The Human Mitochondrial Genome Encodes for an Interferon-Responsive Host Defense Peptide

Reviewer #1 (Public Review):

In this work, the authors examine the mechanism of action of MOTS-c and its impact on monocyte-derived macrophages. In the first part of the study, they show that MOTS-c acts as a host defense peptide with direct antibacterial activity. In the second part of the study, the authors aim to demonstrate that MOTS-c influences monocyte differentiation into macrophages via transcriptional regulation.

Major strengths. Methods used to study the bactericidal activity of MOTS-c are appropriate and the results are convincing.

Major weaknesses. Methods used to study the impact on monocyte differentiation are inappropriate and the conclusions are not supported by the data shown. A major issue is the use of the THP-1 cell line, a transformed monocytic line which does not mimic physiological monocyte biology. In particular, THP-1 differentiation is induced by PMA, which is a completely artificial system and conclusions from this approach cannot be generalized to monocyte differentiation. The authors would need to perform this series of experiments using freshly isolated monocytes, either from mouse or human. The read-out used for macrophage differentiation (adherence to plastic) is also not very robust, and the authors would need to analyze other parameters such as cell surface markers. It is also not clear whether MOTS-c could act in a cell-intrinsic fashion, as the authors have exposed cells to exogenous MOTS-c in all their experiments. The authors did not perform complementary experiments using MOTS-c deficient monocytes. The authors have also analyzed the transcriptomic changes induced by MOTS-c exposure in macrophages derived from young or old mice. While the results are potentially interesting, the differences observed seem independent from MOTS-c and mainly related to age, therefore the conclusions from this figure are not clear. Another concern is the reproducibility of the experiments, as the authors do not indicate the number of biological replicates analyzed nor the number of independent experiments performed.

The different parts of the manuscript do not appear well connected and it is not clear what the main message from the manuscript would be. The physiological relevance of this study is also unclear.

Reviewer #2 (Public Review):

The research study presented by Rice et al. set out to further profile the host defense properties of the mitochondrial protein MOTS-c. To do this they studied i. the potential antimicrobial effects of MOTS-c on common bacterial pathogens E.coli and MRSA, ii. the effects of MOTS-c on the stimulation and differentiation of monocytes into macrophages. This is a well performed study that utilizes relevant methods and cell types to base their conclusions on. However, there appear to be a few weaknesses to the current study that hold it back from more broad application.

Comment 1: From reading the manuscript methods and results, it is unclear exactly what the synthetic MOTS-c source is. Therefore it is hard to determine whether there may be any impurities in the production of this synthetic protein that may interfere with the results presented throughout the manuscript. Though, the data presented in Supplemental Figure 4F, where E.coli expressing intracellular MOTS-c inhibited bacterial growth certainly support MOTS-c specific effects. Similarly with the experiments showing endogenous MOTS-c levels rising in stimulation and differentiated macrophages (Figure 3).

Comment 2: It is interesting that the mice receiving bacteria coupled with MOTS-c lost about 10% of their body weight. It would have been interesting to demonstrate the cause of this weight loss since the effect appears to be separate from mere PAMPs as shown by using heat-killed MRSA in Supplemental Figure 5. Was inflammation changed? Is this due to changes in systemic metabolism? Would have been interesting to have seen CRP levels or circulating liver enzymes.

Despite these concerns, the data are well suited to answering their research question, and they open up the door to studying how mitochondrial peptides like MOTS-c could have roles outside of the mitochondria.