Creating an In Vitro Model for Acute Inflammation Using Tissue-Engineered Blood Clots
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Leonard, Julia
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Abstract
This work served to determine whether inflammatory responses induced in a disease state, such as Acute Respiratory Distress Syndrome (ARDS), can be modeled in vitro by tissue engineered whole blood clots. ARDS is characterized by hypoxia, immune cell dysfunction, sepsis, and mortality. Blood clots cultured at 37°C mimic sterile inflammation by exposing white blood cells WBC to damage-associated molecular patterns (DAMPs) that induce an innate immune response. Clots were cultured at 37ºC under hypoxia to determine whether hypoxia increases the expression of inflammatory mediators that mimic an ARDS disease state, including IL-8/CXCL8 and HIF-1α inducible genes. Transcriptomics of cultured clots and fresh blood was assessed using RT-PCR and RNA-sequencing while serum proteomics was carried out by ELISA. We used IL-8 as a marker of DAMP-induced inflammation and added LPS and IL-6 induction as a mimetic of sepsis. Whole blood (WB) was drawn under IRB-approved protocols on different occasions from 6 healthy consenting donors. Blood clots cultured at 37ºC elicited high IL-8 mRNA expression at 45m that was sustained over 4h. IL-8 was undetectable in baseline plasma and progressively increased at 45m (60pg/mL), 2h (185pg/mL), and 4h (3000 pg/mL). Hypoxia elevated serum IL-8 (4700 pg/mL), but this was not significant compared to normoxia (N =6, p = 0.23). LPS had the greatest average IL-8 induction, at 7800pg/mL, which was significantly upregulated compared to normoxia (p = 0.03). Only LPS-spiked 4h cultured clots expressed IL-6 at the RNA and protein level. RNA-sequencing revealed over 2300 genes differentially expressed in the clot conditions compared to FB. All 3 cultured clot sample types showed HIF-1α and NFκB pathway inductions. The top expressed genes (p < 0.05) included the chemokines CXCL2, CXCL3, IL-8/CXCL8, VEGF and the chemokine receptor CXCR4. RT-PCR confirmed that VEGF expression was induced in most cultured blood clots compared to fresh blood. Only 28 genes were differentially expressed between cultured clots and cultured hypoxia clots. In a drug-screening model, cultured clots showed variable anti-inflammatory responses to aspirin and dexamethasone between N=2 healthy donors. Dexamethasone outperformed aspirin on reducing IL-8 and IL-6 levels in LPS-stimulated blood clots. This study showed that healthy blood from 6 donors was successfully engineered to induce an acute innate immune response within 4 hours using the cultured blood clot model. Cultured blood clots showed elevated VEGF, which is a pro-angiogenic factor induced by hypoxic environments. These results suggest the clots were inherently hypoxic and is consistent with the low number of differentially expressed genes between the cultured clot without and with hypoxia incubation. The cultured clot model also showed variable responses between 2 donors when treated with common anticoagulant medications. This has positive implications in studying disease states such as ARDS and studying current medications on an individualized basis.
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This thesis has been embargoed for 2 years. It will not be available until April 2023 at the earliest.
Keywords
Acute inflammation, Blood clots, RNA sequencing, White blood cells, Tissue-engineered