视黄醇结合蛋白4在非酒精性脂肪肝发病机制中的作用

非酒精性脂肪肝(NAFLD)是一种与血脂异常、高胰岛素血症、2型糖尿病以及遗传-环境-代谢应激密切相关的临床病理综合征,胰岛素抵抗(IR)与其发病关系密切.视黄醇结合蛋白(RBP)4是新近发现的一种调节肝脏和肌肉组织中胰岛素作用的脂肪因子,与IR发病相关.目前RBP4与NAFLD的关系正逐渐受到人们的重视,现就RBP4在NAFLD发病机制中的作用作一综述.     

视黄醇结合蛋白4与胰岛素抵抗

视黄醇结合蛋白4（RBP4）是一种主要来源于肝脏和脂肪细胞的转运蛋白。过去认为其作用是转运血液中的视黄醇（维生素A）。而近期的研究发现,RBP4作为一种脂质因子,可能具有引起胰岛素抵抗（IR）的作用,参与一系列与IR相关疾病,包括糖尿病、代谢综合征、非酒精性脂肪肝的发生发展。除外受到IR的影响,不同个体RBP4血清水平的影响因素各不相同。     

视黄醇结合蛋白4与胰岛素抵抗

脂肪组织是一个重要的内分泌器官.肥大的脂肪细胞分泌过量脂肪细胞因子,促进了胰岛素抵抗和糖尿病的发生、发展.视黄醇结合蛋白4(RBP4)主要由肝脏合成,负责维生素A的转运.新近研究发现,RBP4是一个新的脂肪细胞因子,与胰岛素抵抗相关疾病、体脂分布和糖尿病均有密切联系,它的发现为明确胰岛素抵抗发生机制提供了新的论据.     

视黄醇结合蛋白4与多囊卵巢综合征及妊娠糖尿病

视黄醇结合蛋白4主要由肝脏合成,是血液中一种运送视黄醇的结合蚩白,亦是脂肪组织分泌的一种脂肪细胞因子.小鼠实验发现,其在组织中的过度表达可使磷脂酰肌醇3激酶活性下降,胰岛素受体底物1酪氨酸磷酸化降低.可能与胰岛素抵抗的发生有关.肥胖、糖耐量减低、胰岛素抵抗、多囊卵巢综合征、妊娠糖尿病患者血清中视黄醇结合蛋白4含量与正常者相比升高,其可能与此类疾病的发病机制有关.     

2型糖尿病合并冠心病患者血清内脂素和视黄醇结合蛋白4的临床意义

目的探讨血清内脂素(visfatin)和视黄醇结合蛋白4(RBP4)水平与2型糖尿病(T2DM)合并冠心病(CHD)的临床意义。方法选取单纯T2DM(T2DM组)患者61例、T2DM合并CHD(T2DM+CHD组)患者58例、单纯CHD(CHD组)患者60例、门诊健康体检者(对照组)60例,检测各组血清内脂素、RBP4水平、空腹血糖(FBG)、总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平,HbA1c和空腹血清胰岛素(FINS),计算胰岛素抵抗指数(HOMA-IR)、体重指数(BMI)及腰臀比(WHR),并作相关性分析。结果 T2DM+CHD组血清内脂素和RBP4水平均明显高于T2DM组、CHD组和对照组,差异均有统计学意义(q=2.56~3.13,均P<0.05)。T2DM+CHD组内脂素水平与WHR、RBP4、TG和HOMA-IR呈正相关(r=0.27~0.52,均P<0.05);T2DM与RBP4和HbA1c密切相关(OR=2.09~3.67,P=0.05),T2DM合并CHD与内脂素、RBP4和TG密切相关(OR=2.13~3.81,P=0.05)。结论血清内脂素和RBP4与T2DM合并CHD的发病有一定联系。     

视黄醇结合蛋白4和心血管疾病危险因素

脂肪组织不仅是一个储备能量、提供能量的器官,更是重要的内分泌器官。2005年美国哈佛大学医学中心鉴定出一种新的参与胰岛素抵抗的脂肪细胞因子——视黄醇结合蛋白4（RetinolBindingprotein,RBP4）。随后多项研究表明其可诱导胰岛素抵抗,并且发现血浆RBP4水平在2型糖尿病、肥胖、代谢综合征以及心血管疾病中均有升高。同时国内外一些研究报道了它与脂质代谢、动脉粥样硬化、糖代谢、胰岛素抵抗、高血压、心力衰竭等心血管疾病相关危险因素的相关性。本文综述RBP4的结构、功能、测定及其在心血管疾病中的研究进展。     

Caspase-1 autoproteolysis is differentially required for NLRP1b and NLRP3 inflammasome function

Inflammasomes are caspase-1–activating multiprotein complexes. The mouse nucleotide-binding domain and leucine rich repeat pyrin containing 1b (NLRP1b) inflammasome was identified as the sensor of Bacillus anthracis lethal toxin (LT) in mouse macrophages from sensitive strains such as BALB/c. Upon exposure to LT, the NLRP1b inflammasome activates caspase-1 to produce mature IL-1β and induce pyroptosis. Both processes are believed to depend on autoproteolysed caspase-1. In contrast to human NLRP1, mouse NLRP1b lacks an N-terminal pyrin domain (PYD), indicating that the assembly of the NLRP1b inflammasome does not require the adaptor apoptosis-associated speck-like protein containing a CARD (ASC). LT-induced NLRP1b inflammasome activation was shown to be impaired upon inhibition of potassium efflux, which is known to play a major role in NLRP3 inflammasome formation and ASC dimerization. We investigated whether NLRP3 and/or ASC were required for caspase-1 activation upon LT stimulation in the BALB/c background. The NLRP1b inflammasome activation was assessed in both macrophages and dendritic cells lacking either ASC or NLRP3. Upon LT treatment, the absence of NLRP3 did not alter the NLRP1b inflammasome activity. Surprisingly, the absence of ASC resulted in IL-1β cleavage and pyroptosis, despite the absence of caspase-1 autoprocessing activity. By reconstituting caspase-1/caspase-11^−/− cells with a noncleavable or catalytically inactive mutant version of caspase-1, we directly demonstrated that noncleavable caspase-1 is fully active in response to the NLRP1b activator LT, whereas it is nonfunctional in response to the NLRP3 activator nigericin. Taken together, these results establish variable requirements for caspase-1 cleavage depending on the pathogen and the responding NLR.Anthrax is a zoonotic disease caused by the Gram-positive bacterium Bacillus anthracis. B. anthracis provokes a shock-like syndrome that can prove fatal to the host (1) and has recently gained notoriety as a potential bioterrorism agent. Anthrax pathogenicity relies on its ability to secrete three virulence proteins, which combine with each other to form two toxins. The protective antigen (PA) combines with the edema factor (EF) to form the edema toxin (2, 3). EF is an adenylate cyclase that causes edema of the infected tissue. The binary combination of PA with lethal factor (LF) gives rise to the most virulent factor, called lethal toxin (LT), responsible for the systemic symptoms and death of the infected animal. To escape the host immune response, LT impairs the host innate immunity by killing macrophages (4–6). The PA protein interacts with LF and binds to cell surface receptors, enabling endocytosis of the LT complex. In the acidic compartment, PA forms pores allowing the delivery of LF to the cytosol. LF is a zinc metalloprotease that was shown to cleave the N-terminal region of many MAP kinase kinases and to induce apoptosis of macrophages. LT also triggers pyroptosis through the formation of a caspase-1–activating platform, named “inflammasome” (6–8).Inflammasomes are multiprotein complexes of the innate immune response that control caspase-1 activity and pro–IL-1β and pro–IL-18 maturation. Most inflammasomes are composed of specific cytosolic pathogen recognition receptors (PRRs), as well as the apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (CARD) (ASC) adaptor protein that enables the recruitment and activation of the caspase-1 protease. Once caspase-1 is oligomerized within an inflammasome platform, the enzyme undergoes autoproteolysis to form heterodimers of active caspase-1 (9–12). In the mouse, at least five distinct inflammasomes have been described, distinguished by the PRR that induces the complex formation. The PRRs capable of participating in inflammasome platform formation are either members of the nod-like receptor (NLR) family (e.g., NLRP1, NLRP3, or NLRC4) or of the PYrin and HIN (PYHIN) family (e.g., AIM2) (13, 14). ASC is composed of a pyrin domain (PYD) and a caspase activation and recruitment domain (CARD). ASC interacts with a PYD-containing PRR via its PYD domain and recruits the CARD domain of caspase-1 via its CARD domain. Thus, ASC is essential to the formation of the inflammasome by receptors such as NLRP3 or AIM2 (15–18). However, its presence is dispensable for NLRC4, which contains a CARD in place of a PYD, allowing direct interaction with the CARD domain of caspase-1 (19, 20).Past studies have determined that certain mouse strains are more sensitive than others to LT cytotoxicity, and genetic studies identified NLRP1b as the factor conferring mouse strain susceptibility to anthrax LT (21). The mouse genome contains three different NLRP1 isoforms (a, b, and c) and a functional NLRP1b was found to be expressed by the mouse strains sensitive to LT (e.g., BALB/c or 129 background). Expression of NLRP1b was shown to mediate IL-1β release and caspase-1–mediated cell death in response to LT (7, 21, 22). Mouse NLRP1b differs structurally from human NLRP1 in that it lacks the N-terminal PYD (23). The absence of the PYD suggests that NLRP1b can directly engage caspase-1 without a requirement for ASC. However, studies dissecting the mechanism of NLRC4 inflammasome activation demonstrated that ASC is required for the amplification of caspase-1 autoprocessing and IL-1β secretion but not for pyroptosis (19, 20). Cell lysis mediated by LT was shown to be dependent on sodium and potassium fluxes (24), and high extracellular potassium inhibited IL-1β secretion upon LT treatment, suggesting a role for the NLRP3 inflammasome in LT sensing (22, 25). Therefore, we investigated whether NLRP3 and/or ASC were required for caspase-1 activation in response to LT. The NLRP3, ASC, and caspase-1 mouse knockout strains were backcrossed into the BALB/c background and the response of macrophages and dendritic cells (DCs) to LT intoxication was studied. Our data reveal that (i) in response to LT, ASC is dispensable for caspase-1 activation, but uncleavable caspase-1 is fully active; and (ii) upon activation of the NLRP3 inflammasome, uncleavable caspase-1 is inactive.     

新诊断2型糖尿病视黄醇结合蛋白4的变化及影响因素研究

目的研究新诊断2型糖尿病(T2DM)血清视黄醇结合蛋白4(RBP4)变化规律并探讨其影响因素。方法对2002年4月至10月卫生部北京医院95例新诊断2型糖尿病患者,根据体重指数(BMI)分为肥胖糖尿病组(BMI≥25)50例、非肥胖糖尿病组(BMI<25)45例,并将30名正常体重非糖尿病者设为对照组。用ELISA方法检测各组空腹血清RBP4浓度,同时测定血糖、血脂、血压、胰岛素等,用胰岛素抵抗指数(HOMA-IR)评价各组胰岛素敏感性。结果肥胖糖尿病组RBP4明显高于非肥胖糖尿病组和对照组(P<0.05),而后两组RBP4比较差异无统计学意义;单因素相关分析显示,血清RBP4与腰围(W)、HOMA-IR、BMI、三酰甘油(TG)呈正相关;多元逐步回归分析发现,腰围、HOMA-IR为影响RBP4的最显著因素。结论肥胖2型糖尿病患者血清RBP4显著升高;腰围和胰岛素抵抗是影响血清RBP4的主要因素。     

Retinol-binding protein 4 as a plasma biomarker of renal dysfunction and cardiovascular disease in type 2 diabetes

OBJECTIVE: The retinol-binding protein 4 (RBP4) has been linked to the insulin resistance state in obesity and type 2 diabetes in animal studies. Data in humans are controversial and their relationship with organ damage in diabetic patients is lacking. We studied the association of plasma RBP4 with organ complications in type 2 diabetic patients. SETTING: Sant Joan University Hospital, Reus, Spain. SUBJECTS: 165 nonsmoker type 2 diabetic subjects according to American Diabetes Association criteria, aged 36-79 years, without proteinuria or severely decreased glomerular filtration rates (MDRD-GFR <30 mL min(-1) 1.73 m(-2)), were included in the study. MAIN OUTCOME MEASURE: Plasma RBP4 concentrations were the primary outcome variable. Statistics were performed in relation to clinical and subclinical arteriosclerosis, renal function parameters and biochemical data. RESULTS: Plasma RBP4 concentrations were positively correlated with serum creatinine levels (r = 0.322, P < 0.001) and inversely correlated with MDRD-GFR (r = -0.468, P = 0.009). Patients with moderately renal dysfunction (MDRD-GFR <60 mL min(-1) 1.73 m(-2)) had higher plasma RBP4 concentrations than those with normal to mildly decreased GFR (55.3 +/- 24.6 vs. 40.8 +/- 15.4, P <0.001). Patients in the top quartile of RBP4 concentrations had an increased adjusted odds ratio for moderately renal dysfunction compared with lower quartiles (4.68; 95% CI: 1.52-14.36, P = 0.007). The presence of microalbuminuria was not associated with RBP4. Plasma RBP4 concentrations were higher in those subjects with previous clinical arteriosclerosis than in event-free subjects (48.8 +/- 24.2 vs. 40.6 +/- 13.9, P = 0.045). The presence of retinopathy or polyneuropathy did not differ across RBP4 quartiles. CONCLUSIONS: Plasma RBP4 concentration might be a biomarker of nephropathy and cardiovascular disease in type 2 diabetic subjects.     

Toll-like receptors activate programmed necrosis in macrophages through a receptor-interacting kinase-3-mediated pathway

We report here that mouse macrophages undergo receptor-interacting kinase-3 (RIP3)-dependent but TNF-α-independent necrosis when Toll-like receptors (TLR) 3 and 4 are activated by poly(I:C) and LPS, respectively. An adaptor protein, Toll/IL-1 receptor domain-containing adapter inducing IFN-β (TRIF/TICAM-1), which is dispensable for TNF-α-induced necrosis, forms a complex with RIP3 upon TLR3/TLR4 activation and is essential for TLR3/TLR4-induced necrosis. Mice without RIP3 or functional TRIF did not show macrophage loss and elevation of inflammatory cytokines when they were exposed to LPS. Necrosis in mouse macrophages induced by either TNFR or TLR3/TLR4 is executed by reactive oxygen species. Taken together, these data indicate that there are multiple upstream necrosis-initiating signaling pathways converging on the RIP3 during an innate immune response to viral and bacterial infections in mammals.     

Flagellin-induced NLRC4 phosphorylation primes the inflammasome for activation by NAIP5

The Nlrc4 inflammasome contributes to immunity against intracellular pathogens that express flagellin and type III secretion systems, and activating mutations in NLRC4 cause autoinflammation in patients. Both Naip5 and phosphorylation of Nlrc4 at Ser533 are required for flagellin-induced inflammasome activation, but how these events converge upon inflammasome activation is not known. Here, we showed that Nlrc4 phosphorylation occurs independently of Naip5 detection of flagellin because Naip5 deletion in macrophages abolished caspase-1 activation, interleukin (IL)-1β secretion, and pyroptosis, but not Nlrc4 phosphorylation by cytosolic flagellin of Salmonella Typhimurium and Yersinia enterocolitica. ASC speck formation and caspase-1 expression also were dispensable for Nlrc4 phosphorylation. Interestingly, Helicobacter pylori flagellin triggered robust Nlrc4 phosphorylation, but failed to elicit caspase-1 maturation, IL-1β secretion, and pyroptosis, suggesting that it retained Nlrc4 Ser533 phosphorylating-activity despite escaping Naip5 detection. In agreement, the flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas deletion of the S. Typhimurium flagellin carboxy-terminus prevented caspase-1 maturation only. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation prepares Nlrc4 for subsequent activation by the flagellin sensor Naip5.Inflammasomes contribute critically to immunity and antimicrobial host defense of mammalian hosts. Their activation is tightly controlled because aberrant inflammasome signaling is harmful to the host, and results in inflammatory diseases (1, 2). Inflammasomes are a set of cytosolic multiprotein complexes that recruit and activate caspase-1, a key protease that triggers secretion of the inflammatory cytokines interleukin (IL)-1β and IL-18. In addition, caspase-1 induces pyroptosis, a proinflammatory and lytic cell death mode that contributes to pathogen clearance (3, 4). Several inflammasomes respond to a distinctive set of microbial pathogens (5). Activating mutations in the nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) member Nlrc4 were recently shown to induce autoinflammation in patients (6–8). Moreover, the inflammasome assembled by Nlrc4 is critically important for clearing a variety of bacterial infections, including Salmonella enterica serovar Typhimurium (S. Typhimurium), Shigella flexneri, Pseudomonas aeruginosa, Burkholderia thailandensis, and Legionella pneumophila (3, 9–17). These intracellularly-replicating bacteria have in common that they propel themselves with flagella (18) and/or express bacterial type III secretion systems (T3SS) to translocate effector proteins into infected host cells (19). Members of the NLR apoptosis-inhibitory protein (Naip) subfamily recognize the cytosolic presence of the building blocks of these evolutionary conserved bacterial structures, and trigger Nlrc4 to assemble an inflammasome (20–25). C57BL/6J mice express four Naip proteins, Naip1, -2, -5, and -6, which are expressed from a multigene cluster located on chromosome 13qD1 (26). Mouse Naip1 and human NAIP bind T3SS needle proteins, Naip2 interacts with the T3SS basal rod component PrgJ, and Naip5 and Naip6 recognize flagellin (20, 22–25).In addition to these Naip sensors, recent work showed that phosphorylation of Nlrc4 at Ser533 is critical for activation of the Nlrc4 inflammasome following infection with S. Typhimurium and L. pneumophila, or transfection of purified S. Typhimurium flagellin (27). Reconstitution of immortalized Nlrc4^−/− macrophages with wild-type Nlrc4 restored S. Typhimurium- and L. pneumophila-induced inflammasome activation, whereas cells reconstituted with Nlrc4 S533A mutant were specifically defective in maturation of caspase-1, secretion of IL-1β, assembly of ASC (apoptosis-associated speck-like protein containing a CARD) specks and induction of pyroptosis by these pathogens (27). However, a central outstanding question is how these upstream events (i.e., bacterial recognition by Naip members and Nlrc4 phosphorylation) relate to each other. Naip binding of bacterial components may trigger Nlrc4 phosphorylation to induce inflammasome activation. Alternatively, Nlrc4 phosphorylation and Naip sensing of flagellin and T3SS may converge independently onto Nlrc4 inflammasome activation.Here, we approached this question by breeding Nlrc4^Flag/Flag mice that express Nlrc4 fused to a carboxy-terminal 3× Flag tag from both Nlrc4 alleles (27) with Naip5-deficient mice (22, 28). We found S. Typhimurium infection and cytosolic delivery of S. Typhimurium flagellin, S. Typhimurium PrgJ and Yersinia enterocolitica flagellin to induce Nlrc4 phosphorylation at Ser533 independently of Naip5. Interestingly, Helicobacter pylori (H. pylori) flagellin induced robust Nlrc4 Ser533 phosphorylation without caspase-1 activation, suggesting that Nlrc4 Ser533 phosphorylation and caspase-1 activation are molecularly decoupled. In agreement, the S. Typhimurium flagellin D0 domain was required and sufficient for Nlrc4 phosphorylation, whereas caspase-1 activation required the flagellin carboxy-terminus. Collectively, this work suggests a biphasic activation mechanism for the Nlrc4 inflammasome in which Ser533 phosphorylation primes Nlrc4 for subsequent activation by the flagellin sensor Naip5.     

Role of sweet taste receptors in the activation of NLRP3 inflammasome signaling in diabetic kidney disease

正Objective To investigate the role of sweet taste receptors (STRs) in the activation of reactive oxygen species (ROS)-NLRP3 inflammasome signaling in diabetic kidney disease (DKD).Methods DKD mice were established by high-fat diet and streptozotocin. After mouse glomerular mesangial cells (GMCs) were exposed to high glucose, STRs (T1R2/T1R3) and associated signaling components and NLRP3 inflammasome signaling compo-     

Melatonin alleviates acute lung injury through inhibiting the NLRP3 inflammasome

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinically severe respiratory disorders, and there are currently no Food and Drug Administration–approved drug therapies. Melatonin is a well‐known anti‐inflammatory molecule, which has proven to be effective in ALI induced by many conditions. Emerging studies suggest that the NLRP3 inflammasome plays a critical role during ALI. How melatonin directly blocks activation of the NLRP3 inflammasome in ALI remains unclear. In this study, using an LPS‐induced ALI mouse model, we found intratracheal (i.t.) administration of melatonin markedly reduced the pulmonary injury and decreased the infiltration of macrophages and neutrophils into lung. During ALI, the NLRP3 inflammasome is significantly activated with a large amount of IL‐1β and the activated caspase‐1 occurring in the lung. Melatonin inhibits the activation of the NLRP3 inflammasome by both suppressing the release of extracellular histones and directly blocking histone‐induced NLRP3 inflammasome activation. Notably, i.t. route of melatonin administration opens a more efficient therapeutic approach for treating ALI.     

Retinol binding protein 4, low birth weight-related insulin resistance and hormonal contraception

It has been recently reported that increased serum levels of retinol binding protein 4 (RBP4), a molecule secreted by adipocytes and liver, could be an early marker of insulin resistance (IR). We determined whether serum RBP4 was increased in low birth weight (LBW)-young women as a model of early-onset IR, through a historical prospective study. The study-population included 35 LBW and 35 born at term appropriate for gestational age (term AGA) young women. Metabolic evaluations included the composite-insulin sensitivity index (composite ISI). Serum RBP4 was measured with a competitive enzyme-linked immunosorbent assay (ELISA). RBP4 levels were similar in LBW and term AGA women, while composite ISI was significantly lower in the former group. With multivariate logistic regression analysis hormonal contraception (HC) use but not birth weight, diabetes in either parents and body mass index was significantly associated with higher RBP4 levels: odds ratio = 10.6; 95% confidence interval (CI) = 2.4–76.6. In spite of higher RBP4 levels in women under HC, composite ISI was similar in women with or without HC. Women under HC also exhibited significantly higher levels of sex hormone binding globulin (SHBG), triglycerides, cholesterol, and C-reactive protein (CRP), and all of them, but not composite ISI, were significantly correlated with RBP4 levels. In conclusion, RBP4 serum level was not a marker of IR but, for the first time, it is documented a sustained increase of serum RBP4 under HC. Pathophysiological and clinical significance of this novel finding requires further investigations     

Activation of the NLRP3 inflammasome by CCl4 exacerbates hepatopathogenic diet-induced experimental NASH

Introduction and objectivesAdministration of carbon tetrachloride (CCl₄), along with an hepatopathogenic diet, is widely employed as a chemical inducer to replicate human nonalcoholic steatohepatitis (NASH) in rodents; however, the role of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (NLRP3) inflammasome in this model remains unclear. We aimed to determine the relevance of NLRP3 inflammasome activation in the development of NASH induced by CCl₄ along with an hepatopathogenic diet in male Wistar rats.Materials and methodsAnimals were fed either a high fat, sucrose, and cholesterol diet (HFSCD) or a HFSCD plus intraperitoneal injections of low doses of CCl₄ (400 mg/kg) once a week for 15 weeks. Liver steatosis, inflammation, fibrosis, and NLRP3 inflammasome activation were evaluated using biochemical, histological, ultrastructural, and immunofluorescence analyses, western blotting, and immunohistochemistry.ResultsOur experimental model reproduced several aspects of the human NASH pathophysiology. NLRP3 inflammasome activation was induced by the combined effect of HFSCD plus CCl₄ and significantly increased levels of both proinflammatory and profibrogenic cytokines and collagen deposition in the liver; thus, NASH severity was higher in the HFSCD+CCl₄ group than that in the HFSCD group, to which CCl₄ was not administered. Hepatic stellate cells, the most profibrogenic cells, were activated by HFSCD plus CCl₄, as indicated by elevated levels of α-smooth muscle actin. Thus, activation of the NLRP3 inflammasome, triggered by low doses of CCl₄, exacerbates the severity of NASH.ConclusionsOur results indicate that NLRP3 inflammasome activation plays a key role and may be an important therapeutic target for NASH treatment.     

Galectin-3 promotes noncanonical inflammasome activation through intracellular binding to lipopolysaccharide glycans

Cytosolic lipopolysaccharides (LPSs) bind directly to caspase-4/5/11 through their lipid A moiety, inducing inflammatory caspase oligomerization and activation, which is identified as the noncanonical inflammasome pathway. Galectins, β-galactoside–binding proteins, bind to various gram-negative bacterial LPS, which display β-galactoside–containing polysaccharide chains. Galectins are mainly present intracellularly, but their interactions with cytosolic microbial glycans have not been investigated. We report that in cell-free systems, galectin-3 augments the LPS-induced assembly of caspase-4/11 oligomers, leading to increased caspase-4/11 activation. Its carboxyl-terminal carbohydrate-recognition domain is essential for this effect, and its N-terminal domain, which contributes to the self-association property of the protein, is also critical, suggesting that this promoting effect is dependent on the functional multivalency of galectin-3. Moreover, galectin-3 enhances intracellular LPS-induced caspase-4/11 oligomerization and activation, as well as gasdermin D cleavage in human embryonic kidney (HEK) 293T cells, and it additionally promotes interleukin-1β production and pyroptotic death in macrophages. Galectin-3 also promotes caspase-11 activation and gasdermin D cleavage in macrophages treated with outer membrane vesicles, which are known to be taken up by cells and release LPSs into the cytosol. Coimmunoprecipitation confirmed that galectin-3 associates with caspase-11 after intracellular delivery of LPSs. Immunofluorescence staining revealed colocalization of LPSs, galectin-3, and caspase-11 independent of host N-glycans. Thus, we conclude that galectin-3 amplifies caspase-4/11 oligomerization and activation through LPS glycan binding, resulting in more intense pyroptosis—a critical mechanism of host resistance against bacterial infection that may provide opportunities for new therapeutic interventions.

Lipopolysaccharides (LPSs) are pathogen-associated molecular patterns that can elicit a host defense response through binding to cell-surface Toll-like receptor 4 (TLR4). Systemic inflammatory response syndrome is induced by overstimulation of the innate immune response via LPSs, resulting in severe multiple organ failure, which is a major cause of death worldwide in intensive care units (1). LPS-induced dimerization of TLR4 initiates signal transduction involving the NF-κB– and MyD88-dependent and -independent pathways, thereby contributing to various inflammatory responses (2). Another set of the immune repertoire, which resides in the cytosol and comprises NLRP1, NLRP3, NAIP/NLRC4, and AIM2, is known as the inflammasome. Inflammasomes can be activated in response to a number of well-defined pathogen-derived ligands and physiological aberrations, which in turn trigger caspase-1–mediated pyroptotic death (3, 4). This process has been associated with strengthening the host defense program to eliminate intracellular bacteria.Recently, a cytosolic LPS-sensing pathway involving caspase-4/5 in humans and caspase-11 in mice was termed the noncanonical inflammasome pathway, and this pathway is independent of TLR4 (5–8). LPSs from extracellular bacteria can enter the cytoplasm and trigger caspase-4/5/11–dependent responses. LPSs can be delivered into the cytosol when LPS-containing outer membrane vesicles (OMVs) from gram-negative bacteria are taken up by the cells or when intracellular bacteria escape from the phagosomes that are damaged by host resistant factors such as guanylate-binding protein and HMGB1 or microbe-derived hemolysins (9–12). LPSs comprise three regions: lipid A, core oligosaccharide, and O-polysaccharide (also termed O-antigen). The lipid A moiety binds directly to the caspase-4/5/11 caspase activation and recruitment domain (CARD, also known as prodomain), leading to caspase oligomerization and activation (7). This event likely mimics the proximity-induced dimerization model of initiator caspase activation (13). Furthermore, caspase-4/5/11 executes downstream signaling events via gasdermin D. Activated inflammatory caspase proteolytically cleaves gasdermin D to create an N-terminal fragment that self-oligomerizes and then inserts into the cell membrane to form pores, causing lytic cell death (14–17). Various stimuli have been identified in the caspase-1–mediated canonical-inflammasome signaling pathway (3, 4), but the detailed mechanism underlying noncanonical inflammasome activation mediated by caspase-4/5/11 remains unclear.Galectins, a family of β-galactoside–binding proteins, can decode host-derived complex glycans and are involved in various biological responses (18–23). Galectins are nucleocytoplasmic proteins synthesized without a classical signal sequence, although they can be secreted through unconventional pathways (19, 21, 23, 24). Recent studies have revealed prominent roles of cytosolic galectins in host defense programs (12, 25, 26). The proposed molecular mechanisms involve the binding of galectins to host glycans exposed to the cytosolic milieu upon endosomal or phagosomal membrane damage. In addition to binding host glycans, galectins also recognize microbial glycans, particularly LPSs (27–30). However, the contribution of galectins to the host response through binding to cytosolic LPSs is unknown.Galectin-3 is an ∼30-kDa protein that contains a carbohydrate-recognition domain (CRD) connected to N-terminal proline, glycine, and tyrosine-rich tandem repeats. Upon binding to multivalent glycoconjugates through its CRD, the protein forms oligomers, which is attributable to the self-association property of its N-terminal region (31, 32). Galectin-3 binds to LPSs of various gram-negative bacteria by recognizing their carbohydrate residues (33–36).Although structural information is scarce (37), existing information suggests that ligand-induced oligomerization of caspase CARD is necessary for the activation of inflammatory caspases (7, 38). Therefore, we hypothesized that galectin-3 may be an intracellular LPS sensor that participates in LPS-induced CARD-mediated inflammatory caspase activation. Specifically, highly ordered arrays of LPS–galectin-3 complexes may amplify caspase-4/5/11 oligomerization and activation. Here, we investigated the formation of galectin-3–LPS–caspase-4/11 complexes in cell-based and cell-free systems. Our findings provide evidence regarding a role of galectin-3 as an intracellular mediator in noncanonical inflammasome activation through LPS glycan recognition.     

TLR4信号传导通路与胰腺炎

胰腺炎的发病机制长期以来一直是基础和临床研究的一个重要课题,然而至今尚不完全明确.研究证实TLRs(Toll-like receptors)家族成员中TLR4可与G-菌内毒素脂多糖(lipopolysaccharide,LPS)结合,通过NF-κB信号通路激发多种炎症因子的合成进而参与多种器官疾病的发病过程.在鼠类模型和临床研究中已经显示TLR4信号通路在急性胰腺炎(acute pancreatitis,AP)的发病过程中起着重要的作用;上调TLR4信号通路可诱导致炎细胞因子大量释放参与重症急性胰腺炎(severe acute pancreatitis,SAP)病程中多器官功能障碍综合征的形成.因此,进一步明确TLR4信号通路在胰腺炎发病机制的作用,有可能通过阻断TLR4信号通路使胰腺炎获得疗效.