ITGAM+S100A9+ Extracellular Vesicles Derived From Innate Immune Cells Promoted CD8 T cell Activation Via Mitochondrial Metabolic Regulation In Acute Kidney Injury

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ITGAM+S100A9+ Extracellular Vesicles Derived From Innate Immune Cells Promoted CD8 T cell Activation Via Mitochondrial Metabolic Regulation In Acute Kidney Injury

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Co-author 1

Yuqi FU shahcdimash@163.com Southeast University School Institute of Nephrology Nanjing China *

Co-author 2

Anran Shen 230229056@seu.edu.cn Southeast University School Institute of Nephrology Nanjing China -

Co-author 3

Yan Bao baoyannn@163.com Southeast University School Institute of Nephrology Nanjing China -

Co-author 4

Bicheng Liu liubc64@163.com Southeast University School Institute of Nephrology Nanjing China -

Co-author 5

Linli Lv lvlinli@seu.edu.cn Southeast University School Institute of Nephrology Nanjing China -

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Introduction

This study aims to characterize EV subpopulations from renal tissue after acute kidney injury (AKI) and delineate the contributions of principle EVs subsets to AKI pathogenesis.

Methods

Single-vesicle proteomic analysis using proximity barcoding assay (PBA) was applied to characterize renal tissue EVs in a murine ischemia-reperfusion injury (IRI) model. Differential proteomic and enrichment analyses revealed distinct EV subpopulations following validation. Biological function of principle EV subset was traced and investigated in EV tracing mouse model of ITGAM-Cre;CAG-CD63-PAGFP(CD63-PAGFPITGAM/KI). The molecular mechanism was then explored through multiple proteomics analysis and validation study.

Results

Single-vesicle proteomic analysis revealed a significant expansion of immune cell-derived EVs at day 1 after AKI, with macrophages, dendritic cells, and neutrophils accounting for the predominant proportions, followed by T cells and B cells. Matrix profiling delineated eight distinct EV subpopulations, wherein ITGAM/HAVCR1-expressing EVs constituted 33.63% of the total EVs. Impressively, differential proteomics and pathway analysis identified ITGAM as the central mediator orchestrating T cell cytotoxicity, antigen presentation, and immune response amplification, which underscores the significance of ITGAM+ EV subsets.

ITGAM+EVs were further purified from EVs mixture from renal tissue of IRI mice through immunomagnetic enrichment, subsequently characterized via nanoscale flow cytometry, revealing dendritic cells (CD11c+, 11.7%), macrophages (F4/80+, 7.68%), and neutrophils (Ly6G+, 3.09%) as the main source of this EV subpopulation. Functional study demonstrated that IRI kidney-derived ITGAM+ EVs promoted CD8+ T cell expansion with elevated TNF-α and IFN-γ production as well as suppressed IL-10. Proteomic analysis of ITGAM+EVs identified S100A9 as the top differential protein enriched in IRI kidney-derived ITGAM+EVs compared to that from healthy kidney. S100A9 neutralization reversed the activation of T cells induced by ITGAM+ EVs. To demonstrate the role of ITGAM+EVs in vivo, CD63-PAGFPITGAM/KI transgenic mice were constructed. It was observed that ITGAM+ PA-GFP+ EV production was enhanced, which colocalized with CD8+ T cells around renal lymphatics as demonstrated by monitoring live kidney sections, resulting in amplified renal inflammation and CD8+ T cell activation following AKI.

Further study identified mitochondrial SLC25A12 as the protein interacting with S100A9 on EV surfaces through IP-MS screening and validation. Correspondingly, S100A9 inhibitor and SLC25A12 siRNA treatment ameliorated T cell activation induced by ITGAM+ EVs. Interestingly, S100A9 carried by ITGAM+ EV coordinately enhanced mitochondrial aspartate/glutamate transportation by SLC25A12, promoting mitochondrial metabolic reprogramming and ultimately augmented CD8+ T cell activation. Impressively, S100A9 inhibitors ameliorated CD8+ T cell activation, accompanied by reduced mitochondrial SLC25A12 function after IRI model in CD63-PAGFPITGAM/KI transgenic mice.

Figure 1. Overview of the AKI immune microenvironment: Renal ischemia-reperfusion injury model in mice, with comprehensive profiling of AKI-associated immune microenvironment using proximity barcoding assay of extracellular vesicles marked by distinct surface markers Figure 2. Single-EV analysis identified eight major and distinct EV subpopulations; among them, the most prominently expressed ITGAM+ EV subset was validated by nanoparticle flow cytometry for cellular origin tracing and further confirmed by tissue localization assays. Figure 3. Proteomic analysis of EV membrane proteins shows that ITGAM is the most significantly different protein, primarily involved in immune regulation, T cell cytotoxicity, and antigen presentation pathways.Immunomagnetic isolation of ITGAM+ EVs. IRI-derived ITGAM+ EVs can promote T cell proliferation and enhance the secretion of cytokines such as TNF-α Figure 4. Construction and identification of ITGAM-CD63-PA-GFP transgenic mice, as well as in vivo functional validation. Figure 5. Colocalization and dynamic interactions of ITGAM+ EVs with CD8+T cells near renal lymphatic vessels. Figure6. Identification of the key protein S100A9 in ITGAM+ EVs and validation experiments Figure7. SLC25A12 was identified as the effector target protein of S100A9 in CD8+T cells. Exosome uptake experiments and validation of S100A9 and SLC colocalization were performed

Conclusion

This study identified the principle subpopulation of ITGAM+ EVs derived from innate immune cells after AKI through single EV proteomic analysis. ITGAM+ EVs promoted CD8T cell activation through S100A9/SLC25A12 mediated mitochondrial metabolic reprogramming with enhanced aspartate/glutamate transporting, providing a new insight into EV mediated communication between innate and adaptive immune cells.

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