Secretory Functions of Macrophages in the Human Pancreatic Islet Are Regulated by Endogenous Purinergic Signaling
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. In this study, we show that the human islet contains macrophages in perivascular regions that are the main local source of the anti-inflammatory cytokine interleukin-10 (IL-10) and the metalloproteinase MMP9. Macrophage production and secretion of these homeostatic factors are 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 β-cell activity due to increased insulin demand and increased cell death produce high levels of ATP that downregulate purinergic receptor expression. Loss of ATP sensing in macrophages may reduce their secretory capacity.
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 noninflamed, 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 β-cell proliferation (4–7). We recently established that islet macrophages act as sentinels of β-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 subjects without diabetes (9) and patients with type 1 diabetes (10). There is a limited number of articles describing in biopsies how macrophage numbers change in type 2 diabetes (T2D) (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 h (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.
In this study, we used an experimental strategy that allowed us to overcome these technical 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 changes in intracellular free Ca2+ concentration ([Ca2+]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 T2D.
Research Design and Methods
Experimental Model and Subject Details
Human Organ Donors
All human tissues that were obtained are from de-identified cadaveric donors. We obtained human pancreatic tissue for islet isolation from seven individuals without diabetes and four individuals with T2D for analysis of gene expression and cultured cytokine secretion experiments, which were obtained from Prodo Labs (Aliso Viejo, CA), as well the Human Islet Cell Processing Facility at the Diabetes Research Institute, University of Miami (see Supplementary Fig. 9 for information on donors).
Method Details
Preparation of Living Pancreatic Tissue Slices
Tissue blocks were obtained and imbedded in 3.9% low-gelling-temperature agarose (1.2%; dissolved in HEPES solution as described below) (catalog number 39346-81-1; Sigma-Aldrich). Tissue blocks were solidified (4°C) for 15 min. Living slices were then cut (100 μm) on a vibroslicer (1000S; Leica Biosystems). Slices were incubated in HEPES solution (125 mmol/L NaCl, 5.9 mmol/L KCl, 2.56 mmol/L CaCl2, 1 mmol/L MgCl2, 25 mmol/L HEPES, 0.1% BSA, pH 7.4, and aprotinin 10 μg/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 using vascular scaffolds for transport within the islet (Supplementary Video 8).
Immunohistochemistry
Blocks of human pancreas (0.5 cm3) were fixed in 4% paraformaldehyde, cryoprotected (30% sucrose), and tissue sections (40 μm) cut on a cryostat. After permeabilization (PBS–Triton X-100 0.3%), sections were incubated in blocking solution (BioGenex, Fremont, CA). Primary antibodies were diluted in blocking solution. To visualize macrophages, we used antibodies against Iba1 (Wako Chemicals USA, Corp., Richmond, VA) and CD206 (1:100) (catalog number 141721; BioLegend, San Diego, CA). Cell nuclei were stained with DAPI. Slides were mounted with ProLong Antifade (Invitrogen). See Supplementary Material for additional methods.
Ca2+ Imaging of Living Pancreatic Tissue Slices
To visualize macrophages in situ, we used fluorescence-conjugated antibodies for CD45 (1:50) (catalog number 304011; BioLegend) and CD14 (1:50) (catalog number 301805; BioLegend). Glucose was added to the buffered solution to give a basal glucose concentration of 3 mmol/L, unless otherwise specified. All stimuli were bath applied. Throughout the study, we used the nonhydrolyzable ATP agonist ATPγS (Tocris Bioscience, Bristol, U.K.). Antagonists were allowed to equilibrate with receptors for 5 min before stimulation with an agonist. For [Ca2+]i imaging, a Z-stack of ∼15–30 confocal images was acquired every 8 s using a Leica SP5 confocal laser-scanning microscope. [Ca2+]i responses in pancreatic macrophages were quantified as the areas under the curve of individual traces of Fluo-4 fluorescence intensity during stimulus application. To be included in the analyses, [Ca2+]i responses had to be reproducible in greater than or equal to three pancreatic slices.
Confocal Imaging
Confocal images (pinhole = airy 1) of randomly selected islets were acquired on a Leica SP5 confocal laser-scanning microscope with ×40 magnification (numerical aperture 0.8). Macrophages were reconstructed in Z-stacks of 15–30 confocal images (step size of 2.5–5.0 μm) 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 Labs as well the Human Islet Cell Processing Facility at the Diabetes Research Institute, University of Miami Miller School of Medicine using the Ricordi chamber. In all cases, islets were shipped on the same day of isolation, and islet leukocytes were isolated the next day. No differences could be detected in macrophage gene expression between islets from Prodo Labs in California and the local facility at the Diabetes Research Institute (Supplementary Fig. 7). Islet macrophages were sorted based on viability for CD45+CD14+. For nonmacrophage internal controls, islet cells were also sorted based on the viable CD45−CD14− population. See additional methods for TaqMan probes used for RT-PCR in Supplementary Fig. 11. RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA), and cDNA was prepared using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) from FACS-sorted islet macrophages and nonmacrophage internal controls. cDNA products were preamplified 10 cycles using the TaqMan PreAmp Master Mix (Applied Biosystems). PCR reactions were run using the TaqMan gene expression assays (Applied Biosystems) in a StepOnePlus Real-Time PCR System (Applied Biosystems). Relative copy number quantification of gene expression was done based on the equation: relative quantification = 2−ΔCt × 100, in which ΔCt is the difference between the threshold cycle (Ct) value (number of cycles at which amplification for a gene reaches a threshold) of the target gene and the Ct value of the ubiquitous housekeeping gene GAPDH.
We reanalyzed an islet macrophage single-cell RNA-sequencing (RNA-seq) data set of mice kept on a high-fat diet (HFD) (21). In those studies, macrophages were labeled with mAbs against CD11b, CD11c, and F4/80 and sorted using an FACSAria II. CD11c+ intraislet macrophages were compared between control and HFD mice as well as CD11c− peri-islet macrophages.
Cytokine Secretion of Human Pancreatic Islets
Islets were obtained from Prodo Labs as well the Human Islet Cell Processing Facility at the Diabetes Research Institute using the Ricordi chamber. In all cases, islets were shipped the day of isolation and arrived a day later in Miami (in the case of islets from the University of Miami, a simulated shipping day). During this time, islets recovered from the islet isolation process (24 h; the recovery time is included in the shipping for the Prodo Labs islets). Culture supernatants from islets that received no treatment (control) or treatment with purinergic agonists or antagonists were collected after overnight exposure (∼40 h after isolation). Cell lysates were collected for normalization. Cultured islet supernatants were tested for cytokine secretion using the Bio-Plex 200 system (Bio-Rad Laboratories, Hercules, CA) as well as for MMP9 using the Human MMP9 Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). Limit of detection for cytokines was as low as 1 pg/mL. Human islet leukocytes do not stay within the pancreatic islet for long after islet isolation (19). We found similar results, as 72 h of islet culture (after 24 h of shipping, for a total time of 96 h) depleted most leukocytes (Supplementary Fig. 7).
Quantification and Statistical Analyses
Quantification of Cytosolic Ca2+ Levels
To quantify changes in intracellular Ca2+ levels, we selected regions of interest around individual islet macrophages, lymphocytes, and endocrine cells. Fluorescence intensity was measured using ImageJ. Changes in fluorescence intensity are expressed as percentage changes over baseline (ΔF/F). We measured changes in total cytosolic Ca2+ levels by computing the area under the curve above baseline using Prism software (Prism 7; GraphPad Software, La Jolla, CA). Areas under the curve were determined before, during, and after each stimulus for the same time period and compared with statistical tests.
Data Analyses and Statistics
For quantification of [Ca2+]i responses, we calculated the areas under the curve of the fluorescence intensity traces of Fluo-4. Our criteria for accepting [Ca2+]i responses for analyses were 1) that responses could be elicited greater than or equal to two times by the same stimulus, and 2) the peak signal was greater than or equal to two times the baseline fluctuation. Statistical comparisons were performed using Student t test or one-way ANOVA followed by multiple-comparison procedures with the Tukey or Dunnett tests. Data are shown as mean ± interquartile range. Transcriptome sequencing data were obtained from the Gene Expression Omnibus (murine islet macrophages, accession number GSE112002 [21]; nondiabetic macrophages and stellate cells [Supplementary Fig. 2], accession number GSE84133 [22]).
Data and Resource Availability
Further information and requests for resources, reagents, and data should be directed to and will be fulfilled by J.R.W.
Results
Human Islet Tissue Macrophages Share Similar Features With Mouse Peri-islet Macrophages, Including Their Expression of Surface Markers and Tissue Niche
In the mouse pancreas, macrophages can be classified into two major populations: those residing inside the islet and those in the peri-islet regions (3). We found that macrophages within the human islet occupied a different tissue niche and had a unique CD profile. Human macrophages, regardless of whether they were located in the periphery or inside the islet, stained for Iba1 and CD206 at proportions similar to those of macrophages in the mouse peri-islet region (∼50 and ∼60%, respectively; Fig. 1A–C). By contrast, mouse intraislet macrophages did not stain for CD206 (Fig. 1B and C). Mouse macrophages located in the islet periphery stained for CD206 in a pattern that clearly delineated the islet border (Fig. 1B) (8).
Human intraislet macrophages share features of mouse islet stromal macrophages and rarely enter the islet parenchyma. A: Maximum projection of confocal images from a fixed-frozen pancreatic tissue section from a human showing CD206 (green) and Iba1 (red) immunostaining. DAPI (blue) shows nuclei. Scale bar, 50 μm. Islet border denoted by white dotted line. A’: Zoomed image of human macrophages in A, with Iba1+ (red) macrophages (*) corresponding to those inside of the islet border. A’’: Zoomed image of human macrophages in A, with CD206+ (green) macrophages (*) corresponding to those inside of the islet border. Scale bar, 20 μm, also applies to A’. B: Maximum projection of confocal images from a fixed-frozen C57BL/J6 mouse pancreatic tissue section showing CD206 (green), Iba1 (red), and insulin (white) immunostaining. DAPI (blue) shows nuclei. Scale bar, 50 μm. Islet border denoted by white dotted line. C: Quantification of the percentage of CD206+ macrophages found within the islet parenchyma in human and mouse islets (Intra) and in the peri-islet (Peri) area. D: Maximum projection of confocal images from a fixed-frozen human pancreatic tissue section showing macrophages labeled with Iba1 (red) and the vessel marker CD31 (cyan). Islet border denoted by white dotted line. Scale bar, 50 μm. E: Maximum projection of confocal images from a fixed-frozen C57BL/J6 mouse pancreatic tissue section showing macrophages labeled with Iba1 (red) and the vessel marker CD31 (cyan). Islet border denoted by white dotted line. Scale bar, 50 μm. Zoomed images of human macrophages (F) and mouse macrophages (G), with Iba1+ macrophages inside of the intraislet vessels (CD31). Scale bar, 25 μm. H: Quantification of the percentage of Iba1+ immunostaining found outside the islet CD31-labeled area in human and mouse peri-islet (Peri) and intraislet (Intra) regions. I and J: Maximum projection of confocal images from a fixed-frozen human pancreatic tissue section showing macrophages labeled with Iba1 (red) and the stromal marker PDGFRβ (cyan). Islet border denoted by white dotted line. Scale bar, 25 μm. I’: Zoomed image of human macrophages in I, with Iba1+ (red) macrophages inside of the islet stroma labeled for PDGFRβ (cyan). J: Maximum projection of confocal images from a fixed-frozen C57BL/J6 mouse pancreatic tissue section showing macrophages labeled with Iba1 (red) and the stromal marker PDGFRβ (cyan). Islet border denoted by white dotted line. Scale bar, 25 μm. K: Quantification of the percentage of Iba1+ immunostaining found outside the islet stroma (PDGFRβ-labeled) area in human and mouse peri-islet (Peri) and intraislet (Intra) areas. *P < 0.05, ANOVA.
In both human and mouse islets, macrophages were intimately associated with the vasculature (Fig. 1D–H), but macrophages in the human islet were usually close to the vasculature, whereas those in mouse islets often entered the islet parenchyma (Fig. 1D–G). We therefore quantified how far human and mouse islet macrophages reach into the islet parenchyma. We defined the parenchyma as those regions that are not labeled for the vascular and stromal makers CD31 and platelet-derived growth factor β (PDGFβ). We found that Iba1 immunostaining in the human islet was mostly confined to the PDGF receptor β (PDGFRβ)–labeled stroma and was rarely seen in CD31− regions (Fig. 1H, J, and K). Iba1 staining of mouse peri-islet macrophages was more confined to the PDGFRβ-labeled stroma than that of intraislet macrophages (Fig. 1K). Iba1 staining of intraislet macrophages was more likely to be found in CD31− regions (Fig. 1H). These results indicate that macrophages in the human islet penetrate less into the endocrine parenchyma. Because human islet macrophages and mouse peri-islet macrophages share similar surface markers and occupy a homologous stromal niche, it is conceivable that many of their functions are conserved between species.
Islet Macrophages Express a Unique Repertoire of Cytokines and Genes Involved in Tissue Remodeling
We next sought to assess the gene expression profiles of local islet leukocyte populations. Macrophages were not the only major leukocyte population found within the human islet, in line with recent findings (9,14). The different populations of leukocytes contrasts with studies in mouse islets in which ∼98% of the islet-resident CD45+ cells were classified as macrophages (2,3). For this reason, we included nonmacrophage leukocytes in addition to macrophages in our flow cytometry, gene expression, and physiological studies (Figs. 2–5). We sorted macrophages (M; CD45+CD14+), nonmacrophage leukocytes (L; CD45+CD14−), and other nonleukocyte islet cells (I; CD45−CD14−, contain mostly endocrine cells, but also endothelial, ductal, acinar, and other cell types typically found in isolated islet preparations) by flow cytometry and evaluated gene expression by RT-PCR (Fig. 2A). The endocrine fraction (I; CD45−CD14−) expressed high levels of INS, while the macrophage fraction (M; CD45+CD14+) selectively expressed the myeloid-specific gene CSF1R (Fig. 2B and C). The leukocyte populations could be further distinguished by cell size and by differential expression of the CD45 gene PTPRC and the T-cell marker CD3D (Supplementary Fig. 1). We examined the expression profile of genes involved in inflammatory processes and tissue remodeling in the three distinct populations (Fig. 2D–G). In macrophages, we detected mRNA of cytokines involved in inflammatory processes, such as IL1B (Fig. 2D), TNFα (Fig. 2E), and IL6 (Fig. 2F), as well as of cytokines involved in the resolution of inflammation, such as IL10 (Fig. 2G). IL1B and IL10 expression was almost exclusive to islet macrophages (Fig. 2D and G), while lymphocytes also expressed TNFα (Fig. 2E).
Gene expression profiles of distinct human islet leukocyte populations. A: Flow cytometry analysis of isolated human islets. Macrophages were sorted based on viable, singlet, CD14+CD45+ cells (top black box; M); nonleukocyte cells, including mixed endocrine cells based on viable, singlet, CD45−CD14− (middle black box; I); and hemopoietic, nonmyeloid cells based on viable, singlet, CD45+CD14− cells (bottom black box; L). B and C: Validation of expression from FACS-sorted mixed endocrine cells (CD45−CD14−), macrophages (CD45+CD14+), and lymphocytes (CD45+CD14−) shown in A. Quantification of mRNA levels used for validation of individual population identity shown in B and C (endocrine INS and macrophage CSF1R). Values are from n = 6 independent donor isolations (median ± interquartile range. D–I: Differences in gene expression among endocrine cells (I), macrophages (M), and lymphocytes (L) for the following genes: IL1B, TNF, IL6, IL10, MMP9, and MMP2. *P < 0.05, ANOVA followed by Tukey test for multiple comparisons.
[Ca2+]i imaging of human leukocytes in the pancreatic tissue microenvironment reveals islet macrophages respond to local signals from β-cells. A: Confocal image taken during a live video recording of a living pancreatic tissue slice. The pancreatic tissue slice was incubated with the [Ca2+]i indicator Fluo-4 (green), as well as CD14 (blue) and CD45 (red) antibodies. Scale bar, 100 μm. Arrows denote macrophage/myeloid cells (CD14+CD45+), while asterisks denote nonmacrophage leukocytes (CD14−CD45+). B: Zoomed images with split individual channels of A showing CD14+ (blue, top), CD45+ (red, middle), and merged image of CD14 and CD45 and islet endocrine cells (islet backscatter, bottom). C and D: Sequential images in pseudocolor scale of Fluo-4 intensity taken before (3G; C) and during (ATP; D) stimulation of the pancreatic tissue slice with ATP. Macrophages were identified a priori by colabeling of the antibodies CD45 and CD14 (see Supplementary Video 4 for ex vivo image and video). The horizontal scale indicates a pseudocolor scale of Fluo-4 intensity values in C, which also applies to D. Scale bar, 20 μm, also applies to C. E: Traces of [Ca2+]i responses of macrophages exposed to low (3G; 3 mmol/L) and high glucose concentration (16G; 16 mmol/L). F: Traces of [Ca2+]i responses of macrophages during low glucose (3G; 3 mmol/L), high glucose (16G; 16 mmol/L), high glucose with suramin (16G + Sur; 10 μmol/L), and ATPγS stimulation (ATP; 100 μmol/L). G: Traces of [Ca2+]i responses of endocrine cells (gray lines; Endocrine) and macrophages (black lines; Macro) during high glucose stimulation (16G; 16 mmol/L), high glucose with nifedipine (16G + Nif; 10 μmol/L), and ATPγS stimulation (ATP; 100 μmol/L). H: Quantification of macrophage [Ca2+]i responses in traces as shown in C and D. Macrophages showed increased [Ca2+]i responses during high glucose and ATPγS stimulation. In the presence of nifedipine (NIF; 10 μmol/L) during high glucose stimulation (16 mmol/L), no significant changes to the baseline (3 mmol/L) were observed. N = 8 macrophages from four donor slices. *P < 0.05, ANOVA followed by Dunnett test for multiple comparisons. AU, arbitrary units.
Purinergic signals regulate macrophage physiology and secretion. Quantification of mRNA levels of the purinergic receptors P2X7 (A), P2X4 (B), P2Y6 (C), and P2Y2 (D). mRNA was isolated from FACS-sorted cells from pancreatic islets (islet mixed endocrine cells, macrophages, and lymphocytes). Data are presented as median ± interquartile range from n = 6 control donors without diabetes. E: Individual pancreatic macrophages (black lines; Macro) showed [Ca2+]i increases when exposed to the P2X agonist BzATP (10 μmol/L), as well as general P2 agonist ATPγS (100 μmol/L), while endocrine cells (gray lines) only responded to ATPyS (100 μmol/L). F: Individual pancreatic macrophages (black lines) showed [Ca2+]i increases when exposed to the P2Y2 agonist MRS2768 (2768; 1 μmol/L), the P2Y6 agonist MRS2693 (2693; 1 μmol/L), and ATPγS (100 μmol/L). Endocrine cells (gray) only responded to ATPγS (100 μmol/L). G: Quantification of [Ca2+]i responses of human islet macrophages to purinergic stimuli, as shown in E and F. Responses are expressed as areas under the curve from n = 5–12 macrophages from at least two donor slices. Quantification of cytokine secretion from MMP9 (H), IL-10 (I), IL-1β (J), and TNF (K) from isolated islets from two biological replicates in n = 3–6 islet preparations in response to manipulating purinergic input with ATPγS (100 μmol/L), oATP (10 μmol/L), or control (5 mmol/L) glucose. Data are shown as total amount of cytokine released normalized to DNA content per islet preparations. *P < 0.05, ANOVA followed by Dunnett test for multiple comparisons. L and M: Cytokine secretion from isolated islets in response to manipulating purinergic input with ATPγS (100 μmol/L, H), oATP (10 μmol/L, I), or baseline control (5 mmol/L) glucose. *P < 0.05, one-sample t test normalized to the control with a hypothetical value of 1, followed by Wilcoxon signed-rank test. Data are presented as mean ± interquartile range. Independent preparations pooled from two biological replicates from n = 2–6 islet preparations. AU, arbitrary units.
Changes in mRNA expression for purinergic receptors IL-10 and MMP9 in obese and diabetic states. A: Quantification of the significance (P value) vs. fold change of transcripts per million (TPM) levels of purinergic receptors expressed by isolated murine peripheral islet macrophages. TPM analysis was performed using a reanalysis of a previously published RNA-seq data set (21). Data are presented as median ± interquartile range from n = 2 independent control mice preparations (chow diet) and n = 2 to 3 independent replicates for HFD mouse preparations (two for peripheral islet macrophage HFD). Quantification of RNA-seq TPM values of mmp9 (B) and il10 (C) expressed by isolated murine islet macrophages. n = 2 independent control mice preparations (chow diet) and n = 2–3 independent replicates for HFD mice preparations (two for peripheral islet macrophage HFD). Quantification of mRNA levels of MMP9 (D), IL10 (E), P2X4 (F), P2X7 (H), IL1β (J), TNFα (K), IL6 (L), and IFNγ (M) expressed by isolated human islet macrophages vs. islet macrophages from donors with T2D. Data are presented as median ± interquartile range; n = 6 control donors without diabetes and n = 4 donors with T2D. Expression of P2X4 and P2X7 is also shown relative to donor BMI (G and I). *P < 0.05, Student t test. II, intraislet macrophage; PI, peripheral islet macrophage.
In addition to a unique pattern of cytokine expression, islet macrophages expressed mRNA transcripts for molecules involved in tissue remodeling, such as MMP9 (Fig. 2H), MMP2 (Fig. 2I), MMP14 (Supplementary Fig. 2), and CD36 (Supplementary Fig. 1). We found that MMP9 was selectively expressed by macrophages and that MMP9 was the most highly expressed MMP in islet macrophages (Fig. 2H and Supplementary Fig. 2). The only other cell population found in the pancreas to express MMP9 was a small fraction of activated stellate cells (Supplementary Fig. 2). Importantly, MMP9 activity is required for degrading islet amyloid and remodeling the extracellular matrix and is downregulated in islets in T2D (23–25). The cellular source of MMP9, however, was not identified in previous studies. Our results indicate that local macrophages are the only source of MMP9 in the islet and hence may play a role in tissue remodeling in the human pancreatic islet.
Functional Characterization of Islet Macrophages in Living Pancreatic Human Tissue Slices
Tissue macrophages receive both genetic and environmental cues, which are necessary for programming their niche-required function (26). Culturing tissue macrophages outside their native environment can induce changes in as little as 12 h (17). We therefore studied islet macrophage physiology in situ by preparing living pancreatic slices for [Ca2+]i imaging from cadaveric pancreases without diabetes (8,20). In this organotypic preparation, the islet cytoarchitecture, islet vasculature, local innervation, and the islet localization within the acinar tissue are maintained (Fig. 3A). Of relevance to our studies, the pancreas slice retained immune cells (Fig. 3A). To specifically locate macrophages within organotypic slices, we used CD14 and CD45 antibodies with conjugated fluorophores, which we had previously validated for islet leukocytes (Fig. 3A and B). Colabeling of macrophages with antibodies and the Ca2+ indicator Fluo-4 allowed for real-time imaging of Ca2+ responses in macrophages (Fig. 3 and Supplementary Video 4) as well as lymphocytes (Supplementary Fig. 3).
Islet-resident macrophages respond to purinergic signals (ATP and ADP) in the murine pancreas (8) as well as in other tissues such as the brain (27). We found that human islet macrophages also responded to ATP (Fig. 3C, D, F, and H). We recently determined that stimulating murine β-cells elicited [Ca2+]i responses in islet macrophages via paracrine ATP signaling (8). Raising the glucose concentration from 3 mmol/L to 16 mmol/L increased [Ca2+]i in macrophages in the human islet (Fig. 3E). This increased activity was diminished in the presence of the ATP receptor antagonist suramin (10 μmol/L) (Fig. 3F), indicating that the effects were mediated by ATP. Moreover, the [Ca2+]i responses elicited by high glucose concentration were inhibited in macrophages as well as in endocrine cells by nifedipine (10 μmol/L), an L-type Ca2+ channel blocker (Fig. 3G and F) that inhibits endocrine cell activity and secretion.
Purinergic Signaling Control of Islet Tissue Macrophage Function
Because islet macrophages responded to ATP, we next sought to identify the purinergic receptors expressed by macrophages. We sorted islet macrophages, islet cells (mostly endocrine), and nonmyeloid leukocytes (mostly lymphocytes) by flow cytometry to determine the expression profile of purinergic receptors in each of the cellular subsets. Of 25 purinergic receptor subtypes examined, P2X7 and P2Y6 were selectively expressed in macrophages (Fig. 4A and C). P2X4 expression was highest in macrophages (twofold), but was also expressed by other islet cells and lymphocytes (Fig. 4B). Not all purinergic receptor mRNAs were expressed in macrophage populations (Supplementary Fig. 5). Lymphocytes expressed P2Y2 at higher levels than macrophages (Fig. 4D). To confirm the presence of functional purinergic receptors, we used a physiological approach using pancreas slices. Macrophages responded with increases in [Ca2+]i to the P2X receptor agonists BzATP (20 μmol/L) and ATPγS (100 μmol/L) (Fig. 4E–G). In addition, islet tissue macrophages responded with increases in [Ca2+]i to MRS2693 (5 μmol/L), an agonist with preference for P2Y6 receptors, as well as to MRS2768 (10 μmol/L), an agonist with preference for P2Y2 receptors (Fig. 4F). These results indicate that islet macrophages are exquisite sensors of ATP.
To investigate the functional consequences of purinergic activation, we cultured freshly isolated human islets overnight in the presence of purinergic agonists and antagonists and measured cytokine secretion in the culture supernatant (Fig. 4H–M and Supplementary Fig. 6). Overnight exposure to ATPγS (100 μmol/L) increased the secretion of MMP9, interleukin-1β (IL-1β), and IL-10 in all human preparations tested (Fig. 4L). Moreover, macrophage-specific cytokines such as MMP9 (Fig. 4H) and IL-10 (Fig. 4I) were found to be highly released from pancreatic islets. Other cytokines such as TNF-α were detected at relatively lower levels (Fig. 4K). Secretion of other nine cytokines did not increase consistently (IL-6, G-CSF, GM-CSF, MCP-1, TNF-α, MIP-1β, IL-17, IL-12, and IL-13). These findings indicate that ATPγS increases the secretion of molecules that are selectively expressed in macrophages (Fig. 2). To address the role of endogenous purinergic signaling in macrophage secretion, we incubated islets in the presence of the P2X receptor antagonist oxidized ATP (oATP) (10 μmol/L). Overnight exposure to oATP reduced the secretion of most cytokines, including the macrophage-specific ones, as well as release of MMP9 (Fig. 4H–J and M). Other purinergic agonists that we tested, including adenosine, which activate P1 receptors, and α,β-methylene ATP, which predominantly activates P2X1 and P2X3, had only minor effects on cytokine secretion from human islet preparations (Supplementary Fig. 6). These results indicate that endogenous purinergic signaling modulates cytokine secretion as well as the secretion of matrix remodeling enzymes from islet macrophages.
Purinergic Receptors and Purinergic-Sensitive Secretory Products Are Downregulated in High Metabolic Demand and Diabetic States
An early hallmark of HFD treatment in mice and T2D progression is increased insulin demand (28). During these states, β-cells adapt to prevent elevated levels of glucose by releasing more insulin (reviewed in Alejandro et al. [29]; for studies showing that human subjects at risk for development of diabetes [e.g., relatives of subjects with T2D] and those with impaired glucose tolerance exhibit reduced insulin release, see Weyer et al. (30)]. In the diabetic state, β-cells ultimately fail. Because ATP is cosecreted from the insulin granule (31,32) and is also released from dying cells (33), it is likely that exacerbated β-cell activity chronically increases extracellular ATP levels in the islet. We hypothesized that the prolonged activation of purinergic receptors leads to receptor desensitization, rundown, or downregulation (34,35). To test this notion, we reanalyzed an islet macrophage single-cell RNA-seq data set of mice kept on an HFD (18–20 weeks [21]). We quantified changes in purinergic receptor transcript levels and found that numerous purinergic receptors were downregulated in mouse peri-islet macrophages after an HFD (Fig. 5A). Moreover, we found that the secretory products IL10 and MMP9, which depended on purinergic signaling in isolated human islets (Fig. 4), were also downregulated in mouse peri-islet macrophages (Fig. 5B and C).
We examined the expression of purinergic receptors in macrophages from donors without diabetes and donors with T2D and found that P2X4 and P2X7 were downregulated in three out of four preparations from donors with diabetes (Fig. 5F and H; means, however, were not significantly different). We also found that MMP9 was significantly downregulated (Fig. 5D), while IL10 expression was lower in two preparations (Fig. 5E). Based on our data, we propose that in the diseased state, endogenous purinergic signaling is disrupted, thus diminishing the macrophage production of homeostatic anti-inflammatory cytokines and MMPs.
Discussion
Macrophages represent ∼40% of the islet CD45+ leukocytes in the human pancreatic islet. This population expressed bona fide macrophage markers such as Iba1, CD14, CSF1R, CD206, and CD163 (present study) (see also Supplementary Fig. 10). This is in stark contrast to the mouse pancreas where ∼98% of islet leukocytes are resident macrophages (3). Because the human and mouse mononuclear phagocyte systems lack overlapping phenotypic markers, it is difficult to identify homologous populations between species (36). Nevertheless, intraislet human macrophages share features with murine macrophages that reside in stromal regions (PDGFβ+) of the peri-islet border (3,8). The intraislet human and mouse peri-islet macrophage populations partially express CD206 (50-60%), mostly occupy a stromal vascular niche, and less frequently penetrate into the islet endocrine parenchyma. The stromal niche includes perivascular cells (e.g., vascular smooth muscle cells, pericytes, and fibroblasts) that produce and secrete extracellular matrix proteins such as collagen and laminin. The structure of the microvasculature and its associated extracellular matrix in the human islet differs dramatically from that of the mouse islet and is more continuous with that of blood vessels surrounding the islet (37,38). This may explain why the phenotype of the human islet macrophage is closer to that of the peri-islet macrophage of the mouse. Because the human islet macrophage shares anatomical and functional markers with the mouse peri-islet macrophage, we consider it prudent for future work using the mouse model to extend studies to the biology of peri-islet macrophages.
To participate in a homeostatic circuit, macrophages need to monitor the environment to adjust their function and thus prevent deviations from the steady state. We found that human islet macrophages express purinergic receptors that make them exquisite sensors of ATP. Activating ATP (purinergic) receptors stimulated intracellular signaling and altered secretory activity in islet macrophages. We found that blocking endogenous intraislet purinergic signaling had profound effects on macrophage secretion. Within the pancreas, β-cells are a major source of releasable ATP during glucose stimulation. Because ATP is cosecreted with insulin from human (39) and mouse β-cell granules (31,32), it is possible that local macrophages detect interstitial ATP levels as a proxy signal for the activation status of β-cells. In line with this notion, we found that human islet macrophage responses during high glucose stimulation could be blocked by suramin or the Ca2+ channel inhibitor nifedipine, suggesting that macrophages responded to ATP released from β-cells. It is important to note, however, that high glucose not only affects β-cells, but also has pleiotropic effects in the islet and pancreas, that suramin may have many targets, and that nifedipine may inhibit other endocrine cells. ATP released from β-cells may also affect other endocrine cells, which in turn may stimulate macrophages. Moreover, other cell types within the pancreas may release ATP (40) (e.g., during acinar stimulation or cell death) and may activate local macrophages. Therefore, demonstrating that there is a signaling axis between β-cells and macrophages mediated by ATP in the human islet requires further experimentation.
In addition to our anatomical and physiological studies, we found that human islet macrophages also expressed and secreted high levels of MMP9, IL1β, and IL10, but relatively low levels of TNF (Fig. 4H–M). Because macrophages sense endogenous ATP, we sought to investigate whether these cytokines were ATP dependent. In response to ATP stimulation, macrophages produce and secrete cytokines, including IL-10 and IL-1β. Due to the lack of consensus in the available literature, the role of IL-10 on islet function remains inconclusive (reviewed in Russell and Morgan [41]). Several previous studies have investigated the function of IL-1β on the β-cell. Although high concentrations of IL-1β for long periods of time induce apoptosis and necrosis in islet β-cells (42), acute stimulation of islets with IL-1β has beneficial effects on glucose-stimulated insulin secretion (43). These beneficial effects thus appear to be concentration dependent (44). In the context of this study, macrophages may acutely promote β-cell function during insulin demand via IL-1β signaling. However, chronic release of IL-1β by macrophages may be detrimental to islet health.
In addition to cytokine secretion, islet macrophages also release MMPs (MMP9) in an ATP-dependent manner. In this study, we show that macrophages are the major source of MMP9 expression in the human islet. While it has been reported that MMP function is dispensable for islet morphogenesis in the mouse (45), other groups have shown that MMP9 activity is important for β-cell function, islet vascularization, reduction of cellular inflammation, and degradation of islet amyloid (23,25,46). As such, the macrophage may also be a controller in a homeostatic circuit that regulates the composition of the extracellular matrix. Given that the conformation of the extracellular matrix is more complex in the human islet (47), including toxic deposits of the amyloidogenic form of islet amyloid polypeptide (48), macrophage regulation of matrix composition may be important to prevent pathological changes in humans. In line with these findings, purinergic receptor expression and MMP9 production and secretion by macrophages are downregulated in the obese and diabetic state, suggesting that during pathophysiological conditions, endogenous purinergic signaling is disrupted. Due to the known role of MMP9 in degradation of islet amyloid (23), this could affect deposition of extracellular matrix proteins and amyloid, a frequent lesion in the islets of people with diabetes (Fig. 6). However, the direct contribution of purinergic signaling control over amyloidosis remains to be tested.
Proposed model for the role of macrophages in tissue homeostasis. Human islet macrophages reside in the stromal compartment of the microvasculature. These macrophages sense ATP released from β-cells during insulin secretion (Physiology). In response to ATP sensing by purinergic receptors, healthy islet macrophages produce and secrete anti-inflammatory cytokines (IL-10) and metalloproteinases (MMP9). Purinergic receptor expression of macrophages and MMP9 and cytokine production and secretion by macrophages are downregulated in the obese and diabetic state (Pathology), probably because of increased ATP secretion from β-cells and ATP release from injured cells. This likely affects islet homeostasis (e.g., extracellular matrix turnover and β-cell function).
Insulin demand increases early during the progression to obesity and T2D, thus chronically elevating β-cell activity (reviewed in Alejandro et al. [29]; for studies showing that human subjects at risk for development of diabetes [e.g., relatives of subjects with T2D] and those with impaired glucose tolerance exhibit reduced insulin release, see Weyer et al. [30]). We postulate that the associated persistent high levels of interstitial ATP downregulate purinergic receptor expression, which undermines β-cell control of macrophage homeostatic function. This interpretation is based on our results showing that HFP-elicited changes in gene expression of purinergic receptors as well as MMP9 and IL-10 in murine peri-islet macrophages (21) were remarkably similar to diabetes-induced changes in gene expression in human islet macrophages (Fig. 5). The resulting loss of MMP9 production and secretion may impair tissue remodeling in the islet.
Article Information
Acknowledgments. The authors thank the Network for Pancreatic Organ Donors with Diabetes Slice Group at the University of Florida (Gainesville, FL), University of Miami Miller School of Medicine (Miami, FL), and Paul Langerhans Institut Dresden (Dresden, Germany) for the efforts to obtain human donor material and distribute living pancreatic tissue slices.
Funding. This work was supported by the Diabetes Research Institute Foundation, National Institute of Diabetes and Digestive and Kidney Diseases grants R56-DK-084321, R01-DK-084321, R01-DK-111538, R01-DK-113093, U01-DK-120456 (all to A.C.), National Institute of Environmental Health Sciences grants R33-ES-025673 and R21-ES-025673 (to A.C.), the Leona M. and Harry B. Helmsley Charitable Trust grants G-2018PG-T1D034 and G-1912-03552, and American Heart Association grant 19POST34450054 (to J.R.W.).
Duality of Interest. No potential conflicts of interest relevant to this article were reported.
Author Contributions. J.R.W. contributed to the study’s design, collection of data, and analysis and interpretation of results; wrote the original draft of the manuscript; and approved the final version of the manuscript. C.J.-S. contributed to the collection of data and analysis of cytokine secretion from human islets. M.M.F.Q. and J.D.-B. contributed by optimizing and maintaining the human slice culture. O.U. contributed to expert advice on flow cytometry, sample sorting, and data analysis. E.P., F.Q., and A.T. contributed to the collection of the data, including immunohistochemical experiments and cytokine secretion studies. R.R.-D. contributed to the conception of the study and expert advice on immunohistochemical experiments. J.A. contributed to the conception of the study, provided expert advice for imaging of human pancreatic slices, edited the original manuscript, and approved the final version of the manuscript. A.C. contributed to the conception of the design of the study and data interpretation, reviewed the original draft of the manuscript, and edited and approved the final version of the manuscript. All authors approved the final version of the manuscript. A.C. is the guarantor of this work and, as such, had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.
Footnotes
This article contains supplementary material online at https://doi.org/10.2337/db20-4567/suppl.12040752.
- Received July 12, 2019.
- Accepted March 26, 2020.
- © 2020 by the American Diabetes Association
Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. More information is available at https://www.diabetesjournals.org/content/license.
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