Review Reports - TRPV4-Mast Cell Interactions in Neurogenic Inflammation and Chronic Diseases: A Narrative Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This work describes the role of MCs, and TRPV4 in conditions such as rosacea, asthma, visceral hypersensivity, migraine, pain, and osteoarthritis. The manuscript is well organized and well written. Though very pertinent research field, I have some concerns:

Fig. 1 Please illustrate with different colors or symbols the molecules released by activated mast cells.

Table 1 should be included in the main text and not as supplemental material.

Author Response

Reviewer 1

Reviewer’s comment 1: Fig. 1 Please illustrate with different colors or symbols the molecules released by activated mast cells.

Authors’ Reply: Thank you for the comment. We have revised the figure to illustrate mast cell derived mediators using distinct colors .

Reviewer’s comment 2: Table 1 should be included in the main text and not as supplemental material.

Authors’ Reply: We appreciate the reviewer’s comment. In response, Table 1 has been relocated to the main text.

Reviewer 2 Report

Comments and Suggestions for Authors

The title should indicate that this is a narrative review.
In asthma, the authors broadly describe TRPV4’s roles across pulmonary cells and nerves, but the the narrative then slides into a “TRPV4–ATP–P2X axis” involving mast cells and it is not consistently clear which findings are mast-cell intrinsic vs mast-cell dependent vs mast-cell adjacent. Please make this explicit.
Even for a narrative review, it would be preferable to provide a description of search strategy, inclusion/exclusion logic, how evidence was weighted, or how contradictory findings were handled etc.
“TRPV4 activation consistently induces MC degranulation… driving disease progression” reads as universal, but the manuscript itself notes context dependence (“can vary across experimental models and tissues”). Language used should also be tempered.
Pain is not a condition. Pain is a symptom, phenotype, or outcome, not a disease entity. Listing “pain” as a condition in Table 1 is conceptually incorrect.
Table 1 is too superficial and should include references to key studies/evidence and indicate their actual contributions/findings.
Large portions recap asthma/IBS/OA basics rather than focusing on what specifically is known about TRPV4 in mast cells, what experiments were done, and what remains unknown. For experimental medicine value, readers need: pathways, readouts, and discriminating experiments.
Please standardize the citation style throughout.

Comments on the Quality of English Language

Moderate edits required.

Author Response

Reviewer 2

Reviewer’s comment 1: The title should indicate that this is a narrative review.

Authors’ Reply: We thank the reviewer for this insightful comment. The title has been revised to explicitly indicate that this manuscript is a narrative review.

Reviewer’s comment 2: In asthma, the authors broadly describe TRPV4’s roles across pulmonary cells and nerves, but the narrative then slides into a “TRPV4–ATP–P2X axis” involving mast cells and it is not consistently clear which findings are mast-cell intrinsic vs mast-cell dependent vs mast-cell adjacent. Please make this explicit.

Authors’ Reply: We thank the reviewer for this insightful comment. In response, we have revised the manuscript to explicitly distinguish mast cell–intrinsic, mast cell–dependent, and mast cell–adjacent components of TRPV4 signaling in asthma. We have also added a new figure specific to the asthma section to clarify cellular causality. This figure illustrates that TRPV4 activation occurs in airway smooth muscle (ASM) cells, leading to intracellular Ca²⁺ elevation and ATP release from ASM. Extracellular ATP then acts on P2X4 receptors expressed on mast cells within ASM bundles, promoting mast cell degranulation and cysteinyl leukotriene release, which in turn drives ASM contraction and contributes to airway narrowing and remodeling.

Reviewer’s comment 3: Even for a narrative review, it would be preferable to provide a description of search strategy, inclusion/exclusion logic, how evidence was weighted, or how contradictory findings were handled etc.

Authors’ Reply: We thank the reviewer for this valuable suggestion. In response, we have added a detailed description in the first part of the review outlining the literature search strategy, inclusion and exclusion criteria, and the approach used to evaluate and interpret the evidence. We also clarified how conflicting findings were considered and discussed to ensure a balanced and transparent narrative synthesis. We believe these additions strengthen the methodological rigor and clarity of the review.

Reviewer’s comment 4: “TRPV4 activation consistently induces MC degranulation… driving disease progression” reads as universal, but the manuscript itself notes context dependence (“can vary across experimental models and tissues”). Language used should also be tempered).

Authors’ Reply: We thank the reviewer for highlighting this important point. We agree that the original wording may have implied a universal effect of TRPV4 activation on mast cell degranulation. The text has now been revised to temper the language.

Reviewer’s comment 5: Pain is not a condition. Pain is a symptom, phenotype, or outcome, not a disease entity. Listing “pain” as a condition in Table 1 is conceptually incorrect.

Authors’ Reply: We thank the reviewer for this important conceptual clarification. We agree that acute pain is classically considered a symptom or outcome of an underlying pathology. However, in the context of chronicity, pain is increasingly recognized as more than a mere symptom. Specifically, chronic pain is considered a condition characterized by persistent maladaptive changes in nociceptive and neuroimmune pathways, leading to sustained sensory and affective disturbances. This conceptual shift is supported by modern pain classifications, including the International Classification of Diseases (ICD-11), which recognizes chronic pain as a distinct disease entity rather than solely a symptom.

Treede RD, Rief W, Barke A, Aziz Q, Bennett MI, Benoliel R, Cohen M, Evers S, Finnerup NB, First MB, Giamberardino MA, Kaasa S, Korwisi B, Kosek E, Lavand'homme P, Nicholas M, Perrot S, Scholz J, Schug S, Smith BH, Svensson P, Vlaeyen JWS, Wang SJ. Chronic pain as a symptom or a disease: the IASP Classification of Chronic Pain for the International Classification of Diseases (ICD-11). Pain. 2019 Jan;160(1):19-27. doi: 10.1097/j.pain.0000000000001384. PMID: 30586067.

Reviewer’s comment 6: Table 1 is too superficial and should include references to key studies/evidence and indicate their actual contributions/findings.

Authors’ Reply: We thank the reviewer for this insightful comment. We have revised Table 1 accordingly and substantially improved its content and structure. The table has been reconstructed to include the following columns: Tissues/Cells Affected, Experimental Model, Experimental Method, and Roles of TRPV4, providing a more detailed and mechanistic summary of the findings. In addition, we have now referenced each study cited in the table to clearly indicate the original evidence and specific contributions of each work. We believe that these revisions significantly enhance the scientific depth, clarity, and usefulness of Table 1.

Reviewer’s comment 7: Large portions recap asthma/IBS/OA basics rather than focusing on what specifically is known about TRPV4 in mast cells, what experiments were done, and what remains unknown. For experimental medicine value, readers need: pathways, readouts, and discriminating experiments.

Authors’ Reply: We sincerely thank the reviewer for this insightful comment. We fully acknowledge that direct experimental evidence specifically addressing TRPV4 function in mast cells remains limited in several disease contexts, including asthma, IBS, and osteoarthritis. This limitation reflects the current state of the field rather than an oversight in the review. Accordingly, we deliberately structured the review to clearly distinguish between conditions in which TRPV4 has been directly investigated in mast cells and conditions in which TRPV4 has been studied in other cell types, and the critical pathogenic role mast cells play. In these latter cases, we aimed to highlight the emerging and biologically plausible neuroimmune crosstalk between mast cells, TRPV4-expressing cells, and the surrounding tissue microenvironment. We believe that emphasizing this intercellular communication is essential, as it likely represents a key mechanism driving disease initiation and progression. We have revised the manuscript to better emphasize known signaling pathways, experimental readouts, and mechanistic evidence where available, while explicitly identifying knowledge gaps and unresolved questions. We now clarify that although mast cell activation is well established in disorders such as IBS, the precise molecular intersections between mast cell signaling and TRPV4 pathways remain incompletely defined. This gap underscores the urgent need for future discriminating experiments to delineate how TRPV4-mediated signaling integrates with mast cell–driven inflammation and neuroimmune communication in chronic pain and inflammatory diseases.

Reviewer’s comment 8: Please standardize the citation style throughout.

Authors’ Reply: We thank the reviewer for pointing this out. We have carefully reviewed the manuscript and standardized the citation style throughout to ensure consistency with the journal’s formatting requirements.

Reviewer 3 Report

Comments and Suggestions for Authors

This review addresses a broad and timely topic and reflects substantial familiarity with the literature on TRPV4 signaling and mast cell–mediated neuroimmune interactions. By drawing on studies across multiple disease contexts, the manuscript has the potential to serve as a useful entry point for readers new to this area.
However, in its current form, the review is overly descriptive and lacks sufficient conceptual synthesis. Many sections read as sequential summaries of individual studies, and it is often unclear how these findings collectively advance a unifying framework. While the manuscript proposes a TRPV4–mast cell axis as a common mechanism across diverse pathologies, this concept is largely implied through accumulation of examples rather than explicitly developed, critically examined, or tested against alternative interpretations.
Although the authors include a summary table (Table 1), it primarily catalogs reported associations and does not adequately distinguish between mast cell–specific evidence, indirect tissue-level observations, and more speculative links. Additional analytical structure is needed—for example, tables that stratify evidence by cell type, experimental system, disease context, and strength of support—to help readers assess where the evidence is strong, limited, or indirect. More importantly, the manuscript would benefit from a dedicated conceptual model figure that integrates TRPV4 signaling, mast cell activation, and disease-specific outcomes into a coherent mechanistic framework. This figure should go beyond the current graphical abstract by explicitly illustrating proposed signaling pathways, cell-type–specific interactions, and points of divergence across disease contexts, rather than simply summarizing the review’s structure.
In several sections, mechanistic claims appear stronger than the underlying data support, particularly when mast cell involvement is inferred indirectly from neuronal or whole-tissue studies. A clearer distinction between mast cell–specific findings and broader TRPV4 signaling effects would improve balance and accuracy. The therapeutic discussion would also benefit from deeper critical analysis, including feasibility, cell specificity, and potential off-target effects, given the widespread physiological roles of TRPV4.
Overall, the manuscript would require substantial restructuring to move beyond a broad literature survey. Significant condensation of descriptive sections, clearer prioritization of key disease examples, introduction of an explicit model framework, and more critically organized summary tables would be necessary to strengthen the conceptual impact. With these major changes, a revised version could offer a more authoritative and integrative contribution to the field.

Comments on the Quality of English Language

The English is generally clear and understandable, but the manuscript would benefit from editing to improve concision and flow.

Example 1
Original: “TRPV4 has been reported to play an important role in mast cell activation in multiple disease contexts, and this role has been increasingly recognized in recent years by many studies.”
Revised: “Recent studies suggest that TRPV4 contributes to mast cell activation across multiple disease contexts.”

Example 2
Original: “TRPV4 is widely expressed in many tissues, and its widespread expression suggests that TRPV4 may play diverse roles in different tissues.”
Revised: “TRPV4 is widely expressed across tissues, indicating that it may have context-dependent functions.”

Author Response

Reviewer 3

Reviewer’s comment 1: This review addresses a broad and timely topic and reflects substantial familiarity with the literature on TRPV4 signaling and mast cell–mediated neuroimmune interactions. By drawing on studies across multiple disease contexts, the manuscript has the potential to serve as a useful entry point for readers new to this area.

However, in its current form, the review is overly descriptive and lacks sufficient conceptual synthesis. Many sections read as sequential summaries of individual studies, and it is often unclear how these findings collectively advance a unifying framework. While the manuscript proposes a TRPV4–mast cell axis as a common mechanism across diverse pathologies, this concept is largely implied through accumulation of examples rather than explicitly developed, critically examined, or tested against alternative interpretations.

Although the authors include a summary table (Table 1), it primarily catalogs reported associations and does not adequately distinguish between mast cell–specific evidence, indirect tissue-level observations, and more speculative links. Additional analytical structure is needed—for example, tables that stratify evidence by cell type, experimental system, disease context, and strength of support—to help readers assess where the evidence is strong, limited, or indirect. More importantly, the manuscript would benefit from a dedicated conceptual model figure that integrates TRPV4 signaling, mast cell activation, and disease-specific outcomes into a coherent mechanistic framework. This figure should go beyond the current graphical abstract by explicitly illustrating proposed signaling pathways, cell-type–specific interactions, and points of divergence across disease contexts, rather than simply summarizing the review’s structure.

In several sections, mechanistic claims appear stronger than the underlying data support, particularly when mast cell involvement is inferred indirectly from neuronal or whole-tissue studies. A clearer distinction between mast cell–specific findings and broader TRPV4 signaling effects would improve balance and accuracy. The therapeutic discussion would also benefit from deeper critical analysis, including feasibility, cell specificity, and potential off-target effects, given the widespread physiological roles of TRPV4.

Overall, the manuscript would require substantial restructuring to move beyond a broad literature survey. Significant condensation of descriptive sections, clearer prioritization of key disease examples, introduction of an explicit model framework, and more critically organized summary tables would be necessary to strengthen the conceptual impact. With these major changes, a revised version could offer a more authoritative and integrative contribution to the field.

Authors’ Reply:

We thank the reviewer for this thoughtful and constructive critique and for recognizing the timeliness and breadth of the topic. We agree that the initial version of the manuscript was overly descriptive in places and did not sufficiently articulate a unifying conceptual framework for TRPV4–mast cell (MC) signaling across disease contexts. In response, we have substantially revised and restructured the manuscript to strengthen conceptual synthesis, analytical clarity, and critical interpretation.

We condensed and reorganized multiple sections to reduce sequential summaries of individual studies and instead emphasize recurring mechanistic themes. Descriptive content has been streamlined, and representative disease contexts have been prioritized where evidence for TRPV4–MC interactions is strongest, while more speculative or indirect observations are now explicitly identifiedand framed accordingly.
Toaddress the need for an explicit unifying framework, we added multiple new conceptual figures (Figures 2–6), while retainingand refining Figure 1 as a foundational overview. Together, these figures integrate TRPV4 signaling, mast cell activation, and downstream neuroimmune outcomes across distinct disease contexts, including airway disease, visceral hypersensitivity, bladder pain, osteoarthritis, and mechanically driven inflammatory conditions. These figures extend well beyond the original graphical abstract by explicitly illustrating proposed signaling pathways, cell-type–specific interactions (mast cells, sensory neurons, and epithelial or stromal cells), as well as points of convergence and divergence across systems. The accompanying text now explicitly develops and critically examines the proposed TRPV4–MC axis, including alternative interpretations and remaining knowledge gaps.
We revised and expanded the summary table to improve analytical structure. In addition to the original overview, evidence is now stratified by cell type (mast cell–specific versus indirect tissue-level data), experimental system (in vitro, ex vivo, in vivo, human, mouse, guinea pig), disease context, and TRPv4 relevant evidence.This organization enables readers to more readily assess where evidence is strong, limited, or inferential.
4. Weclarified and moderated mechanistic claims throughout the manuscript. Statements implying direct mast cell involvement have been revised where evidence is indirect, and we now clearly distinguish mast cell–specific findings from broader TRPV4 signaling effects observedat the neuronal or tissue level. These changes improve balance and align conclusions more closely with the available data.
The therapeuticdiscussion has been substantially revisedto provide a deeper and more critical assessment of feasibility, cell specificity, and potential off-target effects, particularly given the widespread physiological roles of TRPV4. Rather than presenting TRPV4 modulation as uniformly beneficial, the revised text emphasizes context dependence, disease-specific mechanisms, and the need for cell-targeted or pathway-selective therapeutic strategies.

Collectively, these revisions represent a major restructuring of the manuscript, shifting it from a broad literature survey toward a more integrative, concept-driven review. By incorporating multiple disease-focused conceptual figures and a more critically organized table, the revised version more clearly delineates where evidence for TRPV4–mast cell interactions is strong, where it remains limited or indirect, and how these pathways may be differentially engaged across disease states. We thank the reviewer for their insightful guidance, which has significantly strengthened the conceptual rigor and impact of the manuscript.

Reviewer 4 Report

Comments and Suggestions for Authors

Dear authors,

My comments are attached.

Regards.

Comments for author File: Comments.pdf

Author Response

Reviewer 4

Reviewer’s comment 1: The authors did not address the inclusion and exclusion criteria for articles cited in the manuscript. I recommend you do so.

Authors’ Reply: We thank the reviewer for this valuable suggestion. In response, we have added a detailed description of the inclusion and exclusion criteria at the beginning of the review. This section now outlines the rationale for article selection, the types of studies considered, and the criteria used to include or exclude relevant literature. We believe that this addition improves the transparency and methodological rigor of the manuscript.

Reviewer’s comment 2: The authors did not specify the period covered by the selected articles in the manuscript. Was it the last 5 or 10 years?

Authors’ Reply: From year 2000-current

Reviewer’s comment 3: The authors did not indicate the software used to develop Figure 1 and the graphical abstract. I recommend you do this.

Authors’ Reply: We thank the reviewer for this suggestion. The graphical abstract and Figure 1 were created using BioRender.com, which has now been indicated in the figure legends as the software used for illustration.

Reviewer’s comment 4: The authors presented only one figure in the manuscript. I recommend including more figures.

Authors’ Reply: We thank the reviewer for this constructive suggestion. In response, we have substantially expanded the visual content of the manuscript by adding six additional figures. These figures were designed to correspond to the major sections of the review and to provide clear schematic representations of the crosstalk between TRPV4 signaling and mast cell activation across the different pathological conditions discussed. We believe that these figures significantly enhance the clarity of complex neuroimmune interactions and offer readers an intuitive framework to better understand the mechanisms and disease-specific roles of TRPV4–mast cell signaling highlighted in this review.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

1. Please remove all methods-like text from the Introduction and move all search content to a proper Methods section.

2. What is the difference between TRPV4 in “any cell” vs TRPV4 specifically in mast cells? This can be stated more explicitly.

3. In allergy/immunology, “mast cells” are not one entity. Tissue- and context-specific phenotypes (connective tissue vs mucosal MCs, human vs mouse differences, FcεRI density, MRGPRX2 expression patterns, mediator content, protease profiles) matter for any mechanistic narrative.

4. "The hallmark of irritable bowel syndrome (IBS)IBS is visceral hypersensitivity (VH), which arises from neuro-immune interactions. TRPV4 channels and MCs are central to this process, as MC-derived mediators sensitize sensory neurons, exacerbating pain and gut dysfunction [82, 83]." - In addition to ref [82,83], suggest also to cite evidence of low-grade inflammation in IBS (citation: pmc.ncbi.nlm.nih.gov/articles/PMC6159811).

5. Include in Supplementary Table 1 information on the model type, key outcome(s) etc.

Comments on the Quality of English Language

Moderate edits required.

Author Response

Reviewer’s comment 1: Please remove all methods-like text from the Introduction and move all search content to a proper Methods section.

Authors’ Reply: We thank the reviewer for this valuable suggestion. We have revised the manuscript accordingly by removing methodological and search-related content from the Introduction and relocating it to a newly structured Materials and Methods section. This revision improves the clarity and organization of the manuscript by clearly distinguishing background information from methodological aspects of the review.

Reviewer’s comment 2: What is the difference between TRPV4 in “any cell” vs TRPV4 specifically in mast cells? This can be stated more explicitly.

Authors’ Reply: We thank the reviewer for this important comment and agree that the distinction between TRPV4 function in general cellular contexts and in mast cells should be clarified. The manuscript has been revised to make this distinction more explicit where possible. At present, there is no definitive evidence demonstrating that TRPV4 possesses intrinsically different properties in mast cells compared with other cell types. TRPV4 is a broadly expressed polymodal calcium-permeable channel whose functional contribution appears to depend strongly on cellular context, upstream receptor signaling, and local regulatory mechanisms.

Functional expression of TRPV4 in mast cells has been demonstrated in human mast cell lines, where TRPV4 activation induces membrane currents and increases intracellular calcium levels, indicating that TRPV4 can contribute to calcium influx under certain experimental conditions (Kim et al., 2010). These findings establish functional coupling of TRPV4 to calcium signaling in mast cells but do not support a universal role in mast cell activation across mast cell subtypes.

Additional evidence indicates that TRPV4 can contribute to mast cell activation in specific disease contexts rather than serving as a general activation pathway. In rosacea models, TRPV4 mediates mast cell activation in cathelicidin (LL37)-driven inflammation, where LL37 signaling through MRGPRX2 increases TRPV4 expression and TRPV4 activity is required for full mast cell degranulation and inflammatory responses (Mascarenhas et al., 2017). These findings support a model in which TRPV4 functions downstream of disease-relevant receptor signaling to facilitate calcium influx and amplify mast cell activation under inflammatory conditions.

In contrast, studies in primary mouse peritoneal mast cells show that although TRPV channels, including TRPV4, are expressed, TRPV4 does not participate in FcεRI- or MRGPR-mediated activation pathways or in responses to thermal or osmotic stimulation (Solís-López et al., 2017). This indicates that TRPV4 expression alone does not predict functional involvement and highlights substantial heterogeneity among mast cell populations.

Taken together, current evidence indicates that the role of TRPV4 in mast cells remains context-dependent and incompletely defined. It remains unclear whether differences in TRPV4 signaling between mast cells and other cell types reflect distinct regulatory mechanisms, cell-specific signaling environments, or differences in experimental models, and further mechanistic studies are required to resolve this question.

Reviewer’s comment 3: In allergy/immunology, “mast cells” are not one entity. Tissue- and context-specific phenotypes (connective tissue vs mucosal MCs, human vs mouse differences, FcεRI density, MRGPRX2 expression patterns, mediator content, protease profiles) matter for any mechanistic narrative.

Authors’ Reply: We thank the reviewer for this important comment and agree that mast cells represent a heterogeneous population with tissue- and context-specific phenotypes that influence receptor expression, mediator content, and functional responses. As discussed in our response to Comment 2, the manuscript has been revised where appropriate to acknowledge mast cell heterogeneity and to clarify that TRPV4-dependent signaling and mast cell activation are context-dependent and may vary across mast cell subtypes, tissues, and experimental models. We have incorporated this clarification throughout the revised text to avoid implying that mast cells function as a single uniform entity and to emphasize that mechanistic conclusions may differ between connective tissue and mucosal mast cells, as well as between human and mouse systems.

Reviewer’s comment 4: “The hallmark of irritable bowel syndrome (IBS)IBS is visceral hypersensitivity (VH), which arises from neuro-immune interactions. TRPV4 channels and MCs are central to this process, as MC-derived mediators sensitize sensory neurons, exacerbating pain and gut dysfunction [82, 83]." - In addition to ref [82,83], suggest also to cite evidence of low-grade inflammation in IBS (citation: pmc.ncbi.nlm.nih.gov/articles/PMC6159811).

Authors’ Reply: We thank the reviewer for this helpful suggestion. We have revised the text to additionally acknowledge evidence of low-grade inflammation in IBS, which further supports the role of immune activation in visceral hypersensitivity. The citation addressing low-grade inflammation in IBS has now been added to the manuscript.

Reviewer’s comment 5: Include in Supplementary Table 1 information on the model type, key outcome(s) etc.

Authors’ Reply: We thank the reviewer for this helpful suggestion. Information regarding experimental model type is already included in Supplementary Table 1 through the existing columns detailing the tissues or cells studied, experimental model, and experimental methodology for each cited study. In response to the reviewer’s comment, we have expanded the “Roles of TRPV4” column to also summarize the key outcomes of each study. By incorporating key outcomes alongside model and methodological information, the revised table provides a clearer and more comprehensive overview of how TRPV4 contributes to neuroimmune signaling and inflammatory processes across different conditions, while remaining aligned with the central focus and aims of the review.

Reviewer 3 Report

Comments and Suggestions for Authors

The revision makes limited progress toward addressing the central concerns raised previously. While additional literature has been incorporated and some sections expanded, the manuscript remains largely descriptive and does not achieve the level of conceptual integration expected of a high-impact review.
The principal issue persists: the proposed TRPV4–mast cell axis is not developed into a coherent mechanistic framework. The text continues to present disease contexts sequentially, with emphasis on individual findings rather than synthesis across systems. As a result, the central premise remains associative rather than explanatory. The revision does not clearly articulate how TRPV4 signaling in mast cells converges on shared pathways, nor does it critically evaluate where this model holds, where it breaks down, or how it compares with alternative interpretations.
Table 1 remains primarily a catalog of reported associations. It does not differentiate mast cell–specific evidence from indirect tissue-level observations, nor does it convey the relative strength or limitations of the underlying studies. This limits its utility as an analytical summary. Similarly, the graphical abstract continues to function as a thematic overview rather than a true conceptual model. It does not integrate signaling pathways, cell-type specificity, or disease-dependent divergence in a way that advances mechanistic understanding.
Concerns regarding interpretive balance also remain. In several sections, mast cell involvement is inferred from neuronal or whole-tissue data without sufficient qualification, and the distinction between direct mast cell signaling and broader TRPV4-mediated effects is not consistently maintained. The therapeutic discussion remains superficial and does not engage meaningfully with issues of feasibility, specificity, or off-target effects, which are essential given the pleiotropic roles of TRPV4.
Although the language has been modestly improved for concision and clarity, these changes are editorial rather than substantive. The revision does not reflect the level of restructuring, critical synthesis, or conceptual sharpening that was requested.

Author Response

Reviewer’s comment 1: The revision makes limited progress toward addressing the central concerns raised previously. While additional literature has been incorporated and some sections expanded, the manuscript remains largely descriptive and does not achieve the level of conceptual integration expected of a high-impact review.

The principal issue persists: the proposed TRPV4–mast cell axis is not developed into a coherent mechanistic framework. The text continues to present disease contexts sequentially, with emphasis on individual findings rather than synthesis across systems. As a result, the central premise remains associative rather than explanatory. The revision does not clearly articulate how TRPV4 signaling in mast cells converges on shared pathways, nor does it critically evaluate where this model holds, where it breaks down, or how it compares with alternative interpretations.

Table 1 remains primarily a catalog of reported associations. It does not differentiate mast cell–specific evidence from indirect tissue-level observations, nor does it convey the relative strength or limitations of the underlying studies. This limits its utility as an analytical summary. Similarly, the graphical abstract continues to function as a thematic overview rather than a true conceptual model. It does not integrate signaling pathways, cell-type specificity, or disease-dependent divergence in a way that advances mechanistic understanding.

Concerns regarding interpretive balance also remain. In several sections, mast cell involvement is inferred from neuronal or whole-tissue data without sufficient qualification, and the distinction between direct mast cell signaling and broader TRPV4-mediated effects is not consistently maintained. The therapeutic discussion remains superficial and does not engage meaningfully with issues of feasibility, specificity, or off-target effects, which are essential given the pleiotropic roles of TRPV4.

Although the language has been modestly improved for concision and clarity, these changes are editorial rather than substantive. The revision does not reflect the level of restructuring, critical synthesis, or conceptual sharpening that was requested.

Authors’ Reply: We thank the reviewer for the careful evaluation and for highlighting the need for stronger conceptual integration and clearer distinction between associative and mechanistic interpretations. In response, we have substantially revised the manuscript to improve conceptual clarity, strengthen mechanistic synthesis across disease contexts, and explicitly address the limitations of the current evidence base while preserving the original organizational structure of the review.

We introduced an explicit conceptual framework in the Introduction that defines how TRPV4–MC interactions are interpreted throughout the manuscript. Rather than presenting TRPV4 activities as a uniform mast cell–specific pathway, the revised text now distinguishes between (i) disease settings in which direct TRPV4-dependent MC activation has been experimentally demonstrated (e.g., LL-37–driven rosacea and microbubble-induced mast cell activation in decompression sickness), and (ii) disease contexts in which TRPV4 activity in adjacent epithelial, neuronal, or stromal cells influences MC function indirectly through neuroimmune crosstalk. This distinction is now maintained consistently across all sections to avoid inferring mast cell–specific mechanisms from tissue-level or neuronal data.
Individual disease sections have been revised to improve mechanistic continuity by linking findings to shared signaling processes, including Ca²⁺-dependentactivation, purinergic signaling,and neuroimmune amplification, while clarifying where evidence remains indirect or context-dependent. These revisions were intended to improve conceptual synthesis without overstating mechanistic conclusions.
The Supplementary Table 1 has also been expanded to include key experimental outcomes and to better distinguish between studies demonstrating direct MC involvement now shown in bold type face those reflecting broader tissue-level TRPV4 effects, improving its function as an analytical summary of the available evidence.
The therapeutic discussion has been revised to address feasibility, specificity, and the pleiotropic biology of TRPV4. The revised text emphasizes that TRPV4 represents a context-dependent therapeutic target and that successful translation will likely require tissue-specific delivery strategies, pathway-selective modulation, or combination approaches that account for MC heterogeneity and non–mast cell TRPV4 functions.
The revised graphical abstract is now divided into two clearly defined components: a. Direct TRPV4 signaling in mast cells (left panel):This section isolates mast cell–intrinsic TRPV4 activation, depicting receptor inputs (e.g., TRPV4, GPCRs, purinergic signaling), PLC/IP₃ pathways, Ca²⁺ influx, and degranulation. b. mast cell – TRPV4 crosstalk across chronic diseases (right panel): This panel shows how mast cell mediator release influences neighboring cell types (e.g., sensory neurons, epithelial, smooth muscle, chondrocytes), producing tissue-specific outcomes such as neurogenic inflammation or visceral hypersensitivity. By structurally separating cell-autonomous TRPV4 signaling from downstream tissue-level effects, the revised graphical abstract distinguishes direct mast cell mechanisms from broader TRPV4 biology and presents a unified, mechanistic framework rather than a sequential list of disease associations.

Importantly, the disease-based organizational structure was intentionally retained. Our primary objective is to synthesize and contextualize existing evidence supporting a mast cell–centric role for TRPV4, particularly in disease states where neuroimmune crosstalk is prominent (e.g., rosacea, decompression sickness, acupuncture-induced analgesia), and to extend this discussion to chronic conditions (asthma, IBS, bladder pain, osteoarthritis, migraine) in which TRPV4 signaling is implicated but MC-specific mechanisms remain incompletely defined. Given the heterogeneity of disease models and the limited availability of direct MC-specific mechanistic data in several of these contexts, a single unifying molecular pathway cannot yet be asserted without overinterpretation. Presenting evidence within disease contexts allows the reader to evaluate where the proposed TRPV4–MC framework is strongly supported, where it remains indirect, and where further mechanistic investigation is required.

Overall, the revisions were intended to transform the manuscript into a conceptually integrated review that defines shared mechanisms, delineates evidentiary boundaries, and highlights unresolved questions while maintaining a structure that accurately reflects the current state of the field.

Round 3

Reviewer 2 Report

Comments and Suggestions for Authors

I am fine with this.

Comments on the Quality of English Language

Moderate edits required.

Author Response

Thank you! We have revised the manuscript and performed the required edits.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript addresses a timely and biologically intriguing topic. The intersection of TRPV4 signaling with mast cell–mediated neuroimmune communication is an area of growing interest, and the authors demonstrate clear familiarity with a wide body of literature spanning pain biology, airway inflammation, dermatologic disease, and visceral hypersensitivity. The breadth of coverage and the effort to connect mechanotransduction with immune activation reflect thoughtful engagement with an emerging field.

That said, several substantive issues remain. The review continues to function primarily as a sequential compilation of disease-specific findings rather than as a conceptually integrated synthesis of the TRPV4–mast cell axis. The accumulation of examples does not yet crystallize into a coherent mechanistic framework.

The graphical abstract remains thematic rather than mechanistic. It illustrates TRPV4-dependent Ca²⁺ influx and mast cell degranulation across multiple conditions, but it does not delineate upstream triggers, downstream signaling divergence, or tissue-specific modifiers. A stronger model would explicitly define shared signaling modules (e.g., Ca²⁺-dependent exocytosis, ATP–purinergic amplification, neuropeptide feedback loops) and clarify where this logic holds or diverges across disease contexts.

Table 1 remains largely descriptive. It catalogs associations but does not stratify evidence strength or distinguish mast cell–intrinsic findings from broader tissue-level observations. For example, in asthma and osteoarthritis, TRPV4 upregulation and mast cell presence are discussed in parallel, but the causal architecture is not clearly defined. A revised table that categorizes evidence (mast cell–specific genetic data, pharmacologic modulation, correlative expression studies, inferred associations) would substantially strengthen the review’s analytical value.

The distinction between mast cell–specific TRPV4 signaling and TRPV4 activity in other cell types also requires greater precision. In several sections (e.g., asthma, migraine), neuronal or smooth muscle TRPV4 signaling is discussed alongside mast cell activation without consistently clarifying directionality or autonomy. This blending weakens interpretive clarity.

Finally, the therapeutic discussion remains somewhat aspirational. Given the pleiotropic roles of TRPV4 in vascular tone, mechanosensation, and osmoregulation, a more critical assessment of feasibility, specificity, and potential off-target effects is warranted. Without this, the translational narrative risks overstating readiness.

In summary, the manuscript has a solid foundation and covers a relevant field with breadth. To reach the level of an authoritative review, however, it would benefit from deeper conceptual integration, clearer evidence stratification, and a more rigorously defined mechanistic framework.

Author Response

Reviewer 3

Reviewer’s comment 1: The manuscript addresses a timely and biologically intriguing topic. The intersection of TRPV4 signaling with mast cell–mediated neuroimmune communication is an area of growing interest, and the authors demonstrate clear familiarity with a wide body of literature spanning pain biology, airway inflammation, dermatologic disease, and visceral hypersensitivity. The breadth of coverage and the effort to connect mechanotransduction with immune activation reflect thoughtful engagement with an emerging field.

The graphical abstract remains thematic rather than mechanistic. It illustrates TRPV4-dependent Ca²⁺ influx and mast cell degranulation across multiple conditions, but it does not delineate upstream triggers, downstream signaling divergence, or tissue-specific modifiers. A stronger model would explicitly define shared signaling modules (e.g., Ca²⁺-dependent exocytosis, ATP–purinergic amplification, neuropeptide feedback loops) and clarify where this logic holds or diverges across disease contexts.

Authors’ Reply: We thank the reviewer for the thoughtful and constructive evaluation of our manuscript and for recognizing the relevance of the TRPV4–mast cell axis in neuroimmune communication. We appreciate the concern that the review may read as a sequential compilation of disease-specific findings rather than a fully integrated mechanistic synthesis. Our intent was to synthesize the literature within disease contexts while highlighting shared biological themes, particularly TRPV4-dependent Ca²⁺ signaling, mast cell activation, and neuroimmune amplification, without imposing a unified mechanistic framework that is not yet consistently supported across all models. Given the heterogeneity of experimental systems and the variable depth of mast cell–specific mechanistic data, we sought to balance integration with appropriate caution.

Regarding the graphical abstract, it was designed to distinguish mast cell–intrinsic TRPV4 signaling from TRPV4 activity in adjacent cell types while emphasizing Ca²⁺-dependent signaling as the central unifying mechanism. The schematic highlights both cell-autonomous mast cell signaling and bidirectional interactions with TRPV4-expressing neurons, epithelial cells, and smooth muscle, reflecting shared signaling modules such as Ca²⁺-dependent exocytosis and ATP–purinergic amplification. We believe this structure provides a mechanistic framework consistent with the current state of the evidence.

In response to concerns about Table 1, we have substantially revised and reorganized the evidence. The new primary Table 1 (“Stratified Evidence for TRPV4–Mast Cell Signaling Across Disease Contexts”) categorizes studies by evidence type (genetic, pharmacologic, functional), cellular context, and degree of mast cell specificity (direct, indirect, unresolved, or not tested). This structure clearly distinguishes mast cell–intrinsic TRPV4 signaling from broader tissue-level TRPV4 activity and clarifies where causal relationships are established versus inferred. The prior detailed disease-specific table has been moved to the Supplementary Materials to preserve experimental depth while allowing the main text to emphasize evidence hierarchy and mechanistic strength.

Finally, we have refined the therapeutic discussion to provide a more balanced and context-dependent assessment. The revised section differentiates mast cell–intrinsic TRPV4 signaling from multicellular TRPV4 activity and explicitly addresses TRPV4’s pleiotropic physiological roles and feasibility considerations, including systemic versus localized targeting and short-term versus prolonged exposure. Rather than presenting TRPV4–mast cell signaling as a universal therapeutic switch, we now frame it as a context-dependent modulatory network requiring disease-specific validation.

We believe these revisions strengthen mechanistic clarity, improve stratification, and provide a more rigorous and balanced translational perspective