Lentiviral shRNA against KCa3.1 inhibits allergic response in allergic rhinitis and suppresses mast cell activity via PI3K/AKT signaling pathway

Calcium-activated potassium ion channel-3.1 (KCa3.1) plays a pivotal role in the potassium-calcium exchange involved in atopy. This study aimed to explore the impact of lentiviral-mediated shRNA silencing KCa3.1 on allergic response in a murine allergic rhinitis (AR) model. The BALB/c mice were divided into four groups: untreated AR group, negative control AR group, lentiviral KCa3.1-shRNA treated AR group and normal control group. Concentrations of ovalbumin (OVA)-specific IgE, histamine and leukotrienes C4 (LTC4) in serum, and IL-4, IL-9 and IL-17 in nasal lavage fluid (NLF) were analyzed. Goblet cells and mast cells were counted. KCa3.1 positive cells were counted after immunolabelling by immunofluorescence method. KCa3.1, Mucin 5AC (MUC5AC), and tryptase mRNA levels were determined using real-time polymerase chain reaction. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells. The results showed that LV-KCa3.1-shRNA intervention effectively attenuated allergic responses in LV-KCa3.1-shRNA treated mice. LV-KCa3.1-shRNA intervention effectively suppressed KCa3.1 levels and phosphorylation of AKT in P815 cells, leading to the downregulation of tryptase, IL-6 and IL-8 levels. LV-KCa3.1-shRNA intervention effectively attenuated the allergic responses in AR and suppressed mast cell activity by inhibiting PI3K/AKT signaling pathway.

RNA interference (RNAi) is a gene-specific RNA degradation process mediated by short interfering RNA (siRNA) and is a promising therapeutic tool directed against a potential therapeutic target in the allergic disorders, infectious diseases and cancers [9][10][11] . A short hairpin RNA (shRNA) is a precursor of siRNA and is composed of siRNA sequence and hairpin loop sequence 12,13 . It has been established that shRNA was more stable than siRNA and was more readily integrated into the plasmid or viral vectors 14 . Lentivirus-mediated shRNA using lentiviral vector yielded an efficient and stable response of the target gene silencing both in vitro and in vivo 15 .
It is well established that calcium ion (Ca 2+ ) is essential for regulating most cell activities involving cell growth, transformation, and migration. There is ample evidence that upregulation of intracellular Ca 2+ participates in the proliferation of airway epithelial cells, smooth muscle cells and lymphocytes involved in asthma [16][17][18] . It has been illustrated that sustained Ca 2+ influx through Ca 2+ release-activated Ca 2+ channel (CRAC) requires sufficient electrical driving force which is conserved by efflux of potassium ion (K + ) and intermediate Ca 2+ -activated K + channel (KCa3.1, also named as KCNN4) has been proven to mainly modulate the efflux of K + and plays a pivotal role in the potassium-calcium exchange involved in the allergic and inflammatory responses 19 . Nevertheless, whether KCa3.1 is involved in the pathophysiology of AR and the inhibition of KCa3.1 could suppress the allergic response in AR are unclear. Hence, the present study was aimed to explore the expression and role of KCa3.1 in the pathogenesis of AR and to further assess the therapeutic effect of lentivirus-mediated shRNA silencing of KCa3.1 in a murine AR model. Furthermore, P815 cell line was used to explore the role and mechanism of lentiviral KCa3.1-shRNA on mast cells.

Results
Effects of LV-KCa3.1-shRNA on nasal symptoms in a murine AR model. As indicated in Fig. 1a,b, significant elevation of frequencies of sneezes and nasal rubbing were found in untreated AR mice compared with normal control mice, respectively (P < 0.01), and LV-KCa3.1-shRNA group showed Concentrations of OVA-specific IgE, histamine and LTC4 in serum, and IL-4, IL-9 and IL-17 in NLF assayed by ELISA (c-h). One-way ANOVA test was employed for intergroup comparison. *P < 0.01 vs UnT-AR group. #P < 0.01 vs NeC-AR group. ※ P > 0.05 vs UnT-AR group.

Effects of LV-KCa3.1-shRNA on OVA-specific IgE, histamine and LTC4 in serum and cytokine levels in NLF.
Goblet cell hyperplasia, mast cell infiltration and KCa3.1 immunoreactivity in nasal tissues.
As indicated in Fig. 2 and Fig. 3b,c, significant elevation of goblet cell and mast cell numbers were found in untreated AR mice compared with normal control mice (P < 0.01), and LV-KCa3.1-shRNA group showed less goblet cell and mast cell numbers compared with untreated AR or negative control group, respectively (P < 0.01), indicating that LV-KCa3.1-shRNA treatment reduced goblet cell hyperplasia and mast cell infiltration in AR mice.
As depicted by immunofluorescence staining (Fig. 4), KCa3.1 positive cells were mainly epithelial cells among mucosal surface and surrounding basal lamina, and sub-mucosal inflammatory cells in the nasal mucosa. KCa3.1 was significantly overexpressed in AR group compared with control group; notably, LV-KCa3.1-shRNA treatment resulted in the reduction of KCa3.1 protein levels in LV-KCa3.1-shRNA treated group compared with untreated AR or negative control group, respectively (P < 0.01). These results were consistent with the changes in the nasal symptoms and cytokine levels following LV-KCa3.1-shRNA treatment.
Effects of LV-KCa3.1-shRNA on KCa3.1, MUC5AC and tryptase mRNA levels in AR mice. To further illuminate the roles of LV-KCa3.1-shRNA on goblet cell and mast cell in AR mice, mRNA levels of KCa3.1, MUC5AC, which is mainly secreted by goblet cell 20 , and tryptase, known as a key mast cell marker which mainly exists in the secretory granules of mast cell 21 , were assayed by real-time PCR.

Discussion
It has been proved that goblet cell hyperplasia, mast cell infiltration, Th-2, Th-9 and Th-17 polarization are recognized as important features of AR 3,5,22,23 . In the present study, significantly increased goblet cell hyperplasia, mast cell infiltration, IL-4, IL-9 and IL-17 levels were found in AR mice compared with normal control mice, which is consistent with previous reports 3,5,22,23 . In addition, MUC5AC and tryptase mRNA levels were significantly upregulated in AR mice compared with normal control mice, further confirming that goblet cell hyperplasia and mast cell infiltration are involved in the pathogenesis of AR.
Our previous reports demonstrated that Orai1, an essential component of the CRAC was upregulated and led to elevation of intracellular Ca 2+ concentration in a murine AR model and target inhibition of Orai1 could effectively alleviate murine AR through reducing Ca 2+ influx 24,25 . Recently, there is emerging evidence that KCa3.1 has been implicated as an amplifier of CRAC current and could sustain sufficient membrane potential and electrochemical driving force for Ca 2+ influx by modulating K + efflux 26,27 . Previous studies have demonstrated that targeted inhibition of KCa3.1 gene could effectively suppress the allergic responses in asthma and inhibit cell proliferation in cancers 28,29 . In our present study, as indicated in Figs 1-3 and Fig. 3a-f, LV-KCa3.1-shRNA could effectively alleviate allergic symptoms of AR mice by reducing goblet cell hyperplasia, mast cell infiltration, OVA-specific IgE, histamine, LTC4, IL-4, IL-9, IL-17, KCa3.1, MUC5AC and tryptase levels. These results show that LV-KCa3.1-shRNA intervention may suppress goblet cell hyperplasia and mast cell infiltration, and inactivate plasma cells, Th2 ,Th9 and Th17 cells.
Considerable data indicate that mast cells play central roles in the pathologic change of nasal mucosal tissue and pathophysiologic process in AR [30][31][32] . Accumulating evidence has shown that upregulation of intracellular Ca 2+ concentration is essential for mast cell activation 33,34 and treatment of mast cells with carbon dioxide suppresses degranulation via repression of intracellular calcium levels 35 . KCa3.1 channels are required for mast cell recruitment and activation 36 . To date, there are many lines of evidence indicating that PI3K/AKT signaling pathway plays an essential role in the maturation and activation of mast cells [37][38][39] and PI3K/AKT signaling pathway inhibitor LY294002 suppresses mast cells degranulation and activation 40,41 . In the present study, to further illustrate the roles and mechanism of LV-KCa3.1-shRNA on mast cell, P815 cell line was treated with LV-KCa3.1-shRNA and PI3K/AKT signaling pathway inhibitor LY294002. As depicted in Fig. 5a-f, LV-KCa3.1-shRNA group showed lower levels of KCa3.1, AKT phosphorylation, tryptase, IL-6 and IL-8 compared with blank control or negative control group, respectively and LY294002 enhanced these effects. These results indicate that the roles of LV-KCa3.1-shRNA on mast cells may be mediated via inactivation of the PI3K/AKT signaling pathway in mast cells. Our findings are in agreement with previous findings demonstrating that PI3K/AKT signaling pathway inhibitor LY-294002 exerted inhibitory effects on the mast cell activation [41][42][43] .
Some weaknesses and limitations of our study should be interpreted. First, relatively small sample size was included in this study and studies with large sample sizes are warranted to further clarify these findings. Second, it is necessary to perform whole genome microarray to assess the off target effects of LV-KCa3.1-shRNA in future studies.
In conclusion, LV-KCa3.1-shRNA could effectively alleviate murine allergic rhinitis by suppressing KCa3.1, leading to the reduction of goblet cell hyperplasia and mast cell infiltration, as well as attenuation of allergic responses and inflammatory factors. The role of LV-KCa3.1-shRNA on mast cells may be mediated via inactivation of the PI3K/AKT signaling pathway in mast cells. The targeted inhibition of KCa3.1 gene mediated by lentiviral vector may be a novel promising approach in AR gene therapy.     group. The murine AR model was established as described previously 24 . Briefly, mice were sensitized with 0.2 mL suspension comprising 0.5 mg/mL ovalbumin (OVA, Sigma-Aldrich, USA) and 20 mg/mL aluminum hydroxide (Sinopharm Chemical Reagent Co. Ltd., China) by intraperitoneal injection on Day 1, 8, and 15, respectively. Then, the mice were challenged daily with 20 μ l OVA solution (40 mg/ml in normal saline) by intranasal instillation on Days 22 to 29 (Fig. 6d). To improve gene transfection efficiency, the mice were instilled intranasally with 4μ l lysophosphatidylcholine (1% w/v in PBS, Sigma-Aldrich, USA) prior to delivery of the lentiviral vector system, as described previously 44 . Mice in lentiviral KCa3.1-shRNA treated group were instilled intranasally with lentivirus mediated KCa3.1-shRNA (20 μ l, 1 × 10 8 TU/ml) and the mice in negative control group were given with the same dose of lentivirus mediated negative-shRNA.Mice in normal control group were administrated with normal saline.

Construction of lentiviral
Nasal symptoms evaluation and sample preparation. On Day 29, immediately after the final OVA provocation, nasal symptoms involving frequency of sneezes and nasal rubbing were assessed for 10 minutes. The mice were observed and reviewed independently by three blinded reviewers whose inter-interviewer variability was not statistically significant. After 24 hours, the mice were killed and the blood samples were harvested by cardiac puncture and were centrifuged to obtain serum for enzyme-linked immunosorbent assay (ELISA). Tracheas of the mice were partially resected and a 22-gauge catheter was inserted via the tracheal opening to the nasopharynx in order to perfuse the nasal passages from choana to nostril with 1 ml PBS. Nasal lavage fluid (NLF) was collected from the nares and centrifuged, and the supernatants were collected and stored at − 80 °C for ELISA. Nasal mucosas were assayed by Periodic Acid-Schiff (PAS) stainning, toluidine blue staining, immunofluorescence staining, real-time PCR or western blotting methods.
ELISA assay. Serum OVA-specific immunoglobulin E (IgE), histamine and leukotrienes C4 (LTC4) levels were assayed using specific ELISA kit (BlueGene Biotech, Shanghai, China) and concentrations of IL-4, IL-9 and IL-17 in NLF or IL-6 and IL-8 in the cell culture supernatants were assayed with specific ELISA kits (eBioscience Inc., USA). Each sample was analyzed in triplicate.
PAS, toluidine blue and immunofluorescence staining. The mice were decapitated and the heads were fixed with 4% paraformaldehyde for 2 days and decalcified for 21 days in 10% ethylene diamine tetra acetic acid (EDTA).Then the samples were paraffin-embedded and serially sectioned. The sections were stained with PAS for counting goblet cell numbers, stained with toluidine blue for assessing mast cell infiltration, and stained with immunofluorescence for assessing KCa3.1 immunolabelling. Following deparaffinization and rehydration, the sections were rinsed using PBS for three times and then microwaved for antigen retrieval in EDTA buffer (PH8.0). The sections were incubated with primary antibody (rabbit anti-mouse KCa3.1, 1:100 dilution, Abcam) at 37 °C for 60 min, rinsed for three times and incubated with secondary antibody (cy3-labeled goat anti-rabbit IgG, 1:300 dilution, Abcam) for 30 min. Then, 4,6-diamidino-2-phenylindole (DAPI) was added for nuclear staining. Goblet cell, mast cell and KCa3.1 positive cell numbers were microscopically counted under high-powered field (HPF, × 400), and five fields of each section were randomly selected and mean value per HPF was calculated. All pathology slides images were reviewed independently by three blinded reviewers whose inter-interviewer variability was not statistically significant.

RNA extraction and reverse transcription.
After homogenization of the tissues or cells, TRIzol reagent was added to extract total RNA. The purity and integrity were assessed by measuring absorbance ratios at 260/280 nm (1.8 ~ 2.0 was considered eligible) and agarose gel electrophoresis, respectively. For reverse transcription, PrimeScript ™ 1st Strand cDNA Synthesis Kit (TaKaRa) was applied to reversely transcribe two micrograms total RNA into cDNA as a template for real-time PCR.
Western blotting. The cells were homogenized and lysed in RIPA buffer (Pierce) and then centrifugated at 14000 rpm for 15 min at 4 °C. The supernatant involving the total protein were collected. Following quantification of protein concentration, the samples were loaded on SDS-PAGE gels (Invitrogen) for protein separation and subsequently electroblotted onto PVDF membranes (Millipore) under the same experimental conditions. After blocking of excess protein-binding sites with 5% skimmed milk for 1 h, the PVDF membranes were incubated with the primary antibody (rabbit anti-mouse KCa3.1, 1:1000 dilution, Abcam; rabbit anti-mouse p-AKT and AKT, 1:1000 dilution, Cell Signaling Technology) at 4 °C overnight. After TBST washing, the secondary goat anti-rabbit IgG-HRP antibody (Abcam) was added for 1 h incubation. Lastly, immunoreactions were visualised by the ECL kit (Pierce). β -actin was used as an internal control. Densitometric analyses were performed using Gel-Pro Analyzer 4.0 software (Media Cybernetics).
Statistical analysis. Statistical analysis softwares (GraphPad Prism 5.0 and SPSS version 20.0 software) were applied to conduct the analyses. One-way ANOVA test followed by Dunnett's tests were employed for intergroup comparison of all the continuous variables for them had normal distribution. Correlations were assessed by Spearman's test. Values were shown as mean ± standard error (SE). Two-sided P value < 0.05 was deemed statistically significant.