RNF213 Variants, Vasospastic Angina, and Risk of Fatal Myocardial Infarction.

Importance: Vasospastic angina (VSA) is vasospasm of the coronary artery and is particularly prevalent in East Asian populations. However, the specific genetic architecture for VSA at genome-wide levels is not fully understood. Objective: To identify genetic factors associated with VSA. Design, Setting, and Participants: This was a case-control genome-wide association study of VSA. Data from Biobank Japan (BBJ; enrolled patients from 2002-2008 and 2013-2018) were used, and controls without coronary artery disease (CAD) were enrolled. Patients from the BBJ were genotyped using arrays or a set of arrays. Patients recruited between 2002 and 2005 were classified within the first dataset, and those recruited between 2006 and 2008 were classified within the second dataset. To replicate the genome-wide association study in the first and second datasets, VSA cases and control samples from the latest patients in the BBJ recruited between 2013 and 2018 were analyzed in a third dataset. Exposures: Single-nucleotide variants associated with VSA. Main Outcomes and Measures: Cases with VSA and controls without CAD. Results: A total of 5720 cases (mean [SD] age, 67 [10] years; 3672 male [64.2%]) and 153 864 controls (mean [SD] age, 62 [15] years; 77 362 male [50.3%]) in 3 datasets were included in this study. The variants at the RNF213 locus showed the strongest association with VSA across the 3 datasets (odds ratio [OR], 2.34; 95% CI, 1.99-2.74; P = 4.4 × 10-25). Additionally, rs112735431, an Asian-specific rare deleterious variant (p.Arg4810Lys) experimentally shown to be associated with reduced angiogenesis and a well-known causal risk for Moyamoya disease was the most promising candidate for a causal variant explaining the association. The effect size of rs112735431 on VSA was distinct from that of other CADs. Furthermore, homozygous carriers of rs112735431 showed an association with VSA characterized by a large effect estimate (OR, 18.34; 95% CI, 5.15-65.22; P = 7.0 × 10-6), deviating from the additive model (OR, 4.35; 95% CI, 1.18-16.05; P = .03). Stratified analyses revealed that rs112735431 exhibited a stronger association in males (χ21 = 7.24; P = .007) and a younger age group (OR, 3.06; 95% CI, 2.24-4.19), corresponding to the epidemiologic features of VSA. In the registry, carriers without CAD of the risk allele rs112735431 had a strikingly high mortality rate due to acute myocardial infarction during the follow-up period (hazard ratio, 2.71; 95% CI, 1.57-4.65; P = 3.3 × 10-4). As previously reported, a possible overlap between VSA and Moyamoya disease was not found. Conclusions and Relevance: Results of this study suggest that vascular cell dysfunction mediated by variants in the RNF213 locus may promote coronary vasospasm, and the presence of the risk allele could serve as a predictive factor for the prognosis.

Activation of NOTCH signaling impedes cell proliferation and survival in acute megakaryoblastic leukemia.

The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis.

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Unfavorable switching of skewed X chromosome inactivation leads to Menkes disease in a female infant.

Menkes disease is an X-linked disorder of copper metabolism caused by mutations in the ATP7A gene, and female carriers are usually asymptomatic. We describe a 7-month-old female patient with severe intellectual disability, epilepsy, and low levels of serum copper and ceruloplasmin. While heterozygous deletion of exons 16 and 17 of the ATP7A gene was detected in the proband, her mother, and her grandmother, only the proband suffered from Menkes disease clinically. Intriguingly, X chromosome inactivation (XCI) analysis demonstrated that the grandmother and the mother showed skewing of XCI toward the allele with the ATP7A deletion and that the proband had extremely skewed XCI toward the normal allele, resulting in exclusive expression of the pathogenic ATP7A mRNA transcripts. Expression bias analysis and recombination mapping of the X chromosome by the combination of whole genome and RNA sequencing demonstrated that meiotic recombination occurred at Xp21-p22 and Xq26-q28. Assuming that a genetic factor on the X chromosome enhanced or suppressed XCI of its allele, the factor must be on either of the two distal regions derived from her grandfather. Although we were unable to fully uncover the molecular mechanism, we concluded that unfavorable switching of skewed XCI caused Menkes disease in the proband.

Caspase-11 deficiency attenuates neutrophil recruitment into the atherosclerotic lesion in apolipoprotein E-deficient mice.

Caspase-11 is an inflammatory caspase that triggers an inflammatory response by regulating non-canonical NLRP3 inflammasome activation. Although the deficiency of both caspase-11 and caspase-1, another inflammatory caspase that functions as an executor of the inflammasome, prevents the development of atherosclerosis, the effect of caspase-11 deficiency alone on the development of atherosclerosis has not been fully evaluated. In the present study, we found that caspase-11 deficiency prevented the formation of the necrotic core, whereas it did not affect the development of atherosclerosis in Apoe-deficient mice. Notably, the infiltration of neutrophils into atherosclerotic lesions was attenuated by caspase-11 deficiency. RNA-seq analysis of stage-dependent expression of atherosclerotic lesions revealed that both upregulations of caspase-11 and neutrophil migration are common features of advanced atherosclerotic lesions. Furthermore, similar expression profiles were observed in unstable human plaque. These data suggest that caspase-11 regulates neutrophil recruitment and plaque destabilization in advanced atherosclerotic lesions.

NLRP3 inflammasome-driven IL-1ß and IL-18 contribute to lipopolysaccharide-induced septic cardiomyopathy.

Sepsis is a life-threatening syndrome, and its associated mortality is increased when cardiac dysfunction and damage (septic cardiomyopathy [SCM]) occur. Although inflammation is involved in the pathophysiology of SCM, the mechanism of how inflammation induces SCM in vivo has remained obscure. NLRP3 inflammasome is a critical component of the innate immune system that activates caspase-1 (Casp1) and causes the maturation of IL-1ß and IL-18 as well as the processing of gasdermin D (GSDMD). Here, we investigated the role of the NLRP3 inflammasome in a murine model of lipopolysaccharide (LPS)-induced SCM. LPS injection induced cardiac dysfunction, damage, and lethality, which was significantly prevented in NLRP3-/- mice, compared to wild-type (WT) mice. LPS injection upregulated mRNA levels of inflammatory cytokines (Il6, Tnfa, and Ifng) in the heart, liver, and spleen of WT mice, and this upregulation was prevented in NLRP3-/- mice. LPS injection increased plasma levels of inflammatory cytokines (IL-1ß, IL-18, and TNF-α) in WT mice, and this increase was markedly inhibited in NLRP3-/- mice. LPS-induced SCM was also prevented in Casp1/11-/- mice, but not in Casp11mt, IL-1ß-/-, IL-1α-/-, or GSDMD-/- mice. Notably, LPS-induced SCM was apparently prevented in IL-1ß-/- mice transduced with adeno-associated virus vector expressing IL-18 binding protein (IL-18BP). Furthermore, splenectomy, irradiation, or macrophage depletion alleviated LPS-induced SCM. Our findings demonstrate that the cross-regulation of NLRP3 inflammasome-driven IL-1ß and IL-18 contributes to the pathophysiology of SCM and provide new insights into the mechanism underlying the pathogenesis of SCM.

A Myb enhancer-guided analysis of basophil and mast cell differentiation.

The transcription factor MYB is a crucial regulator of hematopoietic stem and progenitor cells. However, the nature of lineage-specific enhancer usage of the Myb gene is largely unknown. We identify the Myb -68 enhancer, a regulatory element which marks basophils and mast cells. Using the Myb -68 enhancer activity, we show a population of granulocyte-macrophage progenitors with higher potential to differentiate into basophils and mast cells. Single cell RNA-seq demonstrates the differentiation trajectory is continuous from progenitors to mature basophils in vivo, characterizes bone marrow cells with a gene signature of mast cells, and identifies LILRB4 as a surface marker of basophil maturation. Together, our study leads to a better understanding of how MYB expression is regulated in a lineage-associated manner, and also shows how a combination of lineage-related reporter mice and single-cell transcriptomics can overcome the rarity of target cells and enhance our understanding of gene expression programs that control cell differentiation in vivo.

dsDNA-induced AIM2 pyroptosis halts aberrant inflammation during rhabdomyolysis-induced acute kidney injury.

Rhabdomyolysis is a severe condition that commonly leads to acute kidney injury (AKI). While double-stranded DNA (dsDNA) released from injured muscle can be involved in its pathogenesis, the exact mechanism of how dsDNA contributes to rhabdomyolysis-induced AKI (RIAKI) remains obscure. A dsDNA sensor, absent in melanoma 2 (AIM2), forms an inflammasome and induces gasdermin D (GSDMD) cleavage resulting in inflammatory cell death known as pyroptosis. In this study using a mouse model of RIAKI, we found that Aim2-deficiency led to massive macrophage accumulation resulting in delayed functional recovery and perpetuating fibrosis in the kidney. While Aim2-deficiency compromised RIAKI-induced kidney macrophage pyroptosis, it unexpectedly accelerated aberrant inflammation as demonstrated by CXCR3+CD206+ macrophage accumulation and activation of TBK1-IRF3/NF-κB. Kidney macrophages with intact AIM2 underwent swift pyroptosis without IL-1ß release in response to dsDNA. On the other hand, dsDNA-induced Aim2-deficient macrophages escaped from swift pyroptotic elimination and instead engaged STING-TBK1-IRF3/NF-κB signalling, leading to aggravated inflammatory phenotypes. Collectively, these findings shed light on a hitherto unknown immunoregulatory function of macrophage pyroptosis. dsDNA-induced rapid macrophage cell death potentially serves as an anti-inflammatory program and determines the healing process of RIAKI.

Cryo-sensitive aggregation triggers NLRP3 inflammasome assembly in cryopyrin-associated periodic syndrome.

Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3 gene encoding cryopyrin. Familial cold autoinflammatory syndrome, the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1ß. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1ß release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was necessary for mutated NLRP3-mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation.

Depth-targeted intracortical microstroke by two-photon photothrombosis in rodent brain.

Significance: Photothrombosis is a widely used model of ischemic stroke in rodent experiments. In the photothrombosis model, the photosensitizer rose bengal (RB) is systemically introduced into the blood stream and activated by green light to induce aggregation of platelets that eventually cause vessel occlusion. Since the activation of RB is a one-photon phenomenon and the molecules in the illuminated area (light path) are subject to excitation, targeting of thrombosis is unspecific, especially in the depth dimension. We developed a photothrombosis protocol that can target a single vessel in the cortical parenchyma by two-photon excitation. Aim: We aim to induce a thrombotic stroke in the cortical parenchyma by two-photon activation of RB to confine photothrombosis within a vessel of a target depth. Approach: FITC-dextran is injected into the blood stream to visualize the cerebral blood flow in anesthetized adult mice with a cranial window. After a target vessel is chosen by two-photon imaging (950 nm), RB is injected into the blood stream. The scanning wavelength is changed to 720 nm, and photothrombosis is induced by scanning the target vessel. Results: Two-photon depth-targeted single-vessel photothrombosis was achieved with a success rate of 84.9 % ± 1.7 % and an irradiation duration of < 80 s . Attempts without RB (i.e., only with FITC) did not result in photothrombosis at the excitation wavelength of 720 nm. Conclusions: We described a protocol that achieves depth-targeted single-vessel photothrombosis by two-photon excitation. Simultaneous imaging of blood flow in the targeted vessel using FITC dextran enabled the confirmation of vessel occlusion and prevention of excess irradiation that possibly induces unintended photodamage.

Calciprotein Particles Induce IL-1ß/α-Mediated Inflammation through NLRP3 Inflammasome-Dependent and -Independent Mechanisms.

Calciprotein particles (CPPs) are nanoparticles composed of calcium phosphate crystals and fetuin-A and have been implicated in diseases associated with inflammation. In the current study, we investigated the molecular mechanisms underlying CPP-induced inflammation in mice. CPPs predominantly upregulated IL-1ß and IL-1α and provided priming and activation signals for the NLRP3 inflammasome in murine macrophages. Pharmacological and genetic inhibition of the NLRP3 inflammasome revealed that CPPs induced the release of IL-1ß and IL-1α via NLRP3 inflammasome-dependent and -independent mechanisms, respectively. CPPs also induced necrotic cell death, but gasdermin D was dispensable for CPP-induced IL-1ß release and necrotic cell death. Although phagocytosis of CPPs was required for CPP-induced IL-1ß/α release and necrotic cell death, lysosomal dysfunction and K+ efflux were mainly involved in CPP-induced NLRP3 inflammasome activation and subsequent IL-1ß release but not in CPP-induced IL-1α release and necrotic cell death. In vivo experiments showed that CPP administration evoked acute inflammatory responses characterized by neutrophil accumulation via both IL-1ß and IL-1α. In particular, CPP-induced neutrophil inflammation was mediated predominantly through an IL-1α-induced CXCL1/CXCR2 signaling pathway. These results provide new insights into the mechanism underlying CPP-induced inflammation and suggest that targeting both IL-1ß and IL-1α is necessary to regulate the CPP-induced inflammatory response and to treat CPP-associated inflammatory disorders.

Mitochondria transfer from early stages of erythroblasts to their macrophage niche via tunnelling nanotubes.

Adult erythropoiesis entails a series of well-coordinated events that produce mature red blood cells. One of such events is the mitochondria clearance that occurs cell-autonomously via autophagy-dependent mechanisms. Interestingly, recent studies have shown mitochondria transfer activities between various cell types. In the context of erythropoiesis, macrophages are known to interact closely with the early stages of erythroblasts to provide a specialized niche, termed erythroblastic islands (EBI). However, whether mitochondria transfer can occur in the EBI niche has not been explored. Here, we report that mitochondria transfer in the EBI niche occurs in vivo. We observed mitochondria transfer activities from the early stages of erythroblasts to macrophages in the reconstituted in vitro murine EBI via different modes, including tunnelling nanotubes (TNT). Moreover, we demonstrated that Wiskott-Aldrich syndrome protein (WASp) in macrophages mediates TNT formation and mitochondria transfer via the modulation of F-actin filamentation, thus promoting mitochondria clearance from erythroid cells, to potentially enhance their differentiation. Taken together, our findings provide novel insight into the mitochondria clearance machineries that mediate erythroid maturation.

Autophagy is dispensable for the maintenance of hematopoietic stem cells in neonates.

Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to sustain lifelong hematopoiesis. HSCs are preserved in quiescence with low mitochondrial activity. Recent studies indicate that autophagy contributes to HSC quiescence through suppressing mitochondrial metabolism. However, it remains unclear whether autophagy is involved in the regulation of neonatal HSCs, which proliferate actively. In this study, we clarified the role of autophagy in neonatal HSCs using 2 types of autophagy-related gene 7 (Atg7)-conditional knockout mice: Mx1-Cre inducible system and Vav-Cre system. Atg7-deficient HSCs exhibited excess cell divisions with enhanced mitochondrial metabolism, leading to bone marrow failure at adult stage. However, Atg7 deficiency minimally affected hematopoiesis and metabolic state in HSCs at neonatal stage. In addition, Atg7-deficient neonatal HSCs exhibited long-term reconstructing activity, equivalent to wild-type neonatal HSCs. Taken together, autophagy is dispensable for stem cell function and hematopoietic homeostasis in neonates and provide a novel aspect into the role of autophagy in the HSC regulation.

Prolonged maintenance of hematopoietic stem cells that escape from thrombopoietin deprivation.

Hematopoietic stem cells (HSC) rarely divide, rest in quiescence, and proliferate only upon stress hematopoiesis. The cytokine thrombopoietin (Thpo) has been perplexingly described to induce quiescence and promote self-renewal divisions in HSCs. To clarify the contradictory effect of Thpo, we conducted a detailed analysis on conventional (Thpo-/-) and liver-specific (Thpofl/fl;AlbCre+/-) Thpo-deletion models. Thpo-/- HSCs exhibited profound loss of quiescence, impaired cell cycle progression, and increased apoptosis. Thpo-/- HSCs also exhibited diminished mitochondrial mass and impaired mitochondrial bioenergetics. Abnormal HSC phenotypes in Thpo-/- mice were reversible after HSC transplantation into wild-type recipients. Moreover, Thpo-/- HSCs acquired quiescence with extended administration of a Thpo receptor agonist, romiplostim, and were prone to subsequent stem cell exhaustion during competitive bone marrow transplantation. Thpofl/fl;AlbCre+/- HSCs exhibited similar stem cell phenotypes but to a lesser degree compared with Thpo-/- HSCs. HSCs that survive Thpo deficiency acquire quiescence in a dose-dependent manner through the modification of their metabolic state.

Hematopoietic stem cells acquire survival advantage by loss of RUNX1 methylation identified in familial leukemia.

RUNX1 is among the most frequently mutated genes in human leukemia, and the loss or dominant-negative suppression of RUNX1 function is found in myelodysplastic syndrome and acute myeloid leukemia (AML). How posttranslational modifications (PTMs) of RUNX1 affect its in vivo function, however, and whether PTM dysregulation of RUNX1 can cause leukemia are largely unknown. We performed targeted deep sequencing on a family with 3 occurrences of AML and identified a novel RUNX1 mutation, R237K. The mutated R237 residue is a methylation site by protein arginine methyltransferase 1, and loss of methylation reportedly impairs the transcriptional activity of RUNX1 in vitro. To explore the biologic significance of RUNX1 methylation in vivo, we used RUNX1 R233K/R237K double-mutant mice, in which 2 arginine-to-lysine mutations precluded RUNX1 methylation. Genetic ablation of RUNX1 methylation led to loss of quiescence and expansion of hematopoietic stem cells (HSCs), and it changed the genomic and epigenomic signatures of phenotypic HSCs to a poised progenitor state. Furthermore, loss of RUNX1 R233/R237 methylation suppressed endoplasmic reticulum stress-induced unfolded protein response genes, including Atf4, Ddit3, and Gadd34; the radiation-induced p53 downstream genes Bbc3, Pmaip1, and Cdkn1a; and subsequent apoptosis in HSCs. Mechanistically, activating transcription factor 4 was identified as a direct transcriptional target of RUNX1. Collectively, defects in RUNX1 methylation in HSCs confer resistance to apoptosis and survival advantage under stress conditions, a hallmark of a preleukemic clone that may predispose affected individuals to leukemia. Our study will lead to a better understanding of how dysregulation of PTMs can contribute to leukemogenesis.

TUBB1 dysfunction in inherited thrombocytopenia causes genome instability.

Inherited thrombocytopenia is a genetically heterogeneous disease characterized by varying degrees of thrombocytopenia and risk of haematological malignancy, and the genetic cause of many cases remains unknown. We performed whole-exome sequencing of a family with thrombocytopenia and myeloid malignancy and identified a novel TUBB1 variant, T149P. Screening of other thrombocytopenia pedigrees identified another TUBB1 variant, R251H. TUBB1 encodes the tubulin ß-1 chain, a major component of microtubules abundant in megakaryocytes. Variant TUBB1 disrupted the normal assembly of microtubules and impaired proplatelet formation in vitro. In addition, DNA damage response was severely attenuated by loss of TUBB1. We found that the nuclear accumulation of p53 (also termed TP53) and the expression of pro-apoptotic genes triggered by genotoxic stress were blocked in TUBB1-deficient cells and, accordingly, apoptosis after DNA damage was diminished by knockdown of TUBB1. Thus, we have demonstrated that microtubule dysfunction confers resistance to apoptosis, even in DNA damage-accumulated cells, which explains genome instability in the affected individuals. These studies will lead us to a better understanding of how microtubule dysfunction can contribute to the accumulation of DNA damage, genetic instability and leukaemogenesis.

Genetic determinants and an epistasis of LILRA3 and HLA-B*52 in Takayasu arteritis.

Takayasu arteritis (TAK) is a systemic vasculitis with severe complications that affects the aorta and its large branches. HLA-B*52 is an established susceptibility locus to TAK. To date, there are still only a limited number of reports concerning non-HLA susceptibility loci to TAK. We conducted a genome-wide association study (GWAS) and a follow-up study in a total of 633 TAK cases and 5,928 controls. A total of 510,879 SNPs were genotyped, and 5,875,450 SNPs were imputed together with HLA-B*52. Functional annotation of significant loci, enhancer enrichment, and pathway analyses were conducted. We identified four unreported significant loci, namely rs2322599, rs103294, rs17133698, and rs1713450, in PTK2B, LILRA3/LILRB2, DUSP22, and KLHL33, respectively. Two additional significant loci unreported in non-European GWAS were identified, namely HSPA6/FCGR3A and chr21q.22. We found that a single variant associated with the expression of MICB, a ligand for natural killer (NK) cell receptor, could explain the entire association with the HLA-B region. Rs2322599 is strongly associated with the expression of PTK2B Rs103294 risk allele in LILRA3/LILRB2 is known to be a tagging SNP for the deletion of LILRA3, a soluble receptor of HLA class I molecules. We found a significant epistasis effect between HLA-B*52 and rs103294 (P = 1.2 × 10-3). Enhancer enrichment analysis and pathway analysis suggested the involvement of NK cells (P = 8.8 × 10-5, enhancer enrichment). In conclusion, four unreported TAK susceptibility loci and an epistasis effect between LILRA3 and HLA-B*52 were identified. HLA and non-HLA regions suggested a critical role for NK cells in TAK.

Thrombopoietin Metabolically Primes Hematopoietic Stem Cells to Megakaryocyte-Lineage Differentiation.

During acute myelosuppression or thrombocytopenia, bone marrow (BM) hematopoietic cells respond rapidly to replenish peripheral blood platelets. While the cytokine thrombopoietin (Thpo) both regulates platelet production and maintains HSC potential, whether Thpo controls megakaryocyte (Mk)-lineage differentiation of HSCs is unclear. Here, we show that Thpo rapidly upregulates mitochondrial activity in HSCs, an activity accompanied by differentiation to an Mk lineage. Moreover, in unperturbed hematopoiesis, HSCs with high mitochondrial activity exhibit Mk-lineage differentiation in vitro and myeloid lineage-biased reconstitution in vivo. Furthermore, Thpo skewed HSCs to express the tetraspanin CD9, a pattern correlated with mitochondrial activity. Mitochondria-active HSCs are resistant to apoptosis and oxidative stress upon Thpo stimulation. Thpo-regulated mitochondrial activity associated with mitochondrial translocation of STAT3 phosphorylated at serine 727. Overall, we report an important role for Thpo in regulating rapid Mk-lineage commitment. Thpo-dependent changes in mitochondrial metabolism prime HSCs to undergo direct differentiation to an Mk lineage.

Single-Nucleotide Polymorphism of the MLX Gene Is Associated With Takayasu Arteritis.

BACKGROUND: Takayasu arteritis (TAK) is an autoimmune systemic arteritis of unknown pathogenesis. Genome-wide association studies revealed that single-nucleotide polymorphisms in the MLX gene encoding the MLX (Max-like protein X) transcription factor are significantly associated with TAK in Japanese patients. MLX single-nucleotide polymorphism rs665268 is a missense mutation causing the Q139R substitution in the DNA-binding site of MLX. METHODS: To elucidate the hypothesis that the single-nucleotide polymorphism of the MLX gene plays a critical role in the development of TAK, we conducted clinical and laboratory analyses. RESULTS: We show that rs665268 significantly correlated with the severity of TAK, including the number of arterial lesions and morbidity of aortic regurgitation; the latter may be attributed to the fact that MLX mRNA expression was mostly detected in the aortic valve. Furthermore, the Q139R mutation caused structural changes in MLX, which resulted in enhanced formation of a heterodimer with MondoA, upregulation of TXNIP (thioredoxin-interacting protein) expression, and increase in the activity of the NLRP3 (NACHT, LRR, and PYD domains-containing protein 3) inflammasome and cellular oxidative stress. Furthermore, autophagy, which negatively regulates inflammasome activation, was suppressed by the Q139R mutation in MLX. The MLX-Q139R mutant significantly induced macrophage proliferation and macrophage-endothelium interaction, which was abolished by the treatment with SBI-477, an inhibitor of MondoA nuclear translocation. Our findings suggest that the Q139R substitution in MLX plays a crucial role in the pathogenesis of TAK. CONCLUSIONS: MLX-Q139R mutation plays a crucial role in the pathogenesis of TAK through promoting inflammasome formation.