SHIP-1 differentially regulates IgE-induced IL-10 and antiviral responses in human monocytes

Abstract

IgE-mediated stimulation of monocytes regulates multiple cellular functions including cellular maturation, cytokine release, antiviral responses, and T cell priming and differentiation. The high affinity IgE receptor, FcεRI, is closely linked to serum IgE levels and atopic disease. The signaling molecules which regulate effector functions of this receptor have been well studied in mast cells and basophils, however, less is known about the signaling components, regulatory molecules, and mechanisms downstream of receptor activation in monocytes. This study sought to identify regulators of IgE-mediated cytokine release in human monocytes. SHIP-1 was identified as a negative regulator of IgE-induced IL-10 production. It was also determined that IgE-mediated stimulation and SHIP-1 inhibition decreased antiviral IP-10 production after liposomal poly(I:C) stimulation, indicating differential regulation by SHIP-1 in IgE-driven and antiviral response pathways. Both SHIP-1 and NF-κB were activated following IgE-mediated stimulation of primary monocytes, and NF-κB activation was related to both SHIP-1 and FcεRIα expression levels in monocytes. To our knowledge this is the first study to identify a role for SHIP-1 in regulating IgE-driven responses and antiviral responses in human monocytes. Given the importance of monocytes in inflammation and immune responses, a better understanding of the signaling and regulatory mechanisms downstream of FcεRI receptor could lead to new therapeutic targets in allergic disease.

Keywords: IL-10; IP-10; IgE; SHIP-1; monocyte.

Figure 1.. IgE-crosslinking activates NF-κB and SHIP-1 in monocytes.

Primary human monocytes were pre-treated with SHIP-1 inhibitor, 3AC (iSHIP), for 1 hour then IgE crosslinking was performed (αIgE) along with media alone and IgG isotype controls for 30 minutes. (A & B) Whole cell lysates were analyzed by western blot analysis for phosphorylated forms of signaling molecules: p-Lyn, p-Syk, p-SHIP-1, and the p65 subunit of NF-kB (p-p65). GAPDH was used as a loading control for quantification. N=3 biological replicates were performed and mean with SEM are shown in (B). p values were obtained by one-way ANOVA with post-hoc comparisons. (C & D) Flow cytometry analysis was performed for phosphorylated p65 NF-κB (p-NF-κB). (C) Flow cytometry gating strategy for p-NF-κB+ cells in purified monocytes. Flow plots shown are a concatenation of 4 biological replicates to demonstrate the range of cellular data. (C & D) p-NF-κB positive cells were measured across the various treatment conditions; the mean with SEM shown of 4 biological replicates are shown in (D). p values obtained by ANOVA with Fishers LSD test post-hoc comparisons; *p<0.05 and **p<0.01.

Figure 2.. IgE-mediated activation of NF-κB is related to MHC II, SHIP-1, and FcεRIα expression.

Primary human monocytes were pre-treated with SHIP-1 inhibitor, 3AC (iSHIP), for 1 hour then IgE crosslinking was performed (αIgE) along with media alone and IgG isotype controls for 30 minutes. Flow cytometry analysis was performed for phosphorylated p65 NF-κB (p-NF-κB), and total MHC II (HLA-DR), FcεRIα, and SHIP-1. Flow plots are a concatenation of all biological replicates to demonstrate the range of cellular data. (A & B) Under IgE crosslinking conditions (αIgE), MHC II, FcεRIα, and SHIP-1 levels were compared in the p-NF-κB positive population (purple) as compared to total monocytes (grey). (C & D) MHC II and FcεRIα were analyzed in the SHIP-1 high (blue) and low (grey) populations in media alone and IgE crosslinking conditions. (E & F) p-NF-κB levels were compared in SHIP-1 high and low cell populations. N=5; p values were obtained by paired two-tailed t test; *p<0.05 and **p<0.01.

Figure 3.. SHIP-1 inhibition enhances IgE-induced IL-10 production.

Primary human monocytes were purified and treated with inhibitors to (A & C) SHIP-1 (3AC, iSHIP, n=23) or (B & D) Syk (R406, iSyk, n=10) for 1 hour followed by IgE crosslinking (αIgE) or IgG isotype or media (carrier) alone controls. Supernatants were harvested at 24 hours and (A & B) IL-10 and (C & D) IL-6 measured by ELISA. Bar graphs show mean with SEM with individual subject data points overlaid. p values obtained by ANOVA with mixed effects analysis and post-hoc comparisons; *p<0.05, **p<0.01, and ***p<0.001.

Figure 4.. IgE-mediated stimulation and SHIP-1 regulate antiviral IP-10 in human monocytes.

Primary human monocytes were pre-treated with or without SHIP-1 inhibitor, 3AC (iSHIP), for 1 hour then followed by IgE-crosslinking (with media or IgG isotype controls) for 1 hour, and subsequent stimulation with liposomal poly(I:C). Supernatants were harvested at 24 hours and analyzed for IP-10 and IL-10 release. (A & B) IP-10 production after liposomal poly(I:C) treatment with and without IgE crosslinking or IgG isotype control and (B) combined with SHIP inhibition. (C) IL-10 production after IgE crosslinking and liposomal poly(I:C) treatment. (D) Linear regression analysis of IL-10 versus IP-10 in the αIgE + liposomal polyI:C condition. Pearson coefficient (R²) and p value noted. Mean with SEM and individual subjects shown for n=15 biological replicates. ANOVA with mixed effects analysis and post-hoc comparison was performed; *p<0.05, **p<0.01 and “ns” p>0.05.

Figure 5.. Model of SHIP-1-driven regulation of IgE receptor signaling and antiviral responses in monocytes.

IgE-mediated activation of FcεRI leads to activation of downstream signaling molecules including receptor tyrosine kinases Lyn and Syk, and transcription factor, NF-κB via phosphorylation. SHIP-1 is also activated, serving as a negative regulator of IgE-induced IL-10. Simultaneously, IgE-mediated stimulation and SHIP-1 have differential effects on the regulation of liposomal poly(I:C) induced antiviral responses (IP-10 release).