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Essential Role of Canonical NF-Κb Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs

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Essential Role of Canonical NF-Κb Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs Essential Role of Canonical NF-κB Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs This information is current as Dana Bogdanova, Arata Takeuchi, Madoka Ozawa, of September 24, 2021. Yasuhiro Kanda, M. Azizur Rahman, Burkhard Ludewig, Tatsuo Kinashi and Tomoya Katakai J Immunol published online 5 November 2018 http://www.jimmunol.org/content/early/2018/11/02/jimmun ol.1800539 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/11/03/jimmunol.180053 Material 9.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 by guest on September 24, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 5, 2018, doi:10.4049/jimmunol.1800539 The Journal of Immunology Essential Role of Canonical NF-kB Activity in the Development of Stromal Cell Subsets in Secondary Lymphoid Organs Dana Bogdanova,* Arata Takeuchi,* Madoka Ozawa,* Yasuhiro Kanda,* M. Azizur Rahman,* Burkhard Ludewig,† Tatsuo Kinashi,‡ and Tomoya Katakai* Organized tissue structure in the secondary lymphoid organs (SLOs) tightly depends on the development of fibroblastic stromal cells (FSCs) of mesenchymal origin; however, the mechanisms of this relationship are poorly understood. In this study, we specifically inactivated the canonical NF-kB pathway in FSCs in vivo by conditionally inducing IkBa mutant in a Ccl19-IkBSR mouse system in which NF-kB activity is likely to be suppressed in fetal FSC progenitors. Given that NF-kB activation in fetal FSCs is essential for SLO development, the animals were expected to lack SLOs. However, all SLOs were preserved in Ccl19-IkBSR mice. Instead, Downloaded from the T cell area was severely disturbed by the lack of CCL21-expressing FSCs, whereas the follicles and associated FSC networks were formed. Fate mapping revealed that IkBSR-expressing cells constituted only a small fraction of stromal compartment outside the follicles. Taken together, our findings indicate an essential role of the canonical NF-kB pathway activity in the development of three FSC subsets common to SLOs and suggest transient or stochastic CCL19 expression in FSC progenitors and a compensatory differentiation program of follicular FSCs. The Journal of Immunology, 2018, 201: 000–000. http://www.jimmunol.org/ econdary lymphoid organs (SLOs) develop through com- for the expression of chemokines such as CXCL13, CCL19, and plex processes of intimate interactions between lymphoid CCL21, which recruit lymphoid cells to SLOs (11–16). However, S cells and nonhematopoietic stromal cells (1, 2). The in- in a strict sense, it has not been proven directly that NF-kB terplay of lymphoid tissue inducer cells, an innate lymphoid cell activity in LTo cells or mesenchymal progenitors itself is es- population, with mesenchymal lymphoid tissue organizer (LTo) sential for the development and tissue organization of SLOs cells in fetal anlagen is necessary for the formation of lymph in vivo. Recent studies have suggested the presence of other nodes (LNs) and Peyer’s patches (PPs) (3–6), whereas the orga- complicated cellular interactions involving endothelial com- nization of white pulps in the spleen involves postnatal collabo- partments in LN formation and distinct organ-specific develop- ration of mature lymphocytes and perivascular stromal cells (7, 8). ment programs (8, 17). by guest on September 24, 2021 In these cellular processes, lymphotoxin (LT) a1b2 and LTbR, In adult SLOs, B and T cells are clearly segregated in the follicles which are displayed on lymphoid cells and mesenchymal cells, re- and T cell area. Different types of fibroblastic stromal cells (FSCs) spectively, and the activation of NF-kB transcription factors via the that form intricate networks support each of the regions by pro- canonical (RelA/p50 complex) and noncanonical (RelB/p52 com- viding a structural backbone (18). Follicular dendritic cells (FDCs) plex)pathwaysarebelievedtoplaythekeyroles;genedefi- and marginal reticular cells (MRCs) in the center and outer part of ciencies of these signaling mediators lead to the absence or the follicle, respectively, produce CXCL13 that regulates B cell malformation of SLOs (1, 9–12). In particular, NF-kB is required localization and motility (19–21). MRCs, which display charac- teristics similar to those of LTo cells, such as the expression of RANKL and MAdCAM-1, have been shown to be FDC precursors *Department of Immunology, Niigata University Graduate School of Medical and (21–23). In T cell area, T zone reticular cells (TRCs), also known Dental Sciences, Niigata 951-8510, Japan; †Institute of Immunobiology, CH-9007 St. Gallen, Switzerland; and ‡Department of Molecular Genetics, Institute of Biomedical as fibroblastic reticular cells, produce CCL19 and CCL21, af- Science, Kansai Medical University, Hirakata, Osaka 573-1010, Japan fecting the localization and migration of T cells and dendritic cells ORCIDs: 0000-0003-0412-533X (A.T.); 0000-0001-5026-2306 (M.O.); 0000-0001- (24–27). These three stromal cell subsets are common to all types 8131-8130 (Y.K.); 0000-0002-7685-573X (B.L.); 0000-0002-4290-7028 (T. Katakai). of SLOs and are considered to originate from a common mesen- Received for publication April 16, 2018. Accepted for publication October 11, 2018. chymal progenitor (17, 28, 29). This work was supported by Grant-in-Aid for Scientific Research on Innovative The factors required for the development of FDCs are well Areas 24111005 and Grant-in-Aid for Scientific Research Scientific Research (B) studied, as this stromal cell type is absent in mice engineered to lack 16H05204 from the Ministry of Education, Culture, Sports, Science and Technology– Japan Society for the Promotion of Science (both to T. Katakai). some immune system–related genes (10, 30). In particular, signals b k Address correspondence and reprint requests to Prof. Tomoya Katakai, Department through LT R and TNFR together with subsequent NF- B acti- of Immunology, Graduate School of Medical and Dental Sciences, Niigata Univer- vation play critical roles in the differentiation and maintenance of sity, 1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan. E-mail address: FDC network in the follicles (16, 31, 32). In contrast, the devel- [email protected] opmental processes of MRCs and TRCs remain poorly under- The online version of this article contains supplemental material. stood. Moreover, the mechanisms that regulate the generation of Abbreviations used in this article: Ccl19-EYFP, Ccl19-Cre/R26-stopflox-EYFP; FDC, follicular dendritic cell; FSC, fibroblastic stromal cell; HEV, high endothelial venule; FSC subsets may possibly differ between SLO types, although it LN, lymph node; LT, lymphotoxin; LTo, lymphoid tissue organizer; MRC, marginal could not be verified in animals that completely lack LNs and PPs reticular cell; PP, Peyer’s patch; SLO, secondary lymphoid organ; TRC, T zone (e.g., in the cases of deficiencies in NF-kB components). reticular cell. Ccl19-Cre BAC transgenic mouse strain is a useful tool for Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 conditional gene targeting and fate mapping in FSC lineages www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800539 2 NF-kB CONTROLS THE DEVELOPMENT OF STROMAL SUBSETS during SLO development because LTo cells in fetal anlagen or at 280˚C. Frozen sections (10 mm) were stained with Abs and mounted postnatal progenitors express Cre recombinase driven by the with Permafluor (Thermo Fisher Scientific). The specimens were examined Ccl19 gene (8, 33). A mutant form of IkBa,IkBSR, is resistant to using an LSM710 (Carl Zeiss) or an FV1200 (Olympus) confocal micro- scope. Digital images were prepared using Adobe Photoshop CS6 soft- the degradation upon signaling; it strongly inhibits the activation ware. of the canonical NF-kB pathway (34), which in turn suppresses For whole-mount immunohistochemistry, excised organs were fixed with the expression of noncanonical pathway components. Thus, 1% paraformaldehyde in PBS overnight at 4˚C, and endogenous peroxidase IkBSR potentially inhibits both pathways (15). In this study, to activity was blocked with 0.3% H2O2 and 40% methanol in PBS overnight at 4˚C. The specimens were permeabilized with 0.3% Triton X-100 in PBS assess the consequences of in vivo inactivation of the canonical at 4˚C for 1.5 h followed by blocking with 1% BSA in PBST (0.05% NF-kB pathway specific to FSCs in SLOs, we generated a mouse Tween-20 in PBS) at 4˚C for 2 h. The specimens were then incubated with in which both IkBSR and EGFP are conditionally induced under 2 mg/ml biotin–anti-B220 Ab (eBioscience) in 1% BSA in PBST overnight the control of the Ccl19-Cre locus. Characteristic structural al- at 4˚C. Biotinylated Ab was detected using Vectastain ABC Kit and DAB terations of SLOs in these animals prompted us to address the Substrate Kit (Vector). cause of these abnormalities. Fate mapping using bicistronic Quantitative RT-PCR analysis EGFP expression permitted the identification of IkBSR-expressing Total RNA was extracted from SLOs using an RNeasy Mini kit (QIAGEN) cells in situ, which allowed us to elucidate the indispensable or TRIzol reagent (Invitrogen), and cDNA was synthesized using the Su- role of the canonical NF-kB pathway activity in the development perscript III First-Strand Synthesis System (Invitrogen).
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