Critical Appraisal Of Human Microglia and Astrocytes Express cGAS-STING Viral

Sensing Components

Roy Bagus Kurniawan

011711133003
A Class

Faculty Of Medicine
Universitas Airlangga
Surabaya
 Critical Appraisal of a Journal Article

Name : Roy Bagus Kurniawan

NIM : 011711133003
Title of Paper : Human microglia and astrocytes express cGAS-STING viral sensing
components

Published on : Neuroscience Letters 658 (2017) 53–56

Date of publish : August 19, 2017

1. FORMAT PAPER :

Item A/NA
 Title A (Page 53)
 Abstract and or Summary A (Page 53)
 Introduction, background A (Page 53 - 54)
 Method A (Page 54)
 Result A (Page54 - 55)
 Discussion A (Page 55 - 56)
 Acknowledgement NA (In the page 56, it is stated
that acknowledgement is not
applicable)
 Reference A (Page 56)

A: Available
NA: Not Available

Conclusion :Complete/Not Complete

2. VALIDTY OF RESEARCH

The objectives of Study : to show that human microglia and astrocytes functionally respond to
cytosolic B form DNA and that these cell types constitutively express robust levels of cGAS and
STING.
Methodology
Item Explanation Pages
Design From the journal we have appraised, 54
specifically in the methods (subpoint 2.2)
explanation, it is implied that the
research used Randomized Controlled
Trials (RCTs).
Hierarchy of Evidence Second level
Sample 1. U87 MG, an immortalized human 54
astrocytic cell line which is (subpoint 2.1)
obtained from the ATCC (HTB-
14).
2. The human microglial cell line,
hμglia, a kind gift from
Dr.Jonathan Karn (Case Western
Reserve University).
3. Primary human astrocytes and
microglia which were purchased
from ScienCell Research
Laboratories (Carlsbad, CA) and
were cultured in medium supplied
by the vendor.

Sample Size Sample size is not mentioned because 54

the samples are several cells which (subpoint 2.1)
 are cultivated in several different
medium.
Inclusion Criteria Researchers take the human glial cells 54
(astrocytes and hμglia) and primary (subpoint 2.1)
cells as the samples.
Sampling Frame U87 MG, an immortalized human 54
astrocytic cell line, was obtained from (subpoint 2.1)
the ATCC (HTB-14). Cells were
maintained in Eagle's Minimum
Essential Medium supplemented with
10% FBS and
penicillin/streptomycin. The human
microglial cell line, hμglia, was a kind
gift from Dr. Jonathan Karn (Case
Western Reserve University). These
cells were derived from primary
human cells transformed with
lentiviral vectors expressing SV40 T
antigen and hTERT, and have been
classified as microglia due to their
microglia-like morphology,
migratory and phagocytic activity,
presence of the microglial cell surface
markers CD11b, TGFβR, and
P2RY12, and characteristic
microglial RNA expression profile.
This cell line was maintained in
Dulbecco's Modified Eagle Medium
supplemented with 5% FBS and
penicillin/streptomycin. Primary
human astrocytes and microglia were
 purchased from ScienCell Research
Laboratories (Carlsbad, CA) and
were cultured in medium supplied by
the vendor.

Methodology Firstly, the researchers determined 54

the samples and their source, then, (Methods)
were continued by propagating the
samples in several mediums. In this
research, the samples were U87 mg
(immortalized human astrocytic cell
line), Hμglia, and primary human
atrocytes and microglia.

Secondly, all of samples were given

in vitro stimulation with B-DNA. For
comparison purpose, cells were
exposed to transfection reagent alone
or were untreated.

Thirdly, at the indicated time points

following transfection, whole cell
protein isolates were collected and
RNA was isolated for immunoblot
analysis, semi-quantitative RT-PCR,
and performed quantification of IFN-
β in glial cell culture supernatants,
respectively.

Lastly, data was presented as the

mean ± standard error of the mean
(SEM). Statistical analyses were
performed using Student’s t-test or
 one-way analysis of variance
(ANOVA) with Tukey’s post hoc test
using commercially available
software (GraphPad Prism, GraphPad
Software, La Jolla, CA). In all
experiments, results were considered
statistically significant when a P-
value of less than 0.05 was obtained.

Measurement and or Immunoblot analysis for phospho- 54

Assessment IRF3 (pIRF3), cGAS, and STING (Methods)

Whole cell protein isolates were

collected from microglial and
astrocytic cells using Triton lysis
buffer (10mM Tris-HCl pH 10.5,
5mM MgCl2, and 1% (v/v) Triton X-
100) and analyzed by immunoblot
analysis. Bound enzyme was detected
with the Super Signal system
(Thermo Fisher Scientific).

To assess total protein loading in each

well, immunoblots were reprobed
with a goat anti-mouse β-actin
antibody (Santa Cruz Biotechnology,
Santa Cruz, CA).

RNA extraction and semi-quantitative

reverse transcription PCR (RT- PCR)

Total RNA was isolated from

cultured glial cells using Trizol
Reagent (Thermo Fisher Scientific)
according to the manufacturer’s
instructions and quantified using a
Nanodrop ND-1000 spectro-
photometer. Semiquantitative RT
PCR was performed on 5% of the
reverse-transcribed cDNA product

To assess the relative levels of

expression of mRNA-encoding IFN-
β and the housekeeping gene product
glyceraldehyde 3-phosphate
dehydrogenase (GAPDH).

Quantification of IFN-β in glial cell

culture supernatants A

A specific capture ELISA was

performed to quantify concentrations
of human IFN-β using a polyclonal
rabbit anti-human IFN-β capture
antibody and a biotinylated
polyclonal rabbit anti-human IFN-β
detection antibody (Abcam,
Cambridge, MA)

A standard curve was constructed

using varying dilutions of
recombinant human IFN-β (Abcam)
and the cytokine content of culture
supernatants determined by extra-
 polation of absorbances to the
standard curve.

Finally the outcomes from this study

are below.

1. Human microglia and

astrocytes functionally
respond to B-form DNA.
2. Human microglia and
astrocytes constitutively
express robust levels of the
viral DNA sensor cGAS.
3. Human microglia and
astrocytes constitutively
express robust levels of the
critical downstream adaptor
protein STIN.

Instrument Eagle’s Minimum Essential Medium, 54

Dubelco’s Modified Eagle Medium, (methods)
Lipofectamine 2000 transfecion
reagen, Triton lysis buffer, 12% SDS-
polycrylamide gel, Immobilon-P
transfer membranes, Bio-Rad
ChemiDoc imaging system,
ImageLab software, Trizol reagen,
Nanodrop ND-1000
spectrophotometer, TMB substrate,
 H2SO4 stop solution, and GraphPad
Prism software.
Randomization Randomization was performed in this 54
research. It is indirectly stated in the (subpoint 2.2)
methods explanation that there hadnt
had any specific selection in exposing
B-DNA to the samples in this
research. The intervention was
arbitrarily exposed to the samples to
know the effect of intervention,
respectively.
Intervention In vitro stimulation of human 54
microglia and astrocytes with B-DNA (subpoint 2.2)
Analysis Method Data collected from experiment were 54
analyzed by Statistical analysis to be (subpoint 2.6)
furtherly drawn a reliable conclusion.

a) Was the instruments suitable for the data that the researchers wants to measure?
Yes, it was. Chemical and statistical instrument used in this study is suitable with the
purpose and design of this study itself.
b) Was the study design appropriate for the research question or objectives of the study?
Yes, it was. It is states in the introduction that the researchers want to show that human
microglia and astrocytes functionally respond to cytosolic B form DNA and these cell types
constitutively express robust levels of cGAS and STING. Therefore, to reach this purpose,
the researchers conducted Randomized Control Trials (RCTs) and this design is suitable
enough to prove their pre-assumption (implied in methods, page 54)
c) Did the study methods address the most important potential sources of bias?
Yes, it did. All of the research sequences performed in the controlled environment (in vitro)
to ensure that the effect generated is merely because of intervention given. Besides, they
also use statistical analysis to ensure that bias is surely minimalized as the all experiments,
 results were considered statistically significant when a P-value of less than 0.05 was
obtained. (implied in subpoint 2.2 and stated in data analysis, page 54)
d) Was the study performed according to the original protocol?
Yes, it was. All of the research were performed based on the protocol mentioned in method
explanations and kept in track based on the purpose of the study (implied in methods
explanation, page 54)

e) Were the statistical analyses performed correctly?

Yes, it were. They use many statistical software to give an imaging about all of the data
gotten from this research and it is so much helpful in the analyzing and accurately
interpreting the findings (stated in data analysis, page 54)
f) Do the data justify the conclusions?
Yes, they do. All of the data gotten from this study justify the conclusions- human
microglia and astrocytes functionally respond to B-form DNA, human microglia and
astrocytes constitutively express robust levels of the viral DNA sensor cGAS, and human
microglia and astrocytes constitutively express robust levels of the critical downstream
adaptor protein STIN (stated in results, pages 54-55)
g) Are there any conflicts of interest?
No, there are not. The authors declare that they have no competing interests (clearly stated
in pages 56)

Consclusion : Valid / Not Valid (based on item a,b,c,d,e,f,g?)

3. The Importance of the research

Does the study add anything new?
Yes, it does. We know that this research proves human microglia and astrocytes
functionally respond to B-form DNA, human microglia and astrocytes constitutively
express robust levels of the viral DNA sensor cGAS, and human microglia and astrocytes
constitutively express robust levels of the critical downstream adaptor protein STIN.
Besides, these findings clearly contribute in exploring our understanding in neuroscience
field. Besides, The present finding that human glia express the principle components of the
cGAS-STING pathway provides a foundation for future studies to investigate the relative
importance of these molecules in clinically relevant viral CNS infections (stated in abstract
and results).