asthma is an allergic disease characterized by airway inflammation , mucin hypersecretion , and airway hyperresponsiveness ( ahr ) .
infiltration of cd4 t cells , eosinophils , mast cells , and b cells and their interaction with airway resident cells contribute to airway inflammation . il-17-producing ( th17 )
numerous studies have shown an increase of th17 cells in inflammatory airway [ 5 , 6 ] . therefore , suppressing th17 response may be a novel therapeutic strategy for treating asthma .
notch signaling pathway is evolutionarily conserved . in mammalian , there are four notch receptors ( notch1 , notch2 , notch3 , and notch4 ) and five notch ligands ( jagged1 , jagged2 , delta - like ligand ( dll)1 , dll3 , and dll4 ) .
notch plays a crucial role in a broad spectrum of cellular activities such as proliferation , differentiation , and regulation of cell function .
a series of enzymatic reactions lead to the cleavage of the notch receptor intracellular domain ( nicd ) which is translocated to the nucleus , where it binds with csl / rbp - jk to recruit mastermind - like 1 protein .
previously , we showed that notch signal pathway regulates the proliferation and differentiation of cd4 t lymphocytes in a mouse model of asthma , indicating that notch may be a potential target for treating asthma .
others have reported that pharmacologic inhibitor of notch signaling can reduce allergic pulmonary inflammation by modulating th1 and th2 responses [ 10 , 11 ] .
the present study aims to test whether gsi has therapeutic effects on the development of asthma through regulating th17 mediated immune response .
male balb / c mice , 4 to 6 weeks old , weighing 2022  g , were purchased from shanghai laboratory animal center ( shanghai , china ) and bred in pathogen - free environment in the animal center of wenzhou medical university .
animal experimental protocol was approved by institutional animal care and use committee ( iacuc ) of wenzhou medical university .
experimental animals were divided into three groups : sham group , ova + dmso ( vehicle ) group , and ova + gsi ( 0.3  mg / kg ) group .
injection of 10  g ovalbumin ( ova ) ( sigma , usa ) emulsified in 20  mg al ( oh)3 gel in 0.1  ml normal saline ( ns ) on days 1 and 13 .
they were then challenged with ova ( 1  mg / ml ) aerosol for 30  min daily for eight consecutive days from day 25 by jet nebulizer ( pari is-2 jet nebulizer ; pari respiratory equipment ) .
gsi l685,458 ( calbiochem , ca ) was administered intranasally 30 minutes before each ova challenge at 0.3  mg / kg as previously described .
the sham mice were sensitized and challenged with normal saline ( ns ) and treatment with dimethylsulfoxide ( dmso ) as a control for gsi .
mice were sacrificed within 24  h after the last allergen challenge . at the time of sacrifice ,
the left lung tissue was first fixed with 4% paraformaldehyde for 4  h. it was then dehydrated in ethylic alcohol , embedded with paraffin , sectioned in 4  m , and stained with haematoxylin and eosin ( he ) .
tissue slices were evaluated through light microscope ( nikon ) by trained technician in a blind fashion .
the degree of allergic airway inflammation was scored according to the following histologic grading system ( scored 04 ) : absence of peribronchial inflammatory cells ; a few scattered peribronchial inflammatory cells involving less than 25% of the circumference of the bronchus ; focal peribronchial inflammatory cells infiltration not completely surrounding a bronchus ( i.e. , involving approximately 25%75% of the circumference of the bronchus ) ; one definite layer of peribronchial inflammatory cells completely surrounding a bronchus ; 2 or more layers of peribronchial inflammatory cells completely surrounding a bronchus . in each lung section
the mean peribronchial inflammatory score was determined by the sum of scores of all individual bronchioles in the section divided by the number of bronchioles .
the spleen tissue was fragmented into small pieces that were then pressed against nylon mesh with a plunger of a disposable syringe .
cd4 t cells from splenic single - cell suspension were isolated by magnetic cell sorting by positive selection method using mouse cd4 t cell isolation kit ( macs , miltenyi biotec , germany ) according to the manufacturers ' instruction .
the purity of the cells was 92.04  5.18% , as confirmed by flow cytometry analysis .
fitc - labeled anti - mouse cd4 and pe - labeled anti - mouse il-17a were used to detect th17 cells .
for th17 cell analysis , cd4 t cells ( 1  10/ml ) from the spleen tissue were stimulated for 4.5  hr with phorbol myristate acetate ( pma ) at 100  ng / ml and ionomycin at 1  g / ml in the presence of 1.6  g / ml monensin ( all from beyotime , china ) .
cells were collected , washed , and surface - stained with fitc - labeled anti - cd4 antibody at 4c for 20  min in the dark and resuspended in fix / perm solution according to the manufacturer 's instruction ( invitrogen , usa ) .
after washing , cells were resuspended in fixation solution and subjected to facscalibur flow cytometer ( bd facscanto ii , usa ) analysis .
lung tissue was fragmented and lysed in ripa buffer with protease inhibitor mixture ( beyotime , china ) .
a total protein of 20  g was loaded into each well of a sds - page gel for separation by electrophoresis and then transferred onto nitrocellulose membrane .
the resulting blots were blocked for 1  h with tbs tween 20 containing 5% powder skim milk and then probed overnight at 4c with anti - nicd ( abcam , uk ) .
blots were then washed three times and probed for 1  h with anti - rabbit hrp - conjugated antibody .
rna was extracted from lung tissue using trizol ( invitrogen , usa ) according to the manufacturer 's instruction .
the quantitative real - time rt - pcr was performed using an abi step one plus system ( applied biosystems , usa ) with quantifast sybr green pcr kit ( qiagen , germany ) .
primers used were as follows : gapdh , 5-tggccttccgtgttcctac-3 ( forward ) and 5-gagttgctgttgaagtcgca-3 ( reverse ) ; notch1 , 5-tgccacaatgagatcggctc-3 ( forward ) and 5-gagttgctgttgaagtcgca-3 ( reverse ) .
delta - delta ct method was used to express the fold induction of target mrna after gapdh normalization .
the concentration of cytokines il-17 in serum was assessed by standardized sandwich elisa according to the manufacturer 's protocol .
the il-17 kit was purchased from ebioscience , san diego , usa . all data were expressed as mean  sem . differences between groups were analyzed for statistical significance by one - way analysis of variance using spss 13.0 software ( spss inc . ,
to explore the effect of gsi on ova - induced asthma , babl / c mice were sensitized and challenged with ova ( or ns for sham mice ) . those mice received gsi , a highly selective inhibitor of -secretase or vehicle ( dmso ) , during the challenge phase .
mice were sacrificed within 24  h after the last allergen challenge and lung tissue was fixed , embedded , and sectioned for he staining .
the degree of airway inflammation of he - stained lung tissue was scored as described in materials and methods . as shown in figure 2(a ) , ova + dmso group demonstrated significant infiltration of eosinophils and lymphocytes with marked thickening of airway wall and epithelial goblet cell metaplasia , as compared with the sham group .
ova - challenged mice showed an inflammation score of 3.25  0.46 as compared to sham control ( 0.38  0.52 ) .
gsi treated group showed a score of 1.88  0.64 that is significantly lower than ova - dmso group ( figure 2(b ) , p < 0.01 ) . to investigate the blockage effects of gsi on notch signaling , mrna expression of notch1 , a receptor of notch signaling , was examined .
as shown in figure 3(a ) , ova - challenged mice revealed enhanced notch1 mrna expression , as compared with the sham group ( 1.31  0.13 versus 0.84  0.13 , p < 0.01 )
. on the other hand , gsi treatment led to the reduction of notch1 mrna expression ( 0.92  0.088  p < 0.01 comparing to ova group )
. consistent with this observation , ova - challenged mice revealed increased nicd generation as compared to sham group ( 0.18  0.02 versus 0.09  0.01 , p < 0.01 ) .
gsi treatment decreased nicd generation ( 0.06  0.03 ) comparing to ova group ( figure 3(b ) , p < 0.01 ) .
results presented here suggest that gsi can effectively block notch signaling . to evaluate the effect of gsi treatment on th17 cell expansion , splenic cd4 t cells were isolated by magnetic cell sorting .
sham group expressed a baseline th17 cell frequency of 0.30  0.16% of total splenic cd4 t cells .
ova - induced asthma mice revealed a significant increase of th17 cells ( 2.43  0.69% , p < 0.01 , comparing to sham group ) .
gsi treatment reduced th17 cell frequency to 1.26  0.85% which is statistically significant from ova group ( p < 0.05 , figure 4 ) .
th17 cells are the main source of il-17 . to further examine the function of such th17 cells , serum levels of il-17 were measured from ova - induced asthma mice . as illustrated in figure 5 , sham group expressed a baseline level of il-17 in serum at 48.07  5.73 
the il-17 level was significantly elevated in ova - induced asthma mice ( 120.09  5.73  pg / ml , p < 0.01 ) .
gsi administration during challenge phase significantly reduced the il-17 level to 81.82  8.95  pg / ml , p < 0.01 .
the notch signaling pathway is involved in many aspects of organ formation and cell function .
the effect of notch signaling inhibition on the development of asthma has been addressed in several recent studies .
jin and colleagues reported that inhibition of notch signal pathway by gsi alleviated the airway inflammation in ova - induced asthma model and it was through the regulation of th1 and th2 responses .
knockdown of the notch l gene by small interfering rna led to overproduction of il-4 and ifn- , which played an important role in the pathogenesis of asthma .
however , the exact underlying mechanism is yet to be fully elucidated . in the present study , we used gsi to block notch signaling in a mouse model of asthma and demonstrated that in vivo administration of gsi effectively attenuated eosinophilic and lymphocyte infiltration in the airways and decreased goblet cell metaplasia .
furthermore , notch inhibition by gsi reduced the frequency of th17 cells in spleen and the serum levels of il-17 .
taken together , these findings demonstrate the effectiveness of gsi in animal models and strongly suggest that inhibition of notch signaling could be an effective strategy for the treatment of asthma .
t helper 17 ( th17 ) cells play a critical role in adaptive immune responses through the production of cytokines , namely , il-17a , il-17f , and il-22 .
recent studies indicate that th17 cells are active players in acute airway inflammation of allergic asthma . marked elevation of il-17a
li et al .   suggested that the ratio of th17 cells / cd3 t cells in peripheral blood was significantly increased in asthma patients compared with nonasthma individuals .
similar change was found in the present study ; that is , the proportion of th17 cells in isolated spleen cd4 t cells was significantly increased in ova - induced asthma mice compared to the sham group .
reported that adaptive transferring of th17 cells to an asthma mouse led to neutrophils infiltration and airway hyperresponsiveness , which is resistant to corticosteroid therapy .
consistent with these findings , our investigation revealed that treatment with gsi markedly reduced serum il-17 levels of asthma mice .
of note , doe et al .   found that il-17a was elevated in mild to moderate human asthma compared with healthy control , while it was not increased in severe asthma .
therefore , further research is needed to characterize the exact relationship between il-17 and the severity of asthma .
notch can directly regulate retinoic acid - related orphan receptor t , an important transcription factor for th17 differentiation .
inhibition of notch signaling by notch3 antibody attenuates th17-type responses , while treatment with notch ligand delta - like 1 promotes th17 response . in vitro knockdown of notch and in vivo administration of gsis result in reduced il-17 production and
gsis have been actively tested in clinical trials for alzheimer disease for their potential in blocking the generation of a peptide .
mrk003 , a -secretase inhibitor , exhibits promising in vitro preclinical activity in multiple myeloma and non - hodgkin 's lymphoma . after blocking notch signaling in our mouse asthma model
, we noticed decreased level of nicd , ameliorated airway inflammation , reduced serum il-17 level , and improved clinical signs .
our data strongly suggest that inhibition of notch signaling could be considered as an effective therapy for asthma .
of course , further preclinical and clinical research is needed to address such potential . in conclusion
, the current study proves that gsi administration inhibits th17 differentiation , decreases il-17 production , and alleviates airway inflammation in ova - sensitized and ova - challenged balb / c mice .
these results support the idea of considering gsi as a novel , effective antiasthma agent .
 t helper 17 ( th17 ) cells play an important role in the pathogenesis of allergic asthma . 
 th17 cell differentiation requires notch signaling . -secretase inhibitor ( gsi ) 
 blocks notch signaling ; thus , it may be considered as a potential treatment for allergic asthma . 
 the aim of this study was to evaluate the effect of gsi on th17 cell differentiation in a mouse model of allergic asthma . 
 ova was used to induce mouse asthma model in the presence and absence of gsi . 
 gsi ameliorated the development of ova - induced asthma , including suppressing airway inflammation responses and reducing the severity of clinical signs . 
 gsi also significantly suppressed th17-cell responses in spleen and reduced il-17 levels in serum . 
 these findings suggest that gsi directly regulates th17 responses through a notch signaling - dependent pathway in mouse model of allergic asthma , supporting the notion that gsi is a potential therapeutic agent for the treatment of allergic asthma . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion