Inhaled Inactivated-mycobacterium Phlei Modulates γδT Cell Function And Alleviates Airway Inflammation In A Mouse Model Of Asthma

Part1Effects of inhaled inactivated-Mycobacterium phlei on airway inflammation in mouse asthmatic modelsObjective We use a murine model of OVA-induced asthma to study whether nebulized inhalation of inactivated Mycobacterium phlei can alleviate asthmatic airway inflammation through influencing cytokine production and determine whether it can prevent and treat asthma.Methods Fifty male Balb/c mice were randomly divided into4groups:normal control group (A), asthma model group (BO, B3, B4, B5), the treatment group (CO, C3, C4,C5), prevention group (D).Mice were sensitisated and challenged with Ovalbumin to make a murine asthma model. Group C were given treatment of aerosol mycobacterium phlei once daily after OVA challenge.Group C3, C4, C5were treated for3days,4days,5days respectively. Group D inhaled the solution of inactivated-Mycobacterium phlei daily before each time’s OVA challenge. All the animals were killed and lung tissue and bronchoalveolar lavage fluid(BALF) were harvested.Pathological HE staining, AB-PAS staining made to measure lung inflammation and mucus production.Total cell numbers and differential cell count in BALF were performed.Cytokines IL-4, IL-10, IFN-y levels in BALF were quantified by ELISA.Results In groupC4, C5and group D, IL-4production in BALF was decreased and IL-10, IFN-γ were increased (P<0.05).The number of total inflammatory cells and the mean percentage of eosinophils in the BALF of groupD, groupC4and group C5was lower than in the corresponding groupB(P<0.05).Histological examination of the lungs showed Airway inflammation of groupD and groupC5were attenuated.Conclusion The inhalation of Mycobacterium phlei can reduce airway inflammation in asthmatic mice.This ability was associated with its immunomodulatory effect on regulating IL-4, IL-10and IFN-y secretion.Aerosol administration of inactivated-Mycobacterium phlei may accepted as an alternative method with less risk of adverse reactions in treatment of asthma. Part2Inhaled inactivated-Mycobacterium phlei modulates γδT cell function and alleviates airway inflammation in a mouse model of asthmaObjective:We use an OVA-induced murine asthma model to study the effect of inhaled inactivated-Mycobacterium phlei therapy on attenuating asthmatic airway inflammation and influencing cytokine profile of y8T cell and Vy1,Vy4mRNA expression in lung.Methods:Male Balb/c mice were randomly divided into3groups of6mice each:normal control group (A), asthma model group (B), the treatment group (C).Mice were sensitisated and challenged with Ovalbumin to make a murine asthma model. Mice in group C were given treatment of aerosol mycobacterium phlei once daily for5days after OVA challenge.After the last inactivated-Mycobacterium phlei treatment,all the animals were killed and lung tissue were harvested.Pathological HE staining made to measure lung inflammation.Immunohistochemistry examination of ySTCR to detect γδT cells migrated into the lung.Flow Cytometric Analysis for intracellular cytokines of γδT cell,Quantitative Real-Time PCR for Vγ1mRNA, Vy4mRNA expression were performed.Results:In group C,airway inflammation were attenuated by treatment of inhaled inactivated-Mycobacterium phlei. IL-10+γδT cells,IFN-y+γδT cells,Vy4mRNA expression were significantly increased.Whereas,Vyl mRNA expression were significantly decreased.Conclusion:The inhalation of Mycobacterium phlei can modulate γδT cell function,the proportion of different γδT cell subsets and reduce airway inflammation in asthmatic mice. Part3Effects of inhaled inactivated-Mycobacterium phlei on airway inflammation and expression of TLR2in asthmatic miceObjective Studies have shown Mycobacterium bovis Bacille Calmette-Guerin (BCG) and other mycobacterial infections suppress airway hyperresponsiveness and inflammation in asthma.We use a murine model of asthma to study whether nebulized inhalation of inactivated Mycobacterium phlei can alleviate asthmatic airway inflammation through influencing cytokine production and TLR2.Methods Male Balb/c mice were randomly divided into3groups of8mice each:normal control group (A), asthma model group (B), the intervention group (C).Male Balb/c mice were made a murine asthma model with OVA. The mice in group C inhaled the solution of inactivated-Mycobacterium phlei daily before each time’s OVA challenge. Lung tissue and bronchoalveolar lavage fluid(BALF) were harvested.Results In group C,IL-4production in BALF was decreased and IL-10, IFN-y were increased;The levels of TLR2mRNA were increased compared to the model group.Inflammatory cells count and Histological examination of the lungs showed airway inflammation were attenuated because of using Mycobacterium phlei.Conclusion The inhalation of Mycobacterium phlei can alleviate airway inflammation in mouse asthmatic models.It has an immunomodulatory effect on regulating IL-4, IL-10and IFN-y secretion and expression of TLR2. Part4Therapeutic effects of inhaled Inactivated-mycobacterium phlei in adult patients with moderate persistent asthmaObjective:To observe the clinical therapeutic effects of inhaled inactivated-mycobacterium phlei in adult patients with moderate persistent asthma by way of atomizing inhalation via mucosa.Methods:This was a single-centre, randomized, controlled study. Twenty-eight patients with newly diagnosed uncontrolled moderate persistent bronchial asthma were randomly divided into two groups:group A (treatment group)and group B(control group),14in each group.The patients in group A were treated with solution of inhaled inactivated-mycobacterium phlei once daily for5days.The treatment was repeated once after a month.The patients in group B were treated with salmeterol xinafoate and fluticasone propionate powder for inhalation twice daily for constant use.Spirometry and Bronchial Provocation Test were carried out on the Day0,6,30and90. FVC,FEV1,PEF were collected.Airway hyperresponsiveness was assessed using MCh challenge test,FEV1-PD2o was calculated.Level of asthma control was evaluated with the ACT.Results:There was a improvement trend on FEV1of the patients in group A after treatment, but no significantly difference compared to pre-treatment. FEV1of the patients in group B increased significantly compared to pre-treatment on day90(P<0.05).PEF of the patients in the two groups increased significantly on Day6,day30and day90after treatment (P<0.05).There in no significantly difference between group A and group B. The increase of FEV1%of predicted in group B was significantly higher than in group A on day30,day90(P<0.05). FEVI-PD20of the patients increased significantly on Day6,day30and day90after treatment in both group A and group B(P<0.05),with no difference between the two groups.There was no significant difference in negative conversion rates of Bronchial Provocation Test between the two groups by chi-square test.ACT scores of the patients in the two groups were significantly increased on day30and day90(P<0.01).Conclusions:Inhaled inactivated-Mycobacterium phlei to a certain extent improve asthma symptoms, reduce need for rescue medication and reduce acute exacerbation of asthma.It plays the same role as inhaled Seretide in reducing airway hyperresponsiveness. Inactivated Mycobacterium phlei inhalation may accepted as an alternative method with less risk of adverse reactions,more convenience and better compliance in treatment of asthma.