VRAC coordinates the trade-off between nutrient absorption and antimicrobial defense in enterocytes against inflammation

Domain: chemistry
Published: 2026-02-25
Editorial outcome: minor_revision

Abstract
Enterocytes mediate both nutrient absorption and antimicrobial peptide (AMP) secretion while constantly exposed to osmotic fluctuations. However, the role of volume-regulated anion channel (VRAC), a key osmo-sensitive ion channel, in enterocytes and inflammatory bowel disease (IBD) remains unclear. Here, we show that intestinal epithelial cell (IEC)-specific knockout of LRRC8A, the essential VRAC subunit, exacerbates colitis and inflammation-associated colorectal cancer. VRAC deficiency specifically disrupts enterocyte maturation and zonation, expanding AMP-producing enterocytes while reducing enterocytes responsible for nutrient absorption. Retinoic acid metabolism emerges as the most affected nutrient pathway in VRAC-deficient IECs, with reduced Adh1, Aldh1a2 expression and aldehyde dehydrogenase activity. Supplementation with retinoic acid reversed inflammation caused by VRAC deficiency. Conversely, VRAC deficiency induced gut microbiota dysbiosis, while administration of Lactobacillus species effectively restored microbial balance and alleviated inflammation. This study delineates the role of VRAC in balancing nutrient absorption and antimicrobial defense in enterocytes to safeguard gut homeostasis.

Introduction
Inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic, relapsing disorder of the gastrointestinal tract and represents key risk factors for inflammation-associated colorectal cancer (CAC)1,2. Although the precise pathogenesis of IBD remains unclear, multiple factors have been implicated, including dysregulated immune responses, alterations in the gut microbiota, genetic predisposition, and environmental triggers.

Intestinal epithelial cells (IECs)—comprising stem cells, enterocytes, goblet cells, enteroendocrine cells, Paneth cells, and tuft cells—form a physical and functional barrier that not only separates luminal contents from the underlying mucosa but also regulates nutrient absorption and barrier integrity3. A growing body of evidence highlights the critical role of intestinal epithelial dysfunction in the initiation and progression of IBD, serving as a key interface that links luminal insults to aberrant immune activation and chronic inflammation4–6. Dysfunction in IEC differentiation, IEC-microbe interactions, or IEC-immune crosstalk plays a critical role in the pathogenesis of IBD or colorectal cancer7,8.

Among different types of IECs, enterocytes, which constitute approximately 90% of IECs, are the most prominent type and are responsible for nutrient and water absorption, and antimicrobial defense9,10. Importantly, enterocytes in both the small intestine and colon exhibit a spatially organized transcriptional and functional zonation pattern along the crypt–surface axis11–14. At the crypt base, enterocytes predominantly express genes involved in host defense, such as antimicrobial peptides (AMPs) and metal-ion transporters. As cells migrate upward, mid-zone enterocytes specialize in amino acid, vitamin, and carbohydrate absorption, while surface-located enterocytes are primarily responsible for lipid uptake11,12. Although nutrient malabsorption has been closely linked to the development and progression of IBD, the molecular mechanisms that coordinate enterocyte function in nutrient absorption and antimicrobial defense—especially under inflammatory stress—remain poorly defined.

Mammalian cells need to regulate their volume by transporting ions and small organic osmolytes via ion channels and transporters to counteract osmotic swelling or shrinkage13,14. The volume-regulated anion channel (VRAC) plays a pivotal role in restoring normal cell volume by mediating the efflux of chloride in response to cellular swelling14. VRAC is a heteromeric channel composed of subunits from the LRRC8 protein family (LRRC8A-E), with LRRC8A (also named SWELL1) being essential and required to heteromerize with at least one other subunit (LRRC8B–E) to form a functional channel15,16. In addition to transporting Cl−, VRAC is capable of transporting a wide variety of substances, including inositol, taurine, amino acids, and immune metabolites17–21. Notably, the specific heteromeric composition of VRAC subunits determines the channel’s distinct biophysical properties and physiological roles22,23. Serving as the largest barrier surface in the body, IECs are constantly exposed to significant osmolarity alterations caused by dietary intake, digestion, and various pathophysiological conditions24,25. However, little is known about the role of VRAC in regulating IEC function and its involvement in intestinal inflammation. In this study, we systematically investigated VRAC function in IECs using a combination of IEC-specific Lrrc8a conditional knockout mice and multiple inflammation models, including dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis, as well as the azoxymethane (AOM)/DSS-induced inflammation-associated cancer model. We reveal that epithelial VRAC plays a critical role in preserving intestinal homeostasis under inflammatory conditions. Furthermore, deletion of Lrrc8d, encoding another key subunit of VRAC, also aggravated colitis, underscoring the functional importance of LRRC8A/8D-containing VRAC complexes in IECs. Mechanistically, we demonstrate that VRAC is specifically required for enterocyte maturation and zonation, as shown by scRNA-seq and organoid analyses. VRAC deficiency results in enterocyte reprogramming toward antimicrobial defense at the expense of nutrient absorption. Retinoic acid metabolism was the most impaired nutrient absorption pathway in VRAC-deficient IECs, and its supplementation alleviated inflammation. Conversely, VRAC deficiency induced gut microbiota dysbiosis, notably reducing Lactobacillus species, while Lactobacillus administration mitigated inflammation caused by IEC VRAC loss. Collectively, our findings identify VRAC as a critical rheostat that coordinates nutrient absorption and antimicrobial defense in enterocytes, thereby maintaining intestinal homeostasis during inflammation.

Results
Intestinal VRAC loss exacerbates colitis and inflammation-associated colorectal cancer
To investigate the potential role of VRAC in IEC homeostasis and intestinal inflammation, we analyzed the expression levels of VRAC subunits in healthy individuals and IBD patients using published RNA-seq data from the GEO database. The data demonstrate that the expression of LRRC8A, the essential subunit of VRAC, was increased in the colon of IBD patients compared to healthy donors (Fig. S1A, B). LRRC8D, another key VRAC subunit, also exhibited high expression relative to other VRAC components, which indicates a potential link between VRAC and the development of IBD. Besides, immunohistochemistry showed slightly enhanced epithelial LRRC8A staining in CD biopsies, and this was accompanied by a modest increase in overall LRRC8A protein levels (Fig. S1C, D).

To determine the effects of VRAC in IECs and IBD, we generated IEC-specific Lrrc8a-knockout mice by crossing Lrrc8afl/fl mice with Villin1 (Vil1)-Cre transgenic mice26. These mice were hereinafter referred to as Lrrc8aΔIEC mice, whereas littermate Lrrc8afl/fl mice were used as WT controls (Fig. S2A). The loss of Lrrc8a in IECs of Lrrc8aΔIEC mice was validated by qPCR and Western blot (Fig. S2B, C). The body weight, water intake, and spleen weight were similar between WT and Lrrc8aΔIEC mice (Fig. S2D–F). Besides, the deletion of LRRC8A in IECs had no significant effect on fecal weight or water content (Fig. S2G). Histology and tight-junction markers of the colon and distal ileum also showed no overt abnormalities between WT and Lrrc8aΔIEC mice (Fig. S2H–K). These suggest VRAC in IECs is dispensable for the functionality of intestinal epithelia in the steady state. To elucidate the potential role of VRAC in IECs upon inflammatory stress, WT and Lrrc8aΔIEC mice were then subjected to DSS-induced colitis27. Of note, greater body weight loss and higher disease activity index (DAI) upon challenge with 1.5% or 2.5% DSS was observed in Lrrc8aΔIEC mice compared to WT mice (Fig. 1A, B). The colon length of Lrrc8aΔIEC mice was much shorter than that of WT mice upon DSS challenge, indicating more severe inflammatory damage (Fig. 1C). Moreover, the lethality in the Lrrc8aΔIEC mice subjected to 3% DSS was significantly increased (Fig. 1D). Furthermore, Lrrc8aΔIEC mice exhibited heightened epithelial erosion and inflammation, along with a pronounced reduction in mucus-secreting goblet cells after DSS treatment (Fig. 1E, F). To further validate the role of VRAC in intestinal inflammation, we established a TNBS-induced colitis model, which closely mimics the immunepathology of CD. Lrrc8aΔIEC mice also exhibited reduced colon length and enhanced inflammation, together with greater body weight loss (Fig. S3). Taken together, these findings suggest that intestinal VRAC plays a vital protective role in the development and progression of colitis.

VRAC and cell volume regulation have been shown to transport specific immune metabolites such as cGAMP and itaconate, thereby playing key roles in both innate and adaptive immunity28,29. Thereby, Lrrc8afl/fl mice were also crossed with LysM-Cre mice or CD4-Cre mice, to achieve conditional ablation of Lrrc8a in myeloid cells (Lrrc8aΔLysM) or T cells (Lrrc8aΔCD4), respectively (Fig. S2A). However, the results demonstrate that mice lacking Lrrc8a in either myeloid cells or T cells exhibited comparable weight loss, DAI, colon shortening, and histopathological features compared to control littermates upon DSS challenge (Fig. 1G–L). These findings emphasize that it is intestinal epithelial VRAC, rather than immune cell-intrinsic VRAC, that plays a critical role in protecting against intestinal inflammation.

IBD is a well-established risk factor for the development of inflammation-associated colorectal cancer30. We further explored the role of intestinal VRAC in the development of inflammation-associated colorectal cancer by using AOM combined with low concentrations of DSS (1%), given that Lrrc8aΔIEC mice tended to succumb to DSS exposure. Lrrc8aΔIEC mice exhibited significantly more body weight loss during DSS treatment (Fig. 2A). At day 80 post-AOM-DSS treatment, colons of Lrrc8aΔIEC mice developed a markedly increased tumor burden, including higher tumor numbers and larger tumor sizes (Fig. 2B–D). Histological analysis revealed a greater extent of inflammation and epithelial hyperplasia in Lrrc8aΔIEC mice (Fig. 2E–G). Taken together, our data demonstrate a crucial role of intestinal VRAC in protecting the intestine against inflammatory stimuli, including colitis and inflammation-associated colorectal cancer.

Lrrc8aΔIEC mice exhibited enhanced inflammatory responses and intestinal epithelial damage
Next, we performed bulk RNA-seq analysis using colon RNAs from WT and Lrrc8aΔIEC mice after DSS challenge. In Lrrc8aΔIEC mice, 110 genes were notably downregulated, while 355 genes were significantly upregulated (|log FC|>1; adjusted P<0.05) (Fig. 3A). Gene ontology (GO) analysis by Enrichr showed a significant upregulation associated with inflammatory response genes, neutrophil migration, and antimicrobial response. Whereas, genes involved in lipid transport and ion transport exhibited a noticeable reduction in Lrrc8aΔIEC mice (Fig. 3B). Inflammatory genes (e.g., Cxcl10, Il6) were significantly increased, as confirmed via qPCR, in Lrrc8aΔIEC mice subjected to DSS or AOM-DSS treatment (Fig. 3C, E). Besides, the expression levels of AMPs such as calprotectin subunits S100a8 and S100a9, were markedly elevated in Lrrc8aΔIEC mice (Fig. 3D). Moreover, increased apoptosis and reduced epithelial cell proliferation in Lrrc8aΔIEC mice were observed by enhanced staining for TUNEL along with decreased staining for the proliferation marker Ki67, demonstrating impaired intestinal healing (Fig. 3F). Thus, the loss of VRAC results in enhanced inflammatory and antimicrobial responses while decreasing ion and lipid transport under intestinal inflammatory stress.

Given the central role of intestinal barrier dysfunctions in intestinal inflammation, we examined whether IEC VRAC deficiency compromised the barrier integrity by analyzing the distribution or expression of tight-junction protein zonula occludens-1 (ZO-1), Occludin, and Claudin-2. Consistently, colons from Lrrc8aΔIEC mice exhibited notably disrupted tight junctions (Fig. 3G), suggesting a compromised mucosal barrier in Lrrc8aΔIEC mice. This was further confirmed by the elevated fluorescence in the serum of Lrrc8aΔIEC mice fed with FITC-labeled dextran (MW 4000), indicative of colonic leakage (Fig. 3H). The data reveal that VRAC in IECs is crucial for maintaining the intestinal barrier and provides significant protection against colitis and CAC.

Intestinal VRAC is specifically required for enterocyte maturation and zonation
To explore the underlying mechanism by which IEC VRAC protects against intestinal inflammation, we analyzed different VRAC subunits in IECs using single-cell RNA sequencing (scRNA-seq) data of human colon epithelial cells reported by Smillie et al.31. The data demonstrate that LRRC8A was predominantly expressed in enterocytes, as well as in goblet cells (Fig. 4A, B). In line with our analysis of published GEO data, LRRC8A and LRRC8D were the primary subunits of VRAC in human IECs (Fig. 4B). Besides, the expression of LRRC8A and LRRC8D in enterocytes was increased in colitis patients compared to healthy donors (Fig. 4C). We also induced DSS colitis in Lrrc8d−/− mice to assess the role of LRRC8D in intestinal inflammation. Compared to controls, Lrrc8d−/− mice exhibited significantly worsened colitis symptoms (Fig. S4A–D). This suggests that while LRRC8A serves as the essential subunit of IEC VRAC, LRRC8D may act as another subunit critical for IEC VRAC functions. Notably, compared to heteromers containing LRRC8A combined with LRRC8C or LRRC8E, LRRC8A/8D-containing VRAC exhibits significantly higher permeability to large organic anions or uncharged osmolytes32,33. This unique biophysical property underscores the importance of LRRC8A/8D heteromers in mediating organic osmolytes transport and maintaining IEC homeostasis, positioning them as critical regulators of IEC physiology. However, Lrrc8d−/− mice exhibited increased consumption of DSS-containing water, although the difference was not statistically significant (Fig. S4E). This observation may reflect the essential role of VRAC in renal function34. We therefore focused on LRRC8A’s role in regulating IEC function in the present study. In WT and Lrrc8aΔIEC mice post-DSS challenge, the expression of mature enterocyte markers (Slc26a3, Alpi) was significantly decreased in colons isolated from Lrrc8aΔIEC mice (Fig. 4D). In contrast, there were no notable expression changes in markers associated with stem cells (Lgr5, Ephb3), goblet cells (Muc2, Tff3), tuft cells (Dclk1, Trpm5), or enteroendocrine cells (Chga), supported by qPCR and bulk RNA-seq data (Fig. S5A, B). We further investigated the impact of epithelial VRAC on intestinal proliferation and turnover by measuring crypt Ki67 expression and tracking 12 h and 24 h 5-Ethynyl-2-deoxyuridine (EdU)+ labeling under homeostatic conditions. The number of Ki67+ cells in the crypts, as well as epithelial turnover rate, were compared between WT and Lrrc8aΔIEC mice (Fig. S5C–F), suggesting that epithelial LRRC8A deficiency does not affect intestinal stemness or general differentiation, but specifically impairs the maturation and function of enterocytes.

We also generated LRRC8A-knockout Caco-2 cells (Caco-2ΔLRRC8A) and verified loss of VRAC function by electrophysiology (Fig. S6A, B). Swelling-activated currents displayed the characteristic voltage-dependent inactivation of VRAC at positive potentials, with tail currents upon repolarization to −80 mV reflecting recovery from inactivation. Loss of LRRC8A led to a marked reduction in TEER (Fig. S6C). In parallel, ZO-1 staining revealed increased junctional tortuosity and reduced signal intensity, indicative of tight-junction disruption (Fig. S6D). Notably, re-expression of LRRC8A partially rescued these barrier defects (Fig. S6E, F).

Along the colonic crypt–surface axis, enterocytes (clusters C1–C5) exhibit a spatially organized zonation program characterized by antimicrobial peptide (AMP) secretion and metal-ion handling at the crypt base, followed by progressive acquisition of metabolic and absorptive functions toward the luminal surface (Fig. 4E)11,35. Given the loss of IEC VRAC, specifically decreased enterocyte markers, we were prompted to explore the impact of VRAC on enterocyte zonation following DSS exposure, using enterocyte zonation genesets derived from the Moor et al.11 dataset. Our results indicated that the absence of IEC VRAC disrupts enterocyte zonation, causing a shift away from middle-top zone enterocytes toward a bottom-associated program characterized by elevated expression of AMP and metal-ion transporters (Fig. 4E, F).

To mimic the regenerative capacity of intestinal tissue and its ability to restore homeostasis following damage in vitro36, organoids were derived from the small intestine of WT and Lrrc8aΔIEC mice. The surface area measurements and budding structure counts revealed no significant differences between WT and Lrrc8aΔIEC organoids samples, indicating that the proliferation or complexity of organoids was unaltered upon loss of VRAC in IECs (Fig. S7A, B). Immunofluorescence analysis of organoids for lineage-specific markers revealed a significant reduction in the enterocyte marker Alpi in Lrrc8aΔIEC organoids compared to WT controls, whereas expression of the stem cell marker Lgr5 and goblet cell marker Muc2 remained comparable between genotypes (Fig. 4G). Overexpression of LRRC8A in organoids suppressed Alpi expression, consistent with the reduction of VRAC currents caused by LRRC8A overexpression in WT cells15. Notably, re-expression of LRRC8A in Lrrc8aΔIEC organoids restored Alpi expression, as shown by immunofluorescence and qPCR (Fig. 4H). These results indicate that while IEC VRAC is not essential for stemness, proliferation, or lineage allocation in intestinal organoids, it plays a specific role in promoting enterocyte differentiation and functional identity.

In addition, scRNA-seq was conducted using colonic epithelial cells pooled from 2 pairs of WT and Lrrc8aΔIEC mice at 8 weeks of age. We profiled 2787 Lrrc8afl/fl cells and 4461 Lrrc8aΔIEC cells. Based on gene expression profiles, the cells were clustered into 10 distinct clusters (Clusters 1–10), which were categorized into six major cell populations.

Methods

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_2": "In this study the authors investigate the role of a volume-regulated anion channel (VRAC) in intestinal epithelium. They\ncleary demonstrate that knock-out of subunits of this channel abolish (as it should) volume-activated anion currents. KO mice\nwith an intestinal-specific knock-out of the LRRC8A subunit seemed to have a normal physiology, but were much more\nsensitive to inflammatory damage in classical IBD models. Changes in the expression of genes involved in retinoic acid\nmetabolism and beneficial effects of retinoic acid supplementation support the idea that this metabolite may play a central\nrole in the changes observed.\nWhile this key message is clearly supported by the (quite large) number of different methodological approaches, there\nremain some points to be clarified/rewritten to increase the impact of the paper.\nIn detail:\n- p.23, line 666: Why did the hypotonic solution contain CsCl, whereas the isotonic solution contained KCl? With 140 mM\nCsCl in the pipette, I would not assume that you need in addition an extracellular K+ channel blocker.\n- Fig. S2: What was the reversal current of the swelling-induced current? It cannot be recognized from your ramp experiment.\nThe reversal potential would give you some hint about the selectivity of the conductance. In addition, the tail currents at the\nend of your voltage-ramp should be discussed.\n- Figure 3: Main components determining the permeability of the tight junctions are claudins. Did you observe any changes\nin the claudin expression pattern?\n- p.12, line 343: I do not think that the conclusion \"... crucial role of gut microbiota in the protective role of VRAC...\" is correct.\nIn all classical IBD models, you need gut microbiota to induce a colitis.\n- Fig. 4E: You show here on the left enterocyte distribution from crypt to villus in the SMALL intestine (and refer to the paper\nfrom Moor et al., working with proximal jejunum). However, the legend describes that in the right part of Fig. 4E your data\nwere collected from colon. There are no villi in the colon, there are only crypts and surface epithelium and transporters\nexpressed strongly differ between small and large intestine. In the whole manuscript, you must indicate whether your data\nrefer to small or large intestine.", "reviewer_3": "Yi and colleagues report in their manuscript # NCOMMS-25-28940 that knocking out the essential volume-regulated 30\nanion channel (VRAC) unit LRRC8A in epithelial cells of mice aggravates colitis and colitis-associated cancer. The\nexacerbated colitis phenotype in these mice is associated with a disturbed enterocyte maturation and an altered gut\nmicrobiota. Retinoid acid supplementation or lactobacillus administration partially reversed the observed phenotype in these\nmice.\nMajor points\nTo better understand the role of VRAC in immunomodulatory metabolite transport, the authors should quantify extracellular\nlevels of known VRAC-exported metabolites such as cGAMP, itaconate, and glutamate. These measurements should be\nconducted in conditioned media from WT and Lrrc8aΔIEC organoids or colon explants, both under basal conditions and\nafter DSS exposure, as well as in serum and fecal samples from DSS-treated WT and knockout mice. If VRAC facilitates the\nexport of anti-inflammatory metabolites, such as itaconate, its absence may cause intracellular retention, thereby\nexacerbating inflammation. To functionally validate this mechanism, treating Lrrc8aΔIEC mice with exogenous itaconate or\ncGAMP could be used to assess whether the inflammatory phenotype can be rescued.\nTo further substantiate the link between VRAC and epithelial-immune crosstalk, co-culture experiments should be performed\nusing WT or Lrrc8aΔIEC intestinal epithelial cells (IECs) with bone marrow-derived macrophages (BMDMs), in the presence\nor absence of DSS. The impact on immune modulation can be assessed by evaluating macrophage polarization (e.g., M1\nvs. M2 markers) and cytokine secretion profiles. Additionally, neutrophil chemotaxis assays should be conducted to\ndetermine whether supernatants from Lrrc8aΔIEC IECs enhance neutrophil migration, potentially contributing to the\nheightened immune infiltration observed in vivo.\nThe authors should also investigate whether VRAC upregulation is protective in human IBD. This could be achieved by\nperforming immunohistochemical (IHC) staining for LRRC8A in colonic biopsies from IBD patients and correlating\nexpression levels with disease severity, tight junction integrity (e.g., ZO-1), and epithelial apoptosis (e.g., TUNEL staining).\nTo determine whether barrier loss in Lrrc8aΔIEC mice is linked to osmotic imbalance, transepithelial electrical resistance\n(TEER) should be measured in WT and knockout colonoids under both isotonic and hypotonic conditions. A rescue\nexperiment using hypertonic media could help clarify whether restoring osmotic balance ameliorates tight junction disruption\nand improves barrier integrity.\nThe observation that VRAC loss leads to increased tumor burden despite elevated epithelial apoptosis raises an important\nmechanistic question. How can the authors explain these observations?\nTo explore the mechanistic link between VRAC and enterocyte maturation, rescue experiments should be performed using\nLrrc8aΔIEC IEC organoids. Specifically, re-expression of LRRC8A and LRRC8D via lentiviral transduction could help\ndetermine whether the expression of mature enterocyte markers, such as Alpi and Slc26a3, can be restored. Additionally,\nmeasuring the transport of organic osmolytes, such as taurine uptake, would provide a functional readout for enterocyte\nmaturation and VRAC activity.\nTo investigate VRAC-dependent zonation mechanisms, spatial transcriptomics could be applied to colon sections from WT\nand Lrrc8aΔIEC IEC mice to map changes in epithelial zonation with high spatial resolution. Complementary experiments\nusing intestinal organoids treated with modulators of the Wnt and BMP pathways could reveal whether VRAC deficiency\nalters responsiveness to these critical developmental gradients.\nTo assess the functional consequences of disrupted epithelial zonation, nutrient absorption capacity should be quantified by\nmeasuring glucose and fatty acid uptake in Lrrc8aΔIEC enteroids. These findings can be further validated using Ussing\nchamber assays on WT versus knockout colon explants. Metabolomic profiling of luminal contents via LC-MS could then be\nused to correlate epithelial functional impairments with alterations in intestinal metabolic output.\nThe authors show impaired RA synthesis based on reduced expression of biosynthetic enzymes and RA-responsive genes.\nHowever, direct quantification of RA levels in IECs and colon tissues from WT and Lrrc8aΔIEC mic using LC-MS or ELISA\nwould provide crucial biochemical validation.\nTo confirm that the observed defects in RA metabolism are epithelial-intrinsic, organoid cultures from WT and Lrrc8aΔIEC\nmice should be treated with retinol, and the capacity to generate RA (via ALDH activity or RA levels) should be compared in\nvitro.\nThe authors should assess RAR/RXR transcriptional activity directly (e.g., using RA response element–luciferase reporters)\nin WT and VRAC-deficient IECs ± atRA to demonstrate the functional consequences of impaired RA signaling.\nWhile the authors demonstrate reduced IL-17A/IL-22 production, they should investigate whether this defect is rescued by\nconditioned media from atRA-treated Lrrc8aΔIEC organoids, thereby supporting an epithelial-immune signaling axis.\nThe role of the microbiota would be more rigorously validated by transferring fecal microbiota from WT or Lrrc8aΔIEC mice\ninto germ-free recipients and assessing the severity of colitis after DSS treatment. This would test whether the microbiota\nalone is sufficient to transfer susceptibility or protection.\nTo isolate the impact of RA-modified microbiota, fecal transplants from RA-treated Lrrc8aΔIEC mice should be tested for\ntheir ability to mitigate DSS-induced colitis when transplanted into antibiotic-pretreated WT or KO hosts.\nThe rescue effects of L. murinus and L. reuteri should be compared\nmechanistically. For instance, do both strains restore ALDH activity, suppress AMP expression, and modulate immune responses in a similar manner? Do they influence RA\nlevels directly or via a secondary mechanism (e.g., bile acid modulation, SCFA)?\nMinor points\nSpecify which IBD subtypes (UC, CD, or both) were analyzed in Fig. S1.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We sincerely thank all the Reviewers for their insightful and constructive comments.\nAs detailed in the point-by-point responses below, we have performed new experiments\nto address the specific concerns raised by each Reviewer. We believe that these\nadditional data have substantially strengthened the manuscript. All newly generated\nresults have been incorporated into the revised manuscript.\nReviewer #2\nIn this study the authors investigate the role of a volume-regulated anion\nchannel (VRAC) in intestinal epithelium. They cleary demonstrate that knock-\nout of subunits of this channel abolish (as it should) volume-activated anion\ncurrents. KO mice with an intestinal-specific knock-out of the LRRC8A subunit\nseemed to have a normal physiology, but were much more sensitive to\ninflammatory damage in classical IBD models. Changes in the expression of\ngenes involved in retinoic acid metabolism and beneficial effects of retinoic acid\nsupplementation support the idea that this metabolite may play a central role in\nthe changes observed.\nWhile this key message is clearly supported by the (quite large) number of\ndifferent methodological approaches, there remain some points to be\nclarified/rewritten to increase the impact of the paper.\nResponse: We thank the Reviewer for the time and effort dedicated to evaluating our\nmanuscript, and we sincerely appreciate the positive and encouraging comments.\n1. - p.23, line 666: Why did the hypotonic solution contain CsCl, whereas the\nisotonic solution contained KCl? With 140 mM CsCl in the pipette, I would not\nassume that you need in addition an extracellular K+ channel blocker.\nResponse: We thank the Reviewer for this thoughtful comment. We agree that the use\nof a CsCl-based pipette solution is sufficient to block the major potassium currents\nactivated under hypotonic conditions. The additional replacement of extracellular KCl\nwith CsCl was intended to further eliminate any potential residual contribution of\npotassium currents during VRAC recordings. Similar intra- and extracellular CsCl-\nbased solutions have been used in previous publications 1,2.\n2. - Fig. S2: What was the reversal current of the swelling-induced current? It\ncannot be recognized from your ramp experiment. The reversal potential would\ngive you some hint about the selectivity of the conductance. In addition, the tail\ncurrents at the end of your voltage-ramp should be discussed.\nResponse: The reversal potential of the swelling-induced current was approximately -\n6.56 mV (Fig. S2A), which is consistent with our previous studies2,3 and also confirms\nthat the current is predominantly mediated by Cl⁻ ions.\nTime- and voltage-dependent inactivation at membrane potentials above +40 mV\nis a characteristic electrophysiological feature of VRAC 4. Recovery from inactivation\nis facilitated by negative membrane potentials, and the tail currents observed at –80 mV\nreflect the recovery from inactivation. Similar tail-current behaviors have been reported\nin multiple electrophysiological studies of VRAC 5,6. A brief explanation of the tail\ncurrents has been incorporated into the revised manuscript.\n1\n3. - Figure 3: Main components determining the permeability of the tight\njunctions are claudins. Did you observe any changes in the claudin expression\npattern?\nResponse: Thank you for this valuable suggestion. We performed additional\nimmunofluorescence staining for Claudin-2. Under homeostatic conditions, Claudin-2\nexpression and localization were comparable between WT and Lrrc8aΔIEC mice\n(revised Fig. S2K). DSS treatment reduced Claudin-2 expression in control mice, and\nthis loss was markedly intensified in Lrrc8aΔIEC mice (revised Fig. 3G), demonstrating\nthat VRAC deficiency exacerbates inflammation-induced tight-junction damage.\n4. - p.12, line 343: I do not think that the conclusion \"... crucial role of gut microbiota in the protective role of VRAC...\" is correct. In all classical IBD\nmodels, you need gut microbiota to induce a colitis.\nResponse: We thank the Reviewer for this suggestion. In accordance with this comment,\nthe text has been revised to: “…these findings demonstrate that the heightened colitis\nsusceptibility in Lrrc8aΔIEC mice is microbiota-dependent…”.\n5. - Fig. 4E: You show here on the left enterocyte distribution from crypt to villus\nin the SMALL intestine (and refer to the paper from Moor et al., working with\nproximal jejunum). However, the legend describes that in the right part of Fig.\n4E your data were collected from colon. There are no villi in the colon, there are\nonly crypts and surface epithelium and transporters expressed strongly differ\nbetween small and large intestine. In the whole manuscript, you must indicate\nwhether your data refer to small or large intestine.\nResponse: We thank the Reviewer for this comment. Although the small intestine and\ncolon differ in anatomy, they share a broadly conserved epithelial maturation gradient\nthat progresses from the crypt base toward the luminal surface. For example, Lorenzo-\nMartín and colleagues applied villus-axis zonation markers, originally defined in the\nsmall intestine, to classify bottom, middle, and top colonocytes in ex vivo “mini-colon”\ncultures, demonstrating that these small-intestine zonation modules can reliably\nannotate spatial differentiation states in the colon 7,8. Besides, Niec and colleagues also\nshowed that both the small intestine and colon follow a comparable directional\nmaturation program. Using the SpaceFold framework, they reconstructed the epithelial\ndifferentiation continuum for the small-intestinal crypt–villus axis and the colonic\ncrypt–surface axis within a unified pseudo-space. Their analysis indicated that small-\nintestine zonation markers reflect conserved maturation patterns across the intestinal\nepithelium 9. Collectively, these studies provide a well-established basis for applying\nsmall-intestine zonation signatures to interpret colonic epithelial differentiation. In\naccordance with the Reviewer’s suggestion, we have clarified this in the revised\nmanuscript and have corrected our terminology from “crypt–villus” to “crypt–axis”\nwhen referring to colonic data.\nReviewer #3\nYi and colleagues report in their manuscript # NCOMMS-25-28940 that\nknocking out the essential volume-regulated 30 anion channel (VRAC) unit\ncancer. The exacerbated colitis phenotype in these mice is associated with a\ndisturbed enterocyte maturation and an altered gut microbiota. Retinoid acid\nsupplementation or lactobacillus administration partially reversed the observed\nphenotype in these mice.\nResponse: We are truly grateful for the Reviewer’s thoughtful and detailed feedback\non our manuscript. In response to the Reviewer’s comments, we have performed new\nexperiments to address the concerns raised. These new experiments include\ndemonstrating that VRAC governs enterocyte zonation through a BMP-dependent\nmechanism; defining epithelial–immune crosstalk by assessing how conditioned media\nfrom WT and LRRC8A-KO Caco-2 cells to immune cell cultures; and performing\ncomplementary metabolomic analyses to delineate the metabolic consequences of\nepithelial VRAC deficiency across multiple biological matrices. Collectively, these\nresults provide clearer mechanistic insight and reinforce the robustness of our\nconclusions.\nMajor points\n1. To better understand the role of VRAC in immunomodulatory metabolite\ntransport, the authors should quantify extracellular levels of known VRAC-\nexported metabolites such as cGAMP, itaconate, and glutamate. These\nmeasurements should be conducted in conditioned media from WT and\nLrrc8aΔIEC organoids or colon explants, both under basal conditions and after\nDSS exposure, as well as in serum and fecal samples from DSS-treated WT\nand knockout mice. If VRAC facilitates the export of anti-inflammatory\nmetabolites, such as itaconate, its absence may cause intracellular retention,\nthereby exacerbating inflammation. To functionally validate this mechanism,\ntreating Lrrc8aΔIEC mice with exogenous itaconate or cGAMP could be used\nto assess whether the inflammatory phenotype can be rescued.\nResponse: We sincerely appreciate the Reviewer’s insightful comments. The main\nfinding of our study is that intestinal VRAC is essential for protecting against colitis\nand inflammation-associated colorectal cancer. Mechanistically, we showed that VRAC\ndeficiency impaired enterocyte maturation and disrupted epithelial zonation, shifting\nepithelial programs from nutrient absorption toward antimicrobial defense. We agree\nthat identifying the specific ions or metabolites whose permeability is altered would\nprovide deeper mechanistic insight into how VRAC regulates epithelial homeostasis\nand inflammatory responses. However, pinpointing a single causative ion or metabolite\nremains technically challenging and is beyond the scope of the present study. This will\nbe an important direction for future investigation.\nAlthough organoid systems effectively recapitulate epithelial regeneration and\ndifferentiation potential in vitro, their enclosed architecture—with the apical surface\nfacing the lumen and the basal surface embedded in Matrigel—renders them poorly\nsuited for permeability studies. Moreover, intestinal organoids rapidly deteriorate upon\nDSS exposure (response Fig. 1), and very few studies have applied DSS directly to\norganoid cultures due to their limited viability under such conditions.\nmetabolomic analyses between wild-type and Lrrc8aΔIEC mice, including: (i) targeted\nmetabolomic analyses of colon tissues (steady-state and post-DSS) and plasma samples\n(post-DSS); (ii) untargeted metabolomic analysis of fecal samples (post-DSS) (revised\nFig. S7E–H). Notably, our metabolomic datasets revealed no significant alterations in\nthe levels of metabolites previously reported to be VRAC-permeable, including ATP,\nglutamate, glutathione, glycine, taurine, lactate, and itaconate (revised Fig. S13). We\nalso analyzed colonic explant supernatants between wild-type and Lrrc8aΔIEC mice,\nhowever, metabolomics showed poor sample clustering and minimal metabolite\nchanges (response Fig. 2A–D). Therefore, these explant data were not included in the\nrevised manuscript.\nRegarding cGAMP, the role of cGAS-STING signaling in IECs remains far less\nunderstood than its well-established functions in immune cells. Previous studies\nshowed that cGAS deficiency, but not STING deficiency, results in intestinal stem cell\nloss and impaired barrier integrity after DSS injury10. In our study, deletion of Lrrc8a\nin myeloid cells did not affect the severity of DSS-induced colitis, suggesting that other\ntransporters such as SLC46A2 and ABCC1, which are known to mediate cGAMP\ntransport in immune cells, may compensate for the loss of VRAC in this context. GSEA\ncomparing wild-type and Lrrc8aΔIEC mice showed a clear upregulation of the cGAS-\nSTING pathway in the absence of IEC VRAC, indicating that VRAC loss does not\ndampen this signaling cascade in IECs (response Fig. 2E). Taken together, we consider\nthat IEC VRAC deficiency likely does not regulate enterocytes zonation by inhibiting\ncGAMP transport or dampening cGAS-STING signaling. Nevertheless, we measured\ncGAMP levels in the supernatants of organoid and Caco-2 cell, but the concentrations\nwere extremely low and fell below the lower limit of detection of the standard curve\n(data not shown). Regarding itaconate, its levels did not differ significantly between\nWT and Lrrc8aΔIEC mice in our metabolomic analyses (revised Fig. S13). Collectively,\nthese findings indicate that epithelial VRAC does not regulate intestinal inflammation\nthrough the transport of a specific metabolite previously reported to be VRAC-\npermeable.\nResponse Figure 1. DSS treatment leads to the death of the organoids. Bright-field images of\norganoids treated with varying DSS concentrations for 24 hours, with Propidium Iodide (PI) staining\nindicating cell death.\n(A) Principal coordinate analysis depicting the metabolite composition in colonic explant\nsupernatant. (B) Volcano plot of differential metabolites between Lrrc8aΔIEC compared with WT\nmice. (C) Heatmap demonstrating representative metabolites associated with VRAC substrates. (D)\nKEGG enrichment analysis of differentially metabolites. (E) GSEA of cytosolic DNA sensing\npathway in colonic transcriptome.\n2. To further substantiate the link between VRAC and epithelial-immune\ncrosstalk, co-culture experiments should be performed using WT or Lrrc8aΔIEC\nintestinal epithelial cells (IECs) with bone marrow-derived macrophages\n(BMDMs), in the presence or absence of DSS. The impact on immune\nmodulation can be assessed by evaluating macrophage polarization (e.g., M1\nvs. M2 markers) and cytokine secretion profiles. Additionally, neutrophil\nchemotaxis assays should be conducted to determine whether supernatants\nfrom Lrrc8aΔIEC IECs enhance neutrophil migration, potentially contributing to\nthe heightened immune infiltration observed in vivo.\nResponse: Thank you for the insightful suggestion. Our flow cytometry analysis\nshowed a marked increase in neutrophil infiltration in Lrrc8aΔIEC mice, whereas IL-17+\nand IL-22+ Th17 and ILC3 populations were significantly reduced. Macrophage\n5\npercentages, in contrast, remained largely unchanged. Given the minimal changes in\nmacrophage abundance, we focused our functional assays on epithelial–neutrophil and\nepithelial–ILC3/Th17 interactions. To assess whether epithelial-derived factors directly\nmodulate neutrophil migration, we performed chemotaxis assays using supernatants\nfrom WT (Caco-2WT) and LRRC8A-knockout Caco-2 (Caco-2ΔLRRC8A) cells. Caco-\n2ΔLRRC8A supernatants had no effect on neutrophil migration compared with those from\nWT cells (revised Fig. S11E). In contrast, supernatants from Caco-2ΔLRRC8A cells\nsignificantly reduced the frequencies of IL-17A⁺ and IL-22⁺ Th17 and ILC3 subsets\n(revised Fig. 6E, F). Given the established roles of IL-17A- and IL-22-producing Th17\ncells and ILC3s in epithelial repair and mucosal regeneration, these findings support\nthat epithelial VRAC deficiency impaired mucosal healing by disrupting epithelial-\nimmune crosstalk with these populations.\n3. The authors should also investigate whether VRAC upregulation is protective\nin human IBD. This could be achieved by performing immunohistochemical\n(IHC) staining for LRRC8A in colonic biopsies from IBD patients and correlating\nexpression levels with disease severity, tight junction integrity (e.g., ZO-1),\nand epithelial apoptosis (e.g., TUNEL staining).\nResponse: Thank you for this valuable suggestion. We agree that assessing VRAC in\nhuman IBD samples could provide additional context. However, as VRAC is formed\nby LRRC8 heterohexamer, its activity cannot be inferred from LRRC8A abundance\nalone. Channel function depends on the precise subunit composition, and altering this\nstoichiometry—such as by overexpressing LRRC8A in WT cells—has been reported to\nreduce, rather than enhance, VRAC currents1. Thus, expression analyses in human\ntissues provide suggestive but not definitive insights into VRAC function. To address\nthe Reviewer’s question, we examined LRRC8A expression in intestine biopsies from\napproximately nine Crohn’s disease (CD) patients. Immunohistochemistry revealed\nstrong epithelial staining of LRRC8A with a slight enhancement in CD samples, and\nWestern blot showed a modest increase in LRRC8A protein levels in CD patients\ncompared with controls (revised Fig. S1C, D). Because the number of clinical samples\nwas limited, detailed correlation analyses could not be reliably performed.\n4. To determine whether barrier loss in Lrrc8aΔIEC mice is linked to osmotic\nimbalance, transepithelial electrical resistance (TEER) should be measured in\nWT and knockout colonoids under both isotonic and hypotonic conditions. A\nrescue experiment using hypertonic media could help clarify whether restoring\nosmotic balance ameliorates tight junction disruption and improves barrier\nintegrity.\nResponse: We appreciate the Reviewer’s insightful suggestion. While intestinal\norganoids faithfully recapitulate the 3D architecture and regenerative capacity of the\nintestinal epithelium, their enclosed three-dimensional structure makes TEER\nmeasurement technically challenging. Our literature review also indicates that TEER\nassessments in organoid systems are rarely reported, likely due to these technical\nlimitations. Therefore, we employed Caco-2 cells, a more suitable in vitro model for\nassessing epithelial barrier function, to examine the impact of VRAC deficiency.\ngenerated Caco-2ΔLRRC8A cell line and found that TEER was markedly reduced\ncompared to control cells (revised Fig. S6C). Furthermore, ZO-1 immunofluorescence\nstaining revealed increased junctional tortuosity and reduced fluorescence intensity,\nindicating tight junction disruption (revised Fig. S6D). Re-expression of LRRC8A\nrestored VRAC currents and partially rescued ZO-1 continuity and barrier integrity\n(revised Fig. S6E, F).\nWe also attempted to stimulate VRAC activity under hypotonic conditions to\nassess its acute effects on tight junctions. Short-term exposure (≤ 6 h) to hypotonic\nsolution have been reported to promote cGAMP transport and NLRP3 inflammasome\nactivation by activating VRAC activity, however, prolonged incubation led to cell\nswelling and death3,11,12. Within short exposure to hypotonic solution, Caco-2 cells\nbecame noticeably enlarged, but no obvious changes in tight junction morphology were\nobserved, possibly due to the limited duration (response Fig. 3).\nRegarding the Reviewer’s suggestion of applying hypertonic conditions as a\npotential rescue strategy, we consider this approach biologically inappropriate for\nprobing VRAC function. Hypertonic and hypotonic responses involve fundamentally\ndistinct osmotic programs: hypertonic stress triggers regulatory volume increase via\nNKCCs, NHEs, and Cl⁻/HCO₃⁻ exchangers coordinated by WNK signaling, whereas\nVRAC mediates regulatory volume decrease specifically under hypotonic conditions13–\n15. Thus, hypertonic medium would not be expected to restore VRAC-dependent\nprocesses or provide mechanistic insight into VRAC-mediated barrier regulation.\nResponse Figure 3. Effects of hypotonic stress on Caco-2 cells (A) Representative\nimages of Caco-2WT or Caco-2ΔLRRC8A cells post isotonic (320 mOsm) or hypotonic\n(240 mOsm) treatment for 3 h. (B) Cell viability after 3/6 h hypotonic treatment (vs.\nisotonic) by CCK-8 assay. (C) ZO-1 staining of Caco-2WT or Caco-2ΔLRRC8A cells post\nisotonic or hypotonic treatment for 6 h. Data were represented as mean ± SEM.\n5. The observation that VRAC loss leads to increased tumor burden despite\nelevated epithelial apoptosis raises an important mechanistic question. How\ncan the authors explain these observations?\nResponse: The increased epithelial apoptosis and the enhanced tumor burden arise at\ndifferent stages of inflammation-driven carcinogenesis. In our study, epithelial\napoptosis increases during acute DSS-induced injury as a downstream consequence of\nsevere mucosal damage. In contrast, under chronic or repetitive inflammatory\nconditions, impaired epithelial regeneration and dysregulated repair create a\nmicroenvironment that promotes the expansion and survival of transformed cells,\n7\nultimately resulting in increased tumor burden. This progression is well supported by\nprevious reviews 16,17. Consistent with this framework, several experimental models\nshow that increased apoptosis during acute injury can coexist with enhanced\ntumorigenesis during chronic inflammation. For example, cGAS-deficient mice exhibit\nreduced Lgr5⁺ stem cells, increased TUNEL⁺ apoptotic cells, and simultaneously\nelevated proliferation within neoplastic lesions in the AOM-DSS model—illustrating\nhow impaired regeneration, rather than reduced apoptosis, governs tumor progression\n10. Together, these findings support the concept that elevated apoptosis in the acute\nphase and increased tumor burden in the chronic phase are mechanistically compatible\ncomponents of inflammation-associated carcinogenesis.\n6. To explore the mechanistic link between VRAC and enterocyte maturation,\nrescue experiments should be performed using Lrrc8aΔIEC IEC organoids.\nSpecifically, re-expression of LRRC8A and LRRC8D via lentiviral transduction\ncould help determine whether the expression of mature enterocyte markers,\nsuch as Alpi and Slc26a3, can be restored. Additionally, measuring the\ntransport of organic osmolytes, such as taurine uptake, would provide a\nfunctional readout for enterocyte maturation and VRAC activity.\nResponse: We thank the Reviewer for this important suggestion. In the present study,\nwe focused on the essential VRAC subunit LRRC8A. In Lrrc8aΔIEC intestinal organoids,\nre-expression of LRRC8A restored the expression of mature enterocyte markers such\nas Alpi, as demonstrated by both immunofluorescence and qPCR (revised Fig. 4H).\nSimilarly, in Caco-2ΔLRRC8A cells, LRRC8A re-expression rescued tight-junction\nintegrity, as evidenced by improved ZO-1 organization (revised Fig. S6F). As\nmentioned previously, we examined taurine levels across multiple metabolomic\ndatasets. Taurine abundance did not differ significantly in colon tissues, fecal samples,\nand plasma between WT and Lrrc8aΔIEC mice (revised Fig. S13). These results suggest\nthat altered taurine transport is unlikely to account for the impaired enterocyte zonation\nobserved in the absence of epithelial VRAC.\n7. To investigate VRAC-dependent zonation mechanisms, spatial\ntranscriptomics could be applied to colon sections from WT and Lrrc8aΔIEC\nIEC mice to map changes in epithelial zonation with high spatial resolution.\nComplementary experiments using intestinal organoids treated with modulators\nof the Wnt and BMP pathways could reveal whether VRAC deficiency alters\nresponsiveness to these critical developmental gradients.\nResponse: Given the established roles of Wnt/BMP signaling in orchestrating intestinal\nepithelial lineage differentiation18,19, we systematically analyzed transcriptomic\nalterations in these pathways. RT-qPCR analysis of canonical Wnt signaling markers\n(Ascl2 and Axin2) in colon tissues from DSS-treated mice and in vitro organoid cultures\nrevealed no significant differences between WT and Lrrc8aΔIEC groups (revised Fig.\nS7I and Fig. 4L). Meanwhile, β-catenin immunostaining in organoids demonstrated\ncomparable levels of both total and nuclear-localized β-catenin across both genotypes\n(revised Fig. S7J). In contrast, BMP2, one of the major BMP ligands in the intestine,\nwas significantly reduced in Lrrc8aΔIEC colon tissues and organoids (revised Fig. S7I\n8\nand Fig. 4L). Moreover, exogenous BMP2 supplementation substantially restored Alpi\nexpression in Lrrc8aΔIEC organoids (revised Fig. 4M). These findings indicate that\nVRAC regulates intestinal epithelial zonation through a BMP2-dependent mechanism.\n8. To assess the functional consequences of disrupted epithelial zonation,\nnutrient absorption capacity should be quantified by measuring glucose and\nfatty acid uptake in Lrrc8aΔIEC enteroids. These findings can be further\nvalidated using Ussing chamber assays on WT versus knockout colon explants.\nMetabolomic profiling of luminal contents via LC-MS could then be used to\ncorrelate epithelial functional impairments with alterations in intestinal\nmetabolic output.\nResponse: To evaluate the functional consequences of disrupted epithelial zonation, we\nperformed glucose and lipid uptake assays in WT and Lrrc8aΔIEC organoids. The data\ndemonstrates that VRAC deficiency significantly impaired lipid absorption. The\nglucose uptake was also reduced, the magnitude of inhibition was weaker compared\nwith the defect in lipid absorption (revised Fig. S7C, D). Together with our\ntranscriptomic and metabolomic data, these findings indicate that loss of epithelial\nVRAC causes broad defects in enterocyte zonation and maturation, leading to\nsubstantial impairment of nutrient-absorptive functions. Our data demonstrates that\nimpaired retinoic acid (RA) metabolism is a key factor contributing to the exacerbated\ninflammation observed in the absence of epithelial VRAC. RA has been shown to\nregulate enterocyte differentiation and modulate both T cell and ILC functions, thereby\nshaping intestinal inflammatory responses20,21. Consistent with these roles, our in vivo\nand in vitro experiments indicate that disrupted RA metabolism provides a mechanistic\nexplanation for the defective enterocyte zonation and compromised nutrient absorption\nin Lrrc8aΔIEC mice.\n9. The authors show impaired RA synthesis based on reduced expression of\nbiosynthetic enzymes and RA-responsive genes. However, direct quantification\nof RA levels in IECs and colon tissues from WT and Lrrc8aΔIEC mic using LC-\nMS or ELISA would provide crucial biochemical validation.\nResponse: RA levels in colonic tissue are physiologically very low, typically on the\norder of ~10 pmol per gram of tissue, which makes their direct quantification\ntechnically challenging22,23. During our study, we attempted to measure RA in colon\nsamples using LC-MS; however, the signal intensity was below the threshold required\nto generate reliable peak identification. We also surveyed all available commercial kits\nand found that no validated ELISA assays exist for RA detection in tissue samples.\nGiven these technical limitations, we assessed RA activity through established\nindirect readouts. Measuring the expression of Aldh enzymes, the rate-limiting\nregulators of RA synthesis, is widely used to surrogate measure for RA content24–26. In\naddition, both in vivo and in vitro experiments demonstrated that atRA supplementation\nrestored the expression of RA-responsive genes and key differentiation markers. These\nresults confirm that RA signaling is indeed impaired in VRAC-deficient intestinal\nepithelial cells, and that this pathway remains functionally responsive when exogenous\nRA is provided.\n9\n10. To confirm that the observed defects in RA metabolism are epithelial-\nintrinsic, organoid cultures from WT and Lrrc8aΔIEC mice should be treated\nwith retinol, and the capacity to generate RA (via ALDH activity or RA levels)\nshould be compared in vitro.\nResponse: Retinol enhanced Alpi expression and ALDH activity in Lrrc8aΔIEC\norganoids, although these effects were significantly attenuated compared to direct atRA\nadministration (revised Fig. S10E, F), suggesting impaired retinol uptake in the\nabsence of VRAC.\n11. The authors should assess RAR/RXR transcriptional activity directly (e.g.,\nusing RA response element–luciferase reporters) in WT and VRAC-deficient\nIECs ± atRA to demonstrate the functional consequences of impaired RA\nsignaling.\nResponse: We agree that direct assessment of RAR/RXR transcriptional activity using\nRARE-luciferase reporters would provide valuable mechanistic insight. However, in\nintestinal epithelial cell systems such as Caco-2 cells, reporter-based assays are difficult\nto interpret due to consistently low transfection efficiency. Our organoid and in vivo\natRA supplementation studies demonstrate that epithelial VRAC is functionally\nconnected to RA signaling. atRA treatment effectively rescued Alpi expression, ALDH\nactivity, and other RA-responsive markers in VRAC-deficient models, demonstrating\nthat RA signaling impairment underlies the defects caused by VRAC loss. We therefore\nconsider these rescue experiments to provide a physiologically relevant readout of RA\npathway activity.\n12. While the authors demonstrate reduced IL-17A/IL-22 production, they\nshould investigate whether this defect is rescued by conditioned media from\natRA-treated Lrrc8aΔIEC organoids, thereby supporting an epithelial-immune\nsignaling axis.\nResponse: As noted above and in our response to Comment #2, we consider organoid\nsupernatants unsuitable for directly stimulating immune cells. Organoid cultures often\nrelease endotoxins and contain complex, incompletely defined components derived\nfrom Matrigel, making it difficult to attribute immunological effects to specific\nepithelial-derived factors. While organoid conditioned medium has influenced immune\ncells like DCs and CTLs, direct evidence for its role in stimulating T and ILC cells to\nsecrete IL-17A/IL-22 is limited27. Accordingly, most high-quality studies rely on direct\nimmune cell–organoid co-culture rather than immune stimulation with organoid\nsupernatants. As mentioned earlier, we collected conditioned media from Caco-2WT and\nCaco-2ΔLRRC8A cells and applied them to in vitro-differentiated splenic Th17 and ILC3\ncultures. Our results showed that supernatants from Caco-2ΔLRRC8A cells markedly\nreduced the proportions of IL-17A⁺ and IL-22⁺ Th17 and ILC3 cells (revised Fig. 6E,\nF). These findings demonstrate that epithelial VRAC deficiency alters epithelial\nmetabolism that modulate innate lymphoid and T-cell effector functions, supporting a\ncritical epithelial-immune signaling axis disrupted by the epithelial loss of VRAC.\n13. The role of the microbiota would be more rigorously validated by transferring\nfecal microbiota from WT or Lrrc8aΔIEC mice into germ-free recipients and\n10\nassessing the severity of colitis after DSS treatment. This would test whether\nthe microbiota alone is sufficient to transfer susceptibility or protection.\nResponse: To further validate the role of the microbiota, we transferred fecal\nmicrobiota collected from DSS-treated WT and Lrrc8aΔIEC donor mice into antibiotic-\npretreated recipient mice. Our results showed that recipients receiving microbiota from\nDSS-treated Lrrc8aΔIEC donors developed markedly more severe colitis following DSS\nchallenge (revised Fig. 7E–H). These findings demonstrate that the dysbiotic\nmicrobiota arising in Lrrc8aΔIEC mice under inflammatory condition is sufficient to\nconfer increased susceptibility to colitis.\n14. To isolate the impact of RA-modified microbiota, fecal transplants from RA-\ntreated Lrrc8aΔIEC mice should be tested for their ability to mitigate DSS-\ninduced colitis when transplanted into antibiotic-pretreated WT or KO hosts.\nResponse: We appreciate the Reviewer’s thoughtful suggestion. However, DSS-\ninduced colitis is strictly microbiota-dependent. In our ABX-pretreated recipients, the\noverall microbial load is greatly reduced, and under these conditions both WT and\nLrrc8aΔIEC mice develop little to no colitis, which is consistent with previous reports\n(revised Fig. S12A–D). Similarly, RA-treated Lrrc8aΔIEC mice exhibit minimal disease\neven without transfer. Therefore, transferring microbiota from RA-treated Lrrc8aΔIEC\ndonors into antibiotic-pretreated recipients would not be informative, as both donor and\nrecipient conditions result in negligible disease, precluding a meaningful assessment of\nmicrobiota-mediated protection.\n15. The rescue effects of L. murinus and L. reuteri should be compared\nmechanistically. For instance, do both strains restore ALDH activity, suppress\nAMP expression, and modulate immune responses in a similar manner? Do\nthey influence RA levels directly or via a secondary mechanism (e.g., bile acid\nmodulation, SCFA)?\nResponse: We performed additional experiments to more directly compare the effects\nof different Lactobacillus strains. Our new data show that L. reuteri supplementation,\nsimilar to L. murinus, restored epithelial ALDH activity, normalized tissue AMP\nexpression, and recovered IL-17A⁺ and IL-22⁺ lymphocyte populations in the lamina\npropria of Lrrc8aΔIEC mice (revised Fig. 8F–H; revised Fig. S12J–L). In addition,\nupdated metabolomic analyses revealed alterations in several bile acid species under\nepithelial VRAC deficiency (revised Fig. S7G, H). We consider these bile acid\nperturbations to be a consequence of disrupted epithelial zonation under IEC VRAC\ndeficiency. Although we cannot exclude the possibility that VRAC may directly\ninfluence the transport of certain bile acids or related lipid metabolites, clarifying\nwhether VRAC contributes through direct permeability or indirect secondary\nmechanisms will require future investigation.\nTogether, these new findings support a coherent mechanistic model in which\nLactobacillus supplementation enhances RA metabolism and restores enterocyte\nzonation, thereby ameliorating epithelial–immune dysregulation in IEC VRAC-\ndeficient mice.\nSpecify which IBD subtypes (UC, CD, or both) were analyzed in Fig. S1.\nResponse: We thank the Reviewer for this comment. We have now specified the IBD\nsubtypes in the main text and figure legend that the samples analyzed in revised Fig.\nS1.\nReviewer #4\nI co-reviewed this manuscript with one of the reviewers who provided the listed\nreports.\nResponse: We appreciate the insightful suggestions from the Reviewer regarding our\nmanuscript.\n12"}, {"round": 2, "reviewer_comments": {"reviewer_3": "Thank you for the revision.\nI do not have any additional suggestions for the manuscript anymore.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": ""}]

Decision letter