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.

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 activity is required for UVB-induced apoptosis of human keratinocytes.

Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1ß. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

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.

Caspase-4 is required for activation of inflammasomes.

IL-1ß and IL-18 are crucial regulators of inflammation and immunity. Both cytokines are initially expressed as inactive precursors, which require processing by the protease caspase-1 for biological activity. Caspase-1 itself is activated in different innate immune complexes called inflammasomes. In addition, caspase-1 activity regulates unconventional protein secretion of many other proteins involved in inflammation and repair. Human caspase-4 is a poorly characterized member of the caspase family, which is supposed to be involved in endoplasmic reticulum stress-induced apoptosis. However, its gene is located on the same locus as the caspase-1 gene, which raises the possibility that caspase-4 plays a role in inflammation. In this study, we show that caspase-4 expression is required for UVB-induced activation of proIL-1ß and for unconventional protein secretion by skin-derived keratinocytes. These processes require expression of the nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 inflammasome, and caspase-4 physically interacts with its central molecule caspase-1. As the active site of caspase-4 is required for activation of caspase-1, the latter most likely represents a substrate of caspase-4. Caspase-4 expression is also essential for efficient nucleotide-binding domain leucine-rich repeat containing, Pyrin domain containing-3 and for absent in melanoma 2 inflammasome-dependent proIL-1ß activation in macrophages. These results demonstrate an important role of caspase-4 in inflammation and innate immunity through activation of caspase-1. Therefore, caspase-4 represents a novel target for the treatment of (auto)inflammatory diseases.