A Novel Function for P2Y2 in Myeloid Recipient−Derived Cells during Graft-versus-Host Disease

This information is current as Verena Klämbt, Sebastian A. Wohlfeil, Lukas Schwab, Jan of September 29, 2021. Hülsdünker, Korcan Ayata, Petya Apostolova, Annette Schmitt-Graeff, Heide Dierbach, Gabriele Prinz, Marie Follo, Marco Prinz, Marco Idzko and Robert Zeiser J Immunol 2015; 195:5795-5804; Prepublished online 4

November 2015; Downloaded from doi: 10.4049/jimmunol.1501357 http://www.jimmunol.org/content/195/12/5795

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Novel Function for P2Y2 in Myeloid Recipient–Derived Cells during Graft-versus-Host Disease

Verena Kla¨mbt,*,1 Sebastian A. Wohlfeil,*,1 Lukas Schwab,* Jan Hulsd€ unker,*€ ,†,‡ Korcan Ayata,x Petya Apostolova,* Annette Schmitt-Graeff,{ Heide Dierbach,* Gabriele Prinz,* Marie Follo,* Marco Prinz,‖,# Marco Idzko,x and Robert Zeiser*,#

Acute graft-versus-host disease (GvHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. During the initiation phase of acute GvHD, endogenous danger signals such as ATP are released and inform the innate immune system via activation of the purinergic P2X7 that a noninfectious damage has occurred. A second ATP-activated involved in inflammatory diseases is P2Y2. In this study, we used P2y22/2 mice to test the role of this receptor in GvHD. P2y22/2 recipients experienced reduced GvHD-related mortality, IL-6 levels, enterocyte apoptosis, and histopathology scores. Chimeric mice with P2y2 deficiency restricted to hematopoietic tissues survived longer after GvHD induction than did wild-type mice. P2y2 Downloaded from deficiency of the recipient was connected to lower levels of myeloperoxidase in the intestinal tract of mice developing GvHD and a reduced myeloid cell signature. Selective deficiency of P2Y2 in inflammatory monocytes decreased GvHD severity. Mechanisti- cally, P2y22/2 inflammatory monocytes displayed defective ERK activation and reactive oxygen species production. Compatible with a role of P2Y2 in human GvHD, the frequency of P2Y2+ cells in inflamed GvHD lesions correlated with histopathological GvHD severity. Our findings indicate a novel function for P2Y2 in ATP-activated recipient myeloid cells during GvHD, which could be exploited when targeting danger signals to prevent GvHD. The Journal of Immunology, 2015, 195: 5795–5804. http://www.jimmunol.org/

cute graft-versus-host disease (GvHD) occurs after al- leads to tissue injury with a subsequent release of pathogen- logeneic hematopoietic cell transplantation (allo-HCT) associated molecular patterns (PAMPs) or noninfectious damage- A and is dependent on T cell recognition of host alloanti- associated molecular patterns (DAMPs) (4). The maturation status gens displayed at the surface of hematopoietic (1) and non- of APCs determines the strength of the T cell response (5), whereas hematopoietic (2) APCs (3). Additionally, the conditioning regimen maturation itself is induced by the recognition of PAMPs or DAMPs (4).

Myeloid cell populations including dendritic cells (DCs) (1), by guest on September 29, 2021 *Department of Hematology and Oncology, University Medical Center Freiburg, neutrophils (6–8), macrophages (9), and certain monocyte subsets 79106 Freiburg, Germany; †Spemann Graduate School of Biology and Medicine, University of Freiburg, 79104 Freiburg, Germany; ‡Faculty of Biology, University (10, 11) were also found to enhance or reduce GvHD. Recently, x of Freiburg, 79104 Freiburg, Germany; Department of Pneumology, University we described that neutrophils were recruited into the gastroin- Medical Center Freiburg, 79106 Freiburg, Germany; {Institute of Pathology, Univer- sity Medical Center Freiburg, 79106 Freiburg, Germany; ‖Institute of Neuropathol- testinal tract during GvHD (7) where they contribute to the disease ogy, University Medical Center Freiburg, 79106 Freiburg, Germany; and #BIOSS via the production of reactive oxygen species (ROS). Other innate Centre for Biological Signalling Studies, Albert Ludwigs University of Freiburg, immune cells, which are recruited to inflammation sites, are 79104 Freiburg, Germany monocytes. Monocytes are separated into two subsets in mice (12) 1V.K. and S.A.W. contributed equally to this work. and are equipped with different chemokine receptors and surface ORCIDs: 0000-0001-7043-6692 (K.A.); 0000-0002-8263-5768 (G.P.). molecules. Mouse monocytes are separated into an inflammatory Received for publication June 15, 2015. Accepted for publication October 11, 2015. (Ly-6Chigh, CCR2high, and CX3CR1low) and a patrolling subset This work was supported by Wilhelm Sander-Stiftung Grant 2008.046.3 (to R.Z.), (Ly-6Clow, CCR2low, and CX3CR1high). Deutsche Forschungsgemeinschaft, Germany, Heisenberg Professorship ZE 872/3-1 (to R.Z.), the Mildred-Scheel-Doktorandenprogramm of the Deutsche Krebshilfe (to Previously, we have shown that extracellular ATP released from S.A.W.), and the Excellence Initiative of the German Research Foundation (GSC-4, dying or stressed cells serves as an endogenous DAMP in GvHD Spemann Graduate School) (to J.H.). (13, 14). A genetic lack of the ecto-59-nucleotidase CD73, which V.K. and S.A.W. helped design the studies, performed most of the experiments, and metabolizes AMP, enhances GvHD (15). Extracellular helped write the manuscript; L.S., K.A., P.A., and J.H. performed experiments and helped analyze data; A.S.-G. analyzed murine GvHD histopathology and human metabolites can be sensed by the P2X and P2Y receptors. P2X tissue staining for P2Y2; H.D., G.P., M.F., M.P., and M.I. helped design and perform receptors are nonselective cation channels that open after the experiments; and R.Z. developed the overall concept and wrote the manuscript. All authors have read and agreed to the final version of the manuscript. binding of extracellular ATP. Whereas P2Y receptors are coupled Address correspondence and reprint requests to Prof. Robert Zeiser, Department of to heterotrimeric –binding proteins (G proteins) Hematology and Oncology, University Medical Center Freiburg, Hugstetterstrasse and get activated by various such as ADP, ATP, UDP, 55, 79106 Freiburg, Germany. E-mail address: [email protected] UTP, or UDP-glucose. Recently, a proinflammatory role for P2X7 The online version of this article contains supplemental material. has been observed in cardiac allograft survival (16) and islet allograft Abbreviations used in this article: allo-HCT, allogeneic hematopoietic cell transplan- rejection(17).Similarly,theactivationofP2Y2wasshowntopro- tation; BLI, bioluminescence imaging; BM, bone marrow; BMDC, BM-derived DC; DAMP, damage-associated molecular pattern; DC, dendritic cell; GvHD, graft- mote tissue damage in airway inflammation (18, 19) and acute liver versus-host disease; HCT, hematopoietic cell transplantation; MPO, myeloperoxi- injury (20), whereas P2Y2 also showed protective effects in a model dase; PAMP, pathogen-associated molecular pattern; ROS, reactive oxygen species; of lung infection induced by pneumonia virus of mice (21). A TBI, total body irradiation; WT, wild-type. functional role for the P2Y2 ligand ATP was found in different in- Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 flammatory diseases, including inflammatory bowel disease (22), www.jimmunol.org/cgi/doi/10.4049/jimmunol.1501357 5796 P2y2 DEFICIENCY REDUCES GvHD glomerulonephritis (23), asthma (24), and diabetes (22). However, described (30). BLI for the detection of active myeloperoxidase was per- all these models differ from the situation that occurs after allo-HCT, formed as follows: 200 mg/kg body weight luminol was dissolved in when donor T cells with a suitable TCR are activated via foreign distilled water and injected i.p. Ten minutes after application the mice were imaged in the bioluminescence camera with 5 min of luminescent expo- MHCs. sure as previously described (31). In the light of previous findings indicating a role for both P2Y2 and ATP in inflammation in various model systems, the goal of this Cytokine measurements in serum of mice with GvHD study was to delineate the role of P2Y2 in acute GvHD. Our data IL-6, IL-10, IFN-g, TNF-a, IL-12, and MCP-1 were measured in serum clearly indicate that P2Y2 plays a major role for myeloid innate collected from transplant recipients 7–8 d after allo-HCT using a bead immune cells, such as inflammatory monocytes, with respect to their array kit (BD Biosciences). contribution to GvHD, migratory potential, and production of ROS. Generation of BM-derived DCs BM-derived DCs (BMDCs) were prepared as described (32), except not Materials and Methods adding IL-4. These BMDCs were used on days 7–9 of culture. Human subjects Generation of monocytes All samples were collected after approval by the Ethics Committee of the Albert Ludwigs University of Freiburg (protocol no. 267/11) and after The generation of mature murine monocytes from BM was performed with written informed consent. Intestinal tissue biopsies were collected in a slight modifications as previously described (33). Murine BM cells were 6 prospective manner from individuals undergoing allo-HCT with or without cultured at 8 3 10 cells/ml in an uncoated, nonadherent 10-cm petri dish GvHD. GvHD grading was performed on the basis of histopathology (Greiner Bio-One) to inhibit adherence-mediated maturation. M-CSF according to a published staging system (25). (PeproTech) was added at a final concentration of 20 ng/ml. On day 3 supernatant was taken off containing BM-derived monocytes and macro- Downloaded from Mice phages. Up to 15% of all harvested cells were characteristically described as inflammatory monocytes (CD11b+, CD115+, Ly-6Chigh). When applied C57BL/6 (H-2Kb) and BALB/c (H-2Kd) mice were purchased from the local in vivo, inflammatory monocytes were enriched by cell sorting. stock of the animal facility at University of Freiburg or obtained from The 2/2 + Jackson Laboratory. The P2y2 mice (20) and the luciferase (luc )trans- Cell migration assay genic mice (26) (both C57BL/6) have been previously described. We gen- erated luc+ P2y22/2 mice by crossing luc+ mice and P2y22/2 mice. The cell migration assay has been described previously (34). After incu- http://www.jimmunol.org/ Genotypes were confirmed by PCR. All mice were raised under specific bation for 3 h at 37˚C with 5% CO2, the number of cells migrating into the pathogen-free conditions. Mice were used between 6 and 12 wk of age. All bottom of the Transwells and remaining in the insert were quantified by animal protocols were approved by the University Committee on the Use and flow cytometry for 40 s at high pressure. Care of Laboratory Animals at University of Freiburg (protocol no. G-12/34). pERK detection by phospho-flow Allo-HCT model and induction of GvHD Prior to phosphoflow analysis, cells were stimulated with 2 mM ATP. After Allo-HCT experiments were performed as previously described (27). centrifugation (1200 rpm, 8–10 min, 20˚C) the cells were fixed in fixation Briefly, recipients received myeloablative total body irradiation (TBI) (10 medium (Fix and Perm cell fixation and cell permeabilization kit, Life Gy for C57BL/6, 9 Gy for BALB/c) in two equally split doses, 4 h apart, Technologies) at 20˚C for 30 min. After permeabilization, the cells were followed by i.v. injection of 5 3 106 bone marrow (BM) cells. For in- washed with PBS. Phospho-ERK Ab (Cell Signaling Technology) was + + duction of acute GvHD, CD4 and CD8 splenic T cells were enriched added at a dilution of 1:50 in permeabilization medium and cells were by guest on September 29, 2021 with CD4/CD8 MACS microbeads and LS columns (Miltenyi Biotec). The incubated 2 h at 20˚C protected from light. To get rid of excess Ab, the number of injected T cells varied depending on the transplant model: cells were washed twice with PBS. Bound primary Ab was detected using C57BL/6 → BALB/c (0.3 3 106 T cells) and BALB/c → C57BL/6 (0.3 3 an Alexa Fluor 647–conjugated anti-rabbit secondary Ab (Invitrogen), 106, 0.8 3 106, and 1 3 106 T cells). Baytril (enrofloxacin) was added to which was added and then incubated for 1 h at 20˚C in darkness diluted in the drinking water at 0.5 mg/ml for 3–4 wk after allo-HCT. permeabilization medium to a final concentration of 5 mg Ab/ml solution B. Cells were then washed carefully three times with PBS. Finally, ex- Generation of chimeric recipients with P2y2 deficiency of tracellular staining was performed and mean fluorescence intensity was hematopoietic or nonhematopoietic tissues quantified as a readout. All Abs were used at optimized concentrations. 6 +/+ Wild-type (WT) (C57BL/6) recipients were given 5 3 10 P2y2 or CellROX staining for ROS P2y22/2 BM cells (C57BL/6) i.v. after TBI with 10 Gy (2 3 5 Gy). The following donor/recipient pairs were combined: P2y22/2 into P2y2+/+ CellROX Deep Red reagent (Life Technologies) is highly specific for the (hematopoietic system, P2y22/2), P2y2+/+ into P2y22/2 (nonhematopoietic detection of ROS. Cells were stimulated with 100 mM ATP before the level system, P2y22/2), P2y22/2 into P2y22/2 (both systems, P2y22/2), and of ROS was quantified. Staining for ROS with CellROX dye was per- P2y2+/+ into P2y2+/+ (no deficiency). The second HCT (allogeneic) (TBI formed directly after stimulation according to the manufacturer’s recom- with 4.5 Gy; BALB/c T cell dose, 8 3 105) was performed 30 d after the first mendation and evaluated by flow cytometry. HCT (syngeneic). When indicated, chimeric mice with hematopoietic systems deficient in Flow cytometry and mAbs 2/2 3 6 2/2 P2y2 received WT or P2y2 DCs (2 10 )orWTorP2y2 in- All Abs were purchased from BD Pharmingen (Heidelberg, Germany), 3 5 flammatory monocytes (1.5 10 ) on day 0 after allo-HCT. BioLegend, eBioscience (Frankfurt, Germany), Abcam, AbD Serotec, and Histopathology scoring of acute GvHD and histology of human Jackson Immunoresearch Laboratories and used as FITC, Alexa Fluor 488, PE, PE-Cy5, PE-Cy7, PerCP-Cy5.5, Pacific Blue, Alexa Fluor 647, allo- tissues phycocyanin, or allophycocyanin-Cy7 conjugates. The following Abs were Sections of liver and small and large intestine collected 10 d after allo-HCT used for cell surface molecules and flow cytometric analysis: CD4 (RM4-5), were stained with H&E and scored by an experienced pathologist (A.S.G.) CD8a (53-6.7), TNF-a (MP6-XT22), IL-17A (TC11-18H10), IL-4 (11B11), blinded to the treatment groups as previously described (28). Immuno- IL-1b (JK1B-1), IFN-g (XMG1.2), Ly6G (1A8), CD62L (MEL-14), CD44 histochemical staining of the human intestinal biopsies was performed for (IM7), H2kb (AF6-88.5), CD11b (M1/70), CD11c (HL3/N418), CD45 (30-F11), Ly6C (AL-21), CD115 (AFS98), pERK primary Ab (phospho- P2Y2 according to standard protocols (P2Y2 polyclonal Ab PA1-451, 202 204 Thermo Scientific) and counterstained with hematoxylin. p44/42 MAPK [Erk1/2] [Thr /Tyr ], Cell Signaling Technology), and secondary Ab [F(ab9)2 fragment of goat anti-rabbit IgG, Alexa Fluor 647, In vivo bioluminescence imaging Invitrogen]. To exclude dead cells we used the Live/Dead Fixable Aqua dead cell stain kit (Thermo Fisher Scientific). For TNF-a, IL-4, IL-17, For in vivo bioluminescence imaging (BLI), 10 min after i.p. injection and INF-g staining, we cultured and restimulated cells for 4 h using a with 150 mg/g body weight luciferin (D-luciferin 1-(4,5-dimethoxy-2- cell stimulation cocktail (5003; eBioscience). Cells were fixed and nitrophenyl)ethyl ester, Biosynth), mice were imaged using an IVIS100 permeabilized using a Foxp3 kit (eBioscience) or BD cytokine staining charge-coupled device imaging system (Calipers) with an exposure time of (BD Biosciences; for intracellular cytokines) according to the manufacturers’ 5 min (29). In vivo BLI for firefly luciferase was performed as previously instructions. Samples were acquired on a CyAn ADP (Beckman Coulter, The Journal of Immunology 5797

Brea, CA) or LSRFortessa (BD Biosciences) flow cytometer and analyzed Quantitative real-time PCR with FlowJo v7.6.5 (Tree Star, Ashland, OR). To assess Ccl5, Ccl22, and Mmp9 mRNA expression, total RNA was Leukocyte isolation from small intestine for flow cytometry– isolated from the intestines with TRIzol (Invitrogen) and then reverse based analysis and FACS sorting transcribed into cDNA using the SuperScript III first-strand synthesis system (Invitrogen). Quantitative PCR was performed according to stan- The procedure of leukocyte isolation from the small intestine after digestion dard methods on an ABI 7500 sequence detection system (Applied Bio- was described before by our group (35). systems). The reaction mixture consisted of cDNA, SYBR Green super RNA isolation from small intestines mix (Bio-Rad), and 0.5 mM primers (36). Relative expression of the genes for b-actin or b2-microglobulin was determined in duplicate samples The terminal ileum was isolated, homogenized, and the RNA was extracted. according to the method of Pfaffl (37). A melting curve performed at the RNA was isolated according to a standard protocol (Qiagen RNA isolation end of each run and agarose gel electrophoresis verified a single, specific kit) from mice that were untreated or on day 3 after allo-HCT. amplification product for each of the target genes. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. P2y2 deficiency of the recipient causes reduced GvHD mortality. (A–C) Allo-HCTs were performed on lethally irradiated mice as described for the BALB/c into C57BL/6 combination (0.8 3 106 T cells). (A) Follow-up of P2y2+/+ (n =16)andP2y22/2 (n = 16) recipients after allo-HCT is shown. Survival is significantly improved in P2y22/2 mice compared with P2y2+/+ mice (p , 0.0001). The experiment was performed three times, and the results were pooled. (B) The small intestine, large intestine, and liver were isolated on day 8 after allo-HCT, and histopathological changes were scored as described in Materials and Methods. The mean score 6 SD is shown for four animals per group, and the experiment was performed twice with comparable results. The Mann–Whitney U test was applied. (C) IL-6 was determined in the serum of the indicated groups on day 8. The mean score 6 SD is shown; the experiment was performed twice with comparable results. (D) TUNEL staining of the small bowel isolated on day 8 is shown. The number of TUNEL+ (apoptotic) cells is significantly reduced in P2y22/2 mice as compared with P2y2+/+ mice developing GvHD (n =6/group,p = 0.0003). The experiment was performed three times; the mean 6 SD is shown. Representative stains for both groups are shown on the right (red arrow, DAPI+TUNEL+ nucleus of an apoptotic cell; white arrow, unspecific green fluorescence). Scale bars, 50 mm. (E and F) Allo-HCTs were performed on lethally irradiated mice as described for the C57BL/6 into BALB/c combination. (E) Follow-up of mice receiving T cells from P2y2+/+ (n = 11) or P2y22/2 (n = 11) donors after allo-HCT is shown. No difference between mice receiving T cells from P2y22/2 mice compared with P2y2+/+ mice was observed. The experiment was performed twice, and the results were pooled. (F) Serial luciferase-specific imaging was performed with BALB/c WT mice that had undergone allo-HCT with 5 3 106 BM cells and 0.3 3 106 T cells from C57BL/6 luc+ P2y22/2 mice compared with luc+ P2y2+/+ donors. The experiment was performed twice, and the mean 6 SD for each time point is shown, with n = 11 for both groups. 5798 P2y2 DEFICIENCY REDUCES GvHD

FIGURE 2. P2y2 deficiency of the recipient leads to reduced transcription of Ccl5, Ccl22, and Mmp9 in the inflamed small intestine, and P2y2 deficiency in the hematopoietic system reduces GvHD. (A and B) RNA was isolated from the small intestines from mice 3 d after TBI (10 Gy) and allo-HCT (BALB/c into C57BL/6) (A) or from untreated C57BL/6 mice (B) as shown for P2y22/2 or P2y2+/+ recipients. RNA expression levels of Ccl5, Ccl22, and Mmp9 in the small bowel were analyzed using quantitative PCR–based analysis. The mean 6 SD of four to six individual mice for each group is shown, with p values indicated for the respective comparisons. Downloaded from http://www.jimmunol.org/ Immunohistochemistry/fluorescence ples allocated to their genotypes/treatment group. Statistical analyses were performed with GraphPad Prism 4 or 5 software (GraphPad Software). Paraffin-embedded sections of 5-mm thickness were mounted on micro- Data are reported as means 6 SD. When normality was given, an unpaired scope slides (SuperFrost Plus; R. Langenbrinck). Evaluation of tissue t test (two-sided) was applied. When the data did not meet the criteria of sections was performed on a Zeiss Axioplan 2 microscope (Zeiss, Jena, normality, the Mann–Whitney U test was applied. A p value ,0.05 was 3 Germany). The standard objectives used were 20 (numerical aperture of considered to be statistically significant. 0.45) and 340 (numerical aperture of 0.60). Digital photos from the mi- croscope were obtained using a Spot digital camera. For immunoenzymatic staining, the tissue was fixed for 10 min in acetone (Sigma-Aldrich) and Results the primary Ab was applied (anti-P2Y2 1:50; Abcam). Sections were then

P2y2 deficiency of the recipient leads to reduced GvHD by guest on September 29, 2021 incubated with a specific secondary biotinylated Ab. Streptavidin HRP and 3,39-diaminobenzidine were used according to the manufacturer’s in- severity structions (Ventana Medical Systems), and sections were counterstained To study the role of P2Y2 in acute GvHD, an allo-HCT model with hematoxylin. (BALB/c → C57BL/6) with MHC class I and II mismatches was In situ detection of dead cells in the intestines by TUNEL assay used. Transfer of allogeneic WT BM and T cells into P2y22/2 recipients reduced GvHD-related mortality compared with trans- Staining for apoptotic cells by TUNEL assay was performed on tissue +/+ sections that had been embedded in paraffin as recommended by the fer into allogeneic WT mice (P2y2 ) (Fig. 1A, p , 0.0001). The 2 2 manufacturer (in situ cell death detection kit, Roche Applied Sciences). reduction in mortality observed in P2y2 / recipients of alloge- Images were acquired and analyzed with a Scan^R high content screening neic BM and T cells was accompanied by histopathological evi- 3 station (Olympus), using a 20 (numerical aperture of 0.75) UPLSAPO dence of reduced GvHD severity. Histopathological evaluation of objective. liver, small intestine, and colon was carried out according to a Statistical analysis previously published scoring system (28) and demonstrated sig- 2/2 For the sample size in the mouse GvHD survival experiments, power nificantly lower scores in P2y2 recipients (Fig. 1B). analysis was performed. A sample size of at least n = 10 per group was The proinflammatory cytokine IL-6 is known to contribute to determined by 80% power to reach a statistical significance of 0.05 to GvHD development (38). Comparison of allo-HCT recipients detect an effect size of at least 1.06. Differences in animal survival (BALB/c → C57BL/6) revealed 6- to 7-fold lower IL-6 serum (Kaplan–Meier survival curves) were analyzed by log-rank test. To obtain 2 2 levels collected 8 d after transplantation in P2y2 / recipients unbiased data, the histopathology scoring of GvHD severity was per- +/+ formed by a pathologist blinded to the genotype or treatment group. Only compared with P2y2 recipients (Fig. 1C), whereas other cyto- after finalization of the quantitative GvHD severity scores were the sam- kines revealed no difference in serum levels (Supplemental Figs.

FIGURE 3. P2Y2 is required in the hematopoietic and nonhematopoietic system. Syngeneic HCT was performed as described (see Materials and Methods) for the generation of syngeneic bone morrow chi- meras prior to the secondary allo-HCT. The number of mice is indicated for the respective groups. The experiment was performed three times, and the results were pooled. The Journal of Immunology 5799

1, 2). Additionally, the T cell phenotype did not differ in P2y22/2 (39, 40), were found to be significantly downregulated in P2y22/2 recipients compared with P2y2+/+ recipients, except for reduced mice that developed GvHD (Fig. 2A). This finding was specific for levels of TNF-a in recipient-derived CD4 and CD8 T cells the allogeneic situation and not observed in the untreated control (Supplemental Fig. 2). Compatible with a reduction in tissue (Fig. 2B), suggesting a role for P2Y2 in recipient hematopoietic damage, the frequency of TUNEL+ apoptotic cells in the intestines cells that survive TBI and produce CCL5, CCL22, and MMP9. was reduced in P2y22/2 mice compared with P2y2+/+ mice, P2Y2 is required in the hematopoietic and nonhematopoietic which developed GvHD (Fig. 1D). system In contrast, there was no role for P2Y2 in the donor T cell compartment because GvHD-related mortality was comparable To clarify which recipient tissues require P2Y2 for the full phe- when P2y2+/+ or P2y22/2 T cells were used (Fig. 1E). Consistent notype of acute GvHD, we created BM chimera with either the with this observation, expansion of luc+ transgenic P2y22/2 C57BL/ recipient hematopoietic system or the nonhematopoietic system 6 T cells in BALB/c mice did not differ from expansion of luc+ being P2Y2 deficient as previously described (13). Lack of P2y2 in transgenic P2y2+/+ C57BL/6 T cells (Fig. 1F). These data indicate the hematopoietic system led to improved survival compared with that P2Y2 in the recipient tissues is required for the full phenotype of WT mice (Fig. 3). These observations support the concept that acute GvHD, whereas it is dispensable for alloreactive donor T cells. hematopoietic myeloid recipient–derived cells are responsible for P2Y2-mediated contribution to GvHD severity. Interestingly, allo- P2y2 deficiency is linked to reduced MMP9, CCL5, and CCL22 HCT in mice lacking P2y2 in nonhematopoietic cells also led to production in inflamed small intestine improved survival compared with WT mice (Fig. 3). Consistent +/+ To determine which factors were different in P2y2 compared with these findings, we had also seen lower levels of apoptotic Downloaded from with P2y22/2 mice undergoing allo-HCT, we analyzed the small nonlymphoid cells in P2y2-deficient recipients (Fig. 1D). These bowel by quantitative PCR. The genes Ccl5, Ccl22, and Mmp9, findings support the concept that P2Y2 mediates cell death in which are characteristic for different inflammatory myeloid cells nonhematopoietic cells during GvHD. http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. P2y2 deficiency of the recipient reduced DC migration in vivo and in vitro. (A and B) Serial luciferase-specific imaging was performed with C57BL/6 WT mice that had undergone allo-HCT with 5 3 106 BM cells from BALB/c mice and 4 3 106 DCs from C57BL/6 luc+ P2y22/2 mice, or DCs from luc+ P2y2+/+ C57BL/6 mice. (A) Representative images of mice are shown for the indicated time points after allo-HCT. (B) The signal due to the luc+ DCs is significantly lower for the group receiving luc+ P2y22/2 DCs. The p values are indicated for every time point. The experiment was performed twice with n = 3 in each group with comparable results. One representative experiment with mean 6 SD is shown. (C) P2y22/2 chimeric mice underwent allo- HCT with addition of 2 3 106 BMDCs (P2y22/2 or P2y2+/+), and survival was studied. This experiment was performed twice, and the results were pooled. 5800 P2y2 DEFICIENCY REDUCES GvHD

P2y2 deficiency in the DC compartment is not responsible for when GvHD developed (Fig. 5A, 5B). On day 3 after allo-HCT, GvHD protection neutrophils infiltrating the small intestines were not different in 2/2 +/+ Recipient-type hematopoietic cells and innate immune cells that P2y2 mice compared with P2y2 mice (data not shown). Conversely, inflammatory monocytes were reduced in P2y22/2 survive TBI for several days include DCs, neutrophils, and in- +/+ flammatory monocytes. To further address the question whether the mice compared with P2y2 mice (Fig. 5C), indicating that P2Y2 P2Y2 receptor defect has a functional role in DC migration, we could mediate migratory activity of inflammatory monocytes early used the newly generated luc+ P2y22/2 mice. BMDCs were after allo-HCT. + 2/2 + cultivated from luc P2y2 mice and luc WT mice and Functional role of P2Y2+ inflammatory monocytes after transferred into the syngeneic recipient on the day of transplan- allo-HCT tation to monitor migration of recipient-type DCs following con- ditioning and allo-HCT. We observed reduced accumulation of Based on the observation that host inflammatory monocytes may luc+ P2y22/2 DCs in secondary lymphoid organs, and later also in contribute to acute GvHD, we next aimed to evaluate the role of GvHD target organs, particularly in the skin and the intestinal tract P2Y2 on this effect. Therefore, chimeric mice (C57BL/6) with a 2/2 (Fig. 4A). This correlated with a significant reduction in luc+ hematopoietic system deficient in P2y2 received WT or P2y2 P2y22/2 DC signals on days 20–42 after allo-HCT (Fig. 4B). inflammatory monocytes (C57BL/6), which were highly enriched + + high Chimeric mice with P2y2 deficiency in the hematopoietic sys- by sorting for CD11b , CD115 ,and Ly-6C on day 0 after allo- tem received WT or P2y22/2 DCs on day 0 after allo-HCT. HCT. 2/2 Replenishing with DCs led to equivalent levels of GvHD-related Survival was improved in the group receiving P2y2 in- mortality when P2y22/2 DCs were given compared with WT DCs flammatory monocytes compared with the group receiving WT Downloaded from (Fig. 4C). Consistent with this finding, there was no difference in inflammatory monocytes (Fig. 6A). Together with improved sur- the ability of DCs derived from WT compared with P2y22/2 mice vival, the GvHD histopathological scoring was lower in mice that to stimulate allogeneic T cells (Supplemental Fig. 3) or in the had received P2y2-deficient monocytes (Fig. 6C). IL-6 and MCP- expression of costimulatory molecules (Supplemental Fig. 4). 1 are related to GvHD and inflammatory monocyte function, and These findings indicate that P2Y2 is dispensable for the DC- both were reduced when P2y2-deficient inflammatory monocytes mediated contribution to acute GvHD. were transferred on the day of transplantation (Fig. 6B). http://www.jimmunol.org/ + Migration of myeloid immune cells toward sites of inflam- P2Y2 mediates chemotaxis of MPO cells during acute GvHD mation is a crucial step in GvHD development. Therefore, we Because quantitative PCR–based analysis of the intestines had studied the impact of P2Y2 deficiency on migration of this cell shown a reduction in Ccl5 and Ccl22 in P2y2-deficient mice after type. P2y22/2 inflammatory monocytes migrated significantly allo-HCT, and because both factors are produced by inflammatory less than did WT monocytes toward an ATP gradient (Fig. 6D). monocytes and neutrophils, we next investigated the activity of A central signaling event for cell motility in monocytes is ERK active MPO, a key enzyme for both cell types, by BLI. MPO activation. We found a reduction in ERK phosphorylation in activity was lower in P2y22/2 mice compared with P2y2+/+ mice response to ATP in P2y22/2 inflammatory monocytes com- by guest on September 29, 2021

FIGURE 5. P2y2 deficiency of the recipient reduced inflammatory mono- cyte migration during GvHD. (A–C) C57BL/6 WT mice and C57BL/6 P2y22/2 mice that had undergone allo- HCT with 5 3 106 BM cells and 3 3 105 T cells (A)or83 105 T cells (B and C)fromBALB/cmiceareanalyzed.(A) Serial MPO-specific imaging was per- formed. MPO imaging of representative mice is shown for the indicated time points. (B) The MPO signal is signifi- cantly lower in C57BL/6 P2y22/2 mice compared with the signal in P2y2+/+ mice. The mean 6 SD is shown for each time point. The experiment was per- formed twice with n = 3 in each group each time and showed similar results. One representative experiment is shown. (C) The percentage of inflammatory monocytes (CD11b+,CD115+, Ly6Chigh ) in the small intestine was quantified in C57BL/6 P2y22/2 mice compared with P2y2+/+ mice on day 3 after allo-HCT. The mean 6 SD is shown, with six mice present per group. The experiment was performed twice. The Journal of Immunology 5801 pared with WT monocytes at different time points after stim- P2y2-deficient monocytes showed a decrease in the intracel- ulation (Fig. 6E). To understand how monocytes could exert a lular ROS levels in response to ATP (Fig. 6F). proinflammatory function after being activated by P2Y2, we These data indicate a crucial role for P2Y2 in the inflammatory next analyzed their ability to produce ROS. We observed that monocyte compartment during GvHD induction. Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. Inflammatory monocytes that contribute to GvHD are dependent on P2Y2. (A) Survival of P2y2-deficient chimeric mice shows enhanced survival for the chimera receiving P2y2-deficient inflammatory monocytes (p , 0.0001). First, syngeneic HCT was performed to create the chimera, followed by secondary allo-HCT. BM-derived monocytes were cultured as described and underwent sorting for inflammatory monocytes prior to transplantation. Recipient chimeric mice underwent allo-HCT and received 1.5 3 105 inflammatory monocytes (either P2y2 deficient or P2y2 suffi- cient) on day 0 of BM transplantation. The experiment was repeated three times, and the results were pooled (n = 12 mice/group). (B) Cytometric bead array analysis of the serum showed a significant reduction in MCP-1 and IL-6 levels in the chimera group, which received P2y2-deficient inflammatory monocytes (p , 0.0001). MCP-1 and IL-6 were determined in the serum of the indicated groups on day 7. The mean 6 SD is shown of three animals per group, and the experiment was performed twice with similar results. (C) On day 7 after allo-HCT the small bowel, large bowel, and liver were isolated and their histological score was determined. The mean 6 SD is shown of three animals per group, and the experiment was performed twice with similar results. (D) Transwell migration assay showed a significant reduction of migration ability in P2y2-deficient inflammatory monocytes toward ATP (p , 0.0001). The experiment was performed four times with similar results. The mean 6 SD is shown. (E) ERK phosphorylation was measured by flow cytometry staining as mentioned in Materials and Methods. P2y2-deficient inflammatory monocytes show significantly lower levels of phosphorylated ERK1/2 protein compared with WT inflammatory monocytes. Prior to flow cytometry, the monocytes were stimulated with 2 mM ATP for 2.5, 5, and 10 min. Pervanadate was used as a positive control. This experiment was repeated three times with similar results. The mean 6 SD is shown. (F) To quantify production of ROS by inflammatory monocytes, cells were first stimulated with 100 mM ATP for 30, 60, 120, and 190 min and then stained with CellROX. Inflammatory monocytes lacking the P2Y2 receptor showed lower levels of ROS after stimulation with ATP and failed to upregulate them later, indicating a deficiency in ROS production or signaling. The experiment was repeated three times with duplicate values. One representative experiment with mean 6 SD is shown. 5802 P2y2 DEFICIENCY REDUCES GvHD

P2Y2 expression on inflammatory cell infiltrates in human P2Y2, this was not an absolute requirement for acute GvHD be- GvHD lesions cause transfer of P2y2-deficient and WT DCs was equally effec- To evaluate whether the findings in the mouse model could also tive in inducing GvHD. This can be explained by the ability of play a role in the human situation, intestinal tissue biopsies were DCs to prime T cells in different tissues, independent of their analyzed. The frequency of P2Y2+ cells correlated with intestinal ability to sense extracellular ATP by P2Y2 and the functional GvHD severity (Fig. 7A, 7B). The major subset of P2Y2+ cells in redundancy of other purinergic receptors in DC chemotaxis (19). the inflamed human GvHD lesions were myeloid cells, including Our results show that P2y2 deficiency led to reduced monocyte monocytoid and polymorphonuclear cells (Fig. 7C). migration in response to ATP, which could be functionally linked to reduced ERK phosphorylation, an essential signaling event in Discussion cellular motility (48). Besides the migratory defect, P2y2 defi- Activation of the innate immune system in the initiation phase of ciency led to impairment of ROS production in inflammatory acute GvHD is promoted via translocation of PAMPs (41, 42) or monocytes when stimulated with ATP. This finding is consistent DAMPs (13, 43), which has already been shown by different in- with a recent study showing that P2Y2 activation by ATP leads to vestigators. The release of endogenous molecules deriving from ROS production in endothelial cells (49). Because monocytes the cytosol of stressed cells, such as ATP (13) and mitochondria need ROS to participate in microbial defense mechanisms (50), it (44), or even damaged extracellular matrix (14, 45), is a sign for is possible that P2y2-deficient inflammatory monocytes, which the innate immune system that noninfectious damage has occurred. produce ROS to a lower extent, induce less tissue damage com- In the present study, we show that the ATP-sensing receptor pared with WT monocytes.

P2Y2 is required for the development of acute GvHD in recipient However, the impact of different monocyte subsets on GvHD is Downloaded from hematopoietic cells. Recipient-derived inflammatory myeloid cells, still discussed controversially. G-CSF was found to mobilize a including DCs and inflammatory monocytes, were recruited by the so-far-uncharacterized population of CD34+ donor monocytes ATP/P2Y2 axis. However, in contrast to P2X7, the role of P2Y2 with a suppressive effect during GvHD (10). The authors attrib- was more diverse because P2y2 deficiency in both the hemato- uted principal monocytic characteristics to these unique cells, with poietic as well as the nonhematopoietic system led to reduced differences on the transcriptional level when compared with

GvHD-related mortality compared with the WT group. known monocyte subsets. Conversely, there is also evidence for a http://www.jimmunol.org/ In accordance with the results obtained in hematopoietic cell proinflammatory role of monocytes, as the induction of Th17 cells types, an essential role for P2Y2 on neutrophil chemotaxis exerted by activated monocytes and the stimulatory effects of proin- via ATP (46) has been described and neutrophils were shown to flammatory S100 proteins were shown to be connected to the contribute to GvHD (7). We extend these findings with the dem- development of acute GvHD (11). Additionally, high monocyte onstration that other myeloid cells, such as DCs and inflammatory levels in the blood were reported to be connected to greater se- monocytes, can be recruited via P2Y2 both in vitro and in vivo verity of GvHD (51). during GvHD. This observation is supported by a study of Elliott Consistent with our results showing that recipients of P2y2- et al. (47) showing that monocytes and macrophages employ deficient monocytes survived better after GvHD induction, a de- P2Y2 to sense nucleotides, which are released as “find me” signals ficiency of P2y2 in myeloid cells was also associated with im- by guest on September 29, 2021 by dying cells. Although recipient-type DC migration toward proved survival in models of sepsis (52), emphysema (53), and inflamed GvHD target tissues was reduced when the cells lacked hepatitis (20).

FIGURE 7. Expression of P2Y2 is increased in inflammatory cell infiltrates in human GvHD le- sions. (A) Human intestinal tissue samples were analyzed for the frequency of P2Y2+ cells. The frequency of P2Y2+ cells is significantly increased in patients with histologically confirmed GvHD (p = 0.0002); n = 3. The mean 6 SD is shown. (B) Representative intestinal biopsies from a patient without and a patient with grade IV intestinal GvHD are shown, with arrows indicating staining for P2Y2 (brown). Scale bars, 50 mm(upper panels), 20 mm (lower panels). (C) Polymorphonuclear neutrophils found in intestinal GvHD lesions express P2Y2 (red stain). Scale bars, 20 mm. The Journal of Immunology 5803

Our findings that a lack of P2Y2 in nonhematopoietic cells target to prevent DAMP-induced recipient myeloid cell activation in protects against GvHD, and that the enterocyte apoptosis rate in the early phase of GvHD. P2y2-deficient mice was significantly reduced, support the con- cept that P2Y2 activation in nonhematopoietic cells may promote Disclosures cell death. This hypothesis is in accordance with reports showing The authors have no financial conflicts of interest. that P2Y2 mediates cell death by activation of the cell death–in- ducing signaling complex. Furthermore, cellular apoptosis is prevented by inhibition of P2Y2 with or genetic P2y2 References deficiency (20, 54). Because we found P2Y2 to be of vast im- 1. Shlomchik, W. D., M. S. Couzens, C. B. Tang, J. McNiff, M. E. Robert, J. Liu, M. J. Shlomchik, and S. G. Emerson. 1999. Prevention of graft versus host portance for inflammatory monocytes in the hematopoietic disease by inactivation of host antigen-presenting cells. 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