Macrophage Phenotype Orchestrates Mammalian Tissue Regeneration

The concept of controlling fibrosis and inducing organ regeneration is an awe-inspiring one, and one that would influence nearly every branch of medicine. Despite clinical advances in regenerative medicine the fact remains that humans do not regenerate injured tissue. Instead, tissue trauma stimulates inflammation, which in turn induces fibrosis and ultimately the formation of scar tissue. Inflammation is primarily regulated by macrophages which are key regulators of fibrosis. Although current therapies to control human fibrotic diseases include immunosuppressive and anti-inflammatory agents that target macrophages, new data indicates these treatments negatively impact regenerative ability. This stems from our complete lack of knowledge about how macrophages regulate endogenous tissue regeneration. What is necessary to address this knowledge gap is a mammalian model of tissue regeneration to directly study how macrophages control inflammation and effect local fibroblasts. Our discovery that African spiny mice (Acomys) can regenerate complex tissues of the external ear including skin, hair follicles, cartilage, adipose tissue and muscle, provides such a model. Furthermore, because laboratory mice produce scar tissue in response to an identical injury, this comparative system provides a unique opportunity to directly study how macrophage phenotypes regulate regeneration and scarring. The long-term goal of our research is to develop clinical interventions that inhibit fibrosis while promoting tissue regeneration. The objective of this proposal is to identify regenerative macrophage sub-populations in spiny mice and manipulate macrophage phenotypes in outbred laboratory mice (Mus) to be pro-regenerative. The central hypothesis we will test is that specific macrophage sub-populations stimulate a regenerative extracellular environment and that the timing of phenotypic switching from one state to another is critical to the outcome of injury. This hypothesis is based upon our published and preliminary studies indicating: (1) certain macrophage phenotypes are associated with regeneration, (2) Acomys and Mus exhibit different cytokine and inflammatory gene profiles and (3) production of a pro-fibrotic or pro-regenerative extracellular environment corresponds to specific macrophage phenotypes observed in Mus and Acomys respectively. To test the central hypothesis and accomplish our objective we will pursue the following two specific aims: AIM 1: identify regenerative macrophage phenotypes. The working hypothesis is that specific macrophage sub-populations are temporally enriched during regeneration (relative to scarring) and these stimulate production of a pro-regenerative extracellular matrix (ECM). Using FACS to isolate macrophages over a regeneration time course we will (1) determine the temporal expression of macrophage phenotypes, (2) identify the contribution of recruited vs. resident macrophages, (3) transcriptionally characterize macrophage phenotypes and (4) test the ability of Acomys and Mus macrophages to stimulate a regenerative vs. fibrotic phenotype respectively in fibroblasts. AIM 2: test the role of macrophage-produced Arginase 1 to regulate tissue regeneration. The working hypothesis for this aim is that high levels of Arginase1 (Arg1) are produced early during scarring and this contributes to a pro-fibrotic extracellular environment. To test the effect of precocial arginase expression on regeneration we will drive Arg1 in vivo using azithromycin. In an attempt to reduce fibrosis and induce regeneration in Mus, we will (1) completely ablate Arg1 expression in macrophages (LysMcre;Arg1flox/flox) prior to wounding and (2) temporally ablate macrophage Arg1 expression (CSF1Rcremercre;Arg1flox/flox) at specific time points post injury. In both experimental settings we will use genomic, proteomic and cellular analyses to quantify a proregenerative ECM, macrophage sub-populations and tissue regeneration. Completion of the above specific aims will transform our understanding of how macrophage sub-populations regulate tissue regeneration and fibrosis in mammals. How macrophages regulate inflammation and fibrosis during endogenous tissue regeneration is completely unknown. Results from this study will determine specific macrophage activities that inhibit fibrotic pathologies and promote tissue regeneration and this represents a potentially transformative approach towards developing novel clinical therapies for use in humans.