The Heat Shock Protein 90 (HSP90) Is Required for the IL-33-Induced Cytokine Production in Mast Cells (MCs)

Abstract

The alarmin interleukin-33 (IL-33) is released upon cell stress and damage in peripheral tissues. The receptor for IL-33 is the Toll/Interleukin-1 receptor (TIR)-family member T1/ST2 (the IL-33R), which is highly and constitutively expressed on MCs. The sensing of IL-33 by MCs induces the MyD88-TAK1-IKK2-dependent activation of p65/RelA and MAP-kinases, which mediate the production of pro-inflammatory cytokines and amplify FcεRI-mediated MC-effector functions and the resulting allergic reactions. Therefore, the investigation of IL-33-induced signaling is of interest for developing therapeutic interventions effective against allergic reactions. Importantly, beside the release of IL-33, heat shock proteins (HSPs) are upregulated during allergic reactions. This maintains the biological functions of signaling molecules and/or cytokines but unfortunately also strengthens the severity of inflammatory reactions. Here, we demonstrate that HSP90 does not support the IL-33-induced and MyD88-TAK1-IKK2-dependent activation of p65/RelA and of mitogen-activated protein (MAP)-kinases. We found that HSP90 acts downstream of these signaling pathways, mediates the stability of produced cytokine mRNAs, and therefore facilitates the resulting cytokine production. These data show that IL-33 enables MCs to perform an effective cytokine production by the upregulation of HSP90. Consequently, HSP90 might be an attractive therapeutic target for blocking IL-33-mediated inflammatory reactions.

Keywords: 17AAG; HSP90; IL-33; mast cells.

Figure 1

IL-33 upregulates HSP90 expression. (a) BMMCs were stimulated with IL-33 (50 ng/mL) for 24 h. Afterwards, cells were analyzed by flow cytometry (one representative experiment is shown). (b,c) BMMCs were stimulated with IL-33 (50 ng/mL) as indicated. Cells were lysed and analyzed by Western blotting (one representative Western blot experiment is shown), (b) or the supernatants were collected and analyzed by ELISA (c) (the summary of n = 4 biological replicates is shown; unpaired Student’s t-test; ns: not significant, ** p ≤ 0.01, *** p ≤ 0.001).

Figure 2

HSP90 mediates IL-33-induced cytokine production. (a) BMMCs treated with DMSO (vehicle) or 17AAG (1 µM) were stimulated with IL-33 (50 ng/mL) as indicated. Supernatants were collected and analyzed by ELISA (the summary of n = 3 biological replicates is shown; unpaired Student’s t-test; ns: not significant, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). (b) The fold inhibition of the produced cytokines in the presence of 17AAG is shown. The concentrations of the produced cytokines in vehicle- or 17AAG-treated and IL-33-stimulated BMMCs as shown in (a) were used to calculate the DMSO−IL-33/17AAG−IL-33 rate (the summary of n = 3 biological replicates is shown; unpaired Student’s t-test; ns: not significant, * p ≤ 0.05, *** p ≤ 0.001). (c) BMMCs were treated with 17AAG (1 µM) for 24 h. BMMCs were lysed and analyzed by Western blotting. (d–g) BMMCs were treated with 17AAG (1 µM) for 24 h. Afterwards, cells were analyzed by flow cytometry (one representative experiment is shown) or were lysed and analyzed by Western blotting (one representative experiment is shown) (g). (h,i) BMMCs were treated with DMSO (vehicle) or 17AAG (1 µM) and were stimulated with IL-33 (50 ng/mL). After 24 h, cells were analyzed by flow cytometry (one representative experiment is shown).

Figure 3

HSP90 mediates cytokine transcript stability. (a) BMMCs treated with DMSO (vehicle) or 17AAG (1 µM) were stimulated with IL-33 (50 ng/mL) as indicated. Cells were lysed and analyzed by Western blotting (one representative Western blot experiment is shown). (b) P65/RelA−eGFP MC/9 cells were treated with DMSO (vehicle) or 17AAG (1 µM) and were stimulated with IL-33 (50 ng/mL) for 24 h. Supernatants were collected and analyzed by ELISA (the summary of n = 3 replicates is shown; unpaired Student’s t−test; *** p ≤ 0.001). (c) P65/RelA−eGFP MC/9 cells were treated with DMSO (vehicle) or 17AAG (1 µM) and were stimulated with IL-33 (50 ng/mL) for 24 h. Cells were harvested and analyzed for eGFP expression by flow cytometry (one representative experiment is shown). (d) Shown is the statistical analysis of the experiment shown in (c) (the summary of n = 5 replicates is shown; unpaired Student’s t-test; ns: not significant). (e) BMMCs were treated with DMSO (vehicle) or 17AAG (1 µM) and were stimulated with IL-33 (50 ng/mL) as indicated. Cells were lysed and analyzed by Western blotting (one representative Western blot experiment is shown). (f) BMMCs were treated with DMSO (vehicle) or 17AAG (1 µM) and were stimulated with IL-33 (50 ng/mL) for 6 h. Cells were harvested and analyzed by qPCR (the summary of n = 3 biological replicates is shown; unpaired Student’s t−test; * p ≤ 0.05, ** p ≤ 0.01).