Synergism between IL-33 and MRGPRX2/FcεRI Is Primarily Due to the Complementation of Signaling Modules, and Only Modestly Supplemented by Prolonged Activation of Selected Kinases

Abstract

Skin mast cells (MCs) express high levels of MRGPRX2, FcεRI, and ST2, and vigorously respond to their ligands when triggered individually. IL-33/ST2 also potently synergizes with other receptors, but the molecular underpinnings are poorly understood. Human skin-derived MCs were stimulated via different receptors individually or jointly in the presence/absence of selective inhibitors. TNF was quantified by ELISA. Signaling cascades were studied by immunoblot. TNF was stimulated by FcεRI ≈ ST2 > MRGPRX2. Surprisingly, neither FcεRI nor MRGPRX2 stimulation elicited NF-κB activation (IκB degradation, p65 phosphorylation) in stark contrast to IL-33. Accordingly, TNF production did not depend on NF-κB in FcεRI- or MRGPRX2-stimulated MCs, but did well so downstream of ST2. Conversely, ERK1/2 and PI3K were the crucial modules upon FcεRI/MRGPRX2 stimulation, while p38 was key to the IL-33-elicited route. The different signaling prerequisites were mirrored by their activation patterns with potent pERK/pAKT after FcεRI/MRGPRX2, but preferential induction of pp38/NF-κB downstream of ST2. FcεRI/MRGPRX2 strongly synergized with IL-33, and some synergy was still observed upon inhibition of each module (ERK1/2, JNK, p38, PI3K, NF-κB). IL-33's contribution to synergism was owed to p38 > JNK > NF-κB, while the partner receptor contributed through ERK > PI3K ≈ JNK. Concurrent IL-33 led to slightly prolonged pERK (downstream of MRGPRX2) or pAKT (activated by FcεRI), while the IL-33-elicited modules (pp38/NF-κB) remained unaffected by co-stimulation of FcεRI/MRGPRX2. Collectively, the strong synergistic activity of IL-33 primarily results from the complementation of highly distinct modules following co-activation of the partner receptor rather than by altered signal strength of the same modules.

Keywords: ERK; FcεRI; IL-33; JNK; MRGPRX2; NF-κB; PI3K; TNF-α; mast cells; p38.

Figure 1

Distinct modules contribute to TNF stimulation in dependence of the stimulus. Skin MCs were stimulated by IgER-CL (crosslinking, AER-37 at 0.2 µg/mL), or SP (30 µM), or c48/80 (10 µg/mL), or IL-33 (20 ng/mL) for 24 h, TNF was quantified in the resulting supernatants by ELISA. (a) control; vehicle was used instead of inhibitor. b-f, cells were pretreated with the respective inhibitors for 15 min and then stimulated as in (a). (b) ERK1/2 inhibitor SCH772984 at 10 µM, (c) JNK inhibitor SP600125 at 10 µM, and (d) p38 inhibitor SB203580 at 10 µM. (e) NF-κB inhibitor BAY11-7082 at 1 µM and (f) PI3K inhibitor Pictisilib at 10 µM. Mean ± SEM of n = 8–10 separate experiments; individual values are given as dots. The y-axis is consistent across inhibitors to allow direct comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. Friedman test with Dunn’s multiple comparisons test. i = inhibitor.

Figure 2

IL-33 elicits the NF-κB pathway in human skin MCs, while FcεRI and MRGPRX2 do not. MCs were stimulated by IL-33 (20 ng/mL), IgER-CL (crosslinking, AER-37 at 0.2 µg/mL), or SP (30 µM) or c48/80 (10 µg/mL) for the times given and degradation of IκBα and phosphorylation of p65 were detected by immunoblot. α-actinin served as the loading control. And 1 out of 3 experiments with comparable outcome is shown.

Figure 3

IL-33 potently activates p38, while FcεRI and MRGPRX2 elicit pERK and pAKT. Skin MCs were stimulated by IL-33 (20 ng/mL), IgER-CL (crosslinking, AER-37 at 0.2 µg/mL), or SP (30 µM), or c48/80 (10 µg/mL) for the times given and phosphorylations of ERK1/2, p38 and AKT were detected by immunoblot. Cyclophilin B (CyclB) served as the loading control. Note that pERK activation is only occasionally visible after IL-33 stimulation. And 1 out of 3 experiments with largely comparable outcome is shown.

Figure 4

Synergism between IL-33 and FcεRI aggregation relies on IL-33 and FcεRI-stimulated modules with a dominance of ERK1/2. Skin MCs were stimulated by IgER-CL (crosslinking, AER-37 at 0.2 µg/mL), IL-33 (20 ng/mL), or both combined for 24 h; TNF was quantified in the resulting supernatants by ELISA. (a) control; vehicle was used instead of inhibitor. b-f, cells were pretreated with the respective inhibitors for 15 min and then stimulated as in (a). (b) ERK1/2 inhibitor SCH772984 at 10 µM, (c) JNK inhibitor SP600125 at 10 µM, and (d) p38 inhibitor SB203580 at 10 µM. (e) NF-κB inhibitor BAY11-7082 at 1 µM and (f) PI3K inhibitor Pictisilib at 10 µM. Mean ± SEM of n = 10 separate experiments; individual values are given as dots. The y-axis is consistent across inhibitors to allow direct comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Friedman test with Dunn’s multiple comparisons test. i = inhibitor.

Figure 5

The synergism between IL-33 and c48/80 most strongly relies on ERK activity followed by other modules. Skin MCs were stimulated by c48/80 (10 µg/mL), IL-33 (20 ng/mL), or both combined for 24 h; TNF was quantified in the resulting supernatants by ELISA. (a) control; vehicle was used instead of inhibitor. (b–f), cells were pretreated with the respective inhibitors for 15 min and then stimulated as in (a). (b) ERK1/2 inhibitor SCH772984 at 10 µM, (c) JNK inhibitor SP600125 at 10 µM, and (d) p38 inhibitor SB203580 at 10 µM. (e) NF-κB inhibitor BAY11-7082 at 1 µM and (f) PI3K inhibitor Pictisilib at 10 µM. Mean ± SEM of n = 10 separate experiments; individual values are given as dots. The y-axis is consistent across inhibitors to allow direct comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant. i = inhibitor.

Figure 6

The modest synergism between SP and IL-33 relies most strongly on p38, followed by ERK. Skin MCs were stimulated by SP (30 µM), IL-33 (20 ng/mL), or both combined for 24 h; TNF was quantified in the resulting supernatants by ELISA. (a) control, vehicle was used instead of inhibitor. b–f, cells were pretreated with the respective inhibitors for 15 min and then stimulated as in (a). (b) ERK1/2 inhibitor SCH772984 at 10 µM, (c) JNK inhibitor SP600125 at 10 µM, and (d) p38 inhibitor SB203580 at 10 µM. (e) NF-κB inhibitor BAY11-7082 at 1 µM and (f) PI3K inhibitor Pictisilib at 10 µM. Mean ± SEM of n = 10 separate experiments; individual values are given as dots. The y-axis is consistent across inhibitors to allow direct comparison. * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant. Friedman test with Dunn’s multiple comparisons test. i = inhibitor.

Figure 7

IL-33, FcεRI, and MRGPRX2 triggered signaling events only modestly influence each other. Skin MCs were stimulated by IL-33 (20 ng/mL), IgER-CL (crosslinking, AER-37 at 0.2 µg/mL), SP (30 µM), or c48/80 (10 µg/mL), or the specified combinations for 30 min. The degradation of phosphorylation of signaling components were detected by immunoblot. Cyclophilin B (CyclB) served as the loading control. (a) Mean ± SEM of n = 6–11. (b) Representative individual blots. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant. RM one-way ANOVA with Holm-Šídák’s multiple comparisons test.