Disruption of the Novel Nested Gene Aff3ir Mediates Disturbed Flow-Induced Atherosclerosis in Mice

Reviewer #1 (Public review):

Summary:

The authors report the role of a novel gene Aff3ir-ORF2 in flow-induced atherosclerosis. They show that the gene is anti-inflammatory in nature. It inhibits the IRF5-mediated athero-progression by inhibiting the causal factor (IRF5). Furthermore, the authors show a significant connection between shear stress and Aff3ir-ORF2 and its connection to IRF5 mediated athero-progression in different established mice models which further validates the ex vivo findings.

Strengths:

(1) An adequate number of replicates were used for this study.
(2) Both in vitro and in vivo validation was done.
(3) The figures are well presented.
(4) In vivo causality is checked with cleverly designed experiments.

Weaknesses:

(1) Inflammatory proteins must be measured with standard methods e.g ELISA as mRNA level and protein level does not always correlate.

(2) RNA seq analysis has to be done very carefully. How does the euclidean distance correlate with the differential expression of genes. Do they represent the neighborhood? If they do how does this correlation affect the conclusion of the paper?

(3) The volcano plot does not indicate the q value of the shown genes. It is advisable to calculate the q value for each of the genes which represents the FDR probability of the identified genes.

(4) GO enrichment was done against the Global gene set or a local geneset? The authors should provide more detailed information about the analysis.

(5) If the analysis was performed against a global gene set. How does that connect with this specific atherosclerotic microenvironment?

(6) What was the basal expression of genes and how did the DGE (differential gene expression) values differ?

(7) How was IRF5 picked from GO analysis? was it within the 20 most significant genes?

(8) Microscopic studies should be done more carefully? There seems to be a global expression present on the vascular wall for Aff3ir-ORF2 and the expression seems to be similar to AFF3 in Figure 1.

Reviewer #2 (Public review):

Summary:

The authors recently uncovered a novel nested gene, Aff3ir, and this work sets out to study its function in endothelial cells further. Based on differences in expression correlating with areas of altered shear stress, they investigate a role for the isoform Aff3ir-ORF2 in endothelial activation and development of atherosclerosis downstream of disturbed shear stress. Using a knockout mouse model and in vivo overexpression experiments, they demonstrate a strong potential for Aff3ir-ORF2 to alleviate atherosclerosis. They find that Aff3ir-ORF2 interacts with the pro-inflammatory transcription factor IRF5 and retains it in the cytoplasm, hence preventing upregulation of inflammation-associated genes. The data expands our knowledge of IRF5 regulation which could be relevant to researchers studying various inflammatory diseases as well as adding to our understanding of atherosclerosis development.

Strengths:

The in vivo data is solid using immunofluorescence staining to assess AFF3ir-ORF2 expression, a knockout mouse model, overexpression and knockdown studies, and rescue experiments in combination with two atherosclerotic models to demonstrate that Aff3ir-ORF2 can lessen atherosclerotic plaque formation in ApoE-/- mice.

Weaknesses:

While the in vivo data is generally convincing, a few data panels have issues and will need addressing. Also, the knockout mouse model will need to be described, since the paper referred to in the manuscript does not actually report any knockout mouse model. Hence it is unclear how Aff3ir-ORF2 is targeted, but Figure S2B shows that targeting is partial, since about 30% expression remains at the RNA level in MEFs isolated from the knockout mice.

While the effect on atherosclerosis is clear, the conclusion that this is the result of reduced endothelial cell activation is not supported by the data. The mouse model is described as a global knockout and the shRNA knockdowns (Figure 5) and overexpression data in Figure 2 are not cell type-specific. Only the overexpression construct in Figure 6 uses an ICAM-2 promoter construct, which drives expression in endothelial cells, though leaky expression of this promoter has been reported in the literature. Therefore, other cell types such as smooth muscle cells or macrophages could be responsible for the effects observed.

The weakest part of the manuscript is the in vitro experiments. While they are solidly executed, all experiments are performed in MEFs, and results are interpreted as being equivalent to endothelial cell responses. There is also an RNA-seq experiment performed on MEFs from the Aff3ir-ORF2 knockout and control mice, but the data is not disclosed other than showing some non-identifiable expression differences. The data is used to hypothesise on a role for IRF5 in the effects observed with Aff3ir-ORF2 knockout.

Overall, the paper succeeds in demonstrating a link between Aff3ir-ORF2 and atherosclerosis, but the cell types involved and mechanisms remain unclear. The study also shows a functional interaction between Aff3ir-ORF2 and IRF5 in embryonic fibroblasts, but any relevance of this mechanism for atherosclerosis or any cell types involved in the development of this disease remains largely speculative.

Reviewer #3 (Public review):

This study is to demonstrate the role of Aff3ir-ORF2 in the atheroprone flow-induced EC dysfunction and ensuing atherosclerosis in mouse models. Overall, the data quality and comprehensiveness are convincing. In silico, in vitro, and in vivo experiments and several atherosclerosis were well executed. To strengthen further, the authors can address human EC relevance.

Major comments:

(1) The tissue source in Figures 1A and 1B should be clarified, the whole aortic segments or intima? If aortic segment was used, the authors should repeat the experiments using intima, due to the focus of the current study on the endothelium.

(2) Why were MEFs used exclusively in the in vitro experiments? Can the authors repeat some of the critical experiments in mouse or human ECs?

(3) The authors should explain why AFF3ir-ORF2 overexpression did not affect the basal level expression of ICAM-1, VCAM-1, IL-1b, and IL-6 under ST conditions (Figure 2A-C).

(4) Please include data from sham controls, i.e., right carotid artery in Figure 2E.

(5) Given that the merit of the study lies in the effect of different flow patterns, the legion areas in AA and TA (Figure 3B, 3C) should be separately compared.

(6) For confirmatory purposes for the variations of IRF5 and IRF8, can the authors mine available RNA-seq or even scRNA-seq data on human or mouse atherosclerosis? This approach is important and could complement the current results that are lacking EC data.

(7) With the efficacy of using AAV-ICAM2-AFF3ir-ORF2 in atherosclerosis reduction (Figure 6), the authors are encouraged to use lung ECs isolated from the AFF3ir-ORF2-/-mice to recapitulate its regulation of IRF5.