NLRP1 haplotypes associated with vitiligo and autoimmunity increase interleukin-1β processing via the NLRP1 inflammasome

Nuclear localization leucine-rich-repeat protein 1 (NLRP1) is a key regulator of the innate immune system, particularly in the skin where, in response to molecular triggers such as pathogen-associated or damage-associated molecular patterns, the NLRP1 inflammasome promotes caspase-1–dependent processing of bioactive interleukin-1β (IL-1β), resulting in IL-1β secretion and downstream inflammatory responses. NLRP1 is genetically associated with risk of several autoimmune diseases including generalized vitiligo, Addison disease, type 1 diabetes, rheumatoid arthritis, and others. Here we identify a repertoire of variation in NLRP1 by deep DNA resequencing. Predicted functional variations in NLRP1 reside in several common high-risk haplotypes that differ from the reference by multiple nonsynonymous substitutions. The haplotypes that are high risk for disease share two substitutions, L155H and M1184V, and are inherited largely intact due to extensive linkage disequilibrium across the region. Functionally, we found that peripheral blood monocytes from healthy subjects homozygous for the predominant high-risk haplotype 2A processed significantly greater (P < 0.0001) amounts of the IL-1β precursor to mature bioactive IL-1β under basal (resting) conditions and in response to Toll-like receptor (TLR) agonists (TLR2 and TLR4) compared with monocytes from subjects homozygous for the reference haplotype 1. The increase in basal release was 1.8-fold greater in haplotype 2A monocytes, and these differences between the two haplotypes were consistently observed three times over a 3-mo period; no differences were observed for IL-1α or TNFα. NLRP1 RNA and protein levels were not altered by the predominant high-risk haplotype, indicating that altered function of the corresponding multivariant NLRP1 polypeptide predisposes to autoimmune diseases by activation of the NLRP1 inflammasome.     

Vitamin D differentially regulates Salmonella-induced intestine epithelial autophagy and interleukin-1β expression

AIM To investigate the effects of active vitamin D3 on autophagy and interleukin(IL)-1β expression in Salmonella-infected intestinal epithelial cells(IECs).METHODS Caco-2 cells, NOD2 siR NA-, Atg16L1 siR NA- or vitamin D receptor(VDR) siR NA-transfected Caco-2 cells were pretreated with 1,25-dihydroxyvitamin D3(1,25D3), and then infected by wild-type S. typhimurium strain SL1344. The conversion of LC3-I to LC3-II was detected by Western blot analysis and LC3+ autophagosome was analyzed by immunofluorescence. Caco-2 cells or VDR si RNA-transfected cells were pretreated with 1,25D3, and then infected by SL1344. Membrane protein and total RNA were analyzed by Western blot and RT-PCR for VDR and Atg16L1 protein and m RNA expression, respectively. Atg16L1 si RNA-transfected Caco-2 cells were pretreated by 1,25D3 and then infected with SL1344. Total RNA was analyzed by RT-PCR for IL-1β mR NA expression.RESULTS The active form of vitamin D, 1,25D3, showed enhanced VDR-mediated Atg16L1 mR NA expression, membranous Atg16L1 protein expression leading to autophagic LC3 II proteins expression and LC3 punctae in Salmonella-infected Caco-2 cells which was counteracted by Atg16L1 and VDR si RNA, but Atg16L1 mediated suppression of IL-1β expression. Thus, active vitamin D may enhance autophagy but suppress inflammatory IL-1β expression in Salmonella-infected IECs.CONCLUSION Active vitamin D might enhance autophagic clearance of Salmonella infection, while modulation of inflammatory responses prevents the host from detrimental effects of overwhelming inflammation.     

Inflammasome activation and IL-1β target IL-1α for secretion as opposed to surface expression

Interleukin-1α (IL-1α) and -β both bind to the same IL-1 receptor (IL-1R) and are potent proinflammatory cytokines. Production of proinflammatory (pro)-IL-1α and pro-IL-1β is induced by Toll-like receptor (TLR)-mediated NF-κB activation. Additional stimulus involving activation of the inflammasome and caspase-1 is required for proteolytic cleavage and secretion of mature IL-1β. The regulation of IL-1α maturation and secretion, however, remains elusive. IL-1α exists as a cell surface-associated form and as a mature secreted form. Here we show that both forms of IL-1α, the surface and secreted form, are differentially regulated. Surface IL-1α requires NF-κB activation only, whereas secretion of mature IL-1α requires additional activation of the inflammasome and caspase-1. Surprisingly, secretion of IL-1α also required the presence of IL-1β, as demonstrated in IL-1β-deficient mice. We further demonstrate that IL-1β directly binds IL-1α, thus identifying IL-1β as a shuttle for another proinflammatory cytokine. These results have direct impact on selective treatment modalities of inflammatory diseases.     

Targeting IL-1β in disease; the expanding role of NLRP3 inflammasome

NLRP3 inflammasome activation and IL-1β secretion have recently emerged as a central mechanism in the pathogenesis of disease. Genetically defined syndromes like cryopyrin-associated periodic syndromes (CAPS, cryopyrinopathies) and familial Mediterranean fever (FMF) or diseases associated with NLRP3 activation by danger signals like gout, pseudogout, Alzheimer's disease or type 2 diabetes are included in this group of diseases. The contribution of anakinra, a recombinant, nonglycosylated human IL-1 receptor antagonist, in both the identification and treatment of such syndromes was considerable. Recently, rilonacept, a long-acting IL-1 receptor fusion protein, and canakinumab, a fully humanized anti-IL-1β monoclonal antibody, have been developed, with the intention to further extent IL-1β inhibition treatment strategies to a broader spectrum of disorders beyond the characterized autoinflammatory syndromes, offering a more favorable administration profile. On the other hand, the developed caspase-1 inhibitors, even though effective in experimental models, were not proven efficient in the treatment of inflammatory diseases.     

FXR1P is a GSK3β substrate regulating mood and emotion processing

Inhibition of glycogen synthase kinase 3β (GSK3β) is a shared action believed to be involved in the regulation of behavior by psychoactive drugs such as antipsychotics and mood stabilizers. However, little is known about the identity of the substrates through which GSK3β affects behavior. We identified fragile X mental retardation-related protein 1 (FXR1P), a RNA binding protein associated to genetic risk for schizophrenia, as a substrate for GSK3β. Phosphorylation of FXR1P by GSK3β is facilitated by prior phosphorylation by ERK2 and leads to its down-regulation. In contrast, behaviorally effective chronic mood stabilizer treatments in mice inhibit GSK3β and increase FXR1P levels. In line with this, overexpression of FXR1P in the mouse prefrontal cortex also leads to comparable mood-related responses. Furthermore, functional genetic polymorphisms affecting either FXR1P or GSK3β gene expression interact to regulate emotional brain responsiveness and stability in humans. These observations uncovered a GSK3β/FXR1P signaling pathway that contributes to regulating mood and emotion processing. Regulation of FXR1P by GSK3β also provides a mechanistic framework that may explain how inhibition of GSK3β can contribute to the regulation of mood by psychoactive drugs in mental illnesses such as bipolar disorder. Moreover, this pathway could potentially be implicated in other biological functions, such as inflammation and cell proliferation, in which FXR1P and GSK3 are known to play a role.Glycogen synthase kinase 3α and β (GSK3α/β) are two serine threonine kinases involved in myriads of biological functions ranging from metabolism to immunity and behavior (1, 2). In particular, several single nucleotide polymorphisms (SNPs) in the GSK3B locus or in genes involved in GSK3β signaling have been identified as genetic risk factors for bipolar disorder and schizophrenia (3, 4). In addition, GSK3β is inhibited—either directly or following its phosphorylation on Ser9 by AKT—in response to three major classes of psychiatric drugs that are mood stabilizers, antidepressants, and antipsychotics (5–10). Furthermore, pharmacological or genetic inhibition of GSK3β replicates behavioral effects of these drugs in rodents (11).Because of its potential implication in mental disorders and their treatment, pharmacological inhibition of GSK3β has become an attractive therapeutic strategy for mental illnesses (12). However, several limitations associated to the selectivity and long-term toxicity of GSK3β inhibitors have remained serious obstacles (13, 14). One interesting alternative would be to target GSK3β substrates that are involved in the regulation of behavior by this kinase. However, the nature of these substrates has remained elusive, and evidence for their role in regulating behavior in humans is scarce.An important layer of complexity for the identification of substrates involved in the regulation of behavior by GSK3β comes from the fact that GSK3β displays a 500–1,000-fold preference toward phosphoproteins (2, 15, 16). Indeed, most GSK3β substrates require prior phosphorylation to be phosphorylated by this kinase. This phenomenon, called “priming,” results from the nature of the consensus amino acid sequence S/T₁XXXpS/T₂ that is recognized and phosphorylated by GSK3β. In this sequence, the amino-terminal S/T₁ corresponds to the serine or threonine that is phosphorylated by GSK3β, X is any amino acid, and pS/T₂ is the serine or threonine that acts as a priming site for GSK3β. Because of this need for priming, phosphorylation by GSK3β may therefore be highly context dependent and vary with changes of cell signaling landscapes that could be permissive or nonpermissive for the priming of a specific substrate.Among the psychoactive drugs that affect GSK3β activity, mood stabilizers are a heterogeneous class of pharmacological agents used for the management of bipolar disorder as well as adjunct therapy for depression and schizophrenia. Lithium is the prototypical member of this class of psychiatric drugs that also includes the antiepileptic drugs lamotrigine and sodium valproate. Chronic administration of lithium, valproate, or lamotrigine has been shown to increase the inhibitory phosphorylation of GSK3β as a result of AKT activation (8, 17–20).Regulation of GSK3β in response to mood stabilizers has several behavioral readouts that are not presently explained by the action of this kinase on any of its known substrates. Inhibition of exploratory locomotion is a known behavioral effect of GSK3β inhibition that is used to model “antimanic” effects (21, 22). Furthermore, lithium treatments and inhibition of GSK3β have been shown to exert an effect similar to antidepressants by reducing immobility in the tail suspension test (TST) (8, 23–25). Finally, lithium exerts GSK3β signaling-dependent effects similar to antidepressants and anxiolytics in the dark–light emergence test (DLET) (8, 24, 25). In this test, mice are placed in a darkened compartment and allowed free exploration of darkened and adjoining lighted compartments. Reduced latency to explore the light compartment as well as an increase in time and activity in this compartment are general indices of drug effect on mood-related behavior (26).We identified two chronic treatments with sodium valproate (low-dose VAL 10 and high-dose VAL 25), which exhibit different behavioral effects in mice while resulting in comparable GSK3β inhibition. We have used this experimental paradigm to identify a substrate of GSK3β involved in the regulation of mood and emotions. This substrate, the fragile X mental retardation-related protein 1 (FXR1P), belongs to a small family of RNA binding proteins that also includes the fragile X mental retardation protein (FMRP) (27). Interestingly, SNPs in the FXR1 gene have recently been identify as genetic risk factors for schizophrenia (28).Our results indicate that FXR1P is down-regulated by GSK3β. Furthermore FXR1P is involved in the regulation of mood-related behaviors in mice, whereas genetic, brain imaging, and behavioral evidence obtained from healthy human subjects revealed a role of GSK3B/FXR1 gene interactions in regulating emotional control. Taken together, these findings support a role of a GSK3β/FXR1P signaling pathway in regulating behavioral dimensions relevant to mood disorders in humans. Furthermore identification of a functional regulation of FXR1P by GSK3β suggests that this pathway may also be important for other biological processes such as inflammation in which involvement of these two proteins has been shown (29, 30).     

Glucocorticoids facilitate astrocytic amyloid-β peptide deposition by increasing the expression of APP and BACE1 and decreasing the expression of amyloid-β-degrading proteases

In most cases, the molecular mechanism underlying the pathogenesis of sporadic Alzheimer's disease (AD) is unknown. Elevated basal cortisol levels in AD patients suggest that glucocorticoids (GC) may contribute to the development and/or maintenance of AD. Amyloid plaques are the hallmark of AD, and they are considered to play an early role in the AD process. However, little is known about how their formation is regulated by stress and GC. Astrocyte accumulation is one of the earliest neuropathological changes in AD. Here, we report that GC elevated amyloid-β (Aβ) production in primary cultures of astrocytes by increasing amyloid precursor protein (APP) and β-site APP-cleaving enzyme 1 gene expression. Notably, GC administered to normal, middle-aged mice promoted the expression of APP and β-site APP-cleaving enzyme 1 in astrocytes, as determined by double immunofluorescence. Additionally, confocal microscopy and ELISA revealed that GC markedly reduced Aβ degradation and clearance by astrocytes in vitro, indicating a decreased neuroprotective capacity of the astrocytes. This may have been due to the decrease of several Aβ-degrading proteases, such as insulin-degrading enzyme and matrix metalloproteinase-9. These effects occurred through the activation of GC receptors. Taken together, our results demonstrate that GC can enhance the production of Aβ, reduce its degradation in astrocytes, and provide a molecular mechanism linking stress factors to AD. Our study suggests that GC can facilitate AD pathogenesis and that reducing GC in the elderly and early AD patients would be beneficial.     

4-Hydroxynonenal differentially regulates adiponectin gene expression and secretion via activating PPARγ and accelerating ubiquitin-proteasome degradation

Although well-established, the underlying mechanisms involved in obesity-related plasma adiponectin decline remain elusive. Oxidative stress is associated with obesity and insulin resistance and considered to contribute to the progression toward obesity-related metabolic disorders. In this study, we investigated the effects of 4-hydroxynonenal (4-HNE), the most abundant lipid peroxidation end product, on adiponectin production and its potential implication in obesity-related adiponectin decrease. Long-term high-fat diet feeding led to obesity in mouse, accompanied by decreased plasma adiponectin and increased adipose tissue 4-HNE content. Exposure of adipocytes to exogenous 4-HNE resulted in decreased adiponectin secretion in a dose-dependent manner, which was consistent with significantly decreased intracellular adiponectin protein abundance. In contrast, adiponectin gene expression was significantly elevated by 4-HNE treatment, which was concomitant with increased peroxisome proliferator-activated receptor gamma (PPAR-γ) gene expression and transactivity. The effect was abolished by T0070907, a PPAR-γ antagonist, suggesting that PPAR-γ activation plays a critical role in this process. To gain insight into mechanisms involved in adiponectin protein decrease, we examined the effects of 4-HNE on adiponectin protein degradation. Cycloheximide (CHX)-chase assay revealed that 4-HNE exposure accelerated adiponectin protein degradation, which was prevented by MG132, a potent proteasome inhibitor. Immunoprecipitation assay showed that 4-HNE exposure increased ubiquitinated adiponectin protein levels. These data altogether indicated that 4-HNE enhanced adiponectin protein degradation via ubiquitin-proteasome system. Finally, we demonstrated that supplementation of HF diet with betaine, an antioxidant and methyl donor, alleviated high-fat-induced adipose tissue 4-HNE increase and attenuated plasma adiponectin decline. Taken together, our findings suggest that the lipid peroxidation product 4-HNE can differentially regulates adiponectin gene expression and protein abundance and may play a mechanistic role in obesity-related plasma adiponectin decline.     

Glucocorticoid ameliorates early cardiac dysfunction after coronary microembolization and suppresses TGF-β1/Smad3 and CTGF expression

Objectives

This study was designed to evidence the protective effect of glucocorticoid therapy on cardiac dysfunction after coronary microembolization (CME), and to clarify its mechanism with the expression of transforming growth factor-beta 1 (TGF-β1)/Smad3 and connective tissue growth factor (CTGF).

Methods

Eighteen mini-pigs were studied, including Sham-operation group (n = 4), CME group (n = 8) and Glucocorticoid therapy group (n = 6, received methylprednisolone 25 mg/kg intravenously 30 min before CME). Magnetic resonance imaging (3.0-T) was performed at baseline, 6th hour and one week after operation to evaluate cardiac function. Serum TGF-β1, CTGF and troponin T were also detected. Myocardial expressions of TGF-β1, CTGF and Smad3 were detected by western blot and immunohistochemistry. Total collagen expression was demonstrated by Masson Trichrome stain.

Results

Compared with Sham-operation group, left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV) in CME group were increased at 6th hour after CME, while left ventricular ejection fraction (LVEF) was decreased significantly. Compared with CME group, methylprednisolone greatly improved LVEF after CME (6th hour: 56.0 ± 3.2% vs. 51.8 ± 3.8%, P = 0.030; one week: 57.8 ± 3.2% vs. 54.6 ± 2.6%, P = 0.053). We found that methylprednisolone not only significantly decreased serum TGF-β1, CTGF and troponin T, but also reduced myocardial expressions of TGF-β1, CTGF and Smad3 after CME. In addition, collagen volume fraction in glucocorticoid therapy group was markedly lower than that in CME group.

Conclusions

Glucocorticoid therapy could improve early cardiac function after CME, and its mechanism could be associated with TGF-β1/Smad3 and CTGF suppression.     

The fusion proteins TEL-PDGFRβ and FIP1L1-PDGFRα escape ubiquitination and degradation

Background

Chimeric oncogenes encoding constitutively active protein tyrosine kinases are associated with chronic myeloid neoplasms. TEL-PDGFRβ (TPβ, also called ETV6-PDGFRB) is a hybrid protein produced by the t(5;12) translocation, FIP1L1-PDGFRα (FPα) results from a deletion on chromosome 4q12 and ZNF198-FGFR1 is created by the t(8;13) translocation. These fusion proteins are found in patients with myeloid neoplasms associated with eosinophilia. Wild-type receptor tyrosine kinases are efficiently targeted for degradation upon activation, in a process that requires Cbl-mediated monoubiquitination of receptor lysines. Since protein degradation pathways have been identified as useful targets for cancer therapy, the aim of this study was to compare the degradation of hybrid and wild-type receptor tyrosine kinases.

Design and Methods

We used Ba/F3 as a model cell line, as well as leukocytes from two patients, to analyze hybrid protein degradation.

Results

In contrast to the corresponding wild-type receptors, which are quickly degraded upon activation, we observed that TPβ, FPα and the ZNF198-FGFR1 hybrids escaped down-regulation in Ba/F3 cells. The high stability of TPβ and FPα hybrid proteins was confirmed in leukocytes from leukemia patients. Ubiquitination of TPβ and FPα was much reduced compared to that of wild-type receptors, despite marked Cbl phosphorylation in cells expressing hybrid receptors. The fusion of a destabilizing domain to TPβ induced protein degradation. Instability was reverted by adding the destabilizing domain ligand, Shield1. The destabilization of this modified TPβ reduced cell transformation and STAT5 activation.

Conclusions

We have shown that chimeric receptor tyrosine kinases escape ubiquitination and down-regulation and that their stabilization is critical to efficient stimulation of cell proliferation.     

Genetic modulation of islet β-cell iPLA2β expression provides evidence for its impact on β-cell apoptosis and autophagy

β-cell apoptosis is a significant contributor to β-cell dysfunction in diabetes and ER stress is among the factors that contributes to β-cell death. We previously identified that the Ca²⁺-independent phospholipase A₂β (iPLA₂β), which in islets is localized in β-cells, participates in ER stress-induced β-cell apoptosis. Here, direct assessment of iPLA₂β role was made using β-cell-specific iPLA₂β overexpressing (RIP-iPLA₂β-Tg) and globally iPLA₂β-deficient (iPLA₂β-KO) mice. Islets from Tg, but not KO, express higher islet iPLA₂β and neutral sphingomyelinase, decrease in sphingomyelins, and increase in ceramides, relative to WT group. ER stress induces iPLA₂β, ER stress factors, loss of mitochondrial membrane potential (?Ψ), caspase-3 activation, and β-cell apoptosis in the WT and these are all amplified in the Tg group. Surprisingly, β-cells apoptosis while reduced in the KO is higher than in the WT group. This, however, was not accompanied by greater caspase-3 activation but with larger loss of ?Ψ, suggesting that iPLA₂β deficiency impacts mitochondrial membrane integrity and causes apoptosis by a caspase-independent manner. Further, autophagy, as reflected by LC3-II accumulation, is increased in Tg and decreased in KO, relative to WT. Our findings suggest that (1) iPLA₂β impacts upstream (UPR) and downstream (ceramide generation and mitochondrial) pathways in β-cells and (2) both over- or under-expression of iPLA₂β is deleterious to the β-cells. Further, we present for the first time evidence for potential regulation of autophagy by iPLA₂β in islet β-cells. These findings support the hypothesis that iPLA₂β induction under stress, as in diabetes, is a key component to amplifying β-cell death processes.     

血清NLRP3、IL-1Ra、IL-1水平与冠心病相关性的临床研究

目的 探讨血清NOD样受体蛋白3(NLRP3)、白细胞介素（IL）-1Ra、IL-1水平与冠心病的相关性。方法 选取义乌稠州医院2019年1月至2021年1月收治的149例冠心病患者为观察组,同期体检的100名健康志愿者为对照组,比较两组对象血清NLRP3、IL-1Ra、IL-1等炎症因子水平;比较不同病变支数、Gensini评分冠心病患者血清炎症因子水平,采用ROC曲线分析血清炎症因子水平预测冠心病患者病变支数的效能,Pearson相关分析血清炎症因子水平与Gensini评分的相关性。结果 观察组患者血清NLRP3、IL-1Ra、IL-1水平均明显高于对照组,差异均有统计学意义（均P<0.05）。不同病变支数、Gensini评分冠心病患者血清炎症因子水平比较,差异均有统计学意义（均P<0.05）。血清NLRP3、IL-1Ra、IL-1水平预测冠心病患者三支病变的AUC分别为0.822、0.813、0.783 （均P<0.05）。冠心病患者血清NLRP3、IL-1Ra、IL-1水平与Gensini评分均呈正相关（r=0.374、0.411、0.317,均P<0....