Extracellular Vesicles Derived from Human Umbilical Cord Mesenchymal Stem Cells Protect Liver Ischemia/Reperfusion Injury by Reducing CD154 Expression on CD4+ T Cells via CCT2

Abstract As a cause of postoperative complications and early hepatic failure after liver transplantation, liver ischemia/reperfusion injury (IRI) still has no effective treatment during clinical administration. Although the therapeutic potential of mesenchymal stem cells (MSCs) for liver IRI has been previously shown, the underlying mechanisms are not completely clear. It is accepted that MSC‐derived extracellular vesicles (MSC‐EVs) are newly uncovered messengers for intercellular communication. Herein, it is reported that umbilical cord‐derived MSCs (UC‐MSCs) improve liver IRI in mice through their secreted EVs. It is also visualized that UC‐MSC‐EVs mainly concentrate in liver after 6 h of reperfusion. Furthermore, UC‐MSC‐EVs are found to significantly modulate the membranous expression of CD154 of intrahepatic CD4+ T cells, which is an initiation of inflammatory response in liver and can aggravate liver IRI. Mechanistically, protein mass spectrum analysis is performed and it is revealed that Chaperonin containing TCP1 subunit 2 (CCT2) enriches in UC‐MSC‐EVs, which regulates the calcium channels to affect Ca2+ influx and suppress CD154 synthesis in CD4+ T cells. In conclusion, these results highlight the therapeutic potential of UC‐MSC‐EVs in attenuating liver IRI. This finding suggests that CCT2 from UC‐MSC‐EVs can modulate CD154 expression of intrahepatic CD4+ T cells during liver IRI through the Ca2+‐calcineurin‐NFAT1 signaling pathway.

To identify the characterizations of UC-MSCs and UC-MSCs shCCT2 , the potentials of adipogenesis and osteogenesis were also detected to assess their multipotential differentiation according to previously described. [2] Harvest of UC-MSC conditioned medium and EV-depleted conditioned medium Complete conditioned medium (CM) from UC-MSC (UC-MSC-CM) was prepared as previous described. [3] In brief, since grown to 70-80%, the cells were washed twice with PBS and cultured in low-glucose DMEM with 10% exosome-depleted FBS for another 48 h. The UC-MSC-CM was harvested and centrifugated at 3,200 g 4℃ for 10 min to remove debris. To collect EV-depleted condition medium from UC-MSC (UC-MSC-ECM), the UC-MSC-CM was ultra-centrifugated at 100,000 for 2 h to deplete human EVs. [1] Finally, the supernatant was obtained and filtered through a filter (0.22 μm).

Transmission Electron Microscope
The size and morphology of UC-MSC-EVs were detected by transmission electron microscope (TEM). The specimens were prepared by placing UC-MSC-EVs on a copper mesh and stained with 3% phosphotungstic acid (Ph 7.0) at 2 min. The specimens were observed and photographed under a H-7650 TEM (HITACHI High-Technologies Corp., Tokyo, Japan) at an accelerating voltage of 80 kV.

Detection of liver function
Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH), which are indicators of liver injury, were measured by a 7180 Biochemical Analyzer (Hitachi, Japan).

Histological evaluation of liver section
4 μm thick liver paraffin sections were prepared and stained with hematoxylin-eosin (H&E) to determine liver injury. The severity of IRI in liver tissues was scored blindly according to the Suzuki's criteria which was classified 0-4 scale. [4] The scale was evaluated by three pathological indicators, including hepatocyte necrosis, ballooning degeneration and sinusoidal congestion. No centrilobular ballooning, hepatocellular necrosis and congestion is given 0, while the area of necrosis >60% and severe ballooning degeneration and congestion is given 4.

Enzyme-Linked Immunosorbent Assay (ELISA)
The concentrations of IFN-γ and TNF-α in serum and liver tissue in vivo study and in supernatant in vitro study were measured by enzyme-linked immunosorbent assay kits following to the manufacturer's protocols (EIAAB Science Company, Wuhan, China). The optical density (OD) was determined at 450 nm by an automatic microplate reader (Biotek Vermont, USA).
The membranes were immersed in 5% non-fat milk on oscillating table at room temperature for 1 h to block non-specific antigen and respectively treated with the primary antibodies at 4 ℃ overnight. Subsequently, the membranes were incubated with the secondary antibody (anti-rabbit IgG, 1:5000, Sigma-Aldrich) at room temperature for 1 h. After treated with an enhanced chemiluminescence (ECL) substrate, the blots were visualized by a FluorChem System imager (ProteinSimple, CA, USA). The intensities of the blots were analyzed using an image analyzer (ImageJ software, USA).

Total RNA extraction and quantitative real-time polymerase chain reaction (RT-qPCR)
Total RNA extraction and qRT-PCR were performed according to the previous described. [5] In brief, total RNA extractions from the both UC-MSCs and liver tissue were used TRIzol (Invitrogen) following to the manufacturer's instruction.
After checking the amount and purity of total RNA according to the absorbance at 260 nm and 280 nm wavelengths using Biophotometer plus (Eppendorf, Germany), cDNA was reverse transcribed using Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, USA). cDNA amplification was performed with heating at 65℃ for 10 min firstly, incubating at 55℃ for 30min sequentially, then deactivating at 85℃ for 5min and storage at 4℃ 5 min, at last, using the PCR Thermal Cycler (Bio-Rad, USA). Finally, RT-PCR was performed with Roche Applied Science SYBR Master Mix using reverse transcription system (LC-480, Roche, USA). β -actin, as a housekeeping gene, were used to normalization. Specific primer sequences used to amplify in this study are listed in Supplemental Table 1.

Gene Symbol Sequence direction Sequence
Mouse-IFN-γ performed as previous described. [6] In simple terms, protein sample was suspended in Liquid Chromatograph coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific, USA) was conducted for 120 min. Peptides were separated on a RP-C18 analytical column at a flow rate of 300 nl/min over 120 min. After the capillary separation, digested samples were analyzed using a Q Exactive mass spectrometer. interested by groups, and the interesting expressed proteins were used for bioinformatics analysis. Gene Ontology (GO) and KEGG were annotated and enriched with the R-package 'clusterprofier' [7] , and Reactome was annotated and enriched with the R-package 'ReactomePA' [34] . For functional annotation and enrichment analysis of Biocarta, Biocarta database was extracted from the R-package 'pathfinder' [35] and analyzed using 'clusterProfier' package`.

Supplemental Figure 2. CD4+ T cells are pivotal for the initiation of inflammatory response in liver IRI
Mice underwent liver IRI were pre-treated with CD4 depleting Ab (GK1.5) and anti-IgG antibody (as control), and then were sacrificed at 6 h after reperfusion.