House dust mite-treated PAR2 over-expressor mouse: A novel model of atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a complex skin disease involving causative effects from both intrinsic and extrinsic sources. Murine models of the disease often fall short in one of these components and, as a result, do not fully encapsulate these disease mechanisms. OBJECTIVE: We aimed to determine whether the protease-activated receptor 2 over-expressor mouse (PAR2OE) with topical house dust mite (HDM) application is a more comprehensive and clinically representative AD model. METHODS: Following HDM extract application to PAR2OE mice and controls, AD clinical scoring, itching behaviour, skin morphology and structure, barrier function, immune cell infiltration and inflammatory markers were assessed. Skin morphology was analysed using haematoxylin and eosin staining, and barrier function was assessed by transepidermal water loss measurements. Immune infiltrate was characterised by histological and immunofluorescence staining. Finally, an assessment of AD-related gene expression was performed using quantitative RT-PCR. RESULTS: PAR2OE mice treated with HDM displays all the characteristic clinical symptoms including erythema, dryness and oedema, skin morphology, itch and inflammation typically seen in patients with AD. There is a significant influx of mast cells (P < .01) and eosinophils (P < .0001) into the dermis of these mice. Furthermore, the PAR2OE + HDM mice exhibit similar expression patterns of key differentially expressed genes as seen in human AD. CONCLUSION: The PAR2OE + HDM mouse presents with a classic AD pathophysiology and is a valuable model in terms of reproducibility and overall disease representation to study the condition and potential therapeutic approaches.

Interleukin-4 and interleukin-13 evoke scratching behaviour in mice.

Persistent and relapsing itch commonly manifests in inflammatory skin disorders such as atopic dermatitis (AD). AD pathogenesis is driven by interleukin-4 (IL-4) and interleukin-13 (IL-13). Dupilumab, a monoclonal antibody blocking the action of IL-4 and IL-13 effectively reduces the symptoms of AD and itch. Little is known whether IL-4 and IL-13 directly contribute to itch transduction. A recently published study (Oetjen et al, Cell, 2017, 171, 217) found IL-4 and IL-13 to directly activate itch-sensory neurons in vitro. Surprisingly, they found no significant increase in scratching after intradermally injecting high doses (2.5 ug/ml) of IL-4 and IL-13 into mice. Similar experiments in our lab, however, suggested that both IL-4 and IL-13 contribute to acute itch in vivo. We intradermally injected lower doses (1 ug/ml) of IL-4 and IL-13 into mice and found a significant increase of scratching bouts compared to vehicle. Interestingly, the combined treatment of IL-4 and IL-13 produced additive increase of scratching and acute pruritus at an earlier time point compared to each cytokine administered alone. In summary, our study shows a rapid and significant increase of scratching after intradermal injection of IL-4, IL-13 or combined IL-4/ IL-13 compared to vehicle in mice 5-10 minutes after injection. Our data suggest that IL-4 and IL-13 alone and combined directly act as potent acute pruritogens on sensory nerves. This finding expands our understanding of cytokines as pruritogens, how targeted anticytokine medications act in AD, and about neuroimmune communication in the skin.

Pharmacologic inhibition of hypoxia-inducible factor (HIF)-hydroxylases ameliorates allergic contact dermatitis.

BACKGROUND: When an immune cell migrates from the bloodstream to a site of chronic inflammation, it experiences a profound decrease in microenvironmental oxygen levels leading to a state of cellular hypoxia. The hypoxia-inducible factor-1α (HIF-1α) promotes an adaptive transcriptional response to hypoxia and as such is a major regulator of immune cell survival and function. HIF hydroxylases are the family of oxygen-sensing enzymes primarily responsible for conferring oxygen dependence upon the HIF pathway. METHODS: Using a mouse model of allergic contact dermatitis (ACD), we tested the effects of treatment with the pharmacologic hydroxylase inhibitor DMOG, which mimics hypoxia, on disease development. RESULTS: Re-exposure of sensitized mice to 2,4-dinitrofluorobenzene (DNFB) elicited inflammation, edema, chemokine synthesis (including CXCL1 and CCL5) and the recruitment of neutrophils and eosinophils. Intraperitoneal or topical application of the pharmacologic hydroxylase inhibitors dymethyloxalylglycine (DMOG) or JNJ1935 attenuated this inflammatory response. Reduced inflammation was associated with diminished recruitment of neutrophils and eosinophils but not lymphocytes. Finally, hydroxylase inhibition reduced cytokine-induced chemokine production in cultured primary keratinocytes through attenuation of the JNK pathway. CONCLUSION: These data demonstrate that hydroxylase inhibition attenuates the recruitment of neutrophils to inflamed skin through reduction of chemokine production and increased neutrophilic apoptosis. Thus, pharmacologic inhibition of HIF hydroxylases may be an effective new therapeutic approach in allergic skin inflammation.

Altered expression of mir-222 and mir-25 influences diverse gene expression changes in transformed normal and anaplastic thyroid cells, and impacts on MEK and TRAIL protein expression.

Thyroid cancer is the most common endocrine malignancy and accounts for the majority of endocrine cancer-related deaths each year. Our group and others have previously demonstrated dysfunctional microRNA (miRNA or miR) expression in the context of thyroid cancer. The objective of the present study was to investigate the impact of synthetic manipulation of expression of miR-25 and miR-222 in benign and malignant thyroid cells. miR-25 and miR-222 expression was upregulated in 8505C (an anaplastic thyroid cell line) and Nthy-ori (a SV40-immortalised thyroid cell line) cells, respectively. A transcriptomics-based approach was utilised to identify targets of the two miRNAs and real-time PCR and western blotting were used to validate a subset of the targets. Almost 100 mRNAs of diverse functions were found to be either directly or indirectly targeted by both miR-222 and miR-25 [fold change ≥2, false discovery rate (FDR) ≤0.05]. Gene ontology analysis showed the miR-25 gene target list to be significantly enriched for genes involved in cell adhesion. Fluidigm real-time PCR technologies were used to validate the downregulation of 23 and 22 genes in response to miR-25 and miR-222 overexpression, respectively. The reduction of the expression of two miR-25 protein targets, TNF-related apoptosis­inducing ligand (TRAIL) and mitogen-activated protein kinase kinase 4 (MEK4), was also validated. Manipulating the expression of both miR-222 and miR-25 influenced diverse gene expression changes in thyroid cells. Increased expression of miR-25 reduced MEK4 and TRAIL protein expression, and cell adhesion and apoptosis are important aspects of miR-25 functioning in thyroid cells.

Platelet adhesion and degranulation induce pro-survival and pro-angiogenic signalling in ovarian cancer cells.

Thrombosis is common in ovarian cancer. However, the interaction of platelets with ovarian cancer cells has not been critically examined. To address this, we investigated platelet interactions in a range of ovarian cancer cell lines with different metastatic potentials [HIO-80, 59M, SK-OV-3, A2780, A2780cis]. Platelets adhered to ovarian cancer cells with the most significant adhesion to the 59M cell line. Ovarian cancer cells induced platelet activation [P-selectin expression] in a dose dependent manner, with the most significant activation seen in response to the 59M cell line. The platelet antagonists [cangrelor, MRS2179, and apyrase] inhibited 59M cell induced activation suggesting a P2Y12 and P2Y1 receptor mediated mechanism of platelet activation dependent on the release of ADP by 59M cells. A2780 and 59M cells potentiated PAR-1, PAR-4, and TxA2 receptor mediated platelet activation, but had no effect on ADP, epinephrine, or collagen induced activation. Analysis of gene expression changes in ovarian cancer cells following treatment with washed platelets or platelet releasate showed a subtle but valid upregulation of anti-apoptotic, anti-autophagy pro-angiogenic, pro-cell cycle and metabolic genes. Thus, ovarian cancer cells with different metastatic potential adhere and activate platelets differentially while both platelets and platelet releasate mediate pro-survival and pro-angiogenic signals in ovarian cancer cells.