The Effect Of IRAK-M On Airway Inflammation Induced By Exposure To LPS Or Cigarette Smoking

Background:Cigarette smoking(CS)triggers airway inflammation by activating innate and adaptive immune cells.IRAK-M,expressed by airway epithelial cells and macrophages,negatively regulates Toll-like receptor(TLR)-mediated NFB activation.IRAK-M has been shown the dual properties in the various disease contexts.Object:In this investigation,we studied the effect of IRAK-M on airway inflammation induced by CS under acute and sub-acute conditions using IRAK-M knockout(IRAK-M-/-)mice.Method:1.Mice were exposed to CS in a whole-body exposure system.Briefly,mice were placed in a closed plastic box connected to a smoke generator.The mice were exposed to tobacco smoke of five cigarettes(Reference Cigarette 3 R4F,University of Kentucky,USA four times a day with 30-min smoke-free intervals between each smoke exposure for 3 days or for 7 weeks.Control mice were exposed to room air(RA).On The fifth day,some mice were challenged with PBS or LPS(100μg/ml)by inhalation for 30mins according to previously described with minor modification.Mice were sacrificed after last challenge for furTher analysis.2.To detect the airway resistance(Rn)of mice by using the machine of Flexivent.3.To evaluate the lung inflammation by HE staining.4.To count the number and classification of inflammatory cells in BALF.5.To determine the cytokines production by me multiplex analysis.6.To analyze the percentage of T cells subpopulation in the lung.7.To evaluate the expression of co-stimulatory molecules on lung DCs and macrophages.Results:Part I The effect of IRAK-M on The pulmonary inflammation in mice induced by 3-day cigarette smoke exposure or LPS insult associated mechanism1.Airway resistanceCompared with the control group,the airway resistance was increased in mice exposed to CS or LPS with no significant difference.And airway resistance was not significantly elevated in IRAK-M-/-mice relative to WT mice after exposed to CS and LPS.2.Lung morphology We observed epithelial damage,inflammatory cell inflLtration,squamous cell metaplasia,in The CS,LPS and CS+LPS groups.IRAK-M-/-mice showed more serious lung inflammation after 3-day CS exposure and LPS insult.3.Inflammatory cells in BALF Inflammatory cells,including the macrophages,neutrophILs and lymphocytes,were increased in in The CS,LPS and CS+LPS groups when compared with the control group.But the BAL inflammatory cells were comparable in CS or LPS group between IRAK-M deficient mice and WT mice.Meanwhile,the inflammatory cells were increased in The IRAK-M-/-mice Than their WT conterpart(1812±131×103/ml vs 1149±132×103/ml;p<0.01)in CS+LPS group.4.The protein expressions of chemokines or cytokines in lung tissue Compared with The control group,the CS+LPS groups were significantly increased in The concentration of following cytokines in The lung tissue:Th1-secreting cytokines(including TNF-α,IFN-β,IL-12p70,IFN-y),Th17-related cytokines(including IL17A and IL6),epithelium-derived pro-inflammatory cytokines and chemokines(including GM-CSF,IL-1β,IL-1α).Meanwhile,significantly elevated concentrations of Th1-secreting cytokines(TNF-α,IFN-β,IL-12p70,IFN-γ),Th17-related cytokines(including IL17A and IL6),epithelium-derived pro-inflammatory cytokines and chemokines(including GM-CSF,IL-1β,IL-1α)were found in lung homogenates from IRAK-M-/-mice compared wiTh WT mice.5.T cells differentiation in lung tissue The percentage of Tregs(represented by CD3+CD4+Foxp3+T)was significantly lower in lungs from IRAK-M-/-mice than that from WT mice(4.99%±0.46%vs 6.65%±0.42%)in the CS+LPS group,conversely,the percentage of Th 17(CD3+CD4+Th17A+T)cells was significantly higher in lungs from IRAK-M-/-mice than that from WT mice(4.32%± 0.28%vs 2.54%±0.32%)after 3-day CS exposure and LPS insult.Under the stimulation with LPS following CS short exposure,we also observed a significant decrease in the percentage of Tregs in the spleen of IRAK-M-/-mice compared to WT mice.A mild elevation of the percentage of Th7 in the spleen of IRAK-M-/-mice challenged with LPS and acute CS.6.surface expression of co-stimulatory molecules by DCS and macrophages There was significantly increased up-regulation of costimulatory molecules CD40 and CD86 on lung DCs from IRAK-/-mice compared to WT mice challenged with LPS and acute CS.In addition,significantly increased expression of CD40 by lung macrophages was seen in IRAK-M-/-mice following LPS inhalation after 3-day CS insult compared with WT littermates treated under the same exposure condition.Part Ⅱ The effect of IRAK-M on The pulmonary inflammation in mice under sub-acute exposure to CS and associated mechanism1.Airway resistance Compared with the control group,the airway resistance was increased in mice exposed to 7-week CS.And airway resistance was significantly decreased in IRAK-M-/-mice relative to WT mice after 7-week CS exposure.2.Lung morphology We observed epithelial damage,inflammatory cell infiltration,squamous cell metaplasia,after7-week CS exposure.IRAK-M-/-mice showed more serious lung inflammation compared with WT counterpart after sub-acute CS exposure.3.Inflammatory cells in BALF The number of inflammatory cells in BALF was increased after 7-week CS exposure compared with the control group.Meanwhile,the inflammatory cells,including macrophages and lymphocyte were decreased in the IRAK-M-/-mice than their WT conterparts(122.8±9.2×103/ml vs 202.8±22×103/ml;p<0.01)after sub-acute CS exposure.4.The protein expressions of chemokines or cytokines in lung tissue Compared with the control group,the protein expressions of chemokines or cytokines in lung tissue were significantly increased in the concentration of following cytokines in the lung tissue:Thl-secreting cytokines(including TNF-α,IFN-β,IL-12p70,IFN-γ),Th17-related cytokines(including IL17A and IL6),epithelium-derived pro-inflammatory cytokines and chemokines(including GM-CSF,IL-1β,IL-1α)after 7-week CS exposure.However,significantly decreased concentrations of Th1-secreting cytokines(including TNF-α,IFN-β,IL-12p70,IFN-γ),Thl7-related cytokines(including IL17A and IL6),epithelium-derived pro-inflammatory cytokines and chemokines(including GM-CSF,IL-1β,IL-la)were found in lung homogenates from IRAK-M-/-mice compared with WT mice after sub-acute CS exposure.5.T cells differentiation in lung tissue After exposed to sub-acute CS,we found elevated percentage of Tregs and Th 17 cells in lungs and spleen.There were significantly increased percentage of Tregs in lungs from subacute CS-exposed-IRAK-M-/-mice compared with those from similarly treated-WT mice(15.0%±0.9%vs 10.5%±1.0%,p<0.001),however,there were significantly less percentages of Th17 cells in IRAK-M-/-mice than those from WT mice(7.6%±0.6%vs 9.8%±0.4%,p<0.05).Compared to WT mice,IRAK-M-/-mice showed a significant elevation of Tregs,not Th17,in spleen after subacute CS exposure.6.surface expression of co-stimulatory molecules by DCs and macrophages Compared to air-exposed mice,expression of costimulatory molecule CD11b was lower on lung DCs from both IRAK-/-and WT mice after 7-week CS exposure.There were no significant differences in expression of other costimulatory molecules by DCs between two types of mice.Chronic exposure to CS constitutively activates alveolar macrophages.However,our FACS analysis showed that expression of CD11b and CD86 by lung macrophages was significantly lower in IRAK-M-/-mice than that in WT mice after 7-week CS exposure.Conclusions:IRAK-M signaling plays protection or damage in airway inflammation induced by CS depending on the duration of CS in a mouse model.IRAK-M attenuated airway inflammation insulted by LPS/acute CS exposure by influencing Treg/Th17 imbalance skewing to Th17 and upregulating expression of costimulatory molecules CD40 and CD86 by DCs and macrophages.In contrast,IRAK-M showed a pro-inflammatory role in airway pathology after 7-week CS exposure by influencing Treg/Th17 imbalance skewing to Treg and down-regulating expression of costimulatory molecules CD11b and CD86 by DCs or macrophages.A better understanding of IRAKM effect in airway inflammation induced CS under the various phases of insult might be helpful in appropriately modulating CS-associated airway pathology.