The Response of Tissue Mast Cells to TLR3 Ligand Poly(I:C) Treatment

Abstract

Mast cells (MCs) are found mainly at the anatomical sites exposed to the external environment; thus, they are localized close to blood vessels, lymphatic vessels, and a multitude of immune cells. Moreover, those cells can recognize invading pathogens through a range of surface molecules known as pathogen recognition receptors (PRRs), mainly Toll-like receptors (TLRs). MCs are extensively engaged in the control and clearance of bacterial infections, but much less is known about their contribution to antiviral host response as well as pathomechanisms of virus-induced diseases. In the study, we employed in vivo differentiated mature tissue mast cells freshly isolated from rat peritoneal cavity. Here, we demonstrated that rat peritoneal mast cells (rPMCs) express viral dsRNA-specific TLR3 molecule (intracellularly and on the cell surface) as well as other proteins associated with cellular antiviral response: IRF3, type I and II IFN receptors, and MHC I. We found that exposure of rPMCs to viral dsRNA mimic, i.e., poly(I:C), induced transient upregulation of surface TLR3 (while temporarily decreased TLR3 intracellular expression), type II IFN receptor, and MHC I. TLR3 ligand-stimulated rPMCs did not degranulate but generated and/or released type I IFNs (IFN-α and IFNβ) as well as proinflammatory lipid mediators (cysLTs), cytokines (TNF, IL-1β), and chemokines (CCL3, CXCL8). We documented that rPMC priming with poly(I:C) did not affect FcεRI-dependent degranulation. However, their costimulation with TLR3 agonist and anti-IgE led to a significant increase in cysLT and TNF secretion. Our findings confirm that MCs may serve as active participants in the antiviral immune response. Presented data on modulated FcεRI-mediated MC secretion of mediators upon poly(I:C) treatment suggests that dsRNA-type virus infection could influence the severity of allergic reactions.

Figure 1

rPMCs exhibit phenotype engaged in response to viral infection. Constitutive expression of TLR3, IRF3, IFNAR1, IFNGR1, and MHC I proteins in cell lysates analyzed by Western blot. β-Actin was used as a loading control (a). Constitutive expression of TLR3 (surface and intracellular), IRF3, IFNAR1, IFNGR1, and MHC I molecules analyzed by flow cytometry. Shaded areas indicate staining with isotype-matched control antibodies (b). Data are representative of 3 independent experiments.

Figure 2

rPMCs change their phenotype upon stimulation with TLR3 ligand. rPMCs were treated with poly(I:C) at 10 μg/mL or medium alone (constitutive expression) for 6 or 12 h. Expression of intracellular TLR3 (a), surface TLR3 (b), IFNAR1 (c), IFNGR1 (d), and MHC I (e) was analyzed by flow cytometry. Results are shown as percentage changes in MFI values to constitutive expression (100%). Representative flow cytometry histograms demonstrate protein expression on poly(I:C)-stimulated (white) and nonstimulated (shaded) rPMCs. Data are presented as the mean ± SD of 5 independent experiments (n = 5). ^∗p < 0.05 and ^∗∗p < 0.01; n.s.: not significant.

Figure 3

rPMCs release de novo synthesized mediators upon stimulation with TLR3 ligand. rPMCs were challenged with poly(I:C) at the indicated concentrations, PGN from S. aureus (10 μg/mL), or LPS from E. coli (100 ng/mL) for 2 (a) or 12 h (b–f). CysLT (a), IFN-α (b), IFN-β (c), TNF (d), CCL3 (e), and CXCL8 (f) were measured in cell supernatants using ELISA. Results are presented as the mean ± SD of 4 independent experiments, and each experiment was done in duplicate (n = 8). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; n.s.: not significant.

Figure 4

rPMCs produce a wide array of cytokine mRNAs upon stimulation with TLR3 ligand. rPMCs were challenged with poly(I:C) at 10 μg/mL or medium alone (control) for 2 h. Cytokine mRNA expression was established by qRT-PCR and demonstrated as a fold increase above the value of cytokine mRNA expression in untreated cells after normalization with the transcript level of the housekeeping gene of rat Actb. Results are presented as the mean ± SD of at least 3 separate experiments performed in duplicates (n ≥ 6).

Figure 5

TLR3 ligand-induced release of de novo synthesized mediators from rPMCs is TLR3- and NF-κB-dependent. rPMCs were preincubated with medium alone (control), anti-TLR3 antibodies (40 μg/mL), or isotype control antibodies for 1 h and after rinsing exposed to poly(I:C) at 10 μg/mL for 2 h (cysLT release) or 12 h (cytokine release) (a). rPMCs were preincubated with medium alone (control) or MG-132 (3 μM) for 15 min and after rinsing treated with poly(I:C) at 10 μg/mL for 2 h (cysLT release) or 12 h (cytokine release) (b). Results are reduced by the value of the spontaneous release. Data are expressed as the mean ± SD of 4 independent experiments, and each experiment was done in duplicate (n = 8). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; n.s.: not significant.

Figure 6

TLR3 ligand-induced release of type I IFNs from rPMCs is TBK1/IKKε-dependent. rPMCs were preincubated with medium alone (control) or BX-795 (1 μM) for 15 min and then exposed to poly(I:C) at 10 μg/mL for 12 h. IFN-α (a) and IFN-β (B) concentration was measured in cell supernatants using ELISA. Data are presented as the mean ± SD of 4 independent experiments, and each experiment was done in duplicate (n = 8). ^∗∗∗p < 0.001.

Figure 7

FcεRI-mediated release of de novo synthesized mediators upon rPMC stimulation with TLR3 ligand. rPMCs were treated with medium alone, poly(I:C) at 10 μg/mL, anti-IgE at 5 μg/mL, or both poly (I:C) and anti-IgE for 2 (a) or 12 h (b–f). CysLT (a), IFN-α (b), IFN-β (c), TNF (d), CCL3 (e), and CXCL8 (f) were measured in cell supernatants using ELISA. Bars for the poly(I:C) at 10 μg/mL and medium alone demonstrate the same data set as in Figure 3. Results are presented as the mean ± SD of 4 independent experiments, and each experiment was done in duplicate (n = 8). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001; n.s.: not significant.