RE Simmonds presents a study on the role of LPS on human monocyte-derived macrophages in the context of regulation of miRNA abundance. The author first analysed relative levels of miRNA during LPS stimulation by microarray technology. This resulted in identification of several miRNAs that were upregulated more than 1.5 fold upon LPS treatment. The most upregulated was miR-155-3p, derived from a pri-miR-155 (B cell integration cluster RNA - BIC). Absolute quantification of miR-155-3p. miR-155-5p and BIC showed intricate, time-dependent regulation of their abundance upon LPS stimulation. Intriguingly, other pathogen-associated molecular patterns also influenced miR-155-3p. miR-155-5p and BIC abundance but with various profiles. Finally, the author wanted to see if one of the predicted miR-155-3p target (TNF) was affected by the miR-155-5p or miR-155-3p inhibition. In general, it's an interesting study that shows fast and substantial response from the miRNAs to LPS stimulation. That said, few important controls are missing, the research into miRNA biogenesis is not adequately cited, some figure panels are not referenced in the text and most importantly there are other possible explanations for the observed phenotypes that have to be considered.

My specific comments are as follow:

1. Please expand the CD14 and MD2 abbreviations.

2. It would be good to provide GO term analysis of miR-155-5p and miR-155-3p predicated and validated targets. This might help to see a broader picture of what these miRNAs regulate in the context of innate immune response.

3. "...miR-155-3p was >100-fold induced at 2 hours, after which the levels began to reduce again (Figure 1D)..." I am pretty sure this is a reference to Figure 1E.

4. "...following exposure of macrophages to LPS were monitored alongside miR-155-5p and miR-155-3p in 10 human donors, taking a more detailed look at either early or later timepoints following stimulation (compiled data, Figure 1E)..." This is a reference to Figure 1F.

5. The main conclusion of the paper is that the biogenesis of miR-155-5p and miR-155-3p is altered during LPS stimulation. Unfortunately, there is no evidence for that. One possible way to get this information would be to perform in vitro pri-miRNA and pre-miRNA processing assays (before and after LPS stimulation). Also, one could analyse the levels of pre-miR-155 upon LPS stimulation. This could add additional layer of information about which biogenesis step (if any) is regulated.

6. There is another possible explanation for the observed phenotypes. It is possible that while miR-155-3p is upregulated in line with the BIC levels, the stability of miR-155-5p is compromised after LPS stimulation. To assay that, one would have to inhibit pol II (by actinomycin D) and trace the levels of miR-155-5p and miR-155-3p to calculate their half-life with and without LPS stimulation.

7. I don't understand the use of cycloheximide? I would rather see inhibition of pol II by actinomycin D (as described before) to see if the regulation of miR-155 is uncoupled from the regulation of transcription.

8. The reference to Figure 3D is missing.

9. The references to papers in miRNA biogenesis filed are completely missing. There is a mention of the KHSRP protein regulating miRNA biogenesis but no reference provided. My advice is to at least cite some recent comprehensive reviews on this subject (Treiber et al. 2019 (1), Michlewski and Caceres 2019 (2) and Creugny et al. 2018 (3)).

10. The author reports that there were failed attempts to validate predicted mRNA targets for miR-155-3p function. This is a wrong approach. The focus should be on the proteins that these mRNAs code as the effects of miR-155-3p could be mostly visible at the level of protein translation.

Is the work clearly and accurately presented and does it cite the current literature? Partly

Is the study design appropriate and is the work technically sound? Partly

Are sufficient details of methods and analysis provided to allow replication by others? Yes

If applicable, is the statistical analysis and its interpretation appropriate? Yes

Are all the source data underlying the results available to ensure full reproducibility? Yes

Are the conclusions drawn adequately supported by the results? Partly

References 1. Treiber T, Treiber N, Meister G: Regulation of microRNA biogenesis and its crosstalk with other cellular pathways.Nat Rev Mol Cell Biol. 2019; 20 (1): 5-20 PubMed Abstract | Publisher Full Text 2. Michlewski G, Cáceres JF: Post-transcriptional control of miRNA biogenesis.RNA. 25 (1): 1-16 PubMed Abstract | Publisher Full Text 3. Creugny A, Fender A, Pfeffer S: Regulation of primary microRNA processing.FEBS Lett. 592 (12): 1980-1996 PubMed Abstract | Publisher Full Text

Competing Interests No competing interests were disclosed.

Reviewer Expertise microRNA biogenesis, RNA-binding proteins, innate immunity.

I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.