Peer review of: Triacylglycerol synthesis enhances macrophage inflammatory function (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

 

In this paper, Castoldi et al find evidence for a role for triglycerides in inflammatory macrophage 
activation. Inhibiting triglyceride synthesis blocked the production of cytokines and PGE2 and 
phagocytic activity. PGE2 production with downstream consequences such as IL1beta production 
seems especially important. These findings are interesting but the following issues need to be 
addressed. 

1. What is the precise link between TGs and PGE2? This is missing here and is an important 
consideration. Do TGs lead to phospholipid synthesis with arachidonic then being released (which 
is likely) or is the arachidonic acid coming directly from the TGs? Without these kinds of data the 
link to PGE2 is somewhat indirect. 
2. Is COX2 also being regulated? This might also contribute to the production of PGE2. 
3. The link to IL1beta is clear here but why is IL6 also being regulated? Is that downstream of IL1? 

Reviewer #2 (Remarks to the Author): 

This manuscript by Castoldi and colleagues investigates the function of DGAT1-mediated 
triglyceride synthesis in macrophages during inflammation. The authors find that a 
proinflammatory stimulus upregulates lipid synthesis pathway genes, including DGAT1. Most 
intriguingly, they report that systemic inhibition of DGAT1 lowers inflammation, for instance 
measured as hypothermic response to LPS injection. They also find that the DGAT1 effect can be 
rescued in some assays by supplementation with prostaglandins. 
Overall, this area of research is interesting and some of the data are quite intriguing. However, 
there are at least two major problems that make it difficult to support publication of this study. 
First, throughout the manuscript, the treatment of cells or mice with a DGAT1 inhibitor is equated 
with lowering triglyceride synthesis and second there is no mechanistic understanding of the 
effects that are reported. Below these, and a number of less dominant, yet important points are 
explained in more detail:. 

1) In this paper, T863 treatment is equated with inhibiting TG synthesis. There are at least three 
problems with this assumption: 
• While it is reasonable to assume that inhibiting DGAT1 with this compound lowers TG synthesis, 
there are other effects and TG synthesis in many systems can be redundant (catalyzed by DGAT2). 
For instance, it is possible that specific lipid synthesis intermediates accumulate, triggering ER 
stress or mitochondrial dysfunction (independent of triglyceride synthesis per se). AS for the 
redundancy, it is puzzling why the authors have not included DGAT2 and DGAT1 /DGAT2 double 
inhibition in their studies (as this gene appears also upregulated). 
• Moreover, although T863 is well characterized, it is unclear whether it might have “off-target” 
effects. There are well characterized genetic models for DGAT1 deficiency (in cells, and animals) 
that could have been used to complement these studies. 
• It is hard to see how systemic administration of a drug can be linked to macrophages 
specifically. So, while I find the experiments presented e.g. in Fig. 2n very interesting, without a 
cell line-specific inactivation of DGAT1, they are next to impossible to interpret. 

2) The mechanism of action for DGAT1 inhibition remains unclear. Is it through changes in gene 
expression, alterations of LD-localized biochemical reactions, or some other mechanism? The 
paper provides some correlations but does not really probe any mechanism. This would need to be 
clarified greatly in my opinion. In this regard, the experiments with PGE2 are interesting and 
Figure 2f is suggestive- but the link between DGAT1 inhibition and PGE2 synthesis remains unclear 
(and alternative explanations, not linked to TG synthesis but for instance ER or mito stress can be 
imagined). 

3) there are some problems with the data presented: gene expression changes are not well 
statistically analyzed and the reported changes seem quite small. They also are somewhat 

selective. For instance, the authors report all the GPAT genes, except GPAT1, which is thought to 
provide most substrate for triglyceride synthesis. 
Similarly, the lipidomic and metabolomic data are somewhat weak: “peak area” is very arbitrary 
and hard to evaluate. Ideally, this is normalized to internal standards. For TG and some of the 
other lipids, other quantitative assays are easy to perform as well (in case they authors do not 
have access to lipidomics with standards). 
For the point that IFNg increases TG synthesis, ideally a pulse chase/experiment would be good to 
support the data in figure 1. 
Moreover, cellular changes with LPS and DGAT1i are hard to evaluate (e.g. 3h), more data, and 
quantitative evaluation would need to be provided to make more clear insights about these 
phenotypes.