ERK signalling may be responsible for LGALS1 increase by anti-HLA-I antibody stimulation in glomerular endothelial cells
Alex Boshart1,2,3, Sofia Farkona1,2, Sergi Clotet-Freixas1,2, Caitriona McEvoy1,2,4, Julia Murphy1,2,5, Sarah Crome1,2,5, Ana Konvalinka1,2,3,4.
1Toronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada; 2Ajmera Transplant Centre, University Health Network, Toronto, ON, Canada; 3Institute of Medical Science, University of Toronto, Toronto, ON, Canada; 4Division of Nephrology, Department of Medicine, University Health Network, Toronto, ON, Canada; 5Department of Immunology, University of Toronto, Toronto, ON, Canada
Background: Antibody-mediated rejection (AMR) accounts for >50% of premature kidney graft loss1. AMR is caused by donor specific antibodies (DSA) against human leukocyte antigens (HLA) on the graft. DSA can directly injure the endothelium via extracellular signal-regulated kinase (ERK) and mTOR signaling2. Since HLA does not have a signalling domain, direct injury by DSA remains poorly understood. Using unbiased proteome analysis, we have previously identified the immunomodulatory protein LGALS1 to be significantly increased in AMR glomeruli3. We hypothesized that glomerular endothelial cells (GMECs) are the primary kidney cells expressing LGALS1 and that anti-HLA antibody stimulation of GMECs induces LGALS1 through ERK activation.
Methods: We examined LGALS1 gene expression in our single cell RNAseq (scRNAseq) map of kidney biopsies from 19 living donors and validated expression using immunostaining. We assessed LGALS1 secretion after stimulation with anti-HLA Class I antibody, isotype control, or vehicle and assessed the effect of pre-treatment with Temuterkib, a phospho-ERK inhibitor, prior to stimulation with anti-HLA Class I antibody.
Results: LGALS1 expression was observed in endothelial and immune cells in healthy human kidneys (Fig.1A)4 and immunostaining validated LGALS1 protein expression in cytoplasm and membranes of GMECs (Fig.1B) LGALS1 secretion is significantly increased upon stimulation with anti-HLA Class I (Fig.1C), with corresponding ERK phosphorylation observed that peaked at 30 minutes post-stimulation. Preliminary studies support inhibition of ERK signaling decreases anti-HLA Class I antibody-induced LGALS1 secretion (Fig.1D).
Conclusion: GMECs express LGALS1, and this expression increases with anti-HLA Class I antibody stimulation via a mechanism that requires ERK signalling. Future studies will explore the consequence of ERK inhibition and LGALS1 secretion on GMECs as well as peripheral blood mononuclear cells.
We would like to acknowledge the support from members of both the Konvalinka and Crome labs. Work on this project was funded in part thanks to salary support from the Canadian Institute of Health Research's Canadian Graduate Scholarship - Master's Award presented to Alex Boshart..
References:
[1] Salehi S, Sosa RA, Jin Y-P, Kageyama S, Fishbein MC, Rozengurt E, et al.: Outside-in HLA class I signaling regulates ICAM-1 clustering and endothelial-monocyte interactions via mTOR in transplant antibody-mediated rejection. Am J Transplant 18: 1096–1109, 2018
[2] Sellarés J, Freitas DG de, Mengel M, Reeve J, Einecke G, Sis B, et al.: Understanding the Causes of Kidney Transplant Failure: The Dominant Role of Antibody-Mediated Rejection and Nonadherence. Am J Transplant 12: 388–399, 2012
[3] Clotet-Freixas S, McEvoy CM, Batruch I, Pastrello C, Kotlyar M, Van JAD, et al.: Extracellular Matrix Injury of Kidney Allografts in Antibody-Mediated Rejection: A Proteomics Study. JASN 31: 2705–2724, 2020
[4] McEvoy CM, Murphy JM, Zhang L, Clotet-Freixas S, Mathews JA, An J, et al.: Single-cell profiling of healthy human kidney reveals features of sex-based transcriptional programs and tissue-specific immunity. Nat Commun 13: 7634, 2022
Lectures by Alex Boshart
| When | Session | Talk Title | Room |
|---|---|---|---|
|
Tue-02 16:15 - 17:20 |
Abstracts Session 5 | Anti-HLA-I antibody stimulates glomerular endothelial cells to increase galectin-1 protein secretion via ERK | Grand Georgian |