A positive feedback loop between mTORC1 and cathelicidin promotes skin inflammation in rosacea

Abstract Rosacea is a chronic inflammatory skin disorder whose pathogenesis is unclear. Here, several lines of evidence were provided to demonstrate that mTORC1 signaling is hyperactivated in the skin, especially in the epidermis, of both rosacea patients and a mouse model of rosacea‐like skin inflammation. Both mTORC1 deletion in epithelium and inhibition by its specific inhibitors can block the development of rosacea‐like skin inflammation in LL37‐induced rosacea‐like mouse model. Conversely, hyperactivation of mTORC1 signaling aggravated rosacea‐like features. Mechanistically, mTORC1 regulates cathelicidin through a positive feedback loop, in which cathelicidin LL37 activates mTORC1 signaling by binding to Toll‐like receptor 2 (TLR2) and thus in turn increases the expression of cathelicidin itself in keratinocytes. Moreover, excess cathelicidin LL37 induces both NF‐κB activation and disease‐characteristic cytokine and chemokine production possibly via mTORC1 signaling. Topical application of rapamycin improved clinical symptoms in rosacea patients, suggesting mTORC1 inhibition can serve as a novel therapeutic avenue for rosacea.

30th Oct 2020 1st Editorial Decision 30th Oct 2020 Dear Prof. Li, Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now received feedback from the three reviewers who agreed to evaluate your manuscript. As you will see from the reports below, the referees acknowledge the interest of the study and are overall supporting publication of your work pending appropriate revisions.
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EMBO Molecular Medicine has a "scooping prot ect ion" policy, whereby similar findings that are published by ot hers during review or revision are not a crit erion for reject ion. Should you decide to submit a revised version, I do ask that you get in touch aft er three mont hs if you have not complet ed it , to updat e us on the st at us. In this manuscript, Deng et al provide evidence that suggests the upregulated mTOR signaling may play a role in rosacea. Specifically, the authors suggest a mechanisms whereby cathelicidin stimulates mTOR signalling via the Toll-like receptor (TLR) pathway. In turn, it is proposed that the effects of mTOR in rosacea are mediated by NFkappaB and its target genes. To this end, it was found that the work is significant inasmuch as it links mTOR with previously recognized players in rosacea, and suggests a positive feedback loop between mTOR and cathelicidins. In my opinion, the major weaknesses of the study are the lack of mechanistic evidence explaining observed correlations between cathelicidin, mTOR and NFkappaB and insufficient dissection of the implied role of immune cells in described phenotypes. My specific comments are listed below: Major comments: 1. Notwithstanding that correlative experiments were appreciated, it remains largely unclear how mTOR affects cathelicidin levels as well as how it activates NFkappaB (the relationship between mTOR and NFkappaB is quite complex and context dependent). Are these effects direct or indirect? What are the mediators? Are the effects of mTORC1 on NFkappaB in the context of rosacea IKK-dependent? In Fig 4G the induction of cathelicidin appears to be reversed by rapamycin. At which level does mTOR regulate cathelicidin levels? 2. It is implied that the immune system plays a major role: "Since chemokines and cytokines orchestrate inflammatory response by recruiting and activating distinct immune cells, thus induce the histopathological characteristics of rosacea (10, 14)" but this does not appear to be directly tested. I find that the authors should either directly test the role of mTOR in immune responses in skin in their model or tone down conclusions related to immune compartment. 3. Readouts in addition to phopsho-rpS6 should be included when monitoring mTOR activity. This is particularly important as the Abs that were employed (at least according to the provided catalogue numbers) recognize Ser235/236 on rpS6, which can also be phosphorylated by RSKs and these residues are in fact phosphorylated even when S6K1/2 are ablated (Pende at al MCB 2004). To this end, rpS6 phosphoacceptor sites uniquely affected by mTORC1/S6K axis are Ser 240/244, and thus these pAbs should have been used. Moreover, the phosphosites should be noted in figures throughout the manuscript. 4. In multiple places number of biological replicates for mouse experiments are not indicated (Figs 1I, 2B, 2C, S2H, 2H-J, 6E). If the statement "data are representative of at least 3 independent experiments" indicates that mouse experiments were done at a N=3, this may not be sufficient to reach adequate statistical power. What was the statistical power in these experiments? Quantifications of IHC are also in large part absent. 5. Fig. 5D shows induction of phopspho-rpS6 even in an apparent absence of TLR2. Was the overexpression done in a knock-out cell line? What is the explanation for the absence of TLR2 here when compared to Scr controls from Fig. 5C.
Other comments: 1. Figs 1G/1H -Out of possible noted triggers (heat, spicy foods, UV, chemical and physical stimuli, bacteria) heat and spicy foods were chosen seemingly arbitrarily. The experiments were done only in tissue culture and not on mice. Furthermore, it is noted that capsaicin causes a dose-dependent activation of mTORC1. This may be problematic since the 100uM concentration is close to inducing cell death. In addition, blots monitoring mTOR activity should be shown pS6. 2. In order to eliminate mTORC2 only a single figure with p-Akt (Ser473) immunostaining was presented (Supp Fig 1E). Further evidence is recommended. 3. Figs 3B, 3C -Description of how quantifications were performed should be included. 4. The dynamics of phospho-rpS6 induction by LL37 in Fig. 4E are quite different from Fig. 1J. This should be commented on. 5. Fig. 5E -PKH26 labeling (as in Fig. 5B) would be helpful. 6. A number of typos were noted and thus it was thought that the article may benefit from some careful editing.
I hope that the authors will find my comments constructive and of sufficient pathos The manuscript by Deng el al describes a positive feedback loop between mTORC1 and cathelicidin expression, which promotes skin inflammation in rosacea, a chronic inflammatory skin disease whose pathogenesis is unclear. mTORC1 signaling is found hyper-activated in both rosacea patients and in a LL37-induced mouse model of rosacea-like skin inflammation, whereas deletion or inhibition of mTORC1 blocks the development of rosacea-like skin inflammation. The authors also show that LL37 activates mTORC1 by binding to TLR2 in HaCat cells thereby establishing a positive feedback loop via NFkB and increased cytokine expression. Furthermore, topical application of rapamycin improved the clinical symptoms in rosacea patients suggesting a novel therapeutic route for rosacea through mTORC1 inhibition. This is an interesting paper with many in vivo data, which in principle should be suitable for publication in EMM. However, there are several major points, which should be considered in a revised version of the manuscript: Major points: 1. The paper lacks clarity and is hard to read. Often the choice of tools e.g. individual mouse strains is not described at all. Furthermore, the logistics of experiments is unclear: human data are mixed with in vitro data of HaCat cells and various mouse models in one chapter. The manuscript should be re-written in a concise manner and the English wording must be improved.
2. One main message from the manuscript is that mTORC1 is specifically activated in rosacea, but there is no discussion what causes the specificity and whether possibly upstream-regulators such as expression of TSC1/2 are affected and being causal to upregulation of mTORC1. Along the same line, TLR-2 is specifically upregulated but not TLR4 or other receptors. What is the molecular explanation for the TLR-2 specificity? 3. Re the data analyses, most of the Figures/data are convincing. However, the data using HaCat cells in Figures 1, 5 and 7 are highly questionable. The authors refer to this aneuploid human immortalized cell line as 'keratinocytes' or 'keratinocyte cells', which is misleading and unacceptable. Some of these mechanistic data need to be repeated in primary mouse or human keratinocytes, even though these might have a limited passage number. 4. Regarding the link between mTORC1 hyperactivation and exacerbation of rosacea-like features, it is not clear whether mTOR hyperactivation without injection of LL37 is sufficient to promote skin inflammation/rosacea-like features, since there are no statistically significant differences between control and TSC2+/-mice without LL37. However, authors state that their results demonstrate that mTORC1 signaling promotes the development of rosacea. 5. Is pS6 overexpression directly correlated with an increase in mTOR1 protein levels in patients/rosacea-like mouse models? A Western blot for both proteins should be shown. 6. How do the authors reconcile their positive feedback loop with the observation that only human, but not murine LL37 injection induces rosacea-like disease in mice? This point should be discussed.
Minor points: 1. Rapamycin should be added to the list of Keywords 2. Page numbers and Figures must be labelled at the bottom of the pages 3. Are there differences in severity of rosacea features or are lesional skin areas different in different human populations? The authors only mention references that take into account the Chinese population. 4. Change "healthy individuals" to HS in Fig 6H, as HS is previously used in the manuscript. 5. Could cold/freezing temperatures affect/exacerbate rosacea? The authors mention several times that heat could exacerbate rosacea, but nothing is said about other extreme stresses e.g. cold temperatures. 6. The authors did not mention what is the blue staining in Figure 4B. The reader needs to guess that the blue color is the nuclear staining dye, which could be DAPI, Hoechst? 7. Figure 4C and Figure 6E: Red and blue colors of "EPI" and "Der" words are wrongly used for the proteins of the images, because authors used same colors. 8. Which cells produce LL37? Please, clarify if it is made in keratinocytes and/or other cells and show the data.
Referee #3 (Remarks for Author): In this paper, Deng Z, Chen M and their colleagues found mTORC1 hyperactivation occur in both rosacea patient and LL37-indueced mouse model. In a series of elegant experiments with mouse genetics and human keratinocytes studies, the authors demonstrated mTORC1 signaling is responsible for rosacea symptoms, via enhancing cathelicidin production, NFκB activation, and chemokines upregulation. Most importantly, this study provided a novel and effective clinical therapeutic method, which will be beneficial for rosacea patients. In all, I think the main conclusion from this work is based on solid experimental data, they revealed novel biology in a pathological condition, and offered potential treatment. I recommend it for publication. Minor comments are listed below: 1. Units of length in quantification data are absent in following figure panels: Fig We would like to express our deepest appreciation for your time and effort in handling our manuscript and providing the opportunity to revise our paper. To strengthen the manuscript, we carefully went through the constructive and thoughtful comments by the reviewers and the editor, and performed a series of additional experiments to fully address their concerns. The revised manuscript has now been substantially rewritten with the new data.
Corresponding modifications have also been made to the figures and supplementary files. The changes in the manuscript have been highlighted in yellow color. To address the concerns raised by the reviewers, we have also prepared additional data in Supplementary file figure S8-9 (also included in the "point to point response letter"), which is for review process only.
Please find our point to point responses to the reviewers' comments as detailed below. We are hopeful that all of the additional data, analyses and information we have provided satisfy the reviewers' concerns.

Referee #1
Comments on Novelty/Model System for Author Comment 1:

It is not clear what was the number of the mice (that's why I marked down
technical quality).

Response 1:
Thank you for your careful reviewing and helpful suggestions. We apologize for not indicating the number of the mice in multiple places. In the revised manuscript, we have indicated the number of the mice used in mouse experiments, and we also detailedly answered this question in the "response to major comment 5".

th Jan 2021 1st Authors' Response to Reviewers
Comment 2: 2. Novelty is there, most of the described components have already been linked to rosacea but the role of mTOR as far as I know was unknown.

Response 2:
We thank the reviewer for the recognition of our work.

Comment 3:
3. These findings can be swiftly translated into clinical practice

Response 3:
We thank the reviewer for the recognition of our work, and we will keep trying to translate our findings into clinical practice.

Response 4:
We thank the reviewer for the recognition of our work.

Remarks for Author
Overall comments: In this manuscript, Deng et al provide evidence that suggests the upregulated mTOR signaling may play a role in rosacea. Specifically, the authors suggest a mechanisms whereby cathelicidin stimulates mTOR signalling via the Toll-like receptor (TLR) pathway. In turn, it is proposed that the effects of mTOR in rosacea are mediated by NFkappaB and its target genes. To this end, it was found that the work is significant inasmuch as it links mTOR with previously recognized players in rosacea, and suggests a positive feedback loop between mTOR and cathelicidins. In my opinion, the major weaknesses of the study are the lack of mechanistic evidence explaining observed correlations between cathelicidin, mTOR and NFkappaB and insufficient dissection of the implied role of immune cells in described phenotypes. My specific comments are listed below: Overall response: We thank the reviewer for the recognition of our work and the insightful comments, all of which have been addressed, as detailed below.

Major comment 1:
1. Notwithstanding that correlative experiments were appreciated, it remains largely unclear how mTOR affects cathelicidin levels as well as how it activates NFkappaB (the relationship between mTOR and NFkappaB is quite complex and context dependent). Are these effects direct or indirect? What are the mediators? Are the effects of mTORC1 on NFkappaB in the context of rosacea IKK-dependent? In Fig 4G the induction of cathelicidin appears to be reversed by rapamycin. At which level does mTOR regulate cathelicidin levels?

Response 1:
We thank the reviewer for the recognition of our work and the insightful comments. suggesting that mTORC1 regulates cathelicidin in a VDR-independent manner.
As for other experiments, due to the influence of COVID-19, the materials for the related experiments are relatively difficult to be obtained, resulting in a slow proceeding. However, we will continue to explore this issue, and we hope to publish the outcomes in the future.
As for "How mTOR activates NF-kappaB", we admit that the relationship between mTOR and NFkappaB might be quite complex and context dependent. To follow the reviewer's suggestion, we also performed additional experiments to investigate the mechanism. Unexpectedly, we found that inhibition of mTORC1 signaling could reverse the upregulation of TNF-α (a typical NFkappaB activator) induced by LL37 in human primary keratinocytes in vitro (shown in Revised Supplementary Figure 6D). In the meantime, our data showed that deletion of mTORC1 in epithelial cells could decline the expression of TNF-α in the skin of rosacea mouse model (shown in Revised Figure 2F). Therefore, we speculate that mTORC1 might indirectly activate NFkappaB via upregulation of TNF-α, and we aslo discussed this issue in the Discussion part of the revised manuscript.

Figure for reviewers removed
2. It is implied that the immune system plays a major role: "Since chemokines and cytokines orchestrate inflammatory response by recruiting and activating distinct immune cells, thus induce the histopathological characteristics of rosacea (10, 14)" but this does not appear to be directly tested. I find that the authors should either directly test the role of mTOR in immune responses in skin in their model or tone down conclusions related to immune compartment.

Response 2:
We thank the reviewer for the constructive suggestion. As suggested, we have toned down the conclusions related to immune compartment in the revised manuscript.

Major comment 3:
3. Readouts in addition to phopsho-rpS6 should be included when monitoring mTOR activity. This is particularly important as the Abs that were employed (at least according to the provided catalogue numbers) recognize Ser235/236 on rpS6, which can also be phosphorylated by RSKs and these residues are in fact phosphorylated even when S6K1/2 are ablated (Pende at al MCB 2004).
To this end, rpS6 phosphoacceptor sites uniquely affected by mTORC1/S6K axis are Ser 240/244, and thus these pAbs should have been used. Moreover, the phosphosites should be noted in figures throughout the manuscript.

Response 3:
We thank the reviewer for the constructive suggestion. Our in vivo and in vitro data both showed that phopsho-rpS6 (Ser235/236) can be blocked by mTORC1 specific inhibitor rapamycin, suggesting that the increased phopsho-rpS6 (Ser235/236) in rosacea can monitor mTORC1 activation. In the meantime, as suggested, we also used phopsho-rpS6 (Ser240/244) antibody

Major comment 4:
In multiple places number of biological replicates for mouse experiments are not indicated (Figs 1I, 2B, 2C, S2H, 2H-J, 6E). If the statement "data are representative of at least 3 independent experiments" indicates that mouse experiments were done at a N=3, this may not be sufficient to reach adequate statistical power. What was the statistical power in these experiments?
Quantifications of IHC are also in large part absent.

Response 4:
We thank the reviewer for the careful reviewing and apologize for not

Response 5:
We thank the reviewer for the careful reviewing. In fact, TLR2 was overexpressed in a normal cell line, not a knock-out cell line ( Figure 5D).
Because the overexpressing efficiency of TLR2 is quite high in keratinocyte cells, the intensity of TLR2 immunoblot band of overexpressing-cells will be much stronger than that of control cells. Therefore, when the exposure time is Furthermore, it is noted that capsaicin causes a dose-dependent activation of mTORC1. This may be problematic since the 100uM concentration is close to inducing cell death. In addition, blots monitoring mTOR activity should be shown pS6.

Response 1:
We thank the reviewer for the insightful comments. To address the concerns, we added the pS6 (Ser240/244) to monitor mTORC1 activity in addition to pS6 (Ser235/236). First, we repeated the heatshock experiment in human primary keratinocytes, which showed that heatshock increased mTORC1 activity, which was consistent with the result in HaCaT keratinocytes (shown in Revised Supplementary Figure 1K). To address the concern that the increased pS6 may be problematic since the 100uM concentration is close to 2. In order to eliminate mTORC2 only a single figure with p-Akt (Ser473) immunostaining was presented (Supp Fig 1E). Further evidence is recommended.

Response 2:
We thank the reviewer for the constructive suggestion. As suggested, by immunostaining we detected the expression of pAkt (Ser473) in the skin of rosacea mouse model, which demonstrated that mTORC2 is not changed in rosacea-like mice skin compared to control mice (shown in Revised Supplementary Figure 1J). Moreover, by immunoblotting we also assessed the expression of pAkt (Ser473) in human primary keratinocytes treated with LL37. Our results showed that LL37 stimulation does not affect mTORC2 signaling in keratinocytes (shown in Revised Figure 1H). Based on these findings, we focused on mTORC1 rather than mTORC2 in this study.

Other comment 3:
3. Figs 3B, 3C -Description of how quantifications were performed should be included.

Response 3:
Thank you for the kind suggestion. As suggested, the description of how to quantify the redness score and area in figure 3B and C was included in materials and methods section of the revised manuscript (see in the part of "LL37-induced rosacea-like mouse model").

Other comment 4:
4. The dynamics of phospho-rpS6 induction by LL37 in Fig. 4E are quite different from Fig. 1J. This should be commented on.

Response 5:
Thank you for the helpful suggestion. As suggested, we repeated this experiment in primary human keratinocytes and PKH26 labeling was added (shown in Revised Figure 5E).

Other comment 6:
6. A number of typos were noted and thus it was thought that the article may benefit from some careful editing.

Response 6:
Thank you for the kind suggestion. We have carefully edited the whole manuscript and improved the English wording with the help from a native English speaker as suggested.

Referee #2
Comments on Novelty/Model System for Author Comment 1:

Response 1:
We thank the reviewer for the constructive suggestion. As suggested, we have confirmed our main conclusions in primary human keratinocytes to fully address the reviewer's concerns, which was also detailedly answered in "response to Major comment 3".

Remarks for Author
Overall comments: The manuscript by Deng el al describes a positive feedback loop between mTORC1 and cathelicidin expression, which promotes skin inflammation in rosacea, a chronic inflammatory skin disease whose pathogenesis is unclear. This is an interesting paper with many in vivo data, which in principle should be suitable for publication in EMM. However, there are several major points, which should be considered in a revised version of the manuscript:

Overall response:
We thank the reviewer for the recognition of our work and insightful comments, all of which have been addressed, as detailed below.

Major comment 1:
1. The paper lacks clarity and is hard to read. Often the choice of tools e.g.
individual mouse strains is not described at all. Furthermore, the logistics of experiments is unclear: human data are mixed with in vitro data of HaCat cells and various mouse models in one chapter. The manuscript should be re-written in a concise manner and the English wording must be improved.

Response 1:
We thank the reviewer for the helpful suggestion. As suggested, the choice of tools e.g. individual mouse strains has been described in the material and methods of revised manuscript. We readjusted the combination of data to avoid the mix of human data with in vitro data of human primary keratinocytes (added as suggested) and various mouse models in one chapter. We have re-written the manuscript and improved the English wording with the help from a native English speaker as suggested.

Major comment 2:
2. One main message from the manuscript is that mTORC1 is specifically activated in rosacea, but there is no discussion what causes the specificity and whether possibly upstream-regulators such as expression of TSC1/2 are affected and being causal to upregulation of mTORC1. Along the same line, TLR-2 is specifically upregulated but not TLR4 or other receptors. What is the molecular explanation for the TLR-2 specificity?

Response 2:
We thank the reviewer for the insightful comments. As suggested, we detected the expression of TSC1 or TSC2 in rosacea. Our data demonstrated that TSC2 rather than TSC1 is significantly decreased (shown in Revised supplementary Figure 1D), which might be responsible for the hyperactivation of mTORC1 in rosacea. And, we discussed this issue in the discussion part of revised manuscript, but more evidence is needed to elucidate why mTORC1 is specifically hyperactivated in the epidermis of rosacea. In fact, besides TLR2, we found that TLR4 and other TLRs (eg. TLR7 and TLR8) were also increased in rosacea (shown in Supplementary table 3), but knockdown of these TLRs (eg. TLR4) did not reverse the activation of mTORC1 induced by LL37 in keratinocytes (data not shown). We also discussed this issue in the discussion part of revised manuscript. To be mentioned, we are now carrying out a project to study the role of these TLRs in the pathogenesis of rosacea, and these findings will be published before long.

Major comment 3:
3. Re the data analyses, most of the Figures/data are convincing. However, the data using HaCat cells in Figures 1, 5 and 7 are highly questionable. The authors refer to this aneuploid human immortalized cell line as 'keratinocytes' or 'keratinocyte cells', which is misleading and unacceptable. Some of these mechanistic data need to be repeated in primary mouse or human keratinocytes, even though these might have a limited passage number.

Response 3:
We thank the reviewer for the helpful suggestion.

Major comment 4:
4. Regarding the link between mTORC1 hyperactivation and exacerbation of rosacea-like features, it is not clear whether mTOR hyperactivation without injection of LL37 is sufficient to promote skin inflammation/rosacea-like features, since there are no statistically significant differences between control and TSC2+/-mice without LL37. However, authors state that their results demonstrate that mTORC1 signaling promotes the development of rosacea.

Response 4:
We thank the reviewer for the insightful comments. Indeed, this is a very interesting question. We really did not observe rosacea-like features in TSC2+/-mice without LL37 injection (the mice used for establishment of rosacea mouse model are 8 weeks old, referred to as young mice). However, we observed obvious inflammatory features (similar to rosacea-like features) in the back skin of some TSC2+/-mice without LL37 injection when older than half a year, and the symptoms were increasingly obvious with the increasing of age. We speculate that in young TSC2+/-mice, despite the activation of mTORC1 signaling, the inflammatory responses do not break out due to the protection of homeostasis, but the homeostasis might be disrupted with age increasing, the activation of mTORC1 signaling might be enough to induce skin inflammation in old TSC2+/-mice. On the other hand, when the homeostasis in the skin is disrupted (eg. the excess expression of TLR2 or LL37), the activation of mTORC1 might exacerbate the development of rosacea.

Major comment 5:
5. Is pS6 overexpression directly correlated with an increase in mTOR1 protein levels in patients/rosacea-like mouse models? A Western blot for both proteins should be shown.

Response 5:
We thank the reviewer for the helpful suggestion. As suggested, we performed a western blot in rosacea patients and healthy individuals. Our results showed that the upregulation of pS6 was not directly correlated with an increase in mTOR protein levels (shown in Revised Supplementary Figure 1C).

Major comment 6:
How do the authors reconcile their positive feedback loop with the observation that only human, but not murine LL37 injection induces rosacea-like disease in mice? This point should be discussed.

Response 6:
We thank the reviewer for the insightful comments. Previous study demonstrated that the mouse GLL34 (homologous to human LL37) was showed that mouse cathelicidin (CRAMP), the precursor of GLL34, was increased in rosacea mouse skin, and deficiency of mTORC1 could significantly reverse this increase (shown in Revised Figure 4C and D, Figure 4A and B). These results suggested that mouse LL37 (GLL34) might also play an important role in the formation of rosacea-like skin inflammation in LL37-induced rosacea mouse model.

Revised Supplementary
However, further evidence is needed to figure out this question, and we also discussed this issue in the discussion part of revised manuscript.

Minor comment 1:
1. Rapamycin should be added to the list of Keywords

Response 1:
Thank you for the kind suggestion. Rapamycin has been added to the list of Keywords in the revised manuscript as suggested.

Response 2:
As suggested, the page numbers and figures were labeled at the bottom of the pages in the revised manuscript.

Minor comment 3:
3. Are there differences in severity of rosacea features or are lesional skin areas different in different human populations? The authors only mention references that take into account the Chinese population.

Response 3:
It has been reported that humans with fair skin are more likely to be affected,

Minor comment 4:
4. Change "healthy individuals" to HS in Fig 6H, as HS is previously used in the manuscript.

Response 4:
Thank you for the kind suggestion. We have changed "healthy individuals" to HS in Fig 6H   Minor comment 5: 5. Could cold/freezing temperatures affect/exacerbate rosacea? The authors mention several times that heat could exacerbate rosacea, but nothing is said about other extreme stresses e.g. cold temperatures.

Response 5:
To my knowledge, there has been no published evidence showing whether cold/freezing temperatures could affect/exacerbate rosacea. However, according to our clinical experience, it seems that cold/freezing temperatures can exacerbate rosacea in some patients.

Minor comment 6:
6. The authors did not mention what is the blue staining in Figure 4B. The reader needs to guess that the blue color is the nuclear staining dye, which could be DAPI, Hoechst?

Response 6:
Thank you for the kind suggestion. We have indicated the blue staining in the Revised Figure 4B as suggested.

Minor comment 7:
7. Figure 4C and Figure 6E: Red and blue colors of "EPI" and "Der" words are wrongly used for the proteins of the images, because authors used same colors.

Response 7:
Thank you for the kind suggestion. We have corrected the red and blue colors of "EPI" and "Der" words in the corresponding places of the revised figures.

Minor comment 8:
8. Which cells produce LL37? Please, clarify if it is made in keratinocytes and/or other cells and show the data.

Response 8:
Thank you for the helpful suggestion. Previous study showed that cathelicidin (the precursor protein of LL37) was mainly upregulated in the epithelial cells (keratinocytes) in rosacea, and the increased cathelicidin was proteolytically processed to generate biologically active LL37 peptide (a 37 amino acids peptide) by KLK5 (Yamasaki K, et al. Nat Med. 2007;13:975-980). Our data also showed that human cathelicidin or mouse CRAMP (homologous to human cathelicidin) was significantly increased in epithelial cells (keratinocytes) and certain inflammatory cells in rosacea patients or mouse model, which was also indicated (shown in Revised Figure 4B and C, Revised supplementary Figure 4A).

Referee #3
Overall comments: In this paper, Deng Z, Chen M and their colleagues found mTORC1 hyperactivation occur in both rosacea patient and LL37-indueced mouse model. In a series of elegant experiments with mouse genetics and human keratinocytes studies, the authors demonstrated mTORC1 signaling is responsible for rosacea symptoms, via enhancing cathelicidin production, NFκB activation, and chemokines upregulation. Most importantly, this study provided a novel and effective clinical therapeutic method, which will be beneficial for rosacea patients. In all, I think the main conclusion from this work is based on solid experimental data, they revealed novel biology in a pathological condition, and offered potential treatment. I recommend it for publication.

Overall response:
We thank the reviewer for the recognition of our work and helpful comments, all of which have been addressed, as detailed below.

Response 1:
Thank you for the kind suggestion. We have added the units of length in quantification data of corresponding figure panels in the revised manuscript as suggested.

Response 2:
Thank you for the helpful suggestion. We have repeated the results of Fig. 4E and Fig. S4C in primary human keratinocytes suggested by another reviewer, and these figures were included in the revised figure 1H as suggested.

Response 3:
Thank you for the kind suggestion. We have quantified the expression of cathelicidin in Fig. 4G as suggested (shown in Revised Figure 4F).

Comment 4:
4. mTORC2 activation data in vivo is needed in LL37-induced mouse model.

Response 4:
Thank you for the helpful suggestion. We have performed additional experiment to detected the mTORC2 activation in the skin of LL37-induced mouse model as suggested. Our results showed that mTORC2 activation was not altered in the skin of rosacea mouse model compared to control mouse (shown in Revised Supplementary Figure 1J).
Again, we appreciate the constructive and insightful suggestions from the reviewers. We hope that with the revisions the manuscript will be acceptable for publication. Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the two referees who re-reviewed your manuscript. As you will see, they are both supportive of publication, and I am therefore pleased to inform you that we will be able to accept your manuscript, once the following minor points will be addressed: 1) Referees' comments: -Referee #1, comment on the number of mice and statistics: please address this comment in writing, and justify the number of mice used in the experiments.
-Referee #1, comment on the mechanistic understanding: if you have data at hand, we will be happy for you to include it. Alternatively, please discuss the limitations along the lines suggested by this referee. -Referee #2: please make sure that all changes mentioned in your point-by-point letter have indeed been addressed.
2) Main manuscript text -Please answer/correct the changes suggested by our data editors in the main manuscript file (in track changes mode). This file will be sent to you in the next few days. Please use this file for any further modification.
-Please remove the highlighted text.
-We can accommodate a maximum of 5 keywords, please adjust accordingly.
-Please reformat the references so that they are listed in alphabetical order, and with 10 authors only listed before et al.  Table S3-S8 should be renamed Table EV1-6, and a legend should be added to the respective file. The other tables should be added to the appendix files.
-Please add a table of content to the appendix, and update the names to Appendix Figure S1 etc.
-Please add/define scale bars in all Appendix figures. 4) Source Data: you submitted a file labeled Source Data and containing Figure S8 and Figure S9. Could you please clarify if these are supplemental figures or Source Data? Source data should be uploaded as 1 file per figure. 5) Checklist: Please add more information throughout, in particular for section B/1.a, sections D/8 and D/9, section E and section F (including F/20). 6) Thank you for providing The Paper Explained section. I added minor edits, please let me know if you agree with the following: Problem Rosacea is a common chronic inflammatory skin disorder of uncertain etiology. It mainly occurs in the central face, which greatly affects the quality of life, and is associated with multiple systemic diseases (such as cancer). Although this cutaneous syndrome has been described centuries ago, its pathophysiological mechanism remains unclear. Multiple therapies have been used for the management of rosacea, including oral tetracycline and isotretinoin, topical application of azelaic acid, metronidazole, and vascular lasers, etc. However, no specific therapeutic target has been defined, and most therapies are unsatisfactorily symptom-based treatments.

Results
Our study demonstrates that mTORC1 signaling is hyperactivated in the skin of rosacea patients. Ablation or specific inhibition of mTORC1 blocked the development of rosacea-like skin inflammation in a rosacea mouse model. Conversely, hyperactivation of mTORC1 signaling aggravated rosacea-like features. Mechanistically, mTORC1 signaling regulates cathelicidin in keratinocytes through a positive feedback loop, in which cathelicidin LL37 activates mTORC1 signaling by binding to Toll-like receptor 2 (TLR2), which in turn increases the expression of cathelicidin itself. Moreover, excess cathelicidin LL37 induces both NF-κB activation, and diseasecharacteristic cytokines and chemokines via mTORC1 signaling. Importantly, topical application of rapamycin significantly improved rosacea symptoms in patients.

Impact
Our data suggest an essential role for mTORC1 signaling in the pathogenesis of rosacea and reveal a promising therapeutic target for rosacea treatment. 7) As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329), EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts. In the event of acceptance, this file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript. Let us know whether you agree with the publication of the RPF and as here, IF YOU WANT TO REMOVE OR NOT ANY FIGURES from it prior to publication. Please note that the Authors checklist will be published at the end of the RPF.
I look forward to receiving your revised manuscript.

Yours sincerely,
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Captions
For animal studies, the sample size was included in the figure legend for every mouse experiment.
For human skin biopsies, inclusion/exclusion criteria：1. Female; 2. aged 20-50; 3. no other facial diseases or systemic diseases; 4. no treatment within 2 weeks. The criteria was pre-established. No animals were excluded from the study.
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Data
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