Cell-type differential targeting of TLR5 and TLR7 agonists and their synergistic effect as nasal mucosal adjuvants

Abstract Objective To compare the differential activation characteristics of Toll-like receptor 5 (TLR5) agonist KFD and Toll-like receptor 7 (TLR7) agonist R837 on nasal epithelial cells (NECs) and bone marrow-derived dendritic cells (BMDCs), and to determine whether their combination produces synergistic enhancement as nasal mucosal adjuvants. Methods Mouse NECs and BMDCs were stimulated with 200 ng/mL KFD or R837 alone or in combination (n=3). At 3 h after stimulation, transcriptome sequencing was performed to analyze gene expression profiles, with key gene expression validated by qPCR. IL-6 levels in the supernatants were measured by ELISA at 12 h post-stimulation. Female C57BL/6 mice (6 to 8 weeks old, weighing 18 to 20 g) were randomly divided into negative control (normal saline), KFD alone (2 μg) group, R837 alone (2 μg) group, and KFD+R837 combination (2 μg KFD+2 μg R837) groups (n=5). At 18 h after intranasal administration, nasal lavage fluid (NALF) and bronchoalveolar lavage fluid (BALF) were collected to detect IL-6 levels, and the expression of costimulatory molecules CD80/CD86 on dendritic cells (DCs) in cervical lymph nodes (CLNs) was analyzed by flow cytometry. Using ovalbumin (OVA) as a model antigen for intranasal immunization, mice were randomly divided into normal saline (negative control), OVA alone (25 μg), OVA+KFD (25 μg OVA+2 μg KFD), OVA+R837 (25 μg OVA+2 μg R837), and OVA+combination adjuvant (25 μg OVA+1 μg KFD+1 μg R837) groups (n=5). Mice were immunized intranasally at weeks 0, 4, and 8. Serum and saliva samples were collected at 2 weeks after the final immunization, and OVA-specific IgG/IgA antibody levels were detected by ELISA. Results Transcriptomic analysis revealed that KFD primarily activated NECs, with differentially expressed genes enriched in innate immune pathways such as TNF and NF-κB. In contrast, R837 potently activated BMDCs, with differentially expressed genes involved in TNF and IL-17 signaling pathways. In vitro BMDC experiments confirmed that R837 upregulated Tnf and Cxcl10 mRNA levels more strongly than KFD (P<0.01). The combination synergistically enhanced the expression of Tnf, Cxcl10, and Cd40 (P<0.001) and IL-6 secretion (KFD+R837 combination vs single agent, P<0.000 1). After intranasal administration, both KFD and R837 significantly upregulated CD80/CD86 expression on CLN DCs, with the combination group showing the most significant CD80 upregulation (KFD+R837 combination vs single agent, P<0.05). KFD alone significantly elevated IL-6 levels in NALF, which was not further enhanced by the combination. In the OVA immunization model, the combination adjuvant group exhibited significantly higher levels of serum OVA-specific IgG and salivary OVA-specific IgA than the OVA group (P<0.000 1), which were superior to either single adjuvant (serum IgG: OVA+ combined adjuvants vs OVA+R837, P<0.05; salivary IgA: OVA+ combined adjuvants vs OVA+KFD, OVA+ combined adjuvants vs OVA+R837, both P<0.05). Conclusion TLR5 and TLR7 agonists differentially activate NECs and DCs. Their combination synergistically activates DCs and significantly enhances both systemic and local antigen-specific immune responses to mucosal vaccines.