Proximal regulation of innate immune signals in ischemia reperfusion injury

This proposal evaluates how the obligate TLR coreceptor cluster of differentiation 14 (CD14) regulates NLRP3 signaling pathways relevant for ischemia/reperfusion injury (IRI). IRI occurs commonly in the setting of vascular compromise. In substantial work from our lab and others it is now well-established that signals generated by molecules released from IR injured renal tuble epithelial cells (RTECS) in the kidney activate innate immune signaling pathways that in turn trigger a robust inflammatory/cell death cascade. Injured RTECs release damage activated molecule patterns (DAMPs) that potently activate injurious inflammatory signaling pathways by binding to pattern recognition receptors (PRRs) constitutively expressed on parenchymal cells in the kidney. Work from our lab, and others, has established that blocking specific PRRs (namely toll-like receptors 2 or 4 or the cytoplasmic NOD like receptor NLRP3) can prevent kidney IRI. Clinical therapeutics are not yet available to target these TLRs or NLRP3 but a promising approach is monoclonal antibody blockade of CD14, as it is the key proximal regulator of these pathways. Broad/long-term objectives: The long-term goals of the proposed research are to define how CD14 contributes to injurious tissue responses in the setting of IRI and how it’s signaling can be effectively and selectively targeted. Specific Aims: The overall hypothesis is that CD14 regulates NLRP3 inflammasome activation in RTECs. Elucidating how CD14 regulates NLRP3 inflammasome activation, including whether its triggering of NLRP3 depends on TLR engagement, is essential hypothesis will be tested in two specific aims. Aim 1 tests whether CD14 must engage with TLR2 or TLR4 to trigger NLRP3 signaling in RTECs. Aim 2 tests whether CD14 can bypass TLR engagement and directly activate NLRP3 signaling pathways in RTECs. Research Design and Methods for Achieving the Stated Goals: Aim 1 employs a combination of knockout mouse models and small-molecule inhibitors to dissect the key signaling pathways linking CD14 to NLRP3 activation to determine whether CD14 engagement with TLR2 (canonical pathway) or TLR4 (noncanonical pathway) drives NLRP3 activation in RTECs and to assess whether CD14 blockade alone is sufficient to attenuate both pathways or if additional interventions, such as TLR inhibitors, are necessary. Aim 2 uses RTECs that express CD14 but lack TLRs, stimulating them with ligands known to induce CD14/TLR-independent NLRP3 activation. We will then determine whether CD14 directly activates NLRP3 in RTECs by examining DAMP/CD14 endocytosis and direct cytosolic activation of NLRP3. Health Relatedness of Project: If the aims of this proposal are met, our studies will provide a platform on which to discover the link between IRI and CD14-mediated signals and will set the stage for future clinical studies directed at pharmacologic blockade of this master regulator of PRR signaling. This knowledge is crucial for the development of rational targeted therapies for prevention or amelioration of IRI, a syndrome that causes significant morbidity and enormous healthcare expenditures. Project Number: 1R03AI196996-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Dianne Mckay | Institution: SCRIPPS RESEARCH INSTITUTE, THE, LA JOLLA, CA | Award Amount: $184,000 | Activity Code: R03 | Study Section: Pathobiology of Kidney Disease Study Section[PBKD] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R03AI19699601