Allergy Asthma Immunol Res.  2020 Mar;12(2):338-358. 10.4168/aair.2020.12.2.338.

Epithelial PI3K-δ Promotes House Dust Mite-Induced Allergic Asthma in NLRP3 Inflammasome-Dependent and -Independent Manners

• Kim SR
• Park HJ
• Lee KB
• Kim HJ
• Jeong JS
• Cho SH
• Lee YC

• KMID: 2468466
• DOI: http://doi.org/10.4168/aair.2020.12.2.338

Abstract

PURPOSE
Phosphoinositide 3-kinase (PI3K)-δ-dependent Akt activation is known to play critical roles in various immune responses of white blood cells in which PI3K-δ isoform is mostly expressed in contrast to the classes IA PI3Ks p110α and p110β. However, the immunological role of PI3K-δ isoform is still controversial in airway epithelium under house dust mite (HDM)-induced allergic response. This study aimed to evaluate the role of PI3K-δ isoform in HDM-induced allergic responses, focusing on NLRP3 inflammasome activation in airway epithelium.
METHODS
We used wild-type mice and PI3K-δ knock-out (KO) mice for HDM-induced asthma animal model and also performed in vitro experiments using primary cultured murine tracheal epithelial cells and human airway epithelial cells.
RESULTS
PI3K-δ activated HDM-induced NLRP3 inflammasome and epithelial cell-derived cytokines in the lung including airway epithelial cells. PI3K-δ KO mice or knock-down of PI3K-δ using siRNA exhibited the significant reduction in allergic asthmatic features and the suppression of NLRP3 inflammasome assembly as well as epithelial cell-derived cytokines. Interestingly, significantly increased expression of PI3K-δ isoform was observed in stimulated airway epithelial cells and the increases in epithelial cell-derived cytokines were markedly suppressed by blocking PI3K-δ, while these cytokine levels were independent of NLRP3 inflammasome activation.
CONCLUSIONS
The results of this study suggest that PI3K-δ-isoform can promote HDM-induced allergic airway inflammation via NLRP3 inflammasome-dependent response as well as via NLRP3 inflammasome-independent epithelial cell activation.

Keyword

Epithelial cell; asthma; house dust mite; inflammasomes; kinases

MeSH Terms

Animals
Asthma*
Cytokines
Dust*
Epithelial Cells
Epithelium
Humans
In Vitro Techniques
Inflammasomes
Inflammation
Leukocytes
Lung
Mice
Models, Animal
Phosphotransferases
Pyroglyphidae
RNA, Small Interfering
Cytokines
Dust
Inflammasomes
Phosphotransferases
RNA, Small Interfering

Figure

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  Fig. 1 Expression of PI3K-δ isoform, TLR4, phosphorylation of Akt and the levels of PIP3 were evaluated in HDM-instilled WT, PI3K-δ KO mice or airway epithelial cells. (A) Representative Western blots of p-Akt in lung tissues from SAL + VEH, HDM + VEH, HDM + IC 0.1 and HDM + IC 1.0 and (B) the densitometric analysis. (C) Enzyme immunoassay of PIP3 in lung tissue extracts. (D) Western blotting of p110δ protein in lung tissues from WT + HDM and δPKO + HDM. (E, F) Representative Western blots of p-Akt in lung tissues and the densitometric analysis. (G) Real-time reverse transcription polymerase chain reaction data showing the levels of PI3K-δ mRNA expression in the primary cultured tracheal epithelial cells. (H) Representative Western blots of p110δ in NHBE cells pre-treated with TAK-242 or vehicle and the densitometric analysis. (I, J) Representative Western blots of NF-κB in NHBE cells pre-treated with TAK-242 or vehicle and the densitometric analysis. (K, L) Representative Western blots of TLR4 (K) and KC (L) in NHBE cells pre-treated with IC87114 or vehicle and the densitometric analyses. (M) Representative Western blots of TLR4 in lung tissues from HDM-instilled mice and the densitometric analysis. Bars represent mean ± standard error of the mean from 4 or 6 mice per group. In vitro experimental data was obtained from 3 independent experiments. PI3K, phosphoinositide 3-kinase; TLR, toll-like receptor; HDM, house dust mite; WT, wild type; KO, knock-out; NHBE, normal human bronchial epithelial; SAL + VEH, saline-instilled mice with administered with vehicle; HDM + VEH, HDM-instilled mice administered with vehicle; HDM + IC 0.1, HDM-instilled mice administered with 0.1 mg/kg of IC87114; HDM + IC 1.0, HDM-instilled mice administered with 1.0 mg/kg of IC87114; WT + HDM, WT mice instilled with HDM; PKO + HDM, PI3K-δ KO mice instilled with HDM; NF-κB, nuclear factor-κB; LPS, lipopolysaccharide; CON siRNA, control scrambled siRNA; R.D., relative density. *P < 0.05 vs. HDM + VEH, HDM + CON siRNA, LPS + VEH or WT + HDM; †P < 0.05 vs. SAL + VEH or Control.

• 

  Fig. 2 PI3K-δ increases protein levels of NLRP3, cleaved caspase-1 or mature IL-1β in the HDM-exposed lung. The protein levels were evaluated in HDM-instilled WT and PI3K-δ KO mice. Representative Western blots of NLRP3 (A, D, O), cleaved caspase-1 (B, E, P) and mature IL-1β (C, F, Q) in lung tissues and the densitometric analyses. Representative Western blots of NLRP3 in primary cultured tracheal epithelial cells treated with IC87114 and drug vehicle (G) or transfected with δPI3KD siRNA or CON siRNA (H). (I) Representative confocal laser immunofluorescence photomicrography of primary cultured tracheal epithelial cells shows the expression of NLRP3. Blue fluorescent DAPI stain was used for nuclear localization. Bars indicate a scale of 20 μm. Representative Western blots of cleaved caspase-1 (J) and mature IL-1β (K, L, R) in BAL fluids and the densitometric analysis. Enzyme immunoassay of cleaved caspase-1 (M) and mature IL-1β (N) in BAL fluids of HDM-instilled mice. (S) Representative Western blots for mature IL-1β in cell lysates of primary cultured tracheal epithelial cells treated with IC87114 or drug vehicle. Arrow indicates each target band. (T) Enzyme immunoassay of mature IL-1β in the supernatants of primary cultured tracheal epithelial cells from HDM-instilled mice. Bars represent mean ± standard error of the mean from 4 or 6 mice per group. In vitro experimental data was obtained from 3 independent experiments. PI3K, phosphoinositide 3-kinase; IL, interleukin; HDM, house dust mite; WT, wild type; KO, knock-out; PI3KD siRNA, siRNA targeting PI3K-δ isoform; CON siRNA, control scrambled siRNA; BAL, bronchoalveolar lavage; HDM + VEH, HDM-instilled mice administered with vehicle; WT + HDM, WT mice instilled with HDM; SAL + VEH, saline-instilled mice with administered with vehicle; SAL, saline-instilled mice; HDM + IC 0.1, HDM-instilled mice administered with 0.1 mg/kg of IC87114; HDM + IC 1.0, HDM-instilled mice administered with 1.0 mg/kg of IC87114; PKO + HDM, PI3K-δ KO mice instilled with HDM; R.D., relative density. *P < 0.05 vs. HDM + VEH, WT + HDM, or HDM + CON siRNA; †P < 0.05 vs. SAL + VEH or SAL + SAL.

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  Fig. 3 Role of PI3K-δ in the assembly of NLRP3 inflammasome in the lung of HDM-instilled mice. (A, B) Representative confocal laser immunofluorescence photomicrography shows the expression of NLRP3 (red), ASC (green in A) and cleaved caspase-1 (green in B) in BAL cells from SAL+VEH, HDM+VEH, HDM+IC 0.1 and HDM+IC 1.0. (C, D) Primary cultured tracheal epithelial cells show the immunofluorescence of NLRP3 (red), ASC (green in C) and cleaved caspase-1 (green in D). The cells were obtained from saline-instilled CON and HDM-instilled mice and were treated with IC87114 (10 μmol/L) or drug VEH. The blue fluorescent DAPI stain was used for nuclear localization. Bars indicate a scale of 20 μm. Merge means the merger views and the square inside of merger view indicate the magnified fields on the right panels. Representative Western blots for ASC oligomer, dimer and monomer in lung tissues (E), BAL cells (F), and in primary cultured tracheal epithelial cells (G) from saline- or HDM-instilled mice. Representative Western blots for caspase-1 (H) and ASC (I) immuno-precipitated with NLRP3 in lung tissues from SAL + VEH, HDM + VEH, HDM + IC 0.1, and HDM + IC 1.0. These data is representative of 6 independent experiments. Arrowheads indicate the target bands. PI3K, phosphoinositide 3-kinase; HDM, house dust mite; BAL, bronchoalveolar lavage; CON, control mice; VEH, vehicle; DAPI, 4′,6-diamidino-2-phenylindole; SAL + VEH, saline-instilled mice with administered with vehicle; HDM + VEH, HDM-instilled mice administered with vehicle; HDM + IC 0.1, HDM-instilled mice administered with 0.1 mg/kg of IC87114; HDM + IC 1.0, HDM-instilled mice administered with 1.0 mg/kg of IC87114.

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  Fig. 4 Changes in NF-κB activation and mitochondrial ROS generation in HDM-instilled mice. (A) Representative Western blots of NF-κB in lung tissues from SAL + VEH, HDM + VEH, HDM + IC 0.1 and HDM + IC 1.0, and the densitometric analysis. Bars represent mean ± SEM from 6 mice per group. (B) Representative Western blots of NF-κB in lung tissues from WT + HDM and PKO + HDM, and the densitometric analysis. Bars represent mean ± SEM from 4 mice per group. Representative confocal laser immunofluorescence photomicrographs of BAL cells (C) and primary cultured tracheal epithelial cells (D) show the localization of mitochondrial ROS. The blue fluorescent DAPI stain was used for nuclear localization. Bars indicate a scale of 10 μm (C) or 20 μm (D). (E) Schematic diagram of the role of PI3K-δ isoform in the assembly of the NLRP3 inflammasome and the production of epithelial cell-derived cytokines in the bronchial epithelium of HDM-induced allergic asthmatic mice. NF-κB, nuclear factor-κB; ROS, reactive oxygen species; HDM, house dust mite; SEM, standard error of the mean; BAL, bronchoalveolar lavage; DAPI, 4′,6-diamidino-2-phenylindole; PI3K, phosphoinositide 3-kinase; SAL + VEH, saline-instilled mice with administered with vehicle; HDM + VEH, HDM-instilled mice administered with vehicle; HDM + IC 0.1, HDM-instilled mice administered with 0.1 mg/kg of IC87114; HDM + IC 1.0, HDM-instilled mice administered with 1.0 mg/kg of IC87114; WT + HDM, WT mice instilled with HDM; PKO + HDM, PI3K-δ KO mice instilled with HDM; R.D., relative density; IL, interleukin; PAMP, pathogen-associated molecular pattern molecule. *P < 0.05 vs. HDM + VEH; †P < 0.05 vs. SAL + VEH; ‡P < 0.05 vs. WT + HDM.

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  Fig. 5 Effects of IC87114 or genetic KO of PI3K-δ on HDM-induced allergic asthmatic features. (A) The number of inflammatory cells in BAL fluids of saline-instilled or HDM-instilled mice. (B) Representative H&E stained sections (upper panel) and PAS-stained sections (lower panel) of the lung from SAL + VEH, HDM + VEH, HDM + IC 0.1, and HDM + IC 1.0. Bars indicate 50 μm. (C) Inflammation scores of the lung. Total lung inflammation was defined as the average of the peribronchial and perivascular inflammation scores. (D) Quantitation of airway mucus expression. (E, F) Airway responsiveness assessed by invasive (Rrs) measurement in HDM-instilled mice. (G-L) Representative Western blots for IL-4, IL-5, IL-13, KC, IL-17 and TNF-α in lung tissues, and the densitometric analysis. (M) The number of inflammatory cells in BAL fluids of WT + HDM and PKO + HDM. (N) Representative H&E stained sections (upper panel) and PAS-stained sections (lower panel) of the lung from WT + HDM and PKO + HDM. Bars indicate 50 μm. (O) Inflammation scores of the lung. (P) Quantitation of airway mucus expression. (Q, R) Airway responsiveness assessed by invasive (Rrs) measurement. (S-U) Representative Western blots for IL-4, IL-5 and IL-13 in lung tissues, and the densitometric analysis. Bars represent mean ± standard error of the mean from 4 or 6 mice per group. PI3K, phosphoinositide 3-kinase; HDM, house dust mite; BAL, bronchoalveolar lavage; H&E, haematoxylin and eosin; IL, Interleukin; TNF-α, tumor necrosis factor-α; SAL + VEH, saline-instilled mice with administered with vehicle; HDM + VEH, HDM-instilled mice administered with vehicle; HDM + IC 0.1, HDM-instilled mice administered with 0.1 mg/kg of IC87114; HDM + IC 1.0, HDM-instilled mice administered with 1.0 mg/kg of IC87114; WT + HDM, wild type mice instilled with HDM; PKO + HDM, PI3K-δ knock-out mice instilled with HDM; R.D., relative density; MAC, macrophage; EOS, eosinophil; NEU, neutrophil; LYM, lymphocyte. *P < 0.05 vs. HDM + VEH or WT + HDM; †P < 0.05 vs. SAL + VEH.

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  Fig. 6 Changes in asthma features of HDM-instilled mice administered with PI3K-δ targeting siRNA or MCC950. (A, K) The number of inflammatory cells in BAL fluids of HDM-instilled mice. (B-E, O) Representative H&E-stained sections of the lung from HDM-instilled mice. Bars indicate 50 μm. (F, G, P, Q) Airway responsiveness assessed by invasive (Rrs) measurement in HDM-instilled mice. Representative Western blots for IL-4 (H, L), IL-5 (I, M), IL-13 (J, N) and mature IL-1β (R) in lung tissues, and the densitometric analysis. (S) Protein expression of mature IL-1β in BAL fluids quantified by Western blotting. Bars represent mean ± standard error of the mean from 5 or 6 mice per group. HDM, house dust mite; PI3K, phosphoinositide 3-kinase; BAL, bronchoalveolar lavage; H&E, haematoxylin and eosin; IL, interleukin; CON siRNA, control scrambled siRNA; HDM + VEH, HDM-instilled mice administered with vehicle; SAL, saline-instilled mice; SAL + VEH, saline-instilled mice with administered with vehicle; PI3KD siRNA, siRNA targeting PI3K-δ isoform; R.D., relative density; MAC, macrophage; EOS, eosinophil; NEU, neutrophil; LYM, lymphocyte. *P < 0.05 vs. HDM + CON siRNA or HDM + VEH; †P < 0.05 vs. SAL + SAL or SAL + VEH.

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  Fig. 7 Effects of IC87114 or MCC950 on the expression of epithelial cell-derived cytokines in lung tissues and primary cultured tracheal epithelial cells from HDM-instilled mice. Lung tissues were obtained from SAL + VEH, HDM + VEH, HDM + IC 0.1, HDM + IC 1.0 and HDM + MCC950. The cells were obtained from saline-instilled control mice (Control) and HDM-instilled mice, and they were treated with IC87114 (10 μmol/L), MCC950 (50 nmol/L) or drug vehicle. Representative Western blots for IL-25, IL-33 and TSLP in lung tissues (A-C, G-I) and primary cultured tracheal epithelial cells (D-F, J-L), and the densitometric analysis. Bars represent mean ± standard error of the mean from 6 mice per group. In vitro data was obtained from 3 independent experiments. HDM, house dust mite; SAL + VEH, saline-instilled mice with administered with vehicle; HDM + VEH, HDM-instilled mice administered with vehicle; HDM + IC 0.1, HDM-instilled mice administered with 0.1 mg/kg of IC87114; HDM + IC 1.0, HDM-instilled mice administered with 1.0 mg/kg of IC87114; TSLP, thymic stromal lymphopoietin; IL, interleukin; R.D., relative density. *P < 0.05 vs. HDM + VEH or Vehicle.; †P < 0.05 vs. SAL+VEH or Control.

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  Fig. 8 Expression of IL-25, IL-33, and TSLP in lung tissues of HDM-instilled WT and PI3K-δ KO mice (A-C) and in LPS- or Der-p1-stimulated NHBE cells transfected with control or NLRP3 targeting siRNA (D-I). Western blotting of IL-25 (A), IL-33 (B) and TSLP (C) in lung tissues from WT + HDM and δPKO + HDM, and its densitometric analysis. Bars represent mean ± standard error of the mean from 6 mice per group. Representative Western blots for IL-25 (D, G), IL-33 (E, H), and TSLP (F, I) in LPS or Der-p1-stimulated NHBE cells transfected with control or NLRP3 targeting siRNA and the densitometric analyses. Data were from 3 independent experiments. IL, interleukin; LPS, lipopolysaccharide; HDM, house dust mite; WT, wild type; KO, knock-out; TSLP, thymic stromal lymphopoietin; NHBE, normal human bronchial epithelial; WT + HDM, WT mice instilled with HDM; PI3K, phosphoinositide 3-kinase; PKO + HDM, PI3K-δ KO mice instilled with HDM; CON siRNA, control scrambled siRNA; R.D., relative density. *P < 0.05 vs. WT + HDM; †P < 0.05 vs. Control.

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