GPR15LG regulates psoriasis-like inflammation by down-regulating inflammatory factors on keratinocytes

Abstract Psoriasis is a common chronic inflammatory skin disease characterized by aberrant proliferation of keratinocytes and infiltration of immune cells. We previously found that GPR15LG protein is highly expressed in psoriasis lesional skin and it positively regulates psoriatic keratinocyte proliferation. Our data also showed that GPR15LG could regulate the activity of NF-κB pathway, which is associated with psoriatic inflammation. In the present study, we demonstrated that Gpr15lg (ortholog of GPR15LG) knockdown attenuated the severity of imiquimod (IMQ)-induced psoriasis-like inflammation in mice. Such an effect was achieved by down-regulating the expression of inflammatory cytokines interleukin (IL)-1α, IL-1β, tumor necrosis factor (TNF)-α and S100A7. Consistently, GPR15LG knockdown in vitro significantly downgraded the expression of inflammatory factors in the cellular model of psoriasis. These results suggested that GPR15LG could be involved in the development of psoriasis by regulating inflammation.


Introduction
Psoriasis is a chronic immune-mediated skin disorder, affecting 2-4% of the world's population [1,2].Psoriasis manifests as scaly erythematous plaques [1,2].Patients with psoriasis are at an increased risk of developing several comorbidities [3,4].They experience a reduction in the life quality with substantial economic burden and psychological burden [5].It is characterized by epidermal hyperplasia and intense inflammation [6].The exact pathogenesis of psoriasis is still not fully understood and available treatments are not absolutely effective.Therefore, more research is need to further elucidate the pathogenesis of psoriasis.
GPR15LG is a human antimicrobial peptide expressed in epithelial tissues [7,8].GPR15LG has been shown to modulate a variety of cellular functions and several functions of GPR15LG have been found in the context of psoriasis.However, to our knowledge, the capability of GPR15LG on regulating psoriasis-like skin inflammation remains largely unknown.
In the present study, the IMQ-induced psoriasis-like mouse model and M5-induced cellular model of psoriasis were employed to investigate the role of GPR15LG in psoriatic inflammation in vivo and in vitro.

Lentivirus transduction
The oligonucleotides of shRNAs were listed in Table 1.HaCaT cells were incubated with virus suspension for 48 h and puromycin was used to screen the stable infected cells for 14 days.

RNA extraction and qRT-PCR
Total RNA was extracted from cells or tissues using TRIzol (CWBIO) following the manufacturers' instructions.cDNA was synthesized with the kit (R223-01, Vazyme).qRT-PCR was done using SYBR qPCR Master Mix (Vazyme).
The primer sequences used in the experiment were shown in Table 2.

Mice
Female BALB/c mice (8 weeks of age) were acclimatized for 1 week with free access to food and water.The study was approved by the Ethics Committee of Fujian Provincial Hospital (approval number: K2019-03-056) and all experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Animal experiments
Mice were randomly divided into the following four groups: Control group (Ctr, n=6), IMQ group (IMQ, n=6), IMQ + sh-NC group (IMQ+sh-NC, n=6), IMQ + shRNA-1 group (IMQ+sh-1, n=6).Mice in IMQ + sh-NC group and mice in IMQ + sh-1 group were injected intradermally with lentivirus particles (1.0 × 10 9 TU, 50 μl) encoding negative control shRNA or Gpr15lg shRNA.The oligonucleotides of shRNAs were listed in Table 3.Three days after adenovirus particles treatment, groups, except the control group, were topically administered with 62.5 mg of 5% IMQ cream on the shaved back for 7 days.After treatment with IMQ, the mice were killed and we collected the dorsal skin samples.Half samples was fixed in formalin prepared for histological evaluation and immunohistochemistry and other tissues were frozen in liquid nitrogen for further detection.

Evaluation severity of skin inflammation
Psoriasis Area Severity Index (PASI) was used to score the mice skin inflammation severity.Scales, erythema and thickness were scored independently from 0 to 4. The cumulative score was obtained from the sum of the above three parameters.

Histological evaluation and Immunohistochemistry
The skin samples from each group were fixed in formalin for 24 h and 5 μm-thickness paraffin sections were stained with H&E.The cell layers of the epidermis and inflammatory cells were counted under high-power fields.Immunohistochemistry (IHC) was performed according to standard methods.For immunohistochemical staining, sections were incubated with specific primary antibodies against IL-1α (YT232), IL-1β (YT2322) and S100A7 (YT6273).

Statistical analysis
All data were presented as mean + − SEM from at least three independent experiments.Statistical analysis was carried out with GraphPad Prism 5.0.Student's t test was used to compare differences.P<0.05 was considered as statistically significant.

Gpr15lg knockdown ameliorates IMQ-induced psoriatic inflammation in mice
To explore the function of GPR15LG in IMQ-induced psoriatic inflammation, we locally knocked down Gpr15lg (the mouse ortholog of GPR15LG) expression in mouse back skin by injecting the adenoviral particles expressing shRNAs.The knockdown efficiencies of shRNAs were first investigated in C2C12 cells.As shown in Figure 1A, shRNA-1 showed the best knockdown efficiency among three shRNAs and it was chosen for the following animal experiment.IMQ treatment induced typical psoriasis-like lesions (Figure 1B).However, compared with IMQ+sh-NC group, Gpr15lg knockdown ameliorated the IMQ-induced mice skin condition (Figure 1B).In addition, we scored the severity of lesions on days 2, 4, 6 and 8 based on PASI.The PASI score of the IMQ group was significantly higher than that of control mice and the mice in IMQ+sh-1 group had lower score than IMQ+sh-NC group (Figure 1C).These results suggest that Gpr15lg knockdown could significantly attenuate the IMQ-induced psoriasis-like inflammation in mice.

Gpr15lg knockdown alleviates the morphologies of psoriatic skin inflammation in histopathological analysis
We carried out HE staining to further analyzed the lesions.Histopathological analysis showed the mice treated with IMQ had epidermal hyperplasia and inflammatory cells accumulation (Figure 2A-H).The number of cell layers and the number of inflammatory cells were further calculated.The data demonstrated that sh-1 treatment resulted in significant alleviation in the above two indexes (Figure 2I,J).These data showed that Gpr15lg knockdown alleviated the histopathological morphologies of psoriasis-like inflammation.

Gpr15lg knockdown reduced levels of inflammatory cytokines in mice psoriatic lesion
To further investigate whether Gpr15lg can regulate immune response in psoriasis, we detected the level of IL-1α, TNF-α, IL-1β and S100A7 by qRT-PCR and IHC.As shown in Figure 3, IMQ significantly up-regulated levels of IL-1α, TNF-α, IL-1β and S100A7.However, Gpr15lg knockdown attenuated the up-regulation of those inflammatory cytokines.These results indicated that Gpr15lg knockdown could effectively ameliorate psoriasis-related inflammatory micro-environment.

GPR15LG knockdown inhibited M5-induced inflammation in HaCaT cells
M5 cytokines cocktail (IL-17A, IL-22, oncostatin M, IL-1α and TNF-α) was widely used to establish psoriatic cell model, and we chose this model to investigate the role of GPR15LG on the regulation of psoriasis-related cytokines in vitro.First, we confirmed the effective down-regulation of GPR15LG mRNA by shRNAs (Figure 4A).Our data showed that M5 increased the expressions of IL-1α, TNF-α, IL-1β and S100A7 (Figure 4B).However, they were all down-regulated by GPR15LG knockdown in M5-treated HaCaT cells (Figure 4C).These findings, which were consistent with the results in vivo, suggest a pivotal role for GPR15LG in keratinocyte-mediated inflammation in psoriasis.

IL-1α and TNF-α alone promoted GPR15LG expression in psoriatic keratinocytes
We previously showed that GPR15LG expression was greatly elevated in M5-treated HaCaT cells, while we do not know which cytokines or cytokines could promote the expression of GPR15LG.In the present study, HaCaT cells were stimulated with 10 ng/ml recombinant IL-1α, TNF-α, OSM, IL-17A and IL-22 alone and we found that IL-1α and TNF-α alone could increased the expression level of GPR15LG mRNA in HaCaT cells (Figure 5A).

Discussion
In the present study, we investigated the role of GPR15LG in psoriatic inflammation.We found that the knockdown of Gpr15lg, the mouse ortholog of GPR15LG, is capable of ameliorating the severity of IMQ-induced psoriatic inflammation in mice.In addition, Gpr15lg knockdown significantly down-regulated levels of IL-1α, TNF-α, IL-1β and S100A7 in vivo.Furthermore, GPR15LG knockdown inhibited M5-induced inflammation in HaCaT cells in vitro.
The present study provided evidences that GPR15LG might participate in the progress of psoriasis via regulating keratinocyte-mediated inflammation.GPR15LG is a multifunctional protein implicated in the pathogenesis of several diseases.GPR15LG exhibits potent wide-spectrum antimicrobial activity and it could promote cutaneous wound healing [8,9].The role of GPR15LG in the regulation of inflammation has been previously reported.A study showed GPR15LG knockout mouse exhibits a decreased serum IgM level and an increased ratio of CD4+/CD8+ cells [10].Several groups independently showed GPR15LG is a ligand for GPR15 [11][12][13].It was found that GPR15LG is significantly elevated in psoriatic lesions [14,15].We previously showed that GPR15LG is involved in the proliferation of psoriatic keratinocytes [14].Recently, a study revealed a new role for GPR15LG in the inflammation and differentiation of keratinocytes [16].Furthermore, it is an epithelial inflammation-derived pruritogen in psoriasis [17].This line of evidence indicates that GPR15LG is critical for psoriasis development, and it may has proinflammation effect in psoriasis.However, the role it plays in psoriatic inflammation is largely unknown.
In this study, we evaluated the effects of Gpr15lg knockdown on IMQ-induced psoriatic inflammation in mice.The mice treated with IMQ exhibited typical psoriatic symptoms.While Gpr15lg knockdown significantly relieved those symptoms and improved both individual and cumulative PASI scores, and remarkably reduced the epidermal layers and inflammatory cells infiltration.Proinflammatory cytokines IL-1α, TNF-α, IL-1β and S100A7 have been reported to be up-regulated in psoriatic skins and they are involved in the psoriasis pathogenesis [6,18,19].In our study, the strong increase of IL-1α, TNF-α, IL-1β and S100A7 was observed in psoriasis-like lesions.While Gpr15lg knockdown exerted an inhibitory effect on the production of these cytokines.Results indicated that Gpr15lg knockdown could improve IMQ-induced psoriasis-like inflammation in mice.
Evidence demonstrated that epidermal keratinocytes play crucial roles in psoriasis [20][21][22].A study showed GPR15LG transfection increases the expression of TSLP, IL-1β, β-defensin 4, IL-6 and CXCL1 and reduces barrier gene expression in keratinocytes [16].M5 cytokines cocktail induces keratinocytes manifesting features of psoriatic keratinocyte in vitro [23].Our previous study showed that M5 cocktail greatly increased GPR15LG expression in HaCaT cells [14] and we chose this cell model to investigate the role of GPR15LG on psoriatic inflammation in vitro.We found GPR15LG knockdown down-regulated the expressions of IL-1α, TNF-α, IL-1β and S100A7 in M5-treated HaCaT cells, suggesting a pivotal role of GPR15LG in keratinocyte-mediated inflammation in psoriasis.
In the present study, we noted that IL-1α and TNF-α alone could induce the expression of GPR15LG in HaCaT cells, while more research is needed to clarified the mechanism for the induction of GPR15LG expression in future.
In summary, at the present study, we demonstrated that GPR15LG exhibited potent proinflammatory in psoriasis in vivo and in vitro.These results provide us with a deeper understanding of the role of GPR15LG in the pathogenesis of psoriasis at the fundamental level.