Female C57BL/6N mice (7-10 weeks old) were procured from Orient Bio, and ovalbumin (OVA)-specific OT-II transgenic mice on a C57BL/6 background were obtained from Jackson Laboratory. All mice were maintained under specific pathogen-free conditions. Mice were euthanized by gradually introducing CO₂ at a flow rate that displaced 30-50% of the chamber vol/min, following the AVMA Guidelines for the Euthanasia of Animals (2020 Edition). Following the cessation of respiration, CO₂ exposure was continued for at least 1 min, and the absence of both breathing and cardiac activity was used to confirm death. Femurs, tibias, and lymph nodes were then collected for further analysis. All animal experiments were approved by the Institutional Animal Care and Use Committee of Korea University (KUIACUC-2021-0094) and were performed in accordance with institutional guidelines.

Bone marrow-derived dendritic cells (DCs) were obtained from C57BL/6N mice and cultured according to the protocol outlined by Inaba et al (27). Briefly, bone marrow cells were isolated from the femurs and tibias of mice and cultured in RPMI-1640 medium (Corning, 10-043-CVR) supplemented with 10% fetal bovine serum (Gibco, 16000-044), 20 ng/ml granulocyte/macrophage colony-stimulating factor (GM-CSF), HEPES (Corning, 25-060-CI), penicillin/streptomycin solution (Corning, 30-002-CI), and 50 µM 2-mercaptoethanol (Sigma, M6250-10M). The culture medium was refreshed by replacing half of it every alternate day. On day 7, the semi-adherent cells were gently pipetted and harvested for use as immature GM-CSF-derived dendritic cells. To generate Flt3L-derived dendritic cells, bone marrow (BM) cells were resuspended at 6x10⁶ cells/ml in RPMI-1640 medium containing 200 ng/ml of human recombinant FMS-like tyrosine kinase 3 ligand (Flt3L, BioLegend, 550602). Subsequently, the cell suspensions were seeded at 5 ml per well in 6 well plates and incubated for 9 days without adding additional media.

For in vitro syngeneic co-culture of CD4⁺ T cells and DCs, the DCs (5x10⁵ cells/well) cultured for 7 days were pulsed with 20 ng/ml OVA_323-339 peptide for 2 h. Following this, the DCs were treated for 6 h with imiquimod (IMQ, 1 µg/ml), lipopolysaccharide (LPS, 100 ng/ml) or not. Subsequently, CD4⁺ T cells (1x10⁶ cells/well) were co-cultured with OVA_323-339 peptide-pulsed DCs or OVA_323-339 peptide-pulsed IMQ- or LPS-stimulated DCs at a 1:10 ratio. After 3 days, the cells were harvested to analyze the T cell population, and the supernatant was collected to detect levels of IFN-γ, IL-17A, IL-9, and IL-10. CD4⁺ T cells were isolated from the lymph nodes of OT-II mice. The whole lymph nodes were minced using a slide glass and then filtered using a 40 µm cell strainer (Falcon, 352340). Subsequently, CD4⁺ T cells were purified using a CD4 (L3T4) microbead purification kit (Miltenyi Biotec, 130-117-043), ensuring a purity level of over 95%. Purified CD4⁺ T cells were then stimulated with anti-CD3 Ab (1 µg/ml) and anti-CD28 Ab (1 µg/ml) for 3 days.

The dendritic cells (1x10⁶ cells/well) were washed with PBS and harvested using FACS buffer (0.5% FBS and filtered 0.05% NaN₃ in PBS). The purity of BMDCs was assessed by flow cytometry using CD11c staining, with >90% of cells gated as CD11c⁺. Subsequently, DCs were blocked with mouse IgG (Sigma, I5381) for 20 min at 4˚C to minimize nonspecific binding. Following this, the cells were stained with APC-conjugated anti-mouse CD11c antibody (eBioscience, 17-0114-82), PE-conjugated anti-mouse CD40 (eBioscience, 12-0401-81) and CD80 (BD PharmingenTM, 553769), FITC-conjugated anti-mouse CD86 (eBioscience, 11-0862-82) and MHCII (eBioscience, 11-5322-82) for 20 min at 4˚C while protected from light. For intracellular staining, CD4⁺ T cells (1x10⁶ cells/well) were re-stimulated with PMA (50 ng/ml), ionomycin (1 µg/ml), and GolgiPlug containing Brefeldin A (BD sciences, 555029) for 5 h. The cells were harvested using FACS buffer and incubated with FITC-conjugated anti-mouse CD4 RM4-5 (eBioscience, 11-0042-82) for 20 min at 4˚C. The T cells were fixed and permeabilized using Cytofix/Cytoperm solution (BD sciences, 51-2090KZ). Intracellular cytokines were stained with PE-conjugated anti-IFN-γ XMG1.2 (eBioscience, 12-7311-82) and anti-IL-17A eBio17B7 (eBioscience, 12-7177-81), APC-conjugated anti-mouse IL-9 RM9A4 (BioLegend, 514105) and anti-mouse Foxp3 FJK-16s (eBioscience, 17-5773-82) for 1 h at 4˚C. The labeled cells were analyzed using a FACSymphony^™ A1 flow cytometer (BD Sciences), and the FCS data were analyzed using FlowJo software (BD Sciences).

Total mRNA was extracted from DCs (1x10⁶ cells/well) using RiboEX reagent (GeneAll, 301-001), following the manufacturer's protocol. RNA purity was assessed using a NanoDrop^™ 2000/2000c spectrophotometer (Thermo Fisher Scientific, Inc.), with A260/A280 ratios above 1.8 considered pure. The isolated total RNA was then reverse transcribed into cDNA using RocketScript Reverse Transcriptase (Bioneer). The cDNA was subsequently amplified by PCR. Following PCR amplification, the products were resolved on 1.5% agarose gels and visualized with ethidium bromide staining. The sequences of the RT-PCR primers used in this study were as follows: mouse TNF-α forward, 5'-GGCAGGTCTACTTTGGAGTC-3' and reverse, 5'-ACATTCGAGGCTCCAGTGAA-3'; mouse IL-12p40 forward, 5'-TGTGGAATGGCGTCTCTGTC-3' and reverse, 5'-CCTTTGCATTGGACTTCGGTAG-3'; mouse IL-12p35 forward, 5'-TCAGCGTTCCAACAGCCTC-3' and reverse, 5'-CCAAGGCACAGGGTCATCATCA-3'; mouse IL-1β forward, 5'-CTGAAGCAGCTATGGCAACT-3' and reverse, 5'-ACAGGACAGGTATAGATTC-3'; mouse IL-6 forward, 5'-TGAACAACGATGATGCACTT-3' and reverse, 5'-CGTAGAGAACAACATAAGTC-3'; mouse TGF-β forward, 5'-TATAGCAACAATTCCTGGCGT-3' and reverse, 5'-TCCTAAAGTCAATGTACAGCT-3'; mouse GAPDH forward, 5'-ACATCAAGAAGGTGGTGAAG-3' and reverse, 5'-ATTCAAGAGAGTAGGGAGGG-3'.

The levels of IL-12p40, IL-12p70, IL-6, TNF-α, IL-1β, IL-17A, IL-9, and IL-10 in the culture supernatants were detected using ELISA kits (eBioscience). The concentration of IFN-γ was measured using a specific mouse anti-IFN-γ R4-6A2 (BD sciences, 554430) capture antibody and biotinylated anti-IFN-γ XMG1.2 (BD sciences, 554410) detection antibody. A standard curve was established using recombinant mouse IFN-γ (BD sciences, 554587). The wells were washed with PBST (0.05% Tween-20 in PBS), and then o-phenylenediamine (OPD) containing citrate and H₂O₂ was added to each well and incubated for 15 min at room temperature. To stop the reaction, 2 N H₂SO₄ was added to each well. Detection limits for all cytokines were as specified by the manufacturers' protocols.

The DCs (1x10⁶ cells/well) were gently washed with serum-free medium. Then, the DCs were fixed with 4% paraformaldehyde (PFA) and permeabilized using 0.1% Triton X-100. After permeabilization, the cells were washed with 0.1% BSA in PBS and then blocked with 0.5% BSA in PBS for 30 min at room temperature. The cells were incubated with rabbit anti-mouse NF-κB p65 (1:100 dilution) at 4˚C overnight, and subsequently stained with Alexa Fluor 488-conjugated anti-rabbit IgG Ab (1:300 dilution) for 1 h at room temperature. Nuclei were counterstained with DAPI (Molecular Probes, 3 µM) for 3 min and imaged using a confocal laser scanning microscope (LSM 800, Carl Zeiss).

Statistical analyses were performed using GraphPad Prism 8.4.3 (GraphPad Software, Inc.). One-way ANOVA followed by the Bonferroni post hoc test was used to compare multiple groups. Results with P-values <0.05 were considered statistically significant. Data are presented as mean ± SD from three independent experiments.

When immature dendritic cells (iDCs) undergo maturation, they acquire the capacity to activate and direct the differentiation of CD4⁺ T cells through various mechanisms, including co-stimulatory molecules, MHC class molecules, and the secretion of cytokines (28,29). To investigate whether imiquimod (IMQ) affects the activation and maturation of bone marrow-derived dendritic cells in a dose-dependent manner, iDCs were cultured for 20 h in the presence or absence of IMQ. The expression levels of cell surface molecules were subsequently analyzed using flow cytometry to confirm the impact of IMQ on the modulation of crucial DCs maturation markers such as CD40, CD80, CD86, and MHC class II. As illustrated in Fig. 1, IMQ markedly increased the expression of co-stimulatory molecules CD40, CD80, and CD86 on CD11c⁺ BMDCs in a dose-dependent manner. Additionally, MHC class II expression also increased upon treatment with IMQ (Fig. 1). The high expression of these key surface markers suggests that IMQ significantly enhances the activation and maturation processes of DCs. CD40, CD80, CD86, and MHC class II are critical surface molecules involved in the interaction between DCs and CD4⁺ T cells. Overall, these results indicate that IMQ significantly enhanced the activation and maturation of GM-CSF-DCs.

The activation and maturation of DCs is intricately linked to the modulation of pro-inflammatory cytokines, presenting a crucial aspect of immune response orchestration. Upon activation, DCs undergo a transformation that involves heightened expression of co-stimulatory molecules and the secretion of various cytokines, including pro-inflammatory mediators (29). Therefore, to evaluate whether IMQ induces an elevation in the cytokine gene expression levels of DCs, DCs were treated with IMQ, and the cytokine expression levels were assessed at both mRNA and protein levels. The mRNA levels of pro-inflammatory cytokines, such as IL-12p35, IL-12p40, IL-6, TNF-α, and IL-1β were upregulated in IMQ-treated DCs (Fig. 2A and B). IMQ treatment generally increased the secretion of IL-12p40, IL-12p70, IL-6, TNF-α, and IL-1β, with varying degrees of responsiveness among cytokines (Fig. 2C). Collectively, these results suggest that IMQ increases the mRNA levels and secretion levels of pro-inflammatory cytokines by activating and maturing DCs, similar to the increase observed in surface marker data.

Recent studies have highlighted the distinct strategies for generating DCs using GM-CSF or Flt3L, resulting in different DC subsets. GM-CSF-derived DCs, often referred to as inflammatory DCs, lack the cDC1 subset and are characterized by their robust inflammatory responses. In contrast, Flt3L-derived DCs represent a steady-state DC population that includes both pDCs and cDCs, playing critical roles in maintaining immune homeostasis (30-33). In our study, we examined the effects of IMQ on both GM-CSF-derived DCs and Flt3L-derived DCs to comprehensively understand its impact on various DC subsets. Consistent with our findings in GM-CSF-derived DCs, IMQ treatment of Flt3L-derived DCs also resulted in a significant dose-dependent increase in the expression of co-stimulatory molecules (CD40, CD80, and CD86) and MHC class II, as well as the secretion levels of pro-inflammatory cytokines, except for IL-12p40 (Fig. S1).

Nuclear factor-kappa B (NF-κB) signaling plays a central role in dendritic cell (DC) activation by regulating the expression of cytokines, co-stimulatory molecules, and immune response mediators. This pathway is closely associated with the induction of adaptive immunity via T cell activation and polarization (34-36). Although IMQ is known to activate the NF-κB pathway and promote DC maturation, the precise molecular components involved in this process remain poorly characterized. In particular, no prior study has evaluated the expression of DC surface molecules using flow cytometry in vitro following NF-κB inhibition and subsequent IMQ stimulation.

To investigate this gap in knowledge, we utilized two NF-κB pathway inhibitors, Caffeic Acid Phenethyl Ester (CAPE) and Bay 11-7082 (BAY), each targeting distinct molecular steps within the NF-κB cascade. CAPE inhibits NF-κB activation by preventing the phosphorylation and degradation of IκB, thereby blocking the nuclear translocation of NF-κB (37). In contrast, BAY acts upstream by inhibiting the IκB kinase (IKK) complex, which is responsible for IκB phosphorylation and subsequent degradation, effectively preventing NF-κB signaling (38). To assess the role of this pathway in IMQ-induced DC maturation, DCs were pre-treated with CAPE or BAY and then stimulated with IMQ. The expression levels of DC surface markers and pro-inflammatory cytokines were subsequently analyzed using flow cytometry and ELISA. The surface molecules associated with DC activation and maturation, including co-stimulatory molecules CD40, CD80 and CD86, were significantly suppressed by CAPE. Conversely, BAY significantly reduced CD40 expression on DCs compared with the IMQ group, while showing a non-significant trend toward decreased expression of CD80 and CD86. Neither treatment significantly affected MHC class II expression on DCs (Fig. 3A and B). Additionally, both inhibitors significantly decreased the secretion levels of IL-12p40 and IL-6 in a dose-dependent manner, which are two key cytokines involved in the polarization of CD4⁺ T cells into Th1 and Th17 subsets (Fig. 3C and D). In conclusion, these data demonstrate that IMQ induces the activation and maturation of DCs via the NF-κB-dependent signaling pathway.

NF-κB p65 nuclear translocation is a key step in pathway activation and serves as a hallmark of immune stimulus-induced transcriptional activity. As shown in Fig. 3, the expression of DC surface molecules and secretion of IL-12p40 and IL-6 were substantially diminished when NF-κB signaling was inhibited during IMQ treatment, supporting a central role for NF-κB p65. To directly confirm whether IMQ-induced DC activation is accompanied by NF-κB p65 nuclear translocation, we performed an immunofluorescence (IF) assay. Following IMQ exposure, NF-κB p65 was detected in the nucleus of DCs, confirming translocation (Fig. 4). Taken together, our findings provide a deeper understanding of the molecular mechanisms underlying IMQ-induced DC activation, clarifying the roles of NF-κB pathway components targeted by CAPE and BAY, and revealing the direct activation of DCs by IMQ in vitro.

DCs play a crucial role in the immune system by capturing and presenting antigens to T cells, thereby initiating adaptive immune responses. Upon activation and maturation, DCs upregulate surface and co-stimulatory molecules, enhancing their antigen-presenting capabilities and enabling more effective interactions with T cells. As a result, the interaction between mature DCs and naïve CD4⁺ T cells leads to increased T cell activation and differentiation into various Th cell subsets, including Th1 and Th17 (22-25,39-43). This enhanced antigen-presenting cell (APC) function of DCs is essential for mounting a robust and targeted immune response. As previously shown in Fig. 2C, IMQ-treated DCs increased the secretion of IL-12p40 and IL-6, which are critical for Th1 and Th17 polarization. To investigate whether IMQ-stimulated DCs polarize naïve CD4⁺ T cells into various effector Th subsets, including Th1, Th17, Th9, and Treg, DCs were incubated with ovalbumin (OVA) (10 ng/ml) for 2 h. Subsequently, they were treated with IMQ (1 µg/ml) for 6 h and then co-cultured with naïve CD4⁺ T cells isolated from OT-II mice for 3 days. The population of Th subsets was assessed by flow cytometry. As depicted in Fig. 5A and B, IMQ-treated DCs notably increased the percentage of CD4⁺ IFN-γ⁺ T cells (Th1) and CD4⁺ IL-17A⁺ T cells (Th17).

Recent studies have highlighted the involvement of other Th subsets, such as Th9 cells, in autoimmune diseases like psoriasis and rheumatoid arthritis. Th9 cells, characterized by IL-9 production, have been implicated in the pathogenesis of several autoimmune diseases (44-46). Building on these studies, we observed a significant increase in the population of CD4⁺ IL-9⁺ T cells (Th9) when IMQ-treated DCs were co-cultured with naïve OT-II CD4⁺ T cells. IL-9 secretion tended to increase but was not statistically significant. Additionally, IMQ-treated DCs induced an increase in the population of CD4⁺ Foxp3⁺ Tregs and IL-10 secretion; however, these changes were not statistically significant (Fig. S2).

IFN-γ and IL-17A are well-recognized signature cytokines predominantly secreted by Th1 and Th17 cells, respectively. To confirm whether IMQ-stimulated DCs enhance the production of IFN-γ and IL-17A in co-cultured CD4⁺ T cells, cytokine production levels in the supernatants of the co-cultures were determined using ELISA. IMQ-stimulated DCs increased IFN-γ and IL-17A production in co-cultured OT-II CD4⁺ T cells (Fig. 5C). Additionally, IMQ-treated DCs notably tended to increase IL-9 secretion in co-cultured OT-II CD4⁺ T cells, with IL-10 exhibiting a similar trend (Fig. S2C). These results are the first to demonstrate that IMQ-treated DCs can polarize naïve CD4⁺ T cells into specific effector T helper cell subsets, such as Th1, Th17, and Th9. The substantial increase in IL-9 secretion, along with elevated Th9 population levels, suggests a key role for IL-9 in Th9 differentiation. This novel finding highlights the unique capability of IMQ-activated DCs to influence T cell differentiation, providing a new perspective on the immunomodulatory effects of IMQ.

DCs secrete cytokines essential for T cell polarization. Specifically, IL-12 and IL-6 are key drivers of Th1 and Th17 cell differentiation, respectively. These cytokines direct the differentiation of naïve CD4⁺ T cells and shape adaptive immune responses. To determine whether IL-12 and IL-6 secreted from IMQ-stimulated DCs contribute to the enhancement of Th1 and Th17 responses, neutralizing antibodies against IL-12 and IL-6 were added to co-cultures of IMQ-treated DCs and naïve CD4⁺ T cells isolated from OT-II transgenic mice. The frequencies of Th1 and Th17 cells, along with IFN-γ and IL-17A production, were subsequently analyzed. As illustrated in Fig. 6A and B, blockade of IL-12 or IL-6 led to a significant reduction in the frequencies of CD4⁺ IFN-γ⁺ (Th1) and CD4⁺ IL-17A⁺ (Th17) cells. Consistent with these findings, IFN-γ and IL-17A levels in culture supernatants were also decreased (Fig. 6C). In contrast, IgG isotype control antibodies had no effect on these responses. These data demonstrate that IMQ-activated DCs promote Th1 and Th17 polarization via IL-12 and IL-6 secretion, underscoring the critical role of these cytokines in mediating the immunomodulatory effects of IMQ. This novel finding highlights the therapeutic potential of targeting DC-derived IL-12 and IL-6 pathways to modulate pathogenic T cell responses.