Session Time: 9:57am-10:40am
Presentation Time: 10:10am-10:17am
Location: Main Channel
*Purpose: Transplant recipients developing donor specific HLA antibodies (DSA) are at risk for acute and chronic antibody-mediated rejection (AMR). DSA contribute to the process of AMR by binding to HLA molecules expressed on endothelial cells (EC) and eliciting EC activation and leukocyte recruitment. HLA I Abs elicit endothelial exocytosis of P-selectin leading to monocyte adherence, identifying P-selectin as a putative therapeutic target to prevent macrophage infiltration during AMR. HLA molecules lack signal motifs and kinase activity in their short cytoplasmic tail. Previous studies showed HLA I molecules engage in crosstalk with TLR signaling. We therefore postulated that HLA class I (HLA I) forms a complex with Toll-like Receptor 4 (TLR4) to induce P-selectin expression and monocyte recruitment.
*Methods: Primary human aortic HAEC were stimulated with human HLA-I mAb and protein phosphorylation and expression were detected by Western Blot. For HLA I-induced protein complex formation, HAEC were stimulated with F(ab’)2 fragment of anti-HLA I mAb W6/32 or mouse IgG2a as a control and cell lysates were immunoprecipitated with HLA-I Ab. Complex formation between class I and TLR4 was detected by Western Blot. Gene silencing was determined with siRNA transfection. P-selectin expression was detected by cell-based ELISA. Monocyte adhesion was assessed by CFSE labeling, measured by fluorescence microscopy and analyzed with CellProfiler.
*Results: Western blot results identified expression of TLR1, TLR3, TLR4, TLR5, and TLR10 on HAEC. Co-immunoprecipitation experiments revealed complex formation between HLA I and TLR4, MD2 and MyD88, but not TRIF, TLR1, TLR3, TLR5, nor TLR10 in HAEC. Confocal immunofluorescence confirmed their molecular association demonstrating co-localization between HLA I and TLR4 on antibody-activated HAEC. Knockdown of TLR4 or MyD88 by siRNA abrogated the ability of HLA-I to stimulate phosphorylation of PI3K, Akt, NF-kB, I-kBα, JNK, and ERK; P-selectin expression, and monocyte adhesion to HAEC.
*Conclusions: These results indicate a dependency between HLA I and TLR4 to stimulate the P-selectin expression and monocyte adhesion in HAEC, which may be important in promoting leukocyte recruitment during AMR. Our results provide novel mechanisms of HLA-I Ab-mediated alteration in HAEC and suggest that disrupting complex formation between HLA-I and TLR4 may provide a novel therapeutic target to prevent AMR.
To cite this abstract in AMA style:Jin Y, Valenzuela NM, Sosa RA, Reed EF. Crosstalk between TLR4 and HLA Class I Mediates P-Selectin Expression and Monocyte Capture to Endothelial Cells [abstract]. Am J Transplant. 2020; 20 (suppl 3). https://atcmeetingabstracts.com/abstract/crosstalk-between-tlr4-and-hla-class-i-mediates-p-selectin-expression-and-monocyte-capture-to-endothelial-cells/. Accessed March 1, 2021.
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