3.1 NK-1R expression was elevated in patients with psoriasis, and NK-1R agonist aggravated IMQ-induced psoriatic skin lesions in mice, while NK-1R inhibitors improved skin lesions
Studies reported abnormal neuroimmune modulation in psoriasis and increased expression of neurotransmitters such as SP, NKA, and corresponding neurotransmitter receptors in psoriatic skin lesions. Immunofluorescence assay was used to detect the expression of the neurotransmitter SP and its receptor NK-1R in psoriatic lesions. The results showed that the expression of SP and NK-1R in psoriatic lesions increased compared with that in healthy lesions, which was consistent with previous studies (Fig. 1). We further observed the effects of NK-1R agonists and inhibitors on the skin lesions in IMQ-induced psoriasis-like lesions in mice. The results showed that NK-1R agonists aggravated the skin lesions in mice; especially, the PASI score increased significantly (Fig. 2A and 2B). After the application of the NK-1R inhibitor, the skin lesions of mice were alleviated, especially after intraperitoneal injection of NK-1R inhibitor, showing a decrease in PASI score and epidermal thickness (Fig. 2C and 2D).
3.2 IMQ-induced psoriasiform dermatitis was alleviated in NK-1R KO mice
IMQ was applied on the back skin and right ear of mice continuously for 7 days to compare psoriasiform dermatitis between NK-1R KO and WT control mice. We found that the manifestations of psoriasiform dermatitis in mice were less severe in the KO/IMQ group (Fig. 3A). The PASI scores, infiltration, and erythema, as well as the total scores, decreased in the KO/IMQ group (Fig. 3B). We also compared the epidermal hyperproliferation and thickening conditions. H&E staining showed hyperkeratosis and acanthosis after treatment with IMQ; the epidermal thickness of back skin and ears were measured and found to be reduced in NK-1R KO mice compared with WT control mice (Fig. 3C, 3D, 3G, and 3H). We also examined the number of CD3+ T lymphocytes in the dermis. The immunohistochemical results showed that the number of CD3+ lymphocytes increased in the dermis of both WT and KO mice after IMQ application; however, the number of CD3+ cells was decreased in KO mice than in WT mice after IMQ application (Fig. 3E and 3F).
3.3 Expression levels of SP and NK-1R reduced in IMQ-induced NK-1R KO mice compared with WT mice
SP is a tachykinin peptide whose receptor is NK-1R. The interaction between them triggers intracellular signaling in some immune cells, which affects the function of immune cells. Clinical studies reported that the expression levels of SP were higher in the psoriasiform skin than in the healthy skin. In this study, we compared the expression levels of SP and NK-1R between WT and NK-1R KO mice. Immunohistochemistry (IHC) results showed the immunoreactivity of SP on nerve fibers after IMQ treatment, which extended from the epidermis to the dermis, but the number of SP-positive fibers decreased in NK-1R KO mice, which appeared as discontinuity of SP-positive nerve fibers (Fig. 4A). The expression level of SP was also detected using Western blotting. The results showed that the expression level of SP decreased after IMQ treatment in NK-1R KO mice compared with WT mice (Fig. 4C). We also detected the expression level of NK-1R and found that NK-1R expression significantly increased after IMQ treatment in the WT/IMQ group, but only slightly increased in the KO/IMQ group, which was consistent with Western blot results (Fig. 4B and 4D).
3.4 NK-1R KO mice showed decreased production of cytokines in both skin and peripheral blood after IMQ treatment
It was believed that inflammatory cytokines played a crucial role in the pathogenesis of psoriasis, and biologics against cytokines, such as TNF-α, IL-12, IL-17, and IL-23, were recommended as the first-line treatment of moderate-to-severe plaque psoriasis [1]. Therefore, the expression levels of some cytokines associated with the development of psoriasiform lesions were determined in this study. We found that the secretion of some cytokines, such as interferon (INF)-γ, IL-12p70, IL-1β, IL-4, TNF-α, GM-CSF, IL-17A, IL-23, IL-13, and IL-22, in the supernatants of skin tissue increased in the WT/IMQ group compared with the WT group, while only the secretion of IL-1β, TNF-α, GM-CSF, and IL-13 was upregulated in the KO/IMQ group compared with the KO group. It was observed that the expression levels of INF-γ, IL-12p70, IL-4, IL-17A, and IL-23 did not increase after IMQ treatment in NK-1R KO mice. Furthermore, the expression levels of the aforementioned cytokines were significantly higher in the WT/IMQ group than in the KO/IMQ group (Fig. 5A–5J).
Besides, we also determined the secretion levels of cytokines in serum. In serum, the expression levels of IL-13, IL-22, IL-23, IL-1β, TNF-α, IL-27, IL-9, IL-4, MIP-1α, MIP-1β, MIP-2, and IL-1α significantly increased in WT mice after IMQ treatment, while only the secretion of IL-13 and TNF-α was upregulated in the KO/IMQ group compared with the KO group (Fig. 5H–5S).
3.5 SP/NK-1R and RND3/VEGF pathways were involved in reducing psoriatic lesions in NK-1R KO mice
Next, we wanted to explore the mechanisms underlying the reduction of inflammation in skin lesions of NK-1R KO mice. Studies showed that the binding of NK-1R to SP triggered guanosine triphosphate (GTP) to Guanosine diphosphate (GDP) conversion on its Gα subunit, which dissociated Gαq from Gβγ and subsequently activated downstream effector molecules, such as phospholipase C (PLC) and Phosphoinositide-3 kinase (PI3K). PLC could directly activate Raf by activating protein kinase C (PKC), leading to the activation of mitogen-activated protein kinase (MAPK) pathway, and could also activate the MAPK pathway through kinase protein Shc. PI3K could activate Akt, leading to the activation of the nuclear factor kappa-B (NF-κB) pathway and the synthesis of inflammatory cytokines [12]. Therefore, we examined the expression levels of Akt, p-Akt, NF-κB, and p-NF-κB in mouse skin lesions. The expression level of p-Akt was upregulated after IMQ treatment in WT mice, and no changes were observed in NK-1R KO mice (Fig. 6A–6C).
We further examined the expression level of VEGF, which is important in psoriasis. The result showed that the level of VEGF was higher after IMQ treatment in WT mice, while no change was observed after IMQ treatment in KO mice. We also determined the content of RND3, the upstream of VEGF, we found that the expression level of RND3 was higher after IMQ treatment in WT mice, while it was almost the same after IMQ treatment in KO mice (Fig. 6D–6F). These results revealed that RND3/VEGF pathway was involved in reducing psoriatic lesions in NK-1R KO mice.
3.6 Function of DCs was restrained in IMQ-induced NK-1R KO mice
We examined the cell types in the ears and skin by flow cytometric analysis to investigate why psoriasiform dermatitis was alleviated in NK-1R KO mice. This study found that the number of CD11c+ DCs in the ear decreased after IMQ treatment, and was lower in KO/IMQ mice compared with WT/IMQ mice (Fig. 7A). The number of CD11c+ DCs in the skin also decreased after IMQ treatment, but no differences were observed between WT/IMQ and KO/IMQ mice (Fig. 8A). Furthermore, we analyzed the expression levels of I-A/I-E, CD80, and CD86, which represented the function of CD11c+ DCs. The result showed that the expression level of I-A/I-E in the ear was decreased in KO/IMQ mice compared with WT/IMQ mice; CD86 was upregulated after IMQ treatment, while no significant differences were observed in the expression level of CD80 (Fig. 7B–7D). However, the results were not the same in the skin. We found that the expression level of I-A/I-E decreased after IMQ treatment in both WT and NI-1R KO mice. Furthermore, KO/IMQ mice showed a lower expression level of I-A/I-E than WT/IMQ mice. The expression levels of CD80 and CD86 were upregulated after IMQ treatment in both WT and NK-1R KO mice but were downregulated in KO/IMQ mice compared with WT/IMQ mice (Fig. 8B–8D). We next analyzed the number of CD11b+F4/80+ macrophages and CD11b+F4/80- cells in the ears and skin. The number of macrophages and CD11b+F4/80- cells increased in both WT and NK-1R KO mice after IMQ treatment, the number of macrophages was almost the same in WT/IMQ and KO/IMQ mice, and the number of CD11b+F4/80– cells decreased in KO/IMQ mice compared with WT/IMQ mice (Fig. 7E).
We also investigated the numbers of CD3+ T cells, CD3+TCRγδ+ T cells, CD3+CD4+ cells, and CD3+CD8+ cells. The results indicated that the number of CD3+ T cells decreased after IMQ treatment in the ears in NK-1R KO mice, and the number of CD3+TCRγδ+ T cells decreased in both WT and NK-1R KO mice (Fig. 7F and 7G). However, the number of CD3+ T cells in the skin increased only in WT mice after IMQ treatment. The number of CD3+TCRγδ+ T cells increased in the skin of both WT and NK-1R KO mice and was even higher in KO/IMQ mice compared with WT/IMQ mice (Fig. 8F and 8G). We found that, in the ear, the number of CD3+CD4+ and CD3+CD8+ cells was unchanged after IMQ treatment (Fig. 7H and 7I), while in the skin, only the number of CD3+CD8+ cells increased in WT mice after IMQ treatment (Fig. 8H and 8I).
We also detected the expression levels of immune cells in spleens. The results showed that only the expression levels of CD80 and CD11c+ MHC II were lower in the KO/IMQ group than in the IMQ group (Fig. 9A–9D). These results suggested that the inhibition of CD11c+ DC function rather than the reduction in their numbers might result in the reduction of skin lesions in NK-1R KO mice.
3.7 Inhibition of NK-1R reduced the proinflammatory function of BMDCs
We used mouse BMDCs and stimulated cells with Toll-like receptor (TLR) 7/8 agonist R848 to observe the changes in cytokines secreted by mouse BMDCs so as to further verify the effect of NK-1R on the function of DCs. The results showed that IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12P70, IL-13, IL-22, IL-27, IL-23, and TNF-α were secreted by BMDCs of WT mice after R848 stimulation. However, the secretion of only IL-2, IL-4, IL-5, IL-6, IL-10, IL-12P70, and TNF-α increased in BMDCs of NK-1R KO mice after R848 stimulation. The secretion of IL-2, IL-4, IL-5, IL-6, IL-10, IL-12P70, and TNF-α in BMDCs of KO mice was significantly lower than that in WT mice, suggesting that NK-1R affected the proinflammatory function of R848 in DCs (Fig. 10A–10M).