Background/Purpose: Neuropsychiatric manifestations are observed in 20-40% of systemic lupus erythematosus patients, including cognitive deficits and affective behaviors such as anxiety and depression. Neuropsychiatric lupus (NPSLE) involves complex mechanisms that are not yet completely understood. Inflammatory mediators such as cytokines and brain-specific autoantibodies, together with compromised brain barriers including the blood brain barrier and the blood-cerebrospinal fluid barrier (formed by the choroid plexus) as the site of entry of these mediators, are thought to play an important pathogenic role.  Furthermore, a leukocytic infiltration into the brain is observed in both mouse NPSLE models and in some human NPSLE patients, and the resolution of this infiltration with treatment correlates with improved neuropsychiatric manifestations. Additional characterization of the choroid plexus and its cellular infiltrate was undertaken to better understand the potential contributions of B cells, T cells, and the choroid plexus to NPSLE.

Methods: The choroid plexus from 17-week old MRL/lpr mice (n=3) was excised and digested with 0.5% trypsin-EDTA. The single cell suspension was stained and subsequently sorted for live cells. Single cell immune profiling was performed with the 10X Genomics platform and the reads were sequenced with Illumina High-Seq instrument. The transcripts were aligned using reference genome, mm10, using the CellRanger pipeline (10X Genomics). The Seurat R package and the 10X Genomics Loupe Browser were used for analysis.

Results: MRL/lpr choroid plexuses clustered into 18 unique cell clusters as depicted in Figure 1A. Preliminary characterization revealed four T cell clusters and multiple B cell subsets including germinal center B and plasma cells. Furthermore, the top 5 significant markers for each cluster is depicted by a heatmap in Figure 1B. Previous bulk RNA-sequencing revealed the upregulation of a number of germinal center-associated genes in the MRL/lpr choroid plexus including chemokines such as Cxcl9, Cxcl10, Cxcl11, and Cxcl13. We found that border-associated macrophages (BAM) and the additional macrophage cluster were the predominant sources of recruiting chemokines, particularly Cxcl9. Correspondingly, immune cells infiltrating the choroid plexus including T cells, B cells, and myeloid cells expressed the cognate receptors, Cxcr3 and Cxcr5. Further profiling of the T and B cells revealed the top BCR and TCR clones in the plasma cell and T cell clusters. The top BCR clones formed IgG2c and IgA antibodies and ranged between 15.3% to 41.1% of all B cells present in the libraries.

Conclusion: The extensive immune infiltration observed in the choroid plexus of neuropsychiatric lupus mice are potential contributors in the pathogenesis of NPSLE. Preliminary analysis reveals that border-associated macrophages contribute to the recruitment of immune cells into the choroid plexus by secreting CxCl9. Understanding the heterogeneity of brain resident and infiltrating immune cells and their transcriptomes may help identify potential therapeutic targets in NPSLE.

Figure 1: A) Uniformed Manifold Approximation and Projection of three 17 week old MRL/lpr choroid plexuses. B) Heatmap of top 5 significant markers in each cluster. C) Dot plot demonstrating both percentage and average expression of recruiting chemokine and cognate receptor expression. CP EPI = choroid plexus epithelial cells, Treg = T regulatory cells, GC B = germinal center B cells, Fibro = stromal fibroblast-like cells, BAM = border-associated macrophages, Endo = endothelial cells, cDC = circulating dendritic cells, Prolif B = proliferating B cells, Peri = pericytes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/border-associated-macrophages-drive-recruitment-of-immune-cells-into-the-choroid-plexus-in-mice-with-neuropsychiatric-lupus/