Pathophysiology of Lung Disease and Wound Repair in Cystic Fibrosis

Round 1

Reviewer 1 Report

Compromised wound healing has been associated with cystic fibrosis. Even though the exact role of CFTR in the wound healing process is complicated, the review provides a nice overview of the molecular and cellular events that may offer therapeutic insights to treat CF.

Author Response

We thank the Reviewer for her/his appraisal and kind consideration of our effort.

Reviewer 2 Report

The topic addressed by Conese and Di Gioia in this review is important for the pathophysiology of cystic fibrosis. It is worth to mention that many aspects described here have been already discussed in a recent in depth review of the “role of CFTR in Development, Differentiation, Regeneration and Cancer” by Amaral and collaborators. Overall, this review is interesting but needs important improvement to be useful for the readers.

 

General comments:

The review is mostly descriptive and misses many relevant references to the topic, mostly those dealing with primary cultures of airway epithelial cells grown at ALI. Some sections are lacking details (the part on EMT for instance) and others could be reduced since they are not directly relevant to the topic (the role of CFTR in skin cell migration). Importantly, the authors did not discuss strengths and limitations of the different cell models used to study wound repair, which to my opinion is critical. There is little attempt to integrate literature or to provide assumptions on why some data on the very same cell model appear contradictory. It would be useful for example to provide a Table that includes the different models (cell lines, primary cells, monolayer, polarized or ALI…), the wounding method used (Scratch, 2D migration...), the observed effect on proliferation and migration or other important phenotype, and the appropriate references. To my opinion, the study of proliferation and migration after wounding on an immortalized cell line grown as a monolayer is hardly comparable to well-differentiated primary cells grown at ALI, where re-differentiation from progenitor cells occurs. Another suggestion would be to summarize each section in a graphical format, making it easier to understand for the readers. Detailed analyses linking the different aspects from the different sources would be good too. Some sentences were also formulated poorly making them difficult to understand.

 

Major comments:

Figure 1: the figure is not very nice with heteroclite images obviously taken from the web without proper referencing. Instead, a scheme that summarizes the different cell models used/experiments done to tackle the question of wound repair would be more helpful.

p.3, l.88: additional relevant references on the effects of quorum sensing molecules on epithelial repair should be cited (ie PMID: 2556267).

P.5, section 2: not enough detail explaining the different pathological processes occurring in CF. Why focusing only on the EGFR/ADAM17 axis while its role is uncertain. How the authors relate this axis to goblet and basal cell hyperplasia, squamous metaplasia, increase in epithelial height and cell shedding? The link is unclear.

p.6, l.185-198. This paragraph is very important since it deal with repair (re-differentiation from progenitor cells) but only two references are provided. There are earlier or completing studies that need to be cited here. It may be also important to define precisely repair, healing, regeneration… according to the cell model/methods of injury used.

p.6, following paragraphs: The authors are mixing data from different cell models that could not be compared (immortalized cells vs primary cells, monolayer vs polarized, presence or not of basal progenitor cells). For non-familiar researchers in the CF field, this is hard to understand. Importantly, the different steps of wound repair described in l.185-197 do not occur in cell lines. For example, l.227-228, the authors state : ”no differences were found in cell proliferation and migration rates among CFBE, corrected CFBE and 16HBE cells questioning the usefulness of immortalized cell lines for these studies. If these cell lines are considered by the authors as not useful, what information should be retained from EMT studies, the role of klf4 or the modulation by cell-cell contact with mesenchymal cells discussed later? In addition, in case of a wound repair process, it is very important to mention how the injury was induced in these experiments. Can the authors discuss in more details how CFTR regulates these processes, via its “channel” function or protein/protein interaction? What is known, what is your assumption?

p.6, l.225-229: Another example showing the descriptive nature of the text without discussion of the data! CFSME and Calu-3 cells are from different background and cannot be directly compared to evaluate wound healing rates!  A better comparison between the effect of CFTR inhibitors, mutations, silencing, KO or CFTR correction would be interesting. It is strongly suggested to the authors to discuss what type of information can be obtained from these simpler models!

The paragraph on keratinocyte migration is interesting but to my opinion, more discussion should be provided on the literature dealing with airway epithelial cells rather than skin cells.

p.7, l.281-289: more recent papers than reference 76 show, on the contrary, that b1-intergrin is overactive in CF airway epithelial cells! it could be important to discuss here the relationship between b1-integrin, fibronectin (a ligand of b1-integrin) and MMPs. This phenotype may have implications for EMT! This paragraph (l.278-289) needs to be improved.

p.8, l.307-309: How does delayed wound closure promote wound healing? Klf4 is enhanced in CF: it is expected to increase wound healing, not decrease. Could you elaborate on this?

p.8, l.312: what the authors mean by “CF cells are already in the basal state more mesenchymal than wt-CFTR cells”. Could they define what basal state is? Fibronectin is considered as a mesenchymal marker during EMT. Why its expression is decreased in CF if we have partial EMT? KLF4 is a known negative regulator of TGFb-mediated inflammation but not fibronectin expression. Mesenchymal cells are generally described as cells with enhanced migratory capacity. If CF cells display a partial EMT and are over-sensitive to EMT, why do they have lower wound repair capacity and decreased migration? This paragraph on EMT needs some thoughtful revision.

The figure 3a is not necessary (just label scheme in Fig.3b accordingly). Fig. 3b is not clear. It would be better to show a comparison between CF vs. NCF epithelium repair after injury and to integrate the pathways discussed in this review. Is TGFb only produced by the macrophages? No evidence in the text that it is produced from macrophages or epithelial cells themselves. There is also a crosstalk between TGFb and MMPs, since TGFb is secreted in a latent form and needs to be activated by MMPs. Is there a relation between MMPs and TGFb in CF? This part was not very clear and should be mentioned since remodeling of the extracellular matrix is very important for wound healing.

Another aspect that was not discussed in the review is cytoskeleton remodeling and its modulation by CFTR. This is also important for the establishment of cell polarity during the migration process and directionality (front vs back). Is there any known link between CFTR defect, cytoskeleton remodeling and wound healing in CF?

 

Minor comments:

The text should be read by a native English speaker. Below are examples of sentences that need to be corrected:

Abstract:

“eventually leading to airway epithelium injury and remodelling” - remove eventually!

“In vitro and in vivo studies point to a dysregulated wound closure in CF airways, whose feature are to be traced back to epithelial CFTR lack/dysfunction and altered ion/fluid fluxes and/or signaling, ensuing in reduced cell migration”. This sentence is hard to understand!

“Although of wealth of in vivo and in vitro models have obtained insight into the pathophysiology of wound healing in CF”.  Again, hard to understand the meaning.

Introduction:

“Cystic fibrosis (CF) is the commonest autosomal recessive disorder…”. Replace by most common!

 “Class I comprises mutations that lead to a premature stop codon and the protein is not expressed at all”.  Is the protein not expressed at all or is a truncated/non-functional protein expressed?

“CFTR transports chloride ions and it is involved in sodium concentration in the ASL”. Add sodium “ions”!

“have been implied in the matrix remodelling”. Change to implicated!

 “HGF and KGF acts as chemotactic and growth-stimulating factors as well as stimulate of the synthesis…”. Delete “of”!

Wound Healing in CF:

“After verified that CFTR plays an important role in wound closure…”. Change to verifying!

“CFBE showed slower wound repair in comparison to its corresponding plasmid-corrected CFBE41o-pCep4, overexpressing wild type-CFTR, but not towards non-CF 16HBE14o- cells.” Sorry, it’s not understandable.

“Also CFSME cells (CF submucosal gland) showed a significant reduced wound healing rate…”. Change to significantly!

 “In these models it was shown that CFTR activation is implied…”. I don’t understand, do they show that CFTR is activated or not…?

Cellular and Molecular Event in CF Wound Healing: 

“Overall, these data implicate an important role of ion transporters in airway epithelial wound closure and the positive role of CFTR”. Change to imply!

“KLF4 knockout significantly increased TEER of F508del CFTR cells (indicating a less leaky epithelium), suggesting that, in the context of CF airways, KLF4 promotes wound healing”.  Insert after the parentheses “and delayed wound closure”!

Modulation of Wound Healing in CF:

“As about the rescue of mutant CFTR (i.e, F508del) contrasting results have been obtained [87]…” – Hard to understand!

Conclusion:

“Continuous injury by bacteria and their exoproducts, as well chronic exposure to inflammatory cytokines may render CF cells less susceptible to the migratory machine…”….“as well as”!

“The dara published so far support the model that is depicted in Figure 3”. Correct to data!

“Other issues have to further studied. For example, the complex role of CFTR: does its positive effect on wound closure is dependent on its functional activity as an ion channel or on other intracellular functions?”…. to “be” further studied; …wound closure “depends” on its…!

Author Response

The topic addressed by Conese and Di Gioia in this review is important for the pathophysiology of cystic fibrosis. It is worth to mention that many aspects described here have been already discussed in a recent in depth review of the “role of CFTR in Development, Differentiation, Regeneration and Cancer” by Amaral and collaborators. Overall, this review is interesting but needs important improvement to be useful for the readers.

 

General comments:

The review is mostly descriptive and misses many relevant references to the topic, mostly those dealing with primary cultures of airway epithelial cells grown at ALI. Some sections are lacking details (the part on EMT for instance) and others could be reduced since they are not directly relevant to the topic (the role of CFTR in skin cell migration). Importantly, the authors did not discuss strengths and limitations of the different cell models used to study wound repair, which to my opinion is critical. There is little attempt to integrate literature or to provide assumptions on why some data on the very same cell model appear contradictory. It would be useful for example to provide a Table that includes the different models (cell lines, primary cells, monolayer, polarized or ALI…), the wounding method used (Scratch, 2D migration...), the observed effect on proliferation and migration or other important phenotype, and the appropriate references. To my opinion, the study of proliferation and migration after wounding on an immortalized cell line grown as a monolayer is hardly comparable to well-differentiated primary cells grown at ALI, where re-differentiation from progenitor cells occurs. Another suggestion would be to summarize each section in a graphical format, making it easier to understand for the readers. Detailed analyses linking the different aspects from the different sources would be good too. Some sentences were also formulated poorly making them difficult to understand.

We agree with the Reviewer and we have tried at our best to fill the gaps about topics of interest and related reference works in the field. The part on EMT has been considerably expanded and detailed, whereas the role of CFTR in skin cell migration has been deleted (consequently also the part on curcumin). Moreover, to reconcile all the models and different outcomes we have integrated these results throughout the manuscript. To this end, main features of wound repair and regeneration have been presented for both non-CF and CF airway epithelia (Figure 2 and Figure 5, respectively). We consider very fruitful the suggestion of a comprehensive table that is now provided (as Table 2). As to well-differentiated primary cells grown at ALI, we agree with their relevance and we have expressed their importance in the novel paragraph describing this culture method, i.e. that the ALI cultures mimic the in vivo situation better than 2D monolayers. As suggested, we have included two novel figures about the epithelial regeneration occurring in the xenograft model (Figure 3) and about the cell models and wound repair (Figure 4). Moreover, we have done our best to connect all the different aspects from the different sources. Finally, we also re-formulated some sentences to make them more readable.

Major comments:

Figure 1: the figure is not very nice with heteroclite images obviously taken from the web without proper referencing. Instead, a scheme that summarizes the different cell models used/experiments done to tackle the question of wound repair would be more helpful.

We have deleted Figure 1, while we have added a figure summarizing different cell models used and different models of regeneration and wound repair after injury.

p.3, l.88: additional relevant references on the effects of quorum sensing molecules on epithelial repair should be cited (ie PMID: 2556267).

We have added the reference indicated.

P.5, section 2: not enough detail explaining the different pathological processes occurring in CF. Why focusing only on the EGFR/ADAM17 axis while its role is uncertain. How the authors relate this axis to goblet and basal cell hyperplasia, squamous metaplasia, increase in epithelial height and cell shedding? The link is unclear.

In the revised version, we have clarified that MMPs and ADAMs are involved in the activation of EGFR pathway after TNF-a stimulation, by shedding EGFR ligands. ADAM17 is now described to be involved in this axis, due to ADAM17-mediated shedding of the EGFR ligands, among which amphiregulin (AREG). AREG autocrine signaling affects mucus expression and cytokine secretion, whereas its paracrine signaling has been linked to TGF-β-induced fibrosis. Moreover, CF airway epithelial cells have been shown to have an increased activity of AREG as compared with wt-CFTR expressing cells. Since the EGFR is involved in mucin expression and secretion in the CF airways, we thought it was appropriate to mention the putative role of this axis above all in mucin secretion, inflammation, and fibrosis. We do not claim that ADAM17-AREG-EGFR axis is related to all the other pathological hallmarks of CF lung disease, but that, given its properties, it would be worth to study it in this context.

p.6, l.185-198. This paragraph is very important since it deal with repair (re-differentiation from progenitor cells) but only two references are provided. There are earlier or completing studies that need to be cited here. It may be also important to define precisely repair, healing, regeneration… according to the cell model/methods of injury used.

To comply with the Reviewer’s concern, we have considerably expanded the paragraphs about the use of xenografts to understand molecular and cellular mechanisms involved in regeneration and wound repair, also citing other relevant works in this field. Moreover, we have added a paragraph explaining more precisely the terms “regeneration” and “wound repair”. Although many authors use “healing” and “repair” interchangeably, we have avoided to use the term “healing” throughout the manuscript since it is referred to various complex processes, that completely restore the original architecture and structure of an organ or anatomic part as it was before injury.

p.6, following paragraphs: The authors are mixing data from different cell models that could not be compared (immortalized cells vs primary cells, monolayer vs polarized, presence or not of basal progenitor cells). For non-familiar researchers in the CF field, this is hard to understand. Importantly, the different steps of wound repair described in l.185-197 do not occur in cell lines. For example, l.227-228, the authors state : ”no differences were found in cell proliferation and migration rates among CFBE, corrected CFBE and 16HBE cells questioning the usefulness of immortalized cell lines for these studies. If these cell lines are considered by the authors as not useful, what information should be retained from EMT studies, the role of klf4 or the modulation by cell-cell contact with mesenchymal cells discussed later? In addition, in case of a wound repair process, it is very important to mention how the injury was induced in these experiments. Can the authors discuss in more details how CFTR regulates these processes, via its “channel” function or protein/protein interaction? What is known, what is your assumption?

We agree with the Reviewer and to comply with her/his observation, we have now divided this Section in subsections related to xenografts, submerged monolayer cultures, and polarized/ALI cultures. Sometimes, primary cultures have been used as monolayers and we have referred to them in the relevant subsection. Table 2 presents all these models and the results obtained thereof. If the Reviewer agree, we have listed studies in chronological order, since some studies used both immortalized and primary airway epithelial cells.

In the Section 3.4 we have more detailed the assumption that the role of CFTR in regeneration and wound repair is due to its channel function and relationship, either direct or indirect, with other ion channels. To the best of our knowledge, we are not aware that this role can be attributed to protein/protein interactions.

p.6, l.225-229: Another example showing the descriptive nature of the text without discussion of the data! CFSME and Calu-3 cells are from different background and cannot be directly compared to evaluate wound healing rates!  A better comparison between the effect of CFTR inhibitors, mutations, silencing, KO or CFTR correction would be interesting. It is strongly suggested to the authors to discuss what type of information can be obtained from these simpler models!

The work to which these lines are referred is indeed problematic and we have now better described and criticized its outcomes, concluding that these simpler models can take advantage only when isogenic cell lines are compared.

We have also clearly written in the Conclusion Section that all CFTR manipulations clearly lead to the undisputable role of CFTR in airway epithelium regeneration and wound repair.

The paragraph on keratinocyte migration is interesting but to my opinion, more discussion should be provided on the literature dealing with airway epithelial cells rather than skin cells.

Accordingly, we have removed this paragraph.

p.7, l.281-289: more recent papers than reference 76 show, on the contrary, that b1-intergrin is overactive in CF airway epithelial cells! it could be important to discuss here the relationship between b1-integrin, fibronectin (a ligand of b1-integrin) and MMPs. This phenotype may have implications for EMT! This paragraph (l.278-289) needs to be improved.

We thank the Reviewer for this interesting observation and we have added the reference by Grassmé et al. (Beta1-Integrin Accumulates in Cystic Fibrosis Luminal Airway Epithelial Membranes and Decreases Sphingosine, Promoting Bacterial Infections. Cell Host Microbe 21: 707-18 e8.2017) about the presence of high levels of b1-integrins on the surface of airway epithelial cells in vivo and in vitro. Moreover, we have added a more recent study (Badaoui M, Zoso A, Idris T, Bacchetta M, Simonin J, et al. (2020) Vav3 Mediates Pseudomonas aeruginosa Adhesion to the Cystic Fibrosis Airway Epithelium. Cell Rep 32: 107842) which shows that b1-integrin overexpression on the apical side of CF cells is linked to overexpression of Vav3, a nucleotide exchange factor. However, the same study showed that Vav3 silencing did not affect cell migration in a wound healing assay, questioning if b1 integrin, Vav3, and fibronectin at this location are involved in any process associated with EMT. We have discussed these issues at pages 21-22.

p.8, l.307-309: How does delayed wound closure promote wound healing? Klf4 is enhanced in CF: it is expected to increase wound healing, not decrease. Could you elaborate on this?

Sousa and colleagues (Int J Mol Sci 2020) showed that KLF4 KO decreases wound closure even further in F508del-CFTR cells (although not significantly), suggesting that KLF4 promotes wound healing. On the other hand, the wound closure in non-CF cells was not altered by KLF4 KO. The authors speculated that since CF cells start from a more mesenchymal state, this may influence the outcome of KLF4 KO. It has been shown a high expression of KLF4 in the same CF cell line as compared with wt-CFTR expressing cells (Sousa, Cells 2020). However, KLF4 downregulation promoted expression of wt-CFTR but not of F508del–CFTR, indicating a specific impact of KLF4 on normal CFTR. Moreover, in primary CF airway epithelial cells grown at ALI, the mRNA levels for KLF4 remained unchanged during wound repair (Crespin et al., The International Journal of Biochemistry & Cell Biology 2014). Overall, these data indicate that if KLF4 have an impact on epithelial wound healing in CF this is subtle, and the reason could be that  there might be some compensatory mechanism between KFL4 and other KLFs (KLF5 and KlF2), as well as some degree of redundancy.

 

We have referred to all these issue in Section 3.3.

p.8, l.312: what the authors mean by “CF cells are already in the basal state more mesenchymal than wt-CFTR cells”. Could they define what basal state is? Fibronectin is considered as a mesenchymal marker during EMT. Why its expression is decreased in CF if we have partial EMT? KLF4 is a known negative regulator of TGFb-mediated inflammation but not fibronectin expression. Mesenchymal cells are generally described as cells with enhanced migratory capacity. If CF cells display a partial EMT and are over-sensitive to EMT, why do they have lower wound repair capacity and decreased migration? This paragraph on EMT needs some thoughtful revision.

This paragraph has been re-written and we hope that the Reviewer will find it more specific and appropriate about the fact that CF cells start from a more mesenchymal state. Indeed, Quaresma and colleagues have demonstrated that native lung tissue from CF patients have increased transcript levels of epithelial markers, such as those of TJs (occludin and ZO-1), GJs (connexin 43, connexin 26), and cytokeratin 18 (CK18) as well as have increased mesenchymal marker expression (vimentin) as compared with non-CF tissues. However, other epithelial markers (claudin, E-cadherin, beta-catenin, connexin 31 and desmoplakin) were not changed, as also other mesenchymal markers (N-cadherin and fibronectin). These phenotypic characteristics are appropriate for a partial EMT activation in CF. In the partial CF EMT, cells still migrate mostly as monolayers as observed by live cell imaging of wound closure, like epithelial cells do. Thus, it is likely that the CF TJ defects could account for the observed CF delay in wound closure, given the importance of cell-to-cell contacts in cell migration. In parallel, mutant CFTR can cause increased proliferation while being detrimental to tissue regeneration, as reported. Finally, increased migration does not necessary occur in EMT. Thus, the partial CF EMT behavior, characterized by TJ dysfunction and low migratory phenotype, may explain the defect in wound healing.

We have added all these considerations in Section 3.4.

The figure 3a is not necessary (just label scheme in Fig.3b accordingly). Fig. 3b is not clear. It would be better to show a comparison between CF vs. NCF epithelium repair after injury and to integrate the pathways discussed in this review. Is TGFb only produced by the macrophages? No evidence in the text that it is produced from macrophages or epithelial cells themselves. There is also a crosstalk between TGFb and MMPs, since TGFb is secreted in a latent form and needs to be activated by MMPs. Is there a relation between MMPs and TGFb in CF? This part was not very clear and should be mentioned since remodeling of the extracellular matrix is very important for wound healing.

We have generated two different figures for regeneration and repair in non-CF and CF airway epithelial, Figure 2 and Figure 5 respectively. They are more detailed in molecules and processes involved.

TGFb is produced by immune and nonimmune cells (including epithelial cells) and this has been mentioned at page 10. The crosstalk between TGFb and MMPs, with TGFb activation by MMPs has been reported in Section 1.1 (page 6). At the best of our knowledge, we are not aware of studies concerning the relation between MMPs and TGFb in CF.

Another aspect that was not discussed in the review is cytoskeleton remodeling and its modulation by CFTR. This is also important for the establishment of cell polarity during the migration process and directionality (front vs back). Is there any known link between CFTR defect, cytoskeleton remodeling and wound healing in CF?

The issue of cytoskeleton remodeling in CF has been reported in Section 1. We acknowledge that a recent study (Badaoui M. et al. (2020) Vav3 Mediates Pseudomonas aeruginosa Adhesion to the Cystic Fibrosis Airway Epithelium. Cell Rep 32: 107842) demonstrated that primary CF airway epithelial cells and CFTR-silenced Calu-3 cells showed Vav3 overexpression associated with increased fibronectin and b1 integrin expression at their apical side. However, despite an altered rearrangement of actin cystoskeleton, cell migration in a wound healing assay was not affected. We have thus discussed these findings at pages 21-22.

Minor comments:

The text should be read by a native English speaker. Below are examples of sentences that need to be corrected:

Abstract:

“eventually leading to airway epithelium injury and remodelling” - remove eventually!

We have removed “eventually”.

“In vitro and in vivo studies point to a dysregulated wound closure in CF airways, whose feature are to be traced back to epithelial CFTR lack/dysfunction and altered ion/fluid fluxes and/or signaling, ensuing in reduced cell migration”. This sentence is hard to understand!

We have divided this hard-to-understand sentence in two shorter ones.

“Although of wealth of in vivo and in vitro models have obtained insight into the pathophysiology of wound healing in CF”.  Again, hard to understand the meaning.

We have rephrased this sentence.

Introduction:

“Cystic fibrosis (CF) is the commonest autosomal recessive disorder…”. Replace by most common!

It was replaced.

 “Class I comprises mutations that lead to a premature stop codon and the protein is not expressed at all”.  Is the protein not expressed at all or is a truncated/non-functional protein expressed?

Accordingly to the classification by Marson et al. (2016). which includes a previous proposal of De Boeck and Amaral (2016), class I comprises those of class 1A (no mRNA transcription) and class 1B (stop-codon mutations), both having the same outcome, i.e. absence of the CFTR protein (in case of class IB due to degradation of truncated mRNA by nonsense-mediated decay).
We have now included this statement in Section 1.

“CFTR transports chloride ions and it is involved in sodium concentration in the ASL”. Add sodium “ions”!

It was added.

“have been implied in the matrix remodelling”. Change to implicated!

It was changed.

 “HGF and KGF acts as chemotactic and growth-stimulating factors as well as stimulate of the synthesis…”. Delete “of”!

This sentence was modified.

Wound Healing in CF:

“After verified that CFTR plays an important role in wound closure…”. Change to verifying!

It has been changed.

“CFBE showed slower wound repair in comparison to its corresponding plasmid-corrected CFBE41o-pCep4, overexpressing wild type-CFTR, but not towards non-CF 16HBE14o- cells.” Sorry, it’s not understandable.

We have rephrased this sentence.

“Also CFSME cells (CF submucosal gland) showed a significant reduced wound healing rate…”. Change to significantly!

It has been changed.

 “In these models it was shown that CFTR activation is implied…”. I don’t understand, do they show that CFTR is activated or not…?

This sentence was deleted since it was part of the role of CFTR in skin cell migration.

Cellular and Molecular Event in CF Wound Healing: 

“Overall, these data implicate an important role of ion transporters in airway epithelial wound closure and the positive role of CFTR”. Change to imply!

It has been changed to “support”, as requested by the Reviewer 3.

“KLF4 knockout significantly increased TEER of F508del CFTR cells (indicating a less leaky epithelium), suggesting that, in the context of CF airways, KLF4 promotes wound healing”.  Insert after the parentheses “and delayed wound closure”!

It has been added.

Modulation of Wound Healing in CF:

“As about the rescue of mutant CFTR (i.e, F508del) contrasting results have been obtained [87]…” – Hard to understand!

This sentence has been deleted since it belonged to the role of CFTR in cutaneous wound repair.

Conclusion:

“Continuous injury by bacteria and their exoproducts, as well chronic exposure to inflammatory cytokines may render CF cells less susceptible to the migratory machine…”….“as well as”!

It has been corrected.

“The dara published so far support the model that is depicted in Figure 3”. Correct to data!

It has been corrected.

“Other issues have to further studied. For example, the complex role of CFTR: does its positive effect on wound closure is dependent on its functional activity as an ion channel or on other intracellular functions?”…. to “be” further studied; …wound closure “depends” on its…!

We have deeply changed the formulation of these sentences to highlight that wound closure depends on CFTR, although all the mechanisms have not been completely disclosed yet.

Reviewer 3 Report

I found this manuscript to be interesting and comprehensive in its review of the literature.  However there are a significant number of language issues that will require editing and I have some specific comments that the authors need to address: Also, I have sent an edited copy of the manuscript pdf to the section managing editor, Ms. Milica Tanasic, that identifies many additional suggested edits that should be helpful in making revisions to the manuscript. 

Major Comments:

Lines 32-39: This listing of the CFTR mutation classification system is awkward. I recommend creating a table that can be referenced in the text. This will be much easier for the reader to follow. 

Lines 41-45: Figure 1 does not clearly show these structural features of CFTR. If the authors wish to reference figure 1 here, they need to modify the figure so that these structural properties are explicitly identified.

Line 47: What is meant by disposal? do the authors mean "internalization" or "ubiquitination"? More clarity is needed.

Lines 50-52: This sentence is unclear. If the authors are suggesting that ENaC activity is being regulated by extracellular [Cl] then they should cite Collier and Snyder, "Extracellular Chloride Regulates the Epithelial Sodium Channel", J. Biol. Chem. 284(43)29320-29325, 2009. 

Lines 65-70: Run-on sentence. Revise by making this two sentences.

Figure legend 1, Line 72: What is meant by "unbalanced"? Be specific.

Lines 112-114:  Why? The abundance of these cells is very low and their role (as pointed out in this sentence) is essentially unknown. Just because they express high levels of CFTR is not a very compelling reason to think that they significantly contribute to mucociliary clearance, particularly considering their low abundance. 

Line 172: What is meant by "developed epithelial cells"? Do you mean progenitor cells or basal cells?

Line 199: replace with: "The first in vitro study"

Line 202-203: replace with: "CFTR expression was silenced"

Line 280: replace with: "CFTR silenced Calu-3 cells"

Line 377: "less susceptible to the migratory machine" Unclear. What is meant by this phrase? Revise. 

Lines 394-395: Let's not forget that there is evidence supporting CFTR channel function as having a critical role in wound healing. Specifically the wound current measurements reported in native rhesus monkey tracheal epithelium and the in vitro cell migration studies where the rate of wound closure was reduced by treatment with the CFTR blocker CFTRinh-172. There is also evidence indicating an indirect role for CFTR, for example silencing CFTR reduces GM1 and cholesterol expression which alters beta1 integrin mediated cell adhesion, which presumably affects the ability of cells to achieve sufficient traction to support migration.  This sentence makes it sound like these data are not relevant and that the issue is a completely open question. 

Comments for author File: Comments.pdf

Author Response

Reviewer 3

I found this manuscript to be interesting and comprehensive in its review of the literature.  However there are a significant number of language issues that will require editing and I have some specific comments that the authors need to address: Also, I have sent an edited copy of the manuscript pdf to the section managing editor, Ms. Milica Tanasic, that identifies many additional suggested edits that should be helpful in making revisions to the manuscript. 

We would like to thank the Reviewer for her/his kind appraisal of our manuscript. We have done our best to consider all the editorial changes required by the Reviewer in the file that was sent to the attention of Ms. Milica Tanasic.

Major Comments:

Lines 32-39: This listing of the CFTR mutation classification system is awkward. I recommend creating a table that can be referenced in the text. This will be much easier for the reader to follow. 

We have included a Table with CFTR mutation classes. However, we would like to maintain the listing of CFTR mutation classes in the text, which was nevertheless emended, otherwise the Table would not be easily readable. We hope that the Reviewer agrees on this.

Lines 41-45: Figure 1 does not clearly show these structural features of CFTR. If the authors wish to reference figure 1 here, they need to modify the figure so that these structural properties are explicitly identified.

As also the Reviewer 2 questioned the quality of Figure 1, this figure has been removed.

Line 47: What is meant by disposal? do the authors mean "internalization" or "ubiquitination"? More clarity is needed.

We agree with the Reviewer that the sentence was not clear and thus we have substituted it with the following: “Low or null CFTR protein expressed at the plasma membrane or its dysfunction…”

Lines 50-52: This sentence is unclear. If the authors are suggesting that ENaC activity is being regulated by extracellular [Cl] then they should cite Collier and Snyder, "Extracellular Chloride Regulates the Epithelial Sodium Channel", J. Biol. Chem. 284(43)29320-29325, 2009. 

The aim of this sentence was to expose the fact that wt-CFTR acts as a tonic inhibitor of ENaC in the airways and thus we have modified it accordingly, adding also two related references.

Lines 65-70: Run-on sentence. Revise by making this two sentences.

We have made two sentences from one. However, since the review was re-organized, we have moved these sentences in the Section 2. We hope that the Reviewer agrees on this.

Figure legend 1, Line 72: What is meant by "unbalanced"? Be specific.

This Legend has been deleted along with Figure 1.

Lines 112-114:  Why? The abundance of these cells is very low and their role (as pointed out in this sentence) is essentially unknown. Just because they express high levels of CFTR is not a very compelling reason to think that they significantly contribute to mucociliary clearance, particularly considering their low abundance. 

The aim of this assertion on ionocytes was not to be intended that these rare cells contribute to mucociliary clearance but that they should be considered in next studies concerning regeneration and wound repair of the CF airway epithelium. Basically, so far we know only that basal cells are the main cell type involved in these processes when studied with well-differentiated primary cultures at ALI (air-liquid interface). Interestingly, a very recent studies have considered ionocytes in a transcriptomic profile study of human airway epithelial cells from study applied to wound repair (Zoso et al., Sci Data 2019, 6, 240), which have been commented in Section 3.3.

Line 172: What is meant by "developed epithelial cells"? Do you mean progenitor cells or basal cells?

It was meant “differentiated airway epithelial cells” and it was modified accordingly.

Line 199: replace with: "The first in vitro study"

Since the study by Schiller et al. was not really the first in vitro study, it now reads: “one of the first works”.

Line 202-203: replace with: "CFTR expression was silenced"

We have modified accordingly.

Line 280: replace with: "CFTR silenced Calu-3 cells"

We have modified accordingly.

Line 377: "less susceptible to the migratory machine" Unclear. What is meant by this phrase? Revise.

We have modified with the following: “less amenable to migration”. 

Lines 394-395: Let's not forget that there is evidence supporting CFTR channel function as having a critical role in wound healing. Specifically the wound current measurements reported in native rhesus monkey tracheal epithelium and the in vitro cell migration studies where the rate of wound closure was reduced by treatment with the CFTR blocker CFTRinh-172. There is also evidence indicating an indirect role for CFTR, for example silencing CFTR reduces GM1 and cholesterol expression which alters beta1 integrin mediated cell adhesion, which presumably affects the ability of cells to achieve sufficient traction to support migration.  This sentence makes it sound like these data are not relevant and that the issue is a completely open question. 

We agree with the Reviewer and thus we have deeply reconsidered the critical role of CFTR in wound repair, considering that the use of CFTR-deficient cells, CFTR inhibitors, CFTR gene silencing, and CFTR modulators that CFTR plays a key role in the regeneration and repair of the airway epithelium. Moreover, in the Conclusion Section we have added the consideration about the effects on CFTR silencing on GM1, cholesterol, and beta 1 integrin.

Reviewer 4 Report

The ms requires few english corrections and a more attention to uptodate references

Author Response

We have done our best to correct the English language and to cite updated references.

Round 2

Reviewer 2 Report

This is an outstanding review. There are not many on this particular aspect of the CF pathology. The new Table 2 is very informative and useful. Thank you for compelling these references.

The authors should verify that all references mentioned in their responses to the Reviewers are indeed cited in the revised manuscript. Few typos remain. Finally, legends should be verified to ascertain that they described appropriately the Figures. 

Author Response

This is an outstanding review. There are not many on this particular aspect of the CF pathology. The new Table 2 is very informative and useful. Thank you for compelling these references.

We thank the Reviewer for her/his appraisal of our effort.

We have verified that all references mentioned in our responses to the Reviewers were indeed cited in the revised manuscript. If the Reviewer refers to the article cited to as PMID: 2556267, this appears in PubMed as: “Sambucetti LC, Cherrington JM, Wilkinson GW, Mocarski ES. NF-kappa B activation of the cytomegalovirus enhancer is mediated by a viral transactivator and by T cell stimulation. EMBO J. 1989 Dec 20;8(13):4251-8”, that has no relation with the topic discussed. Instead we have included the following novel reference: “Maillé É, Ruffin M, Adam D, Messaoud H, Lafayette SL, McKay G, Nguyen D, Brochiero E. Quorum Sensing Down-Regulation Counteracts the Negative Impact of Pseudomonas aeruginosa on CFTR Channel Expression, Function and Rescue in Human Airway Epithelial Cells. Front Cell Infect Microbiol. 2017 Nov 10;7:470. doi: 10.3389/fcimb.2017.00470. PMID: 29177135”.

We ask apologies to be not precise on this issue.

The authors should verify that all references mentioned in their responses to the Reviewers are indeed cited in the revised manuscript. Few typos remain. Finally, legends should be verified to ascertain that they described appropriately the Figures. 

We have done our best to correct all the remained typos. Finally, we have verified thet the legends faithfully describe the Figures and that the text references correspond to Figures. Accordingly, we provide new Figures 2 and 5.

Reviewer 3 Report

Doublecheck text references to figures.

Author Response

Doublecheck text references to figures.

We have verified that the legends faithfully describe the Figures and that the text references correspond to Figures. Accordingly, we provide new Figures 2 and 5.