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Single-cell RNA sequencing (scRNA-seq) enables specific profiling of cell populations at single-cell resolution. The osteoimmunology microenvironment in the occurrence and development of periodontitis remains poorly understood at the single-cell level. In this study, we used single-cell transcriptomics to comprehensively reveal the complexities of the molecular components and differences with counterparts residing in periodontal tissues. Methods: We performed scRNA-seq to identify 51248 single cells from healthy controls (n=4), patients with severe chronic periodontitis (n=5), and patients with severe chronic periodontitis after initial periodontal therapy within 1 month (n=3). Uniform manifold approximation and projection (UMAP) were further conducted to explore the cellular composition of periodontal tissues. Pseudotime cell trajectory and RNA velocity analysis, combined with gene enrichment analysis were used to reveal the molecular pathways underlying cell fate decisions. CellPhoneDB were performed to identify ligand-receptor pairs among the major cell types in the osteoimmunology microenvironment of periodontal tissues. Results: A cell atlas of the osteoimmunology microenvironment in periodontal tissues was characterized and included ten major cell types, such as fibroblasts, monocytic cells, endothelial cells, and T and B cells. The enrichment of TNFRSF21 + fibroblasts with high expression of CXCL1, CXCL2, CXCL5, CXCL6, CXCL13 , and IL24 was detected in patients with periodontitis compared to healthy individuals. The fractions of CD55 + mesenchymal stem cells (MSCs), APOE + pre-osteoblasts (pre-OBs), and IBSP + osteoblasts decreased significantly in response to initial periodontal therapy. In addition, CXCL12 + MSC-like pericytes could convert their identity into a pre-OB state during inflammatory responses even after initial periodontal therapy confirmed by single-cell trajectory. Moreover, we portrayed the distinct subtypes of monocytic cells and abundant endothelial cells significantly involved in the immune response. The heterogeneity of T and B cells in periodontal tissues was characterized. Finally, we mapped osteoblast/osteoclast differentiation mediators to their source cell populations by identifying ligand-receptor pairs and highlighted the effects of Ephrin-Eph signaling on bone regeneration after initial periodontal therapy. Conclusions: Our analyses uncovered striking spatiotemporal dynamics in gene expression, population composition, and cell-cell interactions during periodontitis progression. These findings provide insights into the cellular and molecular underpinning of periodontal bone regeneration.

NLRP3 inflammasome plays an important role in the pathogenesis of rheumatoid arthritis (RA). However, the post-transcriptional regulation of NLRP3 expression by miRNA in synovial macrophages is still not well understood. The aim of the study is to elucidate the mechanisms of RA with the focus on miRNAs mediated post-transcriptional regulation of the NLRP3 inflammasome. Here, we used NLRP3-deficient mice (NLRP3KO) to cross with TNFα-transgenic mice (TNFTG) to generate NLRP3KO/TNFTG mice, and compared their joint phenotypes with those of their TNFTG and wild-type (WT) littermates at 5 months of age. In comparison to WT mice, articular bone volume and cartilage area are decreased, whereas inflammed area, eroded surface, ALP+ osteoblast number, TRAP+ osteoclast number, and the areas of RelA+F4/80+, Caspase-1+F4/80+, IL-1β+F4/80+ synoviocytes are increased in the TNFTG mice. Knockout of NLRP3 ameliorates joint inflammation and bone damage in TNFTG mice. Further, in TNFα-primed BMDMs, RelA positively regulates NLRP3 expression, but negatively regulates miR-30a. Additionally, miR-30a negatively mediates NLRP3 expression by directly binding to its 3ʹ UTR, suggesting a miR-30a-mediated feedforward loop acting on NLRP3. Finally, intra-articular injection of AAV-miR-30a inhibits NLRP3 inflammasome activation, reduces joint inflammation, and attenuates bone damage in TNFTG mice. Thus, RelA/miR-30a/NLRP3 signal axis is involved in RA through regulating NLRP3 Inflammasome in macrophages.

The lymphatic system plays a crucial role in the maintenance of tissue fluid homeostasis and the immunological response to inflammation. The effects of lymphatic drainage dysfunction on periodontitis have not been well studied. Here we show that lymphatic vessel endothelial receptor 1 (LYVE1)+/podoplanin (PDPN)+ lymphatic vessels (LVs) are increased in the periodontal tissues, with accumulation close to the alveolar bone surface, in two murine periodontitis models: rheumatoid arthritis (RA)‐associated periodontitis and ligature‐induced periodontitis. Further, PDPN+/alpha‐smooth muscle actin (αSMA)− lymphatic capillaries are increased, whereas PDPN+/αSMA+ collecting LVs are decreased significantly in the inflamed periodontal tissues. Both mouse models of periodontitis have delayed lymph flow in periodontal tissues, increased TRAP‐positive osteoclasts, and significant alveolar bone loss. Importantly, the local administration of adeno‐associated virus for vascular endothelial growth factor C, the major growth factor that promotes lymphangiogenesis, increases the area and number of PDPN+/αSMA+ collecting LVs, promotes local lymphatic drainage, and reduces alveolar bone loss in both models of periodontitis. Lastly, LYVE1+/αSMA− lymphatic capillaries are increased, whereas LYVE1+/αSMA+ collecting LVs are decreased significantly in gingival tissues of patients with chronic periodontitis compared with those of clinically healthy controls. Thus, our findings reveal an important role of local lymphatic drainage in periodontal inflammation‐mediated alveolar bone loss. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Calcium modulates bone cell recruitment, differentiation, and function by binding to the calcium‐sensing receptor (CaSR). However, the function of CaSR induced by high extracellular calcium (Ca2+e) in the regulation of osteoclast formation in rheumatoid arthritis (RA) remains unknown. Here, we used TNFα‐transgenic (TNFTG) RA mice and their wildtype (WT) littermates fed a normal or a rescue diet (high calcium, high phosphorus, and high lactose diet, termed rescue diet) to compare their joint bone phenotypes. In comparison to TNFTG mice fed the normal diet, articular bone volume and cartilage area are increased, whereas inflamed area, eroded surface, TRAP+ surface, and osteoclast‐related genes expression are decreased in TNFTG mice fed the rescue diet. Besides, TNFTG mice fed the rescue diet were found to exhibit more CaSR+ area and less NFATc1+/TRAP+ area. Furthermore, at normal Ca2+e concentrations, osteoclast precursors (OCPs) from TNFTG mice formed more osteoclasts than OCPs from WT mice, but the number of osteoclasts gradually decreased when the Ca2+e concentration increased. Meanwhile, the expression of CaSR increased responding to a high level of Ca2+e, whereas the expression of NF‐κB/NFATc1 signaling molecules decreased. At last, the knockdown of CaSR blocked the inhibition of osteoclast differentiation attributed to high Ca2+e. Taken together, our findings indicate that high Ca2+e inhibits osteoclast differentiation in RA mice partially through the CaSR/NF‐κB/NFATc1 pathway.

NLRP3 has been involved in several physiological and pathological processes. However, the role and mechanism of NLRP3 activation in mandibular healing remain unclear. Here, a full-thickness mandibular defect model by osteotomy was established in wild-type (WT) and Prx1-Cre/ROSA nTnG mice to demonstrate the NLRP3 inflammasome activation in mandibular healing. We found that NLRP3 was activated in mesenchymal stem cells (MSCs)-mediated mandibular healing and was prominent in Prx1 + cells. Inhibition of NLRP3 exerted a positive effect on bone formation without changing the number of Prx1-cre + cells in the defect areas. In addition, NLRP3 deficiency promoted osteoblast differentiation. We next screened for the deubiquitinating enzymes that were previously reported to be associated with NLRP3, and identified UCHL5 as a regulator of NLRP3 activation in mandibular healing. Mechanistically, NLRP3 directly bound to UCHL5 and maintained its stability through reducing ubiquitin-proteasome pathway degradation in mandibular MSCs. At last, UCHL5 inhibition enhanced osteoblast differentiation by promoting NLRP3 ubiquitination and degradation. Thus, our results provided the proof that NLRP3 acted as a negative modulator in mandibular healing and extended the current knowledge regarding posttranslational modification of NLRP3 by UCHL5.

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