ABSTRACT
Pathogenic autoreactive antibodies that may be associated with life-threatening coronavirus disease 2019 (COVID-19) remain to be identified. Here, we show that self-assembled genome-scale libraries of full-length proteins covalently coupled to unique DNA barcodes for analysis by sequencing can be used for the unbiased identification of autoreactive antibodies in plasma samples. By screening 11,076 DNA-barcoded proteins expressed from a sequence-verified human ORFeome library, the method, which we named MIPSA (for Molecular Indexing of Proteins by Self-Assembly), allowed us to detect circulating neutralizing type-I and type-III interferon (IFN) autoantibodies in five plasma samples from 55 patients with life-threatening COVID-19. In addition to identifying neutralizing type-I IFN-α and IFN-ω autoantibodies and other previously known autoreactive antibodies in patient plasma, MIPSA enabled the detection of as yet unidentified neutralizing type-III anti-IFN-λ3 autoantibodies that were not seen in healthy plasma samples or in convalescent plasma from ten non-hospitalized individuals with COVID-19. The low cost and simple workflow of MIPSA will facilitate unbiased high-throughput analyses of protein-antibody, protein-protein and protein-small-molecule interactions.
Subject(s)
Autoantibodies , COVID-19 , COVID-19/therapy , Gene Library , Humans , Immunization, Passive , Interferon-alpha , COVID-19 SerotherapyABSTRACT
OBJECTIVE: Vascular endothelial cells (ECs) confer atheroprotection at locations of the arterial tree where pulsatile laminar flow (PS) exists with a high shear stress and a large net forward direction. We investigated whether the PS-induced expression of the transcription factor Krüppel-Like Factor 2 (KLF2) in cultured ECs and its expression in the mouse aorta is regulated by AMP-activated protein kinase (AMPK). METHODS AND RESULTS: AMPK inhibition by Compound C or siRNA had a significant blocking effect on the PS-induced KLF2 expression. The induction of KLF2 by PS led to the increase in eNOS and the suppression of ET-1, which could be reversed by KLF2 siRNA. In addition, PS induced the phosphorylation of ERK5 and MEF2 which are necessary for the KLF2 expression. These mechanotransduction events were abrogated by the blockade of AMPK. Furthermore, the phosphorylation levels of ERK5 and MEF2, as well as the expression of KLF2, were significantly reduced in the aorta of AMPKalpha2 knockout mice when compared with wild-type control mice. CONCLUSIONS: The flow-mediated AMPK activation is a newly defined KLF2 regulatory pathway in vascular endothelium that acts via ERK5/MEF2.
Subject(s)
AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/metabolism , Endothelial Cells/enzymology , Kruppel-Like Transcription Factors/metabolism , Analysis of Variance , Animals , Aorta, Abdominal/cytology , Cells, Cultured , DNA, Complementary/metabolism , Endothelial Cells/cytology , Humans , Immunoblotting , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Phosphorylation , Probability , Pulsatile Flow/physiology , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Stress, Mechanical , Transfection , Up-RegulationABSTRACT
Chemotaxis, cell migration directed by spatial concentration gradients of chemoattractant molecules, is critical for proper function of the immune system. Materials capable of generating defined chemoattractant gradients via controlled release may be useful for the design of improved vaccines and immunotherapies that draw specific cells to an immunization site. To this end, we encapsulated formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fN'LFN'YK) peptides or macrophage inflammatory protein-3alpha (MIP-3alpha or CCL20) in degradable poly(lactide-co-glycolide) microspheres that provided sustained release for more than 2 weeks in vitro. fN'LFN'YK and MIP-3alpha chemoattract dendritic cells (DCs), the key antigen-presenting cells involved in generation of primary immune responses, and their precursors, monocytes. Using an in vitro videomicroscopy migration assay, we detected strong chemotaxis of human monocytes and monocyte-derived DCs through 3D collagen gels toward microspheres releasing fN'LFN'YK. Similarly, microparticles releasing MIP-3alpha were able to attract mouse bone marrow-derived dendritic cells. Strikingly, prolonged attraction of DCs from distances up to 500 microm from the source to the point of contact with individual microspheres was observed. Such microspheres could be of general interest for the design of vaccines that promote adaptive immunity and as a platform for studying the biology of chemotaxis in vitro and in vivo.
