Intravascular Infusible Extracellular Matrix for the Mitigation of Severe Systemic Inflammation Relevant to Sepsis and COVID-19 Pathology

Abstract

Rationale: Multi-organ failure (MOF), associated with conditions such as acute respiratory distress syndrome, sepsis, and the recent SARS-CoV-2 virus, often exacerbates disease severity by the overwhelming systemic inflammation, thus, highlighting the need for alternative therapeutics. To address this need, intravascularly delivered infusible extracellular matrix (iECM) material was evaluated in a sepsis mouse model using lipopolysaccharide (LPS). Immunomodulatory properties of decellularized ECM that have demonstrated endogenous tissue repair promotion could favorably mitigate systemic inflammation pathology related to MOF. Compared to decellularized ECM or injectable ECM hydrogels where deployment is limited to a specific tissue per procedure, intravascular iECM delivery targets leaky vasculature in areas of inflammation. We hypothesized iECM could simultaneously alleviate systemic inflammation across multiple vulnerable tissues, thus, demonstrating potential to mitigate MOF in systemic inflammatory conditions. Methods: iECM was generated as previously described1. Porcine left ventricular tissue was decellularized, lyophilized, milled into fine powder, and 2-day pepsin digested. Insoluble ECM proteins were pelleted by high-speed centrifugation, supernatant was dialyzed in 100- 500 Da tubing, and dialyzed ECM was lyophilized, resuspended with 1x PBS, and sterile filtered. Resulting iECM was aliquoted and lyophilized for storage until resuspended with deionized water. A dual LPS-LPS mouse was utilized for modeling severe systemic inflammation. Nine-week female C57BL6/J mice underwent the following procedures: 1) Intraperitoneal (IP) injection of LPS (10 mg/kg), 2) IP LPS injection (1 mg/kg) at 6 hours for further prolonged and severe inflammation, 3) 200 μL tail vein injection of saline or iECM (10 mg/mL) at 10 hours (n = 6 per group), and 4) euthanasia and harvesting of lungs, heart, brain, and kidneys for gene expression analysis by qRT-PCR and Nanostring nCounter® Immunology Panel (Mouse). Results: Il1b and Il6 trended (p < 0.1) lower for the lungs while both were significantly decreased for the heart following iECM delivery. Additionally, Il6 was significantly decreased in the brain and kidney (Fig 1A). Multiplex profiling of lungs demonstrated inflammatory marker downregulation, including Il6, Il1α, and various chemokines (Fig 1B). Gene enrichment determined modulation of inflammatory cytokine profiles along with immune cell activation, proliferation, migration, and differentiation. Similar results were determined for the heart, brain, and kidney (data not shown). Conclusion: Systemic immunomodulation was observed among multiple affected organs, demonstrating iECM's potential for mitigating systemic inflammatory responses. Future work will investigate infiltrating immune cell subpopulations, validate results through proteomic analysis, and complete long term tissue morphology and functional assessments.