Gelsolin Counteracts ER Stress-Driven Inflammatory Circuits in Psoriasis-like Dermatitis

Reviewer #2 (Public review):

Summary:

This paper shows that imiquimod, a compound often used to induce a psoriasis-like skin inflammation in mice, has a TLR7-independent effects that induce the unfolded protein response and amplify cytokine expression in dendritic cells. Although these effects of imiquimod have been described in the literature before, this study provides more detailed evidence and different contexts to this observation. These findings add to existing literature that imiquimod has a pleotropic mechanism of action involving changes in mitochondrial functions and cellular stress responses. Specifically, the authors show that imiquimod can induce calcium signaling in immune cells and potentiate two branches of the unfolded protein response in a TLR7-independent and MyD88-independent manner. They also show that some of these effects might be partially mediated by direct binding of imiquimod to Gelsolin. These findings expand our understanding of imiquimod-mediated inflammation and are useful for the field of experimental skin immunology and mouse models of psoriasis. However, molecular and cellular mechanisms connecting Gelsolin to the unfolded protein response and skin inflammation presented in this paper requires further investigation in the context of TLR-mediated inflammation.

Strengths:

(1) TLR7-independent effects of imiquimod to the expression of genes and proteins involved into the unfolded protein response are well demonstrated.

(2) Gelsolin is identified as a new imiquimod-binding protein in mouse cells.

Weaknesses:

(1) Effects of imiquimod on mitochondrial Ca signaling are not clear form the presented data.

(2) The mechanism of action connecting imiquimod to Gelsolin on the unfolded protein response and cytokine production remains not fully explained.

(3) It remains unclear if Gelsolin contributes to regulating TLR7 (or other types of TLR-mediated) inflammation in vivo.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

The study is technically extensive and employs a wide range of experimental approaches, including in vivo analyses, cell-based assays, and transcriptomic data integration. The authors provide a detailed characterization of inflammatory and stress-related pathways activated following IMQ exposure in mouse skin. These datasets may be informative for researchers specifically interested in IMQ-induced dermatitis or in stress responses triggered by chemical skin irritants.

We sincerely thank the reviewer for this positive assessment of our study. We appreciate the recognition of the breadth of our experimental approaches, including the integration of in vivo analyses, cell-based experiments, and transcriptomic analyses, as well as the acknowledgement that our findings provide useful insights into IMQ-induced dermatitis and stress responses triggered by chemical skin irritants. We have carefully considered the reviewer's comments and have substantially revised the manuscript to address the concerns regarding the interpretation and disease relevance of our findings. Detailed responses to the specific points raised are provided below.

Strengths:

The study is technically extensive and employs a wide range of experimental approaches, including in vivo analyses, cell-based assays, and transcriptomic data integration. The authors provide a detailed characterization of inflammatory and stress-related pathways activated following IMQ exposure in mouse skin. These datasets may be informative for researchers specifically interested in IMQ-induced dermatitis or in stress responses triggered by chemical skin irritants.

We sincerely thank the reviewer for this positive assessment and for recognizing the breadth of our experimental approaches and the potential value of our findings for researchers studying IMQ-induced dermatitis and stress responses.

Weaknesses:

A major limitation of the manuscript is its exclusive reliance on the IMQ model, which does not adequately represent the immunological drivers, cellular interactions, or therapeutic responsiveness of human psoriasis, despite the manuscript's framing. IMQ-induced inflammation is dominated by innate immune activation and mouse-specific pathways, whereas human psoriasis is driven primarily by IL-23/IL-17-mediated interactions between keratinocytes and Th17/Tc17 cells. As a result, conclusions drawn entirely from IMQ-based experiments have limited relevance to human disease biology.

Consistent with this issue, the manuscript places strong emphasis on pathways such as TLR signaling, inflammasome activation, and IL-1-associated responses, none of which are established as central drivers of plaque psoriasis in patients. Therapeutic strategies targeting these pathways have failed to achieve clinical efficacy comparable to IL-23 or IL-17 blockade, yet this translational gap is not adequately addressed.

The in vitro keratinocyte experiments further limit interpretability. Stimulation of keratinocytes with IMQ is not an accepted model of psoriasis-relevant keratinocyte activation, and the study does not demonstrate induction of well-established psoriasis signature gene programs. Without this validation, it is difficult to assess the relevance of the observed cellular stress responses to human disease.

The RNA-sequencing analyses raise additional concerns regarding rationale and interpretation. The basis for selecting specific mouse and human datasets is unclear, including the use of unpublished or non-psoriasis inflammatory datasets. Key methodological details related to data processing, normalization, cross-species comparison, and statistical analysis are insufficiently described. In addition, the limited number of differentially expressed genes identified does not align with the extensive psoriasis transcriptomic literature, raising concerns about analytical rigor.

Finally, the manuscript emphasizes a small number of genes described as "psoriasis-associated" while failing to demonstrate regulation of widely accepted psoriasis signature genes known to correlate with disease activity and therapeutic response in patients.

We thank the reviewer for this thoughtful and comprehensive assessment of our study. We carefully considered each of the concerns raised regarding the interpretation and translational relevance of our findings and have substantially revised the manuscript accordingly.

Specifically, we have tempered our interpretation throughout the manuscript to clearly distinguish IMQ-induced dermatitis from human plaque psoriasis and to emphasize that our conclusions should be interpreted primarily within the context of the IMQ-induced inflammation model. To improve the relevance of our findings to psoriasis biology, we performed additional experiments using IL-17A- and TNF-α-stimulated primary keratinocytes and incorporated these data into the revised manuscript. We also expanded the description of our RNA-seq analyses, including dataset selection, analytical procedures, and cross-species comparison, and clarified the rationale for selecting representative UPR-responsive genes for validation. In addition, we revised the Introduction and Discussion to more appropriately position the roles of inflammasome signaling, IL-1-associated pathways, and Gelsolin in the context of IMQ-induced inflammation and human psoriasis.

We believe that these revisions substantially strengthen the manuscript and address the reviewer's major concerns. Detailed responses to each specific point are provided below.

Reviewer #2 (Public review):

Summary:

This paper shows that imiquimod, a compound often used to induce a psoriasis-like skin inflammation in mice, has a TLR7-independent effects that induce the unfolded protein response and amplify cytokine expression in dendritic cells. Although these effects of imiquimod have been described in the literature before, this study provides more detailed evidence and different contexts to this observation. These findings add to existing literature that imiquimod has a pleotropic mechanism of action involving changes in mitochondrial functions and cellular stress responses. Specifically, the authors show that imiquimod can induce calcium signaling in immune cells and potentiate two branches of the unfolded protein response in a TLR7-independent and MyD88-independent manner. They also show that some of these effects might be partially mediated by direct binding of imiquimod to Gelsolin. These findings expand our understanding of imiquimod-mediated inflammation and are useful for the field of experimental skin immunology and mouse models of psoriasis. However, the molecular and cellular mechanisms connecting Gelsolin to the unfolded protein response and skin inflammation presented in this paper require further investigation in the context of TLR-mediated inflammation.

We sincerely thank the reviewer for this thoughtful and balanced assessment of our study. We greatly appreciate the recognition that our work provides additional mechanistic insight into the TLR7-independent actions of IMQ and expands current understanding of IMQ-induced inflammation, mitochondrial dysfunction, and unfolded protein response signaling. We also appreciate the recognition that our findings may be valuable for the fields of experimental skin immunology and mouse models of psoriasis. In response to the reviewer's comments, we have further revised the manuscript to clarify the rationale for investigating Gelsolin, to better distinguish IMQ-induced inflammation from human psoriasis, and to more appropriately discuss the limitations of our mechanistic interpretation. Detailed responses to the specific points raised are provided below.

Strengths:

(1) TLR7-independent effects of imiquimod on the expression of genes and proteins involved in the unfolded protein response are well demonstrated.

(2) Gelsolin is identified as a new imiquimod-binding protein in mouse cells.

We sincerely thank the reviewer for these positive comments. We greatly appreciate the recognition that our study provides clear evidence for the TLR7-independent effects of IMQ on unfolded protein response signaling and identifies Gelsolin as a novel IMQ-binding protein. We are grateful for the reviewer's positive assessment of these findings.

Weaknesses:

(1) Effects of imiquimod on mitochondrial Ca signaling are not clear from the presented data.

(2) The mechanism of action connecting imiquimod to Gelsolin on the unfolded protein response and cytokine production remains not fully explained.

(3) It remains unclear if Gelsolin contributes to regulating TLR7 (or other types of TLR-mediated) inflammation in vivo.

We thank the reviewer for these thoughtful comments. We have carefully considered each of the concerns raised regarding the interpretation of mitochondrial Ca²⁺ signaling, the mechanistic link between Gelsolin and UPR signaling, and the role of Gelsolin in TLR-mediated inflammatory responses. In the revised manuscript, we have clarified the interpretation of our mitochondrial Ca²⁺ data, strengthened the discussion regarding the mechanistic role of Gelsolin based on the additional experiments performed in this revision, and more clearly defined the scope of our conclusions regarding Gelsolin in IMQ-induced inflammation. We believe these revisions substantially strengthen the manuscript while avoiding conclusions that extend beyond the experimental evidence. Detailed responses to each point are provided below.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The following points outline specific issues that would need to be addressed to substantially strengthen the manuscript. Several of these represent fundamental limitations of the current study design and interpretation.

We thank the reviewer for this overall assessment. We have carefully addressed each of the points raised and have revised the manuscript accordingly. Detailed point-by-point responses are provided below.

(1) Model limitations

- The manuscript relies exclusively on imiquimod (IMQ), which does not model the core immune drivers or treatment responsiveness of human psoriasis.

- Claims regarding relevance to human psoriasis should therefore be substantially tempered, or alternative models more reflective of human disease biology should be incorporated.

We thank the reviewer for this important comment. We agree that the IMQ-induced dermatitis model does not fully recapitulate the immunopathology, chronicity, or therapeutic responsiveness of human plaque psoriasis. Accordingly, we have substantially tempered our interpretation throughout the manuscript. In the revised Introduction and Discussion, we explicitly state that our findings should be interpreted primarily in the context of IMQ-induced skin inflammation rather than human psoriasis, and that further validation in human psoriasis-relevant systems will be required. We have also revised statements throughout the manuscript to avoid overgeneralization regarding human psoriasis. In addition, to improve the disease relevance of our mechanistic findings, we have performed additional keratinocyte experiments using IL-17A and TNF-α stimulation, which better reflect the canonical inflammatory pathways implicated in human psoriasis. Together with the revised interpretation throughout the manuscript, we believe these changes appropriately position our findings within the context of the IMQ-induced dermatitis model while improving their relevance to human psoriasis.

Changes made in the manuscript: See Figure S6

- The Introduction and Discussion were revised to clarify the limitations of the IMQ-induced dermatitis model and to distinguish IMQ-driven inflammation from human plaque psoriasis.

- Statements implying direct relevance to human psoriasis were revised to more appropriately reflect the scope of the study.

- Additional experiments using IL-17A and TNF-α-stimulated keratinocytes were incorporated to strengthen the relevance of the findings to established psoriasis biology.

(2) Immune context

- gdT cells should be removed from the Introduction as contributors to IL-17A production in human psoriasis, as this is a feature of mouse models rather than human disease (PMID: 28945199).

- The roles of Th17 and Tc17 cells should be emphasized, with appropriate acknowledgment that IMQ-driven inflammation differs substantially from human psoriasis immunopathology.

We thank the reviewer for this valuable comment. We agree that gdT cells are major IL-17A-producing cells in the IMQ-induced mouse model but are not considered the principal source of IL-17A in human plaque psoriasis. Accordingly, we have revised the Introduction to remove statements implying that gdT cells are major contributors to IL-17A production in human psoriasis and have instead emphasized the established roles of Th17 and Tc17 cells. In addition, we have revised the Discussion to explicitly acknowledge that the immunopathology of the IMQ-induced dermatitis model differs substantially from that of human psoriasis and that our findings should therefore be interpreted within the context of IMQ-induced skin inflammation.

Changes made in the manuscript:

- Statements describing gdT cells as major IL-17A-producing cells in human psoriasis were removed from the Introduction.

- The roles of Th17 and Tc17 cells in human psoriasis were emphasized.

- Additional text was added to the Discussion clarifying the immunological differences between the IMQ-induced dermatitis model and human plaque psoriasis.

(3) Keratinocyte stimulation

- IMQ stimulation of keratinocytes is not an accepted model of psoriasis-relevant keratinocyte activation.

- To support disease relevance, experiments should incorporate stimulation with IL-17A (100 ng/ml) {plus minus} TNF-a (10 ng/ml) and appropriate time points consistent with established psoriasis biology.

We thank the reviewer for this constructive suggestion. We agree that stimulation of keratinocytes with IL-17A and TNF-α more closely reflects cytokine-mediated keratinocyte activation in human psoriasis than stimulation with IMQ alone.

We would like to clarify that our IMQ stimulation experiments were not intended to serve as a model of psoriasis-associated keratinocyte activation. Rather, these experiments were designed to investigate the molecular mechanisms by which IMQ directly induces UPR signaling in keratinocytes, as IMQ is the primary experimental stimulus used throughout this study.

To address the reviewer's concern regarding disease relevance, we performed additional experiments using primary keratinocytes stimulated with IL-17A and TNF-α. As expected, IL-17A and TNF-α robustly induced canonical psoriasis-associated genes, including S100a8, S100a9, and Defb14, thereby providing complementary evidence supporting the relevance of our findings to psoriasis-associated inflammatory signaling. These new data have been incorporated as Figure S6.

We believe that these additional experiments appropriately complement our mechanistic studies by linking the identified pathways to established psoriasis-associated inflammatory signaling while preserving the original objective of the IMQ stimulation experiments.

Changes made in the manuscript: See Figure S6

- New experiments using IL-17A and TNF-a-stimulated primary keratinocytes were performed.

- A time-course analysis was conducted to evaluate cytokine-induced gene expression.

- The new data have been included as Figure S6.

(4) Inflammasome and IL-1-related claims

- The manuscript should acknowledge that IL-1 and inflammasome targeting have failed to demonstrate clinical efficacy in plaque psoriasis.

- Observations in inflammasome-, MyD88-, or TLR-deficient mice should not be interpreted as evidence of underexplored human psoriasis pathways, but rather as reflecting IMQ-specific biology.

We thank the reviewer for this important comment. We agree that the contribution of IL-1 signaling and inflammasome activation to IMQ-induced skin inflammation should not be directly extrapolated to human plaque psoriasis. Accordingly, we have revised the Discussion to acknowledge that clinical studies targeting IL-1 signaling have not demonstrated consistent therapeutic efficacy in plaque psoriasis. We have also tempered our interpretation of findings obtained from inflammasome-, MyD88-, and TLR-deficient mouse models, emphasizing that these observations likely reflect mechanisms specific to the IMQ-induced dermatitis model rather than underexplored pathways in human psoriasis. Throughout the revised manuscript, statements implying direct relevance to human psoriasis have been modified accordingly.

Changes made in the manuscript:

- The Introduction and Discussion now acknowledge the limited clinical efficacy of IL-1-targeted therapies in plaque psoriasis.

- Statements regarding inflammasome-, MyD88-, and TLR-dependent mechanisms have been revised to clarify that they primarily reflect IMQ-specific biology.

- Interpretations linking these pathways directly to human psoriasis have been moderated throughout the manuscript.

(5) Cathelicidin and defensin biology

- Statements suggesting that LL37 or b-defensins "exacerbate psoriasis" should be revised to reflect their contributory roles in disease pathogenesis rather than disease worsening.

- It should be acknowledged that CRAMP- or defensin-deficient mice do not show improvement in IMQ-induced dermatitis, consistent with IMQ bypassing these pathways.

We thank the reviewer for this valuable comment. We agree that LL37 and b-defensins should not be described as factors that directly exacerbate psoriasis, but rather as multifunctional mediators that contribute to disease pathogenesis in a context-dependent manner. Accordingly, we have revised the relevant statements throughout the manuscript to more accurately reflect their proposed roles.

In addition, we have expanded the Discussion to acknowledge previous studies demonstrating that CRAMP- or b-defensin-deficient mice do not exhibit substantial improvement in IMQ-induced dermatitis. We now explicitly state that IMQ-driven inflammation can proceed independently of these pathways, highlighting the distinction between mechanisms contributing to human psoriasis pathogenesis and those operating in the IMQ-induced dermatitis model.

Changes made in the manuscript:

- Statements describing LL37 or b-defensins as exacerbating psoriasis were revised to indicate that they contribute to disease pathogenesis.

- The Discussion was expanded to acknowledge that CRAMP- or b-defensin deficiency does not substantially attenuate IMQ-induced dermatitis, consistent with the IMQ model bypassing these pathways.

- The limitations of extrapolating these observations to human psoriasis were clarified.

(6) Gelsolin and IMQ-binding experiments

- Experiments focused on proteins that bind IMQ have limited translational relevance.

- If gelsolin is to be emphasized, the rationale should be grounded in human psoriasis data demonstrating altered gelsolin levels rather than IMQ binding (PMIDs: 36902587, 32002760).

We thank the reviewer for this important comment. We agree that the translational relevance of proteins identified solely through their ability to bind IMQ is limited. Accordingly, we have revised the manuscript to clarify that our rationale for focusing on Gelsolin is supported not only by its interaction with IMQ but also by independent evidence demonstrating altered Gelsolin expression in patients with psoriasis and psoriatic arthritis. We have incorporated these human studies into the Introduction and Discussion and have revised the relevant text to emphasize the potential clinical relevance of Gelsolin beyond its interaction with IMQ.

Changes made in the manuscript:

- Statements emphasizing IMQ binding as the primary rationale for studying Gelsolin were revised.

- Additional references reporting altered Gelsolin expression in patients with psoriasis and psoriatic arthritis were incorporated.

- The Discussion now clarifies that the rationale for investigating Gelsolin is supported by both its interaction with IMQ and independent observations from human disease.

(7) Vehicle controls

- Appropriate vehicle controls for IMQ treatment (Aldara alone) should be included and clearly described, particularly given the known inflammasome-activating properties of isostearic acid (PMID: 23463003).

- The use of DMSO or ethanol as vehicle controls is not justified in this context.

We thank the reviewer for this important comment. We agree that inclusion of an appropriate vehicle control for Aldara cream would have strengthened the interpretation of the in vivo experiments. Unfortunately, the vehicle formulation of Aldara was not commercially available to us and therefore could not be included in the present study.

Because the in vivo experiment assessing mtDNA accumulation in ear tissue following topical IMQ cream application could not be appropriately controlled, we have removed these data from the revised manuscript. We believe this is the most appropriate approach to avoid overinterpretation of results that may potentially be influenced by the cream formulation.

Importantly, our in vitro experiments were performed using purified IMQ rather than IMQ cream. Thus, our conclusion that IMQ induces mtDNA release is supported by experiments performed with purified IMQ, independent of the topical cream formulation.

Changes made in the manuscript: See Figure 4

- The in vivo experiment assessing mtDNA accumulation in ear tissue following topical IMQ cream application has been removed from the revised manuscript.

- The Results and corresponding figure have been revised accordingly.

- The Discussion has been updated to acknowledge the limitation associated with the absence of an Aldara vehicle control.

(8) RNA-sequencing and bioinformatics

- The rationale for selecting specific mouse and human RNA-sequencing datasets should be clearly articulated.

- All datasets analyzed should be explicitly identified, with clarification of their relevance and publication status.

- Methods describing normalization, differential expression analysis, false-discovery correction, and cross-species gene mapping should be expanded to ensure transparency and reproducibility.

We thank the reviewer for this constructive comment. We have substantially expanded the description of the RNA-sequencing and bioinformatics analyses to improve transparency and reproducibility.

First, we now explicitly describe the rationale for selecting the public RNA-seq datasets. GSE289485 was selected because it contains transcriptomic profiles from an IMQ-induced mouse model of psoriasis-like dermatitis, whereas GSE117405 contains transcriptomic data from human psoriasis lesions and healthy control skin, enabling comparison between the mouse model and human disease. We also clarify that both datasets were publicly available at the time of analysis.

Second, detailed information regarding all GEO datasets analyzed, including accession numbers, sample descriptions, publication status, and the specific samples included in the present study, has been provided in Supplementary Table 14.

Finally, the bioinformatics workflow has been described in substantially greater detail. The revised Methods now include descriptions of raw data retrieval from the SRA, quality trimming, sequence alignment, rRNA read removal, read counting, normalization, differential expression analysis, multiple-testing correction, and the orthology-based cross-species comparison used to identify conserved transcriptional responses between mouse and human datasets.

Changes made in the manuscript: See Table S14

- The rationale for selecting GSE289485 and GSE117405 was added.

- Detailed information for all analyzed datasets was summarized in Supplementary Table 14.

- The RNA-seq Methods were expanded to describe quality control, alignment, read counting, normalization, differential expression analysis, false-discovery correction, and orthology-based cross-species comparison.

(9) Gene selection and validation

- Validation should include canonical psoriasis signature genes known to correlate with disease activity and therapeutic response (e.g., S100A8/A9, IL-17C, IL-36g, IL-23, TNF; see PMID: 21085185).

- The current emphasis on Ccl20, Nr4a3, and Defb14 does not adequately capture established psoriasis biology and should be better justified.

We thank the reviewer for this valuable suggestion. We agree that validation using canonical psoriasis-associated genes strengthens the disease relevance of our findings. Accordingly, we performed additional experiments using primary keratinocytes stimulated with IL-17A and TNF-α and evaluated the expression of established psoriasis signature genes, including S100a8 and S100a9. As expected, both genes were robustly induced by IL-17A and TNF- a stimulation. In addition, Defb14, which was also induced by IMQ stimulation, was similarly upregulated by IL-17A and TNF-α. These results further support the relevance of our findings to psoriasis-associated inflammatory signaling.

We have also clarified the rationale for selecting Ccl20, Nr4a3, and Defb14 for further validation. These genes were not intended to represent canonical psoriasis biomarkers but were selected because our transcriptomic analyses identified them as representative UPR-responsive genes that were strongly induced by IMQ stimulation. Accordingly, they were used to investigate the mechanistic contribution of UPR signaling rather than to represent the overall psoriasis transcriptional signature. This rationale has now been clarified in the revised Results section.

Changes made in the manuscript: See Figure S6, and S7

- Additional validation experiments were performed using IL-17A/TNF-a-stimulated keratinocytes.

- The rationale for selecting Ccl20, Nr4a3, and Defb14 was clarified in the Results section.

(10) Histology and data presentation

- Ear skin sampling procedures should be standardized and clearly described.

- All relevant control groups should be shown histologically.

- Figures require clearer labeling, consistent terminology (e.g., ear skin vs. ear lobe), appropriate white balancing, and improved quantitative presentation (e.g., dot plots where appropriate).

- qRT-PCR data should be presented using clearly defined raw or normalized values rather than scaled or ambiguous controls.

We thank the reviewer for these helpful suggestions. We have revised the manuscript to improve the description of histological procedures and the presentation of histological and quantitative data.

First, the Methods section has been expanded to clearly describe the ear skin sampling procedure. We now specify that approximately the distal 2 mm of the ear was collected and processed for histological analysis, thereby ensuring consistent sampling among all animals.

Second, we carefully reviewed the histological figures and confirmed that the relevant control groups are included in the revised manuscript.

Third, figure labeling and terminology have been standardized throughout the manuscript. In addition, quantitative histological data are now presented with individual data points overlaid on the graphs where appropriate, thereby improving data visualization and transparency. The representative histological images are intended to illustrate epidermal thickening, whereas the conclusions are based on quantitative measurements rather than qualitative assessment of image appearance.

Finally, the presentation of the RT-qPCR data has been clarified. The Y-axis labels now explicitly indicate the reference condition used for normalization, and the Methods section has been expanded to describe the DDCt method, including the reference genes used for normalization.

Changes made in the manuscript: See Figure 4D, and 5C

- The ear skin sampling procedure was described in greater detail in the Methods.

- Histological figures and figure labels were reviewed and terminology was standardized throughout the manuscript.

- Individual data points were added to quantitative graphs where appropriate.

- The RT-qPCR y-axis labels were revised to clearly indicate the normalization reference, and the DDCt analysis was described in the Methods.

Reviewer #2 (Recommendations for the authors):

(1) The effects of IMQ in Figure 1B are very small. I recommend adding an orthogonal approach to support that mitochondrial Ca is involved.

We sincerely thank the reviewer for this thoughtful and constructive comment. We agree that the increase in the Rhod-2 signal observed following IMQ stimulation is quantitatively modest. However, we respectfully believe that the biological significance of this observation should not be judged solely by the magnitude of the fluorescence change. Rather, the increase in mitochondrial Ca²⁺ should be interpreted in the context of the multiple independent functional assays presented throughout Figure 1, all of which consistently support a role for mitochondrial Ca²⁺ in IMQ-induced inflammatory responses.

First, we demonstrated that IMQ stimulation significantly increased intracellular ROS production, as assessed by CellROX staining (Figure 1C), and that this increase was significantly suppressed by pretreatment with 2-APB, suggesting that Ca²⁺ dysregulation functionally contributes to ROS generation. In addition, 2-APB markedly reduced IL-1b secretion (Figure 1D), further supporting a functional link between Ca²⁺ signaling and inflammasome activation.

Second, IMQ stimulation induced mitochondrial dysfunction, as demonstrated by JC-1 staining (Figure 1G), and significantly increased mitochondrial ROS production, as measured by MitoSOX (Figure 1H). These findings indicate that IMQ triggers a cascade of mitochondrial events extending beyond the modest increase in mitochondrial Ca²⁺, ultimately resulting in oxidative stress and mitochondrial damage.

Furthermore, treatment with the mitochondria-targeted antioxidant MitoQ, as well as the antioxidants GSHEE, NAC, and PDTC, significantly suppressed IMQ-induced IL-1b secretion (Figures 1I and 1K), supporting the conclusion that mitochondrial ROS is a critical downstream mediator of IMQ-induced inflammatory signaling.

Taken together, these complementary findings provide multiple independent lines of evidence supporting a role for mitochondrial Ca²⁺-associated signaling in IMQ-induced inflammation. Although the increase in Rhod-2 fluorescence was relatively modest, it was consistently accompanied by mitochondrial ROS generation, mitochondrial dysfunction, and inflammasome activation. We therefore believe that the biological significance of mitochondrial Ca²⁺ is best evaluated in the context of these convergent functional outcomes rather than by the magnitude of the Rhod-2 signal alone.

We agree that an independent approach for measuring mitochondrial Ca²⁺ would further strengthen this conclusion. However, because several complementary functional assays consistently support the involvement of mitochondrial Ca²⁺-associated mitochondrial dysfunction in IMQ-induced inflammatory responses, we believe that the current data collectively provide strong evidence for the proposed mechanism.

(2) Figure 7E-G would benefit from testing effects of IMQ versus RSQ on observed phenotypes to confirm that they are mediated by Gelsolin-IMQ interaction but not an effect of Gelsolin on TLR7-mediated inflammation.

We sincerely thank the reviewer for this valuable suggestion. We agree that it is important to determine whether the observed phenotypes reflect a specific interaction between Gelsolin and IMQ rather than a general role of Gelsolin in TLR7-mediated signaling.

In response to this comment, we performed additional experiments using resiquimod (RSQ), a TLR7/8 agonist. Gelsolin-deficient MEFs were stimulated with RSQ, and the same parameters analyzed in the IMQ experiments were evaluated.

Unlike the phenotypes observed following IMQ stimulation, RSQ stimulation did not reveal significant differences between wild-type and Gelsolin-deficient MEFs. Specifically, the Xbp1s/Xbp1 ratio was comparable between the two genotypes following RSQ stimulation. Similarly, mitochondrial ROS production, assessed by MitoSOX staining, and MAM formation, evaluated by proximity ligation assay (PLA), were not significantly altered by Gelsolin deficiency under RSQ stimulation.

Although the IMQ and RSQ experiments were performed independently and were not intended as a direct side-by-side quantitative comparison, the absence of genotype-dependent effects in the RSQ experiments indicates that Gelsolin deficiency alone does not generally enhance TLR7-mediated responses. Instead, these findings support the interpretation that the enhanced UPR activation, mitochondrial ROS production, and MAM formation observed in Gelsolin-deficient cells are specific to IMQ stimulation, consistent with the interaction between Gelsolin and IMQ.

Accordingly, the Results section has been revised to include these additional experiments, and the corresponding data have been incorporated into the revised Figure S11.

Changes made in the manuscript: See Figure S12

- Additional experiments using RSQ-stimulated wild-type and Gelsolin-deficient MEFs were performed.

- The Xbp1s/Xbp1 ratio, mitochondrial ROS production, and MAM formation were evaluated following RSQ stimulation.

- No significant genotype-dependent differences were observed under RSQ stimulation, supporting the specificity of the phenotypes observed following IMQ stimulation.