Prolyl endopeptidase remodels macrophage function as a novel transcriptional coregulator and inhibits fibrosis

Macrophages are immune cells crucial for host defense and homeostasis maintenance, and their dysregulation is involved in multiple pathological conditions, such as liver fibrosis. The transcriptional regulation in macrophage is indispensable for fine-tuning of macrophage functions, but the details have not been fully elucidated. Prolyl endopeptidase (PREP) is a dipeptidyl peptidase with both proteolytic and non-proteolytic functions. In this study, we found that Prep knockout significantly contributed to transcriptomic alterations in quiescent and M1/M2-polarized bone marrow-derived macrophages (BMDMs), as well as aggravated fibrosis in an experimental nonalcoholic steatohepatitis (NASH) model. Mechanistically, PREP predominantly localized to the macrophage nuclei and functioned as a transcriptional coregulator. Using CUT&Tag and co-immunoprecipitation, we found that PREP was mainly distributed in active cis-regulatory genomic regions and physically interacted with the transcription factor PU.1. Among PREP-regulated downstream genes, genes encoding profibrotic cathepsin B and D were overexpressed in BMDMs and fibrotic liver tissue. Our results indicate that PREP in macrophages functions as a transcriptional coregulator that finely tunes macrophage functions, and plays a protective role against liver fibrosis pathogenesis.


RNA-seq library preparation and sequencing
Total RNA was isolated via TRIzol reagent according to the manufacturer's protocol and used for subsequent RNA-seq analysis. 500 ng RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were subsequently constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. Paired-end sequencing was carried out with the Illumina NovaSeq 6000 with read length of 150 bp. About 50 million raw reads for each sample were generated.
The bedGraph files containing signal per million reads were then compared to derive differential ChIP-seq peaks by using command bdgdiff from macs2. Command plotProfile and plotHeatmap in deeptools were used to visualize the H3K27ac and PU.1 distribution over regions centered by H3K27ac and PU.1 peaks respectively.

scRNA-seq data analysis pipeline
scRNA-seq data of liver from mice on either standard diet or western diet were generated by a previous research 2 (GSE192742) and are available for additional analysis and download at www.livercellatlas.org. The preprocessed data with annotated cell-identity information generated by the original research were downloaded at www.livercellatlas.org. DotPlot made by package Seurat (4.3.0) was used to visualize the gene expression pattern in different cell clusters.

Serum biochemical analysis
The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by ALT activity assay kit (MAK052, Sigma Aldrich) and AST activity assay kit (MAK055, Sigma Aldrich), respectively.

RT-qPCR
Total RNA was isolated via TRIzol reagent according to the manufacturer's protocol.
RNA was reverse transcribed to cDNA using the PrimeScript™ RT Master Mix kit (TaKaRa, RR036A). The cDNA was diluted 10 times and used as RT-qPCR template.
cDNA was amplified and quantified in QuantStudio3 Real-Time PCR system using the qPCR SYBR® Green Master Mix (Yeasen, 11199ES08). The relative expression levels

Immunoblotting and immunoprecipitation
The liver tissue samples were homogenized in RIPA buffer containing 1% PMSF. The protein samples were separated on 10% SDS-PAGE gel, and electroblotted onto PVDF membranes. The membranes were blocked for 1 h at RT in 5% w/v non-fat dry milk in TBST buffer, and incubated overnight at 4°C with the following primary antibodies: For PREP immunoprecipitation, cells were lysed in cold RIPA buffer supplemented with Protease inhibitor cocktail (Beyotime, P1010), sonicated and centrifuged.
Supernatants were then incubated with primary antibody of PREP (Abcam, ab58988) for 1 hour at RT. Protein A magnetic beads (Beyotime, P2102) were added and rotated at RT for 1 hour. The beads were then washed 3 times with TBST, resuspended in reducing SDS gel loading buffer, and heated at 95º C for 10 minutes. The immunoprecipitated proteins were subsequently analyzed by western blotting with the primary antibodies for PREP (Abcam, ab58988) and PU.1 (Abcam, ab227835).

Histological analysis
For general histological pathology and fibrosis detection, formalin-fixed liver tissue samples were embedded in paraffin and 4 μm sections and stained with Hematoxylin and Eosin (HE) and Sirius Red according to standardized protocols. Images were captured by using Olympus BX51 microscope. Quantification of Sirius red positive staining of liver sections was performed by using ImageJ (v1.53c).
For histological immunofluorescence, paraffin-embedded mouse liver tissue sections were deparaffinized and rehydrated, followed by antigen retrieval in citrate solution for 15 min. Then the sections were covered with 5% BSA in PBS at room temperature (RT) for 30 min before antibody incubation.