Thymic Stromal Lymphopoietin Enhances Th2/Th22 and Reduces IL-17A in Protease-Allergen-Induced Airways Inflammation

Abstract

Background. Thymic stromal lymphopoietin (TSLP) is induced in allergic skin and lung inflammation in man and mice. Methods. Allergic lung inflammation induced by two proteases allergens HDM and papain and a classical allergen ovalbumin was evaluated in vivo in mice deficient for TSLPR. Eosinophil recruitment, Th2 and Th17 cytokine and chemokine levels were determined in bronchoalveolar lavage fluid, lung homogenates and lung mononuclear cells ex vivo. Results. Here we report that mice challenged with house dust mite extract or papain in the absence of TSLPR have a drastic reduction of allergic inflammation with diminished eosinophil recruitment in BAL and lung and reduced mucus overproduction. TSLPR deficient DCs displayed diminished OVA antigen uptake and reduced capacity to activate antigen specific T cells. TSLPR deficient mice had diminished proinflammatory IL-1 β , IL-13, and IL-33 chemokines production, while IL-17A, IL-12p40 and IL-10 were increased. Together with impaired Th2 cytokines, IL-17A expressing TCR β (+) T cells were increased, while IL-22 expressing CD4(+) T cells were diminished in the lung. Conclusion. Therefore, TSLPR signaling is required for the development of both Th2 and Th22 responses and may restrain IL-17A. TSLP may mediate its effects in part by increasing allergen uptake and processing by DCs resulting in an exacerbated asthma.

Figure 1

Reduced eosinophils influx in TSLPR^−/− mice during HDM induced allergic asthma lung inflammation. HDM sensitized WT and TSLPR^−/− mice (C57BL/6 background) were challenged three times with HDM inhalation. 24 h after the third challenge, the number of eosinophils (a), the lymphocytes (b), macrophages (c), and neutrophils (d) were determined in BALF and EPO activity in lung tissue (e). These experiments were performed twice (n = 8 mice per group). One representative experiment is shown. Values are the mean ± SEM of 8 mice per group.

Figure 2

Decreased pulmonary Th2 cytokine and chemokine responses in TSLPR^−/− mice in response to HDM. Mice were immunized and challenged with HDM as before. IL-1β, IL-13, IL-33, TSLP, CCL11 (Eotaxin-1), CCL17 (TARC), CCL22 (MDC), and CCL24 (Eotaxin-2) were measured in the lung homogenate by ELISA (a–h) from HDM treated WT and TSLPR^−/− mice at 24 h after the third challenge. These experiments were performed twice (n = 8 mice per group). One representative experiment is shown. Values are the mean ± SEM of 8 mice per group. **P ≤ 0.01; ***P ≤ 0.001.

Figure 3

Reduced lung inflammation in TSLPR^−/− mice in response to HDM. The formalin-fixed lung sections were stained with periodic acid Schiff reagent (PAS) to visualize mucus (a). Magnification ×20. Representative sections from WT saline control, HDM treated WT, and TSLPR^−/− mice are shown. A semi-quantitative histological assessment of cell infiltration and mucus hypersecretion was performed by two independent observers (b). A scale from 0 to 3 is given on the axis. These experiments were performed twice (n = 8 mice per group). One representative experiment is shown. Values are the mean ± SEM of 8 mice per group.

Figure 4

TSLPR is essential for the development of innate type airway inflammation induced by papain. Mice were exposed daily to 25 μg papain for 3 days and analyzed 24 after the last intranasal instillation. The number of eosinophils (a), lymphocytes (b), macrophages (c), and neutrophils (d) were determined in BALF as well as EPO activity in lung tissue (e) was determined 24 h after the last papain or saline control administration in wild-type (WT) and TSLPR^−/− mice. Lung sections stained with Hematoxylin-Eosin (HE) (20x magnification) and score of the severity of inflammation and mucus production at 24 h after the last papain or PBS inhalation are shown (f-g). These experiments were performed twice (n = 8 mice per group). One representative experiment is shown. Values are the mean ± SEM of 8 mice per group.

Figure 5

Diminished cytokine and chemokine expression in TSLPR^−/− mice in innate type of lung inflammation induced by papain. IL-1β, IL-13, IL-33, TSLP, CCL11 (Eotaxin-1), CCL17 (TARC), CCL22 (MDC), and CCL24 (Eotaxin-2) in the lung homogenate after papain exposure were determined by ELISA (a–h). These experiments were performed twice (n = 8 mice per group). One representative experiment is shown. Values are the mean ± SEM of 8 mice per group.

Figure 6

Reduced antigen uptake and eosinophils influx in TSLPR^−/− mice upon OVA induced allergic asthma model. Dendritic cells were differentiated in vitro from naive bone marrow derived cells. Uptake of OVA-FITC by dendritic cells after 2 h was analyzed by FACS (100 μg/mL). The data are given as the mean fluorescence intensity (MFI). OVA peptide specific T cell proliferation was assessed by coculture of DC from WT or TSLPR^−/− mice loaded with OVA peptide (10 μg/mL) with CFSE labelled CD4⁺ OT2 T cells (b). Critical role of TSLPR signalling for allergic inflammatory cell recruitment in BALF in OVA immunized and challenged mice (c). OVA sensitized WT and TSLPR^−/− mice were challenged three times with OVA instillation. 24 h after the third challenge, eosinophil, lymphocyte, macrophage, and neutrophil recruitment in BAL was determined. These experiments were performed twice (n = 8 mice per group). One representative experiment is shown. Values are the mean ± SEM. of 8 mice per group.

Figure 7

Increased pulmonary IL-17A⁺ cell populations in the absence of TSLPR. Lung mononuclear cells from OVA or HDM sensitized and challenged WT and TSLPR^−/− mice were isolated and restimulated for 4 h with PMA (50 ng/mL) and ionomycin (750 ng/mL) followed by membrane staining of TCRαβ and TCRγδ. Representative dot plot, frequency and absolute numbers of IL-17A⁺ producing T cells gated either on TCRαβ ⁺ or TCRγδ ⁺ T cell populations (a–e) are shown for OVA model. Representative dot plot and the frequency of IL-17A⁺ producing cells gated on TCRαβ ⁺ T cell populations (f, g), IL-17A (h), IL-12p40 (i), IL-10 (j) and IFNγ (k) levels in lung supernatant from HDM treated WT and TSLPR^−/− mice are shown. Values are the mean ± SEM. of 6–8 mice per group.

Figure 8

Reduced pulmonary IL-5⁺ and IL-22⁺ cell populations in the absence of TSLPR. Lung mononuclear cells from OVA sensitized and challenged WT and TSLPR^−/− mice were isolated and restimulated for 4 h with PMA (50 ng/mL) and ionomycin (750 ng/mL) followed by extracellular staining of TCRγδ and CD4. Representative dot plot, the frequency and the absolute numbers of IL-5⁺ and IL-22⁺ producing cells gated on TCRαβ ⁺CD4⁺ T cell populations (a–e) are shown. Values are the mean ± SEM of 6 mice per group.