Trimannose-coupled antimiR-21 for macrophage-targeted inhalation treatment of acute inflammatory lung damage.

Recent studies of severe acute inflammatory lung disease including COVID-19 identify macrophages to drive pulmonary hyperinflammation and long-term damage such as fibrosis. Here, we report on the development of a first-in-class, carbohydrate-coupled inhibitor of microRNA-21 (RCS-21), as a therapeutic means against pulmonary hyperinflammation and fibrosis. MicroRNA-21 is among the strongest upregulated microRNAs in human COVID-19 and in mice with acute inflammatory lung damage, and it is the strongest expressed microRNA in pulmonary macrophages. Chemical linkage of a microRNA-21 inhibitor to trimannose achieves rapid and specific delivery to macrophages upon inhalation in mice. RCS-21 reverses pathological activation of macrophages and prevents pulmonary dysfunction and fibrosis after acute lung damage in mice. In human lung tissue infected with SARS-CoV-2 ex vivo, RCS-21 effectively prevents the exaggerated inflammatory response. Our data imply trimannose-coupling for effective and selective delivery of inhaled oligonucleotides to pulmonary macrophages and report on a first mannose-coupled candidate therapeutic for COVID-19.

Novel intein-based self-cleaving affinity tag for recombinant protein production in Escherichia coli.

We evaluated several intein-based self-cleaving affinity tags for expression and single-step affinity chromatography purification of recombinant proteins produced in Escherichia coli. We used human growth hormone (hGH) as target protein that contains two internal disulfide bridges and an N-terminal phenylalanine. Use of N-terminal thiol-induced Sce VMA1 intein affinity tag resulted in purified hGH deficient in disulfide bonds. Inteins with self-cleavage inducible by pH and/or temperature shift were analyzed. N-terminal Ssp DnaX intein affinity tag resulted in a completely cleaved cytosolic protein, whereas N-terminal Ssp DnaB intein affinity tag resulted in a cytosolic fusion protein incapable of releasing hGH. Periplasmic expression of target protein was analyzed using an N-terminal signal peptide and C-terminal Ssp DnaX pH-inducible self-cleaving affinity tag. The fusion protein was properly expressed in pH 8 buffered culture medium. Fusion of a periplasmic signal peptide to the N-terminus of the POI allowed secretion to the periplasmic region and presence of the natural N-terminal amino acid of the POI following cleavage. Periplasmic expression of hGH fused to this novel C-terminal DnaX intein-based self-cleaving affinity tag made possible expression and purification of hGH protein containing disulfide bonds and free of extra amino acids.

MicroRNA-21-Dependent Macrophage-to-Fibroblast Signaling Determines the Cardiac Response to Pressure Overload.

BACKGROUND: Cardiac macrophages (cMPs) are increasingly recognized as important regulators of myocardial homeostasis and disease, yet the role of noncoding RNA in these cells is largely unknown. Small RNA sequencing of the entire miRNomes of the major cardiac cell fractions revealed microRNA-21 (miR-21) as the single highest expressed microRNA in cMPs, both in health and disease (25% and 43% of all microRNA reads, respectively). MiR-21 has been previously reported as a key microRNA driving tissue fibrosis. Here, we aimed to determine the function of macrophage miR-21 on myocardial homeostasis and disease-associated remodeling. METHODS: Macrophage-specific ablation of miR-21 in mice driven by Cx3cr1-Cre was used to determine the function of miR-21 in this cell type. As a disease model, mice were subjected to pressure overload for 6 and 28 days. Cardiac function was assessed in vivo by echocardiography, followed by histological analyses and single-cell sequencing. Cocultures of macrophages and cardiac fibroblasts were used to study macrophage-to-fibroblast signaling. RESULTS: Mice with macrophage-specific genetic deletion of miR-21 were protected from interstitial fibrosis and cardiac dysfunction when subjected to pressure overload of the left ventricle. Single-cell sequencing of pressure-overloaded hearts from these mice revealed that miR-21 in macrophages is essential for their polarization toward a M1-like phenotype. Systematic quantification of intercellular communication mediated by ligand-receptor interactions across all cell types revealed that miR-21 primarily determined macrophage-fibroblast communication, promoting the transition from quiescent fibroblasts to myofibroblasts. Polarization of isolated macrophages in vitro toward a proinflammatory (M1-like) phenotype activated myofibroblast transdifferentiation of cardiac fibroblasts in a paracrine manner and was dependent on miR-21 in cMPs. CONCLUSIONS: Our data indicate a critical role of cMPs in pressure overload-induced cardiac fibrosis and dysfunction and reveal macrophage miR-21 as a key molecule for the profibrotic role of cMPs.