Page 1 of 55 Diabetes SECRETORY FUNCTIONS OF MACROPHAGES IN THE HUMAN PANCREATIC ISLET ARE REGULATED BY ENDOGENOUS PURINERGIC SIGNALING Jonathan R. Weitz1, Carol Jacques-Silva2, Mirza Muhammed Fahd Qadir2,3, Oliver Umland2, Elizabeth Pereira1, Farhan Qureshi1,3, Alejandro Tamayo1, Juan Dominguez-Bendala2,3,5, Rayner Rodriguez-Diaz1, Joana Almaça1, Alejandro Caicedo1,2,3,4,6 1Division of Endocrinology, Diabetes and Metabolism, Department of Medicine, University of Miami Miller School of Medicine, Miami, FL, USA 2Diabetes Research Institute, University of Miami Miller School of Medicine, Miami, FL, USA 3Molecular Cell and Developmental Biology, University of Miami Miller School of Medicine, Miami, FL, USA 4Program in Neuroscience, University of Miami Miller School of Medicine, Miami, FL 33136, USA 5Dept. of Surgery, University of Miami Miller School of Medicine, Miami, FL, USA 6Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL 33136, USA Corresponding authors: [email protected] (J.W.), [email protected] (J.A) [email protected] (A.C). Address: Department of Medicine, 1580 NW 10th Ave, Miami Fl 33136, USA Telephone: +1 (305) 243 6025 Fax: +1 (305) 243 7268 For Peer Review Only Diabetes Publish Ahead of Print, published online April 20, 2020 Diabetes Page 2 of 55 ABSTRACT Endocrine cells of the pancreatic islet interact with their microenvironment to maintain tissue homeostasis. Communication with local macrophages is particularly important in this context, but the homeostatic functions of human islet macrophages are not known. Here we show that the human islet contains macrophages in perivascular regions that are the main local source of the anti-inflammatory cytokine Il-10 and the metalloproteinase MMP9. Macrophage production and secretion of these homeostatic factors is controlled by endogenous purinergic signals. In obese and diabetic states, macrophage expression of purinergic receptors, MMP9, and Il-10 is reduced. We propose that in those states exacerbated beta cell activity due to increased insulin demand and increased cell death produces high levels of ATP that downregulate purinergic receptor expression. Loss of ATP sensing in macrophages may reduce their secretory capacity. 2 For Peer Review Only Page 3 of 55 Diabetes INTRODUCTION Macrophages of the pancreatic islet have been studied mainly in the context of immunological responses associated with diabetes pathogenesis. However, macrophages in nearly all tissues also have a homeostatic function in the non-inflamed, undamaged steady state. Resident macrophages such as Kupffer cells of the liver or microglia of the brain participate in a variety of housekeeping functions, including removal of cellular debris, remodeling of the extracellular matrix, and tissue repair (1). If these functions are impaired it can lead to pathological conditions (e.g. fibrosis). It was not until 2015 that it was determined that the islet contains its unique bona fide tissue resident macrophage (2,3). These islet macrophages have been shown to contribute to tissue homeostasis by promoting beta cell proliferation (4-7). We recently established that islet macrophages act as sentinels of beta cell activity (8), but the factors and mechanisms through which macrophages impact islet homeostasis remain mostly unexplored. While these recent studies are starting to unveil new roles for the macrophage in the mouse islet, the biology of the macrophage in the human islet has barely been investigated. Previous studies on human islet leukocytes focused on lymphocytes in both non-diabetic subjects (9) and patients with type 1 diabetes (10). There is a limited amount of papers describing in biopsies how macrophage numbers change in type 2 diabetes (11-16). There are no physiological studies of human islet macrophages, likely because studying resident macrophages is challenging. Removal and culture of tissue macrophages causes loss of tissue resident identity in as little as 12 hours (17). In addition, islets are inflamed immediately after isolation (18), and culturing islets depletes leukocytes (19). Consequently, islet macrophage biology has to be studied in situ and within a narrow temporal window. Here we used an experimental strategy that allowed us to overcome these technical 3 For Peer Review Only Diabetes Page 4 of 55 limitations. We first conducted immunohistochemical analyses to determine the anatomical properties and distribution of macrophages in human pancreas tissue sections. To examine gene expression, we used RT-PCR of macrophages sorted from isolated human islets. We then recorded 2+ 2+ changes in intracellular free Ca concentration ([Ca ]i) of islet macrophages by adapting the ex vivo pancreas slice technique (20). For these recordings in living pancreas slices, macrophages were manipulated with pharmacological tools and identified with fluorescence-conjugated antibodies. We also measured changes in cytokine secretion from isolated islets in response to purinergic agonists and antagonists. Using these approaches, we established that endogenous purinergic signaling regulates resident macrophage function, which comprises secretion of metalloproteinases that regulate the islet extracellular matrix. Our findings further show that these purinergic-dependent macrophage functions are compromised in a mouse model of obesity as well as in human type 2 diabetes. 4 For Peer Review Only Page 5 of 55 Diabetes RESEARCH DESIGN AND METHODS Experimental model and subject details Human organ donors All human tissues which were obtained are from de-identified cadaveric donors. We obtained human pancreatic tissue for islet isolation from n = 7 non-diabetic individuals and n = 4 type 2 diabetic individuals for analysis of gene expression and cultured cytokine secretion experiments, which were obtained from PRODO laboratories, as well the Human Islet Cell Processing Facility at the Diabetes Research Institute, University of Miami. See additional methods for information on donors (Supplementary Fig. S9). Method details Preparation of Living Pancreatic Tissue Slices Tissue blocks were obtained and imbedded in 3.9% low gelling temperature agarose (1.2%, Sigma Aldrich cat. no 39346-81-1, dissolved in HEPES solution as described below). Tissue blocks were solidified (4°C) for 15 minutes. Living slices were then cut (100 m) on a vibroslicer (Leica 1000S). Slices were incubated in HEPES solution (125 mM NaCl, 5.9 mM KCl, 2.56 mM CaCl2, 1 mM MgCl2, 25 mM HEPES, 0.1% BSA, pH 7.4, Aprotinin 10ug/ml). Based on our functional readouts, we have reason to believe that the different cellular components of the pancreas are functional. Indeed, we observed Ca2+ responses in acinar, endocrine, immune, and vascular cells. It is important to note that we avoided the injured cut surface of the slice in our imaging studies. Thus, to image intact islets we focused on smaller islets. Although there is no blood flow, we observed immune cells utilizing vascular scaffolds for transport within the islet (Movie S8). Immunohistochemistry 5 For Peer Review Only Diabetes Page 6 of 55 Blocks of human pancreas (0.5 cm3) were fixed in 4% paraformaldehyde, cryoprotected (30% sucrose), and tissue sections (40 m) cut on a cryostat. After permeabilization (PBS-Triton X-100 0.3%), sections were incubated in blocking solution (Biogenex, San Ramon, CA). Primary antibodies were diluted in blocking solution. To visualize macrophages we used antibodies against Iba1 (Wako Chemicals USA, Richmond, VA), CD206 (1∶100 Biolegend San Diego, CA cat No. 141721). Cell nuclei were stained with DAPI. Slides were mounted with ProLong Anti Fade (Invitrogen). See ESM for additional methods. Ca2+ Imaging of Living Pancreatic Tissue Slices To visualize macrophages in situ we used fluorescence conjugated antibodies for CD45 (1:50 Biolegend San Diego, CA cat No. 304011) and CD14 (1:50 Biolegend San Diego, CA cat No. 301805). Glucose was added to the buffered solution to give a basal glucose concentration of 3 mM, unless otherwise specified. All stimuli were bath applied. Throughout the study we used the nonhydrolyzable ATP agonist ATPS (Tocris Biosciences, Bristol, UK). Antagonists were 2+ allowed to equilibrate with receptors for 5 min before stimulation with an agonist. For [Ca ]i imaging, a Z stack of ~15-30 confocal images was acquired every 8 s using a Leica SP5 confocal 2+ laser-scanning microscope. [Ca ]i responses in pancreatic macrophages were quantified as the areas under the curve of individual traces of Fluo-4 fluorescence intensity during stimulus 2+ application. To be included in the analyses, [Ca ]i responses had to be reproducible in ≥ 3 pancreatic slices. Confocal Imaging Confocal images (pinhole = airy 1) of randomly selected islets were acquired on a Leica SP5 confocal laser-scanning microscope with 40x magnification (NA = 0.8). Macrophages were 6 For Peer Review Only Page 7 of 55 Diabetes reconstructed in Z-stacks of 15-30 confocal images (step size = 2.5-5.0 m) and analyzed using ImageJ. Using confocal images, we established the location of macrophages within islets (endocrine) or acinar regions (exocrine). To prevent bias, we used an automated method in ImageJ to segment the pancreas regions based on DAPI staining before determining macrophage position. Flow Cytometry and RT-PCR Islets were obtained from PRODO laboratories (Aliso Viejo, CA) as well the Human Islet Cell Processing Facility at the Diabetes Research Institute, University of Miami (Miami, FL) using the Ricordi Chamber. In all cases,