Linear ubiquitin chains: NF-κB signalling, cell death and beyond.

Ubiquitylation is a versatile post-translational modification. Met1-linked linear ubiquitin chains are involved in nuclear factor-κB signalling and cell death, and dysfunctions in linear ubiquitylation underlie chronic inflammation. Recent identification of deubiquitylating enzymes and binding domains that are specific for linear ubiquitin chains suggests new physiological roles for linear ubiquitin chains. Moreover, the ligase required for linear ubiquitylation has a crucial role in the pathogenesis of some malignancies. Structural and functional analyses of the conjugation and deconjugation of linear ubiquitin chains have enabled the development of new probes to study the roles of linear chain ubiquitylation.

The cell-cycle regulator c-Myc is essential for the formation and maintenance of germinal centers.

Germinal centers (GCs) are sites of intense B cell proliferation and are central for T cell-dependent antibody responses. However, the role of c-Myc, a key cell-cycle regulator, in this process has been questioned. Here we identified c-Myc(+) B cell subpopulations in immature and mature GCs and found, by genetic ablation of Myc, that they had indispensable roles in the formation and maintenance of GCs. The identification of these functionally critical cellular subsets has implications for human B cell lymphomagenesis, which originates mostly from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the c-Myc(+) GC subpopulations may be at a particularly high risk for malignant transformation.

PI3 kinase signals BCR-dependent mature B cell survival.

Previous work has shown that mature B cells depend upon survival signals delivered to the cells by their antigen receptor (BCR). To identify the molecular nature of this survival signal, we have developed a genetic approach in which ablation of the BCR is combined with the activation of specific, BCR dependent signaling cascades in mature B cells in vivo. Using this system, we provide evidence that the survival of BCR deficient mature B cells can be rescued by a single signaling pathway downstream of the BCR, namely PI3K signaling, with the FOXO1 transcription factor playing a central role.

LUBAC accelerates B-cell lymphomagenesis by conferring resistance to genotoxic stress on B cells.

The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death and is a suitable therapeutic target for B-cell lymphomas.

Crucial Role of Linear Ubiquitin Chain Assembly Complex-Mediated Inhibition of Programmed Cell Death in TLR4-Mediated B Cell Responses and B1b Cell Development.

Linear ubiquitin chain assembly complex (LUBAC)-mediated linear polyubiquitin plays crucial roles in thymus-dependent and -independent type II Ab responses and B1 cell development. In this study, we analyzed the role of LUBAC in TLR-mediated B cell responses. A mouse strain in which LUBAC activity was ablated specifically in B cells (B-HOIPΔlinear mice) showed defective Ab responses to a type I thymus-independent Ag, NP-LPS. B cells from B-HOIPΔlinear mice (HOIPΔlinear B cells) underwent massive cell death in response to stimulation of TLR4, but not TLR9. TLR4 stimulation induced caspase-8 activation in HOIPΔlinear B cells; this phenomenon, as well as TLR4-induced cell death, was suppressed by ablation of TRIF, a signal inducer specific for TLR4. In addition, LPS-induced survival, proliferation, and differentiation into Ab-producing cells of HOIPΔlinear B cells were substantially restored by inhibition of caspases together with RIP3 deletion, but not by RIP3 deletion alone, suggesting that LPS stimulation kills HOIPΔlinear B cells by apoptosis elicited via the TRIF pathway. Further examination of the roles of cell death pathways in B-HOIPΔlinear mice revealed that deletion of RIP3 increased the number of B1 cells, particularly B1b cells, in B-HOIPΔlinear mice, indicating that B1b cell homeostasis is controlled via LUBAC-mediated suppression of necroptosis. Taken together, the data show that LUBAC regulates TLR4-mediated B cell responses and B1b cell development and/or maintenance by inhibiting programmed cell death.

Immobile BCRs: the safety on the signal trigger.

B cell receptor (BCR)-mediated tonic signals are important for B cell survival and development. In this issue of Immunity, Treanor et al. (2010) show that triggering of downstream responses is kept in check with BCR immobilization by actin.

Defective immune responses in mice lacking LUBAC-mediated linear ubiquitination in B cells.

The linear ubiquitin chain assembly complex (LUBAC) plays a crucial role in activating the canonical NF-κB pathway, which is important for B-cell development and function. Here, we describe a mouse model (B-HOIP(Δlinear)) in which the linear polyubiquitination activity of LUBAC is specifically ablated in B cells. Canonical NF-κB and ERK activation, mediated by the tumour necrosis factor (TNF) receptor superfamily receptors CD40 and TACI, was impaired in B cells from B-HOIP(Δlinear) mice due to defective activation of the IKK complex; however, B-cell receptor (BCR)-mediated activation of the NF-κB and ERK pathways was unaffected. B-HOIP(Δlinear) mice show impaired B1-cell development and defective antibody responses to thymus-dependent and thymus-independent II antigens. Taken together, these data suggest that LUBAC-mediated linear polyubiquitination is essential for B-cell development and activation, possibly via canonical NF-κB and ERK activation induced by the TNF receptor superfamily, but not by the BCR.

Roles of the NF-κB Pathway in B-Lymphocyte Biology.

NF-κB was originally identified as a family of transcription factors that bind the enhancer of the immunoglobulin κ light-chain gene. Although its function in the regulation of immunoglobulin κ light-chain gene remains unclear, NF-κB plays critical roles in development, survival, and activation of B lymphocytes. In B cells, many receptors, including B-cell antigen receptor (BCR), activate NF-κB pathway, and the molecular mechanism of receptor-mediated activation of IκB kinase (IKK) complex has been partially revealed. In addition to normal B lymphocytes, NF-κB is also involved in the growth of some types of B-cell lymphomas, and many oncogenic mutations involved in constitutive activation of the NF-κB pathway were recently identified in such cancers. In this review, we first summarize the function of NF-κB in B-cell development and activation, and then describe recent progress in understanding the molecular mechanism of receptor-mediated activation of the IKK complex, focusing on the roles of the ubiquitin system. In the last section, we describe oncogenic mutations that induce NF-κB activation in B-cell lymphoma.

IFN-γ or IFN-α ameliorates chronic proliferative dermatitis by inducing expression of linear ubiquitin chain assembly complex.

The linear ubiquitin chain assembly complex (LUBAC) ubiquitin ligase complex, composed of HOIL-1L-interacting protein (HOIP), heme-oxidized IRP2 ubiquitin ligase-1L (HOIL-1L), and SHANK-associated RH domain protein, specifically generates linear polyubiquitin chains and is involved in NF-κB activation. Lack of SHANK-associated RH domain protein, which drastically reduces the amount of HOIP and HOIL-1L, causes chronic proliferative dermatitis (cpdm) in mice. Impaired NF-κB activation and augmented apoptosis have been implicated in the pathogenesis of cpdm in mice. In this study, we found that IFN-γ increased the amount of LUBAC by inducing HOIP and HOIL-1L mRNA transcription and enhanced the signal-induced NF-κB activation in embryonic fibroblasts, keratinocytes, and bone marrow-derived macrophages from wild-type and/or cpdm mice; however, IFN-γ failed to augment NF-κB activation in mouse embryonic fibroblasts lacking linear polyubiquitination activity of LUBAC. Moreover, s.c. injection of IFN-γ for 3 wk into the skin of cpdm mice increased the amount of HOIP, suppressed apoptosis, and ameliorated the dermatitis. Inhibition of keratinocyte apoptosis by IFN-γ injection suppressed neutrophil, macrophage, and mast cell infiltration and the amount of TNF-α in the skin of cpdm mice. Similarly, IFN-α also enhanced the amount of HOIP as well as NF-κB activation, inhibited apoptosis, and ameliorated cpdm dermatitis. These results indicate that the IFNs enhance NF-κB activation and ameliorate cpdm dermatitis by augmenting expression of HOIP and HOIL-1L and linear polyubiquitination activity of LUBAC.

Suppression of LUBAC-mediated linear ubiquitination by a specific interaction between LUBAC and the deubiquitinases CYLD and OTULIN.

Linear ubiquitin chains generated by the linear ubiquitin chain assembly complex (LUBAC) play an important role in NF-κB activation. However, the regulation of linear ubiquitin chain generation by LUBAC is not well characterized. Here, we identified two deubiquitinating enzymes (DUBs), ovarian tumor DUB with linear linkage specificity (OTULIN/Gumby/FAM105B) and cylindromatosis (CYLD) that can cleave linear polyubiquitin chains and interact with LUBAC via the N-terminal PNGase/UBA or UBX (PUB) domain of HOIP, a catalytic subunit of LUBAC. HOIP interacts with both CYLD and OTULIN even in unstimulated cells. The interaction of CYLD and OTULIN with HOIP synergistically suppresses LUBAC-mediated linear polyubiquitination and NF-κB activation. Moreover, introduction of a HOIP mutant unable to bind either deubiquitinase into HOIP-null cells augments the activation of NF-κB by TNF-α stimulation. Thus, the interactions between these two deubiquitinases and the LUBAC ubiquitin ligase are involved in controlling the extent of TNF-α-induced NF-κB activation in cells by fine-tuning the generation of linear ubiquitin chains by LUBAC. The interaction of HOIP with OTULIN is also involved in OTULIN suppressing the canonical Wnt signaling pathway activation by LUBAC. Our observations provide molecular insights into the roles of ligase-deubiquitinase interactions in regulating molecular events resulting from linear ubiquitin conjugation.

IgG1 B cell receptor signaling is inhibited by CD22 and promotes the development of B cells whose survival is less dependent on Ig alpha/beta.

We describe a mouse strain in which B cell development relies either on the expression of membrane-bound immunoglobulin (Ig) gamma1 or mu heavy chains. Progenitor cells expressing gamma1 chains from the beginning generate a peripheral B cell compartment of normal size with all subsets, but a partial block is seen at the pro- to pre-B cell transition. Accordingly, gamma1-driven B cell development is disfavored in competition with developing B cells expressing a wild-type (WT) IgH locus. However, the mutant B cells display a long half-life and accumulate in the mature B cell compartment, and even though partial truncation of the Ig alpha cytoplasmic tail compromises their development, it does not affect their maintenance, as it does in WT cells. IgG1-expressing B cells showed an enhanced Ca(2+) response upon B cell receptor cross-linking, which was not due to a lack of inhibition by CD22. The enhanced Ca(2+) response was also observed in mature B cells that had been switched from IgM to IgG1 expression in vivo. Collectively, these results suggest that the gamma1 chain can exert a unique signaling function that can partially replace that of the Ig alpha/beta heterodimer in B cell maintenance and may contribute to memory B cell physiology.

B-cell depletion reactivates B lymphopoiesis in the BM and rejuvenates the B lineage in aging.

Aging is associated with a decline in B-lymphopoiesis in the bone marrow and accumulation of long-lived B cells in the periphery. These changes decrease the body's ability to mount protective antibody responses. We show here that age-related changes in the B lineage are mediated by the accumulating long-lived B cells. Thus, depletion of B cells in old mice was followed by expansion of multipotent primitive progenitors and common lymphoid progenitors, a revival of B-lymphopoiesis in the bone marrow, and generation of a rejuvenated peripheral compartment that enhanced the animal's immune responsiveness to antigenic stimulation. Collectively, our results suggest that immunosenescence in the B-lineage is not irreversible and that depletion of the long-lived B cells in old mice rejuvenates the B-lineage and enhances immune competence.

Hyperactivation of nuclear factor of activated T cells 1 (NFAT1) in T cells attenuates severity of murine autoimmune encephalomyelitis.

Nuclear factor of activated T cells (NFAT) proteins are a group of Ca(2+)-regulated transcription factors residing in the cytoplasm of resting cells. Dephosphorylation by calcineurin results in nuclear translocation of NFAT and subsequent expression of target genes; rephosphorylation by kinases, including casein kinase 1 (CK1), restores NFAT to its latent state in the cytoplasm. We engineered a hyperactivable version of NFAT1 with increased affinity for calcineurin and decreased affinity for casein kinase 1. Mice expressing hyperactivable NFAT1 in their T-cell compartment exhibited a dramatically increased frequency of both IL-17- and IL-10-producing cells after differentiation under Th17 conditions-this was associated with direct binding of NFAT1 to distal regulatory regions of Il-17 and Il-10 gene loci in Th17 cells. Despite higher IL-17 production in culture, the mice were significantly less prone to myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalomyelitis than controls, correlating with increased production of the immunomodulatory cytokine IL-10 and enhanced accumulation of regulatory T cells within the CNS. Thus, NFAT hyperactivation paradoxically leads to decreased susceptibility to experimental autoimmune encephalomyelitis, supporting previous observations linking defects in Ca(2+)/NFAT signaling to lymphoproliferation and autoimmune disease.

Store-operated Ca2+ entry through ORAI1 is critical for T cell-mediated autoimmunity and allograft rejection.

ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.

Requirement for balanced Ca/NFAT signaling in hematopoietic and embryonic development.

NFAT transcription factors are highly phosphorylated proteins residing in the cytoplasm of resting cells. Upon dephosphorylation by the phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. NFAT is rephosphorylated and inactivated through the concerted action of at least 3 different kinases: CK1, GSK-3, and DYRK. The major docking sites for calcineurin and CK1 are strongly conserved throughout vertebrate evolution, and conversion of either the calcineurin docking site to a high-affinity version or the CK1 docking site to a low-affinity version results in generation of hyperactivable NFAT proteins that are still fully responsive to stimulation. In this study, we generated transgenic mice expressing hyperactivable versions of NFAT1 from the ROSA26 locus. We show that hyperactivable NFAT increases the expression of NFAT-dependent cytokines by differentiated T cells as expected, but exerts unexpected signal-dependent effects during T cell differentiation in the thymus, and is progressively deleterious for the development of B cells from hematopoietic stem cells. Moreover, progressively hyperactivable versions of NFAT1 are increasingly deleterious for embryonic development, particularly when normal embryos are also present in utero. Forced expression of hyperactivable NFAT1 in the developing embryo leads to mosaic expression in many tissues, and the hyperactivable proteins are barely tolerated in organs such as brain, and cardiac and skeletal muscle. Our results highlight the need for balanced Ca/NFAT signaling in hematopoietic stem cells and progenitor cells of the developing embryo, and emphasize the evolutionary importance of kinase and phosphatase docking sites in preventing inappropriate activation of NFAT.

BAFF activates Akt and Erk through BAFF-R in an IKK1-dependent manner in primary mouse B cells.

B cell activating factor (BAFF) signals through BAFF-R to promote mature B cell survival. Recent analyses of BAFF-induced signaling revealed direct association between augmented B cell metabolic fitness and activation of Akt, one of the key regulators of cell survival. The strongest and most reproducible induction of Akt occurs with significant delay (24 h) after BAFF treatment, where it precedes activation of anabolism. It was also recently shown that BAFF induces sustained Erk activation and increased turnover of the proapoptotic molecule Bim. Here we show that these BAFF-induced signaling pathways are mediated by BAFF-R and represent previously unknown arms of I kappa B kinase (IKK)1-dependent signaling. In combination with the known role of IKK1 in regulating transcription of prosurvival genes, our data underscore the central role of IKK1 in coordinating multiple BAFF-R-mediated signaling pathways controlling mature B cell homeostasis.

NIK overexpression amplifies, whereas ablation of its TRAF3-binding domain replaces BAFF:BAFF-R-mediated survival signals in B cells.

BAFF-R-dependent activation of the alternative NF-kappaB pathway plays an essential role in mature B cell survival. Mutations leading to overexpression of NIK and deletion of the TRAF3 gene are implicated in human multiple myeloma. We show that overexpression of NIK in mouse B lymphocytes amplifies alternative NF-kappaB activation and peripheral B cell numbers in a BAFF-R-dependent manner, whereas uncoupling NIK from TRAF3-mediated control causes maximal p100 processing and dramatic hyperplasia of BAFF-R-independent B cells. NIK controls alternative NF-kappaB signaling by increasing the protein levels of its negative regulator TRAF3 in a dose-dependent fashion. This mechanism keeps NIK protein levels below detection even when they cause B cell hyperplasia, so that contributions of NIK to B cell pathologies can easily be overlooked.

Neutrophil-to-Lymphocyte and Platelet-to-Lymphocyte Ratios as Noninvasive Predictors of the Therapeutic Outcomes of Systemic Corticosteroid Therapy in Ulcerative Colitis.

INTRODUCTION: Predictive biomarkers for the therapeutic outcome of induction therapy with systemic corticosteroid for active ulcerative colitis (UC) have not been established. This study aimed to investigate whether neutrophil-to-lymphocyte ratio (NLR) and/or platelet-to-lymphocyte ratio (PLR) can be predictive biomarkers for the therapeutic outcomes of systemic corticosteroid therapy in UC. METHODS: This was a single-center retrospective cohort study. In total, 48 patients with UC who received induction therapy with systemic corticosteroid were enrolled. Based on the achievement of clinical remission after 8 weeks of treatment, the patients were divided into the remission group (n = 28) and the nonremission group (n = 20). Clinical characteristics, NLR, and PLR at baseline between the remission and nonremission groups were compared via a univariate analysis. The independent risk factors of nonremission were identified via a multivariate analysis. RESULTS: The baseline Mayo score, platelet count, lymphocyte count, C-reactive protein (CRP) levels, NLR, and PLR between the 2 groups significantly differed. The nonremission group had higher NLR and PLR than the remission group (4.70 [3.04-11.3] vs. 3.10 [1.36-16.42]; p < 0.05, and 353.6 [220.3-499.8] vs. 207.2 [174.4-243.6]; p < 0.001, respectively). A multivariate analysis revealed that a Mayo score of ≥9, CRP level of ≥1.26 mg/dL, and PLR of ≥262 (hazard ratio: 23.1, 95% confidence interval: 1.29-413.7, p = 0.033) were considered independent risk factors for nonremission. CONCLUSION: This report first identified the efficacy of NLR and PLR as candidate biomarkers for predicting the therapeutic outcomes of systemic corticosteroid therapy in UC.

Suggestive Diagnostic Process in a Case of Multiple Myeloma with Gastrointestinal Immunoglobulin Light-Chain Amyloidosis Accompanied by Protein-Losing Enteropathy.

Multiple myeloma is a type of plasma cell neoplasm that produces monoclonal immunoglobulin. Multiple myeloma is known to cause immunoglobulin light-chain (AL) amyloidosis, which frequently involves the kidney and heart. Bone pain or fractures caused by osteolytic lesions and physical disorders related to renal or cardiac AL amyloidosis are major initial symptoms in multiple myeloma. Multiple myeloma diagnosed from the gastrointestinal symptoms is rare. We report a case of an 80-year-old man with multiple myeloma accompanied by gastrointestinal AL amyloidosis and secondary protein-losing enteropathy. The diagnostic process was suggestive, in that diarrhea and refractory leg edema related to protein-losing enteropathy were the primary symptoms and the trigger for making a sequential diagnosis of gastrointestinal AL amyloidosis and underlying multiple myeloma. This case is highly suggestive, in that multiple myeloma with gastrointestinal AL amyloidosis should be considered one of the background diseases of protein-losing enteropathy.

Presymptomatic Crohn's Disease in a Young Patient Diagnosed Just After the Onset of Idiopathic Acute Pancreatitis.

Acute pancreatitis is an extraintestinal manifestation of inflammatory bowel disease. There have been few reports describing acute pancreatitis preceding a diagnosis of inflammatory bowel disease. We herein report a rare case of a 16-year-old boy with presymptomatic Crohn's disease that was newly diagnosed just after the onset of idiopathic acute pancreatitis. Crohn's disease of any stage, much less in the presymptomatic stage, is rarely diagnosed just after the development of acute pancreatitis. The present case suggests that acute pancreatitis without an apparent cause in young or pediatric population can precede a diagnosis of presymptomatic Crohn's disease.