IL-17a induces age-related olfactory dysfunction by impairing regeneration and promoting respiratory metaplasia in mice

Domain: neuroscience
Published: 2025-12-29
Editorial outcome: minor_revision

Abstract
The olfactory epithelium (OE) undergoes life-long renewal and regeneration. This process is supported by the globose basal cells (GBC) during the homeostatic state, as well as horizontal basal cells (HBC) during severe damage. Inflamm-aging refers to the low-grade, chronic and progressive state of heightened pro-inflammation associated with aging. However, the impact of inflamm-aging on OE homeostasis, regeneration, and the inflammatory microenvironment is not fully understood. In this study using mouse models, we elucidate the role of interleukin-17a (IL-17a) in OE regeneration and olfactory function. Our findings implicate that inflamm-aging in aged OE promotes the recruitment and activation of immune cells, accompanied by crosstalk between HBC and T cells. Elevated expression of IL-17a in aged OE triggers inflammatory signals and impairs olfactory function. Administration of IL-17a inhibitor Y-320 or neutralizing antibody promotes sensory neuronal regeneration and reverses age-related respiratory metaplasia in OE. Co-culturing mouse OE organoids with Th17 cells impairs neuronal generation and enhances the transformation towards respiratory cells, while neutralizing antibody against IL-17a alleviates neuronal loss and respiratory transformation. Additionally, conditional knockout of IL-17a in T cells facilitates OE regeneration by promoting HBC recruitment and differentiation into GBC. Collectively, our study identifies a function of IL-17a in OE regeneration and age-related deficits in olfactory function, providing evidence for further investigation of IL-17a as a possible therapeutic target against presbyosmia.

Introduction
Olfaction is a crucial sensory function in mammals, and profoundly influences human physiology, cognition, and emotions. Olfactory dysfunction (OD) refers to the deterioration or loss of smell function, serving as an early indicator of neurodegenerative diseases, a hallmark symptom of viral infections, and a common manifestation of aging1. Due to accelerated population aging, the incidence of olfactory dysfunction is rising annually, affecting up to 50% of the elderly. This condition significantly impairs quality of life and can even become life-threatening, particularly in older adults, due to the loss of ability to detect warning odors such as gas leaks, smoke, or spoiled food2–4. Impaired regeneration of the olfactory epithelium (OE) is a key factor in the prevalence of age-related OD5. However, the pathogenesis and the key regulatory mechanisms underlying aging-induced deficits in the OE regeneration remain largely unknown.

Inflammation is a complex physiological process involving the recruitment of immune cells and the secretion of pro-inflammatory factors. Inflamm-aging refers to the low-grade, chronic, and progressive state of heightened pro-inflammation associated with aging that occurs in the absence of infection6. Studies indicated the involvement of inflammatory factors in the progression of inflamm-aging was often concurrent with various chronic age-related diseases7. Thus, there is a possibility of immune dysfunction-mediated inflamm-aging in elderly individuals with chronic diseases.

The olfactory epithelium is a pseudostratified epithelial structure primarily composed of horizontal basal cells (HBC), globose basal cells (GBC), sustentacular cells (SUS), and olfactory sensory neurons (OSN), among others. Olfactory function is mainly mediated through sensory neurons, which detect odors and transmit signals to the olfactory bulb and higher brain regions8. Under homeostatic conditions, mitotically active GBC continuously produce new neurons throughout life, whereas quiescent HBC adhering to the basal lamina do not participate in maintaining normal tissue turnover. Upon severe damage, HBC are activated and transformed into GBC, which then differentiate into sensory neurons and non-sensory sustentacular cells; HBC may also directly generate sustentacular cells, thereby completing epithelial regeneration9,10. However, under the state of inflamm-aging, the accumulation of immune cells releases more inflammatory factors, leading to an imbalance in the inflammatory microenvironment. This imbalance impedes the activation and differentiation of basal cells, resulting in attenuated neuronal renewal and subsequently causing olfactory disorders11.

In the process of inflamm-aging, T cells play a pivotal role in tissue renewal, homeostasis, and repair12,13. Multiple signaling pathways and inflammatory factors are involved in tissue aging, such as IL-6, TNF-α, IFN-α, etc. Among them, IL-17a was reported to be increased in aged tissue and organs14,15. In this study, based on the published single-cell sequencing data from the OE of both young and aged mice16, we observed a notable elevation of IL-17a, secreted by CD4+ Th cells, in the OE of aged mice. This increase of IL-17a was found to be associated with olfactory function. Using mouse models, we revealed that inhibiting IL-17a secretion from CD4+ Th cells via Y-320 or the specific neutralizing antibody facilitated OE regeneration following injury. OE organoids co-cultured with Th17 cells or incubated with IL-17a hindered the generation of olfactory neurons, while a neutralizing antibody against IL-17a alleviated neuronal loss and respiratory transformation. Furthermore, using conditional knockout mice, we found that conditional IL-17a knockout in T cells accelerated OE regeneration through promoting HBC activation and differentiation towards GBC.

In this work, our findings support the hypothesis that IL-17a acts as a critical inhibitor of neuronal regeneration in the olfactory epithelium, thereby driving age-related regenerative decline and olfactory dysfunction. This study aims to investigate how IL-17a perturbs HBC-mediated regeneration within the context of inflamm-aging and to evaluate the therapeutic potential of targeting IL-17a signaling. Moreover, given our observation that the IL-17a-associated inflammatory microenvironment emerges as early as middle age, we propose that timely intervention against IL-17a signaling could prevent the onset of olfactory decline during aging. We anticipate that this work will not only elucidate key mechanisms of olfactory aging but also provide a rationale for promising immunomodulatory strategies to restore olfactory function in the elderly.

Results
Loss of olfactory sensory neurons in aged mice is accompanied by changes in the immune microenvironment
In our previously published study, we drew an atlas of OE aging at single-cell resolution16. To further investigate the impact of T cells in OE aging, we conducted a detailed analysis focusing on the major OE cell types, including HBC, GBC, immature and mature OSN (iOSN and mOSN), SUS, and T cells. The two-dimensional representation through uniform manifold approximation and projection (UMAP) provided the integrated and clustered view of various OE cell types and T cells in both young (3 months) and aged (24 months) mice (Fig. 1a, b). The proportions of cell types were displayed in Fig. 1c, highlighting substantial decreases in GBC, mOSN, and SUS, along with significant increases in T and HBC cells in aged mice. Feature plots in Fig. 1d depicted the expression levels of marker genes specific to each cell type: Omp for mOSN, Tubb3 (Tuj1) and Gap43 for iOSN, Sox2 for SUS and GBC, Krt5 for HBC, and Cd3e for T cells. Immunostaining and subsequent statistical analysis of OE tissues per unit length (200 μm) unveiled a significant reduction in the number of Tuj1+, OMP+ and GAP43+ cells by 56.44 ± 8.48% (p < 0.0001), 40.76 ± 5.32% (p < 0.0001), and 56.75 ± 6.36% (p < 0.0001) in aged mice compared with young ones, respectively (Fig. 1e, f). The number of Krt5+ HBC increased by 16.40 ± 6.98% (p = 0.0317) in aged mice. Notably, we observed that the morphology of Krt5+ cells transitioned from a flattened shape in young mice to an irregular triangular shape in aged mice, suggesting a transition of HBC from a dormant to an activated state. This was consistent with our previous finding that more activated HBC were present in the aged OE16. Additionally, aging also led to thinning of OE by 41.69 ± 4.65%, further coinciding with a reduced number of sensory neurons and potential defects in recognition and transmission of odor signals (Fig. S1a, b).

In contrast, the number of immune cells exhibited a notable increase in aged mice. Immunostaining and statistical analysis revealed increased numbers of CD3 (a marker for T cells), Ptprc (CD45, a pan immune cell marker), Ly6g (a marker for neutrophils), and Adgre1 (F4/80, a marker for macrophages) positive cells in aged mice compared to young ones, with approximate fold increases of 11.40 (p < 0.0001), 1.52 (p < 0.0001), 2.48 (p < 0.0001), and 9.00 (p < 0.0001), respectively (Fig. 1e, f and Fig. S1c, d). The majority of these immune cells were localized in the lamina propria of OE, suggesting an accumulation of immune cells within the aged tissue.

To investigate the alteration of intercellular interactions in the aged OE, we conducted cell-cell communication analysis (CellChat) focusing on T cells and various OE cell types. Our data revealed an obvious increase in the macrophage migration inhibitory factor (MIF) signaling pathway in aged mice, which is a pleiotropic pro-inflammatory cytokine involved in various functions related to aging, such as leukocyte recruitment, inflammation, immune response, and tumorigenesis17. As shown in Fig. 1g, Mif-(Cd74+Cd44) was the most prominent ligand/receptor pair between T cell and HBC in aged OE. Immunostaining and subsequent statistical analysis demonstrated a significant age-related increase in MIF+, CD74+, and CD44+ cells within the OE. The number of positive cells per 200 μm of OE increased by 899 ± 223.0% (p = 0.0024) for MIF+ cells, 343 ± 57.1% (p < 0.0001) for CD74+ cells, and 158 ± 30.6% (p < 0.0001) for CD44+ cells in aged mice compared to young controls. Notably, the increased number of CD74+ cells in aged tissue were predominantly located near the basal cell layer (marked by Icam1), whereas CD44+ cells were distributed both in the basal cell layer and the lamina propria (Fig. 1h, i). Violin plots further supported these observations, showing an accumulation of Mif in T cells and markedly elevated expression of Cd74 and Cd44 in both HBC and T cells of aged OE (Fig. 1j, p < 0.0001). Collectively, these findings indicated an age-associated enhancement of immune response within the OE stem cell niche.

NicheNet analysis was conducted to further reveal the ligand/receptor pairs and the predicted target genes upregulated in aged HBC18. We found that Il17a exhibited high ligand activity in T cells, binding with the corresponding receptor Il17ra expressed in HBC (Fig. S1e). The GO enrichment analysis indicated predicted target genes upregulated in aged HBC were involved in response to molecules of bacterial origin, cytokine-mediated signaling pathway and the canonical NF-κB signaling transduction (Fig. S1f). KEGG enrichment analysis suggested that these genes were associated with Th17 cell differentiation, IL-17 signaling pathway, and NF-κB signaling pathway (Fig. S1g). The expression levels of Il17a, Il17ra, Il17rc, Il1b, Tgfb1, Cxcl10 were increased in aged HBC (Fig. S1h).

To identify the specific cell types expressing IL-17ra within OE, we performed immunostaining using IL-17ra-specific antibody on OE tissues from young and aged mice, with Tuj1 and Icam1 labeled iOSN and HBC, respectively. Based on established anatomical criteria, we analyzed OE sections from distinct regions: dorsal, medial, and lateral area, as well as respiratory epithelium (RE) (Fig. 1k)19. Our results uncovered pronounced spatial and cell type-specific alterations in IL-17ra expression between age groups. In the dorsal OE region of young mice, IL-17ra was predominantly localized to the neuronal layer, with approximately 87.6% of signals co-localizing with Tuj1. This pattern underwent a marked shift in aged mice, where IL-17ra expression was increasingly observed in the basal cell layer, evidenced by ~60% co-localization with Icam1. In the medial region, IL-17ra co-localized more frequently with neurons in aged tissue (~45.7% co-stained with Tuj1) compared to young OE (~32.8%). In the RE region, IL-17ra was almost exclusively restricted to the basal layer in both groups (Fig. 1l, m). We further analyzed Il17ra expression using our single-cell RNA sequencing data. Analysis of Il17ra-expressing cells across HBC, GBC, iOSN, mOSN, SUS, and T cells demonstrated that in young mice, Il17ra was predominantly expressed in mOSN (55.1%) and iOSN (24.2%). In aged OE, however, expression shifted notably to HBC (40.1%) and T cells (28.4%) (Fig. S1j).

To elucidate the mechanism underlying Th17 cell accumulation in aged OE, we analyzed the expression of genes associated with Th17 recruitment, polarization, and activation20. Our data revealed a profoundly pro-inflammatory microenvironment in aged OE. We observed significant upregulation of key cytokines essential for Th17 cell differentiation and maintenance, including Il6, Il23a, Il1b, Tgfb1, and Tnf, along with elevated expression of the critical transcription factors Stat3, Rorc, and Batf (Fig. S1k). Furthermore, the aged OE exhibited enhanced expression of chemokines and their receptors crucial for Th17 cell recruitment. Specifically, Ccl20, Cxcl10, Ccl2, and Ccl7, as well as their cognate receptors (Ccr6, Cxcr3, and Ccr2), were significantly upregulated. This result suggested a strengthened chemotactic gradient that promotes Th17 cell recruitment to aged tissue. Moreover, molecules involved in T cell co-stimulation, such as Cd80, Cd86, Icos, and Cd40, were also increased, indicating an enhanced capacity for local T cell activation within aged OE. Additionally, several members of the TNF receptor and ligand superfamily, including Tnfrsf1a, Tnfsf10, Tgfbr2, Tnfsf8, and Tnfrsf1b, were also found to be upregulated in aged OE21,22.

Collectively, these findings suggest the presence of inflamm-aging in aged OE and the promotion of immune cell recruitment and activation. Our data also imply a potential role for HBC in mediating crosstalk with T cells during this process.

Elevated expression of IL-17a in the aged OE is related to olfactory function
To delve deeper into the impact of T cells on the OE aging, we categorized T cells into 7 sub-clusters based on the expression of known marker genes, namely Naïve T, CD4+ Th, Treg T, γδT, CD8+ T, NKT, and Cycling T23, as depicted in Fig. 2a. Notably, the proportions of sub-clusters differed significantly between young and aged tissues. CD4+ Th, Treg, and γδT were predominantly present in the aged group (Fig. 2b), and these cell types are important regulators during the process of immune response24. Feature plots illustrated the expression of Il17a and known marker genes for CD4+ Th (Ctla4), γδT (Trdc), and Treg (Foxp3) (Fig. 2c). The selected marker genes for each sub-cluster were represented in the dot plot (Fig. 2d). With the progression of immune response, CD4+ Th cells are activated and subsequently differentiate into various subsets, including Th1, Th2, Th9, Th17, Th22, and Tfh25. To gain insight into the differentiation pathway of CD4+ Th cells, we evaluated the differentiation index scores according to the marker genes for various subsets (Fig. 2e). Specifically, the accompanying violin plots demonstrated the expression patterns of representative marker genes (Fig. 2f). Our findings revealed that Th17 cells exhibited the highest score, indicating a predominant polarization of CD4+ Th cells toward the Th17 lineage (Fig. 2g, p < 0.0001). Subsequent analysis of our scRNA-seq data was performed to characterize the cellular sources of Il17a in aged tissue. A pie chart illustrating this distribution showed that among all IL-17a-expressing cells, CD4+ Th cells constituted the majority (76.7%), followed by γδT cells (13.7%) and other immune populations (Fig. 2h).

We subsequently validated the expression of IL-17a in the OE tissues. Compared with young controls, ELISA results showed that both IL-17a and IL-23 concentrations were significantly elevated in the OE tissue and plasma of aged mice (Fig. 2i). Consistent with these results, Western blot and qPCR analyses further demonstrated a notable increase in IL-17a protein and mRNA expression levels in aged OE tissues (Fig. 2j, k). A violin plot also revealed a pronounced upregulation of Il17a transcript in aged mice (Fig. 2l). Additionally, immunostaining of OE sections showed a substantially greater presence of CD3+, CD4+, and IL-17a+ cells in aged mice, where IL-17a signal was nearly undetectable in young tissue (Fig. 2m). Accordingly, analysis of scRNA-seq data demonstrated an approximately 32-fold increase in IL-17a+ cell count in aged OE relative to young mice (Fig. 2n).

To evaluate the olfactory function, we conducted a buried food test, and observed that aged mice required significantly more time to locate hidden cookies than young controls (Fig. 2p). Correlation analysis revealed a negative association between Il17a mRNA expression levels and olfactory performance, suggesting that higher Il17a expression correlates with longer food-finding latency (Fig. 2o). Together, these findings imply that elevated expression of IL-17a within the aged OE triggers inflammatory signals, thereby contributing to the decline in olfactory function.

Administration of IL-17 inhibitor Y-320 promotes OE regeneration
Given the potential association of elevated IL-17a in the OE with olfactory dysfunction, we sought to investigate whether IL-17a plays a role in OE regeneration. We constructed an OE injury model by injecting methimazole (Met)26. Il17a-EGFP mice were used to directly visualize the in vivo expression of intrinsic Il17a. As depicted in Fig. 3a, a substantial number of OMP+ cells were present in the uninjured OE. On day 1 post-injection (1 dpi), only a single layer of basal cells (delimited by a white dashed line) remained, with a significant influx of CD45+ inflammatory cells into the lamina propria. Meanwhile, intrinsic Il17a-EGFP expression images revealed a marked increase in the number of IL-17a+ cells after injury. In contrast, a small number of Tuj1+ newborn immature olfactory neurons emerged on 2 dpi. By 7 dpi, the presence of Tuj1+ cells was evident, while the numbers of IL-17a+ cells were decreased, compared to 3 dpi (Fig. 3a, b).

Y-320, a specific IL-17 inhibitor, was used to suppress IL-17 production secreted by CD4+ T cells27. It was reported that treatment with Y-320 accelerated wound healing at earlier time points28. To investigate the role of IL-17a in the regeneration of OE after injury, Y-320 was gavaged 7 days prior to Met administration, and the mice were sacrificed at 7 or 14 dpi (Fig. 3c). Mice treated with DMSO were used as vehicle control group. To ensure that olfactory function was not affected by Y-320, we conducted the olfactory behavior test before the Met injection. We found that the aged mice spent much longer finding cookies, showing impaired smell function when compared to young mice. However, Y-320 treatment did not affect olfactory function compared to DMSO-injected controls in either young or aged animals (Fig. 3d), suggesting that inhibition on IL-17a did not affect olfactory function under normal conditions.

Following Met administration, the buried food test revealed a stark impairment in olfactory function. On 1 dpi, mice were unable to locate cookies within the allotted timeframe, indicating a pronounced decline in olfactory function and affecting both young and aged mice similarly. Olfactory assessments were subsequently repeated on 3, 7, and 14 dpi. In the young mice receiving Y-320, a significant improvement in olfactory function was present when compared to the DMSO group on 3 dpi, and this improvement persisted until 14 dpi. Meanwhile, aged mice in the Y-320 group also exhibited an improvement in olfactory function compared to the DMSO group on 3 dpi to 14 dpi, as depicted in Fig. 3e. These findings implied that inhibiting IL-17a production expedites the recovery of olfactory function in both young and aged mice.

To examine the production level of IL-17a, we collected plasma samples from aged mice at 7 dpi and subsequently measured the concentrations of IL-17a using ELISA. It was shown that Y-320 administration led to a significant reduced levels of IL-17a in peripheral blood plasma (Fig. 3f), supporting the efficacy of Y-320 in inhibiting the IL-17a production.

Furthermore, we performed immunostaining against IL-17a and CD45 in both young and aged mice at 14 dpi, with or without Y-320 administration. Both IL-17a+ and CD45+ cells were found to be located in the lamina propria of the mucosal tissues (Fig. 3h). The number of double positive cells was elevated in aged mice compared to young ones, while administration of Y-320 reduced the percentage of IL-17a+ cells within CD45+ cells population in both young and aged group (Fig. 3g). These data indicated that Y-320 ameliorates the inflammatory activation in the injured OE.

As OSN were primarily concentrated in the dorsal and medial regions29, we measured the OE thickness of these two regions in both young and aged mice at 7 and 14 dpi. As illustrated in Fig. 3i, the OE thickness of aged mice was significantly reduced, compared to that of young mice treated with DMSO. Furthermore, administration of Y-320 significantly restored OE thickness in both young and aged mice. We then performed immunostaining using specific antibodies against mature (OMP) and immature OSN (Tuj1, Gap43). As shown in Fig. 3j–m, a certain degree of recovery in the number of mature and immature neurons was observed in the dorsal OE of young mice receiving DMSO at 7 dpi. With the administration of Y-320, there was a significant increase by 49.61 ± 6.93% (p < 0.0001) and 49.97 ± 8.26% (p < 0.0001) in the number of Gap43+ and Tuj1+ newborn neurons at 7 dpi, respectively. At 14 dpi, the effect of Y-320 on the recovery of neurons in young mice was more significant, with a notable increase of 81.67 ± 13.91% in OMP+ mature neurons (p < 0.0001). For aged mice, neuronal recovery was limited at both 7 and 14 dpi, with a smaller number of Tuj1+ and Gap43+ cells in OE. By contrast, application of Y-320 led to a significant increase by 316.67 ± 58.03% and 128.92 ± 27.61% in Gap43+ and Tuj1+ immature neurons compared to DMSO-injected controls, and the number of OMP+ mature neurons was increased significantly by 228.48 ± 36.10% (p < 0.0001) at 14 dpi. Similar results were observed in the medial region, as shown in Fig. S2a–d. These data indicated that although the repair process in aged mice was much slower than in young mice, the use of Y-320 inhibited the secretion of IL-17a and facilitated neuronal regeneration in the OE post injury.

Inhibition of IL-17 reverses the age-related respiratory metaplasia in the OE
It has been reported that OE damage can result in respiratory metaplasia and subsequent olfactory dysfunction30,31. To determine whether Y-320 administration could reverse metaplasia changes in the injured OE, we performed immunostaining for Krt19, a specific marker for respiratory basal cells, on OE tissues from both young and aged mice treated with either DMSO or Y-320; untreated mice served as controls. As shown in Fig. 4a, d, Krt19+ cells in untreated control mice were predominantly localized to the RE region (refer to Fig. 1k). In contrast, following Met-induced injury, DMSO-treated mice exhibited widespread Krt19+ metaplasia throughout the dorsal, medial and lateral regions of OE, outlined as the “patch area” within the designated frame (green dashed line, Fig. 4b). In young mice, the metaplasia remained relatively limited, with many regions retaining Tuj1+ iOSN. In aged mice, however, the metaplastic area encompassed nearly the entire OE. Notably, Y-320 administration resulted in a substantial reduction in the extent of metaplasia, an effect particularly pronounced in aged mice. The metaplasia length was significantly reduced by 158.70 ± 9.83 μm (p < 0.0001) in the dorsal region and 150.20 ± 12.18 μm (p < 0.0001) in the medial region (Fig. 4c). Quantitative analysis revealed that the Krt19+ metaplasia area accounted for 77.97% of the total OE in aged mice, which was significantly higher than that in the young group (43.5%). Following Y-320 treatment, the metaplastic area decreased by 44.99% and 21.65% in aged and young groups, respectively (Fig. 4d). These results suggested that under identical injury conditions, the aged OE was more susceptible to metaplastic transformation and exhibited a markedly impaired regenerative capacity compared to young OE, possibly due to age-related declines in intrinsic repair mechanisms.

To further validate the functional role of IL-17a in age-related olfactory decline, we administered an IL-17a-neutralizing antibody to aged mice and assessed olfactory function at 3 and 7 days following Met-induced injury (Fig. 4e). Two concentrations of the neutralizing antibody were administered: 105 µg/mL (low) and 150 µg/mL (high)15,32. Mice receiving the higher dose exhibited markedly shorter food-finding times, indicating accelerated functional recovery (Fig. 4f). Immunostaining and quantitative analysis revealed that the neutralizing antibody reduced the area of Krt19+ respiratory metaplasia and significantly increased the density of Tuj1⁺ neuronal cells (Fig. 4g, i, j). Additionally, CD74+ cell numbers were substantially decreased following treatment, while CD44+ cell numbers remained unchanged (Fig. 4h, k). Notably, the spatial distribution of both CD74+ and CD44+ cells was altered: in DMSO control and low dose groups, these cells were present in the lamina propria (LP), neural epithelial layer (NE) and basal layer (BL), while high-dose treatment confined CD74 and CD44 expression predominantly to the LP and NE (Fig. 4l). The results demonstrated that neutralization of IL-17a significantly improved olfactory recovery in a dose-dependent manner.

Together, these findings revealed that both Y-320 and IL-17a neutralization effectively counteract aging-associated pathology in the OE, promoting tissue repair and functional recovery.

IL-17a neutralization counteracts Th17-induced metaplasia to restore neuronal integrity in olfactory organoids
We have previously established OE organoids with mature sensory neurons and functional response to odor stimulation33. To elucidate the regulatory role of IL-17a in OE regeneration, we conducted an in vitro model using organoids derived from OE tissues of both young and aged mice. Single-cell suspensions from OE tissues were cultured in a 3D system, yielding organoids containing key OE cell types, including basal cells, SUS, iOSN, and mOSN (Fig. S3a). Aged organoids exhibited smaller size, slower proliferation, and decreased relative growth efficiency across multiple passages over the culture period (Days 1, 3, 7, and 14 post culture) (Fig. S3b–d). qPCR analysis over time revealed increasing mRNA expression levels of olfactory neuronal markers Gap43 and Omp after 10 days of culture, accompanied by a decline in Mki67, consistent with reduced proliferative activity (Fig. S3e). Sox2, an essential transcription factor in olfactory regeneration, and a marker for SUS, GBC and other olfactory progenitor/stem cells34, showed progressively elevated expression with longer culture duration. Notably, Gap43 expression was significantly suppressed in aged organoids, compared to young controls. Furthermore, on day 14, aged organoids showed a 5.32 ± 0.47 folds increase (p = 0.0004) in the expression of P16 (Cdkn2a), a canonical senescence marker35 (Fig. S3f). These findings suggested that the in vitro organoid model faithfully recapitulated age-related cellular senescence, providing a representative system for investigating OE regeneration.

To investigate the influence of IL-17a on olfactory neuronal differentiation, we co-cultured OE organoids with in vitro-derived Th17 cells to mimic the inflammatory microenvironment observed in vivo. Based on previous research36, naïve CD4+ T cells were isolated from mouse spleens and then subjected to in vitro differentiation into Th17 cells prior to co-culture with olfactory organoids (Fig. 5a). Following magnetic bead sorting of the single-cell suspension derived from mouse spleens, the proportion of CD3+ CD4+ naïve T cells was 91.6%, compared to 17.5% in unsorted controls (Fig. S3g). These cells were subsequently polarized into Th17 cells in vitro. Flow cytometric analysis confirmed successful differentiation, with 17.8% of cells expressing IL-17a, and 86.2% exhibiting activation marker CD25 (Fig. 5b).

To ascertain the optimal co-culture conditions, we conducted mixed-culture experiments using varying medium ratios for both cell types. We found that a blend of 20% Th17 cell culture medium and 80% organoid-conditioned medium best supported organoid expansion and Th17 cell viability (Fig. 5c). After 14 days of co-culture, marked morphological changes were observed. Young organoids co-cultured with Th17 cells exhibited increased size and predominantly adopted a cystic morphology (~60%). In contrast, aged organoids, which were initially smaller than young controls (Fig. S3b), showed no significant change in overal dimensions under co-culture; however, the proportion of cystic structures reached about 80%. This Th17-induced cyst formation was significantly suppressed by the addition of an IL-17a neutralizing antibody, which also reduced organoid size. The proportion of cystic structures decreased to approximately 18% in young organoids and 40% in aged organoids, with the majority of organoids reverting to a solid and filled morphology similar to the controls (Fig. 5d, e; Fig. S3h, i).

Immunostaining analysis revealed a pronounced reduction in Tuj1+ and OMP+ cells following Th17 co-culture (Fig. 5f–i). Quantification showed significant decrease of OMP+ cells in both young (p < 0.0001) and aged (p = 0.0012) organoids. Concurrently, Krt19+ cells increased markedly and displayed intense intracellular staining, indicative of a metaplastic shift. Muc5ac+ mucus cells and secretory content were also elevated in both young and aged co-cultured organoids (Fig. 5f). Notably, administration of IL-17a neutralizing antibody restored neuronal marker expression of OMP and Tuj1, and suppressed Krt19 and Muc5ac levels, with organoids regaining a near-normal morphology. Both immunostaining-based cell quantification and qPCR analysis consistently confirmed these findings (Fig. 5h, i). As summarized in Fig. 5j, the bar graph demonstrated the relative alterations in the proportions of OMP+, Tuj1+ and Krt19+ cells under two experimental conditions: co-cultured with Th17 cells alone, and Th17 co-culture supplemented with IL-17a NAb, each compared to the control group. Values above zero reflected an increase in cell proportion relative to controls, where values below zero indicated a reduction. Notably, the effects of Th17 co-culture and NAb intervention on lineage differentiation were more pronounced in aged organoids.

Moreover, the expression level of P16 mRNA was more substantially altered in aged organoids. Specifically, co-culture with Th17 cells led to a marked increase in P16 expression in aged organoids, while the addition of NAb significantly reduced its level to near control baseline. In contrast, young organoids showed no significant differences under the same conditions (Fig. 5k). To verify that Th17 exposure induced metaplasia, we isolated cells from mouse respiratory mucosa (RE region) and generated 3D respiratory organoids. Following proliferation and differentiation, these respiratory organoids consistently exhibited cystic morphology, with high expression of Krt19 and Muc5ac, and minimal Tuj1 signal, confirming their respiratory identity (Fig. S3j, k).

Collectively, these results indicated that Th17 cells disrupt olfactory organoids differentiation and drive them toward a respiratory epithelial phenotype at the expense of sensory neurogenesis. Importantly, IL-17a neutralization effectively prevented this lineage transition and preserved olfactory differentiation, highlighting the pivotal role of IL-17a in impairing cell fate specification. Furthermore, the disruptive effects of Th17 cells were more pronounced in aged organoids, suggesting that the aging microenvironment is even more susceptible to Th17-mediated impairment.

IL-17a directly alters olfactory epithelial lineage specification and triggers an inflammatory transcriptional program in organoids
IL-17a acts as the primary effector molecule mediating Th17 cell functions. Previous studies have shown that in vitro IL-17a stimulation modulates the proliferation and differentiation patterns of intestinal organoids37. Given the technical limitations associated with precise quantification using primary T cells, we sought to further elucidate the specific role of IL-17a in regulating the proliferation and differentiation of basal cells. To this end, we treated young organoids with three concentrations of recombinant IL-17a (10, 20 and 40 ng/mL). Immunostaining results showed that addition of IL-17a markedly inhibited neuronal differentiation, as indicated by a reduction in OMP+ cells (Fig. S4a). qPCR analysis further demonstrated concentration-dependent downregulation of Krt14, Lgr5, and Omp expression. Additionally, elevated expression of inflammatory cytokine Il6 was observed, suggesting that IL-17a induced an inflammatory state in organoids (Fig. S4b).

Based on these findings, a concentration of 40 ng/mL was selected for subsequent experiments in both young and aged organoids. Immunostaining and quantitative analysis revealed that IL-17a suppressed the generation of Tuj1+ iOSNs by 80.24 ± 7.85% (p < 0.0001) in young organoids and 77.89 ± 21.20% (p = 0.0015) in aged ones. Concurrently, IL-17a significantly increased the number of Krt19+ respiratory cells by 217.81 ± 37.7% (p < 0.0001) and 93.11 ± 15.65% (p < 0.0001), respectively (Fig. S4c, e). Furthermore, the number of OMP+ mOSNs was reduced, whereas no discernible effect was observed on Sox2+ cells (Fig. S4d, e). qPCR analysis further confirmed that, in addition to the reduction in neuronal cell marker (Gap43), IL-17a treatment significantly downregulated mRNA expression of Krt14 and Krt5. Notably, the expression level of the senescence-associated marker P16 in aged organoids was markedly increased, while no significant change was detected in the young group (Fig. S4f).

To further explore the effects and mechanisms of IL-17a on olfactory organoids, we performed transcriptome sequencing on organoids treated with (IL-17a) or without IL-17a (Control). The results revealed significant Differentially Expressed Genes (DEGs) between the two groups. A heatmap was generated to display the expression levels of selected DEGs, among which inflammation-related genes such as Cxcl1, Cxcl2, Il1a, Csf2, Csf3 and Tnf were up-regulated in response to IL-17a stimulation. Conversely, the basal cell marker Krt14, stem/progenitor cell marker Lgr5, as well as olfactory sensory neuron marker Omp were down-regulated in IL-17a treated group (Fig. S4g). Volcano plot illustrated that, compared to the control group, the IL-17a treated organoids exhibited 301 up- and 194 down-regulated genes (Fig. S4h). The biological process category of GO enrichment analysis showed that DEGs were enriched in categories such as regulation of immune effector response, innate immune response, epithelium cell migration, among others (Fig. S4i). Furthermore, KEGG signaling pathway enrichment analysis revealed that pathways such as cytokine-cytokine receptor interaction, NF-kappa B signaling pathway, cellular senescence, and IL-17 signaling pathway were up-regulated in IL-17a treated organoids. In contrast, pathways including Notch, Wnt, Cell cycle, MAPK were down-regulated (Fig. S4j). These results further suggested that changes in the micro-environment altered the differentiation trajectories of basal cells within olfactory organoids.

Collectively, our findings from the in vitro olfactory organoids further substantiate the inhibitory effect of IL-17a on neuronal generation. This co-culture model effectively mimics the inflammatory microenvironment and potentially provides a reliable tool to reflect the real in vivo conditions.

Conditional knockout of Il17a facilitates the regeneration of OE
To further investigate the role of IL-17a in the regeneration process following OE injury, we conditionally ablated Il17a allele (Il17aflox/flox) in T cells using Lck-Cre driver (named CKO thereafter). Littermate Il17aflox/flox mice (named Flox thereafter) were used as controls (Fig. S5a). As shown in Fig. S5b, flow cytometry analysis of peripheral lymph node cells from both CKO and Flox mice confirmed a successful reduction in the percentage of IL-17a+ cells in CKO mice.

At Day 7 and 14 post Met injection, OE tissues were collected and analyzed by single-cell RNA sequencing (Fig. 6a). After filtering, 40,731 single cells were subjected to subsequent analysis. The UMAP visualization depicted the clustering of various cell types, including HBC, GBC, SUS, iOSN, mOSN, microvilli cells (MV), olfactory ensheathing glia (OEG), respiratory basal cells (Res BC), respiratory ciliated cells (Res C), as well as immune populations such as Neutrophils (Neu), CD4+ Th, γδT, Cycling T, B cells, macrophages (Mac), dendrite cells (DC), and plasmacytoid dendritic cells (pDC) (Fig. 6b). The dot plot showed the typical marker genes for each cell type (Fig. 6c). The proportion plot and grouped UMAP plots (Fig. 6d and Fig. S5c) revealed significant alterations in cell type distributions between CKO and Flox groups. Notably, compared to Flox mice, CKO mice exhibited a substantial increase in the ratios of GBC and iOSN, while the percentage of CD4+ Th cells decreased at both 7 and 14 dpi. Feature plots for specific marker genes were presented in Fig. 6e. Violin plots illustrated the differential expression levels of key genes. Among them, Omp in mOSN, Tubb3 in iOSN, and Ascl1 in GBC were upregulated in CKO mice, compared to Flox controls. Conversely, the expression of Il17a was markedly reduced in CKO mice (Fig. 6f).

We then conducted olfactory behavioral experiments to assess the recovery of olfactory functions post-injury in CKO and Flox mice. Behavioral buried-food test revealed that CKO mice exhibited a more rapid recovery of olfactory function compared to control mice (Fig. 6g). The olfactory habituation and dishabituation experiments demonstrated that CKO mice displayed enhanced exploratory behavior towards new odors, and elevated thresholds for detected odors (Fig. 6h and Fig. S5d). Furthermore, immunostaining on OE tissues at 7 and 14 dpi showed that the number of mOSN (OMP+) in CKO mice was significantly increased by 66.18 ± 9.92% (p < 0.0001) at 14 dpi, and iOSN (Tuj1+) in CKO mice was increased by 54.13 ± 14.32% (p = 0.0013) and 54.11 ± 9.69% (p < 0.0001) at 7 and 14 dpi, respectively (Fig. 6i, j). These data indicated that OE regeneration was improved in Il17a CKO mice, highlighting the important role of IL-17a in modulating OE regeneration following injury.

We then performed CellChat analysis to gain a deeper understanding of the intercellular communication between T cells (including CD4+ Th, γδT, Cycling T) and the key olfactory epithelial cells (HBC, GBC, iOSN, mOSN, and SUS) in the four groups. Pathways related to immune response, such as CXCL, GALECTIN, GRN, TGF-β, and MIF, were upregulated in Flox mice, while those involved in neuronal development and cell differentiation, including MK, PTN, KIT, GAS, FGF38 and SEMA339, exhibited increased activity in CKO mice (Fig. S5e). Among the ligand-receptor (L-R) pairs from T cells to HBC, MIF-(CD74+CD44) was significantly downregulated in CKO mice (Fig. 6k). This observation was consistent with our previous finding that MIF signaling was enhanced in aged mice (refer to Fig. 1g). Next, our analysis of L-R pairs from HBC to GBC and SUS revealed that Ptn-Ncl was upregulated in CKO mice. Meanwhile, among the L-R pairs from GBC to iOSN and mOSN, Mdk-Ncl was upregulated in CKO mice (Fig. S5g, h). It was reported that both cytokines pleiotrophin (PTN) and midkine (MDK) signaling pathways are crucial for tissue regeneration and differentiation40. Genes related to immune response, such as Mif, Cd74, Cd44, Tgfb1, Cxcl2, Tgfbr2, were increased in Flox mice. In contrast, olfactory neuronal markers Omp, Tubb3, Gap43, Gng13, GBC marker Neurod1 and Ascl1, and genes associated with MDK and Sema3a pathways, such as Mdk, Plxna2, Nrp1, Sema3a and Cxcr4 were upregulated in CKO mice (Fig. S5i). These results further implied that conditional knockout of Il17a modifies the chemotactic ability of immune cells on HBC, thereby altering their inflammatory microenvironment. This, in turn, facilitates the regeneration of OE tissue.

Conditional knockout of Il17a accelerates the recruitment of horizontal basal cells
To delve into the mechanism underlying Il17a CKO, we compared gene expression patterns of individual OE cell types between CKO and Flox groups, and identified a total of 2520 and 4702 DEGs (|ave_Log2FC| > 0.5 and adjusted p-val < 0.05) across various cell types in CKO mice, at 7 and 14 dpi, respectively. Some DEGs exhibited widespread changes across different cell subtypes, whereas others were unique to particular cell types. Notably, HBC displayed the greatest number of DEGs at the two time points, suggesting the most profound changes in CKO mice (Fig. S6a). Consequently, we classified the marker genes of HBC into six sub-clusters, based on their expression patterns (Supplementary Data 4). GO enrichment analysis revealed that genes upregulated in CKO mice at both time points (C6) were linked to dendrite development, Wnt signaling pathway, regulation of developmental growth and neurogenesis. Genes consistently downregulated in CKO mice (C1) were associated with innate immune response, regulation of leukocyte activation and chromosome segregation (Fig. S6b). Among the DEGs on Day 7 and 14 post injury in HBC of CKO mice, 123 up-regulated genes were overlapped, including Nrcam, Ogn, Ctnnd2, Ncam1, Ncam2, Cxcl14, while 302 down-regulated genes were overlapped, such as Krt6a, Sprr1a, Cxcl10 and Gbp4 (Fig. S6c–f, Supplementary Data 5 and 6).

Based on our single-cell RNA sequencing analysis, we re-clustered the HBC population into 7 distinct subgroups (Fig. 7a)41. Under homeostatic conditions, HBC are typically maintained in a quiescent state with the expression of Trp63, Krt14, or Krt5, identified as quiescent HBC. Within the subgroup of Trp63+ cells, we observed high expression level of Ctnnd2 and Nrcam. Nrcam, also known as neural glial-related cell adhesion molecule, belongs to the L1 family of immunoglobulin superfamily, and plays a wide variety of roles in neural development, including cell proliferation and differentiation, axon growth and guidance42. It was reported to activate quiescent stem cells for regeneration and identified as a novel marker for resting HBC43. Ctnnd2 (Delta-catenin) was reported to play a critical role in neuronal development, particularly in the formation and maintenance of dendritic spines and synapses44.

Under injured conditions, resting HBC become activated and differentiate into GBC and SUS, GBC then further generate OSN. Methimazole-induced injury has been shown to prompt HBC to transition into transient states HBC*1 and HBC*241,45. Activated HBC were identified by the expression of Sprr1a, Krt6a, and Krtdap, and further divided into HBC*1 (with low expression of cell cycle genes) and HBC*2 (with high expression of cell cycle genes). Additionally, cells with low levels of resting HBC markers but positive for Sox2 were split into two subgroups: ▲HBC1, marked by Reg3g+, and ▲HBC2, characterized by Cyp2g1+, both of which are markers for sustentacular cells. Clusters expressing high levels of resting HBC markers but low levels of Krtdap were renamed as HBC1, while those with low expression of Reg3g were termed as HBC2 (Fig. 7b).

The expression of specific marker genes for each subgroup was displayed in the UMAP and violin plots (Fig. S6g, h). Nrcam and Ctnnd2, which serve as marker genes for resting HBC, exhibited elevated expression levels in CKO mice, at both 7 and 14 dpi. Conversely, activated HBC markers Krt6a and Sprr1a were upregulated in Flox mice, particularly at 7 dpi. The distinct expression patterns of these marker genes suggested different states of HBC in CKO and Flox mice following OE injury. From the proportion of each subcluster, it was evident that the ratio of resting HBC was highly increased in CKO mice, whereas activated HBC were predominantly observed in Flox mice, at both 7 and 14 dpi (Fig. 7c).

Furthermore, RNA velocity analysis confirmed the activated status of HBC in Flox mice, with arrows originating from the HBC*1 subcluster being longer at both 7 and 14 dpi, indicative of more activated conditions of HBC in Flox mice (Fig. 7d). Monocle 2 trajectory inference analysis of HBC subclusters colored by pseudotime (Fig. 7e) and cell type (Fig. 7f) in each sample, further indicated that HBC in CKO mice transitioned into a relatively stable state. Specifically, resting HBC emerged at 7 dpi, while HBC1 was present at 14 dpi. The expression of marker genes for each subcluster of HBC were displayed along the pseudotime trajectory (Fig. S6i). We then hypothesized that CKO mice complete the recruitment of HBC at 7 dpi, whereas Flox mice are still in the process of HBC activation following injury.

To support this hypothesis, we collected OE from Flox and CKO mice at 1, 2 and 3 dpi. Krt5+ HBC in OE exhibited distinct morphologies from 1 dpi to 14 dpi between Flox and CKO mice (Fig. 7g). At 1 dpi, the OE of Flox mice was detached from the lamina propria, leaving only a thin layer of flat Krt5+ cells. In contrast, CKO mice exhibited a thicker vertical layer of Krt5+ cells. In the subsequent days at 2 dpi and 3 dpi, the thickness of HBC in CKO mice consistently exceeded that in Flox mice, indicating a more activated status of HBC in CKO mice. However, the status of HBC switched between CKO and Flox mice at 7 dpi. HBC in Flox mice displayed triangular morphology, while those in CKO mice tended to be flat at 7 and 14 dpi. The number of Krt5+ HBC was significantly increased by 328.57 ± 54.53% (p < 0.0001), 55.77 ± 17.6% (p = 0.0123), and 51.76 ± 10.77% (p < 0.0001) at 1, 2, 3 dpi with no statistical difference at 7 (p = 0.0616) and 14 dpi (p = 0.9955) in the OE of CKO mice compared to Flox mice (Fig. 7h).

To validate that Il17a CKO mice undergo rapid HBC activation following injury, we stained OE tissues with antibodies against Mki67 and Icam1. The number of Mki67+ cycling HBC was highly increased by 105.56 ± 29.92% (p < 0.01) and 52.38 ± 25.64% (p = 0.0912) in CKO mice at 1 and 2 dpi, compared to Flox mice (Fig. 7i, k). By combining immunostaining with in situ hybridization, we confirmed the increased number of Krt6a-mRNA+/Krt5+ activated HBC by 65.16 ± 9.50% (p = 0.0025) in CKO mice than Flox mice at 1 dpi (Fig. 7j, l).

Furthermore, to validate the differentiation capacity of HBC in CKO and Flox mice at 7 and 14 dpi, we performed RNA velocity analysis focusing on HBC subclusters, in conjunction with GBC and SUS. As expected, HBC in Flox mice at 7 dpi were actively differentiating towards GBC and SUS, whereas HBC in CKO mice remained in a relatively homeostatic status (Fig. S6j). To provide further context, integrated RNA velocity results were displayed, organized by cell type (Fig. S6k) and by sample group (Fig. S6l), for both the HBC subcluster analysis (corresponding to Fig. 7d) and the combined HBC+GBC+SUS analysis (corresponding to Fig. S6j). These results were consistent with the trajectory inferences across all samples and strengthen the robustness of our conclusions.

Immunostaining with resting HBC marker Trp63 and GBC marker Mash1 revealed an increase in the number of Mash1+ cells and Trp63+ cells by 5.7-fold (p < 0.0001) and 3.5-fold (p = 0.0005) in CKO mice (Fig. S6m, n), further confirming our hypothesis that Il17a CKO facilitates HBC recruitment and differentiation into GBC.

To delve deeper into the underlying mechanisms driving the changes in regeneration observed in Il17a CKO mice, we performed immunostaining on OE tissue at 7 and 14 dpi using markers for CD45, Icam1 and IL-17a, in both Flox and CKO mice. Our findings revealed that in Flox mice, at both 7 dpi or 14 dpi, there were obvious CD45+ cells infiltrating the olfactory epithelium. In contrast, in CKO mice, the majority of CD45+ cells were confined to the lamina propria, just beneath the Icam1+ HBC. Notably, the total number of CD45+ cells and the number of CD45+/Icam1+ double-positive cells were significantly reduced by 23.16 ± 5.44% (p = 0.0016) and 50.91 ± 6.68% (p < 0.0001) in CKO mice, particularly at 7 dpi. Furthermore, the number of IL-17a+/CD45+ double-positive cells was also decreased by 63.33 ± 12.02% (p = 0.0004) at 7 dpi (Fig. S7a, b). These results indicated that immune cells infiltrated the olfactory epithelium in Flox mice, whereas this infiltration was prevented by T cell-specific knockout of Il17a in CKO mice.

Next, we focused on the HBC and T cells for further analysis. Combined and grouped UMAPs showcased the subclusters of HBC and T cells, based on our prior analysis with young and aged mice (Fig. S7c, d). Consistent with immunostaining results, feature plots depicting the expression of Krt5 and Ptprc (the gene encoding CD45) revealed that some Krt5+ cells also co-expressed Ptprc, which further confirmed the infiltration of immune cells into olfactory basal cells (Fig. S7e). Subsequently, we conducted CellChat analysis to gain deeper insights into the interactions between the subclusters of HBC and T cells, primarily focusing on the 7 dpi time point. To investigate how CD4+ Th cells regulate HBC regeneration, we designated CD4+ Th as the source and all types of HBC as the targets. The dot plots in Fig. S7f showed the ligand-receptor pairs increased or decreased in CKO mice, compared to Flox mice. Notably, the MIF signaling pathway was decreased in CKO mice, particularly in the process originating from CD4+ Th cells and targeting to HBC2, HBC*2, and ▲HBC1. The Cd6-Alcam pathway, which is crucial for modulating T cell activation, proliferation, and trafficking, showed increased expression from CD4+ Th cells to resting HBC, but a notable decrease in expression from CD4+ Th cells to HBC*2 in CKO mice46. Furthermore, several other ligand-receptor pairs that increased in CKO were mostly from CD4+ Th to resting HBC, including Sema4d-Plxnb2, Lamc1-Cd44, Lamc1-(Itga6+Itgb1), and others. To validate this finding, we performed immunostaining on OE tissues from Flox and CKO mice at 14 dpi (Fig. S7g, h). The analysis revealed a marked alteration in the spatial distribution and a significant reduction in the number of CD74⁺ and CD44⁺ cells in CKO mice. In Flox mice, CD74 and CD44 signals were readily detectable within the basal and neuronal layers, whereas in CKO mice, the remaining positive cells were largely confined to the lamina propria. This pattern of immune cell redistribution aligns with our previous observation in young and aged OE tissues (Fig. 1h, i) and also control versus IL-17a NAb-treated conditions (Fig. 4h, i).

Collectively, these findings demonstrate IL-17a as a critical negative regulator of the pace and resolution of HBC-driven tissue regeneration following injury.

IL-17a-associated inflammatory microenvironment emerges in the OE during middle-age
To investigate the onset of IL-17a accumulation during aging, we examined the OE of middle-aged mice (12-month-old). IL-17a expression revealed olfactory neuron markers OMP and Tuj1 remained strongly expressed, and HBC morphology labeled by Krt5 was comparable to that in young mice (Fig. S8a, refer to Fig. 1e). However, a small number of CD4+ and IL-17a+ inflammatory cells was observed within the OE and lamina propria (Fig. S8b). Furthermore, we assessed the infiltration marked by CD74 and CD44. Compared with young and aged OE (refer to Fig. 1h), CD74 and CD44 expression in middle-aged mice exhibited an intermediate pattern, with regional heterogeneity. Some regions displayed low CD74 and CD44 levels, resembling the young phenotype (Fig. S8c, upper); whereas other regions showed clear CD74 infiltration into the epithelial layer, an abundance of CD44+ cells in the lamina propria, and co-localization with Icam1 in basal cells (Fig. S8c, lower). Similarly, IL-17ra expression also showed heterogeneity, localized to both the basal layer co-localizing with Icam1 (Fig. S8d, upper), and in the neuronal layer (Fig. S8d, lower).

Thus, although olfactory function remained largely intact at this stage, cellular alterations were already apparent, including mild immune infiltration and shifts in the inflammatory milieu, accompanied by elevated IL-17a, suggesting the onset of inflamm-aging.

We next established in vitro OE organoids from middle-aged (12-month-old) Il17a-CKO and Flox mice. The two genotypes exhibited marked differences in organoid morphology and cellular composition. After 14 days of culture, Flox mice-derived organoids frequently developed cystic structures, whereas CKO organoids remained predominantly solid and were larger throughout the culture period (Fig. S8e, f). Furthermore, Flox organoids showed a reduction in OMP+ and Tuj1+ neurons, an increase in Krt19+ cells, and elevated expression of the respiratory epithelial marker Muc5ac. In contrast, CKO organoids displayed increased neuronal numbers (OMP and Tuj1), higher mRNA expression of neuronal markers (Omp and Gap43), and reduced Krt19 expression and positive cell numbers (Fig. S8e–j).

Based on these findings, we propose that timely intervention targeting IL-17a production during aging may help prevent the onset of olfactory dysfunction.

Methods
Animals
C57BL/6JCya-Tg (Lck-Cre), C57BL/6JCya-Il17aem1flox, C57BL/6JCya-Il17atm2(EGFP) mice were purchased from Cyagen Biologics (Suzhou, China). Il17aflox/flox and Lck-Cre mice were crossbred to achieve conditional knockout of Il17a in T cells. Il17a-EGFP mice were constructed by inserting an enhanced green fluorescent protein (EGFP) expression cassette downstream the termination codon of the endogenous Il17a gene. Wild-type C57BL/6J mice of 2 months (young), 12 months (middle-aged), and 19-22 months (aged) were purchased from Shanghai Bikai Laboratory Animal Company Ltd (Shanghai, China). To avoid the impact of estrogen on aging, we used male mice for the behavioral tests, and both male and female mice were randomly assigned to the other experimental groups. The number of mice involved in experiments has been added in the figure legends. All mice were housed under standard procedures, with ad libitum access to water and food, maintained on a 12:12 light-dark cycle, and were kept at a temperature of 18~22°C. Following all experimental procedures, mice were euthanized by slow-fill (30~70% volume/min) carbon dioxide (CO₂) inhalation, and death was confirmed by cervical dislocation. The animal handling procedures were approved by the Ethics Committee of Laboratory Animals in Naval Medical University (Permit Number: SYXK2022-0011).

Olfactory epithelium lesion
Young, aged, Il17a flox/flox and LckCre/Il17a flox/flox mice were intraperitoneally injected with methimazole (100μg/g, #60560, Sigma-Aldrich), and were sacrificed on Day 7 and 14 post-methimazole treatment.

Y-320 treatment
Mice received Y-320 (#HY-15898, MCE) by intragastric gavage 1 week prior to methimazole treatment at a dose of 3 mg/kg once a day. Y-320 was dissolved in corn oil containing 10% DMSO. Control group mice were treated with the same dose of corn oil with DMSO.

Neutralization treatment in aged mice
Aged mice were administered anti-mouse IL-17a neutralizing antibody (InVivoMAb, clone 17F3, BioXcell, #BE0173) intravenously at doses of 105 µg or 150 µg per mouse15,32,53. Control mice received an equivalent volume of IgG1 isotype control (InVivoMAb, clone MOPC-21, BioXcell, #BE0083) at 150 µg per mouse via the same route. Preventive treatment was initiated 7 days prior to methimazole-induced OE injury and continued three times on alternate days for one week. Following model establishment (designated as day 0), the same dosing regimen (intravenous injection, three times per week) was maintained for an additional two weeks, resulting in a total treatment duration of three weeks.

Buried food test
Tests were performed based on the previously described11. Mice were trained to locate cookies for two days prior to the test and starved for 24h beforehand, with water provided ad libitum. The mice were placed in clean experimental cages (35 cm × 25 cm × 20 cm) with 5 cm thick bedding for 10 min before the test. Then the mice were placed in the same test cage with the cookies buried 3 cm under the bedding, and the time taken to find the cookies was recorded, with a cut-off time of 360 seconds (6 min).

Olfactory habituation-dishabituation test
Mice were exposed to two different scents to test olfactory habituation and discrimination. Each mouse was placed in an isolated testing cage (45 cm × 30 cm × 20 cm) and habituated to a non-odorant cotton tip for 30 min prior to the test. The odors used were geraniol and citronitrile, diluted 1:20 in mineral oil. Following four trials with cotton swabs soaked in mineral oil, the mice were exposed to four presentations of one scent, followed by four exposures to a different scent. The number of odor explorations by the mice within one minute after each exposure was recorded and compared to the responses to mineral oil.

Olfactory threshold test
After inducing injury in the olfactory epithelium, olfactory threshold tests were conducted at 7 or 14 days post-injury (dpi). Concentrated geraniol extract was diluted to various concentrations (1:160, 1:80, 1:40, 1:20, 1:10) using mineral oil. Following a habituation period, the mice were tested four times with cotton sticks soaked with mineral oil. The odorant was then dissolved in mineral oil, and a series of tests were performed using cotton sticks dipped in the odor diluent, ranking from the weakest to the strongest concentration. Simultaneously, control cotton sticks soaked in mineral oil were also placed alongside. The lowest concentration of odorant detected was determined when the mice exhibited significantly longer sniffing duration towards the cotton sticks dipped in diluted odor compared to those soaked in mineral oil.

RT-qPCR analysis
Total RNA was extracted from mice tissues or organoids using the RNA Isolation Kit (#RC112-01, Vazyme). cDNA was synthesized by reverse transcription using the RT SuperMix (#R333, Vazyme), followed by quantitative PCR (qPCR) using SYBR qPCR MasterMix (#Q711, Vazyme) on the real-time fluorescence PCR system (7900HT, ABI). Relative expression levels of target genes were calculated normalized to the expression of β-Actin by 2−ΔΔCt cycle threshold method. The primer sequences used in this study were synthesized by Shanghai Tsingke Biotechnology Co. Ltd., and are listed in Supplementary Data 1.

Olfactory organoid culture
The olfactory mucosa was extracted from both young and aged mice and digested in 0.25% trypsin with EDTA for 15 min. Single cell suspensions were obtained through filtration and then cultured in organoid medium containing 6% Matrigel (#356231, Corning). The organoid medium was prepared as follows: DMEM with 1% GlutaMAX (#10566016, Gibco) as the basic medium, supplemented with HEPES (1 mM, #15630080, Gibco), Wnt3a (50 ng/mL, #5036-WN-500, R&D Systems), R-Spondin (200 ng/mL, #CF001, Longbiotech), Noggin (100 ng/mL, #CF002, Longbiotech), Y27632 (10 μM, #Y0503, Sigma-Aldrich), EGF (50 ng/mL, #HZ-1326, PeproTech), N2 (1%, #17502048, Gibco) and B27 (2%, #17504044, Gibco).

Respiratory organoids culture
The dissociated respiratory mucosal cells from mice were resuspended in airway expansion medium and plated into 24-well plates according to a previous report54. After reaching sufficient cell expansion, the cells were resuspended in airway expansion medium supplemented with 6~8% Matrigel and transferred to ultra-low attachment 24-well plates. Upon spheroid formation (approximately 2~3 days), the medium was replaced with airway differentiation medium to induce differentiation. Organoids were harvested after 12 days of culture.

Preparation of single cells from mouse spleen and Th17 cell culture
Experimental mice were anesthetized and perfused with sterile PBS, and single-cell suspension was prepared from spleens by mechanical disruption. Naive CD4+ T cells were isolated from 2-month-old mice spleens using the CD4+ T cell isolation kit (#19765, STEMCELL) and seeded at a density of 1×106 cells/mL using the CellXVivo Mouse Th17 Differentiation Kit (#CDK017, R&D Systems) or CytoBox Th17-mouse (#130-107-758, Miltenyi Biotec). After 5 days of induction culture using the kit, the percentage of Th17 cells was detected by flow cytometry and used for subsequent related experiments.

Construction of olfactory organoids co-culture with Th17 cells
Once the Th17 cell induction was complete and olfactory organoids were in good growth condition on Day 4 after passaging, we added isolated Th17 cells to the upper chamber to achieve non-contact co-culture with olfactory organoids. The co-culture of Th17 cells was conducted in 24-well plates, with approximately 500 organoids and 20,000 Th17 cells per well. Mixed cells were cultured in a medium consisting of 20% Th17 differentiation medium and 80% organoid culture medium. After 7 days, the transwell upper chamber was removed, and cells in the lower chamber were collected for subsequent analysis. For neutralized IL-17a, 1 µg/mL of IL-17a Monoclonal Antibody (#16-7173-85, Thermo Fisher) was added to the medium.

Flow cytometry
To distinguish T cell types from the single-cell suspension of spleen, we used Fixable viability stain 510 (#564406, Biosciences), FITC anti-mouse CD3 (#100203, BioLegend), APC anti-mouse CD4 (#100411, BioLegend), PE/Cy7 anti-mouse CD25 (#102015, BioLegend) antibodies for flow cytometry analysis. To detect the endogenous IL-17a, fixation was performed after labeling the cell membrane markers, followed by intracellular staining with PE anti-mouse IL-17a (#506903, BioLegend) or BV421 anti-mouse IL-17a (#506925, BioLegend). A representative gating strategy can be found in Source Data file.

ELISA analysis
For serum collection, mice were anesthetized and venous blood was drawn. The blood samples were allowed to clot at room temperature for 30 min, followed by centrifugation at 1200×g for 10 min at 4°C to obtain serum.

For tissue analysis, mice were euthanized and the nasal mucosa was dissected. Tissues were mechanically homogenized on ice in saline at a ratio of 9 µL per milligram of tissue weight. The homogenates were centrifuged at 1000×g for 10 min, and the resulting supernatants were collected for analysis. IL-17a and IL-23 levels were quantified using the following commercial ELISA kits: Mouse IL-17a (#U96-3240E, YOBIBIO) and Mouse IL-23 (#U96-3252E, YOBIBIO).

Immunostaining
For tissue immunostaining, mice were sacrificed under deep anesthesia with intraperitoneal injection of ketamine-xulazine (200 and 15 mg/kg body weight). The hair and muscle tissues of the head were carefully removed under a microscope, and the whole nasal cavity was excised and fixed with 4% PFA (#G1101, Servicebio) for 6 hours. Tissues were then dehydrated and decalcified using 15%, 30% sucrose (#A502792, Sangon) and EDTA decalcification solution (#G1105, Servicebio). Nasal cavity tissues were embedded in O.C.T. compound (#4583, Sakura) and sectioned into 10 μm thick coronal slices using a frozen sectioning platform (CM1950, Leica). For immunostaining, tissue sections were infiltrated with PBS to remove the O.C.T. compound and then repaired using EDTA antigen retrieval solution (#P0086, Beyotime) at 95°C. The sections were blocked in 5% bovine serum albumin (BSA) in PBS with 0.3% Triton X-100 (#20107ES20, Yeasen) for 1 h, followed by incubation with primary antibodies at 4°C overnight.

For immunostaining of organoids, olfactory organoids cultured for varying durations were harvested and fixed in 4% paraformaldehyde for 30 min at 4°C. Subsequently, organoids were blocked with 5% BSA containing 0.3% Triton X-100. Primary antibodies were applied and incubated overnight at 4°C. Details of the primary antibodies used were provided in Supplementary Data 2. Afterwards, sections or organoids were washed with PBST and incubated with secondary antibody for 1.5 hours at room temperature. The fluorescence secondary antibodies used were listed in Supplementary Data 3. For all immunostaining, Hoechst 33342 (#ab228551, Abcam) was employed to counterstain the cell nuclei. Fluorescent images were captured under a Leica SP8 confocal microscope equipped with LAS AF Lite software.

In situ RNAscope hybridization
In situ RNAscope hybridization for Krt6a was performed according to the manufacturer’s instructions for RNAscope™ Multiplex Fluorescent Reagent Kit v2 (#323110, Advanced Cell Diagnostics, ACD). Frozen sections were fixed in 4% PFA for 30 min, and then rinsed in 50%, 70% and 100% ethanol for 5 min each. Subsequently, slides were pretreated with RNAscope Hydrogen Peroxide, Target Retrieval Reagent, and Protease Plus. The Krt6a probe (#464441, ACD) was applied to the slides and hybridization was carried out using the HybEZ™ oven. To amplify the signal, AMP1, AMP2, and AMP3 were sequentially applied and the C1 channel was selected for Krt6a signal detection. Finally, a freshly diluted fluorescent working solution was applied and incubated for 30 min (Opal™ 570 Reagent Pack, #FP1488001KT, Akoya, diluted 1:1000 in TSA Buffer). Tissue immunostaining was then performed after HRP blocker.

Bulk RNA sequencing for organoids
Organoid suspensions were lysed in 1 mL TRIzol (#9101, Takara) for RNA isolation. The concentration was determined using a NanoDrop spectrophotometer (Thermo Scientific). The Read Count values for each gene were compared using HTSeq (v0.9.1) as the basis for original gene expression levels, which were subsequently normalized using FPKM to standardize the expression. Differential gene expression was analyzed using DESeq (v1.38.3) with the screened criteria of |log2FoldChange| > 1 and a significant p-value < 0.05. GO enrichment analysis was performed on the differential genes (including all DEGs, up- and down-regulated DEGs) using top GO (v2.50.0). Additionally, ClusterProfiler (v4.6.0) software was utilized to conduct enrichment analysis of the KEGG pathway for the differential genes, focusing on pathways with a significant p-value < 0.05. The sequencing library was then sequenced on the NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Co., Ltd.

Single-cell RNA sequencing
Sample preparation. For sample preparation, OE tissues were collected from Il17aflox/flox and LckCre/Il17aflox/flox mice (2 months, n=3). The olfactory mucosa of mice was extracted and digested using a mixture of enzyme solutions, which includes DNase I (75 U/mL, #HY-108882, MedChemExpress), Hyaluronidase (250 U/mL, #H3506, Sigma), and Collagenase type I (100 U/mL, #1148089, Sigma). Following a 15-20 min incubation at 37°C, 0.25% trypsin with EDTA was added to obtain a single-cell suspension.

scRNA-Seq raw data processing
Single-cell RNA-Sequencing was conducted by Guangzhou Genedenovo Company Ltd. Approximately 20,000 cells were loaded, and the scRNA-seq library was prepared using the Chromium Next GEM Single Cell 3’ Reagent Kits v3.1. The samples were then processed using the 10x Genomics system and sequenced on the Illumina NovaSeq 6000 System. Cells with unusually high number of UMIs (≥8000), and those with more than 10% mitochondrial gene expression were filtered out. Cell clustering and identification of marker genes were carried out after scaling and normalizing the UMI values using the NormalizeData function.

Cell type identification
Multiple cell-type-specific marker genes were used to determine each cell type and conduct subgroup analyses. The list of marker genes includes: HBC (Krt5, Trp63); GBC (Neurod1, Ascl1); iOSN (Gap43, Tubb3); mOSN (Omp, Gng13); MV1 (Cftr, Galnt13); OEG (Plp1, Cldn5); SUS (Rgs5, Clu); Res BC (Krt19, Gsto1); Res S (Bpifb1, Muc5b); Res C (Cd177, Plet1); Neu (S100a8); CD8+ Th (Gzma); CD4+ Th (Ctla4); γδT (Cxcr6, Cd3d); Cycling T (Top2a, Mki67); Treg (Areg); B (Iglc2); Mac (Adgre1, C1qa); DC (Wdfy4, Cd83) and pDC (Siglech, Ccr9).

scRNA-Seq analysis
Preliminary analysis of single-cell data from OE samples was conducted using the OmicShare online platform and R (version 4.1.0). Gene Ontology (GO) analysis was performed by the clusterProfiler R package (version 3.15, https://bioconductor.org/packages/clusterProfiler/) and the results were visualized with the ggplot2 R package (https://github.com/tidyverse/ggplot2). Intercellular communication networks were analyzed using CellChat (https://github.com/sqjin/CellChat). For RNA velocity analysis, velocity estimates were generated using Seurat’s RunVelocity function, following the guidelines provided on github (https://github.com/hamidghaedi/scRNA_velocity_analysis). Monocle 2 (version 2.22.0) package was employed to construct single-cell trajectories of each HBC subcluster. The DDRTree algorithm was used for dimensional reduction.

Statistical analysis
IBM SPSS version 24.0 and Graphpad Prism version 8.0 (Graphpad Software, La Jolla, CA, USA) were used for the statistical analysis. For normally distributed data, we used the unpaired Student t test (two-sided) to compare the differences between two groups and the analysis of variance (ANOVA) with Tukey post hoc test or Sidak’s multiple comparison test (one-sided) to adjust multiple comparisons. For non-normally distributed data, the two-tailed Mann-Whitney U test was used for comparisons between two groups, and the Kruskal-Wallis test with the Dunn post hoc test was employed for multiple comparisons. Correlations were assessed using the Spearman rank correlation. The p-values were shown in figures. Each experiment was independently performed at least three times, with similar results across replicates.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this research, Wang et al. reported that upregulated IL-17 in inflamm-aging inhibited alfactory epithelium (OE) regeneration. This mechanism possibly contributed to aging-related olfactory dysfunction. Using scRNA-seq analysis, the authors found that IL-17-producing cells, predominantly the Th17 cells, were accumulated in aged OE. Mif/CD74 and CD44 interaction were the most prominent ligand-receptor crosstalk possibly facilitated by IL-17 signaling. Functionally, IL-17 suppressed the generation of sensory neurons and prohibited the recruitment of horizontal basal cells. The following concerns need to be addressed.\nMajor:\n1. Mif/CD74 and CD44 was identified to be the most dominant ligand-receptor interaction pair that was increased in OE of aged mice compared with young mice (Figure 1G). Further, the interaction activity of this ligand-receptor pair was also changed in IL-17-deficient mice compared with controls (Figure S7F). Therefore, this ligand-receptor pair seems be to a strong indicator of OE inflammation and dysfunction. However, the expression level of these markers in representative cell populations need to be confirmed at the protein level.\n2. Figure 3: Y-320 may have off-target effects. Y-320 has been reported to affect protein synthesis and modulate cell apoptosis. The authors should use IL-17 neutralization antibody to perform the same experiment in vivo to demonstrate the role of IL-17 in OE injury. I understand that experiments in Figure 5 and 6 using IL-17 cKO mice provided convincing evidence that IL-17 worsens OE damage and contributes to impaired olfactory function. Nevertheless, IL-17 cKO mice lost IL-17 expression throughout development, which does not represent a treatment efficacy of IL-17 neutralization. Therefore, IL-17 neutralization experiment in vivo is still necessary.\n3. Figure 4G-4K: The authors should perform IL-17 blockade in the experiments of Figure 4G and 4H, rather than in the experiments of Figure 4I-4K.\n4. Figure 4I-4K, since the authors added exogenous recombinant IL-17 in these experiments, IL-17 neutralization was not necessary any more. Instead, the authors could test 2 more different titrations of IL-17 to examine the effect of IL-17 on OE organoids to see if there was a dose dependent effect of IL-17 on OE.\n5. Figure S3H-S3J: EL4 cell line is a tumor cell line that has high cell heterogeneity. EL4 cells have numerous different properties compared with Th17 cells. I suggest that the experiments performed using EL4 cell line be removed from this manuscript.\n6. Figure 4A: The authors should show the methimazole-untreated mice as a control, as illustrated in Figure 4B. Is there a way to quantify the percentage of the patchy area?\n7. Figure 1C, In mice, gene for CD3 is Cd3e, but not Cd3d. Please show the expression profile for Cd3e.\n8. Figure 2I： As scRNA-seq analysis suggested that Th17 cells were the predominant cell population expressing IL-17, CD3 and CD4 needs to be co-stained with IL-17 to analyze if these cells were Th17 cells.\n9. A group without Met injection, needs to be provided as a control for the staining in figure S2B. Number of CD45+ cells should be quantified between the 2 groups to conclude that there was an “influx” of CD45+ cells in the injury group.\n10. Figure S2C-D: Also, these panels need to include a day 0 control side by side to quantitatively analyze the level of IL-17-expressing expressing cells and Tuj1+ cells proliferation.\nMinor:\nFigure 4: It should be clarified in the figure legends that the mice were subjected to methimazole treatment.\nFigure S3E, S4D was not described anywhere in the text.\nFigure 6C, colored bars representing different clusters of cell subsets need to be annotated.\nLine 564, figure S7E.", "reviewer_2": "This study investigates the impact of age-related inflammation (inflamm-aging) on the homeostasis and regeneration of the olfactory epithelium (OE), focusing on the role of interleukin-17a. The results indicate that IL-17a expression increases in aged OE, activating immune cells and promoting interactions between horizontal basal cells (HBCs) and T cells, leading to inflammatory signaling and olfactory dysfunction. Administration of the IL-17a inhibitor Y320 enhanced olfactory nerve regeneration and suppressed age-related abnormal changes in OE, such as respiratory epithelialization. Additionally, co-culturing OE organoids with Th17 cells inhibited neurogenesis and facilitated the transformation of OE into respiratory epithelium, an effect that was alleviated by IL-17a-neutralizing antibodies. T cell-specific knockout of IL-17a promoted HBC mobilization and differentiation into globose basal cells, enhancing OE regeneration. These findings suggest that IL-17a plays a crucial role in age-related olfactory dysfunction and highlight the potential of IL-17a-targeted therapies for treating olfactory impairments in aging.\nWhile the study suggests the importance of Th17 cells in olfactory impairments in aging, several important experiments are required to strengthen the hypothesis. There were several concerns regarding the support for the authors’ hypothesis as described below:\nComments in no specific order:\n・Why do Th17 cells increase in OE as aging? The molecular mechanism should be mentioned. For example, cytokines such as IL-23 and IL-6 and chemokines to recruit Th17 should be examined in detail.\n・What antigens activate Th17 cells in the olfactory epithelium? Are there differences in TCR repertoire as compared to Th17 cells in other tissues?\n・Which cells express IL-17Ra in the olfactory epithelium? Is it expressed only in certain cells?\n・Obesity has also been reported to increase immune system aging and augment Th17 cells. Does obesity also hasten olfactory epithelial dysfunction?\n・Regarding Figure 2C, the authors used Areg as a marker gene for Treg cells. However, Foxp3 is required for the definition of Treg cells.\n・Regarding the scRNA-seq in Figure 2, the authors need to include the proportions of αβ T cells, γδ T cells, and other cell types among the Il17a-expressing cells.\n・Regarding Figure 2H, the authors define the marker genes for each T-cell subset. The violin plot should also be included for representative genes.\n・Regarding Figure 5F, T cells in 7_dpi_CKO exhibits Il17a expression. However, in Lck conditional Il17a knock out mouse, since T cells express Lck during thymic maturation, peripheral T cells should defect Il17a genes. Authors needs to investigate whether the cells defined as 'Il17a in T cells' are contaminated with other Il17a producing cell such as ILC3. Another possibility is that this KO mice express immature transcripts of Il17a, but not express intact transcripts of Il17a. These points need clarification.\n・Regarding to above question, authors need to define γδ T cell in Figure 5B.\n・Figure 5D shows that the proportion of T cells is also greatly reduced in IL-17 cKO mice. What is the reason for this?\n・With regard to Figures 6D to F, the Monocle2 Trajectory analysis determines the cell type in each sample group from a single integrated figure (Figure 6E).\nIn this case, it seems to be better to perform the Velocity analysis (Figure 6D) based on a single integrated figure. Otherwise, it would be better to state the advantages of analysing them individually as shown in current Figure 6D.", "reviewer_3": "In the paper by Wang and coworkers the role of IL17 signaling in olfactory epithelium regeneration is studied in the context of elevated IL17 signaling with age. The main findings of the authors are that with ageing, there is an increase in IL17 signaling, and that inhibition of IL17 signaling using either the drug Y320 or conditional knockout of IL17a in T cells, leads to enhanced regeneration of olfactory epithelium in vitro and in vivo.\nMajor comments\n1. The main comment that comes to mind after evaluating the manuscript is that the 2nd component of the title “alleviates age-related respiratory metaplasia and olfactory dysfynction” is not fully supported by the data. The increase in IL-17 signaling with age is not questioned and is very convincingly demonstrated. Equally, the authors have convinced me that there is a key role for IL-17 signaling with CD4+ T cells as the cellular source based on the elegant experiments using the conditional KO mice. However, those experiments specifically did not involve a comparison between young and old mice. The comparison between young and old is mainly provided for the experiments using the drug Y320. These experiments show a functional effect of the drug, but this appears to be a completely independent phenomenon from age. Thus, both in Figure 3C, 3I, 3J, 3K there is no apparent interaction between drug treatment and age. The drug has an effect, age has an effect and the combination of these two leads to an expected additive effect rather than an interactive effect. For this reason, the experiments shown in Figure 3 do not demonstrate that the effect of Y320 is age-dependent which contrasts the hypothesis.\n- (lines 596-599): “In both young and aged mice, inhibiting the secretion of IL-17a through Y320 effectively alleviated OE damage, respiratory epithelial metaplasia, and olfactory dysfunction caused by Met injury. These changes were even more pronounced in aged mice”. The statement is not justified by the research findings, in the case of “even more pronounced in aged mice”. Research findings suggest instead a similar effect on aged and young mice.\n2. Equally, in the organoid studies reported in Figure 4, there is an age effect and there is an effect of the Th17 coculture but without an obvious interaction, leading to additive functional effects rather than age specific effects.\n- (lines 608-609): “we effectively restored neuronal differentiation and inhibited the metaplasia of Krt19+ respiratory epithelial cells in aged organoids”. This Is just partially justified, since the effect on young organoids is similar (fig 4G-I).\n3. An additional concern is related to the compound Y320. The authors justify the use of this compound as a selective tool to reduce IL17 secretion by T cells, but the available literature indicates a much broader mode of action of this drug.\n4. In Figure 1, why were the data not analysed as one dataset? It appears that the young and old datasets are analysed separately. This is in contrast to the data shown in Figure 2, in which the data are analysed as one pooled cluster of cells. The approach in Figure 2 appears to be the right one.\nMinor comments\n1. Page 4 the sentence “Due….condition” (line 50-52) is missing a reference. How can loss of smell be fatal?\n2. The 3rd paragraph of the introduction contains many abbreviations. An example is the sentence “When…regeneration” (line 71-74). To the broader scientific readership this will be very difficult to read and it is recommended that these abbreviations are written in full to avoid confusion.\n3. Introduction last paragraph. Suggest to write as hypothesis/aim rather than a summary of findings.\n4. Out of curiosity, did the authors also analyse olfactory receptor mRNA expression as a function of age and IL17 interventions?\n5. Figure 3G and 3I: were there any changes in organoid size or morohology?"}, "author_response": "Point-by-PointResponses\nREVIEWERCOMMENTS\nReviewer#1(Remarks totheAuthor):\nIn this research, Wang et al. reported that upregulated IL-17 in inflamm-aging\ninhibited olfactory epithelium (OE) regeneration. This mechanism possibly\ncontributed to aging-related olfactory dysfunction. Using scRNA-seq analysis,\nthe authors found that IL-17-producing cells, predominantly theTh17 cells, were\naccumulated in aged OE. Mif/CD74 and CD44 interaction were the most\nprominent ligand-receptor crosstalk possibly facilitated by IL-17 signaling.\nFunctionally, IL-17 suppressed the generation of sensory neurons and prohibited\nthe recruitment of horizontal basal cells. The following concerns need to be\naddressed.\nMajor:\n1. Mif/CD74 and CD44 was identified to be the most dominant ligand-receptor\ninteraction pair that was increased in OE of aged mice compared with young\nmice (Figure 1G). Further, the interaction activity of this ligand-receptor pair\nwas also changed in IL-17-deficient mice compared with controls (Figure S7F).\nTherefore, this ligand-receptor pair seems be to a strong indicator of OE\ninflammation and dysfunction. However, the expression level of these markers in\nrepresentativecell populations need to beconfirmed attheprotein level.\nResponse:\nWe thank the reviewer for this insightful suggestion. As recommended, we have\nperformed immunofluorescence staining to validate the protein expression and\nlocalizationof MIF,CD74,and CD44 inOE of young and aged mice.\nOur results confirmed a marked increase in the protein levels of all three markers in\naged OE compared to young controls. Quantitative analysis showed a significant\nincrease in the percentages of MIF+, CD74+, and CD44+ cells. Cell-type-specific\nanalysis indicated that the CD74 and CD44 expression were enhanced near the basal\ncell layer, showing co-localization with Icam1+ basal cells. These markers were also\nabundantly expressed in the lamina propria, consistent with increased immune\ninfiltration. The protein-level findings align perfectly with our transcriptomic data,\nproviding direct validation of enhanced immune activity in the aged OE (revised\nFigure 1g-i; pages 8-9, lines 152-160 in revised manuscript). For clarity, the violin\nplot from original Figure 1H has been relocated and visually enhanced in revised\nFigure 1i.\nFurthermore, immunostaining of Flox and CKO mice at 14 dpi demonstrated a\nsubstantial reduction and spatial redistribution of CD74+ and CD44+ immune cells in\nIL-17a CKO mice. In Flox controls, these signals were readily detectable in the basal\nand neuronal layer, whereas in CKO mice, the remaining positive cells were largely\nconfined to the lamina propria. This redistribution pattern aligns with observations in\nyoung versus aged OE (revised Figure 1g-i). These results provided direct\nexperimental validation of the CellChat-predicted downregulation of the\nMif/CD74-CD44 interaction axis unpon IL-17a ablation (revised Figure S7g-h; page\n32-33,lines 683-691).\nTogether, these new data strengthened our conclusion that IL-17a deficiency\nattenuates olfactory inflammation partly by suppressing this key ligand-receptor\npathway, thereby helping preserve olfactory function in aging. Corresponding images\nare presented inResponseFigure1below.\nResponseFigure1\n2. Figure 3: Y-320 may have off-target effects. Y-320 has been reported to affect\nprotein synthesis and modulate cell apoptosis. The authors should use IL-17\nneutralization antibody to perform the same experiment in vivo to demonstrate\nthe role of IL-17 in OE injury. I understand that experiments in Figure 5 and 6\nusing IL-17 cKO mice provided convincing evidence that IL-17 worsens OE\ndamage and contributes to impaired olfactory function. Nevertheless, IL-17 cKO\nmice lost IL-17 expression throughout development, which does not represent a\ntreatment efficacy of IL-17 neutralization. Therefore, IL-17 neutralization\nexperimentinvivo is stillnecessary.\nResponse:\nWe sincerely thank the reviewer for raising this important point regarding the\npotential off-target effects ofY320 and thevalueof IL-17 neutralization in vivo.\nFollowing the reviewer’s suggestion, we performed in vivo intervention using an\nIL-17a neutralizing antibody (NAb) to further validate the specific role of IL-17a in\nOE injury. Aged mice subjected to Met-induced injury were treated with two\nconcentrations of the antibody, NAblow (105 µg/ml) and NAbhigh (150 µg/ml),\naccording to established protocols from recent studies 1, 2, 3. The results clearly\ndemonstrated that IL-17a neutralization promoted the recovery of olfactory function,\nreduced Krt19+ respiratory metaplasia, and increased Tuj1+ neuronal density in a\ndose-dependent manner. Moreover, high-dose antibody treatment modulated the\nexpression and distribution of CD74 and CD44 within OE, shifting their profiles\ntoward a pattern resembling that of young mice. These findings align with the\nprotective effects observed with Y-320, confirming that specific inhibition of IL-17a\nsignaling enhanced olfactory regeneration, and suppressed injury-induced respiratory\nmetaplasia (revised Figure 4e-l; page 17, lines 347-365). Corresponding images are\nalso presented in ResponseFigure2below.\nTogether with the IL-17 CKO model, which established the causal role of IL-17a in\nOE pathogenesis, the antibody-mediated neutralization approach provided\ncomplementary and compelling evidence that the protective effects were specifically\nmediated by IL-17a blockade. We believe that combing genetic and pharmacological\nstrategies strongly supports the conclusion that IL-17a plays a key pathogenic role in\nage-related olfactory dysfunction.\nResponseFigure2\n3. Figure 4G-4K:The authors should perform IL-17 blockade in the experiments\nofFigure4Gand 4H,ratherthanin theexperiments ofFigure4I-4K.\nResponse:\nWe thank the reviewer for the valuable suggestion. Following the guidance, we have\nnow performed IL-17a neutralization in the experiments corresponding to the original\nFigures 4G and 4H (now revised as Figure 5 and Figure S3) and obtained clear and\nsupportiveresults.\nAs detailed in the updated Results section (pages 20-21, lines 401-428),\nadministration of an IL-17a neutralizing antibody (NAb) during Th17 co-culture\neffectively restored the expression of neuronal markers (Tuj1 and OMP) and\nsuppressed Th17-induced increase in respiratory metaplasia markers (Krt19 and\nMuc5ac), while also reversed the cystic morphological changes in co-cultured\norganoids. Both quantification and qPCR analysis confirmed that IL-17a blockade\nprevented the disruptive impact of Th17 cells on cell fate specification, enabling\norganoids to recover a near-normal morphological and molecular phenotype (revised\nFigure 5d-j and Figure S3h, i). Notably, Th17 co-culture led to a significant increase\nin P16 expression in aged olfactory organoids, which was attenuated by IL-17a NAb.\nIn contrast, no significant differences in P16 levels were observed among young\norganoid groups, suggesting heightened susceptibility to IL-17a-mediated effects in\naged organoids (revised Figure 5k; page 21, lines 429-433). Furthermore, to validate\nrespiratory epithelial features, we established murine respiratory organoids (Methods,\npage 42, lines 890-897), which closely resembled Th17-exposed organoids in both\nmorphology and cellular composition (revised Figure S3j, k; page 21, lines 433-438).\nRepresentativeimage are presented inResponseFigure3below.\nThese new results strongly reinforced our conclusion that IL-17a serves as the key\ncytokine underlying Th17-induced impairment of olfactory neurogenesis and\nmetaplastic transformation. We are grateful for the reviewer’s insightful comment,\nwhich has significantly strengthened themechanistic evidence presented inourstudy.\nResponseFigure3\n4. Figure 4I-4K, since the authors added exogenous recombinant IL-17 in these\nexperiments, IL-17 neutralization was not necessary any more. Instead, the\nauthors could test 2 more different titrations of IL-17 to examine the effect of\nIL-17 on OE organoids to see if there was a dose dependent effect of IL-17 on\nOE.\nResponse:\nWe sincerely thank the reviewer for this constructive suggestion. In response, we\nwould like to clarify that the concentration of 40 ng/ml recombinant IL-17a used in\nour original submission was selected based on a preliminary dose-response\nassessment involving three concentrations (10, 20 and 40 ng/ml).As shown in revised\nFigure S4a-b, immunostaining and qPCR analyses demonstrated that IL-17a induced\na clear dose-dependent suppression of neuronal differentiation, as reflected by\nprogressively decreased expression of OMP, Krt14 and Lgr5. In addition, IL-6\nexpression increased in a dose-dependent manner, indicating a graded inflammatory\nresponse. Based on these results, 40 ng/ml was selected for all subsequent organoid\nexperiments. At this concentration, IL-17a significantly suppressed the generation of\nTuj1+ and OMP+ neurons, downregulated mRNA expression of Krt14, Krt5 and\nGap43, and upregulated the respiratory marker Krt19. Notably, IL-17a also increased\nexpression of the senescence marker P16 in aged organoids (revised Figure S4c-f).\nThese new findings strongly support a dose-dependent role for IL-17a in suppressing\nneurogenesis, promoting proliferation, and promoting metaplastic transformation in\nolfactory organoids. The results have been incorporated into the Results section\n(pages 22-23, lines 453-472). Representativeimage are presented inResponse Figure\n4below.\nIn accordance with the reviewer’s suggestion, we have removed the IL-17a\nneutralization data from this figure panel to improve logical consistency and\nexperimental focus.\nWe are grateful to the reviewer for this valuable suggestion, which has significantly\nstrengthened therigorand comprehensiveness ofour study.\nResponseFigure4\n5. Figure S3H-S3J: EL4 cell line is a tumor cell line that has high cell\nheterogeneity.EL4 cells have numerous different properties compared withTh17\ncells. I suggest that the experiments performed using EL4 cell line be removed\nfromthis manuscript.\nResponse:\nWe thank the reviewer for raising the valid concern. We agree that the EL4 cell line,\nas a tumor-derived model with inherent heterogeneity and functional properties\ndistinct from primary Th17 cells, is not ideal for supporting the conclusions of our\nstudy. Accordingly, we have removed all data and text related to the EL4 cell line\nfrom the revised manuscript. We believe this revision improves the biological\nrelevance and overall clarity of ourwork.\n6. Figure 4A: The authors should show the methimazole-untreated mice as a\ncontrol, as illustrated in Figure 4B. Is there a way to quantify the percentage of\nthepatchy area?\nResponse:\nWesincerely appreciatethe reviewer’s constructivesuggestions.\nIn response to the first point, we have now included representative images from\nmethimazole-untreated control mice in revised Figure 4a. These images clearly\nillustrated that under physiological conditions, Krt19+ cells were confined to the\nrespiratory epithelium (RE)region, in sharp contrast to theextensivemetaplasitcareas\nobserved after methimazole-induced injury (revised Figure 4a; page 16, lines\n326-328).\nTo address the second point regarding quantification, we have now provided\ncomprehensive overview images of OE staining with clear annotation of metaplastic\nregions. As illustrated in revised Figure 4b (page 17, lines 339-346), the metaplastic\nareas were outlined in green, based on Krt19 staining in DMSO group. We\nsubsequently performed detailed morphometric analyses to quantify the percentage of\nthe Krt19+ patchy area relative to the total OE. Our results demonstrated that the\nmetaplastic area accounted for 77.97% of the total OE in aged mice, which was\nsignificantly higher than that in young mice (43.5%). Treatment with Y-320\nsignificantly reduced this proportion, leading to a decrease of 44.99% in aged and\n21.65% in young mice. These quantitative data are now provided an objective\nevaluation of both injury-induced metaplasia expansion and the therapeutic efficacy\nof Y-320 in attenuating this process. The relevant images are also presented in\nResponseFigure5below.\nWe thank the reviewer for the insightful comments, which have greatly improved the\nclarity and rigor ofourmanuscript.\nResponseFigure5\n7. Figure 1C, In mice, gene for CD3 is Cd3e, but not Cd3d. Please show the\nexpressionprofile forCd3e.\nResponse:\nWe appreciate the reviewer's careful attention to this important detail regarding the\nappropriate marker for T cells in mice. In response, we have replaced the expression\nprofileof Cd3d withthat ofCd3e in revised Figure 1d(page 7,line126),to accurately\nrepresent T cell presence. This correction has been displayed in the Response Figure\n6below.\nResponseFigure6\n8.Figure 2I ： As scRNA-seq analysis suggested that Th17 cells were the\npredominant cell population expressing IL-17, CD3 and CD4 needs to be\nco-stained with IL-17 to analyzeifthese cells wereTh17cells.\nResponse：\nWe thank the reviewer for the insightful suggestion. As recommended, we have\nperformed triple immunofluorescence co-staining for IL-17a, CD3 and CD4 on young\nand aged OE tissues to identifyTh17 cells in situ.\nOur results demonstrated a marked increase in IL-17a+, CD3+, and CD4+ signals in\naged tissue, compared to young controls. These findings were highly consistent with\nand complementary to our scRNA-seq data, confirming enhanced T cell infiltration\nand IL-17a production inaged OE.\nIn the triple-staining analysis, we observed that although CD3 and CD4 showed\nextensive co-localization as expected, only a minority of IL-17a⁺ signals co-localized\nwith both CD3 and CD4 (indicated by yellow arrowheads). We attributed this limited\nco-localization primarily to the fact that IL-17a is a rapidly secreted cytokine. Upon\nactivation, Th17 cells quickly synthesize and release IL-17a rather than retaining it\nintracellularly. As a result, standard immunofluorescence captures only a small\nfraction of cells at the exact moment of cytokine accumulation, making consistent\nco-localization challenging to detect in static images 4. Additionally, beyond CD4⁺ T\ncells, IL-17a is also produced by other cell types, such as γδ T cells, as described in\nrevised Figure 2h. The corresponding images have been included in revised Figure\n2m without the arrowheads (page 12, lines 240-244), and are also presented in\nResponseFigure7below.\nResponseFigure7\n9.A group without Met injection, needs to be provided as a control for the\nstaining in figure S2B. Number of CD45+ cells should be quantified between the\n2 groups to conclude that there was an “influx” of CD45+ cells in the injury\ngroup.\nResponse:\nWe appreciate the reviewer’s valuable suggestion. In response, we have performed\nCD45 immunostaining on both uninjured control and methimazole-injured mice at 1,\n2, 3, and 7 dpi. Quantitative analysis revealed a significant influx of CD45+ immune\ncells as early as 1dpi,which was sustained throughout the7-day period.The modified\nresults are now displayed in revised Figure 3a and b (page 13, lines 258-265), as well\nas in Response Figure 8 below. These results confirmed a robust injury-induced\ninflammatory response and provided clear support for our conclusion regarding\nimmunecell recruitment following olfactory epithelial damage.\n10. Figure S2C-D:Also, these panels need to include a day 0 control side by side\nto quantitatively analyze the level of IL-17-expressing expressing cells and Tuj1+\ncells proliferation.\nResponse:\nWe sincerely thank the reviewer for the constructive suggestion. In response, we have\nnow performed a comprehensive time-course analysis comparing uninjured controls\n(Day 0) with injury mice at 1, 2, 3, and 7 dpi to quantitatively evaluate\nIL-17a-expressing cells and Tuj1+ neurogenesis. Our results showed that\nIL-17a-EGFP+ cells, nearly absent in uninjured tissue, rapidly accumulated in the\nbasal cell layer and lamina propria by 1 dpi and persisted through 7 dpi. In terms of\nneuronal regeneration, mature OMP+ OSNs were completely lost by 1 dpi, with\nlimited recovery by 7dpi, while immatureTuj1+neurons began toemerge at 2 dpi and\nincreased progressively through day 7. These quantitative findings, now presented in\nrevised Figure 3a and b (page 13, lines 258-265), clearly delineated the temporal\ndynamics of IL-17a+ cell infiltration and subsequent neuro-regenerative responses\nfollowing injury. The corresponding images are presented in Response Figure 8\nbelow.\nWe are grateful to the reviewer for this suggestion, which has significantly\nstrengthened the temporal resolution and mechanistic insight of our injury-repair\nmodel.\nResponseFigure8\nMinor:\n11. Figure 4: It should be clarified in the figure legends that the mice were\nsubjected to methimazole treatment.\nResponse:\nWe thank the reviewer for pointing this out. We have now clarified in the figure\nlegend that all mice included in Figure 4 underwent methimazole-induced injury\n(page 61,line1305).\n12. FigureS3E, S4Dwasnotdescribed anywherein thetext.\nResponse:\nWeappreciate the reviewer’s careful reading.The descriptions of Figure S3Eand S4D\nwere originally included in the text, but their citations were inadvertently omitted.\nThis has been corrected in the revised manuscript, with revised Figure S3e (original\nFigure S3E) now cited in page 18, line 376-378, and revised Figure S4f (original\nFigure S4D)in page 23,line470-472.\n13. Figure 6C, colored bars representing different clusters of cell subsets need to\nbeannotated.\nResponse:\nWe thank the reviewer for this helpful suggestion. We have now added clear\nannotations for the colored bars representing different cell subset clusters in revised\nFigure 7c (original Figure 6C). The revised elements are also presented in Response\nFigure9below.\nResponseFigure9\n14. Line564,figureS7E.\nResponse: We sincerely appreciate the reviewer's careful reading and for bringing the\ncitation error Figure S7Eto our attention.This has nowbeen corrected (revised Figure\nS7e;page 32,line670).\nReviewer#2(Remarks totheAuthor):\nThis study investigates the impact of age-related inflammation (inflamm-aging)\non the homeostasis and regeneration of the olfactory epithelium (OE), focusing\non the role of interleukin-17a. The results indicate that IL-17a expression\nincreases in aged OE, activating immune cells and promoting interactions\nbetween horizontal basal cells (HBCs) and T cells, leading to inflammatory\nsignaling and olfactory dysfunction.Administration of the IL-17a inhibitorY320\nenhanced olfactory nerve regeneration and suppressed age-related abnormal\nchanges in OE, such as respiratory epithelialization. Additionally, co-culturing\nOE organoids with Th17 cells inhibited neurogenesis and facilitated the\ntransformation of OE into respiratory epithelium, an effect that was alleviated\nby IL-17a-neutralizing antibodies. T cell-specific knockout of IL-17a promoted\nHBC mobilization and differentiation into globose basal cells, enhancing OE\nregeneration. These findings suggest that IL-17a plays a crucial role in\nage-related olfactory dysfunction and highlight the potential of IL-17a-targeted\ntherapies fortreating olfactory impairments inaging.\nWhile the study suggests the importance of Th17 cells in olfactory impairments\nin aging, several important experiments are required to strengthen the\nhypothesis. There were several concerns regarding the support for the authors’\nhypothesisas described below:\nComments inno specificorder:\n1. Why doTh17 cells increase in OE as aging?The molecularmechanism should\nbe mentioned. For example, cytokines such as IL-23 and IL-6 and chemokines to\nrecruitTh17shouldbeexamined in detail.\nResponse:\nWe sincerely thank the reviewer for raising this critical question regarding the\nmechanism underlying the age-associated accumulation ofTh17 cells in OE.\nThe increase in Th17 cells in aged OE is driven by a pro-inflammatory\nmicroenvironment resulting from both systemic immunosenescence and local tissue\nalterations. Systemically, aging is associated with a chronic, low-grade systemic\ninflammation state known as inflammaging, which senescent cells release factors like\nIL-6 and IL-1β via the senescence-associated secretory phenotype (SASP).Within the\nlocal OE tissue, resident innate immune cells such as macrophages and dendritic cells\nbecome persistently activated, leading to sustained production of key polarizing\ncytokines, including IL-23, IL-6, IL-1β, and TGF-β. This cytokine milieu promotes\nTh17 differentiation and maintenance. Furthermore, IL-17 secretion from established\nTh17 cells can induce chemokines like CCL20, creating a self-amplifying\ninflammatory loop that recruits moreimmunecells and reinforces the response5,6.\nOur initial data (Figure S1E) confirmed a general upregulation of inflammatory\nmediators, including T cell recruitment (Cxcl10, Cxcl16), NF-κB targets\n(Tnf, Il6, Cxcl5), as well as TNF superfamily members (Tnfrsf1a, Tnfsf10) in the\naged OE.\nGuided by the reviewer’s suggestion, we have now expanded investigation into key\npathways involved in Th17 chemotaxis recruitment, co-stimulatory molecules,\nmaintenance, and polarization 7. Our new analysis revealed that the aged OE\nmicroenvironment exhibits upregulated expression of several chemokines, including\nCcl20, Cxcl10, Ccl2, and Ccl7, along with their corresponding receptors (Ccr6, Cxcr3,\nCcr2), which are critical for Th17 recruitment. In addition, cytokines central to Th17\npolarization and maintenance, such as Il6, Il23a, Il1b, Tgfb1, and Tnf, were\nsignificantly increased in aged OE tissues, along with elevated expression of key\ntranscription factors (Stat3, Rorc, Batf). Several of these are NF-κB target genes, and\nmembers of TNF receptor/ligand superfamily remained upregulated (revised Figure\nS1k; pages 10-11,lines 193-208).\nTo further validate these findings at the protein level, we performed ELISA and\nconfirmed that both IL-17a and IL-23 were significantly elevated in aged OE tissue\nhomogenates and plasma(revised Figure 2i; page12,lines 234-236).\nTogether, these new results delineated a model in which the aged OE exhibits a\npro-inflammatory microenvironment characterized byenhanced chemotacticsignaling,\nantigen presentation, and polarizing cytokine production, collectively facilitating the\nrecruitment, local expansion,and stabilization ofTh17 cells.\nThe corresponding images are presented in Response Figure 10 below. We are\ngrateful to the reviewer for this insightful suggestion, which has substantially\nstrengthened themechanisticscope of ourstudy.\nResponseFigure10\n2. What antigens activate Th17 cells in the olfactory epithelium? Are there\ndifferences inTCRrepertoireascompared toTh17cells in othertissues?\nResponse:\nWe sincerely appreciate the reviewer for raising these critical questions regarding the\nantigenicdrivers andTCRrepertoire ofTh17 cells intheolfactory epithelium.\nFor the first question, the OE represents a unique immunological interface due to its\ndirect and continuous exposure to the external environment. Previous studies have\ndemonstrated that intranasal infections preferentially induce Th17 cells responses 8 .\nTh17 cells in OE are primarily activated by airborne microbial antigens derived from\ncommensal bacteria, fungi, and pathogens, such as Streptococcus pyogenes present in\nthe nasal environment 9.Antigen presentation in the OE is mediated by dendritic cells\nand other immune cells such as eosinophils. These activated Th17 cells produce\ncytokines including IL-17,which contributes to host defense against pathogens.\nTo systematically evaluate the potential antigenic drivers in our model, we analyzed\nscRNA-seq data from the OE of young and aged mice. We first subset CD4+ cells\nfrom the T cell population and subsequently identified Th17 cells based on gene set\nscoring. The Th17 cells were predominantly abundant in aged tissues (Response\nFigure 11a, b). We then manually curated four effector gene modules representing\ndistinct immunological pathways: Fungal-Th17 Axis, Bacterial-TLR/NOD Axis,\nantiviral Type I Interferon ISGAxis, as well as Th2-STAT6Axis.The related genes in\neach axis were displayed in Response Figure 11e 10, 11, 12. Module scoring analysis\nrevealed that the Fungal-Th17 pathway was the most highly enriched in these cells,\nfollowed by theViral module, while the Bacterial andTh2 modules showed lower and\nmore restricted activity (Response Figure 11c). Therefore, we speculated that due to\nthe direct exposure of the OE to air containing abundant fungal spores, aging may\nlead to reduced mucus secretion, impaired ciliary function, or diminished immune\nclearance capacity. This could result in increased exposure to fungal antigens or\ninadequate clearance. It was also reported that fungal and bacterial biofilms are\ncommonly found in CRS and may support chronic and recalcitrant CRS infection 13.\nThus, the persistent activation of Th17 cells and related inflammatory pathways\n(particularly the Fungal-Th17 Axis) in the OE of aged mice is likely closely\netiologically linked to age-related olfactory dysfunction. Based on the analysis of data\nfrom CKO and Flox mice at 7 and 14 dpi, we observed high expression of\nfungi-related genes in Flox mice at 7 dpi (Response Figure 11d), further\ndemonstrating that nasal mucosa becomes more susceptible to fungal invasion under\ninjury conditions.\nFor the second question, based on the literature and our findings, we discovered\nsignificant differences exist in the TCR repertoire of Th17 cells in the OE compared\nto other tissues, such as gut and skin. Primarily driven by tissue-specific antigen\nexposure, unique cytokine and dendritic cell microenvironments, and functional\nspecialization, OE Th17 TCR are shaped by airborne antigens (e.g., fungal spores and\ncommensal/pathogenic bacteria like Streptococcus pyogenes), while gut Th17 TCR\nare selected by intestinal commensals (e.g., segmented filamentous bacteria) and skin\nTh17 TCR recognize cutaneous microbes (e.g., Staphylococcus epidermidis). These\nantigenic differences are reinforced by distinct cytokine signals and DC subsets: OE\nTh17 differentiation depends on TGF-β1 and IL-6 from CD301b⁺ DCs in cervical\nlymph nodes, whereas gut Th17 development relies on IL-1β and IL-23 (often\nTGF-β1-independent) from CX₃CR1⁺ macrophages or CD103⁺ DC, and skin Th17\npolarization requires IL-1β , IL-6, and IL-23 from dermal DC or Langerhans cells.\nConsequently, TCR divergence correlates with tissue-specific homing receptors and\neffector functions 14, 15, 16. Our single-cell data further showed enrichment of\nFungal-Th17 Axis genes (Clec7a, Syk, Il17a/f) in OE Th17 cells, suggesting clonal\nexpansions distinct from other tissues. Thus, the TCR repertoire of OE Th17 cells is\nuniquely adapted to airborne challenges, highlighting the compartmentalization of\nmucosal immunity. However, due to the technical limitation, we couldn’t get more\ndetailed information aboutTCR repertoire.Weappreciated the reviewer that provide a\nnew direction for our future studies, and will directly compare TCR sequences across\ntissues usingsingle-cell RNA/TCRsequencing to validatethesedifferences.\nResponseFigure11\n3. Which cells express IL-17Ra in the olfactory epithelium? Is it expressed only\nincertain cells?\nResponse:\nWe sincerely appreciate the reviewer's insightful comment regarding the cellular\nspecificity of IL-17ra expressionpatterns in OE.\nTo precisely identify which cell types express Il17ra in OE, we performed extensive\nimmunofluorescence co-staining using an IL-17ra-specific antibody on OE tissues\nfrom young and aged mice, in combination with neuronal marker Tuj1 and basal cell\nmarker Icam1. Our findings revealed that Il17ra expression exhibits distinct cell-type\nspecificity and spatiotemporal dynamics. In young mice, IL-17ra was primarily\nexpressed in neurons, with approximately 87.6% of the signal co-localizing with\nTuj1+ neurons in the dorsal region. In aged mice, however, the expression pattern\nshifted markedly, showing strong enrichment in the basal cell layer of the dorsal\nregions, where about 60% co-localized with Icam1+ basal cells. Further regional\nanalysis indicated that in the medial OE of aged mice, a higher proportion of Il7ra\nco-localized with neurons, while in the respiratory epithelium (RE) region, expression\nremained predominantly inthebasal layer across bothage groups.\nQuantitative analysis confirmed that Il17ra was most abundant in neurons of young\nmice but became significantly elevated in HBC of aged OE, demonstrating an\nage-dependent redistribution of IL-17 signaling potential within the tissue (revised\nFigure S1i, j, pages 9-10, lines 175-192). Representative images are also present in\nResponseFigure12.\nIn summary,the cellular landscape of IL-17ra expression is bothspecific and dynamic,\ntransitioningfrom neuronal dominanceto basal cells prevalence withage.\nResponseFigure12\n4. Obesity has also been reported to increase immune system aging and augment\nTh17cells. Does obesity alsohasten olfactory epithelialdysfunction?\nResponse:\nWe sincerely thank the reviewer for raising this insightful question concerning the\npotential link between obesity, accelerated immune aging, and olfactory dysfunction.\nWe agree that obesity, as a state of chronic systemic inflammation and metabolic\ndysregulation, represents aplausiblefactorin hastening age-related olfactory decline.\nPrevious studies have established that obesity-associated chronic inflammation\ncontributes significantly to the pathogenesis of metabolic diseases. Among T helper\nsubsets, Th17 cells are known to promote autoimmune disorders 17. Furthermore,\nclinical evidence indicated impaired insulin signaling, common in obesity, can affect\nodor perception at the level of olfactory epithelium. Indeed, olfactory dysfunction,\nranging from mild impairment to complete anosmia, has been reported in patients\nwithobesity and type 2diabetes, substantiallyimpacting quality oflife18,19.\nWhile direct mechanistic studies linking obesity and OE dysfunction remain limited,\nour data provided compelling preliminary support for this connection.\nSpecifically, single-cell RNA sequencing analysis of young and aged OE revealed\nprofound dysregulation of genes related to adipocyte differentiation in aged tissue.\nWe observed significant upregulation of both positive (GO: 0045600) and negative\n(GO: 0045599) regulators of fat cell differentiation, along with elevated expression of\nkey adipogenesis-related marker genes in aged OE (Response Figure 13a-d). These\nfindings suggested that the aged OE underwent a form of metabolic reprogramming\nresembling aspects ofthesystemic metabolicdisturbance occured inobesity.\nBased onthese findings, we propose a hypothesis that obesity-related systemic factors,\nsuch as elevated free fatty acids and pro-inflammatory cytokines, may accelerate the\nformation of pro-adipogenic and pro-inflammatory microenvironment within the OE.\nThis process could exacerbate Th17-driven inflammation and promote premature\nolfactory functional decline, suggesting that aging and obesity may converge through\nshared pathogenic pathways in OE.\nWe once again thank the reviewer for this valuable suggestion. Inspired by the\nreviewer’s question, we consider this an important new direction for future research.\nWe plan to directly test this hypothesis using a diet-induced obesity mouse model to\nevaluate whether obesity accelerates the expansion of Th17 cells, impairs olfactory\nepithelial regeneration, and compromises olfactory function.\nResponseFigure13\n5. Regarding Figure 2C, the authors used Areg as a marker gene for Treg cells.\nHowever,Foxp3 is required forthedefinitionofTreg cells.\nResponse:\nWe thank the reviewer for this important comment. In response, we have updated the\nrevised Figure 2c by replacing Areg with Foxp3 as the canonical marker for\nregulatory T cells (page 11, line 220). This adjustment ensures accurate definition of\nTreg populations and strengthens the validity of our analysis. The corresponding\nrevised images are presented in ResponseFigure14below.\nResponseFigure14\n6. Regarding the scRNA-seq in Figure 2, the authors need to include the\nproportions of αβ T cells, γδ T cells, and other cell types among the\nIl17a-expressingcells.\nResponse:\nWe thank the reviewer for this valuable suggestion. In response, we have now\nperformed additional analysis on our scRNA-seq data to quantify the cellular\ncomposition of IL-17a-expresing cells in aged OE. The result revealed that among all\nIL-17a-expressing cell, the majority were CD4+ Th (76.7%), followed by γδ T cells\n(13.7%) and other immune populations. The data has been included in revised Figure\n2h(page 12,lines 229-233), and also presented in ResponseFigure15below.\nResponseFigure15\n7. Regarding Figure 2H, the authors define the marker genes for each T-cell\nsubset.Theviolinplotshould alsobeincluded forrepresentativegenes.\nResponse：\nWe thank the reviewer for this suggestion. In accordance with this comment, we have\nnow included violin plots showing the expression patterns of representative marker\ngenes for each T-cell subset in revised Figure2f (page 12, lines 225-227), also shown\ninResponseFigure16below.\nResponseFigure16\n8. Regarding Figure 5F, T cells in 7_dpi_CKO exhibits Il17a expression.\nHowever, in Lck conditional Il17a knock out mouse, since T cells express Lck\nduring thymic maturation, peripheral T cells should defect Il17a genes. Authors\nneeds to investigate whether the cells defined as 'Il17a in T cells' are\ncontaminated with otherIl17a producing cell such as ILC3.Anotherpossibility is\nthat this KO mice express immature transcripts of Il17a, but not express intact\ntranscripts of Il17a.These points need clarification.\nResponse:\nWe sincerely thank the reviewer for raising this critical point regarding the\nspecificity of Il17a ablation inT cells.We agree that this is essential for the correct interpretation\nof our data in Figure 5F. We have addressed these concerns through additional\nanalyses andclarifications as detailed below.\nFirst, the residual IL-17a signal in CKO T cells reflects highly efficient, but not\nabsolute, gene ablation. Our genotyping and functional data confirmed that the\nLck-Cre-mediated knockout was highly effective. Flow cytometry analysis of CD4+T\ncells revealed that the frequency of IL-17a+ cells was dramatically reduced in the\nCKO mice, ranging from 0.77% to 0.95%, compared to 5.07-6.29% in Flox controls\n(original and revised Figure S5b). This represented an approximately 85% reduction\nin the proportion of IL-17a-producing T cells, providing strong functional evidence\nfor theefficacy of theTcell-specific knockout.The lowbut detectable residual IL-17a\nsignal in CKO mice may be attributed to technical and biological factors, such as\nincomplete Cre recombination, delayed turnover of pre-existing IL-17a protein, or\nlow-level background staining in flow cytometry. Importantly, these findings\nconfirmed that the vast majority of IL-17a production in T cells was abolished,\nthereby validating the utility of our model. Moreover, the analysis mentioned above\nwas performed on gated CD4+ T cells. Since ILC3s are innate lymphoid cells that do\nnotexpress CD4,they would notbe captured withinthis gating strategy 20,21.\nSecond, we attribute the low residual IL-17a expression in 7 dpi CKO mice to the\nacute inflammatory environment following injury. Methimazole-induced damage\ntriggers a rapid and robust inflammatory response, which may include IL-17a\nproduction from non-T cell sources that are not targeted by the Lck-Cre system. This\nwas consistent with the dramatic reduction of IL-17a in CKO mice compared to Flox\ncontrols. Furthermore, the near-complete absence of IL-17a signal by 14 dpi in CKO\nmice indicated that as the acute inflammation resolves and tissue repair progresses,\nthe dependency on IL-17a signaling diminishes, thereby validating the efficacy of the\nknockout model during the recovery phase. Furthermore, we analyzed the expression\npatterns of canonical ILC3 marker genes in our scRNA-seq data from both CKO and\nFlox groups. Our results demonstrated that these genes were expressed at very low\nlevels, indicating the absence of an ILC3 population in our dataset (Response Panel\nin Response Figure 17). The relative images are presented in Response Figure 17\nbelow.\nResponseFigure17\n9. Regarding toabovequestion,authors need to define γδTcell in Figure5B.\nResponse:\nWe thank the reviewer for this insightful suggestion. In response, we have now\nperformed a refined sub-clustering analysis of the T cell population, as shown in\noriginal Figure 5B (revised Figure 6b; page 25, line 510). Using canonical marker\ngenes (Cd3d and Ctla4 for CD4+ Th cells, Trdc and Cxcr6 for γδ T cells, and Mki67\nand Top2a for cycling T cells), we have clearly delineated the three distinct subsets.\nThe revised images are also presented in Response Figure 18 below. This updated\nanalysis providedimproved resolution ofTcell heterogeneity inourmodel.\nResponseFigure18\n10. Figure 5D shows that the proportion of T cells is also greatly reduced in\nIL-17 cKO mice.Whatis thereason forthis?\nResponse:\nWe thank the reviewer for raising this important question. This observed reduction in\nT cell proportion in IL-17a CKO mice was likely a consequence of the improved\ntissuerepair and attenuated inflammatory environment during OE regeneration.\nSpecifically, IL-17a is a key pro-inflammatory cytokine known to promote immune\ncell recruitment and sustains local inflammation. In Flox mice, persistent IL-17a\nsignaling likely amplifies inflammatory responses, leading to sustained infiltration\nand retention of T cells and other immune populations. In contrast, the absence of T\ncell-derived IL-17a in CKO mice results in a milder inflammatory milieu, which\nreduces the overall recruitment and retention of T cells in the injury OE tissue.\nThis interpretation aligns with the accelerated tissue recovery and diminished metaplastic\nchanges observed in CKO mice compared to Flox controls. Therefore, the decrease in\nT cell proportion reflects a return toward immune homeostasis resulting from the\ndampened inflammatory response in CKO mice, rather than a direct effect of IL-17a\ndeletion onTcell development orsurvival.\n11. With regard to Figures 6D to F, the Monocle2 Trajectory analysis determines\nthecell type ineach samplegroupfrom asingleintegrated figure(Figure6E).\nIn this case, it seems to be better to perform the Velocity analysis (Figure 6D)\nbased on a single integrated figure. Otherwise, it would be better to state the\nadvantagesof analysing themindividually asshown incurrent Figure6D.\nResponse:\nWe are grateful to the reviewer for raising the important point regarding the\nintegration of RNAvelocity and trajectory analysis. Wewould like to clarify that both\nthe RNA velocity and Monocle2 trajectory inference were performed on the same\nintegrated dataset. To allow clearer visualization of condition-specific dynamics, we\nchose to present sample-wise RNAvelocity plots in original Figure 6 (revised Figure\n7). This approach facilitated a more direct and rigorous comparison of cell-state\ntransitionsunder each experimental condition.\nIn addition, to fully address the reviewer’s concern and provide a comprehensive view,\nwe have now included the integrated RNA velocity analysis, stratified by both cell\ntype and sample group (revised Figure S6j-l; pages 30-31, lines 638-647, and also\nshown in Response Figure 19 below). These data consistently support the trajectory\ninferences across all samples and further reinforce therobustness ofour conclusions.\nResponseFigure19\nReviewer#3(Remarks totheAuthor):\nIn the paper by Wang and coworkers the role of IL17 signaling in olfactory\nepithelium regeneration is studied in the context of elevated IL17 signaling with\nage. The main findings of the authors are that with ageing, there is an increase in\nIL17 signaling, and that inhibition of IL17 signaling using either the drug Y320\nor conditional knockout of IL17a in T cells, leads to enhanced regeneration of\nolfactory epitheliumin vitroand invivo.\nMajorcomments\n1. Themain comment that comes to mind afterevaluating the manuscript is that\nthe 2nd component of the title “alleviates age-related respiratory metaplasia and\nolfactory dysfynction” is not fully supported by the data. The increase in IL-17\nsignaling with age is not questioned and is very convincingly demonstrated.\nEqually, the authors have convinced me that there is a key role for IL-17\nsignaling with CD4+ T cells as the cellular source based on the elegant\nexperiments using the conditional KO mice. However, those experiments\nspecifically did not involve a comparison between young and old mice. The\ncomparison between young and old is mainly provided forthe experiments using\nthe drug Y320. These experiments show a functional effect of the drug, but this\nappears to be a completely independent phenomenon from age. Thus, both in\nFigure 3C, 3I, 3J, 3K there is no apparent interaction between drug treatment\nand age. The drug has an effect, age has an effect and the combination of these\ntwo leads to an expected additive effect rather than an interactive effect. For this\nreason, the experiments shown in Figure 3 do not demonstrate that the effect of\nY320 is age-dependentwhich contrasts the hypothesis.\n- (lines 596-599): “In both young and aged mice, inhibiting the secretion of\nIL-17a through Y320 effectively alleviated OE damage, respiratory epithelial\nmetaplasia, and olfactory dysfunction caused by Met injury. These changes were\neven more pronounced in aged mice”. The statement is not justified by the\nresearch findings, in the case of “even more pronounced in aged mice”. Research\nfindings suggestinstead asimilareffect on aged andyoungmice.\nResponse:\nWe sincerely thank the reviewer for this insightful and critical comment. We agree\nthat our data did not demonstrate a statistically significant interactive effect between\nage and Y320 treatment in the classical sense, and we appreciate the reviewer for\nhighlighting thisimportant distinctionbetween additive and interactive effects.\nWe have changed the manuscript title to: “IL-17a drives age-related olfactory\ndysfunction byimpairing regeneration and promoting respiratory metaplasia”.\nWe also would like to clarify that the central premise of our study is not that Y320’s\nmechanism of action is age-dependent per se, but rather that the age-dependent\nexacerbation of metaplasia and olfactory dysfunction is primarily mediated by\nelevated IL-17 signaling. Our data, supported by previous literature 22, 23,\ndemonstrated that olfactory function declines with age, accompanied by a significant\nreductionin thenumber of olfactory sensory neurons (revised Figure 1), along withan\nincrease in Th17 cells. We therefore identified elevated IL-17a as a key age-related\nmechanism (revised Figure 2). Since young mice inherently exhibit low baseline\nIL-17a expression, we used methimazole to induce olfactory epithelial injury, thereby\nelevating IL-17a under pathological conditions and establishing a comparable model\nfor aged mice. As a pharmacological inhibitor of the IL-17 pathway, Y320 produced\nconsistent therapeutic effects in both young and aged mic. However, a greater\nabsoluteimprovement was observed in aged mice duetothe heightened activity of the\ndriving factor (IL-17).\nIn the revised manuscript, we have further substantiated this finding through multiple\nanalytical approaches. For example, in revised Figure 4d (page 17, lines 339-346), we\ncompared the relative proportions of metaplastic and reparative areas between young\nand aged mice (as shown in Response Figure 5). Furthermore, in revised Figure 5j\n(page 21, lines 422-428), we quantified the differential changes in the number of\nOMP+andTuj1+neurons, as well as Krt19+ cells in organoids derived from young and\naged mice (as shown in Response Figure 3). Additionally, we administered\nIL-17a-specific neutralizing antibodies to aged mice following injury (revised Figure\n4e-l; pages 17-18, lines 347-365), as shown in Response Figure 2. The resulting\nreparative effects showed a clear dose-dependent response and effectively promoted\nthe recovery of olfactory function in aged mice. Finally, we also analyzed the\nolfactory epithelium of middle-aged mice (12 months old) and found that IL-17a\naccumulation begins as early as midlife. Using olfactory organoids derived from\nmiddle-aged CKO and Flox mice, we directly demonstrated that IL-17a conditional\nknockout enhances the differentiation of olfactory sensory neurons (revised Figure S8,\npages 33-34, lines 694-726).\nThe sentence on lines 596-599 was modified as below (line 749-761 in revised\nmanuscript):\n“In both young and aged mice, inhibiting IL-17a secretion via Y320 effectively\nalleviated Met injury-induced OE damage, respiratory epithelial metaplasia, and\nolfactory dysfunction. However, under identical injury conditions, aged mice\nexhibited more severe OE damage and a markedly slower repair process compared to\nyoung mice, underscoring the heightened therapeutic relevance of IL-17a blockade in\naged individuals. Moreover, considering the complex action of Y320, we performed\nadditionally intervention experiments in aged mice using an IL-17a neutralizing\nantibody. The results demonstrated that the antibody promoted OE recovery in a\nconcentration-dependent manner and reduced inflammatory infiltration into basal\ncells, confirming the pivotal role of IL-17a. Our work thus demonstrated that both\nY320 and an IL-17a neutralization constitute viable strategies to mitigate OE\ndegeneration, revealing a promising therapeutic window for clinical intervention in\nage-related olfactory dysfunction.”\n2. Equally, in the organoid studies reported in Figure 4, there is an age effect and\nthere is an effect of the Th17 coculture but without an obvious interaction,\nleadingto additivefunctionaleffects ratherthanagespecificeffects.\n-(lines 608-609): “weeffectively restored neuronal differentiation\nand inhibited the metaplasia of Krt19+ respiratory epithelial cells in aged\norganoids”. This Is just partially justified, since the effect on young organoids is\nsimilar(fig4G-I).\nResponse:\nWe thank the reviewer for this critical observation. We agree that the Th17 co-culture\ninduced significant disruption in both young and aged organoids. Our interpretation is\nnot that the mechanism of Th17-mediated impairment is exclusive to aged organoids,\nbut rather that the baseline state of aged organoids is inherently more vulnerable,\nleading to a quantitatively distinct and more severe pathological outcome. As\nsystematically compared in revised Figure 5e, aged organoids exhibited a higher\nproportion to form cystic structures following Th17 co-culture and showed a reduced\ncapacity to revert to solid morphology upon IL-17 NAb, compared to young\norganoids. Quantitative analyses in Figure 5j further confirmed that the Th17-induced\nreductions in neuronal markers (Tuj1+/OMP+) and increases in Krt19+ cell abundance\nwere significantly more pronounced in aged organoids. Furthermore, the baseline P16\nexpression levels were substantially higher in aged organoids, indicating an inherently\nheightened senescence-associated state that may underlie their increased susceptibility.\nRepresentativeimage are provided in ResponseFigure 3.\nIn light of these findings, we have revised the statement in lines 608–609 to more\naccuratelyreflect thedata (page 36,lines768-771 in revised manuscript):\n\"Notably, IL-17a neutralization in aged organoids effectively restored neuronal\ndifferentiation and suppressed Krt19⁺ metaplasia, rescuing them from a more severely\ncompromised baseline state and a heightened susceptibility to Th17-induced\ndisruption.\"\n3. An additional concern is related to the compound Y320. The authors justify\nthe use of this compound as a selective tool to reduce IL17 secretion by T cells,\nbuttheavailable literature indicates amuch broadermodeof action ofthis drug.\nResponse：\nWe sincerely thank the reviewer for raising this important concern regarding the\npotential multi-target nature of Y320. We fully acknowledge that, beyond modulating\nIL-17a, Y320 has been reported to exhibit a broader biological activities, including\ninhibition of IFN-γ and TNF-α, regulation of Th17/Treg balance, enhancement of\ntranslational readthrough for certain genes, and sensitization of multidrug-resistant\ntumors in combined with chemotherapeutic agents 24,25. Nevertheless, Y320 has been\nsuccessfully employed as an IL-17A inhibitor in the context of anti-inflammatory\ntherapy across multiple studies, including those related to skin inflammation and\nchronic wound healing 26,27.\nIn our study, Y320 was selected based on previous reports demonstrating its efficacy\nin suppressing Th17 cell differentiation and IL-17 production 27, 28. Consistent with\nthese findings, we observed that application of Y320 effectively improved the\ncapacity ofolfactory regenerative(revised Figure 3-4,Figure S2-3).\nHowever, in light of the reviewer’s valid concern, we sought to rigorously confirm\nthat the observed regenerative benefits were specifically attributable to IL-17a\nsignalinginhibition.\nIn our original manuscript, we employed a genetic approach using IL-17a conditional\nknockout (CKO) mice. Results showed that conditional deletion of IL-17a similarly\npromoted functional recovery after olfactory injury, closely mirroring the effects of\nY320 treatment (revised Figure 6-7, Figure S5-7). Furthermore, to strengthen the\nrobustness of our conclusions, we performed additional experiments using an IL-17a\nneutralizing antibody, which also yielded consistent improvements in olfactory\nepithelial regeneration (revised Figure 4e-n).\nThe convergence of the results across three distinct intervention strategies strongly\nsuggested that the regenerative benefits were specifically mediated through IL-17a\npathway inhibition,thereby substantially reducing thepossibility that off-target effects\nofY320 account for theobserved phenotype.\n4. In Figure 1, why were the data not analysed as one dataset? It appears that\nthe young and old datasets areanalysed separately.This is in contrast to the data\nshown in Figure 2, in which the data are analysed as one pooled cluster of cells.\nTheapproachin Figure2appears to bethe rightone.\nResponse:\nWe sincerely thank the reviewer for this thoughtful comment regarding data\nintegration and visualization. We would like to clarify that in our analysis, the young\nand aged datasets were in fact integrated into a unified dataset following batch effect\ncorrection. In Figure 1, the visual separation was intentionally achieved using the\nparameter split.by = \"group\" within the same UMAPembedding, in order to facilitate\nadirect side-by-sidecomparison ofthecellular landscapes between thetwo groups.\nIn response to the reviewer’s comment, we have further improved the figure in the\nrevised manuscript by including both the integrated UMAP plot alongside the\nsplit-view UMAPs (revised Figure 1a, b; page 7, line 121). This addition offers a\ncomprehensive visualization of both the over structure and group-specific\ndistributions, ensuring clarity and alignment with the analytical approach used in\nFigure 2.\nMinorcomments\n1. Page 4 the sentence “Due….condition” (line 50-52) is missing a reference.\nHowcan lossof smell befatal?\nResponse:\nWe thank the reviewer for pointing out this omission. The statement is indeed crucial\nand requires a citation for support. The fatal consequence of smell loss is primarily\nrelated to the inability to detect environmental hazards, such as gas leaks, smoke from\nfires, or spoiled food, which poses a particularly life-threatening for older individuals\nliving alone. Researches also showed a correlation between objective olfactory\nimpairment and increased mortality in older adults. Beyond these immediate safety\nrisks, olfactory loss also affects quality of life, nutrition intake, and may serve as an\nearly sign of neurodegenerative disorder such asAlzheimer’s and Parkinson’s disease\n29,30,31.Wehave nowadded thereferences tosupport this statement:\nThe sentence has now been revised as follows (page 4, lines 55-59 in revised\nmanuscript),:\n“Due to accelerated population aging, the incidence of olfactory dysfunction is rising\nannually, affecting up to 50% of the elderly. This condition significantly impairs\nquality of life and can even become life-threatening, particularly in older adults, due\nto the loss of ability to detect warning odors such as gas leaks, smoke, or spoiled\nfood”.\nWe appreciate the reviewer’s suggestion, which has helped enhance the accuracy and\nscholarly rigor ofourmanuscript.\n2. The 3rd paragraph of the introduction contains many abbreviations. An\nexample is the sentence “When…regeneration” (line 71-74). To the broader\nscientific readership this will be very difficult to read and it is recommended that\ntheseabbreviations arewritten infull to avoidconfusion.\nResponse:\nWe sincerely thank the reviewer for this thoughtful comment. We have now revised\nthe third paragraph by spelling out all abbreviations upon their first use and\nminimizingtheirsubsequent use (page 5,lines 72-81in revised manuscript).\n2. Introduction last paragraph. Suggest to write as hypothesis/aim rather than a\nsummary of findings.\nResponse:\nWe thank the reviewer for the constructive suggestion. Accordingly, we have now\nrevised the paragraph to clearly state our central hypothesis and subsequent research\nobjectives (page 6,lines 101-110inthe revised manuscript).\n3. Out of curiosity, did the authors also analyse olfactory receptor mRNA\nexpressionas afunctionof ageandIL17 interventions?\nResponse:\nWethankthe reviewer for their insightful suggestion regarding olfactory receptor (OR)\nexpression. In response, we would like to note that the effect of aging on OR\nexpression has been previously characterized in our published work 22 (Response\nFigure 20a). Building on this foundation and using the same analytical approach, we\nhave now extended this investigation to the four groups of CKO and Flox mice in this\nstudy. Given that the OE was still undergoing repair at the 7 and 14 dpi, we didn’t\nobserved specific or significant differences in OR expression across groups.\nNevertheless, our analysis consistently indicated a slight but reproducible\nupregulation of OR expression in the CKO group relative to the Flox controls\n(ResponseFigure20b).\nThis finding suggests that the absence of the IL-17a signaling pathway has a mildly\nprotective effect on OR expression. It further supports the concept that IL-17a not\nonly affects tissue regeneration but also directly impairs the functional maturation or\nspecialization ofolfactory neurons.\nResponseFigure20\n5.Figure3G and3I:werethere any changesin organoidsizeormorohology?\nResponse：\nWe sincerely thank the reviewer for this insightful comment. Since no organoid data\nwas presented in Figure 3, we believe the reviewer may be referring to Figure 4G and\n4I.\nIn our study,organoid morphology and sizewere systematically monitored throughout\nthe culture period. For example, morphological and size comparisons between young\nand aged OE organoids were conducted on days 1, 3, 7, and 14, as shown in original\nand revised Figure S3b-d. Under co-culture conditions, consistent morphological\nchanges were observed in both young and aged organoids. However, these results\nwere not included in theoriginal manuscript dueto space limitations.\nIn response to Reviewer #1’s suggestion, we modified the culture conditions in the\nrevised manuscript. The original Figure 4G has been updated and is now presented as\nrevised Figures 5d-k and S3h-k (refer to Response Figure 3). Specifically, we\nperformed co-culture experiments of organoids with Th17 cells and incorporated\nneutralizingantibodies into theco-culture system.Theseexperiments revealed notable\nalterations in organoid morphology and size: control organoids largely maintained\nsolid structures, with young organoids generally larger than aged ones. Upon Th17\nco-culture, organoids underwent a structural transition from solid to cyst-like forms,\naccompanied by a significant increase in size. These effects were attenuated by the\naddition of neutralizing antibodies. Corresponding quantitative analyses of\nmorphology and size are now included in Figure 5d-e and S3h-i of the revised\nmanuscript.\nFurthermore, to compare the morphology of organoids derived from OE and\nrespiratory epithelium (RE), we also cultured RE organoids, which exhibited a\npredominantly cystic morphology. This finding supports our interpretation that the\nTh17 cells induce a metaplasia change of OE organoids and suppress neuronal\ngeneration (revised Figure S3j).\nRegarding to original Figure 4I, organoid sizewas quantified under control conditions,\nIL-17a treatment, and IL-17a with neutralizing antibody (NAb) conditions in the\noriginal study. Due to space constraints, these data were not initially included. The\norganoids size measurements are presented the Response Panel below. Also in\nresponse to Reviewer #1’s comment, Figure 4I has been revised and is now presented\nas revised Figure S4a-f (refer to Response Figure 4). We treated organoids with\ngraded concentrations of IL-17a and observed clear dose-dependent effects on both\norganoid morphology and size. Representative data shown in Response Figure 21\nbelow.\nResponseFigure21\nReferences\n1. Sola P, et al. Targeting lymphoid-derived IL-17 signaling to delay skin aging. Nat Aging 3,\n688-704(2023).\n2. Wang X, et al. Candida albicans Promotes Oral Cancer via IL-17A/IL-17RA-Macrophage Axis.\nmBio14,e0044723(2023).\n3. YangWL,etal.Nsun2couplingwithRoRgammatshapesthefateofTh17cellsandpromotes\ncolitis.NatCommun14,863(2023).\n4. Lyakh L, Trinchieri G, Provezza L, Carra G, Gerosa F. Regulation of\ninterleukin-12/interleukin-23 production and the T-helper 17 response in humans. Immunol\nRev226,112-131(2008).\n5. Li Q, et al. Age-associated alteration in Th17 cell response is related to endothelial cell\nsenescenceandatheroscleroticcerebralinfarction.AmJTranslRes9,5160-5168(2017).\n6. Merino KM, Jazwinski SM, Rout N. 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Olfactory Dysfunction Mediates Adiposity in Cognitive Impairment of Type 2\nDiabetes: Insights From Clinical and Functional Neuroimaging Studies. Diabetes Care 42,\n1274-1283(2019).\n20. Simoni Y, et al. Human Innate Lymphoid Cell Subsets Possess Tissue-Type Based\nHeterogeneityinPhenotypeandFrequency.Immunity46,148-161(2017).\n21. Colonna M. Innate Lymphoid Cells: Diversity, Plasticity, and Unique Functions in Immunity.\nImmunity48,1104-1117(2018).\n22. LiW,etal.Asingle-celltranscriptomiccensusofmammalianolfactoryepitheliumaging.Dev\nCell59,3043-3058e3048(2024).\n23. Wu T, et al. Deficiency of Aging-Related Gene Chitinase-Like 4 Impairs Olfactory Epithelium\nHomeostasis.CellProlif58,e70055(2025).\n24. Hong J, et al. Y-320, a novel immune-modulator, sensitizes multidrug-resistant tumors to\nchemotherapy.AmJTranslRes12,551-562(2020).\n25. Feng Z, et al. Loss of A20 in BM-MSCs regulates the Th17/Treg balance in Rheumatoid\nArthritis.SciRep8,427(2018).\n26. Zhang J, et al. IL-17 Promotes Scar Formation by Inducing Macrophage Infiltration. Am J\nPathol188,1693-1702(2018).\n27. Hadian Y, Bagood MD, Dahle SE, Sood A, Isseroff RR. Interleukin-17: Potential Target for\nChronicWounds.MediatorsInflamm2019,1297675(2019).\n28. Ushio H, et al. A new phenylpyrazoleanilide, y-320, inhibits interleukin 17 production and\nameliorates collagen-induced arthritis in mice and cynomolgus monkeys. Pharmaceuticals\n(Basel)7,1-17(2013).\n29. SchaferL, Schriever VA,CroyI.Human olfactorydysfunction:causesandconsequences. Cell\nTissueRes383,569-579(2021).\n30. Masurkar AV, Devanand DP. Olfactory Dysfunction in the Elderly: Basic Circuitry and\nAlterationswithNormalAgingandAlzheimer'sDisease.CurrGeriatrRep3,91-100(2014).\n31. Choi JS, Jang SS, Kim J, Hur K, Ference E, Wrobel B. Association Between Olfactory\nDysfunctionandMortalityinUSAdults.JAMAOtolaryngolHeadNeckSurg147,49-55(2021)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the previous concerns, and the manuscript has been significantly improved.", "reviewer_2": "The authors have addressed most of my concerns.", "reviewer_3": "The authors have provided excellent responses to the questions that I raised. I have no further comments to consider."}, "author_response": ""}]

Decision letter