Deletion of Biliverdin Reductase a in Myeloid Cells Promotes Chemokine

Deletion of Biliverdin Reductase A in Myeloid Cells Promotes Chemokine Expression and Chemotaxis in Part via a Complement C5a−C5aR1 Pathway This information is current as of September 26, 2021. Kavita Bisht, Giacomo Canesin, Tasneem Cheytan, Mailin Li, Zsuzsanna Nemeth, Eva Csizmadia, Trent M. Woodruff, David E. Stec, Andrew C. Bulmer, Leo E. Otterbein and Barbara Wegiel

J Immunol 2019; 202:2982-2990; Prepublished online 5 Downloaded from April 2019; doi: 10.4049/jimmunol.1701443 http://www.jimmunol.org/content/202/10/2982 http://www.jimmunol.org/

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Deletion of Biliverdin Reductase A in Myeloid Cells Promotes Chemokine Expression and Chemotaxis in Part via a Complement C5a–C5aR1 Pathway

Kavita Bisht,*,†,1 Giacomo Canesin,*,1 Tasneem Cheytan,* Mailin Li,* Zsuzsanna Nemeth,* Eva Csizmadia,* Trent M. Woodruff,‡ David E. Stec,x Andrew C. Bulmer,{ Leo E. Otterbein,* and Barbara Wegiel*

Biliverdin reductase (BVR)-A is a pleotropic enzyme converting biliverdin to bilirubin and a signaling molecule that has cytoprotective and immunomodulatory effects. We recently showed that biliverdin inhibits the expression of complement activation fragment 5a receptor one (C5aR1) in RAW 264.7 macrophages. In this study, we investigated the role of BVR-A in deter- mining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like Downloaded from macrophage markers, including chemokines (RANTES, IP-10), as well as chemotaxis in response to LPS and C5a in bone marrow–derived macrophages from BVRfl/fl and LysM-Cre:BVRfl/fl mice (conditional deletion of BVR-A in myeloid cells). In response to LPS, macrophages isolated from LysM-Cre:BVRfl/fl showed significantly elevated levels of C5aR1 as well as chemokines (RANTES, IP10) but not proinflammatory markers, such as iNOS and TNF. An increase in C5aR1 expression was also observed in peritoneal macrophages and several tissues from LysM-Cre:BVRfl/fl mice in a model of endotoxemia. In addition, knockdown of BVR- A resulted in enhanced macrophage chemotaxis toward C5a. Part of the effects of BVR-A deletion on chemotaxis and RANTES http://www.jimmunol.org/ expression were blocked in the presence of a C5aR1 neutralizing Ab, confirming the role of C5a–C5aR1 signaling in mediating the effects of BVR. In summary, BVR-A plays an important role in regulating macrophage chemotaxis in response to C5a via modulation of C5aR1 expression. In addition, macrophages lacking BVR-A are characterized by the expression of M1 polarization–associated chemokines, the levels of which depend in part on C5aR1 signaling. The Journal of Immunology, 2019, 202: 2982–2990.

iliverdin reductase (BVR)-A is a multifunctional protein, BVR-A present on the surface of macrophages is crucial for which mediates the reduction of biliverdin (BV) to bili- mediating anti-inflammatory effects of BV through Akt–IL-10 B rubin (BR) and regulates intracellular signaling by acting signaling (5). Our previous work showed that knockdown of by guest on September 26, 2021 as a kinase and transcriptional regulator (1–3). The conversion of BVR-A also leads to the development of a proinflammatory phe- BV to BR occurs in many cellular compartments; however, the notype in macrophages, which is characterized by elevated pro- majority of BVR-A reactivity is detected in the endoplasmic reduction of TNF because of increased basal expression of TLR-4 (6). ticulum and cell membrane (4). We have previously showed that Deletion of BVR-A by RNA interference promotes cell death and oxidative stress in response to H2O2 in a similar manner observed with depletion of glutathione (7, 8). Similarly, BVR-A2/2 mice *Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Med- ical School, Boston, MA 02215; †Cancer Care and Biology Program, Mater demonstrate greater oxidative damage to blood components because Research Institute, The University of Queensland, Translational Research Institute, of lower levels of circulating BR (9). Recent studies using condi- Woolloongabba, Queensland 4102, Australia; ‡School of Biomedical Sciences, The x tional deletion of BVR-A in the mouse liver show the importance of University of Queensland, Queensland 4072, Australia; Department of Physiology and Biophysics, The University of Mississippi Medical Center, Jackson, MS 39216; BVR-A in protecting against hepatic steatosis by inhibiting glyco- { and School of Medical Science, Griffith University, Queensland 4222, Australia gen synthase kinase 3b (GSK3b) and activating the peroxisome 1K.B. and G.C. contributed equally. proliferator-activated receptor a (PPARa) (10, 11). Further, BVR-A ORCIDs: 0000-0002-4073-0804 (K.B.); 0000-0002-3318-4550 (G.C.); 0000-0003- deficient animals are more prone to proximal tubular injury in a 2586-6639 (Z.N.); 0000-0003-1382-911X (T.M.W.); 0000-0001-8359-4008 (D.E.S.); model of saturated fatty acid–induced lipotoxicity (12). 0000-0002-7900-0825 (L.E.O.); 0000-0003-0679-8765 (B.W.). The complement system plays a key role in immunity by fa- Received for publication October 13, 2017. Accepted for publication March 11, 2019. cilitating pathogen elimination by opsonization, augmenting Ab production and inflammatory responses (13). It also facilitates This work was supported by National Institutes of Health/National Institute of Dia- betes Digestive and Kidney Diseases Grant R01 DK104714 and start-up funds from clearance of apoptotic and necrotic cells in tissues (14, 15). Ac- the Department of Surgery at Beth Israel Deaconess Medical Center and the Eleanor tivation of complement occurs by one of four different pathways: Shore Harvard Medical School Foundation (to B.W.). classical, lectin, alternative, or extrinsic, and all pathways lead to Address correspondence and reprint requests to Dr. Barbara Wegiel, Department of the cleavage of the central fragments C3 and C5 to generate the Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, 3 Blackfan Circle CLS 613, Boston, MA 02215. E-mail address: [email protected] anaphylatoxins C3a and C5a (16, 17). These small protein frag- The online version of this article contains supplemental material. ments act primarily through their cellular receptors C3aR, com- Abbreviations used in this article: BMDM, bone marrow–derived macrophage; BR, plement activation fragment 5a receptor (C5aR1), and C5aR2, bilirubin; BV, biliverdin; BVR, biliverdin reductase; C5aR1, complement activation respectively (17–19). The C5a–C5aR1 axis has been identified as fragment 5a receptor; qPCR, quantitative PCR; RT, room temperature; shRNA, short a crucial player in inflammation-associated pathologies, such as hairpin RNA; shRNAmir, microRNA-adapted short hairpin RNA. ischemia reperfusion injury, neurodegenerative disorders, athero- Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 sclerosis, rheumatoid arthritis, and sepsis (13, 20–26). We have www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701443 The Journal of Immunology 2983 previously shown that BV inhibits the expression of C5aR1 in mouse anti–b-actin (Sigma-Aldrich), rabbit anti-iNOS (Santa Cruz Bio- RAW 264.7 macrophages in part via mTOR signaling (27). technology, Santa Cruz, CA), anti-mouse IgG (Cell Signaling Technology, In the current study, we sought to determine a cross-talk between Beverly, MA), or anti-rabbit IgG (Cell Signaling Technology). For flow cytometry, PE anti-mouse CD88 and PE Rat IgG2a (BioLegend), FITC immunomodulatory BVR protein and the C5a–C5aR1 axis in anti-mouse F4.80 and FITC Rat IgG2a (BioLegend), and allophycocyanin macrophages, which are key cells involved in mediating responses anti-mouse CD11b and allophycocyanin Rat IgG2a (BioLegend) were during septic shock. used. For immunohistochemistry, Rat anti-mouse CD88/C5aR1 Ab (clone 10/92; LifeSpan Biosciences, Seattle, WA) was applied. Materials and Methods Animal treatment Animals fl/fl fl/fl BVR controls and LysM-Cre:BVR mice were administrated LPS BVRfl/fl mice were described before (10–12). We replicated most of the (5 mg/kg, i.p.), and tissues were harvested 24 h after injection. Peritoneal experiments in these mice based on our original observation in mice cells were isolated by flushing the mouse peritoneum with 10 ml of ice- generated in house on the 129 background. In-house generated BVRfl/fl cold PBS and spinning them down at 1200 3 g for 5 min. Cells were mice (conditional knockout mice) were based on a targeting construct that stained with selected Abs for 30 min on ice or at room temperature was designed based on PGK Neo FRT/loxP vector. A targeted sequence of (RT) and, thereafter, were immediately analyzed by flow cytometry. exons IV and V of the mouse BVR-A gene was inserted into the SacII site, located upstream of neomycin-resistance gene and flanked by two lox RNA extraction and RT-PCR sites. The same exons were targeted in BVRfl/fl mice from Dr. D.E. Stec’s Total RNA was isolated from cultured cells using RNeasy Plus Mini colony. The fragment of 39 (part of intron IV) arm and 59 (exon VI) arm of Kits (QIAGEN, Valencia, CA), and qPCR was performed as previ- homology were inserted outside the loxP sites between the Hpa-I and Sal-I ously described (6). Primers were purchased from Thermo Fisher sites, respectively. Blunt-end cloning was applied for all inserts. The con- Downloaded from Scientific. The following oligonucleotides were used: b-actin: forward, struct was linearized with Not-I and electroporated into embryonic stem cells 59-CCACGGATTCCATACCCAAGA-39 and reverse, 59- TAGACTTCG- (Children’s Hospital Core Facility, Harvard Medical School, Boston, MA). AGCAGGAGATGG-39; BVR: forward, 59-ATTCTGCCACCATGGAAA- Homozygotes BVRfl/fl mice (129S background) were crossed with LysM-Cre 39 and reverse, 59-CTCCAAGGACCCAGATTTGA-39; C5aR1: forward, mice (C57Bl6) to generate myeloid linage-specific knockout of BVR (mixed 59-CATTGCTCCTCACCATTCCA-39 and reverse, 59-CACCACTTTCG- background). These mice are no longer available in our colony because of TTGG-39; iNOS: forward, 59-CAGCTGGGCTGTACAAACCTT-39 and difficulty with breeding. reverse, 59-CATTGGAAGTGAAGCGGTTCG-39; COX-2: forward, 59-

Isolation and differentiation of bone CAAAAGAAGTGCTGGAAAAGGTT-39 and reverse, 59-TCTACCTGA- http://www.jimmunol.org/ marrow–derived macrophages GTGTCTTTGACTGTG-39; IP10: forward, 59-CTTGAAATCATCCCTG- CGAGC-39 and reverse, 59-TAGGACTAGCCATCCACTGGG-39; RANTES: Male and female C57BL/6 (The Jackson Laboratory, Bar Harbor, ME), BVRfl/fl forward, 59-CATATGGCTCGGACACCA-39 and reverse, 59-ACACACTT- controls, and LysM-Cre:BVRfl/fl mice were used at 7–10 wk of age. GGCGGTTCCT-39;IL10:forward,59-CCAAGCCTTATCGGAAATGA-39 Animals were held under specific pathogen–free conditions, and all ex- and reverse, 59-TTTTCACAGGGGAGAAATCG-39;IL1b:forward,59-TG- periments were approved by the Beth Israel Deaconess Medical Center GGCCTCAAAGGAAAGA-39 and reverse, 59-GGTGCTGATGTACCAGTT- Animal Care and Use Committee. Bone marrow–derived macrophages 39;CD206:forward,59-TCTTTGCCTTTCCCAGTCTCC-39 and reverse, 59- (BMDMs) were isolated as previously described (5). BMDMs were TGACACCCAGCGGAATTTC-39; arginase: forward, 59-CTCCAAGCCAA- isolated from the femurs by crushing and washing the femurs with RPMI AGTCCTTAGAG-39 and reverse, 59-AGGAGCTGTCATTAGGGACATC-39; medium (Thermo Fisher Scientific, Logan, UT) supplemented with TNF: forward, 59-TCCCAGGTTCTCTTCAAGGGA-39 and reverse, 59-GG- Antibiotic-Antimycotic (Thermo Fisher Scientific). Isolated cells were dif- TGAGGAGCACGTAGTCGG-39. by guest on September 26, 2021 ferentiated with murine rM-CSF (ProSpec, East Brunswick, NJ) at a final Briefly, RNA was reverse transcribed using iScript cDNA Synthesis Kit concentration 20 ng/ml in RPMI medium supplemented with 15% FBS (Bio-Rad Laboratories, Hercules, CA), and qPCR was performed with (Atlanta Biologicals, Flowery Branch, GA) and Antibiotic-Antimycotic for Mx3000P qPCR System (Agilent Technologies, Santa Clara, CA). Ex- 5 d (M-CSF medium). The medium was changed to fresh M-CSF medium pression levels of BVR were quantified by using SYBR Select Master Mix on the third day of culture. Macrophages were harvested after 5 d and then (Life Technologies, NY). The relative quantification of gene expression was cultured in RPMI medium supplemented with 15% FBS and Antibiotic- analyzed using Δ cycle threshold method, normalized to the housekeeping 2ΔΔ CT Antimycotic prior to experimentation. For macrophage polarization experi- gene and expressed as 2 . ments, cells were treated with LPS (100 ng/ml; Escherichia coli Serotype 0127:B8, Sigma-Aldrich, St. Louis, MO) and IFN-g (20 ng/ml; PeproTech, Isolation of cells from the tissues Rocky Hill, NJ) to induce M1 polarization for 24 h. Fragments of tissues were digested in 500 ml Liberase solution (200 mg/ml) Blocking experiments with anti-mouse C5aR1 (also known as CD88) at 37˚C for 1 h, vortexing the samples every 10 min. After digestion, were performed by preincubating cells with LEAF anti-mouse C5aR1 tissues were further disrupted by pipetting and by using a 25-gauge needle (1 mg/ml; clone 20/70, BioLegend, San Diego, CA) or anti-mouse IgG to obtain a single cell suspension. Cells were then washed twice in PBS, (Cell Signaling Technology, Beverly, MA) for 30 min prior to filtered through a 70-mm strainer, and then incubated with the corre- experimentation. sponding Abs for further analysis. Stable transfection of RAW 264.7 cells with microRNA-adapted Flow cytometry analysis of C5aR1 short hairpin RNA–BVR After harvesting and washing BMDM cells with PBS, cells were stained RAW 264.7 cells were stably transfected as described previously (6). with PE-labeled anti-mouse C5aR1 (CD88) Ab or PE-labeled IgG (1 mg/106 Briefly, microRNA-adapted short hairpin RNA (shRNAmir) against BVR cells) and myeloid markers for 30 min at RT or on ice. Cells were analyzed was subcloned from a pSM2 vector (Open Biosystems) into the MSCV- immediately using a FACSCalibur flow cytometer (Becton Dickinson, San LTRmir30-PIG (LMP) vector (Open Biosystems) with XhoI and EcoRI Jose, CA). The number of positive cells (percentage) was derived and restriction enzymes (Life Technologies). Cloning was verified by re- analyzed using CellQuest ProTM software (Becton Dickinson). striction site analysis and sequencing. For retroviral production, HEK293T cells were transiently transfected with shRNAmir–BVR-1a-LMP, Immunohistochemistry VSVG, and Gag-Pol plasmids using Lipofectamine 2000 (Life Tech- nologies). Media with viruses were collected at 12 h posttransfection, Liver, lung, and spleen tissue samples were formalin-fixed followed and the supernatants were used for transduction of RAW 264.7 cells. by paraffin embedding and immunostaining of 5-mm sections as previ- After 14 h incubation with viruses, RAW 264.7 cells were selected with ously described (5). Mouse Ab against C5aR1 was used at a concen- 5 mg/ml puromycin (Sigma-Aldrich) for 1 wk. Knockdown of BVR was tration 5 mg/ml. Secondary Ab was used as negative control. Briefly, confirmed by Western blot and quantitative PCR (qPCR). sections were processed for Ag retrieval with high-pressure cooking in citrate buffer for 1 h. Sections were then blocked for 30 min in 7% horse Source of Abs serum (Vector Laboratories, Burlingame, CA). Primary Ab against C5aR1 was then applied to the sections overnight at 4˚C. The following The following Abs were used for Western blot: rabbit anti-BVR (Enzo Life day, sections were incubated with biotin-labeled secondary Ab (1.5 mg/ml Sciences), rabbit anti-IkB and anti-GAPDH (Cell Signaling Technology), in PBS; Vector Laboratories) for 1 h at RT, followed by VECTASTAIN 2984 BVR-A IMPACTS CHEMOTAXIS VIA C5a–C5aR1 SIGNALING

Elite ABC kit and detection with ImmPACT DAB (Vector Laboratories). in 13 TBS buffer and membranes were incubated with HRP- The images were captured using a Nikon Eclipse E600 microscope (Nikon conjugated secondary Abs at a dilution of 1:5000 in 13 TBS with 5% Instruments, Melville, NY). nonfat milk for 1 h at RT. Membranes were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) or Cell migration assay Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), followed by exposure to the autobioradiography film (BioExpress, Kaysville, UT). Chemotaxis was evaluated in 24-well Transwell plates (Corning, Corning, NY) using polycarbonate membranes (8-mm pore size). BMDM from ELISA analysis BVRfl/fl and LysM-Cre:BVRfl/fl were resuspended in serum-free RPMI 6 TNF cytokine concentrations were measured in supernatants using medium at 1 3 10 cells/ml. One hundred microliters of cell suspension was added to the upper chamber, and 500 ml of serum-free RPMI medium Quantikine Immunoassays (R&D Systems) according to the manufac- containing recombinant mouse C5a (100 nM, R&D Systems, Minneapolis, turer’s protocol. MN), SDF-1 (50 ng/ml), or MCP-1 (15 ng/ml) was added to the lower NO measurement chamber. Cells were incubated for 24 h. Thereafter, the cells from the upper chamber were removed, and cells on the lower side of the chamber were Supernatants from BMDM cultures were harvested at 24 h after treatment. A stained with Crystal Violet (Sigma-Aldrich) for 10 min, followed by ex- Griess Reagent Kit for nitrite determination (Life Technologies) was used to tensive washing with water. Cells were dried and visualized at 403 mag- measure nitrate in the supernatants. A total of 150 ml of the supernatant was nification. Cells were then dissolved in 10% acetic acid, followed by incubated with 20 ml of Griess Reagent and 130 ml of deionized water for measurement of absorbance at 562 nm on the ELISA plate reader. For 30 min at RT following manufacturer’s protocol. The reference samples blocking experiments with anti-mouse C5aR1, cells were preincubated with were prepared at the same time. Absorbance was measured at 548 nm. LEAF anti-mouse C5aR1 (1 mg/ml) or IgG (1 mg/ml) for 30 min. Statistical analysis

Immunoblotting All data are reported as average 6 SD. Statistical analysis was performed Downloaded from U Cell lysates were prepared in ice-cold RIPA buffer (50 mM Tris-HCl [pH using one-way ANOVAwith post hoc Tukey test or Mann–Whitney test using GraphPad Prism unless otherwise stated. 7.4], 50 mM sodium ﬂuoride, 150 mM NaCl, 1% Nonidet P-40, 0.5 M EDTA [pH 8]) supplemented with the protease inhibitor mixture Complete Mini (Roche, Indianapolis, IN). Samples were centrifuged at 14,000 3 g at Results 4˚C for 20 min, and supernatants were collected. Protein concentrations of Knockdown of BVR-A in RAW 264.7 or deletion of BVR-A in supernatants were measured using a BCA Protein Assay Kit (Thermo

BMDM increases C5aR1 expression in vitro http://www.jimmunol.org/ Fisher Scientific, Tewksbury, MA). Forty micrograms of each protein sample was then electrophoresed on NuPAGE 4–12% Bis-Tris gel (Life We first established whether C5aR1 expression is associated with Technologies) in NuPAGE MES SDS Running Buffer (Life Technologies) macrophage differentiation. Bone marrow–derived myeloid cells for 90 min at 100 V. The membranes were blocked with 5% nonfat dry milk in 13 TBS (Boston Bio Products, Ashland, MA) for 1 h and then stimulated with M-CSF were used. We show significantly increased probed with the appropriate primary Abs (diluted at 1:1000 in 13 TBS C5aR1 surface expression starting as early as 1 d after addition with 5% nonfat milk) overnight at 4˚C. Membranes were then washed of M-CSF and reaching plateau at day 4–5 (Fig. 1A, 1B). by guest on September 26, 2021

FIGURE 1. Increased expression of C5aR1 in RAW 264.7 macrophages upon knockdown of BVR-A. (A and B) C5aR1 cell surface expression on BMDM harvested from C57BL/6 mice was measured at day 0–5 of their differentiation with M-CSF by ﬂow cytometry. Data are representative of three independent experiments. (B) ***p , 0.001, ANOVA with post hoc Tukey test at day 1–5 compared with untreated control cells. (C–F) RAW 264.7 cells were stably transfected with shRNA against BVR-A (shRNA–BVR-A) or shRNA control (Control). Gene (C) and protein expression (D) of BVR were analyzed using RT-PCR and Western blot, respectively. Results are representative of three independent experiments (n = 3 per group). (C)*p , 0.05, Mann–Whitney U test, control versus shRNA–BVR-A. (E and F) Gene expression (E) and cell surface expression (F) of C5aR1 were measured by RT-PCR and ﬂow cytometry, respectively, in RAW 264.7 shRNA control and shRNA–BVR-A cells. The data are representative of three independent experiments (n = 3 per group). (E and F)**p , 0.01, ***p , 0.001, Student t test, p = 0.1 using Mann–Whitney U test, control versus shRNA–BVR-A. The Journal of Immunology 2985

To assess the role of BVR-A in regulating C5aR1 expression, we weobservednochangeingeneexpressionofC5aR1(Supplemental employed RAW 264.7 macrophages with stable knockdown of Fig. 1A). BVR (short hairpin RNA[shRNA]–BVR-A) and control cells (28). We confirmed lower expression of BVR-A in shRNA–BVR-A Deletion of BVR in BMDM increases chemotaxis toward C5a cells compared with control cells by RT-PCR and Western blot- C5a is a potent chemoattractant and the ligand for C5aR1 that ting (Fig. 1C, 1D). Knockdown of BVR-A in these cells resulted mediates macrophage chemotaxis (29). Therefore, we next in significantly higher expression of C5aR1 as compared with assessed the effect of BVR deletion on BMDM migration to- control cells (shRNA control) (Fig. 1E, 1F). ward C5a. BMDM isolated from mice lacking BVR showed To study the role of BVR-A on C5aR1 expression in primary significantly increased migration toward C5a at 24 h compared macrophage culture, we deleted BVR-A by crossing BVRfl/fl with control cells (Fig. 2D). Similarly, BMDM M1-polarized mice to LysM-Cre mice. We show that BVR protein levels were with LPS/IFN-g and lacking BVR-A showed greater chemo- significantly decreased in BMDM isolated from LysM-Cre:BVRfl/fl taxis toward C5a (Fig. 2E). Interestingly, the effect of BVR-A mice as compared with BMDM isolated from BVRfl/fl control mice on chemotaxis was specific toward C5a, as SDF-1– or MCP-1– (Fig. 2A). This corresponded with a significant increase in C5aR1 induced chemotaxis was not associated with BVR-A deletion protein expression in BMDM isolated from LysM-Cre:BVRfl/fl (Supplemental Fig. 1B) fl/fl mice compared with the control BMDM isolated from BVR fl/fl mice (Fig. 2B). These results were confirmed by incubation of BMDM from LysM-Cre:BVR mice express greater cells with Ab against C5aR1 at RT or on ice to prevent in- levels of chemokines RANTES and IP10, markers of an ternalization of Ab (Fig. 2B). Additionally, there was a sig- M1-like phenotype Downloaded from nificant increase in the number of C5aR1+/F4.80+ cells in the Having shown that BMDM from LysM-Cre:BVRfl/fl mice have BMDM population at 5 d compared with3dafterM-CSFand increased expression of C5aR1 and enhanced C5a-mediaed che- a further increase in the number of C5aR1+/F4.80+ cells motaxis, we next assessed the phenotype of BMDM isolated from within the BMDM isolated from LysM-Cre:BVRfl/fl mice BVRfl/fl and LysM-Cre:BVRfl/fl mice in response to LPS, which compared with the control BMDM isolated from BVRfl/fl mice polarizes macrophages toward an M1 phenotype (30). Stimulation

at 5 d (Fig. 2C). However, in BMDM, unlike in RAW 264.7 cells, of BMDM with LPS and IFN-g leads to increased expression of http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. BMDM isolated from LysM-Cre:BVRfl/fl mice are characterized by increased C5aR1 levels and chemotaxis toward C5a. (A) The deletion of BVR in BMDM isolated from LysM-Cre:BVRfl/fl versus BVRfl/fl mice was evaluated by Western blot. (B and C) Surface expression of C5aR1 was assessed by flow cytometry in BMDM from BVRfl/fl and LysM-Cre:BVRfl/fl. Percentage of C5aR+ cells is shown with results representative for (n = 4–6 mice per group). Staining was performed either at RT or on ice (ICE). The kinetics of C5aR1 expression at 3 and 5 d is shown in (C). (B and C)***p , 0.001, *p , 0.05, ANOVA with Tukey post hoc test, LysM-Cre:BVRfl/fl versus BVRfl/fl.(D and E) Representative images (original magnification 340) and absorbance of unpolarized (D) or M1-polarized (E)BMDMfromBVRfl/fl and LysM-Cre:BVRfl/fl mice that migrated through to the lower part of Transwell chamber in response to C5a after 24 h culture in serum-free media. Results are representative of three independent experiments (n = 3 per group). (D and E)***p , 0.001, Mann–Whitney U test, LysM-Cre:BVRfl/fl versus BVRfl/fl. 2986 BVR-A IMPACTS CHEMOTAXIS VIA C5a–C5aR1 SIGNALING

M1 markers including the following: iNOS and TNF, IP-10 Increased expression of C5aR1 in vivo after LPS (CXCL10), IL-1b, IL-6, COX-2, RANTES (CCL5), or MCP-1 administration in LysM-Cre:BVRfl/fl mice among others (31, 32). We next evaluated C5aR1 protein expression in vivo in various Treatment with LPS/IFN-g for24hledtoincreasedmarkers organs basally or after treatment with LPS (5 mg/kg, i.p., 24 h) of of M1 polarization (Fig. 3A–G). We assessed expression of LysM-Cre:BVRfl/fl and BVRfl/fl mice. Total staining of the C5aR1 iNOS (by RT-PCR or activity of iNOS by NO production) and in the spleen, liver, and lungs in untreated (Fig. 4A) and LPS- TNF (by RT-PCR and ELISA); however, no change was observed treated (Fig. 4B) mice was assessed by immunohistochemistry. fl/fl fl/fl in BMDM from LysM-Cre:BVR compared with BVR mice The number of C5aR1+ cells in analyzed organs was increased upon treated with LPS/IFN-g (Fig. 3A–D). Interestingly, M1-polarized instillation of LPS and further elevated in LPS-treated LysM-Cre: BMDM (LPS/IFN-g–treated) from LysM-Cre:BVRfl/fl mice BVRfl/fl compared with LPS-treated BVRfl/fl mice (Fig. 4C–E). showed significantly enhanced expression level of RANTES and There was no difference in expression of C5aR1 in any of the IP-10 compared with M1-polarized BMDM from BVRfl/fl mice organs tested at basal conditions. (Fig. 3E, 3G). A proinflammatory cytokine, IL-1b was signifi- We next evaluated the effects of BVR on C5aR1 expression fl/fl fl/fl cantly induced in M1-polarized BMDM from LysM-Cre:BVRfl/fl in peritoneal macrophages isolated from BVR and LysM-Cre:BVR mice compared with untreated BMDM but not in BMDM from mice with and without LPS administration (Fig. 5A). We found significant upregulation of C5aR1 in peritoneal cells isolated BVRfl/fl mice (Fig. 3F). No difference in COX2 or the anti- from LysM-Cre:BVRfl/fl mice upon administration of LPS com- inflammatory M2 marker IL-10 was observed (Supplemental pared with the control groups analyzed (Fig. 5A). However, in Fig. 2A, 2B). Similarly, we also measured arginase and Downloaded from contrast to the increased number of C5aR1+ cells in the total pop- CD206 by RT-PCR and demonstrated no difference in the ex- ulation of peritoneal isolates, we detected a lower number of pression of these M2 markers between the BMDM isolated C5aR1+/F.480+ residential macrophages in the peritoneal cavity from these strains (Supplemental Fig. 2C, 2D). upon administration of LPS (Fig. 5B). There was a slight but Many of these proinflammatory markers are regulated by nonsignificant increase in the number of C5aR1+/F4.80+ perito- NF-kB signaling in response to LPS, and, therefore, we assessed neal macrophages in LysM-Cre:BVRfl/fl compared with BVRfl/fl mice

fl/fl http://www.jimmunol.org/ the levels of IkB in BMDM from LysM-Cre:BVR mice treated (Fig. 5B). with LPS (Fig. 3H). The levels of IkB degradation did not differ We also performed an extensive analysis of a surface expression of between the strains. C5aR1 in organs by flow cytometry (Fig. 5C–H). LPS administration by guest on September 26, 2021

FIGURE 3. Induction of RANTES, IP-10, and IL-1b expression in M1-polarized BMDM from LysM-Cre:BVRfl/fl mice. (A, C,andE–G) BMDM isolated from LysM-Cre:BVRfl/fl or BVRfl/fl mice were incubated in the presence or absence of LPS/IFN-g (M1) for 24 h. Expression of iNOS (A), TNF (C), RANTES (E), IL-1b (F), and IP-10 (G) was measured by RT-PCR. Data are representative of three independent experiments (n = 4–6 per group). *p , 0.05, **p , 0.01, ***p , 0.001, ANOVA with Tukey post hoc test. (B) Nitrate levels were measured in the supernatants of BMDM from BVRfl/fl and LysM-Cre:BVRfl/fl mice incubated with LPS/IFN-g for 24 h (n = 5–6 per group). *p , 0.05, ANOVA with Tukey post hoc test, LPS/IFN-g–treated versus controls. (D)ELISAwas applied to measure TNF levels in the supernatant of BMDM from BVRfl/fl and LysM-Cre:BVRfl/fl mice incubated with LPS/IFN-g for 24 h. Data are representative of three independent experiments (n = 3 per group). **p , 0.01, control versus LPS. (H) Immunoblot with Abs against IkB, total S6, and GAPDH in the lysates of BMDM treated with LPS for 15 min to 1 h. Data are representative for two to three independent experiments. The Journal of Immunology 2987 Downloaded from http://www.jimmunol.org/

FIGURE 4. C5aR1-expressing cells are increased in LysM-Cre:BVRfl/fl mice basally and in response to LPS. (A and B) BVRfl/fl and LysM-Cre:BVRfl/fl mice were treated with LPS (5 mg/kg, i.p.) and livers, lungs, and spleens were harvested at 24 h. C5aR1 expression was analyzed by immunohistochemistry. Representative pictures of migration assay are shown in (A and B). Pictures were taken at original magnification 3100 and insets at original magnification

3400. (C–E) Semiquantitative analysis for C5aR1 positive cells in multiple fields of view (n = 4–6 mice per group, two to three sections per group) is by guest on September 26, 2021 shown. **p , 0.01, ***p , 0.001, ANOVA with Tukey post hoc test, LysM-Cre:BVRfl/fl versus BVRfl/fl mice. increased the number of C5aR1+ and C5aR1+/F4.80+ populations in Discussion both strains in the spleen (Fig. 5C, 5D). The size of the spleen was In the current study, we show that BVR is a regulator of increased after LPS administration but did not differ between the macrophage chemotaxis toward C5a and its effects are par- strains (Supplemental Fig. 3). There was no difference in the basal tially dependent on C5aR1. Conditional deletion of BVR not only + + + + + number of C5aR1 ,C5aR1/F4.80 ,orC5aR1/CD11b in the augmented C5aR1 expression in primary macrophages but also fl/fl fl/fl organs between LysM-Cre:BVR compared with BVR mice enhanced C5a-mediated chemotaxis. We showed similar effects of (Fig. 5, Supplemental Fig. 4A). We did not see any difference in the BVR deletion on C5aR1 in an endotoxemia model in vivo. Further, + numbers of C5aR1 cells in the strains or after LPS treatment in the LPS-induced chemokine RANTES was elevated in the absence the liver (Fig. 5E, 5F). There was, however, a significant elevation in + of BVR-A and was subsequently blunted by C5aR1 blockade. We the number of C5aR1 cells in the lungs of LPS-treated animals, but suggest that the remarkable effects of BVR on C5a–C5aR1 sig- there was no difference between the strains (Fig. 5G, 5H). naling and macrophage polarization are crucial in the negative The effects of BVR on chemotaxis and chemokine expression regulation of innate immune responses. are in part mediated by C5aR1 We have previously shown that BV inhibits TLR-4 expression To evaluate the role of C5aR1 in the effects seen upon deletion of in part via BVR binding to AP-1 and GATA-4 sites (6). BV is also BVR, BMDM were preincubated with neutralizing Ab against known to block C5aR1 expression in part via the mTOR pathway C5aR1 or control IgG. Confirmation of neutralization of C5aR1 (27); however, whether the effects are dependent on the activity of was performed by flow cytometry (Fig. 6A). C5a-induced che- BVR has not been studied before. Silencing of surface BVR with motaxis in LysM-Cre:BVRfl/fl BMDM was significantly inhibited RNA interference abrogated BV-induced Akt (protein kinase B) by this neutralizing Ab against C5aR1 (Fig. 6B, 6C), suggesting phosphorylation (5) and IL-10 expression and thus was implicated a functional role of this receptor in BVR-mediated effects in in the induction of an anti-inflammatory phenotype of macro- BMDM. phages (5, 33). The anti-inflammatory effects of BV in models of Next, we assessed whether the BVR-modulated RANTES and sepsis (5, 34), transplantation, and ischemic injury (35) have also IP-10 levels were dependent on C5aR1 signaling (Fig. 6D, 6E). been demonstrated; however, the effects of BVR deletion on LPS/IFN-g–induced RANTES expression in BMDM from complement receptor and macrophage polarization status have not LysM-Cre:BVRfl/fl was blunted after treatment with the C5aR been studied before. neutralizing Ab. However, a similar effect was not noted for IP-10 Increased expression of C5a and its receptor C5aR1 are strongly expression (Fig. 6D, 6E). associated with acute and chronic inflammation and inflammatory 2988 BVR-A IMPACTS CHEMOTAXIS VIA C5a–C5aR1 SIGNALING

C5aR1+ cells in the spleens, lungs, and livers upon administration of LPS. However, the number of residential macrophages expressing C5aR1 was not different between strains, although they were increased by LPS treatment. C5aR1 is expressed on multiple cell types beside myeloid cells (38). It is possible that other myeloid cells, such as granulocytes in which Cre is expressed may exhibit changes in C5aR1 expression upon deletion of BVR. The complexity in expression of C5aR1 in different cell types within the tissue has been observed. In the asthma model, C5aR was increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells and monocyte-derived dendritic cells (39). Further, deletion of BVR in myeloid cells may impact tissue microenvironment and expression of C5aR1 in other cells (38). The number of cells expressing C5aR1 is increased in BVRflfl:LysM-Cre mice compared with BVRflfl mice at average of∼10%. It is important to note that this is a baseline expression of C5aR on the surface that changes upon deletion of

only one metabolic gene, BVR-A. Even small changes in number Downloaded from of C5aR1-expressing cells or the levels of C5aR1 are highly relevant to the physiology of myeloid cells, as they have large consequences (40–43). Further, we might not be detecting all cells mediating an increase in levels of C5aR1 at speciﬁc time points, as this is not a synchronized population of cells.

Hu et al. (33) reported that BVR overexpression or ablation had http://www.jimmunol.org/ no effect on TNF expression and release in nonpolarized macrophages; however, overexpression of BVR with adenovirus in nonpolarized BMDM increased both iNOS and arginine-1 expression, suggesting that BVR had no involvement in macrophage polarization (33). C5aR1 regulates the LPS-induced production of proinflammatory cytokines such as IL-6 and IL-12p40 (44). We show, in this study, that incubation of macrophages lacking BVR with LPS and IFN-g stimulated higher expression of RANTES, IP-10, and IL-1b in macrophages. Indeed, C5a stimulated by guest on September 26, 2021 RANTES and IL-1b expression in endothelial cells (45), and the effects of BVR on RANTES were, indeed, partially dependent on C5aR in this study. C5aR1 also plays a key role in disrupting blood–brain barrier integrity via regulating mRNA expression of iNOS in brain endothelial cells (46). We did not see an effect of BVR on TNF or iNOS expression. However, our previous work indicates that long-term depletion of BVR by shRNA in RAW 264.7 macrophages increases TNF levels basally and upon LPS stimulation (28). Interestingly, as much as RNA levels of C5aR1 were elevated in RAW 264.7 shRNA–BVR cells compared with control cells, we demonstrated only changes in C5aR1 protein expression in BMDM. The differences between BMDM and RAW FIGURE 5. Lack of BVR promotes C5aR1 expression in peritoneal 264.7 cells may be due to typical variation between the primary fl/fl A B infiltrates in LysM-Cre:BVR in response to LPS. ( and ) Peritoneal versus cultured cell lines; however, this may also emphasize the BVRfl/fl LysM-Cre:BVRfl/fl cells were isolated from and mice 24 h after LPS importance of BVR deletion for extended periods of time or be injection. The number of C5aR1+ cells was measured by flow cytometry + linked to secondary effects of BVR deletion in RAW 264.7 in the total and F4.80 cell populations. Results are expressed as average (n = 6–10 mice per group). *p , 0.05, **p , 0.01, ANOVA with Tukey macrophages. fl/fl post hoc test. (C–H) The flow cytometry analyses of C5aR1+ cells and Lack of BVR in BMDM from LysM-Cre:BVR mice resulted C5aR1+/F4.80+ cells in the populations isolated from spleens (C and D), in higher protein expression of C5aR1. Our in vitro findings are livers (E and F), or lungs (G and H)ofLysM-Cre:BVRfl/fl and BVRfl/fl supported by in vivo studies, in which we report higher total mice. Data are representative for (n = 6–10) mice. *p , 0.05, **p , 0.01, C5aR1 expression in the spleen, liver, and lung from LysM-Cre: ***p , 0.001, ANOVA with Tukey post hoc test. BVRfl/fl mice and surface C5aR1 in peritoneal macrophages from LysM-Cre:BVRfl/fl mice treated with LPS. Transcriptional acti- disorders (20, 21, 23). C5aR has also been implicated in the control vation of the C5aR1 promoter basally and in the presence of of M2 polarization of macrophages in the tumor microenvironment LPS, requires NF-Y (47), which was induced upon treatment (36). Our studies indicate the importance of C5aR1 in regulation of with heme, a precursor of BV (48). We cannot exclude the M1-like chemokine release upon deletion of BVR. C5aR1 is a G possibility that C5aR1 levels are regulated on the cell surface protein–coupled receptor and is expressed in both myeloid and in addition to mRNA levels. Further, BMDM isolated from nonmyeloid cells, and increased expression of C5aR1 has been LysM-Cre:BVRfl/fl mice and treated with LPS did not show any observed in inflamed tissues (21, 37). Indeed, we observed more difference in NF-kB signaling, which indicates that other signaling The Journal of Immunology 2989 Downloaded from http://www.jimmunol.org/

FIGURE 6. BVR effects on chemotaxis and chemokine expression are in part dependent on C5aR1. (A) BMDM from BVRfl/fl and LysM-Cre:BVRfl/fl mice incubated with anti-mouse IgG or C5aR1 for 30 min, and cell surface expression of C5aR1 was analyzed by flow cytometry. Results are representative of three independent experiments (n = 3 per group). *p , 0.05, ***p , 0.001, ANOVAwith post hoc Tukey test. (B and C) Representative images at original magnification 340 (B) and absorbance of BMDM (C) from BVRfl/fl and LysM-Cre:BVRfl/fl mice that migrated through to the lower part of Transwell chamber in response to C5a after 24 h incubation in the presence or absence of anti-mouse IgG or anti-mouse C5aR1. Data are expressed as mean 6 S.E. of three independent experiments (n = 3 per group). ***p , 0.001, ANOVA with post hoc Tukey test. (D and E) BMDM isolated from LysM-Cre:BVRfl/fl or fl/fl D BVR mice were incubated with anti-mouse IgG or C5aR1 for 30 min and then LPS/IFN-g (M1) was added for 24 h. Expression of IP-10 ( ) and by guest on September 26, 2021 RANTES (E) were measured by RT-PCR. Data are representative of three independent experiments (n = 4–6 mice per group). **p , 0.01, ***p , 0.001, ANOVA with Tukey post hoc test. pathways are implicated in BVR-regulated chemokine and cytokine expression. We identified, in this study, BVR as a target for reg- expression in response to LPS. ulating responses to LPS and complement activation products. Activation of C5aR1 promotes recruitment of neutrophils and Alteration in BVR expression may, therefore, modulate macro- macrophages at the site of infection, trauma, and inflammation phage polarization and contribute to the development of an in- (49, 50). Indeed, higher levels of C5aR1 in BMDM depleted of flammatory pathologic condition. BVR corresponded to higher chemotaxis activity. Soruri et al. (29) showed that the blockade of C5aR1 with neutralizing Ab against Disclosures C5aR1 (clone 20/70) inhibited the migration of rat basophilic The authors have no financial conflicts of interest. leukemia (RBL-2H3) cells toward C5a. A more recent study by Staab et al. (50) also demonstrated that inhibition of C5aR1 with PMX205 (peptidomimetic antagonist) reduced influx of eosino- References phils and neutrophils as well as production of proinflammatory 1. Miralem, T., N. Lerner-Marmarosh, P. E. Gibbs, J. L. Jenkins, C. Heimiller, and M. D. Maines. 2016. Interaction of human biliverdin reductase with Akt/protein cytokines in response to OVA allergen sensitization and chal- kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen syn- lenge, indicating the immunomodulatory potential of C5a–C5aR1 thase kinase 3 activity: a novel mechanism of Akt activation. FASEB J. 30: signaling (50). 2926–2944. 2. Wegiel, B., and L. E. Otterbein. 2012. Go green: the anti-inflammatory effects of Collectively, our data demonstrate that BVR is an important biliverdin reductase. Front. Pharmacol. 3: 47. molecule required for regulating macrophage chemotaxis toward 3. Gibbs, P. E., T. Miralem, N. Lerner-Marmarosh, C. Tudor, and M. D. Maines. 2012. Formation of ternary complex of human biliverdin reductase-protein ki- C5a, and deletion of BVR in macrophages promotes chemotaxis nase Cd-ERK2 protein is essential for ERK2-mediated activation of Elk1 pro- toward C5a. We report that the increased cell migration of BMDM tein, nuclear factor-kB, and inducible nitric-oxidase synthase (iNOS). J. Biol. toward C5a in LysM-Cre:BVRfl/fl mice was suppressed after Chem. 287: 1066–1079. 4. Salim, M., B. A. Brown-Kipphut, and M. D. Maines. 2001. Human biliverdin treatment with a C5aR1 neutralizing Ab, suggesting a partial role reductase is autophosphorylated, and phosphorylation is required for bilirubin for C5aR1 in BVR-mediated modulation of chemotaxis. More- formation. J. Biol. Chem. 276: 10929–10934. over, we translated our findings to an in vivo model, where we 5. Wegiel, B., C. J. Baty, D. Gallo, E. Csizmadia, J. R. Scott, A. Akhavan, fl/fl B. Y. Chin, E. Kaczmarek, J. Alam, F. H. Bach, et al. 2009. Cell surface bili- observed that peritoneal cells from LPS-treated LysM-Cre:BVR verdin reductase mediates biliverdin-induced anti-inflammatory effects via mice expressed more C5aR1 compared with BVRfl/fl mice. phosphatidylinositol 3-kinase and Akt. J. Biol. Chem. 284: 21369–21378. 6. Wegiel, B., D. Gallo, E. Csizmadia, T. Roger, E. Kaczmarek, C. Harris, In summary, we show that BVR modulates C5a–C5aR1 sig- B. S. Zuckerbraun, and L. E. Otterbein. 2011. Biliverdin inhibits Toll-like naling and macrophage phenotype in part via regulation of C5aR1 receptor-4 (TLR4) expression through nitric oxide-dependent nuclear 2990 BVR-A IMPACTS CHEMOTAXIS VIA C5a–C5aR1 SIGNALING

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