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Nitric Oxide Regulates BAFF Expression and T Cell −Independent Antibody Responses This information is current as of June 18, 2017. References Subscription Permissions Email Alerts J Immunol 2014; 193:1110-1120; Prepublished online 20 June 2014; doi: 10.4049/jimmunol.1303158 http://www.jimmunol.org/content/193/3/1110 http://www.jimmunol.org/content/suppl/2014/06/19/jimmunol.130315 8.DCSupplemental This article cites 69 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/193/3/1110.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html 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 © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Supplementary Material Daniela Giordano, Kevin E. Draves, Chang Li, Tobias M. Hohl and Edward A. Clark The Journal of Immunology Nitric Oxide Regulates BAFF Expression and T Cell–Independent Antibody Responses Daniela Giordano,* Kevin E. Draves,* Chang Li,* Tobias M. Hohl,†,1 and Edward A. Clark* N itric oxide produced by innate immune cells plays an important role in regulating T cell immune responses (1, 2). For instance, NO produced by inducible NO synthase 2 (NOS2/iNOS) inhibits Th1 cell responses, silences autoreactive T cells, and restricts autoimmune reactions (3–6). NO affects differentiation and maturation of Ly6Chi inflammatory monocytes (MOs) into MO-derived dendritic cells (Mo-DCs) and DC programming of T cells (7, 8). NOS2 also plays a role in Th17/Th22 cellular immunity (2). To what extent NOS2-derived NO plays a role in T cell– dependent (TD) and T cell–independent (TI) humoral immune *Department of Immunology, University of Washington, Seattle, WA 98109; and † Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109 1 Current address: Department of Medicine, Infectious Disease Service, Memorial Sloan-Kettering Cancer Center, New York, NY. Received for publication November 22, 2013. Accepted for publication May 23, 2014. This work was supported by National Institutes of Health Grants AI44257 and AI52203 (to E.A.C.). Address correspondence and reprint requests to Dr. Daniela Giordano, Department of Immunology, University of Washington, 750 Republican Street, SLU3.1, Building E, Lab E348, Box 358059, Seattle, WA 98109. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: AFC, Ab-forming cell; alum, aluminum hydroxide; APRIL, a proliferation-inducing ligand; B6, C57BL/6; BM, bone marrow; BMDC, bone marrow–derived DC; cDC, conventional DC; CGG, chicken g globulin; CSR, class switch recombination; DC, dendritic cell; DT, diphtheria toxin; DTR, diphtheria toxin receptor; FO, follicular; iNOS, inducible NO synthase; MFI, mean fluorescence intensity; MO, monocyte; Mo-DC, MO-derived dendritic cell; Mph, macrophage; MZ, marginal zone; Nph, neutrophil; NOS2, inducible NO synthase 2; NP, 4-hydroxy3-nitrophenyl acetyl; PC, plasma cell; pDC, plasmacytoid DC; T1, transitional 1; T2, transitional 2; TD, T cell–dependent; TI, T cell–independent; Tip-DC, TNF-a/inducible NO synthase 2–producing DC; WT, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1303158 responses has not been fully investigated. On the one hand, NOS22/2 mice had elevated levels of viral-specific IgG2a Abs after infection with influenza virus (9). On the other hand, NOproducing TNF-a/iNOS–producing DCs (Tip-DCs) are required for Ag-induced IgA production in the intestinal mucosa (10). This study suggested that NO produced by Tip-DCs in mucosal lymphoid tissues regulates TI IgA class switch recombination (CSR) through production of the TNF family ligands BAFF and a proliferation-inducing ligand (APRIL), both of which were reduced in NOS22/2 Tip-DCs. The differences in the requirement for NO in promoting IgA responses and for NO in inhibiting IgG responses could be due to factors including the type and source of Ag and/or the localization and type of cells producing NO. Given the importance of the spleen in TI-2 Ab responses (11), we decided to investigate the role NOS2 plays in Ab responses to TI-2 Ags. BAFF and APRIL play important roles in B cell responses to Ags (12, 13). Whereas APRIL is a major player in inducing IgA Ab responses, BAFF is required for TI-2 B cell responses (14–16). BAFF-deficient and BAFFR-deficient mice have a block in the maturation of B cells from immature transitional 1 (T1) to transitional 2 (T2) B cells and almost entirely lack mature follicular (FO) and marginal zone (MZ) B cells. Neutralization of BAFF reduces TI-2 Ab responses (17, 18), whereas BAFF overexpression enhances TI-2 B cell responses (19). BAFF is expressed in MOs, macrophages (Mphs), conventional DCs (cDCs), and neutrophils (Nphs) (20–24). BAFF produced by MOs and cDCs induces CSR in a CD40-independent manner and signals MZ B cells to initiate TI-2 Ab responses (16, 25). Earlier studies showed that a subset of myeloid DCs is associated with plasma cell (PC) survival and growth in extrafollicular foci triggered by a TI-2 Ag (26, 27). Balázs et al. (16) showed that the DC subset responsible for transporting Ags to the MZ and supporting PC survival through BAFF production is a blood-derived Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Whereas NO is known to regulate T cell responses, its role in regulating B cell responses remains unclear. Previous studies suggested that inducible NO synthase 2 (NOS2/iNOS) is required for normal IgA Ab responses but inhibits antiviral IgG2a Ab responses. In this study we used NOS22/2 mice to determine the role of NO in T cell–dependent and T cell–independent (TI)-2 Ab responses. Whereas T cell–dependent Ab responses were only modestly increased in NOS22/2 mice, IgM and IgG3 Ab responses as well as marginal zone B cell plasma cell numbers and peritoneal B1b B cells were significantly elevated after immunization with the TI-2 Ag 4-hydroxy-3-nitrophenyl acetyl (NP)–Ficoll. The elevated TI-2 responses in NOS22/2 mice were accompanied by significant increases in serum levels of BAFF/BLyS and by increases in BAFF-producing Ly6Chi inflammatory monocytes and monocyte-derived dendritic cells (DCs), suggesting that NO normally inhibits BAFF expression. Indeed, we found that NOS22/2 DCs produced more BAFF than did wild-type DCs, and addition of a NO donor to NOS22/2 DCs reduced BAFF production. Bone marrow chimeric mice that lack NOS2 in either nonhematopoietic or hematopoietic cells had intermediate IgM and IgG3 Ab responses after NP-Ficoll immunization, suggesting that NOS2 from both hematopoietic and nonhematopoietic sources regulates TI-2 Ab responses. Similar to NOS22/2 mice, depletion of Ly6Chi inflammatory monocytes and monocytederived DCs enhanced NP-specific IgM and IgG3 responses to NP-Ficoll. Thus, NO produced by inflammatory monocytes and their derivative DC subsets plays an important role in regulating BAFF production and TI-2 Ab responses. The Journal of Immunology, 2014, 193: 1110–1120. The Journal of Immunology Materials and Methods Mice C57BL/6 (WT or B6) and B6.NOS22/2 (B6.129P2-Nos2tm1Lau/J, stock No.002609) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). CCR2 depleter mice (C2D; CD45.2+C57BL/6-BAC-Tg [pCCR2–diphtheria toxin receptor (DTR)-2A-CFP]) are described in Hohl et al. (37). CD11c-DTR mice were a gift from Dr. Pam Fink (University of Washington, Seattle, WA). Mice were housed in specific pathogen-free conditions according to institutional guidelines and used at 8–12 wk of age. The University of Washington’s Institutional Animal Care and Use Committee approved all animal protocols. NP-Ficoll and NP–chicken g globulin/aluminum hydroxide immunizations, serum collection, and spleen and peritoneal cell isolation Age-matched 8- to 12-wk-old mice were administered i.p. with 20 mg/ mouse NP-Ficoll (NP conjugated to aminoethyl carboxymethyl–Ficoll) or 50 mg/mouse NP–chicken g globulin (CGG) (Biosearch Technologies, Petaluma, CA) in Imject Alum (Thermo Scientific, Rockford, IL). For TD Ab responses, 35 d following primary immunization with NP-CGG/alum, mice were rechallenged with 20 mg/mouse NP-CGG. At the indicated time points mice were bled and/or spleens were removed and dissociated into a single-cell suspension by carefully and thoroughly mincing and grinding the tissue between the ends of two frosted microscope glass slides. Splenic cells were then washed and the erythrocytes (RBCs) were lysed and the remaining mononuclear splenic cells were filtered and processed for FACS analysis as described below. In some experiments peritoneal cells were isolated by peritoneal lavage with 10 ml PBS buffer containing 2% FCS and processed for FACS analysis as described below. Ablation of CCR2+ cells in CCR2-DTR mice and BM chimeras The previously described CCR2-DTR mice (37) express a simian DTR under the control of the CCR2 promoters that allows the selective depletion of CCR2+ cells. For depletion experiments, mice were injected i.p. with 250 ng DT at various times before and after immunization of CCR2DTR mice. To generate BM chimeric mice, recipient mice were sublethally irradiated and reconstituted i.v. with 3 3 106 donor BM cells and housed for at least 6–8 wk before immunization. Generation of BMDCs and cell culture BMDCs were generated as described (38, 39). Briefly, BM cells were isolated by flushing femurs and tibiae, erythrocytes were lysed, and the remaining cells were seeded in 24-well plates at 1 3 106 cells/ml in RPMI 1640 supplemented with 10% FBS, 20 ng/ml GM-CSF, and 10 ng/ml IL-4 (Fitzgerald Industries, Acton, MA) at 37˚C in 5% CO2. Nonadherent cells were removed at day 2 and medium was replaced with fresh medium supplemented with cytokines at days 2 and 4 (50%). After 6–7 d, loosely adherent cells were collected; 80–99% cells were CD11c+. In some experiments BMDCs were seeded at 2.0 3 106 cells/2 ml in 12-well plates in RPMI 1640 with and without the NOS inhibitor L-NAME (1 mM) (Enzo Life Sciences/Biomol International, Farmingdale, NY) for WT BMDCs or with the NO donor NOR4 (100 mM) (Calbiochem, San Diego, CA) or the vehicle DMSO (Sigma-Aldrich, St. Louis, MO) for NOS22/2 BMDCs. In all experiments where NOS22/2 BMDCs were treated with NOR4, the control samples with DMSO vehicle control gave the same results as samples without NOR4. ELISA and ELISPOT assay Serum NP Ab titers were measured by modification of an ELISA assay as previously described (40). Ninety-six–well microplates were coated with a solution of 20 mg/ml NP-BSA (Biosearch Technologies) overnight at 4˚C. Plates were blocked for 1 h at 37˚C using a solution of PBS containing 1% BSA (Sigma-Aldrich). Serum samples were applied and allowed to react at room temperature for 1.5 h. Anti-mouse Abs specific for IgG or IgM isotypes coupled to HRP (SouthernBiotech, Birmingham, AL) were applied and allowed to incubate for 2 h at room temperature. Plates were developed with tetramethylbenzidine substrate and read at 450 nm absorbance. Values were compared with known dilutions of IgG or IgM to calculate Ab concentrations. ELISPOT assays were performed as described (41) with the exception that 96-well nitrocellulose plates (Millipore, Billerica, MA) were coated overnight with 20 mg/ml NP-BSA in 100 ml PBS. Spots were visualized and counted using an ImmunoSpot imaging analyzer system (Cellular Technology, Cleveland, OH). Concentrations of BAFF in sera for in vivo experiments and in media from in vitro cultures were determined by specific ELISA, performed in triplicate using a matched pair of cytokine-specific mAbs and recombinant cytokines as standards using the mouse BAFF ELISA kit from Abcam (Cambridge, MA) according to the manufacturer’s instructions. BAFF detection by quantitative PCR BMDCs from WT and NOS22/2 mice were frozen at 280˚C. RNA was isolated using an RNAeasy Plus Micro kit (Qiagen, Valencia, CA) and converted into cDNA by reverse transcriptase with the high-capacity cDNA reverse transcription kit (Applied Biosystems, Foster City, CA). PCR was performed using the 7300 real-time PCR system (Applied Biosystems) using the Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. Mouse GAPDH was used as housekeeping internal control. All primers were designed using Primer3 software (Whitehead Institute for Biomedical Research, Cambridge, MA). All PCR analyses were done in triplicates. The primer sequences used were as follows: mBAFF, forward, 59-AGGCTGGAAGAAGGAGATGAG-39, reverse, 39-CAGAGAAGACGAGGGAAGGG-59. Flow cytometric analyses RBC-lysed BMDCs or splenic cell populations were incubated with antiCD16/CD32 blocking Ab (2.4G2) for 10 min at room temperature and then stained with various Ab mixtures on ice. Cells were stained with mAbs conjugated to FITC, PE, allophycocyanin, eFluor 450, allophycocyanin– eFluor 780, PerCP-Cy5.5, PE-Cy7, Pacific Orange, or Alexa Fluor 647. For analysis of splenic and peritoneal B cell subsets (gating strategy in Supplemental Fig. 1A, 1C), four- or five-color flow cytometry was performed by staining the cells with combinations of mAbs against B220 (RA3-6B2), IgM (eB121-15F9), and CD5 (53-7.3) from eBioscience (San Diego, CA); CD21/CD35 (7G6) and IgD (11-26c.27) from BioLegend (San Diego, CA); CD23 (B3B4) from Invitrogen Life Technologies (Grand Island, NY); and CD24 (M1/69) and CD138 (281-2) from BD Biosciences (San Jose, CA). For analysis of other myeloid splenic cell subsets (gating strategy in Supplemental Fig. 2), seven- or eight-color flow cytometry was performed by staining the cells with combinations of mAbs against B220 (RA3-6B2), CD11b (M1/70), CD8a (53-6.7), CD11c (N418), CD209a/ DCSIGN (LWC06), and Mac3 (M3/84) from eBioscience; Ly6C (AL21) and Ly6G (1A8) from BD Biosciences; NOS2 (C11) from Santa Cruz Biotechnology (Santa Cruz, CA); and F4/80 (CI:A31) from AbD Serotec (Raleigh, NC). Myeloid splenic cell subsets were defined as follows: eosinophils, CD11bhiLy6CintSSChiLy6Glo/2; Nphs, CD11bhiLy6Cint SSCintLy6Ghi; Ly6Chi MOs, CD11bhiLy6ChiCD11clo/2CD209a/DCSIGN2 Mac3lo; Ly6Chi Mo-DCs, CD11bhiLy6ChiCD11cint/hiCD209a/DCSIGN+ Mac3hi; Ly6Clo MOs, CD11bintCD11c2Ly6Clo; Mphs, CD11b+CD11clo Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 CD11cloCD11bhi DC subset, probably derived from MOs. BAFF produced by MOs and Mo-DCs is also essential for secondary Ab responses under conditions with limited T cell help (28). Ly6Chi inflammatory Mo-DCs, which are also characterized by the expression of high levels of CCR2, play an important role in protective immunity to a variety of pathogens; however, their role in TI-2 Ab responses has not been widely investigated (29–33). Because we found that NOS22/2 mice have dysregulated Ly6Chi inflammatory Mo-DCs (8), we investigated the role of NOS2 and inflammatory MOs and Mo-DCs in TI-2 immune responses. We found that NOS22/2 mice have enhanced Ab responses to 4-hydroxy-3-nitrophenyl acetyl (NP)–Ficoll, a widely used TI-2 Ag (26, 34–36). The elevated TI-2 Ab responses in NOS22/2 mice were accompanied by significant increases in serum levels of BAFF and BAFF-producing Ly6Chi inflammatory MOs and Mo-DCs. This suggested that NO may negatively regulate BAFF expression. Indeed, NOS22/2 bone marrow–derived DCs (BMDCs) produced more BAFF than did wild-type (WT) BM DCs. Depletion of CCR2+Ly6Chi inflammatory MOs and Mo-DCs also enhanced NP-specific IgM and IgG3 responses to NP-Ficoll, suggesting that inflammatory MOs and Mo-DCs may be sources of NO that regulate TI-2 Ab responses. Collectively, our data show that NO produced by inflammatory Mo-DCs as well as nonhematopoietic cells regulates TI-2 Ab responses via inhibition of BAFF production. 1111 1112 SSChiF4/80+; plasmacytoid DCs (pDCs), CD11b2CD11cloB220+; cDCs, CD11chiCD11bint/2 or CD11chiB2202; CD8+ cDCs, CD11chiB2202CD8+; CD82 cDCs, CD11chiB2202CD82. A mAb against BAFF (121808) or a rat IgG2a isotype control (R&D Systems, Minneapolis, MN) was added to the multicolor flow cytometry analysis of all splenic cell populations. For intracellular staining cells were stained with mAbs for surface markers and fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) or 0.1% saponin in staining buffer followed by anti-BAFF or anti-NOS2 staining for 20 min at room temperature. Fluorescence acquisition was done on LSR II FACScan analyzer (Becton Dickinson, Franklin Lakes, NJ) using FACSDiva software, and data analysis was performed with FlowJo software. Statistical analyses All statistical analysis was performed with Prism software (GraphPad Software). A p value of ,0.05 was considered significant. For in vitro experiments, the statistical significance of differences in the means 6 SEM of BAFF mRNA detected by quantitative PCR or BAFF protein detected by ELISA of various groups was calculated with the two-tailed paired Student t test. For in vivo experiments the two-tailed Mann–Whitney nonparametric test was performed for all experiments comparing two groups, and the Kruskal–Wallis followed by a Dunnett post hoc test was performed for BM chimera experiments comparing more than two groups. NOS22/2 mice have enhanced TI-2 Ab responses A previous study showed that naive WT and NOS22/2 mice do not differ in serum levels of IgM or IgG subclasses (10). In the present study, we examined whether NO regulates Ab responses to TI-2 Ags and found that compared with WT control mice, NOS22/2 mice had a 2- to 3-fold increase in serum NP-specific IgM (NP-IgM) and IgG3 (NP-IgG3) Ab levels after immunization with NP-Ficoll (Fig. 1A). The modest IgG1, IgG2a, and IgG2b Ab responses to NP-Ficoll were not different between WT and NOS22/2 mice (data not shown). NOS22/2 mice immunized with NP-Ficoll also had increased percentages and numbers of splenic B220loCD138+ PCs (Fig. 1B and data not shown) and a significant increase in NP-specific IgM and IgG3 Ab-forming cells (AFCs) as determined by ELISPOT (Fig. 1C). The increase in PCs in NPFicoll–immunized NOS22/2 mice was not due to an overall increase in total B cell numbers (Fig. 1D). Thus, the absence of NOS2 enhances TI-2 Ag-induced PC differentiation and Ab production, suggesting that NO produced by NOS2 normally restrains TI-2 Ab responses. We also analyzed whether the upregulation in PCs in NOS22/2 mice after NP-Ficoll immunization was accompanied by increases in B cell subset numbers compared with WT mice. MZ B cells (B220+CD232CD21hiCD24+) were significantly higher in immunized NOS22/2 mice, but other B cell subsets including T1 (B220+CD232CD212/loCD24hi), T2 (B220+CD23+CD21loCD24hi), and FO (B220+CD23+CD21/35intCD24lo) B cells were not significantly increased (Fig. 1D, Supplemental Fig. 1A) (42). The increase in MZ B cells was also evident when B cell subsets were analyzed using other markers and gating strategies (43, 44) (data not shown). Total spleen cell numbers and percentages and absolute cell numbers of B cells or B cell subsets in nonimmunized WT and NOS22/2 mice were not different (Supplemental Fig. 1B and data not shown). After immunization with NP-Ficoll, peritoneal B cells were also increased in NOS22/2 mice but not in WT mice. In particular, peritoneal B1b (CD19+, IgMhi, IgDlo, B220lo, CD52) and B2 (CD19+, IgMlo, IgDhi, B220hi, CD52) (45) cell numbers were elevated in NOS22/2 mice compared with WT mice, whereas B1a B cell numbers were not altered significantly (Fig. 1E, Supplemental Fig. 1C) (45, 46). No significant differences were found in B1 B cells in the spleens of naive or immunized WT and NOS22/2 mice (data not shown). The increases in splenic MZ B cells and peritoneal B1b B cells in NOS22/2 mice after NP-Ficoll immunization are in accord with the fact that MZ B cells and B1 B cells are the major B cell subsets that respond to TI-2 Ags (12, 23, 42). Splenic DCs, Mphs, and Nphs also can play important roles in TI-2 Ab responses (23). Thus, we assessed whether the frequency and numbers of these and other splenic cell subsets were dysregulated in NOS22/2 mice upon NP-Ficoll immunization. Using multicolor flow cytometry, we analyzed myeloid splenic populations based on their relative expression of CD11b, CD11c, and of other markers 4 d after immunizing mice with NP-Ficoll (Supplemental Fig. 2) (30, 47–50). Before immunization there was no difference in the percentage or numbers of myeloid cell subsets in WT and NOS22/2 mice (data not shown), and splenic myeloid cell populations did not change in WT mice after NPFicoll immunization (data not shown). However, in immunized NOS22/2 mice, compared with WT mice, the numbers of Ly6Chi inflammatory MOs and Mo-DCs, Nphs, and CD8a+ cDCs were significantly increased (Fig. 1F). In contrast, eosinophils, Mphs, Ly6Clo MOs, CD8a2 cDCs, and pDCs were not increased significantly in NOS22/2 mice (Fig. 1F), and, as expected, CD8 T cell numbers did not change (data not shown). Similar results were obtained analyzing spleens of WT and NOS22/2 mice 24 h after NP-Ficoll immunization (data not shown). Thus, in the absence of NOS2 together with the enhancement of TI-2 Ab production there was an expansion of some peritoneal and splenic B cell populations that may be involved in TI-2 Aginduced PC differentiation. TD Ab responses are increased in NOS22/2 mice NOS22/2 mice have elevated Th1 immune responses (4, 8). Therefore, we analyzed whether Ab responses to TD Ags were also dysregulated in NOS22/2 mice. We immunized WT and NOS22/2 mice with NP-CGG in alum and measured NP-specific Ab responses 7–35 d later (Fig. 2A). NP-specific IgM Ab responses were not different between WT and NOS22/2 mice; however, NP-specific IgG1 Ab responses were elevated in NOS22/2 mice, particularly after a second challenge with soluble NP-CGG (Fig. 2A). Furthermore, the spleens from NOS22/2 versus WT mice evaluated 7 d after rechallenge with soluble NP-CGG had increased levels of CD138+ PCs (Fig. 2B) and NP-specific IgG1 AFCs (Fig. 2C). There were no major changes in the number of B cell subsets or CD8 T cells or other myeloid cell populations in the spleens of WT and NOS22/2 mice boosted with NP-CGG (data not shown). However, there was a significant increase in the numbers of all splenic B cell subsets and Nphs in NOS22/2 versus WT mice 7 d after a primary NP-CGG/alum immunization (Fig. 2D, 2E). Ly6Clo MOs, CD8a2 DCs, and pDCs were also modestly increased in NP-CGG/alum–immunized NOS22/2 mice (Fig. 2E). Although the total numbers of some splenic cell populations were increased in NOS22/2 mice during the primary TD Ab response, these changes did not translate into major differences in overall Ab levels (Fig. 2A). We conclude that in the absence of NO produced by NOS2, TI-2 Ab responses are upregulated and peritoneal B1 B cells, MZ B cells, and some splenic myeloid cell populations known to play roles in TI-2 immune responses are elevated. TD immune responses are also dysregulated in NOS22/2 mice, but to a lesser extent. Thus, we subsequently focused our analyses on the regulation of TI-2 Ab responses by NOS2-derived NO. NO inhibits BAFF production Given the important role of BAFF in TI-2 Ab responses, we decided to test whether BAFF was increased in NOS22/2 mice. Serum BAFF levels were significantly higher in NOS22/2 mice immu- Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Results NO REGULATES BAFF AND Ab RESPONSES The Journal of Immunology 1113 nized with NP-Ficoll compared with immunized WT mice (Fig. 3A). Interestingly, nonimmunized NOS22/2 mice also had slightly higher serum BAFF levels compared with naive WT mice (Fig. 3A). Using flow cytometry to monitor spleen cells that produce intracellular BAFF, we detected a significant increase in intracellular BAFF expression (by mean fluorescence intensity [MFI]) and in the percentages and numbers of BAFF+ spleen cells in NOS22/2 mice versus WT mice after immunization with NPFicoll (Fig. 3B and data not shown). The numbers of BAFF+ spleen cells were the same in WT and NOS22/2 mice prior to immunization and did not change in WT mice after immunization (Fig. 3B). Several of the myeloid cell subsets that increased significantly after immunizing NOS22/2 mice with NP-Ficoll (Fig. 1F) also are known to produce BAFF, including Ly6Chi inflammatory MOs, Ly6Chi inflammatory DCs, and Nphs (22, 23). Thus, these subsets could be responsible for the increased BAFF and ensuing Ab responses in NOS22/2 mice. To test whether there was a selective expansion of BAFF+ myeloid spleen cell subsets in NOS22/2 mice after immunization with NP-Ficoll, we used the gating strategy described above (Supplemental Fig. 2). Although cDCs, Nphs, and Mphs all expressed BAFF to a moderate level, Ly6Chi inflammatory Mo-DCs expressed the highest BAFF levels (Fig. 3C). To our knowledge, differential BAFF expression by MO subsets has not been described. We found that Ly6Chi inflammatory MOs expressed BAFF at levels comparable to cDCs, whereas Ly6Clo MOs expressed lower levels (Fig. 3C). The levels of these BAFF-producing cells increased after immunization of NOS22/2 mice with NP-Ficoll (Fig. 3D), including a notable selective expansion in the number of BAFF+Ly6Chi inflammatory MOs, Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 1. NOS22/2 mice have enhanced TI-2 Ab responses. (A) C57BL/6 (WT) mice and NOS22/2 mice were immunized i.p. with 20 mg NP-Ficoll, then bled at the indicated time points, and NP-specific IgM (upper panel) and NP-specific IgG3 (lower panel) were analyzed by ELISA. The data presented are from three independent experiments (n = 14). (B and C) Spleens from WT and NOS22/2 mice unimmunized and 4 d after NP-Ficoll immunization were analyzed for PCs by flow cytometry (B) and for NP-specific AFCs by ELISPOT (C). (B) Bar graphs summarize the frequencies of B220loCD138+ PCs from three independent experiments (n = 14). (C) Graphs show NP-specific IgM and NP-specific IgG3 spot numbers. Graphs summarize data from two independent experiments using four mice per group. (D and F) Spleens from WT and NOS22/2 mice were harvested 4 d after NP-Ficoll immunization and analyzed for B cell subset (D) and other splenic cell population (F) frequencies and total numbers by flow cytometry. The gating strategy used to identify splenic B cell subsets (D) is shown in Supplemental Fig. 1A, whereas the gating strategy to identify other splenic cell populations (F) is shown in Supplemental Fig. 2. (E) Peritoneal cavity cells from WT and NOS22/2 mice were harvested 2 d after i.p. immunization with NP-Ficoll and B cells and other cell subsets were analyzed by flow cytometry. The gating strategy used to identify peritoneal B cell subsets is shown in Supplemental Fig. 1C, 1D. Absolute numbers of total splenic B cells (TotB), T1, T2, FO, and MZ B cells are shown. (E) Peritoneal B1b and B2 B cell absolute numbers are shown. (F) Absolute numbers are shown for splenic Ly6Clo MOs, Ly6Chi MOs, Ly6Chi Mo-DCs, eosinophils (Eosph), Nphs, Mphs, CD8+ cDCs, CD82 cDCs, and pDCs. (D and F) Graphs summarize data from three independent experiments (n = 16). (E) Graphs are from one representative of two independent experiments using four mice per group. (D–F) The cell number was calculated based on the total cell number per spleen (D and F) or peritoneal lavage (E). (A–F) Bar graphs show means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, as determined by two-tailed Mann–Whitney nonparametric test. 1114 NO REGULATES BAFF AND Ab RESPONSES Ly6Chi inflammatory Mo-DCs, and Nphs. The myeloid subsets that had the highest levels of BAFF in NOS22/2 mice (Fig. 3D) also have the highest frequency of NOS2-expressing cells in WT mice (Fig. 3E). In particular, Ly6Chi inflammatory Mo-DCs that are a major source of NO (30, 31) (Fig. 3E) are also a major source of BAFF both before (Fig. 3C) and after TI-2 Ag immunization (Fig. 3D). B cell subsets including splenic mature B cells and peritoneal B1 B cells express BAFF (46). After immunization with NP-Ficoll, the number of BAFF-expressing splenic T2 and MZ B cells and peritoneal B1b were greater in NOS22/2 mice than in WT mice (Fig. 3F). However, the number of large and small peritoneal Mphs (51) expressing BAFF did not change after immunization and was not different between NOS22/2 and WT mice (data not shown). Direct regulation of BAFF expression by NO We and others have shown that BMDCs are an in vitro counterpart of Ly6Chi inflammatory DCs (8, 52). Thus, to assess whether NO directly regulates BAFF we compared BAFF production by BMDCs derived from WT and NOS22/2 mice. Compared to WT BMDCs, NOS22/2 BMDCs constitutively expressed substantial levels of BAFF mRNA (Fig. 4A) and released significant BAFF protein into the culture medium (Fig. 4B). To directly assess whether NO regulates BAFF expression, we incubated WT BMDCs for 24 h in the presence or absence of a NOS2 inhibitor (L-NAME) and incubated NOS22/2 BMDCs for 24 h in the presence or absence of a slow-release NO donor (NOR4) (Fig. 4C). The NO inhibitor upregulated BAFF expression in WT BMDCs and the NO donor downregulated BAFF expression in NOS22/2 BMDCs (almost) to the level of WT BMDCs (Fig. 4C). We conclude that NO directly inhibits BAFF expression. We examined intracellular BAFF expression in WT and NOS22/2 BMDCs using flow cytometry; the BAFF MFI and the percentage of BAFF+ cells were elevated in NOS22/2 BMDCs compared with WT BMDCs (Fig. 4D). Both the mRNA expression data (Fig. 4A, 4C) and flow cytometry data of BAFF protein expression (Fig. 4D) Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 2. NOS22/2 mice have enhanced TD Ab responses after a second challenge with NP-CGG. (A) WT mice and NOS22/2 mice immunized i.p. with 50 mg NP-CGG/alum and rechallenged i.p. with 20 mg soluble NP-CGG were bled at the indicated time points and NP-specific IgM (upper panel) and NP-specific IgG1 (lower panel) were analyzed by ELISA. Data presented are from three independent experiments (n = 12). (B and C) Spleens from WT and NOS22/2 mice immunized with NP-CGG/alum and then boosted with NP-CGG were analyzed at day 7 for PCs using flow cytometry (B) and for NPspecific AFCs by ELISPOT (C). (B) Bar graph summarizes the frequencies of B220loCD138+ PCs from two independent experiments (n = 10). (C) Graphs represent NP-specific IgM and NP-specific IgG1 spot numbers. Data are representative of two independent experiments using five mice per group. (D and E) Spleens from WT and NOS22/2 mice were harvested 7 days after NP-CGG/alum immunization and analyzed for splenic B cell subset and myeloid cell subset frequencies and total numbers by flow cytometry (gating strategies shown in Supplemental Figs. 1A, 2). (D) Data reported are absolute numbers of total B cells (TotB), T1, T2, FO, and MZ B cells. (E) Bar graphs report absolute numbers of Ly6Clo MOs, Ly6Chi MOs, Ly6Chi Mo-DCs, Eosphs, Nphs, Mphs, CD8+ cDCs, CD82 cDCs, and pDCs. (D and E) Graphs summarize data from two independent experiments (n = 10). (D and E) The cell number was calculated based on the total cell number per spleen. (A–E) Bar graphs show means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, as determined by twotailed Mann–Whitney nonparametric test. The Journal of Immunology 1115 indicate that BAFF is upregulated on a per cell basis and is not simply due to an increase in BMDC survival. Thus, BMDCs lacking NO production have both more Ly6Chi inflammatory-like Mo-DCs and increased expression of BAFF (Fig. 4D). After immunization of WT mice with TI-2 Ags, Ly6Chi inflammatory Mo-DCs that normally produce NO may be responsible for the NO-dependent inhibition of autocrine or paracrine BAFF and control of Ab responses. In NOS22/2 mice Ly6Chi inflammatory Mo-DCs lacking NO after TI-2 immunization may produce more BAFF that helps to drive PC differentiation and Ab production. Inflammatory MOs/DCs are not required for initiating TI-2 Ab responses but regulate Ag-specific Ab production We next assessed whether circulating Ly6Chi inflammatory MoDCs were indeed the cells producing NO that is responsible for the enhanced TI-2 Ab responses in NOS22/2 mice. To test this, we used CCR2 depleter mice to conditionally deplete CCR2+ Ly6Chi Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 3. BAFF is upregulated in NOS22/2 mice. (A) WT mice and NOS22/2 mice were immunized i.p. with 20 mg NP-Ficoll, bled at the indicated time points, and BAFF titers in the serums were analyzed by ELISA. Representative data are from one of three independent experiments using four mice per group in each experiment. (B) Spleens from WT and NOS22/2 mice were harvested 4 d after NP-Ficoll immunization, and intracellular BAFF levels in splenocytes were analyzed by flow cytometry. Total number of BAFFexpressing cells was calculated based on the total cell number per spleen. Graph summarizes data from three independent experiments using four mice per group. (C) Splenic cell populations from WT mice were analyzed for their intracellular BAFF expression using multicolor flow cytometry. The bar graph shows BAFF MFI for each splenic cell population. (C) Bar graph summarizes data from three independent experiments using four mice per group. (D and F, left panel), Spleens from WT and NOS22/2 mice were harvested 4 d after NP-Ficoll immunization and analyzed for the frequencies and total numbers of splenic cell populations expressing intracellular BAFF by flow cytometry. (D) Data show absolute numbers of BAFF+ Ly6Clo MOs, Ly6Chi MOs, Ly6Chi Mo-DCs, eosinophils (Eosph), Nphs, Mphs, CD8+ cDCs, CD82 cDCs, and pDCs. (F, left panel) Data show absolute numbers of BAFF+ splenic B cells (TotB) and T1, T2, FO, and MZ B cells. (D and F, left panel) Graphs summarize data from three independent experiments (n = 16). (E) Splenic cell populations from WT mice before and 4 d after NP-Ficoll immunization were analyzed for their intracellular NOS2 expression using multicolor flow cytometry. The bar graph shows the percentage of NOS2+ cells in each splenic cell population. Bar graph summarizes data from two independent experiments using four mice per group. (F, right panel) Peritoneal cells (PEC) were harvested 2 d after NP-Ficoll immunization and analyzed for the frequencies and total numbers of PECs expressing intracellular BAFF by flow cytometry. (F, right panel) Data show absolute numbers of BAFF+ PEC B1a, B1b, and B2 cells. (F, right panel) Bar graph data are from one representative of two independent experiments using four mice per group. (A–F) Bar graphs show means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, as determined by twotailed Mann–Whitney nonparametric test. inflammatory MOs and their derivative DC subsets (37, 53) prior to immunizing mice with NP-Ficoll. Previous studies suggested that a circulating DC subset is essential for delivering Ag to B cells and supporting their differentiation into PCs by, for example, producing BAFF (16, 27). Alternatively, circulating Ly6Chi Mo-DCs are a major source of NO, which our data suggest would function to inhibit TI-2 Ab responses. Thus, if inflammatory MoDCs are required to initiate TI-2 Ab responses, then Ab responses to NP-Ficoll should be reduced in mice lacking inflammatory Mo-DCs. However, if inflammatory Mo-DCs are not required for initiating TI-2 Ab responses but rather for regulating TI-2 responses through NO production, then depletion of NO-producing inflammatory Mo-DCs should result in enhanced TI-2 Ab responses. Before immunizing the CCR2 depleter mice, we first verified that 24 h after treating them with DT that Ly6Chi inflammatory monocytes were selectively depleted (37); as expected, Ly6Chi 1116 MOs and Mo-DCs were profoundly reduced 1 d after DT treatment of the CCR2 depleter mice but returned to normal levels by day +4 (Supplemental Fig. 3A, 3B, and data not shown) (37). To ensure longer depletion of MO-derived populations after immunization with NP-Ficoll, we injected DT at day 21 prior to immunization and day +1 after immunization, as previously described (37). Depleting Ly6Chi inflammatory MOs and Mo-DCs in CCR2 depleter mice significantly enhanced TI-2 Ab responses compared with WT control mice treated with DT (Fig. 5A). NP-specific IgM production by DT-treated CCR2 depleter mice was significantly higher than in DT-treated control mice, already evident earlier during the immune response (Fig. 5A, upper panel). In particular, serum NP-specific IgG3 levels of DT-treated CCR2 depleter mice were substantially higher than in DT-treated controls (Fig. 5A, lower panel). Consistent with this finding, both the percentages of PCs (Fig. 5B) and numbers of NP-specific IgM and NP-specific IgG3 AFCs (Fig. 5C) were increased in the spleens of DT-treated CCR2-DTR mice 4 d after NP-Ficoll immunization compared with their nontransgenic controls. We conclude that ablation of NO-producing inflammatory MOs and Mo-DCs is sufficient to remove inhibition of TI-2 Ab responses in WT mice, that is, sufficient to convert mice to a phenotype seen in NOS22/2 mice. These results suggest that the enhancement in NP-specific Ab responses in NOS22/2 mice may be mainly due to the lack of NO production by inflammatory Mo-DCs. Although inflammatory Mo-DCs express high levels of BAFF and their BAFF expression is regulated by NO in vitro, the absence of inflammatory Mo-DCs did not impair the Ab response to NP-Ficoll but rather enhanced it. Thus, this result left unresolved what the cell source of BAFF is that drives elevated TI-2 Ab responses in mice missing NOS2 or inflammatory Mo-DCs. One possibility is that NO produced by inflammatory Mo-DCs normally regulates BAFF in other cells that in turn help initiate or sustain TI-2 Ab responses. Consistent with this hypothesis, the absence of NO-producing inflammatory Mo-DCs in DT-treated CCR2-DTR mice led some other splenic cell subsets such as Ly6Clo MOs, Nphs, and cDCs to express higher levels of BAFF compared with those from nontransgenic controls (Fig. 5D, 5E). Furthermore, we found elevated levels of BAFF in the serum of DT-treated CCR2-DTR mice versus their littermate controls after NP-Ficoll immunizations (data not shown). We conclude that inflammatory Mo-DCs are not involved in the initiation of TI-2 Ab responses but rather can play an important role in regulating Ab production, most likely by producing NO that regulates BAFF production by other cells. NO made by either hematopoietic or nonhematopoietic cells contributes to inhibition of TI-2 Ab responses Gorelik et al. (54) showed that BAFF produced by radiationresistant cells is required for mature B cell survival/maturation and homeostasis. The same study also showed that either stromal cell– or hematopoietic cell–derived BAFF is sufficient for B cell Ab responses to TD Ags. The relative contribution of stromal cell–derived versus hematopoietic cell–derived BAFF in TI-2 Ab responses is unclear. Lymph node stromal cells express NOS2 and help control T cell responses in specific niches (55, 56). To test whether NO responsible for the regulation of TI-2 Ab responses was produced by hematopoietic cells and/or by a nonhematopoietic source, we generated reciprocal BM chimeras using WT B6 and NOS22/2 mice. We injected BM from B6 or NOS22/2 mice to sublethally irradiated B6 and NOS22/2 recipients. Six weeks later we immunized the BM chimeric mice with NP-Ficoll and monitored Ab responses. The NOS22/2 → NOS22/2 chimeras, as expected, had significantly enhanced NP-specific IgM and IgG3 production compared with B6 → B6 chimeric mice (Fig. 6A). NP-specific IgM serum levels from NOS22/2 → B6 chimeric mice were similar to B6 → B6 chimeric mice but were significantly lower than NP-specific IgM levels in NOS22/2 → NOS22/2 chimeras, suggesting that lack of NOS2 in the hematopoietic compartment did not affect NP-specific IgM responses (Fig. 6A, upper panel). Interestingly, at days 7 and 14 after NP-Ficoll immunization, chimeric mice lacking NOS2 in nonhematopoietic cells (B6 → NOS22/2) showed elevated serum levels of NP-specific IgM similar to those found in NOS22/2 → NOS22/2 chimeras and higher than those from B6 → B6 chimeras (Fig. 6A, upper panel). Thus, lack of NO production by radiationresistant nonhematopoietic cells was sufficient to enhance NPspecific IgM serum levels in the initial phase of the TI-2 response. However, at later time points, NP-specific IgM production Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 4. NO inhibits BAFF production in BMDCs. (A and C) Quantitative PCR analysis of expression level of BAFF mRNA in WT and NOS22/2 BMDCs cultured for 24 h in medium only (A and C) or (C) in the presence of a NOS inhibitor L-NAME (WT) or the NO donor NOR4 (NOS22/2). (A and C) Fold increase in BAFF mRNA relative expression (arbitrary units relative to GAPDH) was calculated comparing all the samples to the expression level in WT BMDCs. Graphs summarize data from six (A) and three (C) independent BMDC experiments. (B) BAFF released in the medium of BMDC cultures from WT and NOS22/2 mice was analyzed by ELISA. Graphs summarize data from four independent BMDC culture experiments. (A–C) Bar graphs show means 6 SEM; *p , 0.05, **p , 0.01, ***p , 0.001, as determined by two-tailed paired Student t test. (D) Intracellular concentrations of BAFF (blue) and NOS2 (red) in WT and NOS22/2 BMDCs were detected by flow cytometry. All panels show relative expression of CD11c versus Ly6C. Left panels show total cells; numbers indicate percentage of cells in each gate expressing CD11c versus Ly6C. Middle panels show BAFF+ cells (blue dots) overlaid on total cells; numbers indicate percentage of BAFF+ cells versus total cells in each gate expressing CD11c versus Ly6C. Right panels show BAFF+ cells (blue dots) and NOS2+ cells (red dots) overlaid on total cells; numbers indicating percentage of NOS2+ cells versus total cells in each gate expressing CD11c versus Ly6C are shown in red. Data shown are from one representative experiment out of six independent experiments. NO REGULATES BAFF AND Ab RESPONSES The Journal of Immunology 1117 by B6 → NOS22/2 chimeric mice, although enhanced, was not as high as NOS22/2 → NOS22/2 chimeras (Fig. 6A, upper panel). In contrast to anti-NP IgM responses, NP-specific IgG3 serum levels in NOS22/2 → B6 chimeric mice (lacking NOS2 in the hematopoietic compartment) were significantly enhanced compared with B6 → B6 chimeric mice, but were not as high as IgG3 Ab levels in NOS22/2 → NOS22/2 chimeras, especially at earlier time points (Fig. 6A, lower panel). NP-specific IgG3 production by B6 → NOS22/2 chimeric mice were similar to NOS22/2 → B6 chimeric mice. These data suggest that NO released only by hematopoietic cells is sufficient to control NP-specific IgG3 responses, but not to the levels observed in WT B6 mice. Furthermore, although hematopoietic cells may play a role in the NO-dependent control of NP-specific IgG3 production, a nonhematopoietic source of NO apparently also can contribute. Taken together, these data suggest that the lack of NOS2 in both hematopoietic and nonhematopoietic cells contributes to the elevated TI-2 Ag-specific Ab responses in NOS22/2 mice. We also analyzed BAFF levels in B6 and NOS22/2 chimeric mice after immunization with NP-Ficoll. The lack of NOS2 by hematopoietic cells (NOS22/2 → B6) was sufficient to enhance serum BAFF levels to the levels of NOS22/2 → NOS22/2 chimeric mice, especially at earlier time points (Fig. 6B). In contrast, both B6 → B6 and chimeric mice lacking NOS2 in the nonhematopoietic compartment (B6 → NOS22/2) had somewhat lower BAFF levels in their sera (Fig. 6B). These data suggest that NOS2 expression by hematopoietic cells, most likely the NOS2hi Ly6Chi inflammatory Mo-DCs (Fig. 3E), is responsible for the lower serum levels of BAFF in WT mice immunized with NPFicoll compared with immunized NOS22/2 mice (Fig. 3A). Discussion In this study, we show that NOS2-derived NO regulates TI-2 Ab responses, with a major contribution by the NO-producing inflammatory MOs and Mo-DCs. Previous studies have shown that NOS22/2 mice have either enhanced specific Ab responses to viral infection or reduced TD and TI IgA production by mucosal lymphoid tissues (9, 10). The differences in requirement for NO for IgA and inhibition of IgG could be due to factors including the type and source of Ag and/or the localization and type of cells producing NO. Similar to a study showing that the absence of NOS2 enhances virus-specific IgG2a Abs (9), we found that NOS2 deficiency enhances TI-2 Ag-specific IgM and IgG3 Ab responses. NP-Ficoll–immunized NOS22/2 mice had enhanced levels of PCs and AFCs compared with WT controls, consistent with the increase in plasmablasts observed in vitro in NOS22/2 spleen cells stimulated with B cell mitogens (9). Interestingly, MZ B cells and peritoneal B1 B cells, the major B cell subsets that respond to TI-2 Ags (42, 57), were both increased in NP-Ficoll– immunized NOS22/2 mice. The peritoneal B1b B cell subset enhanced in immunized NOS22/2 mice contributes to the generation of adaptive Ab responses to TI-2 Ags, whereas B1a B cells contribute mainly to innate immune responses by producing natural Abs (58, 59). Our data suggest that NO restricts MZ B cells, Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 5. Depletion of inflammatory MOs/MoDCs enhances TI-2 Ab responses. (A) WT B6 mice and B6.CCR2-DTR mice injected i.p. with DT at day 21 and day 1 after NP-Ficoll immunization were bled at the indicated time points, and NP-specific IgM (upper panel) and NP-specific IgG3 (lower panel) were analyzed by ELISA. Representative data are from two independent experiments using four mice per group (n = 8). (B and C) Spleens from B6 and CCR2-DTR mice injected with DT only (as in (A)) and immunized or not with NP-Ficoll were harvested 4 d after immunization and analyzed for PCs by flow cytometry (B) and for NPspecific AFCs by ELISPOT (C). (B) Graphs summarize the frequencies of B220loCD138+ PCs from two independent experiments using four mice per group. (C) Graphs show NP-specific IgM and NP-specific IgG3 spot numbers and summarize data from two independent experiments using four mice per group. (D and E) Spleens from DT-treated (as in (A)) WT and CCR2DTR mice were harvested 4 d after NP-Ficoll immunization and analyzed for intracellular BAFF expression in different splenic cell populations by flow cytometry (gating strategy shown in Supplemental Fig. 2). (D) Data show BAFF expression in Ly6Clo MOs and are from one representative experiment out of two using 4 mice per group. (E) Data show percentages of BAFF+ cells in each subset, including Ly6Clo MOs, Nphs, Mphs, CD8+ cDCs, CD82 cDCs and pDCs. (E) Graphs summarize data from two independent experiments using four mice per group. In (A)–(C) and (E), bar graphs show means 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001, as determined by two-tailed Mann– Whitney nonparametric test. 1118 B1 B cells, and humoral immune responses generated in the spleen and peritoneal cavity (11, 42). The NO regulation of splenic and peritoneal-associated TI-2 Ab responses is different from that reported by Tezuka et al. (10), who found that in MALTs NOS2 is required for both TD and TI IgA production. BAFF plays a major role in TI-2 Ag-driven B cell responses (14– 16). Our findings suggest a possible mechanism through which NOS2-derived NO restricts TI-2 Ab responses; that is, NO may limit the expansion of certain BAFF-producing cell populations and may directly inhibit BAFF expression by cells. Consistent with this model, serum levels of BAFF were elevated in NOS22/2 mice and in NOS22/2 → B6 chimeric mice, suggesting that the principal cell population responsible for BAFF production and regulation by NO may be of hematopoietic origin. Furthermore, NO has a direct inhibitory effect on BAFF mRNA and protein expression in BMDC cultures. NOS22/2 BMDCs constitutively express higher levels of BAFF compared with WT BMDCs; additionally, a NOS2 inhibitor increased BAFF expression in WT BMDCs, whereas an NO donor inhibited BAFF expression in NOS22/2 BMDC cultures (Fig. 4). The facts that dysregulation of BAFF can lead to lupus-like autoimmune disease (19) and that many patients with autoimmune diseases have elevated serum levels of BAFF (13, 24, 52) underscore the importance of regulating BAFF expression in TI Ab responses (14). Thus, our finding that NO can be a negative regulator of BAFF and TI Ab responses may help in developing strategies to control harmful Ab responses. Furthermore, inhibiting NOS2 in specific cell types may help enhance and sustain protective humoral immune responses. Further studies are needed to assess whether NOS2 inhibitors are useful as adjuvants and in vaccine development. Some of the BAFF-expressing cell populations expanded in NOS22/2 mice after NP-Ficoll injection, such as DCs and Nphs, have been shown to play a role in TI-2 Ab responses via production of BAFF (12, 23, 60, 61). Nphs induce CSR, somatic hypermutation, and Ab production by activating MZ B cells through a mechanism that involved BAFF, APRIL, and IL-21 (62). Garcia de Vinuesa et al. (26, 27) showed that a subset of myeloid DCs is associated with PC survival and growth triggered by NP-Ficoll in extrafollicular foci. BAFF produced by MOs and myeloid DCs induces CSR in a CD40-independent manner and signals MZ B cells to initiate TI-2 Ab responses (25). Balázs et al. (16) showed that a blood-derived CD11cloCD11bhi DC subset was responsible for transporting Ag to the MZ and supported PC survival through BAFF. They also found that similar to bloodderived DCs, Nphs and Mphs pick up Ag and accumulate in the spleen, but in contrast to blood-derived DCs they are not responsible for the induction of the TI-2 Ab response (16). Thus, it remains unclear which BAFF-expressing cell types contribute to the induction of TI-2 Ab responses. Interestingly, BAFF produced by MOs and Mo-DCs is essential for secondary Ab responses when T cell help is limited (28). MacLennan and Vinuesa (15) proposed that both DC subsets described in the earlier studies implicating DCs in TI-2 immune responses may in fact correspond to monocyte-derived DCs, more recently defined as Ly6Chi inflammatory Mo-DCs (29–33). Interestingly, BAFF+ inflammatory MOs and Mo-DCs were expanded in NOS22/2 mice. In addition to BAFF expression, inflammatory Mo-DCs are also specialized NO producers. Using the CCR2 depleter mouse model we found that inflammatory MoDCs are not required to initiate TI-2 Ab responses, but rather they negatively regulate Ab responses to TI-2 Ags. In agreement with this result, experiments with WT and NOS22/2 reciprocal BM chimeras confirmed that a cell type of hematopoietic origin, likely Ly6Chi inflammatory Mo-DCs, may play a major role in controlling IgG3 responses to NP-Ficoll. Thus, our findings suggests that inflammatory Mo-DCs may be an important source of NO that restricts TI-2 immune responses, most likely by regulating BAFF production by other cells, such as cDCs, Nphs, or Mphs. Further studies are in progress to determine the major source of BAFF implicated in the initiation of TI-2 Ab responses. Our results with CCR2-DTR mice differ from a recent study where inflammatory MOs were depleted using CD11b-DTR mice (63). Chen and Snapper (63) concluded that inflammatory MOs are critical for the induction of a polysaccharide-specific Ab response to an intact bacterium. This apparent difference with our results could be due to the fact that a different kind of Ag was used, or more likely that a different depletion system was used resulting in different subsets being depleted. In Chen and Snapper Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 6. NOS2 expressed by either hematopoietic cells or nonhematopoietic cells regulates BAFF and TI-2 Ab responses. (A and B) BM chimeras were prepared by inoculating sublethally irradiated WT B6 and NOS22/2 mice with either WT B6 or NOS22/2 BM. BM chimeric mice were immunized with NP-Ficoll 6–8 wk after irradiation and BM reconstitution. Shown are NP-specific IgM (A, upper panel) and NP-specific IgG3 (A, lower panel) titers and BAFF levels (B) from sera collected at the indicated time points after immunization and detected by ELISA. (A) Graphs summarize data from two independent experiments using five mice per group. (B) Bar graph data are from one representative of two independent experiments using five mice per group. (A and B) Bar graphs show means 6 SEM; *p , 0.05, **p , 0.01, ***p , 0.001, as determined by Kruskal–Wallis nonparametric test, followed by a Dunnett post hoc test. (A and B) In each graph, gray statistics bars show the p value significance of the overall multiple comparisons test, whereas black statistics bars show the p value significance of the post hoc test. NO REGULATES BAFF AND Ab RESPONSES The Journal of Immunology Although BAFF apparently plays an important role, other factors may contribute to the increased TI-2 Ab responses in NOS22/2 mice and CCR2 depleter mice. Nevertheless, our finding that NO regulates BAFF and TI-2 Ab responses may help in the development of therapies to control exaggerated Ab responses in patients with autoimmune diseases or allergies. Future studies are required to define the BAFF-producing cell types activated during TI-2 Ab responses and just how, when, and where NO regulates humoral immune responses. Acknowledgments We thank Natalia Giltiay, Craig Chappell, Marianne Bryan, and Christiane Dresch for helpful comments. Disclosures The authors have no financial conflicts of interest. References 1. Niedbala, W., B. Cai, and F. Y. Liew. 2006. Role of nitric oxide in the regulation of T cell functions. Ann. Rheum. Dis. 65(Suppl. 3): iii37–iii40. 2. M€uhl, H., M. Bachmann, and J. Pfeilschifter. 2011. Inducible NO synthase and antibacterial host defence in times of Th17/Th22/T22 immunity. Cell. Microbiol. 13: 340–348. 3. Kolb, H., and V. Kolb-Bachofen. 1998. Nitric oxide in autoimmune disease: cytotoxic or regulatory mediator? Immunol. Today 19: 556–561. 4. Bogdan, C. 2001. Nitric oxide and the immune response. Nat. Immunol. 2: 907– 916. 5. Shi, F. D., M. Flodström, S. H. Kim, S. Pakala, M. Cleary, H. G. 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Craxton, A., D. Magaletti, E. J. Ryan, and E. A. Clark. 2003. Macrophage- and dendritic cell–dependent regulation of human B-cell proliferation requires the TNF family ligand BAFF. Blood 101: 4464–4471. 22. Mackay, F., W. A. Figgett, D. Saulep, M. Lepage, and M. L. Hibbs. 2010. B-cell stage and context-dependent requirements for survival signals from BAFF and the B-cell receptor. Immunol. Rev. 237: 205–225. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 (63) the use of intact Streptococcus pneumoniae induced both TI IgM and TD IgG responses, whereas in this study we used NPFicoll that invokes only TI-2 Ab responses. Lymph node stromal cells express NOS2 and help control T cell responses in specific stromal niches (55, 56). It is not known whether NOS2 expression by stromal cells is induced by TI-2 Ags and affects B cell responses. By generating reciprocal BM chimeras between WT and NOS22/2 mice, we found that both hematopoietic and nonhematopoietic sources of NO contribute to keeping Ab responses to TI-2 Ags under control. In line with this model, in an experimental model of autoimmune myocarditis, using WT and NOS22/2 chimeric mice, Kania et al. (64) found that NO produced by both hematopoietic and stromal cells is required to negatively regulate T cell responses and protect against the autoimmunity. Interestingly, in this study Ag-activated inflammatory Tip-DCs promoted NO production by stromal fibroblasts in an inflammatory environment. The tight interplay between inflammatory Mo-DCs and stromal cells was also evident in a study showing that stromal NOS2 attenuates T cell responses induced by Ag-presenting inflammatory DCs, but not when the Ag is targeted to other resident DC populations (56). BAFF produced by radiation-resistant stromal cells is required for mature B cell survival/maturation and homeostasis (54). This study also showed that either stromal cell– or hematopoietic cell– derived BAFF is sufficient for B cell Ab responses to TD Ags. Our results suggest that NO produced by stromal cells contributes to TI-2 Ab responses. Furthermore, hematopoietic cell–derived BAFF regulated by NO is likely to play a significant role in TI-2 Ab responses as well. Further studies are needed to determine more clearly the cell source of BAFF regulated by NO. Based on our results using both NOS22/2 mice and CCR2 depleter mice, possible targets for the NO produced by inflammatory Mo-DCs include stromal cells, Nphs, Mphs, cDCs, and Ly6Clo MOs. Randolph et al. (65) proposed that the cell type described by Balázs et al. (16) may correspond to Ly6Clo MOs, which drive TI Ab responses by acquiring particulate Ags in the blood, crawling across blood vessels, presenting them to B cells in the bridging channels of the spleen, and at the same time providing BAFF to B cells. Our results are consistent with this model. In both CCR2 depleter mice and NOS22/2 mice after NP-Ficoll immunization Ly6Clo MOs upregulate BAFF, implying they are a target for NO regulation. However, Ly6Clo MOs are dramatically decreased after DTRinduced depletion of CCR2-expressing cells, whereas Mph levels are unaffected and Nphs increase in number. Furthermore, in DT-treated CCR2 depleter mice, Mphs and Nphs are more numerous and express more BAFF than do Ly6Clo MOs. Thus, it is more likely that Ly6Clo MOs are not the predominant cell type involved in the regulation of TI-2 Ab via BAFF. However, we cannot exclude that this subset might play a role in a specific microenvironment where Ag presentation to B cells takes place and BAFF sustains B cell survival and Ab production. NOS2 deficiency (Fig. 1A) and eliminating cells in CCR2 depleter mice (Fig. 5A) had a more significant and more sustained effect on TI-2 Ag-induced IgG3 Ab responses than IgM responses. Why this is the case requires further investigation. One possible reason for this difference is that the half-life of IgG3 Abs is longer than that of IgM Abs (66). Another possibility is that B1b cells, which can sustain IgG3 Ab production for months after TI-2 Ag stimulation (67) and are elevated in immunized NOS22/2 mice, sustain IgG3 production to a greater extent in NOS22/2 mice and in the absence of regulatory myeloid cells. Alternatively, factors implicated in the regulation of class switching to IgG3, such as IFN-g and IL-10 (68, 69), may be dysregulated in NOS22/2 mice. 1119 1120 47. Waskow, C., K. Liu, G. 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