Interferon Kappa Is Important for Keratinocyte Host Defense against Herpes Simplex Virus-1

Type I interferon kappa (IFNκ) is selectively expressed in human keratinocytes. Herpes simplex virus-1 (HSV-1) is a human pathogen that infects keratinocytes and causes lytic skin lesions. Whether IFNκ plays a role in keratinocyte host defense against HSV-1 has not been investigated. In this study, we found that IFNκ mRNA expression was induced by addition of recombinant IFNκ and poly (I:C); and its expression level was significantly greater than IFNa2, IFNb1, and IFNL1 in both undifferentiated and differentiated normal human epidermal keratinocytes (NHEKs) under resting and stimulation conditions. Although IFNe was expressed at a relatively higher level than other IFNs in resting undifferentiated NHEK, its expression level did not change after stimulation with recombinant IFNκ and poly (I:C). HSV-1 infection inhibited gene expression of IFNκ and IFNe in NHEK. Silencing IFNκ in NHEK led to significantly enhanced HSV-1 replication in both undifferentiated and differentiated NHEK compared to scrambled siRNA-transfected cells, while the addition of recombinant IFNκ significantly reduced HSV-1 replication in NHEK. In addition, we found that IFNκ did not regulate protein expression of NHEK differentiation markers. Our results demonstrate that IFNκ is the dominant type of IFNs in keratinocytes and it has an important function for keratinocytes to combat HSV-1 infection

Correction: Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation.

[This corrects the article DOI: 10.1371/journal.pone.0167392.]

Forkhead Box C1 Regulates Human Primary Keratinocyte Terminal Differentiation

<div><p>The epidermis serves as a critical protective barrier between the internal and external environment of the human body. Its remarkable barrier function is established through the keratinocyte (KC) terminal differentiation program. The transcription factors specifically regulating terminal differentiation remain largely unknown. Using a RNA-sequencing (RNA-seq) profiling approach, we found that forkhead box c 1 (FOXC1) was significantly up-regulated in human normal primary KC during the course of differentiation. This observation was validated in human normal primary KC from several different donors and human skin biopsies. Silencing FOXC1 in human normal primary KC undergoing differentiation led to significant down-regulation of late terminal differentiation genes markers including epidermal differentiation complex genes, keratinization genes, sphingolipid/ceramide metabolic process genes and epidermal specific cell-cell adhesion genes. We further demonstrated that FOXC1 works down-stream of ZNF750 and KLF4, and upstream of GRHL3. Thus, this study defines FOXC1 as a regulator specific for KC terminal differentiation and establishes its potential position in the genetic regulatory network.</p></div

ZNF750 and KLF4 bind to the promoter region of FOXC1 gene.

<p>A) ChIP assays were performed using Ca²⁺ -driven differentiated keratinocytes. 9 pairs of primers covering 2 kilobase region of the upstream of FOXC1 start codon. Increased binding activity was found in the regions of -873bp to -643bp and -663bp to -493bp up-stream of the start codon. Data represented as mean ±SEB. B) The Schematic diagram of FOXC1 promoter region. KLF4 and ZNF750 binding sites are indicated.</p

FOXC1 is induced in differentiated keratinocytes.

<p>A) mRNA levels of FOXC1 is increasing during the course of KC differentiation. Data presented as mean ±SEB. “undiff” is the abbreviation of undifferentiated KC. * p<0.05; ** p<0.01; *** p<0.001. The data are representative of four independent experiments. B) FOXC1 protein is increasing along with KC differentiation degree. The data represent one of the three independent experiments. C) Immunohistochemistry staining of FOXC1 protein expression in human epidermis, hair follicle and the differentiated inner hair root sheath. FOXC1 positive staining is presented as brown color in cell nuclear (Scale bar, 50 μm). The arrows point to examples of typical positive staining.</p

Down-regulated Keratinocytes differentiation markers in FOXC1 silenced KC.

<p>Down-regulated Keratinocytes differentiation markers in FOXC1 silenced KC.</p

Silencing FOXC1 leads to deceased gene expression of KC differentiation markers.

<p>A) mRNA expression of FLG, IVL and LOR in FOXC1 silenced differentiated KC and control cells evaluated by real-time PCR. Data represented as mean ±SEB. The data are representative of four independent experiments. B) Protein expression of FLG, IVL, LOR, DSC1 and Cyclin D in FOXC1 silenced differentiated KC and control cells evaluated by western -blot. The data represent one of the three independent experiments.</p

FOXC1 acts down-stream of ZNF750 and KLF4, and up-stream of GRHL3.

<p>A) FOXC1 expression in ZNF750 and KLF4 silenced KC. B) ZNF750 expression in FOXC1 and KLF4 silenced KC. C) KLF4 expression in ZNF750 and FOXC1 silenced KC. D) GRHL3 expression in ZNF750, KLF4 and FOXC1 silenced KC. Gene expression was evaluated by real time PCR. Data represented as mean ±SEB. The data are representative of three independent experiments.</p

Whole genome sequencing identifies novel genetic mutations in patients with eczema herpeticum

BackgroundEczema herpeticum (EH) is a rare complication of atopic dermatitis (AD) caused by disseminated herpes simplex virus (HSV) infection. The role of rare and/or deleterious genetic variants in disease etiology is largely unknown. This study aimed to identify genes that harbor damaging genetic variants associated with HSV infection in AD with a history of recurrent eczema herpeticum (ADEH+).MethodsWhole genome sequencing (WGS) was performed on 49 recurrent ADEH+ (≥3 EH episodes), 491 AD without a history of eczema herpeticum (ADEH-) and 237 non-atopic control (NA) subjects. Variants were annotated, and a gene-based approach (SKAT-O) was used to identify genes harboring damaging genetic variants associated with ADEH+. Genes identified through WGS were studied for effects on HSV responses and keratinocyte differentiation.ResultsEight genes were identified in the comparison of recurrent ADEH+to ADEH-and NA subjects: SIDT2, CLEC7A, GSTZ1, TPSG1, SP110, RBBP8NL, TRIM15, and FRMD3. Silencing SIDT2 and RBBP8NL in normal human primary keratinocytes (NHPKs) led to significantly increased HSV-1 replication. SIDT2-silenced NHPKs had decreased gene expression of IFNk and IL1b in response to HSV-1 infection. RBBP8NL-silenced NHPKs had decreased gene expression of IFNk, but increased IL1b. Additionally, silencing SIDT2 and RBBP8NL also inhibited gene expression of keratinocyte differentiation markers keratin 10 (KRT10) and loricrin (LOR).ConclusionSIDT2 and RBBP8NL participate in keratinocyte's response to HSV-1 infection. SIDT2 and RBBP8NL also regulate expression of keratinocyte differentiation genes of KRT10 and LOR