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. 2023 May 28;12(11):1493.
doi: 10.3390/cells12111493.

IRF3 Activation in Mast Cells Promotes FcεRI-Mediated Allergic Inflammation

Young-Ae Choi  1 , Hima Dhakal  1 , Soyoung Lee  2 , Namkyung Kim  1 , Byungheon Lee  3 , Taeg Kyu Kwon  4 , Dongwoo Khang  5 , Sang-Hyun Kim  1
Affiliations

IRF3 Activation in Mast Cells Promotes FcεRI-Mediated Allergic Inflammation

Young-Ae Choi et al. Cells. .

Abstract

(1) Background: This study aims to elucidate a novel non-transcriptional action of IRF3 in addition to its role as a transcription factor in mast cell activation and associated allergic inflammation; (2) Methods: For in vitro experiments, mouse bone-marrow-derived mast cells (mBMMCs) and a rat basophilic leukemia cell line (RBL-2H3) were used for investigating the underlying mechanism of IRF3 in mast-cell-mediated allergic inflammation. For in vivo experiments, wild-type and Irf3 knockout mice were used for evaluating IgE-mediated local and systemic anaphylaxis; (3) Results: Passive cutaneous anaphylaxis (PCA)-induced tissues showed highly increased IRF3 activity. In addition, the activation of IRF3 was observed in DNP-HSA-treated mast cells. Phosphorylated IRF3 by DNP-HSA was spatially co-localized with tryptase according to the mast cell activation process, and FcεRI-mediated signaling pathways directly regulated that activity. The alteration of IRF3 affected the production of granule contents in the mast cells and the anaphylaxis responses, including PCA- and ovalbumin-induced active systemic anaphylaxis. Furthermore, IRF3 influenced the post-translational processing of histidine decarboxylase (HDC), which is required for granule maturation; and (4) Conclusion: Through this study, we demonstrated the novel function of IRF3 as an important factor inducing mast cell activation and as an upstream molecule for HDC activity.

Keywords: allergic inflammation; histamine; histidine decarboxylase; interferon regulatory factor 3; mast cells.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Expression of IRF3 in PCA-induced tissue and activated mast cells. Mice (n = 3/group) were sensitized with IgE and challenged with DNP-HSA to induce a PCA reaction. (A) Images of Toluidine blue-stained tissue obtained in ×200 magnification. Scale bar, 50 μm. The number of mast cells counted in five random sites. (B) The expression of p-S396-IRF3, IRF3, and β-actin in PCA-induced ear tissues. Anti-DNP-IgE-sensitized mBMMCs and RBL-2H3 were treated with DNP-HSA for the indicated time. (C) The expression of p-S396-IRF3, IRF3, and lamin B1 in the total and nuclear fraction of (C) mBMMCs and (D) RBL-2H3. (E) Immunostaining images for p-S396-IRF3 and tryptase of mBMMCs stimulated with DNP-HSA for the indicated time. Images were visualized at ×400 magnification. Scale bar, 20 μm. Blue; DAPI, Red; Alexa 594, Green; Alexa 488. The graph represents the means ± standard error of the mean. **** Significant difference at p < 0.0001.
Figure 2
Figure 2
The effect of FcεRI-mediated signaling pathway in the activation of IRF3. Anti-DNP-IgE-sensitized mBMMCs and RBL-2H3 were pre-treated for 1 h with each inhibitor, including PP2 (5 µM), LY 294002 (LY, 5 µM), U 73122 (U7, 5 µM), and Akt inhibitor IV (Akt in, 1 µM), and then stimulated with DNP-HSA for 5 min for p-S396-IRF3 and 15 min for N-IRF3. The expression of p-S396-IRF3, IRF3, and lamin B1 in the total and nuclear fraction of (A) mBMMCs and (B) RBL-2H3.
Figure 3
Figure 3
The effect of Irf3 knockdown and overexpression in DNP-HSA-stimulated mast cells. siRNA or plasmid DNA-transfected cells were sensitized overnight with anti-DNP-IgE and stimulated with DNP-HSA for 30 min for mBMMCs and 4 h for RBL-2H3 cells. Histamine levels were measured using a fluorometric histamine assay. β-hexosaminidase level was measured using β-hexosaminidase substrate buffer. ELISA was used to determine the levels of TNF-α and IL-6. For ELISA, Irf3 knockdown mBMMCs were stimulated with DNP-HSA for 8 h, and Irf3-knockdown RBL-2H3 cells were stimulated with DNP-HSA for 6 h. (A) Level of inflammatory mediators in Irf3-knockdown mBMMCs and RBL-2H3 cells. (B) Levels of inflammatory mediators in Irf3-overexpressing mBMMCs and RBL-2H3. Each dataset presents as the means ± standard deviation of the mean (n = 3). Significant difference at p ** < 0.01, p *** < 0.001, and p **** < 0.0001. ND: not detected.
Figure 4
Figure 4
The effect of Irf3-deficiency in DNP-HSA-induced mast cell activation. Mouse bone marrow cells were isolated and differentiated into matured mast cells for 4 weeks as described in Materials and Methods. (A) Analysis of mast cell phenotypes using flow cytometric analysis. The color changes (blue>green>yellow< red) display the distribution intensity. (B) The gene and protein expression of IRF3 in primary cultured mBMMCs. WT and Irf3 KO-derived mBMMCs were sensitized with anti-DNP-IgE and then stimulated with DNP-HSA. (C) Histamine and β-hexosaminidase levels at 30 min after stimulation. (D) The levels of TNF-α and IL-6 at 8 h after stimulation. (E) The gene expression of TNF-α and IL-6 at 30 min after stimulation. Each dataset presents as the means ± standard deviation of the mean (n = 3). **** Significant difference at p < 0.0001. ND: not detected.
Figure 5
Figure 5
The effect of Irf3-deficiency in the anaphylaxis model. Passive cutaneous anaphylaxis reaction was induced in WT and Irf3 KO mouse ear skin (n = 5/group) as described in Materials and Methods. (A) The representative photographic images of the ear, absorbance values of extracted dye from the ear, and ear thickness. (B) Images of Toluidine blue-stained tissue obtained in ×200 magnification. Scale bar, 50 μm. Active systemic anaphylaxis was induced in WT and Irf3 KO mice (n = 5/group) as described in Materials and Methods. (C) The changes in rectal temperature of OVA-challenged mice for 90 min. (D) Serum histamine levels. (E) Serum IL-4 levels. (F) Serum total IgE levels. (G) Serum OVA-specific IgE levels. Each dataset presents as the means ± standard error of the mean (n = 5). **** Significant difference at p < 0.0001. ND: not detected.
Figure 6
Figure 6
The effect of IRF3 in HDC post-translational processing. (A) The expression of HDC and β-actin in the passive cutaneous anaphylaxis-induced tissue. (B) Anti-DNP-IgE-sensitized mBMMCs (2 × 106 cells/well in 6-well plates) and RBL-2H3 (1.5 × 106 cells/well in 6-well plates) were stimulated with DNP-HSA. The expression of HDC and β-actin after the stimulation for the indicated time. (C) siRNA or (D) plasmid DNA-transfected mBMMCs and RBL-2H3 cells were sensitized with anti-DNP-IgE overnight and then stimulated with DNP-HSA. The expression of HDC and β-actin (upper images) and relative band intensities of HDC (lower graph).
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Grants and funding

This work was supported by the National Research Foundation of Korea grants funded by the Korean government (2022M3A9G8018189, 2020R1A2C1010962, 2020M3A9D3038894, 2019R1A2B5B01069444, and 2021R1A5A2021614).