Peer review of: MG53 suppresses interferon-β and inflammation via regulation of ryanodine receptor- (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The authors are the leaders on the TRIM72 area of research. TRIM72 is also known as MG53. The 
new findings are truly exciting. 

TRIM72 is essential for cell membrane repair and is believed to be a muscle specific. The group 
now has convincing data to demonstrate demonstrate that human macrophages express MG53, 
and that MG53 protein is reduced following viral infection. Knockdown of MG53 in macrophages 
leads to increased type I interferon (IFN) production upon infection, implicating MG53 as a 
negative regulator of IFN response. They further demonstrate a specific interaction into of MG53 
signaling in activation of NFκB signaling, which is linked to an increase in intracellular calcium 
oscillation mediated by ryanodine receptor (RyR). Last, they show that MG53 interacts with RyR 
and inhibits IFNβ transcription in an RyR-dependent manner. 

This study establishes MG53 as a new macrophage target for control of virus-induced disease and 
tissue injury. It can have broad implications for public healthcare. 

I only have very minor comments: 

a) Add a couple of sentences about the overall role of macraphages on inflammation and the 
switch in M1 to M2 for resolution of inflammation. 

b) Item a above, do the same in the discussion. Could speculate whether MG53 interaction occurs 
with M1 or M2, likely M2. 

c) The data is very robust and it is excellent to see all data points plotted for transparency. We do 
recommend that for some of the data, the authors further check the statistics with non parametric 
tests and ad hoc adjustments. 

d) The authors could test using either Pearson or Spearman whether there is a direct correlation 
between MG53 levels and immune activation or disease state. 

Reviewer #2 (Remarks to the Author): 

In their paper the Authors describe a novel role for the skeletal muscle protein MG53 as a 
mediator of innate immune response. The Authors show that MG53 is expressed in human 
macrophages where it regulates the expression of interferon. Knocking down MG53 leads to 
increased interferon production via NFkB signaling. The increased nuclear localization of NFkB is 
mediated by RyR-dependent calcium oscillations. 
These observations are novel and interesting, especially because until now the primary role of 
MG53 was thought to be primarily tissue (skeletal muscle) repair. 
In my opinion additional experiments need to be performed in order to be convincing. 
Major comments 
1) In Figure 1 the Authors show by IP that macrophages express MG53. Can they see an 
immunopositive band by simple Western blot and not only after immunoprecipitation? No 
information regarding the total amount of protein loaded is indicated. How many cells were lysed? 
How many micrograms of proteins were loaded on the gels? They state that the levels are much 
lower than in skeletal muscle, but this is not quantified. Quantification by RT-qPCR or western blot 
comparing the intensity of the immunopositive band in skeletal muscle, cardiac muscle, 
macrophages and several other tissues is necessary. No explanation for panel 1D is included. How 

were the values normalized? 
2) In figure 2 and figure 3 they show that knocking down/out MG53 leads to an increase in IFN 
production, yet mice infected with influenza virus do not show a difference in viral load. How 
come? IFN is released by cells to counterbalance viral infections and thus higher levels of IFN 
should be associated with a decreased viral load and lower morbidity. Mice should recover faster 
and not slower if they have an enhanced inflammatory response. Production of additional cytokines 
should be assessed including IL6, TNFa, IL12, IL23 as well as chemokines. Phenotypically distinct 
classes of macrophages exist, M1 which are pro-inflammatory and microbicidal and M2 which are 
immunosuppressive. Does the absence of MG53 affect all macrophage types equally? Please also 
add serum levels of CRP as this is a good indication of the inflammatory response. 
3) The fact that lungs and heart show increased levels of IFN and IL1 may simply be a 
consequence of the higher number of macrophages present in those tissues. Can the levels of 
these cytokines be normalized to number of macrophages present? As shown in figure 3 levels of 
the cytokines are not normalized to anything. 
4) The involvement of RyR in calcium homeostasis in THP cells is not convincing. Extremely high 
levels (2.5 mM) of 4-CmC were used. The Authors need to show that there is an effect on calcium 
release following the addition of 300-600 µM 4-CmC or 5-10 mM caffeine. They should also show 
that this effect can be blocked with dantrolene, procaine or by pre-incubating the cells with high 
levels of ryanodine as well as in cells silenced for RyR1 and RyR2. 
5) The qPCR results show that RYR1 and RYR2 are expressed in THP cells and that their levels of 
expression are not affected by MG53 levels. What are the levels of RYR1 and RYR2 expression in 
THP cells compared to heart, skeletal muscle, brain, cerebellum, smooth muscle cells and other 
immune cells? 
6) It is puzzling that MG53 and RyR1 form a complex since to my understanding MG53 is in a 
different compartment (either cytosolic or sarcolemmal) while the RyR1 is in the sarcoplasmic 
reticulum. Does MG53 form a complex with the DHPR? and junctophilin? Because MG53 is a TRIM 
protein, it may be sticky and bind non-specifically to many proteins in vitro but this may never 
happen in vivo. 

Minor 
1) Statistical analysis tests are not always defined (see for example figure 4, 5, 7, which post hoc 
test was used after the ANOVA? What was used in figure 5F?). 
2) Many panels in the figures are not described. Please add the missing information to panels 1D, 
2C. 
3) For all experiments, details of how many micrograms of protein were loaded per gel lane need 
to be added. 

Reviewer #3 (Remarks to the Author): 

The manuscript submission by Jianjie Ma and colleagues reports on the role of MG53/TRIM72 in 
regulating innate antiviral responses by controlling inflammatory responses to virus infection. 
Results from cell culture experiments and infection studies with MG53-knockout mice revealed that 
inflammatory responses are elevated in the absence of MG53 and following virus infection; 
however, the absence or presence of MG53 did not alter viral titers. Additional in vitro studies 
indicated that virus infection decreased MG53 protein levels by affecting translation, but not 
transcription. In general, the study presents findings for a novel role and potential mechanism by 
which MG53 regulates innate antiviral responses; however, the manuscript requires modifications 
to improve clarity before it could be consider for acceptance for publication. 

Item 1. Considering the current findings, it would be relevant to describe whether MG53 
expression and/or function is altered in patients diagnosed with type I interferonopathies or other 
inflammatory conditions? 

Item 2. The scientific premise for the study is not adequately developed. The introduction 
summarizes that MG53 is generally a muscle-specific factor that functions in cell membrane repair. 
The rationale for suspecting MG53 would be involved in regulating inflammatory responses must 
be improved. The introduction would also benefit from inclusion of relevant references that 
strengthens the connection between innate immunity and tissue repair, especially by what was 
perceived to be a muscle-specific factor. As the in vitro studies included THP-1 cells, the 
connection of wound repair and inflammatory responses in macrophages, and thus the subject of 
investigation in Figure 1, is unclear. 

Item 3. The authors demonstrated by in vitro studies that Sendai virus infection suppressed 
translation of MG53, which resulted in increased expression of interferon and IL-1beta. These in 
vitro studies were then followed by in vivo studies in which wild-type or MG53-deficient mice were 
infected by PR8 to examine alterations in morbidity and host responses. It is unclear if PR8 alters 
MG53 proteins levels in the same manner as by Sendai virus infection. 

Item 4. Is the expression of other genes regulated in a calcium-dependent manner? 

Item 5. For Figure 1C, was PMA included during the duration of the experiment? PMA treatment 
increased MG53 protein levels; however, it is unclear if MG53 protein levels decreased due to lack 
of stimulation by PMA or if virus infection actively decreased MG53 protein levels. For Figure 1D, 
were only two mock samples analyzed for the 48-hour time point? The figure legend should be 
revised to indicate what D1 and D2 designate. 

Item 6. The title of the legend for Figure 6 indicated that MG53 regulated the activity of the 
ryanodine receptor. However, the data presented demonstrates an interaction between MG53 and 
the ryanodine receptor calcium channel protein. The figure does not provide data that MG53 
specifically regulates the function of the ryanodine receptor calcium channel protein. The data 
presented in Figure 5 indicated that MG53 regulated intracellular calcium dynamics, and inclusion 
of the agonist 4-CmC indicated the involvement of the ryanodine receptor calcium channel protein. 
However, there is no data that indicated MG53 specifically targeted the function of the ryanodine 
receptor calcium channel.