In Vivo Signaling through the Neurokinin 1 Receptor Favors Transgene Expression by Langerhans Cells and Promotes the Generation of Th1- and Tc1-Biased Immune Responses

Abstract

The proinflammatory capacities of the skin and the presence of high numbers of resident dendritic cells (DCs) constitute an ideal microenvironment for successful immunizations. Regardless of the ability of DCs to respond to local inflammatory signals in an immunostimulatory fashion, the immune functions of skin-resident DCs remain controversial, and epidermal Langerhans cells (LCs) have been referred to recently as anti-inflammatory/protolerogenic APCs. Substance P (SP), released by skin nerve fibers, is a potent proinflammatory neuropeptide that favors development of skin-associated cellular immunity. SP exerts its proinflammatory functions by binding with high affinity to the neurokinin 1 receptor (NK1R). In this study, we tested whether signaling skin cells via the NK1R promotes humoral and cellular immunity during skin genetic immunizations. We used the gene gun to deliver transgenic (tg) Ag to the skin of C57BL/6 mice and the selective NK1R agonist [Sar9Met (O2) 11]-SP as a potential proinflammatory Th1-biasing adjuvant. Our strategy expressed tg Ag exclusively in the epidermis and induced a preferential migration of activated LCs to skin-draining lymph nodes. Local administration of the NK1R agonist during skin genetic immunizations increased significantly the expression of tg Ag by a mechanism involving the translocation of NF-κB into the nuclei of cutaneous DCs homing to skin-draining lymph nodes. Importantly, our immunization approach resulted in Th1 and T cytotoxic (CTL)-1 bias of effector T cells that supported cellular and Ab-mediated immune responses. We demonstrate that signaling skin cells via the NK1R provides the adjuvant effect which favors the immunostimulatory functions of LCs.

Neurokinin-1 receptor agonists bias therapeutic dendritic cells to induce type 1 immunity by licensing host dendritic cells to produce IL-12

“…Experiments analyzing T-cell proliferation were performed as described. 20 Secretion of IFN-g (BD PharMingen), IL-5, IL-13, and IL-12p70 (eBioscience) were analyzed by ELISA.…”

“…Five days later, mice were killed, and IFN-g, IL-5, and IL-13 secretion by OVA-specific CD4 T cells in response to ex vivo restimulation of splenocytes with OVA was quantified in culture supernatants by ELISA (BD PharMingen, eBioscience) as described. 20 After skin immunization, Ag-specific in vivo killing assays and detection of cytokine secretion by specific CD8 T cells after ex vivo Ag restimulation was conducted as previously described. 20 In vivo depletion of DCs C57BL/6 CD11c-DTR mice were injected with diphtheria toxin (DT) (4 ng/g body weight) or phosphate-buffered saline (PBS) (control), 1 day before immunization (footpad) with Ag-DCs, NK1R-DCs, or Ag-NK1R-DCs.…”

“…23 Briefly, cells were surface-labeled with PE-Cy7-CD11c (BD PharMingen), Pacific Blue-CD11b (Biolegend), Alexa Fluor 700-CD8a, and PerCP-Cy5.5-Ly6C Ab (eBioscience). After fixation/permeabilization, cells were labeled with PE-IL-12p40/70, tumor necrosis factor-a (TNF-a) ( 20 In vitro Ag-presentation assays Splenic OT-I-CD8 T cells were purified by negative selection (R&D Systems). Ag-DCs, NK1R-DCs, or Ag-NK1R-DCs generated form WT or CCR7 KO C57BL/6 mice were cocultured with OT-I CD8 T cells (1:20 DC:T-cell ratio, for 3 days).…”

“…Luciferase was analyzed by luminometry as previously described. 20,22 Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed with commercially available primers specific for CREB1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (SABioscience). 21 Relative quantitation of expression was determined by extrapolation to standard curve of complementary DNA from mouse brain.…”

“…18,19 In a previous publication, we demonstrated that skin Langerhans cells and bone marrow (BM)-derived DCs (BMDCs) express functional NK1R, and respond to NK1R agonists with increased cell survival and migration to skin-draining lymph nodes (sDLNs). 20,21 These immunologic effects suggest that NK1R agonists could be used as adjuvants for enhancing the efficiency of DC vaccines. Here, we demonstrate that conditioning BMDCs with NK1R agonists, leads to their maturation and abrogation of interleukin-10 (IL-10) release without inducing IL-12p70 secretion.…”