Placating death eaters: Tuning macrophage metabolism and phenotype via nanoparticles - Project Summary: The immune system plays a pivotal role in tissue healing after traumatic injury and in chronic inflammation associated with autoimmune diseases. Macrophages are professional phagocytes (cell-eating cells) of the innate immune system that ingest apoptotic cells (ACs) for nutrient recycling and cellular turnover to maintain tissue-level homeostasis. Internalization of ACs (known as efferocytosis) has long been known to be a trigger for the phenotypic switch of macrophages into a reparatory state. Furthermore, dysregulation of efferocytosis has been shown to contribute to pathologies such as atherosclerosis, cancer, and rheumatoid arthritis. Interestingly, the processing of apoptotic cellular cargo by macrophages releases metabolites (such as nucleotides, amino acids, polyamines, lactate, and fatty acids) that alter the metabolic status of the phagocytes— promoting fatty acid oxidation (FAO) and pro-reparatory phenotype. Here, we will test the hypothesis that this physiological pro-healing response can be triggered in macrophages via nanoparticle-based delivery of AC- mimetic metabolite payloads. We have developed a simplified AC-mimetic cargo-delivery system based on cyclodextrin nanoparticle (CDNP), which is known to be rapidly and preferentially uptaken by macrophages. Here, we will (a) sequester fatty acid payloads within CDNPs (providing “the fuel” for FAO), (b) encode immunomodulatory diamines (such as putrescine and spermidine) directly within the CDNP core, (c) test the uptake of loaded CDNPs by macrophages in vitro, and (d) examine the temporal changes in the transcriptome and functional phenotype of macrophages that result from CDNP uptake. Using primary murine and human macrophages in vitro, we will test if cell-targeted delivery can produce transcriptomic and translational signatures indicative of reparative phenotype. Macrophages will be monitored via morphometric (fluorescence microscopy), transcriptomic (nanoString), and proteomic (Luminex) tools for phenotyping. Results will demonstrate the propensity for stimuli (diamines as “the spark”) and a metabolic driver (fatty acids as “the fuel”) to act either individually or synergistically to promote a reparative state. We will determine the uptake mechanism of CDNPs loaded with lipid cargo—identifying critical cell-surface receptors, endosomal escape routes of metabolites into the cytosol, and the intracellular signaling mechanisms. Metabolic changes, such as increase in FAO, will be studied using Seahorse assay and mass spectrometry. Finally, we will test whether the metabolism-targeted strategy of macrophage phenotypic modulation can synergize with pharmacologic strategies (e.g., delivery of anti-inflammatory drugs) for developing an effective therapeutic modality to prevent chronic inflammation. Completion of these aims would validate the feasibility of metabolic reprogramming of macrophage phenotype via nanoparticle-mediated intracellular delivery of AC-mimetic metabolites. Although our proposed project is limited in scope to in vitro studies, its success will enable the healing potential of the innate immune system to be harnessed for regenerative medicine, including in the context of traumatic injury or autoimmunity.
