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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.
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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
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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.
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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
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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
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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, 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 in California and the local facility at the Diabetes Research Institute
(Supplementary Fig. S7). Islet macrophages were sorted based on viability, CD45+, CD14+. For
non-macrophage internal controls, islet cells were also sorted based on the viable CD45-, CD14-
population. See additional methods for taqman probes used for RT-PCR (Supplementary Fig. S11).
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 non-macrophage internal controls. cDNA products were pre-
amplified 10 cycles using the TaqMan pre-amp 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 where 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 threshold cycle value of the ubiquitous housekeeping gene
GAPDH.
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We re-analyzed an islet macrophage single-cell RNA-seq dataset of mice kept on a high
fat diet (35). In those studies macrophages were labeled with mAbs against CD11b, CD11c, F4/80
and sorted using FACSAria II. CD11c+ intra-islet macrophages were comparted between control and HFD mice as well as CD11c- peri-islet macrophages.
Cytokine secretion of human pancreatic islets
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, 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 isolation from the islet isolation process (24 hours; the recovery
time is included in the shipping for the Prodo islets). Culture supernatants from islets that received
no treatment (control) or treatment with purinergic agonists or antagonists were collected after
overnight exposure (~40 hours after isolation). Cell lysates were collected for normalization.
Cultured islet supernatants were tested for cytokine secretion using the Bio-plexR-200 system (Bio-
Rad Laboratories, Hercules, CA) as well as for MMP-9 using the Human MMP-9 Quantikine
ELISA (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 hours of islet culture (after 24 hours of shipping, for a total time of 96
hours) depleted most leukocytes (Supplementary Fig. S7).
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
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ImageJ. Changes in fluorescence intensity are expressed as percentage changes over baseline
(F/F). We measured changes in total cytosolic Ca2+ levels by computing the area under the curve
above baseline using Prism software (Prism 7, GraphPad, 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
2+ For quantification of [Ca ]i responses, we calculated the areas under the curve of the fluorescence
2+ intensity traces of Fluo-4. Our criteria for accepting [Ca ]i responses for analyses were (1) that
responses could be elicited ≥ 2 times by the same stimulus and (2) the peak signal was ≥ 2 times
the baseline fluctuation. Statistical comparisons were performed using Student’s t test or one-way
ANOVA followed by multiple-comparison procedures with the Tukey or Dunnett’s tests. Data are
shown as mean ± interquartile range. Transcriptome sequencing data were obtained from the Gene
Expression Omnibus (GEO). Murine islet macrophages GSE112002, non-diabetic macrophages
and stellate cells (Supplementary Fig. S2), GSE84133 (21).
Data and Resource Availability
Further information and requests for resources, reagents and data should be directed to and will be
fulfilled by Jonathan Weitz ([email protected]).
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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 for
CD206 at proportions similar to those of macrophages in the mouse peri-islet region (~50 and ~
60%, respectively; Fig. 1A-1C). By contrast, mouse intra-islet macrophages did not stain for
CD206 (Fig. 1B and 1C). Mouse macrophages located in the islet periphery stained for CD206 in
a pattern that clearly delineated the islet border (Fig. 1B, see also (8)).
In both human and mouse islets, macrophages were intimately associated with the
vasculature (Fig. 1D-1H), but macrophages in the human islet were usually close to the
vasculature, whereas those in mouse islets often entered the islet parenchyma (Fig. 1D-1G). 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 PDGF. We found that Iba-1 immunostaining in the human islet was mostly
confined to the PDGFR-labeled stroma and was rarely seen in CD31-negative regions (Fig. 1H
and 1J-1K). Iba-1 staining of mouse peri-islet macrophages was more confined to the PDGFR- labeled stroma than that of intra-islet macrophages (Fig. 1K). Iba-1 staining of intra-islet macrophages was more likely to be found in CD31-negative 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
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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 where ∼98% of the islet-resident CD45+ cells were classified as macrophages (2,3). For this
reason, we included non-macrophage leukocytes in addition to macrophages in our flow
cytometry, gene expression, and physiological studies (Fig. 2-5). We sorted macrophages (M;
CD45+, CD14+), non-macrophage leukocytes (L; CD45+, CD14-), and other non-leukocyte 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 2C). 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. S1). We examined the expression profile of genes involved in
inflammatory processes and tissue remodeling in the 3 distinct populations (Fig. 2D-2G). In
macrophages, we detected mRNA of cytokines involved in inflammatory processes such as IL1B
(Fig. 2D), TNF (Fig. 2E), 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 2G), while lymphocytes also expressed TNF (Fig. 2E).
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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. S2), and CD36 (Supplementary Fig. S1). We found that MMP9 was selectively expressed by macrophages and that MMP9 was the most highly expressed matrix- metalloproteinase in islet macrophages (Fig. 2H and Supplementary Fig. S2). The only other cell population found in the pancreas to express MMP9 was a small fraction of activated stellate cells
(Supplementary Fig. S2). Importantly, MMP9 activity is required for degrading islet amyloid, remodeling the extracellular matrix, and is down-regulated in islets in type 2 diabetes (22-24). 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 (25). Culturing tissue macrophages outside their native environment can induce changes in as little as 12 hours (17). We therefore studied islet macrophage
2+ physiology in situ by preparing living pancreatic slices for [Ca ]i imaging from non-diabetic cadaveric pancreases (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 for 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
3B). Co-labeling of macrophages with antibodies and the Ca2+ indicator Fluo-4 allowed for real
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time imaging of Ca2+ responses in macrophages (Fig. 3, Movie S4) as well as lymphocytes
(Supplementary Fig. S3).
Islet resident macrophages respond to purinergic signals (ATP, ADP) in the murine
pancreas (8) as well as in other tissues such as the brain (26). We found that human islet
macrophages also responded to ATP (Fig. 3C-3D, and 3F-3H). We recently determined that
2+ stimulating murine beta cells elicited [Ca ]i responses in islet macrophages via paracrine ATP
2+ signaling (8). Raising the glucose concentration from 3 mM to 16 mM increased [Ca ]i in
macrophages in the human islet (Fig. 3E). This increased activity was diminished in the presence
of the ATP receptor antagonist suramin (10 M; Fig. 3F), indicating that the effects were mediated
2+ by ATP. Moreover, the [Ca ]i responses elicited by high glucose concentration were inhibited in
macrophages, as well as in endocrine cells by nifedipine (10 M), an L-type Ca2+ channel blocker
(Fig. 3G and 3F) 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 non-
myeloid 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 4C). P2X4
expression was highest in macrophages (2-fold), but was also expressed by other islet cells and
lymphocytes (Fig. 4B). Not all purinergic receptor mRNAs were expressed in macrophage
populations (Supplementary Fig. S5). Lymphocytes expressed P2Y2 at higher levels than
macrophages (Fig. 4D). To confirm the presence of functional purinergic receptors, we used a
2+ physiological approach using pancreas slices. Macrophages responded with increases in [Ca ]i to
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the P2X receptor agonists BzATP (20 M) and ATPS (100 M) (Fig. 4E-4G). In addition, islet
2+ tissue macrophages responded with increases in [Ca ]i to MRS2693 (5 M), an agonist with preference for P2Y6 receptors, as well as to MRS2768 (10 M), 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-4M, and Supplementary Fig. S6).
Overnight exposure to ATPS (100 M) increased the secretion of MMP9, IL-1b, and IL10 in all human preparations tested (Fig. 4L). Moreover, macrophage specific cytokines such as MMP9
(Fig. 4H) and IL10 (Fig. 4I) were found to be highly released from pancreatic islets. Other cytokines such as TNF were detected at relatively lower levels (Fig. 4K). Secretion of other nine cytokines did not increase consistently (IL6, G-CSF, GM-CSF, MCP-1, TNF, MIP-1, IL-17,
IL-12, IL-13). These findings indicate that ATPS 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 M). 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 4M). Other purinergic agonists that we tested, including adenosine, which activate P1 receptors, and ,-methylene ATP, which predominantly activates P2X1 and P2X3, had only minor effects on cytokine secretion from human islet preparations (Supplementary Fig. S6). These results indicate that endogenous purinergic signaling modulates cytokine secretion as well as the secretion of matrix remodeling enzymes from islet macrophages.
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Purinergic receptors and purinergic sensitive secretory products are downregulated in high
metabolic demand and diabetic states
An early hallmark of high fat diet (HFD) treatment in mice and type 2 diabetes progression is
increased insulin demand (27). During these states, beta cells adapt to prevent elevated levels of
glucose by releasing more insulin [reviewed in (28); for studies showing that human subjects at
risk for development of diabetes (e.g. relatives of T2D subjects) and those with impaired glucose
tolerance exhibit reduced insulin release see (29)]. In the diabetic state, beta cells ultimately fail.
Because ATP is co-secreted from the insulin granule (30,31) and is also released from dying cells
(32), it is likely that exacerbated beta cell activity chronically increase extracellular ATP levels in
the islet. We hypothesized that the prolonged activation of purinergic receptors leads to receptor
desensitization, run-down, or downregulation (33,34). To test this notion, we re-analyzed an islet
macrophage single-cell RNA-seq dataset of mice kept on a high fat diet [18-20 weeks; (35)]. We
quantified changes in purinergic receptor transcript levels and found that numerous purinergic
receptors were downregulated in mouse peri-islet macrophages after a high fat diet (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 5C).
We examined the expression of purinergic receptors in macrophages from non-diabetic and
type 2 diabetic donors and found that P2X4 and P2X7 were downregulated in 3 out of 4
preparations from diabetic donors (Fig. 5F and 5H; 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
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diseased state endogenous purinergic signaling is disrupted, thus diminishing the macrophage production of homeostatic anti-inflammatory cytokines and matrix metalloproteinases.
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DISCUSSION
Macrophages represent ~40 % of the islet CD45+ leukocytes in the human pancreatic islet. This
population expressed bona fide macrophage markers such as Iba-1, CD14, CSF1R, CD206 and
CD163 [present study; see also Supplementary Fig. S10]. This is in stark contrast to the ~98 % of
islet leukocytes as resident macrophages in the mouse pancreas (3). Because the human and mouse
mononuclear phagocyte systems lack overlapping phenotypic markers, it is difficult to identify
homologous populations between species (36). Nevertheless, intra-islet human macrophages share
features with murine macrophages that reside in stromal regions (PDGF+), of the peri-islet border
(3,8). Roughly half (50-60%) of the total intra-islet human and mouse peri-islet macrophage
populations express CD206, mostly occupy a stromal vascular niche, and less frequently penetrate
into the islet endocrine parenchyma. The stromal niche includes peri-vascular cells (e.g. vascular
smooth muscle cells, pericytes, 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
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macrophages. We found that blocking endogenous intra-islet purinergic signaling had profound effects on macrophage secretion. Within the pancreas, beta cells are a major source of releasable
ATP during glucose stimulation. Because ATP is co-secreted with insulin from human (40) and mouse beta cell granules (30,31), it is possible that local macrophages detect interstitial ATP levels as a proxy signal for the activation status of beta 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 beta cells. It is important to note, however, that high glucose not only affects beta cells but 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 beta cells may also affect other endocrine cells, which in turn may stimulate macrophages. Moreover, other cell types within the pancreas may release ATP (39) (e.g. during acinar stimulation or cell death) and may activate local macrophages. Therefore, demonstrating that there is a signaling axis between beta 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, IL1 IL10, but relatively low levels of TNF (Fig. 4H-4M). 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 IL10 and IL-1. Due to the lack of consensus in the available literature, the role of IL10 on islet function remains inconclusive
(reviewed in (41)). Several previous studies have investigated the function of IL-1 on the beta cell. Although high concentrations of IL-1 for long periods of time induces apoptosis and necrosis in islet beta cells (42), acute stimulation of islets with IL-1 has beneficial effects on glucose-
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stimulated insulin secretion (43). These beneficial effects thus appear to be concentration
dependent (44) In the context of this study, macrophages may acutely promote beta cell function
during insulin demand via IL-1 signaling. However, chronic release of IL-1 by macrophages
may be detrimental to islet health.
In addition to cytokine secretion, islet macrophages also release matrix metalloproteinases
(MMP9), whose secretion was ATP-dependent. Here 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 beta cell function, islet vascularization, reducing cellular inflammation,
and degrading islet amyloid (22,24,46). 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 is 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 (22) 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.
Insulin demand increases early during the progression to obesity and type 2 diabetes, thus
chronically elevating beta cell activity [reviewed in (28) for studies showing that human subjects
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at risk for development of diabetes (e.g. relatives of T2D subjects) and those with impaired glucose tolerance exhibit reduced insulin release see (29)]. We postulate that the associated persistent high levels of interstitial ATP downregulate purinergic receptor expression, which undermines beta cell control of macrophage homeostatic function. This interpretation is based on our results that show high fat diet elicited changes in gene expression of purinergic receptors as well as MMP9 and IL10 in murine peri-islet stroma (35) that 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.
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ACKNOWLEDGEMENTS
We would like to thank the nPOD slice group at the University of Florida, University of Miami,
and Paul Langerhans Institut Dresden for their efforts to obtain human donor material and
distribute living pancreatic tissue slices. This work was supported by the Diabetes Research
Institute Foundation and National Institutes of Health grants R56DK084321 (AC), R01DK084321
(AC), R01DK111538 (AC), R01DK113093 (AC), U01DK120456 (AC) R33ES025673 (AC) and
R21ES025673 (AC), the Leona M. and Harry B. Helmsley Charitable Trust grants G-2018PG-
T1D034 and G-1912-03552, and by the American Heart Association 19POST34450054 (JRW).
JRW contributed to the study’s design, collection of data, analysis and interpretation of results.
JRW wrote the original draft of the manuscript and approved the final version of the manuscript.
CJ-S contributed to the collection of data and analysis of cytokine secretion from human islets.
FQ, EP, and AT contributed to the collection of the data including immunohistochemical
experiments, cytokine secretion studies. OU contributed to expert advice on flow cytometry,
sample sorting, and data analysis. MQ and JB-D contributed by optimizing and maintaining the
human slice culture. RR-D contributed to the conception of the study and expert advice on
immunohistochemical experiments. JA 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. AC contributed to the conception of the design of the study, data
interpretation, reviewed the original draft of the manuscript, edited and approved the final version
of the manuscript. AC is the guarantor of this work and has full access to the data and takes
responsibility for the integrity of the work. All authors approved the final version of the
manuscript. JRW, CJ-S, FQ, EP, AT, OU, MQ, JB-D, RR-D, JA, AC declare no conflicts of
interest.
21 For Peer Review Only Diabetes Page 22 of 55
REFERENCES CITED
1. Okabe Y, Medzhitov R. Tissue biology perspective on macrophages. Nat Immunol 2016; 17:9-17 2. Ferris ST, Carrero JA, Mohan JF, Calderon B, Murphy KM, Unanue ER. A minor subset of Batf3-dependent antigen-presenting cells in islets of Langerhans is essential for the development of autoimmune diabetes. Immunity 2014; 41:657-669 3. Calderon B, Carrero JA, Ferris ST, Sojka DK, Moore L, Epelman S, Murphy KM, Yokoyama WM, Randolph GJ, Unanue ER. The pancreas anatomy conditions the origin and properties of resident macrophages. J Exp Med 2015; 212:1497-1512 4. Riley KG, Pasek RC, Maulis MF, Dunn JC, Bolus WR, Kendall PL, Hasty AH, Gannon M. Macrophages are essential for CTGF-mediated adult beta-cell proliferation after injury. Mol Metab 2015; 4:584-591 5. Criscimanna A, Coudriet GM, Gittes GK, Piganelli JD, Esni F. Activated macrophages create lineage-specific microenvironments for pancreatic acinar- and beta-cell regeneration in mice. Gastroenterology 2014; 147:1106-1118 e1111 6. Banaei-Bouchareb L, Gouon-Evans V, Samara-Boustani D, Castellotti MC, Czernichow P, Pollard JW, Polak M. Insulin cell mass is altered in Csf1op/Csf1op macrophage- deficient mice. J Leukoc Biol 2004; 76:359-367 7. Brissova M, Aamodt K, Brahmachary P, Prasad N, Hong JY, Dai C, Mellati M, Shostak A, Poffenberger G, Aramandla R, Levy SE, Powers AC. Islet microenvironment, modulated by vascular endothelial growth factor-A signaling, promotes beta cell regeneration. Cell Metab 2014; 19:498-511 8. Weitz JR, Makhmutova M, Almaca J, Stertmann J, Aamodt K, Brissova M, Speier S, Rodriguez-Diaz R, Caicedo A. Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity. Diabetologia 2018; 61:182-192 9. Radenkovic M, Uvebrant K, Skog O, Sarmiento L, Avartsson J, Storm P, Vickman P, Bertilsson PA, Fex M, Korgsgren O, Cilio CM. Characterization of resident lymphocytes in human pancreatic islets. Clin Exp Immunol 2017; 187:418-427 10. Campbell-Thompson ML, Atkinson MA, Butler AE, Chapman NM, Frisk G, Gianani R, Giepmans BN, von Herrath MG, Hyoty H, Kay TW, Korsgren O, Morgan NG, Powers AC, Pugliese A, Richardson SJ, Rowe PA, Tracy S, In't Veld PA. The diagnosis of insulitis in human type 1 diabetes. Diabetologia 2013; 56:2541-2543 11. Ehses JA, Perren A, Eppler E, Ribaux P, Pospisilik JA, Maor-Cahn R, Gueripel X, Ellingsgaard H, Schneider MK, Biollaz G, Fontana A, Reinecke M, Homo-Delarche F, Donath MY. Increased number of islet-associated macrophages in type 2 diabetes. Diabetes 2007; 56:2356-2370 12. Willcox A, Richardson SJ, Bone AJ, Foulis AK, Morgan NG. Analysis of islet inflammation in human type 1 diabetes. Clin Exp Immunol 2009; 155:173-181 13. Eguchi K, Manabe I. Macrophages and islet inflammation in type 2 diabetes. Diabetes Obes Metab 2013; 15 Suppl 3:152-158 14. Butcher MJ, Hallinger D, Garcia E, Machida Y, Chakrabarti S, Nadler J, Galkina EV, Imai Y. Association of proinflammatory cytokines and islet resident leucocytes with islet dysfunction in type 2 diabetes. Diabetologia 2014; 57:491-501
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15. Kamata K, Mizukami H, Inaba W, Tsuboi K, Tateishi Y, Yoshida T, Yagihashi S. Islet amyloid with macrophage migration correlates with augmented beta-cell deficits in type 2 diabetic patients. Amyloid 2014; 21:191-201 16. Richardson SJ, Willcox A, Bone AJ, Foulis AK, Morgan NG. Islet-associated macrophages in type 2 diabetes. Diabetologia 2009; 52:1686-1688 17. Gosselin D, Skola D, Coufal NG, Holtman IR, Schlachetzki JCM, Sajti E, Jaeger BN, O'Connor C, Fitzpatrick C, Pasillas MP, Pena M, Adair A, Gonda DD, Levy ML, Ransohoff RM, Gage FH, Glass CK. An environment-dependent transcriptional network specifies human microglia identity. Science 2017; 356 18. Cowley MJ, Weinberg A, Zammit NW, Walters SN, Hawthorne WJ, Loudovaris T, Thomas H, Kay T, Gunton JE, Alexander SI, Kaplan W, Chapman J, O'Connell PJ, Grey ST. Human islets express a marked proinflammatory molecular signature prior to transplantation. Cell Transplant 2012; 21:2063-2078 19. Lacy PE, Finke EH. Activation of intraislet lymphoid cells causes destruction of islet cells. Am J Pathol 1991; 138:1183-1190 20. Marciniak A, Cohrs CM, Tsata V, Chouinard JA, Selck C, Stertmann J, Reichelt S, Rose T, Ehehalt F, Weitz J, Solimena M, Slak Rupnik M, Speier S. Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology. Nat Protoc 2014; 9:2809- 2822 21. Baron M, Veres A, Wolock SL, Faust AL, Gaujoux R, Vetere A, Ryu JH, Wagner BK, Shen-Orr SS, Klein AM, Melton DA, Yanai I. A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas Reveals Inter- and Intra-cell Population Structure. Cell Syst 2016; 3:346-360 e344 22. Aston-Mourney K, Zraika S, Udayasankar J, Subramanian SL, Green PS, Kahn SE, Hull RL. Matrix metalloproteinase-9 reduces islet amyloid formation by degrading islet amyloid polypeptide. J Biol Chem 2013; 288:3553-3559 23. Lewandowski KC, Banach E, Bienkiewicz M, Lewinski A. Matrix metalloproteinases in type 2 diabetes and non-diabetic controls: effects of short-term and chronic hyperglycaemia. Arch Med Sci 2011; 7:294-303 24. Meier DT, Tu LH, Zraika S, Hogan MF, Templin AT, Hull RL, Raleigh DP, Kahn SE. Matrix Metalloproteinase-9 Protects Islets from Amyloid-induced Toxicity. J Biol Chem 2015; 290:30475-30485 25. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature 2013; 496:445-455 26. Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K, Shinozaki Y, Ohsawa K, Tsuda M, Joshi BV, Jacobson KA, Kohsaka S, Inoue K. UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature 2007; 446:1091-1095 27. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 2003; 52:102-110 28. Alejandro EU, Gregg B, Blandino-Rosano M, Cras-Meneur C, Bernal-Mizrachi E. Natural history of beta-cell adaptation and failure in type 2 diabetes. Mol Aspects Med 2015; 42:19- 41 29. Weyer C, Bogardus C, Mott DM, Pratley RE. The natural history of insulin secretory dysfunction and insulin resistance in the pathogenesis of type 2 diabetes mellitus. J Clin Invest 1999; 104:787-794
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30. Richards-Williams C, Contreras JL, Berecek KH, Schwiebert EM. Extracellular ATP and zinc are co-secreted with insulin and activate multiple P2X purinergic receptor channels expressed by islet beta-cells to potentiate insulin secretion. Purinergic Signal 2008; 4:393- 405 31. Hazama A, Hayashi S, Okada Y. Cell surface measurements of ATP release from single pancreatic beta cells using a novel biosensor technique. Pflugers Arch 1998; 437:31-35 32. Donath MY, Ehses JA, Maedler K, Schumann DM, Ellingsgaard H, Eppler E, Reinecke M. Mechanisms of beta-cell death in type 2 diabetes. Diabetes 2005; 54 Suppl 2:S108-113 33. Giniatullin R, Nistri A. Desensitization properties of P2X3 receptors shaping pain signaling. Front Cell Neurosci 2013; 7:245 34. Hiken JF, Steinberg TH. ATP downregulates P2X7 and inhibits osteoclast formation in RAW cells. Am J Physiol Cell Physiol 2004; 287:C403-412 35. Ying W, Lee YS, Dong Y, Seidman JS, Yang M, Isaac R, Seo JB, Yang BH, Wollam J, Riopel M, McNelis J, Glass CK, Olefsky JM, Fu W. Expansion of Islet-Resident Macrophages Leads to Inflammation Affecting beta Cell Proliferation and Function in Obesity. Cell Metab 2018; 36. Reynolds G, Haniffa M. Human and Mouse Mononuclear Phagocyte Networks: A Tale of Two Species? Front Immunol 2015; 6:330 37. Brissova M, Shostak A, Fligner CL, Revetta FL, Washington MK, Powers AC, Hull RL. Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes. J Histochem Cytochem 2015; 63:637- 645 38. Cohrs CM, Chen C, Jahn SR, Stertmann J, Chmelova H, Weitz J, Bahr A, Klymiuk N, Steffen A, Ludwig B, Kamvissi V, Wolf E, Bornstein SR, Solimena M, Speier S. Vessel Network Architecture of Adult Human Islets Promotes Distinct Cell-Cell Interactions In Situ and Is Altered After Transplantation. Endocrinology 2017; 158:1373-1385 39. Sorensen CE, Novak I. Visualization of ATP release in pancreatic acini in response to cholinergic stimulus. Use of fluorescent probes and confocal microscopy. J Biol Chem 2001; 276:32925-32932 40. Khan S, Yan-Do R, Duong E, Wu X, Bautista A, Cheley S, MacDonald PE, Braun M. Autocrine activation of P2Y1 receptors couples Ca (2+) influx to Ca (2+) release in human pancreatic beta cells. Diabetologia 2014; 57:2535-2545 41. Russell MA, Morgan NG. The impact of anti-inflammatory cytokines on the pancreatic beta-cell. Islets 2014; 6:e950547 42. Eizirik DL, Mandrup-Poulsen T. A choice of death--the signal-transduction of immune- mediated beta-cell apoptosis. Diabetologia 2001; 44:2115-2133 43. Yelich MR. In vivo endotoxin and IL-1 potentiate insulin secretion in pancreatic islets. Am J Physiol 1990; 258:R1070-1077 44. Spinas GA, Mandrup-Poulsen T, Molvig J, Baek L, Bendtzen K, Dinarello CA, Nerup J. Low concentrations of interleukin-1 stimulate and high concentrations inhibit insulin release from isolated rat islets of Langerhans. Acta Endocrinol (Copenh) 1986; 113:551- 558 45. Perez SE, Cano DA, Dao-Pick T, Rougier JP, Werb Z, Hebrok M. Matrix metalloproteinases 2 and 9 are dispensable for pancreatic islet formation and function in vivo. Diabetes 2005; 54:694-701
24 For Peer Review Only Page 25 of 55 Diabetes
46. Christoffersson G, Walden T, Sandberg M, Opdenakker G, Carlsson PO, Phillipson M. Matrix metalloproteinase-9 is essential for physiological Beta cell function and islet vascularization in adult mice. Am J Pathol 2015; 185:1094-1103 47. Almaca J, Molina J, Arrojo EDR, Abdulreda MH, Jeon WB, Berggren PO, Caicedo A, Nam HG. Young capillary vessels rejuvenate aged pancreatic islets. Proc Natl Acad Sci U S A 2014; 111:17612-17617 48. Westermark P, Wilander E. The influence of amyloid deposits on the islet volume in maturity onset diabetes mellitus. Diabetologia 1978; 15:417-421 49. Jurgens CA, Toukatly MN, Fligner CL, Udayasankar J, Subramanian SL, Zraika S, Aston- Mourney K, Carr DB, Westermark P, Westermark GT, Kahn SE, Hull RL. beta-cell loss and beta-cell apoptosis in human type 2 diabetes are related to islet amyloid deposition. Am J Pathol 2011; 178:2632-2640 50. Huang YC, Rupnik M, Gaisano HY. Unperturbed islet alpha-cell function examined in mouse pancreas tissue slices. J Physiol 2011; 589:395-408
25 For Peer Review Only Diabetes Page 26 of 55
FIGURE LEGENDS
Figure 1- Human intra islet 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), Iba1 (red),
immunostaining. DAPI (blue) = nuclei. Scale bars, 50 m (right). Islet border denoted by white
dotted line. A’: Zoomed images of human macrophages in Fig. 1A, with Iba1+ (red) macrophages
inside of the islet border. A’’: Zoomed images of human macrophages in Fig. 1A, with CD206+
(green) macrophages inside of the islet border. Scale bars, 20 m (left) 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), Insulin (white) immunostaining. DAPI (blue) = nuclei.
Scale bars, 50 m (see panel E). Islet border denoted by white dotted line. C: Quantification of the percentage of CD206 islet positive macrophages found within the islet parenchyma in human and mouse islets, and in the peri-islet area. Islet border denoted by white dotted line. 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. 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 bars, 50 m (right) also applies to (D and B). F-G: Zoomed images of human macrophages (1F) and mouse macrophages
(1G), with Iba1+ macrophages inside of the intra islet vessels (CD31). Scale bar, 25 m (bottom left) also applies to (1F). H: Quantification of the percentage of Iba1+ immunostaining found outside the islet CD31-labeled area in human and mouse peri-islet and intra-islet regions. I-J:
Maximum projection of confocal images from a fixed frozen human pancreatic tissue section
26 For Peer Review Only Page 27 of 55 Diabetes
showing macrophages labeled with Iba1 (red) and the stromal marker PDGFR (cyan). Islet border
denoted by white dotted line Scale bar 25 m, bottom right. I’: Zoomed images of human
macrophages in (1I), with Iba1+ (red) macrophages inside of the islet stroma PDGFR 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 PDGFR (cyan).
Islet border denoted by white dotted line. Scale bar 25 m, in figure (1I) applies to (1J). K:
Quantification of the percentage of Iba1+ immunostaining found outside the islet stroma
(PDGFRlabeled area in human and mouse peri-islet and intra-islet areas.
27 For Peer Review Only Diabetes Page 28 of 55
Figure 2- Gene expression profiles of distinct human islet leukocyte populations. A: Flow cytometry analysis of isolated human islets. Macrophages were sorted based on viable, singlets,
CD14+, CD45+ cells (upper black box; M); non-leukocyte cells, including mixed endocrine cells based on viable, singlet, CD45-, CD14- (middle black box; I); and haemopoietic, non-myeloid cells based on viable, singlet, CD45+, CD14- cells (lower black box, L). B-C: Validation of expression from FACS sorted mixed endocrine cells (CD45-, CD14-), macrophages (CD45+,
CD14+), and lymphocytes (CD45+, CD14-) shown in (2A). Quantification of mRNA levels used for validation of individual population identity shown in; endocrine Ins and macrophage Csf1r.
Values are from n = 6 independent donor isolations (median ± interquartile range; *P < 0.05
ANOVA followed by Tukey’s test for multiple comparisons). D-I: Differences in gene expression between endocrine cells, macrophages, and lymphocytes for the following genes; IL1B, TNF, IL6,
IL10, MMP9, MMP2.
28 For Peer Review Only Page 29 of 55 Diabetes
2+ Figure 3- [Ca ]i imaging of human tissue leukocytes in the pancreatic tissue
microenvironment reveals islet macrophages respond to local signals from beta cells. A:
Confocal image taken during a live video recording of a living pancreatic tissue slice. The
2+ pancreatic tissue slice was incubated with the [Ca ]i indicator Fluo-4 (green), as well as CD14
(blue) and CD45 (red). Scale bar 100 m. Arrows (top) denotes macrophage/myeloid cells
(CD14+, CD45+), while * denotes non-macrophage leukocytes (CD14-, CD45+). B: Zoomed
images with split individual channels of A showing CD14+ (blue), CD45+ (red), and merged
image of CD14, CD45, and islet endocrine cells (islet backscatter). C-D: Sequential images in
pseudocolor scale of fluo-4 intensity taken before (3G - top) and during (ATP - bottom) stimulation
of the pancreatic tissue slice with ATP. Macrophages were identified a priori by co-labeling of the
antibodies CD45 and CD14 (see Movie S4 for ex vivo image and video). The x-axis scale indicates
a pseudocolor scale of the intensity values in figure C, which also applies to figure D. Scale bar
2+ shown in panel D also applies to C. E: Traces of [Ca ]i responses of macrophages exposed to low
2+ (3 mM) and high glucose concentration (16 mM). F: Traces of [Ca ]i responses of macrophages
during low glucose (3 mM), high glucose (16 mM), high glucose with suramin (10 M), and during
2+ ATPyS stimulation (100 M). G: Traces of [Ca ]i responses of endocrine cells (gray) and
macrophages (black) during high glucose stimulation (16 mM), during high glucose, high glucose
with nifedipine (10 M), and during ATPyS stimulation (100 M). H: Quantification of
2+ macrophage [Ca ]i responses in traces as shown in C and D. Macrophages showed increased
2+ [Ca ]i responses during high glucose and ATPyS stimulation. In the presence of nifedipine (10
M) during high glucose stimulation (16 mM) no significant changes to the baseline (3 mM) were
observed. N = 8 macrophages from 4 donor slices. *P < 0.05, ANOVA followed by Dunnett test
for multiple comparisons.
29 For Peer Review Only Diabetes Page 30 of 55
Figure 4- Purinergic signals regulate macrophage physiology and function. A-D:
Quantification of mRNA levels of the purinergic receptors; P2X7 (A), P2X4 (B), P2Y6 (C), P2Y2
(D). mRNA was isolated from FACS sorted cells from pancreatic islets; (Islet mixed Endocrine,
Macrophage, Lymphocytes). Data are presented as median +/- interquartile range; from n = 6
2+ control (non-diabetic donors). E: Individual pancreatic macrophages (black) showed [Ca ]i increases when exposed to the P2X agonist BzATP (10 M), as well as a general P2 agonist
ATPyS (100 M), while endocrine cells (shades of grey) only responded to ATPyS (100 M). F:
2+ Individual pancreatic macrophages (black) showed [Ca ]i increases when the exposed to the P2Y2 agonist MRS2768 (1 M), the P2Y6 agonist MRS2693 (1 M), and ATPyS (100 M), Endocrine
2+ cells (grey) only responded to ATPyS (100 M). G: Quantification of [Ca ]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 2 donor slices. H-K: Quantification of cytokine secretion from MMP9 (H), IL10 (I), IL1 (J) and TNFa (K) from isolated islets from 2 biological replicates from n = 3 – 6 islet preparations in response to manipulating purinergic input with ATPyS (100 uM), oATP (10 uM) or control (5 mM) glucose. Data are shown as total amount of cytokine released normalized to the control condition values of DNA content per islet preparations. *P < 0.05, ANOVA followed by Dunnett test for multiple comparisons. L-M:
Cytokine secretion from isolated islets in response to manipulating purinergic input with ATPyS
(100 uM, H), oATP (10 uM, I), or baseline control (5 mM) 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 2
biological replicates from n = 2-6 islet preparations.
30 For Peer Review Only Page 31 of 55 Diabetes
Figure 5- Changes in mRNA expression for purinergic receptors, IL10 and MMP9 in obese
and diabetic states. A: Quantification of the significance (p-value), versus fold change of TPM
levels of purinergic receptors expressed by isolated murine peripheral islet macrophages. TPM
analysis was performed using a re-analysis of a previously published RNA-seq data set (35). Data
are presented as median +/- interquartile range from n = 2 independent control mice preparations
(chow diet), and n = 2 - 3 independent replicates high fat diet mouse preparations (2 for peripheral
islet macrophage HFD). B and C: Quantification of RNAseq TPM values of mmp9 (5B) and il10
(5C) expressed by isolated murine islet macrophages. PI (peripheral islet macrophages), II (intra
islet macrophages). n = 2 independent control mice preparations (chow diet), and n = 2 - 3
independent replicates high fat diet mice preparations (2 for peripheral islet macrophage HFD). D-
M: Quantification of mRNA levels of MMP9 (D), and IL10 (E) P2X4 (F), P2X7 (H), IL1 (J),
TNF (K), IL6 (L) and IFN (M) expressed by isolated human islet macrophages versus islet
macrophages from T2D donors. Data are presented as median +/- interquartile range; from n = 6
control (non-diabetic donors), and 4 T2D diabetic donors. *P < 0.05 Students t-test. Expression of
P2X4 and P2X7 is also shown relative to donor BMI (5G and 5I).
31 For Peer Review Only Diabetes Page 32 of 55
Figure 6- 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 beta 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 is downregulated in the obese and diabetic state (“pathology”), probably because of increased ATP secretion from beta cells and
ATP release from injured cells. This likely affects islet homeostasis (e.g. extracellular matrix turnover, beta cell function).
32 For Peer Review Only APage 33 of 55 DC Diabetes I Islet Islet
Islet
Iba1 Iba1 Iba1 Human CD206 Human CD31 Human PDGFRβ
A’’ A’’ E I’ Islet parenchyma
* *
Islet * * Iba1 PDGFRβ Iba1 CD206 Mouse
B F J Mouse
Islet Islet Human
G * Iba1 Iba1 PDGFRβ
Mouse CD206 Mouse *
C 80 Peri Peri H 80 K 100 * Intra Intra 80 60 60 Peri
60 Intra -- Area (%) Are a (%) Peri
β 40 40 Intra Peri Peri Intra
40 / Iba1+ Area (%) 20 20 * 20 Intra*
CD206+ Iba1+ / CD31-- For Peer Review Only Iba1+ / PDGF 0 0 0 Human Mouse Human Mouse Human Mouse A B Diabetes C Page 34 of 55
30000 80 * M * 25000 INS 60 CSF1R 20000 I 15000 40 CD14 Number 10000 Number L 20 Copy
5000 Copy 0 0 IML IML CD45 D E F 5000 15000 1500 10000 4000 1000 L1 B IL6 I TNF 500 5000 3000 200 Number Number 30 2000 Number 150 20 1000 100 Copy Copy Copy 10 50 0 0 0 IML IML IML G H I * 600 * 100 100 80 L1 0 I MMP 2 MMP 9 400 60
50
Number 40
200 Number Number 20 Copy Copy For PeerCopy Review Only 0 0 0 IML IML IML _ PageA 35 of 55 Diabetes B CD14 100 μm
Endocrine
* CD45
Exocrine
20 μm _
* CD45 * CD14 Blood vessel Fluo-4 Backscatter Living human pancreas slice Merge + backscatter
C 3G E 3G 16G F 3G 16G 16G + Sur 3G ATP F/F Δ F/F 0.5 Δ 0.5
10 min 10 min 0 97
G 16G 16G + Nif ATP H 250 * D ATP ) 200 * 150 100 Endocrine 50 30 F/F Δ 20 0.5 10 Area Under Curve (AU For Peer Review Only 0 20 μm Macro 3G _ 16G ATP 10 min 16G+NIF A Gene Expression B DiabetesC D Page 36 of 55 20 400 200 300 15 300 100 200 10 200 10 100 5 100 5
Copy Number P2X 7 0 Copy Number P2X 4 0 Copy Number P2Y 6 0 Copy Number P2Y 2 0 IML IML IML IML
E Physiology F G Macrophage Responses 2768 2693 ATP S BzATP ATPγS γ 120
80 Macro Macro 40
F/F 20 Δ F/F Δ 0.5 10 0.5
Area Under Curve (AU ) 0
Endocrine Endocrine 3G 2768 2693 2 min 2 min BzATP ATPγS
H Secretion I J K * * * * * 4 3 g DNA) 20 g DNA ) g DNA ) 1000 g DNA ) 3 2 15 2 10 500 1 1
5 release (pg / μ TNF release (pg / μ MMP9 release (pg / μ IL10 release (pg / μ 0 0 IL1 β 0 0
oATP oATP oATP oATP ControlATPγS ControlATPγS ControlATPγS ControlATPγS L M Exogenous stimulation with ATPγS Block of endogenous signaling with oATP
e) 4 e) 2 3 ***** 2 1 1 Secretion (fold chang Secretion (fold chang 0 *** 0 For Peer Review Only IL6 IL2 IL6 IL2 IL1 IL10 TNF IL17 IL12 IL13 IL1 IL10 TNF IL17 IL12 IL13 MMP9 G-CSF MCP-1 MIP-1 MMP9 G-CSF MCP-1 MIP-1 GM-CSF GM-CSF APage 37 of 55 Mouse (HFD) B Diabetes C D Human (T2D) Downregulated Upregulated 200 15 P2X1 * * 600 * 0.0001 P2Y12 150 500 TP M MM P9
- - 10 0.001 P2X7 P2Y1 400 100 NS valu e P2X4 300 P 0.01 P2Y13 NS il10 - TP M
P2Y6 mmp9 5 200 50 0.1 100 Copy Number 1.0 0 0 0 -8 -4 -2 1 2 4 8 IIPI -16 IIPI T2D Fold Change HFD +- - + HFD +- - + E F G H I p = 0.075 R2 = 0.257 p = 0.077 R2 = 0.370 p = 0.1374 p = 0.061 125 400 400 20 20
100 IL1 0 300 300 15 15 75 200 200 10 10 50 100 100 5 5 25 Copy Number Copy Number P2X 4 Copy Number P2X 4 Copy Number P2X 7 Copy Number P2X 7 0 0 0 0 0 20 25 30 35 40 45 20 25 30 35 40 45 T2D T2D BMI T2D BMI J K L M NS NS NS 10000 10000 100000 1.0
10000 0.8 IL1 β 1000 TNF α 1000 IL6 IFN γ 1000 0.6 100 100 100 0.4 10 10 For Peer Review Only 10 0.2 Not Detected Copy Number Copy Number Copy Number Copy Number 1 1 1 0.0 T2D T2D T2D T2D Diabetes Page 38 of 55
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For Peer Review Only Page 39 of 55 Diabetes
SUPPLEMENTARY MATERIAL
Figure S1
A B C 300 ) 9
PTPRC 200
CD14 100 SSC Area (x 10 Copy Number 0 IML
CD45 CD45 D E F G 80 1.0 40 0.10
0.8 0.08 60 30 IL4 CD36 CD3d IFNg 0.6 0.06 40 20 0.4 0.04 20 ND 10 0.2 0.02 Copy Number Copy Number Copy Number Copy Number ND ND 0 0.0 0 0.00 IML IML IML IML
Sorting of unique human islet leukocytes populations
A: Gating on SSC for granularity difference in all leukocytes CD45+ (black box, right panel). B:
No antibody control. C-D: Validation of expression from FACS sorted mixed endocrine cells
(CD45-, CD14-), macrophages (CD45+, CD14+), and lymphocytes (CD45+, CD14-).
Quantification of mRNA levels used for validation of individual population identity shown in;
leukocytes (macrophages and lymphocytes) PTPRC (ie., CD45) (C) and lymphocytes CD3d (D).
Values are from n = 6 independent donor isolations (median ± interquartile range; *P < 0.05
ANOVA followed by Tukey’s test for multiple comparisons). E-G: Differences in gene expression
between endocrine cells, macrophages, and lymphocytes for the following genes; IFN (E), CD36
(F) and IL4 (G). Values are from n = 6 independent donor isolations (median ± interquartile range;
*P < 0.05 ANOVA followed by Tukey’s test for multiple comparisons).
For Peer Review Only Diabetes Page 40 of 55
Figure S2 A
B
Matrix metalloproteinase expression in human pancreas cell subsets
A and B: Differences in macrophage (A) and activated stellate cell (B) gene counts in a subset of randomized single cells sorted from 4 human donors.
For Peer Review Only Page 41 of 55 Diabetes
Figure S3
20
Imaging of human tissue lymphocytes (CD45+, CD14-) in the pancreatic tissue
microenvironment
A: Confocal image taken during a live video recording of a living pancreatic tissue slice. The
2+ pancreatic tissue slice was incubated with the [Ca ]i indicator Fluo-4 (green), as well as CD14
(blue), and CD45 (red). The reflective cellular structures (islet backscatter) represent endocrine
tissue (grey). Stars (*) denoting lymphocytes (CD45+, CD14-) were loaded with Fluo-4 (green).
2+ B: Representative traces of individual pancreatic lymphocytes showing [Ca ]i increases when
exposed to ATPS (100 M) but not to BzATP (10 M).
For Peer Review Only Diabetes Page 42 of 55
Movie S4
A B
* * * *
* *
CD45 CD14 Fluo-4 Fluo-4 Backscatter Backscatter
2+ Live [Ca ]i imaging of macrophage responses in a living human pancreatic tissue slice.
2+ A: Still frame image of a video recording with the [Ca ]i indicator Fluo-4 (green). The reflective cellular structures (islet backscatter) represent endocrine tissue (grey). Stars (*) denoting macrophages (CD45+, CD14+) were loaded with Fluo-4 (green). Video time starts at 00:00 displayed in minutes where 01:00 = 1 minute. B: Confocal image taken during a live video recording of a living pancreatic tissue slice. The pancreatic tissue slice was incubated with the
2+ [Ca ]i indicator Fluo-4 (green), as well as CD14 (red), and CD45 (blue). The reflective cellular structures (islet backscatter) represent endocrine tissue (grey). Stars (*) denoting macrophages
(CD45+, CD14+) were loaded with Fluo-4 (green).
For Peer Review Only Page 43 of 55 Diabetes
Figure S5
250 r 150
100
60
20 8
Normalized Copy Numbe 4
ND ND 0 1 2 3 4 5 6 7 1 2 4 5 6 8 9 0 1 2 4 1 A B 3 1 1 1 13 1 2 2
P2X P2Y Adora
Purinergic gene expression in human islet macrophages
Gene expression of purinergic (P2RX, P2RY and Adora) receptors found in human
islet macrophages from n = 7 independent donors. Data are presented as mean +/- SEM.
For Peer Review Only Diabetes Page 44 of 55
Figure S6
Changes in macrophages secretory products during stimulation of purinergic signals
A and B: Cytokine secretion from isolated human islets. Concentrations of drugs (Adenosine 100
M), (,-meATP 1 M). *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.
For Peer Review Only Page 45 of 55 Diabetes
Figure S7
p = .36 A p = .76 B C p = .28 D 600 125 p = .34 20 400
500 100 15 300 IL10 P2X7 MMP9 400 P2X4 75 300 10 200 50 200 5 100
Copy Number 25 Copy Number Copy Number 100 Copy Number
0 0 0 0 ) ) ) ) ) ) ) ) A L A L A L A L C (F C (F C F F ( I ( I ( ( (C ( o o o I I d R d R d R o R o D o D o D d D r N r N r N ro P P P P N
A and B: Isolating pancreatic islets induces loss of islet leukocytes. Macrophage (iba1, green) and
leukocyte (CD45, red) markers are present in islets in slices but not in isolated islets cultured for
72 hours (B). Dotted line in A denotes pancreatic islet. Scale bar = 40 m, applies to A and B. C:
No differences could be detected in macrophage gene expression between islets from Prodo in
California and the local facility at the Diabetes Research Institute. There were no differences in
gene expression (CT 2-[CT gene(x) – CT (housekeeping)])) for MMP9, IL10, P2X7 and P2X4. For islets
shipped from California, a “rest” period was added after shipping for a total 16-24 hours. Islets
obtained in Miami were incubated at 22°C for 16-24 hours. Notice that sample sizes were small.
For Peer Review Only Diabetes Page 46 of 55
Movie S8
Backscatter Fluo-4
Live imaging of human islet lymphocyte translational movement via a vasculature scaffold.
Still frame image of a video recording of a maximal projection image taken during a live video
2+ recording of a living pancreatic tissue slice. The [Ca ]i indicator Fluo-4 is displayed in a
2+ pseudocolor scale where the amount of [Ca ]i is related to the fluorescent intensity. The reflective cellular structures (islet backscatter) represent endocrine tissue (grey). Stars (*) denoting lymphocytes (CD45+, CD14-) were identified a priori, based on antibody labeling. Video time starts at 00:00 displayed in minutes where 01:00 = 1 minute.
For Peer Review Only Page 47 of 55 Diabetes
Figure S9
Donor – Islet Isolation Age Gender Height Weight BMI COD Source Diabetic
HP2297 56 F 67 170 26.6 Stroke DRI No HP2298 49 M 70 181 26 ICH/Stroke DRI No HP18232-CTRL 33 F 68 200 30.3 Head Trauma Prodo No HP2299 49 M 65 169.6 28.3 Anoxia DRI No HP2303 48 M 69 165 24.4 Head Trauma DRI No HP-18310-01 38 M 69 190 28 Head Trauma Prodo No HP1695 55 F x x 32 CNS Tumor DRI No HP1697 50 M x x 27 Brain Tumor DRI No HP18212-01T2D 40 M 69 185 27.7 Stroke Prodo Yes HP18243-01T2D 51 M 65 228 37.1 Anoxia Prodo Yes HP-18275-01T2D 30 F 64 233 40.1 Asthma Prodo Yes HP-19051-01T2D 53 M 65 190 30.1 Head Trauma Prodo Yes
Donor - Tissue Slices
HP6462 13 F x x 15.2 Anoxia nPOD No HP2298 49 M 70 181 26 ICH/Stroke DRI No HP2299 49 M 65 169.6 28.3 Anoxia DRI No HP2303 48 M 69 165 24.4 Head Trauma DRI No HP6468 16 M x x 15.8 Anoxia nPOD No
Donor - IHC
NE Organ HP2111A 31 M 75 254 31.7 Head Trauma Bank No NE Organ HP2096A 51 F 63 145 25.7 Head Trauma Bank No Live Share of HP2102A 43 F 62 129.3 23.6 Stoke Carolina No HP2116A 53 M 69 196 28.9 Head Trauma NDRI No HP2117A 28 M 74 165.3 21.1 Head Trauma IIAM No Live Link of HP2119A 69 F 64 204 35 Stroke Florida Yes
For Peer Review Only Diabetes Page 48 of 55
Figure S10
Surface Marker Citation
CD11b Butcher et al., 2014 CD11c Butcher et al., 2014 MHC-II Ehses et al., 2007 CD68 Ehses et al., 2007; Richardson et al., 2009; Kamata et al., 2014, Eguchi et al., 2016 CD14 ** CD45 Butcher et al., 2014, ** CD86 Butcher et al., 2014 CD163 Ehses et al., 2007; Kamata et al., 2014 CD204 Kamata et al., 2014 CD206 ** ** identified in current manuscript
For Peer Review Only Page 49 of 55 Diabetes
Figure S11
Gene ID Probe Gene ID Probe p2rx1 Hs01039860_g1 ADORA2B Hs00386497_m1 p2rx2 Hs04176268_g1 ADORA3 Hs00181232_m1 p2rx3 Hs01125554_m1 IL1B Hs01555410_m1 p2rx4 Hs00902156_g1 TNF Hs00174128_m1 p2rx5 Hs01112471_m1 IFNy Hs00989291_m1 p2rx6 Hs01003997_m1 IL6 Hs00174131_m1 p2rx7 Hs00175721_m1 iNOS Hs01075529_m1 p2ry1 Hs00704965_s1 IL-10 Hs00961622_m1 p2ry2 Hs01856611_s1 IL-4 Hs00174122_m1 p2ry4 Hs00267404_s1 ARG Hs00163660_m1 p2ry5 Hs00271758_s1 PTPRC Hs04189704_m1 p2ry6 Hs00366312_m1 CSF1R Hs00911250_m1 p2ry8 Hs01938524_s1 CD3D Hs00174158_m1 p2ry9 Hs00271072_s1 CD19 Hs01047413_g1 p2ry10 Hs00274326_s1 GAPDH Hs02786624_g1 p2ry11 Hs00267414_s1 ITGAM Hs00167304_m1 p2ry12 Hs01881698_s1 MMP9 Hs01548727_m1 p2ry13 Hs00256749_s1 MMP2 Hs00957562_m1 p2ry14 Hs01848195_s1 AMYLIN Hs00169095_m1 ADORA1A Hs00181231_m1 CD36 Hs00354519_m1 ADORA2A Hs00169123_m1 INS Hs00355773_m1
For Peer Review Only Diabetes Page 50 of 55
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/ DB19-0687
Title: Homeostatic functions of macrophages in the human pancreatic islet are regulated by purinergic signals from beta cells
Author list: 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
Corresponding author: Jonathan Weitz Email address: [email protected]
Islet preparation 1 2 3 4 5 6 7 8a
MANDATORY INFORMATION
Unique identifier HP2297 HP2298 HP18232 HP2299 HP2303 HP18310-01 HP1695 HP1697
Donor age (years) 56 49 33 49 48 38 55 50
Donor sex (M/F) F M F M M M F M
Donor BMI (kg/m2) 26.6 26 30.3 28.3 24.4 28 32 27
For Peer Review Only Page 51 of 55 Diabetes
Donor HbA1c or other measure of blood glucose 8.0 7.2 4.9 6.3 5.6 5.9 6.6 6.2 control
Origin/source of isletsb NDRI NDRI Prodo NDRI NDRI Prodo Life Alliance Life Alliance
Islet Cell Islet Cell Islet Cell Islet Cell Islet Cell Islet Cell Islet isolation centre Resource Resource Prodo Labs Resource Resource Prodo Labs Resource Resource Center Center Center Center Center Center Donor history of No No No No No No No No diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years)
Glucose-lowering therapy
at time of deathc
RECOMMENDED INFORMATION
Head Head Head Donor cause of death ICH/Stroke Stroke Anoxia CNS Tumor Brain Tumor Trauma Trauma Trauma
Warm ischaemia time (h)
Cold ischaemia time (h)
Version 1.0, created 16 Nov 2018 For Peer Review Only Diabetes Page 52 of 55
Estimated purity (%) NA NA 80-85 NA NA 85-90 85-80 95
Estimated viability (%) NA NA 95 NA NA 95 85 95
Total culture time (h)d 12 hours 12 hours 12 hours 12 hours 12 hours 12 hours 12 hours 12 houts
Glucose-stimulated insulin secretion or other functional measuremente Handpicked to purity?
Yes/No Additional notes 3 days 4 days 4 days 4 days 2 days 2 days ventilator ventilator ventilator ventilator ventilator ventilator
aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018 For Peer Review Only Page 53 of 55 Diabetes
Checklist for Reporting Human Islet Preparations Used in Research
Adapted from Hart NJ, Powers AC (2018) Progress, challenges, and suggestions for using human islets to understand islet biology and human diabetes. Diabetologia https://doi.org/10.1007/s00125-018-4772-2.
Manuscript DOI: https://doi.org/10.2337/ DB19-0687
Title: Homeostatic functions of macrophages in the human pancreatic islet are regulated by purinergic signals from beta cells 1 2 2,3 2 1 1,3 Author list: Jonathan R. Weitz , Carol Jacques-Silva , Mirza Muhammed Fahd Qadir , Oliver Umland , Elizabeth Pereira , Farhan Qureshi , Formatted: Line spacing: single Alejandro Tamayo1, Juan Dominguez-Bendala2,3,5, Rayner Rodriguez-Diaz1, Joana Almaça1, Alejandro Caicedo1,2,3,4,6
Corresponding author: Jonathan Weitz Email address: [email protected]
Islet preparation 1 2 3 4 5 6 7 8a
MANDATORY INFORMATION
HP18212- HP18243- HP18275- Unique identifier 19051-01T2D 01T2D 01T2D 01T2D
Donor age (years) 40 51 30 53
Donor sex (M/F) M M F M
Donor BMI (kg/m2) 27.7 37.1 40.1 30.1
For Peer Review Only Diabetes Page 54 of 55
Donor HbA1c or other measure of blood glucose 7.4 6.2 6.5 7.8 control
Origin/source of isletsb Prodo Prodo Prodo Prodo
Islet isolation centre Prodo Labs Prodo Labs Prodo Labs Prodo Labs
Donor history of Yes Yes Yes Yes diabetes? Yes/No
If Yes, complete the next two lines if this information is available
Diabetes duration (years) Recent NA NA NA
Oral Med, Glucose-lowering therapy c Diet Oral Med. Diet Glipizide, at time of death Januvia
RECOMMENDED INFORMATION
Head Donor cause of death Stroke Anoxia Asthma Trauma
Warm ischaemia time (h)
Cold ischaemia time (h)
Version 1.0, created 16 Nov 2018
For Peer Review Only Page 55 of 55 Diabetes
Estimated purity (%) 80 90 85 80
Estimated viability (%) 95 95 95 95
Total culture time (h)d 12 hours 12 hours 12 hours 12 hours
Glucose-stimulated insulin secretion or other functional measuremente Handpicked to purity?
Yes/No Additional notes
aIf you have used more than eight islet preparations, please complete additional forms as necessary bFor example, IIDP, ECIT, Alberta IsletCore cPlease specify the therapy/therapies dTime of islet culture at the isolation centre, during shipment and at the receiving laboratory ePlease specify the test and the results
Version 1.0, created 16 Nov 2018
For Peer Review Only