title: Leptin
title: Pleiotropic roles in amphibian immune regulation, wound healing, and regeneration
titleType: Subtitle
Creators: name: Reeve, Robyn E
Publisher: name: Washington State University
Year: 2023
Subjects: subject: Comparative Endocrinology
subject: Immune function
subject: Leptin
subject: Regeneration
subject: Xenopus
subject: Immunology
subject: FOS: Clinical medicine
subject: Wound Healing

description: The roles of leptin as a nutritional regulator of immunity and a pro-inflammatory cytokine are well established in mammals. However, current knowledge of leptin’s immune roles in other classes is limited. This study demonstrates that many of leptin’s immune roles are conserved in the amphibian Xenopus laevis, including acting as a nutritionally regulated immunomodulator. In the first chapter, leptin is expressed in the skin of juvenile Xenopus and can activate JAK/STAT signaling through its receptor. Food restriction slowed wound closure in vitro and wound closure could be rescued by pre-injection with leptin. In food restricted frog skin, pre-treatment with leptin injection was more effective in restoring wound closure than topical application after injury. The second chapter demonstrates that leptin is a conserved, nutritional immunomodulator in amphibians. Larvae exposed to pathogenic bacteria died to infection less when injected with leptin compared to saline. Leptin injection also rescued developmental speed in infected animals. Leptin receptor is present at all life stages in the spleen and leptin increased splenocyte proliferation in vitro with and without additional mitogens. In vivo, leptin increased inflammation in the forearms of juveniles. In food restricted juveniles, leptin injection increased circulating white blood cells, particularly lymphocytes, compared to saline injection. In all, this data shows that leptin is pro-inflammatory and mitogenic to immune cells as well as directly mediating energy investment into immune function during food restriction. Finally, Chapter 3 describes the roles of leptin signaling in larval tail regeneration. After amputation, leptin protein is increased in the wound epithelium and blastema. Leptin treatment, by injection and by leptin bead implantation at the tail tip, rescues regeneration in food restricted larvae and bead implantation increased the speed of wound closure. In lurp:GFP transgenic larvae, leptin changed the behavior of neutrophil-like cells consistent with the hypothesis that leptin is chemoattractant to these cells. Using RNAseq, we showed that leptin bead implantation changes gene transcription at 6 hours post amputation when compared to saline bead implantation. In particular, genes associated with inflammation and proliferation were upregulated while genes associated with apoptosis were downregulated. The matrix metalloproteinases—mmp8, mmp9, and mmp13—were all upregulated with leptin treatment, which was confirmed by qPCR. Together, this leads to the hypothesis that leptin is regulating extracellular matrix expression both directly and through immune cell modulation to increase wound closure and promote a regenerative wound environment. This dissertation show comprehensively that leptin is involved in the nutritional regulation of immunity in amphibians and is an important factor for wound healing in these animals. These results have exciting applications in veterinary medicine for a endogenous immune factor to ameliorate the effects of disease-induced anorexia or stress as well as furthering the developing field of regenerative endocrinology.