The Pattern Recognition Receptor NOD2 Mediates Staphylococcus aureus –Induced IL-17C Expression in Keratinocytes

Abstract

IL-17C is an important epithelial cell-derived cytokine activating innate immunity by the induction of antimicrobial peptides and cytokines. Here, we investigated the role of the cytosolic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) for the Staphylococcus aureus-mediated induction of IL-17C. Activation of NOD2 in HEK293 cells overexpressing NOD2 induced the IL-17C promoter, an activity that was significantly reduced in cells overexpressing the Crohn's disease-associated NOD2 mutation 3020insC (1007fs) or the Crohn's disease- and atopic dermatitis-associated NOD2-R702W variant. The first NF-κB-binding site in the IL-17C promoter was critical for NOD2-mediated IL-17C induction. Infection of human primary keratinocytes with S. aureus induced NOD2 and IL-17C gene expression. Overexpression of NOD2 in keratinocytes augmented S. aureus-mediated IL-17C gene expression as compared with NOD2-R702W overexpression. S. aureus-induced IL-17C expression was diminished in NOD2 small interfering RNA (siRNA)-treated keratinocytes. Moreover, significantly less S. aureus bacteria survived in keratinocytes overexpressing NOD2 but not in cells overexpressing the NOD2-R702W variant. Finally, S. aureus showed an increased survival in keratinocytes treated with NOD2 or IL-17C siRNA. In summary, our study provides evidence that S. aureus activates NOD2 in keratinocytes, resulting in an increased expression of IL-17C, a mechanism that may be dysregulated in atopic dermatitis.

<b><i>Staphylococcus epidermidis</i></b> Activates Aryl Hydrocarbon Receptor Signaling in Human Keratinocytes: Implications for Cutaneous Defense

“…cDNA corresponding to 10 ng total RNA served as the template in a real-time PCR. Real-time PCR was performed in a StepOne Real-Time PCR System (Applied Biosystem, Carlsbad, CA, USA) using SYBR Premix Ex Taq II (TaKaRa Bio) as previously described [23]. The following intron-spanning primers were used: IL-1β: 5′-AAG CCC TTG CTG TAG TGG TG-3′ (forward primer) and 5′-GAA GCT GAT GGC CCT AAA CA-3′ (reverse primer); CYP1A1: 5′-CAC CAT CCC CCA CAG CAC-3′ (forward primer) and 5′-ACA AAG ACA CAA CGC CCC TT-3′ (reverse primer); CYP1B1: 5′-TAT CAC TGA CAT CTT CGG CG-3′ (forward primer) and 5′-CTG CAC TCG AGT CTG CAC AT-3′ (reverse primer); IL-1α: 5′-TGT GAC TGC CCA AGA TGA AG-3′ (forward primer) and 5′-AAG TTT GGA TGG GCA ACT GA-3′ (reverse primer); hBD-3: 5′-TGT TTG CTT TGC TCT TCC TGT-3´ (forward primer) and 5′-CGC CTC TGA CTC TGC AAT AA-3′ (reverse primer); AhR: 5′-TCA GTT CTT AGG CTC AGC GTC-3′ (forward primer) and 5′-AGT TAT CCT GGC CTC CGTTT-3′ (reverse primer).…”

“…Luciferase activity was calculated by the amount of firefly luciferase activity normalized to the amount of renilla luciferase activity. For determination of NF-κB activity, we performed the same experiment as described above using an NF-κB firefly luciferase reporter plasmid instead of pGUD-LUC6.1 [23].…”

The roles and functional mechanisms of interleukin-17 family cytokines in mucosal immunity

“…165 On a molecular level, signals from the TLR and NOD2 pattern-recognition receptor mediate IL-17C induction in different types of epithelial cells. 25,26,166 Both the TLRMyd88-NF-κB and TLR-TRIF-NF-κB pathways are likely to be required for its induction. 26,167,168 The production of IL-17C is also controlled by inflammatory cytokines (that is, TNFα and IL-1β), which might be induced by PRR signaling during infection.…”

Atopic Dermatitis: Collegium Internationale Allergologicum (CIA) Update 2019

“…Recently it has been demonstrated that activated Langerhans cells extend dendrites through the tight junctions supposedly to capture antigens from the outside, while IDEC are localized in the lower part of the epidermis [79, 80]. Antigens may also be recognized by keratinocytes via Toll-like receptors (TLR), e.g., dsRNA by TLR 3 [81], S. aureus products by TLR2/TLR-6 [82], house dust mites by TLR1/6 [83], PAR 2 [84], and pattern recognition receptor NOD2 [85]. Keratinocytes activated by pathogens or mechanical injury release Th2-driving cytokines such as TSLP [82, 86], IL-25 [83], and IL-33 [87], as well as the proinflammatory cytokines IL-6, IL-8, and TNF-α [88].…”