LSD1 Cooperates with Noncanonical NF-κB Signaling to Regulate Marginal Zone B Cell Development This information is current as Robert R. Haines, Christopher D. Scharer, Jenna L. Lobby of October 2, 2021. and Jeremy M. Boss J Immunol published online 6 September 2019 http://www.jimmunol.org/content/early/2019/08/30/jimmun ol.1900654 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2019/08/30/jimmunol.190065 Material 4.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 2, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 6, 2019, doi:10.4049/jimmunol.1900654 The Journal of Immunology LSD1 Cooperates with Noncanonical NF-kB Signaling to Regulate Marginal Zone B Cell Development Robert R. Haines, Christopher D. Scharer, Jenna L. Lobby, and Jeremy M. Boss Marginal zone B cells (MZB) are a mature B cell subset that rapidly respond to blood-borne pathogens. Although the transcriptional changes that occur throughout MZB development are known, the corresponding epigenetic changes and epigenetic modifying pro- teins that facilitate these changes are poorly understood. The histone demethylase LSD1 is an epigenetic modifier that promotes plasmablast formation, but its role in B cell development has not been explored. In this study, a role for LSD1 in the development of B cell subsets was examined. B cell–conditional deletion of LSD1 in mice resulted in a decrease in MZB whereas follicular B cells and bone marrow B cell populations were minimally affected. LSD1 repressed genes in MZB that were normally upregulated in the myeloid and follicular B cell lineages. Correspondingly, LSD1 regulated chromatin accessibility at the motifs of transcription factors known to regulate splenic B cell development, including NF-kB motifs. The importance of NF-kB signaling was examined through an ex vivo MZB development assay, which showed that both LSD1-deficient and NF-kB–inhibited transitional B cells Downloaded from failed to undergo full MZB development. Gene expression and chromatin accessibility analyses of in vivo– and ex vivo–generated LSD1-deficient MZB indicated that LSD1 regulated the downstream target genes of noncanonical NF-kB signaling. Addition- ally LSD1 was found to interact with the noncanonical NF-kB transcription factor p52. Together, these data reveal that the epigenetic modulation of the noncanonical NF-kB signaling pathway by LSD1 is an essential process during the development of MZB. The Journal of Immunology, 2019, 203: 000–000. http://www.jimmunol.org/ cell progenitors develop through multiple stages to be- Specific signaling mechanisms drive the MZB or FoB cell fate come mature naive B cells capable of generating a hu- decision. When immature B cells enter the periphery, they undergo B moral immune response. In the bone marrow, common positive selection through tonic BCR signaling to promote survival lymphoid progenitors progress through the pro-B and pre-B cell (3). The strength of tonic BCR signaling influences immature stages, during which the BCR is rearranged to generate a func- B cell fate with stronger signals promoting FoB commitment tional yet diverse repertoire of B cells (1). BCR-expressing immature and weaker signals promoting MZB commitment (3). Immature B cells migrate to the spleen, where they undergo transitional B cell B cells must experience two additional signaling pathways to (TrB) development, resulting in the formation of follicular B cells further commit to the MZB fate. The first is NOTCH2 signaling by guest on October 2, 2021 (FoB) and marginal zone B cells (MZB). FoB circulate throughout through interaction with the NOTCH ligand DLL1, which is the periphery and facilitate humoral immune responses to Ag and expressed by splenic venules in the red pulp and marginal zone give rise to memory B cells and long-lived plasma cells (1). MZB (1). The second is BAFFR-dependent activation of noncanonical localize to the splenic marginal sinus and rapidly respond to blood- NF-kB signaling (1). Both pathways are necessary for MZB cell borne pathogens, primarily forming short-lived plasmablasts (1, 2). development and function in a synergistic manner (1). Throughout cell fate commitment, MZB acquire a transcrip- tional identity distinct from FoB that confers specific functional Department of Microbiology and Immunology, Emory University School of Medi- capabilities (1, 2). For example, MZB express high levels of S1pr1 cine, Atlanta, GA 30322 to facilitate homing to the marginal zone (4) and downregulate ORCIDs: 0000-0001-9574-4341 (R.R.H.); 0000-0001-7716-8504 (C.D.S.); 0000- the FoB genes Itgb7, Cxcr4, and Ccr7 that facilitate homing to 0002-1395-2583 (J.L.L.); 0000-0002-2432-1840 (J.M.B.). secondary lymphoid organs (5). Myc is highly expressed in MZB, Received for publication June 13, 2019. Accepted for publication August 5, 2019. providing an enhanced capacity to proliferate in response to Ags This work was supported by the following National Institutes of Health grants: R01 such as bacterial LPS (6). MZB can rapidly respond to other TLR AI123733 and P01 AI125180 to J.M.B., T32 GM0008490 to R.R.H., and F31 AI131532 to R.R.H. agonists (7) and display a concomitant increase in innate immune The sequences presented in this article have been submitted to the National Center sensor molecules relative to FoB, including TLR3, TLR7, TLR9, for Biotechnology Information Gene Expression Omnibus (https://www.ncbi.nlm. NOD1/2/3, and NLRC4 (8). Although the MZB transcriptome is nih.gov/geo/query/acc.cgi?acc=GSE132227) under accession number GSE132227. characterized, the epigenetic modifications acquired during B cell Address correspondence and reprint requests to Dr. Jeremy M. Boss, Emory University, development that establish it are not well studied. Additionally, 1510 Clifton Road, Room 3001, Atlanta, GA 30322. E-mail address: [email protected] the enzymes that facilitate splenic B cell epigenetic remodeling The online version of this article contains supplemental material. are not known. Abbreviations used in this article: ATAC-seq, assay for transposase-accessible fl/fl Cre/+ Lysine-specific demethylase 1 (LSD1) is a histone demethylase chromatin sequencing; CKO, Kdm1a Cd19 conditional knockout; CreWT, Kdm1a+/+ Cd19Cre/+ wild-type; DAR, differentially accessible region; DEG, dif- that targets H3K4me1, H3K4me2, H3K9me1, and H3K9me2 ferentially expressed gene; eMZB, ex vivo–derived MZB; FDR, false discovery through FAD-dependent amine oxidation (9). LSD1-based modi- rate; FoB, follicular B cell; FSC, forward scatter; GSEA, gene set enrichment analysis; IP, immunoprecipitation; LSD1, lysine-specific demethylase 1; MZB, fication of chromatin results in the fine-tuning of target gene ex- marginal zone B cell; NP-40, Nonidet P-40; PCA, principal component analysis; pression, which is critical for driving cellular development (9). qPCR, quantitative PCR; RHD, Rel homology domain; RNA-seq, RNA sequenc- Regarding B cell differentiation, LSD1 promotes plasmablast for- ing; rppm, reads per peak per million; SSC, side scatter; TrB, transitional B cell. mation and decommissions active enhancers at Blimp-1, PU.1, and Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 IRF4 binding sites through H3K4me1 demethylation and repression www.jimmunol.org/cgi/doi/10.4049/jimmunol.1900654 2 LSD1 REGULATES MARGINAL ZONE B CELL DEVELOPMENT of chromatin accessibility (10). LSD1 also promotes germinal 13 penicillin/streptomycin, 10 mM HEPES, and 1 mM sodium pyruvate) center formation by repressing plasma cell genes, such as Prdm1 at 150,000 cells/ml, then 1 ml was plated onto OP9 cells after gently aspirating and Irf4, through enhancer decommissioning facilitated by inter- off OP9 media. Ten nanograms BAFF (8876-BF-010; R&D Systems) was added to the wells. Cells were placed in a 37˚C incubator for 3 d, then un- action with BCL6 (11). Despite evidence highlighting a critical derwent analyses. Where indicated, 800 nM of the IkB kinase inhibitor IKK- role for LSD1 in the epigenetic regulation of B cell differentiation, 16 (SML1138; Sigma-Aldrich) (17) or DMSO was added to the wells. its in vivo role during B cell development has not been explored. Flow cytometry In this study, mice with B cell–conditional deletion of LSD1 6 were used to examine its function throughout B cell development. Cells were resuspended at a concentration of 10 cells/100 mlFACS 3 Phenotyping revealed that LSD1 was dispensable for the devel- buffer (1 PBS, 2 mM EDTA, 1% BSA). Cells were stained with Fc block (2.4G2; BD Biosciences) for 15 min and then followed with opment of bone marrow B cell subsets and FoB but was required specific Ab fluorophores for 1 h at 4˚C, then washed with 10 volumes for MZB formation.