DOI: 10.5061/dryad.w9ghx3g0p
title: Cystic fibrosis alters the structure of the olfactory epithelium and the expression of olfactory receptors affecting odor perception

description: A reduced sense of smell is a common condition in people with cystic
 fibrosis (CF) that negatively impacts 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 CF patients 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.
description: <strong>Experimental design and
 participants</strong> The objective of this study
 was to establish the causes of smell alterations observed in cystic
 fibrosis patients. In a prospective study design, patients who presented
 themselves with CF were recruited from CF outpatient clinic consultations
 in the sense of regular disease-specific follow-up. Healthy subjects were
 recruited for comparison. For the Sniffin’ Sticks test, a total of 3
 healthy participants (aged 23 to 52 years, 1 woman) and 10 CF patients
 (aged 18-52 years, 5 women) participated in the study from 07/04/2023 to
 07/25/2023. For the SNOT-22 test, 17 healthy controls (23-52 years old, 10
 women) and the previous 10 CF were used. For snRNA-seq, samples from 7 CF
 and 9 controls were sequenced. The information on the genetic form of the
 disease comes from the documentation of earlier DNA tests. For healthy
 participants, exclusion criteria were neurological diseases, systemic
 diseases associated with smell disorders like chronic renal failure,
 subjective smell impairment, chronic rhinosinusitis, allergic rhinitis,
 alcohol or drug abuse, and pregnancy. The study was conducted according to
 the declaration of Helsinki and had been approved by the Ethics Committee
 at the TU Dresden (EK 552122022). All participants gave written informed
 consent. Olfactory testing A
 detailed medical history was taken including age, gender, CF symptoms,
 medication, surgery, and questions regarding olfactory function. Further,
 the questionnaire sino-nasal outcome test (SNOT-22) was filled out to
 focus on lists of symptoms and social/emotional consequences of
 rhinosinusitis. Patients underwent olfactory tests, using the Sniffin’
 Sticks test battery (Burghart Messtechnik, Holm, Germany) to categorize
 the olfactory function regarding odor threshold and identification
 (<em>1</em>, <em>2</em>). A nasal endoscopy by an
 ENT specialist was performed to categorize endonasal diseases such as
 polyps or any form of chronic rhinosinusitis according to the Lildholdt
 score (<em>3</em>). Nasal brushing was performed on the
 olfactory cleft on both sides under constant endoscopic control using a
 sterile cotton swab (CLASSIQSwabs™, Brescia, Italy). The tips of the
 brushes were stored in CryoStor® cell cryopreservation media (~1-2 ml) for
 10 min at 4°C and then transferred to a cooling device (Thermo Fisher
 Scientific, Dreieich, Germany) and stored at -80°C until further
 processing. Patient records were assigned to codes and
 anonymized. <strong>Human single-nuclei RNA
 sequencing</strong>
 <strong><em>Sample
 preparation:</em></strong> Cellular heterogeneity of human
 cystic fibrosis olfactory epithelium was analyzed by single-nuclei RNA
 sequencing (snRNA-seq). Frozen individual olfactory epithelium swabs were
 thawed and pooled for nuclei extraction in four pools containing 3
 distinct samples from 3 individuals each (16 total individuals, 9 controls
 and 7 CF). This pooling ensured a sufficient amount of material for nuclei
 isolation. Genetic polymorphisms between samples was then used to
 discriminate individual samples (see below). Nuclei extraction was
 performed according to (<em>4</em>), with some modifications.
 In brief, each cryotube was thawed at 37°C, supplemented with 400 µl
 pre-heated saline medium. Brushes were cleared on ice by pipetting,
 transferred into 15 ml tubes, and centrifuged for 5 min 290xg at 4°C.
 After removal of the supernatant, the cell pellet was resuspended into 1
 ml of a buffer containing 25 mM citric acid and 0.25M sucrose. The
 suspension was homogenized by 15 strokes with loose, then tight pestles,
 interrupted by 2 x 3 min incubation on ice. After a filtration on a 20 µm
 filter (Miltenyi, Paris, France), nuclei were centrifuged for 5 minutes
 500xg, resuspended in 500µl of previous citric buffer, and centrifuged for
 5 minutes at 500xg. Nuclei were resuspended in a buffer containing 25mM
 KCl, 3mM MgCl<sub>2</sub>, 5mM Tris-buffer pH8, 0.4U/µl
 RNAsin® Plus ribonuclease inhibitor, 0.4U/µl SUPERaseIn® RNase inhibitor,
 and 1mM dithiothreitol (DTT). After a control of morphology on a Floid
 cell imaging station (PicoQuant, Berlin, Germany), nuclei were counted
 with a Countess II FL Automated Cell Counter (Thermo Fisher Scientific,
 Waltham, USA). Nuclei were then diluted at a concentration of 1200 nuclei
 /µl to target a capture of ~10,000 nuclei, and the suspension was rapidly
 loaded on a 10x Genomics Next GEM Single Cell 3ʹ v3.1 system (10X
 Genomics, Pleasanton, USA). Libraries were prepared according to the
 constructor specifications, with sequencing on a Nextseq 2000 (Illumina,
 San Diego, USA). <strong><em>snRNA-seq
 </em></strong><strong><em>processing and analysis:
 </em></strong>Human raw data were analyzed using Cell Ranger
 Single-cell software v6.0.0, with inclusion of the intron sequences. The
 raw gene-barcode matrices were aligned to the hg38 genome. Demultiplexing
 was performed using Demuxafy within a Singularity container to accurately
 assign reads to individual samples (<em>5</em>). After
 demultiplexing, each sample data was processed using Seurat package v.4.0
 (<em>6</em>) in R v.4.0.2. Cells with less than 200 genes or
 more than 5% mitochondrial content, and genes expressed in fewer than 3
 cells were excluded from the analysis. Each individual Seurat object was
 normalized and highly variable features were identified using the
 variance-stabilizing transformation (vst) method, selecting the top 2000
 variable features. Integration features were selected from the variable
 features identified in each object. Each Seurat object was then scaled and
 subjected to Principal Component Analysis (PCA) using the selected
 integration features. Integration anchors were identified using Reciprocal
 PCA (rPCA), and the data was integrated based on these anchors. The
 integrated data was further processed by setting the default assay to
 “integrated”, scaling the data, running PCA, and performing Uniform
 Manifold Approximation and Projection (UMAP) dimensionality reduction on
 the first 30 principal components (PCs). A neighbor graph was then
 constructed, and clustering was performed at a resolution of 1.0.
 Differentially expressed genes were identified for each cluster using the
 FindMarkers function. Pseudobulk analysis was conducted using the
 edgeR−LRT from Libra R package (<em>7</em>) to aggregate
 single-cell data into bulk-like samples for differential expression
 analysis between CF and control. Codes used for all analyses can be found
 here: https://github.com/ymbouamboua/Human_Pig_Olfactory_CF. Numbers for
 all cell types are: B cells (n = 106); Bowman's gland (n = 84); Club
 (n = 2600); Deuterosomal cells (n = 405); GBCs (n = 315); Goblet cells (n
 = 1785); Ionocytes/microvillar cells (n = 430); Macrophages (n = 166);
 Monocytes (n = 345); Neutrophils (n = 32); NK cells (n = 308); olfactory
 HBCs (n = 4104); OSNs (n = 31); pDCs (n = 192); Respiratory HBCs (n =
 183); Respiratory multiciliated cells (n = 10298); Sustentacular cells (n
 = 383); T cells (n = 1147); Tuft cells (n = 18).
 <strong>Pig single-nuclei RNA
 sequencing</strong>
 <strong><em>Sample
 preparation:</em></strong> Pig main olfactory epithelia were
 snap frozen in liquid nitrogen and included in OCT. 300 µm sections were
 made using a cryostat, and kept at -80°C. In a glass dounce tissue grinder
 previously cooled on ice, 7 tissue slices and 1ml of citric acid-based
 buffer (0.25 M sucrose, 25 mM citric acid) were added. Tissue samples were
 homogenized with 3-5 strokes of loose pestle and incubated on ice for 5
 min. After 5 more strokes using a loose pestle, samples were incubated on
 ice for 5 min, homogenized again with 3 strokes using a loose pestle and 5
 strokes using a tight pestle (<em>4</em>). The suspension was
 filtered through a 40 µm cell strainer, and 1ml of citric acid-based
 buffer was used to wash the containers. After a centrifugation for 5 min
 at 500xg at 4°C, the supernatant was carefully removed and the sample was
 resuspended in 1ml of Wash buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM
 MgCl<sub>2</sub>, 1% BSA, 0.1%Tween 20, 1 mM DTT, 0.6 U/μl
 RNaseIn®, 0.2 U/μl SuperaseIn®). Sample was filtered through a 5 µm cell
 strainer and centrifuged for 5min at 500 g at 4°C. Nuclei were resuspended
 in 50-100 µl of diluted nuclei buffer (Nuclei Buffer® 1X; Multiome kit,
 10X Genomics), DTT 1 mM, RNaseIn® 0.6U/µl, SuperaseIn® 0.2 U/μL). The
 nuclei morphology was verified using a Floid cell imaging station. Nuclei
 were counted on a Countess II FL Automated Cell Counter, diluted to the
 desired concentration (for a target capture of 10,000 nuclei). An RNA
 quality test was performed for each preparation before the 10x sequencing
 library was made. To do so, an aliquot of purified nuclei was placed in
 QIAzol Lysis Reagent® and RNA was isolated using miRNeasy Micro kit®. The
 RNA integrity number (RIN) was checked using the Agilent Bioanalyzer
 System. snNucSeq was performed according to the protocol provided by 10X
 Genomics. <strong><em>snRNA-seq
 </em></strong><strong><em>processing and analysis:
 </em></strong>Pig raw data were analyzed using Cell Ranger
 Single-cell software v6.0.0. The raw gene-barcode matrices were aligned to
 the Sscrofa11-1‎ genome. Cell Ranger output was analyzed with the Seurat
 package v.4.0(<em>6</em>) using R v.4.0.2. Each individual
 data was filtered to keep cells with 200-7,000 genes, less than 99% of
 dropouts, and less than 5% of mitochondrial sequences. Doublet cells were
 removed with DoubletFinder (<em>8</em>). After normalization
 and variance stabilization with SCTransform, and dimensionality reduction
 by Principal Component Analysis, data was visualized using UMAP embedding
 based on the first 20 principal components. Global integration was
 performed after normalization and identification of the 4000 most variable
 features of each dataset. Features that varied across datasets were
 selected, and experiments were integrated with the matching mutual nearest
 neighbors method as described
 (<em>9</em>)<em>.</em> Cell clusters were
 annotated according to canonical gene markers: Bowman’s gland
 (<em>SOX9, SLC6A11, TMEM163</em>),  Endothelial
 (<em>MMRN1, CLVS1, CCL21</em>), Fibroblast/Stromal
 (<em>DCN, LUM, PTPRD</em>), GBCs (<em>EZH2, CXCR4,
 HES6</em>), Immature neurons (<em>GNG8, GAP43,
 TPD52</em>), Immune (<em>CD163, ARHGAP15, MRC1</em>),
 Mature neurons (<em>GNG13, STXBP5L, PEX5L</em>), Olfactory
 ensheathing glia (<em>SORCS1, ZNF536</em>), Olfactory HBCs
 (<em>CCDC129, CAPN13, MGAM2</em>), Olfactory microvillar
 (<em>STAP1, CLNK, SLC35F3</em>), Pericytes (<em>NOSTRIN,
 LRFN5, CXCL2</em>), Respiratory ciliated (<em>CFAP126,
 FOXJ1</em>), Respiratory epithelial (<em>NDAL, GPS,
 EIF1</em>), Respiratory HBCs (<em>KRT5, MET, TP63</em>),
 Respiratory secretory (<em>TTC6, EIF2AK2</em>), Sustentacular
 (<em>MOCOS, GLDN, ERMN</em>), Vascular smooth muscle
 (<em>TAGLN, TPM2, MYL9</em>). Bowman's gland (n = 460);
 Endothelial cells (n = 167); Fibroblasts/stromal cells (n = 1475); GBCs (n
 = 313); Immature neurons (n = 1241); Immune cells (n = 144); Mature
 neurons (n = 1835); Olfactory ensheathing glia (n = 360); Olfactory HBCs
 (n = 108); Olfactory microvillar/ionocyte cells (n = 128); Pericytes (n =
 129); Respiratory multiciliated cells (n = 314); Respiratory epithelial
 cells (n = 73); Respiratory HBCs (n = 222); Respiratory secretory cells (n
 = 546); Sustentacular cells (n = 1115); Vascular smooth muscle cells (n =
 293). <strong>Bulk RNA-seq processing and
 analysis</strong> Olfactory epithelia from
 <em>CFTR</em>-null and control piglets were dissected and
 immediately frozen on liquid N<sub>2</sub>. RNA was extracted
 using the RNeasy mini kit (Qiagen) with on-column DNAse digestion. mRNA
 was prepared for sequencing using the TruSeq RNA sample preparation kit
 (Illumina). All samples were multiplexed together and sequenced on four
 lanes on the Illumina HiSeq 2500 platform, to generate 100 bp paired-end
 reads. Sequencing reads were mapped using STAR 2.3 to the 11.1
 <em>Sus scrofa</em> reference genome, annotation version 11.1
 in the Ensembl pig genome database. The number of fragments aligned to
 each gene was counted using the HTSeq package, with the script htseq-count
 (mode intersection-nonempty). Any read that maps to multiple locations in
 the genome (also called multireads) was not counted towards the expression
 estimates as it cannot be assigned to any gene unambiguously. To compare
 the expression values across samples, raw count data was normalized to
 account for the depth of sequencing. Size factors were calculated using
 DESeq2’s function estimateSizeFactorsForMatrix, and raw counts were
 divided by the corresponding size factor for each sample. To test for
 differential expression, DESeq2 was used with standard parameters. Genes
 were considered to be differentially expressed if they had an adjusted P
 value of ≤0.05 (equivalent to a false discovery rate of 5 %). A total of
 11 DE expressed genes located on the Y chromosome were excluded from the
 analysis to prevent sex-specific bias. To find terms that are enriched in
 our list of DE genes the over-/under-representation algorithm from
 GeneTrail (http://genetrail.bioinf.uni-sb.de/) was used. The background
 provided were all those genes tested for differential expression. To
 assess whether the DE genes form putative regulatory networks, STRING
 (http://string-db.org/) was used with default settings, for the 212 DE
 genes only. All normalized data and detailed results of the DE and
 enrichment analyses can be found in the Data S1. Heatmaps of genes with
 significantly changed expression were generated using OriginPro 2021b
 program. Functional classification was analyzed by Gene Ontology
 Consortium (Panther Classification System) and UniProt database
 (https://www.uniprot.org) using the biological process terms.
 Classification into OSN- or non-OSN-specific genes in Fig. 4B-C was
 performed by direct comparison with the dataset published by Saraiva et
 al.(<em>10</em>) obtained by RNA-seq of mouse OMP+ mature
 olfactory sensory neurons (mOSNs). Cell type assignments for specific
 marker genes was based on previously published mouse and human expression
 studies (<em>10</em>, <em>11</em>).
 <strong>Human olfactory neuroepithelium cell culture.
 </strong> Human olfactory cell samples were
 obtained independently from snRNAseq samples using a slightly different
 technique. Cells from the olfactory mucosa were collected by nasal
 brushings from 10 healthy control, non-smoker, SARS-CoV-2 negative
 subjects between 18 and 45 years old (both males and females) as
 previously described (<em>12</em>). Every subject gave written
 informed consent for the study and the procedures involved. The study was
 approved by the local institutional ethics committee CEIm Ethical
 Committee Parc de Salut MAR, IMIM-Hospital del Mar Research Institute,
 Barcelona: study no. 2018/7942/I. Samples from the middle and upper
 turbinates were maintained in 250 µl of cold Dulbecco’s Modified Eagle
 Medium/Ham F-12 (DMEM/F12) enriched with 10% fetal bovine serum (FBS), 2%
 glutamine and 1% streptomycin–penicillin (GibcoBRL), as previously
 described(<em>12</em>). At 80% confluence, cells were expanded
 using 0.25% trypsin (GibcoBRL) and replated in 75
 cm<sup>2</sup> flasks. Cells were then expanded until a
 maximum of 5 passages. Cells from 2 healthy individuals were treated with
 0, 10, 20 or 30 µM of the CFTR<sub>inh</sub>172 selective CFTR
 blocker (Tocris) on the culture media and incubated for 24 hours. Cells
 were washed with phosphate buffered saline (PBS), fixed with
 paraformaldehyde (PFA) 4% for 15 min and <em>in situ</em>
 hybridization and/or immunolabeling was performed as described
 below. <strong>Quantitative
 RT-PCR</strong> RNA from cultured human olfactory
 cells from 10 controls was purified with the PureLink<sup>®
 </sup>RNA Mini Kit (Invitrogen) and stored at − 20 °C. Extracted
 total RNA was converted to cDNA by reverse transcription of 20 ng of RNA
 using the Nucleospin® RNA XS kit (Macherey-Nagel, Düren, Germany)
 according to the manufacturer's protocol. The cDNA was applied in
 TakyonTM No ROX SYBR 2X MasterMix blue dTTP (Eurogentec) and the primers
 used are listed in Data S1. All RT-qPCR reactions were performed in
 96-microwell plates using The LightCycler® 480 Real-Time PCR System (Roche
 Applied Science). Three technical replicates were averaged and the
 quantitative RT-PCR data were analyzed using the 2ΔΔCt method. GAPDH and
 RLP-19 genes were used as a control for normalization. The results are
 expressed as relative fold change.
 <strong><em>CFTR</em></strong><strong>-deficient pigs</strong> Male and female <em>CFTR</em><sup>+/−</sup> heterozygous transgenic pigs (<em>13</em>) were provided by the LMU Munich, Germany, transferred to France. Pigs (Large White breed) were housed together and had access to a standard grain-based diet and water <em>ad libitum</em>. Heterozygous pigs were mated to generate <em>CFTR</em><sup>+/+</sup>, <em>CFTR</em><sup>+/−</sup> and <em>CFTR</em><sup>−/−</sup> piglets. Newborn piglets (0.8-1.5 kg) were genotyped within 6 hours after birth and sacrificed for olfactory tissue collection. E75 pig fetuses were collected from amniotic sacs by c-section in pregnant dams. Pig littermates homozygous for <em>CFTR</em> (<em>CFTR</em><sup>+/+</sup>) served as controls. Littermates were randomly assigned to experimental groups keeping the same sex ratio between groups. All experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee at INRAE. All experimental procedures were evaluated by the Ethics Committee of the Val de Loire (CEEA VdL, committee N°19) and approved by the Ministry of Higher Education and Research (APAFIS#1166-2015071615392426 Notification and APAFIS#10125-20170602162555 Notification). <strong>Neonate suckling assay</strong> Newborn piglets were tested for suckling latency right after birth. Delivery was induced after 112 days of gestation by injection of 175 µg of cloprostenol and 20 units (IU) of oxytocin and each piglet was marked right after birth, monitored and latencies to stand up after birth and to suckle were scored (see Movie S1). Testing lasted until the piglet reached the mother’s nipple and started to suckle or for a maximum of 400 min after the birth of each piglet. Each test group contained piglets from one single litter. Each litter was tested only once, to ensure no learning occurred. Time scoring was performed by an experimenter blind to the genotype of the piglets, as biopsies for genotyping were collected after behavior testing. <strong>Calcium imaging</strong> Ca<sup>2+</sup> imaging was performed in freshly dissociated pig olfactory epithelium cells adapting protocols previously developed for mouse olfactory cells (<em>14</em>, <em>15</em>). The olfactory neuroepithelium was detached from the cartilage and minced in PBS at 4°C. The tissue was incubated (20 min at 37°C) in PBS supplemented with papain (0.22 U/ml; Worthington) and DNase I (10 U/ml; Fermentas), urea (40 mM; Sigma) gently extruded in DMEM (Invitrogen) supplemented with 10% FBS, and centrifuged at 100 × g (5 min). Dissociated cells were plated on coverslips previously coated with concanavalin-A type IV (0.5 mg/ml, overnight at 4 °C; Sigma). Cells were used immediately for imaging after loading with fura-2/AM (5 µM; Invitrogen) for 60 min. Coverslips containing cells were placed in a laminar-flow chamber (Warner Instruments) and constantly perfused at 22 °C with extracellular solution Hank’s balanced salt solution (HBSS, Invitrogen) supplemented with 10 mM Hepes (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid). Cells were alternately illuminated at 340 and 380 nm, and light emitted above 510 nm was recorded using a C10600-10B Hamamatsu camera installed on an Olympus IX71 microscope. Images were acquired at 0.25 Hz and analyzed using ImageJ (NIH), including background subtraction, region of interest (ROI) detection and signal analyses. ROIs were selected manually and always included the whole cell body. Peak signals were calculated from the temporal profiles of image ratio/fluorescent values. Results are based on recordings from 5 piglets for each condition and genotype (n = 10–149 activated cells of a total of 18,781 cells analyzed; 10,479 <em>CFTR</em><sup>+/+</sup> and 8,302 <em>CFTR</em><sup>–/–</sup> cells). Cells were stimulated successively and in random order using bath application. The following criteria for stimulus-induced Ca<sup>2+</sup> responses were applied: (1) A response was defined as a stimulus-dependent deviation of fluorescence ratio that exceeded twice the standard deviation of the mean of the baseline fluorescence noise. (2) Cells showing a response to control buffer were excluded from analysis. (3) A response had to occur within 1 min after stimulus application. In time series experiments, ligand application was repeated to confirm the repeatability of a given Ca<sup>2+</sup> response. Chemostimuli were freshly prepared each day and diluted in extracellular solution to give the following final concentrations: 1-octanol 10 µM (Sigma); 2-heptanone 10 µM (Sigma); octanal 10 µM (Sigma); forskolin (Sigma) 50 µM, 3-isobutyl-1-methylxanthine (IBMX; Sigma) 100 µM, and 8-Bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP; Sigma) 500 µM; KCl 90 mM. Volatile odorants were initially prepared in dimethyl sulfoxide (DMSO; Sigma) and further diluted in extracellular solution. <strong>Immunostaining</strong> Human olfactory cells were fixed in PBS containing 4% PFA for 15 min at room temperature, washed 3X in PBS and incubated in blocking solution (PBS solution containing 0.1% Triton X-100 and 3% horse serum) for 30 min at room temperature. For newborn pigs, olfactory tissues were removed, postfixed overnight in 0.1 M phosphate buffer (PB) containing 4% PFA and later cryoprotected in 0.1 M PB buffer containing 30% sucrose. Samples were embedded in Tissue-Tek OCT compound, snap-frozen in cold isopentane and processed on a Leica CM 3050S cryostat. Olfactory epithelia were cut in 16-μm thick coronal sections and were directly mounted on SuperFrost Plus slides glasses (Thermo Scientific). Olfactory bulbs were cut in 30-µm serial free-floating sagittal sections in a PBS solution. Sections were treated with 10mM sodium citrate for 5 min at 95-100 ºC for antigen retrieval, washed (3 × 5 min) with PBS, incubated in blocking solution (PBS solution containing 0.1% Triton X-100 and 5% normal horse serum) for 2 h at room temperature (RT), incubated overnight at 4 °C in blocking solution supplemented with the primary antibody, washed in PBS solution and incubated in blocking solution supplemented with secondary antibody for 1 h at RT. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 0.5 μg/ml; Sigma) for 5 min. We used the following primary antibodies: goat anti-SOX2 (RD Systems AF2018, 1:300), rabbit anti-KRT5 (Biolegend 905501, 1:800), goat anti-OMP (Wako 019-22291, 1:2000) and mouse anti-NGFR (Sigma N5408, 1:1000), mouse anti-PCNA (Sigma P8825, 1:1000), rabbit anti-DCX (Abcam AB18723, 1:2000), rabbit anti-Ki67 (Abcam ab9021, 1:100) mouse anti-Gαolf (Santa Cruz sc-55545, 1:500), rabbit anti-ITPR3 (Millipore AB9076, 1:500), rabbit anti-TRPM5 (Alomone Labs ACC-045, 1:400), rat anti-NES (Santa Cruz sc-33677, 1:600). Secondary antibodies used for the corresponding target species were conjugated with Alexa488, Alexa546, and Alexa647 (Thermofisher, 1:500). Epithelium thickness, cell density, areas and olfactory bulb glomeruli quantifications were preformed from images acquired on a Zeiss LSM-780 confocal laser-scanning microscope. Image regions were analyzed in the entire <em>z</em>-axis with 3 µm step intervals, and images were reconstituted using the Maximum Intensity Projection tool of Zen software. Images were analyzed with Fiji/ImageJ (NIH). Demarcations of epithelium limits and cell-specific layers were based on OMP and NGFR immunoreactivities, and DAPI+ cells were counted using the particle analyzer plug-in of Fiji. Counts of the number of cells were evaluated blindly for each animal. Five slices per animal and genotype were used. Cells were counted from five to seven independent animals. For each sample, images were acquired at least from five different anatomical levels. Olfactory bulb glomeruli demarcation was based on OMP immunoreactivity in 5-17 slices per animal from 6 independent animals per genotype. A total of 819 glomeruli (469 <em>CFTR</em><sup>+/+</sup> and 350 <em>CFTR</em><sup>−/−</sup>) covering all main olfactory bulb topography (dorsal, ventral, lateral and medial) were analyzed.   <strong>In situ hybridization (ISH)</strong> Staining for <em>CFTR</em>, <em>NGFR, OR51E2, OR51E1</em> and <em>NPY</em> mRNAs was performed using multiplex fluorescent ISH. Human olfactory cells and olfactory tissue sections were prepared as described above. RNAscope Fluorescent MultiplexV2 labeling kit (ACDBio 323110) was used to perform the ISH assays according to the manufacturer’s recommendations. Probes used for staining are hs-CFTR (ACDBio 603291), ss-CFTR (ACDBio 541401-C2), ss-NGFR (ACDBio 828841), ss-LOC100625684 (ACDBio 1216061-C2), ss-LOC100737531 (ACDBio 1216070-C3), and ss-NPY (ACDBio 318751). Negative control slides were performed in parallel. After incubation with fluorescent-labeled probes, slides were counterstained with DAPI and mounted with antifade fluorescent mounting medium (Dako). Fluorescent images were captured using sequential laser scanning confocal microscopy (Zeiss LSM-780). <em>CFTR</em>+ cells were counted from four independent human subjects. <strong>Combined ISH and immunohistochemistry</strong> After a standard ISH protocol, cells and tissue sections were directly incubated in blocking solution followed by primary and secondary antibodies for DCX, PCNA, Ki67 and SOX2 as described above. <strong>Quantification and Statistical Analysis</strong> Statistical analyses were performed using the statistical package R version 3.6.0 and the packages ggplot2, drc and ggpubr (R-studio Software, 4.1.1), OriginPro 2021b (OriginLab Corporation, Northampton, MA, USA). Statistical details of experiments can be found in the figure legends, including statistical test used, the exact value of n and dispersion and precision measures. Assumptions of normality and homogeneity of variance were tested before conducting the following statistical approaches. Mann-Whitney <em>U</em> test was used to measure the significance of the differences between two distributions. Multiple groups were compared using a two-way analysis of variance (ANOVA). Kolmogorov–Smirnov test was used to compare cumulative distributions. The probability of error level (alpha) was chosen to be 0.05. P values and the specific statistical test performed for each experiment is included in the appropriate figure legend or main text. Unless otherwise stated, results are presented as individual data points combined with boxplot indicating median lines and 25%-75% ranges. References 1.           T. Hummel, G. Kobal, H. Gudziol, A. 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description: # Cystic fibrosis alters the structure of the olfactory epithelium and the
 expression of olfactory receptors affecting odor perception
 [https://doi.org/10.5061/dryad.w9ghx3g0p](https://doi.org/10.5061/dryad.w9ghx3g0p) ## Description of the data and file structure Bulk and Single-nuclei RNA-seq data generated in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE197187 and available in [https://www.genomique.info//data/public/hts/9258/8646_human_olfactory_cf.h5ad](https://www.genomique.info//data/public/hts/9258/8646_human_olfactory_cf.h5ad). ### **Human olfactory data** * *Mutation*: type of *CFTR* gene mutation * *Kaftrio*: type of cystic fibrosis treatment with kaftrio (Yes=1/No=0) * *Age*: Age range of participants * *m/f*: Gender/sex of participants (male=1/female=0) * *Symptoms*: Whether participants present symptoms of cystic fibrosis (Yes=1/No=0) * *Polyps*: Whether participants present nasal polyps (Yes=1/No=0) * *Subjective smelling ability*: Self-reported smelling performance (0-100) * *SDI threshold*: olfactory function regarding odor threshold using using the Sniffin’ Sticks test battery (Burghart Messtechnik, Holm, Germany). * *SDI identification*: olfactory function regarding odor identification using using the Sniffin’ Sticks test battery (Burghart Messtechnik, Holm, Germany) * *SI*: Threshold plus identification values addition * *SNOT-22*: sino-nasal outcome test (SNOT-22) questionnaire evaluation consisting on a list of symptoms and social/emotional consequences of rhinosinusitis. * *Celltype*: Olfactory epithelium cell type * *Percentage*: relative abundance * *Geneid: *gene names * *reads*: Average expression of the 16 most expressed olfactory receptor genes in the olfactory sensory neuron cluster of the cystic fibrosis sample "n/a" denotes unavailable information to ensure human data is anonymized (age) or subjects were not tested. Statistical tables denote Student’s t test for the parameters subjective smelling ability_0 to 100, SDI threshold, SDI identification, SI andbSNOT-22. ### **Cell cycle scores\_Fig 2b** Cell cycle phase scoring in the GBC cluster using scRNA-seq * *orig.ident*: sequencing sample ID * *nCount_RNA*: transcript number per cell * *nFeature_RNA*: number of genes per cell * *Souporcell_Cluster*: demultiplexing algorithm (0-3) * *Souporcell_Individual_Assignment*: donor assignment clustering * *Barcode*: cell identification tag * *Souporcell_DropletType*: singlet/doublet identification * *Vireo_Individual_Assignment:* donor assignment clustering using Vireo * *Vireo_DropletType:* singlet/doublet identification using Vireo * *scds_score:* computational annotation of doublets * *scds_DropletType:* singlet/doublet identification by scds * *MajoritySinglet_DropletType:* singlet/doublet identification by the three algorithms * *MajoritySinglet_Individual_Assignment:* Final donor assignment clustering * *barcode_seurat:* cell identification tag by seurat * *donor:* sequencing sample ID by donor * *sex:* Gender/sex of sample (male/female) * *kaftrio:* type of cystic fibrosis treatment with kaftrio (Y/N) * *sample:* sequencing sample ID * *percent_mito:* percentage of mitochondrial genes * *integrated_snn_res.0.5:* cell cluster assignment at 0.5 resolution * *seurat_clusters:* cluster type * *integrated_snn_res.1.5:* cell assignment into cluster at 1.5 resolution * *integrated_snn_res.1:* cell assignment into cluster at 1 resolution * *integrated_snn_res.1.2:* cell assignment into cluster at 1.2 resolution * *integrated_snn_res.1.3:* cell assignment into cluster at 1.3 resolution * *sample_order:* sample assignment * *S.Score:* phase S score * *G2M.Score:* phase G2M score * *Phase:* cell cycle phase assignment (S/G1/G2M) * *old.ident:* cell type ID number * *celltype:* cell type (GBC) * *group:* type of cystic fibrosis treatment: KAFTRIO=kaftrio-treated; CF=non-treated; CTRL=control healthy samples * *patient:* CF=cystic fibrosis; control=control samples * *integrated_snn_res.0.1:* cell assignment into cluster at 0.1 resolution * *id:* cell identification tag ### **Cell cycle scores\_2d** Cell cycle phase scoring in the GBC cluster using scRNA-seq * *G1S/G2M:* CFTR expression (counts) in each cell cycle phase * *CTRL/CF:* G2M-scores in control (CTRL) and cystic fibrosis (CF) samples Statistical tables denote Mann-Whitney U tests for comparison of G1S/G2M and CTRL/CF conditions. ### **CFTRinh172 effect\_Fig 2f** Effect of the CFTR inhibitor CFTRinh172 on PCNA and Ki67 immunolabeling expressed as Gaussian probability density functions: * *X*: Cell number * *Area:* Area of the cell (µm2) * *Mean:* Mean intensity fluorescence (arbitrary units; a.u.) * *IntDen:* Integrated density fluorescence (a.u.) * *Median:* Median intensity fluorescence (a.u.) * *RawIntDen:* Raw integrated density fluorescence (a.u.) * *Id:* sample id * *Treatment:* Cells treated with 10, 20, or 30 μM of CFTRinh172 and mock-treatment (control) ### **Latency to suckle\_Fig 3b** Latency-to-suckle in minutes of CFTR–/– (KO) and control (WT) piglets suckling. Statistical table denote a Mann-Whitney U test for comparison of KO/WT conditions. ### **Latency to suckle\_Fig 3c** Percentage of pigs suckling of CFTR–/– (KO) and control (WT) piglets over the 400 min assay. Statistical table denote a Kolmogorov–Smirnov test for comparison of KO/WT conditions. * *pigletsKO:* CFTR–/– piglets * *pigletsWT:* control piglets ### **Latency to suckle\_Fig 3d** Percentage of pigs suckling of CFTR–/– (KO) and control (WT) piglets over the 400 min assay period binned in 25 min windows. ### **Birth weights\_Fig 3e** Weights at birth (in kg) of CFTR–/– (KO) and control (WT) piglets. Statistical table denote a Mann-Whitney U test for comparison of KO/WT conditions. ### **OMP count\_Fig 3f** Immunolabeling for OMP in freshly dissociated pig olfactory epithelium cells of CFTR+/+ (WT) and CFTR–/– (KO) animals * *OMP:* number of cells positive for OMP * *DAPI:* total cells * *Ratio:* relative abundance of OMP+ cells Statistical table denotes a Mann-Whitney U test for comparison of KO/WT conditions. ### **Time courses\_Fig 3h** Time courses of Ca2+ amplitudes (ratio 340/380 nm; arbitrary units) in CFTR+/+ (ko) vs. CFTR–/– (wt) cells together with the Mean, Standard Deviation and standard error (SE) of mean values for each stimulation: 1-octanol, 2-heptanone, octanal, 8-Br-cGMP, forskolin, IBMX and KCl. The first column indicates the time in seconds. ### **Rel. activation\_Fig 3i** Number and proportion of activated cells by 1-octanol, 2-heptanone (2-hep), and octanal relative to the total of cells present (total). ### **Amplitudes\_Fig 3j** Ca2+ peak amplitudes (ratio 340/380 nm; arbitrary units) in CFTR+/+ (WT) vs. CFTR–/– (KO) cells after stimulation with 1-octanol, 2-heptanone (2hep), octanal, 8-Br-cGMP, forskolin, IBMX and KCl (K). Statistical table denotes a 2-way ANOVA test for comparison of KO/WT conditions. ### **DE expression\_Fig 4a** Differential gene (DE) expression of the whole olfactory mucosa transcriptome of CFTR–/–compared to control CFTR+/+ piglets, both non-significant and significantly DE. * *Geneid:* gene name * *baseMean.ko-wt:* base mean gene expression * *log2FoldChange.ko-wt:* fold change in gene expression in CFTR–/– (ko) vs CFTR+/+ (wt). ### **DE class\_Fig 4b** DE genes classified according to previously published mouse RNA-seq datasets in Saraiva et al. as OSN-specific genes (OSN genes), OSN-non-specific genes (non-OSN genes), genes common to all cells (common), olfactory receptor genes (ORs), genes with no known ortholog in the mouse genome (no ortholog). * *Geneid:* gene name (Ensembl) * *Gene name:* gene name (annotated) * *baseMean.ko-wt:* base mean gene expression * *log2FoldChange.ko-wt:* fold change in gene expression in CFTR–/– (ko) vs CFTR+/+ (wt). ### **DRvsUR\_Fig 4c** Number of downregulated (DR) and upregulated (UR) genes specific to OSNs, non-specific (nonOSN), and common genes (other). ### **GO\_Fig 4d** Results of Pathway enrichment analysis of all genes that were significantly DE (P &lt; 0.05) using Panther Classification System. ### **Process\_Fig 4e** Gene ontology analysis of all DE genes (P &lt; 0.05) using UniProt database for classification. * *Process:* type of biological process * *Genes:* number of genes ### **Process\_Fig 4f** Differential expression (DE) analysis of genes related to cell cycle/DNA, development and other processes. * *Geneid:* gene name (Ensembl) * *Gene name:* gene name (annotated) * *baseMean.ko-wt:* base mean gene expression * *log2FoldChange.ko-wt:* fold change in gene expression in CFTR–/– (ko) vs CFTR+/+ (wt). ### **Heatmap\_Fig 4g** DE analysis of genes related to cell cycle/DNA or development markers. * *Geneid:* gene name (Ensembl) * *Gene name:* gene name (annotated) * *Chr:* chromosome * *Start:* gene start location * *End:* gene end location * *Strand:* positive (+) or negative (-) * *Length:* length in base pairs * *baseMean.ko-wt:* base mean gene expression * *log2FoldChange.ko-wt:* fold change in gene expression in CFTR–/– (ko) vs CFTR+/+ (wt). * *pvalue.ko-wt:* p-value of ko vs. wt comparison * *padj.ko-wt:* adjusted p-value * *ko/wt*: sample number of each CFTR–/– (ko) vs CFTR+/+ (wt) sample. ### **Heatmap\_Fig 4h** DE analysis of cell type-specific marker genes: GBCs, HBCs, mOSNs, iOSNs, microvillar, sustentacular cells and Bowman’s gland cells. * *Geneid:* gene name (Ensembl) * *Gene name:* gene name (annotated) * *Chr:* chromosome * *Start:* gene start location * *End:* gene end location * *Strand:* positive (+) or negative (-) * *Length:* length in base pairs * *baseMean.ko-wt:* base mean gene expression * *log2FoldChange.ko-wt:* fold change in gene expression in CFTR–/– (ko) vs CFTR+/+ (wt). * *pvalue.ko-wt:* p-value of ko vs. wt comparison * *padj.ko-wt:* adjusted p-value * *ko/wt:* sample number of each CFTR–/– (ko) vs CFTR+/+ (wt) sample. ### **ORs\_Fig 4i** DE analysis and RNA-seq normalized counts from the 16 DE olfactory receptor genes. * *Geneid:* gene name (Ensembl) * *Gene name:* gene name (annotated) * *Chr:* chromosome * *Start:* gene start location * *End:* gene end location * *Strand:* positive (+) or negative (-) * *Length:* length in base pairs * *baseMean.ko-wt:* base mean gene expression * *log2FoldChange.ko-wt:* fold change in gene expression in CFTR–/– (ko) vs CFTR+/+ (wt). * *pvalue.ko-wt:* p-value of ko vs. wt comparison * *padj.ko-wt:* adjusted p-value * *ko/wt:* sample number of each CFTR–/– (ko) vs CFTR+/+ (wt) sample. ### **Neuroepithelium\_Fig 5b** Quantification of the olfactory epithelium (OE) thickness, cell number and density on CFTR–/– (KO) and CFTR+/+ (WT) piglets. Values are expressed as Thickness (µm), relative abundance (nuclei/1000 µm) and density (nuclei/1000 µm²). Statistical tables denote Mann-Whitney U test for comparison of KO/WT conditions. ### **Neuroepithelium\_Fig 5c** Quantification of the cell number of each layer: OMP+, NGFR+, sustentacular cells (OMP– cells above OMP+ layer) and iOSNs (OMP– NGFR– cells between layers) on CFTR–/– (KO) and CFTR+/+ (WT) piglets. Values are expressed as relative abundance (nuclei/1000 µm). Statistical tables denote Mann-Whitney U test for comparison of KO/WT conditions. ### **OB\_Fig 5e** Quantification of the number and size of the OB glomeruli on CFTR–/– (KO) and CFTR+/+ (WT) piglets. Values are expressed as number of glomeruli over surface (GL/surface) in mm2 and mean glomerular are (mean GL area) in µm². Statistical tables denote Mann-Whitney U test for comparison of KO/WT conditions. ### **GBCs\_Fig 6c** Quantification of SOX2+/KRT5– (GBCs) and SOX2+/KRT5+ (HBCs) cells on CFTR–/– (KO) and CFTR+/+ (WT) piglets. Values are expressed as relative abundance (nuclei/1000 µm). Statistical tables denote Mann-Whitney U test for comparison of KO/WT conditions. ### **snRNA-seq\_Fig 7** snRNA-seq analysis of CFTR–/– (KO) piglet olfactory mucosa. Cell proportions for every cell cluster. Olfactory cell types, as a percentage of the total olfactory cells. Ratio of G2M vs. G1+S cells in the CFTR+/+ (WT) GBC cell sample. Percentage of GBCs that express the genes that are in G2M phase. * *Cluster:* WT/KO or cell type * *Number:* number of cells * *Freq:* frequency * *_%:* percentage of each cell type or condition * *Cell type:* non-CFTR or CFTR -expressing * *G2M/G1S:* ratio G2M/G1S * *Condition:* CFTR+/+ or CFTR-/- * *Genes:* gene name * *Percentage:* percentage of GBCs that express each gene ### **ORs\_Fig 8ab** Specific expression of 33 ORs in individual iOSNs and mOSNs. * *Genes:* gene name (Ensembl nomenclature) * *condition:* CFTR–/– or CFTR+/+ * *celltype:* immature or mature OSNs * *Cells_Expressed*: number of cells ### **OR51E2-1\_Fig 8d** Ratio of OR51E2/OR51E1 positive cells per section after dual fluorescent in situ hybridization with probes for the olfactory receptor genes OR51E2 and OR51E1 in the olfactory epithelium of CFTR+/+ (WT) and CFTR–/– (KO) piglets. Statistical table denotes Mann-Whitney U test for comparison of KO/WT conditions. ### **ORs\_Fig S1** Percentage of cells expressing ORs, excluding OSNs. * *Celltype:* type of cell * *Patient:* cystic fibrosis (CF) or control * *Percentage:* relative abundance of each cell type per condition ### **CFTR\_Fig S2** Quantification of olfactory cells expressing CFTR (&gt; 3 puncta/cell), indicating those with low (4- 7 puncta/cell) and high (&gt; 7 puncta/cell) CFTR expression (n = 4 donors: hs16, hs17, hs18 and hs13). Percentage of cells immunolabeled with PCNA, Ki67, NES, DCX, SOX2, OMP, and Gαolf. Percentage of cells labeled with PCNA, Ki67, NES, and DCX that were also positive for CFTR. Distribution of the number of puncta in olfactory cells labeled with the CFTR probe among 4 different donors (100 cells/donor analyzed). Relative gene expression assessed by quantitative RT-PCR of 10 marker genes (cftr, ascl3, pax6, omp, trpm5, neurod1, tp63, dcx, sox2, krt5) in human olfactory cells. ### **Latency\_Fig S3** Latency to stand-up right after birth expressed in seconds of CFTR–/– (KO) and control (WT) piglets. Statistical table denotes Mann-Whitney U test for comparison of KO/WT conditions. ### **RNA-seq\_Fig S4** Transcriptome analysis of the whole olfactory mucosa of CFTR–/– newborn piglets. Distribution of gene expression of the whole olfactory epithelium transcriptome (normalized counts), for both CFTR+/+ (WT) and CFTR–/– (KO) samples. List of the 20 most differentially expressed genes according to –log10 (p value), and log 2-fold expression change between CFTR+/+ and CFTR–/–. * *Geneid:* gene name (Ensembl) * *normalized counts:* RNA-seq counts after normalization * *Gene name:* gene name (annotated) * *log2FoldChange.ko-wt:* fold change in gene expression in CFTR–/– (ko) vs CFTR+/+ (wt). * *pvalue.ko-wt:* p-value of ko vs. wt comparison * *Log_(pvalue.ko-wt):* p-value expressed as -Log * *Function:* cell function type * *n/a:* unavailable information ### **snRNA-seq\_Fig S7** Percentage of olfactory cells expressing CFTR in CFTR+/+. Phase scoring shows an elevated G2M score in GBCs in both CFTR+/+ and CFTR–/– samples. Relative abundance of CFTR-expressing and CFTR-non-expressing olfactory cells in G2M vs. combined G1+S cycles in the CFTR+/+ cell sample. * *Celltype:* type of cell * *Percentage:* relative abundance of each cell type per condition * *CFTR expression:* CFTR-non expressing or CFTR-expressing * *G2M/G1S:* ratio G2M/G1S * *G2M.Score:* G2M score values * *Condition:* CFTR+/+ or CFTR-/- ### **ORs\_Fig S8** Expression analysis of 69 ORs identified by snRNA-seq found to be expressed in &gt;2 neurons, and expression profiles for each of the 198 identified ORs in both mature OSNs (mOSNs) and immature OSNs (iOSNs) for both *CFTR+/+ *and *CFTR–/– *samples. * *genes:* gene name (Ensembl) * *Condition:* CFTR+/+ or CFTR-/- * *Celltype:* iOSNs or mOSNs * *Cells_Expressed:* number of cells were each gene is expressed ## Code/software R is required to run scripts. Codes used for all analyses can be found in [https://github.com/ymbouamboua/Human_Pig_Olfactory_CF](https://github.com/ymbouamboua/Human_Pig_Olfactory_CF). ## Access information Other publicly accessible locations of the data: * Data Inrae Dataverse portal ([https://data.inrae.fr/](https://data.inrae.fr/)) * Bulk and Single-nuclei RNA-seq data generated in this publication have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE197187 and available in [https://www.genomique.info//data/public/hts/9258/8646_human_olfactory_cf.h5ad](https://www.genomique.info/data/public/hts/9258/8646_human_olfactory_cf.h5ad). Data was derived from the following sources: * n/a