Effects of Blockade of IL-17 in Already Established Asthma of BALB/c Mice
|School||Dalian Medical University|
|Keywords||IL-17 asthma AHR inflammation|
Introduction and objective:Allergic asthma is a complex syndrome which has been characterized by airway obstruction, airway inflammation and airway hyperresponsiveness (AHR) Many investigations have reported that T cells have been considered important in the development of AHR and inflammation, through the production of cytokine. Interleukin (IL)-17 (now synonymous with IL-17A) is an archetype molecule for an entire family of IL-17 cytokines, and is currently believed to be produced mainly by a specific subset of CD4 cells named Th-17 cells. Our previous research shows that the level of IL-17 increases in the already established mice asthma model. Most of the studies investing the effectiveness of blocking IL-17, have been performed in models of primary allergen challenge. The role of IL-17 in secondary challenge models, where airway hyperresponsiveness (AHR) and airway inflammation have already been established, has not been defined. The aim of the present study was to evaluate the role of IL-17 on airway function and lung inflammation in secondary challenge mice models by monitoring AHR both 6h and 48h after secondary challenge.Methods:1 Experimental protocol (Sensitization and Airway Challenge):8-to 10-wk-old mice were sensitized by i.p. injection of 20μg of OVA (Grade V; Sigma-Aldrich, St. Louis, MO) emulsified in 2.25 mg of aluminum hydroxide (Alumlmuject; Pierce, Rockford, IL) in a total volume of 100μl on days 1 and 14. Mice were challenged (20 min) via the airways with OVA (1% in saline) for 3 days (days 28, 29, and 30; primary challenge) using ultrasonic nebulization. For the secondary challenge protocol,6 wk after the primary challenge, mice were exposed to a single OVA challenge (1% in saline; secondary challenge), and airway reactivity and tissues were assessed 6 or 48 after Anti-IL-17antibody or control antibody (IgG) was administered by i.v. injection at 100u/mouse (suspended in 100μl of PBS),24 hs and 2 hs before the secondary challenge.2 Determination of Airway Function: A flexiVent small-animal ventilator (SCIREQ, Montreal, PQ, Canada) was used to assess airway function in anesthetized, mechanically ventilated animals, measuring changes in lung resistance (RL) in response to increasing doses of inhaled methacholine (MCh).3 BALF:Immediately after assessment of AHR, lungs were lavaged via the tracheal tube with Hanks’ balanced salt solution (2×1 ml,37℃). The volume of the collected bronchoalveolar lavage fluid (BALF) was measured in each sample, and the number of cells in the BALF was counted. Cytospin slides were stained and differentiated in a blinded fashion by counting at least 300 cells under light microscopy.4 Histochemistry: Lungs were fixed by inflation (1 ml) and immersion in 10% formalin. For detection of mucus-containing cells in formalin-fixed airway tissue, sections were stained with periodic acid Schiff (PAS), H&E.5 Measurement of cytokines and chemokines:All cytokines and chemokines (R&D Systems, Minneapolis, MN) ELISAs were performed according to the manufacturer’s directions. The limits of detection were 4 pg/ml for IL-4, IL-5, IL-13, KC and MIP-2.Results:1 The mice of asthmatic group demonstrated symtomes of acute asthma when exposed to aerosolized OVA. The airway inflammation and AHR in IL-17 blokade group were also higher than the mice in negative control group.2 Six hours after the secondary challenge:After aerosolizing of an increasing dose of methacholine, IL-17 blockade group showed an significant difference only at the dose of 25 mg/ml (P<0.05). IL-17 blockade group also showed a lower number of total cell and neutrophil in BALF (P<0.05). Th2 related cytokine IL-4, IL-5, IL-13 were not attenuated by treatment of IL-17 antibody, but neutrophil related chemokine KC and MIP-2 were significantly decreased(P<0.05). Number of inflammatory cells infiltrated around the bronchioles and blood vessels were less in IL-17 blockade group than in positive control group.3 Forty eight hours after the secondary challenge:After aerosolizing of an increasing dose of methacholine, IL-17 blockade group showed a slight but not significant difference (P>0.05). IL-17 blockade group showed a lower number of neutrophil in BALF (P<0.05). PAS staining showed lower number of goblet cells..Conclusions:1 We established the secondary challenged mice asthmatic model successfully.2 Six hours after secondary challenge:IL-17 blockade significantly attenuateed airway inflammation and AHR.3 Forty eight hours after secondary challenge:IL-17 blockade attenuate airway inflammation and goblet cell metaplasia.