Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer

Domain: biology
Published: 2025-12-14
Editorial outcome: major_revision

Abstract
Colonic smooth muscle cell (CSMC) hypertrophy and hyperplasia have been described in both human ulcerative colitis (UC) and animal models. The deletion of smooth muscle (SM) 22α induces the phenotypic switching of SMCs. Here, we report that Sm22α-deficient mice develop spontaneous colitis, which is characterized by radical S-adenosyl-methionine domain-containing 2 (RSAD2)-driven mitochondrial dysfunction and inflammation in CSMCs and ferroptosis in the colonic mucosa. Mechanistically, RSAD2 mediates YTH m6A RNA-binding protein 1 (YTHDF1) methylation and activation, thereby increasing the mRNA N6-methyladenosine (m6A) modification and translation of dynamin-related protein 1 (DRP1), resulting in mitochondrial fragmentation in CSMCs. Inflammatory CSMC-derived mitochondrial extracellular vesicles trigger intestinal epithelial ferroptosis by inducing ROS production. The ablation of RSAD2 in mice with SMC-specific Sm22α knockout alleviates colitis severity in this experimental model. Importantly, increases in both RSAD2 expression and the ferroptotic signature are observed in serum and/or colonic samples from UC patients. Overall, this study shows a mitochondrial mechanism underlying the ability of dysfunctional smooth muscle to drive colitis and highlights the potential of targeting the RSAD2-YTHDF1 axis as an innovative therapeutic strategy for colitis.

Introduction
Inflammatory bowel disease (IBD) is a chronic, relapsing, and remitting inflammatory disorder of the gut that includes two main phenotypes, Crohn’s disease (CD) and ulcerative colitis (UC), and significantly disrupts bowel function and patient quality of life1. Known disease risk alleles highlight key pathways in the pathogenesis of IBD, including innate and adaptive immunity, gut barrier function, and pathogen sensing and responses. Epithelial cells, goblet cells, stroma and immune cells in the intestinal mucosa interact with each other or with the intestinal microbiota and the immune system, sending and receiving signals2. Furthermore, an inappropriate and continuing mucosal immune response to commensal microbes in a genetically susceptible host is also involved in intestinal inflammation in the gut and IBD2. However, the underlying genes at risk loci have not been mapped to their cells or pathways of action.

The gastrointestinal tract comprises an epithelium surrounded by layers of smooth muscle cells (SMCs). Fully mature SMCs have a unique ability to reversibly alter their phenotype from a contractile state to a synthetic state to respond to environmental cues3. Smooth muscle (SM) 22α, a marker of mature SMCs, is involved in actin filament assembly and stress fiber formation4, which are required for maintaining the differentiated phenotype of SMCs5,6. Our group and others have demonstrated that disruption of SM22α in vascular SMCs enhances the vascular inflammatory response by inducing the phenotypic switching of SMCs7,8. SMC hypertrophy (enlarged muscle cells) and hyperplasia (increased numbers of muscle fibers) have been described in the inflamed intestine in both human IBD and animal disease models and are being increasingly recognized as the main contributors to increased wall thickness in the fibrotic process of IBD9. Furthermore, aberrant SMC phenotypes, as environmental variables, may contribute to dysregulation of the mucosal immune and commensal ecosystem in the gut, ultimately initiating colitis. However, the importance of the smooth muscle component in IBD has been less investigated. This prompted us to ask whether and how SM22α disruption promotes intestinal inflammatory damage.

Radical S-adenosyl-L-methionine domain-containing 2 (RSAD2) is an interferon-stimulated gene that is renowned for its antiviral properties. RSAD2 is involved in complex interplay with mitochondria, and binding of the iron–sulfur (Fe–S) cluster is necessary for the involvement of RSAD2 in mitochondrial metabolism. RSAD2 influences the proliferation and development of immune cells and inflammation via different signaling pathways10. However, the function of RSAD2 has been shown to vary across studies, and knowledge of this topic is lacking. Although a previous study demonstrated that increasing RSAD2 expression in neutrophils suppressed intestinal inflammation in a mouse model of DSS-induced colitis11,12, very little is known about the crucial role of RSAD2 in the pro-inflammatory phenotypic switching of SMCs.

An increasing number of studies have underscored the new roles of YTH N6-methyladenosine (m6A) RNA-binding protein 1 (YTHDF1) in the gut13. Although YTHDF1 is largely dispensable for intestinal homeostasis under physiological conditions, YTHDF1 is required for intestinal epithelial regeneration and tumorigenesis because it maintains stem cell traits by amplifying Wnt/β-catenin signaling at the translational level14. Ythdf1 mRNA is upregulated in ulcerative colitis (UC) patients15 and downregulated in both LPS-induced macrophages and the colons of IBD model mice, whereas the upregulation of YTHDF1 alleviates the inflammatory response16, suggesting that YTHDF1-mediated RNA methylation events are involved in IBD pathogenesis17. In the current study, we demonstrate that SM22α deletion activates the RSAD2-YTHDF1 axis, leading to mitochondrial fragmentation and a proinflammatory phenotype in colonic SMCs (CSMCs). Mitochondrial extracellular vesicles (MitoEVs) from CSMCs induce intestinal epithelial cell ferroptosis and colitis development.

Results
SM22α deletion leads to spontaneous colitis
We first confirmed that the expression of SM22α was restricted to the muscular layer of the intestinal wall of wild-type (WT) mice (Supplementary Fig. 1a). Three-month-old Sm22α knockout (Sm22α−/−) mice exhibited signs of colitis. Weight loss was evident in these mice (Fig. 1a), which passed paste-like stools with high water contents (Supplementary Fig. 1b–d). Gross and microscopic examinations of Sm22α−/− mice revealed shorter colons (Fig. 1b) and increased histological activity index (HAI) scores, indicating architectural distortion and variation in the size and shape of crypts (Fig. 1c). Moreover, compared with their WT littermates, goblet cell loss, inflammatory cell infiltration and intestinal epithelial apoptosis, which are hallmarks of IBD18, were obvious in Sm22α−/− mice (Fig. 1c–e). Disrupted epithelial hypoxia (Fig. 1f) and increased plasma levels of FITC-conjugated dextran (Fig. 1g) indicated an increase in intestinal barrier permeability in Sm22α−/− mice, which was accompanied by impaired peristaltic contractions (Fig. 1h and Supplementary Fig. 1e, f). At the molecular level, the expression of the proinflammatory cytokines Tnf and Il-1β significantly increased in the colonic tissues of Sm22α-deficient mice, whereas the level of the anti-inflammatory cytokine Il-10 decreased (Fig. 1i), indicating an inflammatory phenotype. In addition, Sm22α−/− mice exhibited decreased proliferation and differentiation of intestinal epithelial cells without evidence of epithelial metaplasia (Fig. 1f and Supplementary Fig. 1g).

To determine the role of CSMCs in colitis development, we generated SMC-specific Sm22α knockout (smSm22α−/−) mice by crossing mice with loxP sites flanking Sm22α (Sm22αfl/fl mice) with SMC-specific Cre-expressing transgenic mice (myh11Cre mice). Similar colitis-related changes, including weight loss (Fig. 1j), shorter colons (Fig. 1k), elevated HAI scores (Fig. 1l), and goblet cell loss (Fig. 1m), were observed in 3-month-old smSm22α−/− mice. Additionally, intestinal permeability and peristaltic contraction were impaired (Fig. 1n, o) in these mice compared with Sm22αfl/fl mice, which was accompanied by increased inflammation (Fig. 1p).

To further investigate whether SM22α deletion in CSMCs plays a unique role in DSS-induced inflammation in mice, we administered DSS to induce colitis. Compared with Sm22αfl/fl control mice, smSm22α−/− mice had significantly shorter survival times (Fig. 2a), lower body weights (Fig. 2b), higher disease activity index (DAI) scores (Fig. 2c), shorter colons (Fig. 2d, e) and more severe tissue damage (Fig. 2f, g) 7d after the administration of DSS. Consistently, higher intestinal permeability and more severe peristaltic dysfunction and inflammation were observed in DSS-treated smSm22α−/− mice than in control mice (Fig. 2h–j). Additionally, we found that DSS impaired the contractility of CSMCs cultured in vitro (Supplementary Fig. 1h, i). Notably, the length of the small intestines shortened and the intestinal transit times of the marker that was orally administered, extended in Sm22α−/− mice compared with WT mice at baseline (Supplementary Fig. 2a-c). The small intestinal wall, especially the ileum, was significantly damaged with higher histological score in Sm22α−/− mice (Supplementary Fig. 2d), suggesting the small intestinal dysfunction. Nevertheless, no difference in the colitis phenotype between male and female Sm22α−/− mice was observed (Supplementary Fig. 2e–j). The rescue of SM22α expression by the administration of an adeno-associated virus 2/9 (AAV) carrying SM22α markedly ameliorated colitis phenotype in Sm22α−/− mice, with less pronounced colon shortening, tissue damage, peristaltic dysfunction and inflammation (Supplementary Fig. 3a–g). Collectively, these results suggest that the loss of SM22α in CSMCs plays a crucial role in colitis development.

SM22α deficiency disrupts mitochondrial homeostasis in CSMCs
To elucidate the molecular mechanisms linking SM22α deficiency to colitis, we performed RNA-seq and microarray analyses on colonic smooth muscle tissues from WT and Sm22α−/− mice. Transcriptomic analysis revealed 2,028 differentially expressed genes (DEGs), including 898 upregulated genes and 1,130 downregulated genes, in Sm22α−/− tissues (Supplementary Fig. 4a), as validated by quantitative PCR (qPCR) (Supplementary Fig. 4b). Gene Ontology (GO) enrichment analysis of the differentially expressed genes revealed that the upregulated genes were significantly associated with immune activation, inflammatory responses, and osteogenic processes. In contrast, the downregulated genes were primarily enriched in negative regulation of ossification, glycolysis, and fatty acid transport, suggesting that the colonic SMCs in Sm22α−/− mice undergo phenotypic switching characterized by proinflammatory and osteoblast-like transdifferentiation (Supplementary Fig. 4c). Notably, mitochondrial material transport, amino acid metabolism, oxidative phosphorylation, and ATP production regulation were significantly suppressed in the CSMCs of Sm22α−/− mice (Fig. 3a).

Our previous study demonstrated that SM22α deficiency impaired mitochondrial structure and function in uterine SMCs, leading to contractility defects and dystocia19. Consistent with these findings, electron microscopy revealed mitochondrial shrinkage, severe cristae loss, and membrane rupture in primary Sm22α−/− CSMCs (Fig. 3b). We next evaluated mitochondrial morphology and discovered that mitochondrial fragmentation was substantially increased in Sm22α−/− cells. The mitochondrial number, mean length, and network branch counts were significantly lower in Sm22α−/− CSMCs than in WT controls (Fig. 3c, d). Moreover, mitochondrial dysfunction was further corroborated by elevated mitochondrial reactive oxygen species (mtROS) levels (Fig. 3c) and reduced mitochondrial DNA (mtDNA) copy numbers and ATP production (Fig. 3e, f). Notably, among mitochondrial fusion and fission markers, only dynamin-related protein 1 (DRP1) was markedly upregulated in Sm22α−/− CSMCs (Fig. 3g, h), which is consistent with a significant increase in mitochondrial fragmentation. Treatment with Mdivi-1, a selective DRP1 inhibitor, mitigated mitochondrial fragmentation in Sm22α−/− CSMCs (Supplementary Fig. 5a, b), suggesting that DRP1 plays an essential role in mitochondrial dysfunction in Sm22α−/− CSMCs.

Our previous research demonstrated that TNF induced the epigenetic silencing of SM22α transcription via H3K27 methylation in the SM22α promoter region in vascular SMCs8 and that the disruption of SM22α further aggravated vascular inflammation20, prompting us to explore whether the synergistic interaction of TNF stimulation and SM22α ablation impaired mitochondrial function in Sm22α−/− CSMCs. In line with our hypothesis, stimulation of CSMCs with TNF induced mitochondrial fragmentation and mtROS production (Supplementary Fig. 6a, b) and led to mitochondrial dysfunction (Supplementary Fig. 6c, d), which were similar to the phenotypic outcomes of Sm22α−/− CSMCs. Furthermore, TNF treatment significantly increased DRP1 protein levels (Supplementary Fig. 6e, f). Notably, TNF treatment exacerbated mitochondrial fragmentation and dysfunction in Sm22α−/− CSMCs (Supplementary Fig. 6g–l). Overall, these data indicate that inflammation signaling contributes to mitochondrial damage through the upregulation of DRP1 in CSMCs.

RSAD2 expression is upregulated during inflammation
To further elucidate the molecular mechanisms underlying mitochondrial dysfunction in Sm22α−/− CSMCs, we compared our RNA-seq data with 1,218 mitochondria-related genes cataloged in the MitoCarta 3.0 database21. This analysis revealed 43 overlapping genes, 9 of which were upregulated and 34 of which were downregulated in Sm22α−/− smooth muscle tissues (Fig. 4a, b). Notably, RSAD2, a free radical S-adenosylmethionine (SAM) enzyme associated with immune, inflammatory, and mitochondrial processes22, was significantly upregulated (Fig. 4b), suggesting its potential role in mediating mitochondrial dysfunction linked to SM22α deletion. Therefore, we examined the expression and distribution of RSAD2 in WT and Sm22α−/− colonic tissues and found that the mRNA and protein levels of RSAD2 were markedly increased throughout the entire intestinal wall (Fig. 4c–g), especially in the smooth muscle layers of Sm22α−/− mice (Fig. 4f). Parallel observations in human samples revealed thickening of the colonic muscularis propria and increased RSAD2 expression in both smooth muscle tissues and serum from patients with UC compared with controls without colitis (Supplementary Tables 1 and 2 and Supplementary Fig. 7a–d). Overall, these results suggest that RSAD2 plays a critical role in colitis pathogenesis.

Next, we extended these findings by performing complementary experiments in CSMCs. Consistent with the in vivo observations, the expression of RSAD2 at both the mRNA and protein levels was significantly elevated (Fig. 4h–j), and RSAD2 colocalized mainly with mitochondria in the cytoplasm of Sm22α−/− CSMCs (Fig. 4k, l). Sm22α knockdown by a specific siRNA increased the expression of the RSAD2 protein (Fig. 4m, n), whereas rescuing Sm22α expression in Sm22α−/− CSMCs abolished the increase in RSAD2 protein expression (Fig. 4o, p). Consistently, TNF treatment significantly increased RSAD2 protein levels in CSMCs (Fig. 4q, r). Together, these results indicated that inflammatory signaling contributes to RSAD2 upregulation.

RSAD2 leads to mitochondrial fragmentation and dysfunction in CSMCs
RSAD2 is an endoplasmic reticulum (ER)-localized IFN-inducible iron-sulfur (Fe–S) cluster-binding antiviral protein22. We next investigated whether RSAD2 is involved in mitochondrial dysfunction in CSMCs. Analysis of mitochondrial morphology revealed that mitochondrial fragmentation was induced by the overexpression of RSAD2 and that the majority of mitochondria were relatively short and punctate (Fig. 5a, b). Furthermore, mtROS levels markedly increased (Fig. 5a), which was accompanied by decreases in mtDNA copy numbers (Fig. 5c) and ATP production (Fig. 5d). Given the significant upregulation of DRP1 in Sm22α−/− CSMCs or upon TNF treatment, we examined the effect of RSAD2 on DRP1 expression. RSAD2 overexpression markedly increased the protein level of DRP1 in CSMCs (Fig. 5e, f), indicating that RSAD2 strongly affects mitochondrial dysfunction.

To further confirm the role of RSAD2 in mitochondrial dysfunction under inflammatory conditions, WT and Rsad2−/− CSMCs were treated with TNF. We showed that RSAD2 deficiency mitigated TNF-induced mitochondrial dysfunction, which was characterized by reduced mitochondrial fragmentation (Fig. 5g, h) and decreased mtROS levels (Fig. 5g). Additionally, compared with those in WT cells, the mtDNA copy number and ATP production in Rsad2−/− CSMCs increased upon TNF treatment (Fig. 5i, j). Consistent with these findings, DRP1 expression was significantly downregulated in Rsad2−/− CSMCs compared with WT controls following TNF treatment (Fig. 5k, l), further indicating that RSAD2 plays a dominant role in DRP1-related mitochondrial dysfunction.

We next investigated whether RSAD2 deficiency plays a unique role in protecting mice against DSS-induced colitis. WT and Rsad2−/− mice were given 2.5% DSS in their drinking water for 7 days. Compared with WT mice, Rsad2−/− mice exhibited significantly less weight loss, lower DAIs, and less colon shortening (Supplementary Fig. 8a–d) after DSS administration, findings that were confirmed by histological analysis (Supplementary Fig. 8e–g). Furthermore, DSS-treated Rsad2−/− mice survived longer (Supplementary Fig. 8h). Overall, our data support the conclusion that RSAD2 plays a crucial role in intestinal inflammation.

RSAD2 facilitates DRP1 translation through m6A deposition
To elucidate the molecular mechanism through which RSAD2 affects DRP1-related mitochondrial dysfunction, we examined the regulatory pathways involved in DRP1 protein expression, including mRNA transcription, stability, and translation, as well as ubiquitination and degradation, in CSMCs from WT and Rsad2−/− mice. We showed that the mRNA level of Drp1 did not change upon TNF treatment (Fig. 6a), and its decay kinetics did not significantly differ between WT and Rsad2−/− CSMCs upon actinomycin D treatment (Fig. 6b), suggesting that RSAD2 does not affect Drp1 mRNA transcription or stability. We then assessed the role of RSAD2 in DRP1 protein stability using ubiquitin immunoprecipitation assays. No significant differences in DRP1 ubiquitination or degradation were detected between WT and Rsad2−/− CSMCs (Fig. 6c), effectively ruling out protein stability as a regulatory mechanism. Taken together, these results indicated that RSAD2 may increase DRP1 expression at the translational level.

The N6-methyladenosine (m6A) RNA modification plays a crucial role in regulating RNA metabolism, including its transport, decay, and translation. Given that RNA methylation activity is associated with RSAD223, we hypothesized that RSAD2 influences Drp1 mRNA translation via m6A deposition. Under inflammatory conditions, global m6A levels were elevated in CSMCs, whereas RSAD2 deficiency significantly attenuated m6A deposition (Fig. 6d). Methylated RNA immunoprecipitation (MeRIP)–qPCR analysis revealed significant enrichment of m6A on Drp1 mRNA in CSMCs following TNF treatment (Fig. 6e). In contrast, Rsad2−/− CSMCs exhibited markedly reduced m6A deposition under the same conditions, suggesting that RSAD2 modulates m6A deposition. However, unexpectedly, we did not detect an interaction between RSAD2 and Drp1 mRNA (Fig. 6f), which excluded the possibility that the enrichment of m6A on Drp1 mRNA was due to RSAD2-associated RNA methylation activity.

Next, we conducted RNA interference (RNAi) targeting key m6A regulatory proteins, including writers (METTL3 and METTL14), erasers (ALKBH5 and FTO), and readers (YTHDF1 and YTHDF3). Among these, knockdown of YTH domain family protein 1 (YTHDF1) significantly reduced m6A enrichment on the mRNA of Drp1 upon TNF treatment, as indicated by MeRIP–qPCR (Fig. 6g, h). m6A was recently shown to mediate the regulation of gene expression at the mRNA level through the direct recruitment of reader proteins to the transcript24. We showed that the expression of the DRP1 protein decreased after YTHDF1 knockdown (Fig. 6i), suggesting that YTHDF1, as a reader, may increase the translation of Drp1 mRNA by recognizing and binding to its m6A modifications.

To verify our speculation, we investigated the binding of YTHDF1 to Drp1 mRNA via RNA immunoprecipitation (RIP) assays with an anti-YTHDF1 antibody. TNF treatment increased the interaction of YTHDF1 with Drp1 mRNA. Drp1 mRNA was strongly enriched in the immunoprecipitated complexes (Fig. 6j). RNA pull-down assays confirmed that YTHDF1 binds to Drp1 mRNA (Fig. 6k), which is consistent with the increase in DRP1 protein expression. Importantly, the binding of YTHDF1 to Drp1 mRNA was markedly suppressed in Rsad2−/− CSMCs (Fig. 6j, k), indicating that YTHDF1 reader activity depends on RSAD2. Together, these data suggest that RSAD2 increases the translation of Drp1 mRNA through YTHDF1-mediated recognition of m6A modification.

RSAD2 mediates YTHDF1 methylation and activation
To determine how RSAD2 modulates YTHDF1 activity, we conducted computational docking analyses using the HDOCK server (Supplementary Fig. 9a). The top-scoring model predicted an interaction between RSAD2 and YTHDF1 (Supplementary Data 1). This interaction was validated by coimmunoprecipitation (CoIP) assays; furthermore, the binding interaction was enhanced following TNF treatment (Fig. 7a–c). Notably, the protein level of YTHDF1 did not change upon TNF treatment or RSAD2 deletion (Fig. 7d).

Posttranslational modifications of readers may also alter their m6A binding affinity, cellular localization and interactions with protein partners25. Although RSAD2 is a radical SAM enzyme, neither the reaction it catalyzes nor its substrate has been identified23. We hypothesized that RSAD2 may mediate YTHDF1 methylation. To validate the physical and functional interaction of RSAD2 with YTHDF1, we performed immunoprecipitation (IP) assays with anti-α-mono- and anti-dimethyllysine antibodies. Western blot analysis revealed that YTHDF1 was indeed methylated in CSMCs (Fig. 7e). Furthermore, TNF treatment increased YTHDF1 methylation, which was abolished by RSAD2 deletion (Fig. 7f). These results have been also validated in vivo. In DSS-induced colitis models, YTHDF1 methylation was increased in smSm22α−/− mice but reduced in Rsad2−/− mice (Fig. 7g, h). Overall, these data indicated that RSAD2 plays a dominant role in YTHDF1 methylation.

To further define the role of RSAD2 in YTHDF1 methylation, we generated a series of RSAD2 truncation mutants, including the SAM domain (GFP-SAM) and a truncated SAM domain (GFP-ΔSAM) (Fig. 7i). Proximity ligation assays (PLAs) in HEK293A cells co-transfected with the HA-YTHDF1 and RSAD2 constructs revealed that both full-length (FL) RSAD2 and its SAM domain interacted with YTHDF1, whereas the SAM domain deletion construct (GFP-ΔSAM) failed to bind (Fig. 7j). CoIP assays further confirmed that the SAM domain mediated the binding of RSAD2 to YTHDF1 (Fig. 7k). Consistently, both full-length RSAD2 and the SAM domain increased YTHDF1 methylation (Fig. 7l). We validated these results in HEK293A cells co-transfected with the HA-YTHDF1 and GFP-SAM constructs, which exhibited strong interactions between the SAM domain and YTHDF1 (Fig. 7m), accompanied by increased YTHDF1 methylation (Fig. 7n). Moreover, overexpression of GFP-SAM markedly increased the effect of YTHDF1 on Drp1 mRNA expression (Fig. 7o), which was accompanied by an increase in DRP1 protein expression in CSMCs (Fig. 7p), indicating that the RSAD2-YTHDF1 axis positively affects DRP1 expression. Together, these results suggest that YTHDF1 is an endogenous substrate of RSAD2.

In addition to recognizing m6A modifications, reader proteins may indirectly regulate m6A deposition. For example, m6A readers can compete with m6A demethylases for binding to m6A sites, resulting in varying degrees of m6A deposition26. As mentioned above, upon TNF treatment, Drp1 mRNA m6A deposition increased in CSMCs but decreased in Rsad2−/− cells (Fig. 6e). The results of the RNA pull-down experiments confirmed that the binding of ALKBH5, an m6A demethylase, to Drp1 mRNA markedly decreased with increased YTHDF1 expression in TNF-treated cells compared with control cells (Supplementary Fig. 10a, b). This decrease was reversed in Rsad2−/− CSMCs, which exhibited increased enrichment of ALKBH5 on Drp1 mRNA, coincident with the reduced m6A levels of Drp1 mRNA, suggesting that YTHDF1 occupancy at the m6A site of Drp1 mRNA suppresses ALKBH5 demethylase activity. Taken together, these findings suggest that RSAD2 promotes the YTHDF1-driven translational upregulation of Drp1 mRNA by mediating YTHDF1 methylation.

Inflammatory CSMC-derived mitochondrial extracellular vesicles induce ferroptosis in intestinal epithelial cells, contributing to colitis pathology
Recent advances have illustrated the functional importance of mitochondrial extracellular vesicle (MitoEV) release in an increasing number of tissue contexts27. Specific mitochondrial stresses or inflammatory signals trigger MitoEV release28. To verify the causal relationship between CSMC dysfunction and colitis, we first identified CD63-marked MitoEVs using immunofluorescence analysis in CSMCs (Supplementary Fig. 11a). We then isolated the MitoEVs from the conditioned medium of the CSMCs using differential centrifugation (Fig. 8a). Western blot analysis confirmed the presence of the mitochondrial outer membrane proteins TOM20 and TOM40 as well as the EV marker CD63 in CSMCs and in their released MitoEVs (Fig. 8b). Using FACS, we revealed that TNF treatment increased MitoEV release from CSMCs, which was significantly reduced in Rsad2−/− cells (Supplementary Fig. 11b). To trace MitoEV-mediated mitochondrial transfer, we first labeled the mitochondria in CSMCs with MitoTracker Deep Red and the mitochondria in intestinal epithelial cells (IECs) with MitoTracker Green. MitoEVs isolated from the conditioned medium of TNF-treated CSMCs were then used to treat IECs (HCT116 cells). Immunofluorescence analysis confirmed that the CSMC-derived MitoEVs were taken up by IECs, a process inhibited by dynasore, a clathrin-mediated endocytosis inhibitor (Fig. 8c), suggesting that intercellular mitochondrial transfer occurred between CSMCs and IECs.

Intercellular mitochondrial transfer not only rescues tissue damage but also contributes to multifunctional cellular activity and the regulation of inflammation29. An elegant study demonstrated that MitoEV-mediated mitochondrial transfer between cells in vivo resulted in increased mitochondrial ROS production in recipient cells30. To examine this possibility, we treated IECs with MitoEVs derived from different cellular contexts, including WT and Rsad2−/− CSMCs with or without TNF treatment. We observed increased lipid peroxidation (Fig. 8d, e) and elevated MDA levels (Fig. 8f) in IECs treated with MitoEVs of TNF-induced CSMCs, accompanied by a decreased GSH/GSSG ratio (Fig. 8g), suggesting increased oxidative stress. Similar results were observed in other colonic epithelial cell lines, including FHC and Caco-2 (Supplementary Fig. 11c, d). In contrast, Rsad2−/− MitoEVs seemed to have a smaller effect on lipid ROS levels (Fig. 8d–g). Together, these results suggested that MitoEVs from inflammatory CSMCs induced lipid peroxidation in the intestinal epithelium.

Lipid peroxidation products are the main drivers of ferroptosis, an iron-dependent form of necrotic cell death31. We showed that TNF-induced MitoEVs increased the levels of total ferrous iron and free Fe2+ in IECs (Fig. 8h–j). Moreover, expression of the ferroptosis marker COX2 significantly increased while GPX4 expression was decreased (Fig. 8k). In contrast, inflammatory Rsad2−/− MitoEVs had a mild effect on ferroptosis in IECs. In vivo, DSS-treated Rsad2−/− mice displayed less epithelial cell ferroptosis than the WT controls did (Fig. 8l, m and Supplementary Fig. 8i–k).

Similar to the findings in IECs, mouse intestinal organoids were shown to internalize CSMC-derived MitoEVs (Supplementary Fig. 12a, b). Exposure to inflammatory MitoEVs resulted in reduced expression of the barrier-associated protein Claudin-1 in colonic organoids, which was accompanied by elevated oxidative stress and the pronounced downregulation of the ferroptosis marker protein GPX4 and the upregulation of COX2 (Supplementary Fig. 12c–e). In contrast, inflammatory Rsad2−/− MitoEVs failed to induce substantial changes in ROS levels or ferroptosis markers in intestinal organoids (Supplementary Fig. 12c–e).

Given that RSAD2 expression is increased in Sm22α−/− mice with colitis, we investigated whether ferroptosis occurs in the intestinal mucosa in vivo. Western blot analysis revealed significant upregulation of COX2, which was accompanied by the downregulation of GPX4 (Supplementary Fig. 13a, b). Immunofluorescence analysis corroborated the increase in COX2 expression and the reduction in GPX4 expression (Supplementary Fig. 13c). Moreover, ferrous iron and MDA assays confirmed the occurrence of ferroptosis in the intestinal mucosa of Sm22α−/− mice (Supplementary Fig. 13d, e). Intriguingly, genetic deletion of RSAD2 in SMC-specific Sm22α knockout mice alleviated colitis severity (Supplementary Fig. 13f–h) and reduced epithelial ferroptosis (Supplementary Fig. 13i–m). Importantly, compared with those from healthy controls, the colonic mucosa from patients with UC exhibited a similar ferroptotic signature, with COX2 upregulation and GPX4 downregulation (Supplementary Fig. 7e). Together, these results further demonstrated that CSMC dysfunction caused intestinal epithelial damage.

Methods
Study approval
Approval for all the experimental procedures was granted by the Institutional Laboratory Animal Care and Use Committee of Hebei Medical University, Permit No. IACUC-Hebmu-P2023006.

Human samples
The study involving humans received approval from the Medical Ethics Committee of Hebei Medical University, and informed consent was obtained from all participants (2023-001). Between May 2023 and May 2024, colonic tissue and blood samples were collected via endoscopic biopsies at the Second Hospital of Hebei Medical University. UC was diagnosed by colonoscopy and pathological examination of colon tissue samples. This research was conducted with the voluntary participation of individuals who were not offered any compensation. Sex or gender information was not collected, and no sex or gender analysis was carried out.

Animals
All animal studies were carried out in accordance with the guidelines sanctioned by the Ethics Committee for Animal Experiments and the Institutional Animal Care and Use Committee at Hebei Medical University. The experimental procedures followed the guidelines set by Directive 2010/63/EU of the European Parliament regarding the protection of animals used in scientific research. Cyagen Biosciences (S-KO-11081) provided C57BL/6J wild-type (WT) mice and Rsad2−/− mice. Sm22α−/− mice were acquired from the Jackson Laboratory (006878), and Myh11-cre/ERT2 mice were obtained from Youdu Bio (YDS0815). All the mice were kept in a pathogen-free facility under controlled conditions, including a temperature ranging from 21 to 23 °C, a humidity ranging from 50 to 60%, and a 12-h light/dark cycle. Mice aged 12–16 weeks were used for the experiments. Tamoxifen was administered intraperitoneally (i.p.) at 100 mg kg−1 in corn oil, either as a single dose or every other day for 14 days, starting at 5–8 weeks of age.

Culture and treatment of CSMCs
After being isolated from the colonic muscular layer of young mice, the CSMCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 20% fetal bovine serum (FBS; Gibco) and 1% penicillin‒streptomycin (PS; Biological Industries). The cells were incubated in a humidified environment at 37 °C with 5% CO2. Only cells from passages 4 through 10 were used in the experiments. The cells were serum-starved by incubation in 0.5% FBS for 24 h before being stimulated with TNF (10 ng mL−1) and transfected with siRNA or plasmids.

Culture and treatment of intestinal organoids
Organoid cultures and crypt cells were isolated using methods detailed in earlier studies56. The procedures involved dissecting the colon lengthwise, washing it with cold DPBS, and incubating the crypts in Gentle Cell Dissociation Reagent (STEMCELL Technologies) at room temperature on a rocking platform at 20 rpm for 20 min. A 70-μm cell strainer was used to filter the crypt suspension, and the crypt fraction was collected by centrifugation at 290 × g for 5 minutes at 4 °C. The crypt pellet were then embedded in Matrigel (BD Biosciences) on ice and subsequently plated in 24-well plates. Once Matrigel polymerization was complete, 750 μL of complete IntestiCult Organoid Growth Medium (STEMCELL Technologies) was added to each well at room temperature. The culture medium was changed every 2 days. The mitoEVs from the CSMCs were used to treat the organoids for 48 h prior to additional experiments.

Methylated RNA immunoprecipitation (MeRIP) assay
In accordance with the manufacturer’s protocol, the m6A modification level of the Drp1 mRNA was determined using an EpiQuik CUT&RUN m6A RNA Enrichment Kit (P-9018; Epigentek). Briefly, m6A-containing RNA fragments were selectively isolated using beads conjugated with an anti-m6A capture antibody. The alterations in m6A methylation of Drp1 mRNA were quantified by qPCR.

Plasmid or siRNA transfection
The cultured CSMCs were grown to 50–60% cofluence and then transfected with overexpression plasmid (General Biosystems, Anhui, China) or siRNA (Gene Pharma, Suzhou, China) using HighGene transfection reagent (ABclonal, Wuhan, China) according to the manufacturer’s instructions. All plasmids were constructed using standard methods and confirmed by DNA sequencing. The siRNA sequences used for siRNA transfection are listed in Supplementary Table.

Statistical analysis
Unless specified in the figure legends, the data are displayed as the means ± SEMs. Statistical analyses were performed using GraphPad Prism (version 10.1.2; GraphPad Software, La Jolla, CA, USA). Comparisons between two groups were evaluated using two-tailed Student’s t tests. For analyses involving more than two groups, one-way ANOVA followed by Tukey’s post hoc multiple comparison test was applied. The data and images represent at least three independent experiments. Statistical significance was defined as P < 0.05.

The Online Data Supplement contains an expanded methods section.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presented by Han et al. described an interesting mechanism by which CSMC contribute to intestinal inflammation. This involves the RSAD2, YtHDF1, DRP1 dependent mitochondrial dysfunction, which then results in the ferroptosis of IECs, due to the communication between SMC and IECs via MitoEvs. This mechanims could, at least partially translated to the human situation in IBD patients. This is of high novelty and provides a mechanims with high potential to be exploited in the context of IBD.\nThe data are sound, generated using stadnard but adequate technologies and clearly support the conclusions drawn by the authors.\nHowever, there are some aspects which have to be further ellaborated before the manuscript can be considered for publication:\n- the language is not optimal, so it should be revised.\n- some available literature should be included in the intro/discussion, such as the link between YTHDF1 and colitis.\n- not all the methods appear in the main text; which is the basis to separate the methods in main text and supplemental information?\n- in some cases, specially but no only in in vitro experiments, the n number is low, or not even described (figure 7, 8); and it is not clear whether the shown results are technical replicates within a single experiment, or the experiment has been reproduced several times.\n- in the RNA seq, tehre is a clear upregulation of pathways related to the response to the microbiota (fig. 3a), and not only the downregulated pathways associated to the mitochondria. Why not considering those?\nwhy the authors focused on RSAD2? there are other overlapping genes with teh MitoCArta.\n- it is not clear why the authors focused on the trasnfer of MitoEVS from the CSMC to the IECs; why not also checking other cells within the intestinal mucosa?\n-RSAD2 upregulation is not restricted to the muscle layer in Sm22α-/- CSMCs; furhter comment on this?\n- the discussion could be further improved by including especulations about mechanisms to regulate RSAD2? What is it known in the literature? Or in other cell types?\nFor more detail, see specific comments below:\nAbstract:\n- Avoid using terms with no introduction, suhc as SM22a, RSAD2, m6A\nIntroduction:\n- L84-85: this sentence is a bit simplistic, it could be replaceded or complemented by another sentence describing the complexity and the interplay between several mechanisms resulting in IBD. This also includes the fact that the gut tissue is composed by several cell types, which might also interact with each other (related to the already existing sentence).\n- Although the introduction contains important and supporting information to frame the study, sometimes the language and the text reads complicated. I would recommend to carefully check the text and try to improve it.\n- To be added: link between YTHDF1 and IBD. You should cite publications, such as PMID: 38944238, PMID: 38925436, PMID: 39888301, PMID: 34968289. What was still unknown after these studies. And in the discussion, how your data complement the current knowledge?\nResults:\n- Validation of the model: SM22a expression and description of the smooth muscle cells\n- 1d: TUNEL signal is hard to see; contrast method?\n- 1d: The disruption of epithelial hypoxia state, how this has been shown? Literature supporting the link between this and epithelial barrier function\n- 1i: the H&E pics look as taken at different magnification for WT and KO mice; are these the right pics?\n- 2c-d: depict the individual data\n- 1d: Day 7, DAI is different or not?\n- 2f: hard to see histological changes (H&E); include high magnification pictures?\n- Supplemental Figure 1 is not mentioned in the text\n- 3b: depict the changes/alterations on the pictures\n- 3f: details about the ATP assay? Method? Technique?\n- How to better assess mitochondrial fragmentation?\n- Suppl.7a-b: show individual data\n- Figure 7: no description on the n number; experiment repetitions?\n- 8b: why no enrichment of CD63 in mitoEVs vs SMC pellet?\n- Fig. 8: no description on the n number; experiment repetitions?\n- SMC plasticity in your models\nDiscussion\n- What do you mean with „similar to IFN response“?\nMinor comments:\n- L50: shortly introduce SM22α\n- L51: delete „also“\n- L52: shortly introduce RSAD2\n- L53: avoid the use of abbreviations in the abstract: SMC has not been introduced before\n- L55: shortly introduce m6A modification\n- L57: do you mean regulation of the DRP1 mRNA translation?\n- L62_63: do you mean upon induction of experimental colitis? DSS? Or spontaneous?\n- L76: delete „systemic“\n- L90: α smooth muscle actin\n- L147: several spaces\n- L311: typo: was not changed\n- L320: typo: which was abolished\n- L512: different font size", "reviewer_2": "Zhang and colleagues report in their manuscript # NCOMMS-25-12347 that deletion of the smooth muscle cell (SMC) marker SM22 in mice results in spontaneous colitis and increases susceptibility to Dextran Sodium Sulfate (DSS) colitis associated with mitochondrial dysfunction. The observed effects are mediated by Radical S-adenosyl methionine domain-containing protein 2 (RSAD2) and methylation of YTHDF1(YTH N6-Methyladenosine RNA Binding Protein F1). This results in the release of extracellular vesicles by SMCs containing dysfunctional mitochondria, ultimately causing colitis after these extracellular vesicles are uptaken by epithelial cells.\nThe authors provide a vast amount of data aiming to tackle an interesting problem of how SMCs affect colitis outcomes. I have the following suggestions.\nMajor points\nThe authors use SM22-deficient animals, as SM22 is a marker for SMCs. However, as these cells are not deleted in SM22-deficient animals, it remains unclear whether this approach will elucidate the function of SMCs or whether the authors aim to study the function of the SM22 gene.\nThe authors applied the DSS colitis to transgenic mice lacking SM22 in SMCs. these deficient animals probably do not develop spontaneous colitis as the global SM22-deficient animals, indicating that other cells expressing SM22 contribute to the phenotype observed in SM22-deficient animals.\nThe data in supplemental Figure 5 indicate that TNF stimulation leads to mitochondrial damage in CSMCs. This effect could be further pronounced in SM22-deficient CSMCs, providing an amplifying loop that contributes to the reported phenotype in mice.\nPatient characteristics (Montreal classification, type of treatment, etc.).\nRSAD2 is also expressed by epithelial cells, raising the possibility that the reported effects observed in RSAD2-deficient animals may not related to SMCs.\nMinor points\nThe abstract is difficult to read, with several abbreviations not written out.", "reviewer_3": "Zhang et al investigated the role of smooth muscle cell in intestinal inflammation. This concept is novel and very interesting, as the function of SMCs and other connective-tissue cells are clearly insufficiently studied in the context of IBD and their contribution to disease progression not understood. Specifically, the authors revealed that defective small muscle cell function caused spontaneous inflammation in the colon and exacerbated colitis in mice, which was driven by a complex mechanism including mitochondrial dysfunction in smooth muscle cells, that was regulated by the enzyme RSAD2, which facilitated translation of DRP1, a protein that plays a crucial role in mitochondrial fission. Moreover, oxidative stress and mitochondrial dysfunction caused the release of extracellular vesicles from smooth muscle cells, that induced oxidative stress, and as the authors claim, also ferroptosis in intestinal epithelial cells. The concept of the paper is interesting, highlighting importance of small muscle cell function and inter-cellular communication in the gut in IBD, but the complexity of the mechanism, a clear rational to study several pathways and a clinical relevance raises several questions and issues:\n1. A clearer rationale to study smooth muscle cells and using these specific models for intestinal inflammation should be included. Does DSS at baseline affect the function of smooth muscle cells? What induces smooth muscle cell dysfunction in this model? Does Sm22a-KO affect contractility of SMCs? Is gut transit time affected in the small intestine?\n2. As a conventional Sm22a-KO mouse is used: what cells express Sm22a? is it a specific marker for intestinal smooth muscle cells or SMC in general? What other phenotypes (metabolic, cardiovascular) are observed in these mice?\n3. I cannot observe immune cells in the HE in fig 1d. Higher resolution and magnification is needed. Also only colon is shown. What about small intestine?\n4. Is the myh11Cre promoter specific for colonic small muscle cells or small muscle cells in general? How can the authors ensure that no other smooth muscle cells, e.g. in vessels, may affect the described phenotype? Also in regard to the pathway with EVs that affect IECs: smooth muscle cells are probably in closer proximity to IECs than smooth muscle cells of the colon wall. Do endothelial SMCs show a similar phenotype/mechanism as colonic SMCs?\n5. In my opinion, physiologic function of small muscle cells in the context of IBD is very interesting. To investigate this, authors performed a bead expulsion assay, but what about gut transit time in general? Are differences observed in carmine red assay? consistency and size of stool pellets?\n6. PAS staining for goblet cells would be great, also Lysozme staining for paneth cells   are there differences and why and how should smooth muscle cells affect proliferation and differentiation of IECs?\n7. TUNEL labeling was performed to define cell death. Quantification of death cells would be great. Moreover, I cannot characterize the intestinal structure. Co-staining with Hematoxylin is needed   are IECs affected or is it other intestinal cells, eg. Immune cells or SMCs?\n8. Fig 8: how do are MitoEVs isolated and used for stimulation of IECs? How do the authors ensure that no TNFa per-se is in the treatment of IECs, inducing the presented phenotype?\n9. How do the MitoEVs induce ferroptosis? Why are the ferroptosis-protecing mechanisms downregulated and IECs unable to cope with the ROS?\n10. How do mitoEVs from smooth muscle cells interact with IECs, considering the anatomical distance of these two cell types in vivo? Are they carried by other cells or is it diffusion?\n11. The authors use levels of GPX4 and ACSL4 that are quantified by IF to claim that RSAD2 KO mice are protected from DSS-induced ferroptosis. I believe that this claim is very bold for several reasons and to me also misleading. Does DSS affect expression of these molecules in IECs? Is a role of ferroptosis published for DSS colitis? Why should RSAD2 affect expression of these molecules in IECs? Do RSAD2 KO mice show decreased amount of IEC cell death? Do RSAD2 KO mice respond to common rescue strategies of ferroptosis, e.g. ferrostatin or a-tocopherol? In my opinion the presented data is by far not sufficient to claim that ferroptosis is involved in the proposed phenotype, and a plethora of key experiments have to be performed to proof this concept.\n12. Do smooth muscle cells produce other factors, e.g. cytokines or fibrosis inducing factors in models of intestinal inflammation?\n13. In my opinion, the vice-versa axis, e.g. how IECs affect smooth muscle cells would be very interesting, considering the well established function of IECs in the pathophysiology of IBD. What is known about this crosstalk, and how IECs affect smooth muscle function?\n14. Function of smooth muscle cells, particularly contraction, is tightly regulated by the enteric nervous system. Do the used models, e.g. SM22a KO mice show changes in ENS function?\n15. What is known about function of SMCs in human IBD? Obviously, diarrhea and increased intestinal motility is a cardinal symptom of active inflammation in the gut, but do patients with IBD show a smooth muscle cell phenotype, for example hypertrophy of muscle layers? Also, are the factors of the proposed pathway affected in human IBD? Data in this regard would clearly improve the clinical relevance of the manuscript.\n16. As mitochondrial dysfunction in SMCs is a central part of the proposed pathway, but mitochondrial function also central for energy metabolism: Is metabolism in the smooth muscle cells affected? Is the concept of immunometabolism, e.g. metabolic changes during inflammation, described for smooth muscle cells?\n17. What is the physiologic benefit of the proposed mechanism? Why should smooth muscle cells interact with IECs to regulate intestinal physiology? What is the evolutionary benefit of such an interaction?\nMinors:\n• Several abbreviations are not explained in the abstract. Also, a brief explanation of the function of each molecule (e.g., SM22a, RSAD2) in the abstract would be helpful\n• Many statements in the introduction part are without references. Was this part generated using AI tools or large language models? References should be added to all these different statements\n• Is the second sentence of the introduction (UC-CD dichotomy) really needed in the context of this manuscript?\n• Quantification (densitometry) of several immunoblots in the manuscript should be added in addition to representative blots\n• How were colonic smooth muscle tissues for Figure 3a collected? I could not find this in the methods sections"}, "author_response": "Reply: Thank you for taking the time to review our manuscript. We appreciate your valuable comments and suggestions. We have read carefully and responded your comments point by point as listed below. All revisions were clearly marked with red. However, there are some aspects which have to be further ellaborated before the manuscript can be considered for publication:\n- the language is not optimal, so it should be revised.\nReply: We apologize for our language defects. The language of manuscript has been improved by a Native English expert.\n- some available literature should be included in the intro/discussion, such as the link between YTHDF1 and colitis.\nReply: Thank you for your suggestion.\nIn the revised manuscript, the link between YTHDF1 and colitis has been included in the Introduction/Discussion and the relevant literatures have been cited, as follow:\nRecent studies point to a role of YTH N6-methyladenosine (m6A) RNA-binding protein 1 (YTHDF1) in the gut. Although YTHDF1 is largely dispensable for intestinal homeostasis under physiological conditions, YTHDF1 is required for intestinal epithelial regeneration and tumorigenesis by maintaining stem cell traits via amplifying Wnt/β-catenin signaling at the translational level [Ref. 7]. The mRNA expression level of YTHDF1 was upregulated in UC patients [Ref. 8] and down-regulated in both LPS-induced macrophage inflammation and the colon of IBD mice, while up-regulation of YTHDF1 expression alleviates the inflammatory response [Ref. 9], suggesting the implication of YTHDF1-mediated RNA methylation events in IBD pathogenesis [Ref. 10]. (As shown in the Introduction)\nIntriguingly, increased YTHDF1 expression has been demonstrated to be associated with the disruption of the intestinal barrier integrity in CD patients and colitis mice [Ref. 46]. Deletion of YTHDF1 induces CD8+ T cells to stimulate high expression of programmed cell death 1 ligand 1, ultimately exacerbating IBD [Ref. 47]. YTHDF1 overexpression enhanced the mRNA expression of immunosuppressive molecules in an m6A-dependent manner and restructures the ecology of inflammation in colitis [Ref. 48]. Additionally, DNA copy number gain and overexpression of YTHDF1 are considered to be a frequent event in UC and inflammatory colorectal cancer [Ref. 49,50]. (As shown in the Discussion).\n- not all the methods appear in the main text; which is the basis to separate the methods in main text and supplemental information?\nReply: Thank you for your question.\nConsidering the limitation of manuscript length, not all methods are presented in the main text to streamline the manuscript. Some common methods were placed into the Supplemental information.\n- in some cases, specially but no only in in vitro experiments, the n number is low, or not even described (figure 7, 8); and it is not clear whether the shown results are technical replicates within a single experiment, or the experiment has been reproduced several times.\nReply: Thank you for your comments. We apologize for our carelessness. In the present study, all experiments were independently repeated at least three times (n ≥ 5 per group), with each n representing a biologically independent replicate. In the revised manuscript, we have described the n values for the Figure 7 and 8, and determined that the data represent the results from independent biological replicates within a single experiment rather than technical repeats, and each experiment was reproduced 3 times.\n- in the RNA seq, there is a clear upregulation of pathways related to the response to the microbiota (fig. 3a), and not only the downregulated pathways associated to the mitochondria. Why not considering those?\nReply: Many thanks for your question.\nAs you said, the RNA seq indeed showed that there is a clear upregulation of pathways related to the response to the microbiota (Fig. 3a), which were being investigated in our other study on the intestinal microbiota metabolism and cardiovascular homeostasis. So, the current study only focused on the downregulated pathways associated to the mitochondria.\nwhy the authors focused on RSAD2? there are other overlapping genes with the MitoCArta.\nReply: It’s a good question.\nAs you said, there are other overlapping genes with the MitoCArta. In the verification experiment, we found that the change of RSAD2 expression has good repeatability and reproducibility in vitro and in vivo. Importantly, the role of RSAD2 in IBD development is unknown. There is the reason why we focused on RSAD2.\n- it is not clear why the authors focused on the transfer of MitoEVS from the CSMC to the IECs; why not also checking other cells within the intestinal mucosa?\nReply: It’s a very good question.\nInflammatory diseases of the intestinal tract commonly affect the mucosa, but also challenge the normal homeostatic balance of all parts of the intestine, including the intestinal SMC in the muscularis externa. Extensive research suggested that aberrant responses to environmental factors, genetic susceptibility, abnormal immune regulation, the intestinal mucosal barrier, and intestinal microecological changes contribute to the occurrence and development of IBD. Epithelial cells, fibroblast, monocytes, B cell, and T cells in the colon mucosa seem to be associated with IBD pathology [DOI: 10.1016/j.cell.2019.06.029; DOI:10.1038/s41586-020-2025-2].\nAbnormal regulation of epithelial cell proliferation and cell death that is frequently found in both UC and CD, is considered to exacerbate intestinal damage, leading to disruption in intestinal barrier integrity and to trigger an inflammatory cascade [DOI:10.1016/j.redox.2022.102469]. This study aimed to investigate how aberrant CSMC phenotypes contribute to dysregulation of the epithelial homeostasis using Sm22α-/- mice that display dysfunction of SMCs and focused on the transfer of MitoEVs from the CSMC to the IECs in IBD pathology. We are going to investigate other cells within the intestinal mucosa in future study.\nThank you for your suggestion.\n-RSAD2 upregulation is not restricted to the muscle layer in Sm22α-/- CSMCs; further comment on this?\nReply: Your comment is very professional.\nIndeed, we also noticed the expression of RSAD2 at the tips of intestinal epithelial villi in Sm22α-/- mice and DSS-induced colitis model, which may be associated with aberrant mucosal immunity. Microbial signals are recognized by the intestinal epithelial cells (IECs) to promote intestinal homeostasis. IEC-derived signals influence both innate and adaptive effector cell function and homeostasis. This allows IECs to locally regulate immune response at the intestinal barrier and promote the maintenance of intestinal homeostasis [DOI:10.1038/nri3608]. Our other study that is in progress showed that the composition of gut microbiota between WT and Sm22α-/- mice was different. Furthermore, elevated plasma LPS level was observed in Sm22α-/- mice, suggesting damaged mucosal barrier. An increasing number of studies have underscored that RSAD2 plays an essential role in immunomodulation and is a potential biomarker in many autoimmune diseases [Ref. 5]. We thought that increased RSAD2 expression in intestinal epithelial cells may be a way of host immune response to altered gut microbiota.\nThe sentence in the Discussion was revised as “Increased expression of RSAD2 was observed in the damaged colonic tissues (the original sentence: the colonic muscular layer) of mice with colitis and the UC patients”.\nDo you agree with our explanation? Please advise again.\n-the discussion could be further improved by including especulations about mechanisms to regulate RSAD2? What is it known in the literature? Or in other cell types?\nReply: Thanks for your suggestion.\nAccumulating studies have underscored that RSAD2 plays an essential role in immunomodulation, mitochondrial metabolism and autoimmune diseases [Ref. 5].\nRSAD2 expression in the immune system can be directly or indirectly induced by various stimuli, including viral infections and interferons, synthetic analogs of double-stranded RNA or B-form DNA, as well as lipopolysaccharide (LPS) via IFN-dependent and -independent pathways [Ref. 33]. IFN-dependent pathway induces the formation of the heterotrimeric complex interferon-stimulated gene factor 3 (ISGF3) via activation of the JAK-STAT pathway, which translocates to the nucleus and binds to the promoter of RSAD2 to drive its transcription [Ref. 33]. While phosphorylated STAT1 levels decrease, unphosphorylated complexes can maintain RSAD2 synthesis upon prolonged IFN exposure. IFN-independent induction of RSAD2 expression can be directly activated by interferon regulatory factor 3 (IRF3), activator protein 1 (AP-1), and IRF1[Ref. 33]. Additionally, RSAD2 has been localized to mitochondria in cells of brown adipose tissue [Ref. 39] and transiently in human foreskin fibroblasts, where it regulates β-oxidation of fatty acids and energy production [Ref. 36]. This discussion has been added in the Discussion.\nFor more detail, see specific comments below:\nAbstract:\n- Avoid using terms with no introduction, suhc as SM22α, RSAD2, m6A\nReply: Thanks for your suggestion. All abbreviations, such as SM22α, RSAD2, m6A, have been defined when they first appear in the revised Abstract.\nIntroduction:\n- L84-85: this sentence is a bit simplistic, it could be replaced or complemented by another sentence describing the complexity and the interplay between several mechanisms resulting in IBD. This also includes the fact that the gut tissue is composed by several cell types, which might also interact with each other (related to the already existing sentence).\nReply: Many thanks for your comments and advice.\nThe sentences describing the complexity and the interplay between several mechanisms resulting in IBD have been complemented in the Introduction, as following:\nKnown disease risk alleles highlight key pathways in the pathogenesis of IBD, including innate and adaptive immunity, gut barrier function, and pathogen sensing and response. The epithelial cells, goblet cells, stroma and immune cells in the intestinal mucosa interact with each other, or with the intestinal microbiota and the immune system, sending receiving signals to and from it [Ref. 2]. Furthermore, inappropriate and continuing mucosal immune response to commensal microbes in a genetically susceptible host is also involved in intestinal inflammation in the gut and IBD [Ref. 2].\n- Although the introduction contains important and supporting information to frame the study, sometimes the language and the text reads complicated. I would recommend to carefully check the text and try to improve it.\nReply: According your suggestion, the Introduction has been refined in the revised manuscript. We have carefully checked the text and removed the content describing the effect of SMC phenotypic switching and SM22α on vascular disease.\n- To be added: link between YTHDF1 and IBD. You should cite publications, such as [Ref. 8,10,46,48]. What was still unknown after these studies. And in the discussion, how your data complement the current knowledge?\nReply: Thank you for your suggestion.\nIn the revised manuscript, the link between YTHDF1 and IBD has been included in the Introduction and the Discussion and the relevant literatures, such as [Ref. 8,10,46,48], have been cited. We also highlighted our data complement the current knowledge, as following:\nRecent studies point to key roles of YTHDF1 in the gut. Although YTHDF1 is largely dispensable for intestinal homeostasis under physiological condition, YTHDF1 is required for intestinal epithelial regeneration and tumorigenesis by maintaining stem cell traits via amplifying Wnt/β-catenin signaling at the translational level [Ref. 7]. The level of YTHDF1 mRNA was upregulated in UC patients [Ref. 8] and down-regulated in both LPS-induced macrophage inflammation and the colon of IBD mice, while up-regulation of YTHDF1 expression alleviates the inflammatory response [Ref. 9], suggesting the implication of YTHDF-mediated RNA methylation events in IBD pathogenesis [Ref. 10]. (As shown in the Introduction).\nFurthermore, increased YTHDF1 expression has been demonstrated to be associated with the disruption of the intestinal barrier integrity in Crohn’s disease patients and colitis mice [Ref. 46]. Deletion of YTHDF1 induces CD8+ T cells to stimulate high expression of programmed cell death 1 ligand 1, ultimately exacerbating IBD [Ref. 47]. YTHDF1 overexpression enhanced the mRNA expression of immunosuppressive molecules in m6A-dependent manner and restructures the ecology of inflammation in colitis [Ref. 48]. Additionally, DNA copy number gain and overexpression of YTHDF1 is considered to be a frequent event in UC and inflammatory colorectal cancer [Ref. 49,50]. The present study demonstrated for the first time a methylation-dependent mechanism of YTHDF1 activity in IBD development. We identified that RSAD2 directly interacted with YTHDF1 via its SAM domain, promoting lysine methylation of YTHDF1, accompanied by increased YTHDF1-driven translation efficiency of m6A-modified DRP1 mRNA, contributing to SMC-driven intestinal epithelial inflammation. It needs further investigation whether targeting YTHDF1 may increase the efficacy of the current IBD therapy regime. (As shown in the Discussion)\nResults:\n- Validation of the model: SM22α expression and description of the smooth muscle cells\nReply: Thank you for your insightful suggestion. We have added the data of SM22α expression in the colonic tissues of mice, as shown in the Supplementary Fig. 1a.\n- 1d: TUNEL signal is hard to see; contrast method?\nReply: We appreciate your observation. In response to the reviewer’s suggestion, TUNEL staining was repeated with Hematoxylin counterstaining, and quantification of apoptotic IECs is now included (Fig. 1e). The results revealed a significant increase in apoptotic IECs in Sm22α-/- mice. These data reinforce our conclusion that SMC dysfunction compromises epithelial integrity and increases IEC death.\n- 1d: The disruption of epithelial hypoxia state, how this has been shown? Literature supporting the link between this and epithelial barrier function\nReply: Many thanks for your important question.\nThe disruption of the epithelial hypoxic state was demonstrated, as shown in the left of the revised Fig. 1f. Mice were intraperitoneally injected with pimonidazole hydrochloride (60 mg·kg-1), a well-established hypoxia marker, and hypoxic regions in the colonic epithelium were assessed by immunofluorescence staining [Supplementary Ref. 2]. The literature has been cited in the SUPPLEMENTAL METHODS\nExisting literature supports that physiological hypoxia is essential for maintaining epithelial barrier integrity by promoting tight junction formation, mucin production, and antimicrobial peptide secretion. Conversely, loss of epithelial hypoxia leads to microbial dysbiosis, increased permeability, and systemic inflammation [Supplementary Ref. 2; DOI: 10.1016/j.it.2021.05.004]. Limited by the article space, the literatures supporting the link between this and epithelial barrier function have not been cited in the text.\n- 1i: the H&E pics look as taken at different magnification for WT and KO mice; are these the right pics?\nReply: We sincerely apologize for this oversight.\nIn the revised Fig. 1l (not 1i), we have replaced the images to ensure consistent magnification between groups, allowing accurate comparison of histological features.\n- 2c-d: depict the individual data\nReply: Thank you for your valuable suggestion. Individual data points have been overlaid in the revised Fig. 2b-c (not 2c-d) to improve data transparency and interpretability.\n- 1d: Day 7, DAI is different or not?\nReply: Thank you for the query.\nAt day 7 (Fig. 2c, not 1d), there was no statistically significant difference in DAI, which may be attributed to a ceiling effect—surviving mice exhibited maximal colitis severity, limiting observable variance between groups.\n- 2f: hard to see histological changes (H&E); include high magnification pictures?\nReply: Thank you for your valuable feedback.\nIn the revised manuscript, high-magnification H&E images in the Fig. 2f have been included to clearly exhibit the histological changes.\n- Supplemental Figure 1 is not mentioned in the text\nReply: Thank you for pointing this out. The data of the revised Supplementary Fig. 2 (original Supplementary Fig. 1) have been mentioned in the Results section. No difference in colitis phenotype between male and female Sm22α-/- mice was observed.\n- 3b: depict the changes/alterations on the pictures\nReply: We appreciate the suggestion. In the revised Fig. 3b, we have highlighted regions of key morphological changes with arrows.\n- 3f: details about the ATP assay? Method? Technique?\nReply: Thanks for your questions.\nATP levels were quantified using a commercially available ATP assay kit (S0026, Beyotime Biotechnology), which utilizes a luciferase-catalyzed luminescent reaction. In this assay, ATP serves as the energy source for luciferin oxidation, producing light proportional to ATP concentration. Briefly, cultured adherent cells were lysed directly in the wells. Tissue samples were homogenized in 100–200 µL lysis buffer per 20 mg tissue, followed by centrifugation at 12,000 × g for 5 min at 4°C. Supernatants were collected and analyzed. A standard curve (0.01–10 µmol/L) was generated from ATP standards diluted in lysis buffer. A 1:9 mixture of detection reagent to buffer was prepared and added (100 µL per well) and incubated for 3–5 min to eliminate background ATP. Subsequently, 20 µL of sample or standard was added, and luminescence was immediately recorded. ATP concentrations were normalized to total protein content (nmol/mg protein). The detail has been shown in the SUPPLEMENTAL METHODS.\n- How to better assess mitochondrial fragmentation?\nReply: We agree that comprehensive evaluation is crucial.\nAs recommended by recent literature [DOI: 10.1038/s41467-025-56494-6], we expanded our morphological assessment to include quantification of mitochondrial number, mean mitochondria length per cell, and network connectivity (number of junctions per mitochondrial network), as shown in the Fig.3d, 5b, 5h, Supplementary Fig. 5b, 6b and 6h.\n- Suppl.7a-b: show individual data\nReply: Thank you for your suggestion. Individual data points have been added to the Supplementary Fig. 8a-b.\n- Figure 7: no description on the n number; experiment repetitions?\nReply: We appreciate this reminder.\nThe relative intensity of the western blot bands has been shown in the Figure 7, and the n number was marked in the revised Figure 7 legend. All experiments were independently repeated at least three times.\n- 8b: why no enrichment of CD63 in mitoEVs vs SMC pellet?\nReply: Thank you for your question.\nAs shown in Fig. 8b, both SMC pellets (CSMC lysate) and MitoEVs (Secreted Mito.) exhibit CD63 enrichment, supporting the presence of mitochondria-containing vesicles within both the intracellular and extracellular compartments.\n- Fig. 8: no description on the n number; experiment repetitions?\nReply: Thank you for pointing this out. The n number was marked in the revised Figure 8 legend (n = 6 biological replicates).\n- SMC plasticity in your models\nReply: Thank you for raising this important aspect.\nIn the present study, transcriptomic analysis of colonic SMCs isolated from adult Sm22α-/- mice revealed a pronounced shift in gene expression profiles indicative of phenotypic plasticity. Gene ontology (GO) enrichment analysis of differentially expressed genes showed that upregulated transcripts were significantly associated with immune activation, inflammatory responses, and osteogenic processes. In contrast, downregulated genes were primarily involved in negative regulation of ossification, glycolysis, and fatty acid transport. These findings suggest that in the absence of SM22α, colonic SMCs undergo phenotypic switching characterized by a pro-inflammatory state and osteoblast-like transdifferentiation. The data were added to the Supplementary Fig. 4c.\nDiscussion\n- What do you mean with „similar to IFN response“?\nReply: We deeply apologize for our inappropriate citation. The sentence has been deleted in the revised manuscript.\nMinor comments:\n- L50: shortly introduce SM22α\nReply: All abbreviations, such as SMC, SM22α, RSAD2, m6A, have been defined when they first appear in the revised manuscript.\n- L51: delete „also“\nReply: Thank you. The word “also” has been deleted.\n- L57: do you mean regulation of the DRP1 mRNA translation?\nReply: We have revised the sentence to explicitly state “YTHDF1 enhances the translation of DRP1 mRNA in an m6A dependent manner”.\n- L62_63: do you mean upon induction of experimental colitis? DSS? Or spontaneous?\nReply: Thank you for your reminder. We have changed the text sentence in the Abstract to “The extracellular vesicles (MitoEVs) containing damaged mitochondria components from CSMCs subsequently trigger oxidative stress and ferroptosis in the intestinal epithelial cells”.\n- L76: delete „systemic“\nReply: We greatly admire your patience and rigor. The word “systemic” has been deleted.\n- L147: several spaces\nReply: Thank you. Extra spaces have been removed.\n- L311: typo: was not changed\nReply: Thank you. The typo has been corrected.\n- L320: typo: which was abolished\nReply: We deeply apologize for our omission. The missing word “was” has been added.\n- L512: different font size\nReply: Thank you for noticing this. The font size has been corrected for consistency throughout the manuscript.\nWe hope that your concerns and questions have been addressed. Thanks again for the time and effort that you have put into our manuscript and we appreciate your kind comments.\nReviewer #2 (Remarks to the Author)\nZhang and colleagues report in their manuscript # NCOMMS-25-12347 that deletion of the smooth muscle cell (SMC) marker SM22 in mice results in spontaneous colitis and increases susceptibility to Dextran Sodium Sulfate (DSS) colitis associated with mitochondrial dysfunction. The observed effects are mediated by Radical S-adenosyl methionine domain-containing protein 2 (RSAD2) and methylation of YTHDF1(YTH N6-Methyladenosine RNA Binding Protein F1). This results in the release of extracellular vesicles by SMCs containing dysfunctional mitochondria, ultimately causing colitis after these extracellular vesicles are uptaken by epithelial cells.\nReply: Thank you for taking the time to review our manuscript. We appreciate your valuable comments and suggestions. We have read carefully and responded your comments point by point as listed below. All revisions were clearly marked with red.\nThe authors provide a vast amount of data aiming to tackle an interesting problem of how SMCs affect colitis outcomes. I have the following suggestions.\nMajor points\n1. The authors use SM22-deficient animals, as SM22 is a marker for SMCs. However, as these cells are not deleted in SM22-deficient animals, it remains unclear whether this approach will elucidate the function of SMCs or whether the authors aim to study the function of the SM22 gene.\nReply: It’s a good question.\nOur previous studies have demonstrated that disruption of SM22α leads to the phenotypic switching of vascular SMCs and enhances vascular inflammatory response. The present study aims to investigate the effect of SM22α deficiency on intestinal SMC function and the role and mechanism of SM22α deletion-induced CSMC dysfunction in colitis development. Thus, Sm22α-/- mice and SMC-specific SM22α knockout (smSm22α-/-) were used in the experiment.\n2. The authors applied the DSS colitis to transgenic mice lacking SM22 in SMCs. these deficient animals probably do not develop spontaneous colitis as the global SM22-deficient animals, indicating that other cells expressing SM22 contribute to the phenotype observed in SM22-deficient animals.\nReply: We appreciate your insightful comment.\nThere was possibility that the phenotype observed in global SM22α-deficient mice may involve contributions from other cell types expressing SM22α. Notably, in the conditional knockout mice, we observed features of spontaneous colitis even in the absence of DSS induction, as shown in Fig. 1j-p. These findings suggest that SM22α deficiency in SMCs alone may be sufficient to initiate colonic inflammation, though the contribution of other SM22α-expressing cell populations in the global knockout model remains an important consideration.\n3. The data in supplemental Figure 5 indicate that TNF stimulation leads to mitochondrial damage in CSMCs. This effect could be further pronounced in SM22-deficient CSMCs, providing an amplifying loop that contributes to the reported phenotype in mice.\nReply: Thanks for your suggestion.\nIn the revised manuscript, we have provided an amplifying loop that contributes to the inflammatory phenotype in mice, as following:\nOur previous researches have demonstrated that TNF-α induced the epigenetic silencing of SM22α transcription via EZH2-mediated H3K27 methylation in the SM22α promoter region in vascular SMCs [Ref. 15], and the disruption of SM22α aggravates vascular inflammation via enhanced activation of NF-κB [Ref. 18], prompting us to explore whether synergistic interaction of TNF-α stimulation and SM22α ablation impairs mitochondrial function in Sm22α-/- CSMCs (as shown in the Results and Supplementary Fig. 6g-l).\n4. Patient characteristics (Montreal classification, type of treatment, etc.).\nReply: Thank you for your suggestion. We have included detailed patient characteristics—such as Montreal classification, Modified Mayo Score, and treatment regimens—in the revised Supplemental Table 2.\n5. RSAD2 is also expressed by epithelial cells, raising the possibility that the reported effects observed in RSAD2-deficient animals may not related to SMCs.\nReply: There is a certain possibility for your inference.\nA recent study demonstrated that IFNλ can modulate the function of neutrophils by upregulating RSAD2, leading to the suppression of intestinal inflammation in mouse model of DSS-induced colitis, which was recapitulated in human neutrophils [Ref. 40,41]. We speculated that the positive or negative effects of RSAD2 on colitis may be associated with the cell types. This has been added in the Discussion.\nMinor points\nThe abstract is difficult to read, with several abbreviations not written out.\nReply: We accept your criticism.\nAll abbreviations, such as SM22α, RSAD2, m6A and YTHDF1, have been defined when they first appear in the revised Abstract.\nThanks again for the time and effort that you have put into our manuscript and we appreciate your kind comments. We hope that your concerns and questions have been addressed.\nReviewer #3 (Remarks to the Author):\nZhang et al investigated the role of smooth muscle cell in intestinal inflammation. This concept is novel and very interesting, as the function of SMCs and other connective-tissue cells are clearly insufficiently studied in the context of IBD and their contribution to disease progression not understood. Specifically, the authors revealed that defective small muscle cell function caused spontaneous inflammation in the colon and exacerbated colitis in mice, which was driven by a complex mechanism including mitochondrial dysfunction in smooth muscle cells, that was regulated by the enzyme RSAD2, which facilitated translation of DRP1, a protein that plays a crucial role in mitochondrial fission. Moreover, oxidative stress and mitochondrial dysfunction caused the release of extracellular vesicles from smooth muscle cells, that induced oxidative stress, and as the authors claim, also ferroptosis in intestinal epithelial cells. The concept of the paper is interesting, highlighting importance of small muscle cell function and inter-cellular communication in the gut in IBD, but the complexity of the mechanism, a clear rational to study several pathways and a clinical relevance raises several questions and issues:\nReply: Thank you for taking the time to review our manuscript. We appreciate your valuable comments and suggestions. We have read carefully and responded your comments point by point as listed below. All revisions were clearly marked with red.\n1. A clearer rationale to study smooth muscle cells and using these specific models for intestinal inflammation should be included. Does DSS at baseline affect the function of smooth muscle cells? What induces smooth muscle cell dysfunction in this model? Does Sm22a-KO affect contractility of SMCs? Is gut transit time affected in the small intestine?\nReply: Thank you for your comments.\nThe contribution of SMCs in IBD fibrotic process has been suggested but less extensively studied [Ref. 4]. SMC hypertrophy and hyperplasia are described in the inflamed intestine in both human IBD and in animal disease models. These findings indicate that aberrant SMC phenotypes, as an environmental variable, may contribute to dysregulation of the mucosal immune and commensal ecosystem in the gut. We and others have demonstrated that disruption of SM22α in vascular SMCs reduces contractility and enhances vascular inflammatory response via modulation of phenotypic switching of SMC [Ref. 14,15]. Given key role of SM22α in maintaining SMC contractile phenotype, the present study investigated the effect of SM22α deficiency on intestinal SMC function and the role and mechanism of SM22α deletion-induced CSMC dysfunction in colitis development. Thus, Sm22α-/- mice and SMC-specific SM22α knockout (smSm22α-/-) were used in the experiment.\nOur supplementary experiments revealed that DSS under baseline conditions affected the contractile function of mouse CSMCs. Specifically, CSMCs were treated with DSS at concentrations of 1%, 3%, and 5% for 24 hours [DOI: 10.1016/j.biopha.2025.118185]. Subsequently, cell images were captured before and after stimulation with 1 mM carbachol for 20 minutes, and contractile function was evaluated by calculating the proportion of contracted cell area [DOI: 10.1016/j.actbio.2020.10.042]. The results demonstrated that baseline exposure to DSS significantly impaired the contractile capacity of mouse colonic smooth muscle cells, with the degree of impairment showing a dose-dependent increase with rising DSS concentrations. These data are presented in the Supplementary Fig. 1h, i.\nOur results suggested that DRP1-mediated mitochondrial fragmentation resulted in SMC dysfunction in this model. Sm22a-KO decreased the contractility of CSMCs and delayed the gut transit time, as shown in Fig. 1h, 1o, and 2i. Additionally, electrophysiological experiments and carbachol-induced contraction assays on colonic SMCs of mice confirmed that Sm22α deficiency resulted in contractile dysfunction as shown below. The data were added into the Supplementary Fig 1e, f.\n2. As a conventional Sm22a-KO mouse is used: what cells express Sm22a? is it a specific marker for intestinal smooth muscle cells or SMC in general? What other phenotypes (metabolic, cardiovascular) are observed in these mice?\nReply: We appreciate the reviewer’s concern regarding cell-type specificity in our global Sm22α-deficient model.\nSM22α is expressed broadly in both vascular and visceral SMCs in the adult animals, including intestinal SMCs. In addition, we found lower expression of SM22α in cardiomyocytes and endothelial cells. As you said, SM22α, as an important cytoskeleton associated protein, is required for maintaining the contractile phenotype of vascular SMCs and is a sensitive and specific marker for identifying the dedifferentiation and phenotypic switching of SMCs. Our previous studies have demonstrated that Sm22α-deficient mice display increased vascular inflammation [Ref. 15,18], oxidative stress [DOI: 10.1161/CIRCRESAHA.112.272013], proliferation [DOI: 10.1161/ATVBAHA.109.200501] and disorder of glucose and lipid metabolism phenotypes in vascular SMCs [DOI: 10.1007/s00109-016-1468-2; DOI: 10.1161/CIRCRESAHA.115.306233], which contributes to the development of atherosclerosis [DOI: 10.1038/s41419-021-04239-w], neointimal formation [DOI: 10.1161/ATVBAHA.109.200501] and abdominal aortic aneurysm [DOI: 10.1155/2021/5564884]. Furthermore, these mice are more prone to heart failure after TAC [DOI: 10.1371/journal.pone.0271578], have an impaired uterine contractility [Ref. 17] and neurocognitive deficits [DOI: 10.3390/ijms24087117].\nLimited by the article space, the above discussion was not shown in the text.\n3. I cannot observe immune cells in the HE in fig 1d. Higher resolution and magnification is needed. Also only colon is shown. What about small intestine?\nReply: We appreciate the Reviewer’s remark on image clarity.\nWe have replaced Figure 1d (the revised Fig. 1c) with high-resolution H&E images and these enhanced images clearly illustrate immune cell infiltration in both the colonic mucosa and muscularis of Sm22α-/- mice, with inflammatory foci marked by arrows. Given that our model primarily mimics ulcerative colitis, which predominantly affects the colon, our focus was on colonic pathology. However, we acknowledge the value of exploring small intestinal involvement and have included this as a future direction.\n4. Is the myh11Cre promoter specific for colonic small muscle cells or small muscle cells in general? How can the authors ensure that no other smooth muscle cells, e.g. in vessels, may affect the described phenotype? Also in regard to the pathway with EVs that affect IECs: smooth muscle cells are probably in closer proximity to IECs than smooth muscle cells of the colon wall. Do endothelial SMCs show a similar phenotype/mechanism as colonic SMCs?\nReply: We thank the reviewer for this thoughtful question. Your concern is necessary.\nIndeed, Myh11-CreER targets both visceral and vascular SMCs [DOI: 10.1038/nm1666]. In the present study, we cannot exclude the potential effects of vascular smooth muscle on the intestinal mucosa and disease phenotype via change of the gut vascular barrier function. We have explicitly acknowledged this limitation in the Discussion.\n5. In my opinion, physiologic function of small muscle cells in the context of IBD is very interesting. To investigate this, authors performed a bead expulsion assay, but what about gut transit time in general? Are differences observed in carmine red assay? consistency and size of stool pellets?\nReply: Thank you for this excellent suggestion.\nIt should be noted that this study mainly focuses on colonic inflammatory lesions, so we did not conduct in-depth research on small intestinal muscle cell function or the carmine red assay. In the evaluation of colonic transit function, we found that compared with WT mice, Sm22α-/- mice exhibited a statistically significant delay in colonic transit time (Fig. 1h). Additionally, stool analysis [Supplementary Ref. 3] revealed pasty feces with increased wet weight and water content in Sm22α-/- mice (Supplementary Fig.1b-d), consistent with the characteristics of impaired intestinal motility. These data further support the role of smooth muscle dysfunction in colonic transit abnormalities. These data have been added to the Results and Supplementary Methods section.\nWe believe that these supplementary colonic-related physiological parameters further strengthen the association between Sm22α deletion, smooth muscle cell dysfunction, and altered colonic transit function, providing more robust support for the research conclusions.\n6. PAS staining for goblet cells would be great, also Lysozme staining for paneth cells are there differences and why and how should smooth muscle cells affect proliferation and differentiation of IECs?\nReply: We thank the Reviewer for this insightful suggestion. We agree that assessing IEC differentiation provides important mechanistic depth. In response, Alcian blue staining was replaced with Alcian blue-Periodic acid Schiff (AB-PAS) staining (Fig. 1d), and Lysozyme staining was added (Supplementary Fig 1g) to assess goblet and Paneth cells, respectively. We observed a significant reduction in goblet cells in Sm22α-/- mice, whereas no significant Lysozyme-positive Paneth cells were detected in colonic tissues of either WT or Sm22α-/- mice, consistent with their physiological absence from the colon [DOI: 10.1038/nature09637]. These findings suggest that colonic smooth muscle dysfunction may alter IEC differentiation, particularly goblet cell lineage commitment, potentially through MitoEV-mediated signaling.\n7. TUNEL labeling was performed to define cell death. Quantification of death cells would be great. Moreover, I cannot characterize the intestinal structure. Co-staining with Hematoxylin is needed are IECs affected or is it other intestinal cells, eg. Immune cells or SMCs?\nReply: In response to the reviewer’s suggestion, we repeated TUNEL staining with Hematoxylin counterstaining and quantified apoptotic cells (Fig. 1e). The results showed a significant increase in apoptotic IECs in Sm22α-/- mice compared with WT controls, confirming that SMC dysfunction compromises epithelial integrity and increases IEC death.\n8. Fig 8: how do are MitoEVs isolated and used for stimulation of IECs? How do the authors ensure that no TNFa per-se is in the treatment of IECs, inducing the presented phenotype?\nReply: Thank you for this important question. MitoEVs from CSMC conditional media were isolated using differential centrifugation, as previous described [Ref. 55; DOI: 10.1016/j.cmet.2024.05.016]. To rule out cytokine contamination, the isolated vesicles were resuspended in PBS, subjected to an additional ultracentrifugation step, and the resulting supernatant was assayed for TNF-α using the Mouse TNF-alpha Quantikine ELISA Kit (R&D Systems, MTA00B). The assay sensitivity is <1.0 pg/mL with a detection range of 15.6-1000 pg/mL. However, we did not detect TNFα in the supernatant using the Kit, indicating that the presented IEC phenotype is not induced by TNFα.\n9. How do the MitoEVs induce ferroptosis? Why are the ferroptosis-protecing mechanisms downregulated and IECs unable to cope with the ROS?\nReply: We appreciate the Reviewer’s interest in this mechanistic aspect.\nMitoEVs secretion, as the cue to release content, is the clear role for specific mitochondrial stress, or inflammatory signaling [Ref. 26]. For example, the adipose-derived MitoEVs containing mitochondrial content prompted an initial burst of ROS of the recipient cardiomyocytes in the heart, which led to tissue remodeling, suggesting that the MitoEVs carrying mitochondrial content as a messenger between adipocytes and the heart have potentially long-range impact on the heart, particularly in conditions of adipocyte stress [DOI: 10.1016/j.cmet.2021.08.002].\nWe observed that MitoEVs derived from dysfunctional CSMCs elevated intracellular ROS in IECs, leading to lipid peroxidation and concomitantly suppressed GPX4 expression, accompanied by increased COX2, a ferroptosis-promoting marker. We considered that the MitoEVs from stressed CSMCs evoked oxidative stress, and thereby induced ferroptosis in IECs. We also added the experiments (e.g., GSH depletion, lipid peroxidation quantification) to confirm this phenotype, as shown in Fig. 8d-j and Supplementary Fig. 11c, d.\n10. How do mitoEVs from smooth muscle cells interact with IECs, considering the anatomical distance of these two cell types in vivo? Are they carried by other cells or is it diffusion?\nReply: We thank the reviewer for this insightful anatomical question. Although CSMCs reside within the muscularis externa, it is generally known that the mucosal integrity is compromised in IBD, contributing to MitoEVs diffusion. Additionally, studies have demonstrated that the EVs can traverse tissue barriers and enter systemic circulation to mediate a long-range effect [DOI：10.1016/j.cmet.2021.08.002]. So, we thought these MitoEVs may interact with IECs via diffusion.\n11. The authors use levels of GPX4 and ACSL4 that are quantified by IF to claim that RSAD2 KO mice are protected from DSS-induced ferroptosis. I believe that this claim is very bold for several reasons and to me also misleading. Does DSS affect expression of these molecules in IECs? Is a role of ferroptosis published for DSS colitis? Why should RSAD2 affect expression of these molecules in IECs? Do RSAD2 KO mice show decreased amount of IEC cell death? Do RSAD2 KO mice respond to common rescue strategies of ferroptosis, e.g. ferrostatin or a-tocopherol? In my opinion the presented data is by far not sufficient to claim that ferroptosis is involved in the proposed phenotype, and a plethora of key experiments have to be performed to proof this concept.\nReply: We appreciate this important critique.\nWe recognized that the levels of GPX4 and ACSL4 that are quantified by IF are insufficient to establish RSAD2 KO mice are protected from DSS-induced ferroptosis. In our revised manuscript, we presented additional data showing that Rsad2-/- mice were protected from DSS-induced ferroptosis, as indicated by significantly increased GPX4 expression, decreased COX2 expression, reduced ferrous iron and lipid peroxidation levels in the intestinal epithelium (Fig. 8l, m and Supplementary Fig. 8i-k), accompanied with decreased amount of IEC cell death and alleviated DSS-induced colitis (Supplementary Fig. 8a-h). These results suggest that RSAD2 may directly or indirectly modulates ferroptosis of IEC.\nAdditionally, increasing evidence have demonstrated a role of ferroptosis for DSS colitis, as evidenced by ferrous iron accumulation, increased ROS production, decreased the expression of GPX-4 and FTH, SOD and GSH depletion in DSS-induced mice [DOI: 10.1016/j.lfs.2022.121312; DOI: 10.1016/j.imlet.2020.06.005; DOI: 10.1038/s41419-020-2299-1; DOI: 10.1016/j.freeradbiomed.2021.06.012]. Collectively, these data strongly suggest that ferroptosis is involved in the proposed phenotype.\nSince Rsad2-/- mice displayed reduced ferroptosis of IEC, these mice may not respond to common inhibitors of ferroptosis, e.g. ferrostatin or a-tocopherol.\n12. Do smooth muscle cells produce other factors, e.g. cytokines or fibrosis inducing factors in models of intestinal inflammation?\nReply: Thank you for raising this valuable point.\nIncreased intestinal wall thickness in IBD is mainly attributed to the expansion of the intestinal smooth muscle layers and to a lesser extent to collagen deposition [DOI: 10.1038/s41575-021-00543-0]. Various factors in the inflamed mucosa could trigger the activation of SMCs mainly in the submucosa, including proinflammatory cytokines and growth factors [DOI: 10.1016/j.crohns.2014.09.008]. Activated SMCs change their phenotype and function, synthesize extracellular matrix (ECM), secrete a range of cytokines and fibrosis-related mediators, as well as growth factors and cytokines that attract more immune cells and increase SMC proliferation via paracrine or autocrine signaling [DOI: 10.1093/ibd/izab251]. The products released from these cells create a positive feedback loop that maintains the inflammatory state, and SMCs might be a promising therapeutic target in the future [DOI: 10.1093/ibd/izae292].\nIn our model, transcriptome analysis revealed upregulation of Il6, Cxcl5, and Ccl5 in Sm22α-/- compared to WT mice (Supplementary Fig. 4b), supporting this concept. In the future, we are going to investigate the potential multifactorial role of SMCs in IBD.\n13. In my opinion, the vice-versa axis, e.g. how IECs affect smooth muscle cells would be very interesting, considering the well established function of IECs in the pathophysiology of IBD. What is known about this crosstalk, and how IECs affect smooth muscle function?\nReply: We agree with the reviewer that the bidirectional communication between IECs and SMCs is of great interest. As already mentioned, various factors in the inflamed mucosa could trigger the activation of SMCs mainly in the submucosa [DOI: 10.1038/s41575-021-00543-0], including proinflammatory cytokines and growth factors, chemokines, endothelin and components of the renin-angiotensin system, lipid mediators as well as damage-associated molecular patterns (DAMPs) derived from damaged epithelial cells [DOI: 10.1016/j.crohns.2014.09.008; DOI: 10.1111/1751-2980.12879]. Upon activation, SMCs exhibit a more synthetic de-differentiated phenotype, with loss of contractile smooth muscle markers (ie, SM22a, smoothelin, αSMA) [DOI: 10.4081/ejh.2014.2457] and significantly impaired colonic smooth muscle contractility [DOI: 10.3389/fphys.2023.1215900]. Further studies are warranted to thoroughly explore the role of smooth muscle growth in IBD stricturing disease together with the molecular mechanisms and signaling pathways that accompany these changes, to design new therapeutic targets addressing this issue.\nAre you satisfied with our explanation? We hope to receive more suggestions from you.\n14. Function of smooth muscle cells, particularly contraction, is tightly regulated by the enteric nervous system. Do the used models, e.g. SM22a KO mice show changes in ENS function?\nReply: Thank you for this insightful comment. That is an interesting question.\nSorry to say, the current study did not concern the colonic nervous system changes, and thus did not examine the expression of key enteric neuron markers in the colon of Sm22α-/- mice. Changes in colonic nervous system function in the colitis model mice have been reported, demonstrating that the ENS undergoes major structural damage during inflammation, with a significant and lasting decrease in neuron number [DOI: 10.1016/S0002-9440(10)65207-8]. We have recognized the importance of the interaction between the enteric nervous system and smooth muscle cells in SM22α KO mice, and may investigate it in the future.\nYour comment has pointed out the direction for our future research, thanks again.\n15. What is known about function of SMCs in human IBD? Obviously, diarrhea and increased intestinal motility is a cardinal symptom of active inflammation in the gut, but do patients with IBD show a smooth muscle cell phenotype, for example hypertrophy of muscle layers? Also, are the factors of the proposed pathway affected in human IBD? Data in this regard would clearly improve the clinical relevance of the manuscript.\nReply: We appreciate the request for translational insights.\nRecent data highlight the crucial role of smooth muscle expansion in stricture formation in inflammatory bowel diseases (IBD) that presumably oversteps that of intestinal fibrosis. SMCs not only participate in the stricturing process but seem to orchestrate this anatomical remodeling considering their synthetic activity, their ability to change their phenotype, and to communicate with other non-immune and immune cells [DOI: 10.1093/ibd/izae292]. In UC patients, collagen deposition in the muscularis mucosa and submucosa together with thickening of the muscularis mucosa has been observed in sites of chronic inflammation and these changes may lead to luminal narrowing and the “lead pipe” appearance of the affected colon [DOI: 10.1053/j.gastro.2016.09.047]. The presence of specific growth factors and cytokines in the muscular microenvironment, either with autocrine or paracrine signaling, can lead to SMC proliferation, migration, and change of phenotype from a contractile to a secretory one with the generation of various mediators, which will subsequently contribute to intestinal fibrosis and inflammation, which suggest SMCs as the main contributors to intestinal wall thickening [DOI: 10.1093/ibd/izae292].\nIn the present study, we provided HE staining data of human colitis, which showed CSMCs were hypertrophic in UC patients compared to non-IBD controls, as shown in Supplementary Fig 7a. Increased RSAD2 expression (Supplementary Fig. 7b, c) and ferroptosis (Supplementary Fig. 7e) were observed in sites of damaged intestinal epithelium for UC patients. These data strengthen the clinical relevance of our proposed pathway.\n16. As mitochondrial dysfunction in SMCs is a central part of the proposed pathway, but mitochondrial function also central for energy metabolism: Is metabolism in the smooth muscle cells affected? Is the concept of immunometabolism, e.g. metabolic changes during inflammation, described for smooth muscle cells?\nReply: Thank you for highlighting this critical link.\nMitochondrial oxidative stress affects the changes of structural and biochemical and apoptosis in the SMCs [DOI: 10.1161/ATVBAHA.111.243121]. Oxidative stress–induced mitochondrial dysfunction drives inflammation and SMC remodeling [DOI: 10.1016/j.jaci.2015.01.046].\nIndeed, our RNA-seq indicated reduced oxidative phosphorylation and increased glycolysis in Sm22α-/- colonic smooth muscle tissue that have pro-inflammation phenotype (Fig. 3a and Supplementary Fig. 4c), consistent with an inflammatory metabolic shift.\n17. What is the physiologic benefit of the proposed mechanism? Why should smooth muscle cells interact with IECs to regulate intestinal physiology? What is the evolutionary benefit of such an interaction?\nReply: We thank the reviewer for this thought-provoking question.\nWhile speculative, we considered that CSMCs may serve as local sensors of tissue stress or barrier compromise. By releasing mitochondrial EVs, they may alert neighboring epithelial cells to initiate protective responses. This paracrine signaling axis may have evolved to coordinate muscular and epithelial responses during injury or infection.\nMinors:\n• Several abbreviations are not explained in the abstract. Also, a brief explanation of the function of each molecule (e.g., SM22a, RSAD2) in the abstract would be helpful\nReply: We have defined key abbreviations (SM22α, RSAD2, SMC, IEC) when they first time appear.\n• Many statements in the introduction part are without references. Was this part generated using AI tools or large language models? References should be added to all these different statements\nReply: In the revised Introduction, key statements have been supported by citations.\n• Is the second sentence of the introduction (UC-CD dichotomy) really needed in the context of this manuscript?\nReply: The sentence (UC-CD dichotomy) has been removed in the revised manuscript.\n• Quantification (densitometry) of several immunoblots in the manuscript should be added in addition to representative blots\nReply: All immunoblots have analyzed by densitometric quantification.\n• How were colonic smooth muscle tissues for Figure 3a collected? I could not find this in the methods sections\nReply: The isolation method of colonic smooth muscle tissues for Figure 3a was shown in the SUPPLEMENTAL METHODS section."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The manuscript presented by Han et al. described an interesting mechanism by which CSMC contribute to intestinal inflammation. This involves the RSAD2, YTHDF1, DRP1 dependent mitochondrial dysfunction, which then results in the ferroptosis of IECs, due to the communication between SMC and IECs via MitoEVs. This mechanism could, at least partially translated to the human situation in IBD patients.\nThis is of high novelty and provides a mechanism with high potential to be exploited in the context of IBD. The data are sound, generated using standard but adequate technologies and clearly\nsupport the conclusions drawn by the authors.\nThe revised version of the manuscript has been improved in several ways: additonal literature has been added to the introduction and the discussion; n numbers or biological replicates are now described in figure legends; new data has been included, such as the validation of the mouse model, the quantification of TUNEL with a corresponding counterstaining, or parameteres depicting mitochondria fragmentation; higher magnification pictures for histology. On the other hand, I still think the language is sometimes not optimal, for instance in new paragraphs from the Introduction, and some sentecnes are not fully correct.\nFrom a more conceptual point of view, there are other points which have not been addressed in the revised version of the manuscript. As an example, we understand that the communication between CSMC to other cells within the intestinal mucosa might be the focus of other studies, sicne this definitely go byonf the scope of the study. However, I still do not see the point of \"ignoring\" the effect on the microbiota shown by the RNAsequencing data, or the focus on RSAD2. Althought the authors mentioned that this is the focus of other studies, I think this should be somehow reflected, maybe in the discussion, and a clear justification of the independence of those mechanisms to the one suggetsed in teh manuscript should be provided.", "reviewer_2": "Thank you for your response to my suggestions. I do not have any additional suggestions anymore.", "reviewer_3": "Zhang et al have revised their manuscript by discussing and experimentally addressing several points raised by the reviewers. The refined manuscript includes a thoroughly adapted abstract and introduction, and new data investigating specific epithelial subtypes, smooth muscle cell function and markers of ferroptosis. The current version of the manuscript is clearly improved in my opinion, following points should still be addressed:\n- While both the abstract and introduction are clearly improved, due to complexity of the proposed mechanism and abbreviations it is still difficult to follow. A common thread and step-wise approach for every new mechanistic link with clear rationales should be included to further improve readability. For example, in the third sentence of the abstract, SM22a is introduced, but no rational why this specific molecule is studied (or important in SMC function). This should be addressed throughout the manuscript, but most importantly in the abstract.\n- As SM22a-deficient mice develop spontaneous colitis, small intestine at baseline should be investigated and included into the current study.\n- The authors now include a discussion of limitations of the models (e.g., promoters that are not specific to colonic smooth muscle cells). I feel like this should be extended.\n- As the key aspect of the paper relates to function of smooth muscle cells in IBD, this section should be expanded in the introduction in my opinion. Probably you can shorten the first paragraph as information included in there appears to be less important for your study in my opinion.\nMinors:\n- Phrasing and grammar should be reviewed carefully throughout the manuscript."}, "author_response": "Response to reviewers:\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe manuscript presented by Han et al. described an interesting mechanism by which CSMC\ncontribute to intestinal inflammation. This involves the RSAD2, YTHDF1, DRP1 dependent\nmitochondrial dysfunction, which then results in the ferroptosis of IECs, due to the\ncommunication between SMC and IECs via MitoEVs. This mechanism could, at least partially\ntranslated to the human situation in IBD patients.\nThis is of high novelty and provides a mechanism with high potential to be exploited in the\ncontext of IBD. The data are sound, generated using standard but adequate technologies and\nclearly support the conclusions drawn by the authors.\n1. The revised version of the manuscript has been improved in several ways: additonal literature\nhas been added to the introduction and the discussion; n numbers or biological replicates are now\ndescribed in figure legends; new data has been included, such as the validation of the mouse\nmodel, the quantification of TUNEL with a corresponding counterstaining, or parameteres\ndepicting mitochondria fragmentation; higher magnification pictures for histology. On the other\nhand, I still think the language is sometimes not optimal, for instance in new paragraphs from the\nIntroduction, and some sentences are not fully correct.\nReply: Thank you for taking the time to review our manuscript. We appreciate your valuable\ncomments and suggestions. We have read carefully and responded your comments point by point\nas listed below. All revisions were clearly marked with blue. The language of the manuscript has\nbeen improved by Springer Nature Author Services (Language Editing: Gold).\n2. From a more conceptual point of view, there are other points which have not been addressed in\nthe revised version of the manuscript. As an example, we understand that the communication\nbetween CSMC to other cells within the intestinal mucosa might be the focus of other studies,\nsicne this definitely go byonf the scope of the study. However, I still do not see the point of\n\"ignoring\" the effect on the microbiota shown by the RNAsequencing data, or the focus on\nRSAD2. Althought the authors mentioned that this is the focus of other studies, I think this should\nbe somehow reflected, maybe in the discussion, and a clear justification of the independence of\nthose mechanisms to the one suggetsed in the manuscript should be provided.\nReply: Thank you for your suggestion.\nIn the third paragraph of the revised Introduction, we stated reason why the focus on RSAD2.\nMeantime, in the discussion, we provided a clear justification of the independence of reported\nmechanisms to the one suggested in the manuscript. Furthermore, the relationship between the\nmicrobiota and inflammation and impaired mucosal barrier function in Sm22α-/- mice was shown\nin the first paragraph of the discussion.\nReviewer #3 (Remarks to the Author):\nZhang et al have revised their manuscript by discussing and experimentally addressing several\npoints raised by the reviewers. The refined manuscript includes a thoroughly adapted abstract and\nintroduction, and new data investigating specific epithelial subtypes, smooth muscle cell function\nand markers of ferroptosis. The current version of the manuscript is clearly improved in my\nopinion, following points should still be addressed:\n- While both the abstract and introduction are clearly improved, due to complexity of the proposed\nmechanism and abbreviations it is still difficult to follow. A common thread and step-wise\napproach for every new mechanistic link with clear rationales should be included to further\nimprove readability. For example, in the third sentence of the abstract, SM22a is introduced, but\nno rational why this specific molecule is studied (or important in SMC function). This should be\naddressed throughout the manuscript, but most importantly in the abstract.\nReply: Thank you for your comments. We apologize for our language defects.\nIn the revised abstract, we stated the important of SM22α in SMC function. The common thread\nand step-wise approach for every new mechanistic link with clear rationales have been addressed\nthroughout the manuscript. Furthermore, the language of the manuscript has been improved by\nSpringer Nature Author Services (Language Editing: Gold).\n- As SM22a-deficient mice develop spontaneous colitis, small intestine at baseline should be\ninvestigated and included into the current study.\nReply: Thank you for your suggestion.\nThe small intestines of Sm22α-deficient mice at baseline have been investigated and included\ninto the current study.\nIntestinal transit time was estimated by the time required for the fecal output and intestinal\ndistribution of the marker, carmine red that was orally administered in mice, as shown in\nSupplementary methods.\nWe found that the length of the small intestines shortened and the small intestinal transit times\nof the marker that was orally administered, extended in Sm22α-/- mice compared with WT mice at\nbaseline (Supplementary Fig. 2a-c). The small intestinal wall, especially the ileum, was\nsignificantly damaged with higher histological score in Sm22α-/- mice (Supplementary Fig. 2d).\n- The authors now include a discussion of limitations of the models (e.g., promoters that are not\nspecific to colonic smooth muscle cells). I feel like this should be extended.\nReply: Thank you for your comments.\nThe revised manuscript has now included a discussion of limitations of the models, as shown\nbelow:\nGiven the importance of the muscular component in IBD, inducible mouse model of IBD by\ncolonic smooth muscle-specific inactivation of the Sm22α gene is necessary in future study.\n- As the key aspect of the paper relates to function of smooth muscle cells in IBD, this section\nshould be expanded in the introduction in my opinion. Probably you can shorten the first\nparagraph as information included in there appears to be less important for your study in my\nopinion.\nReply: We agree with your proposal.\nThe function of smooth muscle cells in IBD has been expanded in the introduction. The\ninformation in the first paragraph was remained according to Reviewer 1#’s suggestion.\nMinors:\n- Phrasing and grammar should be reviewed carefully throughout the manuscript.\nReply: The language of the manuscript has been improved by Springer Nature Author Services\n(Language Editing: Gold)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed my comments by, at least, explaining or justifying the focus on RSAD2 and the relevance of dysbiosis.", "reviewer_3": "The manuscript was adopted according to my suggestions, especially by providing more context and additional rationale for the studies. I don’t have additional comments."}, "author_response": "Reply: Thanks for your acceptance.\nReply: Thanks for your acceptance."}]

Decision letter