen species (ROS) [9,11,12] . rxn), DNA template (0.5 ng), control DNA (0.5 ng), and
Microglial activation has been implicated in numerous water (up to 25 µL). Thermocycler settings were as follows:
neurodegenerative diseases including multiple 96 °C-1 min, (94 °C-10 s, 59 °C-1 min, 72 °C-1 min for 30
sclerosis, Alzheimer’s disease, and Parkinson’s cycles), 60 °C-10 min. Results were analyzed using
disease [13-16] . GeneMapper-ID Software (Applied Biosystems).
220 Neuroimmunology and Neuroinflammation ¦ Volume 4 ¦ October 19, 2017