Toll-Like Receptor 7 (TLR7) Mediated Transcriptomic Changes on Human Mast Cells

Abstract

Background: Mast cells are skin immune sentinels located in the upper dermis, where wheal formation and sensory nerve stimulation take place. Skin inflammation is occasionally accompanied by mast cell-driven responses with wheals, angioedema, or both. Immunoglobulin E (IgE) antibodies are regarded as typical stimuli to drive mast cell activation. However, various causative factors, including microbial infections, can drive IgE-independent mast cell response. When infected, the innate immunity orchestrates an immune response by activating receptor signaling via Toll-like receptors (TLRs).

Objective: In this study, we determined the effect of TLR7 stimulation on mast cells to investigate the possible mechanism of IgE-independent inflammatory response.

Methods: Human mast cell (HMC) line, HMC-1 cells were treated with TLR7 agonist and the morphologic alteration was observed in transmission electron microscopy. Further, TLR7 agonist treated HMC-1 cells were conducted to RNA sequencing to compare transcriptomic features.

Results: HMC-1 cells treated with TLR7 agonist reveals increase of intracellular vesicles, lipid droplets, and ribosomes. Also, genes involved in pro-inflammatory responses such as angiogenesis are highly expressed, and Il12rb2 was the most highly upregulated gene.

Conclusion: Our data suggest that TLR7 signaling on mast cells might be a potential therapeutic target for mast cell-driven, IgE-independent skin inflammation.

Keywords: Mast cell; Skin inflammation; Toll-like receptor 7.

Fig. 1. Human mast cell (HMC)-1 cells show morphologic alterations in response to Toll-like receptor 7 (TLR7) stimulation. (A) Reverse transcription polymerase chain reaction was used to determine TLR7 expression in HMC-1 cells cultured with or without imiquimod (IMQ) for 24 h. The GAPDH gene was the internal control. The band pixel density of the TLR7 amplicon was divided by the pixel density of the corresponding GAPDH band. (B) HMC-1 cells cultured with or without IMQ were visualized by toluidine blue stain (original magnification, ×1,000). (C) Transmission electron microscope images of control HMC-1 cells (left) or IMQ-treated HMC-1 cells (right). The red arrows point to vesicles containing one or more smaller vesicles. Yellow arrows indicate lipid droplets. Black arrows point to ribosomes next to the rough endoplasmic reticulum. Original magnification, ×10,000 (top) and ×20,000 (bottom).

Fig. 2. Transcription differs between control human mast cell (HMC)-1 cells and imiquimod-treated HMC-1 cells. (A) A heatmap of hierarchical clustering indicates differentially expressed gene (rows) between control HMC-1 cells and imiquimod-treated HMC-1 cells (fold-change>2, p<0.05). Red indicates upregulation and blue indicates downregulation. (B) Functional enrichment analysis of highly regulated genes in control HMC-1 cells and imiquimod-treated HMC-1 cells were annotated in the GO category of biological process (left). Distribution of positively regulated genes was annotated in the GO category of biological process (right).

Fig. 3. Validation of genes upregulated in response to imiquimod. The mRNA expression of Muc22, Ern2, Rps17, Epha1, Kcnt1, Nlrp3, Ccl4, and Il12rb2 in control human mast cell (HMC)-1 cells and imiquimod-treated HMC-1 cells was analyzed by quantitative reverse transcription polymerase chain reaction. Values are presented as the mean±standard error of the mean (^*p<0.05).