Optimizing the IMQ model: Deep characterization of the human TLR7 response for early drug development

Optimizing the IMQ model: Deep characterization of the human TLR7 response for early drug development | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Optimizing the IMQ model: Deep characterization of the human TLR7 response for early drug development Juliette Anne van den Noort, Salma Assil, Micha Nathaniel Ronner, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4655296/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Aug, 2024 Read the published version in Inflammation → Version 1 posted 7 You are reading this latest preprint version Abstract Purpose Imiquimod (IMQ; brand name Aldara®) is a registered topical agent that has been proven to induce local inflammation via the Toll-like receptor (TLR)7 pathway. The purpose of this study was to characterize TLR7-mediated inflammation following 7 days (168h) of topical IMQ exposure in healthy volunteers, and to compare the effects of short exposure (48h-72h) with prolonged exposure (120h-168h). Methods IMQ (100mg) was applied under occlusion to 5 different tape-stripped treatment sites on the back of 10 healthy participants for a maximum of 7 consecutive days. Erythema and skin perfusion were measured daily up to 168h. Biopsies for immunohistochemical staining and RNA sequencing were collected at 0h, 48h, 72h, 120h and 168h post IMQ application. Results IMQ triggered an inflammatory response starting at 48h after application, including erythema and perfusion of the skin. At the transcriptomic level, IMQ induced TLR7 signalling, IRF involvement and activation of TNF signalling via NF-κB. Furthermore, an enhanced inflammatory response at the cellular level was observed after prolonged IMQ exposure, with cellular infiltration of dendritic cells, macrophages and T cells which was also corroborated by transcriptomic profiles. No difference was found in the erythema and perfusion response after 168h of IMQ exposure compared to 72h. Conclusion Prolonged IMQ exposure revealed enhanced cellular responses and additional pathways with modulated activity compared to short exposure and can therefore be of interest as a model for investigational compounds targeting innate and adaptive immune responses. TLR7 imiquimod prolonged exposure challenge Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Mouse models are the backbone of the preclinical development of investigational compounds, but in the field of inflammatory diseases, species differences can be pronounced and can hamper the translational step from animals to humans[ 1 , 2 ]. In this context, human innate immune challenge models are a valuable tool to mimic components of the pathophysiology of a disease state in a healthy individual. Innate immune challenge models can thereby unravel underlying human physiological processes and facilitate the evaluation of pharmacological effects of investigational compounds in early clinical development. Imiquimod (IMQ) is primarily an agonist of the Toll-like receptor (TLR)7, an endosomal TLR which recognizes single stranded RNA from viruses. IMQ is marketed as a 5% cream (Aldara®) for the treatment of (pre)malignant and HPV-induced skin lesions because of its antiviral and tumoricidal effects[ 3 ]. The antiviral and tumoricidal effects are attributed to the attraction of TLR7-bearing monocytes, macrophages and plasmacytoid dendritic cells (pDCs), which subsequently produce proinflammatory cytokines and chemokines and attract other immune cells to the application site[ 4 ]. Topical IMQ has been used as a challenge agent to induce psoriasis-like skin inflammation in mice[ 4 – 6 ]. In these studies, mice were exposed to IMQ for 120h to 168h, resulting in a dose-dependent clinical inflammation (i.e. increased ear thickness, erythema and scaling) for the entire duration of the treatment[ 4 , 5 ]. The clinical inflammation was accompanied by a substantial influx of T cells, conventional dendritic cells (DCs) and pDCs, with an essential role for the interleukin (IL) 23/IL-17 axis[ 4 , 6 ]. Additionally, research showed that the murine IMQ response was driven by neutrophil influx and complement factor C3[ 5 ]. IMQ was previously used as a human pharmacological challenge agent in multiple studies, where it was topically applied for 48h or 72h[ 7 , 8 ]. In these studies, IMQ application resulted in a transient, mild to moderate local skin inflammation with a significant increase in skin erythema and perfusion peaking 48h after the first application. Consistent with the working mechanism of IMQ, the response was accompanied by a clearly increased expression of Mx-A, an interferon-driven protein, suggesting engagement of Interferon Regulatory Factor (IRF)7 signalling[ 7 , 8 ]. Interestingly, the cellular and molecular responses after 48h-72h were relatively mild with a moderate influx of monocytes, natural killer (NK) cells and DCs, mild IL-6 production, and no significant deposition of complement. There was almost no involvement of neutrophils in this model after 48h-72h of IMQ application[ 8 ]. The lack of neutrophil involvement is surprising given the molecular signalling of TLR7[ 4 ], and contrasts the preclinical findings in mice, in which neutrophils play a more prominent role. Notably, the duration of the exposure to IMQ in clinical studies has never exceeded 72h, whilst preclinical studies usually span up to 6 days. Characterization of IMQ-induced skin inflammation in healthy participants following extended exposure may therefore elucidate valuable novel aspects of the model and the underlying human immune response. The purpose of this study was to characterize TLR7-mediated inflammation following 7 days (168h) of IMQ exposure in healthy volunteers. We aimed to provide deeper insights into the translational value of the IMQ model for future early-stage clinical studies, particularly for the investigation of the pharmacological activity of innate immune-targeting compounds. Materials and methods This clinical study was a single-centre, open-label, investigator-initiated inflammatory challenge study executed in accordance with the Dutch Act on Medical Research involving Human Subjects (WMO). The study protocol was approved by a Medical Ethics Committee (Stichting Beoordeling Ethiek Biomedisch Onderzoek, Assen, the Netherlands). Written informed consent was obtained from all subjects prior to any study-related procedures. Study design and inclusion We recruited ten healthy male and female volunteers between the ages of 18 and 45 and with Fitzpatrick skin types I-III. Their health status was assessed by means of medical history, physical examination, laboratory tests, and 12-lead electrocardiograms (ECG). Participants were excluded if they had a familial history of psoriasis, pathological skin conditions in the treatment area, prior experience with hypertrophic scarring or keloid, or if they were exposed to IMQ within three months of enrolment. Treatment IMQ was topically applied to five treatment sites on the back for a maximum of seven consecutive days (168h). The back was marked with six squares: one untreated area and five treatment areas (Fig. 1 ). Each treatment area was tape stripped (D-Squame, CuDerm, Dallas, TX) to induce mild skin barrier disruption until a trans-epidermal water loss (AquaFlux, Biox Systems) value of 20g/m 2* h was reached. After tape stripping, a standard daily dose of 5 mg IMQ (100mg Aldara® 5%) was applied under occlusion using a 12mm Finn chamber (Bipharma, Almere, the Netherlands), which was renewed once every 24h. In this article, we define “short exposure” as the application of IMQ for 48h-72h, and “long exposure” as IMQ application in the duration of 120h-168h. Skin assessments To evaluate the inflammatory skin response, subjects underwent sequential assessments prior to IMQ challenge (0h) and at 48h, 72h, 120h and 168h post IMQ challenge, as well as during follow up (14 days after first IMQ application). One treatment site was selected to evaluate endpoints of non-invasive procedures only, throughout the duration of the study (Fig. 1 ). Erythema was assessed by a physician using a 4-point scale ranging from 0 (absent) to 3 (severe). Additionally, erythema and skin perfusion were assessed by means of multispectral imaging analysis (Antera 3D, Miravex, Ireland) and laser speckle contrast imaging (LSCI, PeriCam PSI System, Perimed Jäfälla, Sweden), respectively. All skin assessments were performed under standardized conditions with a room temperature between 20–24 degrees Celsius (°C). Biopsy processing At specified time intervals (Fig. 1 ), 4-millimetre biopsy samples were obtained from the IMQ-treated regions as well as from the untreated region, resulting in a total of 5 biopsies per volunteer. One part of the biopsy was fixed in 4% formaldehyde at 4°C for 24-48h and subsequently transferred to 70% ethanol at room temperature. The other part was rapidly frozen in gelatine capsules containing Tissue Tek OCT medium (Sakura Finetek USA, Inc., Torrance, USA) and stored in liquid nitrogen until immunohistochemistry (IHC) staining at the pathology Laboratory of Erasmus Medical Centre, Rotterdam, the Netherlands. IHC and Direct Immunofluorescence (DIF) IHC staining was performed for the following targets: CD11c (Clone EP157, Bio SB), CD14 (Clone EPR3653, Ventana), CD20 (Clone L26, Ventana), CD1a (Clone EP3622, Cell Marque), CD3 (Clone 2GV6, Ventana), CD4 (Clone SP35, Ventana), CD8 (Clone C8/144B, DAKO), HLA-DR (Clone CR3142, Ventana), MPO (Polyclonal, Ventana), and NF-κB (Clone d14e12, Cell Signaling). Biopsies were scored by a clinical pathologist using a 6-point nominal scale: negative (0), minimal (1), few (2), moderate (3), many (4), excessive (5). DIF was applied for complement C3c (rabbit polyclonal, DAKO, Glostrup, Denmark) and C4d (rabbit polyclonal, Biomedica, Wien, Austria). DIF intensity was scored by a clinical pathologist on a nominal scale of 0–3: none (0), weak (1), moderate (2) and strong (3). RNA isolation, sequencing, and data preprocessing The remaining snap frozen tissue was lysed using RLT lysis buffer with β-mercaptoethanol and extracted using the RNeasy micro plus kit (Qiagen, cat no. 74034). The extracted RNA concentration was assessed using the Molecular Probes Quant iT RNA HS Assay Kit (ThermoFisher Scientific, cat no. Q32852). A set of 45 samples yielded sufficient amounts of RNA for sequencing at Genomescan BV, Leiden. RNA libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit from Illumina (New England BioLabs, Ipswich, MA, USA, cat. no. E7760S/L). Samples were prepared using the NEBNext® Poly(A) mRNA Magnetic Isolation Module. mRNA was isolated using oligo-dT magnetic beads, followed by RNA fragmentation, cDNA synthesis, adapter ligation and PCR amplification of the cDNA library. Bulk RNA sequencing was performed to obtain 40 million reads per sample using the Illumina NovaSeq 6000, yielding 150 bp paired end reads. Raw sequencing reads were processed as follows: adapter trimming and filtering of low-quality bases using fastpv0.23.2, alignment to GRCh38.p13 human reference using STAR2 v2.7.10 and gene level raw count quantification using HTSeq version 2.0.2. RNA sequencing data analyses Data visualization and statistical analyses of the RNA sequencing data were performed using R statistical software (v4.3.1)[ 9 ]. An overview of samples and associated information is provided in Supplemental Table S1 . t-distributed Stochastic Neighbour Embedding (t-SNE) was used as an unsupervised dimensionality reduction approach to visualize the intrinsic structure of the dataset. The algorithm was applied to DESeq2 variance-stabilized (vst) counts of 2000 most variably expressed genes using the Rtsne package (v0.16)[ 10 – 12 ]. Based on transcriptomic profiles, a set of 15 IMQ-treated samples co-clustering with the untreated samples were designated as putative (partial or) non-responders. The molecular response to IMQ was investigated in the transcriptomes of responders, using differential gene expression and subsequent pathway analyses. Differential gene expression analysis was performed using the DESeq2 package (v1.40.2) with subsequent LFC shrinkage using the ‘apeglm’ estimator[ 13 , 14 ]. Gene set enrichment analysis (GSEA) was performed on a curated subset of the Molecular Signatures Database (MSigDB) v2023.2.Hs using the ​ clusterProfiler package (v4.8.2)[ 15 – 18 ]. Databases referenced included: hallmark gene sets, oncogenic signatures Gene Ontology, 3CA, miRNA and transcription factor targets, curated gene sets from Wikipathways, PID, Reactome, Biocarta and KEGG/KEGG Medicus. For the MSigDB hallmark gene set collection[ 18 ], pathway activity was additionally assessed at the single-sample level using Gene Set Variation Analysis (GSVA) applied with the GSVA package (v1.48.3)[ 19 ]. Hierarchically clustered gene expression heatmaps were generated using the pheatmap package (v1.0.12)[ 20 ]. Plots linking core enrichment genes and enriched pathways were drawn using the cnetplot function of the enrichplot package (v1.20.3)[ 21 ]. Gene expression boxplots were produced using the DESeq2 plotCounts function and further modified with ggplot2 (v3.5.0). CIBERSORTx[ 22 ] was used to quantify the abundances of 22 immune cell type populations across all samples in the RNA sequencing dataset. The LM22 signature matrix was used as the reference profile and B-mode batch correction was applied. The Skillings-Mack test was used to assess the differences in absolute scores for each cell type across five sample groups (untreated, IMQ48h, IMQ72h, IMQ120h, IMQ168). The Conover’s all-pairs test was applied with the PMCMRplus package (v1.9.10) as the post hoc test using data from subjects with complete observations. Unless otherwise specified, the statistical significance threshold across all analyses was set at 0.05 and a Benjamini-Hochberg correction was applied to account for multiple testing. Complement analysis in blood samples Blood was collected in a 4 mL Clot activation Tube (CAT) and plasma in a 4 mL K2EDTA collection tube. Concentrations of the complement components C3, C3d, C3d/C3 ratio and the soluble membrane attack complex C5b-9 were measured in plasma at the laboratory of the Department of Nephrology, University Medical Centre Groningen as described earlier[ 23 ]. Statistics All repeatedly measured pharmacodynamic (PD) endpoints were summarised (n, mean, standard deviation (SD)) by area and time. Repeatedly measured continuous PD endpoints were analysed using a mixed model analysis of covariance (ANCOVA) with area (48h IMQ, 72h IMQ, 120h IMQ, 168h IMQ and untreated), hours (0h, 48h,72h,120h and 168h) and area by hours as the fixed factors and subject as the random factor and the covariate baseline measurement (when applicable). A summary table of the analysis results per variable was generated with estimates of the differences between the contrasts and a back transformed estimate of the differences in percentage for log transformed parameters, 95% confidence intervals (in percentages for log-transformed parameters) and Least Square Means (geometric means for log transformed parameters) with corresponding p-values. Results Study population and adverse events Ten healthy volunteers were enrolled, 7 of whom were female and 3 were male. General subject characteristics are provided in Supplemental Table S2. All subjects had Fitzpatrick skin type I-III and a mean age of 25.6 (SD ± 6.7) years. The most frequently reported adverse event was application site pruritus, which was generally transient and disappeared spontaneously after IMQ application was stopped. No serious adverse events were reported. IMQ treatment drives erythema and skin perfusion, but long exposure does not enhance these responses A visual representation of all imaging and biophysical assessments is provided in Fig. 2 a. IMQ application under occlusion for 168h led to a significant increase in erythema (estimated difference (ED): 7.69, 95% CI[5.51, 9.86], p < 0.0001) and blood perfusion (ED: 25.1%, 95% confidence interval (CI)[13.5%, 37.9%], p < 0.0001) compared to untreated (Fig. 2 a-c). Comparison of the response after 168h of IMQ versus 0h revealed a similar significant difference for erythema (11.48, 95% CI[6.75, 16.21], p < 0.0001) and for perfusion (ED: 41.6%, 95% CI[14.2%, 75.6%], p < 0.0018). The erythema and perfusion response peaked at 48h with subsequent decline over time. No significant differences in erythema (ED: 1.07, 95% CI[-3.67, 5.80], p = 0.6567) or perfusion (estimated difference: -12.8%, 95% CI[-29.7%, 8.1%], p = 0.2093) were observed upon long IMQ exposure (168h) compared to short exposure (48h; Fig. 2 b-c). IMQ triggers an inflammatory response at the transcriptomic level Analysis of the RNA sequencing dataset using t-SNE revealed three major clusters predominantly enriched in samples from the untreated group, short exposure time points (48h-72h) and long exposure time points (120h-168h) (Fig. 3 a). Based on transcriptomic profiles, we grouped samples from the short exposure time points (48h-72h) and samples from the long exposure time points (120h-168h) for downstream analysis. The short exposure group (48h-72h) was compared to the long exposure group (120h-168h), and both groups were also compared to the untreated group. Pathway activity for Hallmarks gene sets demonstrated that IMQ application leads to an activation of the following inflammatory pathways: TNF signalling via NF-κB, IFN-α and IFN-γ responses, and complement pathways, which were most prominently activated after long exposure to IMQ (Fig. 3 b, Supplemental Fig.S3). IMQ exposure induces TLR7 signalling and activation of TNF signalling via NF-κB We further investigated the biological processes involved in the different stages of exposure to IMQ using GSEA (Fig. 3 c). Short IMQ exposure led to activation of TLR signalling pathways, interferon-driven responses and TNF signalling via NF-κB (Fig. 3 c). Upon longer IMQ exposure, activation of TLR-induced IRF7 signalling was revealed and TNF signalling via NF-κB became more prominent (Fig. 3 c). Additionally, downstream effects representative of the TLR pathway including induction of type I and type II interferons leading to the activation of the JAK-STAT pathway were identified after long exposure (Fig. 3 c). When comparing long to short IMQ exposure, a positive enrichment of gene sets relating to IFN-α and -γ was found, as well as additional activated pathways including IL-2 signalling (Fig. 3 c). A network plot was generated displaying the linkages between the biological pathways involved in the IMQ response (Fig. 4 a). We next focused on a selection of relevant differentially expressed genes (DEGs) involved in TLR-induced IRF7 signalling, NF-κB signalling and complement activation pathways (Fig. 4 b; full pathways shown in Supplemental Fig. 4). For IRF signalling, a time-dependent increase was found in the expression of transcripts encoding for IRF7, Mx-1 and CXCL10 in IMQ-treated samples compared to untreated samples (Fig. 4 b). RNA sequencing-based NF-κB responses were confirmed by immunohistochemical staining of skin punch biopsies: NF-κB (total) staining was elevated between 120h and 168h post IMQ application, compared to baseline (Supplemental Fig. S5c). Downstream of NF-κB, we found a similar increase in expression for IL-6 and CCL2 (Fig. 4 b). CXCL8 expression was upregulated after IMQ application, but the difference in expression was not significant between long and short exposure. An overview of expression for markers corresponding to the IHC staining is available in Supplemental Fig. S6. IMQ-driven expression of complement genes Transcriptomic analysis at the pathway level showed a positive enrichment of gene sets related to the complement cascade, with stronger enrichment upon prolonged exposure (Fig. 3 c). Complement genes elevated in expression by IMQ application included C1QA, C3 and CFB (Fig. 4 b). Results for the transcripts encoding for complement proteins downstream of C3 were inconsistent, with mixed expression levels between samples (Supplemental Fig. S4b). IHC staining for complement revealed traces of dermal C4d at baseline, but no deposition of C3c or C4d after 168h of exposure to IMQ. Complement proteins showed no systemic elevation of C3, C3d, C3d/C3 or C5b-9 in plasma after 168h of IMQ-exposure (not shown). Increased cellular infiltration after long IMQ exposure Histologically, the general inflammation pattern after IMQ exposure was a lymphohistiocytic perivascular dermatitis with an increasing degree and deeper extension of inflammation over time (Fig. 5 a). In 9/10 individuals the infiltrate showed peri-adnexal (peri-follicular and/or peri-eccrine) involvement and epidermal interface dermatitis (6/10). A rise in general cell infiltration accompanied by a mild increase in acanthosis as well as lymphocytic exocytosis (Supplemental Fig. S5a,b) was evident upon prolonged IMQ exposure (Fig. 5 b). Immunohistochemical staining showed infiltration of monocytes, DCs and macrophages (Fig. 5 b) but no presence of neutrophils (Supplemental Fig. S5e). The cells were present after 48h-72h, with infiltration more elevated after 120h-168h post IMQ application. This pattern was mirrored by the T cell response, which showed a rise in T helper cells and cytotoxic T cells until 168h. (Fig. 5 b). A minimal number of Langerhans cells and B cells was present (Supplemental Fig. S5d,f). The CIBERSORTx algorithm identified a total of 19 out of 22 immmune cell subpopulations (Fig. 6 ). IMQ application generally led to increased absolute scores of several immune cell types compared to the untreated samples. A globally significant difference (using Skillings-Mack test) between groups is observed, among others, for resting NK cells (p = 0.0292), naïve B cells, (p = 0.0051,), M1 Macrophages (p = 0.0003), M2 macrophages (p = 0.0403), activated DCs (p = 0.001,), resting CD4 + memory T cells (p = 0.0003) and activated CD4 + memory T cells (p = 0.0241), and CD8 + T cells (p = 0.0463) depicted in Supplemental Fig. S7. Of these, naïve B cells, M1 macrophages, resting NK cells and both resting and activated CD4 + memory T cells showed significant increase in abundance at 168h vs. 72h after IMQ exposure in the post-hoc analysis. Additionally, a statistically significant reduced abundance was observed in both resting and activated DCs at 168h vs. 72h. M2 macrophages and CD8 + T cells did not show statistically significant changes at individual timepoints. Remarkably, overall low abundance of neutrophils was observed (Supplemental Fig. S7). Discussion This study is the first to elucidate the acute effects of long-term IMQ exposure using a multimodal approach in healthy volunteers. We have shown that compared to short exposure, long exposure to IMQ results in a stronger immunological response as evidenced by additional enriched pathways such as TLR-induced IRF7 signalling, more prominent TNF signalling via NF-κB along with downstream effects such as induction of type I and type II interferons leading to activation of the JAK-STAT pathway. Furthermore, increased complement gene expression was identified upon long exposure to IMQ[ 7 , 22 ]. Although imaging and biophysical measurements showed no significantly enhanced response after long IMQ application compared to short exposure, a strong cellular infiltration boost was observed. At the transcriptomic level, this was demonstrated by an increased abundance of M1 macrophages, resting NK cells, and resting and activated CD4 + memory T cells. While a statistically significant increase was observed in M2 macrophages and CD8 + T cells following IMQ application, no difference was observed between long and short exposure. The increased abundance of naïve B cells and the appearance of T cells is indicative of both innate and adaptive immune responses involvement. The transcriptomic profile partially aligns with the IHC-based cellular infiltration of macrophages, NK cells and CD4 + T cells, demonstrating clear time-dependent effects with increased infiltration after long IMQ exposure. Additionally, IMQ increased the expression of type II interferon-related genes, which aligns with the IHC observation of CD8 + cell influx. These cellular findings are consistent with classical TLR signalling. Activation of TLRs is also known to trigger MyD88, IRAK1 and IRAK4, leading to IRF7 and NF-κB signalling, which is in line with our findings. These pathways result in upregulation of transcription factors for several cytokines including type I interferons, TNF, IL-2, IL-6, IL-8, IL-12, IFN-α and chemokines such as macrophage inflammatory protein (MIP)-1α, MIP-1β and monocyte chemotactic protein-1[ 24 ]. Another challenge agent that we use to effectively induce an in vivo TLR response in men is lipopolysaccharide (LPS)[ 25 – 28 ]. Intradermal injection of LPS triggers an acute inflammatory response via TLR4, leading to increased innate immune cell populations including neutrophils, monocytes and dendritic cells. Furthermore, LPS elicits an adaptive immune response, as evidenced by the presence of B and T cells. Elevated levels of IL-6, IL-8, IL-1β and TNF following LPS injection indicate NF-κB involvement. The current study showed moderate activation of NF-κB signalling after long IMQ application, supported by upregulated expression of NF-κB1, NF-κB2, IL-6, CXCL8, CCL2, IL-17C and IL-23A at the transcriptomic level. This contradicts previous studies, as no significant IL-6 and IL-8 responses were observed 72 hours after IMQ application, suggesting only mild NF-κB involvement. However, the release of Mx-A (a downstream marker indicative of IFN-α activation through IRF7) was evident[ 7 , 8 ]. Our current data reinforces this finding, as it suggests both My-D88 transcript expression and downstream IRF7 and Mx-1 activation, which increases upon prolonged IMQ application. The activation of interferons leads to the engagement of their respective receptors, which in turn triggers the JAK - STAT pathway culminating in the release of proinflammatory cytokines[ 29 , 30 ]. The JAK - STAT pathway was more enriched, with chemokines such as CXCL9, CXCL10 and CXCL11 significantly overexpressed after prolonged IMQ exposure. Recently, Chen et al . provided an overview of studies examining DNA and RNA specific profiles in cutaneous lupus erythematosus (CLE) patients, which indicates an upregulation of innate immune response functions including JAK – STAT signalling, TLR signalling, and pattern recognition receptors. Furthermore, there was a notable increase in the expression of type 1 interferons, along with an upregulated expression of chemokines CXCL9, CXCL10, and CXCL11, which are recognized as characteristic indicators for CLE[ 31 – 34 ]. Although a direct comparison of our data with the existing RNA datasets of CLE patients was not conducted, analysis of pathway activity and overexpressed genes detected upon prolonged IMQ application allows us to conclude that our current model aligns more closely with CLE characteristics than with psoriasiform lesions[ 7 ]. In addition, the histopathological changes of a vacuolar interface dermatitis with adnexal involvement were also reminiscent of CLE. These observations differ from the prevalent use of the model in preclinical studies, where the murine IMQ model is typically used to investigate psoriasis-like conditions[ 35 ]. In contrast to our cellular observations, imaging and biophysical measurements showed no significantly enhanced response after long IMQ application compared to short exposure. This may be because Aldara (besides its role as a TLR7 agonist) may also act as an exogenous mediator by enhancing transient receptor potential vanilloid 1 channel activity on the primary afferent sensory neuron[ 36 ]. Activation of this channel leads to the release of bioactive substances such as nitric oxide (NO). NO can then interact with target cells in the surrounding tissue, including vascular smooth muscle cells. The interaction of NO with smooth muscle cells leads to vasodilation, resulting in increased blood perfusion and erythema[40]. Our observations suggest that the vascular response is independent of the inflammatory process. The role of bioactive, vasodilating substances in the IMQ-induced erythema and perfusion response remains to be further elucidated. Our second objective was to explore the translational value of the IMQ model. In contrast to the mouse data, IHC staining did not show involvement of complement in the human IMQ response. We hypothesize that this may be due to the difference in severity of the hit, as in mice, the entire surface area of the back is challenged, whereas in humans IMQ is applied to a much smaller relative surface area. Therefore, it is currently unknown whether the observed differences are a result of the magnitude of TLR activation or can be contributed to species differences[ 1 , 2 ]. However, at the transcriptome level, classical, and alternative pathway genes were enriched, particularly after prolonged exposure to IMQ, suggesting complement involvement in the human IMQ response. It is unclear how these sequencing results translate to the protein level, or if the transient nature of complement explains the lack of IMQ-driven complement responses in IHC analysis. The same holds true for the observed lack of neutrophils and CXCL8 expression, which emphasizes that the role of neutrophils in the human IMQ response needs to be elucidated further[ 6 ]. The observed differences between preclinical animal models and the human response may partially be explained by species differences in TLR7 expression[ 37 ]. For instance, Bhagchandani et al . described that the expression of TLR7 on neutrophils is higher in mouse than in men[ 37 , 38 ]. Moreover, expression patterns of TLR7 within a given cell type may differ across tissues and across activation status of the cell[ 39 ], further complicating the translational interpretation both across and within species. These findings highlight the complementary value of human challenge models in the development of immune-targeting compound development. In conclusion, our study provides a comprehensive characterization of the cutaneous response to both short and prolonged IMQ exposure in healthy volunteers by using a multimodal approach. We have demonstrated that prolonging the IMQ exposure has added value by enhancing cellular responses and increasing abundance of specific immune cell types along with stronger activation of a diverse set of pathways, particularly those driven by IRF and related to complement. We also argue that prolonged IMQ application results in a CLE-like cutaneous inflammation, both at the transcriptomic level and from a histopathological perspective. Our results suggest that biophysical and vascular responses are not exclusively driven by cutaneous inflammation. The described discrepancies between preclinical and clinical results, most notably the neutrophil response, illustrate the complementary value of human challenge models in the development of compounds targeting the immune system. This in vivo immune challenge model is of value for future early clinical evaluation of topically or systemically applied anti-inflammatory or immunomodulatory compounds, particularly compounds targeting IRF and JAK-STAT signalling. Declarations Acknowledgements: This study was funded by the Centre for Human Drug Research, Leiden. The authors thank all the healthy volunteers who participated in this clinical research. They also extend their thanks to the operational staff members involved in the setup, clinical conduct, and data analysis of the study. Authorship Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Juliette van den Noort, Salma Assil, Micha Ronner, Michelle Osse, Jeffrey Damman and Ingrid Tomljanovic. The first draft of the manuscript was written by Juliette van den Noort and Salma Assil and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Disclosure of Conflicts of Interest: The authors have no relevant financial or non-financial interests to disclose. Statements and declarations: The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Data availability: The datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request. Ethics approval: This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Independent Ethics Committee (Medisch Ethische ToetsingsCommise) of the Foundation ‘Evaluation of Ethics in Biomedical Research’ (Stichting Beoordeling Ethiek Biomedisch Onderzoek), Assen, The Netherlands (Date 15DEC2021/NL79321.056.21). Sources of funding: Centre for Human Drug Research, Leiden Conflict of interest: The authors state no conflict of interest. References Junhee Seok, H. Shaw Warren, Alex GC, et al (2013) Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A 110:3507–3512 Mestas J, Hughes CCW (2004) Of Mice and Not Men: Differences between Mouse and Human Immunology. The Journal of Immunology 172:2731–2738 Hanna E, Abadi R, Abbas O (2016) Imiquimod in dermatology: an overview. Int J Dermatol 55:831–844 van der Fits L, Mourits S, Voerman JSA, et al (2009) Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice Is Mediated via the IL-23/IL-17 Axis. 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Br J Clin Pharmacol 88:680–690 Dillingh MR, Van Poelgeest EP, Malone KE, Kemper EM, Stroes ESG, Moerland M, Burggraaf J (2014) Characterization of inflammation and immune cell modulation induced by low-dose LPS administration to healthy volunteers. Journal of Inflammation (United Kingdom) 11:1–9 van Poelgeest EP, Dillingh MR, de Kam M, Malone KE, Kemper M, Stroes ESG, Burggraaf J, Moerland M (2018) Characterization of immune cell, endothelial, and renal responses upon experimental human endotoxemia. J Pharmacol Toxicol Methods 89:39–46 Majoros A, Platanitis E, Kernbauer-Hölzl E, Rosebrock F, Müller M, Decker T (2017) Canonical and non-canonical aspects of JAK-STAT signaling: Lessons from interferons for cytokine responses. Front Immunol. https://doi.org/10.3389/fimmu.2017.00029 Mahjoor M, Mahmoudvand G, Farokhi S, Shadab A, Kashfi M, Afkhami H (2023) Double-edged sword of JAK/STAT signaling pathway in viral infections: novel insights into virotherapy. Cell Communication and Signaling 21:1–17 Nickles MA, Huang K, Chang YS, Tsoukas MM, Sweiss NJ, Perkins DL, Finn PW (2020) Gene Co-expression Networks Identifies Common Hub Genes Between Cutaneous Sarcoidosis and Discoid Lupus Erythematosus. Front Med (Lausanne) 7:1–8 Solé C, Gimenez-Barcons M, Ferrer B, Ordi-Ros J, Cortés-Hernández J (2016) Microarray study reveals a transforming growth factor-β-dependent mechanism of fibrosis in discoid lupus erythematosus. British Journal of Dermatology 175:302–313 Blomberg S, Rönnblom L, Eloranta ML, Cederblad B, Alm G V., Nordlind K, Nordlind K (2001) Presence of cutaneous interferon-α producing cells in patients with systemic lupus erythematosus. Lupus 10:484–490 Wongpiyabovorn J, Ruchusatsawat K, Onganantapong Y, Sintupak W, Hirankarn N (2011) Interferon Alpha mRNA level and subtypes in lesion and non-lesion from discoid lupus erythematosus patients without systemic lupus erythematosus. Asian Biomedicine 5:643–647 Flutter B, Nestle FO (2013) TLRs to cytokines: Mechanistic insights from the imiquimod mouse model of psoriasis. Eur J Immunol 43:3138–3146 Kittaka H, Tominaga M (2017) The molecular and cellular mechanisms of itch and the involvement of TRP channels in the peripheral sensory nervous system and skin. Allergology International 66:22–30 Bhagchandani S, Johnson JA, Irvine DJ (2021) Evolution of Toll-like receptor 7/8 agonist therapeutics and their delivery approaches: From antiviral formulations to vaccine adjuvants. Adv Drug Deliv Rev. https://doi.org/10.1016/j.addr.2021.05.013 Hayashi F, Means TK, Luster AD (2003) Toll-like receptors stimulate human neutrophil function. Blood 102:2660–2669 Trinchieri G, Sher A (2007) Cooperation of Toll-like receptor signals in innate immune defence. Nat Rev Immunol 7:179–190 Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 26 Aug, 2024 Read the published version in Inflammation → Version 1 posted Editorial decision: Revision requested 29 Jul, 2024 Reviews received at journal 29 Jul, 2024 Reviewers agreed at journal 08 Jul, 2024 Reviewers invited by journal 03 Jul, 2024 Editor assigned by journal 01 Jul, 2024 Submission checks completed at journal 01 Jul, 2024 First submitted to journal 28 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4655296","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328903375,"identity":"1d4f5e5e-5e30-4585-9aa2-6c8c2e802def","order_by":0,"name":"Juliette Anne van den Noort","email":"","orcid":"","institution":"Centre for Human Drug Research","correspondingAuthor":false,"prefix":"","firstName":"Juliette","middleName":"Anne van den","lastName":"Noort","suffix":""},{"id":328903376,"identity":"76166671-7198-4996-8660-c060bdd6b675","order_by":1,"name":"Salma Assil","email":"","orcid":"","institution":"Centre for Human Drug Research","correspondingAuthor":false,"prefix":"","firstName":"Salma","middleName":"","lastName":"Assil","suffix":""},{"id":328903377,"identity":"9cc7c443-cd4d-4143-a182-8aeaae766779","order_by":2,"name":"Micha Nathaniel Ronner","email":"","orcid":"","institution":"Centre for Human Drug Research","correspondingAuthor":false,"prefix":"","firstName":"Micha","middleName":"Nathaniel","lastName":"Ronner","suffix":""},{"id":328903378,"identity":"45f781c0-3e47-4116-9f5b-44fdef114283","order_by":3,"name":"Michelle Osse","email":"","orcid":"","institution":"Centre for Human Drug Research","correspondingAuthor":false,"prefix":"","firstName":"Michelle","middleName":"","lastName":"Osse","suffix":""},{"id":328903379,"identity":"d5278b52-b52f-4eea-9655-42412b7eb898","order_by":4,"name":"Iris Pot","email":"","orcid":"","institution":"Leiden Academic Centre for Drug 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Centre","correspondingAuthor":false,"prefix":"","firstName":"Ingrid","middleName":"","lastName":"Tomljanovic","suffix":""},{"id":328903386,"identity":"570ca3c1-78c8-403d-8b63-0873f744ebd7","order_by":11,"name":"Manon Aleida Adriana Jansen","email":"","orcid":"","institution":"Centre for Human Drug Research","correspondingAuthor":false,"prefix":"","firstName":"Manon","middleName":"Aleida Adriana","lastName":"Jansen","suffix":""},{"id":328903387,"identity":"48a72d0f-402d-412b-9e06-5bcbf837b271","order_by":12,"name":"Matthijs Moerland","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtUlEQVRIiWNgGAWjYNCCAiA+3sDGwAOk+YjTYgDEZw5AtLARr+VGApFadKcdPvi4wsAuj+/m42cP3u5hyCOoxex2WrLhGYPkYsnbaeaGc54xFBOhJcdMssGAOXHD7Rw2aZ4DDIltRGgx/9lgUJ+44eYZ4rWYMTYYHE7ccIOHaC1pyUCHHU+ceSbNTHLOAQlitCQf/NhQUZ3Yd/zwM4k3B2wS+wlpQQcSpGoYBaNgFIyCUYANAAA/VUAyEqd32gAAAABJRU5ErkJggg==","orcid":"","institution":"Centre for Human Drug Research","correspondingAuthor":true,"prefix":"","firstName":"Matthijs","middleName":"","lastName":"Moerland","suffix":""}],"badges":[],"createdAt":"2024-06-28 14:06:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4655296/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4655296/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10753-024-02127-x","type":"published","date":"2024-08-26T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60943891,"identity":"60d90e00-7f6a-4ef2-b9f8-4798c8291fcd","added_by":"auto","created_at":"2024-07-23 22:04:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1806858,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of the study design\u003c/strong\u003e. IMQ was topically applied to five treatment sites on the back of healthy participants for a maximum of seven consecutive days (168h). At specified time intervals (48h, 72h, 120h and 168h), 4-millimetre biopsy samples were obtained from the IMQ-treated regions as well as from the untreated region, resulting in a total of 5 biopsies per volunteer. Endpoints included non-invasive imaging, immunohistochemical staining and RNA sequencing of biopsy material. Abbreviations: cDNA= complement DNA, hrs = hours, IMQ = imiquimod. \u003cem\u003eCreated with BioRender.com\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/8207e534cc194a2623b1e604.png"},{"id":60944600,"identity":"1e2bbb6f-357a-4ba4-acb2-a2ee17957c3c","added_by":"auto","created_at":"2024-07-23 22:12:47","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4531395,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClinical impression and quantification of inflammatory skin response by multispectral imaging and LSCI\u003c/strong\u003e. \u003cstrong\u003ea) \u003c/strong\u003eOverview of 2D imaging, LSCI and multispectral imaging.\u003cstrong\u003e b)\u003c/strong\u003e Erythema measured by multispectral camera, illustrated as CFB. No difference observed between short (48h) and long (168h) IMQ exposure, estimated difference:1.07, 95% CI [-3.67, 5.80], p=0.6567. \u003cstrong\u003ec)\u003c/strong\u003eSkin perfusion by LSCI, illustrated as CFB. No difference observed between short (48h) and long (168h) IMQ exposure, estimated difference: -12.8%, 95% CI [-29.7%, 8.1%], p= 0.2093. Abbreviations: CFB= change from baseline, CI= confidence interval, IMQ = imiquimod, LSCI= laser speckle contrast imaging, LSMeans= Least Squares Mean.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/85c3fcf603ddcd14b66cac15.png"},{"id":60943892,"identity":"f099062d-13f8-428e-ac1a-22861dc8a3f7","added_by":"auto","created_at":"2024-07-23 22:04:47","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1912540,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e t-SNE plot visualizing the clusters present in the full dataset (n=45, top) and a reduced subset of the dataset (n=30) excluding the treated samples which showed similarity to the untreated group (putative non-responders based on transcriptomic profiles). Top figure represents the full dataset (n=45), while the bottom plot excludes the samples (n=15) that show similarity to untreated group based on their transcriptomic profiles. \u003cstrong\u003eb\u003c/strong\u003e Heatmap of GSVA single-sample pathway enrichment scores on 11 representative MSigDB Hallmark gene sets (n=30). IMQ application leads to activation of pathways involved in inflammatory and immune responses, including TNF signalling via NF-κB, IFN-α and IFN-γ responses, and complement pathways. \u003cstrong\u003ec) \u003c/strong\u003eBarplots displaying GSEA normalized enrichment scores of 35 representative pathways upregulated upon IMQ exposure for the following comparisons: short exposure versus untreated (left), long exposure versus short exposure (middle) and long exposure versus untreated (right), based on the selected dataset (n=30). Abbreviations: GSEA= gene set enrichment analysis, GSVA= gene set variation analysis, IFN= interferon, IMQ= imiquimod, NES= normalized enrichment score, NF-κB= nuclear factor kappa-light-chain-enhancer of activated B cells, TNF= tumour necrosis factor, t-SNE= t-distributed Stochastic Neighbour Embedding.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/233433a043404c8f1c134b1c.png"},{"id":60945262,"identity":"28527cd1-a5ce-41b9-b7ca-4fba25fc042d","added_by":"auto","created_at":"2024-07-23 22:20:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2321059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea) \u003c/strong\u003eNetwork plot displaying the key biological pathways involved in the IMQ response and their associated core enrichments genes. Colours correspond to each biological pathway including complement, TNF-signalling via NF-κB, TLR7/9/IRF7sSignalling, Type I interferon to JAK-STAT signalling and TLR signalling. \u003cstrong\u003eb) \u003c/strong\u003eBoxplots of DESeq2 normalized counts for 9 selected DEGs involved in the TLR-induced IRF7 signalling (top row), NF-κB signalling (middle row) and complement activation pathways (bottom row).\u003c/p\u003e","description":"","filename":"Fig4..png","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/9efa083cc9af47e82ebfd87c.png"},{"id":60943896,"identity":"8b6312d7-e52f-45a4-86c0-da8d1f24229d","added_by":"auto","created_at":"2024-07-23 22:04:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3292224,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced inflammatory responses at histological and cellular levels observed upon prolonged IMQ exposure\u003c/strong\u003e. \u003cstrong\u003ea)\u003c/strong\u003e histological sections. Section \u003cem\u003eI)\u003c/em\u003e Untreated skin, shows healthy skin without any signs of inflammation (zoom factor 5). \u003cem\u003eII) \u003c/em\u003eSection of 72h post IMQ application showing an abundant lymphocytic exocytosis in the epidermis and superficial lymphohistiocytic perivascular infiltrate (zoom factor 5). Inset I shows exocytosis of lymphocytes in a sweat gland (zoom factor 5). Inset II shows lymphocytic exocytosis in the epidermis accompanied by an apoptotic/necrotic keratinocyte (arrow, zoom factor 40). \u003cem\u003eIII)\u003c/em\u003e Section of 120h of IMQ application showing profound perifollicular inflammation and influx of lymphocytes in the hair follicle epithelium (zoom factor 20). \u003cem\u003eIV)\u003c/em\u003e Section of 168h of IMQ application resulting in cuffing/lymphocytic vasculopathy of the infiltrate surrounding the deep vascular plexus at the levels of the sweat glands (zoom factor 20). \u003cstrong\u003eb)\u003c/strong\u003e General\u003cstrong\u003e \u003c/strong\u003einfiltration and IMQ-driven immune cells (macrophages, monocytes, T helper cells, dendritic cells and cytotoxic T cells) measured by IHC in skin punch biopsies. Abbreviations: IHC= immunohistochemistry, IMQ= imiquimod.\u003c/p\u003e","description":"","filename":"Fig5..png","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/4479bcdf088874fbf7dde8cd.png"},{"id":60944602,"identity":"14855125-2338-4780-b099-a74934e544b6","added_by":"auto","created_at":"2024-07-23 22:12:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":215213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverview of immune cell type infiltration based on CIBERSORTx. \u003c/strong\u003eStacked barplot displaying the absolute scores for 19 cell types detected by CIBERSORTx across all samples (n=45). \u0026nbsp;An asterisk (*) refers to putative molecular non-responders based on transcriptomic profile similarity to untreated samples. Abbreviations: IMQ= imiquimod, NK= natural killer.\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/f3831575549cd3a448fd3709.png"},{"id":63821375,"identity":"6450e66e-6197-49eb-911e-cffd668dbdb4","added_by":"auto","created_at":"2024-09-02 16:13:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17109942,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/1b37ca58-cc5f-4828-a821-7c5fe1a27604.pdf"},{"id":60944599,"identity":"59272daf-1e49-418f-b133-45e94032c5f4","added_by":"auto","created_at":"2024-07-23 22:12:47","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2607066,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-4655296/v1/48cc0326e7769967947b5cdb.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimizing the IMQ model: Deep characterization of the human TLR7 response for early drug development","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMouse models are the backbone of the preclinical development of investigational compounds, but in the field of inflammatory diseases, species differences can be pronounced and can hamper the translational step from animals to humans[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this context, human innate immune challenge models are a valuable tool to mimic components of the pathophysiology of a disease state in a healthy individual. Innate immune challenge models can thereby unravel underlying human physiological processes and facilitate the evaluation of pharmacological effects of investigational compounds in early clinical development.\u003c/p\u003e \u003cp\u003eImiquimod (IMQ) is primarily an agonist of the Toll-like receptor (TLR)7, an endosomal TLR which recognizes single stranded RNA from viruses. IMQ is marketed as a 5% cream (Aldara\u0026reg;) for the treatment of (pre)malignant and HPV-induced skin lesions because of its antiviral and tumoricidal effects[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The antiviral and tumoricidal effects are attributed to the attraction of TLR7-bearing monocytes, macrophages and plasmacytoid dendritic cells (pDCs), which subsequently produce proinflammatory cytokines and chemokines and attract other immune cells to the application site[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Topical IMQ has been used as a challenge agent to induce psoriasis-like skin inflammation in mice[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In these studies, mice were exposed to IMQ for 120h to 168h, resulting in a dose-dependent clinical inflammation (i.e. increased ear thickness, erythema and scaling) for the entire duration of the treatment[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The clinical inflammation was accompanied by a substantial influx of T cells, conventional dendritic cells (DCs) and pDCs, with an essential role for the interleukin (IL) 23/IL-17 axis[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, research showed that the murine IMQ response was driven by neutrophil influx and complement factor C3[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIMQ was previously used as a human pharmacological challenge agent in multiple studies, where it was topically applied for 48h or 72h[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In these studies, IMQ application resulted in a transient, mild to moderate local skin inflammation with a significant increase in skin erythema and perfusion peaking 48h after the first application. Consistent with the working mechanism of IMQ, the response was accompanied by a clearly increased expression of Mx-A, an interferon-driven protein, suggesting engagement of Interferon Regulatory Factor (IRF)7 signalling[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Interestingly, the cellular and molecular responses after 48h-72h were relatively mild with a moderate influx of monocytes, natural killer (NK) cells and DCs, mild IL-6 production, and no significant deposition of complement. There was almost no involvement of neutrophils in this model after 48h-72h of IMQ application[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The lack of neutrophil involvement is surprising given the molecular signalling of TLR7[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and contrasts the preclinical findings in mice, in which neutrophils play a more prominent role. Notably, the duration of the exposure to IMQ in clinical studies has never exceeded 72h, whilst preclinical studies usually span up to 6 days. Characterization of IMQ-induced skin inflammation in healthy participants following extended exposure may therefore elucidate valuable novel aspects of the model and the underlying human immune response.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to characterize TLR7-mediated inflammation following 7 days (168h) of IMQ exposure in healthy volunteers. We aimed to provide deeper insights into the translational value of the IMQ model for future early-stage clinical studies, particularly for the investigation of the pharmacological activity of innate immune-targeting compounds.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e This clinical study was a single-centre, open-label, investigator-initiated inflammatory challenge study executed in accordance with the Dutch Act on Medical Research involving Human Subjects (WMO). The study protocol was approved by a Medical Ethics Committee (Stichting Beoordeling Ethiek Biomedisch Onderzoek, Assen, the Netherlands). Written informed consent was obtained from all subjects prior to any study-related procedures.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy design and inclusion\u003c/h2\u003e \u003cp\u003eWe recruited ten healthy male and female volunteers between the ages of 18 and 45 and with Fitzpatrick skin types I-III. Their health status was assessed by means of medical history, physical examination, laboratory tests, and 12-lead electrocardiograms (ECG). Participants were excluded if they had a familial history of psoriasis, pathological skin conditions in the treatment area, prior experience with hypertrophic scarring or keloid, or if they were exposed to IMQ within three months of enrolment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTreatment\u003c/h2\u003e \u003cp\u003eIMQ was topically applied to five treatment sites on the back for a maximum of seven consecutive days (168h). The back was marked with six squares: one untreated area and five treatment areas (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Each treatment area was tape stripped (D-Squame, CuDerm, Dallas, TX) to induce mild skin barrier disruption until a trans-epidermal water loss (AquaFlux, Biox Systems) value of 20g/m\u003csup\u003e2*\u003c/sup\u003eh was reached. After tape stripping, a standard daily dose of 5 mg IMQ (100mg Aldara\u0026reg; 5%) was applied under occlusion using a 12mm Finn chamber (Bipharma, Almere, the Netherlands), which was renewed once every 24h. In this article, we define \u0026ldquo;short exposure\u0026rdquo; as the application of IMQ for 48h-72h, and \u0026ldquo;long exposure\u0026rdquo; as IMQ application in the duration of 120h-168h.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSkin assessments\u003c/h2\u003e \u003cp\u003eTo evaluate the inflammatory skin response, subjects underwent sequential assessments prior to IMQ challenge (0h) and at 48h, 72h, 120h and 168h post IMQ challenge, as well as during follow up (14 days after first IMQ application). One treatment site was selected to evaluate endpoints of non-invasive procedures only, throughout the duration of the study (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Erythema was assessed by a physician using a 4-point scale ranging from 0 (absent) to 3 (severe). Additionally, erythema and skin perfusion were assessed by means of multispectral imaging analysis (Antera 3D, Miravex, Ireland) and laser speckle contrast imaging (LSCI, PeriCam PSI System, Perimed J\u0026auml;f\u0026auml;lla, Sweden), respectively. All skin assessments were performed under standardized conditions with a room temperature between 20\u0026ndash;24 degrees Celsius (\u0026deg;C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eBiopsy processing\u003c/h2\u003e \u003cp\u003eAt specified time intervals (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), 4-millimetre biopsy samples were obtained from the IMQ-treated regions as well as from the untreated region, resulting in a total of 5 biopsies per volunteer. One part of the biopsy was fixed in 4% formaldehyde at 4\u0026deg;C for 24-48h and subsequently transferred to 70% ethanol at room temperature. The other part was rapidly frozen in gelatine capsules containing Tissue Tek OCT medium (Sakura Finetek USA, Inc., Torrance, USA) and stored in liquid nitrogen until immunohistochemistry (IHC) staining at the pathology Laboratory of Erasmus Medical Centre, Rotterdam, the Netherlands.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eIHC and Direct Immunofluorescence (DIF)\u003c/h2\u003e \u003cp\u003eIHC staining was performed for the following targets: CD11c (Clone EP157, Bio SB), CD14 (Clone EPR3653, Ventana), CD20 (Clone L26, Ventana), CD1a (Clone EP3622, Cell Marque), CD3 (Clone 2GV6, Ventana), CD4 (Clone SP35, Ventana), CD8 (Clone C8/144B, DAKO), HLA-DR (Clone CR3142, Ventana), MPO (Polyclonal, Ventana), and NF-κB (Clone d14e12, Cell Signaling). Biopsies were scored by a clinical pathologist using a 6-point nominal scale: negative (0), minimal (1), few (2), moderate (3), many (4), excessive (5). DIF was applied for complement C3c (rabbit polyclonal, DAKO, Glostrup, Denmark) and C4d (rabbit polyclonal, Biomedica, Wien, Austria). DIF intensity was scored by a clinical pathologist on a nominal scale of 0\u0026ndash;3: none (0), weak (1), moderate (2) and strong (3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRNA isolation, sequencing, and data preprocessing\u003c/h2\u003e \u003cp\u003eThe remaining snap frozen tissue was lysed using RLT lysis buffer with β-mercaptoethanol and extracted using the RNeasy micro plus kit (Qiagen, cat no. 74034). The extracted RNA concentration was assessed using the Molecular Probes Quant iT RNA HS Assay Kit (ThermoFisher Scientific, cat no. Q32852). A set of 45 samples yielded sufficient amounts of RNA for sequencing at Genomescan BV, Leiden. RNA libraries were constructed using the NEBNext Ultra II Directional RNA Library Prep Kit from Illumina (New England BioLabs, Ipswich, MA, USA, cat. no. E7760S/L). Samples were prepared using the NEBNext\u0026reg; Poly(A) mRNA Magnetic Isolation Module. mRNA was isolated using oligo-dT magnetic beads, followed by RNA fragmentation, cDNA synthesis, adapter ligation and PCR amplification of the cDNA library. Bulk RNA sequencing was performed to obtain 40\u0026nbsp;million reads per sample using the Illumina NovaSeq 6000, yielding 150 bp paired end reads. Raw sequencing reads were processed as follows: adapter trimming and filtering of low-quality bases using fastpv0.23.2, alignment to GRCh38.p13 human reference using STAR2 v2.7.10 and gene level raw count quantification using HTSeq version 2.0.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing data analyses\u003c/h2\u003e \u003cp\u003eData visualization and statistical analyses of the RNA sequencing data were performed using R statistical software (v4.3.1)[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. An overview of samples and associated information is provided in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. t-distributed Stochastic Neighbour Embedding (t-SNE) was used as an unsupervised dimensionality reduction approach to visualize the intrinsic structure of the dataset. The algorithm was applied to \u003cem\u003eDESeq2\u003c/em\u003e variance-stabilized (vst) counts of 2000 most variably expressed genes using the \u003cem\u003eRtsne\u003c/em\u003e package (v0.16)[\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Based on transcriptomic profiles, a set of 15 IMQ-treated samples co-clustering with the untreated samples were designated as putative (partial or) non-responders. The molecular response to IMQ was investigated in the transcriptomes of responders, using differential gene expression and subsequent pathway analyses. Differential gene expression analysis was performed using the \u003cem\u003eDESeq2\u003c/em\u003e package (v1.40.2) with subsequent LFC shrinkage using the \u0026lsquo;apeglm\u0026rsquo; estimator[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Gene set enrichment analysis (GSEA) was performed on a curated subset of the Molecular Signatures Database (MSigDB) v2023.2.Hs using the ​\u003cem\u003eclusterProfiler\u003c/em\u003e package (v4.8.2)[\u003cspan additionalcitationids=\"CR16 CR17\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Databases referenced included: hallmark gene sets, oncogenic signatures Gene Ontology, 3CA, miRNA and transcription factor targets, curated gene sets from Wikipathways, PID, Reactome, Biocarta and KEGG/KEGG Medicus. For the MSigDB hallmark gene set collection[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], pathway activity was additionally assessed at the single-sample level using Gene Set Variation Analysis (GSVA) applied with the \u003cem\u003eGSVA\u003c/em\u003e package (v1.48.3)[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Hierarchically clustered gene expression heatmaps were generated using the \u003cem\u003epheatmap\u003c/em\u003e package (v1.0.12)[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Plots linking core enrichment genes and enriched pathways were drawn using the cnetplot function of the \u003cem\u003eenrichplot\u003c/em\u003e package (v1.20.3)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Gene expression boxplots were produced using the DESeq2 plotCounts function and further modified with \u003cem\u003eggplot2\u003c/em\u003e (v3.5.0). CIBERSORTx[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] was used to quantify the abundances of 22 immune cell type populations across all samples in the RNA sequencing dataset. The LM22 signature matrix was used as the reference profile and B-mode batch correction was applied. The Skillings-Mack test was used to assess the differences in absolute scores for each cell type across five sample groups (untreated, IMQ48h, IMQ72h, IMQ120h, IMQ168). The Conover\u0026rsquo;s all-pairs test was applied with the PMCMRplus package (v1.9.10) as the post hoc test using data from subjects with complete observations. Unless otherwise specified, the statistical significance threshold across all analyses was set at 0.05 and a Benjamini-Hochberg correction was applied to account for multiple testing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eComplement analysis in blood samples\u003c/h2\u003e \u003cp\u003eBlood was collected in a 4 mL Clot activation Tube (CAT) and plasma in a 4 mL K2EDTA collection tube. Concentrations of the complement components C3, C3d, C3d/C3 ratio and the soluble membrane attack complex C5b-9 were measured in plasma at the laboratory of the Department of Nephrology, University Medical Centre Groningen as described earlier[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll repeatedly measured pharmacodynamic (PD) endpoints were summarised (n, mean, standard deviation (SD)) by area and time. Repeatedly measured continuous PD endpoints were analysed using a mixed model analysis of covariance (ANCOVA) with area (48h IMQ, 72h IMQ, 120h IMQ, 168h IMQ and untreated), hours (0h, 48h,72h,120h and 168h) and area by hours as the fixed factors and subject as the random factor and the covariate baseline measurement (when applicable). A summary table of the analysis results per variable was generated with estimates of the differences between the contrasts and a back transformed estimate of the differences in percentage for log transformed parameters, 95% confidence intervals (in percentages for log-transformed parameters) and Least Square Means (geometric means for log transformed parameters) with corresponding p-values.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStudy population and adverse events\u003c/h2\u003e \u003cp\u003eTen healthy volunteers were enrolled, 7 of whom were female and 3 were male. General subject characteristics are provided in Supplemental Table S2. All subjects had Fitzpatrick skin type I-III and a mean age of 25.6 (SD\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7) years. The most frequently reported adverse event was application site pruritus, which was generally transient and disappeared spontaneously after IMQ application was stopped. No serious adverse events were reported.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eIMQ treatment drives erythema and skin perfusion, but long exposure does not enhance these responses\u003c/h2\u003e \u003cp\u003eA visual representation of all imaging and biophysical assessments is provided in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. IMQ application under occlusion for 168h led to a significant increase in erythema (estimated difference (ED): 7.69, 95% CI[5.51, 9.86], p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and blood perfusion (ED: 25.1%, 95% confidence interval (CI)[13.5%, 37.9%], p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to untreated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c). Comparison of the response after 168h of IMQ versus 0h revealed a similar significant difference for erythema (11.48, 95% CI[6.75, 16.21], p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and for perfusion (ED: 41.6%, 95% CI[14.2%, 75.6%], p\u0026thinsp;\u0026lt;\u0026thinsp;0.0018). The erythema and perfusion response peaked at 48h with subsequent decline over time. No significant differences in erythema (ED: 1.07, 95% CI[-3.67, 5.80], p\u0026thinsp;=\u0026thinsp;0.6567) or perfusion (estimated difference: -12.8%, 95% CI[-29.7%, 8.1%], p\u0026thinsp;=\u0026thinsp;0.2093) were observed upon long IMQ exposure (168h) compared to short exposure (48h; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eIMQ triggers an inflammatory response at the transcriptomic level\u003c/h2\u003e \u003cp\u003eAnalysis of the RNA sequencing dataset using t-SNE revealed three major clusters predominantly enriched in samples from the untreated group, short exposure time points (48h-72h) and long exposure time points (120h-168h) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Based on transcriptomic profiles, we grouped samples from the short exposure time points (48h-72h) and samples from the long exposure time points (120h-168h) for downstream analysis. The short exposure group (48h-72h) was compared to the long exposure group (120h-168h), and both groups were also compared to the untreated group. Pathway activity for Hallmarks gene sets demonstrated that IMQ application leads to an activation of the following inflammatory pathways: TNF signalling via NF-κB, IFN-α and IFN-γ responses, and complement pathways, which were most prominently activated after long exposure to IMQ (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, Supplemental Fig.S3).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eIMQ exposure induces TLR7 signalling and activation of TNF signalling via NF-κB\u003c/h2\u003e \u003cp\u003eWe further investigated the biological processes involved in the different stages of exposure to IMQ using GSEA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Short IMQ exposure led to activation of TLR signalling pathways, interferon-driven responses and TNF signalling via NF-κB (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Upon longer IMQ exposure, activation of TLR-induced IRF7 signalling was revealed and TNF signalling via NF-κB became more prominent (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Additionally, downstream effects representative of the TLR pathway including induction of type I and type II interferons leading to the activation of the JAK-STAT pathway were identified after long exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). When comparing long to short IMQ exposure, a positive enrichment of gene sets relating to IFN-α and -γ was found, as well as additional activated pathways including IL-2 signalling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). A network plot was generated displaying the linkages between the biological pathways involved in the IMQ response (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). We next focused on a selection of relevant differentially expressed genes (DEGs) involved in TLR-induced IRF7 signalling, NF-κB signalling and complement activation pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb; full pathways shown in Supplemental Fig.\u0026nbsp;4). For IRF signalling, a time-dependent increase was found in the expression of transcripts encoding for IRF7, Mx-1 and CXCL10 in IMQ-treated samples compared to untreated samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). RNA sequencing-based NF-κB responses were confirmed by immunohistochemical staining of skin punch biopsies: NF-κB (total) staining was elevated between 120h and 168h post IMQ application, compared to baseline (Supplemental Fig. S5c). Downstream of NF-κB, we found a similar increase in expression for IL-6 and CCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). CXCL8 expression was upregulated after IMQ application, but the difference in expression was not significant between long and short exposure. An overview of expression for markers corresponding to the IHC staining is available in Supplemental Fig. S6.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eIMQ-driven expression of complement genes\u003c/h2\u003e \u003cp\u003eTranscriptomic analysis at the pathway level showed a positive enrichment of gene sets related to the complement cascade, with stronger enrichment upon prolonged exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Complement genes elevated in expression by IMQ application included C1QA, C3 and CFB (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). Results for the transcripts encoding for complement proteins downstream of C3 were inconsistent, with mixed expression levels between samples (Supplemental Fig. S4b). IHC staining for complement revealed traces of dermal C4d at baseline, but no deposition of C3c or C4d after 168h of exposure to IMQ. Complement proteins showed no systemic elevation of C3, C3d, C3d/C3 or C5b-9 in plasma after 168h of IMQ-exposure (not shown).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIncreased cellular infiltration after long IMQ exposure\u003c/h2\u003e \u003cp\u003eHistologically, the general inflammation pattern after IMQ exposure was a lymphohistiocytic perivascular dermatitis with an increasing degree and deeper extension of inflammation over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). In 9/10 individuals the infiltrate showed peri-adnexal (peri-follicular and/or peri-eccrine) involvement and epidermal interface dermatitis (6/10). A rise in general cell infiltration accompanied by a mild increase in acanthosis as well as lymphocytic exocytosis (Supplemental Fig. S5a,b) was evident upon prolonged IMQ exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Immunohistochemical staining showed infiltration of monocytes, DCs and macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb) but no presence of neutrophils (Supplemental Fig. S5e). The cells were present after 48h-72h, with infiltration more elevated after 120h-168h post IMQ application. This pattern was mirrored by the T cell response, which showed a rise in T helper cells and cytotoxic T cells until 168h. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). A minimal number of Langerhans cells and B cells was present (Supplemental Fig. S5d,f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CIBERSORTx algorithm identified a total of 19 out of 22 immmune cell subpopulations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). IMQ application generally led to increased absolute scores of several immune cell types compared to the untreated samples. A globally significant difference (using Skillings-Mack test) between groups is observed, among others, for resting NK cells (p\u0026thinsp;=\u0026thinsp;0.0292), na\u0026iuml;ve B cells, (p\u0026thinsp;=\u0026thinsp;0.0051,), M1 Macrophages (p\u0026thinsp;=\u0026thinsp;0.0003), M2 macrophages (p\u0026thinsp;=\u0026thinsp;0.0403), activated DCs (p\u0026thinsp;=\u0026thinsp;0.001,), resting CD4\u003csup\u003e+\u003c/sup\u003e memory T cells (p\u0026thinsp;=\u0026thinsp;0.0003) and activated CD4\u003csup\u003e+\u003c/sup\u003e memory T cells (p\u0026thinsp;=\u0026thinsp;0.0241), and CD8\u003csup\u003e+\u003c/sup\u003e T cells (p\u0026thinsp;=\u0026thinsp;0.0463) depicted in Supplemental Fig. S7. Of these, na\u0026iuml;ve B cells, M1 macrophages, resting NK cells and both resting and activated CD4\u003csup\u003e+\u003c/sup\u003e memory T cells showed significant increase in abundance at 168h vs. 72h after IMQ exposure in the post-hoc analysis. Additionally, a statistically significant reduced abundance was observed in both resting and activated DCs at 168h vs. 72h. M2 macrophages and CD8\u003csup\u003e+\u003c/sup\u003e T cells did not show statistically significant changes at individual timepoints. Remarkably, overall low abundance of neutrophils was observed (Supplemental Fig. S7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study is the first to elucidate the acute effects of long-term IMQ exposure using a multimodal approach in healthy volunteers. We have shown that compared to short exposure, long exposure to IMQ results in a stronger immunological response as evidenced by additional enriched pathways such as TLR-induced IRF7 signalling, more prominent TNF signalling via NF-κB along with downstream effects such as induction of type I and type II interferons leading to activation of the JAK-STAT pathway. Furthermore, increased complement gene expression was identified upon long exposure to IMQ[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Although imaging and biophysical measurements showed no significantly enhanced response after long IMQ application compared to short exposure, a strong cellular infiltration boost was observed. At the transcriptomic level, this was demonstrated by an increased abundance of M1 macrophages, resting NK cells, and resting and activated CD4\u0026thinsp;+\u0026thinsp;memory T cells. While a statistically significant increase was observed in M2 macrophages and CD8\u003csup\u003e+\u003c/sup\u003e T cells following IMQ application, no difference was observed between long and short exposure. The increased abundance of na\u0026iuml;ve B cells and the appearance of T cells is indicative of both innate and adaptive immune responses involvement. The transcriptomic profile partially aligns with the IHC-based cellular infiltration of macrophages, NK cells and CD4\u003csup\u003e+\u003c/sup\u003e T cells, demonstrating clear time-dependent effects with increased infiltration after long IMQ exposure. Additionally, IMQ increased the expression of type II interferon-related genes, which aligns with the IHC observation of CD8\u003csup\u003e+\u003c/sup\u003e cell influx. These cellular findings are consistent with classical TLR signalling. Activation of TLRs is also known to trigger MyD88, IRAK1 and IRAK4, leading to IRF7 and NF-κB signalling, which is in line with our findings. These pathways result in upregulation of transcription factors for several cytokines including type I interferons, TNF, IL-2, IL-6, IL-8, IL-12, IFN-α and chemokines such as macrophage inflammatory protein (MIP)-1α, MIP-1β and monocyte chemotactic protein-1[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnother challenge agent that we use to effectively induce an in vivo TLR response in men is lipopolysaccharide (LPS)[\u003cspan additionalcitationids=\"CR26 CR27\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Intradermal injection of LPS triggers an acute inflammatory response via TLR4, leading to increased innate immune cell populations including neutrophils, monocytes and dendritic cells. Furthermore, LPS elicits an adaptive immune response, as evidenced by the presence of B and T cells. Elevated levels of IL-6, IL-8, IL-1β and TNF following LPS injection indicate NF-κB involvement. The current study showed moderate activation of NF-κB signalling after long IMQ application, supported by upregulated expression of NF-κB1, NF-κB2, IL-6, CXCL8, CCL2, IL-17C and IL-23A at the transcriptomic level. This contradicts previous studies, as no significant IL-6 and IL-8 responses were observed 72 hours after IMQ application, suggesting only mild NF-κB involvement. However, the release of Mx-A (a downstream marker indicative of IFN-α activation through IRF7) was evident[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Our current data reinforces this finding, as it suggests both My-D88 transcript expression and downstream IRF7 and Mx-1 activation, which increases upon prolonged IMQ application. The activation of interferons leads to the engagement of their respective receptors, which in turn triggers the JAK - STAT pathway culminating in the release of proinflammatory cytokines[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The JAK - STAT pathway was more enriched, with chemokines such as CXCL9, CXCL10 and CXCL11 significantly overexpressed after prolonged IMQ exposure. Recently, Chen \u003cem\u003eet al\u003c/em\u003e. provided an overview of studies examining DNA and RNA specific profiles in cutaneous lupus erythematosus (CLE) patients, which indicates an upregulation of innate immune response functions including JAK \u0026ndash; STAT signalling, TLR signalling, and pattern recognition receptors. Furthermore, there was a notable increase in the expression of type 1 interferons, along with an upregulated expression of chemokines CXCL9, CXCL10, and CXCL11, which are recognized as characteristic indicators for CLE[\u003cspan additionalcitationids=\"CR32 CR33\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Although a direct comparison of our data with the existing RNA datasets of CLE patients was not conducted, analysis of pathway activity and overexpressed genes detected upon prolonged IMQ application allows us to conclude that our current model aligns more closely with CLE characteristics than with psoriasiform lesions[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, the histopathological changes of a vacuolar interface dermatitis with adnexal involvement were also reminiscent of CLE. These observations differ from the prevalent use of the model in preclinical studies, where the murine IMQ model is typically used to investigate psoriasis-like conditions[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn contrast to our cellular observations, imaging and biophysical measurements showed no significantly enhanced response after long IMQ application compared to short exposure. This may be because Aldara (besides its role as a TLR7 agonist) may also act as an exogenous mediator by enhancing transient receptor potential vanilloid 1 channel activity on the primary afferent sensory neuron[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Activation of this channel leads to the release of bioactive substances such as nitric oxide (NO). NO can then interact with target cells in the surrounding tissue, including vascular smooth muscle cells. The interaction of NO with smooth muscle cells leads to vasodilation, resulting in increased blood perfusion and erythema[40]. Our observations suggest that the vascular response is independent of the inflammatory process. The role of bioactive, vasodilating substances in the IMQ-induced erythema and perfusion response remains to be further elucidated.\u003c/p\u003e \u003cp\u003eOur second objective was to explore the translational value of the IMQ model. In contrast to the mouse data, IHC staining did not show involvement of complement in the human IMQ response. We hypothesize that this may be due to the difference in severity of the hit, as in mice, the entire surface area of the back is challenged, whereas in humans IMQ is applied to a much smaller relative surface area. Therefore, it is currently unknown whether the observed differences are a result of the magnitude of TLR activation or can be contributed to species differences[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, at the transcriptome level, classical, and alternative pathway genes were enriched, particularly after prolonged exposure to IMQ, suggesting complement involvement in the human IMQ response. It is unclear how these sequencing results translate to the protein level, or if the transient nature of complement explains the lack of IMQ-driven complement responses in IHC analysis. The same holds true for the observed lack of neutrophils and CXCL8 expression, which emphasizes that the role of neutrophils in the human IMQ response needs to be elucidated further[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The observed differences between preclinical animal models and the human response may partially be explained by species differences in TLR7 expression[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. For instance, Bhagchandani \u003cem\u003eet al\u003c/em\u003e. described that the expression of TLR7 on neutrophils is higher in mouse than in men[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Moreover, expression patterns of TLR7 within a given cell type may differ across tissues and across activation status of the cell[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], further complicating the translational interpretation both across and within species. These findings highlight the complementary value of human challenge models in the development of immune-targeting compound development.\u003c/p\u003e \u003cp\u003eIn conclusion, our study provides a comprehensive characterization of the cutaneous response to both short and prolonged IMQ exposure in healthy volunteers by using a multimodal approach. We have demonstrated that prolonging the IMQ exposure has added value by enhancing cellular responses and increasing abundance of specific immune cell types along with stronger activation of a diverse set of pathways, particularly those driven by IRF and related to complement. We also argue that prolonged IMQ application results in a CLE-like cutaneous inflammation, both at the transcriptomic level and from a histopathological perspective. Our results suggest that biophysical and vascular responses are not exclusively driven by cutaneous inflammation. The described discrepancies between preclinical and clinical results, most notably the neutrophil response, illustrate the complementary value of human challenge models in the development of compounds targeting the immune system. This \u003cem\u003ein vivo\u003c/em\u003e immune challenge model is of value for future early clinical evaluation of topically or systemically applied anti-inflammatory or immunomodulatory compounds, particularly compounds targeting IRF and JAK-STAT signalling.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThis study was funded by the Centre for Human Drug Research, Leiden. The authors thank all the healthy volunteers who participated in this clinical research.\u0026nbsp;They also extend their thanks to the operational staff members involved in the setup, clinical conduct, and data analysis of the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contributions:\u0026nbsp;\u003c/strong\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Juliette van den Noort, Salma Assil, Micha Ronner, Michelle Osse, Jeffrey Damman and Ingrid Tomljanovic. The first draft of the manuscript was written by Juliette van den Noort and Salma Assil and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure of Conflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatements and declarations:\u0026nbsp;\u003c/strong\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u0026nbsp;\u003c/strong\u003eThis study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Independent Ethics Committee (Medisch Ethische ToetsingsCommise) of the Foundation \u0026lsquo;Evaluation of Ethics in Biomedical Research\u0026rsquo; (Stichting Beoordeling Ethiek Biomedisch Onderzoek), Assen, The Netherlands (Date 15DEC2021/NL79321.056.21).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSources of funding:\u0026nbsp;\u003c/strong\u003eCentre for Human Drug Research, Leiden\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest:\u0026nbsp;\u003c/strong\u003eThe authors state no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJunhee Seok, H. Shaw Warren, Alex GC, et al (2013) Genomic responses in mouse models poorly mimic human inflammatory diseases. 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Nat Rev Immunol 7:179\u0026ndash;190\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"TLR7, imiquimod, prolonged exposure, challenge","lastPublishedDoi":"10.21203/rs.3.rs-4655296/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4655296/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003e Imiquimod (IMQ; brand name Aldara\u0026reg;) is a registered topical agent that has been proven to induce local inflammation via the Toll-like receptor (TLR)7 pathway. The purpose of this study was to characterize TLR7-mediated inflammation following 7 days (168h) of topical IMQ exposure in healthy volunteers, and to compare the effects of short exposure (48h-72h) with prolonged exposure (120h-168h).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIMQ (100mg) was applied under occlusion to 5 different tape-stripped treatment sites on the back of 10 healthy participants for a maximum of 7 consecutive days. Erythema and skin perfusion were measured daily up to 168h. Biopsies for immunohistochemical staining and RNA sequencing were collected at 0h, 48h, 72h, 120h and 168h post IMQ application.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIMQ triggered an inflammatory response starting at 48h after application, including erythema and perfusion of the skin. At the transcriptomic level, IMQ induced TLR7 signalling, IRF involvement and activation of TNF signalling via NF-κB. Furthermore, an enhanced inflammatory response at the cellular level was observed after prolonged IMQ exposure, with cellular infiltration of dendritic cells, macrophages and T cells which was also corroborated by transcriptomic profiles. No difference was found in the erythema and perfusion response after 168h of IMQ exposure compared to 72h.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eProlonged IMQ exposure revealed enhanced cellular responses and additional pathways with modulated activity compared to short exposure and can therefore be of interest as a model for investigational compounds targeting innate and adaptive immune responses.\u003c/p\u003e","manuscriptTitle":"Optimizing the IMQ model: Deep characterization of the human TLR7 response for early drug development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-23 22:04:42","doi":"10.21203/rs.3.rs-4655296/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-29T13:12:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-29T12:59:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145459273097700715555352059765140438294","date":"2024-07-08T09:38:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-03T12:41:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-01T06:14:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-01T06:13:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Inflammation","date":"2024-06-28T14:03:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"inflammation","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ifla","sideBox":"Learn more about [Inflammation](https://www.springer.com/journal/10753)","snPcode":"10753","submissionUrl":"https://submission.nature.com/new-submission/10753/3","title":"Inflammation","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e08f49d5-a433-4fee-92f0-32c4465c476f","owner":[],"postedDate":"July 23rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-02T16:07:34+00:00","versionOfRecord":{"articleIdentity":"rs-4655296","link":"https://doi.org/10.1007/s10753-024-02127-x","journal":{"identity":"inflammation","isVorOnly":false,"title":"Inflammation"},"publishedOn":"2024-08-26 15:57:36","publishedOnDateReadable":"August 26th, 2024"},"versionCreatedAt":"2024-07-23 22:04:42","video":"","vorDoi":"10.1007/s10753-024-02127-x","vorDoiUrl":"https://doi.org/10.1007/s10753-024-02127-x","workflowStages":[]},"version":"v1","identity":"rs-4655296","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4655296","identity":"rs-4655296","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}