GTS-21 Enhances Regulatory T Cell Development from T Cell Receptor-Activated Human CD4+ T Cells Exhibiting Varied Levels of CHRNA7 and CHRFAM7A Expression.

Immune cells such as T cells and macrophages express α7 nicotinic acetylcholine receptors (α7 nAChRs), which contribute to the regulation of immune and inflammatory responses. Earlier findings suggest α7 nAChR activation promotes the development of regulatory T cells (Tregs) in mice. Using human CD4+ T cells, we investigated the mRNA expression of the α7 subunit and the human-specific dupα7 nAChR subunit, which functions as a dominant-negative regulator of ion channel function, under resting conditions and T cell receptor (TCR)-activation. We then explored the effects of the selective α7 nAChR agonist GTS-21 on proliferation of TCR-activated T cells and Treg development. Varied levels of mRNA for both the α7 and dupα7 nAChR subunits were detected in resting human CD4+ T cells. mRNA expression of the α7 nAChR subunit was profoundly suppressed on days 4 and 7 of TCR-activation as compared to day 1, whereas mRNA expression of the dupα7 nAChR subunit remained nearly constant. GTS-21 did not alter CD4+ T cell proliferation but significantly promoted Treg development. These results suggest the potential ex vivo utility of GTS-21 for preparing Tregs for adoptive immunotherapy, even with high expression of the dupα7 subunit.

NLRP3 Inflammasome Activation in Lung Vascular Endothelial Cells Contributes to Intestinal Ischemia/Reperfusion-Induced Acute Lung Injury.

Intestinal ischemia/reperfusion (I/R) injury is a life-threatening complication that leads to inflammation and remote organ damage. The NLRP3 inflammasome regulates the caspase-1-dependent release of IL-1ß, an early mediator of inflammation after I/R injury. In this study, we investigated the role of the NLRP3 inflammasome in mice with intestinal I/R injury. Deficiency of NLRP3, ASC, caspase-1/11, or IL-1ß prolonged survival after intestinal I/R injury, but neither NLRP3 nor caspase-1/11 deficiency affected intestinal inflammation. Intestinal I/R injury caused acute lung injury (ALI) characterized by inflammation, reactive oxygen species generation, and vascular permeability, which was markedly improved by NLRP3 deficiency. Bone marrow chimeric experiments showed that NLRP3 in non-bone marrow-derived cells was the main contributor to development of intestinal I/R-induced ALI. The NLRP3 inflammasome in lung vascular endothelial cells is thought to be important to lung vascular permeability. Using mass spectrometry, we identified intestinal I/R-derived lipid mediators that enhanced NLRP3 inflammasome activation in lung vascular endothelial cells. Finally, we confirmed that serum levels of these lipid mediators were elevated in patients with intestinal ischemia. To our knowledge, these findings provide new insights into the mechanism underlying intestinal I/R-induced ALI and suggest that endothelial NLRP3 inflammasome-driven IL-1ß is a novel potential target for treating and preventing this disorder.

Inflammasome-Independent and Atypical Processing of IL-1ß Contributes to Acid Aspiration-Induced Acute Lung Injury.

Inflammation plays a pivotal role in the pathophysiology of gastric aspiration-induced acute lung injury (ALI). However, its mechanism remains unclear. In this study, we investigated the role of NLRP3 inflammasome-driven IL-1ß production in a mouse model of acid aspiration-induced inflammation and ALI. Acid aspiration-induced inflammatory responses and ALI in wild-type mice were significantly attenuated in IL-1ß-/- mice, but not NLRP3-/- mice. In vitro experiments revealed that severe acidic stress (pH 1.75) induced the processing of pro-IL-1ß into its 18-kDa mature form (p18-IL-1ß), which was different from the caspase-1-processed 17-kDa form (p17-IL-1ß), in human THP-1 macrophages and primary murine macrophages. Deficiency of NLRP3 and caspase-1 had no effect on acidic stress-produced IL-1ß. The production of IL-1ß by severe acidic stress was prevented by inhibitors of serine proteases [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride], but not of cysteine proteases (E-64), cathepsin G, or inflammasome. The cathepsin D inhibitor pepstatin A inhibited IL-1ß production induced by mild acidic stress (pH 6.2) or lactic acid, but not severe acidic stress. Using mass spectrometry and processing-site mutants of pro-IL-1ß, we identified D109 as a novel cleavage site of pro-IL-1ß in response to severe acidic stress and calculated the theoretical molecular mass of the mature form to be 18.2 kDa. The bioactivity of acidic stress-produced IL-1ß was confirmed by its ability to promote p38 phosphorylation and chemokine upregulation in alveolar epithelial cells. These findings demonstrate a novel mechanism of acid-induced IL-1ß production and inflammation independent of NLRP3 inflammasome and provide new insights into the therapeutic strategies for aspiration pneumonitis and ALI.

Glucose regulates hypoxia-induced NLRP3 inflammasome activation in macrophages.

Although the intimate linkage between hypoxia and inflammation is well known, the mechanism underlying this linkage has not been fully understood. Nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome is an intracellular multiprotein complex that regulates interleukin-1ß (IL-1ß) secretion and pyroptosis, and is implicated in the pathogenesis of sterile inflammatory diseases. Here, we investigated the regulatory mechanism of NLRP3 inflammasome activation in response to hypoxia in macrophages. Severe hypoxia (0.1% O2 ) induced the processing of pro-IL-1ß, pro-caspase-1, and gasdermin D, as well as the release of IL-1ß and lactate dehydrogenase in lipopolysaccharide (LPS)-primed murine macrophages, indicating that hypoxia induces NLRP3 inflammasome-driven inflammation and pyroptosis. NLRP3 deficiency and a specific caspase-1 blockade inhibited hypoxia-induced IL-1ß release. Hypoxia-induced IL-1ß release and cell death were augmented under glucose deprivation, and an addition of glucose in the media negatively regulated hypoxia-induced IL-1ß release. Under hypoxia and glucose deprivation, hypoxia-induced glycolysis was not driven and subsequently, the intracellular adenosine triphosphates (ATPs) were depleted. Atomic absorption spectrometry analysis showed a reduction of intracellular K+ concentrations, indicating the K+ efflux occurring under hypoxia and glucose deprivation. Furthermore, hypoxia and glucose deprivation-induced IL-1ß release was significantly prevented by inhibition of K+ efflux and KATP channel blockers. In vivo experiments further revealed that IL-1ß production was increased in LPS-primed mice exposed to hypoxia (9.5% O2 ), which was prevented by a deficiency of NLRP3, an apoptosis-associated speck-like protein containing a caspase recruitment domain, and caspase-1. Our results demonstrate that NLRP3 inflammasome can sense intracellular energy crisis as a danger signal induced by hypoxia and glucose deprivation, and provide new insights into the mechanism underlying hypoxia-induced inflammation.

Tyrosine-phosphorylated SOCS3 negatively regulates cellular transformation mediated by the myeloproliferative neoplasm-associated JAK2 V617F mutant.

In the majority of myeloproliferative neoplasms (MPNs) patients, a point mutation, V617F has been found in Janus kinase 2 (JAK2) gene, and this JAK2 mutant provoked aberrant signaling pathway. In the current study, we found that suppressor of cytokine signaling proteins 3 (SOCS3) possessed the tumor suppressive activity against the JAK2 V617F mutant-provoked cellular transformation. The knockdown of SOCS3 increased the expression level of the JAK2 V617F mutant, which enhanced the activation of signaling mediators, including signal transducer and activator of transcription 3 and 5 (STAT3, STAT5) and extracellular signal-regulated kinase (ERK), and also increased of the proliferation rate and tumorigenesis activity of Ba/F3 cells expressing the JAK2 V617F mutant and erythropoietin receptor (EpoR). In contrast, the enforced expression of SOCS3 significantly inhibited the JAK2 V617F mutant-induced activation of downstream signaling molecules, cell proliferation, and tumorigenesis by down-regulating the expression level of the JAK2 V617F mutant. SOCS3 interacted with the JAK2V617F mutant through its SH2 domain and was phosphorylated at Tyr-204 and Tyr-221 in its SOCS box by the JAK2V617F mutant. SOCS3 mutants carrying a mutation in the SH2 domain (R71E) and a substitution at Tyr-221 (Y221F) failed to exert inhibitory effects on JAK2V617F mutant-induced cellular transformation and tumorigenesis. Collectively, these results imply that SOCS3 plays a negative role in the JAK2 V617F mutant-induced oncogenic signaling pathway through its SH2 domain and the phosphorylation of Tyr-221 in its SOCS box.

Saturated Fatty Acids Undergo Intracellular Crystallization and Activate the NLRP3 Inflammasome in Macrophages.

OBJECTIVE: Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1ß (interleukin 1ß) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. APPROACH AND RESULTS: The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1ß release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1ß release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1ß release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1ß release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1ß release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1ß-deficient mice. CONCLUSIONS: These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

Interaction of Neutrophils with Macrophages Promotes IL-1ß Maturation and Contributes to Hepatic Ischemia-Reperfusion Injury.

Accumulating evidence suggests that IL-1ß plays a pivotal role in the pathophysiology of hepatic ischemia-reperfusion (I/R) injury; however, the mechanism by which I/R triggers IL-1ß production in the liver remains unclear. Recent data have shown that neutrophils contribute to hepatic I/R injury independently of the inflammasomes regulating IL-1ß maturation. Thus, we investigated the role of neutrophils in IL-1ß maturation and tissue injury in a murine model of hepatic I/R. IL-1ß was released from the I/R liver and its deficiency reduced reactive oxygen species generation, apoptosis, and inflammatory responses, such as inflammatory cell infiltration and cytokine expression, thereby resulting in reduced tissue injury. Depletion of either macrophages or neutrophils also attenuated IL-1ß release and hepatic I/R injury. In vitro experiments revealed that neutrophil-derived proteinases process pro-IL-1ß derived from macrophages into its mature form independently of caspase-1. Furthermore, pharmacological inhibition of serine proteases attenuated IL-1ß release and hepatic I/R injury in vivo. Taken together, the interaction between neutrophils and macrophages promotes IL-1ß maturation and causes IL-1ß-driven inflammation in the I/R liver. Both neutrophils and macrophages are indispensable in this process. These findings suggest that neutrophil-macrophage interaction is a therapeutic target for hepatic I/R injury and may also provide new insights into the inflammasome-independent mechanism of IL-1ß maturation in the liver.

ARIH2 Ubiquitinates NLRP3 and Negatively Regulates NLRP3 Inflammasome Activation in Macrophages.

The nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is a molecular platform that induces caspase-1 activation and subsequent IL-1ß maturation, and is implicated in inflammatory diseases; however, little is known about the negative regulation of NLRP3 inflammasome activation. In this article, we identified an E3 ligase, Ariadne homolog 2 (ARIH2), as a posttranslational negative regulator of NLRP3 inflammasome activity in macrophages. ARIH2 interacted with NLRP3 via its NACHT domain (aa 220-575) in the NLRP3 inflammasome complex. In particular, we found that while using mutants of ARIH2 and ubiquitin, the really interesting new gene 2 domain of ARIH2 was required for NLRP3 ubiquitination linked through K48 and K63. Deletion of endogenous ARIH2 using CRISPR/Cas9 genome editing inhibited NLRP3 ubiquitination and promoted NLRP3 inflammasome activation, resulting in apoptosis-associated speck-like protein containing a caspase recruitment domain oligomerization, pro-IL-1ß processing, and IL-1ß production. Conversely, ARIH2 overexpression promoted NLRP3 ubiquitination and inhibited NLRP3 inflammasome activation. Our findings reveal a novel mechanism of ubiquitination-dependent negative regulation of the NLRP3 inflammasome by ARIH2 and highlight ARIH2 as a potential therapeutic target for inflammatory diseases.

NLRP3 protein deficiency exacerbates hyperoxia-induced lethality through Stat3 protein signaling independent of interleukin-1ß.

Supplemental oxygen inhalation is frequently used to treat severe respiratory failure; however, prolonged exposure to hyperoxia causes hyperoxic acute lung injury (HALI), which induces acute respiratory distress syndrome and leads to high mortality rates. Recent investigations suggest the possible role of NLRP3 inflammasomes, which regulate IL-1ß production and lead to inflammatory responses, in the pathophysiology of HALI; however, their role is not fully understood. In this study, we investigated the role of NLRP3 inflammasomes in mice with HALI. Under hyperoxic conditions, NLRP3(-/-) mice died at a higher rate compared with wild-type and IL-1ß(-/-) mice, and there was no difference in IL-1ß production in their lungs. Under hyperoxic conditions, the lungs of NLRP3(-/-) mice exhibited reduced inflammatory responses, such as inflammatory cell infiltration and cytokine expression, as well as increased and decreased expression of MMP-9 and Bcl-2, respectively. NLRP3(-/-) mice exhibited diminished expression and activation of Stat3, which regulates MMP-9 and Bcl-2, in addition to increased numbers of apoptotic alveolar epithelial cells. In vitro experiments revealed that alveolar macrophages and neutrophils promoted Stat3 activation in alveolar epithelial cells. Furthermore, NLRP3 deficiency impaired the migration of neutrophils and chemokine expression by macrophages. These findings demonstrate that NLRP3 regulates Stat3 signaling in alveolar epithelial cells by affecting macrophage and neutrophil function independent of IL-1ß production and contributes to the pathophysiology of HALI.

Caspase-1 deficiency promotes high-fat diet-induced adipose tissue inflammation and the development of obesity.

Caspase-1 is a cysteine protease responsible for the processing of the proinflammatory cytokine interleukin-1ß and activated by the formation of inflammasome complexes. Although several investigations have found a link between diet-induced obesity and caspase-1, the relationship remains controversial. Here, we found that mice deficient in caspase-1 were susceptible to high-fat diet-induced obesity with increased adiposity as well as normal lipid and glucose metabolism. Caspase-1 deficiency clearly promoted the infiltration of inflammatory macrophages and increased the production of C-C motif chemokine ligand 2 (CCL2) in the adipose tissue. The dominant cellular source of CCL2 was stromal vascular fraction rather than adipocytes in the adipose tissue. These findings demonstrate a critical role of caspase-1 in macrophage-driven inflammation in the adipose tissue and the development of obesity. These data provide novel insights into the mechanisms underlying inflammation in the pathophysiology of obesity.

Hemizygosity of transsulfuration genes confers increased vulnerability against acetaminophen-induced hepatotoxicity in mice.

The key mechanism for acetaminophen hepatotoxicity is cytochrome P450 (CYP)-dependent formation of N-acetyl-p-benzoquinone imine, a potent electrophile that forms protein adducts. Previous studies revealed the fundamental role of glutathione, which binds to and detoxifies N-acetyl-p-benzoquinone imine. Glutathione is synthesized from cysteine in the liver, and N-acetylcysteine is used as a sole antidote for acetaminophen poisoning. Here, we evaluated the potential roles of transsulfuration enzymes essential for cysteine biosynthesis, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH), in acetaminophen hepatotoxicity using hemizygous (Cbs(+/-) or Cth(+/-)) and homozygous (Cth(-/-)) knockout mice. At 4 h after intraperitoneal acetaminophen injection, serum alanine aminotransferase levels were highly elevated in Cth(-/-) mice at 150 mg/kg dose, and also in Cbs(+/-) or Cth(+/-) mice at 250 mg/kg dose, which was associated with characteristic centrilobular hepatocyte oncosis. Hepatic glutathione was depleted while serum malondialdehyde accumulated in acetaminophen-injected Cth(-/-) mice but not wild-type mice, although glutamate-cysteine ligase (composed of catalytic [GCLC] and modifier [GCLM] subunits) became more activated in the livers of Cth(-/-) mice with lower Km values for Cys and Glu. Proteome analysis using fluorescent two-dimensional difference gel electrophoresis revealed 47 differentially expressed proteins after injection of 150 mg acetaminophen/kg into Cth(-/-) mice; the profiles were similar to 1000 mg acetaminophen/kg-treated wild-type mice. The prevalence of Cbs or Cth hemizygosity is estimated to be 1:200-300 population; therefore, the deletion or polymorphism of either transsulfuration gene may underlie idiosyncratic acetaminophen vulnerability along with the differences in Cyp, Gclc, and Gclm gene activities.

Lipopeptides are signaled by Toll-like receptor 1, 2 and 6 in endolysosomes.

Toll-like receptors (TLRs) recognize a variety of microbial products and induce defense responses. Pathogen sensing by TLRs occurs either on the cell surface or in endolysosomes. TLR-dependent responses are greatly influenced by the site of pathogen sensing. TLR heterodimers TLR1/TLR2 and TLR2/TLR6 recognize tri- or diacylated microbial lipopeptides, respectively. Although TLR1, 2 and 6 are believed to localize on the cell surface of immune cells, little is known about where lipopeptides are signaled. In this study, we established mAbs to TLR1, 2 and 6. TLR1, 2 and 6 were expressed on the surface of B cells, monocytes and dendritic cells in a manner dependent on a TLR-specific chaperone PRAT4A (protein associated with TLR4 A). Cell surface localization of TLR1 or TLR6 was not necessarily required for TLR2 response. Furthermore, a dynamin inhibitor 'Dynasore' abolished the lipopeptide responses by preventing lipopeptide internalization into LAMP-1 and LAMP-2 positive compartments. Our findings suggest that lipopeptides elicit TLR1/2 and TLR2/6 signaling in the endolysosomes, but not on the cell surface.

Dynamin isoforms decode action potential firing for synaptic vesicle recycling.

Presynaptic nerve terminals must maintain stable neurotransmission via synaptic vesicle membrane recycling despite encountering wide fluctuations in the number and frequency of incoming action potentials (APs). However, the molecular mechanism linking variation in neuronal activity to vesicle trafficking is unknown. Here, we combined genetic knockdown and direct physiological measurements of synaptic transmission from paired neurons to show that three isoforms of dynamin, an essential endocytic protein, work individually to match vesicle reuse pathways, having distinct rate and time constants with physiological AP frequencies. Dynamin 3 resupplied the readily releasable pool with slow kinetics independently of the AP frequency but acted quickly, within 20 ms of the incoming AP. Under high-frequency firing, dynamin 1 regulated recycling to the readily releasable pool with fast kinetics in a slower time window of greater than 50 ms. Dynamin 2 displayed a hybrid response between the other isoforms. Collectively, our findings show how dynamin isoforms select appropriate vesicle reuse pathways associated with specific neuronal firing patterns.

Neutral aminoaciduria in cystathionine ß-synthase-deficient mice; an animal model of homocystinuria.

The kidney is one of the major loci for the expression of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). While CBS-deficient (Cbs(-/-)) mice display homocysteinemia/methioninemia and severe growth retardation, and rarely survive beyond the first 4 wk, CTH-deficient (Cth(-/-)) mice show homocysteinemia/cystathioninemia but develop with no apparent abnormality. This study examined renal amino acid reabsorption in those mice. Although both 2-wk-old Cbs(-/-) and Cth(-/-) mice had normal renal architecture, their serum/urinary amino acid profiles largely differed from wild-type mice. The most striking feature was marked accumulation of Met and cystathionine in serum/urine/kidney samples of Cbs(-/-) and Cth(-/-) mice, respectively. Levels of some neutral amino acids (Val, Leu, Ile, and Tyr) that were not elevated in Cbs(-/-) serum were highly elevated in Cbs(-/-) urine, and urinary excretion of other neutral amino acids (except Met) was much higher than expected from their serum levels, demonstrating neutral aminoaciduria in Cbs(-/-) (not Cth(-/-)) mice. Because the bulk of neutral amino acids is absorbed via a B(0)AT1 transporter and Met has the highest substrate affinity for B(0)AT1 than other neutral amino acids, hypermethioninemia may cause hyperexcretion of neutral amino acids.

Temporal expression and mitochondrial localization of a Foxp2 isoform lacking the forkhead domain in developing Purkinje cells.

FOXP2, a forkhead box-containing transcription factor, forms homo- or hetero-dimers with FOXP family members and localizes to the nucleus, while FOXP2(R553H), which contains a mutation related to speech/language disorders, features reduced DNA binding activity and both cytoplasmic and nuclear localization. In addition to being a loss-of-function mutation, it is possible that FOXP2(R553H) also may act as a gain-of-function mutation to inhibit the functions of FOXP2 isoforms including FOXP2Ex10+ lacking forkhead domain. Foxp2(R552H) knock-in mouse pups exhibit impaired ultrasonic vocalization and poor dendritic development in Purkinje cells. However, expressions of Foxp2 isoforms in the developing Purkinje are unclear. The appearance of 'apical cytoplasmic swelling' (mitochondria-rich regions that are the source of budding processes) correlates with dendritic development of Purkinje cells. In the present study, we focused on Foxp2 isoforms localizing to the apical cytoplasmic swelling and identified two isoforms lacking forkhead domain: Foxp2Ex12+ and Foxp2Ex15. They partly localized to the membrane fraction that includes mitochondria. Foxp2Ex12+ mainly localized to the apical cytoplasmic swelling in early developing Purkinje cells at the stellate stage (P2-P4). Mitochondrial localization of Foxp2Ex12+ in Purkinje cells was confirmed by immune-electron microscopic analysis. Foxp2Ex12+ may play a role in dendritic development in Purkinje cells.

Sulfatides inhibit adhesion, migration, and invasion of murine melanoma B16F10 cell line in vitro.

Endogenous sulfatide, such as 3-sulfated galactosylceramide (3-sulfatide) has been reported to be involved in neuronal development and regulation of tumor cell metastasis. Recently, a new 6-sulfated glucosylceramide (6-sulfatide) has been isolated from the ascidian, Ciona intestinalis. To determine the antitumor function of the new sulfatide, we examined the effects of synthetic 6-sulfatide and 3-sulfatide on the metastatic features of a murine melanoma cell line, B16F10. Both sulfatides significantly inhibited the adhesion of melanoma cells onto fibronectin-coated tissue plates and, the motility and invasion of the cells, with 6-sulfatide showing stronger inhibitory activities. In addition, both sulfatides inhibited α(5)-, and ß(1)- but not α(v)- or ß(3)-integrin expression. Furthermore, these sulfatides inhibited the activation of focal adhesion kinase, Akt, and extracellular signal-regulated kinase signaling pathways, which are thought to be important for cell migration and invasion. Therefore, these sulfatides may serve as promising drug candidates for the treatment of cancer metastasis.

STAT5 activation is critical for the transformation mediated by myeloproliferative disorder-associated JAK2 V617F mutant.

It has been well established that disruption of JAK2 signaling regulation is involved in various hematopoietic disorders; however, the detailed mechanism by which abnormal activation of JAK2 exhibits transforming activity remains to be elucidated. Here, to clarify the functional role of the erythropoietin receptor (EpoR) and its downstream transcription factor STAT5 in the abnormal activation of JAK2-induced hematopoietic diseases, we generated a stable transfectant of Ba/F3 cells expressing EpoR and analyzed the molecular mechanism of how JAK2 mutation induces cell growth disorder. JAK2 V617F mutant exhibited transforming activity when EpoR was coexpressed. According to a study utilizing several truncated mutants of EpoR, the ability of EpoR to facilitate the transforming activity of JAK2 V617F mutant required the intracellular domain to interact with STAT5. Strikingly, once the truncated EpoR (EpoR-H) was mutated on Tyr-343, the phosphorylation of which is known to be important for interaction with STAT5, JAK2 V617F mutant failed to exhibit transforming activity, suggesting that STAT5 is critical for JAK2 mutant-induced hematopoietic disorder. Furthermore, the expression of the constitutively active STAT5 mutant exhibited transforming activity in Ba/F3 cells, and short hairpin RNA-mediated knockdown of STAT5 significantly inhibited the transforming activity of JAK2 V617F mutant. Taking these observations together, STAT5 plays an essential role in EpoR-JAK2 V617F mutant-induced hematopoietic disorder. Although it remains unclear why the presence of EpoR is required to activate oncogenic signaling via the JAK2 mutant and STAT5, its interacting ability is a target for the treatment of these hematopoietic diseases.

Role of bone marrow-derived monocytes/macrophages in the repair of mucosal damage caused by irradiation and/or anticancer drugs in colitis model.

Mucosal damage is a common side effect of many cancer treatments, especially radiotherapy and intensive chemotherapy, which often induce bone marrow (BM) suppression. We observed that acetic acid- (AA-) induced mucosal damage in the colon of mice was worsened by simultaneous treatment with irradiation or 5-FU. However, irradiation 14 days prior to the AA treatment augmented the recovery from mucosal damage, suggesting that the recovery from BM suppression had an advantageous effect on the mucosal repair. In addition, BM transplantation also augmented the recovery from AA-induced mucosal damage. We further confirmed that transplanted BM-derived cells, particularly F4/80+Gr1+ "inflammatory" monocytes (Subset 1), accumulated in the damaged mucosal area in the early healing phase, and both of Subset 1 and F4/80+Gr1⁻ "resident" monocytes (Subset 2) accumulated in this area in later phases. Our results suggest that monocytes/macrophages contribute to the mucosal recovery and regeneration following mucosal damage by anticancer drug therapy.

GSDME-Dependent Incomplete Pyroptosis Permits Selective IL-1α Release under Caspase-1 Inhibition.

Pyroptosis is a form of regulated cell death that is characterized by gasdermin processing and increased membrane permeability. Caspase-1 and caspase-11 have been considered to be essential for gasdermin D processing associated with inflammasome activation. In the present study, we found that NLRP3 inflammasome activation induces delayed necrotic cell death via ASC in caspase-1/11-deficient macrophages. Furthermore, ASC-mediated caspase-8 activation and subsequent gasdermin E processing are necessary for caspase-1-independent necrotic cell death. We define this necrotic cell death as incomplete pyroptosis because IL-1ß release, a key feature of pyroptosis, is absent, whereas IL-1α release is induced. Notably, unprocessed pro-IL-1ß forms a molecular complex to be retained inside pyroptotic cells. Moreover, incomplete pyroptosis accompanied by IL-1α release is observed under the pharmacological inhibition of caspase-1 with VX765. These findings suggest that caspase-1 inhibition during NLRP3 inflammasome activation modulates forms of cell death and permits the release of IL-1α from dying cells.

Licochalcone A potently inhibits tumor necrosis factor alpha-induced nuclear factor-kappaB activation through the direct inhibition of IkappaB kinase complex activation.

Glycyrrhiza inflata has been used as a traditional medicine with anti-inflammatory activity; however, its mechanism has not been fully understood. Licochalcone A is a major and biogenetically characteristic chalcone isolated from G. inflata. Here, we found that licochalcone A strongly inhibited tumor necrosis (TNF)-alpha-induced nuclear localization, DNA binding activity, and the transcriptional activity of nuclear factor-kappaB (NF-kappaB). Whereas licochalcone A had no effect on the recruitment of receptor-interacting protein 1 and IkappaB kinase beta (IKKbeta) to TNF receptor I by TNF-alpha, it significantly inhibited TNF-alpha-induced IkappaB kinase complex (IKK) activation and inhibitor of nuclear factor-kappaB degradation. It is interesting that we found that the cysteine residue at position 179 of IKKbeta is essential for licochalcone A-induced IKK inhibition, because licochalcone A failed to affect the kinase activity of the IKKbeta (C179A) mutant. In contrast, a structurally related compound, echinatin, failed to inhibit TNF-alpha-induced IKK activation and NF-kappaB activation, suggesting that the 1,1-dimethy-2-propenyl group in licochalcone A is important for the inhibition of NF-kappaB. In addition, TNF-alpha-induced expression of inflammatory cytokines CCL2/monocyte chemotactic protein-1and CXCL1/KC was clearly inhibited by licochalcone A but not echinatin. Taken together, licochalcone A might contribute to the potent anti-inflammatory effect of G. inflata through the inhibition of IKK activation.