CXCR4s in Teleosts: Two Paralogous Chemokine Receptors and Their Roles in Hematopoietic Stem/Progenitor Cell Homeostasis This information is current as of October 2, 2021. Xin-Jiang Lu, Kai Zhu, Hong-Xia Shen, Li Nie and Jiong Chen J Immunol 2020; 204:1225-1241; Prepublished online 20 January 2020; doi: 10.4049/jimmunol.1901100 Downloaded from http://www.jimmunol.org/content/204/5/1225 Supplementary http://www.jimmunol.org/content/suppl/2020/01/17/jimmunol.190110 Material 0.DCSupplemental http://www.jimmunol.org/ References This article cites 65 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/204/5/1225.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on October 2, 2021 • No Triage! 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The Journal of Immunology CXCR4s in Teleosts: Two Paralogous Chemokine Receptors and Their Roles in Hematopoietic Stem/Progenitor Cell Homeostasis Xin-Jiang Lu, Kai Zhu, Hong-Xia Shen, Li Nie, and Jiong Chen Hematopoietic stem/progenitor cells (HSPCs) generate the entire repertoire of immune cells in vertebrates and play a crucial role during infection. Although two copies of CXC motif chemokine receptor 4 (CXCR4) genes are generally identified in teleosts, the function of teleost CXCR4 genes in HSPCs is less known. In this study, we identified two CXCR4 genes from a teleost, ayu (Plecoglossus altivelis), named PaCXCR4a and PaCXCR4b. PaCXCR4b was constitutively expressed in ayu HSPCs, whereas PaCXCR4a was induced by LPS treatment. The stromal-derived factor-1–binding activity of CXCR4b was significantly higher than that of CXCR4a, whereas the LPS-binding activity of CXCR4a was significantly higher than that of CXCR4b in the teleosts + ayu, large yellow croaker (Larimichthys crocea), and tiger puffer (Takifugu rubripes). CXCR4a HSPCs were mobilized into blood Downloaded from by LPS, whereas CXCR4b+ HSPCs were mobilized by leukocyte cell–derived chemotaxin-2. PaSDF-1 and PaCXCR4b, but not PaCXCR4a, inhibited HSPC proliferation by regulating reactive oxygen species levels. Compared with PaCXCR4b+ HSPCs, PaCXCR4a+ HSPCs preferentially differentiated into myeloid cells in ayu by maintaining high stem cell leukemia expression. These data suggest that the two copies of CXCR4s achieve a division of labor in the regulation of teleost HSPC homeostasis, supporting the concept that subfunctionalization after gene duplication in teleosts may stabilize the immune system. The Journal of Immunology, 2020, 204: 1225–1241. http://www.jimmunol.org/ ematopoiesis is a complex and well-orchestrated process progenitor cell (HSPC) homeostasis, including proliferation, mo- that produces all lineages of immune cells in vertebrates bilization, and differentiation (1, 2). Teleosts, as early vertebrates, H (1). In the bone marrow niches of mammals, a variety of constitute a highly successful and diverse group, including half of factors have been reported to be crucial for hematopoietic stem/ vertebrate species (3). The immune system of teleosts shows many differences compared with that of mammals. For example, teleost-specific genome duplication produces two copies of several State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and crucial immune genes that exhibit subfunctionalization (4, 5). by guest on October 2, 2021 Safety of Agro-products, Ningbo University, Ningbo 315211, Zhejiang, People’s Moreover, HSPC composition has been modified for adaptation to Republic of China; Laboratory of Biochemistry and Molecular Biology, School of Marine Sciences, Ningbo University, Ningbo 315211, People’s Republic of China; special environments in some teleosts, such as cavefish (Astyanax and Key Laboratory of Applied Marine Biotechnology of Ministry of Education, mexicanus) (R. Peuß, A.C. Box, Y. Wang, S. Chen, J. Krishnan, Ningbo University, Ningbo 315211, Zhejiang, People’s Republic of China D. Tsuchiya, B. Slaughter, and N. Rohner, manuscript posted on ORCID: 0000-0002-7796-4401 (X.-J.L.). bioRxiv). However, the teleost-specific regulatory mechanism Received for publication September 10, 2019. Accepted for publication December underlying HSPC homeostasis is still unclear. 11, 2019. Although HSPCs are not mature immune cells and are frequently This work was supported by the Program for the Natural Science Foundation of China (31972821; 31772876; 41776151), the Zhejiang Provincial Natural Science dormant, it has been suggested that HSPCs participate in the Foundation of China (LR18C040001; LZ18C190001), the Scientific Innovation Team primary response to infections (6, 7). HSPCs can be mobilized by Project of Ningbo (2015C110018) and the K.C. Wong Magna Fund in Ningbo directly sensing infection through TLR (8). In addition to the di- University. rect sensing of pathogens, HSPC activity in response to infection The sequences presented in this article have been submitted to GenBank (https:// www.ncbi.nlm.nih.gov/genbank/) under accession numbers MN148390, MN148391, is also mediated indirectly by proinflammatory cytokines (9, 10). MN148393, MN158722, MN158723, MN158724, MN156535, and MN156536, Furthermore, infections also affect the lymphoid versus myeloid MN628572, MN628573. fate choice. LPS binds with TLR4 to induce the rapid generation Address correspondence and reprint requests to Prof. Jiong Chen, Ningbo University, of myeloid cells, including macrophages (11). In zebrafish, HSPCs 818 Fenghua Road, Ningbo 315211, Zhejiang, People’s Republic of China. E-mail address: [email protected] can also proliferate and differentiate into required immune cell lineages postinfection (12). HSPCs are mobilized into the pe- The online version of this article contains supplemental material. ripheral blood from the kidney postinfection in another teleost, Abbreviations used in this article: BPI, bacterial/permeability increasing protein; CFU-C, CFU cell; CRU, competitive repopulating unit; DHR, dihydrorhodamine- ayu (Plecoglossus altivelis) (13). HSPC mobilization is induced 123; EGR1, early growth response protein 1; GATA2, GATA-binding protein 2; by bacterial components such as LPS (14), or host cytokines such GATA3, GATA-binding protein 3; HSC, hematopoietic stem cell; HSPC, hematopoi- as G-CSF and leukocyte cell–derived chemotaxin-2 (LECT2) etic stem/progenitor cell; LECT2, leukocyte cell–derived chemotaxin-2; Mef2C, myocyte enhancer factor 2C; MHC I, MHC class I; MO/MF, monocyte/macrophage; (15). However, it is necessary to further investigate the regulatory MPO, myeloperoxidase; NAC, N-acetylcysteine; PaCXCR4a, P. altivelis CXCR4a; mechanism underlying HSPC self-renewal, mobilization, and PaCXCR4b, P. altivelis CXCR4b; PaSRB2a, ayu scavenger receptor class B 2a; PAX5, reduced paired box 5; PU.1, PU box-binding protein; ROS, reactive oxygen differentiation postinfection in teleosts. species; RT-qPCR, real-time quantitative PCR; RUNX1, runt-related transcription CXC chemokines and their receptors play crucial roles in the factor 1; SCL, stem cell leukemia; SDF, stromal cell–derived factor; shRNA, short migration and function regulation of immune cells in both teleosts hairpin RNA; siRNA, small interfering RNA; SP, side population. and mammals (16, 17). Stromal cell–derived factor (SDF)-1 (also Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50 known as CXCL12) is a member of the CXC group of chemokines www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901100 1226 THE ROLES OF TWO CXCR4s IN TELEOST HSPCS (18). The physiological SDF-1 receptor is CXC motif chemokine To inhibit HSPC mobilization in vivo, we neutralized LPS and LECT2 receptor 4 (CXCR4), a heptahelical receptor coupled to hetero- using human bacterial/permeability increasing protein (BPI) peptide and trimeric GTP-binding proteins (19). SDF-1/CXCR4 is involved in anti-LECT2 Ab. The BPI peptide (85–99 aa) was synthesized by GL Biochem (Shanghai, China) and has been demonstrated to inhibit the HSPC homeostasis, development, tumor metastasis, metabolism, binding of LPS to monocytes (33). The anti-LECT2 Ab was prepared by and HIV entry (20–22). CXCR4 normally promotes bone marrow immunizing mice with recombinant LECT2, which was produced in our HSPC mobilization, homing, retention, and quiescence (23). previous work (34). Ayu were treated i.p. with 0.5 mg of BPI peptide/g Moreover, CXCR4 desensitization affects the lymphoid differen- body weight or 0.5 mg of anti-LECT2 Ab/g 30 min postinfection. tiation of HSPCs (24). CXCR4 genes have also been cloned in a Characterization of cDNA gene sequences in teleosts variety of teleosts (16). It has long been known that two copies of CXCR4 genes (CXCR4a and CXCR4b) exist in teleosts, whereas The cDNA sequences of the ayu CXCR4a (P. altivelis CXCR4a [PaCXCR4a]), CXCR4b (P. altivelis CXCR4b [PaCXCR4b]) and P. altivelis