Abstract
LRP-1, low-density lipoprotein receptor-related protein, is a member of the LDL receptor family with multi-faced functions in the immune system (Sizova O, et al., Front Immunol. 2023). Specifically, LRP1 has been studied extensively in macrophages on removal of apoptotic cells and suppression of inflammation (Donnelly S, et al., Arthritis Rheumatol. 2006). We reported that anti-LRP-1 Ab restores T cell proliferation when cocultured with neutrophils displaying membrane bound proteinase 3 (mPR3) (Yang TH, et al., J Immunol. 2018). Using T cell specific deletion of LRP1 (LRP1fx/fxLckCre+) mice generated in our lab, we found LRP1-deficient T cells protect against acute Graft-versus-host disease (GVHD) in MHC-mismatched mice (Sizova O, et al., ASH poster, Blood. 2022). Herein, we report the identification of molecular pathways regulating T cell function by LRP-1 using CRISPR LRP-1 knockout Jurkat T cells, RNA-Seq and lipidomics.
CRISPR LRP-1 knockout Jurkat T cells (LRP-1-KO) were generated by using lentiCRISPR v2 plasmids with three different sgRNA sequences targeting LRP-1, and validated by western blot with over 90% reduction of LRP-1 protein expression compared to non-target control (NT). We found all three LRP-1-KO lines showed a significantly reduced proliferation rate compared to NT control (p<0.05), with 82±3%, 69±2% and 76±2% reduction respectively. Moreover, there is a significantly increased apoptosis (p<0.05) detected with Annexin V in LRP-1-KO cells compared to NT control, with 85±7%, 69±5% and 97±4% increase in three LRP-1-KO lines respectively. The data suggest T cells deficient of LRP-1 expression have marked decrease in cell proliferation and survival.
RNA-Seq data of LRP-1-KO cells showed upregulation of genes in cholesterol biosynthesis and other lipid metabolic pathways compared to NT control, including SREBP (Sterol Regulatory Element-Binding Protein), which regulates de novo lipid synthesis. SREBP is synthesized as precursor proteins embedded in the ER membrane, and SREBP pathway is upregulated when cellular cholesterol levels are low (Brown MS and Goldstein JL, J Lipid Res. 2009). Furthermore, LRP-1-KO cells showed downregulation of genes involved in apoptosis in response to ER stress (e.g. ERN1, CEBPB, and TP53) and ferroptosis (e.g. FTL and FTH1). This is in line with our data that LRP-1-KO Jurkat T cells have increased apoptosis in comparison to NT control.
Lipid metabolism plays an important role in T cell signaling and function (Lim SA, et al., Nat Chem Biol. 2022). Using lipidomic analysis, we found LRP-1-KO cells have altered profile of global lipid network. Acetylcarnitine (AcCa) level reduced in LRP-1-KO cells compared to NT control. AcCa is an important molecule in fatty acid oxidation (FAO) and oxidative phosphorylation (OXPHOS), and regulates T cell activation, proliferation, and cytokine production. Reduced AcCa could limit the metabolic capacity for rapid proliferation, leading to the reduction of cell proliferation rate seen in LRP-1-KO Jurkat T cells. AcCa can also function as pro-inflammatory signaling molecules, activating pro-inflammatory pathways including NF-κB signaling. Reduced AcCa level could directly contribute to the decreased production of inflammatory cytokines, resulting in the amelioration of GVHD in mice that received LRP-1-KO T cells (Sizova O, et al., Blood. 2022).
Several major bioactive inflammatory lipids including sphingolipid, ceramide and hexosylceramide levels are reduced in LRP-1-KO cells compared to NT control. Sphingolipids and ceramides function as second messengers in multiple inflammatory pathways, and both play significant roles in GVHD (Tian L, et al., Front Immunol. 2022; Sofi MH, et al., JCI Insight. 2017). First, LRP-1 appears to directly influence sphingolipid biosynthesis pathways, evidenced by the consistent reduction in various sphingomyelin species in LRP-1 KO cells. Second, the decrease of multiple ceramide species in LRP-1-KO cells indicates LRP-1 plays a crucial role in ceramide homeostasis. Third, the reduction in hexosylceramides demonstrates LRP-1's involvement in glycosphingolipid metabolism. Furthermore, we found decreased phospholipid and membrane lipid in LRP-1-KO cells, which could attenuate T cell receptor signal transduction, resulting in reduced T cell proliferation and activation. In summary, LRP-1 is a novel regulator of T cell proliferation and activation through the global lipid network. Loss of LRP-1 eliminates GVHD in vivo.
