asthma is a complex syndrome , characterized by intermittent reversible obstruction , airway hyper - responsiveness ( ahr ) and pulmonary inflammation in which many cells and cellular elements play a vital role , such as eosinophils , mast cells , t lymphocytes , macrophages , neutrophils and epithelial cells . asthma is usually divided into allergic asthma and non - allergic asthma , and approximately two - thirds of asthma cases are allergic . it is widely accepted that antigen - specific t helper cell type 2 ( th2 ) and their cytokines such as il-5 , il-4 , and il-13 orchestrate the feature of asthma . recently , the classical theory has been expanded to include th17 cells and their associated cytokines . many therapies have been adopted basis on pathogenesis , but there is still a definite increase in prevalence of asthma . cd4 t cells are differentiated into th1 or th2 cells depending on the distinct involved cytokines . recently , a new phenotype of th cell , th17 cell , has been identified . th17 cells , with its secretion il-17a , il-17f and il-22 , play a vital role in host defense and in induction and propagation of autoimmunity in animal models . il-17a and il-17f , augmentative in the inflammation of bronchial submucosa in mild to moderate asthma , contribute to excessive mucus and airway smooth muscle proliferation . house dust mite ( hdm ) , a major source of allergen in house dust , is closely associated with development of asthma . it can lead to prominent and sustained airway eosinophilic inflammation , along with elevated serum levels of th2-associated immunoglobulins and cytokines . hdm directly induces the release of pro - inflammation cytokines and chemokines from bronchial epithelial cells and airway epithelial cells and evokes the direct , non - allergic inflammation . many researchers merely investigate the mechanism of asthmatic mice challenged by hdm alone , the complex hdm exposure is less well defined . the role of th17 response in asthma remains a controversial problem , especially it in hdm - alone - induced asthmatic mice . to know more about the asthma patients who re - expose to hdm allergen , we pay attention to th2 and th17 responses in our complex allergen challenge model , ova - induced asthmatic mice exposed to hdm . more inflammatory cell infiltrations were observed in hdm alone groups than that in control group , which demonstrates that hdm can directly evoke non- allergic inflammatory response . the expression of th17 related cytokines were augmented in ova+hdm group compared with ova alone group . th2 related cytokines were increased in ova+hdm group in comparison with ova alone group at gene level , while at protein level , they were increased with no significant difference . we confirm that the augmented th17 response is linked with the inflammation in ova - induced asthmatic mice exposed to hdm . when the ova - induced asthmatic mice are re - exposed to hdm , the pathomechanism is different from ova alone exposure . thus , hdm is an important interferential factor for asthma therapy in the indoor living environment of asthma patients the 6-week - old male balb / c mice were purchased from the animal center of si chuan university , and were cared for and experimented on under specific , pathogen - free conditions at our animal facilities in the laboratory animal center of west china medical school , sichuan university . hdm ( 50000 bu / ml ) was obtained from allergopharma ( reinbeck , germany ) . the mice were divided into 4 groups : control group , hdm alone group , ova alone group , and ova+hdm group ( table 1 ) with 10 mice in each group . ova ( 100 g / ml , sigma , bornem , belgium ) was mixed with an equal volume of 10% ( w / v ) alum ( sigma , usa ) in distilled water . to establish the ova asthmatic model , the mice were sensitized by intra - peritoneal injections of ova / alum ( 100 g / ml ) for 13 days . then the mice were aerosol inhalation challenged with 10 ml ova / pbs ( 2 mg / ml ) during day 3337 . in ova+hdm group , ova - induced asthmatic mice received nasal drops 100 l hdm 10 bu / ml ( total 1 bu ) 1 hour after aerosol inhalation of ova / pbs , while in control group , mice were sensitized by intra - peritoneal injection of pbs , were aerosol inhalation challenged with pbs solution , and received nasal drops with pbs . the number of sneezing and nasal rubbing movement of mice were counted in an observation cage for 20 min . the observer assessed movements by the double - blinded method . serum , bronchial alveolus lavage fluid , spleen , thymus and lung tissues were obtained for further analysis . the original pathologic specimen of lung tissues were fixed in 10% neutral buffered formalin and processed to paraffin , and 45 m sections were prepared . for histopathology , for immunohistochemistry , sections were incubated with 1:200 diluted anti - il-17 antibody ( boaosen , beijing , china ) overnight at 4c . then the sections were washed with pbs and incubated with 1:1000 diluted biotinylated secondary anti - goat immunoglobulin ( zhongshan , beijing , china ) . the positively stained cells were assessed in 20 randomly selected fields of view on each image at a final magnification of 400 . mrna level of rorc ( retinoic acid - related orphan receptor c ) il-17a , il-23 , t - bet ( t - box transcription factor ) , gata-3 ( gata binding protein ) were estimated by quantitative pcr . total rna was purified using takara kit ( takara , dalian , china ) following manufacture s instructions . cdna was synthesized by the prime script rt reagent kit ( takara , dalian , china ) . cdna , equivalent to 40 ng total rna , was examined by an icycler iq quantitative pcr detection system ( bio - rad , ca , usa ) by their specific primer sequences : ( 1 ) rorc forward : tc tgg gat cca cta cgg g and reverse : aaa ctt gac agc atc tcg gg ; ( 2 ) il-17a forward : ctg tgt ctc tga tgc tgt tgc and reverse : cgt gga acg gtt gag gta gt ; ( 3 ) t - bet forward : gcc agg gaa ccg ctt ata t and reverse : gag tga tct ctg cgt tct ggt ; ( 4 ) gata-3 forward : tga cgg aag agg tgg acg ta and reverse : tgg atg gac gtc ttg gag aa ; ( 5 ) il-23 forward : cca gtt ctg ctt gca aag g and reverse : ggt gat cct ctg gct gga ; ( 6 ) -actin forward : agg gtg tga tgg tgg gaa t and reverse : ctc ggt gag cag cac agg . pcr amplification consisted of 40 cycles of denaturation at 95c for 15 s and annealing and extension at 60c for 45 s. the target product was detected by sybr premix ex taq ii kit ( takara , dalian , china ) and -actin was used to normalize for transcription and amplification variations among samples . the relative expression quantity was assessed by the gene expression macro version 1.1 software ( bio - rad , ca , usa ) . the serum , bronchial alveolus lavage fluid , spleen , thymus and lung tissues were gathered from mice at room temperature and stored at 80c . the levels of inf- , il-5 , il-23 and il-17 cytokines were analyzed by commercial sandwich elisa kits ( r&d systems , minneapolis , usa ) . two - way anova test and post - hoc analysis were used for intra - group comparisons . the 6-week - old male balb / c mice were purchased from the animal center of si chuan university , and were cared for and experimented on under specific , pathogen - free conditions at our animal facilities in the laboratory animal center of west china medical school , sichuan university . hdm ( 50000 bu / ml ) was obtained from allergopharma ( reinbeck , germany ) . the mice were divided into 4 groups : control group , hdm alone group , ova alone group , and ova+hdm group ( table 1 ) with 10 mice in each group . ova ( 100 g / ml , sigma , bornem , belgium ) was mixed with an equal volume of 10% ( w / v ) alum ( sigma , usa ) in distilled water . to establish the ova asthmatic model , the mice were sensitized by intra - peritoneal injections of ova / alum ( 100 g / ml ) for 13 days . then the mice were aerosol inhalation challenged with 10 ml ova / pbs ( 2 mg / ml ) during day 3337 . in ova+hdm group , ova - induced asthmatic mice received nasal drops 100 l hdm 10 bu / ml ( total 1 bu ) 1 hour after aerosol inhalation of ova / pbs , while in control group , mice were sensitized by intra - peritoneal injection of pbs , were aerosol inhalation challenged with pbs solution , and received nasal drops with pbs . the number of sneezing and nasal rubbing movement of mice were counted in an observation cage for 20 min . the observer assessed movements by the double - blinded method . serum , bronchial alveolus lavage fluid , spleen , thymus and lung tissues were obtained for further analysis . the original pathologic specimen of lung tissues were fixed in 10% neutral buffered formalin and processed to paraffin , and 45 m sections were prepared . for histopathology , for immunohistochemistry , sections were incubated with 1:200 diluted anti - il-17 antibody ( boaosen , beijing , china ) overnight at 4c . then the sections were washed with pbs and incubated with 1:1000 diluted biotinylated secondary anti - goat immunoglobulin ( zhongshan , beijing , china ) . the positively stained cells were assessed in 20 randomly selected fields of view on each image at a final magnification of 400 . mrna level of rorc ( retinoic acid - related orphan receptor c ) il-17a , il-23 , t - bet ( t - box transcription factor ) , gata-3 ( gata binding protein ) were estimated by quantitative pcr . total rna was purified using takara kit ( takara , dalian , china ) following manufacture s instructions . cdna was synthesized by the prime script rt reagent kit ( takara , dalian , china ) . cdna , equivalent to 40 ng total rna , was examined by an icycler iq quantitative pcr detection system ( bio - rad , ca , usa ) by their specific primer sequences : ( 1 ) rorc forward : tc tgg gat cca cta cgg g and reverse : aaa ctt gac agc atc tcg gg ; ( 2 ) il-17a forward : ctg tgt ctc tga tgc tgt tgc and reverse : cgt gga acg gtt gag gta gt ; ( 3 ) t - bet forward : gcc agg gaa ccg ctt ata t and reverse : gag tga tct ctg cgt tct ggt ; ( 4 ) gata-3 forward : tga cgg aag agg tgg acg ta and reverse : tgg atg gac gtc ttg gag aa ; ( 5 ) il-23 forward : cca gtt ctg ctt gca aag g and reverse : ggt gat cct ctg gct gga ; ( 6 ) -actin forward : agg gtg tga tgg tgg gaa t and reverse : ctc ggt gag cag cac agg . pcr amplification consisted of 40 cycles of denaturation at 95c for 15 s and annealing and extension at 60c for 45 s. the target product was detected by sybr premix ex taq ii kit ( takara , dalian , china ) and -actin was used to normalize for transcription and amplification variations among samples . the relative expression quantity was assessed by the gene expression macro version 1.1 software ( bio - rad , ca , usa ) . the serum , bronchial alveolus lavage fluid , spleen , thymus and lung tissues were gathered from mice at room temperature and stored at 80c . the levels of inf- , il-5 , il-23 and il-17 cytokines were analyzed by commercial sandwich elisa kits ( r&d systems , minneapolis , usa ) . two - way anova test and post - hoc analysis were used for intra - group comparisons . the classical ova - induced asthmatic mice were generated referencing the method of other investigators . after the final administration , the mice in the hdm alone group , ova alone group and ova+hdm group were slow in movement and showed various degrees of respiratory failure . the increase frequency of nasal rubbing and sneezing were found in ova alone group and ova+hdm group compared with control group . eosinophil infiltrations , epithelial cell hyperplasia and airway obstruction were evident in each experimental group in comparison with control group . more inflammatory cell infiltration was observed in hdm alone group than that in control group ( figure 1a , b ) . it indicated that hdm can directly evoke non - allergic inflammatory response , in accordance with others conclusions . compared with ova alone group , the inflammatory response in ova+hdm group was more evident ( figure 1 c , d ) . hdm can evoke non - allergic inflammatory , we assume that the non - allergic inflammatory is present in ova+hdm group . in spleen tissue , rorc , il-17 , il-23 and gata3 mrna expressions were increased in ova+hdm group , while they were elevated with no significant difference in hdm alone and ova alone group in comparison with control group ( figure 2a ) . in thymus tissue , there was no significant difference in gene mrna expressions among all the groups . th2 and th17 response in spleen tissue was more obvious than that in thymus tissue . we deduced that spleen tissue was easy respone to the stimulation of allergen . in lung tissue , th2 and th17 related gene mrna expressions were not significant increased in hdm alone group as we supposed . they were significant elevated in ova alone group and ova+hdm group compared with control group . in comparison with ova alone group , th2 and th17 responses were still significant augmented in ova+hdm group . th2 and th17 response related gene mrna expressions were augmented when ova - induced asthmatic mice were exposed to hdm . th2 and th17 response related cytokines in serum and bronchoalveolar lavage fluid were detected by elisa . in serum , il-17 was sharply up - regulated only in ova+hdm group compared with other groups , while inf- and il-23 showed no evident increase ( figure 3a ) . both il-17 and il-23 were apparently increased in bronchoalveolar lavage fluid in ova+hdm group , but not in hdm alone group and ova alone group ( figure 3b ) . il-5 was augmented in both ova alone group and ova+hdm group in serum and bronchoalveolar lavage fluid compared to control group ( figure 3a , b ) . in comparison with ova alone group , il-5 was increased with no significant difference in ova+hdm group in bronchoalveolar lavage fluid ( figure 3a ) . in other words , il-17 staining was low to moderate intensity in inflammatory cells in hdm alone group ( figure 4b ) . abundant infiltrating inflammatory cells were moderate positive for il-17 in ova alone group ( figure 4c ) . in accordance with elisa and gene mrna expressions results , il-17 was strongly positive in inflammatory cells in ova+hdm group compared with other groups by immunohistochemistry ( figure 4a , d and table 2 ) ) . the classical ova - induced asthmatic mice were generated referencing the method of other investigators . after the final administration , the mice in the hdm alone group , ova alone group and ova+hdm group were slow in movement and showed various degrees of respiratory failure . the increase frequency of nasal rubbing and sneezing were found in ova alone group and ova+hdm group compared with control group . eosinophil infiltrations , epithelial cell hyperplasia and airway obstruction were evident in each experimental group in comparison with control group . more inflammatory cell infiltration was observed in hdm alone group than that in control group ( figure 1a , b ) . it indicated that hdm can directly evoke non - allergic inflammatory response , in accordance with others conclusions . compared with ova alone group , the inflammatory response in ova+hdm group was more evident ( figure 1 c , d ) . hdm can evoke non - allergic inflammatory , we assume that the non - allergic inflammatory is present in ova+hdm group . in spleen tissue , rorc , il-17 , il-23 and gata3 mrna expressions were increased in ova+hdm group , while they were elevated with no significant difference in hdm alone and ova alone group in comparison with control group ( figure 2a ) . in thymus tissue th2 and th17 response in spleen tissue was more obvious than that in thymus tissue . we deduced that spleen tissue was easy respone to the stimulation of allergen . in lung tissue , th2 and th17 related gene mrna expressions were not significant increased in hdm alone group as we supposed . they were significant elevated in ova alone group and ova+hdm group compared with control group . in comparison with ova alone group , th2 and th17 responses were still significant augmented in ova+hdm group . th2 and th17 response related gene mrna expressions were augmented when ova - induced asthmatic mice were exposed to hdm . th2 and th17 response related cytokines in serum and bronchoalveolar lavage fluid were detected by elisa . in serum , il-17 was sharply up - regulated only in ova+hdm group compared with other groups , while inf- and il-23 showed no evident increase ( figure 3a ) . both il-17 and il-23 were apparently increased in bronchoalveolar lavage fluid in ova+hdm group , but not in hdm alone group and ova alone group ( figure 3b ) . il-5 was augmented in both ova alone group and ova+hdm group in serum and bronchoalveolar lavage fluid compared to control group ( figure 3a , b ) . in comparison with ova alone group , il-5 was increased with no significant difference in ova+hdm group in bronchoalveolar lavage fluid ( figure 3a ) . in other words , il-17 staining was low to moderate intensity in inflammatory cells in hdm alone group ( figure 4b ) . abundant infiltrating inflammatory cells were moderate positive for il-17 in ova alone group ( figure 4c ) . in accordance with elisa and gene mrna expressions results , il-17 was strongly positive in inflammatory cells in ova+hdm group compared with other groups by immunohistochemistry ( figure 4a , d and table 2 ) ) . in a study performed in united states , 80% of allergic asthmatic adults were challenged by more than one aeroallergen . however , when investigating the pathomechanism of asthma , many researchers merely focus on a single allergen challenge model . in recent years hdm , a main allergen in asthma patient living environment , is strongly associated with the development of asthma . the mechanism of asthma patients who re - exposed to hdm is less well defined . to know more about the asthma patient who re - expose to hdm allergen rorc , il-17 and il-23 were chosen as th17 response related genes referencing previous studies . as for th2 response , gata3 and il-5 were selected , while t - beta and inf- were selected for th1 response . in our study , th1 , th2 and th17 response related genes were detected in lung tissue and central immune organs of our model . unexpectedly , th17 response associated genes were significant elevated in spleen tissue in ova+hdm group compared with ova alone group , while the tendency was not observed in thymus tissue . interestingly , th2 and th17 response related gene mrna expressions , not th1 , were significant augmented in ova+hdm group compared with ova alone group in lung tissue . given that the gene mrna expression results were not as we expected , th1 , th2 and th17 responses related cytokines were detected in serum , bronchoalveolar lavage fluid and lung tissue . il-17 and il-23 were sharply augmented in ova+hdm group compared with ova alone group , while il-5 was increased with not significant difference . as the result of transcription regulation , the mrna expressions of th2 response related gene was inconsistent with protein expression in ova+hdm group . we confirm that the augmentation of th17 response is linked to the inflammation in the ova - induced asthmatic mice exposed to hdm . we also conclude that th17 and th2 responses are definitely augmented in the ova - asthmatic model , but when it is re - exposed to hdm , th17 response is dominant . that is to say , if the ova - induced asthmatic mice is re - exposed to hdm , the pathogenesis would be different from ova alone exposure . therefore , hdm is an important interferential factor for asthma therapy , which also has important implications in the development of future asthma therapy . high level of il-23 in lung tissue induces th17 cell to participate in the inflammation of asthma , so that we can widely find il-17 cytokine in balf and serum . the augmented th17 cells also have a particular ability to recruit granulocytes to participate in . hence , the inflammation in ova + hdm group was more serious than that in ova alone group . to our knowledge , this is the first study to investigate the pathogenesis of asthma with complex hdm exposure . recent study has revealed that hdm can evoke the non - allergic asthma , which is similar with the allergic asthma . we observed inflammatory cell infiltrations in lung tissue in hdm alone group , which was consistent with previous findings . thus , we assume that non - allergic inflammatory response is present in ova+hdm group . however , single hdm challenge may not be enough to achieve reliable lung function changes , and it appears that multiple challenges may be necessary in the model . this is the reason we have difficulty in inducing the asthmatic model by a single hdm challenge . from the inflammation in hdm alone group , we infer that the longer people are exposed to hdm , the more risk of developing to asthma they will undertake . the non - allergic inflammatory response may be present in the inflammation in ova- induced asthmatic mice exposed to hdm . the augmented th17 response is linked with the inflammation in ova - induced asthmatic mice exposed to hdm . when the ova - induced asthmatic mice are re - exposed to hdm , the pathomechanism is different from ova alone exposure . thus , hdm is an important interferential factor for asthma therapy in the indoor living environment of asthma patients
summarybackgroundit is widely accepted that t helper 2 ( th2 ) cells , th17 cells and their cytokines orchestrate the feature of asthma 
 . however , most of studies on asthma mechanisms use a single allergen challenge model . 
 actually , humans are concurrently exposed to various allergens , and the mechanism of asthma with complex allergen exposure is less well defined . to explore whether the mechanism would be altered 
 if asthma patients are re - exposed to another allergen , we exposed the chicken egg albumin ( ova ) induced - asthmatic mice to house dust mite ( hdm).material / methodshe staining was used to analyze pathologic variation in lung tissue of mice in each sub - group : control group , hdm alone group , ova alone group and ova+hdm group . 
 th1 , th2 and th17 associated gene mrna expressions were detected by quantitative pcr ; associated cytokines were determined by elisa or immunohistochemistry.resultsthe severe of inflammatory cell infiltration , the augmentation of th17 and th2 related gene mrna expressions and the increase of th17 associated cytokines expression were shown in ova+hdm group in comparison with ova alone group . 
 however , th2 related cytokines were increased with no significant difference in ova+hdm group compared with ova alone group.conclusionswe have found that th17 response is connected with inflammation in the ova - induced asthmatic mice exposed to hdm . when ova - induced asthmatic mice are re - exposed to hdm , 
 the pathomechanism is different from ova alone exposure . 
 hdm , indoor allergen , may be an important interferential factor for asthma therapy . 
 it will give an important direction in the development of future asthma therapy .