Donor-specific antibodies enhance inflammatory immune remodeling associated with TAK1/NF-κB and IL-6/STAT3 signaling in a liver transplantation-related co-culture model.

Abstract

Antibody-mediated rejection (AMR) after liver transplantation remains underrecognized, and how donor-specific antibodies (DSAs) amplify inflammation within the liver immune microenvironment remains unclear. We generated DSA-positive serum in Lewis rats through Brown Norway splenocyte infusion and skin-allograft presensitization and established a co-culture model containing flow cytometry-validated donor-derived liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) with recipient-derived CD4+ T cells. Under four LPS/DSA conditions, quantitative proteomics identified the strongest inflammatory signature in the LPS+DSA+ group, with enrichment of NF-κB-related pathways and MAP3K7 (TAK1) as a highly connected hub. Western blotting showed time-dependent changes in TAK1/NF-κB-related and IL-6/STAT3 signaling. Quantitative immunofluorescence demonstrated early p-p65 nuclear translocation in CD146+ LSECs, with the greatest response under combined stimulation at 6 h. Combined DSA and LPS stimulation also increased IL-6 secretion, promoted pro-inflammatory KC polarization and the M1/M2 ratio, and induced increased Th1/Th17 and reduced Th2/Treg features at 12-24 h. These findings suggest that DSAs amplify endothelial, innate, and adaptive immune remodeling under inflammatory conditions, providing a mechanistic framework for DSA-associated liver allograft injury. Pathway-blocking and in vivo studies remain necessary to establish causality and translational relevance.