Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript investigates the role of lncRNA NR_003508 in Pseudomonas aeruginosa infection and proposes that NR_003508 enhances macrophage autophagy through two ceRNA branches, miR-344i/LC3 and miR-344g-5p/p62, thereby promoting bacterial clearance. The topic is potentially interesting, and the combination of cell-based assays, mouse experiments, and mechanistic assays gives the study some breadth. However, in its current form, the manuscript still has important conceptual and methodological weaknesses, and I believe it requires major revision before it can be considered further.

My main concern is that the manuscript still overstates the mechanistic certainty of its central model. The authors conclude that NR_003508 “boosts autophagy” by facilitating LC3 processing and p62 degradation, but the presented evidence does not fully establish this as a direct and specific mechanism. The data show changes in LC3 and p62, Cyto-ID signal, tandem mRFP-GFP-LC3 puncta, and bacterial burden after NR_003508 knockdown, which is suggestive, but not sufficient to conclude that the lncRNA directly drives productive autophagic flux through the proposed dual ceRNA pathway. In particular, the manuscript would be stronger if the authors used standard pharmacologic autophagy-flux controls or lysosomal blockade experiments to distinguish more clearly between increased autophagosome formation and altered downstream turnover. At present, the conclusions are somewhat stronger than the data allow.

A related issue is that the study does not fully demonstrate that the observed effect on bacterial clearance is specifically mediated through autophagy. NR_003508 knockdown increases bacterial load in vitro and in vivo, but macrophages can influence bacterial killing through many pathways beyond autophagy. The manuscript assumes that the change in colony counts is a direct consequence of altered autophagy, but this is not directly shown. Additional experiments linking the bacterial phenotype to autophagy dependence would substantially strengthen the work.

I also found the use of the GEO dataset somewhat unconvincing in its current form. The authors analyze transcriptomic data from primary human bronchial epithelial cells from cystic fibrosis donors exposed to PA, then use this to nominate LC3 as a “core protein” in PA-infected autophagy, while the rest of the manuscript is centered on mouse macrophages. This cross-system extrapolation is difficult to justify biologically. The manuscript would be stronger if the authors either explained this choice much more carefully or reduced the weight placed on that analysis. As presented, the GEO analysis feels disconnected from the main macrophage-focused story.

The ceRNA claims also need to be interpreted more cautiously. The luciferase and RIP data support interaction, and the localization data are helpful, but the manuscript ultimately proposes a rather elaborate model in which one lncRNA simultaneously regulates two separate miRNA-target branches to control autophagy. That is possible, but for such a model the evidence should be especially rigorous. At present, the data support association and regulatory influence, but the paper should avoid implying that the entire regulatory architecture is fully established.

There are also several presentation and consistency issues that should be corrected. The manuscript uses the phrase “LC3 cleavage”, which is not the clearest or most standard terminology for LC3-I to LC3-II conversion. In addition, the p62 section contains obvious inconsistencies: for example, Section 3.8 refers to miR-346-3p in the heading and figure legends, whereas the text discusses miR-344g-5p. Likewise, the Methods section under bacterial load unexpectedly refers to “Mycobacterium tuberculosis load”, which appears to be a carry-over error from another manuscript. These errors are not trivial, because they reduce confidence in the care with which the manuscript was prepared.

Another weakness is that the translational framing is somewhat overstated. The Discussion suggests that NR_003508 may be a promising biomarker or therapeutic target for PA infection, but the evidence here remains preclinical and largely mechanistic within murine systems. No human infection samples or patient-based validation are presented. That language should therefore be moderated.

The manuscript would also benefit from better contextualization within the broader literature on non-coding RNAs and autophagy. In this regard, the authors should cite and discuss the review by Ghafouri-Fard et al., “Exploring the role of non-coding RNAs in autophagy” (DOI: 10.1080/15548627.2021.1883881), which reviews how lncRNAs and miRNAs regulate autophagy across multiple disease settings. This reference is directly relevant to the conceptual basis of the present study and would strengthen both the Introduction and the Discussion by placing NR_003508 within the larger ncRNA–autophagy field.

In summary, the manuscript contains interesting observations, but the current version still overstates the certainty of the mechanistic model, does not sufficiently prove that the bacterial phenotype is autophagy-dependent, relies on a somewhat disconnected transcriptomic analysis, and contains multiple presentation inconsistencies that need correction. For these reasons, I recommend major revision.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In the present research, Liu et al. demonstrated that the NR_003508 long noncoding RNA promotes autophagy to clear infecting Pseudomonas aeruginosa. Associated molecular players were identified and studied. The topic is highly relevant in the context of rising bacterial infections. The experimental design is correct, and ethical approval is mentioned. Some of the methods lack relevant information, and the presentation or description of some of the data should be improved (please see detailed comments below). Nevertheless, the manuscript is compelling, detailed, consistent, and uncovers molecular mechanisms. Its findings have the potential to contribute to a novel antibiotic strategy against Pseudomonas aeruginosa.

 

The authors should address the following recommendations:

1. On lines 33 and 65, please include the full name of ceRNA. On line 115, please clarify "OD". On line 143, please include the full name of MOI.

2. On line 107, in "the order of treatments is random", "is" should be "was". Please check.

3. In section 2.1, please include a schematic representation of the experimental design described in the last paragraph of this section, with the animal groups and respective treatments.

4. In section 2.1, please mention whether there was (or not) exclusion of any animals or data points.

5. In section 2.4, please include references for the LC3 and p62 Plasmids.

6. In section 2.5, please include the amount of total RNA or miRNA used for cDNA synthesis and the number of qPCR cycles.

7. In sections 2.6, 2.8, and 2.14, please include the antibody dilutions. Please also mention the primary antibodies used in section 2.8.

8. In section 2.7, please include information about the FISH probes.

9. In section 2.10, please mention how many events were used in flow cytometry and please also describe the gating used.

10 Please include the protocol of the subcellular fractionation experiments.

11. On lines 230-231, please rephrase for clarity: "Eluted to gain the enriched RNA for subsequent q-PCR detection of the expression of LncRNA NR_003508, miR-344i, miR-346-3p, LC3 and p62".

12. In the legend of Figure 1H, please explain the meaning of GAPDH, U6, and Lnc.

13. On lines 340-341, please include references for "In addition to LC3 mentioned above, we also found that p62 played a key role in autophagy through a literature review".

14. Please describe the content of Figure 3E in some detail in its legend.

15. In Figures 3G and 8A, please label the horizontal axes.

16. In the legends of Figures 3H and 5D, please describe the histological features and staining observed in the respective micrographs.

17. In the legend of Figure 4J, please describe how the autophagic rate was calculated.

18. In Figure 10, please translate the words in Chinese. In Figure 10, it also mentions "clearance of Pseudomonas aeruginosa" and "PA survival", which appear to be in contradiction. Please check.

19. In the discussion, please mention the limitations of the study.

20. In the ARRIVE checklist, please check the line numbers (some are inaccurate).

21. Please perform a general English language proofreading.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript is ready for publication in this final format. All the concerns were addressed by the authors.