Mouse pancreatic islet macrophages use locally released ATP to monitor beta cell activity.

To investigate how islet-resident macrophages respond to cues from the microenvironment, we generated mice expressing a genetically encoded Ca 2+ indicator in myeloid cells. We produced living pancreatic slices from these mice and used them to monitor macrophage responses to stimulation of acinar, neural and endocrine cells.

Methods Mice

First filial generation (F1) mice for Ca 2+ imaging experiments were bred by crossing mice that contain the fluorescent calcium indicator, GCaMP3 (Rosa-GCaMP3 mice; B6, stock no. 014538; JAX laboratories, Bar Harbor, ME, USA), with mice expressing Cre recombinase in myeloid cell-specific promoters: ( Lyz M)- Cre (B6, stock no. 004781; JAX laboratories); Tg( Csf1r -i Cre )1Jwp (referred to herein as Csf1r - Cre ; FVB, stock no. 021024; JAX laboratories); and Cx3cr1 cre mice (B6, stock no. 025524; JAX laboratories). Mice were housed in a temperature- and lighting-controlled room (lights on for 12 h). F1 offspring expressing GCaMP3 in macrophages (≥ 3 months old, both male and female) were euthanised and pancreatic tissue slices were processed as described below. All experimental protocols using mice were approved by the University of Miami Animal Care and Use Committee.

Preparation of living pancreatic tissue slices

Tissue slices were prepared from 15 young adult mice (25–30 g) as described [ 14 ]. Mice were anaesthetised with isofluorane (2% vol./vol.) and euthanised by cervical dislocation to prepare for pancreatic duct injection of low-gelling-temperature agarose (1.2% [wt/vol.], dissolved in HEPES-buffered solution as described below, without BSA; catalogue no. 39346-81-1; Sigma Aldrich, St. Louis, MO, USA). Syringes (5 ml) were filled with agarose solution, and a 30-gauge needle was used to inject in the common bile duct. After injection, tissue blocks were cut, imbedded and left to solidify (4°C). Living slices were then cut (150 μm) on a vibroslicer (Leica VT1000s, Leica Biosystems, Buffalo Grove, IL, USA). Slices were incubated in HEPES-buffered solution (125 mmol/l NaCl, 5.9 mmol/l KCl, 2.56 mmol/l CaCl 2 , 1 mmol/l MgCl 2 , 25 mmol/l HEPES, 0.1% BSA [wt/vol.], pH 7.4).

Immunohistochemistry

Mice were anaesthetised, exsanguinated and perfused with 4% (wt/vol.) paraformaldehyde. Blocks of mouse pancreas (0.5 cm 3 ) were post-fixed in 4% paraformaldehyde, cryoprotected (30% [wt/vol.] sucrose) and tissue sections (40 μm) cut on a cryostat. After permeabilisation (PBS–Triton X-100 0.3% [wt/vol.]), sections were incubated in blocking solution (Biogenex, San Ramon, CA, USA). Primary antibodies were diluted in blocking solution. To visualise macrophages, we used antibodies against ionised calcium-binding adapter molecule 1 (IBA1) (1:1000; catalogue no. 019-19741, Wako Chemicals, Richmond, VA, USA), F4/80 (1∶200, catalogue no. ab6640; Abcam, Cambridge, MA, USA) and CD206 (1∶100, catalogue no. 141721; Biolegend San Diego, CA, USA). We performed immunostaining for green fluorescent protein (GFP) (1∶200, catalogue no. ab6658; Abcam) to visualise GFP expression under the control of the myeloid promoters (see JAX laboratories mice below). Cell nuclei were stained with DAPI. Slides were mounted with ProLong Anti Fade (Invitrogen, Waltham, MA, USA). All antibodies were validated and used as per manufacturer’s instructions. See electronic supplementary materials (ESM) Methods for additional details. Ca 2+ imaging of living pancreatic tissue slices We generated mice for [Ca 2+ ] i imaging by using F1 mice crossed from mice expressing Cre recombinase in myeloid cell-specific promoters with mice expressing the floxed Ca 2+ indicator GCaMP3 (see above). Mice (≥ 3 months old, both sexes) were euthanised, and pancreatic tissue slices were processed as described above. Living tissue slices containing GCaMP3-labelled macrophages were placed in a perfusion chamber and immersed in HEPES-buffered solution. 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 non-hydrolysable ATP agonist ATPγS (adenosine 5′-(3-thiotriphosphate; Tocris Biosciences, Bristol, UK). Antagonists were left to equilibrate with receptors for 5 min before stimulation with an agonist. For [Ca 2+ ] i imaging, a Z-stack of ~15–30 confocal images was acquired every 8 s using a Leica SP5 confocal laser-scanning microscope. [Ca 2+ ] i responses in pancreatic macrophages were quantified as the AUC of individual traces of GCaMP3 fluorescence intensity (expressed as change in fluorescence intensity compared with baseline fluorescence [ΔF/F]) during the application of stimuli. To be included in the analyses, [Ca 2+ ] i responses had to be reproducible in three or more pancreatic slices. We further analysed and quantified pseudopodia movement and velocity using the ImageJ plugin MTrackJ ( https://imagescience.org/meijering/software/mtrackj/ , accessed 14 October 2016.). For immunohistochemical staining after [Ca 2+ ] i imaging, slices were processed as described in ESM Methods (Immunohistochemistry section).

Confocal imaging

Confocal images (pinhole = 1 airy unit) of randomly selected islets were acquired on a Leica SP5 confocal laser-scanning microscope with 40× magnification (NA = 0.8). Macrophages were reconstructed in Z-stacks of 15–30 confocal images (step size = 2.5–4.0 μm) and analysed using ImageJ (version 1.51h; http://imagej.nih.gov/ij ). Using confocal images, we established the location of macrophages within islets (endocrine) or in acinar regions (exocrine). Experiments were not blinded. To prevent bias, we used an automated method in ImageJ to segment different pancreas regions based on DAPI staining to determine macrophage positions.

Flow cytometry

Islet macrophages were sorted based on the viable, GFP + and F4/80 + labelled cells. For non-macrophage internal controls, islet cells were also sorted based on the viable GFP − , F4/80 − population. See ESM Methods for additional details. RT-PCR RNA was extracted from FACS sorted islet macrophages and non-macrophage internal controls using RNeasy mini kit (Qiagen, Valencia, CA), and cDNA was prepared using the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). cDNA products were pre-amplified 10 cycles using the TaqMan pre-amp master mix (Applied Biosystems). PCR reactions were run using TaqMan gene expression assays (Applied Biosystems) in a StepOnePlus Real-Time PCR System (Applied Biosystems). Relative quantification of gene expression was done based on the equation relative quantification = 2 −ΔCt × 1,000,000 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 18s . Genes analysed: Csf1r , 18s (also known as Rn18s ), Gapdh , P2rx1, P2rx2, P2rx3, P2rx4, P2rx5, P2rx6, P2rx7, P2ry1, P2ry2, P2ry4, P2ry6, P2ry10, P2ry12, P2ry13, P2ry14, Adora1, Adora2a Adora2b, Adora3 . For fold change comparisons, we used the delta delta Ct method, calculated by 2 ΔΔCt . To ensure for consistent qPCR results, 18s normalised samples were compared to that of Gapdh normalised samples. Fold change differences were less than a 1.2-fold between groups when normalised to 18s vs Gapdh . Genes analyzed: Csf1r , 18s , Gapdh, Tnf, Il1b, Tgfb1, Il6, Mmp2, Mmp9, Egf, Vegf (also known as Vegfa ).

Data analyses and statistics

To quantify [Ca 2+ ] i responses we calculated the AUC of the fluorescence intensity traces of GCaMP3. Our criteria for accepting [Ca 2+ ] i responses for analyses were that responses could be elicited two or more times by the same stimulus and that the peak signal was two or more 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 means ± SEM. See ESM Methods for data analysis of immunohistochemical results.

Supplementary Material 125_2017_4416_MOESM1_ESM 125_2017_4416_MOESM2_ESM