Central immune tolerance depends on crosstalk between the classical and alternative NF-κB pathways in medullary thymic epithelial cells.

Medullary thymic epithelial cells (mTECs) contribute to self-tolerance by expressing and presenting peripheral tissue antigens for negative selection of autoreactive T cells and differentiation of natural regulatory T cells. The molecular control of mTEC development remains incompletely understood. We here demonstrate by TEC-specific gene manipulation in mice that the NF-κB transcription factor subunit RelB, which is activated by the alternative NF-κB pathway, regulates development of mature mTECs in a dose-dependent manner. Mice with conditional deletion of Relb lacked mature mTECs and developed spontaneous autoimmunity. In addition, the NF-κB subunits RelA and c-Rel, which are both activated by classical NF-κB signaling, were jointly required for mTEC differentiation by directly regulating the transcription of Relb. Our data reveal a crosstalk mechanism between classical and alternative NF-κB pathways that tightly controls the development of mature mTECs to ensure self-tolerance.

The deubiquitinating enzyme CYLD regulates the differentiation and maturation of thymic medullary epithelial cells.

The cross talk between thymocytes and the thymic epithelium is critical for T-cell development and the establishment of central tolerance. Medullary thymic epithelial cells (mTECs) are located in the thymic medulla and mediate the elimination of self-reactive thymocytes, thereby preventing the onset of autoimmunity. Previous studies identified the deubiquitinating enzyme CYLD as a critical regulator of T-cell development by activating proximal T-cell receptor signaling during the transition of double-positive to single-positive thymocytes. Here we evaluated the impact of the naturally occurring short-splice variant of the cyld gene (sCYLD) on the development and maturation of mTECs. We found that thymi of CYLD(ex7/8) mice, solely expressing sCYLD, displayed a reduced number of mature mTECs caused by a developmental block during the transition of immature to mature mTECs. Further, we could demonstrate an impaired negative selection of thymocytes in these mice. Our data demonstrate that inefficient negative selection in the thymus of CYLD(ex7/8) mice result from a defect in mTEC maturation.

NF-κB2/p100 deficiency impairs immune responses to T-cell-independent type 2 antigens.

Formation of the splenic marginal zone (MZ) depends on the alternative NF-κB signaling pathway. Recently, we reported that unrestricted activation of this pathway in NF-κB2/p100-deficient (p100(-/-) ) knock-in mice alters the phenotype of MZ stroma and B cells. Here, we show that lack of the p100 inhibitor resulted in an expansion of both MZ B and peritoneal B-1 cells. However, these cells failed to generate proliferating blasts in response to T-cell-independent type 2 (TI-2) Ags, correlating with dampened IgM and absent IgG3 responses. This phenotype was in part due to increased activity of the NF-κB subunit RelB. Moreover, p100(-/-) →B6 BM chimeras were more susceptible to infection by encapsulated Streptococcus pneumoniae bacteria, pathogens that induce TI-2 responses. In contrast to the TI-2 defect, p100 deficiency did not impair immune responses to the TI-1 Ag LPS and p100(-/-) MZ B cells showed normal Ag transportation into B-cell follicles. Furthermore, p100(-/-) MZ B and B-1 cells failed to respond to TI-2 Ags in the presence of WT accessory cells. Thus, NF-κB2/p100 deficiency caused a predominant B-cell-intrinsic TI-2 defect that could largely be attributed to impaired proliferation of plasmablasts. Importantly, p100 was also necessary for efficient defense against clinically relevant TI-2 pathogens.

p100 Deficiency is insufficient for full activation of the alternative NF-κB pathway: TNF cooperates with p52-RelB in target gene transcription.

BACKGROUND: Constitutive activation of the alternative NF-κB pathway leads to marginal zone B cell expansion and disorganized spleen microarchitecture. Furthermore, uncontrolled alternative NF-κB signaling may result in the development and progression of cancer. Here, we focused on the question how does the constitutive alternative NF-κB signaling exert its effects in these malignant processes. METHODOLOGY/PRINCIPAL FINDINGS: To explore the consequences of unrestricted alternative NF-κB activation on genome-wide transcription, we compared gene expression profiles of wild-type and NF-κB2/p100-deficient (p100(-/-)) primary mouse embryonic fibroblasts (MEFs) and spleens. Microarray experiments revealed only 73 differentially regulated genes in p100(-/-) vs. wild-type MEFs. Chromatin immunoprecipitation (ChIP) assays showed in p100(-/-) MEFs direct binding of p52 and RelB to the promoter of the Enpp2 gene encoding ENPP2/Autotaxin, a protein with an important role in lymphocyte homing and cell migration. Gene ontology analysis revealed upregulation of genes with anti-apoptotic/proliferative activity (Enpp2/Atx, Serpina3g, Traf1, Rrad), chemotactic/locomotory activity (Enpp2/Atx, Ccl8), and lymphocyte homing activity (Enpp2/Atx, Cd34). Most importantly, biochemical and gene expression analyses of MEFs and spleen, respectively, indicated a marked crosstalk between classical and alternative NF-κB pathways. CONCLUSIONS/SIGNIFICANCE: Our results show that p100 deficiency alone was insufficient for full induction of genes regulated by the alternative NF-κB pathway. Moreover, alternative NF-κB signaling strongly synergized both in vitro and in vivo with classical NF-κB activation, thereby extending the number of genes under the control of the p100 inhibitor of the alternative NF-κB signaling pathway.

VEGFR-3 is expressed on megakaryocyte precursors in the murine bone marrow and plays a regulatory role in megakaryopoiesis.

VEGFR-3 is a transmembrane receptor tyrosine kinase that is activated by its ligands VEGF-C and VEGF-D. Although VEGFR-3 has been linked primarily to the regulation of lymphangiogenesis, in the present study, we demonstrate a role for VEGFR-3 in megakaryopoiesis. Using a human erythroleukemia cell line and primary murine BM cells, we show that VEGFR-3 is expressed on megakaryocytic progenitor cells through to the promegakaryoblast stage. Functionally, specific activation of VEGFR-3 impaired the transition to polyploidy of CD41+ cells in primary BM cultures. Blockade of VEGFR-3 promoted endoreplication consistently. In vivo, long-term activation or blockade of VEGFR-3 did not affect steady-state murine megakaryopoiesis or platelet counts significantly. However, activation of VEGFR-3 in sublethally irradiated mice resulted in significantly elevated numbers of CD41+ cells in the BM and a significant increase in diploid CD41+ cells, whereas the number of polyploid CD41+ cells was reduced significantly. Moreover, activation of VEGFR-3 increased platelet counts in thrombopoietin-treated mice significantly and modulated 5-fluorouracil-induced thrombocytosis strongly, suggesting a regulatory role for VEGFR-3 in megakaryopoiesis.

Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia.

Community-acquired pneumonia presents a spectrum of clinical phenotypes, from lobar pneumonia to septic shock, while mechanisms underlying progression are incompletely understood. In a transcriptomic and metabolomic study across tissues, we examined serotype-specific regulation of signaling and metabolic pathways in C57BL/6 mice intratracheally instilled with either serotype 19F Streptococcus pneumoniae (S19; causing lobar pneumonia), or serotype 2 S. pneumoniae (S2; causing septic pneumococcal disease,) or vehicle (Todd-Hewitt broth). Samples of lung, liver, and blood were collected at 6 and 24 h postinfection and subjected to microarray analysis and mass spectrometry. Results comprise a preferential induction of cholesterol biosynthesis in lobar pneumonia at low-infection doses (10(5) colony forming units/mouse) leading to increased plasma cholesterol (vehicle: 1.8±0.12 mM, S2: 2.3±0.10 mM, S19: 2.9±0.15 mM; P<0.05, comparing S19 to vehicle and S2). This induction was pneumolysin dependent, as a pneumolysin-deficient strain of serotype 19F failed to induce cholesterol biosynthesis (S19ΔPLY: 1.9±0.03 mM). Preincubation of pneumolysin with purified cholesterol or plasma from hypercholesterolemic mice prior to intratracheal instillation protected against lung barrier dysfunction and alveolar macrophage necrosis. Cholesterol may attenuate disease severity by neutralizing pneumolysin in the alveolar compartment and thus prevent septic disease progression.

Impact of monocarboxylate transporter-8 deficiency on the hypothalamus-pituitary-thyroid axis in mice.

In patients, inactivating mutations in the gene encoding the thyroid hormone-transporting monocarboxylate transporter 8 (Mct8) are associated with severe mental and neurological deficits and disturbed thyroid hormone levels. The latter phenotype characterized by high T3 and low T4 serum concentrations is replicated in Mct8 knockout (ko) mice, indicating that MCT8 deficiency interferes with thyroid hormone production and/or metabolism. Our studies of Mct8 ko mice indeed revealed increased thyroidal T3 and T4 concentrations without overt signs of a hyperactive thyroid gland. However, upon TSH stimulation Mct8 ko mice showed decreased T4 and increased T3 secretion compared with wild-type littermates. Moreover, similar changes in the thyroid hormone secretion pattern were observed in Mct8/Trhr1 double-ko mice, which are characterized by normal serum T3 levels and normal hepatic and renal D1 expression in the presence of very low T4 serum concentrations. These data strongly indicate that absence of Mct8 in the thyroid gland affects thyroid hormone efflux by shifting the ratio of the secreted hormones toward T3. To test this hypothesis, we generated Mct8/Pax8 double-mutant mice, which in addition to Mct8 lack a functional thyroid gland and are therefore completely athyroid. Following the injection of these animals with either T4 or T3, serum analysis revealed T3 concentrations similar to those observed in Pax8 ko mice under thyroid hormone replacement, indicating that indeed increased thyroidal T3 secretion in Mct8 ko mice represents an important pathogenic mechanism leading to the high serum T3 levels.

The pivotal role of the alternative NF-kappaB pathway in maintenance of basal bone homeostasis and osteoclastogenesis.

The alternative NF-kappaB pathway consists predominantly of NF-kappaB-inducing kinase (NIK), IkappaB kinase alpha (IKKalpha), p100/p52, and RelB. The hallmark of the alternative NF-kappaB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF-kappaB activation in bone biology, however, is not well understood. To elucidate the role of the alternative pathway in bone homeostasis, we first analyzed alymphoplasic (aly/aly) mice, which have a defective NIK and are unable to process p100, resulting in the absence of p52. We observed increased bone mineral density (BMD) and bone volume, indicating an osteopetrotic phenotype. These mice also have a significant defect in RANKL-induced osteoclastogenesis in vitro and in vivo. NF-kappaB DNA-binding assays revealed reduced activity of RelA, RelB, and p50 and no binding activity of p52 in aly/aly osteoclast nuclear extracts after RANKL stimulation. To determine the role of p100 itself without the influence of a concomitant lack of p52, we used p100(-/-) mice, which specifically lack the p100 inhibitor but still express p52. p100(-/-) mice have an osteopenic phenotype owing to the increased osteoclast and decreased osteoblast numbers that was rescued by the deletion of one allele of the relB gene. Deletion of both allele of relB resulted in a significantly increased bone mass owing to decreased osteoclast activity and increased osteoblast numbers compared with wild-type (WT) controls, revealing a hitherto unknown role for RelB in bone formation. Our data suggest a pivotal role of the alternative NF-kappaB pathway, especially of the inhibitory role of p100, in both basal and stimulated osteoclastogenesis and the importance of RelB in both bone formation and resorption.

RelB-dependent stromal cells promote T-cell leukemogenesis.

BACKGROUND: The Rel/NF-kappaB transcription factors are often activated in solid or hematological malignancies. In most cases, NF-kappaB activation is found in malignant cells and results from activation of the canonical NF-kappaB pathway, leading to RelA and/or c-Rel activation. Recently, NF-kappaB activity in inflammatory cells infiltrating solid tumors has been shown to contribute to solid tumor initiation and progression. Noncanonical NF-kappaB activation, which leads to RelB activation, has also been reported in breast carcinoma, prostate cancer, and lymphoid leukemia. METHODOLOGY/PRINCIPAL FINDINGS: Here we report a novel role for RelB in stromal cells that promote T-cell leukemogenesis. RelB deficiency delayed leukemia onset in the TEL-JAK2 transgenic mouse model of human T acute lymphoblastic leukemia. Bone marrow chimeric mouse experiments showed that RelB is not required in the hematopoietic compartment. In contrast, RelB plays a role in radio-resistant stromal cells to accelerate leukemia onset and increase disease severity. CONCLUSIONS/SIGNIFICANCE: The present results are the first to uncover a role for RelB in the crosstalk between non-hematopoietic stromal cells and leukemic cells. Thus, besides its previously reported role intrinsic to specific cancer cells, the noncanonical NF-kappaB pathway may also play a pro-oncogenic role in cancer microenvironmental cells.

Constitutive alternative NF-kappaB signaling promotes marginal zone B-cell development but disrupts the marginal sinus and induces HEV-like structures in the spleen.

Nuclear factor-kappaB (NF-kappaB) plays a crucial role in B-cell and lymphoid organ development. Here, we studied the consequences of constitutive, signal-independent activation of the alternative NF-kappaB pathway for the splenic marginal zone (MZ). In contrast to nfkb2(-/-) mice, which lack both p100 and p52, mice that lack only the inhibitory p100 precursor but still express the p52 subunit of NF-kappaB2 (p100(-/-)) had markedly elevated MZ B-cell numbers. Both cell-intrinsic mechanisms and increased stromal expression of vascular cell adhesion molecule-1 (VCAM-1) contributed to the accumulation of MZ B cells in p100(-/-) spleens. While migration of p100(-/-) MZ B cells toward the lysophospholipid sphingosine-1 phosphate (S1P) was not affected, CXCL13-stimulated chemotaxis was impaired, correlating with reduced migration of MZ B cells into follicles in response to lipopolysaccharide (LPS). Strikingly, p100 deficiency resulted in the absence of a normal marginal sinus, strongly induced expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) and glycosylated cell adhesion molecule-1 (GlyCAM-1), and the formation of nonfunctional ectopic high endothelial venule (HEV)-like structures in the red pulp. Thus, constitutive activation of the alternative NF-kappaB pathway favors MZ B-cell development and accumulation but leads to a disorganized spleen microarchitecture.

Distinct roles of lymphotoxin-beta signaling and the homeodomain transcription factor Nkx2.3 in the ontogeny of endothelial compartments in spleen.

The formation of peripheral lymphoid tissues is indispensable for the efficient recognition and elimination of external antigens by lymphoid and accessory cells of the adaptive immune system. The spleen is structurally arranged around various vascular beds with distinct endothelial phenotypes. Using immunohistochemistry, we investigated the postnatal developmental characteristics of the marginal sinus and its relationship with various red-pulp sinus subsets. We also determined the importance of the lymphotoxin beta receptor (LT beta R) and the role of the Nkx2.3 transcription factor for the formation of the splenic vasculature. Both the administration of soluble LT beta R-Ig fusion protein to neonates and the deletion of LT beta R or downstream signaling components (RelB and p52) of the NF-kappaB family inhibited the phenotypic maturation of marginal sinus but had no effect on the vascular compartmentalization of the red pulp. The integrity of the marginal sinus and the proper vascular segregation of the red pulp appeared to be controlled by Nkx2.3, as Nkx2.3-deficient mice exhibited an abnormal distribution of IBL-7/1(hi)/IBL-9/2(-) sinuses and a lack of IBL-7/1(lo)/IBL-9/2(+) vessels. Our data suggest that phenotypic heterogeneity among different vascular elements within distinct anatomical regions of the spleen differentially depends on developmental factors such as lymphotoxin signaling or Nkx2.3, whereas the marginal sinus is controlled by both pathways.

RelB is required for Peyer's patch development: differential regulation of p52-RelB by lymphotoxin and TNF.

Targeted disruption of the Rel/NF-kappaB family members NF-kappaB2, encoding p100/p52, and RelB in mice results in anatomical defects of secondary lymphoid tissues. Here, we report that development of Peyer's patch (PP)-organizing centers is impaired in both NF-kappaB2- and RelB-deficient animals. IL-7-induced expression of lymphotoxin (LT) in intestinal cells, a crucial step in PP development, is not impaired in RelB-deficient embryos. LTbeta receptor (LTbetaR)-deficient mice also lack PPs, and we demonstrate that LTbetaR signaling induces p52-RelB and classical p50-RelA heterodimers, while tumor necrosis factor (TNF) activates only RelA. LTbetaR-induced binding of p52-RelB requires the degradation of the inhibitory p52 precursor, p100, which is mediated by the NF-kappaB-inducing kinase (NIK) and the IkappaB kinase (IKK) complex subunit IKKalpha, but not IKKbeta or IKKgamma. Activation of RelA requires all three IKK subunits, but is independent of NIK. Finally, we show that TNF increases p100 levels, resulting in the specific inhibition of RelB DNA binding via the C-terminus of p100. Our data indicate an important role of p52-RelB heterodimers in lymphoid organ development downstream of LTbetaR, NIK and IKKalpha.