Immune-mediated changes in actinic keratosis following topical treatment with imiquimod 5% cream
© Torres et al; licensee BioMed Central Ltd. 2007
Received: 11 June 2006
Accepted: 26 January 2007
Published: 26 January 2007
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.
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 ≥ 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.
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.
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.
Actinic keratosis (AK) are common, cutaneous, precancerous neoplasms appearing as rough, dry, scaly lesions that occur primarily on the sun-exposed skin of middle-aged and elderly people [1–3]. Although the exact mechanism of pathogenesis of AK development is unknown, part of the pathogenesis may involve suppression of the immune response against dysplastic cells . It is believed that prolonged ultraviolet exposure changes the immune surveillance mechanism of the skin, contributing to the tolerance of tumor cells . If left untreated, AK can progress to squamous cell carcinoma, a locally aggressive and occasionally metastatic tumor type . Standard treatment of AK includes various types of surgical and chemical treatments [7, 8], which are often associated with scarring and infection, and may not address sub clinical lesions .
Toll-like receptors (TLR) are pattern recognition receptors that detect pathogen-associated molecular patterns (PAMPs) and play key roles in the activation of innate and adaptive immune responses [9, 10]. Currently, 10 human TLRs have been identified. The natural ligands for all but TLR10 have also been identified . Toll-like receptors are primarily expressed on immune cells such as monocytes, dendritic cells (DCs), and lymphocytes , but some TLRs are also expressed on nonimmune cells, including endothelial cells, epithelial cells, and keratinocytes .
The role of TLRs in the pathogenesis and treatment of dermatological diseases is increasingly recognized . Imiquimod, a member of a class of drugs termed immune response modifiers has been shown to be a selective TLR7 agonist [[14, 15], and unpublished internal data]. Imiquimod is the first TLR-agonist pharmaceutical product approved for human use, and is indicated for the topical treatment of external genital and perianal warts caused by human papilloma virus . Recently, the approved indications have been expanded to include treatment of AK  and superficial basal cell carcinoma [18–20].
The antiviral and anti-tumor activity of imiquimod is believed to be due to the activation of the innate immune response, specifically activation of antigen-presenting cells such as monocytes, macrophages and plasmacytoid and myeloid DCs to induce interferon alpha (IFNα) and other cytokines and chemokines [21, 15]. Imiquimod also enhances co stimulatory molecule expression important for triggering an adaptive immune response . Topical application of the drug has been shown to induce IFNα and interleukin 6 (IL6) in AK lesions and external genital warts [22, 23]. Imiquimod and the chemically related immune response modifier resiquimod have also shown potent vaccine adjuvant effects in mice and man [23–27]. Even though the immune-modulatory activity of imiquimod is well established, the precise molecular changes responsible for the antilesional activity of topically applied imiquimod in AK is not fully understood.
The objective of this study was to explore the molecular processes responsible for the antilesional activity of imiquimod in subjects with actinic keratosis using global gene expression profiling.
Methods and Materials
Institutional review board/informed consent
This study was conducted at Loma Linda University School of Medicine/Medical Center, Department of Internal Medicine, Division of Dermatology, Loma Linda, California. The study protocol, subject informed consent documents, and subject information documents were submitted to and received approval from the study center's Institution Review Board. This study was conducted according to the Code of Federal Regulations of the United States Food and Drug Administration (21 CFR Part 56, Institutional Review Boards, and Part 50, Protection of Human Subjects) and the International Conference on Harmonization Edition 6, Guideline for Good Clinical Practice.
This was a phase II, double-blind, placebo-controlled, randomized parallel group study. Randomized subjects had at least 5 clinically visible AK lesions within a 25-cm2 area on the balding scalp. Subjects were randomized to imiquimod or placebo cream in a 3:1 ratio and applied study cream to the treatment area 3 times per week for 4 weeks. Study cream was applied prior to normal sleeping hours and remained on the skin for approximately 8 hours before it was removed. Safety evaluations were made at all treatment and post treatment visits, and included monitoring of adverse events and local skin reactions, as well as photographing the treatment area and reviewing any concomitant medications.
At the screening visit, AK lesions were assessed clinically and by confocal microscopy and a representative lesion confirmed by histology. Because AK lesions are in general small in size, histology and gene expression analysis could not be performed on the same biopsy. Therefore, confocal microscopy was performed to establish a correlation of the confocal images and their respective non-sun exposed non-lesional skin, sun exposed non-lesional skin, actinic keratoses lesions, and squamous cell carcinoma. All subjects with lesions histologically identified as having a degree of dysplesia suggestive of squamous cell carcinoma were disqualified from the study. All sites identified as AK lesions were marked and a plastic template of their locations made for exact identification at a later time. Thereafter, changes in AK lesions due to treatment with imiquimod were assessed clinically and by confocal microscopy. Lesions were scored as cleared if the skin exhibited normal epidermis as assessed clinically and by confocal microscopy. Assessment of lesion regression and the results of the confocal microscopy as they relate to aberrant gene expression in AK are discussed in a manuscript submitted for publication (Torres et al, 2006. Micro Array Analysis of Aberrant Gene Expression in Actinic Keratosis: Effect of Imiquimod 5% Cream).
At the treatment initiation visit, a shave biopsy was taken for gene expression analysis from an untreated AK lesion, from a sun-exposed non-lesional site on the head and from a non-lesional sun-unexposed site from under the arm area. An additional biopsy was taken at each subsequent study visit, (treatment period weeks 1, 2, and 4) and at 4 weeks post treatment of either a remaining AK, or if no AKs were present, of nonlesional skin at a previously identified lesional site. Thus each biopsy was of a different AK lesion. In an effort to standardize the amount of tissue that was removed at each biopsy, the same size punch was used to score the skin surrounding all the lesions to be biopsied for gene expression studies with an attempt to shave-biopsy the lesion at the papillary dermis level. Biopsies were taken 8 hr to 16 hr after the third treatment of the treatment period. The nonlesional, sun-unexposed site biopsy was used to establish a baseline for comparison of gene expression changes in AK before and after treatment with imiquimod. Shave biopsies were immediately immersed in RNALater (Ambion, Austin, Texas), equilibrated at room temperature for 1 hour, kept at 4°C for 24 hours, and then stored at -20°C prior to RNA extraction.
RNA extraction and purification
Total RNA from the biopsy samples was extracted and purified using Qiagen RNeasy Mini Kit Protocol for the Isolation of Total RNA from Heart, Muscle and Skin Tissue (Qiagen, Valencia, California) according to manufacturer's instructions. RNA yield varied from 1.5 to 14 υg. The purity of the RNA was determined by the 260 nm/280 nm absorbance ratio. The median 260 nm/280 nm ratio value for the 119 samples was 2.0 (range 1.9 to 2.2). RNA integrity was determined using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Assay (Agilant Technologies, Palo Alto, CA). All samples gave 28S/18S ratio between 2 and 3 indicating good quality RNA.
Micro array analysis
Samples for micro array analysis were prepared by 2 rounds of linear target amplification according to the Affymetrix instructions for eukaryotic small sample preparation [28, 29]. Briefly, double-stranded cDNA was synthesized from 100 ng of total RNA with oligo(dT)24T7 primer (Affymetrix, Santa Clara, California), followed by 2 cycles of in vitro transcription of cRNA. The first cycle of invitro transcription was performed using a T7 polymerase (MEGAscript T7 Kit, Ambion, Austin, Texas) and the second cycle using Enzo BioArray High Yield RNA Transcript Labeling Kit (Affymetrix). The median value for the biotinylated cRNA yield over the 119 samples was 90 (range 42 to 121). The biotinylated cRNA was hybridized to Affymetrix U133A and U133B GeneChip arrays containing 22,253 probes sets each. Each array was hybridized for 16 hours, washed, then stained with streptavidin-phycoerythrin conjugate and scanned according to manufacturer's instructions. Images were analyzed using Micro Array Suite Version 5 (MAS5). Chips were normalized to a global average intensity of 150 to allow chip-to-chip comparison. The quality of the images was ascertained by monitoring the noise, background, percent transcript present, and the 3'/5' ratio for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which ranged from 1.5 to 5 and beta actin which ranged from 7 to 24. These values are similar to those reported for double amplification protocols [29, 30].
TaqMan™ real time reverse transcriptase polymerase chain reaction
TaqMan real time RT-PCR was performed for a number of the genes to confirm the micro array results. cDNA was reverse transcribed from total RNA using Invitrogen Superscript First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California). Real time RT-PCR was performed using the Applied Biosystems 7900HT™ sequence detection instrument (Applied Biosystems, Foster City, California), and TaqMan low density custom array micro fluidic cards (Applied Biosystems, Foster City, California) as described previously. The micro fluidic cards consisted of 8 ports with 24 different TaqMan primer pair/probe sets arrayed in duplicate in a 384-well micro plate footprint. Each well contained a gene-specific forward and reverse primer, as well as a gene-specific probe, which is labeled at the 5' position with 6FAM (areporter dye) and at the 3' position with minor groove binder/non-fluorescent quencher. Samples were mixed with TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, California), applied to each port of the card, and analyzed by PCR on the 7900HT instrument using Applied Biosystems Sequence Detection System 2.0 software according to the manufacturer's instructions. A total of 46 selected genes associated with the Toll-like receptor pathway, apoptosis, cell cycle, and immune cell infiltration were analyzed using the TaqMan arrays. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to normalize each sample. The TLR copy number was calculated for 2 ng of RNA.
Analysis of affymetrix gene chip data
Signals from the gene chip images of biopsy samples for untreated normal skin obtained from sun-unexposed sites were used as a control for the calculation of changes in expression in samples of pretreatment AK lesions and AK lesions during and after treatment (treatment weeks 1, 2, and 4 and 4 weeks post treatment). The statistical algorithm in MAS5 evaluates the image for the expression signal, the absent/present call, and the p-value associated with the signal. It also evaluates the fold change of the sample relative to the designated control sample expressed as the signal log2 ratio, the p-value associated with the fold change, and the direction of change (increased, I; decreased, D; or no call, NC). Using the Affymetrix data mining tool software, the expression data from MAS5 were filtered on the basis of specific criteria to identify differentially expressed genes. A given gene was retained if 1sample from the series passed the following criteria: signal detection p-value ≤ 0.01, signal log2 ratio ≤ -2 or ≥ 2, and a change call designation of 'increased' (I) or 'decreased' (D). A total of 1682 genes passed these criteria.
Cluster analysis was performed with Spotfire DecisionSite-8.1 for Functional Genomics (Spotfire Inc, Somerville, Massachusetts), using the Unweighted Pair-Group Method with Arithmetic mean (UPGMA) and the Euclidean similarity measure. Functional categorization of genes was based on gene ontology analysis using the Ontology Browser in Spotfire and gene descriptions at the National Center for Biotechnology Information (NCBI) website . The ontology browser calculates a Fisher's Exact Test p-value, which reflects the chance that the gene ontology category is represented by random chance . P-value < 0.05 is considered significant.
The natural log of the fold change (with respect to sun-unexposed normal skin) from the Affymetrix gene expression and the real time RT-PCR experiments were used in an ANOVA to determine statistically significant changes in expression between sample groups. Due to the small sample size of the vehicle group, 4 subjects, compared to 13 subjects in the imiquimod-treated group, a one way ANOVA comparing vehicle-treated subjects to imiquimod-treated subjects is not expected to yield a reliable determination of imiquimod-response genes. Therefore, a 2-way ANOVA using a blocking factor to account for repeat observations on the same subject was used to compare: (1) the fold change for pretreatment AK (n = 13) and the fold change during treatment with imiquimod (n = 13), and (2) the fold change for pretreatment AK (n = 13) and the fold change for samples taken 4 weeks after the last imiquimod treatment (n = 13). The 'during treatment' fold change is the maximum response fold change value (decreased or increased) selected from week 1, week 2 and week 4 treatment fold changes. Differences between sample groups were considered significant if the p-value for the ANOVA was < 0.05.
Results and Discussion
Demographics and response to treatment
Seventeen white males were randomized to receive treatment (13 to imiquimod and 4 to placebo). The mean age was 75 years (range, 62 to 89 years). All 17 subjects in the study completed the treatment and post treatment portions of the study. The median number of AK lesions at baseline was 10 per subject (range, 6 to 13 lesions). Because of the short duration of the follow-up period (4 weeks for the post treatment period), efficacy was not measured in this study. However, clinical clearance was observed in 25% of the imiquimod-treated subjects 4 weeks after the end of treatment. Imiquimod-treated subjects 1, 2, 4, and 6 and placebo-treated subject 7 were assessed as having clinical clearance of lesions 4 weeks post treatment as determined by return of the lesional site to normal skin. The complete clearance rate in a study where AK subjects were treated for 16 weeks, with a post treatment period of 8 weeks, was 57% .
Analysis of global gene expression using Affymetix GeneChips: Gene Ontology classification
A 2-way analysis of variance (ANOVA) was performed comparing the treatment response gene expression fold change (the maximum response value from week 1, week 2 and week 4 treatments) of samples from the imiquimod-treated subjects to the gene expression fold change values of their respective pretreatment AK samples. This comparison resulted in 530 unique genes that had p-values < 0.05 and a median fold change from pretreatment AK <-2 for suppressed genes and >2 for induced genes. Data for the 530 genes is documented in [Additional file 1]. Two-way ANOVA comparing pretreatment AK samples and samples taken 4 weeks after the last imiquimod treatment resulted in 111 unique genes that differentiated the 2 groups with a p-value < 0.05 [see Additional file 1]. The expression of the rest of the genes returned to basal levels 4 weeks post treatment. Of the total number of differentially regulated genes during imiquimod treatment or 4 weeks post treatment, 87% were up-regulated and 13% were down-regulated.
Gene Ontology Classification of Imiquimod-Induced Genes in AK Lesions
Genes in the Data1
Description of Processes and Specific Genes
Response to stimuli
defense response, response to external biotic stimuli including, pest and pathogens, antimicrobial, anti-fungal, anti-viral response
immune response, inflammatory response
cellular defense response, humoral defense response, antigen binding, pattern binding, cytokine synthesis, chemokine synthesis, antigen presentation processing, inflammatory response
Response to stress
response to pest and pathogen, inflammatory response, response to virus, response to wounding
defensive reaction (by vertebrate tissue) to infection or injury
Response to wounding
defensive reaction (by vertebrate tissue) to physical injury
cytokine signaling, death receptor signaling, G-protein coupled receptor signaling, intigrin binding, MHC protein binding
chemokine receptors, pattern recognition receptors, immunoglobulin receptors, complement receptors, MHC I\MHCII receptors, scavenger receptors, Hematopoietin/interferon class (D200) receptors
SCF1, IGLV2-14, LAG3, LILRA1, SLAMF1, TRA@, TRGC2, TAP2
CCL8, CD69, CLEC4A, FCN1, FGFR2, GALNT7, HMMR, KLRC1/KLRC2, KLRF1, LGALS2, LGALS9, POSTN, PTN, SELL, SELPLG, SN
CCL8, CD14, FGFR2, HMMR, POSTN, PTN, TLR2, TLR7, TLR8, TLR4
CCR5, CCR1, CD74, CSF2RB, CXCR4, IL10RA, IL1RL1, IL21R, TNFRSF1B
CCL3, CCL5, CCL8, CXCL5, CXCL10, CXCL11, CXCL12, CXCL16, TNFSF10, ECGF1, PTN, SLURP1
FCER1A, FCER1G, FCGR3B, 214511_x_at
Cystein type endopeptidase
CASP1, CASP8, CTSB, CTSL, CTSC, STSS, LGMN, TNFAIP3, USP18
Validation of selected genes observed in the Affymetrix experiment using real time reverse transcriptase polymerase chain reaction
Imiquimod increases expression of pattern-recognition receptors of the innate immune system
Imiquimod induces a large number of type I interferon-inducible genes with growth inhibitory and immune-stimulatory activity
Imidazoquinoline TLR7 agonists such as imiquimod and resiquimod are know to induce various cytokines, including interferon-α, IL6, MCP-1 and IL12 as well as the co-stimulatory markers CD80 and CD86 [15, 46]. Type I interferons are known to be powerful regulators of the innate and adaptive immune system through the induction of various genes with antiviral, anti-tumor and immune regulatory functions [43, 44, 47–51]. In this study, we did not detect increased expression of interferon upon treatment with imiquimod, but observed the increased expression of a large number of IFN-inducible genes (114 genes), [see Additional file 3]. The lack of detection of type 1 interferons after imiquimod treatment in this study may be due to the early induction and degradation of their respective mRNA. Biopsies were taken approximately 8 to 16 hr after application of the drug. We have observed that mRNA for type 1 interferons in human blood mononuclear cells treated with imiquimod peaks in 1 to 2 hours and declines to basal levels 6 to 8 hours post treatment (unpublished internal data).
Heterogeneity of pretreatment AK lesions was not documented by clinical assessment in this study. However, the gene expression profiles shown in Figure 5 and Figure 6 indicate heterogeneity in lesions. In addition to AK 11 and Placebo 03 in Figure 6 which cluster with the imiquimod treatment group indicating some level of immune response in these samples, other AK lesions also show low levels of expression of interferon inducible genes. For example, AK 06, AK 03, and AK 07 show low levels of expression of several interferon-inducible genes whereas AK 16, AK 17, AK 12 and AK 15 show normal to slightly depressed levels (Figure 6).
Interferon-inducible genes regulate diverse cellular processes, such as cell growth and differentiation, cell death, and T-cell co stimulation, activation, and migration. These genes have been reported to possess antiviral [52–54], pro-apoptotic [55–57], and anti-proliferative activities [58, 59]. The interferon-inducible genes which increased following imiquimod treatment include those known to be induced by viruses as well as those with known anti-viral activity. These include the 2'5'-oligoadenylate synthetases OAS1, OAS2, OAS3 and OASL, the genes encoding the interferon-inducible proteins with tetratricopepetide repeats, IFIT1, IFIT2, and IFIT3, IFTM1, and other interferon inducible genes such as IFI35, IFI16, MX1, MX2, EIF2AK2 (PRKR), G1P2 (ISG15), G1P3, ISG20, RSAD2 (Cig5), CCL8 (MCP2), CXCL10 (IP10) and CXCL11 (ITAC) [43, 44, 47, 49–52, 54, 60, 61]. Several of the interferon-inducible genes were also increased at statistically significant levels 4 weeks post treatment. These included: IRF7, IFI44, IFIT2, IFIT3, IFITM1, IFI35, RSAD2 (Cig5), G1P2, MX1, OAS1, and OAS2 [see Additional file 1].
Imiquimod induces the expression of chemokines responsible for recruitment of immune cells to AK lesions
Imiquimod increases the expression of genes predictive of infiltrating macrophage and dendritic cells
Of the imiquimod-induced genes classified as immune response in gene ontology, several have receptor activity and (or) are hematopoietic cell surface markers (Table 1). The increased expression of these genes indicates the recruitment of various immune cell types to the lesion sites. Macrophage and/or monocyte infiltration of AK lesions upon treatment with imiquimod was indicated by an increase in CD14, CD163, and CLECSF9 (CLECSF4, MINCLE), a C-type lectin found on activated macrophages . The increase in expression of genes of the classical complement pathway, C1QA, C1QB, C3AR1, and C5R1, also indicates an increase in and/or the activation of macrophages . The data are consistent with histologic observation of macrophage and/or monocyte infiltration after application ofimiquimod in the treatment of AK  and in lentigo maligna [73, 74].
It is interesting to note that CD1C is markedly decreased upon treatment with imiquimod [see Additional file 3]. CD1c is found on Langerhans cells as well as on DCs [79, 80]. The decrease in CD1C expression may reflect (although not exclusively) the migration of CD1C+ Langerhans cells out of the dermis. Migration of Langerhans cells out of mouse dermis was observed after topical treatment with imiquimod [78, 81]. In the case of the Palamara studies  in mouse melanoma, Langerhans cells were observed to return to normal levels in the dermis by day 20. The decrease in CD1C observed upon treatment of AK lesions with imiquimod therefore suggests the activation and migration of Langerhans cells to the lymph nodes and is consistent with previous observations.
Imiquimod increases the expression of genes predictive of infiltrating cytotoxic T cells and natural killer cells
Imiquimod increases the expression of genes predictive of the activation of the adaptive immune system
In summary, the data suggest that the therapeutic effect of imiquimod in the treatment of AK involves the stimulation of both the innate and adaptive immune responses. The role of type 1 interferons in imiquimod's mechanism of action is underscored by the induction of large numbers of IFNα/β-inducible genes with tumor growth-inhibitory and immune stimulatory activity. Data indicate that the antilesional activity of imiquimod is a result of the induction of a strong immune cell-mediated cytolytic and apoptotic gene expression program that leads to destruction of AK lesions and sun-damaged cells. The development of immune memory indicated by this study, as well as the observation of low recurrence in clinical studies, makes imiquimod a unique therapy for AK as well as for other cutaneous neoplasms.
analysis of variance
basal cell carcinoma
interferon regulatory factor
natural killer [cells]
Reverse Transcriptase Polymerase Chain Reaction
Toll like receptors
We thank Rhonda Dick for helping with manuscript preparation, and Dr. Mark Tomai for valuable criticism of the manuscript. Both are employees of 3M Pharmaceuticals.
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