bims-nastce 2022-03-06 papers

Methods Mol Biol. 2022 ;2419 765-778

Single-Cell Antibody Sequencing in Atherosclerosis Research.

Ryosuke Saigusa, Christopher P Durant, Vasantika Suryawanshi, Klaus Ley.

The transcriptomic information obtained by single cell RNA sequencing (scRNA-seq) can be supplemented by information on the cell surface phenotype by using oligonucleotide-tagged monoclonal antibodies (scAb-Seq). This is of particular importance in immune cells, where the correlation between mRNA and cell surface expression is very weak. scAb-Seq is facilitated by the availability of commercial antibodies and antibody mixes. Now panels of up to 200 antibodies are available for human and mouse cells. Proteins are detected by antibodies conjugated to a tripartite DNA sequence that contains a primer for amplification and sequencing, a unique oligonucleotide that acts as an antibody barcode and a poly(dA) sequence, simultaneously detecting extension of antibody-specific DNA sequences and cDNAs in the same poly(dT)-primed reaction. For each cell, surface protein expression is captured and sequenced along with the cell's transcriptome. Here, we list the steps needed to produce antibody sequencing data from tissue or blood cells.

Keywords: AbSeq; Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq); RNA expression and protein sequencing assay (REAP-seq); Surface markers; TotalSeq

DOI: https://doi.org/10.1007/978-1-0716-1924-7_46

STAR Protoc. 2022 Mar 18. 3(1): 101178

LiverClear: A versatile protocol for mouse liver tissue clearing.

Laura M Molina, Yekaterina Krutsenko, Nathaniel E C Jenkins, Megan C Smith, Junyan Tao, Travis B Wheeler, Simon C Watkins, Alan M Watson, Satdarshan P Monga.

Although there are numerous tissue clearing protocols, most are inadequate for clearing liver tissue. Here we present a flexible protocol for mouse liver tissue; we combine strategies from several previously published protocols for delipidation, decolorization, staining, and refractive index matching. LiverClear is sufficiently versatile to allow clearing of healthy and diseased mouse liver followed by immunofluorescence staining and imaging to visualize intact 3D structures such as bile ducts and hepatocyte canaliculi. We also adapted this protocol for clearing human livers. For complete details on the use and execution of this protocol, please refer to Molina et al. (2021).

Keywords: Developmental biology; Microscopy

DOI: https://doi.org/10.1016/j.xpro.2022.101178

Int Immunopharmacol. 2022 Feb 23. pii: S1567-5769(22)00123-0. [Epub ahead of print]107 108639

Targeted inhibition of the immunoproteasome blocks endothelial MHC class II antigen presentation to CD4+ T cells in chronic liver injury.

Yuwei Zhang, Xue Yang, Tao Bi, Xia Wu, Lu Wang, Yafeng Ren, Yangying Ou, Chengliang Xie, Kuangjie Li, Haolong Ran, Jing Wang, Fulan Zhao, Pixian Shui, Jie Qing.

Chronic or overwhelming liver injury is frequently associated with fibrosis, which is the main histological characteristic of non-alcoholic steatohepatitis (NASH). Currently, there is no effective treatment for liver fibrosis. Adaptive immunity is one of the perpetrators of liver inflammation and involves the antigen-specific activation of lymphocytes. Targeting adaptive immunity has been proposed as a novel therapeutic approach for NASH. In this study, we demonstrated that liver endothelial cells contribute to MHC class II (MHC-II) antigen presentation to CD4+ T cells after chronic liver injury. In human cirrhotic liver samples, we observed an increased expression of endothelial MHC-II and of the antigen presentation-associated protein LMP7, which is one of the proteolytically active subunits of the immunoproteasome. In a CCl4-induced chronic injury model or a diet- and chemical-induced NASH model, endothelial MHC-II and LMP7 expression was induced to increase. PR-957, a selective inhibitor of the immunoproteasome, inhibited MHC-II expression in endothelial cells and CD4+ T cell response after chronic liver injury. In vitro experiment demonstrated PR-957 also reversed IFN-γ-induced upregulation of MHC-II in endothelial cells. Furthermore, PR-957 treatment or CD4+ T cell depletion in chronic liver injury alleviated liver fibrosis and reduced inflammation, as indicated by the downregulation of inflammatory response markers (F4/80, IL-1, IL-6 and IL-18). In conclusion, targeted inhibition of the immunoproteasome blocks endothelial MHC-II antigen presentation to CD4+ T cells in chronic liver injury. In this regard, the PR-957 inhibitor is a promising candidate for the development of future therapies against NASH.

Keywords: Antigen presentation; Endothelial cell; Immunoproteasome; Liver fibrosis; PR-957; non-alcoholic steatohepatitis (NASH)

DOI: https://doi.org/10.1016/j.intimp.2022.108639