Cystic fibrosis alters the structure of the olfactory epithelium and the expression of olfactory receptors affecting odor perception

Abstract

A reduced sense of smell is a common condition in people with cystic fibrosis (CF) that negatively affects their quality of life. While often attributed to nasal mucosa inflammation, the underlying causes of the olfactory loss remain unknown. Here, we characterized gene expression in olfactory epithelium cells from patients with CF using single-nuclei RNA sequencing and found altered expression of olfactory receptors (ORs) and genes related to progenitor cell proliferation. We confirmed these findings in newborn, inflammation-free samples of a CF animal model and further identified ultrastructural alterations in the olfactory epithelium and bulbs of these animals. We established that CFTR, the anion channel whose dysfunction causes CF, is dispensable for odor-evoked signaling in sensory neurons, yet CF animals displayed defective odor-guided behaviors consistent with the morphological and molecular alterations. Our study highlights CF's major role in modulating epithelial structure and OR expression, shedding light on the mechanisms contributing to olfactory loss in CF.

Fig. 1.. CF individuals show smell loss and altered gene expression in olfactory cells.

(A) Schematic representation illustrating olfactory testing with the Sniffin’ Sticks test and collection of olfactory epithelium cells by nasal brushing from CF and healthy donors. (B) Scores of the SNOT-22 test (left) and different parameters of the Sniffin’ Sticks test battery showing subjective smelling ability, threshold, identification scores, and the threshold/identification (TI) index (right). Blue-filled circles indicate ETI-treated individuals. *P < 0.05; ns, not significant (Student’s t test); n = 10 CF and 3 controls; n = 10 CF and 17 controls for SNOT-22 test. (C) UMAP dimensionality reduction plot of gene expression in 26,493 integrated CF and control cell nuclei (n = 7 CF and 9 control individuals). (D) UMAP depicting expression of CFTR (top) and violin plots of CFTR normalized expression in the different cell types (bottom). (E) Percentage of each cell type that express CFTR. (F) Volcano plot and heatmap of the 25 most significantly DE genes (up- and down-regulated) in the GBC cell cluster. CF samples correspond to patients not treated with ETI. Two of the control samples contained no GBCs and are not displayed in the analysis. (G) Average expression of the 16 most expressed ORs in the OSN cluster of the CF sample. pDCs, plasmacytoid dendritic cells.

Fig. 2.. CFTR is expressed in proliferating human olfactory cells.

(A) UMAP projection of the computationally assigned cell cycle scores S (left) and G₂M (right). (B) Phase scoring shows an elevated G₂M score in the GBC cluster. (C) UMAP projection of the G₂M, G₁, and S scores (left) and CFTR expression (right) in the GBC cell cluster. (D) CFTR is significantly more expressed in the G₁-S phase in the GBC cluster, and G₂M score is significantly higher in the CF group; **P < 0.005 and ***P < 0.001 (Mann-Whitney test); n = 41 G₁-S and 26 G₂M that express CFTR; n = 214 CF and 101 control. (E) Olfactory cell culture after nasal brushing from healthy donors showing CFTR (green) RNA coexpression with DCX, PCNA, and Ki67 antibodies (magenta) visualized by dual in situ hybridization and immunostaining. Scale bars, 20 μM. (F) Effect of the CFTR inhibitor CFTR_inh172 on PCNA and Ki67 immunolabeling showing an increase on the percentage of positive cells. Gaussian probability density functions show a shift toward an increased PCNA and Ki67 fluorescence intensity in CFTR_inh172-treated cells. Vertical dashed line indicates the background threshold. n = 1298 (mock), 890 (10 μM), 748 (20 μM), and 885 cells (30 μM); P < 0.001 (Kolmogorov-Smirnov test) in all comparisons. a.u., arbitrary unit; NK, natural killer; DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 3.. Newborn CFTR^−/− piglets display deficits in an olfactory-driven behavior.

(A) Litter containing newborn piglets of all three genotypes: CFTR^+/+, CFTR^+/−, and CFTR^−/−. (B) Latency-to-suckle in CFTR^−/− piglets (179.7 min ± 33.9 SEM; n = 17) and CFTR^+/+ (73.9 min ± 18.6 SEM; n = 15) (**P < 0.01, Mann-Whitney U test). (C) Percentage of pigs suckling and their latency to locate the nipple over the 400-min assay period (***P < 0.001, Kolmogorov-Smirnov test). (D) Data from (C) binned in 25-min windows [did not suckle (DNS)]. A total of 23.6% (4 of 17) of CFTR mutants fail to locate the nipple after 400 min. (E) Birth weights (P = 0.26, Mann-Whitney U test). (F) Immunolabeling for OMP in freshly dissociated pig olfactory epithelium cells (28.9% CFTR^+/+ and 26.9% CFTR^−/−; n = 1932 CFTR^+/+ cells and 1917 CFTR^−/− cells from three animals per genotype; P = 1, Mann-Whitney U test). Scale bar, 20 μM. (G) Example of Ca²⁺ imaging showing an activated cell (left, arrow) and the corresponding time course (right) evoked by stimulation with 1-octanol (10 μM). Scale bar, 20 μM. (H) Mean Ca²⁺ peak amplitudes (ΔR/R₀) in CFTR^+/+ (gray) versus CFTR^−/− (blue) cells after stimulation. (I) Proportion of activated OSNs (F_1,14 = 1.64, P = 0.27, two-way ANOVA). (J) Peak amplitudes (F_1,558 = 0.13, P = 0.72, two-way ANOVA). n = 10 to 149 activated cells of a total of 18,781 cells analyzed (10479 CFTR^+/+ and 8302 CFTR^−/− cells from five animals/genotype).

Fig. 4.. Transcriptome analysis of the CFTR^−/− piglet olfactory epithelium shows alterations related to cell cycle and development.

Differential gene expression of the whole olfactory mucosa transcriptome of CFTR^−/−compared to control CFTR^+/+ piglets. (A) Statistically significant DE genes are highlighted in blue. (B) DE genes classified as OSN- and non-OSN–specific according to previously published mouse RNA-seq datasets in Saraiva et al. (30). A majority (48.5%, 63 genes) of the 130 downregulated (DR) genes are non-OSN. (C) Of the 81 up-regulated genes, 55.6% (45 genes) were specific to OSNs. (D) Pathway enrichment analysis of all genes that were significantly DE (P < 0.05) using Panther Classification System. (E) Gene Ontology analysis of all DE genes (P < 0.05) using UniProt database for classification. (F) DE genes related to cell cycle/DNA or development. A total of 41 of 58 (70.7%) were down-regulated. n = 4 per genotype. (G) Heatmap of cell cycle and development-related DE genes. (H) Heatmap of cell type–specific markers: GBCs, HBCs, mOSNs, iOSNs, microvillar cells (microvilli), sustentacular cells (sus) and Bowman’s gland cells (Bow). Significantly DE genes (in green) correspond to GBCs markers. (I) Heatmap and RNA-seq normalized counts from the 16 DE OR genes. DR, down-regulated; UR, up-regulated.

Fig. 5.. Morphological alterations in the CFTR^−/− piglet olfactory epithelium.

(A) Immunostaining for NGFR and OMP in olfactory epithelium sections. Epithelium thickness is delimited by the arrows. (B) Quantification of the olfactory epithelium (OE) thickness, cell number, and density. (C) Quantification of the cell number of each layer: OMP⁺, NGFR⁺, sustentacular cells (OMP⁻ cells above OMP⁺ layer) and iOSNs (OMP⁻ NGFR⁻ cells between layers). (D) Representative images of OB glomeruli immunolabeled for OMP in CFTR^+/+ and CFTR^−/− piglets. (E) Quantification of the number and size of the OB glomeruli. *P < 0.05 and **P < 0.01 (Mann-Whitney U test); n = 6 animals per genotype. Scale bars, 50 μm.

Fig. 6.. CFTR deficiency alters the GBC proliferative phenotype.

Double immunostainings for KRT5/NGFR (A) and KRT5/SOX2 (B). Arrowheads indicate NGFR⁺/KRT5⁻ and basal SOX2⁺/KRT5⁻ labeling specific for GBCs. (C) Quantification of SOX2⁺/KRT5⁻ (GBCs) and SOX2⁺/KRT5⁺ (HBCs) cells. **P < 0.01 (Mann-Whitney U test); ns, not significant. n = 7 CFTR^+/+ and 5 CFTR^−/− animals. (D) Dual fluorescent in situ hybridization with probes for CFTR and NGFR in control CFTR^+/+. CFTR labeling is not only robust in microvillar-like cells in the apical epithelium (white arrowheads, top) but is also detected in other cell types in the OSN cell layer (gray arrowhead, middle), basal cells (black arrowheads, bottom, colocalized with NGFR), and Bowman glands (right panels). (E) Fluorescent in situ hybridization for CFTR and immunostaining for SOX2 in the basal portion of the epithelium in CFTR^+/+ showing colabeling. (F) Dual fluorescent in situ hybridization with probes for CFTR and NGFR in CFTR^−/− showing no evident CFTR labeling. Bottom panels show magnified views of boxed area. Scale bars, 20 μm.

Fig. 7.. snRNA-seq analysis of CFTR^−/− piglet olfactory mucosa.

(A) UMAP dimensionality reduction plot of gene expression in 8923 integrated CFTR^+/+ and CFTR^−/− olfactory and respiratory mucosal cell nuclei (n = 73 to 1835 cells). (B) Cell proportions for every cell cluster. (C) Olfactory cell types, as a percentage of the total olfactory cells. (D) UMAP depicting expression of CFTR in the CFTR^+/+ cell sample. (E) UMAP projection of the computationally assigned cell cycle (G₂M, G₁, S; left) and CFTR expression (right) in the GBC cell cluster from the CFTR^+/+. (F) Ratio of G₂M versus G₁ + S cells in the CFTR^+/+ GBC cell sample. G₂M cells are 3.8 times more abundant among the CFTR-expressing GBCs . (G) DotPlot visualization of the expression of 11 GBC marker genes. (H) Percentage of GBCs that express the genes in (G) that are in G₂M phase.

Fig. 8.. snRNA-seq analysis of CFTR^−/− piglet olfactory mucosa.

(A) Specific expression of 33 ORs in individual iOSNs and mOSNs. (B) Relative OR expression in mature and immature OSNs. (C) Expression map identifying 198 ORs, specific to either CFTR^+/+ (red) or CFTR^−/− (blue) or coexpressed in both (gray). Each row represents an individual cell and each column is a single OR. Gene identities and cell numbers can be found in data S1. (D) Dual fluorescent in situ hybridization with probes for the OR genes OR51E2 [indicated in red in (A)] and OR51E1 in the olfactory epithelium of CFTR^+/+ and CFTR^−/− piglets. Right: Ratio of OR51E2/OR51E1 cells per section. *P < 0.05 (Mann-Whitney U test); n = 6 per genotype. Scale bars, 50 μm.