Interleukin-1β induces autophagy by affecting calcium homeostasis and trypsinogen activation in pancreatic acinar cells

The strong up-regulation of inflammatory mediators has been reported to play a key role in acute pancreatitis (AP). Elevated serum levels of interleukin-1β (IL-1β) are associated with the development of AP. However, the precise effect and mechanism of IL-1β in AP remains obscure. In this study, we investigated the potential role and mechanism of IL-1β in AP. We measured autophagy activation in response to IL-1β in AR42J cells. The disrupting effects of IL-1β on cellular Ca²⁺ were observed. To determine whether the disruption of Ca²⁺ signaling has protective effects in vivo during AP, male C57BL/6 mice were treated with cerulein to induce AP. We found that the treatment of AR42J cells with IL-1β triggered autophagy and that the autophagic flux was impaired. In addition, IL-1β induced Ca²⁺ release from the ER. Furthermore, the expression of the ER stress markers GRP78 and IRE1 also increased. 2APB, an antagonist of the InsP₃ receptor, inhibited increased expression of autophagy markers. Subsequent biochemical assays revealed that co-culture with IL-1β could induce the activation of trypsinogen to trypsin and reduce the viability of acinar cells. Pathological changes of the pancreas were also observed in vivo. We found that the pathological injuries of the pancreas were significantly alleviated in mice co-treated with 2APB. Taken together, our results indicate that IL-1β can induce trypsin activation and decrease cellular viability in pancreatic acinar cells. These effects depend on impaired autophagy via intracellular calcium changes. Ca²⁺ signaling may become a promising therapeutic target in the treatment of pancreatitis.     

Uric acid induces trophoblast IL-1β production via the inflammasome: implications for the pathogenesis of preeclampsia

Citation
Mulla MJ, Myrtolli K, Potter J, Boeras C, Kavathas PB, Sfakianaki AK, Tadesse S, Norwitz ER, Guller S, Abrahams VM. Uric acid induces trophoblast IL‐1β production via the inflammasome: implications for the pathogenesis of preeclampsia. Am J Reprod Immunol 2010; 65: 542–548 Problem  Preeclampsia is associated with hyperuricemia, which correlates with the disease severity. Levels of circulating uric acid increase before the clinical manifestations, suggesting that they may be causally related. Uric acid, or monosodium urate (MSU), activates the Nod‐like receptor, Nalp3, leading to inflammasome activation and IL‐1β processing. Because preeclampsia is associated with placental immune/inflammatory dysregulation, we sought to determine in the trophoblast, the presence of the Nalp3 inflammasome, and the effect of MSU on its activation. Method of study  Isolated first‐ and third‐trimester trophoblasts were assessed for expression of the inflammasome components, Nalp1, Nalp3, and ASC. First‐trimester trophoblast cells were incubated with or without MSU, and after which, IL‐1β secretion and processing and caspase‐1 activation were determined. Results  Trophoblast cells expressed Nalp1, Nalp3, and ASC under basal conditions. Following incubation with MSU, first‐trimester trophoblast IL‐1β secretion was upregulated. This correlated with increased expression levels of active IL‐1β and active caspase‐1. ASC knockdown reduced MSU‐induced IL‐1β secretion. Conclusion  These findings demonstrate that uric acid activates the inflammasome in the trophoblast, leading to IL‐1β production. This may provide a novel mechanism for the induction of inflammation at the maternal–fetal interface leading to placental dysfunction and adverse pregnancy outcome, including preeclampsia.     

Strain- and host species-specific inflammasome activation,IL-1β release,and cell death in macrophages infected with uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTIs). Little is known about interactions between UPEC and the inflammasome, a key innate immune pathway. Here we show that UPEC strains CFT073 and UTI89 trigger inflammasome activation and lytic cell death in human macrophages. Several other UPEC strains, including two multidrug-resistant ST131 isolates, did not kill macrophages. In mouse macrophages, UTI89 triggered cell death only at a high multiplicity of infection, and CFT073-mediated inflammasome responses were completely NLRP3-dependent. Surprisingly, CFT073- and UTI89-mediated responses only partially depended on NLRP3 in human macrophages. In these cells, NLRP3 was required for interleukin-1β (IL-1β) maturation, but contributed only marginally to cell death. Similarly, caspase-1 inhibition did not block cell death in human macrophages. In keeping with such differences, the pore-forming toxin α-hemolysin mediated a substantial proportion of CFT073-triggered IL-1β secretion in mouse but not human macrophages. There was also a more substantial α-hemolysin-independent cell death response in human vs. mouse macrophages. Thus, in mouse macrophages, CFT073-triggered inflammasome responses are completely NLRP3-dependent, and largely α-hemolysin-dependent. In contrast, UPEC activates an NLRP3-independent cell death pathway and an α-hemolysin-independent IL-1β secretion pathway in human macrophages. This has important implications for understanding UTI in humans.     

Caspase-8 functions as a key mediator of inflammation and pro-IL-1β processing via both canonical and non-canonical pathways

Caspase-8 is an apical component of cell death pathways. Activated caspase-8 can drive classical caspase-dependent apoptosis and actively inhibits cell death mediated by RIPK3-driven necroptosis. Genetic deletion of Casp8 results in embryonic lethality as a result of uncontrolled necroptosis. This lethality can be rescued by simultaneous deletion of Ripk3. Recently, caspase-8 has been additionally connected to inflammatory pathways within the cell. In particular, caspase-8 has been shown to be crucially involved in the induction of pro-IL-1β synthesis and processing via both non-canonical and canonical pathways. In this review, we bring together current knowledge regarding the role of caspase-8 in cellular inflammation with a particular emphasis on the interplay between caspase-8 and the classical and non-canonical inflammasomes.     

Interleukin-1β induces apoptosis in annulus fibrosus cells through the extracellular signal-regulated kinase pathway

Purpose: The loss of intervertebral disc (IVD) cells due to excessive apoptosis induced by inflammatory cytokines is a major cause of IVD degeneration. This study aims to explore the mechanism of interleukin-1β (IL-1β)-induced apoptosis of annulus fibrosus cells (AFCs). It's hypothesized that IL-1β induces apoptosis through the extracellular signal-regulated kinase (ERK) pathway in AFCs.

Methods: The mRNA and protein expression levels of apoptosis-associated genes were analyzed by quantitative real-time PCR and Western blotting. The apoptotic rate was measured by flow cytometry. Three experimental groups were established, including Control, IL-1β, and IL-1β+U0126 groups, respectively.

Results: Increase in the expression of apoptosis-associated genes including B-cell lymphoma-2 associated X (Bax), caspase-3, and caspase-9, and meanwhile, decrease in the expression of B-cell lymphoma-2 (Bcl-2) gene were found in patients with degenerative IVDs. In in vitro tests, both apoptosis and phosphorylated ERK expression in rat AFCs decreased in the IL-1β+U0126 group compared with the IL-1β group. The expression levels of Bax, caspase-3, and caspase-9 in AFCs decreased significantly in the IL-1β+U0126 group compared with those in the IL-1β group. The expression level of Bcl-2, on the other hand, significantly increased.

Conclusions: Findings from this study suggest that IL-1β induces apoptosis in AFCs through the ERK pathway, and therefore, ERK inhibition may provide certain protection against the adverse effects of IL-1β.     

NLRP3 inflammasome activation and interleukin-1β release in macrophages require calcium but are independent of calcium-activated NADPH oxidases

Objective and design

We studied the involvement of calcium and calcium-activated NADPH oxidases in NLRP3 inflammasome activation and IL-1β release to better understand inflammasome signaling in macrophages.

Material or subjects

Human volunteer blood donors were recruited to isolate monocytes to differentiate them into macrophages. Wild-type or DUOX1-deficient C57/B6 mice were used to prepare bone marrow-derived macrophages.

Treatment

Murine or human macrophages were treated in vitro with NLRP3 inflammasome agonists (ATP, silica crystals) or calcium agonists (thapsigargin, ionomycin) in calcium-containing or calcium-free medium.

Methods

Intracellular calcium changes were followed by measuring FURA2-based fluorescence. Gene expression changes were measured by quantitative real-time PCR. Protein expression was assessed by western blotting. Enzymatic activity was measured by fluorescence caspase-1 activity assay. IL-1β release was determined by ELISA. ELISA data were analyzed by ANOVA and Tukey’s post hoc test.

Results

Our data show that calcium is essential for IL-1β release in human macrophages. Increases in cytosolic calcium alone lead to IL-1β secretion. Calcium removal blocks caspase-1 activation. Human macrophages express Duox1, a calcium-regulated NADPH oxidase that produces reactive oxygen species. However, Duox1-deficient murine macrophages show normal IL-1β release.

Conclusions

Human macrophage inflammasome activation and IL-1β secretion requires calcium but does not involve NADPH oxidases.     

Sophocarpine attenuates murine lupus nephritis via inhibiting NLRP3 inflammasome and NF-κB activation

Inflammation contributes to the pathogenesis of lupus nephritis (LN), which is the most serious complication that increases mortality of systemic lupus erythematosus (SLE). Sophocarpine is a type of quinolizidine alkaloid that possesses anti-inflammatory property. This study investigated the speculation that sophocarpine might play a beneficial effect in LN. Female MRL/lpr mice received sophocarpine treatment for 8 weeks. Renal function and histopathology were evaluated. The level of immune complex deposition was measured by immunofluorescent staining, and the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and anti-double-stranded DNA (anti-dsDNA) antibodies were measured by ELISA. Western blotting was carried out to evaluate the levels of proteins involved in NLRP3 inflammasome and NF-κB pathway. Sophocarpine treatment reduced urine protein excretion, blood urea nitrogen, and attenuated renal tissue damage. The levels of renal immune complex deposition, serum anti-dsDNA, and serum and renal inflammatory cytokines were significantly reduced by sophocarpine. Sophocarpine treatment reduced the levels of proteins that form NLRP3 inflammasome. Activation of NF-κB in the kidney was inhibited by sophocarpine. Sophocarpine could ameliorate experimental LN in MRL/lpr. These effects might be through suppressing NLRP3 inflammasome and NF-κB pathway.     

Dysregulated production of interleukin-1β upon activation of the NLRP3 inflammasome in patients with familial Mediterranean fever

Familial Mediterranean fever (FMF) is caused by mutations in pyrin, a protein expressed in innate immune cells that interacts with caspase-1 and other inflammasome components to regulate interleukin (IL)-1β maturation. Since NLRP3 inflammasome represents major source of IL-1β, we studied its protein expression and function in FMF. We isolated peripheral white blood cells (WBCs) from 20 symptoms-free FMF patients and 21 healthy individuals. Intracellular protein expression of NLRP3, caspase-1, IL-1β at baseline and after LPS/ATP sequential treatment for NLRP3 activation was assessed by immunoblotting. Secreted IL-1β was quantified by ELISA. THP-1 cells were transfected with wild-type or mutant pyrin and IL-1β secretion was measured. FMF WBCs exhibited lower NLRP3 and active caspase-1 protein expression compared to healthy individuals, and LPS/ATP treatment resulted in significantly lower intracellular IL-1β levels in FMF patients. Likewise, LPS/ATP induced caspase-1-dependent IL-1β release at significantly lower amounts in the FMF group (1182 ± 192 versus 2134 ± 245 pg/mL in controls, p = 0.004). Consistently, THP-1 cells transfected with FMF-associated M694V mutant pyrin displayed lower LPS/ATP-induced IL-1β compared with wild-type pyrin-transfected cells. FMF WBCs demonstrate reduced NLRP3-mediated IL-1β production. Additional studies are needed to define whether this finding represents a compensatory mechanism to control inflammation or is directly linked to disease pathogenesis.     

Ex-vivo α-galactosylceramide activation of NKT cells in humans and macaques

NKT cells are key mediators of antiviral and anticancer immunity. Experiments in mice have demonstrated that activation of NKT cells in vivo induces the expression of multiple effector molecules critical to successful immunity. Human clinical trials have shown similar responses, although in vivo activation of NKT cells in humans or primate models are far more limited in number and scope. Measuring ex vivo activation of NKT cells by the CD1d-restricted glycolipid ligand α-Galactosylceramide (α-GalCer) through cytokine expression profiles is a useful marker of NKT cell function, but for reasons that are unclear, this approach does not appear to work as well in humans and non-human primate macaque models in comparison to mice. We performed a series of experiments on human and macaque (Macaca nemestrina) fresh whole blood samples to define optimal conditions to detect NKT cell cytokine (TNF, IFNγ, IL-2) and degranulation marker (CD107a) expression by flow cytometry. We found that conditions previously described for mouse splenocyte NKT cell activation were suboptimal on human or macaque blood NKT cells. In contrast, a 6h incubation with brefeldin A added for the last 4h, in a 96-well plate based assay, and using an α-GalCer concentration of 1 μg/ml were optimal methods to stimulate NKT cells in fresh blood from both humans and macaques. Unexpectedly, we noted that blood NKT cells from macaques infected with SIV were more readily activated by α-GalCer than NKT cells from uninfected macaques, suggesting that SIV infection may have primed the NKT cells. In conclusion, we describe optimized methods for the ex vivo antigen-specific activation of human and macaque blood NKT cells. These assays should be useful in monitoring NKT cells in disease and in immunotherapy studies.     

Human immunodeficiency virus-1 recombinant gp120 induces changes in protein kinase C isozymes — A preliminary report

Human immunodeficiency virus 1 (HIV-1) and its purified proteins activate target cell functions. Because protein kinase C (PKC) plays a crucial role in signal transduction and there is a molecular heterogeneity of PKC, we compared the effect of recombinant HIV-1 gp 120 and phorbol ester (PMA) on PKC isozymes in monocytic U937 cells, with isozyme-specific antibodies using flow cytometry. All PKC isozymes except PKC-γ were present in U937 cells. Both PMA and HIV-1 gp120 increased levels of calcium-dependent and -independent PKC isozymes. The most striking change was observed in PKC-ξ isozymes levels. This study for the first time demonstrate the HIV-1 gp120 affects calcium-independent PKC isozymes in U937 cells.     

Mast cell chymase in keloid induces profibrotic response via transforming growth factor-β1/Smad activation in keloid fibroblasts

This study was to examine whether mast cell chymase exists in human keloids and exerts its profibrotic effect via transforming growth factor-β₁/Smad signaling pathway. The number of mast cells and the expression levels of chymase in keloids and normal skin were examined by immunohistochemistry assays. The mRNA expression and activity changes of chymase in keloids and normal skin were determined by real-time quantitative PCR and radioimmunoassay. After keloid fibroblasts were treated with different concentrations of chymase (0, 15, 30, 60, and 120 ng/mL) for various time periods, the proliferation of keloid fibroblasts, collagen synthesis, mRNA and protein expression of TGF-β₁, and the protein expression of phosphorylated Smad2/3, Smad2/3 and Smad7 were investigated using MTT assay, ELISA and Western blotting. Mast cells and chymase exist in keloid. Gene expression and activity of mast cell chymase in keloid are significantly higher than those in normal skin. Chymase promotes keloid fibroblast proliferation and collagen synthesis by activating TGF-β₁. The activation of Smad protein signaling pathway by chymase is related to the elevated P-Smad protein expression in keloid fibroblasts. Our data demonstrated that mast cell chymase plays an important role in keloid formation through TGF-β₁/Smad signaling pathway.     

Genomic and sequence variants of protein kinase A regulatory subunit type 1β (PRKAR1B) in patients with adrenocortical disease and Cushing syndrome

PurposeProtein kinase A (PKA) subunit defects (in PRKAR1A and PRKACA) are known to contribute to adrenal tumor pathogenesis. We studied the PRKAR1B gene for any genetic changes in bilateral adrenocortical hyperplasia (BAH) and cortisol-producing adrenal adenomas (CPA).MethodsExome sequencing and PRKAR1B copy-number variant (CNV) analysis were performed in 74 patients with BAH and 21 with CPA. PKA activity was studied in tumors with defects; sequence variants were investigated in vitro.ResultsThree PRKAR1B germline variants (p.I40V, p.A67V, p.A300T) were identified among 74 patients with BAH. PRKAR1B copy-number gains (CNG) were found in 3 of 21 CPAs, one in a tumor carrying a somatic PRKACA “hotspot” pathogenic variant p.L206R. CPAs bearing PRKAR1B CNGs showed higher PRKAR1B messenger RNA (mRNA) levels and reduced PKA activity. Baseline PKA activity was also decreased for p.A67V and p.A300T in vitro, and mutant PRKAR1β bound PRKACα in fluorescence resonance energy transfer (FRET) recordings of cotransfected HEK293 cells stronger than normal.ConclusionPRKAR1B is yet another PKA subunit that may potentially contribute to adrenal tumor formation. Its involvement in adrenocortical disease may be different from that of other subunits, because PRKAR1B variants and PRKAR1B CNGs were associated with decreased (rather than increased) overall PKA activity in vitro.     

α 2-Adrenoceptors activate dihydropyridine-sensitive calcium channels via Gi-proteins and protein kinase C in rat portal vein myocytes

The presence of functional ₂-adrenoceptors was investigated in isolated smooth muscle cells from rat portal vein using the nystatin-perforated patch-clamp technique. The free cytoplasmic calcium concentration ([Ca²⁺]_i) was estimated using emission from the dye Fura-2. Activation of ₂-adrenoceptors by clonidine (an ₂-adrenoceptor agonist) or noradrenaline (a non-selective -adrenoceptor agonist), both in the presence of 0.1 M prazosin to block ₁-adrenoceptors, caused a slow and sustained increase in [Ca²⁺]_i which was inhibited by 0.1 M rauwolscine (an ₂-adrenoceptor antagonist). A similar Ca²⁺ response was obtained with oxymetazoline (a selective _2A-adrenoceptor agonist) suggesting that the increase in [Ca²⁺]_i resulted from activation of the _2A-adrenoceptor subtype. The increase in [Ca²⁺]_i did not occur in calcium-free solution or in the presence of oxodipine (a voltage-dependent calcium channel blocker), indicating that it depended on a calcium influx. The _2A-adrenoceptor-activated calcium influx was unchanged after complete release of the stored calcium induced by applications of ryanodine and caffeine. In addition, no accumulation of inositol trisphosphate was detected in the presence of 0.1 M prazosin. Taken together, these results indicate that _2A-adrenoceptor activation does not stimulate phosphoinositide turnover and subsequent calcium release from intracellular stores. Wholecell patch-clamp experiments showed that _2A-adrenoceptor activation promoted calcium influx through voltage-dependent L-type channels. Concomitant with calcium influx, _2A-adrenoceptor activation induced a 10- to 15-mV depolarization. Similar effects on both calcium channel current and [Ca²⁺]_i were obtained with mastoparan, an activator of Gi-proteins. Activation of calcium influx by both _2A-adrenoceptors and mastoparan was reduced by treatment with pertussis toxin and GF 109203X (a protein kinase C inhibitor). These data suggest that activation of protein kinase C through a transduction pathway involving Gi-proteins phosphorylates voltage-activated L-type calcium channels and thus, increases their opening probability.