Inhibiting Isoprenylation Suppresses FcεRI-Mediated Mast Cell Function and Allergic Inflammation

Abstract

IgE-mediated mast cell activation is a driving force in allergic disease in need of novel interventions. Statins, long used to lower serum cholesterol, have been shown in multiple large-cohort studies to reduce asthma severity. We previously found that statins inhibit IgE-induced mast cell function, but these effects varied widely among mouse strains and human donors, likely due to the upregulation of the statin target, 3-hydroxy-3-methylgutaryl-CoA reductase. Statin inhibition of mast cell function appeared to be mediated not by cholesterol reduction but by suppressing protein isoprenylation events that use cholesterol pathway intermediates. Therefore, we sought to circumvent statin resistance by targeting isoprenylation. Using genetic depletion of the isoprenylation enzymes farnesyltransferase and geranylgeranyl transferase 1 or their substrate K-Ras, we show a significant reduction in FcεRI-mediated degranulation and cytokine production. Furthermore, similar effects were observed with pharmacological inhibition with the dual farnesyltransferase and geranylgeranyl transferase 1 inhibitor FGTI-2734. Our data indicate that both transferases must be inhibited to reduce mast cell function and that K-Ras is a critical isoprenylation target. Importantly, FGTI-2734 was effective in vivo, suppressing mast cell-dependent anaphylaxis, allergic pulmonary inflammation, and airway hyperresponsiveness. Collectively, these findings suggest that K-Ras is among the isoprenylation substrates critical for FcεRI-induced mast cell function and reveal isoprenylation as a new means of targeting allergic disease.

Fig. 1:. Dual isoprenylation inhibitor FGTI-2734 mimics statin inhibitory effects on IgE-mediated mast cell function.

A) Diagram of the isoprenylation pathway indicating where statins, FTI, GGTI, and FGTI act. B) C57BL/6J BMMCs were sensitized with IgE and treated with fluvastatin (5μM), FTI-2717 (5μM), GTI-2418 (5μM), FGTI-2734 (5μM), or vehicle control for 24 hours, then activated by IgE XL for 15 minutes and degranulation markers CD107a and CD63 were measured by flow cytometry. C) BMMCs were treated as in (B) but activated for 16 hours and cytokines were measured in culture supernatant by ELISA. D) BMMCs were treated with increasing doses of FGTI-2734 for 24 hours prior to IgE XL for 16 hours. Cytokines were measured by ELISA. E) BMMCs derived from C57BL/6J, BALB/c, or 129/SvJ were treated as in (D) and IC₅₀ values for suppressing IL-6 production were calculated. F) BMMCs were treated with FGTI-2734 (5μM) for the indicated times before IgE XL for 15 minutes. CD63 expression was measured by flow cytometry and t_1/2 for inhibition was calculated. G) Peritoneal mast cells from 10 mice were treated with FGTI-2734 (5 μM) for 24 hours and activated by IgE XL for 16 hours. Cytokines in culture supernatant were measured by ELISA. Data shown are from 3 independent experiments with at least 3 biological replicates and at least 2 technical replicates. ANOVA was used for statistics. ϕ = unstimulated control cells in DMSO.

Fig. 2. FGTI-2734 can reduced IgE mediated human mast cell function:

Human skin-derived mast cells from 5 donors were treated with either FGTI-2734 (5μM) or vehicle control for 24 hours, cells were then activated by anti-FcεRI for A) 16 hours, and secreted cytokines were measured by ELISA or B) 30 minutes and beta-hexosaminidase was measured by colorimetric assay. Data are from one experiment with each donor’s cells analyzed in quadruplicate. ANOVA was used for statistics.

Fig. 3:. GGT-1 and FT siRNA targeting reduce IgE-mediated degranulation and cytokine production.

C57BL/6J BMMCs were transfected with siRNAs targeting GGT-1, FT, or with scrambled control siRNAs. A) Cell lysates were assessed by Western blot for GGT-1 and FT expression, which was normalized to actin loading control for quantification. B) Transfected cells were activated by IgE XL for 15 minutes and degranulation markers CD63 and CD107a were measured by flow cytometry. C) Transfected cells were activated by IgE XL for 16 hours, and cytokines were measured by ELISA. Data shown are from 2 experiments using 6 samples.

Fig. 4:. FGTI-2734 treatment reduces FcεRI signaling.

A) BMMCs were treated with vehicle or FGTI-2734 (5μM) for 24 hours then activated with IgE XL for 5.5 hours and cytokine production was assessed by flow cytometry. B) BMMCs were treated with FGTI-2734 (5μM) for 24 hours and activated with IgE XL for 4 hours. RT-qPCR was used to measure cytokine mRNA levels. C) BMMCs were treated with FGTI-2734 (5μM) or vehicle for 24 hours and placed in media without growth factors for 2 hours before IgE XL for 5 minutes. Cells were fixed and permeabilized prior to flow cytometry analysis of phospho-protein staining. Data shown are from 3 experiments using 3 samples.

Fig. 5:. K-Ras suppression mimics the effects of FGTI-2734 treatment.

A) BMMCs were treated with either FGTI-2734 or vehicle control for 24 hours and lysed. Membrane and cytosolic fractions were assessed for K-Ras by Western blot. Fyn, a membrane-associated protein that is not isoprenylated, and actin, a cytosolic protein, were used to determine the effectiveness of cell fractionation. B) BMMCs were transfected with siRNA targeting K-Ras as in Fig. 2. Lysates were assessed for K-Ras expression by Western blot. C) Cells from (B) were activated by IgE XL for 15 minutes and degranulation markers CD107a and CD63 were measured by flow cytometry. D) Cells from (B) were activated by IgE XL for 16 hours and cytokines were measured by ELISA. E) BMMCs were transfected with siRNA targeting N-Ras as in Fig. 2. Cell lysates were assessed for N-Ras expression by Western blot. IgE XL-induced degranulation and cytokine production were assessed as described for K-Ras targeting. Data shown are from 3 individual experiments in (A). Parts (B-E) are representative of at least 2 independent experiments. P values were calculated by ANOVA

Fig. 6:. FGTI reduces IgE-induced passive systemic anaphylaxis.

(A-C) Female C57BL/6J mice were treated with vehicle or FGTI-2734 (1mg/kg, i.p.) and subjected to PSA as described in Materials and Methods. (A) Change in core body temperature. (B) Plasma levels of mMCPT-1 10 minutes after antigen injection were measured by ELISA. (C) Plasma levels of IL-6 two hours after antigen injection were measured by ELISA. Data shown are from 3 experiments, N=13. D) Female C57BL/6J mice were treated with vehicle or FGTI-2734 (1mg/kg, i.p.) 20 hours before histamine injection (2mg, i.p.), and core body temperature was measured. Data shown are from one experiment using 3 control mice and 5 mice treated with histamine. E) Female 129/SvJ mice were subjected top PSA as described in (A). Data shown are from 9 control and 12 FGTI 2734-treated mice analyzed in 3 experiments. P values in (A) and (E) were calculated using multiple t-Tests comparing individual time points or comparing AUC values.

Fig. 7:. FGTI reduces Alternaria-induced airway inflammation.

A) Schematic of ALT model. FGTI-2734 was used at 1 mg/kg. B) Total BALF cells and eosinophils were measured by flow cytometry analysis as described in Materials and Methods. C) Lung tissue eosinophils were measured by flow cytometry. D) Scoring of lung inflammation in H&E-stained sections and mucus production in PAS-stained sections was calculated as described in Materials and Methods. E) Plasma mMCPT-1 concentration on day 17 was measured by ELISA. F) Mediastinal lymph node cells were collected on day 17. Cells were restimulated with OVA (300 ng/ml) for 3 days, and cytokines in culture supernatants were measured by ELISA. Data shown are from 4 experiments using N=12 mice.

Fig 8.. FGTI reduces Alternaria-induced airway hyperresponsiveness.

A) Schematic of ALT-induced airway hyperresponsiveness model. FGTI-2734 was used at 5 mg/kg. B) FlexiVent was used to measure lung resistance (Rrs) and elastance (Ers) elicited by increasing doses of methacholine. AUC and ANOVA were used to measure significance. N=8/group from two experiments.