THE NON-CODING RNA, PRINS AFFECTS AIM2 INFLAMMASOME ACTIVATION IN KERATINOCYTES

The non-coding RNA, PRINS was previously described by our research group as a differentially expressed transcript in psoriatic uninvolved and healthy skin. The expression level of PRINS in cultured keratinocytes is altered after exposure to various stress factors and silencing of it decreases the viability of keratinocytes during stress stimulations suggesting its role in stress response of the cells. A potential stress signal in psoriatic skin may be the extracellular DNA, which activates the AIM2 inflammasome. The activated inflammasome cleaves the precursor proIL-1β form into mature, functioning IL-1β. The role of the AIM2 inflammasome and the IL-1β cytokine in psoriasis has been described recently. The aim of our study was to investigate if the PRINS non coding RNA affects the expression and activation of the inflammasome members and IL-1β in normal human epidermal keratinocytes (NHEK) after exposure to extracellular DNA. The expression of PRINS was transiently silenced by a vector based RNA interference method in cultured NHEK cells. Silenced and non-silenced NHEK cells were primed with the cytokines TNF-α and IFN-γ and transfected with the synthetic DNA analogue poly(dA:dT). The expression of PRINS and inflammasome members was detected by real-time RT-PCR and the secreted IL-1β was measured by ELISA. Poly(dA:dT) treatment caused a moderate increase in PRINS expression and IL-1β secretion as well, whereas priming with a combination of TNF-α and IFN-γ before poly(dA:dT) transfection resulted in a highly elevated PRINS expression and higher secreted IL-1β levels. The silencing of PRINS decreased the amount of secreted IL-1β, but did not affect the expression of the proIL-1β or AIM2. Our results suggest that the PRINS non-coding RNA regulates the IL-1β production of NHEK cells, but not through the regulation of proIL-1β expression, rather contributing to inflammasome-activation

Psoriasis-Associated Inflammatory Conditions Induce IL-23 mRNA Expression in Normal Human Epidermal Keratinocytes

Psoriasis is a multifactorial, chronic inflammatory skin disease, the development of which is affected by both genetic and environmental factors. Cytosolic nucleic acid fragments, recognized as pathogen- and danger-associated molecular patterns, are highly abundant in psoriatic skin. It is known that psoriatic skin exhibits increased levels of IL-23 compared to healthy skin. However, the relationship between free nucleic acid levels and IL-23 expression has not been clarified yet. To examine a molecular mechanism by which nucleic acids potentially modulate IL-23 levels, an in vitro system was developed to investigate the IL-23 mRNA expression of normal human epidermal keratinocytes under psoriasis-like circumstances. This system was established using synthetic nucleic acid analogues (poly(dA:dT) and poly(I:C)). Signaling pathways, receptor involvement and the effect of PRINS, a long non-coding RNA previously identified and characterized by our research group, were analyzed to better understand the regulation of IL-23 in keratinocytes. Our results indicate that free nucleic acids regulate epithelial IL-23 mRNA expression through the TLR3 receptor and specific signaling pathways, thereby, contributing to the development of an inflammatory milieu favorable for the appearance of psoriatic symptoms. A moderate negative correlation was confirmed between the nucleic-acid-induced IL-23 mRNA level and the rate of its decrease upon PRINS overexpression

PRINS non-coding RNA regulates nucleic acid-induced innate immune responses of human keratinocytes

Cytosolic DNA fragments are recognized as pathogen- and danger-associated molecular patterns that induce a cascade of innate immune responses. Moreover, excessive cytosolic DNA can enhance chronic inflammation, predominantly by activating inflammasomes, and thereby contributing to the pathogenesis of chronic diseases, such as psoriasis. Psoriasis associated non-protein coding RNA induced by stress (PRINS) is a long non-coding RNA, which has been shown to be associated with psoriasis susceptibility and cellular stress responses; however, the precise mechanism of its action has not been determined. Here, we provide evidence that, in addition to inflammasome activation, cytosolic DNA induces intracellular inflammatory reactions while decreasing PRINS gene expression. Furthermore, PRINS overexpression can ameliorate the inflammatory-mediator production of keratinocytes induced by cytosolic DNA. Overexpression of PRINS resulted in decreased interleukin-6 (IL-6) and chemokine (C-C motif) ligand 5 (CCL-5) expression and secretion. In silico analysis predicted direct binding sites between PRINS and the mRNAs, which was confirmed by an in vitro binding assay and on cellular level. Our results indicated that PRINS binds directly to the mRNAs of IL-6 and CCL-5 at specific binding sites and eventually destabilizes these mRNAs, leading to a decrease in their expression and secretion of the corresponding proteins. These results may indicate a restrictive role for PRINS in inflammatory processes

Genetic Investigation of Inverse Psoriasis

Inverse psoriasis is considered to be a rare variant of plaque-type psoriasis and is associated with significantly impaired quality of life. Clinical manifestations and treatment options are somewhat different for each subtype. Identifying genetic variants that contribute to the susceptibility of different types of psoriasis might improve understanding of the etiology of the disease. Since we have no current knowledge about the genetic background of inverse psoriasis, whole exome sequencing was used to comprehensively assess genetic variations in five patients with exclusively inverse lesions. We detected six potentially pathogenic rare (MAF < 0.01) sequence variants that occurred in all investigated patients. The corresponding mutated genes were FN1, FBLN1, MYH7B, MST1R, RHOD, and SCN10A. Several mutations identified in this study are known to cause disease, but roles in psoriasis or other papulosquamous diseases have not previously been reported. Interestingly, potentially causative variants of established psoriasis-susceptibility genes were not identified. These outcomes are in agreement with our hypothesis that the inverse subtype is a different entity from plaque-type psoriasis

Exosomal long non-coding RNAs as biomarkers in human diseases

The intensive study of extracellular vesicles was started about a decade ago revealing alterations of their amount and content to several cellular stimuli, highly depending on the releasing cell type. Exosomes, a type of extracellular vesicles, are released by every cell type and are present in most body fluids, what makes them attractive targets of biomarker research. Several studies have indicated that their content - including proteins and coding, as well as non-coding nucleic acids - could represent the disease state and serves as specific disease biomarkers. Out of these molecules, a special interest was gained by long noncoding RNAs (lncRNAs). Just as exosomes, lncRNAs are specific to their cell of origin and often specific to diseases, also found extracellularly, mainly contained in extracellular vesicles. Thus, recent efforts in biomarker research has turned to circulating exosomal lncRNAs, which might lead to the development of highly specific disease markers. Here we summarize the current knowledge on disease-associated exosomal long non-coding RNAs. The intensive studies in this area have revealed numerous potential targets for biomarkers, and highlighted the potential of their combination with other exosomal markers to represent a highly sensitive and specific diagnostic tool. However, we believe that additional functional data on both exosomes and lncRNAs are necessary for understanding their deregulation in diseases and developing their use as diagnostic approaches. © 2019 International Federation of Clinical Chemistry and Laboratory Medicine. All rights reserved

Differential inflammatory-response kinetics of human keratinocytes upon cytosolic RNA- and DNA-fragment induction

Keratinocytes are non-professional immune cells contributing actively to innate immune responses partially by reacting to a wide range of molecular patterns by activating pattern recognition receptors. Cytosolic nucleotide fragments as pathogen- or self-derived trigger factors are activating inflammasomes and inducing anti-viral signal transduction pathways as well as inducing expression of inflammatory cytokines. We aimed to compare the induced inflammatory reactions in three keratinocyte cell types-normal human epidermal keratinocytes, the HaCaT cell line and the HPV-KER cell line-upon exposure to the synthetic RNA and DNA analogues poly(I:C) and poly(dA:dT) to reveal the underlying signaling events. Both agents induced the expression of interleukin-6 and tumor necrosis factor alpha in all cell types; however, notable kinetic and expression level differences were found. Western blot analysis revealed rapid activation of the nuclear factor kappaB (NF-kappaB), mitogen activated protein kinase and signal transducers of activator of transcription (STAT) signal transduction pathways in keratinocytes upon poly(I:C) treatment, while poly(dA:dT) induced slower activation. Inhibition of NF-kappaB, p38, STAT-1 and STAT-3 signaling resulted in decreased cytokine expression, whereas inhibition of mitogen-activated protein kinase kinase 1/2 (MEK1/2) signaling showed a negative feedback role in both poly(I:C)- and poly(dA:dT)-induced cytokine expression. Based on our in vitro results nucleotide fragments are able to induce inflammatory reactions in keratinocytes, but with different rate and kinetics of cytokine expression, explained by faster activation of signaling routes by poly(I:C) than poly(dA:dT)