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Macrophages are integral components of the innate immune system, playing a dual role in host defense during infection and pathophysiological states. Macrophages contribute to immune responses and aid in combatting various infections, yet their production of abundant proinflammatory cytokines can lead to uncontrolled inflammation and worsened tissue damage. Therefore, reducing macrophage-derived proinflammatory cytokine release represents a promising approach for treating various acute and chronic inflammatory disorders. However, limited macrophage-specific delivery vehicles have hindered the development of macrophage-targeted therapies. In this study, we screened a pool of 112 lipid nanoparticles (LNPs) to identify an optimal LNP formulation for efficient siRNA delivery. Subsequently, by conjugating the macrophage-specific antibody F4/80 to the LNP surface, we constructed MacLNP, an enhanced LNP formulation designed for targeted macrophage delivery. In both in vitro and in vivo experiments, MacLNP demonstrated a significant enhancement in targeting macrophages. Specifically, delivery of siRNA targeting TAK1, a critical kinase upstream of multiple inflammatory pathways, effectively suppressed the phosphorylation/activation of NF-kB. LNP-mediated inhibition of NF-kB, a key upstream regulator in the classic inflammatory signaling pathway, in the murine macrophage cell line RAW264.7 significantly reduced the release of proinflammatory cytokines after stimulation with the viral RNA mimic Poly(I:C). Finally, intranasal administration of MacLNP-encapsulated TAK1 siRNA markedly ameliorated lung injury induced by influenza infection. In conclusion, our findings validate the potential of targeted macrophage interventions in attenuating inflammatory responses, reinforcing the potential of LNP-mediated macrophage targeting to treat pulmonary inflammatory disorders.
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This content will become publicly available on May 15, 2026
Understanding the Role of Receptor-Interacting-Protein Kinase 2 and X-Linked Inhibitor of Apoptosis Interactions on 5-Lipoxygenase Activation
Inflammation is an essential immune response. However, dysregulated inflammation can result in chronic inflammatory diseases, like Crohn’s disease, Blau syndrome, and early-onset sarcoidosis. Receptor-interacting serine/threonine protein kinase 2 (RIP2) is a kinase that plays a critical role in nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1 and 2) signaling pathways, ultimately triggering NF-kB activation and the secretion of pro- inflammatory cytokines. Our laboratory discovered that RIP2 also promotes the activation of arachidonate 5-Lipoxygenase (ALOX5 or 5LO), an enzyme important for producing lipid mediators. Interaction of RIP2 with the X-linked inhibitor of apoptosis protein (XIAP) has been demonstrated to be crucial for NF-kB activation and cytokine production. The purpose of this project is to determine if and how RIP2:XIAP interactions influence ALOX5 activity. This project utilizes transient transfection, competitive inhibition, fractionation, co-immunoprecipitation, SDS- PAGE, and Western blotting to analyze how disrupting RIP2:XIAP interactions influences ALOX5-activating phosphorylation and ALOX5 association with FLAP (5-Lipoxygenase Activating Protein). This study also discusses the development of a fluorescenct imaging assay to capture ALOX5:FLAP interactions. Our results indicate that RIP2:XIAP interactions are dispensable for RIP2-mediated ALOX5 activation. Understanding the mechanisms by which RIP2 influences ALOX5 will be helpful in the design, development, and testing of drugs treating NOD2-associated inflammatory diseases.
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- Award ID(s):
- 2338587
- PAR ID:
- 10614890
- Publisher / Repository:
- University of Central Florida Showcase of Text, Archives, Research & Scholarship (STARS) Repository
- Date Published:
- Subject(s) / Keyword(s):
- NOD2 RIP2 XIAP ALOX5 FLAP
- Format(s):
- Medium: X
- Institution:
- University of Central Florida
- Sponsoring Org:
- National Science Foundation
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