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

Strittmatter, Gerhard E.; Garstkiewicz, Martha; Sand, Jennifer; Grossi, Serena

doi:10.1007/978-1-4939-3804-9_9

Cited by 9 publications

(11 citation statements)

References 18 publications

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“…34 As noted, release of Caspase-1 through canonical immune pathways results in upregulation of critical inflammatory cytokines such as IL-1β, IL-18, but Caspase-1 can also cleave Pro-IL-1α and Pro-IL-33 through non-canonical immune pathways. 35 Regardless of the pathway, expression of Caspase-1 is linked directly to formation and activation of NLRP Inflammasomes. If the injured cells are creating Caspase-1, it is via an inflammasome-mediated pathway.…”

Section: Caspase-1: a Measure Of Nlrp Inflammasome Activationmentioning

confidence: 99%

“…If the injured cells are creating Caspase-1, it is via an inflammasome-mediated pathway. 35 In 2017, O'Brien et al, reported on a new bioluminescence assay that directly detects the release of ACasp-1 from NLRP inflammasomes. 36 Activation of the NLRP inflammasomes can occur via various agonists such as UV energy or chemical initiators.…”

Section: Caspase-1: a Measure Of Nlrp Inflammasome Activationmentioning

confidence: 99%

“…40 Normal human epidermal keratinocytes (NHEK) have been shown to express Caspase-1 through NLR protein cascades. 35,[41][42][43] However, there is ongoing examination and discussion if the NLRP1 or NLRP3 inflammasome pathways are the most critical to skin health. 42,43 This paper will discuss efforts to examine the expression of Caspase-1 from externally stressed NHEKs.…”

Section: Caspase-1: a Measure Of Nlrp Inflammasome Activationmentioning

confidence: 99%

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<p>In vitro expression of NLRP inflammasome-induced active Caspase-1 expression in normal human epidermal keratinocytes (NHEK) by various exogenous threats and subsequent inhibition by naturally derived ingredient blends</p>

Gruber¹,

Holtz²

2019

JIR

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Background: The discovery of the nod-like receptor protein (NLRP) inflammasomes in 2002 has led to the rapid identification of these unique cellular proteins as key targets for studies on innate inflammation pathways. The NLRP inflammasomes have been shown to be expressed in normal human epidermal keratinocytes (NHEK) and human dermal fibroblasts (HDF). NLRP inflammasomes in keratinocytes are interesting as these skin cells are the first living cells in the skin to contact external exogenous threats such as UV energy, chemicals, physical trauma, and bacteria and viruses. Activation of the NLRP Inflammasomes by exogenous threats results in the release of active Caspase-1 (ACasp-1), a key protease enzyme, which targets inactive forms of IL-1β, IL-18 as well as IL-1α and IL-33. Purpose: This article discusses efforts to examine the release of active Caspase-1 from NHEKs activated by various exogenous threats including UVB energy, ATP, Nigericin and Urban Dust. The work further examines if, after inflammasome activation and Caspase-1 release, certain naturally derived botanical ingredients known to have anti-inflammatory effects can function to inhibit upregulation of active Caspase-1. Methods: NHEK were treated with various doses of UVB, ATP and Nigericin and with a single dose of Urban Dust. ACasp-1 expression was measured after 3 and 20 hours using the Promega Caspase Glo-1 bioluminescent assay. After confirmation that 60 mJ/cm 2 of UVB and 5mM of ATP were effective to activate NHEK ACasp-1 release after 20 hrs, these conditions were employed to examine the influence of three botanical blends of ingredients on their ability to inhibit ACasp-1 expression. Results: Initial results demonstrate that NHEKs can be activated to release active Caspase-1 by ATP and UVB, but not by Nigericin or Urban Dust. In addition, it was unexpectedly found that, while ATP and UVB activated NHEKs, the release of ACasp-1-did not happen within the first 3 hours after exposure but did become significant after 20 hours. Additional results indicate that a blend of polysaccharides and two blends of antioxidants, one oilsoluble and the other water-soluble, known for their anti-inflammatory effects, can reduce expression of active Caspase-1 in activated NHEKs when applied extracellularly. Conclusion: Expression of NLRP activated release of ACasp-1 was found to be influenced by UVB and ATP but not by Nigericin or Urban Dust. The effects were also time dependent. Several botanical extract blends were found to reduce ACasp-1 expression in previously activated NHEKs. Links between these inflammatory effects and processes of cellular inflammaging are discussed.

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Section: Caspase-1: a Measure Of Nlrp Inflammasome Activationmentioning

confidence: 99%

Section: Caspase-1: a Measure Of Nlrp Inflammasome Activationmentioning

confidence: 99%

Section: Caspase-1: a Measure Of Nlrp Inflammasome Activationmentioning

confidence: 99%

See 1 more Smart Citation

<p>In vitro expression of NLRP inflammasome-induced active Caspase-1 expression in normal human epidermal keratinocytes (NHEK) by various exogenous threats and subsequent inhibition by naturally derived ingredient blends</p>

Gruber¹,

Holtz²

2019

JIR

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“…week with 2 mg/ml puromycin. Freshly isolated HPK were cultured in complete keratinocyte medium as described previously (Strittmatter et al, 2016a), transduced for 24 hours and selected for 2 passages with 5 mg/ml puromycin.…”

Section: Crispr/cas9 Gene Deletionmentioning

confidence: 99%

The p38 Mitogen-Activated Protein Kinase Critically Regulates Human Keratinocyte Inflammasome Activation

Fenini

Grossi

Gehrke

et al. 2018

Journal of Investigative Dermatology

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Inflammasomes are key intracellular signaling platforms involved in innate immune responses to micro-organisms and danger signals. Extracellular signal-regulated kinase, Jun N-terminal kinase, and p38 mitogen-activated protein kinase family members are activated by numerous environmental stresses. Recently, it has been reported that Jun N-terminal kinase is involved in inflammasome activation in myeloid immune cells. To date, the role of mitogen-activated protein kinase in inflammasome activity in keratinocytes has not been investigated. Here, we show that, in primary human keratinocytes, p38 mitogen-activated protein kinase is required for inflammasome activation and IL-1β secretion. Using selective small molecule inhibitors, small interfering RNA gene silencing, and CRISPR/Cas9-based deletion, we demonstrate the above and identify p38α and p38δ as critical regulators of ASC oligomerization, inflammasome activation, and IL-1β secretion in keratinocytes. Furthermore, our data suggest that the nature of the mitogen-activated protein kinase regulating inflammasome activity exhibits a certain cell specificity, with p38 playing a predominant role in keratinocytes and Jun N-terminal kinase 1 in cells of myeloid origin.

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“…Hair follicles were collected from patients at least 6 months after resolution of the ADR. Hair follicle extremities containing outer root sheath were cut, washed in complete keratinocyte medium (Strittmatter et al, 2016), and incubated for 3 minutes in 1 mg/ml Dispase II (Roche, Rotkreuz, Switzerland), followed by three washing steps in medium. The hair was then plated on J2 feeder cells in complete medium and cultured until small colonies of keratinocytes became visible.…”

Section: Primary Keratinocyte Culturementioning

confidence: 99%

Culprit Drugs Induce Specific IL-36 Overexpression in Acute Generalized Exanthematous Pustulosis

Meier‐Schiesser

Feldmeyer

Janković

et al. 2019

Journal of Investigative Dermatology

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Acute generalized exanthematous pustulosis (AGEP) is a severe adverse cutaneous drug reaction. Although an involvement of drug-specific T cells has been reported, the physiopathology of AGEP and mechanism of neutrophilic skin inflammation remain incompletely understood. Recently, mutations in IL-36RN, the gene encoding the IL-36 receptor antagonist, have been reported to be more frequent in AGEP patients and pustular psoriasis. Here, we show that IL-36 cytokines, in particular IL-36g, are highly expressed in lesional skin of AGEP patients, keratinocytes and macrophages being a major source of IL-36g. Such an IL-36g overexpression was not observed in patients with drug-induced maculopapular rash. In vitro, the causative drug specifically induced IL-36g release either directly by the patient's peripheral blood monocytes or indirectly by keratinocytes in the presence of autologous peripheral blood mononuclear cells. Such culprit drug induction of IL-36g secretion in vitro was specific for AGEP and involved toll-like receptor 4 sensing the drug/albumin complex as a danger signal. Our results suggest that IL-36g secretion by monocytes/macrophages and keratinocytes in response to culprit drug exposure likely plays a key role in the pathogenesis of AGEP.

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

Cited by 9 publications

References 18 publications

<p>In vitro expression of NLRP inflammasome-induced active Caspase-1 expression in normal human epidermal keratinocytes (NHEK) by various exogenous threats and subsequent inhibition by naturally derived ingredient blends</p>

<p>In vitro expression of NLRP inflammasome-induced active Caspase-1 expression in normal human epidermal keratinocytes (NHEK) by various exogenous threats and subsequent inhibition by naturally derived ingredient blends</p>

The p38 Mitogen-Activated Protein Kinase Critically Regulates Human Keratinocyte Inflammasome Activation

Culprit Drugs Induce Specific IL-36 Overexpression in Acute Generalized Exanthematous Pustulosis

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