Airway epithelial cell necroptosis contributes to asthma exacerbation in a mouse model of house dust mite-induced allergic inflammation (1)
Recent research has highlighted the importance of regulated cell death in tissue homeostasis and inflammation. Necroptosis has been recently identified as a new type of regulated necrotic cell death that stimulates potent inflammatory responses. Receptor interacting protein kinase 3 (RIPK3) induces necroptosis by phosphorylating the pseudokinase Mixed lineage kinase like (MLKL), which executes cell death by damaging the plasma membrane. RIPK1 is a key mediator of necroptosis downstream of death receptors or Toll-like receptors (TLR3 and TLR4).
The Faddfl/fl mice, as expected, developed all the cardinal features of asthma including increased infiltration of immune cells, TH2 cytokine production by Mediastinal Lymph Nodes (MLN) cells and increased titers of IgE antibodies in the serum. MLN cells from FADDAEC-KO mice secreted increased amounts of IL-17A compared to cells from Faddfl/fl mice, indicating that epithelial cell FADD deficiency results in an amplification of TH17 responses. Serum IgE levels were equal between the two genotypes.
RIPK1 kinase activity is required for TNFR1- and TLR3/4-induced necroptosis in cells that lack FADD or caspase-8 or are treated with caspase inhibitors. Assessment of HDM-induced airway inflammation revealed that Faddfl/fl Ripk1D138N/D138N mice developed similar lung pathology compared to Faddfl/fl animals, showing that lack of RIPK1 kinase activity did not affect HDM-induced asthma pathology in mice with intact FADD signaling. Lack of RIPK1 kinase activity strongly attenuated the exacerbated HDM-induced pathology observed in FADDAEC-KO mice as judged by histopathological examination of lung sections, assessment of immune cell infiltration and measurement of serum IgE levels.
RIPK3 deficiency caused a mildly increased HDM-induced pathology, manifesting with slightly elevated numbers of infiltrating eosinophils and T cells, as well as mildly increased amounts of serum IgE and IL-13, IL-5 and IL-17 protein levels in the lung. FADDAEC-KO Ripk3−/− mice showed similarly elevated protein levels of cytokines in the lung compared to Faddfl/fl Ripk3−/− mice.
Phosphorylation of MLKL by RIPK3 is an essential step for the induction of necroptosis. HDM caused the appearance of increased numbers of TUNEL+ AECs in the lungs of FADDAEC-KO mice. PBS instillation caused the death of AECs in FADDAEC-KO and FADDAEC-KORipk3−/− mice, to an extent similar to HDM.
The airways of FADDAEC-KO mice contained overall less mucusproducing goblet cells when compared to their Faddfl/fl littermates 3 days after the last challenge with HDM. RIPK3-dependent mechanisms contribute to the ongoing epithelial cell injury and/or airway remodeling in FADD deficient AECs. Consistent with the immunohistochemistry results, the mRNA levels of Scgb1a1 were significantly downregulated in the lungs of FADDAEC-KO mice when compared to WT littermates.
Lung resistance in HDM-treated Faddfl/fl mice increased considerably upon methacholine exposure. HDM administration in Ripk3−/− mice caused worsening of lung function in a manner similar to Faddfl/fl mice. RIPK3 expression mediated at least to some extent the observed amelioration of AHR in FADDAEC-KO mice, since RIPK3 deficiency partly increased the resistance and elastance parameters in FADDAEC-KORipk3−/− mice.