Morphological reprogramming of primary cilia length mitigates the fibrotic phenotype in fibroblasts across diverse fibrotic conditions

  1. Priyanka Verma
  2. Bharat Yalavarthi
  3. Swati Bhattacharyya
  4. Dinesh Khanna
  5. Johann E. Gudjonsson
  6. Lam C. Tsoi
  7. Rebecca Wells
  8. Rebecca L. Ross
  9. Natalia Riobo-Del Galdo
  10. Francesco Del Galdo
  11. Sean M. Fortier
  12. Maria E. Teves
  13. John Varga
  14. Dibyendu Bhattacharyya

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Abstract

Myofibroblast differentiation, which occurs across fibrotic diseases, is marked by actin polymerization and assembly of α-smooth muscle actin (αSMA) stress fibers. Primary cilia (PC) are solitary membrane-bound organelles present on the majority of cells. PC length regulation is a complex process influenced by actin polymerization. We discovered that fibroblasts from diverse fibrotic conditions display significantly reduced PC length ex vivo. Treatment of healthy fibroblasts (non-fibrotic) with profibrotic TGF-β1 induced PC shortening, whereas silencing ACTA2 in systemic sclerosis (SSc) skin fibroblasts caused PC elongation. Importantly, we found that PC length was negatively correlated with αSMA levels in TGF-β1-treated healthy fibroblasts and pharmacologically dedifferentiated myofibroblasts. Our results suggest that during the fibrotic response, higher-order actin polymerization, along with microtubule destabilization by tubulin deacetylation, drives PC length shortening. In contrast, PC length elongation via stabilization of microtubule polymerization mitigates the fibrotic phenotype in fibrotic fibroblasts. These results reveal a potential link between PC length and fibroblast activation conserved across multiple fibrotic conditions. Our observations suggest that modulation of PC length might represent a novel therapeutic strategy for SSc and other treatment-resistant diseases associated with fibrosis.

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  1. Version published to 10.1242/jcs.264191
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    Manuscript number: RC-2024-02391

    Corresponding author(s): John Varga

    Dibyendu Bhattacharyya

    [Please use this template only if the submitted manuscript should be considered by the affiliate journal as a full revision in response to the points raised by the reviewers.

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    Dear editor,

    We are pleased to submit a full revised version of the manuscript that addresses all the points raised by the reviewers. We have included new experiments and modified the text and figures based on the reviewers’ suggestions. We thank all the reviewers for their insightful feedback, which has significantly enhanced the quality of the manuscript. We are confident and optimistic that our improved manuscript will be accepted by the journal of our choice.

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    Reviewer #1

    • To reliably quantify the ciliary length in different cell types, and in independent ciliary marker needs to be included for comparison and the ciliary base needs to be labeled (e.g., g-TUBULIN). This needs to combined with a non-biased, high-throughput analysis, e.g., CiliaQ, Response: As suggested, we compared primary cilia length measurements using antibodies against Arl13b and γ-tubulin. The comparison between healthy controls (HC) and systemic sclerosis (SSc) is presented in Supplementary Figure S1. No significant differences in primary cilia length were observed compared to our previous measurements. Cilia length was quantified using ImageJ version 1.48v (http://imagej.nih.gov/ij) with the maximum intensity projection (MIP) method and visualized through 3D reconstruction using the ImageJ 3D Viewer.

    • As mentioned in the study, TGFbhas been implicated to drive myofibroblast transition. Thus TGFb stimulate ciliary signaling in the presented primary cells? The authors should provide a read-out for TGFb signaling in the cilium (ICC for protein phosphorylation etc.). Furthermore, canonical ciliary signaling pathways have been suggested to act as fibrotic drivers, such as Hedgehog and Wnt signaling - does stimulation of these pathways evoke a similar effect? Response: Yes, TGF-β1 stimulates ciliary signaling in growth-arrested foreskin fibroblasts. Clement et al. (2013) showed that TGF-β1 induces p-SMAD2/3 at the ciliary base, followed by the nuclear translocation of p-SMAD2/3 after 90 minutes. To assess whether canonical ciliary signaling pathways influence primary cilia length, we treated foreskin fibroblasts with Wnt (#908-SH, R&D) and a Shh agonist (#5036-WN, R&D) at 100 ng/mL each for 24 hours. We did not observe any changes in primary cilia length under either condition. These data are shown here for reference but are not included in the manuscript.

    Clement, Christian Alexandro, et al. "TGF-β signaling is associated with endocytosis at the pocket region of the primary cilium." Cell reports 3.6 (2013): 1806-1814.

    • Does TGFbinduce cell proliferation? If yes, this would force cilium disassembly and, thereby, reduce ciliary length, which is independent of a "shortening" mechanism proposed by the authors. Response: Yes, TGF-β induces cell proliferation in fibroblasts (Lee et al., 2013; Liu et al., 2016). However, we did serum starvation to stop proliferation. In our study, we observed a few percentage of Ki67-positive cells under TGF-β treatment at 24 hours (Supplementary Figure S2C). However, cell proliferation mainly stopped after 48 hours. Typically, proliferating cells rarely display any PC or show very small puncta. In our case, we observe a significantly elongated PC structure (although shorter than that of untreated cells) under TGF-beta-treated conditions. Our results display that a majority of cells are not proliferating but still display PC shortening under TGF-β treatment, suggesting that PC shortening is not due to cell division-induced PC disassembly. TGF beta-induced PC shortening is also reported in another fibroblast type previously (Kawasaki et al., 2024).

    Kawasaki, Makiri, et al. "Primary cilia suppress the fibrotic activity of atrial fibroblasts from patients with atrial fibrillation in vitro." Scientific Reports 14.1 (2024): 12470.

    Lee, J., Choi, JH. & Joo, CK. TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin. Cell Death Dis 4, e714 (2013). https://doi.org/10.1038/cddis.2013.244.

    Liu, Y. et al. TGF-β1 promotes scar fibroblasts proliferation and transdifferentiation via up-regulating MicroRNA-21. Sci. Rep. 6, 32231; doi: 10.1038/srep32231 (2016).

    • As PGE2 has been shown to signal through EP4 receptors in the cilium, is the restoration of primary cilia length due to ciliary signaling? Response: As per your suggestion, we measured cilia length in the presence and absence of the EP4 receptor antagonist (#EP4 Receptor Antagonist 1; #32722; Cayman Chemicals; 500 nM) with PGE2. Interestingly, we did not observe a change in cilia length between the PGE2 and TGFβ (with EP4 receptor antagonist) treatment groups, as shown in supplementary figure S3. We believe that PGE2 works with the EP2 receptor under our experimental conditions. Kolodsick et al., 2003, also observed that PGE2 inhibits myofibroblast differentiation via activation of EP2 receptors and elevations in cAMP levels in healthy lung fibroblasts.

    Kolodsick, Jill E., et al. "Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation." American journal of respiratory cell and molecular biology 29.5 (2003): 537-544.

    • Primary cilia length is regulated by cAMP signaling in the cilium vs. cytoplasm - does cAMP signaling play a role in this context? PGE2 is potent stimulator of cAMP synthesis - does this underlie the rescue of primary cilia length? Response: Yes, cAMP levels are important for both myofibroblast dedifferentiation and cilia length elongation. Kolodsick et al., 2003 observed that PGE2 inhibits myofibroblast differentiation via activation of EP2 receptors and elevations in cAMP levels in healthy lung fibroblasts. In a parallel set of experiments, treatment with forskolin (a cAMP activator) also reduced α-SMA protein levels by 40%. Forskolin is also known to increase PC length.

    Kolodsick, Jill E., et al. "Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation." American journal of respiratory cell and molecular biology 29.5 (2003): 537-544.

    • The authors describe that they wanted to investigate how aSMA impacted primary cilia length. They only provide a knock-down experiment and measured ciliary length, but the mechanistic insight is missing. How does loss of aSMA expression control ciliary length? Response: We measured acetylated α-tubulin levels in ACTA2 siRNA-treated cells compared to control-treated cells. Acetylated α-tubulin levels increased under ACTA2 siRNA-treated conditions, as shown in Figure 4D, and TPPP3 levels were also elevated (Figure S8A). Interestingly, TPPP3 levels negatively correlated with disease severity in SSc fibroblasts (r = -0.2701, p = 0.0183), and TPPP3 expression significantly reduced in SSc skin biopsies, as shown in Figures 6C and 6D. These results strengthen our hypothesis that microtubule polymerization and actin polymerization, while they counterbalance each other, also contrarily affect PC length. We agree that a much more detailed study is needed to extensively delineate the intricate homeostasis of the actin network and microtubule network in conjunction with fibrosis and primary cilia length. We have mentioned this in the discussion.

    • The authors used LiCl in their experiments, which supposedly control Hh signaling. Coming back to my second questions, is this Hh-dependent? And what is the common denominator with respect to TGFbsignaling? And how is this mechanistically connected to actin and microtubule polymerization? Response: We used Shh inhibitor (Cyclopamine hydrate #C4116 Sigma-Aldrich) in both SSc and foreskin fibroblasts (with and without TGFβ). We found that PC length is significantly increased and αSMA intensity is reduced in the Shh inhibitor treated group (data not included in the Manuscript)

    • How was the aSMA Mean intensity determined? Response: We quantified aSMA mean intensity using ImageJ, and the procedure has been added to the respective figure legend and materials and methods section under ‘Quantification of immunofluorescence’ (each point represents mean intensity from three randomly selected hpf/slide was performed using ImageJ).

    • Fig: 1D: Statistical test is missing in Figure Legend and presentation of the p-values for the left graph is confusing Response: We added statistical test information in Figure Legend.

    • Some graphs are presented {plus minus} SD and some {plus minus} SEM, but this is not correctly stated in the Material & Methods Part __Response: __We added information to the figure legend as well as in the Material & Methods section.

      • 4D&E: Statistical test is missing in Figure Legend* Response: We added it now.

    • In general, text should be checked again for spelling mistakes and sentences may be re-written to promote readability. In particular, this applies to the discussion. __Response: __We checked and corrected.

    • Figure Legends are not written consistently, information is missing (e.g., statistical tests, see above). __Response: __We carefully checked and added information accordingly.

    • Figures should be checked again, and all text should be the same size and alignment of images should be improved. __Response: __We checked and corrected.

    Significance

    The authors present a novel connection between the regulation of primary cilia length and fibrogenesis. However, the study generally lacks mechanistic insight, in particular on how TGFb signaling, aSMA expression, and ciliary length control are connected. The spatial organization of the proposed signaling components is also not clear - is this a ciliary signaling pathway? If so, how does it interact with cytoplasmic signaling and vice versa?

    Response: Thank you for your thoughtful and constructive feedback. We appreciate your recognition of the novelty of our study linking primary cilia length regulation to fibrogenesis. In our revised manuscript, we did provide a mechanistic insight, though. Our results suggest that during the fibrotic response, higher-order actin polymerization, along with microtubule destabilization resulting from tubulin deacetylation, drives the shortening of PC length. In contrast, PC length elongation via stabilization of microtubule polymerization mitigates the fibrotic phenotype in fibrotic fibroblasts. We agree that a deeper mechanistic understanding particularly regarding how TGFβ signaling, αSMA expression, and ciliary length control intersect is essential for fully elucidating the pathway. We also acknowledge the importance of clarifying the spatial organization of the signaling components and plan to incorporate such analyses in future studies.

    Reviewer #2

    *I found the paper to be rather muddled and its presentation made if somewhat difficult to follow. For example, the Figures are disorganised (Fig 1 is a great example of this) and there was reference to Sup data that appeared out of order (eg Sup Fig 2 appeared before Sup Fig 1 in the text). *

    Response: We carefully revised the manuscript and arranged the figures.

    *Images in a single figure should be the same size. Currently they are almost random and us different magnifications. Overall, the paper needs to be better organized. *

    Response: We carefully revised the manuscript and figures provided with same magnification.

    *I have some significant concerns about how the PC length data was generated. To my mind the length may be hard to determine from the type of images shown in the paper (which may represent the best images?). Some of the images presented appear to show shorter, fatter PCs in the cells from fibrosis cases. Is this real or is it some kind of artefact? Would a shorter, fatter PCs have a similar or larger surface area? What would be the consequence of this? *

    Response: Primary cilia length was measured with ImageJ1.48v (using maximum intensity projection (MIP) method and visualized by 3D reconstruction with the ImageJ 3D viewer. Each small dot represents the PC length from an individual cell, and each large dot represents the average of the small dots for one cell line.

    *I am confused as to exactly what is meant by matched healthy controls. Age, sex and ethnicity, where stated seem to be very variable? What are CCL210 fibroblasts? *

    Response: We appreciate this comment. This is correct. The age, sex, and ethnicity are not matched for the available healthy controls. We have corrected that in the text. CCL210 is a commercially available fibroblast cell line that was isolated from the lung of a normal White, 20-year-old, female patient.

    *What does a change in PC length signify? DO shot PC foe a cellular transition or are they a consequence of it? What would happen is you targeted PCs with a drug and that influenced the length on all cell types? Is the effect on PC fibroblast specific? *

    __Response: __Significance and regulation of PC length are greatly debated and investigated still. It appears that PC length signify different features in different cell types. Although these are very interesting questions but such experiments are beyond the scope of our present work.

    Minor concerns

    *Page 4 second paragraph. I think it should be clarified that it is this group who have suggested a link between PCs and myofibroblast transition? *

    __Response: __We agree with the reviewer and clarified it.

    *Page 4 second paragraph. The use of the word "remarkably' is a bit subjective. *

    __Response: __We agree with the reviewer and have removed it.

    *Reference 27 is a paper on multiciliogenesis rather than primary ciliogenesis. *

    __Response: __We agree with the reviewer and have removed it.

    Figure 1 panel D. Make the image with the same sized vertical scale

    __Response: __We have replaced it with a new Figure 1.

    Significance

    Reviewer #2 (Significance (Required)):

    To my mind this is a novel paper and the data presented in it may be of interest to the cilia community as well as to the fibrosis field. This could be considered to be a significant advance and I am unaware that other groups are actively working in this area.

    Presentation of the data in the current form does not instil confidence in the work.

    Response: ____Thank you for recognizing the novelty and potential significance of our work. We appreciate your comments and fully acknowledge the concern regarding the presentation of the data. We have carefully revised the manuscript and reorganized the figures to improve clarity and overall presentation.

    Reviewer #3

    Major comments:

    • Need to demonstrate if the fibrotic phenotypes seen are produced through a ciliary-dependent mechanism. For example, to see if LiCl effects on Cgn1 are through ciliary expression or by other mechanisms. To achieve that objective, The authors should repeat the experiments in cells with a knockdown or knockout of ciliary proteins such as IFT20, IFT88, etc. The same approach should be applied to the tubacin experiments. Response: We silenced foreskin fibroblasts with IFT88/IFT20, both in the presence and absence of TGF-β1, followed by treatment with LiCl and Tubacin. Both LiCl and Tubacin can rescue cilia length and mitigate the myofibroblast phenotype in the presence of silenced IFT88/IFT20 gene, as shown in supplementary figure S9. Our result suggests that LiCl and Tubacin functions are both independent of the IFT-mediated ciliary mechanism. Regulation of PC length is still an enigma and highly debated. Moreover, PC length can be affected in multiple ways and is not solely dependent on IFTs (Avasthi and Marshall, 2012). One such method is the direct modification of the axoneme by altering microtubule stability through the acetylation state (Avasthi and Marshall, 2012), a pathway most likely the case for Tubacin. Another mode of PC length regulation is through a change in Actin polymerization. The remodeling of actin between contractile stress fibers and a cortical network alters conditions that are hospitable to basal body docking and maintenance at the cell surface (Avasthi and Marshall, 2012), causing PC length variation. Our results suggest that PC length functions as a sensor of the status of the fibrotic condition, as evidenced by the aSMA levels of the cells.

    Avasthi, P., and W.F. Marshall. 2012. Stages of ciliogenesis and regulation of ciliary length. Differentiation. 83:S30-42.

    • The use of LiCl to increase ciliary length is complicated. What are the molecular mechanisms underlying this effect? It is known that it may be affecting GSK-3b, which can have other ciliary-independent effects. Therefore, using ciliary KO/KD cells (IFT88 or IFT20) as controls may help assess the specificity of the proposed treatments. Response: As explained in the previous paragraph, PC length regulations are dependent on multiple factors and many of them are not IFT dependent. One such method is directly modifying the axoneme by altering microtubule stability/polymerization through the acetylation state(Avasthi and Marshall, 2012), a pathway most likely the case for Tubacin. Another mode of PC length regulation is through a change in Actin polymerization. The remodeling of actin between contractile stress fibers and a cortical network alters conditions that are hospitable to basal body docking and maintenance at the cell surface (Avasthi and Marshall, 2012), causing PC length variation. Higher order microtubule polymerization inhibit actin polymerization. By interrogating RNA-seq data we determined that several PC-disassembly related genes (KIF4A, KIF26A, KIF26B, KIF18A), as well as microtubule polymerization protein genes (TPPP, TPPP3, TUBB, TUBB2A etc), were differentially expressed in LiCl-treated SSc fibroblasts (Suppl. Fig. S6D). Altogether, these findings suggest that microtubule polymerization/depolymerization mechanisms may regulate PC elongation and attenuation of fibrotic responses after either LiCl or Tubacin treatment.

    • Also, assessing the frequency of ciliary-expressing cells is important. That may give another variable important to predict fibrotic phenotypes. Or do 100% of the cultured cells express cilia in those conditions? Response: We carefully checked and observed almost 95% cells express cilia in cultured conditions.

    • Have the authors evaluated if TGF-b1 treatments induce cell cycle re-entry and proliferation in these experimental conditions? This is important to exclude ciliary resorption due to cell cycle re-entry instead of the myofibroblast activation process. __Response:__Yes, TGF-β induces cell proliferation in fibroblasts (Lee et al., 2013; Liu et al., 2016). However, we did serum starvation to stop proliferation. In our study, we observed a few percentage of Ki67-positive cells under TGF-β treatment at 24 hours (Supplementary Figure S2C). However, cell proliferation mainly stopped after 48 hours. Typically, proliferating cells rarely display any PC or show very small puncta. In our case, we observe a significantly elongated PC structure (although shorter than that of untreated cells) under TGF-beta-treated conditions. Our results display that a majority of cells are not proliferating but still display PC shortening under TGF-β treatment, suggesting that PC shortening is not due to cell division-induced PC disassembly. TGF beta-induced PC shortening is also reported in another fibroblast type previously (Kawasaki et al., 2024).

    Kawasaki, Makiri, et al. "Primary cilia suppress the fibrotic activity of atrial fibroblasts from patients with atrial fibrillation in vitro." Scientific Reports 14.1 (2024): 12470.

    Lee, J., Choi, JH. & Joo, CK. TGF-β1 regulates cell fate during epithelial–mesenchymal transition by upregulating survivin. Cell Death Dis 4, e714 (2013). https://doi.org/10.1038/cddis.2013.244.

    Liu, Y. et al. TGF-β1 promotes scar fibroblasts proliferation and transdifferentiation via up-regulating MicroRNA-21. Sci. Rep. 6, 32231; doi: 10.1038/srep32231 (2016).

    • The authors described that they focused on the genes that are affected in opposite ways (supp table 4), but TEAD2, MICALL1, and HDAC6 are not listed in that table. Response: The list in Supplementary Table S3 includes common genes defined as differentially expressed based on a fold change >1 or Minor comments:

    • Figure 1A,B,C should also show lower magnification images where several cells/field are visualized. Response: We have replaced it with a new Figure 1.

    • The number of patients analyzed is not clear. For example, M&M describes 5 healthy and 8 SSc, but only 3 and 4 are shown in the figure. Furthermore, for orbital fibrosis, 2 healthy vs. 2 TAO are mentioned in the figure legend, but only one of each showed. Finally, the healthy control for lung fibroblast seems to be 3 independent experiments of the CCL210 cell line; please show the three independent controls and clarify on the X-axis and in the figure legend that these are CCL210 cells. Response: A total of 5 healthy and 8 SSc skin explanted fibroblast cell lines were used, as described in the Materials and Methods. Since these are patient-derived skin fibroblasts, maintaining equal numbers in each experiment is challenging. Revised graphs for orbital fibroblasts and CCL210 have been added in the new Figures 1B and 1C.

    • For the same set of experiments, please clarify and consistently describe the conditions that promote PC: 12hs serum starvation as described in M&M? Or 24hs as described in the text? Or 16 as described in figure legend 1? Or 24hs as described in supp figure 2? Response: We serum-starved the cells overnight, and this is also mentioned in the manuscript.

    • Please confirm in figure legends and M&M that 100 cells per group were counted. Response: We measured only 100 cells per cell line in Supplementary Figure S1B. To eliminate any confusion, we have now created a superplot for cilia analysis. Each small dot represents the PC length from an individual cell, and each large dot represents the average of the small dots for one cell line. An unpaired two-tailed t-test was performed on the small dots (mean ± SD).

    • Figure 2 should also provide lower magnification to show several cells per field. Response: Foreskin fibroblasts treated with TGF-β1 are added in S2A.

    • How do you explain that the increase in length of primary cilia after siACTA2 doesn't change COL1A1? Wouldn't it be a good approach to also check by Western Blot? Response: We believe that depletion of aSMA was sufficient to reduce the PC length for the reason described earlier (Avasthi and Marshall, 2012), but was not sufficient enough to change *COL1A1 *level. We added the western blot in Supplementary Figure S8B.

    • Once more, figure 5 will benefit from low mag images. How consistent is the effect of LiCl in the cultured cells? What is the percentage of rescued cells? Response: LiCl treatment was consistent for almost all the cells (~95%) as shown below and added in S4A.

    • Figure 5, panels F and G need better explanation in the results text as well as in the figure legend. Response: We added now.

      1. Some figures/supp figures are wrongly referenced in the text. *

    __ Response:__ We carefully revised the manuscript and corrected the references.

    1. Figure 6, panel A is confusing. Is it a comparison between SSC skin fibroblasts and foreskin fibroblasts? Maybe show labels on the panel.

    __ Response:__ We updated the figure legend for Panel A in Figure 6.

    1. Where is Figure 8 mentioned in the text?

    __ Response:__ In the discussion section.

    1. The work will benefit from an initial paragraph in the discussion enumerating the findings and a summary of the conclusion at the end.

    Response: We agree and modified the discussion accordingly.

    1. The nintedanib experiments are not described in the results section at all.

    Response: All nintedanib experiments are now included in Figure S5C-F and are described in the Results section.

    Significance

    Reviewer #3 (Significance (Required)): Beyond the lack of in situ ciliary expression assessment, the work is exciting, and the potential implications of treating/preventing fibrosis with small molecules to modulate ciliary length could be transformative in the field. Furthermore, there are a few HDAC6 inhibitors already in clinical trials for different tumors, which increases the significance of the work.

    Response: Thank you for your encouraging comments regarding the potential impact of our findings. We agree that the therapeutic implications of modulating ciliary length, particularly using small molecules such as HDAC6 inhibitors already in clinical trials, could be transformative in the context of fibrosis. We also acknowledge the importance of in situ assessment of ciliary expression and plan to incorporate such analyses in future studies to further strengthen our findings.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The author's main research topic in this work is the relationship between ciliary length and the level of fibrosis. Fibrotic samples show shorter primary cilia, profibrotic treatment with TGFB decreases the ciliary length, and posterior dedifferentiation of fibroblasts shows longer cilia. Cells with a decrease of αSMA by using siACTA2 siRNA, also show increased ciliary length. Most importantly, inducing the increase of ciliary length with LiCl or Tubacin has an inverse association with fibrosis phenotypes. The modulation of primary cilia length may represent a potential therapeutic strategy for fibrosis-associated diseases.

    The premise is relevant and exciting, and the methods are appropriate. The experiments partially sustain the conclusion. The results open a new potential area for studying fibrosis. The tables and figures aid in understanding the paper. The paper is clear and easy to read for a basic research specialized audience.

    Major comments:

    1. Need to demonstrate if the fibrotic phenotypes seen are produced through a ciliary-dependent mechanism. For example, to see if LiCl effects on Cgn1 are through ciliary expression or by other mechanisms. To achieve that objective, The authors should repeat the experiments in cells with a knockdown or knockout of ciliary proteins such as IFT20, IFT88, etc. The same approach should be applied to the tubacin experiments.
    2. The use of LiCl to increase ciliary length is complicated. What are the molecular mechanisms underlying this effect? It is known that it may be affecting GSK-3b, which can have other ciliary-independent effects. Therefore, using ciliary KO/KD cells (IFT88 or IFT20) as controls may help assess the specificity of the proposed treatments.
    3. Also, assessing the frequency of ciliary-expressing cells is important. That may give another variable important to predict fibrotic phenotypes. Or do 100% of the cultured cells express cilia in those conditions?
    4. Have the authors evaluated if TGF-b1 treatments induce cell cycle re-entry and proliferation in these experimental conditions? This is important to exclude ciliary resorption due to cell cycle re-entry instead of the myofibroblast activation process.
    5. The authors described that they focused on the genes that are affected in opposite ways (supp table 4), but TEAD2, MICALL1, and HDAC6 are not listed in that table.

    Minor comments:

    1. Figure 1A,B,C should also show lower magnification images where several cells/field are visualized.
    2. The number of patients analyzed is not clear. For example, M&M describes 5 healthy and 8 SSc, but only 3 and 4 are shown in the figure. Furthermore, for orbital fibrosis, 2 healthy vs. 2 TAO are mentioned in the figure legend, but only one of each showed. Finally, the healthy control for lung fibroblast seems to be 3 independent experiments of the CCL210 cell line; please show the three independent controls and clarify on the X-axis and in the figure legend that these are CCL210 cells.
    3. For the same set of experiments, please clarify and consistently describe the conditions that promote PC: 12hs serum starvation as described in M&M? Or 24hs as described in the text? Or 16 as described in figure legend 1? Or 24hs as described in supp figure 2?
    4. Please confirm in figure legends and M&M that 100 cells per group were counted.
    5. Figure 2 should also provide lower magnification to show several cells per field.
    6. How do you explain that the increase in length of primary cilia after siACTA2 doesn't change COL1A1? Wouldn't it be a good approach to also check by Western Blot?
    7. Once more, figure 5 will benefit from low mag images. How consistent is the effect of LiCl in the cultured cells? What is the percentage of rescued cells?
    8. Figure 5, panels F and G need better explanation in the results text as well as in the figure legend.
    9. Some figures/supp figures are wrongly referenced in the text.
    10. Figure 6, panel A is confusing. Is it a comparison between SSC skin fibroblasts and foreskin fibroblasts? Maybe show labels on the panel.
    11. Where is Figure 8 mentioned in the text?
    12. The work will benefit from an initial paragraph in the discussion enumerating the findings and a summary of the conclusion at the end.
    13. The nintedanib experiments are not described in the results section at all.

    Significance

    Beyond the lack of in situ ciliary expression assessment, the work is exciting, and the potential implications of treating/preventing fibrosis with small molecules to modulate ciliary length could be transformative in the field. Furthermore, there are a few HDAC6 inhibitors already in clinical trials for different tumors, which increases the significance of the work.

    Expertise: primary cilium functions, cell biology, cancer biology

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    Referee #2

    Evidence, reproducibility and clarity

    This is an interesting paper that appears to show that explanted fibroblasts from a range of fibrotic conditions exhibit a reduction in the length of their primary cilia (PC). The paper employs a number of different experimental approaches that appear to show that the modulation of fibroblast/myofibroblast differentiation is associated with alterations in PC length. The rational for the study is that actin polymerization has previously been associated with PC length. The authors suggest that modulation of PC dynamics may represent a potential theraputic strategy for fibrotic disease. To me that seems like a big jump.

    Major concerns.

    I found the paper to be rather muddled and its presentation made if somewhat difficult to follow. For example, the Figures are disorganised (Fig 1 is a great example of this) and there was reference to Sup data that appeared out of order (eg Sup Fig 2 appeared before Sup Fig 1 in the text). Images in a single figure should be the same size. currently they are almost random and us different magnifications. Overall, the paper needs to be better organised.

    I have some significant concerns about how the PC length data was generated. To my mind the length may be hard to determine from the type of images shown in the paper (which may represent the best images?). Some of the images presented appear to show shorter, fatter PCs in the cells from fibrosis cases. Is this real or is it some kind of artefact? Would a shorter, fatter PCs have a similar or larger surface area? What would be the consequence of this?

    I am confused as to exactly what is meant by matched healthy controls. Age, sex and ethnicity, where stated seem to be very variable? What are CCL210 fibroblasts?

    What does a change in PC length signify? DO shot PC foe a cellular transition or are they a consequence of it? What would happen is you targeted PCs with a drug and that influenced the length on all cell types? Is the effec on PC fibroblast specific?

    Minor concerns

    Page 4 second paragraph. I think it should be clarified that it is this group who have suggested a link between PCs and myofibroblast transition?

    Page 4 second paragraph. The use of the word "remarkably' is a bit subjective.

    Reference 27 is a paper on multiciliogenesis rather than primary ciliogenesis.

    Figure 1 panel D. Make the image with the same sized vertical scale

    Significance

    To my mind this is a novel paper and the date presented in it may be of interest to the cilia community as well as to the fibrosis field. This could be considered to be a significant advance and I am unaware that other groups are actively working in this area.

    Presentation of the data in the current form does not instil confidence in the work.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    The manuscript by Verma et al. describes the involvement of primary cilia length control in driving pro-fibrotic progression of fibroblasts in fibrotic diseases. This is shown in primary cells from several organs from patients, suffering from different fibrotic diseases. They demonstrate that primary cilia are shorter in fibroblasts from different fibrotic conditions and that pro-fibrotic signaling, as exemplified by TGFb stimulation, causes shortening of the cilium. Vice versa, elongation of the cilium via different pharmacological substances reverses the pro-fibrotic phenotype.

    Major comments

    1. To reliably quantify the ciliary length in different cell types, and in independent ciliary marker needs to be included for comparison and the ciliary base needs to be labeled (e.g., -TUBULIN). This needs to combined with a non-biased, high-throughput analysis, e.g., CiliaQ,
    2. As mentioned in the study, TGF has been implicated to drive myofibroblast transition. Thus TGF stimulate ciliary signaling in the presented primary cells? The authors should provide a read-out for TGF signaling in the cilium (ICC fro protein phosphorylation etc.). Furthermore, canonical ciliary signaling pathways have been suggested to act as fibrotic drivers, such as Hedgehog and Wnt signaling - does stimulation of these pathways evoke a similar effect?
    3. Does TGF induce cell proliferation? If yes, this would force cilium disassembly and, thereby, reduce ciliary length, which is independent of a "shortening" mechanism proposed by the authors.
    4. As PGE2 has been shown to signal through EP4 receptors in the cilium, is the restoration of primary cilia length due to ciliary signaling?
    5. Primary cilia length is regulated by cAMP signaling in the cilium vs. cytoplasm - does cAMP signaling play a role in this context? PGE2 is potent stimulator of cAMP synthesis - does this underlie the rescue of primary cilia length?
    6. The authors describe that they wanted to investigate how aSMA impacted primary cilia length. They only provide a knock-down experiment and measured ciliary length, but the mechanistic insight is missing. How does loss of aSMA expression control ciliary length?
    7. The authors used LiCl in their experiments, which supposedly control Hh signaling. Coming back to my second questions, is this Hh-dependent? And what is the common denominator with respect to TGF signaling? And how is this mechanistically connected to actin and microtubule polymerization?
    8. How was the SMA Mean intensity determined?
    9. Fig: 1D: Statistical test is missing in Figure Legend and presentation of the p-values for the left graph is confusing.
    10. Some graphs are presented {plus minus} SD and some {plus minus} SEM, but this is not correctly stated in the Material & Methods Part.
    11. Fig. 4D&E: Statistical test is missing in Figure Legend.

    Minor comments

    • In general, text should be checked again for spelling mistakes and sentences may be re-written to promote readability. In particular, this applies to the discussion.
    • Figure Legends are not written consistently, information is missing (e.g., statistical tests, see above).
    • Figures should be checked again and all text should be the same size and alignment of images should be improved.

    Significance

    The authors present a novel connection between the regulation of primary cilia length and fibrogenesis. However, the study generally lacks mechanistic insight, in particular on how TGF signaling, SMA expression, and ciliary length control are connected. The spatial organization of the proposed signaling components is also not clear - is this a ciliary signaling pathway? If so, how does it interact with cytoplasmic signaling and vice versa?

  6. Version published to 10.1101/2024.01.06.574461 on bioRxiv