ARTICLES nature publishing group See REVIEW page 455 TNFa-dependent development of lymphoid tissue in the absence of RORgt þ lymphoid tissue inducer cells GC Furtado1, ME Pacer1, G Bongers1,CBe´ne´zech2,ZHe1, L Chen1, MC Berin1, G Kollias3, JH Caaman˜o2 and SA Lira1 Lymphoid tissue often forms within sites of chronic inflammation. Here we report that expression of the proinflammatory cytokine tumor necrosis factor a (TNFa) drives development of lymphoid tissue in the intestine. Formation of this ectopic lymphoid tissue was not dependent on the presence of canonical RORgt þ lymphoid tissue–inducer (LTi) cells, because animals expressing increased levels of TNFa but lacking RORgt þ LTi cells (TNF/Rorc(gt) À / À mice) developed lymphoid tissue in inflamed areas. Unexpectedly, such animals developed several lymph nodes (LNs) that were structurally and functionally similar to those of wild-type animals. TNFa production by F4/80 þ myeloid cells present within the anlagen was important for the activation of stromal cells during the late stages of embryogenesis and for the activation of an organogenic program that allowed the development of LNs. Our results show that lymphoid tissue organogenesis can occur in the absence of LTi cells and suggest that interactions between TNFa-expressing myeloid cells and stromal cells have an important role in secondary lymphoid organ formation. INTRODUCTION (PP).4–6 The current model for the development of lymphoid Lymphoid organs are critical for generation of adaptive organs posits that LTi cells originate in the fetal liver from immune response. Secondary lymphoid organs (SLO) are common lymphoid progenitors and that they migrate to the sites formed at predefined areas during embryogenesis, whereas where the LNs are formed (lymph node anlagen).1,7 At these tertiary lymphoid organs (TLO) are formed after birth in tissues sites, binding of the tumor necrosis factor a (TNFa)familyligand with ongoing inflammatory processes.1,2 Both secondary and receptor activator of nuclear factor (NF)-kB (RANKL) to its TLO have lymphocytes that are topologically segregated and receptor RANK induces the differentiation and survival of LTi diverse sets of myeloid and stromal cells. In addition, they have cells and trigger expression of lymphotoxin a1b2(LTa1b2) on specialized vasculature such as high endothelial venules (HEV) their surface.3,8–11 A key step in the development of LNs is the and a lymphatic network. engagement of LTa1b2 expressed by LTi cells to its receptor The two major cell types involved in lymph node organogen- LTbR on mesenchymal organizer cells.12,13 This interaction esis are the hematopoietic lymphoid tissue–inducer (LTi) cells promotes upregulation of intercellular adhesion molecule and non-hematopoietic lymphoid tissue stromal ‘‘organizer (ICAM-1), vascular cell adhesion molecule (VCAM-1) and cells’’ (LTo).1 Clustering of LTi and LTo cells is an essential step mucosal addressin cell adhesion molecule (MAdCAM-1) on the in lymph node development.3 Animals that are deficient in the surface of LTo cells14,15 and the expression of the chemokines nuclear retinoid orphan receptor (ROR)g, encoded by the Rorc C-C motif chemokine ligand 19 (CCL19), CCL21, and C-X-C gene, or the negative regulator of basic helix-loop-helix protein motif chemokine ligand 13 (CXCL13).7 signaling Id2 (inhibitor of DNA-binding 2), lack LTi cells and Animals genetically deficient in LT-alpha and LTbR do not therefore fail to form lymph nodes (LNs) and Peyer’s patches form LNs or PP.10,12,16 Furthermore, genetic deletion of 1Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, New York, USA. 2IBR-MRC Centre for Immune Regulation, College of Medical and Dental Sciences, University of Birmingham, Birmingham, UK and 3Alexander Fleming Biomedical Sciences Research Center, Vari, Greece. Correspondence: GC Furtado ([email protected]) or SA Lira ([email protected]) Received 21 March 2013; revised 28 August 2013; accepted 9 September 2013; published online 16 October 2013. doi:10.1038/mi.2013.79 602 VOLUME 7 NUMBER 3 | MAY 2014 | www.nature.com/mi ARTICLES molecules in the LTbR signaling pathway (NF-kB non- RESULTS canonical pathway) such as NF-kB-inducible kinase (NIK)17 Increased expression of TNFa promotes development of and RelB18 precludes LN formation. Although the role of TLO in the absence of LTi cells LTa1b2/LTbR is firmly established in the process of lymphoid Two types of lymphoid aggregates can be identified in the organogenesis, the role of other members of the TNFa intestine of adult mice: isolated lymphoid follicles and TLO. superfamily is unclear. Isolated lymphoid follicles are genetically programmed clusters Female mice injected in utero with LTbR-immunoglobulin of B cells present at the base of the villi that require RORgt þ LTi (Ig) fusion protein retain cervical and mesenteric LN (mLNs) cells and LTbR signaling for their formation.5,24–26 TLO are but fail to form other LNs.19,20 However, simultaneous composed by large clusters of B220 þ cells that contain CD3 þ treatment of LTbR-Ig fusion protein and anti-TNFR1 antibody, lymphocytes and are formed in response to infection or or LTbR-Ig plus anti-TNFa antibodies, prevents development inflammation.27,28 To further define the role of LTi cells and of all LNs,21 which suggests that TNFa has a role in mLN TNFa in the formation of lymphoid aggregates in the intestine, organogenesis. However, TNFa or TNF-R1-deficient mice we examined the presence of these structures in the ileum of have all LNs, including mLNs, but they fail to form B-cell TNFDARE/ þ mice. The inflammatory infiltrates in the ileum are follicles. These results suggest that TNFa activity in lymphoid composed of neutrophils, macrophages, and T cells that are organogenesis may be secondary to other TNFa members, such distributed throughout the submucosa and muscular layers and as LT. However, simultaneous deficiency of TNFR1 and RelA sometimes reach the serosa. Large mononuclear aggregates abrogates the development of all LNs, despite the presence of a rich in B cells, or TLO, are also found in the terminal ileum of normal complement of LTa1b2 þ LTi cells.22 Thus, the role of the TNFDARE/ þ mice.29 To determine whether the formation TNFa in lymphoid organogenesis remains poorly defined. of these aggregates is dependent on RORgt þ LTi cells, we Here we used TNFDARE/ þ mice, a well-established model of crossed Rorc(gt) À / À mice with TNFDARE/ þ mice to generate human inflammatory disease, to study the role of TNFa in TNF/Rorc(gt) À / À mice. Histological analysis of the terminal lymphoid organogenesis. These animals express increased ileum of age-matched wild-type (WT), Rorc(gt) À / À , TNF/ levels of TNFa under basal conditions, due to mutation in the Rorc(gt) þ / þ and TNF/Rorc(gt) À / À mice at 16–20 weeks of age 30 region of the Tnfa gene that causes higher stability of its showed that TNF/Rorc(gt) þ / þ and TNF/Rorc(gt) À / À mice, but mRNA and, consequently, increased levels of TNFa protein.23 not WT or Rorc(gt) À / À mice, had marked submucosal Intercross of TNFDARE/ þ mice with Rorc(gt) À / À mice led to inflammation, vilus blunting, patchy transmural inflammation, the generation of TNF/Rorc(gt) À / À mice. Surprisingly, and lymphoid aggregates (Figure 1a). The lymphoid aggregates TNF/Rorc(gt) À / À mice developed TLO and several SLO in TNF/Rorc(gt) þ / þ and TNF/Rorc(gt) À / À mice contained (mesenteric, axillary and cervical LN and others) despite large clusters of B220 þ B cells and few CD3 þ T cells the lack of the classical RORgt þ LTi cells. Development of LNs (Figure 1b,c), which were absent in Rorc(gt) À / À mice. was mechanistically linked to activation of stromal cells by These results indicate that TLO can be formed in the ileum TNFa produced by myeloid cells present in the anlagen and in the absence of RORgt þ LTi cells. expression of molecules involved in lymphoid organo- genesis. These results establish that lymphoid organogenesis TNF/Rorc(ct) À / À mice develop SLO can occur in the absence of Rorc if there is increased TNFa Rorc is essential for the development of SLO.5 As expected, no signaling. LNs were found in the Rorc(gt) À / À mice examined Figure 1 Tertiary lymphoid organs are formed in the ileum of TNF/Rorc(gt) À / À mice. (a) Representative hematoxylin and eosin sections of the Ileum of wild-type (WT), Rorc(gt) À / À , TNF/Rorc(gt) þ / þ , and TNF/Rorc(gt) À / À mice at 16 weeks. Notice the presence of inflammatory infiltrates in the ileum of TNF/Rorc(gt) þ / þ and TNF/Rorc(gt) À / À mice. (b) Ileum sections of indicated mice were stained with anti-B220 antibody to visualize B-cell aggregates and 4,6-diamidino-2-phenylindole (DAPI) for nuclear staining. Small B-cell clusters were found in the ileum of WT but were absent in the ileum of Rorc(gt) À / À mice. (c) Overexpression of tumor necrosis factor a (TNFa) induced the formation of large B-cell clusters with few T cells in the ileum of TNF/Rorc(gt) þ / þ and TNF/Rorc(gt) À / À mice. Bars ¼ 250 mm, n ¼ 4/group. MucosalImmunology | VOLUME 7 NUMBER 3 | MAY 2014 603 ARTICLES (Figure 2a). However, we were surprised to find that 100% of To further characterize the structure of the LNs present in the TNF/Rorc(gt) À / À mice had fully developed mLNs that TNF/Rorc(gt) À / À mice, we performed immunostaining. LNs were grossly indistinguishable from those found in WT mice.