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 October 2, 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 published online 5 April 2019 Downloaded from http://www.jimmunol.org/content/early/2019/04/04/jimmun ol.1701443 Supplementary http://www.jimmunol.org/content/suppl/2019/04/04/jimmunol.170144 http://www.jimmunol.org/ Material 3.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published April 5, 2019, doi:10.4049/jimmunol.1701443 The Journal of Immunology 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 activa- tion 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: 000–000. 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 rubin (BR) and regulates intracellular signaling by acting signaling (5). Our previous work showed that knockdown of B by guest on October 2, 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 re- duction 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 2 BVR-A IMPACTS CHEMOTAXIS VIA C5a–C5aR1 SIGNALING 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).