TSLP and IL-33 reciprocally promote each other's lung protein expression and ILC2 receptor expression to enhance innate type-2 airway inflammation

Abstract

Background: The epithelial cell-derived danger signal mediators thymic stromal lymphopoietin (TSLP) and IL-33 are consistently associated with adaptive Th2 immune responses in asthma. In addition, TSLP and IL-33 synergistically promoted group 2 innate lymphoid cell (ILC2) activation to induce innate allergic inflammation. However, the mechanism of this synergistic ILC2 activation is unknown.

Methods: BALB/c WT and TSLP receptor-deficient (TSLPR^-/- ) mice were challenged intranasally with Alternaria extract (Alt-Ext) or PBS for 4 consecutive days to evaluate innate airway allergic inflammation. WT mice pre-administered with rTSLP or vehicle, TSLPR^-/- mice, and IL-33 receptor-deficient (ST2^-/- ) mice were challenged intranasally with Alt-Ext or vehicle once or twice to evaluate IL-33 release and TSLP expression in the lung. TSLPR and ST2 expression on lung ILC2 were measured by flow cytometry after treatment of rTSLP, rIL-33, rTSLP + rIL-33, or vehicle.

Results: Thymic stromal lymphopoietin receptor deficient mice had significantly decreased the number of lung ILC2 expressing IL-5 and IL-13 following Alt-Ext-challenge compared to WT mice. Further, eosinophilia, protein level of lung IL-4, IL-5, and IL-13, and airway mucus score were also significantly decreased in TSLPR^-/- mice compared to WT mice. Endogenous and exogenous TSLP increased Alt-Ext-induced IL-33 release into BALF, and ST2 deficiency decreased Alt-Ext-induced TSLP expression in the lung. Further, rTSLP and rIL-33 treatment reciprocally increased each other's receptor expression on lung ILC2 in vivo and in vitro.

Conclusion: Thymic stromal lymphopoietin and IL-33 signaling reciprocally enhanced each other's protein release and expression in the lung following Alt-Ext-challenge and each other's receptor expression on lung ILC2 to enhance ILC2 activation.

Keywords: Alternaria-extract (Alt-Ext); Group 2 innate lymphoid cells (ILC2); IL-33; TSLP.

Figure 1

TSLPR deficiency decreased Alternaria extract (Alt‐Ext)‐induced type‐2 airway inflammation. A, WT and TSLPR^−/− mice were challenged with Alt‐Ext intranasally for 4 consecutive days. Bronchoalveolar lavage fluid (BALF) and lung were harvested 24 h after the last Alt‐Ext‐challenge. Whole lungs for histological mucus detection were harvested 48 h after the last Alt‐Ext‐challenge. B, Cell differentials in BALF. C, The number and percentage of lung group 2 innate lymphoid cells (ILC2) expressing IL‐5 and IL‐13. D, The protein level of IL‐4, IL‐5, and IL‐13 in the lung homogenates. E, Representative sections and mucus score as determined by periodic acid‐Schiff (PAS) staining. The data are a combination of 2 independent experiments and shown as mean ± SD (n = 4‐9). *P < .05

Figure 2

Endogenous TSLP neutralization decreased Alternaria extract (Alt‐Ext)‐induced type‐2 airway inflammation. A, Anti‐TSLP antibody (α‐TSLP) or the isotype antibody was administered subcutaneously 1 h prior to first Alt‐Ext‐challenge. Bronchoalveolar lavage fluid (BALF) and lung were harvested 24 h after the 4th Alt‐Ext‐challenge. B, Cell differentials in BALF. C, The protein level of IL‐4, IL‐5, and IL‐13 in the lung homogenates. The data are a combination of 2 independent experiments and shown as mean ± SD (n = 4‐9). *P < .05

Figure 3

TSLPR signaling increased IL‐33 release and IL‐33 receptor (ST2) signaling increased TSLP expression in the lung. A, The time course of TSLP and IL‐33 in bronchoalveolar lavage fluid (BALF) and lung homogenates after one Alternaria extract (Alt‐Ext)‐challenge. B, E, G, I, Diagrams of experimental design using mouse model to detect IL‐33 in BALF and TSLP in the lung. C, IL‐33 protein level in the BALF from wild‐type (WT) mice challenged with Alt‐Ext or PBS in the presence or absence of recombinant TSLP (rTSLP) treatment. D, IL‐33 protein level in cell‐free culture supernatant (sup) from human bronchial epithelial cells stably expressing IL‐33 (hBE33) challenged with Alt‐Ext or PBS in the presence or absence of human recombinant TSLP treatment. F, IL‐33 protein level in the BALF from WT and TSLPR^−/− mice challenged with Alt‐Ext or PBS. H, IL‐33 protein level in the BALF from WT mice challenged with Alt‐Ext or PBS in the presence or absence of anti‐TSLP antibody (α‐TSLP) treatment. J, TSLP protein level in the lung homogenates from WT and ST2^−/− mice challenged with Alt‐Ext or PBS. The data are a combination of 2 independent experiments and shown as mean ± SD (n = 3‐9). *P < .05

Figure 4

Recombinant TSLP (rTSLP) and recombinant IL‐33 (rIL‐33) treatment reciprocally increased IL‐33 receptor (ST2) and TSLPR on lung group 2 innate lymphoid cells (ILC2). A, C, Representative histograms of ST2 and TSLPR expression on lung ILC2 from wild‐type (WT) mice treated with rTSLP, rIL‐33 or the vehicle in an in vivo model. B, D, Mean fluorescence intensity (MFI) of ST2 and TSLPR on lung ILC2 in vivo. E, G, Representative histograms of ST2 and TSLPR expression on isolated lung ILC2 treated with rTSLP, rIL‐33, rTSLP + rIL‐33, or the vehicle in an in vitro model. F, H, MFI of ST2 and TSLPR on isolated lung ILC2 in vitro. The data are a combination of 2 independent experiments and shown as mean ± SD. (n = 3‐4). *P < .05

Figure 5

Recombinant IL‐33 (rIL‐33) treatment enhanced TSLP‐induced signal transducer and activator of transcription 5 (STAT5) signaling. A, Representative image of Western blotting. Isolated lung group 2 innate lymphoid cells (ILC2) were stimulated for 24 h with rIL‐33 (10 ng/mL) or vehicle; then, the cells were stimulated for 1 h with recombinant TSLP (rTSLP) (10 ng/mL) or vehicle. Cell lysates were separated by SDS‐PAGE and analyzed by Western blotting with antibodies to phosphorylated STAT5 (pSTAT5), STAT5, and β‐actin. B, Relative intensity of pSTAT5/STAT5 was analyzed by densitometry. C, Isolated lung ILC2 were stimulated with rIL‐33, rTSLP + rIL‐33, or vehicle in presence or absence of pimozide (5 μmol/L) for 40 h. IL‐5 and IL‐13 protein in the ILC2 culture supernatant were measured by ELISA. The data are a combination of 2 independent experiments. *P < .05