A TNF- −α CCL20−CCR6 Axis Regulates Nod1-Induced Responses Maude Paradis, Barbara C. Mindt, Claudia U. Duerr, Olga L. Rojas, Dennis Ng, Bryant Boulianne, Doug D. McCarthy, This information is current as Mingxi Dennis Yu, Leslie E. Summers deLuca, Lesley A. of September 29, 2021. Ward, James B. Waldron, Dana J. Philpott, Jennifer L. Gommerman and Jörg H. Fritz J Immunol 2014; 192:2787-2799; Prepublished online 17

February 2014; Downloaded from doi: 10.4049/jimmunol.1203310 http://www.jimmunol.org/content/192/6/2787

Supplementary http://www.jimmunol.org/content/suppl/2014/02/16/jimmunol.120331 http://www.jimmunol.org/ Material 0.DCSupplemental References This article cites 62 articles, 26 of which you can access for free at: http://www.jimmunol.org/content/192/6/2787.full#ref-list-1

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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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A TNF-a–CCL20–CCR6 Axis Regulates Nod1-Induced B Cell Responses

Maude Paradis,* Barbara C. Mindt,* Claudia U. Duerr,* Olga L. Rojas,† Dennis Ng,† Bryant Boulianne,† Doug D. McCarthy,† Mingxi Dennis Yu,† Leslie E. Summers deLuca,† Lesley A. Ward,‡ James B. Waldron,* Dana J. Philpott,† Jennifer L. Gommerman,†,1 and Jo¨rg H. Fritz*,‡,1

Innate immune responses provoke the accumulation of leukocytes at sites of inflammation. In addition to monocytes and granulocytes, B cells also participate in antimicrobial innate immune responses; however, the mechanisms for accumulation of B cells to sites of inflammation are not well understood. To study B cell accumulation following systemic inflammation, we used a model synthetic ligand

that stimulates a specific pattern recognition molecule, nucleotide-binding oligomerization domain–containing 1 (Nod1). Upon Downloaded from exposure to Nod1 agonists, both B cells and rapidly accumulate within the , and dendritic cells migrate into the periarterial lymphoid sheath. Nod1 stimulation led to a marked increase in several within the spleen, including CXCL13, CCL2, and CCL20. Whereas the pathway was critical for the induction of the B cell chemoattractant CXCL13 in response to Nod1 agonists, B cell accumulation within the spleen following Nod1-induced systemic inflammation was independent of the lymphotoxin pathway. In contrast, a CCR6/CCL20 loop instructed rapid increase of B cells in the spleen in response a to systemic administration of Nod1 agonists in a TNF- –dependent manner. Moreover, CCR6 was required to regulate Nod1- http://www.jimmunol.org/ mediated B cell responses. These results reveal a novel mechanism of B cells during inflammation and shed light on how B cells participate in innate immune responses to microbial stimulation. The Journal of Immunology, 2014, 192: 2787–2799.

he initiation of antimicrobial first-line host defense and cells such as dendritic cells (DC) and granulocytes, and lymphoid Ag-specific immunity is instructed through activation of cells, including NK cells and NKT cells. Such a rapid first-line T a diverse repertoire of germline-encoded pattern recog- response to infection and inflammation requires a change of the nition molecules (PRM), which in mammals comprises several hematopoietic output of the bone marrow (BM), a response often protein families, including TLR and nucleotide-binding oligo- referred to as emergency granulopoiesis, that supports the ex- merization domain (Nod)–like receptors (1, 2). Microbial exposure pansion of short-lived granulocytes (3), which are then rapidly by guest on September 29, 2021 results in the subsequent activation and recruitment of myeloid recruited to sites of inflammation and infection, where they pro- vide microbicidal activity (4). However, infection- or inflammation- *Department of Microbiology and Immunology, McGill University, Complex Traits induced granulopoiesis in the BM occurs at the expense of B Group, Montreal, Quebec H3G 0B1, Canada; †Department of Immunology, Uni- lymphopoiesis (4), and the reduction in BM B cells is accompa- ‡ versity of Toronto, Toronto, Ontario M5S 1A8, Canada; and Department of Phys- nied by increases in the number of B cells in the spleen, indicating iology, McGill University, Complex Traits Group, Montreal, Quebec H3G 0B1, Canada that inflammation mobilizes a large number of B cells from the 1J.L.G. and J.H.F. contributed equally to this work. BM to the periphery (5, 6). This suggests a critical functional Received for publication December 3, 2012. Accepted for publication January 11, 2014. importance for B cells in orchestrating first-line host defense. Indeed, recent evidence underlines significant physiologic roles J.H.F. was supported by the Austrian Academy of Sciences (Austrian Programme for Advanced Research and Technology fellowship), Canadian Institutes of Health Re- for B cells regulating antimicrobial immune responses by diverse search Operating Grant MOP 114972, as well as an infrastructure grant by the Canadian mechanisms ranging from the production of Ab to the secretion of Foundation of Innovation. J.L.G. was supported by Canadian Institutes of Health Re- search Operating Grants MOP 89783 and 67157 and received infrastructure support , chemokines, anti-inflammatory molecules, and micro- from the Ontario Research Fund and the Canadian Foundation for Innovation. C.U.D. bicidal molecules, to Ag presentation and the cross-regulation of was supported by the Canadian Institutes of Health Research and the German National other innate and adaptive cell types (7–13). Although we have made Academy of Sciences Leopoldina. B.B. and L.E.S.d. were supported by doctoral awards from the Canadian Institutes of Health Research, and O.L.R. was supported by a fel- significant progress in unraveling Ab-dependent and Ab-independent lowship award from the Society Society of Canada. M.P. was sup- B cell effector functions during antimicrobial responses, the un- ported by the Faculty of Medicine at McGill University, and M.D.Y. was supported by derlying mechanism that drives B mobilization to and the Canadian Association of Gastroenterology for a Summer-Student award. accumulation at peripheral sites during inflammation and infec- Address correspondence and reprint requests to Dr. Jo¨rg H. Fritz, McGill University, 3649 Promenade Sir William Osler, Bellini Pavilion Room 371, Montreal, QC H3G tion is unresolved. 0B1, Canada. E-mail address: [email protected] At steady state, homeostatic chemokines such as CCL19, The online version of this article contains supplemental material. CCL21, and CXCL13 are constitutively expressed within lymphoid Abbreviations used in this article: BM, bone marrow; DC, ; FSC, for- tissues and regulate lymphocyte homing to and positioning within ward light scatter; GC, germinal center; ILF, isolated lymphoid follicle; im/T1, these organs. CXCR5 is selective for the single chemokine CXCL13, immature transitional 1; KLH, keyhole limpet hemocyanin; LT, lymphotoxin; MF, mature follicular; MZ, marginal zone; Nod, nucleotide-binding oligomerization do- produced by lymphoid tissue-resident stromal cells such as follicular main; Nod1, Nod-containing protein 1; NP, 4-hydroxy-3-nitrophenylacetyl hapten; DCs within the B cell compartment of secondary lymphoid organs PGN, peptidoglycan; PMN, polymorphonuclear ; PRM, pattern recognition (SLO). CXCL13 controls the homing and localization of CXCR5+ molecule; SLO, secondary lymphoid organ; SSC, side light scatter; WT, wild-type. cells, including B cells, constraining them to the B cell follicle Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 (14, 15). Importantly, deficiency in TNF-a, TNFR1, lymphotoxin www.jimmunol.org/cgi/doi/10.4049/jimmunol.1203310 2788 A TNF-a–CCL20–CCR6 AXIS DRIVES B CELL RESPONSES

(LT)a,orLTb leads to defective CXCL13 expression and reduced mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 1 mg/ml collagenase splenic B cell numbers, indicating a key role for LT and TNF D [Roche], and 0.2 mg/ml DNase I [Roche]) and incubated for 30 min at upstream of CXCR5/CXCL13 in the process of B cell follicle 37˚C. Cell suspensions were then homogenized by gentle pipetting before being incubated for an additional 15 min at 37˚C prior to the addition of formation (16, 17). However, because all peripheral B cell subsets EDTA (final concentration 1 mM) and incubation for 10 min at room respond to CXCL13 (18), more selective chemokines other than temperature. Cells were pelleted and resuspended in RBC lysis buffer for CXCL13 may be required for inflammation-induced B cell accu- 1 min at room temperature (150 mM NH4Cl, 100 mM NaHCO3,1mM mulation in SLO. EDTA [pH 8.0]). Furthermore, cells were then resuspended in FACS buffer (PBS supplemented with 2% FBS [PAA]) and pelleted by centrifugation. In this study, we show, using synthetic peptidoglycan (PGN) Finally, cells were filtered through a 70-mm nylon cell strainer (BD Bio- moieties, that the PRM Nod-containing protein 1 (Nod1) directs sciences) and pelleted by centrifugation, resuspended in FACS buffer (PBS neutrophil accumulation and DC positioning within the spleen. In supplemented with 2% FBS [PAA]), and analyzed by flow cytometry. BM conjunction with this inflammatory response, significant numbers from femurs and tibiae was collected, resuspended, and pelleted. Cells of B cells accumulate within the spleen; however, this was found to were pelleted and resuspended in RBC lysis buffer for 1 min at room temperature (150 mM NH4Cl, 100 mM NaHCO3, 1 mM EDTA [pH 8.0]). be independent of the LT/CXCL13/CXCR5 homeostatic axis. In Cells were pelleted by centrifugation, resuspended in FACS buffer (PBS contrast, inflammation-driven accumulation of B cells in the spleen supplemented with 2% FBS [PAA]), and analyzed by flow cytometry. depended on CCL20 expression downstream of TNF-a and re- Peritoneal cavity cells were harvested by injecting 0.5 ml sterile PBS into quired the selective expression of CCR6 on B cells. In addition, the peritoneum of freshly euthanized mice. Cells were pelleted by cen- trifugation, resuspended in FACS buffer (PBS supplemented with 2% FBS CCR6 was required to shape Nod1-mediated Ab responses, un- [PAA]), and analyzed by flow cytometry. covering a novel chemokine axis for the regulation of B cell responses during microbial exposure. In vitro stimulation of splenocytes Downloaded from Splenocytes were cultured for 3 d after stimulation with sterile PBS Materials and Methods (Invitrogen; Carlsbad) as control stimulus, CpG ODN 1826 (Invivogen, San Mice Diego, CA; 10 mg/ml) or FK156 (obtained from Fujisawa; 50 mg/ml). Three days after stimulation, splenocytes were harvested and prolifera- Wild-type (WT) C57BL/6.CD45.2 and C57BL/6.CD45.1 animals were tion of B220+CD19+ B cells was analyzed by Ki67 staining and flow obtained from Charles River Laboratories or The Jackson Laboratory. cytometry.

Nod1-deficient mice (Millenium Pharmaceuticals) have been backcrossed http://www.jimmunol.org/ to the eighth generation to the C57BL/6 background. CCR22/2, CXCR52/2, Chemokine and Ab measurement 2/2 2/2 and CCR6 mice were purchased from The Jackson Laboratory. TNF-a Blood and from animals were harvested at the indicated timepoints. animals were obtained from Taconic. LTb2/2 animals were purchased from 2/2 Blood was allowed to coagulate at room temperature and spun, and sera B&K Universal. JH mice were obtained from S. Fillatreau (DRFZ, were analyzed by ELISA. Spleens were weighed and ground in 300 ml Berlin, Germany). All animals were housed in specific pathogen-free sterile PBS utilizing a tissue grinder (Polytron PY2100; Kinematica AG). conditions and used at 6–8 wk of age. All experiments were performed Tissue homogenates were pelleted by centrifugation, and chemokine con- according to animal use protocols approved by the animal care committee tents from supernatants were determined by ELISA. The concentrations of the University of Toronto and McGill University. of KC/CXCL1, CCL2, CXCL13, CCL19, CCL21, and CCL20 in sera BM chimeras and spleen grind were determined by ELISA, according to the manufacturer’s

recommendations (Duoset; R&D Systems, Minneapolis, MN). PE-specific by guest on September 29, 2021 For the generation of BM chimeras, mice were irradiated with two con- Ab responses from mice immunized with PE were analyzed utilizing secutive doses of 550 rad utilizing a MDS-Nordion Gammacell 40 irradiator platebound PE using standard ELISA procedures (19). NP-specific Ab and subsequently reconstituted by i.v. injection with 2 3 106 RBC-depleted responses from mice immunized with NP-KLH were analyzed using BM cells from sex-matched donors. For mixed chimeras, mice received platebound NP30-BSA or NP8-BSA (both from Biosearch Technologies) a 1:1 mixture of either WT and CCR62/2 BM, or a 1:1 mixture of JH2/2 using standard ELISA procedures (19). and CCR62/2 BM. Mice were then provided 2 mg/ml neomycin-sulfate (Sigma-Aldrich)–supplemented drinking water (sterile filtered) for 2–3 wk CCL20 production by cell lines postirradiation. Mice were further used for experiments following 8–10 wk The murine intestinal epithelial cell line MODE-K (20) (American Type of reconstitution. Chimerism was analyzed by determining CD45.1 and Culture Collection) and the –derived stromal cell line BLS12 CD45.2 expression of the hematopoietic lineage by flow cytometry. Only (21) (obtained from A. Shimizu, Kyoto University, Kyoto, Japan) were animals yielding sucessful chimerism .95% were used for experiments. cultured in T75 culture flasks using high-glucose DMEM supplemented Injections, immunizations, and infections with 2 mM L-glutamine, 1 mM sodium pyruvate, nonessential amino acids (all purchased from Invitrogen, Carlsbad, CA), and 5% FBS (HyClone, The synthetic Nod1 agonist FK156 (D-lactyl-L-Ala-g-D-Glu-meso-DAP- Logan, UT) (complete culture medium). Cells were harvested from flasks Gly) was obtained from Fujisawa. To selectively stimulate Nod1, ani- using trypsin (Invitrogen) and seeded in 24-well plates (0.5 ml per well) in 5 mals were injected i.p. with 0.1 mmol FK156 in endotoxin-free PBS only complete culture medium the day before stimulation (MODE-K, 3 3 10 5 (each time freshly prepared from lyophilized stock) or endotoxin-free PBS cells/well; BLS12, 1.5 3 10 cells/well). Cells were stimulated with sterile only as control in a total volume of 100 ml. For immunization experiments PBS (Invitrogen) as control, TNF-a (50 ng/ml; R&D Systems), or FK156 with PE, animals were injected with PE (R-PE; Prozyme; 15 mg per (100 mM) for 48 h, and the concentrations of CCL20 in cell culture mouse) only or in conjunction with 0.1 mmol FK156 in endotoxin-free supernatants were determined by ELISA (R&D Systems), according to the PBS. For prime-boost (days 0 and 21) immunization experiments with manufacturer’s instructions. 4-hydroxy-3-nitrophenylacetyl hapten (NP) conjugated to keyhole limpet hemocyanin (KLH), animals were injected with NP-KLH (Biosearch Flow cytometry Technologies; 100 mg per mouse) only or in conjunction with 0.1 mmol Cells were washed with ice-cold PBS containing 2% FBS (PAA) (FACS FK156 in endotoxin-free PBS. At day 7 and 28 postinjections, animals buffer), and, prior to Ab staining, cells were incubated with 1 mg/ml rat anti- were sacrificed and blood and tissues were collected and analyzed. Listeria mouse CD16/CD32 Ab (Fc-block, clone 2.4G2) for 15 min at 4˚C. Pre- monocytogenes EGDe (BAA-679; American Type Culture Collection) was determined concentrations of fluorochrome-labeled Abs were added in a grown in brain heart infusion broth media. Animals were infected i.v. with 3 3 total volume of 100 ml, thoroughly mixed with the cells, and incubated for a sublethal dose of 5 10 CFU of a freshly grown culture in a total 30 min at 4˚C. The following Abs were used: mouse anti-mouse CD45.1- volume of 100 ml in sterile PBS. At indicated time points, animals were allophycocyanin (A20), mouse anti-mouse CD45.2-PE (104), rat anti-mouse sacrificed and blood and tissues were collected and analyzed. CD11b-FITC (Mac-1, M1/70), rat anti-mouse CD11b-allophycocyanin Ex vivo isolation of cells (Mac-1, M1/70), Armenian hamster anti-mouse CD11c-PE (N418), rat anti-mouse Ly6G-PE (1A8; BD Biosciences), rat anti-mouse CD19-PerCP- Spleens and lymph nodes were harvested at indicated time points, and fat Cy5.5 (1D3), rat anti-mouse CD4-FITC (GK1.5), rat anti-mouse CD8a- was removed. Tissue was gently homogenized with glass slides in digestion allophycocyanin (53-6.7), rat anti-mouse CD45R (B220)-PE (B220, RA3- buffer (HBSS [Life Technologies] supplemented with 10 mM HEPES, 150 6B2), rat anti-mouse CD45R (B220)-allophycocyanin (B220, RA3-6B2), The Journal of Immunology 2789

Armenian hamster anti-mouse TCRb-PerCP-Cy5.5 (H57-597), and rat Biosciences), and rat anti-mouse mucosal addressin cell adhesion molecule-1 anti-mouse Ly6C-FITC (AL-21; BD Biosciences). For LTab staining, biotin (MECA-367; eBioscience), followed by streptavidin-ALEXA488 we stained with anti-LTb BBF6 (gift of J. Browning, Boston University (Invitrogen). After removal of the staining solution, slides were washed School of Medicine, Boston, MA), followed by FITC-conjugated anti- three times with TBST, once with TBS, and once with PBS. Finally, slides hamster Ig (Jackson ImmunoResearch Laboratories) (22, 23), using spleno- were stained with DAPI nucleic acid stain (Invitrogen) for 30 s and washed cytes from LTb2/2 animals for negative control stainings. Intracellular three times with PBS before being mounted with Gel/Mount (Biomeda). staining for Ki67 (SolA15) was performed using the FoxP3 transcription Images were acquired with a Leica DMRA2 microscope (Leica Micro- factor staining buffer kit (eBioscience, San Diego, CA). PE-specific ger- systems) equipped with a Retiga EXi digital camera (Q Imaging) using minal center (GC) (B220+CD19+GL7+CD382) and memory (B220+CD19+ OpenLab software (Improvision) and Adobe Photoshop. GL72CD38+) B cell numbers in spleens were determined by flow cytom- etry after incubating isolated splenocytes PE, as previously described (24). Statistical analysis The following Abs were used: rat anti-mouse CD19 (1D3), rat anti-mouse Statistical analysis was performed using GraphPad Prism software CD45R (B220, RA3-6B2), rat anti-mouse CD38 (90; BioLegend), and rat (GraphPad Software) with either a Student t test or a Mann–Whitney U test. anti-mouse/ GL7 (GL7). A p value , 0.05 was considered significant. Characterization of splenic B cell subsets Isolated splenocytes were first gated on single cells (forward light scatter Results [FSC]-A versus FSC-H) and then on size (FSC) and granularity (side light Nod1-induced inflammation expands B cell numbers in SLO scatter [SSC]). Dead cells were excluded, and the remaining cells were then divided by CD93 (AA4.1) and B220 (RA3-6B2) expression into com- Nod1 regulates antimicrobial host defense and microbe-specific partments of developing or mature B cells. Both compartments were further T and B cell immunity through sensing of diaminopimelic acid– divided by IgM (II/41) and CD23 (B3B4) expression and refined by CD21 containing PGN of extracellular and intracellular bacteria (1). To Downloaded from (8D9) and IgD (11-26c) expression. By these criteria, we defined marginal zone (MZ) B cells as B220highCD932CD21highCD23lowIgMhighIgDlow, deepen our understanding of the cellular and molecular require- 2/2 mature follicular (MF) B cells as B220highCD932CD21+CD23+IgM+IgD+, ments for Nod1-induced B cell responses, we used Nod1 mice immature transitional 1 (im/T1) B cells as B220lowCD93+CD212CD232 and first compared the spatiotemporal changes of leukocyte pop- IgM+IgD2, and transitional 2 B cells as B220lowCD93+CD21lowCD23+ + low ulations in the spleen to WT animals following systemic admin- IgM IgD . istration of the Nod1 agonist FK156. Injections of FK156 resulted

Characterization of BM B cell subsets in a massive but transient drop of B cell frequency in the blood in http://www.jimmunol.org/ a Nod1-dependent manner (Fig. 1A). The early drop in blood B cell Isolated BM cells were first gated on single cells (FSC-Aversus FSC-H). A lymphocyte gate based on size (FSC) and granularity (SSC) was then frequencyat12hprecededanobservedsplenomegalypeaking applied, and dead cells were excluded. The remaining cells were then between 24 and 48 h postinjection (Fig. 1B). Parallel with the partitioned based on CD93 and B220 expression into compartments of drop in frequency in blood B cells, absolute numbers of B cells developing or mature B cells. Both compartments were further divided by were also observed to be reduced in the blood (Fig. 1C), whereas IgM and CD23 expression and refined by CD21 and IgD expression. By these criteria, we defined MF B cells as B220highCD932CD21+CD23+IgM+ numbers of B cells were observed to increase in the spleen at 36 h IgD+, pro/pre-B cells as B220lowCD93+CD212CD232IgM2IgD2, and im/ postinjection of FK156 (Fig. 1D). In parallel with these events, T1 B cells as B220lowCD93+CD212CD232IgM+IgD2. CD11b+Ly6C+Ly6G+ polymorphonuclear neutrophils (PMN) were found to be increased in frequency and in numbers in the by guest on September 29, 2021 Characterization of peritoneal cavity lavage B cell subsets blood (Fig. 1A, 1C) and the spleen (Fig. 1D). In contrast, FK156 Isolated cells were first gated on single cells (FSC-A versus FSC-H). injection did not lead to significant changes in absolute numbers A lymphocyte gate based on size (FSC) and granularity (SSC) was then of splenic CD4+ or CD8a+ T cells, inflammatory monocytes, and applied, and dead cells were excluded. The remaining cells were then di- vided by CD11b (M1/70) and CD19 (1D3) expression into compartments DC at any timepoints analyzed, suggesting that recruitment of of B2 or B1 B cells. B2 cells were further gated on B220+CD19+ double these cell types to the spleen during inflammation may be driven positive and confirmed B2 according to their undetectable level of CD5 (53- by Nod1-independent signals (data not shown). Therefore, systemic 7.3) expression. B1 cells were gated for their IgM and IgD expression level. injection of FK156 results in an early increase in PMN in the blood These were further divided by their CD5 expression level. B1b cells were defined as CD11b+CD19+IgM+IgDlowCD52 and B1a cells as CD11b+CD19+ andspleenat12h,followedbyareductioninBcellsinthe IgM+IgDlowCD5+. B2 cells were defined as CD11b2CD19+B220+CD52. blood and accumulation of B cells in the spleen at 36 h in a Nod1- Unless otherwise mentioned, all Abs were purchased from eBioscience. dependent manner. In all experiments, cells were labeled with Live/Dead Aqua (Life Tech- We next examined FK156-induced changes in the localization of nologies) prior to staining, and dead cells were excluded. After staining, immune cells within the spleen by immunofluorescence micros- cells were washed twice with FACS buffer prior to analysis by flow + + + cytometry using either a FACSCalibur, a CANTO-II, or a LSR-II (BD copy. The transient increase of CD11b Ly6C Ly6G PMN upon Biosciences). Acquired data were analyzed and processed using FlowJo Nod1 stimulation in the spleen led to the formation of clusters in (Tree Star). the splenic red pulp, found mostly in close proximity to the MZ Immunofluorescence microscopy bordering the white pulp (Fig. 2A). In addition, a rapid and transient redistribution of splenic CD11c+ DC from bridging Spleens were cut out, fat was removed and washed in PBS, and pieces were channels to the periarteriolar lymphoid sheaths rich in T cells was frozen in OCT compound (Sakura Finetek). Sections were cut at 5 mm using observed 12 h postinjection of FK156 in a Nod1-dependent a Leica CM3050 cryostat (Leica Microsystems), mounted on glass mi- + croscope slides, fixed for 7 min in ice-cold acetone, and air dried. Sections fashion (Fig. 2B). Furthermore, the increase of B220 cells in were then rehydrated in TBS, followed by TBST (Sigma-Aldrich). All the spleen 36 h postinjection of FK156 coincided with an elevated blocking and staining steps were then performed in a humidified chamber. signal for BP3 (CD157), known to be expressed on stromal cells First, sections were incubated with TBST supplemented with 10% normal of the B cell zone (15) (Fig. 2C). Taken together, Nod1 stimulation rabbit serum (Jackson ImmunoResearch Laboratories), 10% normal mouse serum (Jackson ImmunoResearch Laboratories), 5% BSA (Sigma-Aldrich), resulted in the rapid accumulation of neutrophils and B cells in the and 2 mg/ml rat anti-mouse CD16/CD32 Ab (Fc-block, clone 2.4G2) spleen, redistribution of DC to the periarteriolar lymphoid sheaths, for 30 min. Fluorochrome-labeled Abs were applied for 60 min in the and changes in the splenic stromal cell compartment. dark. The following Abs were used: rat anti-mouse/human CD45R- As Nod1-mediated PGN recognition has been shown to play a allophycocyanin (B220, RA3-6B2; eBioscience), Armenian hamster anti-mouse CD11c-PE (N418; eBioscience), rat anti-mouse CD11b- critical role in the host’s innate immune response to L. monocytogenes allophycocyanin (Mac-1, M1/70; eBioscience), rat anti-mouse Ly6G- (25–28), we determined the role of Nod1 for the accumulation PE (1A8; BD Biosciences), mouse anti-mouse CD157-PE (BP-3; BD of B cells in the spleen postinfection with L. monocytogenes.As 2790 A TNF-a–CCL20–CCR6 AXIS DRIVES B CELL RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 1. Kinetics of Nod1-mediated leukocyte changes in blood and spleen. (A) Frequencies of CD19+B220+ B cells and CD11b+Ly6C+Ly6G+ neutrophils in blood and (B) weight of spleens from WT (represented by empty circles, n = 4–6) and Nod1-deficient (Nod12/2, represented by filled circles, n = 5) mice either injected with PBS or with the Nod1 agonist FK156 were determined at the indicated times postinjection. The bar represents the mean of each cohort. The asterisk indicates a statistically significant difference (*p , 0.05). (C) Blood and (D) spleens were analyzed for the total counts of + + + + + 2/2 splenocytes, B220 CD19 B cells, and CD11b Ly6C Ly6G neutrophils from WT (represented by empty circles, n = 4–6) and Nod1 (represented by by guest on September 29, 2021 filled circles, n = 5) mice injected with either PBS or the Nod1 agonist FK156 12 and 36 h postinjection. The bar represents the mean of each cohort. In (A)– (D), data are representative of five independent experiments. The asterisk indicates a statistically significant difference (*p , 0.05).

shown in Fig. 3A, Nod1-mediated PGN recognition of L. mono- onist FK156 did not result in B cell proliferation (Supplemental cytogenes is required for the temporary increase of splenic B cells Fig. 2C). Moreover, injection of the Nod1 agonist FK156 did not postinfection. In contrast, and in accordance with earlier reports stimulate proliferation of B lineage cells in spleen, mesenteric (29), Nod1 is dispensable for the numeric changes of neutrophils, lymph node, and BM (Supplemental Fig. 2D). In addition, pro- inflammatory monocytes, and DC after L. monocytogenes infec- liferative BM B cells were found to be reduced 12 h post-Nod1 tion (Supplemental Fig. 1A–C), highlighting the critical role of stimulation (Supplemental Fig. 2D). Nod1 for B cell accumulation in SLO during infection. A detailed analysis of changes of B cell subsets in the spleen Spatiotemporal analysis of chemokine induction in vivo revealed a slight early increase of MZ B cells 2 h post-Nod1 stim- As increases in splenic B cells were preceded by a decrease in ulation (Fig. 3B). Elevated numbers of im/T1 and T2 B cells in B cells in the blood (Fig. 1), we therefore concluded that systemic spleen were found at late time points post-FK156 injection (Fig. administration of a Nod1 agonist provokes rapid B cell accumu- 3B). Similarily, the dominant increase of MF B cells in spleen lation in SLO. To study the immunological mediators required (Fig. 3B) and draining mesenteric lymph node (Supplemental Fig. for infection- and inflammation-induced B cell accumulation in 2A) was found to peak at 36 h post-Nod1 stimulation. Similar SLO, chemokines in blood and spleen upon selective Nod1 stim- results were obtained with injections of the TLR4 agonist LPS ulation were monitored. We observed that the Nod1-specific agonist (Supplemental Fig. 2B). Parallel to the temporary changes in splenic FK156 induced a biphasic release of chemokines. The primary B cell subsets, MF B cells in BM first increase at 2 and 12 h after early wave of mediator release included a rapid induction of KC/ Nod1 stimulation and then drop at 36 and 60 h post-FK156 injection CXCL1 and CCL2 in the bloodstream and the spleen, with highest (Fig. 3C). Similarly, numbers of im/T1 and T2 B cells in BM were levels obtained between 2 and 12 h after injection (Fig. 4). The found to be lower 36 and 60 h post-Nod1 stimulation, respectively induction of both of these chemokines coincides with the observed (Fig. 3C). In contrast, injection of FK156 does not significantly elevation of PMN numbers in blood and spleen (Fig. 1C, 1D), change numbers of B1a, B1b, or B2 cells in the peritoneal cavity suggesting a critical role for these chemokines in neutrophil ac- (Fig. 3D). We next analyzed whether changes in B cell numbers cumulation. Importantly, in accordance with unchanged T cell in SLO are the result of direct activation and proliferation of B numbers, levels of CCL19 and CCL21, CCR7 ligands critical for lineage cells upon Nod1 stimulation. As previously reported (19), T cell migration and positioning, were unaltered poststimulation in vitro stimulation of splenic B lineage cells with the Nod1 ag- of Nod1 (data not shown). Subsequent to this early burst in che- The Journal of Immunology 2791 Downloaded from http://www.jimmunol.org/

FIGURE 2. Nod1 stimulation leads to neutrophil and DC accumulation and changes in the splenic follicular stroma. Sections of spleens from WT mice injected with PBS for 12 h (A and B) and 36 h (A–C) postinjection with the Nod1 agonist FK156 were stained with specific fluorochrome-tagged Abs for (A) by guest on September 29, 2021 CD11b (blue) and Ly6G (red), (B) B220 (blue) and CD11c (red), or (C) B220 (blue) and BP3 (red). Pictures shown are representative of three independent experiments. Scale bars, 50 mm.T and B cell areas are labeled. BC, bridging channels; RP, red pulp; WP, white pulp. mokine production, systemic administration of FK156 induced a the spleen of WT→WT animals was abrogated in LTb2/2→WT second wave of chemokine production, with CXCL13 and CCL20 chimeras (Fig. 5B). In contrast, no difference in the Nod1-dependent peaking in the blood and spleen between 12 and 36 h postinjection induction of splenic CCL20, KC/CXCL1, or CCL2 was observed (Fig. 4). Importantly, induction of both chemokines coincided when comparing WT→WT with LTb2/2→WT animals (Fig. 5C, with elevated numbers of splenic B cells (Fig. 1D), suggesting a 5D). However, despite the reduced levels of CXCL13, Nod1- contribution of these chemokines to Nod1-induced B cell accu- mediated splenomegaly and B cell accumulation (Fig. 5E–G) as mulation to the spleen. well as PMN accumulation and DC repositioning (data not shown) were comparable in WT→WT and LTb2/2→WT chimeric ani- Nod1-induced splenic B cell accumulation does not require the mals. These observations demonstrate that the LTb/CXCL13 axis, LT/CXCL13/CXCR5 axis despite being activated, was dispensable for Nod1-mediated B cell During the steady state, blockade of LTbR signaling leads to accumulation to the spleen. defective CXCL13 expression and reduced splenic B cell num- a bers, identifying a key role for the LT/CXCL13/CXCR5 axis in Nod1-induced splenic B cell accumulation requires TNF- B cell follicle formation and maintenance (16, 17). The striking production by a radio-resistant cell type B cell accumulation and CXCL13 increase observed with Nod1 In the absence of a LT/CXCL13 effect, we explored the possibil- agonist administration led us to determine whether this requires ity that other cytokines may be involved in Nod1-induced B cell activation of the LT axis. Indeed, stimulation of Nod1 leads to accumulation. Nod1 stimulation has been shown to induce TNF-a a rapid upregulation of LTa1b2 on splenic B220+CD19+ cells (19, 31). As TNF-a is involved in the regulation of the expression (Fig. 5A). Because the LT pathway is required for lymph node of chemokines known to be key for lymphocyte positioning (17, 32, development (30), we constructed BM chimeric mice using WT 33), we assessed its role in Nod1-mediated chemokine induction animals as recipients and donor BM from WT or LTb2/2 mice and B cell accumulation. Splenomegaly and B cell accumulation (WT→WT versus LTb2/2→WT) to generate LTb-deficient, LN- induced upon Nod1 stimulation were completely abolished in sufficient mice. In accordance with earlier reports (17), when TNF-a–deficient animals (Fig. 6A–C). The requirement for TNF-a compared with WT→WT animals, LTb2/2→WT mice were found suggested that a TNF-a–dependent chemokine may be involved in to have lower protein levels of splenic CXCL13 at the steady state increasing splenic B cell numbers. We therefore analyzed chemo- in the spleen. Moreover, the Nod1-elicited increase of CXCL13 in kine levels induced upon Nod1 stimulation in WT and TNF-a2/2 2792 A TNF-a–CCL20–CCR6 AXIS DRIVES B CELL RESPONSES Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 3. Kinetics of Nod1-mediated B cell changes in spleen, BM, and peritoneal cavity. (A) Weight, total cellular composition, and total numbers of B220+CD19+ cells of spleens from WT (represented by empty circles, n = 4–6) and Nod1-deficient (Nod12/2, represented by filled circles, n = 5) mice either injected i.v. with PBS or infected with Listeria monocytogenes (L.m.) were determined 12 and 36 h postinjection. The bar represents the mean of each cohort. Data shown are representative of two independent experiments. The asterisk indicates a statistically significant difference (*p , 0.005). (B–D)WT (n = 5) mice were injected with either PBS (represented by empty circles) or the Nod1 agonist FK156 (represented by filled circles), and the total numbers of B cell subsets in (B) spleen, (C) BM, and (D) peritoneal cavity were determined at the indicated times postinjection. The bar represents the mean of each cohort. Data are representative of two independent experiments. The asterisk indicates a statistically significant difference (*p , 0.05). animals. The induction of KC/CXCL1 and CCL2 in the spleen induces CCL20 production to promote Nod1-induced B cell ac- of TNF-a2/2 mice was not significantly affected in the spleen cumulation in SLO. (Fig. 6D), although both chemokines were reduced in the serum (Supplemental Fig. 3A). However, despite these reductions, Nod1- Nod1-induced CCL20 is produced by a radio-resistant cell type dependent PMN accumulation and DC repositioning in the spleen CCL20 is produced by a range of cell types, including some he- were comparable in WT and TNF-a–deficient animals (data not matopoietically derived cells (monocytes, DC) as well as radio- shown). Similarly, the Nod1-mediated increase of CXCL13 in the resistant cell types (epithelial cells such as the follicle-associated spleen (Fig. 6E) and serum (Supplemental Fig. 3A) was somewhat of Peyer’s patches, endothelial cells, fibroblasts) (34– lower in TNF-a2/2 mice when compared with WT animals 36). Furthermore, expression of CCL20 induced by TNF-a–acti- (although not as profoundly reduced as what was observed in the vated endothelial cells contributes to adhesion of B cells to the en- LT-deficient setting). In contrast, the induction of CCL20 in the dothelium (37). To discern which cellular compartment was spleen (Fig. 6F) and serum (Supplemental Fig. 3A) was found to responsible for Nod1-induced CCL20 production, we generated BM be completely abolished in TNF-a2/2 mice. chimeras lacking the Nod1 receptor in either the radio-resistant or We next determined the cellular compartment responsible for radio-sensitive compartments (WT→WT, Nod12/2→WT, Nod1-induced CCL20 induction by creating BM chimeras (WT→WT, WT→Nod12/2). Systemic administration of FK156 Nod1 agonist TNF-a2/2→WT, WT→TNF-a2/2). Nod1-induced splenomegaly, induced the expression of CCL20 in the serum of WT→WT and B cell accumulation, and CCL20 induction were only observed in Nod12/2→WT but not in WT→Nod12/2 chimeric mice (Fig. 7A). WT→WT and TNF-a2/2→WT BM chimeric mice, but not in Therefore, the expression of Nod1 in radio-resistant cells is required for WT→TNF-a2/2 BM chimeras, suggesting that production of FK156-induced CCL20 production. TNF-a by radio-resistant cells is required to induce CCL20 upon To further determine whether radio-resistant cells produce Nod1 stimulation (Supplemental Fig. 3B, 3C). These results CCL20 in response to TNF-a or Nod1 agonists, we tested the suggest that TNF-a derived from the radio-resistant compartment ability of either TNF-a or FK156 to induce CCL20 production in The Journal of Immunology 2793

FIGURE 4. Nod1 stimulation induces two waves of chemokine production. Chemokine concentrations in tissues from WT (represented by filled circles, n =4–6) and Nod1-deficient (Nod12/2, represented by empty circles, n = 5) mice injected with either PBS or the Nod1 agonist FK156 were determined at the indicated times postinjection. The contents of KC/CXCL1, CCL2,

CXCL13, and CCL20 in the serum (A) and the super- Downloaded from natants of spleen homogenates (B) were determined by ELISA. The bar represents the mean of each cohort. Data are representative of three independent experi- ments. The asterisk indicates a statistically significant difference (*p , 0.05). http://www.jimmunol.org/ by guest on September 29, 2021

epithelial and stromal cells. Both TNF-a as well as the Nod1- B cells (38), we tested whether altered inflammation-induced agonist FK156 induced the production of CCL20 in the epithe- B cell recruitment is due to B cell–intrinsic CCR6 expression. lial cell line MODE-K (Fig. 7B) as well as the lymph node–de- For this purpose we generated mixed BM chimeric mice using WT rived stromal cell line BSL12 (Fig. 7C). These results collectively animals as recipients and donor BM from WT, B cell–deficient 2/2 2/2 demonstrate that Nod1 expression in radio-resistant cells induces mice (JH ), or CCR6 animals to generate chimeric mice, 2/2 2/2 CCL20 production to promote B cell accumulation in SLO. whereby B cells selectively lack CCR6 (JH + CCR6 →WT) 2/2→ Nod1-dependent B cell accumulation requires CCR6 and compared these with control chimeras (WT + CCR6 WT). We observed that selective CCR6 deficiency on B cells completely The requirement of TNF-a for the induced expression of CCL20 abrogated Nod1-mediated splenomegaly, increase in leukocytes, and the corresponding accumulation of B cells in SLO led us to and MF B cell accumulation in the spleen (Fig. 7E), demonstrating further explore the role of the CCL20/CCR6 axis in Nod1-induced that CCL20 and CCR6 constitute a critical chemokine axis for the B cell changes, as CCL20 is the only chemokine known to interact rapid accumulation of B cells during early microbial exposure. with CCR6 (38). To genetically verify the involvement of CCR6, we assessed the impact of CCR2, CXCR5, and CCR6 deficiency CCR6 regulates Nod1-induced B cell responses on Nod1-mediated cell accumulation. No differences in PMN Nod1-mediated PGN recognition during mucosal infection with clustering in the red pulp (Supplemental Fig. 4A) and DC repo- Helicobacter pylori has been shown to be required to instruct sitioning (data not shown) were observed in CCR2-, CXCR5-, and efficient microbe-specific Ab levels (19). Moreover, the synthetic CCR6-deficient mice. Furthermore, deficiency of CCR2 or CXCR5 PGN-derived peptide FK156 instructs potent B cell immunity to did not alter the increase in spleen weight, total splenocyte counts, coinjected T cell–dependent Ags in a Nod1-specific manner (19) and B cell numbers observed upon Nod1 stimulation (Supplemental through priming of Ag-specific GC and memory B cells and Fig. 4B, 4C), further demonstrating that the LT/CXCL13/CXCR5 systemic Ag-specific Ab levels (Fig. 8A–C). We therefore inves- axis is dispensable for Nod1-induced B cell recruitment. In contrast, tigated the role of CCR6 for Nod1-mediated priming of Ag- CCR6 deficiency completely abolished Nod1-mediated spleno- specific Ab responses in prime-boost immunization experiments megaly and the Nod1-dependent elevation in total leukocytes and by coinjecting the Nod1 agonist FK156 with the T cell–dependent B cells (Fig. 7D). Ag NP-KLH. Deficiency of CCR6 leads to increased NP-specific As CCR6 has been shown to be expressed on many different Ab titers (Fig. 8D); however, these titres exhibit lower affinity for leukocyte subsets, including PMN, DC, T cells, NK cells, and Ag (Fig. 8E). Specifically, the difference in anti-NP IgG1 titers 2794 A TNF-a–CCL20–CCR6 AXIS DRIVES B CELL RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 5. Nod1-mediated B cell accumulation does not require the LT/CXCL13/CXCR5 axis. (A) B220+CD19+ splenocytes from WT and Nod12/2 mice injected with either PBS (black histograms) or the Nod1 agonist FK156 (red histograms) 36 h previously were analyzed for their expression of by guest on September 29, 2021 LTa1b2 by flow cytometry. Data are representative of two independent experiments. (B–D) Spleens from WT→WT (represented by filled circles, n = 5–7) and LTb2/2→WT (represented by empty circles, n = 5–7) mice injected with either PBS or the Nod1 agonist FK156 12 and 36 h previously were collected. The contents of CXCL13 (B), CCL20 (C), KC/CXCL1, and CCL2 (D) in supernatants of spleen homogenates were determined by ELISA. The bar represents the mean of each cohort. (E–G) Weight (E), total cellular composition (F), and B220+CD19+ B cell content (G) of spleens from WT (WT→WT, represented by filled circles, n = 5) and LTb2/2 (LTb2/2→WT, represented by empty circles, n = 5) chimeric mice injected with either PBS or the Nod1 agonist FK156 were determined after 12 and 36 h. The bar represents the mean of each cohort. Data are representative of two independent experiments. The asterisk indicates a statistically significant difference (*p , 0.05).

between WT and CCR62/2 animals was more pronounced for titres Although recent evidence underscores significant physiologic of high-affinity Abs (NP8) (half-maximal titers: WT, 1/52,678 6 roles for B cells regulating diverse leukocyte subsets to instruct 798; CCR62/2, 1/17,428 6 567) as opposed to total Ab titers innate antimicrobial immune responses in an Ab-dependent and (NP30). Moreover, the difference in high-affinity Ab titers became -independent manner (7–12, 39), the underlying mechanisms that more pronounced upon secondary immunization (half-maximal regulate B lymphocyte mobilization to and accumulation at the titers: WT, 1/453,191 6 4,419; CCR62/2,1/89,1776 2,936), periphery during inflammation and infection are largely unknown. demonstrating that CCR6-mediated accumulation of MF B cells Microbial infections trigger the activation of many different PRM triggered by Nod1-mediated PGN recognition exerts a key role in (40), exerting direct mitogenic and stimulatory capacity of B cells shaping antimicrobial Ab responses. (4). Therefore, we chose to study mechanisms and functions of inflammation-induced B cell accumulation by injecting purified Discussion PGN moieties selectively stimulating only Nod1 (31) because this In this study, we investigated the functional consequences of synthetic PGN moeity does not directly cause proliferation of B Nod1-mediated PGN recognition for B cell responses. Our results lineage cells. We found that systemic stimulation of Nod1 in- show that MF B cells rapidly accumulate in the SLO following structs a biphasic chemokine response. Whereas the first wave systemic inflammation elicited by Nod1 stimulation and that includes the induction of KC and CCL2 accompanied by a marked Nod1-mediated PGN recognition is recquired for B cell accu- increase of neutrophils in blood and spleen and redistribution mulation in the spleen during infection. Surprisingly, this accu- of splenic DC, the second wave involves the induction of CXCL13 mulation was independent of the LT/CXCL13/CXCR5 pathway but and CCL20 concomitant with elevated numbers of splenic B cells. insteadrelieduponTNF-a–induced expression of CCL20 and Importantly, whereas the induction of KC and CCL2 as well as selective expression of CCR6 on B cells. Moreover, CCR6 was the changes within the myeloid compartment were largely inde- required to shape the Nod1-mediated Ag-specific B cell response. pendent of TNF-a, increases in CCL20 and B cells were found The Journal of Immunology 2795 Downloaded from http://www.jimmunol.org/

FIGURE 6. Nod1-mediated B cell accumulation and CCL20 production require TNF-a.(A–C) Weight (A), total cellular composition (B), and B220+ CD19+ B cell content (C) of spleens from WT (represented by filled circles, n = 4–7) and TNF-a–deficient mice (TNF-a2/2, represented by empty circles, n = 4–5) injected with either PBS or the Nod1 agonist FK156 were determined after 12 and 36 h. The bar represents the mean of each cohort. (D–F) Spleens from WT (represented by filled circles, n = 5–6) and TNF-a2/2 (represented by empty circles, n = 5–6) mice injected with either PBS or the Nod1 agonist FK156 12 and 36 h previously were collected. The content of KC/CXCL1 and CCL2 (D), CXCL13 (E), and CCL20 (F) in supernatants of spleen homogenates was determined by ELISA. The bar represents the mean of each cohort. Data are representative of four independent experiments. The asterisk by guest on September 29, 2021 indicates a statistically significant difference (*p , 0.05). to depend on TNF-a expressed by nonhematopoietic stromal play a role in recruiting B cells to the spleen during the course of cells. systemic inflammation, and, indeed, TNF-a expression is essential During the steady state, deficiency of LTbR signaling leads to for Nod1-induced B cell accumulation in the spleen. TNF-a also defective CXCL13 expression and dysregulated splenic B cell orga- affects B lymphopoiesis by lowering expression of CXCL12, lead- nization, demonstrating an essential role for the LT/CXCL13/ ing to decreased retention of B cells in the BM and concomitant CXCR5 axis in B cell follicle formation and maintenance (16, 17). mobilization to the periphery (5, 6). Deficiency of TNF-a,how- Although Nod1 stimulation leads to an increase in LTab expres- ever, did not markedly alter Nod1-mediated KC/CXCL1 and CCL2 sion on B cells and CXCL13 release in a LT-dependent manner, release and concomitant PMN increase in the blood and spleen, LTb and CXCR5 were found to be dispensable for the observed which is in accordance with previous reports demonstrating that increase of splenic B cells. Indeed, accumulation of B cells in the i.v. administration of TNF-a reducesCXCL12inBMand postnatally induced lymphoid tissues such as BALT and nose- mobilizes developing B cells to the spleen, with only a modest effect associated lymphoid tissue in response to microbial stimuli has on granulopoiesis (6). been shown to occur in a LT-independent manner (41, 42). Al- We found that B cell accumulation in SLO was not due to in- though in some contexts the LT pathway is required for optimal creased proliferation of B lineage cells. However, Nod1 stimulation formation of splenic GC (43–45), GC formation occurs normally did not lead to a drop of B lineage cells in the peritoneal cavity; in the mesenteric lymph node (46), and mice deficient in LTbR numbers of MF, pro/pre, im/T1, and proliferating B cells in the BM only exhibit a defect in affinity maturation at low doses of Ag (47). were found to be markedly lower 36 and 60 h post-FK156 injection. We have recently shown that, in the absence of LTab, GC in the This suggests that Nod1-mediated B cell accumulation in SLO is inguinal lymph node form normally, activation-induced deaminase due to repositioning of B cells both from the blood and the BM. is induced in germinal-center Ag-specific B cells, Ag-specific Ab Assuming a total blood volume of 2 ml per mouse, the loss in B cells titres are normal, and B cells can undergo affinity maturation in from blood accounts for approximately an excess of 4 million response to protein Ag emulsified in CFA (48). Because Nod1 B cells in the spleen, whereas calculations from the BM account agonists are present in CFA (19), it is therefore highly unlikely for ∼2 million additional B cells liberated from this location. that Nod1 agonist alone would result in a defect in the GC re- However, we observed a total net increase of ∼30 million B cells sponse in the absence of LTbR signaling, and we would expect per spleen. As we excluded proliferation of B cells as possible that the generation of Ag-specific Abs or affinity maturation of mechanism, it is highly likely that Nod1-mediated accumulation the humoral immune response would occur. Therefore, our data of B cells in SLO is also due to increased B cell retention and collectively suggest that alternative LT-independent chemokines lower egress from the SLO. 2796 A TNF-a–CCL20–CCR6 AXIS DRIVES B CELL RESPONSES Downloaded from http://www.jimmunol.org/

FIGURE 7. Stromal cell–derived CCL20 directs CCR6-dependent splenic B cell accumulation in response to Nod1 agonists. (A) BM chimeric mice by guest on September 29, 2021 WT→WT, Nod12/2→WT, or WT→Nod12/2 were injected with PBS as control or FK156, and 12 h postinjection sera were analyzed for CCL20 by ELISA. The bar represents the mean of each cohort (n = 3–7). Data shown are representative of two independent experiments. The asterisk indicates a statistically significant difference (*p , 0.05). (B) Murine intestinal epithelial cells (MODE-K) or (C) lymph node stromal cells (BLS12) were stimulated with PBS as control, TNF-a, or FK156 for 48 h, and cell culture supernatants were analyzed by ELISA. Data shown are representative of two independent experiments. (D) Weight, total cellular composition, and B220+CD19+ B cell content of spleens from WT (represented by filled circles, n = 5) and CCR6-deficient (CCR62/2, represented by empty circles, n = 5) mice injected with either PBS or the Nod1 agonist FK156 were determined after 12 and 36 h. The bar represents the mean of each cohort. Data shown are representative of two independent experiments. The asterisk indicates a statistically significant dif- 2/2 2/2 ference (*p , 0.05). (E) Spleens derived from mixed BM chimeric mice WT + CCR6 →WT (represented by empty circles, n = 4–6) and JH + CCR62/2→WT (represented by filled circles, n = 5) were analyzed for weight, total cellular composition, total B220+CD19+ B cells, and MF B cell content 12 and 36 h postinjection with PBS or with the Nod1 agonist FK156. The bar represents the mean of each cohort. Data shown are representative of two independent experiments. The asterisk indicates a statistically significant difference (*p , 0.05).

Concomitant with altered B cell accumulation in the spleen, the detailed cellular sources of TNF-a and CCL20 upon Nod1- TNF-a deficiency abolished Nod1-mediated induction of CCL20 mediated PGN recognition. release, suggesting that TNF-a is required for Nod1-mediated The relevance of Nod1-mediated B cell accumulation was fur- CCL20 induction. We have already previously shown that injec- ther highlighted by our finding that Nod1-mediated PGN recognition tion of FK156 leads to a rapid induction of TNF-a in the blood- of L. monocytogenes is required for the temporary increase of stream in a Nod1-dependent manner. In the current study, we splenic B cells postinfection. However, no differences in the in- report that FK156-induced CCL20 levels in the bloodstream and crease of CCL2, CXCL13, and CCL20 production after spleen were found to be highest at 12 h postinjection. As FK156- L. monocytogenes infection were observed between WT and Nod12/2 mediated production of CCL20 is abolished in TNF-a–deficient animals (data not shown), suggesting a redundancy in PRM animals, and induction of TNF-a precedes that of CCL20 in the requirements for optimal chemokine production and implying that blood, we conclude that Nod1-mediated TNF-a production by other chemokines other than CCL2, CXCL13, and CCL20 can nonhematopoietic cells is a prerequisite for CCL20 production. provoke B cell recruitment to the spleen during systemic inflam- TNF-a can exist as both soluble and membrane-bound form. We mation with a complex pathogen. Furthermore, in accordance with do not know which of these forms is acting to induce CCL20 earlier reports (5), Nod1 is dispensable for the observed numeric production. Nevertheless, it is important to address the nuances of changes of neutrophils, inflammatory monocytes, and DC after local versus systemic and membrane-bound versus soluble TNF-a L. monocytogenes infection, highlighting the critical role of Nod1 production in the regulation of lymphoid tissue niches that could for B cell responses during pathologic processes. For these rea- support B cell functions. Thus, future experiments will determine sons, the use of FK156 has proved a useful reductionist means for The Journal of Immunology 2797 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 8. CCR6 regulates Nod1-induced B cell responses. (A–C) WT mice were injected i.p. either with the protein Ag PE only (n = 5, represented by empty circles) or in combination with the Nod1 agonist FK156 (n = 4, represented by filled circles). (A and B) Seven days after immunization, the PE- specific GC (B220+CD19+GL7+CD382) and memory (B220+CD19+GL72CD38+) B cell numbers in spleens were determined by flow cytometry. (C) Three weeks after immunization, sera from mice were analyzed by ELISA for the content of PE-specific IgG1. Immunizations with PE only yielded nondetectable (n.d.) PE-specific IgG1 levels. The bar represents the mean of each cohort. Data shown are representative of two independent experiments. The asterisk indicates a statistically significant difference (*p , 0.05). (D and E)WT(n = 5, represented by filled circles) and CCR62/2 (n = 5, represented by empty circles) mice were injected i.p. either with NP-KLH only or in combination with the Nod1 agonist FK156. Seven days after the first immunization (prime) and 7 d after the second immunization (boost), sera from mice were analyzed by ELISA for (D) the content of NP-specific IgG1. (E) Affinity of NP-specific IgG1 from WT (black lines) and CCR62/2 (red lines) mice was determined using NP30-BSA and NP8-BSA. The bar represents the mean of each cohort. Data shown are representative of two independent experiments. The asterisk indicates a statistically significant difference (*p , 0.05). dissecting Nod1-specific effects that may contribute to the dy- (18, 53–55), suggesting a critical role for positioning to the mantle namics of leukocyte recruitment to the spleen following bacterial zone or corona around the developing GC in the follicle (56). infection. Moreover, recent reports demonstrate a rapid and high acquisition Nod1-induced B cell accumulation did not occur in animals of CCR6 on a precursor B cell population that can develop into deficient for CCR6, the sole receptor for CCL20 (38). CCR6 has switched or unswitched memory B cells in a GC-independent been shown to be expressed on many different leukocyte subsets, fashion (24, 55, 57). Indeed, our data demonstrate that the TNF-a/ including PMN, DC, T cells, NK cells, and B cells (38), exerting CCL20/CCR6 axis is required for the selective accumulation of critical roles for innate and adaptive antimicrobial immune re- MF B cells in SLO. sponses (49–52). Specific elimination of CCR6 expression in B cells Moreover, we found that deficiency of CCR6 leads to enhanced further confirmed a critical role for this in generation of NP-specific Ab titers in the presence of Nod1 ad- Nod1-induced accumulation of MF B cells in the spleen, dem- juvantation, albeit these Abs exhibited reduced affinity for Ag. It is onstrating a specific role for TNF-a/CCL20/CCR6. Importantly, important to note that the increase in titres in CCR62/2 mice is the expression of CCR6 by B lineage cells has been shown to be observed at a time point that is significantly later than what was developmentally regulated. Whereas pre/pro and most immature observed to be the peak time of B cell accumulation post-Nod1 B cells do not express CCR6, loss of CD10 and maturation into agonist administration. Thus, presumably the early influx of B cells the surface IgD+ mature B cell pool leads to its acquisition (53). post-Nod1 agonist administration is required to constrain the GC CCR6 is expressed by all naive follicular and pre-GC B cells in response, thus shaping the quality of the Ab response. Although mice and but is absent from GC B cells and plasma cells we do not yet understand the role of CCR6 in shaping the subsequent 2798 A TNF-a–CCL20–CCR6 AXIS DRIVES B CELL RESPONSES

GC response, our data are in accordance with a recent report 9. Rauch, P. J., A. Chudnovskiy, C. S. Robbins, G. F. Weber, M. Etzrodt, I. Hilgendorf, E. Tiglao, J. L. Figueiredo, Y. Iwamoto, I. Theurl, et al. 2012. demonstrating that CCR6 modulates the GC reaction and affinity Innate response activator B cells protect against microbial sepsis. Science 335: maturation of Ab responses upon immunization using Alum as 597–601. adjuvant (6). Because CCR6 is expressed in other immune cell 10. King, I. L., A. Fortier, M. Tighe, J. Dibble, G. F. Watts, N. Veerapen, A. M. Haberman, G. S. Besra, M. Mohrs, M. B. Brenner, and E. A. Leadbetter. types, B cell–intrinsic deletion of CCR6 would be a useful ap- 2012. Invariant natural killer T cells direct B cell responses to cognate lipid proach for further understanding the effect of CCR6 in shaping the antigen in an IL-21-dependent manner. Nat. Immunol. 13: 44–50. humoral immune response. 11. Puga, I., M. Cols, C. M. Barra, B. He, L. Cassis, M. Gentile, L. Comerma, A. Chorny, M. Shan, W. Xu, et al. 2012. B cell-helper neutrophils stimulate the Our observations are in agreement with recent data suggesting diversification and production of immunoglobulin in the marginal zone of the critical roles of the CCL20/CCR6 axis for B cells at barrier sur- spleen. Nat. Immunol. 13: 170–180. + 12. Kelly-Scumpia, K. M., P. O. Scumpia, J. S. Weinstein, M. J. Delano, faces. CCR6 B cells have recently been shown to enter the A. G. Cuenca, D. C. Nacionales, J. L. Wynn, P. Y. Lee, Y. Kumagai, P. A. Efron, inflamed skin expressing CCL20, suggesting a key role for local et al. 2011. B cells enhance early innate immune responses during bacterial host defense mechanisms (58). In addition, it has been observed sepsis. J. Exp. Med. 208: 1673–1682. 13. Leo´n, B., A. Ballesteros-Tato, J. L. Browning, R. Dunn, T. D. Randall, and that CCR6 deficiency leads to reduced intestinal IgA responses to F. E. Lund. 2012. Regulation of T(H)2 development by CXCR5+ dendritic cells oral Ag (49). This defect might be due to altered intestinal tissue and lymphotoxin-expressing B cells. Nat. Immunol. 13: 681–690. formation and function as CCR6 deficiency results in defective 14. Ansel, K. M., and J. G. Cyster. 2001. Chemokines in lymphopoiesis and lym- phoid organ development. Curr. Opin. Immunol. 13: 172–179. intestinal isolated lymphoid follicle (ILF) formation and reduced 15. Mueller, S. N., and R. N. Germain. 2009. Stromal cell contributions to the ho- numbers of cells in Peyer’s patches (59). Similarly, ILF are lower meostasis and functionality of the immune system. Nat. Rev. Immunol. 9: 618– 629. in number and smaller in size, and intestinal barrier expression of 16. Ansel, K. M., V. N. Ngo, P. L. Hyman, S. A. Luther, R. Fo¨rster, J. D. Sedgwick, CCL20 is reduced in animals deficient in PRM signaling, partic- J. L. Browning, M. Lipp, and J. G. Cyster. 2000. A chemokine-driven positive Downloaded from ularly in Nod1-deficient mice (60), suggesting that the CCL20/ feedback loop organizes lymphoid follicles. Nature 406: 309–314. 17. Ngo, V. N., H. Korner, M. D. Gunn, K. N. Schmidt, D. S. Riminton, CCR6 axis instructed by Nod1-mediated recognition of intestinal M. D. Cooper, J. L. Browning, J. D. Sedgwick, and J. G. Cyster. 1999. Lym- microbes exerts a central role for the regulation of mucosal im- photoxin alpha/beta and are required for stromal cell ex- munity. The role of CCR6 for the spatiotemporal organization of pression of homing chemokines in B and T cell areas of the spleen. J. Exp. Med. 189: 403–412. intestinal leukocytes appears to be important as multiple cell 18. Bowman, E. P., J. J. Campbell, D. Soler, Z. Dong, N. Manlongat, D. Picarella, types, including lymphoid tissue-inducer cells, DC, and B cells, R. R. Hardy, and E. C. Butcher. 2000. Developmental switches in chemokine http://www.jimmunol.org/ response profiles during B cell differentiation and maturation. J. Exp. Med. 191: and subpopulations of innate and adaptive T cells all express this 1303–1318. particular chemokine receptor (61, 62). However, a recent study 19. Fritz, J. H., L. Le Bourhis, G. Sellge, J. G. Magalhaes, H. Fsihi, T. A. Kufer, demonstrated that B cell–specific CCR6 deficiency yields defects C. Collins, J. Viala, R. L. Ferrero, S. E. Girardin, and D. J. Philpott. 2007. Nod1- mediated innate immune recognition of peptidoglycan contributes to the onset of in ILF and Peyer’s patch formation that are similiar to alterations adaptive immunity. Immunity 26: 445–459. observed when CCR6 is lacking in all cell types (59), suggesting 20. Vidal, K., I. Grosjean, J. P. Evillard, C. Gespach, and D. Kaiserlian. 1993. Im- an essential role for CCL20/CCR6 in intestinal B cell positioning mortalization of mouse intestinal epithelial cells by the SV40-large T : phenotypic and immune characterization of the MODE-K cell line. J. Immunol. in response to microbial Ag (59). Our data extend these obser- Methods 166: 63–73. vations and demonstrate that, upon infection and inflammation, 21. Katakai, T., T. Hara, M. Sugai, H. Gonda, and A. Shimizu. 2004. Lymph node

fibroblastic reticular cells construct the stromal reticulum via contact by guest on September 29, 2021 the homeostatic LT/CXCL13/CXCR5 axis is supplanted by the with . J. Exp. Med. 200: 783–795. TNF-a/CCL20/CCR6 axis, key for accumulation of MF B cells to 22. Gommerman, J. L., K. Giza, S. Perper, I. Sizing, A. Ngam-Ek, C. Nickerson- the spleen and shaping the Ag-specific Ab response. Collectively, Nutter, and J. L. Browning. 2003. A role for surface lymphotoxin in experi- mental autoimmune encephalomyelitis independent of LIGHT. J. Clin. Invest. these data provide new insights into mechanisms by which B cells 112: 755–767. participate in immune responses to microbial stimulation. 23. Summers-DeLuca, L. E., D. D. McCarthy, B. Cosovic, L. A. Ward, C. C. Lo, S. Scheu, K. Pfeffer, and J. L. Gommerman. 2007. Expression of lymphotoxin- Acknowledgments alphabeta on antigen-specific T cells is required for DC function. J. Exp. Med. 204: 1071–1081. We thank Dionne White and Nathalie Simard in the Faculty of Medicine 24. Taylor, J. J., K. A. Pape, and M. K. Jenkins. 2012. A germinal center- Flow Cytometry core facility at the University of Toronto for excellent tech- independent pathway generates unswitched memory B cells early in the pri- nical support. mary response. J. Exp. Med. 209: 597–606. 25.Opitz,B.,A.Puschel,W.Beermann,A.C.Hocke,S.Fo€ ¨rster, B. Schmeck, V. van Laak, T. Chakraborty, N. Suttorp, and S. Hippenstiel. 2006. Listeria Disclosures monocytogenes activated p38 MAPK and induced IL-8 secretion in a nucleotide- The authors have no financial conflicts of interest. binding oligomerization domain 1-dependent manner in endothelial cells. J. Immunol. 176: 484–490. 26. Boneca, I. G., O. Dussurget, D. Cabanes, M. A. Nahori, S. Sousa, M. Lecuit, E. Psylinakis, V. Bouriotis, J. P. Hugot, M. Giovannini, et al. 2007. A critical role References for peptidoglycan N-deacetylation in Listeria evasion from the host innate im- 1. Fritz, J. H., R. L. Ferrero, D. J. Philpott, and S. E. Girardin. 2006. Nod-like mune system. Proc. Natl. Acad. Sci. USA 104: 997–1002. in immunity, inflammation and disease. Nat. Immunol. 7: 1250–1257. 27.Kim,Y.G.,J.H.Park,M.H.Shaw,L.Franchi,N.Inohara,andG.Nu´n˜ez. 2. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate 2008. The cytosolic sensors Nod1 and Nod2 are critical for bacterial recog- immunity: update on Toll-like receptors. Nat. Immunol. 11: 373–384. nition and host defense after exposure to Toll-like receptor ligands. Immunity 3. Cain, D. W., P. B. Snowden, G. D. Sempowski, and G. Kelsoe. 2011. Inflam- 28: 246–257. mation triggers emergency granulopoiesis through a density-dependent feedback 28. Mosa, A., C. Trumstedt, E. Eriksson, O. Soehnlein, F. Heuts, K. Janik, A. Klos, mechanism. PLoS One 6: e19957. O. Dittrich-Breiholz, M. Kracht, A. Hidmark, et al. 2009. Nonhematopoietic 4. Cain, D., M. Kondo, H. Chen, and G. Kelsoe. 2009. Effects of acute and chronic cells control the outcome of infection with Listeria monocytogenes in a nucleo- inflammation on B-cell development and differentiation. J. Invest. Dermatol. tide oligomerization domain 1-dependent manner. Infect. Immun. 77: 2908– 129: 266–277. 2918. 5. Ueda, Y., K. Yang, S. J. Foster, M. Kondo, and G. Kelsoe. 2004. Inflammation 29. Kang, S. J., H. E. Liang, B. Reizis, and R. M. Locksley. 2008. Regulation of controls B lymphopoiesis by regulating chemokine CXCL12 expression. J. Exp. hierarchical clustering and activation of innate immune cells by dendritic cells. Med. 199: 47–58. Immunity 29: 819–833. 6. Ueda, Y., M. Kondo, and G. Kelsoe. 2005. Inflammation and the reciprocal 30. Fritz, J. H., and J. L. Gommerman. 2011. /Stromal cell networks and production of granulocytes and lymphocytes in bone marrow. J. Exp. Med. 201: lymphoid tissue environments. J. Cytokine Res. 31: 277–289. 1771–1780. 31.Magalhaes,J.G.,D.J.Philpott,M.A.Nahori,M.Je´hanno,J.Fritz, 7. Lund, F. E., and T. D. Randall. 2010. Effector and regulatory B cells: modulators L. Le Bourhis, J. Viala, J. P. Hugot, M. Giovannini, J. Bertin, et al. 2005. Murine of CD4+ T cell immunity. Nat. Rev. Immunol. 10: 236–247. Nod1 but not its human orthologue mediates innate immune detection of tracheal 8. Fritz, J. H., O. L. Rojas, N. Simard, D. D. McCarthy, S. Hapfelmeier, S. Rubino, cytotoxin. EMBO Rep. 6: 1201–1207. S. J. Robertson, M. Larijani, J. Gosselin, I. I. Ivanov, et al. 2012. Acquisition of 32. Mandik-Nayak, L., G. Huang, K. C. Sheehan, J. Erikson, and D. D. Chaplin. a multifunctional IgA+ plasma cell phenotype in the gut. Nature 481: 199–203. 2001. Signaling through TNF receptor p55 in TNF-alpha-deficient mice alters The Journal of Immunology 2799

the CXCL13/CCL19/CCL21 ratio in the spleen and induces maturation and 47. Futterer,€ A., K. Mink, A. Luz, M. H. Kosco-Vilbois, and K. Pfeffer. 1998. The migration of anergic B cells into the B cell follicle. J. Immunol. 167: 1920–1928. receptor controls organogenesis and affinity maturation in 33. Kunkel, E. J., D. J. Campbell, and E. C. Butcher. 2003. Chemokines peripheral lymphoid tissues. Immunity 9: 59–70. in lymphocyte trafficking and intestinal immunity. Microcirculation 10: 313– 48. Boulianne, B., M. X. Le, L. A. Ward, L. Meng, D. Haddad, C. Li, A. Martin, and 323. J. L. Gommerman. 2013. AID-expressing germinal center B cells cluster nor- 34. Rossi, D. L., A. P. Vicari, K. Franz-Bacon, T. K. McClanahan, and A. Zlotnik. mally within lymph node follicles in the absence of FDC-M1+ CD35+ follicular 1997. Identification through bioinformatics of two new proin- dendritic cells but dissipate prematurely. J. Immunol. 191: 4521–4530. flammatory human chemokines: MIP-3alpha and MIP-3beta. J. Immunol. 158: 49. Cook, D. N., D. M. Prosser, R. Forster, J. Zhang, N. A. Kuklin, 1033–1036. S. J. Abbondanzo, X. D. Niu, S. C. Chen, D. J. Manfra, M. T. Wiekowski, et al. 35. Hromas, R., P. W. Gray, D. Chantry, R. Godiska, M. Krathwohl, K. Fife, 2000. CCR6 mediates dendritic cell localization, lymphocyte homeostasis, and G. I. Bell, J. Takeda, S. Aronica, M. Gordon, et al. 1997. Cloning and charac- immune responses in mucosal tissue. Immunity 12: 495–503. terization of exodus, a novel beta-chemokine. Blood 89: 3315–3322. 50. Wen, H., C. M. Hogaboam, N. W. Lukacs, D. N. Cook, S. A. Lira, and 36.Hieshima,K.,T.Imai,G.Opdenakker,J.VanDamme,J.Kusuda,H.Tei, S. L. Kunkel. 2007. The chemokine receptor CCR6 is an important component Y. Sakaki, K. Takatsuki, R. Miura, O. Yoshie, and H. Nomiyama. 1997. of the innate immune response. Eur. J. Immunol. 37: 2487–2498. Molecular cloning of a novel human CC chemokine and activation- 51. Salazar-Gonzalez, R. M., J. H. Niess, D. J. Zammit, R. Ravindran, A. Srinivasan, regulated chemokine (LARC) expressed in liver: chemotactic activity J. R. Maxwell, T. Stoklasek, R. Yadav, I. R. Williams, X. Gu, et al. 2006. CCR6- for lymphocytes and gene localization on 2. J. Biol. Chem. 272: mediated dendritic cell activation of pathogen-specific T cells in Peyer’s patches. 5846–5853. Immunity 24: 623–632. 37. Meissner, A., O. Zilles, R. Varona, K. Jozefowski, U. Ritter, G. Marquez, 52. Ravindran, R., L. Rusch, A. Itano, M. K. Jenkins, and S. J. McSorley. 2007. R. Hallmann, and H. Korner. 2003. CC chemokine ligand 20 partially controls CCR6-dependent recruitment of blood phagocytes is necessary for rapid CD4 adhesion of naive B cells to activated endothelial cells under shear stress. Blood T cell responses to local bacterial infection. Proc. Natl. Acad. Sci. USA 104: 102: 2724–2727. 12075–12080. 38. Schutyser, E., S. Struyf, and J. Van Damme. 2003. The CC chemokine CCL20 53. Krzysiek, R., E. A. Lefevre, J. Bernard, A. Foussat, P. Galanaud, F. Louache, and and its receptor CCR6. Cytokine Rev. 14: 409–426. Y. Richard. 2000. Regulation of CCR6 chemokine receptor expression and re- 39. Leo´n, B., A. Ballesteros-Tato, R. S. Misra, W. Wojciechowski, and F. E. Lund. sponsiveness to macrophage inflammatory protein-3alpha/CCL20 in human 2012. Unraveling effector functions of B cells during infection: the hidden world B cells. Blood 96: 2338–2345. Downloaded from beyond antibody production. Infect. Disord. Drug Targets 12: 213–221. 54. Dubois, B., C. Massacrier, and C. Caux. 2001. Selective attraction of naive and 40. Serbina, N. V., C. Shi, and E. G. Pamer. 2012. Monocyte-mediated immune memory B cells by dendritic cells. J. Leukoc. Biol. 70: 633–641. defense against murine Listeria monocytogenes infection. Adv. Immunol. 113: 55. Llina`s, L., A. La´zaro, J. de Salort, J. Matesanz-Isabel, J. Sintes, and P. Engel. 119–134. 2011. Expression profiles of novel cell surface molecules on B-cell subsets and 41. Krege, J., S. Seth, S. Hardtke, A. C. Davalos-Misslitz, and R. Fo¨rster. 2009. plasma cells as analyzed by flow cytometry. Immunol. Lett. 134: 113–121. Antigen-dependent rescue of nose-associated lymphoid tissue (NALT) devel- 56. Shen, Y., J. Iqbal, L. Xiao, R. C. Lynch, A. Rosenwald, L. M. Staudt, opment independent of LTbetaR and CXCR5 signaling. Eur. J. Immunol. 39: S. Sherman, K. Dybkaer, G. Zhou, J. D. Eudy, et al. 2004. Distinct gene ex-

2765–2778. pression profiles in different B-cell compartments in human peripheral lymphoid http://www.jimmunol.org/ 42. Rangel-Moreno, J., D. M. Carragher, M. de la Luz Garcia-Hernandez, organs. BMC Immunol. 5: 20. J. Y. Hwang, K. Kusser, L. Hartson, J. K. Kolls, S. A. Khader, and T. D. Randall. 57. Schwickert, T. A., G. D. Victora, D. R. Fooksman, A. O. Kamphorst, 2011. The development of inducible bronchus-associated lymphoid tissue M. R. Mugnier, A. D. Gitlin, M. L. Dustin, and M. C. Nussenzweig. 2011. A depends on IL-17. Nat. Immunol. 12: 639–646. dynamic T cell-limited checkpoint regulates affinity-dependent B cell entry into 43. Matsumoto, M., Y. X. Fu, H. Molina, G. Huang, J. Kim, D. A. Thomas, the germinal center. J. Exp. Med. 208: 1243–1252. M. H. Nahm, and D. D. Chaplin. 1997. Distinct roles of and 58. Geherin, S. A., S. R. Fintushel, M. H. Lee, R. P. Wilson, R. T. Patel, C. Alt, the type I tumor necrosis factor (TNF) receptor in the establishment of follicular A. J. Young, J. B. Hay, and G. F. Debes. 2012. The skin, a novel niche for dendritic cells from non-bone marrow-derived cells. J. Exp. Med. 186: 1997– recirculating B cells. J. Immunol. 188: 6027–6035. 2004. 59. McDonald, K. G., J. S. McDonough, C. Wang, T. Kucharzik, I. R. Williams, and 44. Alimzhanov, M. B., D. V. Kuprash, M. H. Kosco-Vilbois, A. Luz, R. D. Newberry. 2007. CC chemokine receptor 6 expression by B lymphocytes is R. L. Turetskaya, A. Tarakhovsky, K. Rajewsky, S. A. Nedospasov, and essential for the development of isolated lymphoid follicles. Am. J. Pathol. 170:

K. Pfeffer. 1997. Abnormal development of secondary lymphoid tissues in 1229–1240. by guest on September 29, 2021 lymphotoxin beta-deficient mice. Proc. Natl. Acad. Sci. USA 94: 9302–9307. 60. Bouskra, D., C. Bre´zillon, M. Be´rard, C. Werts, R. Varona, I. G. Boneca, and 45. Mackay, F., G. R. Majeau, P. Lawton, P. S. Hochman, and J. L. Browning. 1997. G. Eberl. 2008. Lymphoid tissue genesis induced by commensals through NOD1 Lymphotoxin but not tumor necrosis factor functions to maintain splenic ar- regulates intestinal homeostasis. Nature 456: 507–510. chitecture and humoral responsiveness in adult mice. Eur. J. Immunol. 27: 2033– 61. Eberl, G. 2007. From induced to programmed lymphoid tissues: the long road to 2042. preempt pathogens. Trends Immunol. 28: 423–428. 46. Koni, P. A., and R. A. Flavell. 1999. Lymph node germinal centers form in the 62. Williams, I. R. 2006. CCR6 and CCL20: partners in intestinal immunity and absence of follicular dendritic cell networks. J. Exp. Med. 189: 855–864. lymphorganogenesis. Ann. N. Y. Acad. Sci. 1072: 52–61.