Immune-mediated changes in actinic keratosis following topical treatment with imiquimod 5% cream

Abel Torres, Leslie Storey, Makala Anders, Richard L Miller, Barbara J Bulbulian, Jizhong Jin, Shalini Raghavan, James Lee, Herbert B Slade, Woubalem Birmachu, Abel Torres, Leslie Storey, Makala Anders, Richard L Miller, Barbara J Bulbulian, Jizhong Jin, Shalini Raghavan, James Lee, Herbert B Slade, Woubalem Birmachu

Abstract

Background: The objective of this study was to identify the molecular processes responsible for the anti-lesional activity of imiquimod in subjects with actinic keratosis using global gene expression profiling.

Methods: A double-blind, placebo-controlled, randomized study was conducted to evaluate gene expression changes in actinic keratosis treated with imiquimod 5% cream. Male subjects (N = 17) with > or = 5 actinic keratosis on the scalp applied placebo cream or imiquimod 3 times a week on nonconsecutive days for 4 weeks. To elucidate the molecular processes involved in actinic keratosis lesion regression by imiquimod, gene expression analysis using oligonucleotide arrays and real time reverse transcriptase polymerase chain reaction were performed on shave biopsies of lesions taken before and after treatment.

Results: Imiquimod modulated the expression of a large number of genes important in both the innate and adaptive immune response, including increased expression of interferon-inducible genes with known antiviral, anti-proliferative and immune modulatory activity, as well as various Toll-like receptors. In addition, imiquimod increased the expression of genes associated with activation of macrophages, dendritic cells, cytotoxic T cells, and natural killer cells, as well as activation of apoptotic pathways.

Conclusion: Data suggest that topical application of imiquimod stimulates cells in the skin to secrete cytokines and chemokines that lead to inflammatory cell influx into the lesions and subsequent apoptotic and immune cell-mediated destruction of lesions.

Figures

Figure 1
Figure 1
Variation in the temporal expression of IRF7 during treatment with imiquimod as determined by real time RT-PCR. 'AK,' designates pretreatment AK lesions. 'WK1 IMIQ', 'WK2 IMIQ' and 'WK4 IMIQ' designate treatment times week 1, 2 and 4. '4 WK Post designates' 4 weeks post end of treatment (WK4 treatment). Fold change was calculated with respect to sun-unexposed, non-lesional skin. S-01, S-02, S-06, S-15 and S-16 are samples from subjects 1, 2, 6, 15 and 16.
Figure 2
Figure 2
Comparison of gene expression data obtained by Affymetrix GeneChip analysis and real time RT-PCR. Linear regression analysis for IRF7 comparing fold change values for individual subjects as measured by Affymetrix analysis and real time RT-PCR analysis. The imiquimod response fold change, which is the maximum response from week 1, week 2 and week 4 treatment times was used. Fold change was calculated relative to sun-unexposed, nonlesional skin samples for 13 subjects treated with imiquimod. The R square value for the comparison was 0.83.
Figure 3
Figure 3
Basal TLR, IRF7, and MyD88 gene expression in skin biopsies as determined by real time RT-PCR. White bars represent pretreatment AK, black bars represent during imiquimod treatment (maximum response value from week 1, week 2 and week 4 treatment times), and hatched bars represent 4-weeks post treatment. Relative copy number was determined as outlined in Methods and Materials section. Asterisks indicate those genes that had p-values < 0.05 in the ANOVA, comparing expression in pretreatment AK samples to the maximum response expression in samples from subjects (n = 13) during imiquimod treatment. [See Additional file 2].
Figure 4
Figure 4
Increase in expression of the helicase family of virus-sensing genes upon treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)DDX58 (RIG-I) and (B)IFIH1 (MDA5). Experimental conditions are as described in the Methods and Materials section. Box plots were generated using MINITAB version 14. 'AK,' 'IMIQ,' and 'Post' designate pretreatment AK, imiquimod-treated skin during treatment (maximum response from week 1, week 2 and week 4), and imiquimod-treated skin 4 weeks post treatment, respectively. Fold change was calculated with respect to sun-unexposed skin. Boxes indicate the median 95% confidence intervals, asterisks designate outliers, and the lines connect median values. P values are given for 2-way ANOVA, comparing the fold change values for pretreatment AK samples and imiquimod-treated samples (n = 13).
Figure 5
Figure 5
Cluster analysis of genes regulated by imiquimod treatment. Two-way hierarchical clustering was performed as described in the Methods and Materials section. 'AK,' 'IMIQ,' and 'Placebo' designate the fold change with respect to sun-unexposed, nonlesional skin for pretreatment AK samples; for samples during imiquimod treatment (maximum response from week 1, week 2 and week 4), and samples for vehicle-treatment (maximum response from week 1, week 2 and week 4) respectively. Numbers designate subjects. Hierarchical clustering was performed using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. Red, white, and green indicate up-regulated, unchanged, and down-regulated genes, respectively. The color bar insert shows the corresponding expression levels. The cluster consists of 530 imiquimod-responsive genes whose expression was statistically different when comparing the AK group of samples to the IMIQ group of samples. Expression changes for the 530 genes are documented in [Additional file 1].
Figure 6
Figure 6
Cluster of 46 immune response genes that are also known to be inducible upon treatment with type 1 interferons as reported in [18, 43, 45, 48, 51, 102] Cluster analysis was performed as described in Figure 5. [See Additional file 3].
Figure 7
Figure 7
Imiquimod treatment is associated with an increase in expression of pro-apoptotic genes as determined by Affymetrix GeneChip analysis. (A) TNFSF10 (B)MX1. Experimental conditions and analysis are as described for Figure 4.
Figure 8
Figure 8
Increase in expression of chemokines after treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)CXCL10 (IP10), (B)CXCL11 (ITAC). Experimental conditions and analysis are as described for Figure 4.
Figure 9
Figure 9
Increase in expression of genes indicating the infiltration of DCs upon treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A)CD86 (B) ILT7. Experimental conditions and analysis are as described for Figure 4.
Figure 10
Figure 10
Increase in expression of genes associated with NK cells, and cytotoxic T cells after treatment with imiquimod as determined by Affymetrix gene expression. (A)GZMA (B) NKG7. Experimental conditions and analysis are as described for Figure 4.
Figure 11
Figure 11
Increase in expression of genes associated with lymphocyte function after treatment with imiquimod as determined by Affymetrix GeneChip analysis. (A) SELL (CD62L) (B) NT5E (DC73). Experimental conditions and analysis are as described for Figure 4.

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