Editorial: The Role of Optineurin in Immunity and Immune-Mediated Diseases.

The multifunctional adaptor optineurin has been implicated in an increasing number of protein-protein interactions and cellular functions ever since its first identification as a binding partner for an adenoviral protein (1). Most—if not all—optineurin functions require its ubiquitin-binding domain in its C-terminus, which binds to K63- and/or M1-polyubiquinated proteins, allowing it to act, for example, as an adaptor during inflammatory signaling, autophagy, and vesicle trafficking (2–4). The interest in optineurin intensified after the identification of various mutations and polymorphisms in several human diseases, including primary open-angle glaucoma, amyotrophic lateral sclerosis (ALS), Paget's disease of the bone, and Crohn's disease. With their distinct yet unresolved pathogenesis, and complex genetic and environmental risk factors, these diseases seem unrelated at first. ALS, glaucoma, or Paget's disease are not traditionally regarded as immune-mediated diseases; however, the emerging evidence pinpoints immune system disfunction as their common denominator (5, 6). The aim of this Research Topic was to explore the role(s) of optineurin on a host of diverse cellular pathways that are directly or indirectly linked to the immune response. The articles cover immune signaling, cell death, membrane trafficking, autophagy of intracellular bacteria (xenophagy), damaged mitochondria (mitophagy), and protein aggregates.AS thanks the Medical Research Council (MR/L000261/1) for financial support. FB thanks the Medical Research Council (MR/K000888/1 and MR/N000048/1) and the BBSRC (BB/R001316/1). IM thanks the Croatian Science Foundation (IP-2018-01-8563) and the support of the University of Rijeka (18-211-1369)

Optineurin Dysfunction in Amyotrophic Lateral Sclerosis: Why So Puzzling?

Mutations in optineurin have been linked to amyotrophic lateral sclerosis (ALS) a decade ago, but its exact role in the neurodegenerative process is still unclear. As a lysine 63 (K63) and methionine (M1) poly-ubiquitin binding protein, optineurin has been reported to act as an adaptor in inflammatory signaling pathways mediated via nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and interferon regulatory factor 3 (IRF3), as well as in membrane-associated trafficking events including autophagy, maintenance of the Golgi apparatus, and exocytosis. Other studies have demonstrated its role in other processes such as regulation of mitosis, transcription, necroptosis and apoptosis. However, many of the reported effects in cell models have been proven difficult to reproduce in optineurin animal models, demonstrating the challenges of extrapolation between model systems. Knowing that multifunctional proteins present a “nightmare” for researchers, to help navigating through this field, we address the most common controversies, open questions, and artefacts related to optineurin and its role in pathogenesis of ALS and other neurodegenerative diseases.</p

Investigating the role of optineurin in bone biology and Paget's disease of bone

Paget’s disease of bone (PDB) is a common disease with a strong genetic component. Approaches such as linkage analysis and candidate gene studies have shown that mutations in Sequestosome 1 (SQSTM1) explain up to 40% of familial cases and 10% of sporadic cases, however the majority of PDB patients have no mutations in this gene. Genome-wide association studies (GWAS) have recently identified new susceptibility loci for PDB including variants at CSF1, TNFRSF11A, OPTN, TM7SF4, PML, NUP205 and RIN3 loci. These loci were confirmed to be associated with PDB in various European populations. OPTN encodes optineurin, a widely expressed protein involved in many cellular processes but its role in bone metabolism is yet unknown. The aim of this PhD thesis was to investigate the role of OPTN in bone metabolism and PDB using in vitro and in vivo studies. In chapter 3, the OPTN rs1561570 identified by previous GWAS was examined for its association with the severity and clinical outcome of PDB in patients without SQSTM1 mutations. The results showed that rs1561570 was significantly associated with total disease severity score so that carriers of the risk allele “T” had higher severity score compared to non-carriers (P < 0.05). A trend for reduced quality of life physical scores (SF36) was also associated with the rs1561570 risk allele, but the relationship was not statistically significant. In order to identify functional variants within OPTN, the coding regions as well as the exon-intron boundaries were sequenced in 24 familial PDB cases and 19 controls. No mutation was found that could be predicted as pathogenic suggesting that disease susceptibility could be mediated by regulatory polymorphisms that influence gene expression. In chapter 4, the role of OPTN was investigated in osteoclast development using in vitro knockdown experiments. Optn was expressed in mouse bone marrow derived macrophages (BMDMs) as well as all stages of osteoclast development and it was significantly increased three days post RANKL treatment. Optn expression was knocked down in BMDMs and cells were induced to form osteoclast in the presence of RANKL and M-CSF. Compared to non-targeted cells, Optn depleted cells formed significantly more and larger osteoclasts (P< 0.05). Optn knockdown was also found to enhance osteoclast survival as well as RANKL-induced NFκB activation. In chapter 5, the role of OPTN was investigated in vitro from cells obtained from knock in mice with a loss-of-function mutation in Optn (OptnD477N/D477N). In agreement with the in vitro knockdown experiments, osteoclasts were significantly higher and larger in mutant mice compared to WT and the NF-B activity measured by luciferase reporter assay was significantly higher in cells from OptnD477N/D477N compared to WT during most stages of osteoclast development. OPTN from mutant and WT mice was co-precipitated with its CYLD binding-partner, which acts as a negative regulator to RANK signalling by inhibiting the TRAF6 downstream signalling. The data from this immunoprecipitation (IP) experiment revealed that defective OPTN interacted less with CYLD from mutant mice compared to WT. This study also showed that OPTN was expressed in osteoblasts and the expression rate did not change during osteoblast development. The data obtained from the mineralization assay revealed no significant difference between OptnD477N/D477N and WT. In chapter 6, I investigated the effect of the D477N loss of function mutation in Optn on bone metabolism. Bone Histomorphometrical analysis of OptnD477N/D477N mice showed higher bone resorption parameters (Oc.N/BS and Oc.S/BS) compared to wild type (WT). Osteoid analysis showed evidence of increased bone formation parameters (OS/BS and OV/BV) in mutant mice compared to WT. Calcein labelling showed a significant difference in mineral apposition rate (MAR) from mutant mice compared to WT. Analysis of serum biomarkers of bone turnover showed evidence of enhanced bone turnover in mutant mice compared to WT. Micro computed tomography (μCT) analysis of 4 and 14 months old mice showed no significant differences in bone morphology between WT and OptnD477N/D477N mice of both sexes. In conclusion, this study has shown for the first time that OPTN plays a role in regulating bone turnover by acting as a negative regulator of osteoclast differentiation. The data obtained from this study strongly suggest the crucial role of OPTN in RANK signalling. The effect of OPTN on osteoblast activity may be direct or indirect compensation for increased osteoclast activity. Further detailed studies will be required to explore the underlying mechanism of OPTN including downstream RANK signalling and a complete knockout model to corroborate these findings

A Role for the Immediate Early Gene Product c-fos in Imprinting T Cells with Short-Term Memory for Signal Summation

T cells often make sequential contacts with multiple DCs in the lymph nodes and are likely to be equipped with mechanisms that allow them to sum up the successive signals received. We found that a period of stimulation as short as two hours could imprint on a T cell a “biochemical memory” of that activation signal that persisted for several hours. This was evidenced by more rapid induction of activation markers and earlier commitment to proliferation upon subsequent stimulation, even when that secondary stimulation occurred hours later. Upregulation of the immediate early gene product c-fos, a component of the AP-1 transcription factor, was maximal by 1–2 hours of stimulation, and protein levels remained elevated for several hours after stimulus withdrawal. Moreover, phosphorylated forms of c-fos that are stable and transcriptionally active persisted for a least a day. Upon brief antigenic stimulation in vivo, we also observed a rapid upregulation of c-fos that could be boosted by subsequent stimulation. Accumulation of phosphorylated c-fos may therefore serve as a biochemical fingerprint of previous suboptimal stimulation, leaving the T cell poised to rapidly resume its activation program upon its next encounter with an antigen-bearing DC

CSL–MAML-dependent Notch1 signaling controls T lineage–specific IL-7Rα gene expression in early human thymopoiesis and leukemia

Notch1 activation is essential for T-lineage specification of lymphomyeloid progenitors seeding the thymus. Progression along the T cell lineage further requires cooperative signaling provided by the interleukin 7 receptor (IL-7R), but the molecular mechanisms responsible for the dynamic and lineage-specific regulation of IL-7R during thymopoiesis are unknown. We show that active Notch1 binds to a conserved CSL-binding site in the human IL7R gene promoter and critically regulates IL7R transcription and IL-7R α chain (IL-7Rα) expression via the CSL–MAML complex. Defective Notch1 signaling selectively impaired IL-7Rα expression in T-lineage cells, but not B-lineage cells, and resulted in a compromised expansion of early human developing thymocytes, which was rescued upon ectopic IL-7Rα expression. The pathological implications of these findings are demonstrated by the regulation of IL-7Rα expression downstream of Notch1 in T cell leukemias. Thus, Notch1 controls early T cell development, in part by regulating the stage- and lineage-specific expression of IL-7Rα

Assessing Sustainability in the Shipbuilding Supply Chain 4.0: A Systematic Review

The supply chain is currently taking on a very important role in organizations seeking to improve the competitiveness and profitability of the company. Its transversal character mainly places it in an unbeatable position to achieve this role. This article, through a study of each of the key enabling technologies of Industry 4.0, aims to obtain a general overview of the current state of the art in shipbuilding adapted to these technologies. To do so, a systematic review of what the scientific community says is carried out, dividing each of the technologies into different categories. In addition, the global vision of countries interested in each of the enabling technologies is also studied. Both studies present a general vision to the companies of the concerns of the scientific community, thus encouraging research on the subject that is focused on the sustainability of the shipbuilding supply chain

Transcriptional Repressor Gfi1 Integrates Cytokine-Receptor Signals Controlling B-Cell Differentiation

Hematopoietic stem cell differentiation is specified by cytokines and transcription factors, but the mechanisms controlling instructive and permissive signalling networks are poorly understood. We provide evidence that CLP1-dependent IL7-receptor mediated B cell differentiation is critically controlled by the transcriptional repressor Gfi1. Gfi1-deficient progenitor B cells show global defects in IL7Rα-dependent signal cascades. Consequently, IL7-dependent trophic, proliferative and differentiation-inducing responses of progenitor B cells are perturbed. Gfi1 directly regulates expression levels of IL7Rα and indirectly controls STAT5 signalling via expression of SOCS3. Thus, Gfi1 selectively specifies IL7-dependent development of B cells from CLP1 progenitors, providing clues to the transcriptional networks integrating cytokine signals and lymphoid differentiation

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity

IL-7 Promotes CD95-Induced Apoptosis in B Cells via the IFN-γ/STAT1 Pathway

Interleukin-7 (IL-7) concentrations are increased in the blood of CD4+ T cell depleted individuals, including HIV-1 infected patients. High IL-7 levels might stimulate T cell activation and, as we have shown earlier, IL-7 can prime resting T cell to CD95 induced apoptosis as well. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 (Fas) death receptor pathway and loss of memory B cells. Peripheral B cells are not sensitive for IL-7, due to the lack of IL-7Ra expression on their surface; however, here we demonstrate that high IL-7 concentration can prime resting B cells to CD95-mediated apoptosis via an indirect mechanism. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-γ that T cells secreted in high amounts in response to IL-7. These results suggest that the lymphopenia induced cytokine IL-7 can contribute to the increased B cell apoptosis observed in HIV-1 infected individuals