Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: (a) Representative western immunoblot analysis and quantification of HERV-K envelope (HERV-K env) relative to histone H3 (H3) in PAH vs. Con neutrophils (n=6 Con and n=10 PAH). (b) Immunofluorescence microscopy visualization of HERV-K env protein (green) and nuclei by DAPI staining (blue) in Con and PAH neutrophils. Antibodies used for staining are described in the “Methods”. A range of z-stack images was collected for image analysis. Mean fluorescence intensity (MFI) and cell area were quantified via image j from 3-5 randomly selected visual fields (n=3). Scale bar = 20µm (c-f) HL-60 cells were cultured in RPMI complete media and transfected using HL-60 Cell Avalanche transfection reagent per manufacturer’s protocol. (c) RT-qPCR analysis of HERV-K envelope, RIG-I , and PKR mRNA in HL-60 cells overexpressing HERV-K env (n=3). (d) Representative western immunoblot and quantification of NE relative to H3 in HL-60 cells overexpressing HERV-K env (n=3). (e) Western immunoblot analysis and quantification of HERV-K dUTPase (dUTPase) in plasma collected from Con or PAH patients. Plasma was depleted of albumin, measured by BCA, and run on a gradient gel (n=17). ( f ) Representative western immunoblot analysis and quantification of VCL relative to H3 in dHL-60 cells, treated with 10 µg/mL HERV-K dUTPase or Veh (HERV-K dUTPase elution buffer) for 24 h (n=4). Bars represent mean ±SEM. *p<0.05 and ***p<0.001 by by unpaired Student t-test.

Article Snippet: HL-60 cells were transfected with HERV-K envelope vector (VectorBuilder, Chicago, IL) or EGFP control vector (VectorBuilder) using HL-60 Cell Avalanche™ Transfection Reagent per manufactures instructions (EZ biosystems, College Park, MD).

Techniques: Western Blot, Immunofluorescence, Microscopy, Staining, Fluorescence, Cell Culture, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: Exosomes were isolated from plasma of 17 healthy donor controls and 17 PAH patients, using the ExoTIC device described under ‘Methods’. CD66b positive neutrophil exosomes were pulled down using anti-CD66b beads, from pooled exosomes of Con and PAH pooled plasma. (a) Size distribution of the pooled exosomes, determined using Nanosight. (b) Representative transmission electron microscopy (TEM) images of CD66b positive neutrophil exosomes derived from pooled plasma of PAH vs. Con patients. Scale bar=100 nm (c) Western immunoblot analysis and quantification of NE and HERV-K envelope from pooled PAH vs. Con neutrophil exosomes, relative to the exosome marker CD9. H3 from PAH neutrophil total lysate was used as a negative control. (d) NE activity in PAH vs. Con exosomes after 120 min incubation. NE was assessed by the production of BODIPY FL labeled fluorescent elastin fragments from self-quenching BODIPY FL-conjugated bovine neck ligament elastin. Bars represent mean ± SEM n=3 technical replicates of the pooled exosomes. *p<0.05, **p<0.01 by unpaired Student t-test.

Article Snippet: HL-60 cells were transfected with HERV-K envelope vector (VectorBuilder, Chicago, IL) or EGFP control vector (VectorBuilder) using HL-60 Cell Avalanche™ Transfection Reagent per manufactures instructions (EZ biosystems, College Park, MD).

Techniques: Isolation, Transmission Assay, Electron Microscopy, Derivative Assay, Western Blot, Marker, Negative Control, Activity Assay, Incubation, Labeling

Journal: bioRxiv

Article Title: Endogenous Retroviral Elements Generate Pathologic Neutrophils and Elastase Rich Exosomes in Pulmonary Arterial Hypertension

doi: 10.1101/2021.01.08.426001

Figure Lengend Snippet: The secretion of HERV-K dUTPase from monocytes results in the upregulation of vinculin thereby increasing neutrophil adhesion and reducing migration. An increase in HERV-K envelope (Env) in the neutrophil is likely via double strated (ds) RNA required for the interferon response and heightened neutrophil elastase. The increase in neutrophil elastase promotes neutrophil extracellular traps NETs. Elastase released from granules associates with HERV-K env in exosomes causing pathologic features of pulmonary arterial hypertension.

Article Snippet: HL-60 cells were transfected with HERV-K envelope vector (VectorBuilder, Chicago, IL) or EGFP control vector (VectorBuilder) using HL-60 Cell Avalanche™ Transfection Reagent per manufactures instructions (EZ biosystems, College Park, MD).

Techniques: Migration