DataSheet_2_Heat shock protein 90 inhibition attenuates inflammation in models of atopic dermatitis: a novel mechanism of action.zip

BackgroundHeat shock protein 90 (HSP90) is an important chaperone supporting the function of many proinflammatory client proteins. Recent studies indicate HSP90 inhibition may be a novel mechanism of action for inflammatory skin diseases; however, this has not been explored in atopic dermatitis (AD).ObjectivesOur study aimed to investigate HSP90 as a novel target to treat AD.MethodsExperimental models of AD were used including primary human keratinocytes stimulated with cytokines (TNF/IFNγ or TNF/IL-4) and a mouse model established by MC903 applications.ResultsIn primary human keratinocytes using RT-qPCR, the HSP90 inhibitor RGRN-305 strongly suppressed the gene expression of Th1- (TNF, IL1B, IL6) and Th2-associated (CCL17, CCL22, TSLP) cytokines and chemokines related to AD. We next demonstrated that topical and oral RGRN-305 robustly suppressed MC903-induced AD-like inflammation in mice by reducing clinical signs of dermatitis (oedema and erythema) and immune cell infiltration into the skin (T cells, neutrophils, mast cells). Interestingly, topical RGRN-305 exhibited similar or slightly inferior efficacy but less weight loss compared with topical dexamethasone. Furthermore, RNA sequencing of skin biopsies revealed that RGRN-305 attenuated MC903-induced transcriptome alterations, suppressing genes implicated in inflammation including AD-associated cytokines (Il1b, Il4, Il6, Il13), which was confirmed by RT-qPCR. Lastly, we discovered using Western blot that RGRN-305 disrupted JAK-STAT signaling by suppressing the activity of STAT3 and STAT6 in primary human keratinocytes, which was consistent with enrichment analyses from the mouse model.ConclusionHSP90 inhibition by RGRN-305 robustly suppressed inflammation in experimental models mimicking AD, proving that HSP90 inhibition may be a novel mechanism of action in treating AD.</p

DataSheet_1_Heat shock protein 90 inhibition attenuates inflammation in models of atopic dermatitis: a novel mechanism of action.docx

BackgroundHeat shock protein 90 (HSP90) is an important chaperone supporting the function of many proinflammatory client proteins. Recent studies indicate HSP90 inhibition may be a novel mechanism of action for inflammatory skin diseases; however, this has not been explored in atopic dermatitis (AD).ObjectivesOur study aimed to investigate HSP90 as a novel target to treat AD.MethodsExperimental models of AD were used including primary human keratinocytes stimulated with cytokines (TNF/IFNγ or TNF/IL-4) and a mouse model established by MC903 applications.ResultsIn primary human keratinocytes using RT-qPCR, the HSP90 inhibitor RGRN-305 strongly suppressed the gene expression of Th1- (TNF, IL1B, IL6) and Th2-associated (CCL17, CCL22, TSLP) cytokines and chemokines related to AD. We next demonstrated that topical and oral RGRN-305 robustly suppressed MC903-induced AD-like inflammation in mice by reducing clinical signs of dermatitis (oedema and erythema) and immune cell infiltration into the skin (T cells, neutrophils, mast cells). Interestingly, topical RGRN-305 exhibited similar or slightly inferior efficacy but less weight loss compared with topical dexamethasone. Furthermore, RNA sequencing of skin biopsies revealed that RGRN-305 attenuated MC903-induced transcriptome alterations, suppressing genes implicated in inflammation including AD-associated cytokines (Il1b, Il4, Il6, Il13), which was confirmed by RT-qPCR. Lastly, we discovered using Western blot that RGRN-305 disrupted JAK-STAT signaling by suppressing the activity of STAT3 and STAT6 in primary human keratinocytes, which was consistent with enrichment analyses from the mouse model.ConclusionHSP90 inhibition by RGRN-305 robustly suppressed inflammation in experimental models mimicking AD, proving that HSP90 inhibition may be a novel mechanism of action in treating AD.</p

Table_1_Heat shock protein 90 inhibition attenuates inflammation in models of atopic dermatitis: a novel mechanism of action.xlsx

BackgroundHeat shock protein 90 (HSP90) is an important chaperone supporting the function of many proinflammatory client proteins. Recent studies indicate HSP90 inhibition may be a novel mechanism of action for inflammatory skin diseases; however, this has not been explored in atopic dermatitis (AD).ObjectivesOur study aimed to investigate HSP90 as a novel target to treat AD.MethodsExperimental models of AD were used including primary human keratinocytes stimulated with cytokines (TNF/IFNγ or TNF/IL-4) and a mouse model established by MC903 applications.ResultsIn primary human keratinocytes using RT-qPCR, the HSP90 inhibitor RGRN-305 strongly suppressed the gene expression of Th1- (TNF, IL1B, IL6) and Th2-associated (CCL17, CCL22, TSLP) cytokines and chemokines related to AD. We next demonstrated that topical and oral RGRN-305 robustly suppressed MC903-induced AD-like inflammation in mice by reducing clinical signs of dermatitis (oedema and erythema) and immune cell infiltration into the skin (T cells, neutrophils, mast cells). Interestingly, topical RGRN-305 exhibited similar or slightly inferior efficacy but less weight loss compared with topical dexamethasone. Furthermore, RNA sequencing of skin biopsies revealed that RGRN-305 attenuated MC903-induced transcriptome alterations, suppressing genes implicated in inflammation including AD-associated cytokines (Il1b, Il4, Il6, Il13), which was confirmed by RT-qPCR. Lastly, we discovered using Western blot that RGRN-305 disrupted JAK-STAT signaling by suppressing the activity of STAT3 and STAT6 in primary human keratinocytes, which was consistent with enrichment analyses from the mouse model.ConclusionHSP90 inhibition by RGRN-305 robustly suppressed inflammation in experimental models mimicking AD, proving that HSP90 inhibition may be a novel mechanism of action in treating AD.</p

Correlation between PASI and circPTPRA skin expression in non-lesional (NL) and lesional (L) skin during secukinumab treatment.

(A+B; left) CircTULP4 (A), miR-223-3p (B), and miR-15a-5p (C) log2-transformed expression values plotted against PASI during 84 days of secukinumab treatment. Simple linear regression was used for correlation between log2-transformed normalized counts and PASI. (A+B; right) CircTULP4 (A), miR-223-3p (B), and miR-15a-5p (C) expression in lesional skin during secukinumab treatment and non-lesional skin. Depicted are normalized counts and median expression; n(NL, L D4, L D14, and L D43) = 14, n(L D0) = 13, n(circRNA-L D84) = 12, and n(miRNA-L D84) = 13. (PDF)</p

Correlation between circRNA expression changes and PASI during 84 days of secukinumab treatment.

Correlation between circRNA expression changes and PASI during 84 days of secukinumab treatment.</p

Pathway Analysis of Skin from Psoriasis Patients after Adalimumab Treatment Reveals New Early Events in the Anti-Inflammatory Mechanism of Anti-TNF-α

<div><p>Psoriasis is a chronic cutaneous inflammatory disease. The immunopathogenesis is a complex interplay between T cells, dendritic cells and the epidermis in which T cells and dendritic cells maintain skin inflammation. Anti-tumour necrosis factor (anti-TNF)-α agents have been approved for therapeutic use across a range of inflammatory disorders including psoriasis, but the anti-inflammatory mechanisms of anti-TNF-α in lesional psoriatic skin are not fully understood. We investigated early events in skin from psoriasis patients after treatment with anti-TNF-α antibodies by use of bioinformatics tools. We used the Human Gene 1.0 ST Array to analyse gene expression in punch biopsies taken from psoriatic patients before and also 4 and 14 days after initiation of treatment with the anti-TNF-α agent adalimumab. The gene expression was analysed by gene set enrichment analysis using the Functional Annotation Tool from DAVID Bioinformatics Resources. The most enriched pathway was visualised by the Pathview Package on Kyoto Encyclopedia of Genes and Genomes (KEGG) graphs. The analysis revealed new very early events in psoriasis after adalimumab treatment. Some of these events have been described after longer periods of anti-TNF-α treatment when clinical and histological changes appear, suggesting that effects of anti-TNF-α treatment on gene expression appear very early before clinical and histological changes. Combining microarray data on biopsies from psoriasis patients with pathway analysis allowed us to integrate <i>in vitro</i> findings into the identification of mechanisms that may be important <i>in vivo</i>. Furthermore, these results may reflect primary effect of anti-TNF-α treatment in contrast to studies of gene expression changes following clinical and histological changes, which may reflect secondary changes correlated to the healing of the skin.</p></div

List of targets and probes for custom-designed circRNA NanoString panel.

(XLSX)</p

Estimation of cumulative number of BSs for each considered miRNA using the sum of the product of the respective circRNA expression levels at the time and number of predicted BSs based on circInteractome BS prediction.

(XLSX)</p

Alterations in global circRNA expression in non-lesional (NL) and lesional (L) skin of psoriasis patients before and during secukinumab treatment.

(A) Principal component analysis based on circRNA expression levels in paired lesional and non-lesional patients before treatment and healthy control skin (NN). (B) Volcano plot showing changes circRNA expression in non-lesional psoriasis skin before treatment relative to healthy control skin (NN). Plots depict adjusted (adj.) p-values relative to log2FC. Multiple unpaired t-test with correction for multiple comparisons (FDR-Benjamini-Hochberg). (C) Heatmap with unsupervised hierarchical clustering of circRNA expression (as z-score of log-transformed values) for individual patients from non-lesional and paired lesional psoriasis skin dependent on the day of treatment. (D) Volcano plot showing changes circRNA expression in lesional psoriasis skin before treatment relative to healthy control skin (NN). Plots depict adjusted (adj.) p-values relative to log2FC. Multiple unpaired t-test with correction for multiple comparisons (FDR-Benjamini-Hochberg). (E) Volcano plot showing changes in specific circRNA and mRNA expression in lesional psoriasis skin at day 84 of treatment (L D84) compared to non-lesional skin. Multiple paired t-test with correction for multiple comparisons (FDR-Benjamini-Hochberg). For panels C and E: n(NL, L D4, L D14, and L D43) = 14, n(L D0) = 13, and n(L D84) = 12; for panels A, B, and D: n = 8. (PDF)</p

Changes in circRNA expression and ciRS-7 localization in non-lesional (NL) and lesional (L) skin of psoriasis patients before and during secukinumab treatment.

(A) Volcano plots show changes in specific circRNA and mRNA expression in lesional psoriasis skin before treatment (L D0; left) and during secukinumab treatment on day 42 (middle) and 84 (right). Plots depict adjusted (adj.) p-values relative to log2FC. Multiple paired t-test with correction for multiple comparisons (FDR-Benjamini-Hochberg); n(NL, L D4, L D14, and L D43) = 14, n(L D0) = 13, and n(L D84) = 12. Black points depict circRNAs with a |log2FC| > 1 and adj.-p-value FUS, HNRNPL, DHX9, ADAR, and QKI). (B) Hematoxylin staining combined with RNA CISH for ciRS-7 during treatment with secukinumab. Paired biopsies from non-lesional and lesional skin before (L D0), and 4, 14, 42, and 84 days after treatment initiation. Four micrometer sections of paraffin-embedded biopsies with one representative patient shown. Scale bar: 100 μm and 25 μm in zoom-in. (C) Boxplot showing the median log2FC of individual circRNAs in lesional D84 relative to D0 skin dependent on whether or not they characterize as Alu-mediated circRNA (presence of IAEs within 2300 nucleotide regions flanking the BSJs). Twenty circRNAs out of 48 circRNAs were considered Alu-mediated. Depicted are median values with whiskers extending to min. and max. values. Two-tailed Mann-Whitney test; n = 11. BSJ = backsplicing junction; CISH = chromogenic in situ hybridization; IAE = Inverted Alu elements.</p