Regenerative Cues of Myeloid Cells on Pancreatic Epithelium

Thesis (Ph.D.)--University of Washington, 2016-07

Tissue regeneration is an attractive approach to treating many degenerative diseases including diabetes mellitus, a devastating disease caused by the destruction or dysfunction of insulin-producing β cells in the pancreas. The process of repair and regeneration following injury or disease often recapitulates embryonic development by using similar molecular pathways in the proliferation and maturation of new cells. Nevertheless, the means by which such regenerative events are recapitulated postnatally in pancreatic tissue remain poorly characterized. Macrophages populate the mesenchymal compartment of all organs during embryogenesis and have been shown to support tissue organogenesis and regeneration by regulating remodeling of the extracellular microenvironment. We hypothesized that beyond this conventional role, distinct macrophage subsets or macrophages in distinct functional states, resident in the pancreas or recruited to this organ after injury, may establish a cross-talk with the epithelium to drive developmental/regenerative decisions. Thus, in Chapter 2, using the embryonic pancreatic epithelium as model system, we discovered that macrophages drive the epithelium to execute two developmentally important choices, i.e. the exit from cell cycle and the acquisition of a migratory phenotype. We demonstrate that these developmental decisions are effectively imparted by macrophages activated toward an M2 fetal-like functional state, and involve modulation of the adhesion receptor NCAM and an uncommon "paired-less" isoform of the transcription factor PAX6 in the epithelium. Over-expression of this PAX6 variant in pancreatic epithelia controls both cell motility and cell cycle progression in a gene-dosage dependent fashion. Importantly, induction of these phenotypes in embryonic pancreatic transplants by M2 macrophages in vivo is associated with an increased frequency of endocrine-committed cells emerging from ductal progenitor pools. These results identify M2-like macrophages as key effectors capable of coordinating epithelial cell cycle withdrawal and cell migration, two events critical to pancreatic progenitors' delamination and progression toward their differentiated fates. In addition to their activation states, macrophages may also differ by their developmental origin. Recent lineage tracing experiments have shown that different adult organs harbor distinct pools of two macrophage populations: one originating from primitive hematopoiesis in the yolk sac, referred to as “resident”, and the other one known as “inflammatory-like”, arising from fetal liver or bone marrow progenitors during definitive hematopoiesis 1,2. Recent reports suggest that the propensity of a given tissue to regenerate may be controlled by the prominence of the CCR2+ subset over the resident one. Hence, in chapter 3, I undertook a detailed characterization of resident and inflammatory-like macrophage subsets in the pancreas at mid-gestation and at birth. We observed that resident macrophages are most prevalent and intimately associated with the developing pancreatic epithelium at mid-gestation, a stage characterized by substantial differentiation of islet cells from ductal progenitors. In contrast, a greater number of inflammatory-like cells populate the pancreas at birth, a stage in which islet cells undergo a major wave of expansion. Using CCR2-specific myeloid cell depletion models, we show that loss of this subset in newborn mice leads to a remarkable reduction in  cell proliferation and mass, hypoinsulinemia and inability to maintain glucose homeostasis. Depletion of CCR2+ monocytes and macrophage derivatives results in reduced expression of the transcription factors NKX6.1 and MafA in neonatal islets, a pattern previously associated with hypoproliferative and dysfunctional islet phenotypes. Ablation of CCR2+ cells later in adulthood similarly impairs islet regenerative attempts following streptozotocin-mediated toxic insults. Whole-genome transcriptional profiling of CCR2+ myeloid cells isolated from newborn pancreas identifies several gene clusters potentially relevant to islet cell growth, i.e. most significantly genes related to IGF2 and purinergic signaling. Importantly, replenishment of pancreatic CCR2+ myeloid compartments by adoptive transfer of bone marrow-derived CCR2+ cells restores islet cell proliferation and glucose homeostasis, pointing to the potential use of this myeloid subset as cell-based therapy to enhance  cell regeneration and function. Taken together my studies advance our understanding on the complex network of myeloid cell types contributing regenerative cues to the pancreatic microenvironment during steady state and injury. Our findings provide the ground-work to decipher these critical macrophage-derived cues at the molecular level in future studies.