Review Reports - Hyaluronan Signaling Ameliorates the Epithelial Injury Response and Barrier Disruption After Ozone Exposure

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This study addresses an important question regarding the mechanisms of ozone-induced epithelial injury. The authors provide in vitro evidence that hyaluronan signaling differentially regulates epithelial damage and inflammation through CD44, RHAMM, and TLR5 pathways. The use of both human and murine primary differentiated epithelial cells strengthens the relevance and translational value of the findings. The authors show that high-molecular-weight hyaluronan (HMWHA) confers epithelial protection via TLR5 and suggest that it may have therapeutic potential.

Overall, the study is well designed and clearly written; however, several issues should be addressed before the manuscript can be considered for publication.

1. The in vitro experiments using human and murine cells were designed as a preventive approach, evaluating whether HMWHA can prevent the detrimental effects of ozone exposure. However, it remains unclear whether HMWHA can also exert therapeutic effects once epithelial injury is already established. Can HMWHA attenuate established or progressive epithelial damage? Addressing this point experimentally, or at least discussing it more thoroughly, would strengthen the clinical relevance of the study.

2. Figures 3 and 5: The labels are too small and difficult to read. The font size should be increased to improve clarity and readability.

3. The authors report that the protective effect of HMWHA is mediated by TLR5, but not by the canonical hyaluronan receptors CD44 and RHAMM (CD168). This finding is intriguing, as TLR5 is not traditionally recognized as a receptor for HMWHA. Although Figure 4 demonstrates significant differences between wild-type and Tlr5-/- cells, additional mechanistic evidence would strengthen this conclusion. For example, receptor neutralization experiments, pharmacological inhibition, co-immunoprecipitation, or binding assays could help corroborate a direct or functional interaction between HMWHA and TLR5. While the reviewer acknowledges that hyaluronan can modulate cellular responses through Toll-like receptors, further validation of this novel mechanism is warranted in the present study.

4. In the Discussion, the authors state that administration of HMWHA can ameliorate lung injury in vivo citing previous studies. However, high-molecular-weight hyaluronan may undergo fragmentation in vivo into low-molecular-weight HA under inflammatory conditions. Could this potential fragmentation limit the therapeutic benefit or even exacerbate inflammation? This important point should be discussed, particularly in the context of translational application.

Author Response

Reviewer 1

Overall, the study is well designed and clearly written; however, several issues should be addressed before the manuscript can be considered for publication.

Response: We thank the Reviewer for the overall positive evaluation of our work.

The in vitro experiments using human and murine cells were designed as a preventive approach, evaluating whether HMWHA can prevent the detrimental effects of ozone exposure. However, it remains unclear whether HMWHA can also exert therapeutic effects once epithelial injury is already established. Can HMWHA attenuate established or progressive epithelial damage? Addressing this point experimentally, or at least discussing it more thoroughly, would strengthen the clinical relevance of the study.

Response: We appreciate this insightful comment. We were somewhat constrained by the experimental setup of this study, which necessitated measuring transepithelial resistance (TEER) immediately after termination of ozone exposure. Thus, adding solutions (HMWHA or vehicle) to the apical surface of the culture would distort and potentially falsify TEER measurements. In previous in vivo work, we have shown that HMWHA given after injury (e.g. chlorine gas exposure (PMID: 25747964), viral infection (PMID: 40720792), acid instillation (PMID: 29368549), and house dust mite exposure (PMID: 30188746)) ameliorates adverse phenotypes, i.e., lung inflammation, wet-to-dry lung ratio, histological evidence of lung injury. Although not directly transposable to this study, these results would suggest that therapeutic HMWHA would also have positive impacts. We have added this discussion to the relevant area of the manuscript.

Figures 3 and 5: The labels are too small and difficult to read. The font size should be increased to improve clarity and readability.

Response: We apologize for the mistake. We have reformatted the Figures and hope that they are more legible now.

The authors report that the protective effect of HMWHA is mediated by TLR5, but not by the canonical hyaluronan receptors CD44 and RHAMM (CD168). This finding is intriguing, as TLR5 is not traditionally recognized as a receptor for HMWHA. Although Figure 4 demonstrates significant differences between wild-type and Tlr5-/- cells, additional mechanistic evidence would strengthen this conclusion. For example, receptor neutralization experiments, pharmacological inhibition, co-immunoprecipitation, or binding assays could help corroborate a direct or functional interaction between HMWHA and TLR5. While the reviewer acknowledges that hyaluronan can modulate cellular responses through Toll-like receptors, further validation of this novel mechanism is warranted in the present study.

Response: We appreciate the Reviewer’s concern. We would like to highlight that we have already published on TLR5 mediating HA effects (see Hussain, S., et al., TLR5 participates in the TLR4 receptor complex and promotes MyD88-dependent signaling in environmental lung injury. Elife, 2020). Co-IP or binding assays with hyaluronan are unfortunately beyond the scope of this work, if they are possible at all, given that HA is not a protein and not really amenable to a pull-down assay. Furthermore, absence of binding would not prove absence of signaling: for example TLR4, which mediates HA signaling (PMID: 17400552, PMID: 14764599, PMID: 27694932) has never been shown to directly bind to HA. To demonstrate that HA does indeed lead to TLR5 activation, we utilized a commercial assay using TLR5-reporter cells, available through InvivoGen (HEK-Blue™ TLR5). The cells specifically show activation of NFκB downstream of TLR5 activation. Using this system, we now demonstrate that TLR5 is directly activated by HA, as well as apical wash from human and mouse air-liquid interface cultures. The results are included as new Panel C in Figure 6.

In the Discussion, the authors state that administration of HMWHA can ameliorate lung injury in vivo citing previous studies. However, high-molecular-weight hyaluronan may undergo fragmentation in vivo into low-molecular-weight HA under inflammatory conditions. Could this potential fragmentation limit the therapeutic benefit or even exacerbate inflammation? This important point should be discussed, particularly in the context of translational application.

Response: We appreciate the Reviewer raising this very important question. This has been something we have considered and evaluated in past studies, because we shared this concern. The summary of our past studies is that therapeutically applied HMWHA escapes clinically relevant degradation. As one relevant example, we studied this directly in PMID: 27716205, whereby we treated cystic fibrosis (CF) patients with inhaled HMWHA for 30 days and then evaluated induced sputum HA size and inflammatory cytokines. Although the CF sputum is a highly inflammatory environment, we found that HA size in the sputum increased after HMWHA inhalation. Furthermore, there was no change in sputum inflammatory cytokines before and 30 days after initiation of HMWHA inhalation. In other studies, including human clinical trials with HMWHA nebulization for 5-10 days in patients with significant airway inflammation (COPD exacerbation or severe COVID-19, PMID: 33517896 and PMID: 40720792 respectively) we observed a decline in inflammatory markers in human patients and mouse models of lung injury. Therefore, existing evidence supports that HMWHA does not exacerbate inflammation, on the contrary it suppresses it. We have added this information to the Discussion section.

Reviewer 2 Report

Comments and Suggestions for Authors

Review of the manuscript: “Hyaluronan Signaling Impacts the Epithelial Injury Response After Ozone Exposure”

As a reviewer for this manuscript, I find the topic and the authors' hypothesis to be highly interesting and of significant importance for both environmental health and biology. However, the current version of the manuscript relies too heavily on previous data to support its initial hypothesis. Furthermore, the investigation into hyaluronan (HA) signaling—as promised in the title—is not sufficiently addressed in the results. Given the importance of the subject, I recommend a major revision, including a complete rewriting of certain sections and a reconsideration of the title to better reflect the presented data.

Major Issues:

Methods: Key methodological details are missing. Specifically, the authors should provide a detailed explanation of the TEER procedure, Real-Time PCR parameters, and the ozone treatment protocol. A clear experimental design, including a timeline of treatments and measurements, is essential.
Figure Quality: The overall quality of the figures is sub-optimal.

F1A: The time points must be indicated on the plot.
F1B: Please provide an inset with higher magnification.
F2: The caption should clarify whether the Venn diagram includes both up-regulated and down-regulated genes, or only one set.
F3 and F5: These figures are currently illegible. They are not adequately explained in the text or the captions (e.g., the "circled dots" in F5 are not defined).

Biological Context: In the explanation of Figure 1, the authors should explain why they used murine/human cells throughout the study.
PCA Analysis of fig 3C and 5A : The Principal Component Analysis (PCA) is used to draw conclusions, but the data are not described in the text. Without a description of data and methods, the reader cannot properly interpret these results.
Volcano Plots: The plots in F3 and F5 are significant enough to warrant separate images, explanations, and analyses in the results section.
Section 3.3: The data presented in the text are not fully supported by the figures. The authors state that “HA size was not impacted by ozone exposure,” yet they fail to comment on the total amount/concentration of HA.
Supplementary Data: Supplementary Table 1 should be moved to the main manuscript, as it is the basis for the subsequent analyses. Currently, certain receptors (e.g., LAYN on page 8) are introduced abruptly, while others (TLR5) appear much later without a cohesive introduction.
Figure 3D & Inflammation: This figure is summarized in only three lines at the end of a paragraph. There is no discussion regarding the choice of cytokines measured, which currently appear to be selected at random.
Data Consistency: The concluding sentence (“...in aggregate... Receptors”) is not supported by the data, as it refers to HA molecular weight, which—according to the authors—remained unchanged.
TLR5 Involvement: The role of TLR5 is currently supported only by a citation (Ref 36). It would be more compelling if the authors could demonstrate that TLR5 expression or activity was altered in the treatments shown in Figure 2.
Figures 5B and 5C: These figures are data-dense, yet they are summarized in a single concluding sentence. The authors must provide a detailed analysis, though this is currently difficult due to the illegible font size in the images.
Figure 5 Caption: Please define the meaning of the asterisks. I cannot find it in the images.
Results vs. Methods: In the final lines of the Results, the authors introduce a TLR5 agonist. However, there is no mention of this reagent in the Methods, nor an explanation of its relationship with HA in this context.

Minor Issues:

Acronyms and Formatting: A thorough revision of acronyms and nomenclature is required. Please ensure consistency in capitalization and formatting (e.g., ALI, IL-6 vs. Il6, RHAMM/Hmmr, and TLR5 vs. Tlr5).

Author Response

This manuscript studied the impact of a major component of the extracellular matrix, hyaluronic acid (HA) in human and murine epithelial cells exposed to ozone. High molecular weight HA reestablishes the epithelial barrier that was affected by ozone exposure. And this protected effect was regulated by Tlr5. The hypothesis of this study is interesting however the description of the specific aim is not clear. The manuscript indicates that HA mediates epithelial injury responses to ozone administration, but here the epithelial functions were not deeply studied. However, this manuscript may be accepted after clearly indicates the aims of this study. Concerns and suggestions are listed below.

The title does not reflect the findings performed in this manuscript. Authors do not explore the effects of HA in the epithelial injury, but just in the function of epithelial barrier (TEER). Therefore, the title must be changed to clearly mention what authors found.

Response: We appreciate the Reviewer’s comment but respectfully disagree with the Reviewer’s interpretation of our findings. Epithelial injury is defined as damage-induced derangements that impair normal epithelial structure or function (including barrier integrity). We respectfully submit that original Figures 3 and 5 (new Figures 4-6) offer clear transcriptomic evidence for epithelial injury responses and support the role of HA in epithelial injury. For example, we show transcriptomic changes impacted by the genetic deletion of HA receptors Cd44 and Rhamm, compared to Wildtype cells. As just 2 examples, Rhamm deficiency leads to upregulation of “Inflammatory response” and “Cellular homeostasis” nodes along with cytokine nodes IL1A, IL17A, and Human Epidermal Growth Factor Receptor ERBB2, a transmembrane tyrosine kinase receptor involved in cell growth, division, and survival. Cd44 deficiency leads to downregulation of nodes including MYC, a family of regulator transcription factors controlling cell growth, proliferation, differentiation, and apoptosis, and the above mentioned ERBB2. We also show several Ingenuity pathways and gene sets, respectively, that are directly linked to cell injury and downregulated by HMWHA, including apoptosis, p53 pathway, Kras and Myc signaling, mitogenesis, cell proliferation and movement, amongst many others. New Figure 5 shows upregulation of Cxcl1, Cxcl2 and Il6 after ozone exposure, which is ameliorated by HMWHA. Thus, we respectfully submit that there is ample evidence of epithelial injury and HA effects on the same. We are happy to change the title to “Hyaluronan Signaling Impacts the Epithelial Injury Response and Barrier Disruption After Ozone Exposure”, to highlight the barrier effects the Reviewer mentioned.

The study of the effects of HA on the epithelial barrier should be complemented by measuring the permeability to FITC- dextran.

Response: We appreciate the Reviewer’s request for additional evidence. We have performed these experiments. FITC results mirror TEER results throughout the studies of ozone effects and strengthen our conclusions that ozone impacts epithelial permeability. Unfortunately, FITC dextran is not an appropriate methodology to study HA effects on the epithelial barrier, due to the well-described size-exclusion effect that HA has for FITC-dextran (for example pioneering work by Torvard Laurent in the 1970s, or more recently PMID: 40914464). In agreement with this, see below graph showing the effect of HMWHA in apparent permeability in uninjured, (air exposed) ALI, using FITC-Dextran (left y-axis) and TEER (right y-axis). No effect is seen with TEER (as expected), but an apparent decrease in permeability (lower fluorescent intensity) is observed via the FITC-Dextran method. We believe that this change is not reflective of epithelial biology, but rather an artifact of HMWHA-Dextran interactions as mentioned above.

We analyzed FITC-Dextran permeability on the HMWHA effect after ozone exposure, as the Reviewer suggested. We see a strong improvement in permeability with HMWHA treatment, in fact stronger than is seen by TEER measurement (see figure). However, we are not confident that this is an effect on actual permeability vs simply a physical size exclusion effect from HA on dextran as mentioned above, as we see this effect indiscriminately regardless of genotype. Thus, we respectfully suggest that these data should not be included in the MS, and we have not added them.

The aim is not well-described. Technically authors do not demonstrate epithelial injury since both monolayers look intact by ZO-1 labeling. Therefore, the authors must show monolayer damage and decrease cell viability.

Response: We respectfully disagree with the Reviewer’s interpretation of epithelial injury. Cell death is not the sine qua non of epithelial injury; it is rather the extreme result of severe, irreversible cell injury. We opted to use realistic modeling of ozone exposure at low doses and levels occurring in the real world; therefore, the epithelial injury signs are more subtle than cell death. We maintain that this makes our results more applicable to human health. As stated above, we also respectfully suggest that original Figures 3 and 5 offer ample evidence for the activation of epithelial injury response pathways. To hopefully address the Reviewer’s concerns, we have added HE staining results (new Figure 1D) showing flattening of the epithelial layer and loss of cilia as a result of ozone exposure.

Introduction needs to include a brief description of the importance and functions of epithelial cells.

Response: Thank you for this suggestion. We have added this description as suggested.

Authors may include a brief description of the HA receptors Cd44 or Rham, as well as a brief introduction about the importance of Tlr5.

Response: Thank you for this suggestion. We have added this description as suggested.

Did authors analyze if Tlr4 has the same effect as Tlr5?

Response: We thank the Reviewer for raising this very important question. TLR4 has repeatedly been found to mediate HA signaling (PMID: 17400552, PMID: 14764599, PMID: 27694932). Indeed, we have previously shown that Tlr5 and Tlr4 participate in receptor complexes which mediate HA signaling (Hussain, S., et al., TLR5 participates in the TLR4 receptor complex and promotes MyD88-dependent signaling in environmental lung injury. Elife, 2020, PMID: 31989925). TLR4/5 heteromeric complexes were also described by others (Mizel SB et al, Induction of macrophage nitric oxide production by Gram-negative flagellin involves signaling via heteromeric Toll-like receptor 5/Toll-like receptor 4 complexes, J Immunol 2003, PMID: 12794153). This would suggest that these two receptors may be co-activated. We now tested the effect of HMWHA on TEER using wildtype (WT), Tlr4-deficient and Tlr5-deficient murine epithelia in ALI in addition to Cd44 and Rhamm-deficient cells. We found that Tlr5 deficiency led to decreased epithelial integrity and absent response by HMWHA, while Tlr4 deficiency did not alter the effect to ozone (compared to WT cells), but led to an absent response to HMWHA. We interpret as suggesting that Tlr5 uniquely impacts epithelial permeability, because Tlr5 deficient cells respond to ozone with more barrier disruption (we have another paper in submission, dealing with TLR5 effects on epithelial resiliency which we can share upon request). However, our results support that HMWHA effects are mediated via both Tlr4 and Tlr5. We have added these results to our new Figure 6B.

Authors need to make a clear connection between the transcriptomic results and the experimental approaches presented here. Since the information found by bioinformatic analysis was not experimentally corroborated.

Response: We are somewhat puzzled by this comment, as it may suggest that the reviewer thinks we performed data mining for our analysis. Our results are entirely new data, derived from our experimental exposures as described in the Methods section. Our RNA sequencing results derive from our experiment exposure, and the experimental approach we followed is described in detail in our Methods section. Furthermore, we corroborated the results, for example by performing RTPCR for CXCL1, CXCL2, and IL6, which show the same effect as the RNA sequencing data. Therefore, we are not certain what the Reviewer means by this comment.

Please include in material and methods the information about genetically modified mice (including source, Cat. # and references). Indicate if mice are conditional or total KO?

Response: We have added this information as suggested by the Reviewer.

Authors must corroborate the depletion of the receptors in the tracheal cells by WB or RT-PCR.

Response: We have performed this analysis as requested, and this is now included in the Table.

Add another section (2.5) for the description of RNA isolation and RT-PCR.

Response: We are afraid we do not understand what the Reviewer is requesting. RNA isolation and RTPCR are already described in their own section in Methods. Please note that there is no numbering of sections in the version we submitted, which may have been added by the Journal staff, so we are apparently not seeing the same text the Reviewer is seeing.

Do not us ambiguous words like: “Impacts” and describe exactly the observed effect like: “diminishes the epithelial barrier”.

Response: We have changed the wording as suggested by the Reviewer.

Please explain why authors used different ozone exposure times for functional assays versus transcriptome studies.

Response: Thank you for the opportunity to clarify. We did NOT use different ozone exposure times for functional vs transcriptomic studies. We did use different times for murine vs human cells (1 h vs 2 h, respectively), due to the fact that murine ALI cultures are usually monolayers, while human ALIs are usually pseudostratified, thus more resistant to ozone injury.

The figures must show the TEER values in ohms/cm2, instead of percentage.

Response: We have changed the Figures as suggested by the Reviewer and are now reporting TEER values as Ω x cm². There is no change in results or conclusions from this.

Figure 1B, that shows the image of ZO-1 merge with DAPI, must also show the individual channel for ZO1.

Response: We have made the addition as suggested by the Reviewer.

Minor changes:

Change “ALI” to “ALI cultures”

Response: We have made the changes as suggested by the Reviewer.

In the text (line 282): change 2B to 3B

Response: We apologize for the mistake and have corrected this.

The manuscript is well written but has same typos in the text.

Response: We apologize for the mistakes and have made every effort to correct them.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Manuscript: Hyaluronan Signaling Impacts the Epithelial Injury Response After Ozone Exposure

The title does not reflect the findings performed in this manuscript. Authors do not explore the effects of HA in the epithelial injury, but just in the function of epithelial barrier (TEER). Therefore, the title must be changed to clearly mention what authors found.
The study of the effects of HA on the epithelial barrier should be complemented by measuring the permeability to FITC- dextran.
The aim is not well-described. Technically authors do not demonstrate epithelial injury since both monolayers look intact by ZO-1 labeling. Therefore, the authors must show monolayer damage and decrease cell viability.
Introduction needs to include a brief description of the importance and functions of epithelial cells.
Authors may include a brief description of the HA receptors Cd44 or Rham, as well as a brief introduction about the importance of Tlr5.

Did authors analysis if Tlr4 has the same effect as Tlr5?

Authors need to make a clear connection between the transcriptomic results and the experimental approaches presented here. Since the information found by bioinformatic analysis was not experimentally corroborated.
Please include in material and methods the information about genetically modified mice (including source, Cat. # and references). Indicate if mice are conditional or total KO?
Authors must corroborate the depletion of the receptors in the tracheal cells by WB or RT-PCR.
Please include in material and methods the Cat. # of the Human primary bronchial and MatTek cells. Do authors use Human primary bronchial or tracheal epithelial cells?
Add another section (2.5) for the description of RNA isolation and RT-PCR.
Do not us ambiguous words like: “Impacts” and describe exactly the observed effect like: “diminishes the epithelial barrier”.
Please explain why authors used different ozone exposure times for functional assays versus transcriptome studies.
The figures must show the TEER values in ohms/cm2, instead of percentage.
Figure 1B, that shows the image of ZO-1 merge with DAPI, must also show the individual channel for ZO1.

Minor changes:

Change “ALI” to “ALI cultures”

In the text (line 282): change 2B to 3B

The manuscript is well written but has same typos in the text.

Author Response

Major Issues:

Methods:Key methodological details are missing. Specifically, the authors should provide a detailed explanation of the TEER procedure, Real-Time PCR parameters, and the ozone treatment protocol. A clear experimental design, including a timeline of treatments and measurements, is essential.

Response: We apologize for the missing details and now provide detailed methods as suggested by the Reviewer.

Figure Quality:The overall quality of the figures is sub-optimal.

Response: We apologize for the suboptimal quality and have now amended several figures to improve it.

F1A: The time points must be indicated on the plot.

Response: Respectfully, we do not understand what the Reviewer is asking. There are no timepoints in the plots, which show measurements at one timepoint after air or ozone exposure, as indicated in the methods section.

F1B: Please provide an inset with higher magnification.

Response: We have included this in amended Figure 1B, as per Reviewer’s suggestion.

F2: The caption should clarify whether the Venn diagram includes both up-regulated and down-regulated genes, or only one set.

Response: The Venn diagram includes both up-and down-regulated pathways. We have included this in amended caption, as per Reviewer’s suggestion.

F3 and F5: These figures are currently illegible. They are not adequately explained in the text or the captions (e.g., the "circled dots" in F5 are not defined).

Response: We apologize for the suboptimal quality and have now amended these figures, breaking them up into several figures for improved legibility.

Biological Context:In the explanation of Figure 1, the authors should explain why they used murine/human cells throughout the study.

Response: We are using human ALI cultures to demonstrate applicability of our findings to human biology. For specific gene function analyses, when we need genetically manipulated cells, we have used murine ALI cultures. We have now included this explanation in the text.

PCA Analysis of fig 3C and 5A :The Principal Component Analysis (PCA) is used to draw conclusions, but the data are not described in the text. Without a description of data and methods, the reader cannot properly interpret these results.

Response: We apologize for the omission and have now entered extensive description of these data in our Results section.

Volcano Plots:The plots in F3 and F5 are significant enough to warrant separate images, explanations, and analyses in the results section.

Response: We have changed the Figures as suggested by the Reviewer.

Section 3.3:The data presented in the text are not fully supported by the figures. The authors state that “HA size was not impacted by ozone exposure,” yet they fail to comment on the total amount/concentration of HA.

Response: We are respectfully unsure on what the Reviewer is asking, as we clearly state that “We found that ozone exposure to ALI cultures increases HA expression (Fig. 3A).” and these data are in Figure 3A.

Supplementary Data:Supplementary Table 1 should be moved to the main manuscript, as it is the basis for the subsequent analyses. Currently, certain receptors (e.g., LAYN on page 8) are introduced abruptly, while others (TLR5) appear much later without a cohesive introduction.

Response: We have moved the Table to the main MS, as suggested by the Reviewer. We have added introduction about TLR5 in the text as suggested by the Reviewer.

Figure 3D & Inflammation:This figure is summarized in only three lines at the end of a paragraph. There is no discussion regarding the choice of cytokines measured, which currently appear to be selected at random.

Response: We apologize for the omission and have added this information in the Results section of the MS.

Data Consistency:The concluding sentence (“...in aggregate... Receptors”) is not supported by the data, as it refers to HA molecular weight, which—according to the authors—remained unchanged.

Response: We have removed the statement about HA size.

TLR5 Involvement:The role of TLR5 is currently supported only by a citation (Ref 36). It would be more compelling if the authors could demonstrate that TLR5 expression or activity was altered in the treatments shown in Figure 2.

Response: We have added new experiments, also in Response to critique by Reviewer 1, suggesting TLR5 activation by HA (new Figure 6C).

Figures 5B and 5C:These figures are data-dense, yet they are summarized in a single concluding sentence. The authors must provide a detailed analysis, though this is currently difficult due to the illegible font size in the images

Response: We have changed this Figure and broken it up to multiple. Furthemore, we have added description in the text.

Figure 5 Caption:Please define the meaning of the asterisks. I cannot find it in the images.

Response: We apologize for the omission. We have updated our legends to describe asterisks.

Results vs. Methods:In the final lines of the Results, the authors introduce a TLR5 agonist. However, there is no mention of this reagent in the Methods, nor an explanation of its relationship with HA in this context.

Response: We apologize for the omission. We have now included this information in Methods.

Minor Issues:

Acronyms and Formatting: A thorough revision of acronyms and nomenclature is required. Please ensure consistency in capitalization and formatting (e.g., ALI, IL-6 vs. Il6, RHAMM/Hmmr, and TLR5 vs. Tlr5).

Response: Thank you for the opportunity to clarify. We are using capitalized acronyms for human genes, and non-capitalized acronyms for murine genes as per international convention (https://www.biosciencewriters.com/Guidelines-for-Formatting-Gene-and-Protein-Names.aspx). In instances where both appear, it is because the text references both species, for example: “HMMR/Hmmr expression did not change after ozone exposure in human or murine epithelial cells respectively”, where HMMR refers to the human gene and Hmmr to the mouse gene. To avoid further confusion, we changed all acronyms to capitals, except when expressly discussing genetic deficiency or gene expression of specific genes in mouse cells.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I have no more comments.