Cutting Edge: LL-37−Mediated Formyl Peptide Receptor-2 Signaling in Follicular Dendritic Cells Contributes to B Cell Activation in Peyer's Patch Germinal Centers This information is current as of September 25, 2021. Sae-Hae Kim, Yu Na Kim and Yong-Suk Jang J Immunol 2017; 198:629-633; Prepublished online 14 December 2016; doi: 10.4049/jimmunol.1600886 http://www.jimmunol.org/content/198/2/629 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/12/14/jimmunol.160088 Material 6.DCSupplemental http://www.jimmunol.org/ References This article cites 29 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/198/2/629.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision by guest on September 25, 2021 • No Triage! 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Th eJournal of Cutting Edge Immunology Cutting Edge: LL-37–Mediated Formyl Peptide Receptor-2 Signaling in Follicular Dendritic Cells Contributes to B Cell Activation in Peyer’s Patch Germinal Centers Sae-Hae Kim,*,† Yu Na Kim,† and Yong-Suk Jang*,† Peyer’s patches (PPs) are the major mucosal immune- pathogen infection. However, GCs in PPs are continuously inductive site, and germinal centers (GCs) in PPs de- active as a result of stimulation by commensal bacteria in the termine the quality of the Abs produced. PP GCs are gut; mice treated with antibiotics (ABXs) or raised under continuously induced by the gut microbiota, and their germ-free conditions contain few PP GCs (4). Although the maintenance contributes to the induction of strong IgA formation and maintenance of GC follicles in PPs are poorly defined, pre-existing GCs are closely associated with the in- responses to Ags. In this study, we investigated the role Downloaded from of formyl peptide receptor (FPR)-mediated signaling in duction of strong, highly synchronized, and oligoclonal IgA the maintenance of PP GCs, because FPRs recognize responses dominated by affinity-matured cells against the microbiota and initiate an innate immune response T-dependent Ags following oral immunization (5). by chemotaxis. We found that follicular dendritic cells During GC formation and maintenance, follicular dendritic (FDCs), a key organizer of B cell follicles and GCs in cells (FDCs) derived from perivascular precursors of stromal mucosal immunity, express Fpr2. Additionally, Fpr2- cells have long been regarded as Ag-retaining and Ag-presenting http://www.jimmunol.org/ mediated signaling in PP FDCs promoted Cxcl13 reticular cells. However, recent reports demonstrate that FDCs and B cell activating factor expression, as well as are directly associated with the induction of humoral immunity B cell proliferation and activation. Therefore, we sug- through GC formation and maintenance (6). For example, gest that Fpr2-mediated signaling in FDCs plays a FDCs express CXCL13, which is involved in the maintenance key role in GC maintenance in PPs and results in an of an organized follicular structure in GCs by attracting Ag-specific IgA response in the gut mucosal immune CXCR5-expressing B or T cells, IL-6, and B cell activating compartment. The Journal of Immunology, 2017, factor (BAFF), which enhances B cell survival (7). Addition- 198: 629–633. ally, FDCs matured by lymphotoxin and TNF signaling de- by guest on September 25, 2021 rived from B cells express surface receptors, such as VCAM-1, ICAM-1, MadCAM-1, low-affinity Fc receptor, and TLRs (8). he gut mucosa is exposed to various microorganisms, In pLNs, TLR4 signaling in FDCs enhances FDC activation and and secretory IgA plays a major regulatory role in contributes to Ig class-switched high-affinity plasma and memory T mucosal immune homeostasis through immune ex- B cell formation (9). Although the expression of TLR transcripts clusion and neutralization by binding to pathogenic compo- in gut FDCs is lower than that of pLN FDCs, expression of the nents (1). Class-switch recombination to produce IgA occurs retinoic acid receptor and cosignaling of this receptor with TLRs in germinal centers (GCs) of Peyer’s patches (PPs), sites of directly regulate the increased IgA isotype switching through se- mucosal immune induction (2). Specifically, GCs are com- cretion of TGF-b and BAFF (8). Consequently, we expect that partments within a secondary lymphoid organ and are closely specific receptor-mediated signaling in gut FDCs is closely asso- associated with B cell clonal expansion and affinity matura- ciated with GC maintenance. tion, leading to the production of high-affinity Abs, a hall- The formyl peptide receptors (FPRs) are classic chemo- mark of adaptive immunity, against specific Ags (3). In attractant G protein–coupled receptors that are associated peripheral lymph nodes (pLNs), GC formation is induced by with leukocyte trafficking, cell differentiation, and wound *Department of Molecular Biology and the Institute for Molecular Biology and Genet- Address correspondence and reprint requests to Dr. Yong-Suk Jang, Department of ics, Chonbuk National University, Jeonju 54896, Korea; and †Department of Bioactive Molecular Biology, Chonbuk National University, 567 Baekje-Daero, Dukjin-Gu, Material Sciences and Research Center of Bioactive Materials, Chonbuk National Uni- Jeonju 54896, Korea. E-mail address: [email protected] versity, Jeonju 54896, Korea The online version of this article contains supplemental material. ORCID: 0000-0002-3323-5426 (Y.N.K.). Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; ABX, antibiotic; Received for publication May 26, 2016. Accepted for publication November 14, 2016. BAFF, B cell activating factor; CLSM, confocal laser scanning microscopy; CRAMP, 2 cathelin-related antimicrobial peptide; FDC, follicular dendritic cell; FDC-M1+, B220 This work was supported by Grants 2014K1B1A1073861 (to Y.-S.J.) and 2 CD3 FDC-M1+; FPR, formyl peptide receptor; Fpr, mouse FPR; GC, germinal center; 2014R1A1A3051207 (to S.-H.K.) through the National Research Foundation, which LP,laminapropria;pLN,peripherallymphnode;PP,Peyer’spatch;PPL, is funded by the Korean Ministry of Science, ICT, and Future Planning, as well as by PP lymphocyte; qRT-PCR, quantitative real-time PCR; SPF, specific pathogen-free; Grant HI15C3039 (to Y.-S.J.) through the Korea Health Industry Development Insti- WRW(4), WRWWWW. tute, which is funded by the Korean Ministry of Health and Welfare. Y.-S.J. was supported by the Research Base Construction Fund Support Program funded by Chon- Ó buk National University in 2016, S.-H.K. was supported by the Program of National Copyright 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 University for Innovation and Transformation, and Y.N.K. was supported by the BK21 PLUS program in the Department of Bioactive Material Sciences. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600886 630 CUTTING EDGE: Fpr2 SIGNALING IN FDCs OF PEYER’S PATCHES healing (10, 11). FPRs are also considered innate immune In vivo experiments sensors because various bacteria-derived chemotactic peptides, SPF BALB/c mice were orally administered synthetic LL-37 peptide (50 mg) or such as N-formyl peptide derived from Escherichia coli, Lis- rEGFP conjugated or not with LL-37 (10 mg). After 7 d, B220+IgA+, B220+ teria peptides, and phenol-soluble modulin peptide from IgM+, and B220+GL7+CD95+ PPLs were analyzed by flow cytometry. The numbers of EGFP-specific IgA+ cells in PP and lamina propria (LP) from Staphylococcus aureus, are cognate ligands for these receptors some experiments were determined after 14 d by ELISPOT analysis. (12–14). Additionally, fpr1- and fpr2-deficient mice show increased susceptibility to Listeria monocytogenes and Pneu- Immunofluorescence analysis mococcal meningitis infection (15, 16). The human FPR family The PP specimens prepared from SPF BALB/c mice were stained with a biotin- consists of FPR1, FPR2, and FPR3, and the mouse FPR (Fpr) conjugated anti-FDC–M2 Ab, anti-Fpr2 Ab, and anti-CD45 Ab and then + family is composed of eight members. Among them, Fpr2 is a counterstained with DAPI. Sorted FDC-M1 cells were cultured on fibronectin-coated dishes with TNF-a (5 ng/ml) and anti-LTbRAb(1mg/ml) low-affinity receptor for N-formyl peptide and has several en- for 3 d. After treatment with biotin-conjugated LL-37 peptide (10 mM) for 5 dogenous ligands, such as phosphoenolpyruvate, LL-37, serum or 15 min, cells were fixed with 4% paraformaldehyde, permeabilized with amyloid A, and prion protein 106–126 peptide (11). Notably, 0.2% Triton X-100, stained with an anti-Fpr2 Ab, fluorescent dye–conju- some of these endogenous ligands, such as LL-37 and serum gated streptavidin, and anti-CD21/35 Ab, and counterstained with DAPI. Specimens were analyzed by confocal laser scanning microscopy (CLSM) amyloid A, are expressed in the mucosal compartment as a result (LSM 510 META; Carl Zeiss, Thornwood, NY). of exposure to specific microbes and their metabolites (17, 18). Thus, it is conceivable that Fpr2 is involved in mucosal immune Measurement of calcium influx + regulation, although its precise role is unclear. In this study, we Sorted FDC-M1 cells were cultured for 3 d, treated with 10 mM Fpr2 an- Downloaded from identified the expression of Fpr2 in PP FDCs. We also found tagonistic peptide [WRW(4)] for 1 h, and loaded with Fluo-4 AM using a that Fpr2 signaling via interaction with LL-37 ligand enhances Fluo-4 Calcium Imaging Kit (Molecular Probes), according to the manu- facturer’s recommendations.