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Single-cell sequencing unveils the heterogeneity of nonimmune cells in chronic apical periodontitis

ABSTRACT: Chronic apical periodontitis (CAP) is a unique dynamic interaction between microbial invasions and host defense mechanisms, resulting in bone absorption, infiltration of immune cells and sporadic periapical granuloma. In this study, we constituted a single-cell atlas for 26,737 high-quality cells from hyperplastic periapical tissue using single-cell RNA sequencing. Identifying cell types and signatures at the single-cell level might generate novel insights into the clinical pathogenesis of CAP. A histological analysis to verify the gene signatures of nonimmune cells was combined with immunohistochemistry staining. We then discovered the diversity and heterogeneity of nonimmune cells in regional CAP lesions. The temporal profiling of genomic alterations from common CAP to typical periapical granuloma provided predictions for key transcription factors and biological processes. Our study also inferred that the marked shift of inflammatory cytokines, chemokines, proteases and growth factors enables the initiation of polymorphic cell differentiation, lymphangiogenesis and angiogenesis during CAP.

ORGANISM(S): Homo sapiens

PROVIDER: GSE181688 | GEO | 2021/08/31

REPOSITORIES: GEO

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Project description:Apical periodontitis (AP) is a painful inflammatory disease resulting from tooth infection that affects millions worldwide with a marked impact on quality of life and is accompanied by bone loss surrounding the affected tooth. There is growing evidence that the nociceptive fibers densely innervating teeth regulate the disease progression, in addition to their sensorial function. We hypothesized that nociceptors regulate the transcriptomic profile of the periapical osteolytic lesion in a mouse model of AP. Male control (Nav1.8 cre+/-) and nociceptor-ablated (Nav1.8cre+/- DTAlox+/-) mice were generated and underwent pulp exposure procedures on all 4 first molars and at 8 weeks of age. At either 0, 7, or 14 days after pulp exposure, periapical tissues were dissected from mice (n=3-4/strain/time point). Total RNA was extracted from the pooled periapical lesions from each animal and submitted for total RNA sequencing and bioinformatic analysis. We found that pulp exposure triggers the differential expression of hundreds of genes within the periapical lesion over the course of infection with marked differences between in both control and mice with nociception ablation. Importantly, at 14 days post pulp-exposure, 422 genes were differentially expressed between nociceptor-ablated and control mice with greater enrichment of biological processes related to inflammation, specifically immune cell chemotaxis and migration, compared to control mice. Among these inflammatory markers, TNFα, IL-1α, and IL-1β, that are known to play a crucial role in AP were significantly upregulated in nociceptor-ablated mice. In conclusion, nociceptor-ablation regulates the transcriptomic profile of periapical lesions in a mouse model of AP, shifting the gene expression profile to a greater enrichment of inflammatory genes, suggesting nociceptors play a role in the kinetics of the immune response. This newly uncovered neuro-immune axis and its mechanisms in AP can potentially be an important therapeutic target for the treatment of this prevalent disease.

Project description:OBJECTIVES: To detect the expression levels of MLKL and p-MLKL, and explore its potential roles in inflammatory cell infiltration, angiogenesis, and bone destruction, in human and mouse periapical lesions. METHODS: Forty-six human periapical tissues, including periapical granulomas (PGs, n =26), radicular cysts (RCs, n =20), and eight healthy control tissues were collected. Samples were fixed and analyzed by HE staining, RNA-Seq, western blot, and immunohistochemical staining. A periapical lesion mouse model was induced by pulp exposure in the first lower molars of 15 C57BL/6J mice. After lesion induction, the mice were sacrificed on days 0, 21, and 35. Mandibles were harvested for microcomputed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. Double immunofluorescence was utilized to assess the colocalization of phosphorylated MLKL (p-MLKL) with CD34, matrix metalloproteinase-9 (MMP-9), and Cathepsin K (CTSK) in human and mouse periapical lesions. RESULTS: RNA-Seq analysis showed that, in comparison with healthy gingiva tissues, MLKL was more significantly upregulated in periapical lesions (P-value < 0.05). Immunohistochemistry staining showed that, MLKL and p-MLKL were significantly overexpressed in the RC and PG groups compared with the control group (P-value < 0.05). However, the difference between the RC and PG groups was insignificant (P-value > 0.05). p-MLKL-positive cells were mainly lymphocytes, epithelial cells, and endothelial cells around the vascular wall. In mouse periapical lesions, the expression levels of p-MLKL were positively correlated with the bone defect area and tartrate-resistant acid phosphatase-positive (TRAP+) cell amounts (R2=0.4108, P-value < 0.05; R2=0.5668, P-value < 0.05, respectively). The double-labeling analysis showed that p-MLKL colocalized with CD34 and MMP-9 in human samples, and with CTSK adjacent to the bone in mouse periapical lesions. CONCLUSION: MLKL and p-MLKL were overexpressed in human periapical lesions. p-MLKL exhibited a close relationship with angiogenesis and alveolar bone resorption.

Project description:Cardiac involvement is an important determinant of mortality amongst sarcoidosis patients. While granulomatous inflammation is a hallmark finding in cardiac sarcoidosis, the precise immune cell populations that comprise the granuloma remain unresolved. Furthermore, it is unclear how the cellular and transcriptomic landscape of cardiac sarcoidosis differs from other inflammatory heart diseases. We leveraged spatial transcriptomics (GeoMx DSP) and single nucleus RNA sequencing (snRNAseq) to elucidate the cellular and transcriptional landscape of cardiac sarcoidosis. Using GeoMX DSP technology, we compared the transcriptomal profile of CD68+ rich immune cell infiltrates in human cardiac sarcoidosis, giant cell myocarditis, and lymphocytic myocarditis. We performed snRNAseq of human cardiac sarcoidosis to identify immune cell types and examined their transcriptomic landscape and regulation. Using multi-channel immunofluorescence staining, we validated immune cell populations identified by snRNAseq, determined their spatial relationship, and devised an immunostaining approach to distinguish cardiac sarcoidosis from other inflammatory heart diseases. Despite overlapping histological features, spatial transcriptomics identified transcriptional signatures and associated pathways that robustly differentiated cardiac sarcoidosis from giant cell myocarditis and lymphocytic myocarditis. snRNAseq revealed the presence of diverse populations of myeloid cells in cardiac sarcoidosis with distinct molecular features. We identified GPNMB as a novel marker of multinucleated giant cells and predicted that the MITF family of transcription factors regulated this cell type. We also detected additional macrophage populations in cardiac sarcoidosis including HLA-DR+ macrophages, SYTL3+ macrophages and CD163+ resident macrophages. HLA-DR+ macrophages were found immediately adjacent to GPMMB+ giant cells, a distinct feature compared with other inflammatory cardiac diseases. SYTL3+ macrophages were located scattered throughout the granuloma and CD163+ macrophages, CD1c+ dendritic cells, non-classical monocytes, and T-cells were located at the periphery and outside of the granuloma. Finally, we demonstrate mTOR pathway activation is associated with proliferation and is selectively found in HLA-DR+ and SYLT3+ macrophages. In this study, we identified diverse populations of immune cells with distinct molecular signatures that comprise the sarcoid granuloma. These findings provide new insights into the pathology of cardiac sarcoidosis and highlight opportunities to improve diagnostic testing.

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Single-cell sequencing unveils the heterogeneity of nonimmune cells in chronic apical periodontitis

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