Single Cell RNAseq Analysis of Dendritic Cells in Experimental Periodontitis

Chintan Thakore

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Thakore, Chintan¹, Liu, Min¹, Zhou, Mi¹, Gonzalez², Michael, Garifallou, James², Graves, Dana, T^1
1University of Pennsylvania School of Dental Medicine, Department of Periodontics
²Children's Hospital of Philadelphia, Center for Applied Genomics

Introduction

We tested the hypothesis that the transcription factor FOXO1 plays a key role in the regulation of dendritic cells (DC) in experimental periodontitis in vivo.

Methods

Mice with deletion of FOXO1 in dendritic cells (CD11c.Cre+.FOXO1LL) and matched wild type mice had periodontitis induced by oral inoculation of P. gingivalis and F. nucleatum. After 4 weeks, CD45+ cells were isolated from the gingiva by FACS sorting and subjected to 10x Genomics single cell RNAseq. Transcriptome clustering was accomplished by R/Seraut software and dendritic cell clusters were examined by gene set enrichment analysis (GSEA). Additionally, in vitro DCs from the bone marrow of FOXO1 deleted mice and wild type mice were incubated with P. gingivalis, RNA isolated and examined by RNA-seq and GSEA. P-values and false discovery rates were set at P<0.01 and FDR<25%.

Results

R/Seraut clustering using nearest neighbor unsupervised statistical analysis identified 6 dendritic cell clusters based on their transcriptome profile. In all of the 6 clusters, FOXO1 deletion reduced pathways related to dendritic cell-stimulated immunity, migration of DCs, response to wounding and inflammation (FDR<25%; P-value<0.01). In five of the six clusters, FOXO1 deletion reduced DC activity related to activation of T- or B-cells (FDR<25%; P-value<0.01). In vitro analysis showed that FOXO1 deletion reduced transcripts regulating DC migration and DC stimulation of B- and T-cells (FDR<25%; P-value<0.01).

Conclusion

The impact of FOXO1 deletion on the dendritic cell transcriptome in vivo agrees well with studies demonstrating that FOXO1 reduces DC migration and lymphocyte activation in vitro.