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Background: Atopic dermatitis (AD) is a chronic and complex inflammatory skin disease. At least two AD subtypes have been described: allergic (characterized by Type‐2‐cell‐mediated immunity and increased immunoglobulin E [IgE]) and non‐allergic (characterized by both Type‐2‐cell and Type‐1/17‐cell‐mediated immunity and normal IgE levels). MicroRNA (miR) are small non‐coding RNA molecules involved in genetic regulation. MiR‐146a negatively regulates inflammatory responses during chronic skin inflammation, however, its role in the modulation of immune responses in AD is uncovered. As recently miR‐146a was shown to promote IgE class switch in B cells in mice, we aimed to test whether there is association between miR‐ 146a and increased IgE levels in AD. Method: Serum samples from miR‐146a−/− and wild‐type C57BL/ 6J mice with MC903‐induced AD‐like inflammation were analysed (N = 8 mice/group) for IgE and cytokine levels. Additionally, 32 serum samples from AD patients were also analysed. Subjects were split into allergic (N = 22) and non‐allergic (N = 10) according to IgE threshold of 150 IU/mL. MiR‐146a relative expression was quantified by real‐time PCR, IgE and human IL‐12p40 by ELISA and mouse cytokines by Bioplex. Results: MiR‐146a−/− mice showed decreased IgE and increased IL‐ 12p40 serum levels (P < 0.001), while there were no changes in other detected cytokines. Human miR‐146a expression was not significantly different between allergic and non‐allergic subgroups divided based on serum IgE level. However, we observed a negative correlation of serum miR‐146a and IgE levels (P < 0.05) in allergic AD patients. In the allergic subgroup, miR‐146a expression remained independently negatively associated with IgE (β = −0.488, P < 0.05) after adjusting for confounding variables as gender. Conclusion: Low IgE and high IL‐12p40 serum levels in miR‐ 146a−/− mice indicate that miR‐146a is needed for the production of IgE and associated with the regulation of Type‐1/17‐cell‐mediated immune responses in mice. Negative association of miR‐146a with serum IgE in allergic AD patients suggests that miR‐146a might have capacity to limit Type‐2‐cell‐mediated immune responses in AD. Further studies are needed to elucidate the possible mechanisms of miR‐146a in AD etiopathology.

miR-146a inhibits inflammatory responses in human keratinocytes and in different mouse models of skin inflammation. Little is known about the role of miR-146b in the skin. In this study, we confirmed the increased expression of miR-146a and miR-146b (miR-146a/b) in the lesional skin of patients with psoriasis. The expression of miR-146a was approximately twofold higher than that of miR-146b in healthy human skin, and it was more strongly induced by stimulation of proinflammatory cytokines in keratinocytes and fibroblasts. miR-146a/b target genes regulating inflammatory responses or proliferation were altered in the skin of patients with psoriasis, among which FERMT1 was verified as a direct target of miR-146a. In silico analysis of genome-wide data from >4,000 psoriasis cases and >8,000 controls confirmed a moderate association between psoriasis and genetic variants in the miR-146a encoding gene. Transfection of miR-146a/b suppressed and inhibition enhanced keratinocyte proliferation and the expression of psoriasis-related target genes. Enhanced expression of miR-146a/b-influenced genes was detected in cultured keratinocytes from miR-146a−/− and skin fibroblasts from miR-146a−/− and miR-146b−/− mice stimulated with psoriasis-associated cytokines as compared with wild-type mice. Our results indicate that besides miR-146a, miR-146b is expressed and might be capable of modulation of inflammatory responses and keratinocyte proliferation in psoriatic skin.

Background miR-10a-5p has been shown to regulate cancer cell proliferation and invasiveness and endothelial cell inflammatory responses. The function of miR-10a-5p in the skin has not been previously studied. The aim of the current study was to examine miR-10a-5p expression, regulation, and function in keratinocytes (KCs) in association with atopic dermatitis (AD). Methods The expression of miR-10a-5p and its target genes was analyzed using RT-qPCR, mRNA array analysis, in situ hybridization, and immunofluorescence. The transfection of miRNA mimics, cell cycle distribution analysis, and luciferase assays was used to study miR-10a-5p functions in human primary KCs. Results miR-10a-5p was found to be upregulated in lesional skin from patients with AD and in proliferating KCs. Array and pathway analysis of IL-1β-stimulated KCs revealed that miR-10a-5p inhibited many genes that affect cell cycle progression and only a few inflammation-related genes. Accordingly, fewer cells in S-phase and reduced proliferation were detected as characteristics of miR-10a-5p-transfected KCs. The influence of miR-10a-5p on cell proliferation was also evident in KCs induced by AD-related cytokines, including IL-4, IL-17, and IL-1β, as measured by the capacity to strongly suppress the expression of the proliferation marker Ki-67. Among AD-related putative direct target genes, we verified hyaluronan synthase 3, a damage-associated positive regulator of KC migration and proliferation, as a direct target of miR-10a-5p. Conclusions miR-10a-5p inhibits KC proliferation and directly targets hyaluronan synthase 3 and thereby may modulate AD-associated processes in the skin.

Psoriasis is a chronic inflammatory skin disease with numerous involved factors. miR-146a and miR-146b (miR-146a/b) are anti-inflammatory miRNAs that are increased in psoriatic skin. SERPINB2 has been shown to be upregulated in the inflammation and infections. Here we aimed to study the relationship between miR-146a/b and SERPINB2 and to delineate the role of SERPINB2 in association of plaque psoriasis. We report increased SERPINB2 expression in the skin of psoriasis patients, which was in a positive relationship with psoriasis severity and in a negative relationship with miR-146a/b in psoriatic lesions. In cultured keratinocytes, both cellular and secreted SERPINB2 levels were strongly induced in response to IFN-γ and TNF-α. Interestingly, SERPINB2 mRNA was downregulated by IL-17A and the combination of TNF-α and IL-17A at time points when miR-146a was increased. The predicted binding site for miR-146a/b in 3' untranslated region of SERPINB2 revealed no activity in luciferase assay, while siRNA silencing of miR-146a/b direct targets IRAK1 and CARD10 resulted in reduced expression of SERPINB2, suggesting that miR-146a/b indirectly control SERPINB2 expression in the skin. The siRNA silencing of SERPINB2 increased the expression of IL-8, CXCL5 and CCL5 and migration of neutrophils revealing its anti-inflammatory role in keratinocytes. Our data together suggest that SERPINB2 and miR-146a/b are part of disease-related network of molecules that are coordinately regulated and act in controlling the inflammatory responses in psoriatic skin.

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

Background IL-22- and IL-17-producing T cells have important roles in allergic diseases. MicroRNAs (miRNAs) are posttranscriptional regulators of gene expression and modulate numerous biological processes. Little is known about the functions of miRNAs in IL-22/IL-17-producing T cells. Material and Methods IL-22- and IL-17-positive T cells were sorted from human peripheral blood mononuclear cells (PBMCs) by intracellular staining and dual-secretion assay. miRNA expression profiles were detected with TaqMan array microfluidic cards. T cells were transfected with miRNA mimics. Gene expression was analyzed using RT-qPCR and/or enzyme-linked immunosorbent assay in T-cell subsets and PBMCs from patients with asthma and atopic dermatitis. Results The increased expression of miR-323-3p and noncoding RNA nc886 and reduced expression of miR-93, miR-181a, miR-26a, and miR-874 were detected in IL-22-producing T cells. The pathway analysis of the putative targets suggested that these differentially expressed miRNAs could impact the proliferation, differentiation, and effector functions of T cells. Further analyses showed the highest expression for miR-323-3p in IL-22- and IL-17-double-positive T cells and its capacity to suppress multiple genes from the transforming growth factor-β pathway and the production of IL-22 in T cells. An increased expression of miR-323-3p in PBMCs from patients with asthma and reverse correlation between miR-323-3p levels and IL-22 production in PBMCs cultured in T-cell growth conditions was observed. Conclusions Our data suggest that miR-323-3p acts in a negative feedback loop to control the production of IL-22 in IL-22/IL-17-producing T cells and might thus impact the T-cell responses in asthma.