Cutting Edge: LL-37−Mediated Formyl 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! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 . 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 –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 , 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. Briefly, cells were loaded with Fluo-4 AM at 37˚C the levels of Cxcl13 and Tnfsf13b (BAFF gene) transcripts, as for 30 min, incubated for 30 min at room temperature, washed, and sus- well as in vitro B cell proliferation and activation. pended in live cell imaging solution containing 2 mM glucose. Cells were subjected to CLSM, and calcium influx was recorded by fluorescence emission at 506 nm after stimulation with synthetic LL-37 peptide for 200 s. Data were Materials and Methods analyzed using LSM 501 software. http://www.jimmunol.org/ Mice and experimental materials Syngeneic specific pathogen–free (SPF) BALB/c mice were purchased from Quantitative real-time PCR Charles River Technology through Orient Bio (Sungnam, Korea). ABX- Sorted FDC-M1+ cells were treated or not with synthetic LL-37 peptide (10 treated mice were subjected to oral administration of an ABX mixture (1 g/ mM) for 2 h. Total RNA was prepared using an RNeasy Plus Micro Kit, and l ampicillin, 0.5 g/l vancomycin, and 0.1 g/l polymyxin) in drinking water cDNA was prepared with a QuantiTect Reverse Transcription kit (both from for 30 d. All chemicals were purchased from Sigma Chemical (St. Louis, QIAGEN, Hilden, Germany). Quantitative real-time PCR (qRT-PCR) re- MO), unless otherwise specified. Synthetic LL-37 (LLGDFERKSKE- actions were run on an ABI 7500 (Applied Biosystems, Waltham, MA) with a KIGKEFKRIVQRIKDFLRNLVPRTES) and Biotin-LC–LL-37 peptides and QuantiTect SYBR Green PCR Kit using the following specific primer sets: WRWWWW [WRW(4)]-NH2 were purchased from ANYGEN (Jangseong, Cxcl13-F, 59-TGT CCA AAG CAA AAG TCT GTC T-39 and Cxcl13-R,

a by guest on September 25, 2021 Korea) and AnaSpec (Fremont, CA), respectively. rTNF- was purchased 59-ATA GTG GCT TCA GGC AGC TC-39; Tnfsf13b-F, 59-TGG GAG from R&D Systems (Minneapolis, MN). Collagenase D and DNase I were CAG AGT CCT GAT GT-39 and Tnfsf13b-R, 59-GCT TCT GGG TGA purchased from Roche Applied Science (Mannheim, Germany). GTA CTG CT-39; and Fpr2-F, 59-GAG ACC TCA GCT GGT TGT GC- Anti–FDC-M1, anti-B220 (biotin- or allophycocyanin-Cy7 conjugated), 39 and Fpr2-R, 59-ACC ACC ACT TCT GAT CCA TTC A-39. The relative and anti–VCAM-1 (FITC-conjugated) Abs, purified mouse IgA, purified rat quantity of cDNA was calculated by the DD threshold cycle method using IgG2c (biotin-conjugated), streptavidin-FITC, streptavidin-allophycocyanin, 18S rRNA gene as a reference. Experimental results were acquired from three streptavidin-PE, streptavidin–PE–CF594, and 7-aminoactinomycin D (7- independent experiments, in which each gene was assayed in triplicate. AAD) were purchased from BD Biosciences (Franklin Lakes, NJ). Anti–FDC- M2 Ab was purchased from Amsbio (Cambridge, MA). Anti-TLR2 and Statistical analysis Cy3-conjugated anti-CD45 Abs were purchased from Abcam (Cambridge, MA). FITC- or PE-conjugated secondary Abs were purchased from Santa Statistical analyses were performed using Prism 6 software (GraphPad, La Jolla, Cruz Biotechnology (Dallas, TX). Anti-Fpr2, Alexa Fluor 594–, Alexa Fluor CA). Data are presented as mean 6 SD of triplicates. Differences among the 680– or Alexa Fluor 700–conjugated secondary Abs, and DAPI were pur- means of multiple independent variables were compared between the control chased from Invitrogen (Grand Island, NY). Biotin-conjugated anti-FDC–M2 and each group using one-way ANOVA, followed by the Tukey post hoc test. Ab was purchased from eBioscience (San Diego, CA). FITC-conjugated GL7 Differences in mean values were considered significant at p , 0.05. and anti-IgM Abs were purchased from Bethyl Laboratories (Montgomery, TX). Results and Discussion Cell isolation and flow cytometry PP FDCs express Fpr2 PP FDCs were prepared according to a procedure reported previously with minor modifications (8, 19). Briefly, ∼50 PPs were collected from 10 mice, Regulation between innate and adaptive immunity in PPs is and PP lymphocytes (PPLs) were prepared after tissue digestion with colla- pivotal in host defense, because PP cells are directly or indi- 2 genase. B220 PPLs were collected using an AutoMACS Separator (Miltenyi rectly affected by microorganisms introduced from the in- Biotec, Bergisch Gladbach, Germany) and stained with anti-CD3 and anti– 2 testinal lumen (20). We assumed that Fpr2 contributes to FDC-M1 Abs and 7-AAD. Data were acquired from the collected live B220 2 CD3 FDC-M1+ (FDC-M1+) cells using a FACSAria III (BD Biosciences) immune regulation in PPs to maintain GCs, because it may and analyzed using FlowJo software (TreeStar, Ashland, OR). play a role as an innate receptor linking innate and adaptive immunity. To detect the expression of Fpr2 in PP cells, we B cell analysis investigated PP tissue slices containing GCs prepared from After stimulating the isolated FDCs with 2 mg/ml LPS or 1 mM synthetic LL- SPF mice. GCs primarily consisted of leukocyte lineage cells, 37 peptide for 2 h, 100 FDCs were cocultured with splenic B cells (5 3 103) tagged with CFSE (eBioscience). After 3 d, cells were stained with 7-AAD and such as B and T cells, and stroma-derived cells, such as FDCs; analyzed by flow cytometry. The absolute cell number was calculated using anti-CD45 and anti–FDC-M2 Abs can distinguish these cells. 2 123count eBeads (eBioscience), according to the manufacturer’s instructions. We initially detected the expression of Fpr2 in CD45 FDC- To analyze the level of IgA in the culture supernatant, 100 FDCs, which had M2+ cells (Fig. 1A). FDCs can be further identified using been stimulated with the indicated molecules, were cocultured with splenic or PP B cells. After 5 d, the amount of IgA in the culture supernatant was anti–FDC-M1 or anti–FDC-M2 Ab; anti–FDC-M1 Ab measured by sandwich ELISA. interacts with fat globule epidermal growth factor VIII The Journal of Immunology 631

increased significantly (p , 0.05) by ∼60-fold in response to LL-37, suggesting positive-feedback regulation of Fpr2 in FDCs by an endogenous ligand of Fpr2 (Fig. 2A). We further investigated the direct interaction between LL-37 and Fpr2 in FDC-M1+ cells and found that Fpr2 was distributed in the 2 subcellular compartment of purified B220 FDC-M1+ cells that were not stimulated with LL-37, whereas Fpr2 was lo- calized at the cell periphery, together with LL-37, as long as 5 min after stimulation with LL-37 (Fig. 2C). We next determined whether LL-37–mediated Fpr2 sig- naling triggers intracellular Ca2+ mobilization, because acti- vation of Fpr2 triggers intracellular Ca2+ mobilization by G protein–dependent stimulation, which, in turn, induces chemotaxis (22). When we monitored Ca2+ 2 influx by CLSM during incubation of isolated PP B220 FDC-M1+ cells that were pretreated or not with the Fpr2 antagonist peptide WRW(4) (10 mM), we found that LL-37 + treatment of FDC-M1 cells resulted in an increased level of Downloaded from intercellular Ca2+ that was abrogated in WRW(4)-pretreated cells (Fig. 2B, 2C). These results suggest that the interaction between LL-37 and Fpr2 in FDC-M1+ cells transduces the positive signal into the cells. In fact, LL-37 is continuously FIGURE 1. PP FDCs express Fpr2 with VCAM-1 and TLR2. (A) The ileum PP tissue slide was stained by anti–FDC-M2 (green), anti-CD45 (sky expressed in the ileum, and the finding that its expression is blue), and anti-Fpr2 (red) Abs and counterstained with DAPI (blue) and regulated negatively by Shigella infection and positively by http://www.jimmunol.org/ 2 monitored by CLSM. Scale bars, 50 mm. (B) B220 cells were stained with microbiota-derived metabolites, such as short-chain fatty allophycocyanin-Cy7–conjugated anti-B220, anti-CD3, and anti-FDC M1 acids, suggests that LL-37–mediated FPR-2 signaling in PP Abs, followed by biotin-conjugated anti-rat IgG2c Ab and PE-CF594– conjugated streptavidin. FDC-M1+ cells were sorted by flow cytometry. Sorted cells were stained separately with FITC-conjugated anti–VCAM-1 Ab, anti- TLR2 Ab, or anti-Fpr2 Ab. (C) Cxcl13, tnfsf13b, and fpr2 transcript expression 2 in FDC-M1+ or FDC-M1 cells, sorted as described above, was measured by qRT-PCR. Data represent the mean 6 SD and a representative result from , three independent experiments is shown. *p 0.05. by guest on September 25, 2021 expression, which increases during the GC reaction, whereas anti–FDC-M2 Ab recognizes complement component C4 in naive FDCs (19). For further confirmation of the expression 2 of Fpr2 in PP FDCs, B220 PP cells were enriched, FDCs were sorted using anti–FDC-M1 Ab, and PP FDC-M1+ cells were analyzed. As reported previously, PP FDC-M1+ cells displayed the highest expression of molecules related to the GC reaction, such as VCAM-1 and Cxcl13 and Tnfsf13b transcripts, together with low-level TLR2 expression (Fig. 1B, 1C) (8). Notably, TLR transcript expression was reported to be downregulated in PP FDCs compared with that in pLNs (8), and we found that Fpr2 was expressed in PP FDCs. Collectively, these results show that Fpr2 is expressed in PP FDCs and suggest its role as a mucosal innate immune sensor.

LL-37, an endogenous Fpr2 ligand, promotes the activation of PP FDCs FIGURE 2. LL-37 activates PP FDC-M1+ cells via Fpr2 signaling. (A) We next analyzed whether signals are transduced from Fpr2 Tnfsf13b, Cxcl13, and Fpr2 transcript expression was measured by qRT-PCR in sorted FDC-M1+ cells after LL-37 stimulation. Relative quantitation of into PP FDCs through the interaction with a cognate ligand of DD + cDNA was calculated by the threshold cycle method using 18S rRNA genes. Fpr2. To confirm the activation of Fpr2 on FDCs, FDC-M1 A representative result from three independent experiments is shown. (B) Sorted cells sorted from PPs were stimulated with an endogenous FDC-M1+ cells were cultured for 3 d on fibronectin-coated slides with TNF-a Fpr2 ligand, cathelin-related antimicrobial peptide (CRAMP; and anti-LTbR Ab and then treated with biotin-conjugated LL-37. After 5 or + a mouse ortholog of human cathelicidin LL-37), and the 15 min, FDC-M1 cells were fixed; stained with anti-FPR Ab (green), anti- expression of Tnfsf13b and Cxcl13 transcripts was enhanced CD21/35 Ab (PE), and allophycocyanin-conjugated streptavidin (purple); and counterstained with DAPI. Scale bars, 10 mm. (C) Sorted and cultured FDCs by CRAMP treatment (Supplemental Fig. 1) (21). In addi- + that were treated or not with Fpr2 antagonist peptide [WRW(4)] were loaded tion, LL-37 treatment of FDC-M1 cells resulted in signifi- with Fluo-4 AM and treated with synthetic LL-37 peptide (10 mM); intracellular cantly (p , 0.05) increased expression of the Cxcl13 and calcium influx was monitored by CLSM for 200 s and analyzed using LSM 510 Tnfsf13b transcripts. Additionally, the Fpr2 transcript level software. *p , 0.05. DIC, differential interference contrast. 632 CUTTING EDGE: Fpr2 SIGNALING IN FDCs OF PEYER’S PATCHES

FDCs likely plays a role in GC formation and the mainte- prominent in ABX-treated mice (Supplemental Fig. 2A). It is nance of PPs (18, 23). conceivable that the results from ABX-treated mice are closely associated with the level of Fpr2 expression in PP FDCs, Activation of PP FDCs by Fpr2 signaling promotes B cell proliferation because the level of Fpr2 was downregulated in FDC-M1+ and activation cells prepared from ABX-treated mice compared with that in To confirm the biological function of LL-37–mediated Fpr2 SPF mice and was increased by LL-37 treatment in vitro signaling in PP FDCs, we first monitored B cell prolifera- (Supplemental Fig. 2B). Additionally, LL-37 treatment in- tion, because the Tnfsf13b transcript, whose expression was creased the expression of Cxcl13 and Tnfsf13b transcripts in enhanced by LL-37 treatment in PP FDCs, encodes the FDC-M1+ cells prepared from ABX-treated mice, and the B-lymphocyte stimulator BAFF (24). When CFSE-stained increase was abrogated by WRW(4) pretreatment (Supplemental splenic B cells were cultured for 3 d with isolated PP FDC- Fig. 2C). Consequently, these results support the possibility that M1+ cells that were stimulated or not with LPS or LL-37, the increase in the GC B cell population was dependent on B cells cultured with PP FDCs activated by LPS or LL-37 Fpr2-mediated signaling in FDCs. Given that the frequency of exhibited significantly (p , 0.01) enhanced proliferation GC B cells increased (Fig. 3D), and the interaction between compared with B cells cultured in the absence of FDCs or FDCs and GC B cells triggers the generation of plasma cells cultured with FDCs without additional stimulation with LPS producing Ag-specific Abs (25), it is conceivable that the increase or LL-37 (Fig. 3A). We next measured the amount of IgA in GC B cells positively regulated the plasma cells producing Ag- secreted from B cells that had been cocultured in vitro with specific Abs. Finally, we confirmed this speculation using EGFP Downloaded from FDCs that were stimulated or not with LPS or LL-37, because as an Ag and found that the oral administration of LL-37– + active PP FDCs express a high level of TGF-b1, which is conjugated EGFP evoked an increase in EGFP-specific IgA cells involved in IgA isotype switching (8). The FDCs activated by in PPs, as well as in LP, compared with those of EGFP alone LL-37 promoted efficient expression of IgA from B cells de- (Fig. 3E). Taking these findings together, Fpr2 signaling rived from PPs, as well as from the spleen (Fig. 3B). Addi- potentiated FDC function in PPs via cognate ligand LL-37 http://www.jimmunol.org/ tionally, enhanced IgA class switching was observed in PPs of binding. mice orally administered rEGFP conjugated with LL-37 Under steady-state conditions, gut epithelial cells continu- (Fig. 3C). ously express cathelicidin LL-37 as a result of stimulation by For further investigation of the role of Fpr2 in PP FDCs, GC butyrate, a microbial metabolite. Importantly, LL-37 expres- B cells were analyzed from PPs of SPF mice orally administered sion is low in Crohn’s disease patients (18). We believe that the synthetic LL-37 peptide (50 mg). The recruitment of GC the decreased LL-37 expression causes low-level production of B cells expressing CXCR5, GL7, and CD95, which is de- IgA as a result of reduced FDC activation and is closely as- pendent on CXCL13 secreted from FDCs and follicular Th sociated with disease susceptibility, because IgA binds pref- cells, increased in SPF mice treated with LL-37 (Fig. 3D); this erentially to the colitogenic microbiota (26). In addition, by guest on September 25, 2021 accords well with the increase in CXCL13 transcript expres- CRAMP-knockout mice exhibited increased colonization of sion after LL-37 treatment (Fig. 2A). This LL-37–mediated pathogenic intestinal bacteria (27). Therefore, we believe enhancement of GC B cell recruitment in PPs was more that Fpr2 signaling via LL-37 in FDCs facilitates mucosal

FIGURE 3. FDC-M1+ cells activated with LL-37 modulate B cells in vitro and in vivo. Sorted FDC-M1+ cells were stimulated with the indicated molecules for 2 h and then cocultured with B cells for 5 d. (A) Fluorescence intensity in CFSE-labeled splenic B220+ cells cocultured with FDC-M1+ cells was monitored by flow cytometry. The absolute numbers of live B220+ cells were counted by 123-count eBeads. (B) The amount of IgA in the splenic or PP B cell culture medium cocultured with FDC-M1+ cells was measured by ELISA. The synthetic LL-37 peptide (D) or rEGFP conjugated or not with LL-37 (C and E) was administered orally to SPF mice. After 7 d, the frequency of IgA+ or IgM+ (C) or GL7+CD95+ (D) cells in B220+ PPLs was determined by flow cytometry. (E) After 14 d, the number of EGFP-specific IgA cells in PPs or LP was determined by ELISPOT assay. Data are presented as mean 6 SD, and a representative result from three independent experiments is shown. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 633 homeostasis by contributing to maintenance of GCs in PPs. 11. Bloes, D. A., D. Kretschmer, and A. Peschel. 2015. Enemy attraction: bacterial agonists for leukocyte chemotaxis receptors. Nat. Rev. Microbiol. 13: 95–104. Collectively, these data confirmed that Fpr2 stimulation by 12. Bufe, B., T. Schumann, R. Kappl, I. Bogeski, C. Kummerow, M. Podgo´rska, LL-37 in PP FDCs may support the recruitment of germinal S. Smola, M. Hoth, and F. Zufall. 2015. Recognition of bacterial signal peptides by B and T cells, as well as B cell proliferation and activation. We mammalian formyl peptide receptors: a new mechanism for sensing pathogens. J. Biol. Chem. 290: 7369–7387. believe that this interaction between LL-37 and FDCs sup- 13. Kretschmer, D., A. K. Gleske, M. Rautenberg, R. Wang, M. Ko¨berle, E. Bohn, ports the prolonged induction of PP GCs. Additionally, be- T. Scho¨neberg, M. J. Rabiet, F. Boulay, S. J. Klebanoff, et al. 2010. Human formyl peptide receptor 2 senses highly pathogenic Staphylococcus aureus. Cell Host Microbe cause reutilization of PP GCs promotes highly synchronized 7: 463–473. and affinity-matured gut IgA responses against the orally in- 14. Gao, J. L., E. J. Lee, and P. M. Murphy. 1999. Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor. J. Exp. Med. 189: 657–662. troduced Ags (28), we suggest that a GC-stimulation strategy 15. Liu, M., K. Chen, T. Yoshimura, Y. Liu, W. Gong, A. Wang, J. L. Gao, using Fpr2 ligand LL-37 can be applied for the development P. M. Murphy, and J. M. Wang. 2012. Formylpeptide receptors are critical for of an effective mucosal vaccine adjuvant. Indeed, we reported rapid neutrophil mobilization in host defense against Listeria monocytogenes. Sci. Rep. 2: 786. previously that oral administration of an LL-37–conjugated 16. De Yang, Q., A. P. Chen, G. M. Schmidt, J. M. Anderson, J. Wang, J. J. Wooters, Ag enhanced the Ag-specific immune responses by modulat- Oppenheim, and O. Chertov. 2000. LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a re- ing the mucosal environment (29). ceptor to chemoattract human peripheral blood neutrophils, monocytes, and T cells. J. Exp. Med. 192: 1069–1074. 17. Liang, T. S., J. M. Wang, P. M. Murphy, and J. L. Gao. 2000. Serum amyloid A is Acknowledgments a chemotactic agonist at FPR2, a low-affinity N-formylpeptide receptor on mouse CLSM and flow cytometry were performed at the Center for University-Wide neutrophils. Biochem. Biophys. Res. Commun. 270: 331–335.

18. Doss, M., M. R. White, T. Tecle, and K. L. Hartshorn. 2010. Human defensins Downloaded from Research Facilities of Chonbuk National University. and LL-37 in mucosal immunity. J. Leukoc. Biol. 87: 79–92. 19. Usui, K., S. Honda, Y. Yoshizawa, C. Nakahashi-Oda, S. Tahara-Hanaoka, K. Shibuya, and A. Shibuya. 2012. Isolation and characterization of naı¨ve follicular Disclosures dendritic cells. Mol. Immunol. 50: 172–176. 20. Kim, S. H., K. Y. Lee, and Y. S. Jang. 2012. Mucosal immune system and M cell- The authors have no financial conflicts of interest. targeting strategies for oral mucosal vaccination. Immune Netw. 12: 165–175. 21. Chen, K., Y. Xiang, J. Huang, W. Gong, T. Yoshimura, Q. Jiang, L. Tessarollo, Y. Le, and J. M. Wang. 2014. The formylpeptide receptor 2 (Fpr2) and its en- References dogenous ligand cathelin-related antimicrobial peptide (CRAMP) promote den- http://www.jimmunol.org/ 1. Pabst, O. 2012. New concepts in the generation and functions of IgA. Nat. Rev. dritic cell maturation. J. Biol. Chem. 289: 17553–17563. Immunol. 12: 821–832. 22. Niyonsaba, F., K. Iwabuchi, A. Someya, M. Hirata, H. Matsuda, H. Ogawa, and 2. Craig, S. W., and J. J. Cebra. 1971. Peyer’s patches: an enriched source of precursors I. Nagaoka. 2002. A cathelicidin family of human antibacterial peptide LL-37 in- for IgA-producing immunocytes in the rabbit. J. Exp. Med. 134: 188–200. duces mast cell chemotaxis. Immunology 106: 20–26. 3. Phan, T. G., D. Paus, T. D. Chan, M. L. Turner, S. L. Nutt, A. Basten, and 23. Raqib, R., P. Sarker, P. Bergman, G. Ara, M. Lindh, D. A. Sack, K. M. Nasirul R. Brink. 2006. High affinity germinal center B cells are actively selected into the Islam, G. H. Gudmundsson, J. Andersson, and B. Agerberth. 2006. Improved plasma cell compartment. J. Exp. Med. 203: 2419–2424. outcome in shigellosis associated with butyrate induction of an endogenous peptide 4. Fagarasan, S., M. Muramatsu, K. Suzuki, H. Nagaoka, H. Hiai, and T. Honjo. antibiotic. Proc. Natl. Acad. Sci. USA 103: 9178–9183. 2002. Critical roles of activation-induced cytidine deaminase in the homeostasis of 24. Batten, M., J. Groom, T. G. Cachero, F. Qian, P. Schneider, J. Tschopp, gut flora. Science 298: 1424–1427. J. L. Browning, and F. Mackay. 2000. BAFF mediates survival of peripheral im- mature B lymphocytes. J. Exp. Med. 192: 1453–1466. 5. De Silva, N. S., and U. Klein. 2015. Dynamics of B cells in germinal centres. Nat. by guest on September 25, 2021 Rev. Immunol. 15: 137–148. 25. El Shikh, M. E., R. M. El Sayed, S. Sukumar, A. K. Szakal, and J. G. Tew. 2010. 6. McCloskey, M. L., M. A. Curotto de Lafaille, M. C. Carroll, and A. Erlebacher. Activation of B cells by antigens on follicular dendritic cells. Trends Immunol. 31: 2011. Acquisition and presentation of follicular dendritic cell-bound antigen by 205–211. lymph node-resident dendritic cells. J. Exp. Med. 208: 135–148. 26.Palm,N.W.,M.R.deZoete,T.W.Cullen,N.A.Barry,J.Stefanowski,L.Hao, 7. Vissers, J. L., F. C. Hartgers, E. Lindhout, C. G. Figdor, and G. J. Adema. 2001. P. H. Degnan, J. Hu, I. Peter, W. Zhang, et al. 2014. Immunoglobulin A coating BLC (CXCL13) is expressed by different dendritic cell subsets in vitro and in vivo. identifies colitogenic bacteria in inflammatory bowel disease. Cell 158: 1000– Eur. J. Immunol. 31: 1544–1549. 1010. 8. Suzuki, K., M. Maruya, S. Kawamoto, K. Sitnik, H. Kitamura, W. W. Agace, and 27. Iimura, M., R. L. Gallo, K. Hase, Y. Miyamoto, L. Eckmann, and M. F. Kagnoff. S. Fagarasan. 2010. The sensing of environmental stimuli by follicular dendritic 2005. Cathelicidin mediates innate intestinal defense against colonization with cells promotes immunoglobulin A generation in the gut. Immunity 33: 71–83. epithelial adherent bacterial pathogens. J. Immunol. 174: 4901–4907. 9. Garin, A., M. Meyer-Hermann, M. Contie, M. T. Figge, V. Buatois, M. Gunzer, 28. Bergqvist, P., A. Stensson, L. Hazanov, A. Holmberg, J. Mattsson, R. Mehr, K. M. Toellner, G. Elson, and M. H. Kosco-Vilbois. 2010. Toll-like receptor 4 M. Bemark, and N. Y. Lycke. 2013. Re-utilization of germinal centers in multiple signaling by follicular dendritic cells is pivotal for germinal center onset and affinity Peyer’s patches results in highly synchronized, oligoclonal, and affinity-matured gut maturation. Immunity 33: 84–95. IgA responses. Mucosal Immunol. 6: 122–135. 10. Li, L., K. Chen, Y. Xiang, T. Yoshimura, S. Su, J. Zhu, X. W. Bian, and 29. Kim, S. H., I. Y. Yang, J. Kim, K. Y. Lee, and Y. S. Jang. 2015. Antimicrobial J. M. Wang. 2016. New development in studies of formyl-peptide receptors: critical peptide LL-37 promotes antigen-specific immune responses in mice by enhancing roles in host defense. J. Leukoc. Biol. 99: 425–435. Th17-skewed mucosal and systemic immunities. Eur. J. Immunol. 45: 1402–1413.