ABSTRACT Alveolar macrophages (AMs) are believed to be a self-renewing cell population without a requirement of replenishment from extra-pulmonary sources in healthy adult mice; however, the mechanism(s) involved and whether replenishment occurs by stimulating the proliferation of progenitors or mature AMs are not known. We reported that lung levels of granulocyte/macrophage-colony stimulating factor (GM-CSF) control long-term maintenance of macrophages trans- planted into the lungs (via a reciprocal feedback loop) as well as endogenous AMs. My preliminary data demonstrate that GM-CSF is a critical regulator of AM mitochondrial turnover, integrity and functions and is required for fatty acid oxidation- derived energy production, processes vital to cell proliferation. Prior studies in a leukemia cell line (TF-1) suggest the pleotropic effects of GM-CSF on macrophages may be mediated by biphasic, ligand concentration-dependent receptor signaling, i.e., low levels of GM-CSF promote survival and differentiation (but not proliferation) while high levels also stim- ulate proliferation. Our long-term goal is to determine mechanisms responsible for AM self-renewal. The objective here is to elucidate the mechanism by which GM-CSF regulation of mitochondrial homeostasis controls AM self-renewal. Cen- tral Hypothesis: AMs are maintained by homeostatic self-renewal driven by GM-CSF threshold-triggered/concentration- dependent, niche-limited proliferation of mature (long-lived) AMs (not progenitors). Rationale: This hypothesis was de- veloped based on my reported and preliminary data demonstrating GM-CSF deficiency results in reduced mitochondrial metabolism and integrity despite increased mitochondrial mass. Approach: I will utilize complementary genetic and phar- macologic tools, in vivo and ex vivo studies (with isolated AMs), and pathway-specific inhibitors to pursue two Specific Aims: 1) Ontogeny and transcriptional control of AM renewal, and 2) Role of GM-CSF regulated mitochondrial metabolism in AM renewal. The expected results will inform cellular and molecular mechanism(s) by which GM-CSF regulates AM population size and will lay the foundation for developing novel therapeutic strategies to modulate AM population size. The proposed research is innovative, in my opinion, because it challenges the previously widely-held concept of AMs as short-lived, non-dividing cells replenished from circulating monocytes (regulated by M-CSF) – and instead posits that AM population size is maintained by homeostatic self-renewal mediated by GM-CSF threshold-triggered/concentration-de- pendent, niche-limited proliferation of mature (long-lived) AMs. My novel preliminary data identified GM-CSF dependent regulation of mitochondrial homeostasis as a molecular mechanism for AM self-renewal. In addition, utilization of novel mouse models and novel methodologies will enable determination of how GM-CSF regulation of mitochondrial homeosta- sis regulates AM proliferation and w...