Pyruvate promotes ciliogenesis bypassing IFT88 dependency and attenuates DSS-induced colitis

  1. Maya Sarieddine
  2. Ilaria Cicalini
  3. Damiana Pieragostino
  4. Federica Dimarco
  5. Matthieu Lacroix
  6. Krzysztof Rogowski
  7. Valérie Pinet
  8. Michael Hahne

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Abstract

Primary cilia (PC) are important signaling rheostats, yet their biology in colon remains understudied. We previously reported that the presence of PC on colonic fibroblasts in mice (CF) modulates their susceptibility to colitis. Here, we demonstrate that extracellular pyruvate levels influence both ciliary length and ciliogenesis in CF. Pyruvate supplementation to CF enhanced tubulin and histone acetylation, with the latter promoting MAPK signaling and tubulin detyrosination within PC. MAPK-inhibition reduced tubulin detyrosination and shortened ciliary length, while inhibition of α-tubulin acetylation specifically affected ciliogenesis. Col6a1cre-Ift88flx/flx mice, lacking ciliary Ift88 gene in Col6a1 -expressing CF, displayed reduced ciliogenesis and increased susceptibility to DSS-induced colitis. Surprisingly, in primary CF cultures from these mice, pyruvate supplementation restored PC formation. Moreover, pyruvate administration via drinking water rescued PC formation in Col6a1cre-Ift88flx/flx mice and attenuated DSS-induced colitis. These findings identify pyruvate as a regulator of PC biology in CF and as a therapeutically relevant factor in colitis.

Graphical Abstract

Pyruvate promotes ciliogenesis bypassing IFT88 dependency and attenuates DSS-induced colitis

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  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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    Reply to the reviewers

    Please find our reviewer comments in the submitted revision-plan RC-2025-03356.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    To improve the quality of this study, consider implementing strategic improvements that might enhance the significance of your intriguing findings. The results showing that pyruvate can circumvent IFT88 reliance represent a substantial change in our understanding of ciliary assembly; however, the paper would benefit from a more thorough examination of the mechanisms behind this physical development. Since IFT88 is usually seen as the main "elevator" for ciliary parts, figuring out if other proteins like IFT81 or IFT52 are being reused or increased when pyruvate is present will provide a clearer understanding of how this bypass works.

    While you have successfully outlined the signaling pathways linked to tubulin acetylation and detyrosination, the connection between histone acetylation and MAPK signaling poses a complex question. Figuring out if EP300-mediated acetylation starts the MAPK cascade or works as a feedback loop-possibly through specific inhibition tests-would improve the clarity needed for scientific publications. Furthermore, given the pronounced impact shown in colonic fibroblasts, it would be prudent to investigate if this pyruvate-induced ciliogenesis is a ubiquitous biological phenomenon by doing the same experiment in a conventional model, such as RPE1 cells. This would assist in ascertaining if you have discovered a fundamental metabolic principle of biology or a specific adaptation of the gastrointestinal system.

    Concerning the findings on tubulin detyrosination, there exists a little discrepancy: VASH inhibition influences ciliary length at elevated pyruvate concentrations, but the Western blots do not clearly show the predominant alterations in detyrosination at the same concentrations. To address this discrepancy, one may employ high-resolution immunofluorescence to assess detyrosination selectively within the ciliary axoneme, rather than examining the entire cell. This would likely disclose the localized alterations indicated by your functional data. In the discussion about the DSS-induced colitis model, understanding how pyruvate works as both an energy source for colon cells and an antioxidant, along with its effects on cilia, would strengthen the case for its potential as a treatment. Improving these detailed understandings and clarifying which cell types are involved will elevate the paper from a niche discovery to an important addition to cell biology and mucosal immunology.

    Prospective other Improvement Areas Analyzing the MAPK/Histone Acetylation Feedback Loop:

    1.The findings indicate that histone acetylation and MAPK signaling both play a role in pyruvate-induced ciliogenesis. Comment: As said, it is still unknown if histone acetylation triggers MAPK, or the other way around, or whether they create a feedback loop. Incorporating particular tests, such as assessing MAPK activity while blocking EP300 and vice versa, might elucidate this hierarchy. 2.The article suggests that pyruvate's capability to bypass IFT88 may be exclusive to colonic fibroblasts or certain cell types. Comments: Evaluating this effect in a widely utilized ciliary model such as RPE1 or IMCD3 cells will substantially enhance the paper's significance by ascertaining if this is a universal or specialized biological process. +1

    1. The work demonstrates that PC forms in the absence of IFT88 when pyruvate is available, although it fails to elucidate the mechanism of structure assembly without this essential transport protein. Comment: Examining if additional IFT proteins (such as IFT81 or IFT52) or alternative transport pathways are elevated or repurposed in the presence of pyruvate will significantly enhance the understanding of the "bypass" discovery. 4.The authors noted that VASH inhibition (LV80) decreased PC length at both 2mM and 10mM pyruvate, however bulk detyrosination alterations were only observable at 2mM. Comment: Although the authors explain this to the "higher sensitivity" of PC length measures, including high-resolution immunofluorescence quantification of the ciliary axoneme, rather than overall cell levels, might furnish the necessary visual proof for detyrosination alterations at 10mM. 5.The authors appropriately recognize that pyruvate may have effects on colitis that are independent of PC. Comment: To give a more comprehensive picture of pyruvate's therapeutic advantages, it would be helpful to broaden the interaction to briefly clarify how its ciliary effects could work in conjunction with its recognized functions in antioxidant defense or epithelial energy metabolism.

    Significance

    The study identifies pyruvate as a distinctive environmental regulator of ciliary length and ciliogenesis in colonic fibroblasts (CF).

    A major discovery is that pyruvate can help produce primary cilia in cells lacking IFT88, challenging the earlier belief that IFT88 is essential for making primary cilia.

    The authors clearly explain the signaling pathways, showing that pyruvate affects the amount of primary cilia by changing tubulin acetylation (which involves acetyl-CoA and ATAT1) and influences the length of primary cilia by altering tubulin

    Strong evidence from experiments with Col6a1cre-Ift88flx/flx mice in a DSS-induced colitis model strongly backs the importance of these findings for both biology and potential treatments.

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    The present manuscript demonstrates an important role for pyruvate in ciliogenesis and in the regulation of ciliary length/MT modifications. Previous work by the authors' group showed that primary cilia in colon fibroblasts are critical in experimental colitis: loss of cilia increases susceptibility to colitis. In the current study, the authors not only propose potential mechanisms by which pyruvate affects ciliary length and ciliogenesis, but also show that pyruvate treatment positively impacts cilia number and/or length and ameliorates experimentally induced colitis. Overall, I consider this study highly timely and very carefully executed. The quality of the data is excellent, and the findings will be highly relevant to the cilia research community. I have only a few minor points that could further strengthen the manuscript.

    Minor Comments:

    1. To better assess the generality of the findings beyond colonic fibroblasts and to determine whether the pyruvate axis also plays a role in other cell types, the authors could consider performing analogous experiments in widely used cilia model cell lines, e.g. mIMCD, MCKD, RPE1, or similar. It would also be very interesting to evaluate to what extent differences in commonly used media (e.g., RPMI versus DMEM or others) contribute to differences in cilia number and length. Even if beyond the scope of the current study, the present work will likely inspire such investigations in many laboratories.
    2. It remains unclear how the acetylation level is calculated/determined (Fig. 2B/D). The same applies to detyrosination. Please clarify the quantification method (e.g., normalization strategy, region of interest, background subtraction, and whether the readout is intensity per cilium, per cell, or population-averaged).
    3. Some inhibitors are used at relatively high concentrations compared to their EC50 values (e.g., UK-5099 at 50 µM; LV80 at 100 µM; C646 at 25 µM). At these doses, specificity may become an issue and should be validated experimentally or discussed as a limitation. For example, C646 has been reported to inhibit HDACs at higher concentrations.
    4. Can the authors exclude that β-mercaptoethanol (β-ME) in the medium interferes with the effect of pyruvate? Would it be feasible to culture the colonic fibroblasts without β-ME, at least for the treatment window, to rule out confounding effects?
    5. Are the pictures in Fig. 6C derived taken from in vivo tissue or cultured cells? Quantification would be helpful. Small typo in legend: "10μM" should be "10 µm".
    6. Excluding physiological changes due to sodium pyruvate or osmolarity-matched NaCl in vivo based solely on body weight curves may not be sufficient. Potential effects of the high-salt regimen should be discussed as a limitation, and the difference in the anion component should be discussed. For instance, in addition to renal effects such as polyuria, polydipsia, changes in blood pressure etc. eight weeks of 0.2 M salt in the drinking water could plausibly affect the immune system and, thus, indirectly influence the phenotype.

    Significance

    This study is highly relevant to the cilia-/ciliopathy field. It demonstrates that pyruvate - or, more broadly, the composition of the culture medium - can substantially influence ciliogenesis and ciliary length. Whether the observed effects are specific to colonic fibroblasts or extend to other ciliated cell types remains unclear. Nevertheless, this is a genuinely inspiring piece of work that will likely stimulate follow-up studies across the community.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The paper entitled "Pyruvate promotes ciliogenesis bypassing IFT88 dependency and attenuates DSS-induced colitis" by Maya Sarieddine, Ilaria Cicalini, Damiana Pieragostino, Federica Dimarco, Matthieu Lacroix, Krzysztof Rogowski, Valerie Pinet and Michael Hahne, describes the impact of pyruvate concentration on the number and length of primary cilia in murine colonic fibroblasts. The authors further demonstrate that pyruvate promotes both primary cilium acetylation and the expression of acetyltransferases. These findings appear to correlate with pyruvate's regulation of genes essential for cilia assembly, as well as activation of the MAPK signaling pathway, as revealed by RNA-seq and proteomic analyses. Additionally, pyruvate modulates tubulin detyrosination at primary cilia via MAPK-dependent mechanisms. Notably, pyruvate rescues primary cilium assembly in mice deficient for IFT88, a key protein for cilia assembly and maintenance, and reduces susceptibility to experimentally induced colitis in these mice. These results are interesting and are interesting and open opportunities to explore eventual treatments to colitis. However, the manuscript requires a thorough review, as many sections lack rigor, and several conclusions are drawn based on indirect evidence. The manuscript requires that the authors address the following concerns before publication:

    Major

    • In the different sections, through the text, the authors should clearly state that immunofluorescence microscopy was used to assess the number and length of primary cilia, as well as the intensity of the various markers (including the specific antibodies used). This clarification will allow readers to properly interpret the graphical data.
    • Could the progressive increase in pyruvate and sodium acetate concentrations induce osmotic stress in the cells? If so, the inclusion of an osmotic stress control would be warranted.
    • Regarding the ATAT1 inhibitor data, it is unclear why tubulin acetylation in primary cilia (PC) was not quantified. Since microtubule acetylation is likely affected, this could also impact the proportion of ciliated cells. The authors should address and discuss this point.
    • Regarding the use of MAPK signaling inhibitors, the authors show that only primary cilium length is dependent on this pathway. Inhibition of the pathway does not appear to affect, either the number of primary cilia, or their acetylation status. Therefore, the authors should clarify how acetylation is maintained despite the reduction in primary cilium length observed in Figures 4A and 4C. -It would be nice if authors have investigated if pyruvate increases PC length in control mouse as occurs in cells. In fact, in Figure 6F they only analyzed the PC length in Ift88-deficient mice. The same occurs in experiments reported in figure 7 when they establish a relationship between pyruvate role in cilia and induced colitis.

    Regarding the discussion

    Discussion has a lack of rigor:

    1-pg 15 " This concurs with reports showing that increased tubulin acetylation, catalyzed by ATAT1, can promote cilium assembly 19,20"- Reference 19- The authors describe that ATAT1-depleted cells, following siRNA treatment, display a similar percentage of ciliated cells after 24 h of serum removal compared to control cells, despite the cilia being non-acetylated. This indicates that tubulin acetylation does not promote cilia assembly per se, but rather enhances the efficiency of the biogenesis process, as cilia formation occurs more slowly in its absence. 2-pg 15- "This model aligns with our observation that elevated tubulin acetylation enhances the proportion of ciliated CFs." This is an indirect conclusion because in experiments where aTAT1 is inhibited the authors did not measure the intensity of cilia tubulin acetylation. Additionally, when MAPK signaling is inhibited, they observed that PC acetylation decreases but % of ciliated cells is not affected.

    3-pg16- "Nonetheless, our finding that histone acetylation contributes to pyruvate-driven ciliogenesis is in agreement with previous reports indicating that such modification can modulate transcriptional programs involved in PC formation. For example, depletion of the histone acetyltransferase KAT2B (lysine acetyltransferase 2B) in mouse embryonic fibroblasts impaired ciliogenesis 27." This sentence lacks rigor because the authors forgot that many of the acetyl-transferases have distinct substrates. For example, in the cited paper (27) it is shown that KAT2B directly acetylates tubulin affecting primary cilia assembly. The same critic can be extended to sentence "This concurs with additional reports linking histone acetylation with cilia formation 28,29."

    4-The authors should clarify the consistence between their observations and those described in paper 33- "that Vash deletion affects anterograde IFT train movement, leading to ciliary elongation 33. Consistent with these findings, we observed that treatment of ciliated CF cells with LV80, a potent VASH inhibitor34 did not alter the proportion of ciliated cells but did negatively affect PC length." They observed that Vash inhibition leads to smaller cilia, but in Clamydomonas Vash deletion causes cilia elongation.

    Minor

    Figure 1

    C-The table below Graphic C should be clarified, as the +/- symbols are used simultaneously to indicate both addition and presence/absence, which may cause confusion. In the legend -" after 24h starvation in RPMI, RPMI complemented with either Glucose (Glu, 0.14mM)" - Glucose should be 14 mM??? F- The graphic is not mentioned in the text, and the information overlaps with that of D and E, therefore is redundant.

    Significance

    The results described are interesting and open opportunities to explore eventual treatments to colitis and its relationship with primary cilia. However, the manuscript needs to be profoundly reviewed since in many sites lacks rigor.

    The authors state many conclusions based on indirect evidence.

  5. Version published to 10.64898/2025.12.17.694572 on bioRxiv