Non-canonical IL‑22 receptor signaling remodels the oral mucosal barrier during Candida albicans immunosurveillance

Domain: biology
Published: 2026-04-03
Editorial outcome: major_revision

Abstract
Mucosal barrier integrity is vital for homeostasis with commensal organisms while preventing pathogen invasion. Here we show that fungal-induced immunosurveillance enhances resistance to fungal outgrowth and tissue invasion by remodeling the oral mucosal epithelial barrier in mouse models of adult and neonatal Candida albicans colonization. Epithelial subset expansion and tissue remodeling are dependent on interleukin-22 and signal transducer and activator of transcription 3 signaling, through a non-canonical receptor complex composed of glycoprotein 130 coupled with the interleukin-22 receptor subunit alpha-1 and the interleukin-10 receptor subunit beta. Epithelial proliferation enhanced antifungal host defenses through the upregulation of antimicrobial peptide expression. Immunosurveillance-induced epithelial remodeling is restricted to the oral mucosa, whereas barrier architecture is reset once fungal-specific immunity developed. Collectively, these findings identify fungal-induced transient mucosal remodeling as a critical determinant of resistance to mucosal fungal infection during early stages of microbial colonization.

Introduction
Oral mucosal tissues are continuously exposed to food, airborne antigens, and commensal microbes, including fungi 1. The finely tuned implementation of innate and adaptive immune responses enables the host to maintain homeostasis with commensal species and neutralize invading organisms 2-4. As a central constituent of the mycobiome, the fungus Candida albicans colonizes the oral and gastrointestinal (GI) mucosa of up to 75% of healthy individuals 5. Settings of local or systemic immunosuppression result in oropharyngeal candidiasis (OPC) 6. Since, in healthy individuals C. albicans causes no harm, fungal colonization appears to be evolutionarily selected for appropriate metabolic function and immune priming 7-10. While C. albicans has been extensively studied as a pathogen 11-14, the primary lifestyle of this fungus in the oral cavity is as a commensal 15-17. The natural diversity in C. albicans influences the outcome of the interaction between the fungus and the host 16 implying that the involvement of specific immune pathways to the host defense against C. albicans is modulated in a strain-dependent manner. In mouse models of OPC, C. albicans strains can be grouped into two categories: clearance-biased (CB) and persistence-biased (PB) 16. While CB C. albicans strains induce a strong pro-inflammatory response that leads to rapid clearance in the oral mucosa (acute OPC), PB C. albicans strains instead trigger a tempered inflammatory response that permits long-term fungal colonization, thus mimicking commensal colonization in humans. Still, the regulation of mucosal homeostasis during persistent fungal colonization remains poorly understood.

The epithelial architecture of mucosal surfaces such as the oral cavity is crucial for its host defensive function 18-20, providing structural immunity by initiating and coordinating immune responses 21. However, relatively little is known about how this is coordinated in the oral mucosa. Furthermore, the epithelium balances a multiplicity of roles in early life 22, while the acquired microbiome contributes to the development of immunity in newborns. Following exposure, the mucosal immune system of neonates undergoes successive, non-redundant phases that support the developmental needs of the infant to establish immune homeostasis 23. While tissue remodeling has been associated with pathological features post-injury or disease 24,25, commensal organisms may induce changes in barrier structures to promote homeostasis 26.

Here, we studied mucosal immune responses and tissue homeostasis in mouse models of persistent Candida oral colonization at distinct ranges of age. We show that immunosurveillance-induced epithelial expansion and remodeling mediates resistance against fungal outgrowth during the onset of fungal colonization. IL-22, a critical cytokine in epithelial homeostasis and host defense at mucosal surfaces, has long been associated with signaling through a well-characterized receptor complex 27. Traditionally, IL-22 exerts its protective effects through binding to the IL22RA1-IL10RB receptor complex. However, our study reveals that IL-22 recognition and signaling extend beyond this canonical pathway, expanding the current understanding of its biological roles. Oral mucosal remodeling required IL-22-mediated gp130 activation in non-canonical cytokine receptor complexes with IL-22RA1 and IL-10RB to amplify antimicrobial peptide (AMP) expression. IL-22 mediated oral epithelial remodeling was transitory and a subsequent mucosal remodeling event in later stages of colonization required Candida-specific immunity. Finally, fungal-induced mucosal remodeling prevents tissue invasion in a mouse model of neonatal colonization. These findings provide insight into the molecular mechanisms of IL-22-mediated signaling, identify a pathway that supports antifungal immunity during fungal persistence, and expand our understanding of microbe-induced epithelial remodeling and homeostasis through a non-canonical receptor complex.

Results
Persistent C. albicans colonization induces epithelial remodeling
In a mouse model of OPC 28, acute infection with a prototypic CB C. albicans strain (SC5314) leads to its rapid clearance from the oral cavity, whereas a PB Candida strain (CA101) persists in the oral mucosa (Fig. S1)15. A similar trend was observed in the GI tract following oral infection (Fig. S1). However, oral colonization alone does not result in systemic dissemination in the absence of immunosuppression (Fig. S1). Several host cell types respond to C. albicans encounter in the oral cavity. To develop a single cell transcriptome profile of the oral mucosa, we evaluated gene expression in tissues of PB colonized mice and from mice after pathogenic Candida clearance (Fig. 1a) representing distinct post-infection host states, namely immune and barrier responses post-fungal clearance (CB) and one reflecting ongoing Candida presence and interaction (PB). Our analysis identified 18 distinct cell subpopulations, which were present in both, PB colonized mucosa and mucosal tissue after CB clearance (Fig. 1b). The identified subpopulations expressed cell-type specific marker genes (Fig. S2) consistent with classical, well-established markers for each respective cell population. The scRNA-sequencing analysis revealed that the epithelial proportions increased during persistent fungal colonization (Figure 1c). Accordingly, epithelial cells from colonized mice had higher expression of proliferation marker genes (Mki67, Cenpf, Cenpa) (Fig. 1d). Tissue histology during the onset of PB Candida colonization revealed that, in contrast to CB-infected mice, the epithelial layer expanded (Fig. 1e) and basal epithelial cell proliferation increased indicated by Ki67 staining (Fig. 1f, g). Next, we performed RNA sequencing of epithelial-enriched mucosal tissue (Fig. 2a). Using Gene Set Enrichment Analysis (GSEA), we found that genes involved in keratinization and keratinocyte differentiation were significantly enriched in fungal colonized epithelial tissues (Fig. 2b). Similarly, genes for keratinization were enriched in epithelial cells from colonized mice in the scRNA-sequencing data set (Fig. S3). Keratins influence the epithelial architecture by determining cell compartmentalization and differentiation 29 . Several keratins, including keratin 14 (K14), were highly expressed in the mucosa of colonized mice (Fig. 2c). Basal epithelial cells express K14 in all regions, while the suprabasal epithelial layer (SEL) expresses K13 30. Consistent with these findings the K14 epithelial layer expanded during persistent colonization, while the epithelium had similar architecture after CB encounter and clearance compared to sham infection (Fig.2d-f). Notably, the K13 layer distribution and thickness remained unchanged during fungal persistence (Fig. S4). Time course studies revealed that K14 epithelial expansion occurs after 8 days of fungal colonization (Fig. 2g, SA5). Similar oral epithelial expansion was observed by diverse C. albicans clinical isolates from vaginal, catheter-associated, and oral environments (Fig. S6), indicating that Candida persistence induces conserved epithelial remodeling responses within the oral mucosa. Collectively, these data show that persistent fungal colonization induces distinct epithelial subset expansion; thus, indicating that ongoing Candida presence influences the oral mucosal architecture.

CD4+ T cells are a major source of IL-22 during persistent colonization
T cells play vital roles in the mucosal antifungal immunity 31,32, while some CD4+ T cells reside in the oral mucosa of healthy individuals 20. In fact, T cells can promote stromal cell proliferation through secretion of cytokines 33-35. Thus, we compared protein expression kinetics of various T helper (Th) cell-associated cytokines and chemokines during persistent C. albicans colonization and acute OPC. Acute infection with the CB C. albicans strain led to early IL-1β, TNFα, and IFNγ induction, whereas the PB Candida strain colonized the oral mucosa without inducing canonical pro-inflammatory cytokines (IL-1β, TNFα, IFNγ) and T cell-polarizing cytokines (IL-1β, IL-6, IL-23) (Fig. 3A; S7). In acute OPC, IL-17 and IL-22 are expressed by Type 17 cells with similar kinetics 11,36-38. In contrast, our data show that IL-22 and IL-17 are differentially expressed during persistent colonization (Fig. 3a, b). Mice infected with a CB strain induced IL-17A and IL-22 similarly followed by rapid decline after fungal clearance. However, in PB-colonized mice, mucosal IL-22 levels remained high, whereas IL-17A levels declined to low levels at the onset of colonization (Fig. 3b). Thus, PB colonization induces limited inflammation characterized primarily by a type 17 response (IL-17/IL-22), rather than broad pro-inflammatory activation as seen during the early phase of CB infection. IL-22 neutralization after PB C. albicans colonization (Fig. 3c) resulted in fungal proliferation (Fig. 3d), while antibody neutralization of IL-17A did not alter the fungal burden in colonized mice. This suggested that IL-22 may have a prominent role in controlling fungal outgrowth after mucosal colonization, while IL-17 signaling may be more critical at earlier stages of infection 16, without immediately affecting fungal burden at the onset of oral colonization. Next, we determined the cellular origin of IL-22. Our scRNA-sequencing dataset suggested that CD4+ T cells highly express Il22 during persistent fungal colonization (Fig. 3e). Using IL22TdTomato reporter mice, we confirmed that CD4+ T cells are the major source of IL-22 during colonization with PB Candida (Fig. 3f). Next, we evaluated the localization of CD4 T cells during fungal persistence within the oral mucosa. CD4+ T cells were exclusively found in the epithelial and submucosal layers of PB-infected mice at the onset of colonization, which differed markedly from CB- or Sham-infected mice (Fig. 3g, S8) suggesting that persistent fungal stimulation is required to recruit and retain CD4 T cells in submucosal layers. Within the helper T cells, IL-22 is mainly produced by Th17 and Th22 subsets 39. Ex vivo stimulation of mucosal resident CD4+ T cells from CB- and PB-infected mice showed that during fungal persistence Th17 and Th22 cells are the major source of IL-22, while Th22 cell frequencies increased (Fig. 3h-j).

Epithelial expansion depends on IL-22 signaling via non-canonical gp130 receptor complexes
The IL-17- and the IL-22 receptors, which mediate anti-C. albicans immunity during acute OPC, play distinct and restricted roles in distinct sublayers of the stratified oral epithelium 36. During PB-Candida colonization, IL-22RA1 protein expression extended into the suprabasal layer (Fig. S9) consistent with K14 expression (Fig. 2). IL-22 has been implicated in multiple aspects of epithelial barrier function and wound repair, including regulation of cell growth 40. Accordingly, IL-22 induced proliferation of human oral epithelial cells in a dose-dependent manner, while high cytokine concentrations inhibited oral epithelial cell growth (Fig. 4a, S10). Similarly, IL-22 deficient mice and IL-22 depletion during PB colonization reduced K14 epithelial layer expansion and proliferation (Fig. 4b-d, Fig. S11), as well as resistance against fungal outgrowth (Fig. 3e) in the setting of intact IL-17 signaling (Fig. S12). IL-22-mediated proliferation required JAK-TYK2-STAT3 signaling in human oral epithelial cells (Fig. S13). Importantly, tonic STAT3 activity was required for proliferation in unstimulated oral epithelial cells (Fig. S13). Classical IL-22 signal transduction is mediated by binding of the cytokine to a receptor complex consisting of IL-22RA1 and IL-10RB 27. However, while mice deficient for either receptor, IL-22RA1 and IL-10RB respectively, were more susceptible to PB Candida outgrowth (Fig. 4f), the K14 layer surprisingly still underwent expansion in the absence of either receptor chain (Fig. 4g, S14). Notably, IL-10 signals through a heterotetrameric complex comprising of IL10Rα and IL10RB but was dispensable for fungal control and epithelial remodeling (Fig. S15). Recently, studies in synthetic cytokine biology have challenged the classical view of IL-22 signaling via a heterodimeric IL-22RA1 and IL-10RB complex. Synthetic cytokine receptor chains of IL-22RA1 and IL-10RB form functional heterodimeric receptor signaling complexes with an IL-6 receptor chain of gp130 to induce STAT3 signal transduction 41. Proximity ligation assays and co-immunoprecipitation identified the classical heterodimeric IL-22RA1 and IL-10RB complex, while revealing that IL-10RB, as well as IL-22RA1, form receptor complexes with gp130 in human oral epithelial cells (Fig. 4h, S16). We tested whether antibody blockade of either receptor chain, IL22RA1 and IL-10RB, would abolish STAT3 signal transduction in human oral epithelial cells. While antibodies blocking IL-22RA1 or IL-10RB induced similar STAT3 activation following IL-22 exposure, treatment with both antibodies reduced epithelial activation and IL-22 mediated proliferation (Fig. 4i, j, S17). Next, we determined the contribution of gp130 signaling to IL-22-mediated oral epithelial proliferation. Pharmacological inhibition of gp130 signaling reduced IL-22-mediated oral epithelial proliferation in vitro (Fig. 4K), while reducing STAT3 activation (Fig. 4l, S17). IL-6 binds to membrane-bound IL-6R to induce signaling via gp130 42. However, IL-22-mediated epithelial proliferation was independent of IL-6-gp130 signaling (Fig. S18). Of note, STAT3 activity remained unchanged during inhibition of gp130 in intestinal epithelial cells (Fig. S19), suggestive of organ-specific receptor complex signaling in response to IL-22. Inhibition of gp130 signaling during oral PB persistence increased the fungal burden and inhibited K14 expansion and remodeling (Fig. 4m-p). Thus, IL-22 signals via various receptor chain combinations to provide oral mucosal antifungal immunity during fungal immunosurveillance, while oral epithelial proliferation and expansion depend on non-canonical gp130 signaling receptor complexes. IL-22 cooperates with IL-17 to enhance AMP production, such as defensins, S100 proteins, and small proline-rich proteins (SPRRs) which synergistically protect mucosal surfaces from fungal invasion 12,43-45 . Epithelial-enriched RNA-seq revealed that mice lacking IL-22 or inhibition of gp130 signaling reduced expression of key antimicrobial peptides (Fig. 4q, S20). Thus, oral epithelial expansion mediated by non-canonical IL-22 receptor signaling amplifies antifungal immunity by enhancing AMP expression.

Transient epithelial expansion depends on development of Candida-specific immunity
While oral PB infection leads to epithelial expansion at the onset of colonization, at later time points of fungal persistence the K14 expansion is reset to levels comparable to the baseline cell turnover seen in naïve mice (Fig. 5a-c), suggesting that mucosal remodeling is transitory, regulated, and depends on an auxiliary mechanism. A unifying theme of susceptibility to mucocutaneous candidiasis is seen in both humans and mice with a variety of genetic defects within the IL-17 pathway 3,31,46. The generation of organism-specific adaptive immune responses takes time to generate sufficient cells, due to the inherent demands for extensive proliferation and differentiation of naive cells into effector cells 47. Thus, we tested if Candida-specific immunity is required to remodel the oral mucosal barrier in subsequent phases of colonization. During late stages of fungal persistence, increased numbers of Candida-specific IL-17 secreting cells in cervical lymph nodes (Fig. 5d) inversely correlated with reversion of the K14 barrier thickness (Fig. 5e) suggesting the requirement of Candida-specific immunity to reset barrier architecture. Importantly, Rag1–/– were more susceptible to fungal outgrowth at the onset and late stages of colonization (Fig. 5f), while K14 epithelial expansion remained high (Fig. 5g,h, S21). Despite the absence of mature T cells, IL-22 expression is detected in Rag1–/– mice (Fig. S21), indicating that non-mature T cell sources produce IL-22 48-51. Of note, Cd4–/– mice phenocopied the observed phenotypes of Rag1–/– during the onset of colonization (Fig.S22). In the absence of mature T cells, ILC3s increased IL-22 expression at the onset of Candida colonization, likely contributing to epithelial expansion (Fig. S21). We took advantage of the Rag1–/– model to test if Candida-specific T cell immunity is required to revers epithelial remodeling at later stages of colonization. We adoptively transferred purified CD4+ T cells from PB-colonized (PBCD4 T cells) and Sham-infected WT mice (SCD4 T cells) into PB-colonized Rag1–/– mice (Fig. 5i). PBCD4 T cells transferred into Rag1–/– mice reduced the fungal burden (Fig. 5j) and K14+ epithelial thickness (Fig. 5k, l) suggesting that Candida-primed CD4+ T cells are both required and sufficient of driving subsequent epithelial remodeling that occurs during fungal colonization. Collectively, we show that IL-22-mediated oral epithelial remodeling is transitory and requires Candida-specific immunity to trigger a successive mucosal remodeling event in later stages of colonization.

Epithelial expansion is crucial to prevent fungal tissue invasion in neonates
Neonates exhibit differentially adapted immune responses when compared to adults 52, with important implications for immunity to OPC; indeed, oral thrush is commonly seen in infants 53, though the underlying basis for this is not well defined. In this regard, neonatal umbilical cord blood-derived CD4 T cells are intrinsically less able to differentiate into Th17 cells but rather tend to skew towards an IL-22 phenotype 54. Here, we established a mouse model of neonatal fungal colonization to study early-life interactions between the oral mucosa and Candida, which occurs in humans (Fig. 6a, b). Consistent with our findings in adult animals, oral PB colonization in neonates induced K14 epithelial expansion (Fig. 6c, d) and CD4+ T cell infiltration at the onset of colonization (Fig. S23). Neonatal mice deficient in IL-22 had increased oral fungal burden, abrogated K14 expansion, and decreased fungal-induced epithelial proliferation (Fig. 6f-h). Furthermore, IL-22 deficiency in neonates increased the depth of fungal tissue invasion (Fig. 6I, j) which was not observed in adult Il22–/– mice (Fig. 6j) suggesting that IL-22-mediated immunity and associated tissue remodeling plays a critical role preventing fungal invasion during early life, highlighting the importance of IL-22 in neonatal immune defense and epithelial protection.

Methods
Ethics statement. All animal work was approved by the Institutional Animal Care and Use Committee (IACUC) of the Lundquist Institute at Harbor-UCLA Medical Center and University of Pittsburgh.

Organisms and cell lines. The C. albicans strains SC5314 (clearance-biased; CB), and CA101 (persistence-biased; PB) and 11 clinical isolates (oral isolates71,72 TW1, 529L; catheter-associated isolates 73 SJCA1, SJCA3, SJCA5, SJCA7, SJCA9; vaginal isolates 73,74 JS1, JS3, JS7 and JS26) were used in the experiments. For in vivo experiments, strains were serially passaged three times in YPD broth. The OKF6/TERT-2 cells have been authenticated by RNA-sequencing 12, and have been tested for mycoplasma contamination. The intestinal epithelial cell line C2BBe1 was purchased from ATCC.

Subject details. For in vivo animal studies, age-and sex matched mice were used. Animals were bred and housed under pathogen-free conditions at the Lundquist Institute in ventilated cages at 22 ± 1 °C, 55 ± 10 % relative humidity, 12 h/12 h dark/light cycle, with free access to food and water. Animals were randomly assigned to the different infection or treatment groups. Researchers were not blinded to the experimental groups because the endpoints (fungal burden, histology, cytokine levels) were objective measures of disease severity. Il22ra1E2a-cre (referred to as Il22ra1–/–) mice were provided by Jay K. Kolls 75. IL-22TdTomato 76 mice were bred at the University of Pittsburgh. Il10rb–/– (B6.129S2-Il10rbtm1Agt/J) 77, Il22iCre/iCre (C57BL/6-Il22tm1.1(icre)Stck/J; referred to as Il22–/–) 78, Il10–/– (B6.129P2-Il10tm1Cgn/J) 79, and Rag1–/– (B6.129S7-Rag1tm1Mom/J; non-leaky) 80 mice were purchased from the Jackson Laboratory, and maintained at the Lundquist Institute. C57BL/6 wild type mice were purchased from the Jackson Laboratory and cohoused with breeders for at least 1 week before the experiments.

Mouse model of oropharyngeal candidiasis. For inoculation, immunocompetent animals (7-8 week old male and female mice) were sedated, and a swab saturated with 2 × 107 C. albicans cells was placed sublingually for 75 min. For colony-forming unit (CFU) enumeration the tongues were harvested, weighed, homogenized and quantitatively cultured. Neonatal mice were generated using standard breeding procedures and 8-week-old adult mice. To colonize newborn mice, 2 days after birth neonates were given orally 10 μL of 2 × 107 C. albicans cells (CA101) in HBSS. Mice were euthanized on day 14 and 21 of life. For antibody depletion wild-type mice were treated intraperitoneally with 200 μg of anti-IL-22 (IL22JOP, Invitrogen) and 200μg of anti-mouse IL-17A (17F3, BioXCell), or corresponding concentrations of isotype controls (Rat IgG2a kappa, eBR2a, mouse IgG1 isotype, MOPC-21) on day 11 and 13 post oral PB-infection. For pharmacological gp130 inhibition, mice were treated daily starting one day prior PB colonization with 10mg/kg SC144 (SelleckChem) in veterinary 0.9% Sodium Chloride (Vetivex). In some experiments, fecal pellets were collected, homogenized, and plated on SAB agar supplemented with chloramphenicol (80 μg/ml) and streptomycin (100 μg/ml). To determine dissemination after oral colonization, mice were given a subcutaneous injection of 25 mg/kg triamcinolone (Kenalog-10, Bristol-Myers Squibb Company) on day -1, +1, and+3 relative to infection. On day 4, livers were harvested, weighed, homogenized and quantitatively cultured.

Single cell RNA-sequencing. Mice were orally infected with C. albicans as described above. After 11 days of infection, the animals were administered a sublethal anesthetic mix intraperitoneally. The thorax was opened, and a part of the rib cage was removed to gain access to the heart. The vena cava was transected and the blood was flushed from the vasculature by slowly injecting 10 mL PBS into the right ventricle. The tongue was harvested and cut into small pieces in 100 µL of ice-cold PBS. 1 mL digestion mix (4.8 mg/ml Collagenase IV; Worthington Biochem, and 200 μg/ml DNase I; Roche Diagnostics, in 1x PBS) was added after which the tissue was incubated at 37°C for 45 min. The resulting tissue suspension was then passed through a 100 μm cell strainer. For one sample, single cells of two mice were combined. Single cell library preparation and sequencing were conducted by Singulomics (Bronx, USA). Cryopreserved viable single cell suspensions were thawed, washed, resuspended in cell culture media with 0.04% bovine serum albumin and counted. Viable cell suspensions were then loaded into the Chromium Controller (10x Genomics) to generate gel beads-in-emulsion (GEM), with each GEM containing a single cell as well as barcoded oligonucleotides. We targeted 10,000 cells to be captured per sample. Next, the GEMs were placed in the SimpliAmp 96-well Thermal Cycler (Thermo Fisher Scientific) and reverse transcription was performed in each GEM (GEM-RT). After the reaction, the complementary cDNA was amplified and cleaned using Silane DynaBeads (10x Genomics) and the SPRI select Reagent kit (Beckman Coulter). Amplified full-length cDNAs from poly-adenylated mRNA were then used to generate a 3′ Gene Expression library (Chromium Next GEM 3′ Single Cell Reagent kits v3.1, dual index) following the manufacturer's instructions (10x Genomics). Amplified cDNAs and the libraries were measured using Qubit dsDNA HS assay (Thermo Fisher Scientific) and quality was assessed using BioAnalyzer (Agilent Technologies). The libraries were sequenced with ~200 million PE150 reads per sample on Illumina NovaSeq 6000, and fastq files of two samples were generated using Illumina bcl2fastq. Fastq files were subsequently processed using 10x Genomics Cell Ranger analytical pipeline (v4.0.0) and mouse mm10 reference. Cellranger aggr was used to aggregate outputs from both the libraries and the aggregated filtered processed files were finally taken as input to do a quality control check and further downstream clustering analysis using Cellenics/Trailmaker software (Parse Biosciences). After quality control to remove low-quality cells expressing high mitochondrial gene signatures and exclude doublets, Louvain clustering identified 20 subgroups in our samples (Fig. S2). Cluster 1 and 10 were excluded from further analysis due to enriched lncRNA Gm42418 which is associated with ribosomal RNA contamination. The high-throughput sequencing data from this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession number PRJNA1176644.

Epithelial- enriched RNA-sequencing. The tongue tissue was placed in a 60mm petri dish, and epithelial cells were isolated by gentle mechanical isolation. The isolated cells were placed in lysis buffer reagent and homogenized in Lysing Matrix C tubes (MPBio) containing a ¼” ceramic bead (MPBio). RNA extraction was performed with the RiboPure Kit (Ambion), and RNA sequencing was performed by Novogene Corporation Inc. (Sacramento, USA) 81. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. To generate the cDNA library the first cDNA strand was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads and remaining overhangs of the purified double-stranded cDNA were converted into blunt ends via exonuclease/polymerase. After 3' end adenylation a NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. In order to select cDNA fragments of 150~200 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA). Finally, PCR amplification was performed and PCR products were purified using AMPure XP beads. The samples were read on an Illumina NovaSeq 6000 with ≥20 million read pair per sample. Downstream analysis was performed using a combination of programs including STAR, HTseq, and Cufflink. Alignments were parsed using Tophat and differential expressions were determined through DESeq2. KEGG enrichment was implemented by the ClusterProfiler. Gene fusion and difference of alternative splicing event were detected by Star-fusion and rMATS. The reference genome of Mus musculus (GRCm38/mm10) and gene model annotation files were downloaded from NCBI/UCSC/Ensembl. Indexes of the reference genome was built using STAR and paired-end clean reads were aligned to the reference genome using STAR (v2.5). HTSeq v0.6.1 was used to count the read numbers mapped of each gene. The FPKM of each gene was calculated based on the length of the gene and reads count mapped to it. FPKM, Reads Per Kilobase of exon model per Million mapped reads, considers the effect of sequencing depth and gene length for the reads count at the same time 82. Differential expression analysis was performed using the DESeq2 R package (2_1.6.3). The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the False Discovery Rate (FDR). Genes with an adjusted P-value <0.05 found by DESeq2 were assigned as differentially expressed (cutoff fold change 1.5,).To allow for log adjustment, genes with 0 FPKM are assigned a value of 0.001. Correlation were determined using the cor.test function in R with options set alternative =”greater” and method = “Spearman”. To identify the correlation between the differences, we clustered different samples using expression level FPKM to see the correlation using hierarchical clustering distance method with the function of heatmap, SOM (Self-organization mapping) and kmeans using silhouette coefficient to adapt the optimal classification with default parameter in R. We used clusterProfiler R package to test the statistical enrichment of differential expression genes in KEGG pathways. The high-throughput sequencing data from this study have been submitted to the NCBI Sequence Read Archive (SRA) under accession number PRJNA1176644.

Immunofluorescence. To determine host cell localization in vivo, 15-30-μm-thick sections of OCT-embedded tongues were fixed with cold acetone. Next, the cryosections were rehydrated in PBS and then blocked using BSA. To detect K14 (#Ab181595, Abcam), K13 (#BSM-52053R, Bioss), Ki67 (#9126S, Cell Signaling), or CD4 (#100405, Biolegend) positive cells, the sections were incubated with a primary antibody overnight followed by 1h incubation at room temperature with a secondary antibody either Alexa Fluor 488 or 568 goat anti- rabbit IgG (A11034, #A11011). To detect C. albicans, the sections were also stained with an anti-Candida antiserum (Biodesign International) conjugated with Alexa Fluor 568 (NThermo Fisher Scientific) for 1 hour. To visualize the nuclei, the cells were also stained with DAPI (4′,6-diamidino-2-phenylindole). The sections (z-stack) were imaged by confocal microscopy. To enable comparisons of fluorescence intensities among slides, the same image acquisition settings were used for each experiment.

Histology. Half-tongues were embedded in OCT (Fisher HealthCare, Houston, USA), frozen on dry ice, and stored at -80°C. Sagittal cryosections (5 μm) were air-dried at room temperature, stained with periodic acid-Schiff (Sigma-Aldrich), and counterstained with hematoxylin (Sigma-Aldrich). Sections were mounted with a toluene solution (Permount®; Fisher Chemical). Images were analyzed with a phase-contrast microscope (Zeiss Axiostar) and Gryphax Software. To quantify fungal invasion, PAS-stained sections were randomly selected and imaged by light microscopy, and the depth growth of individual fungi relative to surface of the tongue were determined using PROGRES GRYPHAX® software version 1.1.8.153 (Jenoptik) 83,84.

Cytokine and chemokine measurements in vivo. To determine the whole tongue cytokine and chemokine protein concentrations, mice were infected as described above. The mice were euthanized at various time points, and their tongues were harvested, weighed, and homogenized. The homogenates were cleared by centrifugation and the concentration of inflammatory mediators was measured using the Luminex multiplex bead assay (Invitrogen). In some experiments, IL-17A and IL-22 (R&D Systems) were determined by ELISA according to the manufacturer's instructions.

Immunophenotyping. Mice were orally infected with C. albicans as described above. After different time points, the animals were administered a sublethal anesthetic mix intraperitoneally. The thorax was opened, and a part of the rib cage removed to gain access to the heart. The vena cava was transected and the blood was flushed from the vasculature by slowly injecting 10 ml PBS into the right ventricle. The tongue was harvested and cut into small pieces in 100 μl of ice-cold PBS. 1 ml digestion mix (4.8 mg/ml Collagenase IV; Worthington Biochem, and 200 μg/ml DNase I; Roche Diagnostics, in 1x PBS) was added after which the tissue was incubated at 37°C for 30 min. The resulting tissue suspension was then passed through a 100 μm cell strainer. Cell suspensions were separated by Percoll gradient centrifugation as described before 15,36. To determine IL-17A and IL-22, cell suspensions were stimulated with Pharmingen™ Leukocyte Activation Cocktail (BD Biosciences) for 5 hours. For Th17 and Th22 cells, cells were washed and stained with CD4 antibody (BioLegend), for ILC3 cells, cells were washed and stained with FITC conjugated Gr-1, CD5, CD11b, CD45R (to eliminate unwanted cell populations), and CD3, CD90, Sca-1, CD127, CD95 (BioLegend) antibodies. For intracellular staining, cells were fixed with Cytofix/Cytoperm (BD Biosciences) and stained for 1 hour with IL-17A (BioLegend) and IL-22 (BioLegend) antibodies. The stained cells were analyzed on FACSymphony A5 system (BD Biosciences), and the data were analyzed using FACS Diva and FlowJo software. Th17 cells were identified as singlets CD4+ IL-17A+ IL22+, Th22 cells were identified as singlets CD4+ IL-17A- IL22+ and ILC3 cells were identified as singlets Gr-1- CD5- CD11b- CD45R- CD3- CD90+ CD127+ Sca-1+ CD95+ (Fig. S25).

ELISpot. Cervical lymph nodes (cLN) were isolated from PB-colonized or Sham-infected animals. cLN were digested with 2.4 mg/ml Collagenase I (Thermo Fisher) and 200 μg/ml DNase I (Roche Diagnostics) in 1x PBS. The resulting tissue suspension was then passed through a 70 μm cell strainer, washed, and live cells were determined. 1x105 cLN cells were plated on pre-coated PVDF plate, left unstimulated or were stimulated with 15 μg Candida peptide pool for 24 hrs at 37°C and 5% CO2. ELISpot was performed according to the manufacturer's instructions (ImmunoSpot). Plates were analyzed with a ImmunoSpot® Analyzer (CTL). For Candida- specific IL-17 detection, spots of Candida pool wells were determined and subtracted from spots determined in corresponding unstimulated wells. The peptide pool (lysate) was generated from stationary cultures of PB strain CA101 as described elsewhere 85.

Isolation of T cells and adoptive transfer experiments. CD4 T cells were harvested from WT mice (PB-colonized or Sham-infected 6 weeks after infection) for adoptive transfer into recipient Rag1−/− mice. The spleens and submandibular lymph nodes were harvested, enzymatically digested with PBS containing Collagenase IV (100 U/mL), DNase I (20 U/mL), 1% FBS, and 2.4 mg/ml Collagenase I (240 U/mL) and DNase I (400 U/mL) for 30 minutes. Single cell suspensions were passed through a 70-μm filter. After washing, negative CD4 T cell selection was performed using MojoSort™ Mouse CD4 T Cell Isolation Kit (#480006; BioLegend). Post-enrichment purity of CD4 T cells was >97.1%. 1×106 CD4 T cells were injected intravenously into Rag1−/− recipient mice 21 day after PB-colonization. After 10 days (day 31 post PB-colonization), CFUs and K14 thickness were determined.

Live-cell imaging for cell proliferation. 1.2×104 OKF6/TERT-2 cells were plated in 96-well plates and incubated overnight at 37ºC, allowing them to settle down. Cells were treated without or with 10, 25, 50, 100, and 200 ng/ml of human IL-22 (PeproTech) in keratinocyte serum-free medium (KSF), and immediately placed in the IncuCyte SX5 Live Cell Analysis System (Sartorius, Göttingen, Germany). To identify the impacts of IL-22-mediated receptor complex and downstream signaling pathways, cells were treated with 5µg/ml anti-hIL-22Rα1 (AF2770; R&D Systems), 5µg/ml anti-IL-10RB (90220; R&D Systems) or in a combination, 1µM TYK2 inhibitor (BMS-911543, SelleckChem), 0.5µM (C188-9; SelleckChem), 5µM STAT3 inhibitor (S3I-201; SelleckChem), 50 nM gp130 inhibitor (SC144; SelleckChem), and 5µg/ml tocilizumab (A2012; SelleckChem). Cell proliferation was observed and automatically analyzed from acquired three images per well taken every four hours for a consecutive 72 hours. Data was analyzed with the Incucyte live cell analysis software.

Immunoblotting. OKF6/TERT-2 cells in 24-well tissue culture plates were switched to KSF medium without supplements for 1 h and treated with 50 ng/mL recombinant human IL-22 (PeproTech). Next, the cells were rinsed with cold HBSS containing protease and phosphatase inhibitors and detached from the plate with a cell scraper. After the cells were collected by centrifugation, they were boiled in sample buffer. The lysates were separated by SDS-PAGE, and phosphorylation and total proteins were detected by immunoblotting with specific antibodies against pSTAT3 (Tyr705, D3A7, #9145, Cell Signaling), STAT3 (D3Z2G, #12640, Cell Signaling), and β-actin (8H10D10, #3700, Cell Signaling). Each experiment was performed at least 3 times. The immunoblots were developed using enhanced chemiluminescence and imaged with a C400 digital imager (Azure Biosystems, Dublin, CA, USA). Uncropped immunoblots are presented in Fig. S26-27.

Co-immunoprecipitation. OKF6/TERT-2 cells were grown in KSF medium with supplements to 70% confluence and cells were treated without or with 50ng/ml human IL-22 (PeproTech). After 3 hrs. cells were incubated with fresh media containing 0.1mM DSP-crosslinking (dithiobis(succinimidyl propionate) for 30min. Cells were washed twice with PBS and re-incubated with fresh media containing 25mM Tris-HCl for 15min. Cells were lysed with ice-cold IP lysis buffer with protease/phosphatase inhibitors and protein concentrations were measured by Pierce BCA protein assay (Thermo Scientific). Co-immunoprecipitation was performed using the Pierce Classic Magnetic IP/Co-IP Kit according to the manufacturer's protocol (Thermo Scientific). In brief, 400µg protein samples were added with 15µg IL22RA1 (ASF2770; R&D systems), IL10RB (MAB874; R&D systems), or gp130 antibody (362002; BioLegSend). The protein-antibody mixture was incubated overnight on a rotator at 4 ºC to facilitate immune-complex formation. Following overnight incubation, 25 μL pre-washed Pierce Protein A/G magnetic beads were added to the protein-antibody mixture to capture the immuno-complex. The mixture was incubated for 1h on a rotator at room temperature. Proteins were collected using a magnetic stand and separated by SDS-PAGE.

Proximity ligation assay. 2.5x105 OKF6/TERT-2 cells were seeded onto fibronectin-treated coverslips in a 24-well plate overnight. The next day, cells were fixed with 2% paraformaldehyde for 10 minutes at room temperature. After washing and blocking, the cells were incubated with a goat anti-IL22RA antibody (#AF2770, R&D Systems, 5μg/ml), a mouse anti-IL10RB antibody (#MAB874, Clone 90220, R&D Systems, 5μg/ml), a rabbit anti-gp130 antibody (#37325, Cell Signaling, 5μg/ml), or two of these antibodies in combination overnight at 4°C. The interactions of IL22RA with IL10RB and the interactions of gp130 with either IL22RA or IL10RB were detected using the Duolink In Situ Red Starter Kit (DUO92101-1kit, Sigma-Aldrich) following manufacturer’s instructions. The cells were imaged using a Leica TCS SP8 confocal microscope. Z-stacked images were obtained and overlaid using the Leica image processing software. To calculate the average number of complexes per cell, images were analyzed using ImageJ 86.

Quantification and statistical analysis. At least three biological replicates were performed for all in vitro experiments unless otherwise indicated. Data were compared by Ordinary one-way ANOVA or Mann–Whitney corrected for multiple comparisons were appropriate using GraphPad Prism (V. 10.3) software. P values <0.05 were considered statistically significant.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Millet et al. describes the immune response of mice infected orally with two different strains of Candida albicans.\nThe immune response and the activation of the epithelial barrier were investigated. The main highlighted response is the\nepithelial remodeling mediated by K14 expansion and the IL-22 production, particularly with the 101 Candida albicans\nstrain.\nThe manuscript is well written, and the topic is clinically relevant, but I found essential conceptual and technical limitations\nthat needed to be carefully clarified.\n1.The main limitation of the manuscript is related to the definition of the two strains as 'commensal' and 'pathogenic'.\nConceptually and scientifically, it is profoundly misleading and it needs to be explained or demonstrated in a completely\ndifferent way. The oral model used by the authors is a simple mouse model of an acute infection, it is wrong to consider it as\na 'commensalism' model. The infection is persistent with the 101 strain and this is not identified as a 'symbiotic' relation with\nthe host but conversely as an opportunistic behaviour.\n2.Figure S1 A is very surprising. Authors claim that the CL strain is not inducing inflammation but a non-inflammatory\npathogen can't be associated with such a high persistent mouth colonization. Data should be presented by showing the\nstandard deviation and if there is also dissemination in other tissues.\n3. Authors have to clarify or modify the model, by considering also that Candida albicans is a human gut commensal and not\nproperly a mouse commensal.\n4. It is not clear why in figure 1E it is only shown the histology of mice infected with the CL strain and not the PL strain. Same\nissue for figure 1F, 1G, 1M.\n5. In figure 1C it is surprising that single cell is not focused on Th17 cells but only Tregs are specifically stained, although\nTregs were not the focus of the study.\n6. Statistics is lacking in figure 1C\n7. In figure 2, authors claim that CL is not inducing inflammation. This sentence is profoundly wrong, considering the\npathogenic colonization overtime that the CL strain is inducing and also the evidence that the CL strain is inducing IL-17 and\nIL-22, two pro-inflammatory cytokines. Data coming from the assay in figure 2A should be repeated eventually by using\nconventional ELISA, since it is extremely surprising that IL-17 is induced and there are not T cell polarizing cytokines. In\nother words, do the author know how CD4+IL-22+ T cells were polarized if no inflammation was induced?\n8. Figure 2 E is not showing statistics.\n9. Figure F is showing a wrong statistic test.\n10. Figure H is showing plots not clear. Gating strategy is not presented. CD4+IL-22+T cells should be shown.\n11. Figure 4H is not clear. Why keratin is still there with no T cells?\n12. Conclusions are in apparent contradiction with the main thesis. If you consider 101 a 'commensal' strain, how do you\nconsider essential for survival the induction of IL-17 and IL-22 and keratinization? This is only suggesting an host essential\n(and already shown) response against a fungal pathogen.", "reviewer_2": "The manuscript by Millet et al. demonstrates that non-canonical IL-22 receptor complex (IL-22RA1/IL-10Rb/gp130) mediates\ncommensal Candida-induced remodeling of the oral epithelium, facilitating immunosurveillance of the oral mucosa. Through\ncomparison of pathogenic and commensal strains of Candida, the authors demonstrate that IL-22 is persistently upregulated\nuntil day 11, a process critical for inducing K14+ epithelial thickening via the IL-22RA1/IL-10Rb/gp130 complex, as\nsupported by complementary knockout and inhibition experiments. This study provides valuable insights into non-canonical\nIL-22R signaling in antifungal responses, but certain aspects require further clarification to strengthen the conclusions.\nBelow are my comments and suggestions.\n1.Figures 2A and 2B indicate that IL-22 expression persists in the CL group but not in the PL group, with significantly higher\nlevels at day 11 compared to IL-17A. However, Figure 2I shows that Th17 cells dominate the CD4+ T cell population, with\nan elevated frequency in the CL group, except for a single low outlier, which likely affects statistical significance. Meanwhile,\nTh22 cells are relatively low in frequency despite being significantly upregulated in the CL group. To better assess Th22\ncontributions, I recommend quantifying absolute cell numbers in addition to percentages of CD4+ T cells. Another possible\nexplanation is that the Th17 population in Figure 2H might include IL-22/IL-17A double positive cells, which the authors\nshould clarify or emphasize further.\n2.Schönherr et al. (Mucosal Immunology, 2017) previously reported that IL-17 is critical for responses to both pathogenic and\ncommensal Candida strains in oral infection models, with fungal burden increasing in both Il17ra and Il17rc knockout mice\nfor both SC5314 and CA101 strains. This finding appears to conflict with the results shown in Figure 2D, where no fungal\nload differences were observed after IL-17 neutralization. The authors should discuss or speculate on why these\nobservations differ.\n3.Based on the fungal CFU and K14+ epithelial thickness data in Figure 3, thickening appears to be mediated primarily by\nnon-canonical IL-22 receptor signaling through gp130. However, gp130 inhibition only partially impairs antifungal activity\n(Figure 3M), as CFU levels with gp130 inhibition are similar to those in Il22ra1 KO (Figure 3E), while K14+ thickening\npersists in the latter. This raises questions about the relationship between K14+ thickening and antifungal capacity. Could\nthe authors elaborate on how these two phenomena are interconnected in the CL group?\n4.The inverse correlation between Candida-specific IL-17+ cells and K14+ thickness in wild-type mice at days 11 and 31 is\nintriguing. The authors hypothesize that epithelial resetting is mediated by Th17 expansion, controlling CL Candida, while\nthickening persists in Rag1-/- mice. Since CFU levels in wild-type mice are comparable between days 11 and 31, have the\nauthors investigated CFU levels in Rag1-/- mice at day 11? Additionally, given the IL22TdTomato reporter data indicating\nCD4+ T cells as the primary IL-22 producers during CL colonization (Fig. 2F), what factors might drive epithelial thickening\nin the absence of T cells in Rag1-/- mice which lack IL-22 producing T cells?\n5.The methodology for quantifying invasion depth in Figure 5I&J should be clearly detailed in the Materials and Methods\nsection. Furthermore, the mechanisms underlying the increased fungal invasion observed in IL-22 KO neonates compared\nto adults remain unclear. Are fungal burdens different between neonates and adults? Have the authors examined the roles\nof other immune cells in colonization across these age groups? The findings in Figure 5 are primarily descriptive, and more\nmechanistic insights would strengthen this section.\nMinor comments:\n1.As all molecular analyses were performed on day 11, when Candida is absent in the PL group, how do the authors\ndifferentiate the effects of Candida presence on epithelial remodeling from baseline mucosal epithelial properties? For\nexample, the lack of CD4+ T cells in the submucosal layer of the PL group (Fig. 2G) might simply reflect the absence of\nCandida. Clarification on this point would be helpful.\n2.The statement \"in CL-colonized mice, mucosal IL-22 levels remained high, while IL-17A levels were low at the onset of\ncolonization\" (Lines 114–115) should be revised, as IL-17A levels fluctuate significantly between days 2 and 5.\n3.The legend for Figure S8 refers to treatment with both isotype and anti-IL-22 antibodies. However, the figure only includes\nPL and CL groups without showing data for isotype controls. The authors should ensure all experimental controls are\nrepresented.\n4.Immunofluorescence images of the control group in Figure S6 would be informative. The aggregated IL-22RA1 signal in\nthe PL group appears less pronounced in the CL group. Is this difference due to prior Candida infection, or is it also\nobserved in uninfected controls?\n5.The apparent toxicity or growth inhibition caused by the STAT3 inhibitor (Figure S10C) should be addressed by including\na control group treated with the inhibitor alone. This would rule out potential off-target effects or the possibility that STAT3\nsignaling is essential for epithelial growth or survival.\n6.The function of tocilizumab as an IL-6R-neutralizing antibody should be stated in the text or the legend for Figure S15.\n7.The specificity of gp130 signaling in oral epithelium is an interesting observation. Have the authors examined whether\nsimilar mechanisms are involved in other mucosal tissues, such as the vaginal epithelium, another major colonization site\nfor Candida?\n8.It would be interesting to determine how long CL Candida can colonize the oral mucosa. In Figure 4A, fungal burden\nremains comparable between days 11 and 31, while IL-17+ cell numbers significantly increase by day 31, suggesting\nimmune resetting as proposed by the authors. How do the authors integrate this inverse relationship between IL-17+ cells\nand K14+ thickening in the context of long-term colonization?", "reviewer_3": "In this manuscript, Millet et al. report that oral colonization with a fungal component of the human mouth mycobiota elicits\nremarkable changes in the structure of the oral epithelium in mice. Specifically, the authors show that the tongue’s epithelial\nlayer expands in a process dependent on IL-22 signaling upon colonization with the oral commensal Candida albicans\nisolate 101, a strain that can persist for weeks/months in the murine tongues. Furthermore, Millet et al. demonstrate that IL-22\nsignaling depends on a non-canonical receptor complex composed of gp130 coupled to IL-22RA1 and IL-10RB. Finally,\nusing a mouse model of neonatal fungal colonization, the authors propose that IL-22-mediated immunity and tissue\nremodeling have key roles controlling fungal invasion early in life.\nWhile below I list several weaknesses that I expect should be fully addressed by the authors, I do believe that the main\nfindings reported in the manuscript are generally well supported. Likewise, the work described in this manuscript, in my\nopinion, constitutes a substantial and important step forward in our understanding of fungal-host interactions.\n1) A major issue is the PL-CL comparison done at 11 days post inoculation throughout the manuscript. It is well established\nin the field, and also shown in Fig S1, that at d11 post PL infection, no Candida cells remain in the tissue (PL infection is\ncleared ~72h after infection). CL-inoculated animals, on the other hand, have a stable fungal load at d11 which persists for\nweeks. It is unclear what to make, then, of comparisons like the ones shown in Figs. 1C and 1J-L. As it is, what the authors\nare actually comparing is the effect of post PL infection to active CL colonization. But that’s not how the data are described or\nthe results interpreted. If the purpose is to reveal effects elicited by the CL strain, the control should be ‘sham.’ If the purpose\nis to compare PL-CL effects, the comparison should include an early time point for PL because only then there’s a clear\neffect of fungal presence in the tissue. This is appropriately done only for a few cytokines (Figs. 2A-B) but not for the larger\nscRNA-Seq or epithelial-enriched RNA-Seq included in Fig. 1.\n2) Line 109 and Fig. 2A: “Acute infection with the PL C. albicans strain led to early TNFalpha and IL-1beta induction.” In Fig\n2A, the prominent cytokines up only in PL at day 2 seem TNFalpha and IFNgamma (not IL-1beta).\n3) Lines 122-124 and Fig. 2G: “CD4 T cells were exclusively found in the epithelial and submucosal layers of commensal\ncolonized mice.” The staining in Fig 2G which supports this statement was done with animals 11 days post infection. As\nstated above in point #1, by this time the PL-infected mice have already cleared the fungus. The statement implies that CD4\nT cells are never found during the entire course of infection with PL isolate. But at 2 days post infection, CD4 T cells may\nwell be found in the PL-infected tissue.\n4) Fig 4. While the transitory nature of the epithelial K14 expansion seems clear, the connection of this phenomenon to\n“Candida-specific immunity” seems vague. The increase in Ca-specific IL-17 secreting cells (Fig. 4D) observed at similar\ntimepoints is a correlation but does not indicate causation. The data shown in Rag1-/- animals, while consistent with the\nauthor’s premise, is incomplete (e.g. we don’t know if the epithelial expansion takes place in these animals upon CL\ncolonization) and not very specific (the general lack of mature B/T cells in these animals likely have myriad effects).\nAt the very least, the authors should provide a more nuanced interpretation of the data."}, "author_response": "REVISION#1\nRESPONSE TO REVIEWER COMMENTS\nWe would like to thank the reviewers for the critical reading and constructive comments that\nhelped us to improve the manuscript. We have addressed all comments and provide a detailed\npoint-to-point reply to all comments below with changes shown in yellow highlight in the revised\nmanuscript.\nReviewer #1 (Remarks to the Author):\nThe manuscript by Millet et al. describes the immune response of mice infected orally with two\ndifferent strains of Candida albicans.\nThe immune response and the activation of the epithelial barrier were investigated. The main\nhighlighted response is the epithelial remodeling mediated by K14 expansion and the IL-22\nproduction, particularly with the 101 Candida albicans strain. The manuscript is well written, and\nthe topic is clinically relevant, but I found essential conceptual and technical limitations that\nneeded to be carefully clarified.\n1.The main limitation of the manuscript is related to the definition of the two strains as 'commensal'\nand 'pathogenic'. Conceptually and scientifically, it is profoundly misleading and it needs to be\nexplained or demonstrated in a completely different way. The oral model used by the authors is\na simple mouse model of an acute infection, it is wrong to consider it as a 'commensalism' model.\nThe infection is persistent with the 101 strain and this is not identified as a 'symbiotic' relation with\nthe host but conversely as an opportunistic behaviour.\nResponse: We appreciate the reviewer’s insightful comment regarding the use of the terms\n\"commensal\" and \"pathogenic\" to describe the two C. albicans strains in our study. A commensal\nmicrobe is one that induces no perceptible or only clinically inapparent damage to the host, often\nestablished early in life and resulting in a stable host-microbe relationship without disease\n(PMCID: PMC97744). Thus, labeling the C. albicans strains as \"commensal\" or \"pathogenic\"\nreflects measurable outcomes of their interaction with the host, focusing on host damage and\ninflammatory responses rather than simplistic presence or persistence. Furthermore, the CA101\n(CL) strain’s persistent colonization with curbed inflammation aligns with features interpreted as\ncommensalism, while SC5314 (PL) trigger inflammatory infection and clearance (PMID\n28176789; 30873177). We agree that these classifications can oversimplify the dynamic and\ncontext-dependent nature of host-microbe interactions, particularly in the case of opportunistic\nfungi. Thus, we labeled theses strains as “pathogenic-like (PL)” and “commensal-like (CL)” to\nreflect published (PMID 28176789; 30873177; 32719409; 35404986) and observed phenotypes\nand host responses (this manuscript).\n2.Figure S1 A is very surprising. Authors claim that the CL strain is not inducing inflammation but\na non-inflammatory pathogen can't be associated with such a high persistent mouth colonization.\nData should be presented by showing the standard deviation and if there is also dissemination in\nother tissues.\nResponse: We appreciate the reviewer’s comment. As discussed in comment 1 the CL strain\nused (CA101) has been described initially to fail to induce an acute inflammatory response in the\nhost (PMID 28176789). This phenotype of strain CA101 is well established (PMID 30873177;\n32719409), and our new data strengthens the initial observation by the LeibundGut-Landmann\nlab.\nThe standard deviation is included, and we added statistics for Fig. S1A. Of note, in the\nimmunocompetent mouse model of OPC (used throughout our study), oral Candida infection does\nnot lead to organ dissemination. However, dissemination has been observed during\nimmunosuppression e.g. by using cortisone acetate or mice with defects in barrier immunity\n(PMID: 24442441; 19204111; 24980544). While oral infection in immunocompetent mice does\nnot lead to organ dissemination, oral infection leads to GI tract colonization (Fig. S1B) in which\nthe PL strain is cleared (as described in PMID: 34724818) but the CL strain remained stable\nthroughout the course of our study.\nFigure S1 The commensal-like (CL) isolate CA101 persists in the oral cavity and GI\ntract following oral infection. A Oral fungal burden of wild-type mice infected with\nindicated strains. Results are median ± standard error of the mean (SEM) of four\nindependent experiments (N = 5/group). B Fecal burden of wild-type mice after oral\ninfection with indicated strains. The y-axis is set at the limit of detection (20 CFU/g tissue).\nPL, pathogen-like; CL, commensal-like.\n3. Authors have to clarify or modify the model, by considering also that Candida albicans is a\nhuman gut commensal and not properly a mouse commensal.\nResponse: We have revised the introduction to emphasize that C. albicans colonizes different\nmucosal surfaces. “As a central constituent of the mycobiome, the fungus Candida albicans\ncolonizes the oral and gastrointestinal (GI) mucosa of up to 75% of healthy individuals.”\n4. It is not clear why in figure 1E it is only shown the histology of mice infected with the CL strain\nand not the PL strain. Same issue for figure 1F, 1G, 1M.\nResponse: We thank the reviewer bringing this to our attention. We have added histology (PAS\nstaining; S3) and K14 staining for PL infected mice for day 2, 5, 8, and d11 (Fig. S6). The data\nshows that no epithelial expansion or epithelial remodeling occurs during infection with a PL strain\nor after infection resolution.\nFigure S3. Representative pictures of PAS staining of PL-infected mice over time. N=4-6.\nFigure S6 Time\ncourse studies of\nK14 epithelial\nexpansion.\nRepresentative\nimmunofluorescence\npictures of keratin 14\n(K14) after indicated\ntime points. Mice were\ninfected with CL (A) or\nPL strain (B). Scale bar\n50μm.\n5. In figure 1C it is surprising that single cell is not focused on Th17 cells but only Tregs are\nspecifically stained, although Tregs were not the focus of the study.\nResponse: While scRNA-seq is excellent for measuring RNA transcript levels in individual cells,\nit may not directly reflect the amount or activity of the corresponding protein (PMID: 33414677).\nThus, we determined IL-22 positive immune cell populations (Fig. 2f), as well as the CD4\nsubpopulations Th17 and Th22 (Fig. 2i) via flow cytometry.\n6. Statistics is lacking in figure 1C\nResponse: This dataset served as an unbiased approach to identify broad cell populations in the\noral cavity. Verification of cell numbers and percentages were done separately (Fig. 1g,1l,1m, 2f,\n2i) to confirm this dataset.\n7. In figure 2, authors claim that CL is not inducing inflammation. This sentence is profoundly\nwrong, considering the pathogenic colonization overtime that the CL strain is inducing and also\nthe evidence that the CL strain is inducing IL-17 and IL-22, two pro-inflammatory cytokines. Data\ncoming from the assay in figure 2A should be repeated eventually by using conventional ELISA,\nsince it is extremely surprising that IL-17 is induced and there are not T cell polarizing cytokines.\nIn other words, do the author know how CD4+IL-22+ T cells were polarized if no inflammation\nwas induced?\nResponse: We acknowledge the reviewer’s concern and agree that this statement may have been\noverly broad. Our intention was to convey that, relative to classical inflammatory stimuli, the CL\nstrain does not trigger a robust or classical pro-inflammatory cytokine milieu in the oral mucosa\nsuch as the PL strain. However, we recognize that the induction of IL-17 and IL-22 reflects a\ndegree of mucosal immune activation. We have revised the text to more accurately state that\n\"Thus, CL colonization induces limited inflammation characterized primarily by a type 17 response\n(IL-17/IL-22), rather than broad pro-inflammatory activation as seen during the early phase of PL\ninfection.\" (line 123ff) The reviewer raises an important point about the cytokine milieu. While we\nobserved significant induction of IL-17 and IL-22, the levels of classical upstream polarizing\ncytokines (e.g., IL-1β) were relatively low or unchanged compared to Sham by the multiplex\nassay. This discrepancy might have been due to sensitivity limitations of the Luminex assay used\nor to the transient and localized expression of these cytokines. To address this concern, we have\nrepeated the assay using conventional ELISAs for TNFα and IL-1β on collected samples (included\nin Rebuttal only; Fig. R1). These results confirmed low levels in the CL-colonized mucosa,\nsupporting the notion that a limited inflammatory environment is sufficient to drive type 17\npolarization.\nSham PL CL\n30000 150000\nA Figure R1 Correlation\nS\nIL\nE\ng /g\np\n20000 A\nS IL\nE\nβ\n100000\no\nm\nf\ne a\nT\ns\nN\nu\nF\nre\nα\nd\nand IL-\nb\n1β\ny\nF α 10000\n1\n-L 50000 Luminex and ELISA\nN I\nT after 2 days of\nr 0.8392 r 0.8225\nP<0.0001 P0.0002 infection.\n0 0\n0 10000 20000 30000 40000 50000 0 50000 100000 150000 200000 250000\nTNFα pg/g LUMINEX IL-1β LUMINEX\n8. Figure 2 E is not showing statistics.\nResponse: We added the statistics performed a Mann-Whitney test.\nIl22\nPL Figure 2e Expression\n8\nof Il22 in CD4 T cells\nn\no is 6 identified in the single\ns\ne cell RNA-sequencing\nrp 4\nx E data set. Mann-\n9. Figure F is showing a wrong statistic test.\nResponse: We thank the reviewer to bring this to our attention. Indeed, in the original manuscript\nwe did not correct for multiple comparison. In the revised version, we performed Ordinary one-\nway ANOVA corrected for multiple comparison.\nPL CL\n1×104\nFigure 2f Quantification of IL-\nP0.0077 22 expressing cells after PL\n7.5×103 P0.0286\nand CL infection (day 11)\ns\nlle\nc using IL22TdTomato reporter\n+ 5×103\n2 2 mice. N=4. Ordinary one-way\n-L\nI ANOVA corrected for multiple\n2.5×103\ncomparison.\n10. Figure H is showing plots not clear. Gating strategy is not presented. CD4+IL-22+T cells\nshould be shown.\nResponse: We thank the reviewer for this comment. The gating strategy is presented in figure\nS25. To clarify, Th17 cells were identified as singlets CD4+ IL-17A+ IL22+. Th22 cells were\nidentified as singlets CD4+ IL-17A- IL22+. We revised the plot graph to indicate this.\nFigure 2h Representative flow cytometry\nplots for intracellular IL-17A and IL-22\nlevels in CD4+ cells. Th17 cells were\nidentified as CD4+ IL-17A+ IL-22+. Th22\ncells were identified as CD4+ IL-17A- IL-\n22+.\n11. Figure 4H is not clear. Why keratin is still there with no T cells?\nResponse: We thank the reviewer for this comment. While cytokine expression exhibits a\nsignificant degree of cell-type specificity, compensation by other immune cell types in the absence\nof a particular immune cell can occur due to the plasticity and redundancy within the immune\nsystem. IL-22 expression is not limited to CD4 T cells. Activated NK cells, NK T cells, CD8 T cells,\nγδ T cells, and innate lymphoid cells (ILCs) can also express IL-22 (PMID: 15308104, 17187052,\n19100701, 24448096). Importantly, CD4 T cells are sufficient to reduce IL-22-dependent ILC\nresponses, while ILCs proliferate in the absence of mature CD4 T cells (Rag1 KO mice; PMID\n24448096). In this line, ILCs are required to control fungal outgrowth during acute OPC (PMID:\n23255360). Thus, the immune system demonstrates functional redundancy, where various\ncytokines and immune cells can perform overlapping roles to maintain immune functionality. This\nredundancy allows other immune cell types to partially compensate for the absence of a specific\nimmune cell type by producing cytokines otherwise handled by the missing cell type. We took\nadvantage of this system to analyze if Candida-specific T cell immunity is required to reverse\nepithelial remodeling at later stages of colonization. First, we tested if Rag1 KO mice show\nincreased IL-22 levels at the onset of CL colonization (Fig. S21). This data underlines the\nimportance of the immune system’s plasticity and compensatory actions. Additionally, we added\nfungal burden data as well as K14 thickness of Rag1 KO mice after 11 days of CL colonization\n(Fig. 4f; S21). Of note, the increase in oral burden in Rag1 KO mice is consistent with published\ndata of increased susceptibility of these mice to CL infection (PMID: 32719409). Thus, in the\nabsence of T cells in Rag1 KO, fungal persistent colonization induces upregulation of IL-22 to\ninduce epithelial proliferation and modulation.\nFigure 4f Oral fungal burden of WT and\nRag1–/– mice colonized with CL after 11 and\n31 days . N=4-5; combined data of three\nindependent experiments. Two-tailed Mann–\nWhitney Test. The y-axis is set at the limit of\ndetection (20 CFU/g tissue).\nFigure S21 Figures S17 Rag1–/– mice express IL-22 in the oral mucosa during CL\ncolonization. A IL-22 levels in CL-, and Sham-infected mice; N = 4. Ordinary one-way\nANOVA corrected for multiple comparison. B Representative pictures of K14 staining of\nSham- and CL-infected mice after 11 days. Scale bar 50μm. C Quantification of K14\nthickness in Sham- and CL-infected WT and Rag1–/– mice at day 11. N = 4. Two-tailed\nMann–Whitney Test.\n12. Conclusions are in apparent contradiction with the main thesis. If you consider 101 a\n'commensal' strain, how do you consider essential for survival the induction of IL-17 and IL-22\nand keratinization? This is only suggesting an host essential (and already shown) response\nagainst a fungal pathogen.\nResponse: We appreciate the reviewer’s thoughtful question. While IL-17, IL-22, and epithelial\nremodeling are often associated with responses to pathogens, these mechanisms also play\nessential roles in homeostatic immune surveillance at barrier sites. In our model, the CL strain\ninduces a non-pathological, calibrated response that promotes barrier integrity and prevents\nfungal overgrowth, especially in early life. We do not interpret the involvement of IL-17, IL-22, or\nkeratinization as evidence of pathogenicity, but rather as components of a protective adaptation\nthat maintains host-microbe balance without causing tissue damage. Consistent with this\ninterpretation, we identify a novel host-specific mechanism triggered by persistent oral C. albicans\ncolonization, in which IL-22 signaling promotes barrier protection without inducing inflammation\nor tissue damage. Additionally, to the best of our knowledge this is the first report of oral epithelial\nexpansion mediated by non-canonical Il22 receptor complex signaling to amplify antifungal\nimmunity.\nReviewer #2 (Remarks to the Author):\nThe manuscript by Millet et al. demonstrates that non-canonical IL-22 receptor complex (IL-\n22RA1/IL-10Rb/gp130) mediates commensal Candida-induced remodeling of the oral epithelium,\nfacilitating immunosurveillance of the oral mucosa. Through comparison of pathogenic and\ncommensal strains of Candida, the authors demonstrate that IL-22 is persistently upregulated\nuntil day 11, a process critical for inducing K14+ epithelial thickening via the IL-22RA1/IL-\n10Rb/gp130 complex, as supported by complementary knockout and inhibition experiments. This\nstudy provides valuable insights into non-canonical IL-22R signaling in antifungal responses, but\ncertain aspects require further clarification to strengthen the conclusions. Below are my comments\nand suggestions.\n1.Figures 2A and 2B indicate that IL-22 expression persists in the CL group but not in the PL\ngroup, with significantly higher levels at day 11 compared to IL-17A. However, Figure 2I shows\nthat Th17 cells dominate the CD4+ T cell population, with an elevated frequency in the CL group,\nexcept for a single low outlier, which likely affects statistical significance. Meanwhile, Th22 cells\nare relatively low in frequency despite being significantly upregulated in the CL group. To better\nassess Th22 contributions, I recommend quantifying absolute cell numbers in addition to\npercentages of CD4+ T cells. Another possible explanation is that the Th17 population in Figure\n2H might include IL-22/IL-17A double positive cells, which the authors should clarify or emphasize\nfurther.\nResponse: We thank the reviewer for bringing this to our attention. We added the total numbers\nof Th17 and Th22 cells. In the original manuscript, we identified IL17A+ IL22+ cells as Th17 cells.\nWe now clarified this in the graph. Please see also comment 11 from reviewer 1.\n2.Schönherr et al. (Mucosal Immunology, 2017) previously reported that IL-17 is critical for\nresponses to both pathogenic and commensal Candida strains in oral infection models, with\nfungal burden increasing in both Il17ra and Il17rc knockout mice for both SC5314 and CA101\nstrains. This finding appears to conflict with the results shown in Figure 2D, where no fungal\nload differences were observed after IL-17 neutralization. The authors should discuss or speculate on\nwhy these observations differ.\nResponse: We thank the reviewer for this insightful comment. The discrepancy between the\nconserved requirement of IL-17 signaling for fungal control in IL-17 receptor knockout mice\nreported by Schönherr et al. and the lack of fungal load differences after IL-17 neutralization (this\nmanuscript) can be understood by considering several factors related to experimental design,\ntiming, and the nature of genetic knockout of a receptor versus neutralization of a cytokine.\nSchönherr et al. demonstrated that IL-17 signaling through IL-17RA and IL-17RC receptors is\nessential for controlling fungal burdens of both highly virulent (SC5314) and commensal-like\n(CA101) C. albicans strains in oral infection model. In our manuscript, we depleted IL-17A at the\nonset of colonization after 11 days of oral colonization. This observed difference in IL-17\nrequirement would suggest that the effects of IL-17 signaling may be more critical at earlier stages\nof infection, without immediately affecting fungal burden at the onset of oral colonization as we\nhave analyzed. We revised this in the manuscript line 127 “This suggested that IL-22 may have\na prominent role in controlling fungal outgrowth after mucosal colonization, while IL-17 signaling\nmay be more critical at earlier stages of infection, without immediately affecting fungal burden at\nthe onset of oral colonization.” Notably, while IL-17A signals through the receptor IL-17RA/RC,\nIL-17F signaling is also mediated by this receptor complex (PMID: 19575028). Thus, Schönherr\net al. inhibited IL-17A/F signaling via genetic knockout of a receptor, while we neutralized the\ncytokine IL-17A without targeting IL-17F at the onset of colonization.\n3.Based on the fungal CFU and K14+ epithelial thickness data in Figure 3, thickening appears to\nbe mediated primarily by non-canonical IL-22 receptor signaling through gp130. However, gp130\ninhibition only partially impairs antifungal activity (Figure 3M), as CFU levels with gp130 inhibition\nare similar to those in Il22ra1 KO (Figure 3E), while K14+ thickening persists in the latter. This\nraises questions about the relationship between K14+ thickening and antifungal capacity. Could\nthe authors elaborate on how these two phenomena are interconnected in the CL group?\nResponse: We thank the reviewer for this insightful comment. We added epithelial RNA-seq data\nin which we show that mice lacking IL-22 or inhibition of gp130 signaling during CL colonization\nreduced expression of key antimicrobial peptides (Fig. 3q, S20), known to inhibit fungal\nproliferation (PMID: 24951441.) This data show that epithelial expansion enhances antifungal\nimmunity by amplifying expression of antimicrobial peptides.\nFigure 3q Z-scores of antimicrobial peptide\ngenes in epithelial-enriched tissues in WT and\nIl22–/– mice, and WT mice treated with gp130\ninhibitor (igp130) after CL infection on day 11.\nN = 3.\nWT Il22–/– igp130\n200 200 200 Figure S20 Epithelial\nthickness correlates with\n)m )m )m\nμ150 μ150 μ150 antimicrobial peptide\n(\n+\n(\n+\n(\n+\n4 4 4 expression. Correlation of\n1 1 1\nK100 K100 K100\ns s s K14 thickness and\ns s s\ne e e\nn k c 50 n k c 50 n k c 50 antimicrobial peptide genes\nih\nt r 0.8114\nih\nt r 0.9624\nih\nt r 0.7773 (Defb1, Defb3, Sprr2b). N=3.\n-2 -1 0 1 2 -1 0 1 2 3 -2 -1 0 1 2\nby Pearson.\nDefb1 z-score Defb3 z-score Sprr2b z-score\n4.The inverse correlation between Candida-specific IL-17+ cells and K14+ thickness in wild-type\nmice at days 11 and 31 is intriguing. The authors hypothesize that epithelial resetting is mediated\nby Th17 expansion, controlling CL Candida, while thickening persists in Rag1-/- mice. Since CFU\nlevels in wild-type mice are comparable between days 11 and 31, have the authors investigated\nCFU levels in Rag1-/- mice at day 11? Additionally, given the IL22TdTomato reporter data\nindicating CD4+ T cells as the primary IL-22 producers during CL colonization (Fig. 2F), what\nfactors might drive epithelial thickening in the absence of T cells in Rag1-/- mice which lack IL-22\nproducing T cells?\nResponse: We thank the reviewer for these comments. Please see our reply to comment #11\nfrom reviewer 1 in which we describe that in the absence of T cells in Rag1 KO, fungal persistent\ncolonization induces upregulation IL-22 to induce epithelial proliferation and modulation.\nFurthermore, we added data showing that Cd4 KO mice are more susceptible to infection, while\nproducing IL-22 to expand the epithelial tissue. This data underscores that IL-22 expression is\nnot limited to CD4 T cells during CL infection.\nFigure S22 Cd4–/– mice are more susceptible to CL infection, express IL-22 and\nremodel the oral mucosa. Oral fungal burden of indicated mice colonized with CL. N = 6;\ncombined data of two independent experiments. Two-tailed Mann–Whitney Test. The y-\naxis is set at the limit of detection (20 CFU/g tissue). B Representative pictures of K14\nstaining of CL-infected mice after 11 and 31 days. Scale bar 50μm. C Quantification of K14\nthickness in CL-infected mice at indicated time points. N = 6; combined data of two\nindependent experiments. Two-tailed Mann–Whitney Test. D IL-22 levels in CL -infected\nmice; N = 6. Two-tailed Mann–Whitney Test.\n5.The methodology for quantifying invasion depth in Figure 5I&J should be clearly detailed in the\nMaterials and Methods section. Furthermore, the mechanisms underlying the increased fungal\ninvasion observed in IL-22 KO neonates compared to adults remain unclear. Are fungal burdens\ndifferent between neonates and adults? Have the authors examined the roles of other immune\ncells in colonization across these age groups? The findings in Figure 5 are primarily descriptive,\nand more mechanistic insights would strengthen this section.\nResponse: We have added the description in the methods section (line449) which we published\npreviously (PMID: 28325761). It now reads “To quantify fungal invasion, PAS-stained sections\nwere randomly selected and imaged by light microscopy, and the depth growth of individual fungi\nrelative to surface of the tongue were determined using PROGRES GRYPHAX® software version\n1.1.8.153 (Jenoptik)”. For reference, we added a small graph in this rebuttal letter to visualize\nthis (see below). Furthermore, we acknowledge that Figure 5 is primarily descriptive. However, it\nhighlights a critical phenotype- age-dependent susceptibility to fungal invasion in the absence of\nIL-22 which serves as a foundation for future mechanistic studies. Of note, the experiments are\nongoing.\nFigure R2 Example\nof invasion depth\nmeasurement.\nMinor comments:\n1.As all molecular analyses were performed on day 11, when Candida is absent in the PL group,\nhow do the authors differentiate the effects of Candida presence on epithelial remodeling from\nbaseline mucosal epithelial properties? For example, the lack of CD4+ T cells in the submucosal\nlayer of the PL group (Fig. 2G) might simply reflect the absence of Candida. Clarification on this\npoint would be helpful.\nResponse: We thank the reviewer for raising this important point regarding the interpretation of\nepithelial and immune changes in relation to fungal presence versus baseline mucosal state. We\nagree that the absence of detectable Candida in the PL group at day 11 complicates direct\ncomparisons, as differences in epithelial remodeling and immune cell localization could indeed\nreflect divergent host responses to Candida exposure rather than intrinsic baseline properties.\nHowever, our interpretation is supported by several key considerations. Although Candida is\nundetectable by day 11 in the PL group, mice were colonized earlier in the time course, and thus\nany remodeling or immune changes observed at this time point may represent residual or\nresolving responses to transient Candida exposure. In contrast, the CL group sustains Candida\ncolonization through day 11, allowing us to capture mucosal responses under conditions of\npersistent fungal presence. In some instances, we included uncolonized (naïve or Sham) mice as\na reference for baseline epithelial and immune architecture. These controls showed no distinct\ndifferences from PL and CL mice in epithelial thickness. However, we agree that the PL phenotype\nmay not fully represent the naïve state, but rather a response to prior Candida exposure and\nsubsequent clearance. We added a statement to line 84 ”… representing distinct post-infection\nhost states, namely immune and barrier responses post-fungal clearance (PL) and one reflecting\nongoing Candida presence and interaction (CL)”. As the reviewer notes, CD4 T cells are absent\nfrom the submucosa in the PL group at day 11, while they are enriched in the CL group. We\nperformed experiments in which we stained for CD4 T cells after 2 days post PL and CL infection.\nThese data indicate that during early oral fungal infection no CD4 T cells migrate in the epithelial\nand submucosal layers and this absence is independent of the strain (PL vs. CL). We interpret\nthis as a reflection of ongoing antigenic stimulation by Candida in CL mice, which likely sustains\nlocal CD4 T cell recruitment and retention. To clarify this distinction, we have revised the text in\nmanuscript to “Next, we evaluated the localization of CD4 T cells during fungal persistence within\nthe oral mucosa. CD4 T cells were exclusively found in the epithelial and submucosal layers of\nCL-infected mice at the onset of colonization, which differed markedly from PL- or Sham-infected\nmice (Fig. 2g, S8) suggesting that persistent fungal stimulation is required to recruit and retain\nCD4 T cells in submucosal layers.” (line 134).\nFigure S8 Representative\nimmunofluorescence pictures\nof CD4 expression after 2 days\nof infection. Mice were infected\nwith PL and CL. Scale bar\n50μm.\n2.The statement \"in CL-colonized mice, mucosal IL-22 levels remained high, while IL-17A levels\nwere low at the onset of colonization\" (Lines 114–115) should be revised, as IL-17A levels\nfluctuate significantly between days 2 and 5.\nResponse: We revised the sentence to “However, in CL-colonized mice, mucosal IL-22 levels\nremained high, whereas IL-17A levels declined to low levels at the onset of colonization.” (Line\n121ff.).\n3.The legend for Figure S8 refers to treatment with both isotype and anti-IL-22 antibodies.\nHowever, the figure only includes PL and CL groups without showing data for isotype controls.\nThe authors should ensure all experimental controls are represented.\nResponse: We apologize for providing the wrong figure for the anti-IL-22 depletion experiment.\nWhile preparing the manuscript, we accidently duplicated the K13 distribution of CL and PL\ninfected mice. We have replaced it with the correct data.\nFigure S11 Effect of IL-22 depletion on K14 expansion during oral fungal colonization\n(A) Representative immunofluorescence pictures of keratin 14 (K14) after 11 days of\ninfection. Mice were infected with CL and treated with isotype or α-IL-22 antibody starting\nday -1 every other day. Scale bar 50μm. B Quantification of K14 thickness in indicated mice\nat day 11. N = 6; combined data of two independent experiments. Mann-Whitney Test.\n4.Immunofluorescence images of the control group in Figure S6 would be informative. The\naggregated IL-22RA1 signal in the PL group appears less pronounced in the CL group. Is this\ndifference due to prior Candida infection, or is it also observed in uninfected controls?\nResponse: We thank the reviewer for this comment. We isolated the red channel of the provided\npictures showing that no difference in localization or abundance occurs between Sham-and PL-\ninfected mice. However, the IL22RA1 expression increases in amount as well as expand into the\nupper epithelial layer consistent with our K14 staining in CL-infected mice.\nFigure S9 IL-22RA1 distribution during\nfungal colonization in the oral cavity. A\nRepresentative immunofluorescence\npictures of IL-22RA1 after 11 days of\ninfection. Mice were infected with PL, CL,\nand Sham. Scale bar 50μm. B Red\nchannel IL-22RA1 shown in black.\n5.The apparent toxicity or growth inhibition caused by the STAT3 inhibitor (Figure S10C) should\nbe addressed by including a control group treated with the inhibitor alone. This would rule out\npotential off-target effects or the possibility that STAT3 signaling is essential for epithelial growth\nor survival.\nResponse: We thank the reviewer for bringing this to our attention. Indeed, tonic STAT3 activity\nis required for optimal proliferation of human oral epithelial cells (Fig. S15D). However, the\nconcentrations used in our experiments had no toxic effect on the epithelial cells determined by\nLDH assay (Fig. S15D). We have added this data to Fig S13D, E and discussed this in line 153.\nFigure S13 D Growth curve of human oral epithelial cells in the presence of STAT3 inhibitor.\nN=8. AUC of oral epithelial cells in response to iSTAT3 treatment. Growth was determined by\nconfluence over time. N = 8. Mann-Whitney Test. E Lactate dehydrogenase (LDH) release of\niSTAT3 treated oral epithelial cells after 8 hours. Mann–Whitney Test. N = 3 in duplicate.\n6.The function of tocilizumab as an IL-6R-neutralizing antibody should be stated in the text or the\nlegend for Figure S15.\nResponse: We have added the information to the figure legend. It now reads “Oral epithelial cell\nproliferation in the presence of IL-6 receptor blocker tocilizumab”.\n7.The specificity of gp130 signaling in oral epithelium is an interesting observation. Have the\nauthors examined whether similar mechanisms are involved in other mucosal tissues, such as\nthe vaginal epithelium, another major colonization site for Candida?\nResponse: We thank the reviewer for this insightful comment. We agree that the vaginal\nepithelium represents an important mucosal site for Candida colonization and pathogenesis.\nWhile our current study focused specifically on the oral mucosa to dissect the unique features of\nIL-22/gp130 signaling in this context, we recognize the relevance of investigating whether similar\nnon-canonical pathways are operative in the vaginal mucosa. At this stage, we have not yet\nextended our analyses to the vaginal epithelium. However, we note that prior studies have\nreported tissue-specific differences in IL-22 receptor expression and downstream signaling\ncascades, suggesting that the impact of gp130-mediated signaling may vary across mucosal\nsites. We are actively pursuing follow-up studies to address whether non-canonical IL-22 receptor\nsignaling is conserved or differentially regulated in the vaginal epithelium during Candida\ncolonization and infection.\n8.It would be interesting to determine how long CL Candida can colonize the oral mucosa. In\nFigure 4A, fungal burden remains comparable between days 11 and 31, while IL-17+ cell numbers\nsignificantly increase by day 31, suggesting immune resetting as proposed by the authors. How\ndo the authors integrate this inverse relationship between IL-17+ cells and K14+ thickening in the\ncontext of long-term colonization?\nResponse: We thank the reviewer for this thoughtful comment. While we limited our observations\nto day 31, the LeibundGut-Landmann lab has shown stable colonization for >60 days, and in\nsome mice >1 year (PMID: 32719409). The observed inverse relationship between Candida-\nspecific IL-17 secreting cell expansion and K14 epithelial thickening may reflect an adaptive\nmucosal remodeling process (Fig. S24). We propose that during early and onset colonization, IL-\n22-driven non-canonical signaling via gp130 promotes epithelial proliferation to enhance\nantimicrobial peptide expression. Over time, however, increasing Candida-specific immune cell\ngeneration (IL-17 secreting cells) signal a shift toward immune containment rather than further\ntissue remodeling. One possible interpretation is that Candida-specific IL-17 responses limit the\nneed for continued epithelial remodeling by providing direct specific antifungal activity and\nmaintaining fungal burden at a stable set point. Thus, the reduction in K14⁺ thickening by day 31\nmay represent a homeostatic reset, where epithelial remodeling initiated by IL-22 is gradually\ndownregulated as Candida-specific IL-17-driven immunity becomes the dominant mechanism for\nsustained fungal control. We have included this interpretation in the revised discussion (line 269).\nReviewer #3 (Remarks to the Author):\nIn this manuscript, Millet et al. report that oral colonization with a fungal component of the human\nmouth mycobiota elicits remarkable changes in the structure of the oral epithelium in mice.\nSpecifically, the authors show that the tongue’s epithelial layer expands in a process dependent\non IL-22 signaling upon colonization with the oral commensal Candida albicans isolate 101, a\nstrain that can persist for weeks/months in the murine tongues. Furthermore, Millet et al.\ndemonstrate that IL-22 signaling depends on a non-canonical receptor complex composed of\ngp130 coupled to IL-22RA1 and IL-10RB. Finally, using a mouse model of neonatal fungal\ncolonization, the authors propose that IL-22-mediated immunity and tissue remodeling have key\nroles controlling fungal invasion early in life.\nWhile below I list several weaknesses that I expect should be fully addressed by the authors, I do\nbelieve that the main findings reported in the manuscript are generally well supported. Likewise,\nthe work described in this manuscript, in my opinion, constitutes a substantial and important step\nforward in our understanding of fungal-host interactions.\n1) A major issue is the PL-CL comparison done at 11 days post inoculation throughout the\nmanuscript. It is well established in the field, and also shown in Fig S1, that at d11 post PL\ninfection, no Candida cells remain in the tissue (PL infection is cleared ~72h after infection). CL-\ninoculated animals, on the other hand, have a stable fungal load at d11 which persists for weeks.\nIt is unclear what to make, then, of comparisons like the ones shown in Figs. 1C and 1J-L. As it\nis, what the authors are actually comparing is the effect of post PL infection to active CL\ncolonization. But that’s not how the data are described or the results interpreted. If the purpose is\nto reveal effects elicited by the CL strain, the control should be ‘sham.’ If the purpose is to compare\nPL-CL effects, the comparison should include an early time point for PL because only then there’s\na clear effect of fungal presence in the tissue. This is appropriately done only for a few cytokines\n(Figs. 2A-B) but not for the larger scRNA-Seq or epithelial-enriched RNA-Seq included in Fig. 1.\nResponse: We appreciate the reviewer’s thoughtful comments regarding the PL-CL comparisons\nat day 11 post-inoculation. We agree that by day 11, the PL strain is typically cleared from the\ntissue, as also shown in our Fig. S1 and previously reported in the literature. Our rationale for\nincluding the day 11 comparison was to capture late-phase mucosal remodeling outcomes\nfollowing exposure to either the PL or CL strain. Specifically, we sought to determine whether\nprior transient fungal exposure (PL) elicits lasting epithelial or immune changes that are distinct\nfrom those maintained by persistent colonization (CL). Thus, this comparison reflects divergent\nhost states- one in which fungal clearance has occurred and another in which the fungus remains\nstably colonized. However, we agree that this distinction was not sufficiently emphasized in the\nmanuscript text. To avoid confusion, we revised the manuscript (see lines 84; 106; 109; 133; 136)\nto clarify that the day 11 PL-CL comparison represents distinct post-inoculation host states, one\ninvolving immune and barrier responses post-clearance (PL) and one reflecting ongoing fungal\npresence and interaction (CL). Additionally, we added data for broader comparison, in which we\nshow histology and K14 distribution over the time course if PL infection (Fig. S3; please see\ncomment #4 Reviewer 1), CD4 cell infiltration at early time points for PL and CL infection (day 2;\nFig. S8), which allowed us to distinguish changes that are specifically maintained by CL\ncolonization versus those that may have resolved after PL clearance, as well as compare them\nthe uninfected controls. Of note, the comparison of these strains (CL and PL) to naïve mice during\nearly time points to oral infection/colonization has been published (PMID: 30873177) and were\nconfirmed in our studies with our unbiased cytokine measurements in which we show that the PL\nstrain, in contrast to the CL strain, induces a classical proinflammatory response (Fig. 2A).\n2) Line 109 and Fig. 2A: “Acute infection with the PL C. albicans strain led to early TNFalpha and\nIL-1beta induction.” In Fig 2A, the prominent cytokines up only in PL at day 2 seem TNFalpha and\nIFNgamma (not IL-1beta).\nResponse: We thank to reviewer to bring this to our attention. To highlight the early differences in\nsome classical pro-inflammatory cytokines, we now provide the data in pg/g tissue of Il-1β, TNFα,\nand IFNγ in the supplemental material (Fig. S7) and revised the line 116 to “Acute infection with\nthe PL C. albicans strain led to early TNFα and IL-1β induction, whereas the CL Candida strain\ncolonized the oral mucosa without inducing classical proinflammatory markers (Fig. 2A; S7).”\nIL-1 TNF IFN-\n250000 50000 20000\nP0.0178 P0.006 P0.0322 Sham\nPL\n200000 40000\n15000 Cl\ne u s150000 P0.0568 e u s 30000 e u s\ns it\ng /g100000\ns it\ng /g20000\ns it\ng /g\n10000\np p p\n5000\n50000 10000\n0 0 0\nd2 d5 d11 d2 d5 d11 d2 d5 d11\nFigure S7 Classical proinflammatory cytokines during OPC. Levels of IL-1β, TNFα and\nIFNγ in PL-, CL-, and Sham-infected mice over time; N = 5. Ordinary one-way ANOVA.\n3) Lines 122-124 and Fig. 2G: “CD4 T cells were exclusively found in the epithelial and\nsubmucosal layers of commensal colonized mice.” The staining in Fig 2G which supports this\nstatement was done with animals 11 days post infection. As stated above in point #1, by this time\nthe PL-infected mice have already cleared the fungus. The statement implies that CD4 T cells are\nnever found during the entire course of infection with PL isolate. But at 2 days post infection, CD4\nT cells may well be found in the PL-infected tissue.\nResponse: We thank the reviewer for this comment. We performed experiments in which we\nstained for CD4 T cells after 2 days post PL and CL infection. These data indicate that during\nearly oral fungal infection no CD4 T cells migrate in the epithelial and submucosal layers and this\nabsence is independent of the strain (PL vs. CL). Please see minor comment 1 Reviewer 2.\n4) Fig 4. While the transitory nature of the epithelial K14 expansion seems clear, the connection\nof this phenomenon to “Candida-specific immunity” seems vague. The increase in Ca-specific IL-\n17 secreting cells (Fig. 4D) observed at similar timepoints is a correlation but does not indicate\ncausation. The data shown in Rag1-/- animals, while consistent with the author’s premise, is\nincomplete (e.g. we don’t know if the epithelial expansion takes place in these animals upon CL\ncolonization) and not very specific (the general lack of mature B/T cells in these animals likely\nhave myriad effects).\nAt the very least, the authors should provide a more nuanced interpretation of the data.\nResponse: We thank the reviewer for this comment. We now provide data that Candida-primed T\ncells are required and sufficient to initiate the secondary remodeling event within the oral mucosa.\nCD4 T cells were harvested from WT mice (CL-colonized or Sham-infected 6 weeks after\ninfection) for adoptive transfer into recipient Rag1−/− mice. CD4 T cells were injected intravenously\ninto Rag1−/− recipient mice 21 day after CL-colonization. After 10 additional days (day 31 post CL-\ncolonization), CFUs and K14 thickness were determined.\nFigure 5 i Scheme of CD4 T cell adoptive transfer in CL-colonized Rag1–/– mice. j Oral fungal\nburden of Rag1–/– mice receiving CD4 T cells from Sham-infected (SCD4 T cells) WT mice and\nCL-colonized WT mice (CLCD4 T cells). N = 4. Two-tailed Mann–Whitney Test. The y-axis is\nset at the limit of detection (20 CFU/g tissue). k Quantification of K14 thickness at day 31.\nN = 4 in duplicate; combined data of two independent experiments. Mann-Whitney Test. l\nRepresentative pictures of K14 staining of Rag1–/– mice receiving SCD4 T cells and CLCD4 T\ncells). Scale bar 50μm."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Although the authors revised the manuscript, I found several criticisms that still make the manuscript unsuitable for\npublication.\nHere below some of the concerns highlighted in the first round:\n1. The main limitation of the manuscript is related to the definition of the two strains as 'commensal' and 'pathogenic'.\n1. The authors responded to this criticism by citing previously published manuscripts related to strain 101 and SC5314. The\ncited publications investigated how the two strains are virulent on mouse models of oral candidiasis. These papers suggest\nthat the two strains differentially activate the host response, although Th17 differentiation was indistinguishable after day 7\npost-infections.\nThey also claim that the response (delayed) in the host may be a sign of commensalism.\nBut, the paper here revised is the first where the strains are defined as ‘commensal’ or ‘pathogenic-like’ strains, probably to\noversimplify the already published papers. Therefore, I don’t see the point of not leaving the original names of the strains like\nthey are SC5314- and 101-strains, and as already defined by previous publications.\n2. Figure S1 A is very surprising. Authors claim that the CL strain is not inducing inflammation but a non-inflammatory\npathogen can't be associated with such a high, persistent mouth colonization. Data should be presented by showing the\nstandard deviation and if there is also dissemination in other tissues.\n2. Here, the authors reply by using data and observations collected by already published papers without really supporting\ntheir hypothesis. The ‘persistent’ colonization in a model of oral infection can’t be interpreted as commensalism. Indeed,\nalready published papers claim that the 101 strain is inducing a delayed response, which may only underline a much higher\nvirulence.\nPlus, in Fig. S1B, mice infected with 101 strains start to clear the infection, as do the SC5314-infected mice. Again, I don’t\nsee the point at all in defining this experiment as a commensalism model if mice slowly clear the infection.\n3. Authors have to clarify or modify the model, by considering also that Candida albicans is a human gut commensal and not\nproperly a mouse commensal.\n3. Authors did not clarify why they decide to chose a model of oral mouse infection to study human commensalism. A more\nrealistic approach would be to compare human isolated strain responses in culture with the human oral mucosa by building\n3D cultures.\n4. It is not clear why in Figure 1E it is only shown the histology of mice infected with the CL strain and not the PL strain.\nSame issue for figure 1F, 1G, 1M.\n4. Although I appreciated the data added in Fig.S3, I still would suggest putting the histology of the two strains in the same\nfigure in order to appreciate the underlined differences. To show a quantitative objective increase of epithelial expansion,\nit is required to have higher-resolution images with a quantification score obtained by scanning the whole mouse tongue.\nThe whole scanned picture should be added. In Fig. 1 the histology at D11 is taken from a different portion of the mouse tongue\ncompared to naïve, D2, D5, D8.\n5. In figure 2, authors claim that CL is not inducing inflammation. This sentence is profoundly wrong, considering the\npathogenic colonization overtime that the CL strain is inducing and also the evidence that the CL strain is inducing IL-17 and\nIL-22, two pro-inflammatory cytokines. Data coming from the assay in figure 2A should be repeated eventually by using\nconventional ELISA, since it is extremely surprising that IL-17 is induced and there are not T cell polarizing cytokines. In\nother words, do the author know how CD4+IL-22+ T cells were polarized if no inflammation was induced?\n- Figure R1 should be transferred to the paper.\n- In Figure R1 authors should put the other time points of the infection.\n- The definition: ‘classical cytokine’ is not scientifically recognized in the immunological field. To which group of cytokines do\nthe authors refer?\n- The authors did not explain or give any hypothesis on the unexpected IL-22 expansion in the 101 infection model without\nhaving polarizing cytokines;\n6. Figure 2H is showing plots not clear. Gating strategy is not presented. CD4+IL-22+T cells should be shown.\n6. Fig.2H does not show Th17 cells. Unfortunately, the plot only represents a poor staining. The oral mucosa is full of CD4+T\ncells, which are a well distinct population. Here it is scarcely represented. A major quality of cell staining should be shown.\n7. Figure 4H is not clear. Why is keratin still there with no T cells?\n7. This is a very important point. It is not clear how the first half of the paper supports the idea that T cells are responding to\n101 strain and are fully responsible for IL-22 release, but in Rag1-/-, this response is still there. It is not clarifying the concept\nto state that this is a compensatory response, since they need to understand if other cells (most probably ILCs) are involved,\ntogether with T cells, in responding to the 101 strain. It might be the case to understand if the T memory resident (already\nshown by previous manuscripts) cells are affecting the IL-22 production.", "reviewer_2": "The authors have done extensive work to revise the manuscript and have addressed my questions.\nMinor point: The persistence of IL-22 signal after CD4⁺ T-cell depletion suggests there are additional IL-22–producing cells\nin this model. It would further strengthen the manuscript，though not be strictly required, to characterize these other sources\nusing the IL22-TdTomato reporter mouse (for example, by FACS or imaging analysis of non–CD4⁺ populations).", "reviewer_3": "The authors have satisfactorily addressed all the points I had raised. I believe the manuscript is ready for publication."}, "author_response": "RESPONSE TO REVIEWER COMMENTS (REVISED MANISCRIPT)\nWe would like to thank the reviewers for the critical review and constructive comments that helped\nus to further improve the revised manuscript. We have addressed all comments and provide a\ndetailed point-by-point reply to all comments below with changes highlighted in yellow in the\nrevised manuscript.\nReviewer #1 (Remarks to the Author):\nAlthough the authors revised the manuscript, I found several criticisms that still make the\nmanuscript unsuitable for publication.\nHere below some of the concerns highlighted in the first round:\n1. The main limitation of the manuscript is related to the definition of the two strains as\n'commensal' and 'pathogenic'.\n1. The authors responded to this criticism by citing previously published manuscripts related to\nstrain 101 and SC5314. The cited publications investigated how the two strains are virulent on\nmouse models of oral candidiasis. These papers suggest that the two strains differentially\nactivate the host response, although Th17 differentiation was indistinguishable after day 7 post-\ninfections.\nThey also claim that the response (delayed) in the host may be a sign of commensalism.\nBut, the paper here revised is the first where the strains are defined as ‘commensal’ or\n‘pathogenic-like’ strains, probably to oversimplify the already published papers. Therefore, I\ndon’t see the point of not leaving the original names of the strains like they are SC5314- and\n101-strains, and as already defined by previous publications\nResponse: We appreciate the reviewer’s attention to terminology. As noted in our previous\nresponse, a commensal microbe is defined as one that causes no perceptible or only clinically\ninapparent damage to the host, typically establishing early in life and maintaining a stable host–\nmicrobe relationship without disease (PMCID: PMC97744). We use “commensal-like (CL)” and\n“pathogen-like (PL)” solely as operational descriptors of host responses in the mouse oral model\n(stated in lines 46 - 52). We now include new data showing that 11 independent (randomly\npicked) clinical isolates phenocopy CA101 in colonization kinetics and epithelial remodeling\n(Fig. S7; lines 111 - 113). This demonstrates that the commensal-like category reflects a\nreproducible host-response pattern within the oral mucosa rather than a single-strain artifact.\nOur use of “commensal-like” refers specifically to the behavior of these strains during mucosal\ncolonization rather than an inherent loss of pathogenic capacity. To clarify this point, we\nperformed an additional experiment using a systemic infection model. The CL strain caused\nmortality rates comparable to the PL strain (Figure R1, to be removed from the rebuttal upon\nacceptance), indicating that their mucosal adaptation does not compromise systemic virulence.\nThis supports our interpretation that “commensal-like” describes a lifestyle in the mucosa, not an\noverall attenuation of pathogenic potential. These data are provided here for completeness and\nwill be explored in detail in a future manuscript focused on systemic disease. Ultimately, we\ndefer to the editor’s judgment regarding the most appropriate terminology and are happy to\nrevise if preferred.\nFigure S7 C. albicans clinical isolates show CL phenotype. A Oral fungal burden of wild-\ntype mice infected with indicated strains. Results are median ± standard error of the mean\n(SEM) of a single experiment (N = 3/group). B Representative immunofluorescence pictures\nof keratin 14 (K14) after 11 days of infection with indicated strains. Scale bar 100μm. C K14\nthickness 11 days post infection with indicated strains. N = 3/group.\nCL\n75 strains have similar\n]%\nvirulence during\n[\nla\nv 50 systemic candidiasis.\niv\nru\nS WT mice were infected\n25\nwith 2x105 CA101 (CL)\nor SC5314 (PL) and\n0\n0 5 10 survival was monitored.\nDay\n2. Figure S1 A is very surprising. Authors claim that the CL strain is not inducing inflammation\nbut a non-inflammatory pathogen can't be associated with such a high, persistent mouth\ncolonization. Data should be presented by showing the standard deviation and if there is also\ndissemination in other tissues.\n2. Here, the authors reply by using data and observations collected by already published papers\nwithout really supporting their hypothesis. The ‘persistent’ colonization in a model of oral\ninfection can’t be interpreted as commensalism. Indeed, already published papers claim that the\n101 strain is inducing a delayed response, which may only underline a much higher\nvirulence.\nPlus, in Fig. S1B, mice infected with 101 strains start to clear the infection, as do the SC5314-\ninfected mice. Again, I don’t see the point at all in defining this experiment as a commensalism\nmodel if mice slowly clear the infection.\nResponse: We respectfully disagree with the assertion that high colonization alone equates to\npathogenicity. Our data show that CL (CA101) colonization does not induce canonical pro-\ninflammatory cytokines (IL-1β, TNFα, IFNγ) or T cell-polarizing cytokines (IL-6, IL-23),\ndistinguishing it from acute infection with PL strain (SC5314), while we and others have shown\nthat the CL strain (CA101) does not induce damage (PMID: 28176789, 35404986, 40998980) a\nhallmark of pathogenicity (PMID: 27027296, 33471869). Furthermore, epithelial remodeling is\nIL-22-dependent and reversible, suggesting a regulated host response rather than overt\npathology. The standard deviation bars were added in the previous version and we clarified the\nabsence of dissemination in other tissues in an immunocompetent host (Fig. S1C; lines 84 -85).\nFigure S1C Liver fungal burden of\nimmunocompetent (IC) and\nimmunosuppressed (IS) wild-type\nmice infected with CL after 4 days\nof infection. Results are median ±\nstandard error of the mean (SEM)\nof a single experiment (N =\n4/group). PL, pathogen-like; CL,\ncommensal-like.\n3. Authors have to clarify or modify the model, by considering also that Candida albicans is a\nhuman gut commensal and not properly a mouse commensal.\n3. Authors did not clarify why they decide to chose a model of oral mouse infection to study\nhuman commensalism. A more realistic approach would be to compare human isolated strain\nresponses in culture with the human oral mucosa by building 3D cultures.\nResponse: We respectfully disagree with the assertion that the oral mouse colonization model is\ninappropriate for studying mucosal fungal interactions and commensalism. While C. albicans is\nindeed a human commensal, the mouse oral mucosa provides a well-established and tractable\nin vivo system to study host-fungal interactions in the context of an intact immune system and\nstratified epithelium. This model has been extensively used to investigate both pathogenic and\ncommensal behaviors of C. albicans. Our study builds on this foundation to investigate how\npersistent colonization induces epithelial remodeling and immune adaptation. The oral mucosa\nis a highly relevant site for studying these processes, particularly given the stratified epithelial\narchitecture and immune cell composition that closely resemble human tissues (PMID:\n40751276). To complement the in vivo findings, we also employed 2D tissue culture models\nusing human oral epithelial cells to dissect IL-22–mediated signaling mechanisms. These\nexperiments confirmed the proliferative effects of IL-22 and the involvement of non-canonical\nreceptor complexes, thereby strengthening the translational relevance of our observations.\nThus, our findings provide mechanistic insights into mucosal immunosurveillance that are\nbroadly applicable to human health. Furthermore, current 3D oral mucosa equivalents primarily\nconsist of epithelial and sometimes fibroblast layers. They lack the full repertoire of immune\ncells, such as tissue-resident macrophages, dendritic cells, innate lymphoid cells, and T cells\nthat are essential for maintaining commensalism and orchestrating antifungal immunity. This\ncomplexity is critical because commensalism is not solely an epithelial phenomenon but\ndepends on dynamic immune surveillance and tolerance mechanisms, as we have shown here.\n4. It is not clear why in Figure 1E it is only shown the histology of mice infected with the CL\nstrain and not the PL strain. Same issue for figure 1F, 1G, 1M.\n4. Although I appreciated the data added in Fig.S3, I still would suggest putting the histology of\nthe two strains in the same figure in order to appreciate the underlined differences. To show a\nquantitative objective increase of epithelial expansion, it is required to have higher-resolution\nimages with a quantification score obtained by scanning the whole mouse tongue. The whole\nscanned picture should be added. In Fig. 1 the histology at D11 is taken from a different portion\nof the mouse tongue compared to naïve, D2, D5, D8.\nResponse: We appreciate the reviewer’s thoughtful comments and suggestions. Our study was\nspecifically designed to investigate the epithelial remodeling induced by the CL strain, which is a\ncentral focus of the manuscript. The PL strain, in contrast, does not elicit comparable\nhistopathological changes, and thus was not included in Figures 1E, 1F, 1G, and 1M, which aim\nto highlight the distinct pathological features associated with CL colonization. We agree that\njuxtaposing histological images of both strains could enhance the clarity of the observed\ndifferences. Accordingly, we have included comparative histology in Figure S3 to provide visual\ncontext. However, we respectfully note that our imaging strategy prioritized consistency by\nanalyzing the middle third of longitudinal tongue sections whenever fungal colonization was\nevident, to ensure consistency across groups and to capture areas most relevant to the mucosal\nchanges under investigation.\n5. In figure 2, authors claim that CL is not inducing inflammation. This sentence is profoundly\nwrong, considering the pathogenic colonization overtime that the CL strain is inducing and also\nthe evidence that the CL strain is inducing IL-17 and IL-22, two pro-inflammatory cytokines.\nData coming from the assay in figure 2A should be repeated eventually by using conventional\nELISA, since it is extremely surprising that IL-17 is induced and there are not T cell polarizing\ncytokines. In other words, do the author know how CD4+IL-22+ T cells were polarized if no\ninflammation was induced?\n- Figure R1 should be transferred to the paper.\n- In Figure R1 authors should put the other time points of the infection.\n- The definition: ‘classical cytokine’ is not scientifically recognized in the immunological field. To\nwhich group of cytokines do the authors refer?\n- The authors did not explain or give any hypothesis on the unexpected IL-22 expansion in the\n101 infection model without having polarizing cytokines;\nResponse: We acknowledge the reviewer’s point and have revised the text to clarify that CL\ncolonization induces a limited type 17 response (IL-17/IL-22) without broad pro-inflammatory\nactivation (lines 128 – 129). We have added the ELISA validation (Fig. former R1) to the\nmanuscript (S8B) and clarified that IL-22+ CD4 T cells arise in the absence of classical\npolarizing cytokines, suggesting alternative pathways of activation (lines 265 -270). The term\n“classical cytokines” has been removed and replaced with specific cytokines (lines 122 -123).\n6. Figure 2H is showing plots not clear. Gating strategy is not presented. CD4+IL-22+T cells\nshould be shown.\n6. Fig.2H does not show Th17 cells. Unfortunately, the plot only represents a poor staining. The\noral mucosa is full of CD4+T cells, which are a well distinct population. Here it is scarcely\nrepresented. A major quality of cell staining should be shown.\nResponse: We thank the reviewer for this comment. Our flow cytometry gating for IL-17+ and\nIL-22+ cells was established using a fluorescence-minus-one (FMO) control strategy, where\neach marker was assessed in the absence of one fluorochrome to define positive populations\n(Fig. S.26). While we acknowledge that the flow cytometry plots in Fig. 2H are representative,\nthey were intended to illustrate the presence of IL-22–producing CD4⁺ T cell subsets [Th17\n(IL17+/IL22+) and Th22 (IL17-/IL22+)] during persistent colonization. These data are supported\nby multiple independent experiments and are consistent with our reporter mouse data (Fig. 2E–\nF; gating Fig. S.26), which identify CD4⁺ T cells as the major source of IL-22. Importantly, we\nwould like to clarify that the oral mucosa is not constitutively enriched in CD4+ T cells. As shown\nin Fig. 2G, CD4+ T cells are specifically recruited to the epithelial and submucosal layers in\nresponse to Candida colonization and are largely absent in sham- or PL-infected mice. Thus,\nthe observed CD4+ T cell signal reflects a biologically relevant and colonization-dependent\ninfiltration, not a technical limitation.\n7. Figure 4H is not clear. Why is keratin still there with no T cells?\n7. This is a very important point. It is not clear how the first half of the paper supports the idea\nthat T cells are responding to 101 strain and are fully responsible for IL-22 release, but in Rag1-\n/-, this response is still there. It is not clarifying the concept to state that this is a compensatory\nresponse, since they need to understand if other cells (most probably ILCs) are involved,\ntogether with T cells, in responding to the 101 strain. It might be the case to understand if the T\nmemory resident (already shown by previous manuscripts) cells are affecting the IL-22\nproduction.\nResponse: We thank the reviewer for highlighting this important point. We have clarified the\nmechanism underlying persistent keratin (K14) expansion in Rag1–/– mice and expanded our\ndiscussion to address the role of non-T cell sources of IL-22. As shown in Figure 4H and\ndetailed in the Results (lines 207 - 209), Rag1–/– mice exhibit sustained K14 expansion during\nCL colonization despite lacking mature T cells. This occurs because IL-22 production is not\nexclusively dependent on CD4 T cells. Our new data (Fig. S22D) demonstrate that group 3\ninnate lymphoid cells (ILC3s) significantly upregulate IL-22 in Rag1–/– mice during colonization,\ncompensating for the absence of adaptive immunity and driving epithelial proliferation.\nAdditionally, we discuss this in lines 275 – 278.\nILC3 ILC3 IL22+ ILC3\nFigure S22D %\n40 1×103 1×103 WT\nRag1–/– (left), total (middle),\n+ P 0049 and IL-22+ ILC3s\n7 2 30 7.5×102 7.5×102\n1 (right) in CL-infected\nD C n a P 0.0022 n a P 0.0022\n+ 0 9 D\nC\n20 g ro /s lle\nc\n5×102 g ro /s lle\nc\n5×102 W at T d a a n y d R 11 a . g 1 N –/ – m = ic 6 e .\nfo 10 2.5×102 2.5×102 Two-tailed Mann–\n% Whitney Test.\n0 0 0\nReviewer #2 (Remarks to the Author):\nThe authors have done extensive work to revise the manuscript and have addressed my\nquestions.\nResponse: We thank the reviewer for their positive assessment and for acknowledging our\nrevisions. We appreciate the time and effort invested in reviewing our work and are pleased that\nthe revised manuscript addresses the previous concerns.\nMinor point: The persistence of IL-22 signal after CD4⁺ T-cell depletion suggests there are\nadditional IL-22–producing cells in this model. It would further strengthen the manuscript,\nthough not be strictly required, to characterize these other sources using the IL22-TdTomato\nreporter mouse (for example, by FACS or imaging analysis of non–CD4⁺ populations).\nResponse: Please see comment 7 from Reviewer #1.\nReviewer #3 (Remarks to the Author):\nThe authors have satisfactorily addressed all the points I had raised. I believe the manuscript is\nready for publication.\nResponse: We sincerely thank the reviewer for their thoughtful evaluation and constructive\nfeedback. We greatly appreciate the time and effort dedicated to reviewing our work. We are\npleased that the revisions have addressed the previous concerns and improved the clarity and\nimpact of the manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I sincerely thank the authors for the review but unfortunately, I am feeling very uncomfortable because my vision is\nprofoundly far from the authors and also from the other reviewers. I would only like to share here the content of my view:\n- My concerns are not only related to 'terminology': commensal/pathogenic. My concerns are absolutely science-based. In\nmy view, the authors use a simple model of oral candidiasis challenging mice with two different strains, but in my opinion,\nthis is a model of local infection and not a commensal model.\n- The comparison between CL and PL strains is not often shown without any particular reason. Certain results are\ncomparing the two strains; others are not. This is not acceptable.\n-I was asking for a better quantification of histologic analysis between the two strains, but this was not accomplished. The\nscanning technology on tissues is not that impossible, and I am sure that if you want to demonstrate how commensals\nchange the tongues, you may achieve such a result.\n-I was asking for a better representation of CD4+T cells producing IL-22 by FACS (which is the main focus of the paper)\nthan the poor quality single plot represented in Figure 2h, but this was not modified.\n-The confused interpretation of results using Rag ko mice is still present since the contribution of ILC3 in the first part of the\npaper is not addressed, and not even mentioned.\n-The last figure is hard to understand and contributes to confusing messages.\n-They claim the CL strain induces Th22, but no single hypothesis of a possible polarizing cytokine is exposed, and no one\ncytokine is measured, proving a Th22 polarization.\n-To conclude, I think the authors are only showing that certain strains induce IL-22 just because the CL strain does not\nproduce hyphal invasion compared to the PL strain. Indeed, after 30 days, colonization and keratinization are gone and this\nis because the model only shows a superficial infection.\n-The production of IL-22 and the high K14 expression CAN'T BE SEEN as commensalism; this is very dangerous. We,\nimmunologists, know very well that HIGH IL-22 may represent a chronic stimulation of cancer progression (nature\nimmunology Article https://doi.org/10.1038/s41590-025-02149-z), this is also the reason why Candida is well under\ninvestigation in the cancer microenvironment.", "reviewer_2": "The authors have fully addressed my previous concerns"}, "author_response": "RESPONSE TO REVIEWERS\nRESPONSE TO REVIEWER COMMENTS (REVISED MANISCRIPT#2)\nReviewer #1 (Remarks to the Author):\nI sincerely thank the authors for the review but unfortunately, I am feeling very uncomfortable\nbecause my vision is profoundly far from the authors and also from the other reviewers. I would\nonly like to share here the content of my view:\n- My concerns are not only related to 'terminology': commensal/pathogenic. My concerns are\nabsolutely science-based. In my view, the authors use a simple model of oral candidiasis\nchallenging mice with two different strains, but in my opinion, this is a model of local infection and\nnot a commensal model.\nResponse: We acknowledge this concern and have adopted the terminology “persistence-biased”\nand “clearance-biased” to describe strain-specific tendencies, avoiding the term “commensal-\nlike.” While these strains exhibit classical features of commensal organisms, we agree that the\nOPC model does not fully replicate commensalism. To address this, we have included a\n“Limitations of this study” section at the end of the discussion acknowledging this point. However,\nthe OPC mouse model is widely accepted (over 500 publications) for studying mucosal Candida\ninteractions in vivo. Our study builds on this framework to dissect mechanisms of mucosal\nimmunosurveillance, not to redefine commensalism. Importantly, we have used multiple clinical\nisolates, all of which consistently exhibit a persistence-biased phenotype, reinforcing that this\nobservation is not limited to a single strain but reflects a broader biological mechanism.\n- The comparison between CL and PL strains is not often shown without any particular reason.\nCertain results are comparing the two strains; others are not. This is not acceptable.\nResponse: We have included comparisons between strains in previous versions of the manuscript\nand incorporated additional comparisons based on Reviewer #1’s suggestions. To further improve\nclarity, we will moved the relevant data from the supplemental materials into the main figure (Fig.\n1e) so that these comparisons are presented clearly and systematically. Comparisons were\nperformed only where necessary to illustrate the main phenotypic differences, such as scRNA-\nseq, bulk RNA-seq, K14 expression, histology, CFU counts, and sources of IL-22, while\nminimizing animal use in accordance with ethical guidelines. Using clearance-biased (CB) strains\nfor later-stage analyses would not be informative, as several phenotypes, such as sustained\nepithelial remodeling and immune compensation, only manifest under conditions of fungal\npersistence, which occur with persistence-biased (PB) strains.\n-I was asking for a better quantification of histologic analysis between the two strains, but this was\nnot accomplished. The scanning technology on tissues is not that impossible, and I am sure that\nif you want to demonstrate how commensals change the tongues, you may achieve such a result.\nResponse Quantification of histologic analysis is already presented; however, we understand the\ndesire for whole-tissue imaging. We have now acquired whole-tongue K14 staining images using\na microscope equipped with scanning capabilities and included these in the revised manuscript\n(Fig. 2f) to provide a comprehensive view of epithelial remodeling.\nFigure 2f Representative pictures of K14\nstaining of whole tongues of Sham, CB-\nand PB-infected mice after 11 days.\n-I was asking for a better representation of CD4+T cells producing IL-22 by FACS (which is the\nmain focus of the paper) than the poor quality single plot represented in Figure 2h, but this was\nnot modified.\nResponse: We misunderstood this point previously. We have now provided a higher-quality FACS\nimage to better represent CD4⁺ T cells producing IL-22 (Fig. 3h). However, we would like to\nemphasize that the main focus of the manuscript is on epithelial remodeling, not on demonstrating\nthat IL-22 originates from CD4⁺ T cells (Th17/Th22 cells).\n-The confused interpretation of results using Rag ko mice is still present since the contribution of\nILC3 in the first part of the paper is not addressed, and not even mentioned.\nResponse: Based on Reviewer #1’s queries, we incorporated new data demonstrating IL-22\nproduction by ILC3s in Rag1-/- mice and expanded the discussion to highlight immune\nredundancy and compensatory pathways. We also want to clarify that we initially did not focus on\nILC3s because the IL22 reporter mice did not show differences in TCR-negative cells in the WT\nsetting, suggesting their contribution was not altered under those conditions. However, the\ncontribution of ILC3s is indeed relevant, and was clarified in the revised manuscript's discussion\nthat \"While CD4⁺ T cells dominate IL-22 production in immunocompetent hosts, ILC3s\ncompensate in the absence of T cells, sustaining epithelial proliferation in Rag1–/– mice. These\nfindings underscore the critical role of adaptive immunity in terminating epithelial expansion and\nachieving long-term fungal control.\" Furthermore, we used Rag1–/– mice to determine whether\nCandida-specific T cell immunity is necessary for reversing epithelial remodeling during later\nstages of colonization. Purified CD4+ T cells from Candida-colonized or sham-infected wild-type\nmice were adoptively transferred into Candida-colonized Rag1–/– mice. Adoptive transfer of\nCandida-primed T cells confirmed that Candida-specific immunity was required to reduce fungal\nburden and K14+ epithelial thickness. This data indicates that Candida-primed CD4+ T cells are\nboth required and sufficient to drive secondary epithelial remodeling during colonization.\n-The last figure is hard to understand and contributes to confusing messages.\nResponse: We used neonates because early life represents a critical window for mucosal immune\ndevelopment, where newborns encounter abundant antigenic challenges but have an\ninexperienced adaptive immune system and reduced innate responsiveness. This immunological\ncontext likely explains why oral candidiasis occurs in infants and why compensatory mechanisms,\nsuch as IL-22-mediated epithelial remodeling, are essential. Our data show that neonatal oral\nCandida colonization induces K14 epithelial expansion and CD4⁺ T cell infiltration, and that IL-22\ndeficiency leads to increased fungal burden, loss of epithelial proliferation, all phenotypes\nobserved in adults. However, deeper tissue invasion was not observed in adults. These findings\nsuggest that protective pathways present in adults are underdeveloped or absent in early life,\nmaking epithelial remodeling a critical defense mechanism during this stage. While we\nacknowledge that we do not yet fully understand why remodeling is more pronounced in neonates,\nour data clearly highlights the importance of IL-22 in neonatal immune defense during fungal\nencounter.\n-They claim the CL strain induces Th22, but no single hypothesis of a possible polarizing cytokine\nis exposed, and no one cytokine is measured, proving a Th22 polarization.\nResponse: Based on Reviewer #1’s queries, we expanded the discussion to address this point.\nOur data show that Th22 cells emerge during fungal colonization despite the absence of classical\npolarizing cytokines, which we interpret as evidence for alternative tissue-specific cues driving IL-\n22 production. These cues may arise from persistent fungal antigen exposure rather than\ncanonical inflammatory pathways. We have clarified this in the discussion, emphasizing that this\nfinding challenges the conventional paradigm of Th22 differentiation and suggests a non-\ninflammatory pathway of T cell activation that may be critical for maintaining mucosal homeostasis\nduring commensal colonization. We did not investigate this further since the main focus of the\nmanuscript is epithelial remodeling rather than defining the precise polarization mechanism.\n-To conclude, I think the authors are only showing that certain strains induce IL-22 just because\nthe CL strain does not produce hyphal invasion compared to the PL strain. Indeed, after 30 days,\ncolonization and keratinization are gone and this is because the model only shows a superficial\ninfection.\nResponse: Response: Oropharyngeal candidiasis (OPC) is generally considered a superficial\ninfection of the oral and pharyngeal lining, characterized by Candida overgrowth on mucosal\nsurfaces. While typically localized, OPC involves active fungal adaptation and host\nresponses. We respectfully disagree that IL-22 induction is simply due to the persistent strain\nlacking hyphal invasion. As shown in recent work (Fróis-Martins et al., Nat Microbiol, 2025), even\npersisting strains (CA101 used as we have done) filament and express hyphae-associated genes\nduring colonization. Candidalysin (a fungal toxin) is essential for establishing and sustaining\ncolonization by enabling the fungus to overcome the stratum corneum barrier and resist IL-17-\nmediated clearance. The resolution of colonization and keratinization after ~30 days reflects\nrestoration of mucosal homeostasis once Candida-specific immunity develops, not a limitation of\nthe model. These findings underscore that colonization is an active, dynamic process requiring\nvirulence factors and host responses.\n-The production of IL-22 and the high K14 expression CAN'T BE SEEN as commensalism; this is\nvery dangerous. We, immunologists, know very well that HIGH IL-22 may represent a chronic\nstimulation of cancer progression (nature immunology Article https://doi.org/10.1038/s41590-\n025-02149-z), this is also the reason why Candida is well under\ninvestigation in the cancer microenvironment.\nResonse: We agree that high IL-22 levels have been implicated in certain pathological contexts,\nincluding cancer progression, as noted in the cited Nature Immunology article. However, our study\ndoes not interpret IL-22 production and K14 expansion as indicators of commensalism. Rather,\nthese findings represent protective, transient responses during initial oral Candida colonization,\naimed at preventing fungal overgrowth and tissue invasion until Candida-specific immunity\ndevelops.\nReviewer #2 (Remarks to the Author):\nThe authors have fully addressed my previous concerns\nResponse: We are pleased that the revisions have successfully addressed the previous concerns\nand enhanced the clarity and overall impact of the manuscript."}, {"round": 4, "reviewer_comments": {"reviewer_2": "*Editorial note: This reviewer was asked to comment in the absence of reviewer 1 on the authors response to the prior concerns raised by reviewer 1.\nReviewer 1’s main critique appears to focus on the conceptual framework of defining “commensal” versus “pathogenic” behavior using two distinct Candida strains, rather than assessing a continuous spectrum of infection outcomes within a single strain. In practice, such a continuous infection-gradient experiment may not be readily feasible at this stage. I raised a related point in my initial comments, particularly regarding interpretation across early versus late phases of infection.\nThat said, the authors have made a substantial effort to strengthen both the manuscript and their rebuttal. The additional experiments performed in response to Reviewer 1’s questions are, in my view, adequate and address the major concerns. Importantly, the study limitations are appropriately acknowledged and discussed, which supports a scientifically sound interpretation of the findings."}, "author_response": "Response: We thank the reviewer for their thoughtful evaluation of the revised manuscript. Based\non our responses to Reviewer #1, we appreciate their recognition that the additional experiments\nand revisions adequately address the major conceptual concerns, and that the study limitations\nare appropriately acknowledged."}]

Decision letter