PREreview of Alveolar metabolite availability facilitates secondary infection byPseudomonas aeruginosain acutely injured lungs

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:  

Alveolar metabolite availability facilitates secondary infection by Pseudomonas aeruginosa in acutely injured lungs 

Jennifer M. Baker, Thomas L. Flott, Laura A. McLellan, Ingrid G. Bustos, Jose L. Guerrero, Lina M. Mendez, Annastasia M. Petouhoff, Piyush Ranjan, Joseph D. Metcalf, Roderick A. McDonald, Nicole R. Falkowski, Ying He, Mónica P. Cala, Gary B. Huffnagle, Michael W. Sjoding, Luis F. Reyes, Kathleen A. Stringer, Robert P. Dickson 

bioRxiv 2024.12.18.629020; doi: https://doi.org/10.1101/2024.12.18.629020  

We will adhere to the Universal Principled (UP) Review guidelines proposed in:  

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029  

SUMMARY: Recent advances in our understanding of the microbial communities in the lung have brought forward new questions regarding the lung microenvironment. Baker and colleagues sought to understand how nutrient availability in the lungs might impact secondary bacterial pneumonia during cases of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The authors used a sophisticated experimental design that integrated data from mechanically ventilated human patients and an in vivo mouse model of ALI. In ARDS patients who developed ventilator associated pneumonia (VAP), the two most common bacterial isolates were Staphylococcus aureus and Pseudomonas aeruginosa. To investigate how ARDS supports bacterial colonization the authors used an established murine model of ALI and used the bronchoalveolar lavage fluid (BALF) to culture P. aeruginosa ex vivo. The BALF from mice with ALI enhanced P. aeruginosa growth and this enhanced growth corresponded with increased abundance and diversity of available metabolites in the lung as determined by NMR. The increased metabolites in BALF correlated with metabolites in serum collected at the same timepoints suggesting that lung metabolites are due to alveolar leakage. The onset and severity of alveolar leakage was determined by quantification of alveolar IgM levels. RNA sequencing of P. aeruginosa grown in ALI BALF indicated that genes involved in metabolic pathways were differentially expressed depending on metabolite availability in the lungs. Further supporting this, P. aeruginosa preferentially utilized available metabolites differently between BALF derived from healthy mice versus ALI mice. Finally, the authors sought to understand if ARDS alveolar leakage in ventilated patients supported P. aeruginosa growth. Indeed, they found BALF from ARDS patients enhanced P. aeruginosa growth and this corresponded with the severity of alveolar leak. This study provides novel insight into the ecological factors that contribute to the interaction between P. aeruginosa and host lungs. 

OVERALL ASSESSMENT: This study established a role for host-derived metabolites in the lungs as crucial factors that influence ventilator associated pneumonia caused by P. aeruginosa. The study was well planned and executed and employed a variety of techniques and sample types. Study findings enhance our understanding of the poorly understood lung microenvironment during ALI/ARDS and P. aeruginosa infection. We commend the authors on their data presentation, as they made it accessible to a naïve audience. We have minor critiques regarding scholarship and a few points that should be clarified to better support the authors’ findings. We recommend adding a condition for the mock BALF experiments that would strengthen the paper but is not strictly required for completeness.  

STRENGTHS: The strength of this manuscript comes from the thorough experimental design and inventive ex vivo experiments. Baker, et al. use a patient database to inform their study, apply their findings to a murine model of hyperoxia, and further validate murine model results with patient samples. This solid experimental plan enables a robust study of the impact of acellular factors/metabolites on P. aeruginosa infection during ALI/ARDS. Further, the experimental design adds a significant amount of extensibility to the findings in this study.  

WEAKNESSES: The primary weaknesses relate to scholarship. Overall, we feel that the manuscript could use another proofread to ensure figure captions match the data/labels in figures, that figures are referenced appropriately in the text, and that methodology and data are communicated as clearly as possible. While we like the setup of the “mock BALF” experiment, better description of the rationale and experimental set up would enhance the important conclusions being drawn from this experiment. More detail related to the murine hyperoxia model would also benefit the reader and provide more confidence in the model.  

DETAILED U.P. ASSESSMENT:  

OBJECTIVE CRITERIA (QUALITY)  

1.   Quality: Experiments   

·  Figure by figure, do experiments, as performed, have the proper controls? [note: we use this ‘figure-by-figure' section for broader detailed critiques, rather than only focusing on controls] 

Fig. 1: We suggest the authors revise the figure caption to address inconsistencies between the timepoints and number of patients identified in the figure compared to what is indicated in the figure caption.  

Fig. 2: The authors are missing a critical data point in Figure 2C where they do not show CFU/ml data for day 4 and do not provide explanation in the text. This data point must be added to complete the figure. Authors should be consistent in showing whether data is significant or not throughout the figure set. For example, non-significance is indicated by “ns” in Figure 2C, but in Figures 2A/B, significance is only reported between 2 out of 4 timepoints. The figure caption includes letters E and F, which are not panels in the figure, please revise figure caption to reflect only panels A-D. In the text (line 88), the authors should revise wording to reflect the specific data in Figure 2, which might be something like: “soluble, acellular factors from acutely injured murine lungs promote P. aeruginosa growth”. Additionally, authors should clarify in the figure caption and/or figure panel for Figure 2C what the dashed horizonal line indicates.  

Fig. 3: We would like the authors to provide justification in the text for their choice of only analyzing metabolites at the day 3 timepoint. Providing justification or showing multiple time points would prove to the reader that day 3 is the optimal timepoint for analysis and/or assist in understanding how the metabolite profiles change throughout the course of ALI. 

Fig. 4: In Figure 4A, there is a significant drop in the total protein level in BALF from SPF mice compared to germ free mice. We would like the authors to address this in the text to provide an explanation for the notable difference. We think the “Mock BALF” experiment shown in Figure 4F is a clever experiment, but the manuscript text does not clearly describe the results or how the experiment was performed. We recommend that the authors revise the text to make the data and conclusions clearer. Despite these minor critiques, we would like to commend the authors on the data presented in this figure, especially Figures C/D. Including hints to help the reader interpret the data quickly is a nice touch.  

Fig. 5: No notes.

Fig. 6: We recommend that the authors double check the Figure 6 call-outs in the Results text, as Figure 6E appears to be referred to as 6D and 6F is referred to as 6E. Furthermore, we think the authors would benefit from pulling some of the data from Extended Data Figure 5 into the Main Figures, as it nicely describes the correlation between human metabolites and maximum OD of P. aeruginosa. Including some of this data would further support the authors model, showing that the important metabolites in murine BALF are consistently found in human BALF samples as well.

·  Are specific analyses performed using methods that are consistent with answering the specific question?  Is there appropriate technical expertise in the collection and analysis of data presented? 

We would like the authors to provide more insight into their hyperoxia model. For example, could authors provide more information about positive indicators for ALI induction in the murine model, as we were unsure about the rationale for administering O₂ 95% for a range of 24-96 hours. The publication cited by Baker, et al. reveals that 1 out of 4 of the following measurements are used to determine ALI in animal models: histological evidence, capillary-alveolar barrier alteration, inflammatory response, or physiological dysfunction. It is not clear how the authors used these guidelines to determine that ALI had been induced. We also raise concern around the potential for loss of additional metabolites in the BALF that may be depleted in response to CO₂ asphyxiation. Plasma glucose has been shown to drop rapidly after exposure to CO₂, which may impact the levels of ketones such as 3-Hydroxybutyrate (Nichols et al., 2020; Mierziack et al., 2021).  We would like the authors to provide some literature support for using CO2 asphyxiation in the text. We are curious about why the authors chose CO₂ rather than using isoflurane in combination with cardiac puncture. Additionally, some histology of the alveoli from the mice with ALI could be included as supplemental data to help the reader become more confident and familiar with the model.  

The authors rely heavily on correlative data throughout this manuscript. If the authors would like to make more conclusive or absolute claims, perhaps they could consider further exploring the mock BALF experiments. We suggest using a similar technique but rather than diluting serum, add specific metabolites to PBS and assess P. aeruginosa growth. This would provide a more concrete and direct connection between specific metabolites and P. aeruginosa growth.  

·     Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?   

Yes.

·     Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.   

Yes.

2.   Quality: Completeness   

· Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.  

The data in this manuscript support the abstract and title level conclusions.

· Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.  

No.

3. Quality: Reproducibility   

· Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.? 

Yes

· Is there sufficient raw data presented to assess the rigor of the analysis?  

Yes

· Are methods for experimentation and analysis adequately outlined to permit reproducibility?  

If possible, we would like the authors to disclose where they obtained their PAO1 strain from. It is well known in the Pseudomonas aeruginosaresearch community that PAO1 can differ greatly between labs (Chandler et al 2019; Klockgether et al., 2010). The authors should do their best to identify the source of their PAO1 or if possible, the subline of PAO1 they are working with. Knowing the source/subline of PAO1 will greatly impact the reproducibility of this study.  

· If a ‘‘discovery’ dataset is used, has a ‘‘validation’ cohort been assessed and/or has the issue of false discovery been addressed?   

N/A

4. Quality: Scholarship   

· Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?  

· Yes

· Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?  

· Yes

· Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not be significant basis for decisions.  

We suggest the authors proofread their manuscript again, specifically looking for italicized species names, figure caption details and formatting, and ensuring that experiments/data are adequately explained in both the text and figure caption. A few other specific errors include the reference to the 19^th century in line 274 where we presume the authors meant the 20^th century (1900s). A citation would also be beneficial for the statement in line 274. We also advise the authors to be cautious with the wording of statements in the results such as “serum-derived edema is a major nutrient source for respiratory pathogens” since they acknowledge they have not captured the full range of potential nutrients.  

MORE SUBJECTIVE CRITERIA (IMPACT):  

Impact: Novelty/Fundamental and Broad Interest   

How big of an advance would you consider the findings to be if fully supported but not extended? Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)? The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2) 

This study is impactful for the field of P. aeruginosa infection biology as well as ALI/ARDS and VAP research. The authors have provided novel insight into ecological factors, namely serum-derived metabolites, and how their abundance and diversity change throughout ALI/ARDS and benefit P. aeruginosa infection. The methodology used was sufficient to support the authors claims and provides a framework for future study.  

References:

·  Chandler CE, Horspool AM, Hill PJ, Wozniak DJ, Schertzer JW, Rasko DA, Ernst RK. Genomic and phenotypic diversity among ten laboratory isolates of Pseudomonas aeruginosa PAO1, Jb Bacteriol. 2019 Feb;201(5):e00595-18. doi: 10.1128/JB.00595-18

·  Klockgether J, Munder A, Neugebauer J, Davenport CF, Stanke F, Larbig KD, Heeb S, Schöck U, Pohl TM, Wiehlmann L, Tümmler B. Genome diversity of Pseudomonas aeruginosa PAO1 laboratory strains. J Bacteriol. 2010 Feb;192(4):1113-21. doi: 10.1128/JB.01515-09

·  Mierziak J, Burgberger M, Wojtasik W. 3-Hydroxybutyrate as a Metabolite and a Signal Molecule Regulating Processes of Living Organisms. Biomolecules. 2021 Mar 9;11(3):402. doi: 10.3390/biom11030402.

·  Nichols KE, Holliday-White KL, Bogie HM, Swearingen KM, Fine MS, Doyle J, Tiesma SR. Cardiovascular and Metabolic Responses to Carbon Dioxide Euthanasia in Conscious and Anesthetized Rats. J Am Assoc Lab Anim Sci. 2020 Nov 1;59(6):742-749. doi: 10.30802/AALAS-JAALAS-19-000166

Competing interests

The authors declare that they have no competing interests.