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, 172 (1), 121-8

Intracellular RNA Recognition Pathway Activates Strong Anti-Viral Response in Human Mast Cells

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Intracellular RNA Recognition Pathway Activates Strong Anti-Viral Response in Human Mast Cells

J Lappalainen et al. Clin Exp Immunol.

Abstract

Mast cells have been implicated in the first line of defence against parasites and bacteria, but less is known about their role in anti-viral responses. Allergic diseases often exacerbate during viral infection, suggesting an increased activation of mast cells in the process. In this study we investigated human mast cell response to double-stranded RNA and viral infection. Cultured human mast cells were incubated with poly(I:C), a synthetic RNA analogue and live Sendai virus as a model of RNA parainfluenza virus infection, and analysed for their anti-viral response. Mast cells responded to intracellular poly(I:C) by inducing type 1 and type 3 interferons and TNF-α. In contrast, extracellular Toll-like receptor 3 (TLR)-3-activating poly(I:C) failed to induce such response. Infection of mast cells with live Sendai virus induced an anti-viral response similar to that of intracellular poly(I:C). Type 1, but not type 3 interferons, up-regulated the expression of melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-inducible gene-1 (RIG-1), and TLR-3, demonstrating that human mast cells do not express functional receptors for type 3 interferons. Furthermore, virus infection induced the anti-viral proteins MxA and IFIT3 in human mast cells. In conclusion, our results support the notion that mast cells can recognize an invading virus through intracellular virus sensors and produce high amounts of type 1 and type 3 interferons and the anti-viral proteins human myxovirus resistance gene A (MxA) and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) in response to the virus infection.

Figures

Figure 1
Figure 1
Cytosolic dsRNA induces interferon (IFN) and tumour necrosis factor (TNF)-α expression in human mast cells. Cultured human mast cells were incubated in the presence 10 μg/ml of poly(I:C) for 1–24 h. Poly(I:C) was either added to cell culture media or transfected into cytoplasm. The cells were then sedimented, and total cellular RNA was extracted and analysed for mRNA expression of IFN-α, IFN-β, interleukin (IL)-29 and TNF-α by quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR). The data show relative units (RU), i.e. fold change in gene expression normalized to 18S as an endogenous reference gene, and is calculated relative to a no-template control calibrator. Data are mean ± standard deviation from cells derived from three donors. *P < 0·05; **P < 0·01; ***P < 0·001 compared to non-treated cells at 0 h.
Figure 2
Figure 2
Exposure to live Sendai virus induces anti-viral response in human mast cells. Cultured human mast cells were incubated in the presence of Sendai virus or left untreated for 4–20 h. Thereafter, the cells were sedimented and total cellular RNA was extracted and analysed for interferon (IFN)-α, IFN-β, interleukin (IL)-29 and tumour necrosis factor (TNF)-α mRNA expression by quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR). The data show relative units (RU) as in Fig. 1. Data are mean ± standard deviation from cells derived from three donors. **P < 0·01; ***P < 0·001 compared to non-treated cells at 0 h.
Figure 3
Figure 3
Human mast cells secrete high levels of interleukin (IL)-29 in response to Sendai virus exposure. Cultured human mast cells were incubated in the presence of Sendai virus or left untreated for 4–20 h. Thereafter, the cells were sedimented, the supernatants were collected and analysed for IL-29 and β-hexosaminidase release by enzyme-linked immunosorbent assay (ELISA) and a colorimetric assay, respectively. Data are mean ± standard deviation from cells derived from three donors. ***P < 0·001 compared to non-treated cells at 0 h.
Figure 4
Figure 4
Human mast cells produce the anti-viral proteins myxovirus resistance gene A (MxA) and interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) as a response to Sendai virus infection. Human mast cells were cultured in the presence of Sendai virus or left untreated for 4–20 h. Thereafter, the cells were sedimented, the supernatants were collected and analysed for MxA and IFIT3 mRNA expression by quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR) (a). The data show relative units (RU), as defined in Fig. 1. To analyse the protein levels, mast cells were cultured with Sendai virus up to 48 h, and the MxA and IFIT3 proteins were analysed by Western blot (b). Actin was used as a loading control. *P < 0·05; ***P < 0·001 compared to non-treated cells at 0 h.
Figure 5
Figure 5
Expression of retinoic acid-inducible gene-1 (RIG-1), melanoma differentiation-associated gene 5 (MDA-5) and Toll-like receptor 3 (TLR-3), but not monocyte chemotactic protein-1 (MCP-1), is up-regulated in human mast cells during Sendai virus infection and by type 1 interferon (IFN) stimulation. Cultured human mast cells were incubated in the presence of Sendai virus or left untreated for 4–20 h (a). Alternatively, mast cells were treated for 3, 6 or 24 h with interleukin (IL)-29, IFN-β or a combination of both (b). The cells were then sedimented, and total cellular RNA was extracted and analysed for RIG-1, MDA-5, TLR-3 and MCP-1 mRNA expression by quantitative real-time reverse transcription–polymerase chain reaction (RT–PCR). The data show relative units (RU), as defined in Fig. 1. Data are mean ± standard deviation from cells derived from three donors. **P < 0·01; ***P < 0·001 compared to non-treated cells at 0 h.

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