Debra Weih

Essential Role of RelB in Germinal Center and Marginal Zone Formation and Proper Expression of Homing Chemokines

High levels of the Rel/NF-κB family member RelB are restricted to specific regions of thymus, lymph nodes, and Peyer’s patches. In spleen, RelB is expressed in periarteriolar lymphatic sheaths, germinal centers (GCs), and the marginal zone (MZ). In this study, we report that RelB-deficient (relB−/−) mice, in contrast to nfkb1−/−, but similar to nfkb2−/− mice, are unable to form GCs and follicular dendritic cell networks upon Ag challenge in the spleen. RelB is also required for normal organization of the MZ and its population by macrophages and B cells. Reciprocal bone marrow transfers demonstrate that RelB expression in radiation-resistant stromal cells, but not in bone marrow-derived hemopoietic cells, is required for proper formation of GCs, follicular dendritic cell networks, and MZ structures. However, the generation of MZ B cells requires RelB in hemopoietic cells. Expression of TNF ligand/receptor family members is only moderately altered in relB−/− splenocytes. In contrast, expression of homing chemokines is strongly reduced in relB−/− spleen with particularly low mRNA levels of the chemokine B lymphocyte chemoattractant. Our data indicate that activation of p52-RelB heterodimers in stromal cells downstream of TNF/lymphotoxin is required for normal expression of homing chemokines and proper development of spleen microarchitecture.

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

Targeted disruption of the Rel/NF‐κB family members NF‐κB2, encoding p100/p52, and RelB in mice results in anatomical defects of secondary lymphoid tissues. Here, we report that development of Peyers patch (PP)‐organizing centers is impaired in both NF‐κB2‐ 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. LTβ receptor (LTβR)‐deficient mice also lack PPs, and we demonstrate that LTβR signaling induces p52–RelB and classical p50–RelA heterodimers, while tumor necrosis factor (TNF) activates only RelA. LTβR‐induced binding of p52–RelB requires the degradation of the inhibitory p52 precursor, p100, which is mediated by the NF‐κB‐inducing kinase (NIK) and the IκB kinase (IKK) complex subunit IKKα, but not IKKβ or IKKγ. 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 LTβR, NIK and IKKα.

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.

Constitutive alternative NF-κB 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.

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

The alternative NF‐κB pathway consists predominantly of NF‐κB‐inducing kinase (NIK), IκB kinase α (IKKα), p100/p52, and RelB. The hallmark of the alternative NF‐κB signaling is the processing of p100 into p52 through NIK, thus allowing the binding of p52 and RelB. The physiologic relevance of alternative NF‐κB 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‐κB 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‐κB 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.

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 (105 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.—Weber, M., Lambeck, S., Ding, N., Henken, S., Kohl, M., Deigner, H. P., Enot, D. P., Igwe, E. I., Frappart, L., Kiehntopf, M., Claus, R. A., Kamradt, T., Weih, D., Vodovotz, Y., Briles, D. E., Ogunniyi, A. D., Paton, J. C., Maus, U. A., Bauer, M. Hepatic induction of cholesterol biosynthesis reflects a remote adaptive response to pneumococcal pneumonia. FASEB J. 26, 2424‐2436 (2012). www.fasebj.org

Inhibition of NF-κB in T cells blocks lymphoproliferation and partially rescues autoimmune disease ingld/gld mice

The Fas ligand (FasL)/Fas pathway is crucial for the maintenance of homeostasis of the peripheral immune system. Its importance is illustrated by the spontaneous mouse mutants gld andlpr which lack functional FasL and Fas receptor, respectively. These animals develop lymphadenopathy, splenomegaly, increased serum Ig and autoantibodies, leading to an autoimmune syndromeand premature death. The Rel/NF‐κB family of transcription factors plays an important role in peripheral lymphocyte proliferation and survival. In this report, we studied the consequences of T cell‐specific inhibition of NF‐κB on the development of the gld phenotype. Transgenic gld/gld mice expressing a non‐degradable form of IκBα  under the control of T cell‐specific regulatory elements show dramatically reduced lymphadenopathy, splenomegaly, and an almost complete elimination of Thy‐1+B220+CD4–CD8– abnormal T cells, correlating with reduced proliferative responses and increased apoptosis of peripheral T cells upon TCR triggering. Interestingly, the B cell abnormalities that are characteristic of gld/gld mice, such as the production of autoantibodies, high levels of serum Ig, and the development of glomerulonephritis, are partially corrected. These results suggest that the T cell‐specific inhibition of NF‐κB opens apoptotic pathways distinct from FasL/Fas which, along with a diminished proliferative response, blocks splenomegaly and lymphadenopathy and partially rescues autoimmune disease in gld/gld mice.

RelB-dependent stromal cells promote T-cell leukemogenesis.

Background The Rel/NF-κB transcription factors are often activated in solid or hematological malignancies. In most cases, NF-κB activation is found in malignant cells and results from activation of the canonical NF-κB pathway, leading to RelA and/or c-Rel activation. Recently, NF-κB activity in inflammatory cells infiltrating solid tumors has been shown to contribute to solid tumor initiation and progression. Noncanonical NF-κB 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-κB pathway may also play a pro-oncogenic role in cancer microenvironmental cells.

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.

Distinct roles of lymphotoxin-β 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 β receptor (LTβR) and the role of the Nkx2.3 transcription factor for the formation of the splenic vasculature. Both the administration of soluble LTβR-Ig fusion protein to neonates and the deletion of LTβR or downstream signaling components (RelB and p52) of the NF-κB 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-7/1hi/IBL-9/2− sinuses and a lack of IBL-7/1lo/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.