The mitochondrial DNA common deletion as a potential biomarker of cancer-associated fibroblasts from skin basal and squamous cell carcinomas.

Cancer-associated fibroblasts (CAFs) are components of the tumor microenvironment and represent appealing therapeutic targets for translational studies. Conventional protein-based biomarkers for CAFs have been reported to be limited in their specificity, rendering difficult the identification of CAFs from normal fibroblasts (NFs) in clinical samples and dampening the development of CAF-targeted therapies to treat cancer. In this study, we propose the mitochondrial RNA and the mitochondrial DNA (mtDNA) common deletion (CD) as novel indicators of CAF identity. We found that cancer-activation correlated with decreased levels of the mtDNA CD, a condition not due to altered mitochondria count or cellular redox state, but potentially linked to the generalized overexpression of mtDNA maintenance genes in CAFs. Decreased mtDNA CD content in CAFs was associated with moderate to strong overexpression of mtDNA-encoded genes and to slightly improved mitochondrial function. We identified similar patterns of upregulation of mtDNA-encoded genes in independent single-cell RNA seq data obtained from squamous cell carcinoma (SCC) patients. By using the identified nucleic acids-based indicators, identification of CAFs from NFs could be improved, leading to potential therapeutic benefits in advancing translational and clinical studies.

The gasdermins: a pore-forming protein family expressed in the epidermis.

Gasdermins comprise a family of pore-forming proteins, which play critical roles in (auto)inflammatory diseases and cancer. They are expressed as self-inhibited precursor proteins consisting of an aminoterminal cytotoxic effector domain (NT-GSDM) and a carboxyterminal inhibitor domain (GSDM-CT) separated by an unstructured linker region. Proteolytic processing in the linker region liberates NT-GSDM, which translocates to membranes, forms oligomers, and induces membrane permeabilization, which can disturb the cellular equilibrium that can lead to cell death. Gasdermin activation and pore formation are associated with inflammation, particularly when induced by the inflammatory protease caspase-1 upon inflammasome activation. These gasdermin pores allow the release of the pro-inflammatory cytokines interleukin(IL)-1ß and IL-18 and induce a lytic type of cell death, termed pyroptosis that supports inflammation, immunity, and tissue repair. However, even at the cellular level, the consequences of gasdermin activation are diverse and range from induction of programmed cell death - pyroptosis or apoptosis - to poorly characterized protective mechanisms. The specific effects of gasdermin activation can vary between species, cell types, the membrane that is being permeabilized (plasma membrane, mitochondrial membrane, etc.), and the overall biological state of the local tissue/cells. In epithelia, gasdermins seem to play crucial roles. Keratinocytes represent the main cell type of the epidermis, which is the outermost skin layer with an essential barrier function. Compared to other tissues, keratinocytes express all members of the gasdermin family, in part in a differentiation-specific manner. That raises questions regarding the specific roles of individual GSDM family members in the skin, the mechanisms and consequences of their activation, and the potential crosstalk between them. In this review, we summarize the current knowledge about gasdermins with a focus on keratinocytes and the skin and discuss the possible roles of the different family members in immunity and disease.

Immunophenotyping of Monocyte Migration Markers and Therapeutic Effects of Selenium on IL-6 and IL-1ß Cytokine Axes of Blood Mononuclear Cells in Preoperative and Postoperative Coronary Artery Disease Patients.

Multivessel coronary artery disease (CAD) is characterized by underlying chronic vascular inflammation and occlusion in the coronary arteries, where these patients undergo coronary artery bypass grafting (CABG). Since post-cardiotomy inflammation is a well known phenomenon after CABG, attenuation of this inflammation is required to reduce perioperative morbidity and mortality. In this study, we aimed to phenotype circulating frequencies and intensities of monocyte subsets and monocyte migration markers, respectively, and to investigate the plasma level of inflammatory cytokines and chemokines between preoperative and postoperative CAD patients and later, to intervene the inflammation with sodium selenite. We found a higher amplitude of inflammation, postoperatively, in terms of CCR1high monocytes and significantly increased pro-inflammatory cytokines, IL-6, IL-8, and IL-1RA. Further, in vitro intervention with selenium displayed mitigating effects on the IL-6/STAT-3 axis of mononuclear cells derived from postoperative CAD patients. In addition, in vitro selenium intervention significantly reduced IL-1ß production as well as decreased cleaved caspase-1 (p20) activity by preoperative (when stimulated) as well as postoperative CAD mononuclear cells. Though TNF-α exhibited a positive correlation with blood troponin levels in postoperative CAD patients, there was no obvious effect of selenium on the TNF-α/NF-κB axis. In conclusion, anti-inflammatory selenium might be utilized to impede systemic inflammatory cytokine axes to circumvent aggravating atherosclerosis and further damage to the autologous bypass grafts during the post-surgical period.

High p62 expression suppresses the NLRP1 inflammasome and increases stress resistance in cutaneous SCC cells.

NLRP1 is the primary inflammasome sensor in human keratinocytes. Sensing of UVB radiation by NLRP1 is believed to underlie the induction of sunburn. Although constitutive NLRP1 activation causes skin inflammation and predisposes patients to the development of cutaneous SCCs, the NLRP1 pathway is suppressed in established SCCs. Here, we identified high levels of the autophagy receptor p62 in SCC cells lines and SCC tumors. Increased NF-κB activity in SCC cells causes p62 up-regulation. Suppression of p62 expression rescues UVB-induced NLRP1 inflammasome activation in early-stage SCC cells. p62 expression protects SCC cells from cytotoxic drugs, whereas NLRP1 sensitizes them. In summary, we identify p62 as a novel negative regulator of the NLRP1 inflammasome in human cutaneous SCC cells, in which suppression of NLRP1 by increased levels of p62 supports stress resistance of skin cancer cells.

NLRP1 in Cutaneous SCCs: An Example of the Complex Roles of Inflammasomes in Cancer Development.

Protein complexes termed inflammasomes ensure tissue protection from pathogenic and sterile stressors by induction of inflammation. This is mediated by different caspase-1-induced downstream pathways, including activation of the pro-inflammatory cytokines proIL-1ß and -18, induction of a lytic type of cell death, and regulation of the release of other pro-inflammatory molecules. Aberrant inflammasome activation underlies the pathology of numerous (auto)inflammatory diseases. Furthermore, inflammasomes support or suppress tumor development in a complex cell-type- and stage-dependent manner. In human keratinocytes and skin, NLRP1 is the central inflammasome sensor activated by cellular perturbation induced, for example, by UVB radiation. UVB represents the main inducer of skin cancer, which is the most common type of malignancy in humans. Recent evidence demonstrates that activation of NLRP1 in human skin supports the development of cutaneous squamous cell carcinomas (cSCCs) by inducing skin inflammation. In contrast, the NLRP1 inflammasome pathway is restrained in established cSCCs, suggesting that, at this stage, the protein complex has a tumor suppressor role. A better understanding of the complex functions of NLRP1 in the development of cSCCs and in general of inflammasomes in cancer might pave the way for novel strategies for cancer prevention and therapy. These strategies might include stage-specific modulation of inflammasome activation or its downstream pathways by mono- or combination therapy.

NLRP1 Inflammasome Activation in Keratinocytes: Increasing Evidence of Important Roles in Inflammatory Skin Diseases and Immunity.

In 2007, it was shown that DNA sequence variants of the human NLRP1 gene are associated with autoimmune and autoinflammatory diseases affecting mainly the skin. However, at that time, the underlying cellular and molecular mechanisms were poorly characterized. Meanwhile, increasing evidence suggests that the NLRP1 inflammasome expressed by keratinocytes not only plays a part in the pathology of common inflammatory skin diseases and cancer development but also contributes to skin immunity. Understanding the mechanisms regulating NLRP1 activation in keratinocytes and the downstream events in human skin might pave the way for developing novel strategies for treating patients suffering from NLRP1-mediated skin diseases.

The Pathways Underlying the Multiple Roles of p62 in Inflammation and Cancer.

p62 is a highly conserved, multi-domain, and multi-functional adaptor protein critically involved in several important cellular processes. Via its pronounced domain architecture, p62 binds to numerous interaction partners, thereby influencing key pathways that regulate tissue homeostasis, inflammation, and several common diseases including cancer. Via binding of ubiquitin chains, p62 acts in an anti-inflammatory manner as an adaptor for the auto-, xeno-, and mitophagy-dependent degradation of proteins, pathogens, and mitochondria. Furthermore, p62 is a negative regulator of inflammasome complexes. The transcription factor Nrf2 regulates expression of a bundle of ROS detoxifying genes. p62 activates Nrf2 by interaction with and autophagosomal degradation of the Nrf2 inhibitor Keap1. Moreover, p62 activates mTOR, the central kinase of the mTORC1 sensor complex that controls cell proliferation and differentiation. Through different mechanisms, p62 acts as a positive regulator of the transcription factor NF-κB, a central player in inflammation and cancer development. Therefore, p62 represents not only a cargo receptor for autophagy, but also a central signaling hub, linking several important pro- and anti-inflammatory pathways. This review aims to summarize knowledge about the molecular mechanisms underlying the roles of p62 in health and disease. In particular, different types of tumors are characterized by deregulated levels of p62. The elucidation of how p62 contributes to inflammation and cancer progression at the molecular level might promote the development of novel therapeutic strategies.

Interaction of the NRF2 and p63 transcription factors promotes keratinocyte proliferation in the epidermis.

Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2-p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2-p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.

The NLRP1 Inflammasome in Human Skin and Beyond.

Inflammasomes represent a group of protein complexes that contribute to host defense against pathogens and repair processes upon the induction of inflammation. However, aberrant and chronic inflammasome activation underlies the pathology of numerous common inflammatory diseases. Inflammasome assembly causes activation of the protease caspase-1 which in turn activates proinflammatory cytokines and induces a lytic type of cell death termed pyroptosis. Although NLRP1 (NACHT, leucine-rich repeat and pyrin domain containing 1) was the first inflammasome sensor, described almost 20 years ago, the molecular mechanisms underlying its activation and the resulting downstream events are incompletely understood. This is partially a consequence of the poor conservation of the NLRP1 pathway between human and mice. Moreover, recent evidence demonstrates a complex and multi-stage mechanism of NLRP1 inflammasome activation. In contrast to other inflammasome sensors, NLRP1 possesses protease activity required for proteolytic self-cleavage and activation mediated by the function-to-find domain (FIIND). CARD8 is a second FIIND protein and is expressed in humans but not in mice. In immune cells and AML (acute myeloid leukemia) cells, the anti-cancer drug talabostat induces CARD8 activation and causes caspase-1-dependent pyroptosis. In contrast, in human keratinocytes talabostat induces NLRP1 activation and massive proinflammatory cytokine activation. NLRP1 is regarded as the principal inflammasome sensor in human keratinocytes and UVB radiation induces its activation, which is believed to underlie the induction of sunburn. Moreover, gain-of-function mutations of NLRP1 cause inflammatory skin syndromes and a predisposition for the development of skin cancer. SNPs (single nucleotide polymorphisms) of NLRP1 are associated with several (auto)inflammatory diseases with a major skin phenotype, such as psoriasis or vitiligo. Here, we summarize knowledge about NLRP1 with emphasis on its role in human keratinocytes and skin. Due to its accessibility, pharmacological targeting of NLRP1 activation in epidermal keratinocytes represents a promising strategy for the treatment of the numerous patients suffering from NLRP1-dependent inflammatory skin conditions and cancer.

Genetic activation of Nrf2 reduces cutaneous symptoms in a murine model of Netherton syndrome.

Netherton syndrome is a monogenic autosomal recessive disorder primarily characterized by the detachment of the uppermost layer of the epidermis, the stratum corneum It results from mutations in the SPINK5 gene, which codes for a kallikrein inhibitor. Uncontrolled kallikrein activity leads to premature desquamation, resulting in a severe epidermal barrier defect and subsequent life-threatening systemic infections and chronic cutaneous inflammation. Here, we show that genetic activation of the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2/Nrf2) in keratinocytes of Spink5 knockout mice, a model for Netherton syndrome, significantly alleviates their cutaneous phenotype. Nrf2 activation promoted attachment of the stratum corneum and concomitant epidermal barrier function, and reduced the expression of pro-inflammatory cytokines such as tumor necrosis factor α and thymic stromal lymphopoietin. Mechanistically, we show that Nrf2 activation induces overexpression of secretory leukocyte protease inhibitor (Slpi), a known inhibitor of kallikrein 7 and elastase 2, in mouse and human keratinocytes in vivo and in vitro, respectively. In the Spink5-deficient epidermis, the upregulation of Slpi is likely to promote stabilization of corneodesmosomes, thereby preventing premature desquamation. Our results suggest pharmacological NRF2 activation as a promising treatment modality for Netherton syndrome patients.This article has an associated First Person interview with the first author of the paper.

Electrophiles Against (Skin) Diseases: More Than Nrf2.

The skin represents an indispensable barrier between the organism and the environment and is the first line of defense against exogenous insults. The transcription factor NRF2 is a central regulator of cytoprotection and stress resistance. NRF2 is activated in response to oxidative stress by reactive oxygen species (ROS) and electrophiles. These electrophiles oxidize specific cysteine residues of the NRF2 inhibitor KEAP1, leading to KEAP1 inactivation and, subsequently, NRF2 activation. As oxidative stress is associated with inflammation, the NRF2 pathway plays important roles in the pathogenesis of common inflammatory diseases and cancer in many tissues and organs, including the skin. The electrophile and NRF2 activator dimethyl fumarate (DMF) is an established and efficient drug for patients suffering from the common inflammatory skin disease psoriasis and the neuro-inflammatory disease multiple sclerosis (MS). In this review, we discuss possible molecular mechanisms underlying the therapeutic activity of DMF and other NRF2 activators. Recent evidence suggests that electrophiles not only activate NRF2, but also target other inflammation-associated pathways including the transcription factor NF-κB and the multi-protein complexes termed inflammasomes. Inflammasomes are central regulators of inflammation and are involved in many inflammatory conditions. Most importantly, the NRF2 and inflammasome pathways are connected at different levels, mainly antagonistically.

Inactivation of the Cytoprotective Major Vault Protein by Caspase-1 and -9 in Epithelial Cells during Apoptosis.

Inflammasome activation induces caspase-1-dependent secretion of the proinflammatory cytokine IL-1ß. In addition, caspase-1 activates the protein GSDMD in immune cells, causing pyroptosis, a lytic type of cell death. In contrast, UVB irradiation of human primary keratinocytes induces NLRP1 inflammasome activation, cytokine secretion, and caspase-1-dependent apoptosis, rather than pyroptosis. Here, we addressed the molecular mechanisms underlying the role of caspase-1 in UVB-induced cell death of human primary keratinocytes. We show that GSDMD is a poor substrate of caspase-1 in human primary keratinocytes and that its activation upon UVB irradiation supports secretion of IL-1ß. We screened for novel substrates of caspase-1 by a mass spectrometry-based approach and identified the specific cleavage of the major vault protein (MVP) at D441 by caspase-1 and -9. MVP is the main component of vaults, highly conserved ribonucleoprotein particles, whose functions are poorly understood. Cleavage of MVP is a common event occurring in human primary keratinocytes and fibroblasts undergoing apoptosis induced by different stimuli. In contrast, MVP cleavage could not be detected in pyroptotic cells. Cleavage of MVP by caspase-1 and -9 inactivates this cytoprotective protein. These results demonstrate a proapoptotic activity of caspase-1 and a crosstalk with caspase-9 upon inactivation of the cytoprotective MVP in apoptotic epithelial cells.

IL-36γ drives skin toxicity induced by EGFR/MEK inhibition and commensal Cutibacterium acnes.

Epidermal growth factor receptor (EGFR) and MEK inhibitors (EGFRi/MEKi) are beneficial for the treatment of solid cancers but are frequently associated with severe therapy-limiting acneiform skin toxicities. The underlying molecular mechanisms are poorly understood. Using gene expression profiling we identified IL-36γ and IL-8 as candidate drivers of EGFRi/MEKi skin toxicity. We provide molecular and translational evidence that EGFRi/MEKi in concert with the skin commensal bacterium Cutibacterium acnes act synergistically to induce IL-36γ in keratinocytes and subsequently IL-8, leading to cutaneous neutrophilia. IL-36γ expression was the combined result of C. acnes-induced NF-κB activation and EGFRi/MEKi-mediated expression of the transcription factor Krüppel-like factor 4 (KLF4), due to the presence of both NF-κB and KLF4 binding sites in the human IL-36γ gene promoter. EGFRi/MEKi increased KLF4 expression by blockade of the EGFR/MEK/ERK pathway. These results provide an insight into understanding the pathological mechanism of the acneiform skin toxicities induced by EGFRi/MEKi and identify IL-36γ and the transcription factor KLF4 as potential therapeutic targets.

Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles.

Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1ß by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1ß generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-ΔLysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1ß-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.

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.

Genome Editing of Human Primary Keratinocytes by CRISPR/Cas9 Reveals an Essential Role of the NLRP1 Inflammasome in UVB Sensing.

By forming a protective barrier, epidermal keratinocytes represent the first line of defense against environmental insults. UVB radiation of the sun is a major challenge for the skin and can induce inflammation, aging, and eventually skin cancer. UVB induces an immune response in human keratinocytes resulting in activation and secretion of the proinflammatory cytokines proIL-1ß and -18. This is mediated by an assembly of protein complexes, termed inflammasomes. However, the mechanisms underlying sensing of UVB by keratinocytes, and particularly the types of inflammasomes required for cytokine secretion, are a matter of debate. To address these questions, we established a protocol that allows the generation of CRISPR/Cas9-targeted human primary keratinocytes. Our experiments showed an essential role of the NLRP1 rather than the NLRP3 inflammasome in UVB sensing and subsequent IL-1ß and -18 secretion by keratinocytes. Moreover, NLRP1 but not NLRP3 was required for inflammasome activation in response to nigericin, a potassium ionophore and well-established NLRP3 activator in immune cells. Because the CRISPR/Cas9-targeted cells retained their full differentiation capacity, genome editing of human primary keratinocytes might be useful for numerous research and medical applications.

Nrf2-Mediated Fibroblast Reprogramming Drives Cellular Senescence by Targeting the Matrisome.

Nrf2 is a key regulator of the antioxidant defense system, and pharmacological Nrf2 activation is a promising strategy for cancer prevention and promotion of tissue repair. Here we show, however, that activation of Nrf2 in fibroblasts induces cellular senescence. Using a combination of transcriptomics, matrix proteomics, chromatin immunoprecipitation and bioinformatics we demonstrate that fibroblasts with activated Nrf2 deposit a senescence-promoting matrix, with plasminogen activator inhibitor-1 being a key inducer of the senescence program. In vivo, activation of Nrf2 in fibroblasts promoted re-epithelialization of skin wounds, but also skin tumorigenesis. The pro-tumorigenic activity is of general relevance, since Nrf2 activation in skin fibroblasts induced the expression of genes characteristic for cancer-associated fibroblasts from different mouse and human tumors. Therefore, activated Nrf2 qualifies as a marker of the cancer-associated fibroblast phenotype. These data highlight the bright and the dark sides of Nrf2 and the need for time-controlled activation of this transcription factor.

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.

NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22.

Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1ß, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1ß secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1ß release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1ß. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1ß levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation.

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.