Inhibition of RNase 7 by RNase inhibitor promotes inflammation and Staphylococcus aureus growth: Implications for atopic dermatitis.

BACKGROUND: The antimicrobial ribonuclease RNase 7 is abundantly expressed in the epidermis of lesional skin of atopic dermatitis (AD). Host RNase inhibitor (RI) binds to RNase 7 and blocks its ribonuclease activity. This study aimed to evaluate the impact of RNase 7-RI interactions on AD. METHODS: Cultured human primary keratinocytes, with siRNA-mediated downregulation of RNase 7 and RI, were stimulated with the synthetic RNA polyinosinic-polycytidylic acid (poly I:C). Induction of proinflammatory mediators was analyzed by real-time PCR and ELISA. RI expression in AD non-lesional and lesional skin biopsies and healthy controls was analyzed by real-time PCR and immunostaining. RI protein release in vivo on the AD skin surface was determined by western blot. Antimicrobial and ribonuclease assays were used to investigate the functional role of RI. RESULTS: RNase 7 inhibited the RNA-induced expression of proinflammatory mediators in keratinocytes. Accordingly, downregulation of RNase 7 in keratinocytes enhanced RNA-mediated induction of proinflammatory mediators, whereas downregulation of RI had the opposite effect. RI was released by damaged keratinocytes and epidermis. In vivo expression and release of RI on the skin surface were enhanced in lesional AD skin. Rinsing solution from the surface of lesional AD skin blocked the ribonuclease activity of RNase 7. The anti-Staphylococcus aureus activity of RNase 7 was abrogated by RI. CONCLUSIONS: Our data suggest a novel role of RI as a trigger factor of inflammation in AD by blocking the ribonuclease and antimicrobial activity of RNase 7, thereby enhancing RNA-mediated inflammation and S. aureus growth.

RNase 7 Inhibits Uropathogenic Escherichia coli-Induced Inflammation in Bladder Cells under a High-Glucose Environment by Regulating the JAK/STAT Signaling Pathway.

Antimicrobial peptides (AMPs), which are natural antibiotics, protect against pathogens invading the urinary tract. RNase 7 with antimicrobial properties has rapid and powerful suppressive effects against Gram-positive and Gram-negative bacterial infections. However, its detailed antibacterial mechanisms have not been fully determined. Here, we investigate whether RNase 7 had an impact on bladder cells under uropathogenic Escherichia coli (UPEC) infection in a high-glucose environment using in vitro GFP-UPEC-infected bladder cell and PE-labeled TLR4, STAT1, and STAT3 models. We provide evidence of the suppressive effects of RNase 7 on UPEC infection and UPEC-induced inflammatory responses by regulating the JAK/STAT signaling pathway using JAK inhibitor and STAT inhibitor blocking experiments. Pretreatment with different concentrations of RNase 7 for 24 h concentration-dependently suppressed UPEC invasion in bladder cells (5 µg/mL reducing 45%; 25 µg/mL reducing 60%). The expressions of TLR4, STAT1, and STAT3 were also downregulated in a concentration-dependent manner after RNase 7 pretreatment (5 µg/mL reducing 35%, 54% and 35%; 25 µg/mL reducing 60%, 75% and 64%, respectively). RNase 7-induced decrease in UPEC infection in a high-glucose environment not only downregulated the expression of TLR4 protein and the JAK/STAT signaling pathway but also decreased UPEC-induced secretion of exogenous inflammatory IL-6 and IL-8 cytokines, although IL-8 levels increased in the 25 µg/mL RNase 7-treated group. Thus, inhibition of STAT affected pSTAT1, pSTAT3, and TLR4 expression, as well as proinflammatory IL-6 and IFN-γ expression. Notably, blocking JAK resulted in the rebound expression of related proteins, especially pSTAT1, TLR4, and IL-6. The present study showed the suppressive effects of RNase 7 on UPEC infection and induced inflammation in bladder epithelial cells in a high-glucose environment. RNase 7 may be an anti-inflammatory and anti-infective mediator in bladder cells by downregulating the JAK/STAT signaling pathway and may be beneficial in treating cystitis in DM patients. These results will help clarify the correlation between AMP production and UTI, identify the relationship between urinary tract infection and diabetes in UTI patients, and develop novel diagnostics or possible treatments targeting RNase 7.

RNase 7 downregulates TH2 cytokine production by activated human T cells.

BACKGROUND: The antimicrobial peptide (AMP) RNase 7 is constitutively expressed in the epidermis of healthy human skin and has been found to be upregulated in chronic inflammatory skin diseases such as atopic dermatitis and psoriasis. Activated T cells in lesional skin of patients with atopic dermatitis (AD) and psoriasis (PSO) might be directly exposed to RNase 7. In addition to their antimicrobial activity, immunoregulatory functions have been published for several AMPs. In this study, we investigated immunoregulatory effects of the antimicrobial peptide RNase 7 on activated T cells. METHODS: Isolated human CD3+T cells were stimulated with RNase 7 and screened for possible effects by mRNA microarray analysis. The results of the mRNA microarray were confirmed in isolated CD4+T cells and in polarized TH2 cells using skin-derived native RNase 7 and a recombinant ribonuclease-inactive RNase 7 mutant. Activation of GATA3 was analysed by electrophoretic mobility shift assay. RESULTS: Treatment of activated human CD4+T cells and TH2 cells with RNase 7 selectively reduced the expression of TH2 cytokines (IL-13, IL-4 and IL-5). Experiments with a ribonuclease-inactive recombinant RNase 7 mutant showed that RNase 7 ribonuclease activity is dispensable for the observed regulatory effect. We further demonstrate that CD4+T cells from AD patients revealed a significantly less pronounced downregulation of IL-13 in response to RNase 7 compared to healthy control. Finally, we show that GATA3 activation was diminished upon cultivation of T cells with RNase 7. CONCLUSION: Our data indicate that RNase 7 has immunomodulatory functions on TH2 cells and decreases the production of TH2 cytokines in the skin.

RNase 7 in Cutaneous Defense.

RNase 7 belongs to the RNase A superfamily and exhibits a broad spectrum of antimicrobial activity against various microorganisms. RNase 7 is expressed in human skin, and expression in keratinocytes can be induced by cytokines and microbes. These properties suggest that RNase 7 participates in innate cutaneous defense. In this review, we provide an overview about the role of RNase 7 in cutaneous defense with focus on the molecular mechanism of the antimicrobial activity of RNase 7, the regulation of RNase 7 expression, and the role of RNase 7 in skin diseases.

Integrative RNA-seq and microarray data analysis reveals GC content and gene length biases in the psoriasis transcriptome.

Gene expression profiling of psoriasis has driven research advances and may soon provide the basis for clinical applications. For expression profiling studies, RNA-seq is now a competitive technology, but RNA-seq results may differ from those obtained by microarray. We therefore compared findings obtained by RNA-seq with those from eight microarray studies of psoriasis. RNA-seq and microarray datasets identified similar numbers of differentially expressed genes (DEGs), with certain genes uniquely identified by each technology. Correspondence between platforms and the balance of increased to decreased DEGs was influenced by mRNA abundance, GC content, and gene length. Weakly expressed genes, genes with low GC content, and long genes were all biased toward decreased expression in psoriasis lesions. The strength of these trends differed among array datasets, most likely due to variations in RNA quality. Gene length bias was by far the strongest trend and was evident in all datasets regardless of the expression profiling technology. The effect was due to differences between lesional and uninvolved skin with respect to the genome-wide correlation between gene length and gene expression, which was consistently more negative in psoriasis lesions. These findings demonstrate the complementary nature of RNA-seq and microarray technology and show that integrative analysis of both data types can provide a richer view of the transcriptome than strict reliance on a single method alone. Our results also highlight factors affecting correspondence between technologies, and we have established that gene length is a major determinant of differential expression in psoriasis lesions.

Ag@ZnO Nanoparticles Induce Antimicrobial Peptides and Promote Migration and Antibacterial Activity of Keratinocytes.

Antibacterial activity of silver nanoparticles is often associated with toxicity to the host. We here report that noncytotoxic doses of silver nanoparticles coated with zinc oxide, Ag@ZnO, can stimulate proliferation and migration of human keratinocytes, HaCaT, with increased expression of Ki67 and vinculin at the leading edge of wounds. Interestingly, Ag@ZnO stimulates keratinocytes to produce the antimicrobial peptides hBD2 and RNase7, promoting antibacterial activity against both extracellular and intracellular Staphylococcus aureus isolated from wounds. Overall, these results suggest that Ag@ZnO has the potential to significantly improve treatment outcomes in clearing wound infection.

Involvement of RNase 7 in the malignant potential of oral squamous cell carcinoma cells.

RNase 7 is involved in the innate immunity of the oral epithelium. Variations in the expression levels of RNase 7 have been reported in cutaneous squamous cell carcinoma, but not in oral squamous cell carcinoma (OSCC). The present study investigated the expression levels of RNase 7 in OSCC and its role in the malignant potential of these cells. The localization of RNase 7 in OSCC tissue sections was determined via immunohistochemistry. Positive staining for RNase 7 was observed around the epithelial pearls and spinous cells of the OSCC tissues. Four different types of OSCC cell lines (OSC­19, BSC­OF, SAS, and HSC­2) and a normal keratinocyte (HaCaT) were used. The mRNA and protein expression levels of RNase 7 were significantly higher in the OSCC cells compared to the HaCaT cells. Based on our hypothesis that high levels of RNase 7 expression may be involved in the malignant potential of OSCC cells, the effect of RNase 7 knockdown on both proliferation and invasion were evaluated by transfecting the cells with siRNA. Cell numbers, cell invasion, and MMP 9 expression levels were significantly higher in the siRNA­BSC­OF, ­SAS, and ­HSC­2 cells compared to the BSC­OF, SAS, and HSC­2 cells. The extent of differentiation of the siRNA­OSCC cells was examined using the differentiation and undifferentiation markers involucrin (INV) and K14, respectively. The expression level of K14 was significantly higher in the siRNA­OSCC cells compared to the OSCC cells. Alternatively, HSC­2 and SAS cells demonstrated higher expression levels of INV compared to the siRNA­HSC­2 and ­SAS cells. These findings indicate that RNase 7 may contribute to the suppression of the malignant potential of OSCC.

Host Defense Peptide RNase 7 Is Down-regulated in the Skin of Diabetic Patients with or without Chronic Ulcers, and its Expression is Altered with Metformin.

BACKGROUND: Diabetic foot ulcers (DFUs) are one of the main complications in patients with type 2 diabetes mellitus (DM2), previous studies have reported that DM2 patients have lower production of host defense peptides (HDP). AIM OF THE STUDY: To investigate the expression of RNase 7, cathelicidin, HBD-2, and psoriasin in biopsies obtained from DM2 patients with or without DFU. METHODS: Biopsies from DFU patients grade 3 according to Wagner's classification, from diabetic patients without ulcer and from healthy donors were obtained. qPCR, immunohistochemistry and cell line cultures were performed. To assess whether L-isoleucine, calcitriol, phenyl butyrate, metformin, glyburide or insulin induced RNase 7, keratinocytes were stimulated, and RNase 7 expression was evaluated. RESULTS: Our data showed that RNase 7 levels were decreased in both diabetic groups when were compared with skin from healthy donors. Since most of the DM2 patients are treated with drugs to reduce glycemia, we investigated whether glyburide, metformin or insulin were able to induce any change regarding RNase 7 production. Results showed that metformin reduces the expression of RNase 7 in in vitro treated keratinocytes, suggesting that the chronic use of metformin should be evaluated in DFU patients, whereas calcitriol, phenyl butyrate and L-isoleucine did not increase the RNase 7 production. CONCLUSIONS: Due RNase 7 has antimicrobial activity, its downregulation can make prone to DM2 patients to develop infections and impaired wound healing.

Immunohistochemical Localization of RNase 7 in Normal and Inflamed Oral Epithelia and Salivary Glands.

RNase 7 is a skin-derived antimicrobial peptide expressed in various epithelial tissues. It is upregulated by stimulation with microbes and pro-inflammatory cytokines. Herein, we examined the expression levels of RNase 7 in tissues from normal and inflamed oral epithelia and salivary glands via immunohistochemistry. RNase 7 was expressed mainly in the surface layers of the parakeratinized and orthokeratinized oral epithelium. In addition, it was strongly and weakly expressed in oral lichen planus and radicular cysts, respectively. RNase 7 was constitutively expressed in salivary glands, particularly in the serous and duct cells. In the case of Sjogren's syndrome, RNase 7 was strongly expressed in serous, ductal, and mucous cells in areas with lymphocytic infiltration. The localization patterns of RNase 7 were similar to those of other epithelial antimicrobial peptides, including beta-defensins. Thus, epithelial antimicrobial peptides may act against microbial infections in a coordinated manner in oral epithelia and salivary glands.

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

The Antimicrobial and Immunomodulatory Function of RNase 7 in Skin.

The human ribonuclease RNase 7 has been originally isolated from human skin and is a member of the human RNase A superfamily. RNase 7 is constantly released by keratinocytes and accumulates on the skin surface. The expression of RNase 7 in keratinocytes can be induced by diverse stimuli such as cytokines, growth factors, and microbial factors. RNase 7 exhibits a potent broad spectrum of antimicrobial activity against various microorganisms and contributes to control bacterial growth on the skin surface. The ribonuclease and antimicrobial activity of RNase 7 can be blocked by the endogenous ribonuclease inhibitor. There is also increasing evidence that RNase 7 exerts immunomodulatory activities and may participate in antiviral defense. In this review, we discuss how these characteristics of RNase 7 contribute to innate cutaneous defense and highlight its role in skin infection and inflammation. We also speculate how a potential dysregulation of RNase 7 promotes inflammatory skin diseases and if RNase 7 may have therapeutic potential.