Skin Anti-Inflammatory Potential with Reduced Side Effects of Novel Glucocorticoid Receptor Agonists

Abstract

Glucocorticoids (GCs) are commonly used in the treatment of inflammatory skin diseases, although the balance between therapeutic benefits and side effects is still crucial in clinical practice. One of the major and well-known adverse effects of topical GCs is cutaneous atrophy, which seems to be related to the activation of the glucorticoid receptor (GR) genomic pathway. Dissociating anti-inflammatory activity from atrophogenicity represents an important goal to achieve, in order to avoid side effects on keratinocytes and fibroblasts, known target cells of GC action. To this end, we evaluated the biological activity and safety profile of two novel chemical compounds, DE.303 and KL.202, developed as non-transcriptionally acting GR ligands. In primary keratinocytes, both compounds demonstrated anti-inflammatory properties inhibiting NF-κB activity, downregulating inflammatory cytokine release and interfering with pivotal signaling pathways involved in the inflammatory process. Of note, these beneficial actions were not associated with GC-related atrophic effects: treatments of primary keratinocytes and fibroblasts with DE.303 and KL.202 did not induce, contrarily to dexamethasone-a known potent GC-alterations in extracellular matrix components and lipid synthesis, thus confirming their safety profile. These data provide the basis for evaluating these compounds as effective alternatives to the currently used GCs in managing inflammatory skin diseases.

Keywords: fibroblasts; glucocorticoids; inflammation; inflammatory disease; keratinocytes; skin; skin lipids.

Figure 1

DE and KL did not induce GR transcriptional activation. Western blot analysis and corresponding densitometric analysis of (a) GR and (b) phospho-GR protein expression at cytoplasmic and nuclear levels on NHKs treated with DEX, DE, and KL (1 µM) for 1 h. β-Actin was used as the loading control in the cytoplasmic fraction, and H3 was used for the nuclear one. Representative blots are shown. Results are expressed as the fold change respect to untreated control cells. (c,d) Immunofluorescence analysis of (c) GR and (d) phospho-GR expression in NHKs treated with DEX, DE, and KL (1 µM). Nuclei were counterstained with DAPI. The enlarged view of the selected cells in the white-line-framed areas is shown. Scale bar: 20 µm; enlarged view of the white line-framed areas: 10 µm. (e) Array card heatmap illustrating gene expression analysis in NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Red and blue shadings represent higher and lower relative log2 fold change expression levels, respectively. (f) Real-time RT-PCR analysis of ANGPTL4, DUSP1, ERRF1, FKBP5, GLUL, HAS2, MT2A, RGS2, and TSC22D3 in NHKs treated with DEX, DE, and KL (1 µM) for 24 h. All mRNA values were normalized against the expression of GAPDH and were expressed relative to untreated control cells. The data in the graphs are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control).

Figure 1

DE and KL did not induce GR transcriptional activation. Western blot analysis and corresponding densitometric analysis of (a) GR and (b) phospho-GR protein expression at cytoplasmic and nuclear levels on NHKs treated with DEX, DE, and KL (1 µM) for 1 h. β-Actin was used as the loading control in the cytoplasmic fraction, and H3 was used for the nuclear one. Representative blots are shown. Results are expressed as the fold change respect to untreated control cells. (c,d) Immunofluorescence analysis of (c) GR and (d) phospho-GR expression in NHKs treated with DEX, DE, and KL (1 µM). Nuclei were counterstained with DAPI. The enlarged view of the selected cells in the white-line-framed areas is shown. Scale bar: 20 µm; enlarged view of the white line-framed areas: 10 µm. (e) Array card heatmap illustrating gene expression analysis in NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Red and blue shadings represent higher and lower relative log2 fold change expression levels, respectively. (f) Real-time RT-PCR analysis of ANGPTL4, DUSP1, ERRF1, FKBP5, GLUL, HAS2, MT2A, RGS2, and TSC22D3 in NHKs treated with DEX, DE, and KL (1 µM) for 24 h. All mRNA values were normalized against the expression of GAPDH and were expressed relative to untreated control cells. The data in the graphs are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control).

Figure 2

DE and KL counteracted the TNF-α-induced expression of pro-inflammatory cytokines in NHKs. (a) IL-6 and IL-8 quantitation by ELISA in NHKs pre-treated with DEX, DE, and KL (1 µM) for 1 h and then stimulated with TNF-α (20 ng/mL) for 24 h. The data are expressed as mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01 vs. untreated control; $$ p < 0.05 vs. TNF-α-stimulated cells). (b) IL-6 and IL-8 quantitation by ELISA in NHKs transfected with specific siRNA for the GR gene (siGR) or non-specific siRNA (siCtr) for 24 h, then pre-treated with DEX, DE, and KL (1 µM) for 1 h and stimulated with TNF-α (20 ng/mL) for 24 h. GR protein expression level was evaluated by Western blot. The data are expressed as mean ± SD of three independent experiments (** p < 0.01 vs. TNF-α-stimulated cells).

Figure 3

DE and KL counteracted the NF-κB activation induced by TNF-α in NHKs. (a) Western blot analysis and corresponding densitometric analysis of NF-κB protein expression at cytoplasmic and nuclear level on NHKs pre-treated with DEX, DE, and KL (1 µM) for 1 h and then stimulated with TNF-α (20 ng/mL) for 1 h. β-Actin was used as the loading control in the cytoplasmic fraction, and H3 was used for the nuclear one. Results are expressed as the fold change respect to untreated control cells. (b) Representative immunofluorescence images of NK-kB immunoreactivity in NHKs treated with DEX, DE, and KL and stimulated with TNF-α (20 ng/mL) alone or pre-treated with the compounds (1 µM) for 1 h. The enlarged view of the selected cells in the white line-framed areas is shown. Nuclei were counterstained with DAPI. Scale bar: 50 µm; enlarged view of the white line-framed areas: 20 µm.

Figure 4

DE and KL counteracted the inflammatory pathways induced by TNF-α in NHKs. (a) Western blot analysis and corresponding densitometric analysis of phospho-STAT1, STAT1, phospho-STAT3, STAT3, phospho-c-Jun, c-Jun, phospho-ERK, ERK, phospho-p38, and p38 protein expression in NHKs pre-treated with DEX, DE, and KL (1 µM) for 1 h and then stimulated with TNF-α (20 ng/mL) for 10 min. GAPDH and β-actin were used as endogenous loading control. Results are expressed as the fold change respect to untreated control cells. (b) IL-6 and IL-8 quantitation by ELISA in NHKs pre-treated with TOFA (2 µM), DE (1 µM), and KL (1 µM) for 1 h and then stimulated with TNF-α (20 ng/mL) for 24 h. The data are expressed as mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01 vs. untreated control; $$ p < 0.05 vs. TNF-α stimulated cells).

Figure 5

DE and KL safety profile in NHKs. (a) Real-time RT-PCR analysis of HAS2, HAS3, MMP1, MMP2, and MMP3 in NHKs treated with DEX, DE, and KL (1 µM) for 24 h. All mRNA values were normalized against the expression of GAPDH and were expressed relative to untreated control cells. The data in the graphs are mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01 vs. untreated control). (b) GCMS analysis of FAMEs, FFAs, and CH in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Lipid amounts were expressed as fold change with respect to the untreated control. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). (c) LCMS analysis of total amount of CERs in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. CERa amounts were expressed as nmol normalized to millions of cells. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). (d) LCMS analysis of relative content of non-hydroxy fatty acids (N) ceramide classes in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Each ceramide class was reported as fold change with respect to untreated control. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). Sphingosine (S), dihydrosphingosine (DS), and phytosphingosine (P). (e) LCMS analysis of relative content of α-hydroxy fatty acid (A) ceramide classes in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Each ceramide class was reported as fold change with respect to untreated control. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). Sphingosine (S), dihydrosphingosine (DS), and phytosphingosine (P). (f) The proportion of ceramide families in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Non-hydroxy fatty acids (N), α-hydroxy fatty acids (A), sphingosine (S), dihydrosphingosine (DS), and phytosphingosine (P).

Figure 5

DE and KL safety profile in NHKs. (a) Real-time RT-PCR analysis of HAS2, HAS3, MMP1, MMP2, and MMP3 in NHKs treated with DEX, DE, and KL (1 µM) for 24 h. All mRNA values were normalized against the expression of GAPDH and were expressed relative to untreated control cells. The data in the graphs are mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01 vs. untreated control). (b) GCMS analysis of FAMEs, FFAs, and CH in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Lipid amounts were expressed as fold change with respect to the untreated control. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). (c) LCMS analysis of total amount of CERs in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. CERa amounts were expressed as nmol normalized to millions of cells. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). (d) LCMS analysis of relative content of non-hydroxy fatty acids (N) ceramide classes in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Each ceramide class was reported as fold change with respect to untreated control. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). Sphingosine (S), dihydrosphingosine (DS), and phytosphingosine (P). (e) LCMS analysis of relative content of α-hydroxy fatty acid (A) ceramide classes in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Each ceramide class was reported as fold change with respect to untreated control. The data in the graph are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). Sphingosine (S), dihydrosphingosine (DS), and phytosphingosine (P). (f) The proportion of ceramide families in differentiated NHKs treated with DEX, DE, and KL (1 µM) for 24 h. Non-hydroxy fatty acids (N), α-hydroxy fatty acids (A), sphingosine (S), dihydrosphingosine (DS), and phytosphingosine (P).

Figure 6

DE and KL safety profile in NHFs. (a) Morphological analysis and measurement of cell area of NHFs cells treated with DEX, DE, and KL (1 µM) for 72 h. At least 200 cells were evaluated for each condition (** p < 0.01 vs. untreated control). Scale bar: 100 µm. (b) TRITC-phalloidin staining on NHFs upon treatment with DEX, DE, and KL. Nuclei are counterstained with DAPI. Scale bar: 50 µm (c) Graphs illustrating cell count assay data of NHFs cells treated as above. The data are mean ± SD of three independent experiments (* p < 0.05 vs. untreated control). (d) Immunofluorescence and quantitative analysis of Ki67-positive NHFs treated with DEX, DE, and KL. Arrows point at cells positively stained for the proliferation marker. Nuclei were counterstained with DAPI (* p < 0.05 vs. untreated control). Scale bar: 50 µm. (e) GCMS analysis of FAMEs in NHFs treated with DEX, DE, and KL (1 µM) for 72 h. Lipid amounts were expressed as fold change with respect to untreated control (* p < 0.05 vs. untreated control). Saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). (f) Real-time RT-PCR analysis of HAS2, HAS3, MMP1, MMP2, MMP3, COL1A1, and COL3A1 in NHFs treated with DEX, DE, and KL (1 µM) for 24 h. All mRNA values were normalized against the expression of GAPDH and were expressed relative to untreated control cells. The data in the graphs are mean ± SD of three independent experiments (* p < 0.05, ** p < 0.01 vs. untreated control).