Opposing effects of Nrf2 and Nrf2-activating compounds on the NLRP3 inflammasome independent of Nrf2-mediated gene expression.

The transcription factor Nrf2 regulates the expression of genes required for protection from xenobiotic and oxidative stress. Under normal conditions Nrf2 is constantly degraded upon ubiquitination, mediated by the Nrf2 inhibitor Keap1. Inflammasomes represent stress-induced protein complexes. They are critically involved in acute and chronic inflammation through caspase-1-mediated activation of pro-inflammatory cytokines. Here, we demonstrate that Nrf2 is a positive regulator of the NLRP3 inflammasome. In contrast, Nrf2-activating compounds, including the anti-inflammatory drug dimethyl fumarate (DMF), inhibit inflammasome activation. Both effects are independent of the transcriptional activity of Nrf2 and, at least in part, not interdependent. On the other hand, NLRP3 inflammasome activation induces a rapid and partly caspase-1- and Keap1-independent degradation of Nrf2. These data argue against a simultaneous activation of both stress-related pathways. Finally, we provide evidence that the cross-regulation of both pathways is controlled by a physical interaction between the Nrf2/Keap1 and NLRP3 complexes.

Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

The NLRP1 Inflammasome Pathway Is Silenced in Cutaneous Squamous Cell Carcinoma.

The inflammasome protein NLRP1 is an important innate immune sensor in human keratinocytes, and, together with ASC and caspase-1, it mediates the activation and secretion of the proinflammatory cytokines IL-1ß and IL-18. These cytokines and inflammasomes can have partly opposing roles during tumorigenesis in mice. In contrast, ASC expression is impaired in different types of cancer in humans. In this study, we analyzed inflammasome activation and expression of inflammasome proteins, including their downstream cytokines, in squamous cell carcinomas, a type of nonmelanoma skin cancer derived from keratinocytes. We assessed mRNA and protein levels in human primary keratinocytes and skin carcinoma-derived SCC cell lines and detected a strong down-regulation of expression of NLRP1 inflammasome components, as well as reduced expression of the proinflammatory cytokines proIL-1ß and proIL-1α. Protein levels of NLRP1, ASC, caspase-1, and proIL-1ß were reduced in patient-derived SCC biopsy samples compared with healthy skin. Furthermore, the results suggest that expression of PYCARD (ASC), CASP1, IL1B, and NLRP1 is silenced by methylation in SCC cell lines. In conclusion, the down-regulation of the inflammasome pathway in SCCs might favor late tumor development in human skin.

Expression of inflammasome proteins and inflammasome activation occurs in human, but not in murine keratinocytes.

Inflammasomes are multimeric protein complexes that assemble upon sensing of a variety of stress factors. Their formation results in caspase-1-mediated activation and secretion of the pro-inflammatory cytokines pro-interleukin(IL)-1ß and -18, which induce an inflammatory response. Inflammation is supported by a lytic form of cell death, termed pyroptosis. Innate immune cells, such as macrophages or dendritic cells, express and activate inflammasomes. However, it has also been demonstrated that human primary keratinocytes activate different types of inflammasomes in vitro, for example, upon UVB irradiation or viral infection. Keratinocytes are the main cell type of the epidermis, the outermost layer of the body, and form a protective barrier consisting of a stratified multi-layered epithelium. In human, gain-of-function mutations of the NLRP1 gene cause syndromes mediated by inflammasome activation in keratinocytes that are characterised by skin inflammation and skin cancer susceptibility. Here we demonstrate that murine keratinocytes do not activate inflammasomes in response to stimuli, which induce IL-1ß and -18 secretion by human keratinocytes. Whereas murine keratinocytes produced caspase-1 and proIL-18, expression of the inflammasome proteins Nlrp1, Nlrp3, Aim2, Asc, and proIL-1ß was, compared to human keratinocytes or murine dendritic cells, very low or even undetectable. Priming of murine keratinocytes with cytokines commonly used for induction of proIL-1ß and inflammasome protein expression did not rescue inflammasome activation. Nevertheless, UVB-induced inflammation and neutrophil recruitment in murine skin was dependent on IL-1ß and caspase-1. However, also under these conditions, we did not detect expression of proIL-1ß by keratinocytes in murine skin, but by immune cells. These results demonstrate a higher immunological competence of human compared to murine keratinocytes, which is reflected by stress-induced IL-1ß secretion that is mediated by inflammasomes. Therefore, keratinocytes in human skin can exert immune functions, which are carried out by professional immune cells in murine skin.

Human Primary Keratinocytes as a Tool for the Analysis of Caspase-1-Dependent Unconventional Protein Secretion.

Inflammasomes comprise a group of protein complexes, which activate the protease caspase-1 upon sensing a variety of stress factors. Active caspase-1 in turn cleaves and thereby activates the pro-inflammatory cytokines prointerleukin (IL)-1ß and -18, and induces unconventional protein secretion (UPS) of mature IL-1ß, IL-18, as well as of many other proteins involved in and required for induction of inflammation. Human primary keratinocytes (HPKs) represent epithelial cells able to activate caspase-1 in an inflammasome-dependent manner upon irradiation with a physiological dose of ultraviolet B (UVB) light. Here, we describe the isolation of keratinocytes from human skin, their cultivation, and induction of caspase-1-dependent UPS upon UVB irradiation as well as its siRNA- and chemical-mediated inhibition. In contrast to inflammasome activation of professional immune cells, UVB-irradiated HPKs represent a robust and physiological cell culture system for the analysis of UPS induced by active caspase-1.

IFN-γ Primes Keratinocytes for HSV-1-Induced Inflammasome Activation.

Inflammasomes are immune complexes that induce an inflammatory response upon sensing of different stress signals. This effect is mainly mediated by activation and secretion of the proinflammatory cytokines proIL-1ß and -18. Here we report that infection of human primary keratinocytes with the double-stranded DNA viruses modified vaccinia virus Ankara (MVA) or herpes simplex virus type 1 (HSV-1)-induced secretion of mature IL-1ß and -18. This secretion was dependent on several inflammasome complexes; however, the absent in melanoma 2 (AIM2) inflammasome, which is activated by binding of double-stranded DNA, played the most important role. Whereas prestimulation of keratinocytes with IFN-γ moderately increased MVA-induced IL-1ß and IL-18 secretion, it was essential for substantial secretion of these cytokines in response to herpes simplex virus type 1 infection. IFN-γ partially restored HSV-1 suppressed proIL-1ß expression and was also required for inflammasome activation. Most importantly, IFN-γ strongly suppressed virus replication in keratinocytes in vitro and ex vivo, which was independent of inflammasome activation. Our results suggest that, similar to Herpesviridae infection in mice, HSV-1 replication in human skin is controlled by a positive feedback loop of keratinocyte-derived IL-1/IL-18 and IFN-γ expressed by immune cells.

Caspase-1: the inflammasome and beyond.

Caspase-1 plays a fundamental role in innate immunity and in several important inflammatory diseases as the protease activates the pro-inflammatory cytokines proIL-1ß and proIL-18. Caspase-1 itself is activated in different inflammasome complexes, which assemble in response to a variety of exogenous and endogenous stressors. More recently, pyroptosis, a caspase-1-dependent type of programmed cell death, has been identified that is able to support secreted IL-1 and IL-18 in triggering an inflammatory response. Whereas these 'canonical' activities are well appreciated, this review also highlights less-known pathways and molecules activated by caspase-1. There is evidence that caspase-1 supports cell survival by activation of NF-κB, induction of membrane repair and regulation of unconventional secretion of certain proteins. The physiologic effects of processing of other downstream targets, such as proteins involved in glycolysis or activation of caspase-7, are less well understood. However, there is increasing evidence that caspase-1 contributes to innate and adaptive immunologic defense mechanisms, repair and pathologic conditions by the regulation of several different and partially opposing pathways.