592 - Anti-inflammatory and Immunomodulatory Activity of AVR-48 in Human Cord and Adult Blood Mononuclear Cells

Poster Number: 592
Publication Number: 592.1223

Background: AVR-48 is a small molecule with TLR4 modulating activity that alters macrophage phenotype from pro- to anti-inflammatory. AVR-48 is under development to prevent bronchopulmonary dysplasia (BPD) in at-risk preterm neonates (E-PAS2023:1120.1) and is efficacious in decreasing lung infection in mouse model ( Immuno.2022;2(4):651-670).

Objective: To delineate further the TLR4-mediated mechanism of action (MoA) of AVR-48 in human cord-blood mononuclear cells (CBMCs) and understand the effect of AVR-48 on LPS tolerance in peripheral blood mononuclear cells (PBMCs).

Design/Methods: Cytokine Analysis: CBMCs (1.5 million, StemExpress, N=3) were plated in 6-well plates for 4h, then treated with AVR-48 (100 µM), LPS (25ng/mL), and AVR-48+LPS and incubated for 24h. The supernatant was assessed for IL-6, IL-8, IL-10, IL-1β, TNF-α, and IL-12p40 concentrations using ELISA (Ray biotech). One-way ANOVA.
Western Blot: CBMCs (1.5 million, StemExpress) were plated in 6-well plates for 4h, followed by treatment with AVR-48 (100 µM), LPS (50ng/mL), AVR-48+LPS and incubated for 1h. Using western blotting, cell lysates were assessed for downstream TLR4 signaling molecules: pTIRAP (Abcam), pMyD88 (Abcam), pNF-κβ (ABClonal) and ImageJ was used for densitometry.
LPS-Tolerance Assay: PBMCs (1.5 million, Research Blood Components, N=3) were plated in 6-well plates for 4h, treated with LPS (1 ng/mL) for 72h followed by LPS re-dosing (50 ng/mL) with or without AVR-48 (100 µM) and incubated for additional 24h.The supernatant was assessed for TNF-α using ELISA. One-way ANOVA.

Results: CBMCs treated with AVR-48+LPS show a decrease (~2-4-fold) in cytokine signals in IL-6, IL-8, TNF-α, IL-12, IL-1β, and IL-10, indicating anti-inflammatory efficacy as compared to LPS treated cells (Fig. 1A-F). CBMCs treated with AVR-48 alone while increased the phosphorylation of TIRAP and MyD88, indicating activation of TLR4 pathway, but decreased pNFkB. LPS increased pTIRA, pMyD88, and pNFkB, where AVR-48+LPS decreased all three protein levels, suggesting TLR-4 mediated signaling to inhibit inflammatory response (Fig. 2A-C). Lastly, LPS-tolerant PBMCs exhibit less immune stimulation and appreciably reduced TNF-α signal (~2-fold) after LPS re-challenge, where AVR-48 treatment revived the immune response and increased TNF-α secretion (Fig. 3).

Conclusion(s): AVR-48 decreased LPS-induced inflammatory cytokines via TLR4/NFkB signaling in CBMCs with the potential to reverse LPS tolerance in PBMCs. AVR-48 is a drug candidate to prevent the development of BPD and treat neonatal sepsis.