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

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.

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.

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.

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.

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.

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.

Author response:

We sincerely thank the Reviewing Editor (Dr. Florent Ginhoux), Senior Editor (Dr. Satyajit Rath), and both reviewers for their thoughtful and constructive evaluation of our manuscript. We appreciate the recognition that our study provides a valuable observation regarding the TLR7-independent effects of imiquimod (IMQ) via the unfolded protein response (UPR) and Gelsolin in psoriasis-like dermatitis. Importantly, we acknowledge that the current framing may overemphasize direct relevance to human psoriasis. In the revised manuscript, we will reposition the study to focus on IMQ-induced skin inflammation as a model of chemical- and stress-induced inflammatory responses, rather than a direct representation of human plaque psoriasis. We also acknowledge that the mechanistic link between Gelsolin and skin inflammation remains incomplete, and we are committed to addressing the key concerns raised.

Below, we outline our planned revisions in response to the public reviews. We will submit a revised version after performing the additional experiments and textual improvements.

Reviewer #1 (Public review):

We fully agree that the exclusive use of the IMQ model has limitations in fully recapitulating human plaque psoriasis, which is primarily driven by the IL-23/IL-17 axis involving Th17/Tc17 cells. We will substantially temper our claims regarding direct translational relevance to human psoriasis and clearly discuss the IMQ model as a tool to study innate immune-driven and chemical stress-induced inflammation in the skin (new Discussion section). In addition, we will strengthen the rationale for focusing on Gelsolin by incorporating available human data suggesting altered Gelsolin expression in inflammatory conditions.

(1) We will add a dedicated paragraph in the Introduction and Discussion acknowledging the differences between IMQ-induced dermatitis and human psoriasis (citing key references such as PMID: 28945199).

(2) For keratinocyte experiments, we will revise the text to avoid implying that keratinocytes stimulated with IMQ represent a psoriasis model, and instead position this system more conservatively. Specifically, we will treat keratinocytes as a system to assess AMP and chemokine induction rather than as a direct model of psoriasis. We will therefore incorporate stimulation with IL-17A (100 ng/ml) ± TNF-α (10 ng/ml) to establish AMP/chemokine induction, and additionally examine the effect of UPR activation by co-treatment with DTT (or other UPR inducers). This will allow us to determine whether UPR activation enhances IL-17A/TNF-α-driven AMP and chemokine expression.

(3) We will expand the Methods section with full details on RNA-seq dataset selection, normalization, cross-species mapping, and statistical analysis, and re-evaluate key analyses where necessary to ensure robustness and reproducibility. Canonical psoriasis signature genes (e.g., S100A8/A9, IL-17C, IL-36g) will be validated by qRT-PCR in the revised manuscript.

(4) Vehicle controls (including Aldara-specific effects) will be clearly described and shown in all relevant figures.

Reviewer #2 (Public review):

We thank the reviewer for recognizing the strengths in demonstrating TLR7-independent UPR induction and Gelsolin as an IMQ-binding protein.

(1) To strengthen the mitochondrial Ca²⁺ signaling data (Fig. 1B), we will add an orthogonal approach (e.g., pharmacological inhibition or alternative Ca²⁺ probe) in a new supplementary figure.

(2) For Gelsolin-IMQ interaction specificity (Fig. 7E-G), we will perform additional experiments comparing IMQ versus RSQ (resiquimod) effects on the observed phenotypes, as recommended.

We believe these revisions will substantially address the key concerns raised by the reviewers and strengthen the overall quality of the manuscript.

We again thank the reviewers and editors for their time and valuable feedback, which will significantly improve the manuscript.