Fluoxetine restrains allergic inflammation by targeting an FcɛRI-ATP positive feedback loop in mast cells

Abstract

There is a clinical need for new treatment options addressing allergic disease. Selective serotonin reuptake inhibitors (SSRIs) are a class of antidepressants that have anti-inflammatory properties. We tested the effects of the SSRI fluoxetine on IgE-induced function of mast cells, which are critical effectors of allergic inflammation. We showed that fluoxetine treatment of murine or human mast cells reduced IgE-mediated degranulation, cytokine production, and inflammatory lipid secretion, as well as signaling mediated by the mast cell activator ATP. In a mouse model of systemic anaphylaxis, fluoxetine reduced hypothermia and cytokine production. Fluoxetine was also effective in a model of allergic airway inflammation, where it reduced bronchial responsiveness and inflammation. These data show that fluoxetine suppresses mast cell activation by impeding an FcɛRI-ATP positive feedback loop and support the potential repurposing of this SSRI for use in allergic disease.

Fig. 1.. Fluoxetine suppresses mast cell function in vitro.

(A) BMMC pre-sensitized with dinitrophenyl (DNP)-specific IgE were cultured with fluoxetine, sertraline, fluvoxamine, paroxetine, citalopram or vehicles, for 24 hours. DNP-HSA was added for 16 hours and cytokine concentrations in culture supernatants were determined by ELISA. N=6/group (B) BMMCs were treated with fluoxetine for the indicated time before IgE XL and DNP-HSA was added to crosslink for 16 hours. IL-6 levels in culture supernatant were measured by ELISA. N=4–6/group (C) BMMCs were treated as in (A) with the indicated concentrations of fluoxetine, and IL-6 levels were measured by ELISA. N=6/group (D) BMMCs were treated as in (A) with the indicated concentrations of fluoxetine, and viability as measured by flow cytometry after staining with propidium iodide. N=6/group. (E) BMMCs were treated as in (A), except that IgE XL was performed for 2 hours prior to the addition of monensin for 5 hours, then analyzed by flow cytometry. N= 16–19/group. (F) BMMCs cultured as in (A) were activated with IgE XL for 4 hours and RNA was collected using Trizol. Cytokine mRNA levels were determined by RT-qPCR. N=9/group. Data in A-D are representative of three independent experiments done in biological triplicates or quadruplicates. In (E-F), data are pooled from at least three independent experiments done in biological triplicates. All p values were determined by 1-way ANOVA (Tukeys test). *p<.05; **p<.001; ****p<.0001.

Fig. 2.. Fluoxetine inhibits human mast cell responses to IgE XL.

(A) HuMC were treated with anti-DNP IgE and fluoxetine, crosslinked with DNP-HSA and cytokines were measured by ELISA. Each donor represents an individual subject and were analyzed as technical triplicates, (biological N= 5/group). Statistical values were determined by paired Student’s t-test. *p<.05; **p<.001; ****p<.0001. N.D.= Not detected.

Fig. 3.. Fluoxetine Inhibits the early phase of mast cell activation.

(A) BMMCs were treated with fluoxetine and activated by IgE XL for 30 minutes. Cells were either analyzed by flow cytometry for CD107a surface expression or cell pellets and supernatant were collected for histamine ELISA. N=12/group. (B,C) BMMCs were treated with fluoxetine and activated by IgE XL for 1 hour. Supernatant was analyzed for eicosanoid production by mass spectrometry. N=3/group. Data are pooled from at least three independent experiments with biological triplicates in A. B and C is representative of two independent experiments with biological triplicates, per experiment. Data are pmole/4 million cells from biological triplicate samples analyzed by 1-way ANOVA (Tukey’s test). *p<.05; **p<.001; ****p<.0001. NS, not statistically significant.

Fig. 4.. Fluoxetine inhibits early mast cell signaling events.

(A) BMMCs were treated with fluoxetine and activated by IgE XL. Ca²⁺ flux was measured by FURA-2 staining. N=11–12/group. (B) BMMCs treated with fluoxetine, then crosslinked for the indicated times and flow cytometry was used to measure phosphoprotein expression. N= 3/group. Data are representative of two (A) or (B) three independent experiments performed in biological triplicates (A) or quadruplicates (B). Statistical values were determined by t-Test for (A) and 1-way ANOVA (Tukey’s or Sidak’s test). *p<.05; **p<.001; ****p<.0001.

Fig. 5.. Fluoxetine inhibits ATP-mediated mast cell activation.

(A-E) BMMCs were treated with fluoxetine and activated with ATP (A) prior to measuring Ca²⁺ influx. N= 6/group. (B) BMMCs surface CD107a expression was measured by flow cytometry. N=12/group. (C) BMMC supernatant lipids were measured by mass spectroscopy. Lipids that were significantly reduced by fluoxetine are shown in the bar charts; all others are shown in the heatmap. Data are pmole/4 million cells and analyzed by ANOVA (Tukey’s test). N=4/group. (D) BMMCs NFκB reporter activity was measured by luciferase assay. N=9/group. (E) BMMC IL-6 was measured by ELISA. N=12/group. (F) BMMCs were activated by IgE XL for 60 seconds and supernatant ATP was measured using a luminescent ATP assay. N=6/group from 2 independent experiments. (G) IgE-sensitized BMMCs were treated with fluoxetine, then crosslinked with DNP-HSA for 1 minute and supernatant was used to detect ATP by luminescence. N=9/group. (H) BMMCs were activated by IgE XL in the presence or absence of recombinant mouse CD39. Supernatant cytokines were measured by ELISA. N=6–12/group. All data are pooled from at least 2 independent experiments or a representative of two independent experiments done in quadruplicates. Statistical values were determined by 1-way ANOVA (Tukey’s test) or Student’s t-Test (A, using area under curve values). *p<.05; **p<.001; ****p<.0001.

Fig. 6.. Fluoxetine inhibits passive systemic anaphylaxis.

(A) Mice were sensitized with anti-DNP IgE and treated with PBS or fluoxetine. The next day, anaphylaxis was induced with DNP-has and core body temperature was measured (A, N=9/group). (B) Mice were euthanized 30 minutes after DNP-HSA injection to assess plasma histamine levels by ELISA (N=10/group). (C) Mice were euthanized after 2 hours, and plasma was collected for cytokine analysis by ELISA (N=10/group). (D) Mice (N=5/group) were treated with PBS or fluoxetine as in (A). Histamine was administered and Core body temperature was measured. Data are pooled from two independent repeats. Statistical values were determined by 1-way ANOVA (Tukey’s test) and area under the curve and Student’s t-test in (A). *p<.05; **p<.001; ****p<.0001. n.s.=not statistically significant.

Fig. 7.. Fluoxetine diminishes HDM-induced lung inflammation and airway hyperresponsiveness.

(A) Mice were sensitized and challenged with HDM while receiving fluoxetine or PBS treatments. Animals and tissue were analyzed on day 23. (B) Methacholine-induced lung resistance was measured in anesthetized mice using a Scrieq flexivent instrument. Data are pooled from 2 independent experiments with N=8/group total. (C) Digested lung tissue single cell suspension was analyzed by flow cytometry for mast cells (FcεRI+cKit+), eosinophils (siglecF+CCR3hi) and Th2 cells (CD3+CD4+IL13+IL4+). N=11–14/group. (D) BALF cells were analyzed for T cells (CD90.2+/MHC II-neg), B cells (B220+MHCII+), eosinophils (SiglecF+CCR3+) and neutrophils (CD11b+Ly6G+). Data are pooled from at least 2 independent repeats with each point representing an individual mouse. N=9/group. (E) BALF cytokines were measured by ELISA. Data are pooled from 3 independent experiments. N=10–18/group. (F) Plasma MCPT-1 levels were measured by ELISA from two independent experiments. N=8/group. (G) Lung sections were stained for mucus production with PAS (left) or H&E (right) and blinded samples were scored as described in Materials and Methods. Data are pooled from 2 independent experiments. N=9/group. (H) Anti-HDM IgG and total IgE in plasma were determined by ELISA from two independent experiments. N=11–15/group. All replicates are biological. Statistical values were determined by 1-way ANOVA (Tukey’s test) *p<.05; **p<.001; ****p<.0001 N.D.=not detected.

Figure 8:. Model for fluoxetine-mediated suppression of mast cell function.

The data collectively support a model in which IgE crosslinkage with multivalent antigen elicits rapid ATP release that subsequently activates purinergic receptors and amplifies FcεRI-mediated signaling. Fluoxetine is proposed to inhibit purinergic receptor activation, as the drug does not alter FcεRI-induced ATP secretion but inhibits ATP-mediated mast cell function.