asthma is a common chronic inflammatory disorder , characterised by persistent inflammation of the airways with airway wall remodelling . acute exacerbations of asthma are episodes associated with severe airflow limitation that may require a change in treatment or a visit to an emergency department . exacerbations can be life - threatening and a substantial component of the health care costs of asthma is related to managing these events . they are associated with increased expression of a variety of proinflammatory cytokines and enhanced airway inflammation which extends to the distal airways , with recruitment of large numbers of eosinophils and neutrophils . these drugs are highly effective in reducing chronic inflammation and controlling clinical manifestations in the majority of patients , particularly when combined with long acting 2 agonists . however , long - term use of inhaled steroids is associated with adverse effects , particularly when high doses are used [ 7 , 8 ] . furthermore , a proportion of patients have asthma which is relatively resistant to steroid treatment [ 9 , 10 ] , and these drugs are not always effective during acute exacerbations . to date , there are very few alternatives to glucocorticoids . isu201 , a novel protein drug developed by isu abxis co. , ltd . , may have potential for treating inflammatory diseases including asthma . the active moiety of isu201 is the extracellular domain of the human cell - surface protein bone marrow stromal cell antigen 2 ( bst2 ) , stabilised for delivery by fusion with the fc region of human igg4 . our laboratory has developed a murine model of chronic asthma in which animals are sensitised to ovalbumin and chronically challenged with a low mass concentration of ovalbumin aerosol , to induce background lesions of chronic asthma including airway inflammation and airway wall remodelling . we have also established a model of an allergen - induced acute exacerbation of asthma in which chronically challenged animals are subsequently exposed to an additional moderate - level challenge with allergen . enhanced cytokine production and exaggerated inflammation of the airways and also involves smaller distal airways . these models have been acknowledged to closely replicate key features of the disease in humans   and are useful for evaluating the effects of novel anti - inflammatory compounds . using these models , we have recently demonstrated that long - term administration of isu201 inhibits the progression of inflammation and airway wall remodelling , whereas administration prior to the induction of an exacerbation can limit the development of airway inflammation . resolution of inflammation is an actively regulated process   driven by a variety of endogenous lipid - derived mediators , including resolvins . we recently demonstrated the potential of resolvin e1 ( rve1 ) as a proresolution agent , by administering the drug to animals after the induction of an acute exacerbation . resolution - promoting compounds may have potential for the treatment of chronic inflammatory diseases and may be alternatives to steroids for the treatment of exacerbations of asthma . however , it is unknown whether isu201 can limit the severity of an exacerbation when administered after it has been induced , a setting which more closely resembles that in which the drug would be used to treat patients with an exacerbation of asthma . in this study , we investigated whether isu201 could accelerate the resolution of airway inflammation when administered following induction of an allergen - induced acute exacerbation of mild chronic asthma . in parallel , we characterised its effects on the profile of expression of pro - inflammatory cytokines in the lung . all experimental procedures were approved by the unsw animal care and ethics committee ( acec 11/50a ) . specific pathogen - free female balb / c mice aged 7 - 8 weeks were obtained from australian bio - resources ( sydney , australia ) . animals were housed on autoclaved bedding in individually ventilated cages ( ventirack ) with a 12-hour light / dark cycle and food and water ad libitum . , animals were sensitised by intraperitoneal injection of 50  g of ovalbumin ( grade v ; sigma , australia ) adsorbed to 1  mg of alum in saline , 21 and 7 days prior to commencement of inhalational challenge . mice were challenged via the airways with a low mass concentration of an ovalbumin aerosol ( 3  mg / m ) in a whole body inhalation exposure chamber ( unifab corporation , kalamazoo , mi ) for 30 minutes / day on 3 days / week for 4 weeks to establish background lesions of chronic asthma . the concentration of ovalbumin within the chamber was continuously monitored using a dusttrack 8250 instrument ( tsi , st paul , mn ) . an acute exacerbation was induced by a subsequent single 30-minute challenge with a higher concentration ( 30  mg / m ) of aerosolised ovalbumin , which causes enhanced airway inflammation extending to the distal airways . isu201 was prepared and supplied by isu abxis co. , ltd . , ( seoul , korea ) . experimental groups of 6 mice were treated with isu201 in saline ( 20  mg / kg ; i.p . ) or with water - soluble dexamethasone ( 1  mg / kg ; p.o . ) at 2 and 8 hours after an acute exacerbation . these were compared to mice which received vehicle alone ( saline ; i.p . ) after the exacerbation . samples were collected at 4 , 12 , or 24 hours after the exacerbation ( figure 1(b ) ) . mice were killed by exsanguination following an overdose of sodium pentobarbital and the lungs perfused with saline to removed blood from the pulmonary capillary bed . bronchoalveolar lavage ( bal ) was performed by cannulating the trachea and washing the lungs with 2  1  ml of ice - cold pbs . bal fluid was collected for measurement of cytokine concentrations and a differential count of cells was performed on 300 cells in leishman - stained cytospin preparations . sections ( 5  m ) were cut , stained with haematoxylin and eosin , and assessed for peribronchiolar inflammation . tissue from the middle lobe of the right lung was collected in trireagent ( sigma ) for isolation of total rna . the upper lobe of the right lung was collected for assessment of eosinophil accumulation by assessing eosinophil peroxidase as previously described . the lower lobe of the right lung was lysed in protein lysis buffer ( cell signalling technology , beverly , ma ) and protein concentration in the lysate was determined using a protein assay ( bio - rad , gladesville , new south wales , australia ) . concentrations of proinflammatory cytokines il-6 and tnf- and chemokines cxcl1 and ccl11 were measured in bal fluid and lung tissue lysates using elisa kits ( r&d systems ) according to the manufacturer 's recommendations . total rna ( 5  g ) from lung tissue was treated with dnase ( turbo dnase ; ambion , soresby , australia ) and reverse - transcribed using superscript iii ( life technologies ) . quantitative real - time pcr was performed on a light cycler 480 ( roche ) to assess expression of mrna for a custom panel of 95 genes including cytokines , chemokines , adhesion molecules , growth factors , and housekeeping genes . amplified products were detected using sybr green ( bioline ) and expression was normalised to hprt . a one - way analysis of variance ( anova ) followed by a holm - sidak multiple comparison test was used to identify differences between experimental groups graphpad prism version 6.04 ( graphpad software , san diego , ca ) was used for the analysis of data and preparation of graphs . no adverse events occurred as a result of sensitisation , inhalational challenge , or drug treatment . we investigated the effect of treatment with isu201 , after inducing an acute exacerbation of experimental asthma , on pulmonary inflammation and cytokine production . following an allergen - induced exacerbation , the percentage of neutrophils in bal fluid was increased at 4 hours ( 19.3-fold 3.0 relative to naive mice ) , was maximal after 12 hours ( 22.7-fold 1.7 relative to naive mice ) , and had declined at 24 hours ( 9.5-fold 0.8 relative to naive mice ) . in mice treated with isu201 , the percentage of neutrophils in lavage fluid was significantly reduced relative to vehicle - treated animals after both 4 and 24 hours ( figure 2(a ) ) although numbers recruited at 12 hours were elevated . this contrasted markedly with dexamethasone , which significantly reduced neutrophil recruitment at all time points . eosinophil recruitment to the lung , measured as eosinophil peroxidase , was greatest after 4 hours ( 3.5-fold 0.4 relative to naive mice ) and slowly declined after 12 and 24 hours . isu201 significantly reduced the number of eosinophils in lung tissue at 4 and 12 hours after an acute exacerbation relative to vehicle - treated animals ( figure 2(b ) ) . in this case , the response was similar to suppression seen with dexamethasone at all time points tested . the increases in cytokine concentrations seen in lavage fluid in vehicle - treated animals were markedly suppressed in animals treated with isu201 after an acute exacerbation . isu201 reduced cxcl1 levels in bal fluid by 44% at 12 hours and by 53% at 24 hours compared to vehicle - treated controls ( figure 3(a ) ) . ccl11 levels were also significantly reduced by isu201 ( 45% reduction compared to vehicle - treated animals ) in bal fluid collected at 12 hours ( figure 3(b ) ) . concentrations of il-6 and tnf- in bal fluid were significantly reduced at 4 hours in animals treated with isu201 ( figures 3(c)-3(d ) ) . the suppression of cxcl1 , il-6 , and tnf- by isu201 was equivalent to that achieved with the glucocorticoid dexamethasone . in vehicle - treated mice , concentrations of proinflammatory cytokines in lung tissue lysates were significantly increased 4 hours after exacerbation and remained elevated for up to 24 hours relative to nave animals . levels of the chemokines cxcl1 and ccl11 were 4.9-fold and > 1000-fold higher in lysate than in bal fluid . treatment with isu201 after an acute exacerbation significantly reduced both cxcl1 and ccl11 relative to vehicle - treated animals at all times assessed ( figures 4(a)-4(b ) ) . isu201 also reduced the concentration of il-6 at 4 and 12 hours , while the concentration of tnf- was only reduced at 12 hours ( figure 4 ) . isu201 suppressed cxcl1 , ccl11 , and il-6 in lung lysate as effectively as the control drug dexamethasone . we next examined the effect of treatment with isu201 , after the induction of an exacerbation of asthma , on profiles of mrna expression of a panel of key proinflammatory mediators . in vehicle - treated mice , the expression of mrna for cxcl9 and cxcl10 was elevated 343-fold and 295-fold , respectively , 4 hours after an exacerbation , relative to nave mice . figures 5(a ) and 5(b ) show that isu201 was moderately effective in suppressing mrna levels of these chemokines , relative to vehicle - treated animals , at all times assessed , while dexamethasone causes marked suppression . adhesion of leukocytes to the vascular endothelium , which is critical for their extravasation , is modulated by induction of icam-1 and vcam . genes encoding vcam1 and icam1 were elevated 3.1-fold and 3.2-fold , respectively , in lung tissue from vehicle - treated mice 4 hours after an exacerbation . in animals treated with isu201 , the increase in vcam1 and icam1 mrna was completely suppressed 4 and 24 hours after an exacerbation ( figures 5(c)-5(d ) ) . isu201 also significantly reduced expression of mrna for the proinflammatory cytokines il1b , il12p40 , and csf1 in lungs collected at 4 and 24 hours ( figures 6(a)6(c ) ) . expression of mrna for il6 was significantly reduced by isu201 only at 24 hours ( figure 6(d ) ) . expression of mrna for these mediators was significantly reduced in mice treated with dexamethasone at all time points assessed , to the level seen in nave mice . treatment with isu201 did not significantly reduce expression of mrnas for the chemokines ccl2 , ccl3 , ccl11 , and cxcl1 or for il13 , while ccl4 was reduced only at 24 hours . in contrast , expression of these mrna species was significantly reduced in lungs from animals treated with dexamethasone at every time point examined ( see supplementary figure  1 in supplementary material available online at http://dx.doi.org/10.1155/2015/405629 ) . preclinical investigation of novel therapeutic agents requires not only appropriate animal models but also clinically relevant approaches to administration of drugs . in this study , we used our well - validated murine model to investigate the effects of isu201 on inflammation and cytokine production in an allergen - induced experimental acute exacerbation of chronic asthma . importantly , we assessed the response to treatment when the drug was administered after the induction of the exacerbation , to more closely simulate the clinical setting in which the drug might be used . we showed that treatment with isu201 reduced the accumulation of neutrophils in bal fluid collected at 4 and 24 hours and of eosinophils in lung tissue collected at 4 and 12 hours . assessment of levels of the chemokines cxcl-1 ( gro- , a neutrophil chemoattractant ) and ccl11 ( eotaxin-1 , an eosinophil chemoattractant ) confirmed that these were elevated following exacerbation . in keeping with its negative effect on neutrophil and eosinophil recruitment and in a manner similar to that seen with dexamethasone , isu201 markedly suppressed production of these chemokines . suppression was obvious in bal fluid and particularly in lung lysates , in which levels of chemokines were high and remained elevated for 24 hours or longer . importantly , the effects of isu201 on cytokine expression were apparent at multiple time points and were frequently of a similar magnitude to those of dexamethasone , which in this study was used as a positive control , at a much higher dose ( relative to body weight ) than what would be employed in patients . we also showed that when given after induction of an exacerbation , isu201 potently suppressed induction of chemokine genes important in lymphocyte recruitment . cxcl9 ( known as monokine induced by interferon- ( mig ) ) and cxcl10 ( known as interferon - induced protein 10 ( ip-10 ) ) were significantly reduced in lung tissue at all time points assessed , while the expression of mrna for il1b , il12p40 and csf1 was reduced at 2 of the 3 time points . for several other chemokines , levels of mrna were increased following induction of an exacerbation ( supplementary figure  1 ) but were not significantly altered by isu201 , whereas they were reduced by dexamethasone , indicating diverging pathways of suppression . unsurprisingly , the profile and time course of changes in expression of cytokine mrna did not correlate perfectly with protein levels detected in bal fluid or lysates . this may relate to the secretion of stored protein or the assessment of airway versus peripheral cellular sources of these mediators . there is a significant need for alternatives to steroids for treatment of exacerbations and for management of chronic asthma when steroids are not effective . collectively , our data suggest that isu201 has a potent and broad spectrum of activity , which affects a variety of cells and mediators . thus it seems reasonable to suggest that isu201 may be useful as an alternative to steroids for exacerbations . in this context , it is noteworthy that a number of recently developed treatments which specifically target individual cytokines , including il-4r , il-5 , and tnf- , have been ineffective in clinical trials or at best useful in selected populations of patients [ 2224 ] . this may in part be due to cytokine redundancy , in which cytokines with similar functions are not targeted . we believe that novel agents which target multiple cytokines and chemokines , such as isu201 , are more likely to be clinically effective than therapies which target a single molecule or pathway . the present study confirms and extends our previous work demonstrating that isu201 has potent anti - inflammatory activity . we have shown that administration of isu201 after induction of an exacerbation limits the peak accumulation of neutrophils and eosinophils , as well as the expression of mrna for proinflammatory cytokines . treatment with isu201 also promotes or accelerates the decline in numbers of inflammatory cells and levels of expression of cytokine mrna . the design of our study is in contrast to preclinical evaluations of the potential of novel anti - inflammatory compounds in which the drugs are only given prior to the induction of inflammation . relatively few studies examine the effect of treatment in established inflammation , which is a more clinically relevant setting [ 19 , 26 , 27 ] . typically , anti - inflammatory agents reduce leucocyte recruitment and inhibit cytokine and chemokine production , whereas resolution - promoting agents enhance apoptosis of leucocytes and stimulate phagocytosis . however , some drugs modulate both aspects of the inflammatory process . notably , glucocorticoids are potent anti - inflammatory agents but can also promote phagocytosis   and apoptosis [ 30 , 31 ] . in contrast , rve1 promotes the resolution of inflammation when administered to animals after the induction of an exacerbation but has limited anti - inflammatory activity when administered prior to an exacerbation of asthma . an ideal drug to treat inflammatory disease our previously published data indicate that isu201 functions as an anti - inflammatory agent , while the results of the present study establish that it is also a proresolution agent . currently , the mechanism(s ) of action of isu201 are not completely understood . we recently demonstrated that isu201 suppresses cytokine production by alveolar macrophages and lymphocytes , as well as reducing histone h4 acetylation in airway epithelial cells . the active moiety of isu201 ( bst2 ) has been shown to decrease the adhesion of monocytes to human umbilical vein endothelial cells   so it is also possible that isu201 may inhibit / impede the interaction between leucocytes and adhesion molecules on endothelial cells during inflammation . results from the current study have also demonstrated that treatment of animals with isu201 reduces the expression of mrna for vcam1 and icam1 , which may contribute to inhibiting the adhesion and emigration of inflammatory cells into the lungs . further investigation of the mechanism(s ) of action of isu201 is the focus of ongoing work in our laboratories . we have previously demonstrated that the novel protein drug isu201 can prevent the progression of airway inflammation and remodelling in a murine model of chronic asthma and that pretreatment can suppress the acute inflammatory response associated with an allergen - induced exacerbation . in the current study , we have demonstrated that when administered to animals after the induction of an acute exacerbation of asthma , isu201 effectively reduces the magnitude of the inflammatory response and accelerates the resolution of inflammation . isu201 also suppresses the increase in expression of mrna for a broad range of inflammatory cytokines . thus , this drug may have potential as an alternative to glucocorticoids in the management of asthma and its acute exacerbations .
glucocorticoids are commonly used for treating asthma and its exacerbations but have well - recognised adverse effects and are not always effective . 
 few alternative treatments exist . using a murine model of an acute exacerbation of asthma 
 , we assessed the ability of isu201 , a novel protein drug , to suppress the inflammatory response when administered after induction of an exacerbation . 
 sensitised mice were chronically challenged with a low mass concentration of aerosolised ovalbumin , and then received a single moderate - level challenge to simulate an allergen - induced exacerbation . 
 isu201 was administered to mice 2 and 8 hours later , while pulmonary inflammation and expression of mrna for chemokines and proinflammatory cytokines were assessed after 4 , 12 , and 24 hours . 
 relative to vehicle - treated controls , isu201 suppressed accumulation of pulmonary neutrophils and eosinophils , while accelerating the decline in cxcl1 , tnf- , and il-6 in lavage fluid and lung tissue . isu201 
 significantly reduced peak expression of mrna for the chemokines cxcl9 and cxcl10 , the adhesion molecules icam1 and vcam1 , and the proinflammatory cytokines il1b , il12p40 , and csf1 . 
 the ability of isu201 to promote resolution of inflammation suggests that it may have potential as an alternative to glucocorticoids in the management of asthma , including when administered after the onset of an acute exacerbation .