asthma is a chronic inflammatory disease of the airways affecting over 300 million people worldwide , and its prevalence is rising , especially in children and within developing countries . new studies indicated that in allergic disorders , including asthma , innate immunity is deregulated by allergens that promote sensitization . however , the underlying immunological processes are still not fully understood as asthma is a complex multifactorial disease in which both innate and adaptive immune responses are involved . in addition , asthma is considered as a complex syndrome with different clinical phenotypes in children and adults . eosinophils and neutrophils play a key role in the cellular airway inflammation [ 4 , 5 ] . mild asthma is characterized by chronic inflammation of the airways that is mostly eosinophilic in nature and allergic sensitization . the resulting airway inflammation is thought to be caused by a breakdown of immune tolerance toward environmental antigens and leads to a t helper type 2(th2)-biased immune response . on the contrary , neutrophil accumulation in the bronchial mucosa is an important feature of severe asthma and frequently includes a t helper type 1(th1 ) component as well as a th2 immune response [ 4 , 6 ] . this heterogeneity highlights the importance of more specific treatment approaches based on asthma phenotypes . pattern recognition receptors ( prrs ) , like the toll - like ( tlrs ) and nod - like ( nlrs ) families of receptors , are key components of the innate immune system . these prrs exhibit different cell and stimulus - specific patterns of expression . in the human airways , tlrs are expressed in and on dendritic cells ( dcs ) , epithelial cells , eosinophils , macrophages , and mast cells . nod1 and nod2 are intracellular pattern recognition molecules ( prms ) expressed in various human epithelial cells including lung cells . multiple dc functions are controlled by prrs and , ultimately , modulate the resulting adaptive immune response [ 8 , 10 ] . upon prr activation in the lung , various chemokines and cytokines are produced by mast cells and eosinophils that recruit activated b - lymphocytes and th lymphocytes to the lung , starting the inflammation process in the airways . mast cells express the high - affinity receptors ( fcri ) for immunoglobulin e ( ige ) on their surface , and animal studies have demonstrated that mast cells play an important role in the induction of allergic airway inflammation . in addition , asthmatic patients have been shown to have an increased number of lung mast cells and allergen - specific ige , a phenomenon of allergic asthma shared with mouse models for this disease . in order to reproduce more of the clinical features of severe asthmatic patients , there has been a focus on using ige immune complexes as inducers of immune responses in the murine lung . trinitrophenyl-(tnp- ) ovalbumin-(ova- ) ige immune complexes have been shown to be more potent inducers of immune responses than antigens alone , since challenge of sensitized mice with these complexes resulted in an increased migration of mast cell progenitors to the lung [ 12 , 13 ] . the expression and function of prrs have been linked to susceptibility towards allergic asthma [ 2 , 14 ] . functional genetic variations in tlr1 , tlr6 , and tlr10 genes affecting gene and protein expression have been shown to be associated with increased mrna expression of these tlrs and to protect against atopic asthma in humans . single nucleotide polymorphisms ( snps ) in the tlr2 gene that led to decreased mrna expression were positively associated with asthma susceptibility . cord blood cd34 ( + ) cells from high - atopic - risk infants exhibited low tlr2 , tlr4 , and tlr9 expressions . additionally , amino acid changes in the tlr2 gene have been linked to reduced tlr2 receptor function and to increase in atopy risk in humans . a study in a murine macrophage cell line suggested a proinflammatory role of tlr4 and 5 in the disease . animal studies have demonstrated that the dose of the tlr4 ligand , lipopolysaccharide ( lps ) , determines the type of inflammatory response generated and that lung epithelial cells activation by tlr4 is crucial for induction of airway inflammation via activation of mucosal dcs [ 2022 ] . tlr9 is one of the most extensively studied tlrs in asthma , and it is currently thought to modulate allergic responses by skewing the balance from a th2 towards a th1 response . in addition , snps in the tlr9 gene were associated with increased risk of asthma . tlr11 , 12 , and 13 are not encoded in the human genome , and there are currently no data on associations with asthma in mice . insertion - deletion polymorphisms in the nod1 gene have been associated with increased risk of developing asthma , and genetic variations in nod1 that affected microbial recognition were positively associated with disease susceptibility and pathogenesis [ 26 , 27 ] . polymorphisms in nod2 , that affected lps recognition and tlr4 function , were associated with atopic diseases and were suggested to indirectly increase the severity of asthma .   in asthma , over 50 cytokines have now been identified to determine disease outcome . proinflammatory and th2-associated cytokines , including interleukin-4 ( il-4 ) , il-5 , il-6 , il-13 , and tumor necrosis factor  ( tnf- ) , are reported to enhance the disease . on the other hand , interferon- ( ifn ) , a th1-associated cytokine , in addition , asthmatic patients have been shown to have reduced levels of the anti - inflammatory cytokine il-10 in the sputum . il-10 is produced by macrophages and by a subset of regulatory t cells ( tregs ) and exerts its effects by inhibiting the synthesis of inflammatory cytokines ( including asthma - associated cytokines such as tnf- and il-5 ) and gene presentation . th2 cells play a key role in asthma , and asthmatic subjects have been reported to have th1/th2 imbalances as well as disturbed t helper type 17 ( th17)/treg balances . each th cell type is regulated by a specific transcription factor : tbet for th1 cells , gata-3 for th2 cells , retinoic acid orphan receptor-t ( rort ) for th17 cells , and forkhead box p3 ( foxp3 ) for tregs . animal and human studies have demonstrated that alterations in expression and/or of functions of these transcription factors can contribute to asthma pathogenesis [ 32 , 33 ] . the aim of this current study is to explore the innate and adaptive immune responses and inflammation in allergic asthma by investigation of the mrna expression profiles of the different prrs , t cell - related cytokines , and transcription factors . to this end , we have used a mouse model for both mild allergy and severe asthma with similar pathological characteristics seen in humans . findings from this observational study may contribute to elucidating the underlying mechanisms of mild and severe asthma and the involved inflammatory markers , as a first step in the development of phenotype - directed treatment approaches . male balb / c mice ( 68 weeks ; charles river laboratories , france ) were acclimated to their new environment for at least 1 week before the start of the experiment . all in vivo experiments were approved by and were in accordance with the guidelines of the local dutch committee of animal experimentation . mice were sensitized to ovalbumin ( ova ; chicken egg albumin , grade v , sigma , st . louis , mo , usa ) by intraperitoneal injections of 0.1  ml alum - precipitated antigen , comprising 10  g ova absorbed into 2.25  mg alum ( imject alum ; pierce , rockford , il , usa ) . control animals received 0.1  ml saline only ( nacl 0.9% ; b. braun medical bv , oss , the netherlands ) ( figure 1 ) .   mice were exposed to 10  mg / ml ova aerosol in saline using pari lc star nebulizer ( pari gmbh , starnberg , germany ) in an aerosol cabin for 30  min on days 35 , 38 , and 41 . mice were sensitized with trinitrophenyl-(tnp- ) conjugated ova by intraperitoneal injections of 0.1  ml alum - precipitated antigen , comprising 10  g tnp - ova absorbed into 2.25  mg alum . control animals received 0.1  ml saline only ( figure 1 ) .   from day 14 up to and including day 20 , mice were challenged daily by intranasal administration of a tnp - ovalbumin / ige immune complex ( 2  g tnp - ova plus 20  g dnp - specific ige ( clone h1 26.82 ) ) , as described previously . after sacrifice , on day 42 ( mild model ) or 21 ( severe model ) , lungs were first washed through a tracheal cannula with 1  ml saline containing protease inhibitor cocktail ( complete mini , roche diagnostics , mannheim , germany ) and prewarmed at 37c . cytospin cell preparations were made by cytospinning the cells onto glass for 5  min ( 400  g , 4c ) , and cytospins were stained by diff - quick ( merz and dade ag , ddingen , switzerland ) . numbers of eosinophils , macrophages , neutrophils , and lymphocytes were scored by light microscopy . after mice were sacrificed , on day 42 or 21 , the lungs were dissected , and mrna was isolated from whole lung tissue . messenger rna isolation ( n = 3 mice per group ) was carried out according to the qiagen rneasy mini kit protocol ( qiagen benelux bv , venlo , the netherlands ) . reverse transcriptase pcr was performed using an iscript cdna synthesis kit ( bio - rad laboratories , hercules , ca , usa ) . the reactions were performed in a ptc-100tm programmable thermal controller ( m. j. research inc . , cdna was amplified using iq sybr green supermix in a 96-well pcr plate and run in a cfx96 real - time pcr detection system ( bio - rad ) . primers for tlrs , nlrs , ribosomal protein s13 ( rps13 , reference gene ) , and t - cell transcription factors were purchased by isogen ( isogen life science , de meern , the netherlands ) . the sequences are listed in supplementary table 1 available online at http://dx.doi.org/10.1155/2013/808470 . for mouse t - cell cytokines , the protocol used for amplification was 94c for 3  min , 94c for 10  sec , and specific melt temperature for 45  sec , followed by 39 cycles of 94c for 10  sec and 95c for 10  sec . normalized gene expression ( ct ) was calculated using the built - in gene expression analysis module in cfx manager software ( cfx manager software version 1.6 ) . data analysis was performed using a 1-way analysis of variance ( one - way anova ) with the bonferroni 's post hoc test . in some studies all statistical analyses were performed using the graphpad prism software program ( graphpad prism software version 5.03 ) . mice were rendered asthmatic following the scheme presented in figure 1 . to examine the extent of pulmonary inflammation in the asthmatic mice , bronchoalveolar lavage ( bal ) fluid was examined for leukocyte accumulation ( figure 2 ) . in mild asthma , allergen - sensitized and challenged mice showed a significant increase in the total inflammatory cell number ( figure 2(a ) ) which was due to a relative increase in the number of lymphocytes and eosinophils ( figure 2(c ) ) in bal fluid compared to challenged only mice . in severe asthma , a significantly higher total inflammatory cell number ( figure 2(b ) ) was observed in allergen - sensitized and challenged mice compared to challenged only mice , and this was due to a relative increase in the number of eosinophils ( figure 2(d ) ) . as prrs in the lung can modulate ongoing chronic inflammation during asthma , the mrna expression of tlr1 - 13 and nod1 and 2 was measured ( figure 3 ) . in the mild model , tlr2 expression was significantly increased in control mice when compared to sensitized only , challenged only , and sensitized and challenged mice ( figure 3(a ) ) . in the severe model , tlr2 expression was significantly increased in challenged only mice when compared to control , sensitized only , and allergen - sensitized and challenged mice ( figure 3(a ) ) . in addition , sensitized and challenged mice showed significantly higher tlr1 expression when compared to sensitized only mice and significantly lower nod1 expression in comparison to control mice ( figure 3(a ) ) . in mild asthma , tlr3 expression was significantly decreased in allergen - sensitized and challenged mice when compared to control mice ( figure 3(b ) ) . in severe asthma , tlr3 expression was significantly lower in sensitized and challenged mice but not in control , sensitized only , and challenged only mice ( figure 3(b ) ) . in addition , tlr7 expression was significantly higher in challenged only mice when compared to control and sensitized only mice ( figure 3(b ) ) . the expression of tlr11 and tlr12 remained unchanged in both models ( figures 3(c ) and 3(d ) ) . interestingly , in the severe model , the mrna expression of tlr1 , tlr3 , tlr6 , tlr9 , tlr11 , tlr13 , nod1 , and nod2 was significantly correlated with the total inflammatory cell number in bal fluid ( table 1 ) .   to determine the extent of inflammation in the lung , the mrna expression of various cytokines was measured ( table 2 ) . in the severe model , the expression of almost all cytokines tended to be increased in tnp - ova - sensitized and tnp - ova / ige - challenged mice when compared to ova - sensitized and challenged mice in the mild model . an 8 and 33-times higher il5 and il6 expressions , respectively , was observed in allergen - sensitized and challenged mice but not in control , sensitized only , and challenged only mice .   in mild asthma , il10 showed the largest change in expression followed by ii5 , ii4 , tnf , il6 , il2 , il13 , and ifn , respectively , ( figure 4(a ) ) . in severe asthma , the largest change ( 110-fold ) is observed in ifn expression followed by il13 , il2 , il4 , il10 , il6 , il5 , and tnf , respectively ( figure 4(b ) ) .   to examine the t cell responses in the lung , the mrna expression of t cell - specific transcription factors was measured , and to determine the extent of th response skewing in the lung , ratios for gata3/tbet ( th2/th1 ) , foxp3/rort ( treg / th17 ) , foxp3/gata3 ( treg / th2 ) , and foxp3/tbet ( treg / th1 ) mrna expressions were calculated ( table 3 ) . the expression of tbet and foxp3 was 7 and 2-times higher , respectively , in allergen - sensitized and challenged mice in the severe model when compared to allergen - sensitized and challenged mice in the mild model . in severe asthma , sensitized and challenged mice showed 16 and 17-times higher tbet and foxp3 expression , respectively , when compared to control , sensitized only and challenged only mice . most interestingly , allergen sensitization and challenge resulted in a significant decrease in gata3/tbet ratio as compared to control and sensitized only mice in both models . the ratio of foxp3/rort was 12-times higher in the severe model in comparison to the mild model , and this ratio was significantly increased in allergen - sensitized and challenged mice when compared to control , sensitized only , and challenged only mice in both models . in the mild model , the ratio of foxp3/gata3 in allergen - sensitized and challenged mice was significantly increased when compared to control , sensitized only , and challenged only mice , and this ratio was almost 4-times higher than the mild model . in mild asthma , the largest change ( 9-fold ) was observed in foxp3 expression followed by tbet , gata3 , and rort ( figure 5(a ) ) . tbet showed the largest change in expression in severe asthma followed by foxp3 , gata3 , and rort ( figure 5(b ) ) . as imbalances in t cell responses can also be detected using t cell - specific transcription factors , the correlations between the mrna expression of t cell transcription factors and t cell - specific cytokines were calculated ( table 4 ) . in mild asthma , tbet expression was significantly correlated with il2 , il4 , il5 , il6 , il13 , and tnf expression . rort expression showed a significant correlation with il10 expression , and foxp3 expression was significantly correlated with and il2 , il4 , il5 , il6 , il10 , and tnf expression . in severe asthma , rort expression was significantly correlated with il2 , il4 , il5 , il6 , il10 , and il13 expression . interestingly , foxp3 expression was strongly correlated with il2 , il4 , il5 , il6 , and il13 and significantly correlated with il10 and ifn expression . the aim of this study was to explore the innate and adaptive immune responses and inflammation in mouse models for mild and severe allergic asthma . our results clearly show that pulmonary inflammation is differentially regulated in mild and severe experimental asthma . in the severe asthma model , a higher ( 3-fold ) cell influx in bal fluid was seen compared to the mild model . as expected , tnp - ova - sensitized and tnp - ova / ige - challenged mice showed significantly higher total inflammatory cell number and a relative increase in the number of eosinophils , lymphocytes , and neutrophils as compared to challenged only mice . these findings are in accordance with other animal studies in which ige immune complexes have been used as inducers of airway inflammation [ 12 , 13 , 34 ] .   besides being key components of the innate immunity , prrs are also involved in the activation and shaping of adaptive immunity . the function and expression of prrs have been linked to susceptibility towards allergic asthma . in mild asthma , tlr2 expression was lower in sensitized only , challenged only and ova - sensitized and challenged mice , when compared to the control mice . previous human studies have demonstrated that decreased tlr2 mrna expression and receptor function due to snps in the tlr2 gene are positively associated with asthma susceptibility , and high - atopic - risk infants have been reported to have low tlr2 expression on their cord blood cd34 ( + ) cells [ 1517 ] . in severe asthma , allergen challenge only increased the expression of tlr2 and allergen sensitization and challenge significantly increased tlr1 expression when compared to sensitized only mice . these results are supported by data from animal and human studies which have shown that tlr2/tlr1 heterodimers can play both pro- and anti - inflammatory roles in allergic asthma [ 16 , 35 ] . previous in vitro studies have demonstrated that upon the activation of tlr3 by its ligand , this prr induces upregulation of its own expression as well as expression of other tlrs and various cytokines and chemokines and thereby contributes to exacerbation of inflammation . however , no direct associations between tlr3 expression and function and asthma have been reported yet , and whether the decreased mrna expression of tlr3 caused by the chronic inflammatory status of the animals is proinflammatory or anti - inflammatory is also unknown . in severe asthma , this could be due to a response of plasmacytoid dcs to the inhaled antigen as these cells strongly express tlr7 . in addition , allergen sensitization and challenge decreased nod1 expression when compared to control mice . single nucleotide polymorphisms in the nod1 gene were positively associated with susceptibility towards asthma in children living on farms , and this prr has been reported to be necessary for neutrophil function in mice [ 26 , 27 , 37 ] . however , no direct associations between nod1 expression and function and asthma have been reported yet . interestingly , tlr1 , tlr3 , tlr6 , tlr9 , tlr11 , tlr13 , nod1 , and nod2 expression was significantly correlated with the total inflammatory cell number in bal fluid in the severe model . these correlations might be explained by the increase in the number of inflammatory cells which express these receptors , as is the case for tlr1 and nod1 . additionally , these receptors could also contribute to the increased sensitivity to inflammation / exacerbation since a small trigger can lead to an inflammatory cascade . however , the link between these correlations and asthma pathogenesis remains to be investigated . different mrna expression profiles of t cell - related cytokines are observed in the mild and severe models for allergic asthma . to our knowledge , this is the first report in which cytokine mrna expression is measured in mouse whole lung tissue . in severe asthma , of particular interest , il10 showed the largest change in mrna expression in mild asthma . this profound 13-fold change in il10 expression might be necessary to limit the inflammation in the airways and to counter the effects of the other cytokines on disease progression as previously described . this hypothesis is supported by the findings in the severe model , in which ifn showed the largest change in expression suggesting a th1-skewed response . th2 cells play a key role in the pathogenesis of allergic asthma , and asthmatic patients have been reported to have th1/th2 imbalances as well as disturbed th17 ( th17)/treg balances . in mild asthma , in severe asthma , foxp3 and tbet showed the highest expression in lungs of allergen - sensitized and challenged mice . these findings are in line with our cytokine expression data ( described above ) and are supported by the proposed role of tregs and il10 in the airways . intriguingly , allergen sensitization and challenge resulted in a strong treg response in both models . accordingly , foxp3 showed the largest change in expression in the mild model . in severe asthma , the largest change in expression was found in the th1-related transcription factor , tbet , and this result is in contrast with data obtained from asthmatic patients [ 30 , 32 , 33 ] , possibly due to measurement of mrna expression in mouse whole lung tissue instead of in pbmcs of asthmatics and/or measurement of mrna expression instead of protein expression . it has been reported that both in vivo and in vitro gata3 can inhibit foxp3 gene induction by directly binding to the foxp3 promoter in mice . an opposite action of foxp3 might also be possible , and our results could be explained by a counter - regulatory mechanism of treg / th1 to suppress the th2 immune response . interestingly , the mrna expression of tbet was strongly correlated with the expression of innate cytokines ( il2 , il6 , and tnf ) and th2 cytokines ( il4 , il5 , and il13 ) , but not with th1 cytokine ( ifn ) . in severe asthma , il2 , il4 , il5 , il6 , il13 , and tnfhave been reported to enhance asthma in humans , and th1 cells have been shown to suppress th2 cells through the release of ifn-. additionally , tregs suppress other th cell effector functions through the release of il-10 . no correlations were found between gata3 and th2 cytokines expression suggesting a counter - regulatory mechanism of treg / th1 to suppress the th2 immune response as described previously . to our knowledge , our results demonstrated for the first time that in mild and severe models for experimental asthma , immune and inflammatory responses are regulated differently . we showed that in mild and severe allergic asthma different mrna expression of tlrs , nlrs and t cell - specific cytokines and transcription factors is observed . this study adds to our understanding of the allergic characteristics of mild and severe allergic asthma which can contribute to the identification of phenotype - specific therapeutic targets .