J Immunol 197(11): 4228-4239

PMC: PMC5123825

PMID: 27794000

AIRWAY EPITHELIAL KIF3A REGULATES Th2- RESPONSES TO AEROALLERGENS

Mark Ericksen+4 authors

INTRODUCTION

Asthma is an increasingly common, complex pulmonary disorder causing reversible airflow obstruction, mucus hyperproduction, and inflammation. Both environmental and genetic factors are associated with risk of asthma. While allergic asthma is often associated with atopy and Th2 immune activation, expression of IL-13, IL-4, and IL-5, and eosinophilic pulmonary inflammation (1), there is substantial heterogeneity in asthma phenotypes among patient populations. For example, Th1 activation is associated with obesity and asthma (2). Th2-related asthma patients can be further subdivided on the basis of increased Th2 cytokine and periostin expression, neutrophilic inflammation and responses to inhaled steroids, indicating that the heterogeneity of asthma phenotypes is highly relevant to therapeutic responses (3–5). Environmental exposures to pollutants, respiratory viruses, allergens, bacterial and fungal products are known to play important roles in the pathogenesis of asthma (6, 7, for review). Likewise, a number of genetic factors influence susceptibility and severity of asthma. Extensive genome-wide analyses from diverse populations identified numerous haplotypes and chromosomal loci associated with asthma and atopy, both allergic rhinitis and eczema being closely associated with increased risk of childhood asthma. Genome Wide Association Studies identified increased susceptibility to asthma associated with variations in genes controlling inflammation, including genes regulating Th2 lymphocyte recruitment and activation; for example, IL-33, ST2, TSLP, ORMDL3, GSDML, ADAM33, SPINK5, and others, that were associated with asthma susceptibility (8, 9). Polymorphisms in FCεRI-β, a high affinity receptor for IgE on mast cells, was linked to childhood asthma and allergic dermatitis (10).

The human KIF3A gene locus has been repeatedly implicated in susceptibility to asthma and eczema (11–16). KIF3A is a component of a trimeric motor complex regulating microtubular function and transport and is required for formation and function of both motile cilia and non-motile primary and sensory cilia (17, 18). KIF3A plays pleotropic roles in the regulation of microtubular transport, influencing intracellular protein trafficking, as well as ciliary transport and function (17–19). Genomic deletion of Kif3a in the mouse is embryonic lethal (20–22). In the lung, motile cilia occur as clusters on apical surfaces of ciliated cells that coordinate mucociliary clearance in the airways. Whereas, primary cilia are singular, non-motor organelles present on many cell types, including pulmonary cells, that are known to mediate signal transduction through diverse signaling pathways including Shh, Wnt, Pdgf and others, influencing morphogenesis, homeostasis, and repair of many organs (23–26). In various experimental models, roles for KIF3A in the regulation of cell proliferation, apoptosis, differentiation, intracellular transport, cytoskeletal dynamics, and planar polarity have been demonstrated (27–31).

While KIF3A gene polymorphisms have been correlated with asthma, allergic rhinitis, and eczema, cellular mechanisms underlying this association are unknown. In the present study, we selectively deleted the mouse Kif3a gene in airway epithelial cells. Loss of Kif3a enhanced pulmonary inflammation, airway hyper-responsiveness (AHR), and Th2-mediated inflammation following aeroallergen challenge with Aspergillus fumigatus and house dust mite extracts. KIF3A was required for mucociliary clearance, epithelial cell migration and repair, providing plausible mechanisms by which KIF3A influences susceptibility to asthma.

MATERIALS AND METHODS

Mice

Animal protocols were approved by the Institutional Animal Care and Use Committee in accordance with NIH guidelines. Kif3a^fl/^{mice, generated by Marszaleck et al and Lin et al. (}20, 21), and Ift88^fl/fl mice generated by Haycraft et al. (32) were kindly provided by Samantha A. Brugmann (Department of Plastic Surgery, Cincinnati Children’s Hospital Medical Center). Scgb1a1-Cre mice were kindly provided by Dr. Steven Shapiro (33). Gt(ROSA)26Sor^{tm4(ACTB-tdTomato,-EGFP)Luo}^andShh^/J (34) mice were purchased from Jackson Laboratories.

Naphthalene Injury

Mice were administered naphthalene (Sigma, 30 mg/ml in corn oil) via a single i.p. injection to deliver a dose of 275 μg/g body weight. Control animals were injected with corn oil. Animals were sacrificed at 2, 7, and 10 days post administration.

Aspergillus and House Dust Mite (HDM) Extract Sensitization

Anesthetized 6–8 week old mice were administered a dose of 10 μg Aspergillus fumigatus or house dust mite extract (25μg) (Greer Laboratories, Lenoir, NC) diluted in 50 μl of saline by intratracheal (i.t.) instillation 3 times weekly for 3 weeks. Control animals were dosed with saline. Mice were sacrificed 48 hs following the last exposure.

AHR/flexiVent

Airway responsiveness of anesthetized mice was assessed with a flexiVent apparatus (SCIREQ, Montreal, QC, Canada) using an invasive method. Mice were anesthetized and the tracheas cannulated with an 18-gauge blunt needle. Mice were ventilated at 150 breaths/min, 3.0 cm water positive end-expiratory pressure. Two total lung capacity perturbations were performed for airway recruitment before baseline measurement and subsequent methacholine (MCh) challenges were performed. Acetyl-α-methacholine chloride (Sigma, St. Louis, MO) was administered for 10s (60 breaths/min, 500 μl tidal volume) in increasing concentrations (12.5, 25 and 50 mg/ml) via nebulization via the tracheotomy. Dynamic resistance (R) was determined by fitting the data to a single compartment model of airway mechanics where Ptr = RV + EV+ PO (Ptr, tracheal pressure; V, volume; and PO, constant). Average of three highest R-values with a coefficient of 0.9 or greater was used to plot the dose-response curves.

BALF

Bronchoalveolar lavage fluid (BALF) was collected from the right lobes by lavage (3 times with 0.7 ml of normal saline). BALF was centrifuged and cell pellets were resuspended in HBSS and total cell numbers counted using a hemocytometer. Cytospins were prepared and stained with a Diff-Quik Staining Kit (Polysciences Inc.) to determine differential cell counts.

Histology and immunohistochemistry

Mouse lungs were inflation fixed in 4% paraformaldehyde (PFA) in PBS overnight. Fixed tissue was processed according to standard protocols for paraffin embedding and used for chemical or immunohistochemical staining. Depending on the primary antibody some sections were subjected to antigen retrieval using citrate buffer pH 6.0 or 10 mM Tris-EDTA buffer pH 9.0 with heating. For in vitro studies, cells grown on ibiTreat chambered coverslips (Ibidi Inc) were fixed with 4% PFA/PBS for 15 minutes and permeabilized with 0.2% Triton-X-100 in PBS for 7 minutes at room temperature. Primary antibodies were applied overnight at 4ºC. Primary antibodies used were acetylated tubulin (Sigma T7451, 1:3000), ARL13B (Proteintech 17711-1-AP, 1:200), E-cadherin (Cell Signaling 3195, 1:100), FOXJ1 (eBioscience 1409965-82, 1:200), FOXA3 (Santa Cruz sc-5361, 1:50), MUC5AC (Abcam Ab3649, 1:100), SCGB1A1 (CCHMC, 1:800), ACTA2 (Sigma A5228, 1:2000), TUBB4A (Biogenex MU178-UC, 1:200), Alpha tubulin (Sigma T6199, 1:200), and Phospho-Histone H3 (Santa Cruz, sc-8656-R, 1:100). FOXJ1 and FOXA3 were detected with species-specific biotinylated secondary antibodies and then visualized with a streptavidin-conjugated fluorophore. For all other antibodies, fluorophore-conjugated secondary antibodies were used including Alexa Fluor-488, Alexa Fluor-555, Alexa Fluor-568, Alexa Fluor-594, and Alexa Fluor-647 (Jackson ImmunoResearch and Life Technologies). For fluorescence stains, sections were stained with DAPI and mounted with ProLong Gold anti-fade reagent (Life Technologies). Bright-field images were obtained using a Zeiss Axio ImagerA2 microscope equipped with AxioVision Software. Confocal immunofluorescence images were obtained using a Nikon A1Rsi inverted laser confocal microscope and widefield images were obtained using a Nikon Ti-E inverted microscope equipped with an Andor Zyla 4.2 SCMOS camera. Images were analyzed using NIS Elements (Nikon) or Imaris (Bitplane) software.

Scanning electron microscopy

Three mouse lungs from each genotype were inflation fixed with ice cold 2% paraformaldehyde, 2% glutaraldehyde in 0.1M sodium cacodylate buffer (SCB), pH 7.3, for 30 min, followed by postfixation with fresh fixative at 4°C overnight. Fixed mouse lungs were sliced into 1–2 mm slabs, incubated with 1% osmium and 1.5% potassium ferrocyanide in 0.1M SCB, pH 7.3, for 2 hrs, dehydrated in a graded series of alcohol, washed with hexamethyldisilazane, and air dried in a chemical fume hood for up to 2 days. Mouse lung tissue slabs were mounted on specimen stubs and coated with palladium/gold film using a Denton Vacuum Desk IV sputter coater. Scanning electron micrographs of murine airways were acquired using a Hitachi field emission scanning electron microscope SU8010 at 5 kV.

Mouse trachea and airway sample preparation and video microscopy for ciliary beat and mucociliary clearance

Three mice of each genotype (control, heterozygote, or homozygote) from the Kif3a^Shh or Kif3a^Scg lines were analyzed. Tracheas and lungs from the Kif3a^ScgΔ/Δ mice were prepared as described (35). Tracheas from Kif3a^ShhΔ/Δ mice and controls were removed, washed, and prepared according to Francis and Lo (36). For clearance assays, samples were placed on a glass dish in medium containing 0.20-μm Fluoresbrite microspheres (Polysciences, Inc., Warrington, PA, USA). Ciliary dynamics were captured with a 40X objective using Differential Interference Contrast (DIC) on a Nikon Ti-E inverted microscope (Nikon Microscopy) equipped with a Andor Zyla 4.2 SCMOS camera or with the 60X objective using DIC on an Olympus 1X51 inverted microscope equipped with a Hamamatsu EM-CCD digital camera. Images up to 500 frames/s (fps) were recorded. To quantitate ciliary beat frequency (CBF) and cilia-generated flow, at least two videos were collected from each tracheal sample (n=3/genotype). Using ImageJ software (ImageJ, NIH), a line was marked perpendicular to the cilia captured in each video and a kymograph was created. The number of pixels between each wave peak was measured (one pixel = one movie frame) from which the number of beats per minute (i.e. Hz) was calculated. To measure cilia generated flow, MTrackJ ImageJ software was used to manually track fluorescence beads across the surface of the tracheal epithelia.

RNA isolation and analysis

Total RNA was isolated from snap-frozen left lung lobes using a tissue-homogenizer (OMNI-TH International, Kennesaw, GA) by pulsing the probe for about 20 seconds in 1 ml of TRIzol Reagent (Life Technologies) and extracting the RNA using DirectZOL RNA Miniprep R2072 (Zymo Research, CA, USA). RNA was isolated from purified epithelial cells and cells in culture using the Qiagen RNeasy Micro Kit, 74004 (Valencia, CA). RNA cleanup and on-column DNase digestion (Qiagen RNeasy Micro kit, Valencia, CA) was performed on samples before being reverse transcribed either with the First Strand Superscript Synthesis kit (Invitrogen) or iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR analysis was performed on cDNA samples using TaqMan probes (Applied Biosystems) (Table I) and EUK 18S rRNA (4352930), FoxJ1 (Mm00807215_m1), Arl13b (Mm01349328_m1), Kif3a (Mm01288585_m1), KIF3A (Hs00199901_m1), and CDH1 (Hs01023894_m1) on a ABI 2720 Thermal cycler (Applied Biosystems, NY, USA).

Table I

Increased expression of RNAs related to Th2 inflammation and goblet cell differentiation

qRT-PCR was performed on whole tissue RNA collected from the right lungs of mice sensitized with Aspergillus extract. Fold change in mRNA levels of Kif3a^ScgΔ/+ and Kif3a^ScgΔ/Δ compared to Kif3a^fl/fl and Ift88 gene deletants compared to Ift88^fl/fl control mice is shown after normalization to 18S. Age-matched mice (n=6–7) per group are shown. Il17A RNA was barely detectable at baseline, and was readily detected in both Kif3a deletants.

Gene	Taqman Probe I.D.	Fold Increase Relative to Controls (Mean ± SEM)
		Kif3a^ScgΔ/+	Kif3a^ScgΔ/Δ	Ift88^ScgΔ/Δ
Acta2	Mm00725412_s1	1.7±0.6	3.3±1.2	5.5±3.4
Ccl11	Mm00441238_m1	36.4±21.1^B	171.1±144.1 ^B	0.3±0.1
Ccl17	Mm00516136_m1	7.6±2.8	21.3±17.0	3.2±1.1
Ccl2	Mm00441242_m1	5.5±3.2	2.3±1.0	0.7±0.3
Ccl24	Mm00444701_m1	15.5±9.1	6.0±3.0	2.5±1.1
Cdh1	Mm00480906_m1	1.1±0.3	1.5±0.3	1.3±0.3
Clca1	Mm00777368_m1	2.4±0.6	14.4±11.2 ^A	2.5±0.9
Csf2	Mm01290062_m1	1.2±0.5	2.5±1.2	4.9±2.2
Cxcl1	Mm04207460_m1	1.6±0.7	4.1±1.9	5.0±2.4
Ear11	Mm00519056_s1	18.4±8.0 ^A	26.7±6.5 ^A	4.7±2.7
Foxa3	Mm00484714_m1	2.5±0.5	18.7±14.3 ^A	11.4±4^A
Ifnγ	Mm01168134_m1	3.8±0.6	3.3±1.09	5.3±2.5
Il4	Mm00445259_m1	6.7±1.4 ^A	29.6±21.7 ^A	1.8±0.4
Il13	Mm00434204_m1	8.9±2.6^A	25.4±11.4 ^A	1.9±0.5
Il17A	Mm00439618_m1	78.7±52.6^C	10.3±3.4^D	3.5±1.2
Il33	Mm00434204_m1	2.3±0.6	3.3±0.9	2.1±1.1
Il5	Mm00439646_m1	3.4±0.6	9.5±6.6	2.2±0.7
Il6	Mm00446190_m1	4.4±2.7	2.7±0.7	3.8±1.2
Muc5ac	Mm01276725_g1	4.1±2.1	8.5±5.6	3.1±1.8
Muc5b	Mm00466391_m1	5.3±2.8	6.1±2.1 ^A	6.8±2.9 ^A
Spdef	Mm00600221_m1	4.7±1.1 ^A	10.8±5.9 ^A	7.2±3.7 ^A
Tslp	Mm01157588_m1	2.1±0.8	2.2±0.9	4.3±2.2

^{P<0.05 and}

^{P<0.01by one-way ANOVA followed by Bonferroni’s multiple comparison test.}

^{P=0.024 by ANOVA on Ranks.}

^{P=0.0388 by 2-tailed t-Test.}

Lung cell isolation and EpCAM sorting

Lung tissues were enzymatically digested at 37°C for 1h using Dispase (Corning, Discovery Labware, Inc., Bedford, MA). After 1 hour, DNasel was added prior to passing the solution through a 19-gauge needle to remove tissue aggregates. Cells were re-suspended in MACS buffer and cell counts determined. For every 1X10^{cells of lung suspension, 10 μl of EpCAM-biotin antibody (CD326 EpCAM-biotin, 130-101-859, MACS Miltenyl Biotec) was added. Cells were incubated with primary antibody for 15 min, 4°C, washed and re-suspended in MACS buffer, and incubated with the secondary antibody (streptavidin conjugated microbeads 130-048-102) for selection of epithelial cells. EpCAM positive cells were collected by magnetic separation using an AutoMACS (Miltenyl Biotec).}

In vitro assays

HBEC3 cells (a kind gift of Dr. John D. Minna, UT Southwestern Medical Center) and BEAS2B cells were grown to confluence on 12-well tissue culture plates (TPP plasticware). At 48 hs post-plating, the cells were infected with lentiviral constructs expressing shRNA against KIF3A or scramble controls (MOI of 2) in the presence of polybrene (2 μg/ml, Sigma Aldrich) to determine cell migration and motility. The cells were stained at 72 hours post transduction with di-8-ANEPPS (Biotium) diluted 1:500 in culture-media. A scratch was made in each well of the plate using a p200 pipet tip. The cells were washed with 1X PBS and fed with culture media containing di-8-ANEPPS. For rescue experiments, HBEC3 transduced with lentiviral cells or scramble control shRNA were transfected with a construct expressing the full-length KIF3A cDNA (pCMV3-C-GFPSpark, Sino Biological Inc., Bejing, P.R. China) using Fugene HD (Promega) transfection reagent at 72 hours post transduction. Rescue of cell migration defects was assessed at 48 hours post-transfection. A Nikon A1Rsi inverted laser scanning confocal microscope, equipped with a motorized XY stage, Tokai Hit microplate incubator and Perfect Focus System was used to document cell migration. Cells were imaged at pre-selected XY coordinate points in each well, once every 10 minutes over a period of 16-22 hours. Cells were harvested and mRNA was isolated using Qiagen RNeasy Micro Kit (Qiagen) for qRT-PCR analysis. Cell migration was analyzed using a spot tracking algorithm on Imaris software (Bitplane).

Flow cytometric analysis of lung cells

For flow cytometric analysis, total lung cell suspensions were prepared as previously described (37) from Aspergillus fumigatus extract treated mice injected with Brefeldin A 16 hs prior to sacrifice. Lung tissues were enzymatically digested using Caseinolytic units of Dispase (Corning, Discovery Labware, Inc., Bedford, MA) to obtain single cell suspensions and stained using fluorochrome-labeled antibodies. The gating strategies used are described in Rajavelu et al (38). Eosinophils in lung cell suspensions were detected using SiglecF PE (BD Pharmingen E50-2440) and CCR3 FITC (BioLegend J073E5) positive cells in the CD45 leucocyte gate. For analysis of ILC2 cells the lineage cocktail CD3/Ly-6G(Ly-6C)/CD11b/CD45R/Ter-119 Alexa Fluor 700 (BioLegend 79923), IL-7Rα FITC (BioLegend A7R34), FLT3 APC (BioLegend A2F10), ST2 (BioLegend DIH9), IL-17RB (R&D 752101), and ICOS Pacific Blue (BioLegend C398.4A) were used. T cells were analyzed using CD45 Alexa Fluor 700 (BioLegend 30-F11) and CD3εFITC (BioLegend 145-2C11). For intracellular cytokine detection, mice were i.p. injected with Brefeldin A (Sigma) and lung lobes were processed for single cell suspension as mentioned above followed by surface staining, then permeabilized with Cytofix-Cytoperm solution (BD Pharmingen) and stained for IL-4 PE/Cy7 (BioLegend 11B11) and IL-17A PerCP/Cy5.5 (BioLegend TC11-18H10.1). The stained lung cell samples were acquired and analyzed on a Becton Dickinson FACSCanto III flow cytometer using FACS Diva software.

In vivo capillary-epithelial permeability

Experiments were performed as described by Davidovich, et al (39). In brief, 24 hours after i.t. exposure to Aspergillus extract (100 μg) or saline, mice were anesthetized by isoflurane and 0.3 ml of a 12 mg/ml solution of FITC-conjugated albumin (A9771; Sigma-Aldrich, St. Louis, MO) was injected via tail vein. At the end of 3 hours, blood was collected via direct cardiac puncture and BAL performed with 3 ml of normal saline. Albumin fluorescence in BALF and serum was determined using Biotek Synergy 2, Biotek Instruments Inc., Vermont USA) with absorption/emission wavelengths of 480/520 nm. Epithelial permeability was defined as the ratio of BALF to serum fluorescence.

Statistical analysis

Values are expressed as the mean ± SEM. Statistical analysis was performed using GraphPad Prism 6 software (GraphPad, La Jolla, CA). Data were analyzed using a 2-tailed, unpaired Student’s t test or 2-way ANOVA, and Bonferroni’s correction was used for multiple comparisons. P values of 0.05 or less were considered statistically significant.

Mice

Naphthalene Injury

Aspergillus and House Dust Mite (HDM) Extract Sensitization

AHR/flexiVent

BALF

Histology and immunohistochemistry

Scanning electron microscopy

Mouse trachea and airway sample preparation and video microscopy for ciliary beat and mucociliary clearance

RNA isolation and analysis

Table I

Increased expression of RNAs related to Th2 inflammation and goblet cell differentiation

Gene	Taqman Probe I.D.	Fold Increase Relative to Controls (Mean ± SEM)
		Kif3a^ScgΔ/+	Kif3a^ScgΔ/Δ	Ift88^ScgΔ/Δ
Acta2	Mm00725412_s1	1.7±0.6	3.3±1.2	5.5±3.4
Ccl11	Mm00441238_m1	36.4±21.1^B	171.1±144.1 ^B	0.3±0.1
Ccl17	Mm00516136_m1	7.6±2.8	21.3±17.0	3.2±1.1
Ccl2	Mm00441242_m1	5.5±3.2	2.3±1.0	0.7±0.3
Ccl24	Mm00444701_m1	15.5±9.1	6.0±3.0	2.5±1.1
Cdh1	Mm00480906_m1	1.1±0.3	1.5±0.3	1.3±0.3
Clca1	Mm00777368_m1	2.4±0.6	14.4±11.2 ^A	2.5±0.9
Csf2	Mm01290062_m1	1.2±0.5	2.5±1.2	4.9±2.2
Cxcl1	Mm04207460_m1	1.6±0.7	4.1±1.9	5.0±2.4
Ear11	Mm00519056_s1	18.4±8.0 ^A	26.7±6.5 ^A	4.7±2.7
Foxa3	Mm00484714_m1	2.5±0.5	18.7±14.3 ^A	11.4±4^A
Ifnγ	Mm01168134_m1	3.8±0.6	3.3±1.09	5.3±2.5
Il4	Mm00445259_m1	6.7±1.4 ^A	29.6±21.7 ^A	1.8±0.4
Il13	Mm00434204_m1	8.9±2.6^A	25.4±11.4 ^A	1.9±0.5
Il17A	Mm00439618_m1	78.7±52.6^C	10.3±3.4^D	3.5±1.2
Il33	Mm00434204_m1	2.3±0.6	3.3±0.9	2.1±1.1
Il5	Mm00439646_m1	3.4±0.6	9.5±6.6	2.2±0.7
Il6	Mm00446190_m1	4.4±2.7	2.7±0.7	3.8±1.2
Muc5ac	Mm01276725_g1	4.1±2.1	8.5±5.6	3.1±1.8
Muc5b	Mm00466391_m1	5.3±2.8	6.1±2.1 ^A	6.8±2.9 ^A
Spdef	Mm00600221_m1	4.7±1.1 ^A	10.8±5.9 ^A	7.2±3.7 ^A
Tslp	Mm01157588_m1	2.1±0.8	2.2±0.9	4.3±2.2

^{P<0.05 and}

^{P<0.01by one-way ANOVA followed by Bonferroni’s multiple comparison test.}

^{P=0.024 by ANOVA on Ranks.}

^{P=0.0388 by 2-tailed t-Test.}

Lung cell isolation and EpCAM sorting

In vitro assays

Flow cytometric analysis of lung cells

In vivo capillary-epithelial permeability

Statistical analysis

RESULTS

Kif3a mRNA is widely expressed in many organs and tissue types and is widely expressed during fetal and postnatal lung development (40). In the developing mouse lung, Kif3a is expressed in multiple cell types, including cells of epithelial and mesenchymal origin. Since KIF3A is required for formation and function of both motile and primary cilia, we assessed the immunofluorescence staining of ARL13B and TUBB4A (β-tubulin-IV) during perinatal and postnatal lung development in the mouse. Primary cilia were detected in both mesenchymal and epithelial cells in the fetal lung at E13 and E16.5 as identified by expression of ARL13B and lack of TUBB4A (Figs. 1A, ,2A);2A); whereas, by E18.5 and postnatally, motile cilia co-expressing both proteins were readily detectable (Fig. 1B, 1C). The presence of abundant motile cilia in conducting airways made impossible the detection of potential primary cilia in multi-ciliated cells after E16.5 (Fig. 1C) (41). Primary cilia were not detected in club cells in the normal postnatal lung, consistent with previous findings (41).

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Figure 1

Conditional deletion of Kif3a in the airway epithelium

At E16.5, primary cilia are detected by ARL13B staining in the fetal lung epithelium and mesenchyme (red arrows) and absence of TUBB4A (A). At E18.5 (B) and P07 (C), the presence of motile cilia (staining for ARL13B and TUBB4A) obscures detection of primary cilium. ARL13B staining was not detected in SCGB1A1 stained club cells (scale bars equal 50 μm). eGFP in Rosa-Tomato Red/Green reporter mice (D) were used to assess the extent of recombination by Scgb1a1-Cre at 8 weeks of age. Cilia were stained for TUBA1A (green) in Kif3a^fl/fl control mice (E). Cilia were lacking after deletion of KIF3A in Kif3a^ScgΔ/Δ mice (F). FOXJ1 staining was unchanged (red, E, F). Number and distribution of club cells staining with SCGB1A1 (white) were not altered in Kif3a^ScgΔ/Δ airways. qRT-PCR on whole lung cDNA from adult mice (G–H), n=6 per genotype, demonstrated decreased Arl13b mRNA (**P<0.01 by t-test compared to controls), consistent with loss of Kif3a.

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Figure 2

Loss of primary cilia, ciliary beating, and mucociliary clearance in Kif3a^/^Δ mice

Kif3a was deleted under control of Shh-Cre. At E13, primary cilia, identified by ARL13B staining, were absent in Kif3a^ShhΔ/Δ airway epithelial cells (B, arrows), compared to Kif3a^Shh+/+ embryos (A, arrowheads). Figures are representative of n=3/genotype. Staining of ciliated (FOXJ1 positive nuclei, pink arrows) and club (SCGB1A1, white) cells is shown in the bronchiolar epithelial cells in adult airways (C, D), n=4/genotype. Kif3a mRNA levels were evaluated using exon-2 specific primers. A decrease in Kif3a mRNA was detected in EpCAM (+) but not EpCAM(-) sorted cells from adult Kif3a^/^Δ mice, n=4/genotype, P <0.05 by t-test) (E–F). Motile cilia were identified in control, Kif3a^Shh+ and Kif3a^ShhΔ/Δ airways by scanning electron microscopy (G–I). Uniformly short cilia were observed in Kif3a^ShhΔ/Δ airways (n=3/genotype). Tracheas of adult mice were excised, perfused, and differential interference contrast images viewed longitudinally after introduction of fluorescent microspheres (0.20 μm). Movements of individual beads were followed by video-microscopy. Cilia beat frequency (CBF) was quantified (J). Cilia-generated flow was imaged by tracking the fluorescent microspheres (K). Data represent the mean ± SEM of n=3 mice per group, ***P < 0.001 by one-way ANOVA.

We utilized mice bearing Scgb1a1-Cre or Shh-Cre to selectively delete exon 2 of the Kif3a gene. Scgb1a1-Cre is expressed early during the differentiation of conducting airway epithelial cells (approximately E16–17). As indicated by expression of eGFP in Rosa-Tomato (Red/Green) reporter mice, Scgb1a1-Cre caused extensive recombination in both ciliated and non-ciliated airway epithelial cells at 8 weeks of age (Fig. 1D); recombination was not observed in alveolar regions. Using this driver, the homozygous gene deleted mice Kif3a^;Scgb1a1-Cre⁺ (Kif3a^ScgΔ/Δ) and heterozygous Kif3a^Scgb1a1-Cre⁺ (Kif3a^ScgΔ/+^{mice were generated. Controls were either}Kif3a^fl/fl or Kif3a^;Scgb1a1-Cre⁺ (Kif3a^Scg+/+). Shh-Cre is expressed throughout the developing lung epithelium beginning as early as E9.5 (42) resulting in targeting of the Kif3a^fl alleles in most airway epithelial progenitor cells in proximal and peripheral conducting airways and in the alveoli; henceforth termed Kif3a^ShhΔ^{^Δ} and Kif3a^ShhΔ/+ and the corresponding controls Kif3a^fl/fl or Kif3a^Shh+/+. Both Kif3a^{and Kif3a}^ShhΔ/Δ mice were viable and present in normal Mendelian ratios after birth. The number and distribution of club, goblet, and cells staining for FOXJ1, a transcription factor selectively expressed in ciliated cells, were similar in Kif3a^fl/fl, Kif3a^ScgΔ/+ and Kif3a^ScgΔ/Δ, and Kif3a^ShhΔ/Δ mice (Figs. 1E, 1F, 2C, 2D). QRT-PCR demonstrated that Foxj1 mRNA levels were unchanged, while Arl13b was significantly decreased in adult mice (Fig. 1G, 1H). A marked decrease in staining of acetylated α-tubulin (TUBA1A) was observed in Kif3a^ScgΔ/Δ and Kif3a^ShhΔ/Δ mice while expression of SCGB1A1 persisted in the club cells (Figs. 1E, 1F, 2C, 2D, and data not shown). As an indication of the loss of KIF3A, the presence of primary cilia was evaluated in Kif3a^ShhΔ/Δ mice. ARL13B staining revealed a dramatic reduction in the number of primary cilia in the developing lung epithelial cells, whereas, no changes were detected in the adjacent mesenchyme (Fig. 2A, 2B). Cells disassociated from adult whole lungs isolated from Kif3a^ShhΔ/Δ and Kif3a^Shh+/+ were sorted using the epithelial marker EpCAM. Exon 2 specific primers were used to demonstrate a reduction in Kif3a mRNA in Kif3a^ShhΔ/Δ cells (Fig. 2E, 2F). Consistent with the reduction of acetylated tubulin staining, numbers, size, and shape of motile cilia were reduced in Kif3a^ShhΔ/+ and Kif3a^ShhΔ/Δ mice by scanning EM (Fig. 2G–I). Abnormalities in cilia were most prominent in the homozygous Kif3a^ShhΔ/Δ mice. To assess mucociliary clearance after deletion of Kif3a, video imaging of tracheal and large airway fluid dynamics and ciliary activity were measured in Kif3a^ShhΔ/Δ, Kif3a^ScgΔ/Δ and control mice. Consistent with the extensive loss of motile cilia from the airway epithelium, ciliary beat frequencies were markedly decreased in airways of both Kif3a^ShhΔ/+ and Kif3a^ShhΔ/Δ mice and fluorescent bead movement lost directionality (Fig. 2J, 2K, Video1.mov and Video2.mov). Taken together, these observations indicate that primary cilia are not required for cell fate specification of the airway epithelial cells and that deletion of Kif3a by either Shh-Cre or Scgb1a1-Cre (data not shown) inhibits ciliary function and inhibits mucociliary clearance.

Enhanced Th2 Inflammation and AHR in Kif3a Gene Targeted Mice After Aeroallergen Exposure

To assess the role of Kif3a during aeroallergen sensitization, adult Kif3a^Scg+/+, Kif3a^fl/fl, Kif3a^ScgΔ/+, and Kif3a^ScgΔ/Δ mice were repeatedly treated with relatively low concentrations of Aspergillus fumigatus or HDM extract. At baseline, under our pathogen-free vivarium conditions, no histological evidence of pulmonary inflammation was observed in Kif3a^ScgΔ/Δ and Kif3a^ScgΔ/+ mice, nor were there differences in AHR or inflammatory responses following exposure to saline (Fig. 3). Following intratracheal Aspergillus fumigatus extract sensitization, similar AHR and inflammatory responses were observed in control Kif3a^fl/fl and Kif3a^Scg+/+ mice. In contrast, AHR and the inflammatory responses were significantly increased in Kif3a^ScgΔ/Δ and Kif3a^ScgΔ/+ mice (Fig. 3). AHR was most increased in homozygous Kif3a deleted mice (Fig. 3A). Numbers of inflammatory cells, consisting primarily of eosinophils, were increased in the BALF from both Kif3a^ScgΔ/+ and Kif3a^ScgΔ/Δ mice after aeroallergen challenge (Fig. 3B, C). We observed no differences in plasma levels of Aspergillus specific IgG1 in the exposed mice of each genotype (Suppl. Fig. 1A). Goblet cell metaplasia and mucus hyper-production were observed in mice of all Kif3a genotypes after exposure to Aspergillus fumigatus extract and were more severe in the Kif3a^ScgΔ/Δ and Kif3a^ScgΔ/+ mice, (Fig. 3D–F, Suppl. Fig. 1B). Consistent with these findings, AHR was significantly increased in Kif3a gene deleted mice after house dust mite exposure (Suppl. Fig. 2).

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Figure 3

Increased AHR and eosinophilic inflammation in Kif3a gene deleted mice

Age-matched mice were sensitized with Aspergillus fumigatus antigen or saline. AHR is represented as resistance in response to methacholine. Aspergillus treated mice are represented as solid lines and saline treated mice as dashed lines (A). Total number of cells in BALF was determined (B). Eosinophil numbers were calculated (C). Data represent the mean ± SEM of 6–7 mice per group, *P < 0.05, **P < 0.01 and ***P < 0.001 by 2-way ANOVA. Increased inflammation and mucus were present in both Kif3a gene deletants shown by representative 40X tile scans after Alcian blue staining (D–F).

The increased inflammatory responses to Aspergillus fumigatus extract observed in the Kif3a deleted mice were supported by mRNA expression data, Table I. Histological and immunofluorescence studies demonstrating increased goblet cell metaplasia after exposure to Aspergillus extract were supported by increased expression of Spdef, Foxa3, Clca1, and Muc5b mRNAs in lungs from Kif3a^ScgΔ/Δ mice. Likewise, Il-13, Il-4, Il-17A, Ccl11, and Ear11 mRNAs were significantly increased in lungs of both Kif3a^ScgΔ/Δ and Kif3a^ScgΔ/+ mice, indicating Th2-mediated eosinophilic inflammation (Table I and Fig. 3D–F). We utilized flow analysis to identify the inflammatory cells present following Aspergillus extract administration to Kif3a^ScgΔ/Δ mice. Numbers of SiglecF^/CCR3^{cells in whole lung digests were increased in lungs of}Kif3a^ScgΔ/Δ mice, consistent with the increased eosinophils seen in the BALF; likewise, IL-4^{and IL-17A}^{T cells were increased, consistent with enhanced Th2 and Th17 responses in}Kif3a gene deleted mice (Fig. 4).

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Figure 4

Deletion of Kif3a in airway epithelial cells increased recruitment of IL4^{and IL17}^{T cells}

Flow cytometric analysis of lung cells from Kif3a^fl/^{(■) and}Kif3a^ScgΔ/Δ (●) mice after i.t. administration of Aspergillus fumigatus extract (10 μg, 9 times during a two-week period). Numbers of CD45^{leucocytes (A) and CD3}^{T cells (B). Eosinophilic inflammation was assessed by SiglecF}^/CCR3^{cells (C). Numbers of IL4}^{and IL17}^CD3^{T cells (D and E) were significantly increased in}Kif3a^/^Δ mice. ILC2 (Lin^/IL7Rα^/ICOS^/ST2^/IL17RB^{) cell numbers in}Kif3a^fl/fl and Kif3a^/^Δ were unaltered (F). Graphs represent mean ± S.E.M., * p<0.05, **p<0.005, ns (not significant), compared to controls using one way ANOVA, n= 3 animals per genotype.

Like Kif3a, Ift88 (intraflagellar transporter protein 88) is a microtubule associated transport protein required for both primary and motile cilia formation in airway epithelial cells (43, 44). The Scgb1a1-Cre and Shh-Cre transgenes were bred into the Ift88^fl/fl animals to create Ift88 gene deleted lung epithelium. At baseline, histological findings in the Ift88^ScgΔ/Δ and Ift88^ShhΔ/Δ mice were similar to those seen after epithelial deletion of Kif3a in which specification of lung epithelial cells and absence of motile cilia were observed (Fig. 5A–D, data not shown). Ift88^fl/fl and Ift88^ScgΔ/Δ were exposed to Aspergillus fumigatus or saline using the same sensitization protocol used with the Kif3a gene targeted mice. Pulmonary eosinophilic inflammation and Alcian blue staining of airway cells were similar in Ift88^ScgΔ/Δ and controls after exposure to Aspergillus extract (Fig. 5E–H). While an increase in mRNAs associated with goblet cells was observed in Ift88^ScgΔ/Δ mice after exposure, neither Ear11 or Th2 related cytokine mRNAs were increased to the levels seen in the Kif3a deleted mice (Table I). AHR was not evaluated in these animals since Ift88^ShhΔ/Δ mice were found to have hyper-reactive airways at baseline (43). Taken together, both Kif3a^ScgΔ/+ and Kif3a^ScgΔ/Δ mice were more susceptible to Th2-mediated eosinophilic pulmonary inflammation than the Ift88^ScgΔ/Δ mice indicating a distinct role for Kif3a in asthma-like pulmonary inflammation.

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Figure 5

Response to Aspergillus sensitization in Ift88^/^Δ mice

ac-TUBA1A (green) stained cilia in Ift88^fl/fl controls (A) were nearly absent in Ift88^ScgΔ/Δ mice (B). FOXJ1, a transcription factor expressed in ciliated cells, was unchanged (B). Number and distribution of club cells staining for SCGB1A1 (white) were not altered in Ift88^ScgΔ/Δ airways (A–D). IFT88 staining (green) was nearly absent in Ift88^ScgΔ/Δ mice (C vs D). Age-matched mice (6–8 weeks at initial sensitization) were sensitized 3 times per week for 3 weeks with 10 μg Aspergillus fumigatus antigen or saline by i.t., (n=6–7 mice per genotype). Alcian blue staining of whole left lobes was unchanged (E–F). The number of cells in BALF was not altered (G). Eosinophil numbers were calculated from total numbers of BALF cells and differential cell counts and were not statistically different (H) as determined by one-way ANOVA.

KIF3A is Required for Epithelial Repair

Since primary cilia play important roles in the regulation of proliferation, migration, and differentiation, we assessed airway epithelial repair by treating adult mice with naphthalene. Naphthalene is metabolized to a cellular toxicant by CYP2F2, a p450 enzyme that is selectively expressed in most non-ciliated conducting airway cells in the mouse lung (45, 46). CYP2F2 was normally expressed by club cells in Kif3a deficient airways. After naphthalene exposure, resistant progenitor cells rapidly migrate, proliferate, and differentiate to repair the airways. Seven days after naphthalene exposure, areas of damaged airway were present in both the control and Kif3a gene deleted airways (Fig. 6A). In control mice, the distribution of both ciliated and epithelial club cells was restored 10 days after naphthalene injection (Fig. 6B–D). Repair of the airway epithelium in Kif3a^ScgΔ^{^Δ} mice was incomplete, as indicated by significantly decreased numbers of club cells and squamous cell metaplasia, demonstrating that KIF3A was required for normal repair of the conducting airway epithelium.

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Figure 6

KIF3A is required for repair of the airway epithelium

Mice of each genotype were injected i.p. with one dose of naphthalene. Mice were sacrificed on day 7 (n=3) (A), and day 10 (n=4) (B, C). Lung sections were immunostained for SCGB1A1 (red) and ac-TUBA1A (green in A, B). In panel C, CDH1 (E-cadherin) is shown in (green) and TUBA1A in (white). Restoration of bronchial epithelial cells is shown in controls and persistence of squamous metaplasia and paucity of SCGB1A1 stained cells is shown in the Kif3a^ScgΔ/Δ mice. For quantitation of regenerated cells, 200X magnified images of comparable regions (5 proximal and 5 distal region) from control and mutant mice were evaluated using Image J Software (FIJI, plugin). Percentage of regenerated cells was calculated by counting SCGB1A1 stained cells and the total number DAPI stained epithelial nuclei. The number of club cells were significantly decreased in Kif3a^ScgΔ/Δ mice on day 10 (D). Decreased capillary-epithelial permeability in epithelial barrier function in Kif3a^ShhΔ/Δ mice (E). FITC labeled albumin was injected into the tail vein of 4–5 adult mice of each genotype 24 hours after i.t. treatment with one dose of Aspergillus extract (100 μg). The concentration of the FITC label in the serum and BALF was measured by 3 hours after injection and the ratio calculated, ***P<0.001 by t-test.

KIF3A Mediates Epithelial Cell Migration

To assess the role of KIF3A in cell proliferation and migration, KIF3A mRNA was inhibited in Human Bronchial Epithelial Cells (HBEC3) and BEAS-2B cells with lenti-viral shRNA and cell migration was assessed by video imaging. When KIF3A mRNA was selectively inhibited, cells adhered to the plates, proliferated and reached confluency at 72 hrs post-transduction. Primary cilia were identified by co-staining of ARL13B and acetylated α-tubulin (ac-TUBA4A) in BEAS-2B cells (Fig. 7C). Motile cilia were not detected in these cell lines. Inhibition of KIF3A was confirmed by qRT-PCR, whereas, expression of a non-targeted gene, CDH1 was unchanged (Fig. 7D). KIF3A knockdown suppressed formation of primary cilia and markedly inhibited cell movement and migration in “scratch” assays (Fig. 7A, 7B, see Video3.mov and Video4.mov). KIF3A shRNA did not alter proliferation of HBEC3 cells (Fig. 7E); however, phospho-Histone H3 (pHH3) staining was modestly decreased by KIF3A knockdown in BEAS2B cells at confluency (data not shown). Cytoplasmic staining of ac-TUBA4A was altered by inhibition of Kif3a, indicating a potential role of KIF3A in microtubule assembly (Suppl. Fig. 3A). Cell tracking analysis showed that inhibition of KIF3A did not cause cell death but inhibited cell movement and migration trajectory characteristics, e.g. displacement, directionality, speed, and persistence and expression of the human KIF3A cDNA substantially rescued cell motility and navigation (Suppl. Fig. 3B). Loss of KIF3A was associated with dramatic inhibition of cell movement and disruption of cytoplasmic microtubule organization indicating its important role in bronchial epithelial cell migration, findings consistent with the failure of airway epithelial repair seen after naphthalene induced injury in the Kif3a^ScgΔ/Δ mice in vivo.

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Figure 7

KIF3A is required for cell migration in vitro

HBEC3 and BEAS2B cells were transduced with control and KIF3A shRNA lenti-viruses. Cells were stained with di-8-ANEPPS and the monolayer was wounded by mechanical scratch. Kif3a mRNA was decreased by the shRNAs, (D, n=4). Inhibition of Kif3a inhibited cell migration as observed by videography for 16 hours, data representative of n=3 independent experiments in BEAS2B (A) and HBEC3 (B). Co-staining for ARL13B and ac-TUBA4A showed loss of primary cilia in BEAS-2B cells (C). ARL13B staining was not detected in HBEC3 cells (not shown). Immunofluorescence data are representative of n=2 independent experiments. Average number of pHH3 nuclei detected per 1000 DAPI stained nuclei (E). Proliferation was unaltered by knockdown of Kif3a in HBEC3 cells. Data represent mean ± SEM of nuclei from 6-10 images per group.

Increased Capillary Epithelial Permeability in Kif3a^ShhΔ/Δ mice

Since loss of barrier function is associated with asthma-like pathology and Th2 inflammation (47), we tested whether capillary-epithelial permeability was altered in the Kif3a^ShhΔ/Δ mice in vivo. Twenty-four hours after exposure to one dose of Asperillus extract (i.t.), mice were injected with fluorescein-congugated albumin via tail vein followed by quantification of fluorescein in BALF and serum. Capillary-alveolar permeability was significantly increased in the Kif3a gene deleted mice, Fig. 6E.

Enhanced Th2 Inflammation and AHR in Kif3a Gene Targeted Mice After Aeroallergen Exposure

Figure 3

Increased AHR and eosinophilic inflammation in Kif3a gene deleted mice

Figure 4

Deletion of Kif3a in airway epithelial cells increased recruitment of IL4^{and IL17}^{T cells}

Figure 5

Response to Aspergillus sensitization in Ift88^/^Δ mice

KIF3A is Required for Epithelial Repair

Figure 6

KIF3A is required for repair of the airway epithelium

KIF3A Mediates Epithelial Cell Migration

Figure 7

KIF3A is required for cell migration in vitro

Increased Capillary Epithelial Permeability in Kif3a^ShhΔ/Δ mice

DISCUSSION

Present findings demonstrate the important role of airway epithelial KIF3A in the pathogenesis of aeroallergen-induced inflammation and airway hyper-responsiveness, linking the activities of microtubules to innate immune responses to aeroallergens. Enhanced AHR, goblet cell associated gene expression and Th2-mediated eosinophilic inflammation observed after deletion of Kif3a, provide a plausible mechanistic link between the genetic association of KIF3A alleles, levels of KIF3A protein, microtubule function and asthma (15, 16). While complete deletion of Kif3a is lethal in embryonic development (20–22), present studies demonstrate that loss of Kif3a in airway epithelial cells impairs mucociliary clearance, epithelial repair following injury, and enhances Th2-inflammation that together may influence responses to aeroallergens. Aspergillus fumigatus infection causes Allergic Broncho-Pulmonary Aspergillosis (ABPA) associated with individuals with asthma, cystic fibrosis, and primary ciliary dyskinesia; patients with ABPA are also at risk for eczema, hives, hay fever, and sinusitis (48, 49).

Increased AHR was seen in Kif3a^{^Δ}^andKif3a^{^Δ}^{^Δ} mice following repeated exposures to Aspergillus fumigatus or house dust mite extract. A major Aspergillus fumigataus allergen, ASPF13 promotes airway hyper-responsiveness, recruiting inflammatory cells to the bronchial submucosa and disrupting airway smooth muscle cell-extracellular matrix interactions (50). AHR was assessed by plethysmography, a measure of both smooth muscle hyperplasia and contractility, and mucus hyperproduction. In the present studies, we did not detect differences in alpha-smooth muscle actin (ACTA2) staining in bronchial smooth muscle and Acta2 mRNA was not significantly increased in the Kif3a^ScgΔ/Δ mice after Aspergillus sensitization. Thus, mechanisms involved in the observed AHR are presently unclear. However, expression of Il-13 and Il-4 RNAs were markedly increased in both heterozygous and homozygous Kif3a deleted mice after sensitization, both factors being known to directly activate IL-4Rα receptor signaling in bronchial smooth muscle cells causing AHR (51). Increased secretion of chemokines and cytokines recruiting inflammatory cells including eosinophils has been associated with increased smooth muscle cell activity in asthma (52). Accumulation of mucus in conducting airways seen in both Kif3a^{^Δ}^{^Δ} and Kif3^{^Δ}^{mice may also contribute to AHR.}

Present studies demonstrate diverse functions of KIF3A in airway epithelial cells, including epithelial repair, innate immune responses, and mucociliary clearance, that may influence airway reactivity and Th2-inflammation. There is strong experimental evidence linking the loss of barrier function, epithelial injury, and mucociliary clearance in the pathogenesis of asthma (47, 53, 54). Patients with primary ciliary dyskinesia have recurrent airway infections related to poor mucociliary clearance, (55, 56) and expression of cilia-related genes, including KIF3A, were decreased during acute asthma (57). Reduced mucociliary clearance and increased susceptibility to infection related to motile ciliary dysfunction were proposed to contribute to the pathogenesis of asthma (58). KIF3A is known to play important roles in microtubule assembly and intracellular transport of multiple protein cargos, in addition to its known role in the formation of primary and motile cilia (17–19). It is therefore likely that changes in KIF3A levels or function play diverse roles in respiratory epithelial cell homeostasis. Aspergilla and HDM extracts contain proteases, antigens, and other inflammatory mediators that cause epithelial cell injury (59, 60). Decreased mucociliary clearance, increased uptake of antigen by dendritic cells, and disrupted microtubular transport within the epithelial cells may influence the increased Th2 responses seen following deletion of Kif3a. Changes in barrier function, modulation of cytokine and chemokine signaling from the epithelium, regulate recruitment and activation of dendritic and ILC2 cells in turn recruiting Th2-helper and Th17 cells mediating asthma-like pulmonary inflammation (37, for review, 61, 62). Increased Il-13, Il-4, Ccl11, Il-17A, and Ccl24 RNAs seen in lungs of the Kif3a deleted mice are consistent with the observed Th2-mediated inflammation. Ccl11 (eotaxin1), a potent eosinophil chemoattractant, was markedly induced in the Kif3a but not the Ifit88 deleted mice. Increased expression of Il-13 and Il-4 seen after Aspergilla extract in the Kif3a^ScgΔ/Δ mice is typical of canonical Th2 lymphocytic responses that influence goblet cell metaplasia and mucus hyperproduction (63). Likewise, increased Spdef, Foxa3, Muc5b, and Muc5ac in the Kif3a^ScgΔ/Δ mice is consistent with goblet cell metaplasia being related to the activation of Th2-induced IL-4R signaling and STAT6 activation that occurs following aeroallergen exposure (38, 64, 65).

Repair of the respiratory epithelium was impaired and capillary-epithelial barrier function was decreased, factors that may contribute to the enhanced Th2-inflammation seen in the Kif3a gene deleted mice. Primary cilia influence cell migration, a process critical for repair (66, 67). Decreased expression of KIF3A inhibited migration of both BEAS-2B and HBEC3 cells in vitro, findings consistent with impaired epithelial repair seen in Kif3a^ScgΔ/Δ mice seen after exposure to naphthalene. These findings are supported by previous in vitro studies demonstrating the role of KIF3A in cell migration in kidney epithelial cells (27). Recent in vitro findings demonstrated that the disruption of microtubules seen after inhibition of KIF3A did not occur after inhibition of IFT88 (68). Inflammatory responses to Aspergillus were increased in both Ift88 and Kif3a deleted mice, although Th2-responses were more pronounced in the Kif3a^ScgΔ/Δ mice, supporting the concept that the microtubule associated proteins may have distinct as well as overlapping functions in innate immune regulation in the airway epithelial cells.

Present findings demonstrate that KIF3A was required for suppression of Th2-mediated inflammatory responses, mucus hyperproduction, and AHR following aeroallergen exposure, findings that support the association of KIF3A gene polymorphisms with clinical susceptibility to allergic asthma and rhinitis (11–16). Previous clinical findings in nasal epithelial cells demonstrated decreased expression of KIF3A during acute asthma exacerbations (57). Since in the present study, increased lung inflammation and AHR were observed in haplo-insufficient mice, even a modest decrease in KIF3A expression may influence the susceptibility to Th2-mediated inflammation. Previous observations, that the human KIF3A gene locus is located contiguously with the IL-4/IL-13 genes (69, 70), genes known to regulate Th2 inflammation in asthma, complicate the interpretation of the importance of KIF3A alleles in the pathogenesis of atopy and asthma. While present studies do not exclude the possibility that asthma susceptibility related to this chromosomal region is associated with the other genes in that locus, present findings in our mouse models demonstrate a direct role for KIF3A in the regulation of aeroallergen induced Th2 inflammation, airway epithelial repair, and mucociliary clearance, processes that may influence susceptibility to asthma.

Supplementary Material

Acknowledgments

The authors thank Shawn Grant, Jaymi Semona, Kalpana Srivastava, Brandy Ruff, Mehari E. Mengistu, Gail Macke, and Courtney Stockman for their scientific assistance, Ann Maher for typing and editing on this manuscript, and the CCHMC Viral Vector Core for making the lentiviruses.

^{Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Division of Neonatology, Perinatal and Pulmonary Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039}

^{Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Division of Asthma Research, 3333 Burnet Avenue, Cincinnati, OH 45229-3039}

^{Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Division of Developmental Biology, 3333 Burnet Avenue, Cincinnati, OH 45229-3039}

^{Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Division of Pulmonary Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039}

Correspondence to: Jeffrey A. Whitsett, M.D., Division of Pulmonary Biology, MLC7029, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, Phone: 513-803-2790, FAX: 513-636-7868, gro.cmhcc@ttestihw.ffej

Abstract

KIF3A, the gene encoding Kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. Herein, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and house dust mite, resulting in asthma-like pathology characterized by increased goblet cell metaplasia, airway hyper-responsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, Il-17A) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia, impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyper-responsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.

Keywords: Susceptibility Gene, Asthma, Cilia, Kinesins, Airway Hyper-responsiveness

Abstract

Footnotes

^{This work was supported by Grants HL095580 and HL110964 (JAW) and 2U19AI070235-11 (GKKH)}

Footnotes

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AIRWAY EPITHELIAL KIF3A REGULATES Th2- RESPONSES TO AEROALLERGENS

INTRODUCTION

MATERIALS AND METHODS

Mice

Naphthalene Injury

Aspergillus and House Dust Mite (HDM) Extract Sensitization

AHR/flexiVent

BALF

Histology and immunohistochemistry

Scanning electron microscopy

Mouse trachea and airway sample preparation and video microscopy for ciliary beat and mucociliary clearance

RNA isolation and analysis

Table I

Lung cell isolation and EpCAM sorting

In vitro assays

Flow cytometric analysis of lung cells

In vivo capillary-epithelial permeability

Statistical analysis

Mice

Naphthalene Injury

Aspergillus and House Dust Mite (HDM) Extract Sensitization

AHR/flexiVent

BALF

Histology and immunohistochemistry

Scanning electron microscopy

Mouse trachea and airway sample preparation and video microscopy for ciliary beat and mucociliary clearance

RNA isolation and analysis

Table I

Lung cell isolation and EpCAM sorting

In vitro assays

Flow cytometric analysis of lung cells

In vivo capillary-epithelial permeability

Statistical analysis

RESULTS

Enhanced Th2 Inflammation and AHR in Kif3a Gene Targeted Mice After Aeroallergen Exposure

KIF3A is Required for Epithelial Repair

KIF3A Mediates Epithelial Cell Migration

Increased Capillary Epithelial Permeability in Kif3aShhΔ/Δ mice

Enhanced Th2 Inflammation and AHR in Kif3a Gene Targeted Mice After Aeroallergen Exposure

KIF3A is Required for Epithelial Repair

KIF3A Mediates Epithelial Cell Migration

Increased Capillary Epithelial Permeability in Kif3aShhΔ/Δ mice

DISCUSSION

Supplementary Material

1

2

1

2

Acknowledgments

Abstract

Footnotes

References

Increased Capillary Epithelial Permeability in Kif3a^ShhΔ/Δ mice

Increased Capillary Epithelial Permeability in Kif3a^ShhΔ/Δ mice