A total of 24 healthy male and female Caucasian (Fitzpatrick skin type I–III) volunteers, aged between 18 and 45 years, were enrolled in this study. Health status was confirmed by medical history, physical examination, laboratory tests, and 12-lead electrocardiography (ECG). None of the participants had a family history of psoriasis, no pathological skin conditions at the treatment area, no history of hypertrophic scarring or keloids, no prior use of imiquimod, and no known hypersensitivity to prednisolone.

Participants were equally randomized into two groups to receive oral prednisolone (0.25 mg/kg/dose) or placebo, twice daily with a 10–12-hour interval between doses, over a period of six consecutive days. On the sixth day, the volunteers received only one dose of prednisolone or placebo in the morning. The treatments were administered under supervision at the clinical research unit to ensure treatment compliance. After two days of pre-treatment with oral prednisolone or placebo, challenge with IMQ commenced (6). For this purpose, the upper back was divided into seven rectangles measuring 4 × 3 cm. Each treatment area was tape-stripped 20 times (D-Squame, CuDerm, Dallas, TX) to induce mild barrier skin disruption, whereas the trans-epidermal water loss (TEWL) (AquaFlux, Biox Systems) was used to quantify skin permeability (6, 7). A TEWL between 15 and 20 g/m²/h was considered as mild barrier skin disruption. No IMQ was applied to the first two areas since they represented the non-treated areas, while in the other three to five areas, 5 mg IMQ (100 mg Aldara^®) was applied for either 24 h or 48 h (depending on the assigned cohort) under occlusion by a 12 mm Finn chamber (Bipharma, Almere, The Netherlands) to initiate an inflammatory skin reaction. Subjects were randomized on (pre-)treatment (prednisolone or placebo) and the time point of invasive measurements (blister and biopsy), as illustrated in Figure 1 .

The subjects underwent multiple assessments to evaluate the inflammatory skin response before the IMQ challenge and 24, 48, 72, 168, and 216 h after IMQ application. A single treatment site was selected to evaluate non-invasive endpoints throughout the study period ( Figure 1 ), resulting in N = 12 per time point. An overview of the number of samples per time-point is presented in Table S1 . Erythema was graded in two ways: by a physician using a 4-point scale ranging from 0 (absent) to 3 (severe) and by multispectral photoanalysis (Antera 3D, Miravex, Ireland). Perfusion was quantified using laser speckle contrast imaging (LSCI; PeriCam PSI System, Perimed Jäfälla, Sweden) and Optical Coherence Tomography (OCT; VivoSight, Michelson Diagnostics Maidstone, UK). The latter was also used to measure epidermal thickness. All skin assessments were performed under standardized conditions at room temperature (20–24°C).

Suction blisters and 3 mm biopsy samples were taken from the IMQ-treated areas and control areas at the indicated time points, depending on the cohort and randomization ( Figure 1 ). Three biopsies and three blisters were collected from each healthy volunteer. Suction blisters were induced according to the method described by Buters et al. (8). Biopsies were placed in RNAlater medium directly after harvest and stored at 4°C until analysis at the Immunology Laboratory of Erasmus Medical Center, Rotterdam, Netherlands. Immunohistochemical staining was performed to obtain scoring of markers CD11c (Clone 5D11; Cell Marque), CD14 (Clone EPR3653; Cell Marque), CD1a (Clone EP3622; Cell Marque), CD4 (Clone SP35; Ventana), CD8 (Clone SP57; Ventana), and HLA-DR (CR3/43; Dako) using a 6-point rating scale; 0 = negative, 1 = minimal, 2 = few, 3 = moderate, 4 = many, and 5 =excessive.

Blister fluid was collected in a V-bottom plate containing 50 μl 3% sodium citrate (Sigma) in PBS (Gibco) and kept on ice. The plate was centrifuged, and the supernatant was weighed to estimate the volume and then frozen at −80°C for cytokine analysis (Meso Scale Discovery, Rockville, Maryland, USA). The following cytokines were analyzed: TNF, ASC, IL-1β, IL-6, IL-10, IL-8, IFN-γ, and downstream marker for type 1 interferon Mx-A (V-plex proinflammatory panel of MSD and Mx-A protein ELISA kit from BioVendor). The cell pellet was resuspended in RoboSep buffer (StemCell). A cocktail of fluorescent antibodies against cell surface markers was added to the cells and incubated for 30 min on ice. The stained samples were washed with PBS and measured using MACSQuant 16 (Miltenyi Biotec GmbH). Flow cytometry data were analyzed with Flowlogic 7.2 (Inivai). Parallel to the blister fluid, peripheral blood was collected by venipuncture using a sodium heparin vacutainer (BD). Approximately 100 µl of whole blood was treated with red blood cell lysis buffer (eBioscience), washed with PBS, and resuspended in RoboSep buffer. Staining was similar to that of the previously mentioned blister cells and served as a template for the gating strategy. The following antibodies were used: CD56-PE (cat# 130-113-312, Miltenyi Biotec), CD14-PE-Vio615 (cat# 130-110-526, Miltenyi Biotec), CD16-VioBrighT FITC (cat# 130-119-616, Miltenyi Biotec), CD66b-AF700 (cat# 305114, Biolegend), CD19-BV650 (cat# 302238, Biolegend), CD20-BV650 (cat# 302336, Biolegend), HLA DR-APC (cat# 130-111-790, Miltenyi Biotec), CD4-VioBlue (cat# 130-114-534, Miltenyi Biotec), CD8-BV570 (cat# 301038, Biolegend), CD45-VioGreen (cat# 130-110-638, Miltenyi Biotec), CD1c-PE-Vio770 (cat# 130-110-538, Miltenyi Biotec), CD3-APC-Vio770 (cat# 130-113-136, Miltenyi Biotec), 7AAD (cat# 130-111-568, Miltenyi Biotec). An overview of the gating strategy is shown in Figure S2 . Cell populations (Single live cells) were classified according to the following profile: CD45⁺ HLA-DR⁻ CD66b⁺ CD16⁺ neutrophils, CD45⁺ HLA-DR⁺ CD14⁺ CD16⁻ classical monocytes, CD45⁺ HLA-DR⁺ CD14⁺ CD16⁺ intermediate monocytes, CD45⁺ HLA-DR⁺ CD14⁻ CD16⁺ non-classical monocytes, CD45⁺ HLA-DR⁺ CD19⁻ CD20⁻ CD14⁻ CD16⁻ CD1c⁺ dendritic cells, CD45⁺ HLA-DR⁻ CD56⁺ NK Cells, CD45⁺ HLA-DR⁻ CD3⁺ CD4⁺ CD8⁻ T helper cells, CD45⁺ HLA-DR⁻ CD3⁺ CD4⁻ CD8⁺ cytotoxic T cells, and CD45⁺ HLA-DR⁺ CD19⁺ CD20⁺ B cells.

To investigate the extent of systemic immune suppression with prednisolone (ex vivo drug activity), IMQ whole-blood stimulation was used with cytokine release as a readout. Blood was drawn from healthy volunteers at four time points: pre-dose, 48, 52, and 96 h after the initial prednisolone/placebo dose. Blood samples were drawn 48 h and 96 h after initial dose but before the morning prednisolone/placebo dose. The sample taken 52 h after the first administration was taken 4 h after the previous prednisolone/placebo dose. At these time points, sodium heparinized whole blood was stimulated with 20 µg/ml IMQ (cat# tlrl-imq, InvivoGen) for 24 h. After incubation, the cultures were spun down, and the supernatant was collected and frozen at −80°C for cytokine analysis. The samples were shipped to Ardena (Assen, Netherlands) for analysis. The following cytokines were analyzed: IL-1β, IL-6, IFN-γ (V-plex proinflammatory panel of MSD), IP-10 (V-plex chemokine panel of MSD), and Mx-A (human Mx-A protein ELISA kit from BioVendor).

All repeatedly measured PD endpoints were summarized (n, mean, SD, min, and max values) according to treatment and time. Repeatedly measured continuous PD endpoints were analyzed using a mixed model analysis of covariance with fixed factor treatment, time, and treatment by time, with a random factor subject as a covariate. The baseline for whole blood stimulation was the pre-prednisolone/placebo treatment measurement. A summary table of the analysis results per variable was generated with estimates of the difference of the different contrasts and a back-transformed estimate of the difference in percentage for log-transformed parameters, 95% confidence intervals (in percentage for log-transformed parameters), Least Square Means (geometric means for log transformed parameters), and the p-value of the contrasts. Statistical analyses were performed using SAS for Windows V9.4 M6 (SAS Institute Inc., Cary, NC, USA).

Oral prednisolone or placebo was administered at 0.25 mg/kg per dose b.i.d. for a period of six consecutive days, and the effects on IMQ-driven clinical responses were evaluated. IMQ was applied for a maximum of 48 h under occlusion, after the skin was tape-stripped 20 times. Maximal responses were observed 48 h after IMQ application in the placebo group ( Figures 2A, B ). Treatment with prednisolone resulted in a reduction in the IMQ-driven response from 24 h to 72 h, quantified by imaging of blood perfusion (estimated difference: −16.1%, 95% confidence interval (CI) [−26.4%, −4.3%], p = 0.0111), erythema (estimated difference: −5.04, 95% CI [−7.96, −2.13], p = 0.0016), and epidermal thickness (estimated difference: −0.018, 95% CI [−0.029, −0.006], p = 0.0044), compared to placebo ( Figures 2A-C ). A visual overview of the blood perfusion and erythema is shown in Figure 2D . Application of IMQ (for 24 and 48 h) on the skin resulted in a thickened epidermis, disappearance of the rete ridges and dilatation of the blood vessels ( Figure 2E ). Furthermore, prednisolone treatment shifted the clinically scored erythema response from moderate to mild (at 48 h) and reduced the proportion of clinically scored erythema ( Figure S1 ). All quantified responses (perfusion, erythema, and epidermal thickness) were reversible and returned to baseline during the follow-up phase (168 and 216 h).

In earlier skin challenge research, cellular responses were studied using invasive techniques including suction blisters and skin punch biopsies (6–9). In this study, we implemented the same approach by inducing suction blisters and taking skin punch biopsies at the indicated time points, resulting in a total of three blisters and three biopsies per subject ( Figure 1 ). Biopsy samples were stained for dermal immune cell infiltration and scored by an independent investigator blinded to the treatment.

In addition, immune cells in the blister exudate were evaluated using flow cytometry. A full overview of the analyzed immune cell subsets is shown in Figure 3 . The average time for blister induction was (estimated mean, 95% CI) 86.7 min, 95% CI [71.9, 101.4] in the placebo group and 84.1 min, 95% CI [69.4, 98.8] in the prednisolone group (data not shown). No significant difference in the time required for blister formation was observed between the groups (estimated difference: −2.5, 95% CI [−23.4, 18.3], p = 0.8034). The total number of cells (CD45⁺) increased mildly following IMQ application, with a peak of 624.6 ± 469.5 cells in blister exudate at 48 h (after 2× IMQ application) ( Figure 3A ). IMQ increased the number of CD45⁺ HLA-DR⁻ CD56⁺ (NK cells), reaching a maximum of 84.2 ± 89.76 cells also at 48 h (after 2× IMQ application) ( Figure 3B ). The infiltration was followed by other innate immune cells, such as CD45⁺ HLA-DR⁺ CD19⁻ CD20⁻ CD14⁻ CD16⁻ CD1c⁺ (dendritic cells) and CD45⁺ HLA-DR⁺ CD14⁺ CD16⁻ (“classical monocytes”) peaking at 72 h (24 h after the second IMQ application) with mean ± SD of 26.0 ± 21.06 cells and 25.3 ± 27.7 cells, respectively; however, this resulted in a weaker response compared to the influx of NK cells ( Figures 3C, D ). Although neutrophils are strongly involved in innate immune responses, no infiltration of CD45⁺ HLA-DR⁻ CD66b⁺ CD16⁺ (neutrophils) was observed after IMQ application, resulting in cell counts of <5 cells (data not shown). Similar low cell counts were observed for CD45⁺ HLA-DR⁺ CD14⁺ CD16⁺ (“intermediate monocytes”), CD45⁺ HLA-DR⁺ CD14⁻ CD16⁺ (“non-classical monocytes”), and CD45⁺ HLA-DR⁺ CD19⁺ CD20⁺ (B cells) (data not shown). IMQ treatment did not result in a significant increase in B cells in the blister fluid (data not shown), nor did it substantially attract CD45⁺ HLA-DR⁻ CD3⁺ CD4⁺ CD8⁻ (T helper cells) and CD45⁺ HLA-DR⁻ CD3⁺ CD4⁻ CD8⁺ (cytotoxic T cells) ( Figures 3E, F ). Prednisolone reduced the number of immune cells in blister fluid. This reduction, compared to placebo, was observed for total cells, NK cells, dendritic cells, and classical monocytes (−58.8%, 95% CI [−79.7%, −16.3%], p = 0.0165; −45.5%, 95% CI [−68.7%, −5.2%], p = 0.0333; −55.4%, 95% CI [−76.9%, −13.9%], p = 0.0184; and −58.6%, 95% CI [−76.7%, −26.6%], p = 0.0043, respectively). Although no substantial changes were observed following IMQ challenge, prednisolone significantly reduced the number of T cell subsets (estimated difference: −76.0%, 95% CI [−92.4%, −4.7%], p = 0.0168 for T helper cells and estimated difference: −70.5%, 95% CI [−89.6%, −16.0%], p = 0.0242) for cytotoxic T cells.

Immune cell subsets in biopsies, quantified by IHC, showed a comparable picture to the cells analyzed by flow cytometry in blister fluid. Administration of prednisolone reduced HLA-DR and infiltrating CD11c (dendritic cells), CD4⁺ (T helper cells), CD1a (Langerhans cells), and CD8⁺ cells (cytotoxic T cells) ( Figure 4 ). Time courses of IMQ and prednisolone effects were comparable between biopsy and blister-derived immune cells.

In addition to the dermal cellular response, cytokine levels were analyzed in the blister exudate to evaluate of NF- κB- and IRF7-driven responses. IMQ application induced a mild IL-6 response at 24 and 48 h ( Figures 5A, B ) but had no clear effect on the levels of NF- κB-driven cytokines IL-1β IL-8 and IL-10 ( Figures 5C–E ). IMQ increased the Mx-A concentration in the blister fluid, with a peak at 48 and 72 h ( Figure 5F ), indicating activation of the IRF7 pathway. Prednisolone treatment reduced the levels of NF-κB-driven cytokines (IL-6, IL-8, and TNF), IL-1β, and Mx-A ( Figures 5A–C, F ). No formal statistical analysis for cytokines IL-6, IL-1β, and Mx-A was conducted for the contrast placebo versus prednisolone, because the immune suppression by prednisolone was so strong that most cytokine levels were below the LLOQ. ASC and IFN-γ concentrations in blister fluid are very low, and therefore, have not been reported.

Whole blood samples, drawn from study participants at predefined time points, were stimulated with IMQ for the evaluation of ex vivo prednisolone activity. Stimulation with IMQ led to an increase in IL-1β, IFN-γ, and IL-6 levels ( Figures 6A-C ), but not in a detectable Mx-A response (data not shown). An overview of the ex vivo results, including those of the unstimulated control conditions, is provided in Table S2 . Overall, prednisolone compared to placebo had a significant effect on IFN-γ and IL-1β concentrations (estimated difference: −86.8%, 95% CI [−94.1%, −70.3%, p <0.0001), −55.8%, 95% CI [−78.8%, −8.3%], p = 0.0301, respectively), but not IL-6 (estimated difference: −44.1%, 95% CI [−69.1%, 1.0%], p = 0.0537). However, prednisolone treatment resulted in a statistically significant reduction in IMQ-driven cytokines (IL-1β, IL-6, and IFN-γ), with a maximum inhibitory effect at 52 h post-administration, with an estimated difference of −92.9%, 95% CI [−96.8%, −84.3%], p <0.0001, estimated difference: −87.1%, 95% CI [−93.2%, −75.7%], p <0.0001; estimated difference: −99.0%, 95% CI [−99.6%, 97.4%], p <0.0001, respectively. Interestingly, prednisolone had no effect on IMQ-induced response at time points 48 and 96 h. No statistical analysis was performed on the IP-10 concentration, given that more than 60% of the samples were above the ULOQ.