The hypothetical molecular mechanism of the ethnic variations in the manifestation of age-related macular degeneration; focuses on the functions of the most significant susceptibility genes.

Age-related macular degeneration (AMD) is the leading sight-threatening disease in developed countries. On the other hand, recent studies indicated an ethnic variation in the phenotype of AMD. For example, several reports demonstrated that the incidence of drusen in AMD patients is less in Asians compared to Caucasians though the reason has not been clarified yet. In the last decades, several genome association studies have disclosed many susceptible genes of AMD and revealed that the association strength of some genes was different among races and AMD phenotypes. In this review article, the essential findings of the clinical studies and genome association studies for the most significant genes CFH and ARMS2/HTRA1 in AMD of different races are summarized, and theoretical hypotheses about the molecular mechanisms underlying the ethnic variation in the AMD manifestation mainly focused on those genes between Caucasians and Asians are discussed.

LRBA signalosomes activate vasopressin-induced AQP2 trafficking at recycling endosomes.

Aquaporin-2 (AQP2) water channels are proteins that are recycled between intracellular vesicles and the apical plasma membrane in renal collecting ducts. Lipopolysaccharide-responsive beige-like anchor protein (LRBA) is a protein kinase A (PKA) anchoring protein that creates compartmentalized PKA signalling responsible for AQP2 phosphorylation. In response to increased plasma osmolality, vasopressin/cyclic adenosine monophosphate (cAMP)/PKA signalling phosphorylates AQP2, promoting AQP2 trafficking into the apical plasma membrane and increasing water reabsorption from urine. However, the molecular mechanisms by which LRBA mediates vasopressin-induced AQP2 phosphorylation remain unknown. To investigate AQP2 intracellular localization and phosphorylation status in vivo, a density gradient ultracentrifugation technique was combined with an in situ proximity ligation assay, super-resolution structured illumination microscopy and immunoelectron microscopy. Most of the AQP2 was localized on the recycling endosome in the presence of tolvaptan, a vasopressin type 2 receptor (V2R) antagonist. Desmopressin, a V2R agonist, phosphorylated AQP2, translocating it from the recycling endosome to the apical plasma membrane. In contrast, LRBA was constitutively localized at the recycling endosome. Therefore, LRBA and AQP2 were well colocalized in the absence of vasopressin stimulation. The loss of LRBA/PKA signalling by Lrba knockout impaired vasopressin-induced AQP2 phosphorylation, resulting in AQP2 retention at the recycling endosome. Defective AQP2 trafficking caused low urinary concentrating ability in Lrba-/- mice. The LRBA-PKA complex created compartmentalized PKA signalling at the recycling endosome, which facilitated AQP2 phosphorylation in response to vasopressin. KEY POINTS: Membrane proteins are continuously internalized into the endosomal system via endocytosis, after which they are either recycled back to the plasma membrane or degraded at the lysosome. In T cells, lipopolysaccharide-responsive beige-like anchor protein (LRBA) binds directly to the cytotoxic T lymphocyte antigen 4 (CTLA-4), a checkpoint immune molecule, to prevent CTLA-4 lysosomal degradation and promote its vesicle recycling. LRBA has different physiological functions in renal collecting ducts. LRBA and aquaporin-2 (AQP2) water channels were colocalized on the recycling endosome in vivo in the absence of the anti-diuretic hormone vasopressin. LRBA promoted vasopressin-induced AQP2 trafficking, increasing water reabsorption from urine via AQP2. LRBA determined renal responsiveness to vasopressin at recycling endosomes. LRBA is a ubiquitously expressed anchor protein. LRBA signalosomes might regulate membrane trafficking of several constitutively recycled proteins at recycling endosomes.

Human skin gas profile of individuals with the people allergic to me phenomenon.

Recent studies have shown that some people claim that their skin gases provoke allergy-like reactions in people in their near vicinity. Such a phenomenon or symptom is called 'people allergic to me (PATM)'. Although numerous people suffer from PATM, the actual conditions are unknown. The aim of this study was to investigate the characteristics of human skin profiles in patients with PATM by measuring the dermal emission fluxes of 75 skin gases using passive flux sampler and gas chromatography/mass spectrometry. We found common features in the human skin gas profiles of 20 subjects with PATM, with a significant difference from those of 24 non-PATM subjects: greater emissions of petrochemicals, organosulfur compounds, and some aldehydes and lower emissions of aroma compounds and others. The ratio of toluene to benzaldehyde is considered a vital sign that suggests the fundamental of PATM. These findings indicate that PATM is a medically unexplained phenomenon or symptom worthy of further research, which requires an interdisciplinary approach.

Optineurin deficiency impairs autophagy to cause interferon beta overproduction and increased survival of mice following viral infection.

BACKGROUND: Optineurin (OPTN) is associated with several human diseases, including amyotrophic lateral sclerosis (ALS), and is involved in various cellular processes, including autophagy. Optineurin regulates the expression of interferon beta (IFNß), which plays a central role in the innate immune response to viral infection. However, the role of optineurin in response to viral infection has not been fully clarified. It is known that optineurin-deficient cells produce more IFNß than wild-type cells following viral infection. In this study, we investigate the reasons for, and effects of, IFNß overproduction during optineurin deficiency both in vitro and in vivo. METHODS: To investigate the mechanism of IFNß overproduction, viral nucleic acids in infected cells were quantified by RT-qPCR and the autophagic activity of optineurin-deficient cells was determined to understand the basis for the intracellular accumulation of viral nucleic acids. Moreover, viral infection experiments using optineurin-disrupted (Optn-KO) animals were performed with several viruses. RESULTS: IFNß overproduction following viral infection was observed not only in several types of optineurin-deficient cell lines but also in Optn-KO mice and human ALS patient cells carrying mutations in OPTN. IFNß overproduction in Optn-KO cells was revealed to be caused by excessive accumulation of viral nucleic acids, which was a consequence of reduced autophagic activity caused by the loss of optineurin. Additionally, IFNß overproduction in Optn-KO mice suppressed viral proliferation, resulting in increased mouse survival following viral challenge. CONCLUSION: Our findings indicate that the combination of optineurin deficiency and viral infection leads to IFNß overproduction in vitro and in vivo. The effects of optineurin deficiency are elicited by viral infection, therefore, viral infection may be implicated in the development of optineurin-related diseases.

Involvement of heterologous ubiquitination including linear ubiquitination in Alzheimer's disease and amyotrophic lateral sclerosis.

In neurodegenerative diseases such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS), the progressive accumulation of ubiquitin-positive cytoplasmic inclusions leads to proteinopathy and neurodegeneration. Along with the seven types of Lys-linked ubiquitin chains, the linear ubiquitin chain assembly complex (LUBAC)-mediated Met1-linked linear ubiquitin chain, which activates the canonical NF-κB pathway, is also involved in cytoplasmic inclusions of tau in AD and TAR DNA-binding protein 43 in ALS. Post-translational modifications, including heterologous ubiquitination, affect proteasomal and autophagic degradation, inflammatory responses, and neurodegeneration. Single nucleotide polymorphisms (SNPs) in SHARPIN and RBCK1 (which encodes HOIL-1L), components of LUBAC, were recently identified as genetic risk factors of AD. A structural biological simulation suggested that most of the SHARPIN SNPs that cause an amino acid replacement affect the structure and function of SHARPIN. Thus, the aberrant LUBAC activity is related to AD. Protein ubiquitination and ubiquitin-binding proteins, such as ubiquilin 2 and NEMO, facilitate liquid-liquid phase separation (LLPS), and linear ubiquitination seems to promote efficient LLPS. Therefore, the development of therapeutic approaches that target ubiquitination, such as proteolysis-targeting chimeras (PROTACs) and inhibitors of ubiquitin ligases, including LUBAC, is expected to be an additional effective strategy to treat neurodegenerative diseases.

Pleiotropic Roles of a KEAP1-Associated Deubiquitinase, OTUD1.

Protein ubiquitination, which is catalyzed by ubiquitin-activating enzymes, ubiquitin-conjugating enzymes, and ubiquitin ligases, is a crucial post-translational modification to regulate numerous cellular functions in a spatio-temporal-specific manner. The human genome encodes ~100 deubiquitinating enzymes (DUBs), which antagonistically regulate the ubiquitin system. OTUD1, an ovarian tumor protease (OTU) family DUB, has an N-terminal-disordered alanine-, proline-, glycine-rich region (APGR), a catalytic OTU domain, and a ubiquitin-interacting motif (UIM). OTUD1 preferentially hydrolyzes lysine-63-linked ubiquitin chains in vitro; however, recent studies indicate that OTUD1 cleaves various ubiquitin linkages, and is involved in the regulation of multiple cellular functions. Thus, OTUD1 predominantly functions as a tumor suppressor by targeting p53, SMAD7, PTEN, AKT, IREB2, YAP, MCL1, and AIF. Furthermore, OTUD1 regulates antiviral signaling, innate and acquired immune responses, and cell death pathways. Similar to Nrf2, OTUD1 contains a KEAP1-binding ETGE motif in its APGR and regulates the reactive oxygen species (ROS)-mediated oxidative stress response and cell death. Importantly, in addition to its association with various cancers, including multiple myeloma, OTUD1 is involved in acute graft-versus-host disease and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and ulcerative colitis. Thus, OTUD1 is an important DUB as a therapeutic target for a variety of diseases.

Spinocerebellar ataxia type 17-digenic TBP/STUB1 disease: neuropathologic features of an autopsied patient.

Spinocerebellar ataxia (SCA) type 17-digenic TBP/STUB1 disease (SCA17-DI) has been recently segregated from SCA17, caused by digenic inheritance of two gene mutations - intermediate polyglutamine-encoding CAG/CAA repeat expansions (polyQ) in TBP (TBP41 - 49) and STUB1 heterozygosity - the former being associated with SCA17, and the latter with SCA48 and SCAR16 (autosomal recessive). In SCA17, most patients carry intermediate TBP41 - 49 alleles but show incomplete penetrance, and the missing heritability can be explained by a new entity whereby TBP41 - 49 requires the STUB1 variant to be symptomatic. The STUB1 gene encodes the chaperone-associated E3 ubiquitin ligase (CHIP) involved in ubiquitin-mediated proteasomal control of protein homeostasis. However, reports of the neuropathology are limited and role of STUB1 mutations in SCA17-DI remain unknown. Here we report the clinicopathologic features of identical twin siblings, one of whom was autopsied and was found to carry an intermediate allele (41 and 38 CAG/CAA repeats) in TBP and a heterozygous missense mutation in STUB1 (p.P243L). These patients developed autosomal recessive Huntington's disease-like symptoms. Brain MRI showed diffuse atrophy of the cerebellum and T2WI revealed hyperintense lesions in the basal ganglia and periventricular deep white matter. The brain histopathology of the patient shared features characteristic of SCA17, such as degeneration of the cerebellar cortex and caudate nucleus, and presence of 1C2-positive neurons. Here we show that mutant CHIP fails to generate the polyubiquitin chain due to disrupted folding of the entire U box domain, thereby affecting the E3 activity of CHIP. When encountering patients with cerebellar ataxia, especially those with Huntington's disease-like symptoms, genetic testing for STUB1 as well as TBP should be conducted for diagnosis of SCA17-DI, even in cases of sporadic or autosomal recessive inheritance.

Identification and molecular analysis of RNF31 Q622H germline polymorphism.

The linear ubiquitin chain assembly complex (LUBAC), which is composed of RING finger protein 31 (RNF31), RANBP2-type and C3HC4-type zinc finger containing 1 and SHANK-associated RH domain interactor subunits, is the only ubiquitin ligase to generate Met1-linked linear ubiquitin chains. Linear ubiquitin chains regulate canonical NF-κB activation and cell death. Single nucleotide polymorphisms in RNF31, such as Q584H and Q622L, are known to cause the activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL) because of enhanced LUBAC-mediated NF-κB activation. The present study identified a novel Q622H polymorphism of RNF31 in two patients with lung cancer, one of whom had concurrent ABC-DLBCL. Immunohistochemical analyses revealed that although the expression of RNF31 was elevated in both patients, only the ABC-DLBCL specimen showed increased NF-κB activation. Cancer panel analysis showed that the Q622H-related ABC-DLBCL did not harbor co-mutations that were previously reported in Q584H-/Q622L-related ABC-DLBCL. Furthermore, in contrast to Q584H and Q622L, Q622H showed no enhancement effects on LUBAC and NF-κB activity in vitro compared with wild-type RNF31. The present study's structural prediction suggested that the electrostatic interaction related to the Q622 residue may not have had an important role in LUBAC formation. In conclusion, the molecular mechanism and mutational background of RNF31 Q622H differed from that of RNF31 Q584H or Q622L. Furthermore, RNF31 Q622H appeared not to induce NF-κB activation in lung cancer.

Suppression of Linear Ubiquitination Ameliorates Cytoplasmic Aggregation of Truncated TDP-43.

TAR DNA-binding protein 43 (TDP-43) is a predominant component of inclusions in the brains and spines of patients with amyotrophic lateral sclerosis (ALS). The progressive accumulation of inclusions leads to proteinopathy in neurons. We have previously shown that Met1(M1)-linked linear ubiquitin, which is specifically generated by the linear ubiquitin chain assembly complex (LUBAC), is colocalized with TDP-43 inclusions in neurons from optineurin-associated familial and sporadic ALS patients, and affects NF-κB activation and apoptosis. To examine the effects of LUBAC-mediated linear ubiquitination on TDP-43 proteinopathies, we performed cell biological analyses using full-length and truncated forms of the ALS-associated Ala315→Thr (A315T) mutant of TDP-43 in Neuro2a cells. The truncated A315T mutants of TDP-43, which lack a nuclear localization signal, efficiently generated cytoplasmic aggregates that were colocalized with multiple ubiquitin chains such as M1-, Lys(K)48-, and K63-chains. Genetic ablation of HOIP or treatment with a LUBAC inhibitor, HOIPIN-8, suppressed the cytoplasmic aggregation of A315T mutants of TDP-43. Moreover, the enhanced TNF-α-mediated NF-κB activity by truncated TDP-43 mutants was eliminated in the presence of HOIPIN-8. These results suggest that multiple ubiquitinations of TDP-43 including M1-ubiquitin affect protein aggregation and inflammatory responses in vitro, and therefore, LUBAC inhibition ameliorates TDP-43 proteinopathy.

OTUD1 deubiquitinase regulates NF-κB- and KEAP1-mediated inflammatory responses and reactive oxygen species-associated cell death pathways.

Deubiquitinating enzymes (DUBs) regulate numerous cellular functions by removing ubiquitin modifications. We examined the effects of 88 human DUBs on linear ubiquitin chain assembly complex (LUBAC)-induced NF-κB activation, and identified OTUD1 as a potent suppressor. OTUD1 regulates the canonical NF-κB pathway by hydrolyzing K63-linked ubiquitin chains from NF-κB signaling factors, including LUBAC. OTUD1 negatively regulates the canonical NF-κB activation, apoptosis, and necroptosis, whereas OTUD1 upregulates the interferon (IFN) antiviral pathway. Mass spectrometric analysis showed that OTUD1 binds KEAP1, and the N-terminal intrinsically disordered region of OTUD1, which contains an ETGE motif, is indispensable for the KEAP1-binding. Indeed, OTUD1 is involved in the KEAP1-mediated antioxidant response and reactive oxygen species (ROS)-induced cell death, oxeiptosis. In Otud1-/--mice, inflammation, oxidative damage, and cell death were enhanced in inflammatory bowel disease, acute hepatitis, and sepsis models. Thus, OTUD1 is a crucial regulator for the inflammatory, innate immune, and oxidative stress responses and ROS-associated cell death pathways.

Cloaking the ACE2 receptor with salivary cationic proteins inhibits SARS-CoV-2 entry.

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

LRBA is essential for urinary concentration and body water homeostasis.

Protein kinase A (PKA) directly phosphorylates aquaporin-2 (AQP2) water channels in renal collecting ducts to reabsorb water from urine for the maintenance of systemic water homeostasis. More than 50 functionally distinct PKA-anchoring proteins (AKAPs) respectively create compartmentalized PKA signaling to determine the substrate specificity of PKA. Identification of an AKAP responsible for AQP2 phosphorylation is an essential step toward elucidating the molecular mechanisms of urinary concentration. PKA activation by several compounds is a novel screening strategy to uncover PKA substrates whose phosphorylation levels were nearly perfectly correlated with that of AQP2. The leading candidate in this assay proved to be an AKAP termed lipopolysaccharide-responsive and beige-like anchor protein (LRBA). We found that LRBA colocalized with AQP2 in vivo, and Lrba knockout mice displayed a polyuric phenotype with severely impaired AQP2 phosphorylation. Most of the PKA substrates other than AQP2 were adequately phosphorylated by PKA in the absence of LRBA, demonstrating that LRBA-anchored PKA preferentially phosphorylated AQP2 in renal collecting ducts. Furthermore, the LRBA-PKA interaction, rather than other AKAP-PKA interactions, was robustly dissociated by PKA activation. AKAP-PKA interaction inhibitors have attracted attention for their ability to directly phosphorylate AQP2. Therefore, the LRBA-PKA interaction is a promising drug target for the development of anti-aquaretics.

Cytoglobin attenuates pancreatic cancer growth via scavenging reactive oxygen species.

Pancreatic cancer is a highly challenging malignancy with extremely poor prognosis. Cytoglobin (CYGB), a hemeprotein involved in liver fibrosis and cancer development, is expressed in pericytes of all organs. Here, we examined the role of CYGB in the development of pancreatic cancer. CYGB expression appeared predominately in the area surrounding adenocarcinoma and negatively correlated with tumor size in patients with pancreatic cancer. Directly injecting 7, 12-dimethylbenz[a]anthracene into the pancreatic tail in wild-type mice resulted in time-dependent induction of severe pancreatitis, fibrosis, and oxidative damage, which was rescued by Cygb overexpression in transgenic mice. Pancreatic cancer incidence was 93% in wild-type mice but only 55% in transgenic mice. Enhanced CYGB expression in human pancreatic stellate cells in vitro reduced cellular collagen synthesis, inhibited cell activation, increased expression of antioxidant-related genes, and increased CYGB secretion into the medium. Cygb-overexpressing or recombinant human CYGB (rhCYGB) -treated MIA PaCa-2 cancer cells exhibited dose-dependent cell cycle arrest at the G1 phase, diminished cell migration, and reduction in colony formation. RNA sequencing in rhCYGB-treated MIA PaCa-2 cells revealed downregulation of cell cycle and oxidative phosphorylation pathways. An increase in MIA PaCa-2 cell proliferation and reactive oxygen species production by H2O2 challenge was blocked by rhCYGB treatment or Cygb overexpression. PANC-1, OCUP-A2, and BxPC-3 cancer cells showed similar responses to rhCYGB. Known antioxidants N-acetyl cysteine and glutathione also inhibited cancer cell growth. These results demonstrate that CYGB suppresses pancreatic stellate cell activation, pancreatic fibrosis, and tumor growth, suggesting its potential therapeutic application against pancreatic cancer.

Capacity of extracellular globins to reduce liver fibrosis via scavenging reactive oxygen species and promoting MMP-1 secretion.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) are the primary cell type in liver fibrosis, a significant global health care burden. Cytoglobin (CYGB), a globin family member expressed in HSCs, inhibits HSC activation and reduces collagen production. We studied the antifibrotic properties of globin family members hemoglobin (HB), myoglobin (MB), and neuroglobin (NGB) in comparison with CYGB. APPROACH & RESULTS: We characterized the biological activities of globins in cultured human HSCs (HHSteCs) and their effects on carbon tetrachloride (CCl4)-induced cirrhosis in mice. All globins demonstrated greater antioxidant capacity than glutathione in cell-free systems. Cellular fractionation revealed endocytosis of extracellular MB, NGB, and CYGB, but not HB; endocytosed globins localized to intracellular membranous, cytoplasmic, and cytoskeletal fractions. MB, NGB, and CYGB, but not HB, scavenged reactive oxygen species generated spontaneously or stimulated by H2O2 or transforming growth factor ß1 in HHSteCs and reduced collagen 1A1 production via suppressing COL1A1 promoter activity. Disulfide bond-mutant NGB displayed decreased heme and superoxide scavenging activity and reduced collagen inhibitory capacity. RNA sequencing of MB- and NGB-treated HHSteCs revealed downregulation of extracellular matrix-encoding and fibrosis-related genes and HSC deactivation markers. Upregulation of matrix metalloproteinase (MMP)-1 was observed following MB and NGB treatment, and MMP-1 knockdown partially reversed globin-mediated effects on secreted collagen. Importantly, administration of MB, NGB, and CYGB suppressed CCl4-induced mouse liver fibrosis. CONCLUSIONS: These findings revealed unexpected roles for MB and NGB in deactivating HSCs and inhibiting liver fibrosis development, suggesting that globin therapy may represent a new strategy for combating fibrotic liver disease.

Coordination of retrotransposons and type I interferon with distinct interferon pathways in dermatomyositis, systemic lupus erythematosus and autoimmune blistering disease.

Type I interferon (IFN) plays a crucial role in innate and adaptive immunity, and aberrant IFN responses are involved in systemic autoimmune diseases, such as systemic lupus erythematosus (SLE) and dermatomyositis (DM). Type I IFNs can be induced by transcribed retrotransposons. The regulation of retrotransposons and type I IFN and the downstream IFN pathways in SLE, DM, and autoimmune blistering disease (AIBD) were investigated. The gene expression levels of retrotransposons, including LINE-1, type I-III IFNs, and IFN-stimulated genes (ISGs) in peripheral blood cells from patients with DM (n = 24), SLE (n = 19), AIBD (n = 14) and healthy controls (HCs, n = 10) were assessed by quantitative polymerase chain reaction. Upregulation of retrotransposons and IFNs was detected in DM patient samples, as is characteristic, compared to HCs; however, ISGs were not uniformly upregulated. In contrast, retrotransposons and IFNs, except for type II IFN, such as IFN-γ, were not upregulated in SLE. In AIBD, only some retrotransposons and type I interferons were upregulated. The DM, SLE, and AIBD samples showed coordinated expression of retrotransposons and type I IFNs and distinct spectra of IFN signaling. A positive correlation between LINE-1 and IFN-ß1 was also detected in human cell lines. These factors may participate in the development of these autoimmune diseases.

Physical characteristics of patients with symptomatic lumbar spondylolysis who have recurrent low back pain after returning to sports.

INTRODUCTION: To determine the physical characteristics of patients with symptomatic lumbar spondylolysis (LS) who have recurrent low back pain after returning to sports. METHOD: Fifty-three adolescent patients with symptomatic LS participated in this study. Patients with symptomatic LS were assessed for flexibility, trunk muscle strength, and Functional Movement Screen (FMS) score, and then divided into two groups according to the degree of pain experienced one month after returning to sport. RESULTS: Twenty-four patients returned to their pre-injury sports level without pain (excellent group), while the remaining 29 patients had pain and decreased activity level during sports (pain group). The excellent group had a significantly higher shoulder mobility score on the FMS than the pain group, and the effect size was larger. However, there were no significant differences in body flexibility, muscle tightness, trunk muscle strength, or other FMS items. DISCUSSION: The most important finding of this study is that patients with LS who have recurrent low back pain after returning to sports are characterized by poor functional upper body movement. CONCLUSIONS: Because upper body functional movement pattern may be an important factor in the management of patients with symptomatic LS, preventive rehabilitation to enhance upper body mobility and motor control should be considered for these patients.

Crosstalk Between NDP52 and LUBAC in Innate Immune Responses, Cell Death, and Xenophagy.

Nuclear dot protein 52 kDa (NDP52, also known as CALCOCO2) functions as a selective autophagy receptor. The linear ubiquitin chain assembly complex (LUBAC) specifically generates the N-terminal Met1-linked linear ubiquitin chain, and regulates innate immune responses, such as nuclear factor-κB (NF-κB), interferon (IFN) antiviral, and apoptotic pathways. Although NDP52 and LUBAC cooperatively regulate bacterial invasion-induced xenophagy, their functional crosstalk remains enigmatic. Here we show that NDP52 suppresses canonical NF-κB signaling through the broad specificity of ubiquitin-binding at the C-terminal UBZ domain. Upon TNF-α-stimulation, NDP52 associates with LUBAC through the HOIP subunit, but does not disturb its ubiquitin ligase activity, and has a modest suppressive effect on NF-κB activation by functioning as a component of TNF-α receptor signaling complex I. NDP52 also regulates the TNF-α-induced apoptotic pathway, but not doxorubicin-induced intrinsic apoptosis. A chemical inhibitor of LUBAC (HOIPIN-8) cancelled the increased activation of the NF-κB and IFN antiviral pathways, and enhanced apoptosis in NDP52-knockout and -knockdown HeLa cells. Upon Salmonella-infection, colocalization of Salmonella, LC3, and linear ubiquitin was detected in parental HeLa cells to induce xenophagy. Treatment with HOIPIN-8 disturbed the colocalization and facilitated Salmonella expansion. In contrast, HOIPIN-8 showed little effect on the colocalization of LC3 and Salmonella in NDP52-knockout cells, suggesting that NDP52 is a weak regulator in LUBAC-mediated xenophagy. These results indicate that the crosstalk between NDP52 and LUBAC regulates innate immune responses, apoptosis, and xenophagy.

Th2 cell-derived histamine is involved in nasal Th2 infiltration in mice.

OBJECTIVE: Histamine derived from mast cells and basophils plays important roles in inducing allergic symptoms. Although T cells also produce histamine, the involvement of the histamine produced from T cells has remained enigmatic. We sought to reveal the roles of T helper 2 (Th2) cell-derived histamine in nasal allergic disorders. METHODS: The histamine production from Th2 cells was measured by EIA. The mRNA expression of histidine decarboxylase (HDC) was measured by real-time PCR. To investigate the roles of Th2 cell-derived histamine in vivo, we analyzed an antigen-specific Th2 cell transfer mouse model. RESULTS: Th2 cells produced histamine by T cell receptor stimulation, and these properties were specific for Th2 cells, but not Th1 cells and naïve CD4 T cells. The histamine produced from Th2 cells was involved in the infiltrations of Th2 cells in response to antigen exposure. CONCLUSION: These results suggest that Th2 cell-derived histamine play important roles in nasal allergic disorders.

Th2 cells and macrophages cooperatively induce allergic inflammation through histamine signaling.

Histamine, which is mainly produced by mast cells and basophils, participates in various allergic symptoms, and some studies have reported that macrophages also produce histamine. Moreover, recent studies have revealed that macrophages, especially alternatively activated macrophages (M2) induced by T helper 2 (Th2) cytokines, such as interleukin (IL)-4 and IL-13, participate in the pathogenesis of allergic diseases. The major source of Th2 cytokines is antigen-specific Th2 cells. To elucidate the relationship between histamine, macrophages, and Th2 cells in allergic inflammation, we established a macrophage-Th2 cell co-culture model in vitro and an antigen-specific Th2 cell transfer mouse model of rhinitis. In vitro analyses indicated that macrophages produce histamine by interacting with antigen-specific Th2 cells through the antigen. Furthermore, Th2 cells and macrophages cooperatively elicited rhinitis in the mouse model. We determined that histamine induces Th2- and macrophage-elicited sneezing responses through H1 receptor signaling, whereas it induces nasal eosinophil infiltrations through H4 receptor signaling. Collectively, these results indicate a novel histamine production mechanism by macrophages, in which Th2 cells and macrophages cooperatively induce nasal allergic inflammation through histamine signaling.

Hexa Histidine-Tagged Recombinant Human Cytoglobin Deactivates Hepatic Stellate Cells and Inhibits Liver Fibrosis by Scavenging Reactive Oxygen Species.

BACKGROUND AND AIMS: Antifibrotic therapy remains an unmet medical need in human chronic liver disease. We report the antifibrotic properties of cytoglobin (CYGB), a respiratory protein expressed in hepatic stellate cells (HSCs), the main cell type involved in liver fibrosis. APPROACH AND RESULTS: Cygb-deficient mice that had bile duct ligation-induced liver cholestasis or choline-deficient amino acid-defined diet-induced steatohepatitis significantly exacerbated liver damage, fibrosis, and reactive oxygen species (ROS) formation. All of these manifestations were attenuated in Cygb-overexpressing mice. We produced hexa histidine-tagged recombinant human CYGB (His-CYGB), traced its biodistribution, and assessed its function in HSCs or in mice with advanced liver cirrhosis using thioacetamide (TAA) or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). In cultured HSCs, extracellular His-CYGB was endocytosed and accumulated in endosomes through a clathrin-mediated pathway. His-CYGB significantly impeded ROS formation spontaneously or in the presence of ROS inducers in HSCs, thus leading to the attenuation of collagen type 1 alpha 1 production and α-smooth muscle actin expression. Replacement the iron center of the heme group with cobalt nullified the effect of His-CYGB. In addition, His-CYGB induced interferon-ß secretion by HSCs that partly contributed to its antifibrotic function. Momelotinib incompletely reversed the effect of His-CYGB. Intravenously injected His-CYGB markedly suppressed liver inflammation, fibrosis, and oxidative cell damage in mice administered TAA or DDC mice without adverse effects. RNA-sequencing analysis revealed the down-regulation of inflammation- and fibrosis-related genes and the up-regulation of antioxidant genes in both cell culture and liver tissues. The injected His-CYGB predominantly localized to HSCs but not to macrophages, suggesting specific targeting effects. His-CYGB exhibited no toxicity in chimeric mice with humanized livers. CONCLUSIONS: His-CYGB could have antifibrotic clinical applications for human chronic liver diseases.