Reconstituted Extracellular Vesicles from Human Platelets Decrease Viral Myocarditis in Mice

Abstract

Patients with viral myocarditis are at risk of sudden death and may progress to dilated cardiomyopathy (DCM). Currently, no disease‐specific therapies exist to treat viral myocarditis. Here it is examined whether reconstituted, lyophilized extracellular vesicles (EVs) from platelets from healthy men and women reduce acute or chronic myocarditis in male mice. Human‐platelet‐derived EVs (PEV) do not cause toxicity, damage, or inflammation in naïve mice. PEV administered during the innate immune response significantly reduces myocarditis with fewer epidermal growth factor (EGF)‐like module‐containing mucin‐like hormone receptor‐like 1 (F4/80) macrophages, T cells (cluster of differentiation molecules 4 and 8, CD4 and CD8), and mast cells, and improved cardiac function. Innate immune mediators known to increase myocarditis are decreased by innate PEV treatment including Toll‐like receptor (TLR)4 and complement. PEV also significantly reduces perivascular fibrosis and remodeling including interleukin 1 beta (IL‐1β), transforming growth factor‐beta 1, matrix metalloproteinase, collagen genes, and mast cell degranulation. PEV given at days 7–9 after infection reduces myocarditis and improves cardiac function. MicroRNA (miR) sequencing reveals that PEV contains miRs that decrease viral replication, TLR4 signaling, and T‐cell activation. These data show that EVs from the platelets of healthy individuals can significantly reduce myocarditis and improve cardiac function.

Sex differences in mitochondrial gene expression during viral myocarditis

“…5 μm sections were stained with hematoxylin and eosin (H&E) to detect inflammation. Myocarditis was assessed as the percentage of the heart with inflammation compared to the overall size of the heart section using a microscope eyepiece grid, as previously [ 9 , 77 ]. Sections were scored by two individuals blinded to the treatment group.…”

“…cDNA was generated using the iScript cDNA synthesis kit (Biorad, #1708891). Quantitative real time PCR (qRT-PCR) was assessed with Taqman probes (CD45/ Ptprc Mm01293577_m1; CD11b/ Itgam Mm00434455_m1; F4/80/ Adgre1 Mm00802529_m1; peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1α)/ Ppargc1a Mm01208835_m1; nuclear respiratory factor 1 (NRF1)/ Nrf1 Mm01135606_m1; estrogen-related receptor-a (ERRa)/ Esrra Mm00433143_m1) and normalized against hypoxanthine phosphoribosyltransferase 1 (HPRT) ( Hprt , Mm03024075_m1) to determine relative gene expression (RGE) using ΔΔCt as previously [ 77 , 78 ].…”

“…Whole heart ERRa protein expression was quantified using the Mouse Estrogen-Related Receptor Alpha ELISA Kit from MyBioSource (cat# MBS080310, San Diego, CA). Absorbance was used to calculate concentration relative to a standard curve and normalized to tissue wet weight, as previously [ 46 , 77 , 78 , 81 ]. The lowest detection limit for the ERRa kit was 0.1ng/mL with a detection range of 0.25–8 ng/mL.…”

Mitochondrial extracellular vesicles, autoimmunity and myocarditis

“…When tissue environments are perturbed or cells become damaged as occurs during a viral infection, EV content changes based on cellular reprogramming in response to pathological stress (11,12). EVs can either activate or inhibit innate and adaptive immune cell responses based on their content (13)(14)(15). EVs have been demonstrated to express major histocompatibility complex (MHC) class I or II and directly activate innate antigen presenting cells (APCs) or adaptive T and B cells in an antigen/self-antigenspecific manner (16,17).…”

“…Similar results have been found for other autoimmune diseases like type I diabetes ( 29 ). EVs have also been found to either promote inflammation/remodeling or to inhibit harmful immune responses for a number of autoimmune diseases including RA ( 30 – 33 ), Hashimoto’s thyroiditis ( 34 ), type I diabetes ( 35 ), SLE ( 36 ), and myocarditis ( 15 ). Additionally, EVs have been found with immunoglobulins (Ig) on their surface including IgG or internally in the form of self-antigen-complement-Ig immune complexes (ICs) ( 37 , 38 ) suggesting that EVs may initiate and/or promote autoimmune damage and inflammation via ICs ( 39 – 42 ).…”