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

Shenglei Wang; Xudong Cha; Yingqi Xie; Zengyi Xu; Boyu Cai; Fengzhen Li; Tengfei Li; Zhe Wang; Tianyu Wang; Yiqun Yu; Huanhai Liu; Wenwen Ren

doi:10.1038/s41467-025-67786-2

. 2025 Dec 29;17:1036. doi: 10.1038/s41467-025-67786-2

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

Shenglei Wang ^1,^#, Xudong Cha ^1,^#, Yingqi Xie ^1,^#, Zengyi Xu ^1,^#, Boyu Cai ^1,², Fengzhen Li ¹, Tengfei Li ¹, Zhe Wang ¹, Tianyu Wang ¹, Yiqun Yu ^3,^4,^✉, Huanhai Liu ^1,^✉, Wenwen Ren ^1,^✉

PMCID: PMC12847769 PMID: 41461651

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.

Subject terms: Olfactory system, Regeneration, Mucosal immunology, Respiratory tract diseases, Interleukins

The olfactory epithelium (OE) undergoes changes during aging and this may affect inflammatory responses and immune cell infiltration. Here the authors look at the effect of aging on OE and how this involves cross talk between T cells and horizontal basal cells involving Il17a expression in OE which is increased in aging and inhibition of IL-17a function reduces respiratory metaplasia.

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 food^2–4.

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 regeneration^9,10. 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 repair^12,13. 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 organs^14,15. 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. Featureplots 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).

Fig. 1 — a UMAP visualization of integrated scRNA-seq data from young and aged mice, colored by five major OE cell types and T cells. b UMAP plots split by samples, comparing cellular composition between young and aged mice. c Compositional analysis showing proportional abundance of different cell types in young versus aged OE. d Feature plots displaying expression patterns of canonical cell type markers. Color scale indicates expression levels from low (gray) to high (red/green). e Representative confocal images of OE tissues immunostained for key cell type markers. f Quantification of marker-positive cells for each cell type in young and aged mice (n = 9 biologically independent samples). g Dot plot showing increased ligand-receptor interactions between T cells and OE cell types in aged mice. h Immunofluorescence staining of MIF, Icam1, CD74 and CD44 in OE tissues from young and aged mice. i Quantitative analysis of MIF⁺, CD74⁺ and CD44⁺ cells in OE tissues from young and aged mice (n = 6 for MIF, n = 12 for CD74 and CD44, biologically independent samples). j Violin plots demonstrating the expression levels of *Mif*, *Cd74* and *Cd44* across different OE cell types. k Schematic diagram depicting different regions of mouse nasal cavity (left) and corresponding HE staining of the OE tissue section (right). l Confocal images of IL-17ra, Tuj1 and Icam1 in different regions of young and aged mice. Solid and outlined arrows represent IL-17ra⁺ cells co-localized with HBC and neurons, respectively. m Quantification of IL-17ra⁺ cells in neuronal layer (NL) and basal layer (BL) across different OE regions in young and aged mice (n = 6 for dorsal and medial regions, n = 4 for RE, biological independent samples). Data are presented as mean ± SD (f, i). Statistical comparisons are performed using unpaired two-sided t-test (f, i). Confocal microscopy images are representative of at least three biologically independent experiments with consistent results. All nuclei are counterstained with Hoechst. Scale bars, 50 μm. Source data are provided as a Source Data file.

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 OE^21,22.

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, NK T, 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. Featureplots 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).

Fig. 2 — a UMAP visualization of scRNA-seq data, colored by annotated T cell subtypes. b Compositional bar plot showing the proportional abundance of each T cell subtype. c Feature plots demonstrating expression patterns of canonical markers of representative cell types. d Dot plot showing the expression levels of key marker genes across T cell subtypes. Dot size reflects the percentage of cells expressing the gene; color intensity indicates average expression level. e List of marker genes used to identify CD4⁺ Th cell subsets. f Violin plots showing expression patterns of representative marker genes across CD4⁺ Th cell subsets. g Differentiation index scores for CD4⁺ Th cell subsets, calculated based on marker genes listed in (e). The horizontal solid lines represent the median, and the horizontal dashed lines represent the first/third quartile. h Proportion of IL-17a-expressing cells within each T cell subset. i Concentrations of IL-17a and IL-23 in plasma and OE tissues from young and aged mice (n = 3 biologically independent experiments). j Western blot analysis of IL-17a protein expression in OE tissues from young and aged mice. k qPCR analysis showing *Il17a* mRNA expression in OE tissues of young and aged mice (n = 5 biologically independent experiments). l Violin plot showing *Il17a* expression levels in OE tissues from young versus aged mice based on scRNA-seq data. m Confocal images of OE tissue sections immunostained for CD3, CD4 and IL-17a in young and aged mice. n Quantification of IL-17a⁺ cells from scRNA-seq data in young and aged OE samples. o Correlation analysis between *Il17a* mRNA expression level and time taken to locate hidden cookies in young and aged mice (n = 14 individual mice). The association was evaluated by simple linear regression. p Olfactory behavioral test measuring the time required to locate cookies in young and aged mice (n = 20 biologically independent samples). Data are presented as mean ± SD (g, i, k, l, p). Statistical analyses are performed using the Wilcoxon test (g, l) or an unpaired two-sided t-test (i, k, p). Confocal microscopy images are representative of at least three biologically independent experiments with consistent results. All nuclei are counterstained with Hoechst. Scale bars, 50 μm. Source data are provided as a Source Data file.

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).

Fig. 3 — a Representative confocal images of OE tissues at indicated time points after methimazole-induced injury (1, 2, 3, and 7 dpi), immunostained for CD45, IL-17a-EGFP, OMP and Tuj1. b Quantification of positive cell numbers for markers shown in (a) (n = 6 biologically independent samples). c Experimental scheme of Y-320 administration in young and aged mice. Created in BioRender. Shenglei, W. (2025) https://BioRender.com/o9l4wkk. d, e Olfactory behavioral test measuring time required to locate cookies before (d) and after (e) methimazole-induced injury in young and aged mice treated with Y-320 or vehicle control (n = 9 biologically independent samples). f ELISA measuring the concentration of IL-17a in plasma of aged mice, with or without Y-320 treatment (n = 3 biologically independent replicates). g Quantification of IL-17a⁺/CD45⁺ double-positive cells in OE tissues (n = 9 biologically independent samples). h Representative images of double immunostaining for IL-17a and CD45 in OE of young and aged mice at 14 dpi with or without Y-320 treatment. (i) Measurement of OE thickness in young and aged mice with or without Y-320 treatment (n = 9 biologically independent samples). j Representative confocal images of dorsal OE regions in young and aged mice at 7 and 14 dpi, stained for Tuj1, OMP and Gap43. k–m Statistical analysis of Tuj1⁺ (k), Gap43⁺ (l), and OMP⁺ (m) cells in OE under indicated conditions (n = 9 biologically independent samples). Data are presented as mean ± SD (d–g, i, k–m). Statistical analyses are performed using one-way ANOVA with Sidak’s multiple comparison test (d, g, i, k, l, m), two-way ANOVA with Sidak’s multiple comparison test (e), or an unpaired two-sided t-test (f). Confocal microscopy images are representative of at least three biologically independent experiments with consistent results. All nuclei are counterstained with Hoechst. Scale bars, 50 μm. Source data are provided in the Source Data file.

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 timepoints²⁸. 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 time frame, 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-17a reverses the age-related respiratory metaplasia in the OE

It has been reported that OE damage can result in respiratory metaplasia and subsequent olfactory dysfunction^30,31. 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.

Fig. 4 — a Representative confocal images of dorsal, medial, and patch regions of OE tissues from young and aged mice under control conditions or at 14 dpi with Met-treatment, administered with IL-17a inhibitor (Y-320) or vehicle (DMSO). Sections were immunostained for Krt19, Tuj1, and Icam1. b Schematic representation (left) and corresponding immunostaining results (right) of OE tissue architecture. Green indicates Krt19⁺ area, red denotes Tuj1⁺ area, and boxed regions highlight metaplastic patches in both young and aged OE. c Quantification of Krt19⁺ metaplasia patch length in OE tissues (n = 9 biologically independent samples). d Statistical analysis of metaplastic and recovered area proportions in young versus aged OE tissues (n = 3 biologically independent samples). e Experimental timeline of IL-17a neutralizing antibody (NAb) treatment in aged mice. Created in BioRender. Shenglei, W. (2025) https://BioRender.com/o9l4wkk. f Olfactory behavioral test showing time required for aged mice to locate cookies after Met-induced injury, following treatment with two concentrations of IL-17a NAb (high and low) (n = 3 biologically independent samples). g, h Immunostaining of Krt19, Tuj1 and Icam1 (g), as well as CD74 and CD44 (h) in OE from aged mice, pre-treated with low or high doses of IL-17a NAb before methimazole injury. i Quantification of Krt19⁺ metaplasia patch length under different treatment conditions (n = 6 biologically independent samples). j, k Quantification of Tuj1⁺ (j), CD74⁺ and CD44⁺ (k) cells in aged OE under different treatment conditions (n = 6 for j, and n = 5 for k, biologically independent samples). l Spatial distribution analysis of CD74⁺ and CD44⁺ cells across neuronal layer (NE), basal layer (BL), and lamina propria (LP) in control, low- and high-dose IL-17a NAb conditions (n = 6 for CD74, and n = 5 for CD44, biologically independent samples). Data are presented as mean ± SD (c, f, i, j, k). Statistical analyses are performed using one-way ANOVA with Sidak’s multiple comparison test (c, i, j, k) or two-way ANOVA with Sidak’s multiple comparison test (f). Confocal microscopy images are representative of at least three biologically independent experiments with consistent results. All nuclei are counterstained with Hoechst. Scale bars, 50 μm. Source data are provided in the Source Data file.

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 overall 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. S3 j, 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 (Il17a^flox/flox) in T cells using Lck-Cre driver (named CKO thereafter). Littermate Il17a^flox/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 timepoints, suggesting the most profound changes in CKO mice (Fig. S6a). Consequently, we reclassified 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 timepoints (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*2^41,45. 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 HBCs 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). Monocle2 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 dotplots 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). Immunostaining 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.

Discussion

Chronic inflammation is a defining feature of the aging process, characterized by alterations in T-cell populations, immunodeficiency, and an accumulation of pathogens. This leads to the creation of an immune-cell-induced pro-inflammatory environment within the aging organism, which impairs stem cell renewal and differentiation⁴⁷. Several targeted immunotherapies have been found to decrease the accumulation of senescence-associated secretory phenotype (SASP) factors and senescent cells in mice, thereby improving the pro-inflammatory environment in peripheral organs⁴⁸. As a signature cytokine produced by Th17 cells, IL-17a plays a vital role in tissue inflammation, autoimmunity, and host defense, contributing to both acute and chronic inflammation responses⁴⁹. Alterations in T cells and IL-17a expression have been found to occur during skin aging and aging-induced osteoarthritis. Blocking IL-17 signaling has been shown to delay the process of skin aging and promote skin healing¹⁵.

As shown in Supplementary Fig. 9, this study systematically investigated the impact of the pro-inflammatory cytokine IL-17a on OE aging. We identified IL-17a as a critical driver of age-related olfactory dysfunction. In aged mice, inflammatory accumulation disrupts OE homeostasis, a process to which CD4⁺ Th cell-derived IL-17a contributes pivotally. This phenotype, marked by CD4⁺ T cell infiltration, accumulation of CD44⁺ and CD74⁺ immune cells, and heightened IL-17ra signaling in basal cells, collectively drives the breakdown of OE homeostasis, culminating in neuronal depletion and respiratory metaplasia. In both young and aged mice, inhibiting IL-17a secretion via Y-320 effectively alleviated Met injury-induced OE damage, respiratory epithelial metaplasia, and olfactory dysfunction. However, under identical injury conditions, aged mice exhibited more severe OE damage and a markedly slower repair process compared to young mice, underscoring the heightened therapeutic relevance of IL-17a blockade in aged individuals. Moreover, considering the complex action of Y-320, we performed additional intervention experiments in aged mice using an IL-17a neutralizing antibody. The results demonstrated that the antibody promoted OE recovery in a dose-dependent manner and reduced inflammatory infiltration into basal cells, confirming the pivotal role of IL-17a. Our work thus demonstrated that both Y-320 and IL-17a neutralization constitute viable strategies to mitigate OE degeneration, revealing a promising therapeutic window for clinical intervention in age-related olfactory dysfunction.

Furthermore, we validated this conclusion using in vitro cultured olfactory organoids, which can mimic the regeneration and possess various cell types of OE tissue. Since OE organoids are derived from epithelial stem cells, they cannot fully recapitulate the immune microenvironment in vivo. To address this limitation, we established a co-cultured system employing either primary spleen-derived Th17 cells or the recombinant IL-17a. This multi-faceted approach verified the inhibitory effect of IL-17a on the regulation of basal cell differentiation. Notably, IL-17a neutralization in aged organoids effectively restored neuronal differentiation and suppressed Krt19⁺ metaplasia, rescuing them from a more severely compromised baseline state and a heightened susceptibility to Th17-induced disruption. This in vitro organoids-immune cell co-culturing system can mimic immune responses and disease conditions in vivo, enabling the study of the mechanisms underlying the interaction between immune cells and tissues⁵⁰. For instance, using air-liquid interface (ALI) culture methods, an esophagitis model has been established in esophageal organoids⁵¹. Through co-culturing intestinal organoids with immune cells, it has been discovered that an IL-22 inflammatory environment can promote epithelial regeneration, innate defense, and mucus production⁵². This innovative technique of co-culturing olfactory organoids with immune cells demonstrated its feasibility and expanded the research direction of studying the interaction between OE regeneration and immune cells. It will provide broader approaches for the application of olfactory organoids in diseases in the future.

By generating T cell-specific Il17a conditional knockout mice and performing tissue validation at different time points post injury, we confirmed the crucial role of IL-17a in OE damage and repair. The regenerative process of OE proceeded more rapidly in CKO mice relative to the Flox control group. Single-cell RNA sequencing analysis revealed the most prominent transcriptional differences in HBC between genotypes. Specifically, Il17a ablation altered the distribution of HBC subclusters, reduced immune cell accumulation within HBC niches, and accelerated HBC differentiation activation via signaling pathways involving Cd6 and Laminin.

Moreover, we detected early signs of an IL-17a-associated inflammatory microenvironment in middle-aged mice, at a stage when olfactory function remained largely intact. This finding indicated that IL-17a accumulation may drive the progressive nature of age-related OE degeneration. Supporting this, organoids derived from CKO and Flox middle-aged mice showed that IL-17a directly suppressed basal cell differentiation into olfactory neurons while promoting aberrant Krt19⁺ respiratory epithelial cells. Together, these data revealed that IL-17a-driven pathology begins early and actively shapes the trajectory of olfactory aging, highlighting a precocious window for therapeutic intervention.

Previous studies reported that Th17 cells and their cytokine IL-17a reduce the number of intestinal stem cells through the transcription factor Atoh1 in Lgr5⁺ populations³⁷. The interplay between the local immune system and olfactory HBC is pivotal for regulating endogenous epithelial regeneration. Imbalances in this interaction may lead to tissue damage, inflammation, and functional disorders. Anti-inflammatory treatments and the regulation of inflammatory factors can be employed to improve the regenerative capacity of the OE. Our findings further confirmed the essential role of IL-17a in regulating the interplay between inflammatory cells and HBC within the olfactory system, and provided a potential therapeutic target for treating age-related olfactory dysfunction.

Methods

Animals

C57BL/6 JCya-Tg (Lck-Cre), C57BL/6JCya-Il17a^em1flox, C57BL/6JCya-Il17a^tm2(EGFP) mice were purchased from Cyagen Biologics (Suzhou, China). Il17a^flox/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 Lck^Cre/Il17a ^flox/flox mice were intraperitoneally injected with methimazole (100 μg/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 mouse^15,32,53. 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 24 h 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 Master Mix (#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, ThermoFisher) 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, Hoechst33342 (#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^TM 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^TM 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 Il17a^flox/flox and Lck^Cre/Il17a^flox/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 (75U/mL, #HY-108882, MedChemExpress), Hyaluronidase (250U/mL, #H3506, Sigma), and Collagenase type Ⅰ (100U/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.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information^{(2.5MB, pdf)}

41467_2025_67786_MOESM2_ESM.pdf^{(43.6KB, pdf)}

Description of Additional Supplementary Files

Supplementary Data 1^{(9.9KB, xlsx)}

Supplementary Data 2^{(10.1KB, xlsx)}

Supplementary Data 3^{(9.4KB, xlsx)}

Supplementary Data 4^{(260.8KB, xlsx)}

Supplementary Data 5^{(15.3KB, xlsx)}

Supplementary Data 6^{(24.4KB, xlsx)}

Reporting Summary^{(106.9KB, pdf)}

Transparent Peer Review file^{(5.1MB, pdf)}

Source data

Source Data^{(2.4MB, xlsx)}

Acknowledgements

This work was supported by National Natural Science Foundation of China Grants (82271136 to W.R., U23A20439 to H.L., 82571283 and 32271044 to Y.Y.); Science and Technology Commission of Shanghai Municipality (23ZR1409600 to Y.Y.); Shanghai Municipal Education Commission, the Shanghai Eastern Scholar Program (GZ2022006 to Y.Y.); Shanghai Municipal Health Commission (GWVI-11.2-XD09 to Y.Y.). We thank the staff members of the Integrated Laser Microscopy System at the National Facility for Protein Science in Shanghai (NFPS), Zhangjiang Lab, China, for providing technical support and assistance in data collection and analysis.

Author contributions

W.R., Y.Y., and H.L. conceived and designed the study; S.W., X.C., and Y.X. conducted the experiments; X.C. and Z.X. analyzed the data; B.C. and F.L. contributed to data interpretation; T.L.,Z.W.,and T.W.performed the statistical analysis and W.R.wrote the manuscript.

Peer review

Peer review information

Nature Communications thanks Yusuke Endo, Reinoud Gosens and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

All sequencing data generated in this study have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2025) in the National Genomics Data Center (Nucleic Acids Res 2025), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences. The single-cell RNA sequencing data are available under accession code GSA: CRA033248. The bulk RNA sequencing data are available under accession code GSA: CRA033677. All datasets are publicly accessible as of the date of publication at https://ngdc.cncb.ac.cn/gsa. All other data are available in the article and its Supplementary files or from the corresponding author upon request. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Shenglei Wang, Xudong Cha, Yingqi Xie, Zengyi Xu.

Contributor Information

Yiqun Yu, Email: yu_yiqun@fudan.edu.cn.

Huanhai Liu, Email: liuhuanhaiok@smmu.edu.cn.

Wenwen Ren, Email: wenwenren@smmu.edu.cn.

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-025-67786-2.

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Associated Data

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Description of Additional Supplementary Files

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Supplementary Data 2^{(10.1KB, xlsx)}

Supplementary Data 3^{(9.4KB, xlsx)}

Supplementary Data 4^{(260.8KB, xlsx)}

Supplementary Data 5^{(15.3KB, xlsx)}

Supplementary Data 6^{(24.4KB, xlsx)}

Reporting Summary^{(106.9KB, pdf)}

Transparent Peer Review file^{(5.1MB, pdf)}

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Data Availability Statement

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PERMALINK

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

Shenglei Wang

Xudong Cha

Yingqi Xie

Zengyi Xu

Boyu Cai

Fengzhen Li

Tengfei Li

Zhe Wang

Tianyu Wang

Yiqun Yu

Huanhai Liu

Wenwen Ren

Abstract

Introduction

Results

Loss of olfactory sensory neurons in aged mice is accompanied by changes in the immune microenvironment

Fig. 1. Characterization of olfactory epithelial cell types and T cell interactions in young and aged mice.

Elevated expression of IL-17a in the aged OE is related to olfactory function

Fig. 2. The expression of IL-17a is elevated in aged mice.

Administration of IL-17 inhibitor Y-320 promotes OE regeneration

Fig. 3. Inhibition of IL-17a promotes regeneration of olfactory epithelium in young and aged mice.

Inhibition of IL-17a reverses the age-related respiratory metaplasia in the OE

Fig. 4. Inhibition of IL-17a reverses methimazole-induced respiratory metaplasia in olfactory epithelium.

IL-17a neutralization counteracts Th17-induced metaplasia to restore neuronal integrity in olfactory organoids

Fig. 5. IL-17a neutralization mitigates Th17 cell-induced phenotypic alterations in olfactory organoids.

IL-17a directly alters olfactory epithelial lineage specification and triggers an inflammatory transcriptional program in organoids

Conditional knockout of Il17a facilitates the regeneration of OE

Fig. 6. Characterization of cell types and olfactory function in Il17a CKO mice.

Conditional knockout of Il17a accelerates the recruitment of horizontal basal cells

Fig. 7. Il17a conditional knockout accelerates the recruitment of horizontal basal cells.

IL-17a-associated inflammatory microenvironment emerges in the OE during middle-age

Discussion

Methods

Animals

Olfactory epithelium lesion

Y-320 treatment

Neutralization treatment in aged mice

Buried food test

Olfactory habituation-dishabituation test

Olfactory threshold test

RT-qPCR analysis

Olfactory organoid culture

Respiratory organoids culture

Preparation of single cells from mouse spleen and Th17 cell culture

Construction of olfactory organoids co-culture with Th17 cells

Flow cytometry

ELISA analysis

Immunostaining

In situ RNAscope hybridization

Bulk RNA sequencing for organoids

Single-cell RNA sequencing

Sample preparation

scRNA-Seq raw data processing

Cell type identification

scRNA-Seq analysis

Statistical analysis

Reporting summary

Supplementary information

Source data

Acknowledgements

Author contributions

Peer review

Peer review information

Data availability

Competing interests

Footnotes

Contributor Information

Supplementary information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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