miR-20a Overexpression in Adipose-Derived Mesenchymal Stem Cells Promotes Therapeutic Efficacy in Murine Lupus Nephritis by Regulating Autophagy

Objective. Lupus nephritis is the most common and severe complication of systemic lupus erythematosus. The aim of our study was to investigate the efficacy of miR-20a overexpressing adipose-derived stem cell (ADSC) transplantation in murine lupus nephritis (LN) and explore potential molecular mechanisms. Methods. Mouse ADSCs were transfected with a miR-20a lentiviral vector to obtain miR-20a overexpression ADSCs (miR-20a-ADSCs). We first observed the influence of miR-20a on ADSC viability and apoptosis in vitro. B6.MRL/lpr mice were administered ADSC/miR-20a-ADSC intravenously every week from age 30 to 33 weeks, and the lupus and normal control groups received PBS on the same schedule. Results. miR-20a expression increased in miR-20a-ADSC-derived exosomes, and miR-20a overexpression promoted ADSC proliferation and inhibited apoptosis. Compared with ADSCs, miR-20a-ADSC treatment significantly improved serologic and histologic abnormalities, as evidenced by reduced serum creatinine, anti-dsDNA antibody, 24 h urine protein levels, nephritis scores, and C3/IgG deposits. Furthermore, miR-20a-ADSC treatment resulted in downregulated Akt, mTOR, and p62 expression and upregulated miR-20a, Beclin 1, and LC3 II/I expression compared with ADSC treatment. After treatment with miR-20a-ADSC, a significant increase in the number of autophagosomes within podocytes was observed, along with upregulated expression of podocin and nephrin, compared with the ADSC group. Conclusions. miR-20a-ADSC transplantation prevents the development of lupus nephritis and significantly ameliorates already-established disease, and its mechanism is related to autophagy by targeting the miR-20a-regulated mTOR pathway.

Type I interferon modulates Langerhans cell ADAM17 to promote photosensitivity in lupus

Type I IFN (IFN-I) mediates autoimmune and inflammatory conditions, and better understanding IFN-I-driven pathogenesis could expand therapeutic possibilities. Lupus is an autoimmune disease characterized by photosensitivity, inflammatory skin lesions, and systemic organ damage. Patients have an IFN-I signature in blood and tissues and anifrolumab (anti-IFNAR1), developed for lupus and recently FDA-approved, underscores the importance of IFN-I in pathogenesis. Anifrolumab is especially efficacious for cutaneous disease, but mechanisms are poorly understood. Langerhans cells (LCs) normally limit UVR-induced skin injury via ADAM17, a metalloprotease that clips from the cell membrane and activates skin-protective EGFR ligands. Downregulation of LC ADAM17 mRNA and activity in lupus models contributes to photosensitivity, and here we link IFN-I pathogenesis with LC dysfunction. We show that murine model and human lupus non-lesional skin have IFN-I signatures and that IFN-I reduces ADAM17 sheddase activity in LCs. Furthermore, anti-IFNAR1 in multiple murine lupus models restores LC ADAM17 function and reduces photosensitivity in an EGFR and LC ADAM17-dependent manner. These results suggest that IFN-I contributes to photosensitivity in lupus by downregulating LC ADAM17 function, providing mechanisms for IFN-I pathogenesis and anifrolumab efficacy and highlighting the importance of LCs as a potential therapeutic target.

OP0040 HIPPOCAMPAL IMMUNE CELL TRAFFICKING AND A MYELOID PREDOMINANT INFLAMMATORY RESPONSE WITH ENHANCED ANTIGEN PRESENTATION AND DECREASED LEVELS OF NEUROTRANSMITTERS UNDERLY THE NEUROPSYCHIATRIC PHENOTYPE OF THE NZW/NZB MURINE LUPUS MODEL

Background:Neuropsychiatric events are common in patients with systemic lupus erythematosus (SLE), yet the underlying pathogenesis remains ill-defined, as the access to brain tissue is limited. We have previously shown that NZW/NZB F1 murine lupus model recapitulates the neuropsychiatric lupus phenotype including depressive-like behavior, increased rates of anxiety, cognitive dysfunction and motor disturbances, both at pre-nephritic and nephritic stages of the disease.Objectives:To dissect specific regions in the brain, which account for this phenotype and elucidate inflammatory and non-inflammatory mechanisms involved.Methods:Four distinct brain regions (hippocampus, amygdala, striatum and pre-frontal cortex) were dissected from brains of female C57BL/6 (WT) and NZW/NZB F1 mice at the age of 3 months (pre-nephritic) and 6 months (nephritic stage) (n=5-8/condition/experiment). Since most of the behavioral phenotype corresponds to the hippocampus, we first examined in depth the hippocampal pathology by bulk RNA sequencing, measurements of neurotransmitters levels via high-performance liquid chromatography (HPLC) and by immunophenotyping via flow cytometry analyses. For comparisons, statistical significance was indicated as a two-sided P<0.05.Results:Transcriptomic analysis revealed aberrant immune mediated response in the hippocampus of 6 month-old lupus mice compared to WT. Specifically, inflammatory pathways including both innate and adaptive immune responses, increased cytokine production, increased antigen presentation and immune cell trafficking, along with increased apoptosis and decreased cell proliferation suggest that immune aberrancies may lead to neuronal damage. These aberrancies were present in mice at 3 month-old, yet were progressed with time being more prominent at 6 month of age in lupus hippocampus. The RNA sequencing date were validated by immunophenotyping on lupus hippocampus demonstrating increased reactive GFAP+ astrocytes both at 3 and 6-month old mice. Activated IBA1+ microglia and CD11b+CD45hi CNS myeloid cells were increased only at 6 months of age. Furthermore, increased immune cell infiltration from the periphery including lymphocytes (CD45+CD11b-) mainly T cells (CD4+/CD8+) and monocytes (CD45+CD11b+Ly6G-Ly6C+), was evident only in 6 month-old lupus hippocampus compared to WT. Importantly, microglia cells in lupus hippocampus at 6 but not at 3 month of age, exhibited increased expression of antigen presenting markers including CD80, CD86 and MHC-II indicating that microglia cells may carry out the antigen presentation process seen in transcriptomic data. Low levels of serotonin and noradrenaline were observed at both 3 and 6 months of age in lupus mice; these aberrancies were mainly attributed to decreased serotonin synthesis as evidenced by intact serotonin metabolism (no differences were observed at its metabolite: 5-hydroxyindoleacetic acid). Analysis of the remaining regions of the brain combined with studies of metabolic activities of various brain regions by PET-CT scanning is in progress.Conclusion:Immune cell trafficking from the periphery combined with marked inflammatory response in the hippocampus underlie the neuropsychiatric phenotype in NZW/B murine lupus. Our data indicate increased expression of activated myeloid cells -including microglia- in the hippocampus of lupus mice culminating in increased antigen presentation and decreased neurotransmitter levels.References:[1]Nikolopoulos, D., et al. “THU0223 THE NEUROPSYCHIATRIC PHENOTYPE OF NZB/W LUPUS-PRONE MOUSE MODEL AT PRE-NEPHRITIC AND NEPHRITIC STAGES OF THE DISEASE: MURINE MODEL RECAPITULATES HUMAN DISEASE.” (2020): 334-335.Acknowledgements:This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement No 742390)Disclosure of Interests:None declared

Potential genetic basis of B cell hyperactivation in murine lupus models

Background Lupus B cells not only produce autoantibodies against nuclear antigens but also provide co-stimulation to T cells. However, there is still a lack of comprehensive understanding of the mechanism underlying lupus B cell hyperactivation. Methods This study focuses on the detection of B cell activation status, analysis of early BCR signaling response, DNA sequencing, and quantity determination of BCR signaling regulators in murine lupus models. Results Our result showed that there is a B cell hyperactivation with a significant elevation of B cell activation markers, and a BCR signaling hyperactivity with an abnormal increase of phosphorylated BCR signaling molecules and cytoplasmic calcium in the early response to BCR crosslinking in B6.Sle1/2/3 lupus mouse. Whole exome sequencing identified a multiple point mutation in the exon of many BCR signaling regulators in common murine lupus models, MRL/lpr, NZM2410, BXSB, NZB, and NZW strains. cNDA sequencing confirmed FcγR2b, Ly9, Pirb, Siglecg, and CD22 BCR signaling regulator variants in B6.Sle1/2/3 lupus mouse, but surface protein expression of these regulators on B cells showed an abnormal increase. Conclusion Our findings support that these BCR signaling regulator variants are potential causative genes of B cell hyperactivation in murine lupus models through their possible functional reduction.

mTORC2 contributes to murine lupus

ABSTRACTThe development of systemic lupus erythematosus (SLE) is associated with overactivation of the type I interferon (IFN) pathway, lymphopenia, and increased follicular helper T (Tfh) cell differentiation. However, the cellular and molecular mechanisms of action of type I IFN in SLE remains incompletely understood. Here we show that type I IFN activates the mechanistic target of rapamycin complex 2 (mTORC2) in T cells to promote T cell lymphopenia. mTORC2 also promotes Tfh differentiation and disrupts Treg homeostasis. Inactivation of mTORC2 greatly ameliorated the immunopathology in a lupus-prone mouse model, associated with reduced Tfh differentiation, normalization of Treg homeostasis and reduced T cell glucose metabolism. These data indicate that mTORC2 acts downstream of type I IFN and costimulatory receptor ICOS, to promote T cell lymphopenia and Tfh differentiation in murine lupus development, suggesting that inhibition of mTORC2 could limit lupus disease progression.