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

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 May 15, 2019, 202 (10) 2982-2990; DOI: https://doi.org/10.4049/jimmunol.1701443

Key points

  • Myeloid-specific conditional deletion of BVR-A results in increased response to LPS.

  • Macrophages isolated from LysM-Cre:BVRfl/fl showed elevated levels of C5aR1.

  • Deletion of BVR-A resulted in enhanced macrophage chemotaxis toward C5a.

  • BVR-A–modulated chemotaxis and RANTES levels are dependent in part on C5aR1.

Abstract

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 determining macrophage inflammatory phenotype and function via regulation of C5aR1. We assessed expression of C5aR1, M1-like 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 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.

Introduction

Biliverdin reductase (BVR)-A is a multifunctional protein, which mediates the reduction of biliverdin (BV) to bilirubin (BR) and regulates intracellular signaling by acting as a kinase and transcriptional regulator (1–3). The conversion of BV to BR occurs in many cellular compartments; however, the majority of BVR-A reactivity is detected in the endoplasmic reticulum and cell membrane (4). We have previously showed that BVR-A present on the surface of macrophages is crucial for mediating anti-inflammatory effects of BV through Akt–IL-10 signaling (5). Our previous work showed that knockdown of BVR-A also leads to the development of a proinflammatory phenotype in macrophages, which is characterized by elevated production of TNF because of increased basal expression of TLR-4 (6). 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-A−/− mice demonstrate greater oxidative damage to blood components because of lower levels of circulating BR (9). Recent studies using conditional deletion of BVR-A in the mouse liver show the importance of BVR-A in protecting against hepatic steatosis by inhibiting glycogen synthase kinase 3β (GSK3β) and activating the peroxisome proliferator-activated receptor α (PPARα) (10, 11). Further, BVR-A deficient animals are more prone to proximal tubular injury in a model of saturated fatty acid–induced lipotoxicity (12).

The complement system plays a key role in immunity by facilitating pathogen elimination by opsonization, augmenting Ab production and inflammatory responses (13). It also facilitates clearance of apoptotic and necrotic cells in tissues (14, 15). Activation of complement occurs by one of four different pathways: classical, lectin, alternative, or extrinsic, and all pathways lead to the cleavage of the central fragments C3 and C5 to generate the anaphylatoxins C3a and C5a (16, 17). These small protein fragments act primarily through their cellular receptors C3aR, complement activation fragment 5a receptor (C5aR1), and C5aR2, respectively (17–19). The C5a–C5aR1 axis has been identified as a crucial player in inflammation-associated pathologies, such as ischemia reperfusion injury, neurodegenerative disorders, atherosclerosis, rheumatoid arthritis, and sepsis (13, 20–26). We have previously shown that BV inhibits the expression of C5aR1 in RAW 264.7 macrophages in part via mTOR signaling (27).

In the current study, we sought to determine a cross-talk between immunomodulatory BVR protein and the C5a–C5aR1 axis in macrophages, which are key cells involved in mediating responses during septic shock.

Materials and Methods

Animals

BVRfl/fl mice were described before (10–12). We replicated most of the experiments in these mice based on our original observation in mice generated in house on the 129 background. In-house generated BVRfl/fl mice (conditional knockout mice) were based on a targeting construct that was designed based on PGK Neo FRT/loxP vector. A targeted sequence of 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 sites. The same exons were targeted in BVRfl/fl mice from Dr. D.E. Stec’s colony. The fragment of 3′ (part of intron IV) arm and 5′ (exon VI) arm of homology were inserted outside the loxP sites between the Hpa-I and Sal-I sites, respectively. Blunt-end cloning was applied for all inserts. The construct was linearized with Not-I and electroporated into embryonic stem cells (Children’s Hospital Core Facility, Harvard Medical School, Boston, MA). Homozygotes BVRfl/fl mice (129S background) were crossed with LysM-Cre mice (C57Bl6) to generate myeloid linage-specific knockout of BVR (mixed background). These mice are no longer available in our colony because of difficulty with breeding.

Isolation and differentiation of bone marrow–derived macrophages

Male and female C57BL/6 (The Jackson Laboratory, Bar Harbor, ME), BVRfl/fl controls, and LysM-Cre:BVRfl/fl mice were used at 7–10 wk of age. Animals were held under specific pathogen–free conditions, and all experiments were approved by the Beth Israel Deaconess Medical Center Animal Care and Use Committee. Bone marrow–derived macrophages (BMDMs) were isolated as previously described (5). BMDMs were isolated from the femurs by crushing and washing the femurs with RPMI medium (Thermo Fisher Scientific, Logan, UT) supplemented with Antibiotic-Antimycotic (Thermo Fisher Scientific). Isolated cells were differentiated with murine rM-CSF (ProSpec, East Brunswick, NJ) at a final concentration 20 ng/ml in RPMI medium supplemented with 15% FBS (Atlanta Biologicals, Flowery Branch, GA) and Antibiotic-Antimycotic for 5 d (M-CSF medium). The medium was changed to fresh M-CSF medium on the third day of culture. Macrophages were harvested after 5 d and then cultured in RPMI medium supplemented with 15% FBS and Antibiotic-Antimycotic prior to experimentation. For macrophage polarization experiments, cells were treated with LPS (100 ng/ml; Escherichia coli Serotype 0127:B8, Sigma-Aldrich, St. Louis, MO) and IFN-γ (20 ng/ml; PeproTech, Rocky Hill, NJ) to induce M1 polarization for 24 h.

Blocking experiments with anti-mouse C5aR1 (also known as CD88) were performed by preincubating cells with LEAF anti-mouse C5aR1 (1 μg/ml; clone 20/70, BioLegend, San Diego, CA) or anti-mouse IgG (Cell Signaling Technology, Beverly, MA) for 30 min prior to experimentation.

Stable transfection of RAW 264.7 cells with microRNA-adapted short hairpin RNA–BVR

RAW 264.7 cells were stably transfected as described previously (6). Briefly, microRNA-adapted short hairpin RNA (shRNAmir) against BVR was subcloned from a pSM2 vector (Open Biosystems) into the MSCV-LTRmir30-PIG (LMP) vector (Open Biosystems) with XhoI and EcoRI restriction enzymes (Life Technologies). Cloning was verified by restriction site analysis and sequencing. For retroviral production, HEK293T cells were transiently transfected with shRNAmir–BVR-1α-LMP, VSVG, and Gag-Pol plasmids using Lipofectamine 2000 (Life Technologies). Media with viruses were collected at 12 h posttransfection, and the supernatants were used for transduction of RAW 264.7 cells. After 14 h incubation with viruses, RAW 264.7 cells were selected with 5 μg/ml puromycin (Sigma-Aldrich) for 1 wk. Knockdown of BVR was confirmed by Western blot and quantitative PCR (qPCR).

Source of Abs

The following Abs were used for Western blot: rabbit anti-BVR (Enzo Life Sciences), rabbit anti-IκB and anti-GAPDH (Cell Signaling Technology), mouse anti–β-actin (Sigma-Aldrich), rabbit anti-iNOS (Santa Cruz Biotechnology, Santa Cruz, CA), anti-mouse IgG (Cell Signaling Technology, Beverly, MA), or anti-rabbit IgG (Cell Signaling Technology). For flow cytometry, PE anti-mouse CD88 and PE Rat IgG2a (BioLegend), FITC anti-mouse F4.80 and FITC Rat IgG2a (BioLegend), and allophycocyanin anti-mouse CD11b and allophycocyanin Rat IgG2a (BioLegend) were used. For immunohistochemistry, Rat anti-mouse CD88/C5aR1 Ab (clone 10/92; LifeSpan Biosciences, Seattle, WA) was applied.

Animal treatment

BVRfl/fl controls and LysM-Cre:BVRfl/fl mice were administrated LPS (5 mg/kg, i.p.), and tissues were harvested 24 h after injection. Peritoneal cells were isolated by flushing the mouse peritoneum with 10 ml of ice-cold PBS and spinning them down at 1200 × g for 5 min. Cells were stained with selected Abs for 30 min on ice or at room temperature (RT) and, thereafter, were immediately analyzed by flow cytometry.

RNA extraction and RT-PCR

Total RNA was isolated from cultured cells using RNeasy Plus Mini Kits (QIAGEN, Valencia, CA), and qPCR was performed as previously described (6). Primers were purchased from Thermo Fisher Scientific. The following oligonucleotides were used: β-actin: forward, 5′-CCACGGATTCCATACCCAAGA-3′ and reverse, 5′- TAGACTTCGAGCAGGAGATGG-3′; BVR: forward, 5′-ATTCTGCCACCATGGAAA-3′ and reverse, 5′-CTCCAAGGACCCAGATTTGA-3′; C5aR1: forward, 5′-CATTGCTCCTCACCATTCCA-3′ and reverse, 5′-CACCACTTTCGTTGG-3′; iNOS: forward, 5′-CAGCTGGGCTGTACAAACCTT-3′ and reverse, 5′-CATTGGAAGTGAAGCGGTTCG-3′; COX-2: forward, 5′-CAAAAGAAGTGCTGGAAAAGGTT-3′ and reverse, 5′-TCTACCTGAGTGTCTTTGACTGTG-3′; IP10: forward, 5′-CTTGAAATCATCCCTGCGAGC-3′ and reverse, 5′-TAGGACTAGCCATCCACTGGG-3′; RANTES: forward, 5′-CATATGGCTCGGACACCA-3′ and reverse, 5′-ACACACTTGGCGGTTCCT-3′; IL10: forward, 5′-CCAAGCCTTATCGGAAATGA-3′ and reverse, 5′-TTTTCACAGGGGAGAAATCG-3′; IL1β: forward, 5′-TGGGCCTCAAAGGAAAGA-3′ and reverse, 5′-GGTGCTGATGTACCAGTT-3′; CD206: forward, 5′-TCTTTGCCTTTCCCAGTCTCC-3′ and reverse, 5′-TGACACCCAGCGGAATTTC-3′; arginase: forward, 5′-CTCCAAGCCAAAGTCCTTAGAG-3′ and reverse, 5′-AGGAGCTGTCATTAGGGACATC-3′; TNF: forward, 5′-TCCCAGGTTCTCTTCAAGGGA-3′ and reverse, 5′-GGTGAGGAGCACGTAGTCGG-3′.

Briefly, RNA was reverse transcribed using iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA), and qPCR was performed with Mx3000P qPCR System (Agilent Technologies, Santa Clara, CA). Expression levels of BVR were quantified by using SYBR Select Master Mix (Life Technologies, NY). The relative quantification of gene expression was analyzed using ∆ cycle threshold method, normalized to the housekeeping gene and expressed as 2−∆∆ CT.

Isolation of cells from the tissues

Fragments of tissues were digested in 500 μl Liberase solution (200 μg/ml) at 37°C for 1 h, vortexing the samples every 10 min. After digestion, tissues were further disrupted by pipetting and by using a 25-gauge needle to obtain a single cell suspension. Cells were then washed twice in PBS, filtered through a 70-μm strainer, and then incubated with the corresponding Abs for further analysis.

Flow cytometry analysis of C5aR1

After harvesting and washing BMDM cells with PBS, cells were stained with PE-labeled anti-mouse C5aR1 (CD88) Ab or PE-labeled IgG (1 μg/106 cells) and myeloid markers for 30 min at RT or on ice. Cells were analyzed immediately using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA). The number of positive cells (percentage) was derived and analyzed using CellQuest ProTM software (Becton Dickinson).

Immunohistochemistry

Liver, lung, and spleen tissue samples were formalin-fixed followed by paraffin embedding and immunostaining of 5-μm sections as previously described (5). Mouse Ab against C5aR1 was used at a concentration 5 μg/ml. Secondary Ab was used as negative control. Briefly, 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 serum (Vector Laboratories, Burlingame, CA). Primary Ab against C5aR1 was then applied to the sections overnight at 4°C. The following day, sections were incubated with biotin-labeled secondary Ab (1.5 μg/ml in PBS; Vector Laboratories) for 1 h at RT, followed by VECTASTAIN Elite ABC kit and detection with ImmPACT DAB (Vector Laboratories). The images were captured using a Nikon Eclipse E600 microscope (Nikon Instruments, Melville, NY).

Cell migration assay

Chemotaxis was evaluated in 24-well Transwell plates (Corning, Corning, NY) using polycarbonate membranes (8-μm pore size). BMDM from BVRfl/fl and LysM-Cre:BVRfl/fl were resuspended in serum-free RPMI medium at 1 × 106 cells/ml. One hundred microliters of cell suspension was added to the upper chamber, and 500 μl of serum-free RPMI medium containing recombinant mouse C5a (100 nM, R&D Systems, Minneapolis, MN), SDF-1 (50 ng/ml), or MCP-1 (15 ng/ml) was added to the lower 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 stained with Crystal Violet (Sigma-Aldrich) for 10 min, followed by extensive washing with water. Cells were dried and visualized at 40× magnification. Cells were then dissolved in 10% acetic acid, followed by measurement of absorbance at 562 nm on the ELISA plate reader. For blocking experiments with anti-mouse C5aR1, cells were preincubated with LEAF anti-mouse C5aR1 (1 μg/ml) or IgG (1 μg/ml) for 30 min.

Immunoblotting

Cell lysates were prepared in ice-cold RIPA buffer (50 mM Tris-HCl [pH 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 × g at 4°C for 20 min, and supernatants were collected. Protein concentrations of supernatants were measured using a BCA Protein Assay Kit (Thermo Fisher Scientific, Tewksbury, MA). Forty micrograms of each protein sample was then electrophoresed on NuPAGE 4–12% Bis-Tris gel (Life Technologies) in NuPAGE MES SDS Running Buffer (Life Technologies) for 90 min at 100 V. The membranes were blocked with 5% nonfat dry milk in 1× TBS (Boston Bio Products, Ashland, MA) for 1 h and then probed with the appropriate primary Abs (diluted at 1:1000 in 1× TBS with 5% nonfat milk) overnight at 4°C. Membranes were then washed in 1× TBS buffer and membranes were incubated with HRP-conjugated secondary Abs at a dilution of 1:5000 in 1× TBS with 5% nonfat milk for 1 h at RT. Membranes were visualized using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) or Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific), followed by exposure to the autobioradiography film (BioExpress, Kaysville, UT).

ELISA analysis

TNF cytokine concentrations were measured in supernatants using Quantikine Immunoassays (R&D Systems) according to the manufacturer’s protocol.

NO measurement

Supernatants from BMDM cultures were harvested at 24 h after treatment. A Griess Reagent Kit for nitrite determination (Life Technologies) was used to measure nitrate in the supernatants. A total of 150 μl of the supernatant was incubated with 20 μl of Griess Reagent and 130 μl of deionized water for 30 min at RT following manufacturer’s protocol. The reference samples were prepared at the same time. Absorbance was measured at 548 nm.

Statistical analysis

All data are reported as average ± SD. Statistical analysis was performed using one-way ANOVA with post hoc Tukey test or Mann–Whitney U test using GraphPad Prism unless otherwise stated.

Results

Knockdown of BVR-A in RAW 264.7 or deletion of BVR-A in BMDM increases C5aR1 expression in vitro

We first established whether C5aR1 expression is associated with macrophage differentiation. Bone marrow–derived myeloid cells stimulated with M-CSF were used. We show significantly increased C5aR1 surface expression starting as early as 1 d after addition of M-CSF and reaching plateau at day 4–5 (Fig. 1A, 1B).

FIGURE 1.
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.

To assess the role of BVR-A in regulating C5aR1 expression, we employed RAW 264.7 macrophages with stable knockdown of BVR (short hairpin RNA[shRNA]–BVR-A) and control cells (28). We confirmed lower expression of BVR-A in shRNA–BVR-A cells compared with control cells by RT-PCR and Western blotting (Fig. 1C, 1D). Knockdown of BVR-A in these cells resulted in significantly higher expression of C5aR1 as compared with control cells (shRNA control) (Fig. 1E, 1F).

To study the role of BVR-A on C5aR1 expression in primary macrophage culture, we deleted BVR-A by crossing BVRfl/fl mice to LysM-Cre mice. We show that BVR protein levels were significantly decreased in BMDM isolated from LysM-Cre:BVRfl/fl mice as compared with BMDM isolated from BVRfl/fl control mice (Fig. 2A). This corresponded with a significant increase in C5aR1 protein expression in BMDM isolated from LysM-Cre:BVRfl/fl mice compared with the control BMDM isolated from BVRfl/fl mice (Fig. 2B). These results were confirmed by incubation of cells with Ab against C5aR1 at RT or on ice to prevent internalization of Ab (Fig. 2B). Additionally, there was a significant increase in the number of C5aR1+/F4.80+ cells in the BMDM population at 5 d compared with 3 d after M-CSF and a further increase in the number of C5aR1+/F4.80+ cells within the BMDM isolated from LysM-Cre:BVRfl/fl mice compared with the control BMDM isolated from BVRfl/fl mice at 5 d (Fig. 2C). However, in BMDM, unlike in RAW 264.7 cells, we observed no change in gene expression of C5aR1 (Supplemental Fig. 1A).

FIGURE 2.
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 ×40) and absorbance of unpolarized (D) or M1-polarized (E) BMDM 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 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.

Deletion of BVR in BMDM increases chemotaxis toward C5a

C5a is a potent chemoattractant and the ligand for C5aR1 that mediates macrophage chemotaxis (29). Therefore, we next assessed the effect of BVR deletion on BMDM migration toward C5a. BMDM isolated from mice lacking BVR showed significantly increased migration toward C5a at 24 h compared with control cells (Fig. 2D). Similarly, BMDM M1-polarized with LPS/IFN-γ and lacking BVR-A showed greater chemotaxis toward C5a (Fig. 2E). Interestingly, the effect of BVR-A on chemotaxis was specific toward C5a, as SDF-1– or MCP-1–induced chemotaxis was not associated with BVR-A deletion (Supplemental Fig. 1B)

BMDM from LysM-Cre:BVRfl/fl mice express greater levels of chemokines RANTES and IP10, markers of an M1-like phenotype

Having shown that BMDM from LysM-Cre:BVRfl/fl mice have increased expression of C5aR1 and enhanced C5a-mediaed chemotaxis, we next assessed the phenotype of BMDM isolated from BVRfl/fl and LysM-Cre:BVRfl/fl mice in response to LPS, which polarizes macrophages toward an M1 phenotype (30). Stimulation of BMDM with LPS and IFN-γ leads to increased expression of M1 markers including the following: iNOS and TNF, IP-10 (CXCL10), IL-1β, IL-6, COX-2, RANTES (CCL5), or MCP-1 among others (31, 32).

Treatment with LPS/IFN-γ for 24 h led to increased markers of M1 polarization (Fig. 3A–G). We assessed expression of iNOS (by RT-PCR or activity of iNOS by NO production) and TNF (by RT-PCR and ELISA); however, no change was observed in BMDM from LysM-Cre:BVRfl/fl compared with BVRfl/fl mice treated with LPS/IFN-γ (Fig. 3A–D). Interestingly, M1-polarized BMDM (LPS/IFN-γ–treated) from LysM-Cre:BVRfl/fl mice showed significantly enhanced expression level of RANTES and IP-10 compared with M1-polarized BMDM from BVRfl/fl mice (Fig. 3E, 3G). A proinflammatory cytokine, IL-1β was significantly induced in M1-polarized BMDM from LysM-Cre:BVRfl/fl mice compared with untreated BMDM but not in BMDM from BVRfl/fl mice (Fig. 3F). No difference in COX2 or the anti-inflammatory M2 marker IL-10 was observed (Supplemental Fig. 2A, 2B). Similarly, we also measured arginase and CD206 by RT-PCR and demonstrated no difference in the expression of these M2 markers between the BMDM isolated from these strains (Supplemental Fig. 2C, 2D).

FIGURE 3.
FIGURE 3.

Induction of RANTES, IP-10, and IL-1β expression in M1-polarized BMDM from LysM-Cre:BVRfl/fl mice. (A, C, and E–G) BMDM isolated from LysM-Cre:BVRfl/fl or BVRfl/fl mice were incubated in the presence or absence of LPS/IFN-γ (M1) for 24 h. Expression of iNOS (A), TNF (C), RANTES (E), IL-1β (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-γ for 24 h (n = 5–6 per group). *p < 0.05, ANOVA with Tukey post hoc test, LPS/IFN-γ–treated versus controls. (D) ELISA was applied to measure TNF levels in the supernatant of BMDM from BVRfl/fl and LysM-Cre:BVRfl/fl mice incubated with LPS/IFN-γ 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 IκB, 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.

Many of these proinflammatory markers are regulated by NF-κB signaling in response to LPS, and, therefore, we assessed the levels of IκB in BMDM from LysM-Cre:BVRfl/fl mice treated with LPS (Fig. 3H). The levels of IκB degradation did not differ between the strains.

Increased expression of C5aR1 in vivo after LPS administration in LysM-Cre:BVRfl/fl mice

We next evaluated C5aR1 protein expression in vivo in various organs basally or after treatment with LPS (5 mg/kg, i.p., 24 h) of LysM-Cre:BVRfl/fl and BVRfl/fl mice. Total staining of the C5aR1 in the spleen, liver, and lungs in untreated (Fig. 4A) and LPS-treated (Fig. 4B) mice was assessed by immunohistochemistry. The number of C5aR1+ cells in analyzed organs was increased upon instillation of LPS and further elevated in LPS-treated LysM-Cre:BVRfl/fl compared with LPS-treated BVRfl/fl mice (Fig. 4C–E). There was no difference in expression of C5aR1 in any of the organs tested at basal conditions.

FIGURE 4.
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 ×100 and insets at original magnification ×400. (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 shown. **p < 0.01, ***p < 0.001, ANOVA with Tukey post hoc test, LysM-Cre:BVRfl/fl versus BVRfl/fl mice.

We next evaluated the effects of BVR on C5aR1 expression in peritoneal macrophages isolated from BVRfl/fl and LysM-Cre:BVRfl/fl mice with and without LPS administration (Fig. 5A). We found significant upregulation of C5aR1 in peritoneal cells isolated from LysM-Cre:BVRfl/fl mice upon administration of LPS compared with the control groups analyzed (Fig. 5A). However, in contrast to the increased number of C5aR1+ cells in the total population of peritoneal isolates, we detected a lower number of C5aR1+/F.480+ residential macrophages in the peritoneal cavity upon administration of LPS (Fig. 5B). There was a slight but nonsignificant increase in the number of C5aR1+/F4.80+ peritoneal macrophages in LysM-Cre:BVRfl/fl compared with BVRfl/fl mice (Fig. 5B).

FIGURE 5.
FIGURE 5.

Lack of BVR promotes C5aR1 expression in peritoneal infiltrates in LysM-Cre:BVRfl/fl in response to LPS. (A and B) Peritoneal cells were isolated from BVRfl/fl and LysM-Cre:BVRfl/fl mice 24 h after LPS injection. The number of C5aR1+ cells was measured by flow cytometry 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 post hoc test. (C–H) The flow cytometry analyses of C5aR1+ cells and C5aR1+/F4.80+ cells in the populations isolated from spleens (C and D), livers (E and F), or lungs (G and H) of LysM-Cre:BVRfl/fl and BVRfl/fl mice. Data are representative for (n = 6–10) mice. *p < 0.05, **p < 0.01, ***p < 0.001, ANOVA with Tukey post hoc test.

We also performed an extensive analysis of a surface expression of C5aR1 in organs by flow cytometry (Fig. 5C–H). LPS administration increased the number of C5aR1+ and C5aR1+/F4.80+ populations in both strains in the spleen (Fig. 5C, 5D). The size of the spleen was increased after LPS administration but did not differ between the strains (Supplemental Fig. 3). There was no difference in the basal number of C5aR1+, C5aR1+/F4.80+, or C5aR1+/CD11b+ in the organs between LysM-Cre:BVRfl/fl compared with BVRfl/fl mice (Fig. 5, Supplemental Fig. 4A). We did not see any difference in the numbers of C5aR1+ cells in the strains or after LPS treatment in the liver (Fig. 5E, 5F). There was, however, a significant elevation in the number of C5aR1+ cells in the lungs of LPS-treated animals, but there was no difference between the strains (Fig. 5G, 5H).

The effects of BVR on chemotaxis and chemokine expression are in part mediated by C5aR1

To evaluate the role of C5aR1 in the effects seen upon deletion of BVR, BMDM were preincubated with neutralizing Ab against C5aR1 or control IgG. Confirmation of neutralization of C5aR1 was performed by flow cytometry (Fig. 6A). C5a-induced chemotaxis in LysM-Cre:BVRfl/fl BMDM was significantly inhibited by this neutralizing Ab against C5aR1 (Fig. 6B, 6C), suggesting a functional role of this receptor in BVR-mediated effects in BMDM.

FIGURE 6.
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, ANOVA with post hoc Tukey test. (B and C) Representative images at original magnification ×40 (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 ± 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 BVRfl/fl mice were incubated with anti-mouse IgG or C5aR1 for 30 min and then LPS/IFN-γ (M1) was added for 24 h. Expression of IP-10 (D) and 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.

Next, we assessed whether the BVR-modulated RANTES and IP-10 levels were dependent on C5aR1 signaling (Fig. 6D, 6E). LPS/IFN-γ–induced RANTES expression in BMDM from LysM-Cre:BVRfl/fl was blunted after treatment with the C5aR neutralizing Ab. However, a similar effect was not noted for IP-10 expression (Fig. 6D, 6E).

Discussion

In the current study, we show that BVR is a regulator of macrophage chemotaxis toward C5a and its effects are partially dependent on C5aR1. Conditional deletion of BVR not only augmented C5aR1 expression in primary macrophages but also enhanced C5a-mediated chemotaxis. We showed similar effects of BVR deletion on C5aR1 in an endotoxemia model in vivo. Further, the LPS-induced chemokine RANTES was elevated in the absence of BVR-A and was subsequently blunted by C5aR1 blockade. We suggest that the remarkable effects of BVR on C5a–C5aR1 signaling and macrophage polarization are crucial in the negative regulation of innate immune responses.

We have previously shown that BV inhibits TLR-4 expression in part via BVR binding to AP-1 and GATA-4 sites (6). BV is also known to block C5aR1 expression in part via the mTOR pathway (27); however, whether the effects are dependent on the activity of BVR has not been studied before. Silencing of surface BVR with RNA interference abrogated BV-induced Akt (protein kinase B) phosphorylation (5) and IL-10 expression and thus was implicated in the induction of an anti-inflammatory phenotype of macrophages (5, 33). The anti-inflammatory effects of BV in models of sepsis (5, 34), transplantation, and ischemic injury (35) have also been demonstrated; however, the effects of BVR deletion on complement receptor and macrophage polarization status have not been studied before.

Increased expression of C5a and its receptor C5aR1 are strongly associated with acute and chronic inflammation and inflammatory disorders (20, 21, 23). C5aR has also been implicated in the control of M2 polarization of macrophages in the tumor microenvironment (36). Our studies indicate the importance of C5aR1 in regulation of M1-like chemokine release upon deletion of BVR. C5aR1 is a G protein–coupled receptor and is expressed in both myeloid and nonmyeloid cells, and increased expression of C5aR1 has been observed in inflamed tissues (21, 37). Indeed, we observed more 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 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 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-γ stimulated higher expression of RANTES, IP-10, and IL-1β in macrophages. Indeed, C5a stimulated RANTES and IL-1β 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 264.7 cells may be due to typical variation between the primary versus cultured cell lines; however, this may also emphasize the importance of BVR deletion for extended periods of time or be linked to secondary effects of BVR deletion in RAW 264.7 macrophages.

Lack of BVR in BMDM from LysM-Cre:BVRfl/fl mice resulted in higher protein expression of C5aR1. Our in vitro findings are supported by in vivo studies, in which we report higher total C5aR1 expression in the spleen, liver, and lung from LysM-Cre:BVRfl/fl mice and surface C5aR1 in peritoneal macrophages from LysM-Cre:BVRfl/fl mice treated with LPS. Transcriptional activation of the C5aR1 promoter basally and in the presence of LPS, requires NF-Y (47), which was induced upon treatment with heme, a precursor of BV (48). We cannot exclude the possibility that C5aR1 levels are regulated on the cell surface in addition to mRNA levels. Further, BMDM isolated from LysM-Cre:BVRfl/fl mice and treated with LPS did not show any difference in NF-κB signaling, which indicates that other signaling pathways are implicated in BVR-regulated chemokine and cytokine expression in response to LPS.

Activation of C5aR1 promotes recruitment of neutrophils and macrophages at the site of infection, trauma, and inflammation (49, 50). Indeed, higher levels of C5aR1 in BMDM depleted of BVR corresponded to higher chemotaxis activity. Soruri et al. (29) showed that the blockade of C5aR1 with neutralizing Ab against C5aR1 (clone 20/70) inhibited the migration of rat basophilic 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 eosinophils and neutrophils as well as production of proinflammatory cytokines in response to OVA allergen sensitization and challenge, indicating the immunomodulatory potential of C5a–C5aR1 signaling (50).

Collectively, our data demonstrate that BVR is an important molecule required for regulating macrophage chemotaxis toward C5a, and deletion of BVR in macrophages promotes chemotaxis toward C5a. We report that the increased cell migration of BMDM toward C5a in LysM-Cre:BVRfl/fl mice was suppressed after treatment with a C5aR1 neutralizing Ab, suggesting a partial role for C5aR1 in BVR-mediated modulation of chemotaxis. Moreover, we translated our findings to an in vivo model, where we observed that peritoneal cells from LPS-treated LysM-Cre:BVRfl/fl mice expressed more C5aR1 compared with BVRfl/fl mice.

In summary, we show that BVR modulates C5a–C5aR1 signaling and macrophage phenotype in part via regulation of C5aR1 expression. We identified, in this study, BVR as a target for regulating responses to LPS and complement activation products. Alteration in BVR expression may, therefore, modulate macrophage polarization and contribute to the development of an inflammatory pathologic condition.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health/National Institute of Diabetes Digestive and Kidney Diseases Grant R01 DK104714 and start-up funds from the Department of Surgery at Beth Israel Deaconess Medical Center and the Eleanor Shore Harvard Medical School Foundation (to B.W.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    BMDM
    bone marrow–derived macrophage
    BR
    bilirubin
    BV
    biliverdin
    BVR
    biliverdin reductase
    C5aR1
    complement activation fragment 5a receptor
    qPCR
    quantitative PCR
    RT
    room temperature
    shRNA
    short hairpin RNA
    shRNAmir
    microRNA-adapted short hairpin RNA.

  • Received October 13, 2017.
  • Accepted March 11, 2019.

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