Experimental Research On Effects Of A Murine Interleukin-4 Receptor Antagonist On Mouse Asthmatic Airway Inflammation And Th1/Th2 Cell Disfunction

PART ONEA MODIFIED METHOD IN ESTABLISHING MOUSE ASTHMA MODELAnimal models can offer valuable information in several domains of asthma pathogenesis and treatment,so it is an important and useful tool for asthma research. In this study we choose the BALB/c mice as the target to create asthma model. An obvious advantage is that it is a genetically characterized inbred strain and the relative reagents are at low cost. Through developing the condition of animal feeding and modifying the way of sensitization, we sought to establish a new typical mouse asthma model in order to provide useful tools in the subsequent research of asthma.Objective To establish and assess a modified mouse asthma model.Methods SPF level female BALB/c mice were divided into 2 groups(n=20, 10 for each group).The asthmatic group was sensitized by i.p. and s.c. with ovalbumin(OVA) and aluminum potassium sulfate solution on day 0 and 14. Mice were challenged via the airways with OVA (5% in saline) for 10 days (from day 21 to 30) using high frequency ultrasonic nebulization. The mice of control group received saline as the substitution of OVA.Results Data showed that the mice of asthmatic group demonstrated symptoms of acute asthma, such as breathing deeply and fast, standing still, bowing the back, urinary and fecal incontinence, and so on. There were great increasing of leukocytes and eosinophils in the BALF of asthmatic group. Compared to the mice of control group, the levels of IL-4 and OVA specific IgE were significantly elevated while the levels of IFN-γwere remarkably decreased either in BALF or serum of the asthmatic mice. The airway inflammation, eosinophil infiltration, goblet cell hyperplasia, smooth muscle cell proliferation and pulmonary emphysema were much more serious in asthmatic mice than in control mice.Conclusion This successful modified mouse asthma model provides useful tools in the subsequent research of asthma.PART TWOCONSTRUCTION OF MIL-4RA EXPRESSION VECTOR AND EXPRESSION OF MIL-4RA PROTEIN IN AIRWAY EPITHELIA CELL LINE 9HTEInterleukin(IL)-4 and IL-13 are considered playing crucial roles in the pathogenesis of asthma, and both are key roles in the development and progress of chronic airway inflammation and airway hyperresponsiveness (AHR). To exert biological functions, IL-4 and IL-13 need to bind with their receptor complexes, and both share IL-4R alpha-chain as their mutual functional component. Mutations in the C terminus region of the IL-4 protein produce IL-4 mutants that bind to the IL-4R alpha-chain with high affinity, but do not induce cellular responses. Those IL-4 mutants are competitive antagonists for both IL-4 and IL-13. They have the same capacity to bind with the IL-4R alpha-chain as naive IL-4 and IL-13, but have no signaling activity, so can block the biological function of both IL-4 and IL-13. One of such mutant is a murine IL-4 truncated protein (C118 deletion), in which the terminal amino acids from position 118 onward are deleted. In this study we sought to produce an expression vector of this murine IL-4 receptor antagonist (mIL-4RA) and evaluate the expression of mIL-4RA protein in airway epithelial cell line 9HTE.Objective To construct an expression vector carrying the mIL-4RA (C118 deletion) gene and marker gene, enhanced-green fluorescent protein gene, and evaluate the expression of mIL-4RA protein in airway epithelial cell line 9HTE.Methods The mIL-4RA (C118 deletion) cDNA was amplified by RT-PCR, and subcloned into plasmid pMD18-T simple. After verified by gene sequencing, target gene was amplified by PCR. The 5' oligonucleotide of primer containing a start codon and Kozak sequence, and the 3' oligonucleotide incorporating a stop codon at position 119. PCR fragments were digested by XhoI and EcoRI, and subcloned into plasmid pIRES2-EGFP. Recombinant pIRES2-EGFP/IL-4RA construct was confirmed by XhoI and EcoRI double digestion and dideoxy sequencing, then tansfected into 9HTE cells. The expression of mIL-4RA protein in transfected 9HTE cells was identified by inverted phase fluorescence microscope and ELISA.Results The presence of mIL-4RA cDNA in the recombinant pIRES2-EGFP/IL-4RA construct was verified by PCR and gene sequencing. The 9HTE cells started to express GFP at 6 hours post-transfection and reached a peak at 48 hours post-transfection. Supernatants from the transfected 9HTE cells were harvested at 48 hours post-transfection and analysed by ELISA. The expression levels of mIL-4RA were 30.6±8.2 pg/ml and 2.5±1.2 pg/ml for pIRES2-EGFP/IL-4RA and control pIRES2-EGFP vector transfected 9HTE culture supernatant respectively.Conclusion The mIL-4RA expression vector was constructed successfully, and was able to express mIL-4RA protein in epithelia cell line 9HTE. This lays foundation for further study on mIL-4RA's biological activities and functions.PART THREEEFFECTS OF MIL-4RA EXPRESSION VECTOR ON ASTHMATIC AIRWAY INFLAMMATION AND TH1/TH2 CELL DISFUNCTION THROUGH INTRATRACHEAL ADMINISTRATIONAsthma is a common disease of respiratory system in children, nowadays inhaled corticosteroid is the main effective therapy. But the effect is not satisfied in all patients, so it is urgent to find new therapies. Researches have showed that the immune responses induced by Th2 cytokines played crucial roles in the onset, development, and outcome of asthma, especially the IL-4 and IL-13. Many researchers have regarded those two cytokines as interesting targets for asthma therapies. But because IL-4 and IL-13 play independent roles in the pathogenesis of asthma, researches targeting either IL-4 or IL-13 alone may have limited efficacy. In the study of part two,we have constructed a successful mIL-4RA expression vector. This mIL-4RA has the same capacity to bind with the IL-4R alpha-chain as native IL-4 and IL-13, but has no signaling activities, so can block the biological function of both IL-4 and IL-13. In this part we decided to evaluate the efficacy of this mIL-4RA on asthmatic airway inflammation and Th1/Th2 cell disfunction through intratracheal administration.Objective To investigate the therapeutic effect of mIL-4RA on asthmatic airway inflammation and Th1/Th2 cell disfunction through intratracheal administration.Material and methods SPF level BALB/c mice were randomly divided into 4 groups: healthy naive mice sensitized/challenged with normal saline (NS), OVA sensitized/challenged mice, OVA sensitized/challenged mice intratracheally (i.t.) administrated 10μg mIL-4RA encoding plasmid on the day of first sensitization, and OVA sensitized/challenged mice i.t. administrated 10μg control plasmid on the day of first sensitization. The airway inflammation was determined by histopathological examination. Cytokines were measured by ELISA and immunohistochemistry. The levels of phospho-STAT6 in lung were analyzed by immunofluorescent. Flow cytometry was employed to analyze CD4+ and CD8+ T-lymphocyte subsets in peripheral blood, lung, and spleen cells.Results Our data showed that compared to the mice administrated with control plasmid, intratracheal administration of mIL-4RA encoding plasmid on the sensitization phase protected the mice from the subsequent induction of asthmatic airway inflammation. The eosinophil infiltration in BALF was significantly lower than the control (p<0.01). Compared to the control plasmid treated mice, the levels of IL-13 were significantly decreased (p<0.01) while the levels of IFN-γwere remarkably elevated (p<0.01) in BALF of the mIL-4RA treated mice. Levels of IL-13 in serum were significantly decreased (p<0.01) in mIL-4RA treated mice while the levels of IFN-γin serum were not changed significantly (p>0.05). After treated with mIL-4RA expression plasmid, the levels of phospho-STAT6 in lung were significantly decreased. Our data also suggested that intratracheal administration with mIL-RA encoding plasmid had no influence on the production of CD4+/CD8+ T-lymphocyte subsets either in the peripheral blood,lung or spleen.Conclusions This study demonstrated that intratracheal administration of mIL-4RA encoding plasmid was able to attenuate the asthmatic airway inflamation and eosinophilia, regulate the disorder of Th1/Th2 cytokines in local airway with minor systemic effects, decrease the levels of phospho-STAT6, and had no influence on the production of CD4+/CD8+ T-lymphocyte subsets either in the peripheral blood,lung or spleen. PART FOUREFFECTS OF MIL-4RA EXPRESSION VECTOR ON ASTHMATIC AIRWAY INFLAMMATION AND TH1/TH2 CELL DISFUNCTION THROUGH INTRAPERITONEAL ADMINISTRATIONIn the study of part three, we treated the mouse asthmatic model by intratracheal administration with mIL-4RA expression plasmid and observed obvious therapeutic effects. But intratracheal administration belongs to one of the insult therapies. It needs severely restricted experimental condition, has high risks, and can not be done repeatedly. All those may lay barriers in the experimental research and further clinical study. So in this part we choose a more convenient and cost-effectiveness way to deliver mIL-4RA expression plasmid, that is by intraperitoneal administration (i.p.). We sought to analyze the efficacy of this therapy on asthmatic airway inflammation and Th1/Th2 cell disfunction and compare features of those two kinds of therapies.Objective To investigate the therapeutic effects of mIL-4RA on asthmatic airway inflammation and Th1/Th2 cell disfunction through intraperitoneal administration.Material and methods SPF level BALB/c mice were randomly divided into 4 groups: healthy naive mice sensitized/challenged with NS, OVA sensitized/challenged mice, OVA sensitized/challenged mice i.p. administrated 50μg mIL-4RA encoding plasmid on the day of first and second sensitization, and OVA sensitized/challenged mice i.p. administrated 50μg control plasmid on the day of first and second sensitization. The airway inflammation was determined by histopathological examination. Cytokines was measured by ELISA and immunohistochemistry. The levels of phospho-STAT6 in lung were analyzed by immunofluorescent assay. Flow cytometry was employed to analyze CD4+and CD8+T-lymphocyte subsets in peripheral blood, lung, and spleen cells.Results Our data showed that compared to the mice administrated with control plasmid, intraperitoneal administration of mIL-4RA encoding plasmid on the sensitization phase protected the mice from the subsequent induction of asthmatic airway inflammation. The eosinophil infiltration in BALF was significantly dimilished than the control (p<0.01). Compared to the control plasmid treated mice, the levels of IL-13 in mIL-4RA treated mice were significantly decreased (p<0.01) while the levels of IFN-γwere remarkably elevated (p<0.01) either in BALF or serum. The levels of phospho-STAT6 in lung were significantly decreased after mIL-4RA expression plasmid treated. Our data also suggested that intraperitoneal administration with mIL-RA expression plasmid had no influence on the production of CD4+/CD8+T-lymphocyte subsets either in the peripheral blood,lung or spleen.Conclusions This study demonstrated that intraperitoneal administration of mIL-4RA encoding plasmid was able to attenuate the asthmatic inflamation and eosinophilia, regulate the disorder of Th1/Th2 cytokines in local airway and serum, decrease the levels phospho-STAT6, and had no influence on the production of CD4+/CD8+T-lymphocyte subsets either in the peripheral blood,lung or spleen.