Little is known about how pro-obesity diets regulate tissue stem and progenitor cell function. Here we find that high fat diet (HFD)-induced obesity augments the numbers and function of Lgr5+ intestinal stem-cells (ISCs) of the mammalian intestine. Mechanistically, HFD induces a robust peroxisome proliferator-activated receptor delta (PPAR-d) signature in intestinal stem and (non-ISC) progenitor cells, and pharmacologic activation of PPAR-d recapitulates the effects of a HFD on these cells. Like a HFD, ex vivo treatment of intestinal organoid cultures with fatty acid constituents of the HFD enhances the self-renewal potential of these organoid bodies in a PPAR-d dependent manner. Interestingly, HFD- and agonist-activated PPAR-d signaling endow organoid-initiating capacity to progenitors, and enforced PPAR-d signaling permits these progenitors to form in vivo tumors upon loss of the tumor suppressor Apc. These findings highlight how diet-modulated PPAR-d activation alters not only the function of intestinal stem and progenitor cells, but also their capacity to initiate tumors.

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Background and PurposeCurrent therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response that results in significant reperfusion injury. To advance future therapies, a thorough understanding of the precise spatiotemporal underpinnings of leukocyte extravasation and infiltration is necessary. We describe the evolution of the inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion and the modulation of this response with PECAM blockade.MethodsThe transient Middle Cerebral Artery Occlusion model (90 minutes of ischemia followed by reperfusion) was used to simulate large vessel occlusion stroke and recanalization. We used wide field and confocal immunofluorescence microscopy to examine the exact distribution of neutrophils with close examination of the leukocyte position with regard to the brain vasculature and the perivascular space. Flow cytometry of single cell suspensions was used to confirm cell identity at different time points post‐stroke.ResultsLarge ischemic strokes involving both the subcortex and cortex (over 20% of the ischemic hemisphere) were induced in mice. At 12 and 24 hours, leukocyte recruitment and extravasation was primarily localized to the cortical surface. This contrasts with other organs where there is considerable migration of neutrophils deep into the inflamed tissue by 24 hours. Flow cytometry showed at 24 hours a majority of leukocytes were neutrophils. Over 48 to 72 hours, leukocytes were increasingly found deeper into the subcortex. Throughout the infarct (determined with triphenyl tetrazolium chloride staining), leukocyte recruitment was not uniform but rather organized in clusters. Disrupting leukocyte diapedesis with PECAM function‐blocking monoclonal antibody restricted leukocytes to within 500 microns of the surface when compared to control; and this was still evident at 72 hours (n=3 mice per group, p<0.01, Control 46% ± 4.0 %; PECAM‐1 Ab 62% ± 5.0%). High‐resolution wide‐field microscopy confirmed inhibition of TEM by PECAM‐1 blockade at 24 hours. Flow cytometry showed approximately equal numbers of monocytes and neutrophils at 72 hours.ConclusionsOur findings demonstrate that leukocyte infiltration into a stroke evolves over several days following reperfusion. The use of PECAM blockade modulates the natural progression of leukocytes into the infarcted stroke bed. A better understanding of leukocyte spatiotemporal infiltration and its regulators could help inform the next generation of therapeutic interventions.

Current therapies for ischemic stroke focus on reperfusion but do not address the acute inflammatory response. Previous clinical trials aimed at modulating the inflammatory milieu by disrupting leukocyte infiltration failed to show clinical efficacy. One possible explanation for this unexpected shortcoming is an incomplete understanding of the precise spatio‐temporal underpinnings of leukocyte extravasation and infiltration. Here we describe the pattern of the acute inflammatory response in a mouse transient middle cerebral artery occlusion (tMCAO) stroke model at several time points after reperfusion. We used widefield and confocal immunofluorescence microscopy to examine the distribution of neutrophils with precise examination of the leukocyte position relative to the cerebrovasculature and adjacent perivascular space. The recruitment of neutrophils varied dramatically across the infarcted tissue and surrounding penumbra, especially at early time points. At 12 and 24 hours, neutrophil recruitment and extravasation are predominantly observed at the cortical surface. Over the next few days (48 and 72 hours), neutrophils are increasingly found deeper into the subcortex. Disrupting leukocyte transendothelial migration with PECAM function‐blocking antibodies caused a marked redistribution of neutrophils away from the subcortex with a corresponding increase at the cortical surface at 72 hours. Our findings suggest that the vast majority of infiltrating neutrophils are initially recruited to cortical venules. Furthermore, neutrophils rapidly escape the perivascular compartment and enter the parenchyma. In addition, this initial recruitment to the cortical surface was highly regionalized. In spite of this, over the course of several days neutrophils did redistribute more evenly across the entire infarcted hemisphere. Taken together our findings demonstrate that the infiltration of neutrophils dynamically evolves over several days following reperfusion. The initial heterogeneous pattern of neutrophil recruitment does not correlate well with traditional markers of cellular dysfunction but does become more homogenous as the pathology evolves. A better understanding of the precise spatio‐temporal infiltration of inflammatory cells could help inform the next generation of therapeutic interventions.