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CaSSiDI: novel single-cell "Cluster Similarity Scoring and Distinction Index" reveals critical functions for PirB and
Robert Nechanitzky1,2, Parameswaran Ramachandran3, Duygu Nechanitzky3
1Princess Margaret Cancer Centre, Ontario Cancer Institute, University Health Network, Toronto, ON, Canada. Robert.Nechanitzky@utoronto.ca.
Abstract:
PirB is an inhibitory cell surface receptor particularly prominent on myeloid cells. PirB curtails the phenotypes of activated macrophages during inflammation or tumorigenesis, but its functions in macrophage homeostasis are obscure. To elucidate PirB-related functions in macrophages at steady-state, we generated and compared single-cell RNA-sequencing (scRNAseq) datasets obtained from myeloid cell subsets of wild type (WT) and PirB-deficient knockout (PirB KO) mice. To facilitate this analysis, we developed a novel approach to clustering parameter optimization called "Cluster Similarity Scoring and Distinction Index" (CaSSiDI). We demonstrate that CaSSiDI is an adaptable computational framework that facilitates tandem analysis of two scRNAseq datasets by optimizing clustering parameters. We further show that CaSSiDI offers more advantages than a standard Seurat analysis because it allows direct comparison of two or more independently clustered datasets, thereby alleviating the need for batch-correction while identifying the most similar and different clusters. Using CaSSiDI, we found that PirB is a novel regulator of Cebpb expression that controls the generation of Ly6Clo patrolling monocytes and the expansion properties of peritoneal macrophages. PirB's effect on Cebpb is tissue-specific since it was not observed in splenic red pulp macrophages (RPMs). However, CaSSiDI revealed a segregation of the WT RPM population into a CD68loIrf8+ "neuronal-primed" subset and an CD68hiFtl1+ "iron-loaded" subset. Our results establish the utility of CaSSiDI for single-cell assay analyses and the determination of optimal clustering parameters. Our application of CaSSiDI in this study has revealed previously unknown roles for PirB in myeloid cell populations. In particular, we have discovered homeostatic functions for PirB that are related to Cebpb expression in distinct macrophage subsets.
Insights
PirB receptor deficiency impacts macrophage homeostasis by altering Cebpb expression, affecting monocyte generation and expansion. A new computational tool, CaSSiDI, enabled this discovery by optimizing single-cell RNA sequencing analysis.
Area of Science:
- Immunology
- Computational Biology
- Genetics
Background:
- PirB is an inhibitory receptor on myeloid cells, known to regulate inflammation and tumorigenesis.
- Its specific role in maintaining macrophage homeostasis at steady-state remains largely unexplored.
Purpose of the Study:
- To investigate the functions of PirB in macrophage homeostasis using single-cell RNA sequencing (scRNAseq).
- To develop and validate a novel computational framework, CaSSiDI, for optimizing scRNAseq data analysis and comparison.
Main Methods:
- Generation and comparative analysis of scRNAseq datasets from wild-type and PirB-deficient mice.
- Development and application of the Cluster Similarity Scoring and Distinction Index (CaSSiDI) for parameter optimization and cross-dataset comparison.
- Analysis of myeloid cell subsets, focusing on PirB's role in Cebpb expression and macrophage populations.
Main Results:
- CaSSiDI effectively optimizes clustering parameters and facilitates direct comparison of independent scRNAseq datasets without batch correction.
- PirB deficiency alters Cebpb expression, impacting Ly6C-low patrolling monocyte generation and peritoneal macrophage expansion.
- PirB's influence on Cebpb is tissue-specific, with distinct subpopulations identified in splenic red pulp macrophages.
Conclusions:
- CaSSiDI is a valuable tool for scRNAseq analysis, improving the identification of distinct cell populations and comparative studies.
- PirB plays a previously unrecognized role in myeloid cell homeostasis, regulating Cebpb expression and influencing specific macrophage subsets.
