CD163+ M2 Macrophages Promote Fibrosis in IgG4-Related Disease Via Toll-like Receptor 7/Interleukin-1 Receptor-Associated Kinase 4/NF-κB Signaling.

OBJECTIVE: IgG4-related disease (IgG4-RD) is a fibro-inflammatory condition that can affect multiple organs. We previously demonstrated that TLR7-transgenic C57BL/6 mice showed elevated serum IgG1 levels and inflammation with fibrosis in the salivary glands (SGs), lungs, and pancreas. Moreover, we observed extensive Toll-like receptor 7 (TLR-7)-positive CD163+ M2 macrophage infiltration in SGs from IgG4-RD patients. We undertook this study to examine the fibrotic mechanism via the TLR-7 pathway. METHODS: Gene expression in SGs from human TLR7-transgenic mice and IgG4-RD patients was analyzed using DNA microarrays. We extracted the common up-regulated TLR-7-related genes in SGs from TLR7-transgenic mice and IgG4-RD patients. Finally, we investigated the interaction between CD163+ M2 macrophages and fibroblasts before and after stimulation with the TLR-7 agonist loxoribine. RESULTS: In TLR7-transgenic mice and IgG4-RD patients, IRAK3 and IRAK4 were significantly overexpressed. Real-time polymerase chain reaction validated the up-regulation of only IRAK4 in IgG4-RD patients compared with the other groups (P < 0.05). Interleukin-1 receptor-associated kinase 4 (IRAK4) was strongly detected in and around germinal centers in SGs from patients with IgG4-related dacryoadenitis and sialadenitis alone. Double immunofluorescence staining showed that IRAK4-positive cells were mainly colocalized with CD163+ M2 macrophages in SGs (P < 0.05). After stimulation with loxoribine, CD163+ M2 macrophages exhibited significantly enhanced expression of IRAK4 and NF-κB and increased supernatant concentrations of fibrotic cytokines. Finally, we confirmed that the number of fibroblasts was increased by culture with the supernatant of CD163+ M2 macrophages following stimulation with loxoribine (P < 0.05). CONCLUSION: CD163+ M2 macrophages promote fibrosis in IgG4-RD by increasing the production of fibrotic cytokines via TLR-7/IRAK4/NF-κB signaling.

Hey1 and Hey2 are differently expressed during mouse tooth development.

The Hey family (also known as Chf, Herp, Hesr, and Hrt) is a set of Hairy/Enhancer of Split-related basic helix-loop-helix type transcription factors. Hey1, Hey2, and HeyL have been identified in mammals. Although Hey proteins are known to regulate cardiovascular development, muscle homeostasis, osteogenesis, neurogenesis, and oncogenesis, their roles in tooth development have been largely obscure. Therefore, this study aimed to clarify detailed spatiotemporal expression patterns of Hey1 and Hey2 in developing molars and incisors of mice by section in situ hybridization. Hey1 and Hey2 were not significantly expressed in tooth germs at epithelial thickening, bud, and cap stages during molar development. In the dental epithelium in molars at the bell stage and incisors, Hey2 transcripts were restricted to the undifferentiated inner enamel epithelium and down-regulated in preameloblasts and ameloblasts. On the other hand, Hey1 was mainly expressed in preameloblasts and down-regulated in differentiated ameloblasts. Both genes were not significantly expressed in other dental epithelial tissues, including the outer enamel epithelium, stellate reticulum, and stratum intermedium cells. In the dental mesenchyme, Hey1 was intensely transcribed in the subodontoblastic layer of the dental pulp in both molars and incisors, whereas Hey2 was barely detectable in mesenchymal components. Our data implied that Hey2 function is restricted to transient amplifying cells of the ameloblast cell lineage and that Hey1 plays a role in the composition of the subodontoblastic layer, in addition to ameloblast differentiation. These findings provide novel clues for the better understanding of tooth development.