Overexpression of Bcl-3 inhibits the development of marginal zone B cells.

The transcription factor Bcl-3 functions as a proto-oncogene via regulation of cell proliferation and apoptosis. Bcl-3 is an atypical member of the IκB family and plays a central role in the immune response through interactions with the NF-κB subunits p50 and p52. To investigate the impact of Bcl-3 on B-cell maturation and regulation, we generated mice that overexpress Bcl-3 specifically in B cells. Interestingly, these mice lack marginal zone B cells and exhibit a significant reduction in the number of B-1 B cells. Further, B cells from these mice are impaired in their proliferative capacity. Our data demonstrate that the overexpression of the transcription factor Bcl-3 inhibits germinal c…

Regulation of B cell homeostasis and activation by the tumor suppressor gene CYLD

B cell homeostasis is regulated by multiple signaling processes, including nuclear factor-kappaB (NF-kappaB), BAFF-, and B cell receptor signaling. Conditional disruption of genes involved in these pathways has shed light on the mechanisms governing signaling from the cell surface to the nucleus. We describe a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLD(ex7/8) mice), which is a deubiquitinating enzyme that is integral to NF-kappaB signaling. This shorter CYLD protein lacks the TRAF2 and NEMO binding sites present in full-length CYLD. A dramatic expansion of mature B lymphocyte populations in all peripheral lymphoid organs occur…

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1

Abstract Richter’s transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase–associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurren…

Mutated cylindromatosis gene affects the functional state of dendritic cells

Cylindromatosis gene (CYLD) is a ubiquitously expressed deubiquitinating enzyme, which interacts with members of the NF-κB signaling pathway and attenuates NF-κB and JNK signaling. Here, we report that DC derived from transgenic mice, which solely express a naturally occurring CYLD isoform (CYLD(ex7/8)), display a higher content of nuclear RelB and express elevated levels of NF-κB family members as well as of known NF-κB-target genes comprising costimulatory molecules and pro-inflammatory cytokines, as compared with WT DC. Accordingly, unstimulated CYLD(ex7/8) DC exhibited a significantly higher primary allogenic T-cell stimulatory capacity than WT DC and exerted no tolerogenic activity. Tr…

B Cell Expansion Hinders the Stroma-Epithelium Regenerative Crosstalk During Mucosal Healing

Little is known about the pro-resolution role of immune cells recruited to damaged tissue. Using an experimental model of intestinal epithelial damage and repair, we identified B cells as the dominant cell type in the healing colon. Single-cell RNA-sequencing (scRNAseq) revealed the expansion of an IFN-induced B cell subset during experimental mucosal healing which was associated with colitis severity. In line with this, B cell depletion during mucosal healing resulted in accelerated recovery upon injury, which was associated with enhanced expression of tissue remodeling genes. scRNA-seq from the epithelial and stromal compartment confirmed that lack of B cells during mucosal healing alters…

Apoptosis of oligodendrocytes via Fas and TNF-R1 is a key event in the induction of experimental autoimmune encephalomyelitis.

Abstract In experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, immunization with myelin Ags leads to demyelination and paralysis. To investigate which molecules are crucial for the pathogenesis of EAE, we specifically assessed the roles of the death receptors Fas and TNF-R1. Mice lacking Fas expression in oligodendrocytes (ODCs) were generated and crossed to TNF-R1-deficient mice. To achieve specific deletion of a loxP-flanked fas allele in ODCs, we generated a new insertion transgene, expressing the Cre recombinase specifically in ODCs. Fas inactivation alone as well as the complete absence of TNF-R1 protected mice partially from EAE induced by the imm…

Hepatic B cell leukemia-3 promotes hepatic steatosis and inflammation through insulin-sensitive metabolic transcription factors.

Background & Aims The pathomechanisms underlying non-alcoholic fatty liver disease (NAFLD) and the involved molecular regulators are incompletely explored. The nuclear factor-kappa B (NF-κB)-cofactor gene B cell leukemia-3 ( Bcl-3 ) plays a critical role in altering the transcriptional capacity of NF-κB – a key inducer of inflammation – but also of genes involved in cellular energy metabolism. Methods To define the role of Bcl-3 in non-alcoholic steatohepatitis (NASH), we developed a novel transgenic mouse model with hepatocyte-specific overexpression of Bcl-3 ( Bcl-3 Hep ) and employed a high-fat, high-carbohydrate dietary feeding model. To characterize the transgenic model, deep RNA seque…

Endogenous CD83 Expression in CD4+ Conventional T Cells Controls Inflammatory Immune Responses

Abstract The glycoprotein CD83 is known to be expressed by different immune cells including activated CD4+Foxp3+ regulatory T cells (Tregs) and CD4+Foxp3− conventional T cells. However, the physiological function of endogenous CD83 in CD4+ T cell subsets is still unclear. In this study, we have generated a new CD83flox mouse line on BALB/c background, allowing for specific ablation of CD83 in T cells upon breeding with CD4-cre mice. Tregs from CD83flox/flox/CD4-cretg/wt mice had similar suppressive activity as Tregs from CD83flox/flox/CD4-crewt/wt wild-type littermates, suggesting that endogenous CD83 expression is dispensable for the inhibitory capacity of Tregs. However, CD83-deficient CD…

NF-κB-inducing kinase is essential for B-cell maintenance in mice

NF-κB-inducing kinase (NIK) is a key mediator of the noncanonical NF-κB signaling pathway, which is critical for normal B-cell development and function. It is well established that the complete deletion of NIK in mice results in defective B cells and impaired secondary lymphoid organogenesis. To address the role of NIK deficiency specifically in B cells, we generated a new mouse strain for the conditional deletion of this kinase. Deletion of NIK during B-cell development results in a drastic reduction of mature B cells from the transitional 2 stage on, while B-1 B cells are less affected. Moreover, deletion of NIK in the germinal centers decreases the numbers of germinal center B cells and …

Isolation of T Cells from the Gut

The lymphocytes of epithelial and lamina proprial compartments of the intestine are phenotypically and functionally distinct and serve a wide range of functions in the intestinal mucosa like regulating intestinal homeostasis, maintaining epithelial barrier function as well as regulating adaptive and innate immune responses. To analyze the role of these cells in different disease states, it is necessary to isolate pure cell populations of the intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of the gut. In this protocol we describe a method to isolate T cells from IEL and LPL, which can be used for further investigations like comparative studies of mRNA expression, cell …

A20 deficiency in B cells enhances B-cell proliferation and results in the development of autoantibodies.

A20/TNFAIP3 is an ubiquitin-editing enzyme, important for the regulation of the NF-κB pathway. Mutations in the TNFAIP3 gene have been linked to different human autoimmune disorders. In human B-cell lymphomas, the inactivation of A20 results in constitutive NF-κB activation. Recent studies demonstrate that in mice the germline inactivation of A20 leads to early lethality, due to inflammation in multiple organs of the body. In this report, we describe a new mouse strain allowing for the tissue-specific deletion of A20. We show that B-cell-specific deletion of A20 results in a dramatic reduction in marginal zone B cells. Furthermore, A20-deficient B cells display a hyperactive phenotype repre…

Balanced Bcl-3 expression in murine CD4+T cells is required for generation of encephalitogenic Th17 cells

The function of NF-κB family members is controlled by multiple mechanisms including the transcriptional regulator Bcl-3, an atypical member of the IκB family. By using a murine model of conditional Bcl-3 overexpression specifically in T cells, we observed impairment in the development of Th2, Th1 and Th17 cells. High expression of Bcl-3 promoted CD4+ T-cell survival, but at the same time suppressed proliferation in response to TCR stimulation, resulting in reduced CD4+ T-cell expansion. As a consequence, T cell specific overexpression of Bcl-3 led to reduced inflammation in the small intestine of mice applied with anti-CD3 in a model of gut inflammation. Moreover, impaired Th17-cell develop…

Smad7 in T cells drives T helper 1 responses in multiple sclerosis and experimental autoimmune encephalomyelitis

Autoreactive CD4+ T lymphocytes play a vital role in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis. Since the discovery of T helper 17 cells, there is an ongoing debate whether T helper 1, T helper 17 or both subtypes of T lymphocytes are important for the initiation of autoimmune neuroinflammation. We examined peripheral blood CD4+ cells from patients with active and stable relapsing-remitting multiple sclerosis, and used mice with conditional deletion or over-expression of the transforming growth factor-beta inhibitor Smad7, to delineate the role of Smad7 in T cell differentiation and autoimmune neuroinflammation. We found that Smad…

Generation and functional analyses of hepatocyte-specific type I interleukin-1 receptor (IL-1RI) knockout mice

Alternative Splice Forms of CYLD Mediate Ubiquitination of SMAD7 to Prevent TGFB Signaling and Promote Colitis

Background & Aims The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice. Methods We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from tr…

B Lymphocyte-Deficiency in Mice Causes Vascular Dysfunction by Inducing Neutrophilia

B lymphocytes have been implicated in the development of insulin resistance, atherosclerosis and certain types of hypertension. In contrast to these studies, which were performed under pathological conditions, the present study provides evidence for the protective effect of B lymphocytes in maintaining vascular homeostasis under physiological conditions. In young mice not exposed to any known risk factors, the lack of B cells led to massive endothelial dysfunction. The vascular dysfunction in B cell-deficient mice was associated with an increased number of neutrophils in the circulating blood. Neutrophil depletion in B cell-deficient mice resulted in the complete normalization of vascular f…

Nuclear Translocation of RELB Is Increased in Diseased Human Liver and Promotes Ductular Reaction and Biliary Fibrosis in Mice.

Background & Aims Cholangiocyte proliferation and ductular reaction contribute to the onset and progression of liver diseases. Little is known about the role of the transcription factor nuclear factor-κB (NF-κB) in this process. We investigated the activities of the RELB proto-oncogene NF-κB subunit in human cholangiocytes and in mouse models of liver disease characterized by a ductular reaction. Methods We obtained liver tissue samples from patients with primary sclerosing cholangitis, primary biliary cholangitis, hepatitis B or C virus infection, autoimmune hepatitis, alcoholic liver disease, or without these diseases (controls) from a tissue bank in Germany. Tissues were analyzed by immu…

IL-17A mediated endothelial breach promotes metastasis formation

Abstract The role of the IL23/IL17A axis in tumor–immune interactions is a matter of controversy. Although some suggest that IL17A-producing T cells (TH17) can suppress tumor growth, others report that IL17A and IL23 accelerate tumor growth. Here, we systematically assessed the impact of IL17A-secreting lymphocytes in several murine models of tumor lung metastasis. Genetic fate mapping revealed that IL17A was secreted within lung metastases predominantly by γδ T cells, whereas TH17 cells were virtually absent. Using different tumor models, we found Il17a−/− mice to consistently develop fewer pulmonary tumor colonies. IL17A specifically increased blood vessel permeability and the expression …

Down-regulation of CYLD as a trigger for NF-κB activation and a mechanism of apoptotic resistance in hepatocellular carcinoma cells

The cylindromatosis gene (CYLD) was identified as a tumor suppressor gene, which is mutated in familial cylindromatosis (Brooke-Spiegler syndrome), an autosomal-dominant predisposition to multiple tumors of the skin appendages. CYLD is a deubiquitinating enzyme acting as a negative regulator of the nuclear factor κB (NF-κB) signaling pathway by removing lysine-63-linked polyubiquitin chains from NF-κB activating proteins. In order to investigate the role of CYLD in apoptotic signaling in human hepatocellular carcinoma (HCC) cells, we first studied the expression levels of CYLD in HCC tissues. CYLD expression was lower in HCC both at protein and mRNA levels compared to the surrounding non-ma…

Liver specific deletion of CYLDexon7/8 induces severe biliary damage, fibrosis and increases hepatocarcinogenesis in mice

Background & Aims CYLD is a tumor suppressor gene that is mutated in familial cylindromatosis, an autosomal dominant predisposition to tumors of skin appendages. Reduced CYLD expression has been observed in other tumor entities, including hepatocellular carcinoma. In the present study, we analyzed the role of CYLD in liver homeostasis and hepatocarcinogenesis in vivo . Methods Mice with liver-specific deletion of CYLDexon7/8 ( CYLD FF xAlbCre ) were generated. Liver tissues were histologically analyzed and oval cell activation was investigated. Hepatocarcinogenesis was induced by diethylnitrosamine/phenobarbital (DEN/PB). Microarray expression profiling of livers was performed in untreated …

mTORC1 activation in B cells confers impairment of marginal zone microarchitecture by exaggerating cathepsin activity

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell metabolism and lymphocyte proliferation. It is inhibited by the tuberous sclerosis complex (TSC), a heterodimer of TSC1 and TSC2. Deletion of either gene results in robust activation of mTORC1. Mature B cells reside in the spleen at two major anatomical locations, the marginal zone (MZ) and follicles. The MZ constitutes the first line of humoral response against blood‐borne pathogens and undergoes atrophy in chronic inflammation. In previous work, we showed that mice deleted for TSC1 in their B cells (TSC1(BKO)) have almost no MZ B cells, whereas follicular B cells are minimally affected. To explore potential underl…

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 foun…

Naturally occurring short splice variant of CYLD positively regulates dendritic cell function

Abstract Deubiquitination of NF-κB members by CYLD is crucial in controlling the magnitude and nature of cell activation. The role of the naturally occurring CYLD splice variant in dendritic cell (DC) function was analyzed using CYLDex7/8 mice, which lack the full-length CYLD (flCYLD) transcript and overexpress the short splice variant (sCYLD). Bone marrow–derived DCs from CYLDex7/8 mice display a hyperactive phenotype in vitro and in vivo and have a defect in establishing tolerance with the use of DEC-205–mediated antigen targeting to resting DCs. The combination of sCYLD overexpression and lack of flCYLD in CYLDex7/8 DCs leads to enhanced NF-κB activity accompanied by an increased nuclear…

Aberrant splicing of the tumor suppressor CYLD promotes the development of chronic lymphocytic leukemia via sustained NF-κB signaling

The pathogenesis of chronic lymphocytic leukemia (CLL) has been linked to constitutive NF-κB activation but the underlying mechanisms are poorly understood. Here we show that alternative splicing of the negative regulator of NF-κB and tumor suppressor gene CYLD regulates the pool of CD5+ B cells through sustained canonical NF-κB signaling. Reinforced canonical NF-κB activity leads to the development of B1 cell-associated tumor formation in aging mice by promoting survival and proliferation of CD5+ B cells, highly reminiscent of human B-CLL. We show that a substantial number of CLL patient samples express sCYLD, strongly implicating a role for it in human B-CLL. We propose that our new CLL-l…

Elevated levels of Bcl-3 inhibits Treg development and function resulting in spontaneous colitis

Bcl-3 is an atypical NF-κB family member that regulates NF-κB-dependent gene expression in effector T cells, but a cell-intrinsic function in regulatory T (Treg) cells and colitis is not clear. Here we show that Bcl-3 expression levels in colonic T cells correlate with disease manifestation in patients with inflammatory bowel disease. Mice with T-cell-specific overexpression of Bcl-3 develop severe colitis that can be attributed to defective Treg cell development and function, leading to the infiltration of immune cells such as pro-inflammatory γδT cells, but not αβ T cells. In Treg cells, Bcl-3 associates directly with NF-κB p50 to inhibit DNA binding of p50/p50 and p50/p65 NF-κB dimers, t…

Past, present and future of immunology in Mainz.

The tumor suppressor CYLD controls the function of murine regulatory T cells.

Abstract CYLD was originally identified as a tumor suppressor gene mutated in familial cylindromatosis, an autosomal dominant predisposition to multiple benign neoplasms of the skin known as cylindromas. The CYLD protein is a deubiquitinating enzyme that acts as a negative regulator of NF-κB and JNK signaling through its interaction with NEMO and TNFR-associated factor 2. We have previously described a novel mouse strain that expresses solely and excessively a naturally occurring splice variant of CYLD (CYLDex7/8). In this study, we demonstrate that CYLD plays a critical role in Treg development and function. T cells of CYLDex7/8 mice had a hyperactive phenotype manifested by increased prod…

BAX inhibitor-1 is a Ca(2+) channel critically important for immune cell function and survival.

The endoplasmic reticulum (ER) serves as the major intracellular Ca(2+) store and has a role in the synthesis and folding of proteins. BAX (BCL2-associated X protein) inhibitor-1 (BI-1) is a Ca(2+) leak channel also implicated in the response against protein misfolding, thereby connecting the Ca(2+) store and protein-folding functions of the ER. We found that BI-1-deficient mice suffer from leukopenia and erythrocytosis, have an increased number of splenic marginal zone B cells and higher abundance and nuclear translocation of NF-κB (nuclear factor-κ light-chain enhancer of activated B cells) proteins, correlating with increased cytosolic and ER Ca(2+) levels. When put into culture, purifie…