Up-regulation of Interferon-inducible protein 16 contributes to psoriasis by modulating chemokine production in keratinocytes

Psoriasis is a common chronic inflammatory skin disease characterized by epidermal hyperplasia and dermal inflammation. Keratinocyte activation is known to play a critical role in psoriasis, but the underlying mechanism remains unclear. Interferon-inducible protein 16 (IFI16), an innate immune system sensor, is reported to affect keratinocyte function. We therefore hypothesized that IFI16 promotes psoriasis by modulating keratinocyte activation. In the present study, we cinfirmed that IFI16 was overexpressed in epidermal keratinocytes of psoriasis patients. In addition, psoriasis-related cytokines, including IFN-γ, TNF-α, IL-17 and IL-22, induced IFI16 up-regulation in keratinocytes via activation of STAT3 signaling. We also observed that IFI16 activated the TBK1-NF-κB signaling, leading to the production of CXCL10 and CCL20. Importantly, knocking down p204, which is reported as the mouse orthologous of human IFI16, inhibited epidermal hyperplasia in mice with imiquimod-induced psoriasiform dermatitis. These findings indicate that IFI16 plays a critical role in the pathogenesis of psoriasis and may be a potential therapeutic target.

IFI16 has been implicated in the progression of various inflammatory diseases, including systemic lupus erythematosus (SLE), Sjögren's syndrome (SS) and systemic sclerosis (SSc) [19][20][21] . Recent studies have shown that IFI16 was also expressed in the cytoplasm and nucleus of primary keratinocytes and overexpressed in the epidermis of psoriasis patients 11,22 . However, whether IFI16 plays a pathogenic role in psoriasis and the underlying mechanism remains unknown.
Given that IFI16 is overexpressed in psoriatic keratinocytes and keratinocyte activation is related to psoriasis, we conjectured that IFI16 contributes to the development of psoriasis by modulating keratinocyte function. To test this hypothesis, we assessed IFI16 expression in the lesions of psoriasis patients, and examined the mechanism of IFI16-mediated keratinocyte activation in vitro. Further, we used siRNA to knock down p204 in a mouse model of imiquimod (IMQ)-induced psoriasis-like dermatitis, identifying that IFI16 could promote the development of psoriasis in vivo.

Results
IFI16 is overexpressed in the epidermis of human psoriatic skin. Frist, we examined the expression of IFI16 in the epidermis and dermis of skin samples obtained from psoriasis patients and normal donors. The quantitative (q)PCR results showed that IFI16 mRNA level was significantly increased in both the epidermis and dermis of psoriatic lesions as compared to normal skin samples (Fig.1A,B), while the threshold cycle (CT) value of IFI16 in the dermis of both patients and normal control individuals were extremely high (data not shown), indicating that IFI16 is seldom expressed in the dermis. The western blotting showed that IFI16 protein (three isoforms) was overexpressed in the epidermis of psoriatic lesions (Fig. 1C), but was barely detectable in the dermis (Fig. 1D). Furthermore, the immunohistochemistry and immunofluorescence staining revealed that IFI16 was expressed only in the basal layer of normal skin, but highly expressed in the thickened epidermis of psoriasis patients, primarily in the nuclei of keratinocytes (Fig. 1E,F). These results suggested that IFI16 is overexpressed in the psoriatic lesions, especially in the epidermis.
Cytokines induce the up-regulation of IFI16 expression in keratinocytes via activation of STAT3 signaling. We next examined the mechanism underlying the up-regulation of IFI16 in keratinocytes of psoriatic epidermis. IFN-γ , TNF-α , IL17, and IL22 are known to induce keratinocyte activation in psoriasis 3,4 ; the presence of dsDNA is also an trigger of psoriasis 23 . We therefore investigated the effect of these factors on IFI16 expression in keratinocytes. As shown in Supplementary Fig. S1, IFN-γ , TNF-α , IL-17 and IL-22 could up-regulate IFI16 expression in human primary keratinocytes in a dose-dependent manner ( Supplementary Fig. S1A-D). We then incubated cells with 10 ng/ml IFN-γ , 50 ng/ml TNF-α , 20 ng/ml IL-17A and 20 ng/ml IL-22, and evaluated IFI16 expression at 0 h, 6 h, 12 h, 24 h, 36 h and 48 h. As a result, the mRNA level of IFI16 was up-regulated in the presence of these cytokines in a time-dependent manner and reached to the peak at 24 h (Supplementary Fig. S1E-H).
Then, the IFI16 expression in human primary keratinocytes cultured with the presence of IFN-γ (10 ng/ml), TNF-α (50 ng/ml), IL17A (20 ng/ml), IL22 (20 ng/ml), poly (dA:dT) (2 μ g/ml) or a combination of these cytokines for 24 h was evaluated by qPCR, western blotting, and immunofluorescence. These results showed that IFI16 expression in keratinocytes was up-regulated when treated with each of these components, and most highly expressed with the cytokine cocktail. Besides, cytokines induced IFI16 expression to a greater degree than poly (dA:dT) ( Fig. 2A,B). The immunofluorescence analysis revealed that IFI16 was slightly detected in the nuclei of untreated human primary keratinocytes, but obviously overexpressed and localized in both the nucleus and cytoplasm after cytokine stimulation (Fig. 2C).
To identify the signaling pathway by which cytokines modulate IFI16 expression, we pretreated cells with inhibitors targeting mitogen-associated protein kinase (MAPK), STAT3, or NF-κ B, and then assessed mRNA and protein levels of IFI16. These results showed that only STAT3 inhibition suppressed cytokine-induced IFI16 expression (Fig. 2D,E), suggesting that psoriasis-related cytokines stimulate IFI16 expression in keratinocytes via the activation of STAT3 signaling.
To confirm the effect of IFI16 on CXCL10 and CCL20 expression in keratinocytes, we treated IFI16-deficient cells with poly(dA:dT) or the cytokine cocktail. Unexpectedly, the qPCR results showed that IFI16-knockdown could not suppress the mRNA expressions of CXCL10 and CCL20 induced by dsDNA (P = 0.272, 0.399, respectively; Fig. 3A,E), but significantly decrease the cytokine cocktail-induced expressions of CXCL10 and CCL20 at both the mRNA and protein levels (Fig. 3B,D,F,H). Besides, the mRNA and protein levels of CXCL10 and CCL20 were also increased in cells overexpressing IFI16 (Fig. 3C,D,G,H). Taken together, these data suggest that IFI16 mediates the high expression of CXCL10 and CCL20 in keratinocytes.
Scientific RepoRts | 6:25381 | DOI: 10.1038/srep25381 IFI16 induces the expression of CXCL10 and CCL 20 via the TANK-binding kinase (TBK)1-NF-κB signaling pathway in keratinocytes. Given that IFI16 protein up-regulated the expressions of CXCL10 and CCL20 in vitro, we determined to investigate the pathway through which IFI16 exerted this effect. It has been proved that CXCL10 and CCL20 could be induced by the activation of the NF-κ B signaling 24,25 . We therefore investigated whether IFI16 induces the production of CXCL10 and CCL20 via the NF-κ B signaling pathway. The expression level of phospho-p65 was increased in human primary keratinocytes overexpressing IFI16 and decreased in those deficient in IFI16, as determined by immunofluorescence and western blotting (Fig. 4A,B). Moreover, pre-treating IFI16-overexpressing cells with NF-κ B inhibitor reduced both mRNA and protein levels of CXCL10 and CCL20 ( Fig. 4D-G). These results suggest that IFI16 induces CXCL10 and CCL20 expressions via activation of NF-κ B signaling in keratinocytes.
On the other hand, the TBK1 has been well explored as the downstream molecule of IFI16 11 . Thus, we also explored whether IFI16 activated the NF-κ B signaling through TBK1. We treated IFI16-overexpressed keratinocytes with the siRNA targeting TBK1, and found that the phosphorylation of p65 was restrained (Fig. 4C) and secretion of CXCL10 and CCL20 were reduced ( Fig. 4D-G). These results indicate that IFI16 induces CXCL10 and CCL20 expressions via TBK1-NF-κ B signaling in keratinocytes.
Local depletion of p204 alleviates psoriatic lesions. To determine whether IFI16 plays a critical role in the pathogenesis of psoriasis, we explored the function of p204-the mouse orthologous of the human IFI16 protein as reported in several studies 7 in a mouse model of IMQ-induced psoriasis-like dermatitis. We firstly examined the expression of p204 in the epidermis and dermis in IMQ-induced mice. The p204 transcript was up-regulated in the epidermis after IMQ treatment, reaching a peak on day 1 (Fig. 5A), but was unaltered in the Values represent mean ± SD. **P < 0.01, ***P < 0.001, vs. no treatment; # P < 0.001, vs. dimethylsulfoxide (DMSO). dermis (Fig. 5B). Similar results were observed for p204 protein between day 2 and 6 ( Fig. 5C). The immunohistochemistry analysis also revealed that p204 expression was increased in the epidermis of mice's skin lesions after IMQ treatment (Fig. 5D), implying that IFI16 (p204) contributed to the development of psoriasis not only at a very early stage, but also throughout the whole disease progression.
Subsequently, p204 siRNA was locally applied every 48 h to the ear skin of IMQ-treated mice to suppress p204 levels in the epidermis, with the interference efficiency determined by western blotting and immunohistochemistry ( Supplementary Fig. S5). Notably, p204 knockdown ameliorated psoriatic lesions (Fig. 6A), caused a decrease in ear thickness (Fig. 6B), reversed epidermal hyperplasia (Fig. 6C,D), and reduced the number of inflammatory cells in the dermis (Fig. 6E). Importantly, CXCL10 and CCL20 expressions were decreased in the psoriasis-like lesions in the absence of p204 (Fig. 6F-I), which was accompanied by a decrease in the phospho-p65 level and inhibition of p65 nuclear translocation in epidermal cells (Fig. 7A,B). Taken together, these data receal that local depletion of IFI16 ameliorates psoriatic lesions in vivo.

Discussion
This study demonstrated that IFI16 was highly expressed in the epidermis of lesions from psoriasis patients.
In vitro results showed that several psoriasis-related cytokines, including IFN-γ , TNF-α , IL-17 and IL-22, could up-regulate IFI16 expression in keratinocytes via activation of STAT3 signaling. Moreover, IFI16 induced CXCL10 and CCL20 production in keratinocytes via TBK1-NF-κ B signaling. Importantly, in a mouse model of psoriasis, p204 knockdown improved epidermal hyperplasia, alleviated skin inflammation and reduced the  Keratinocyte activation is thought to be critical for the initiation and acceleration of psoriasis 3,4 . Recent studies have shown that danger-associated molecular patterns (DAMPs) and inflammatory cytokines can activate keratinocytes to produce cytokines and chemokines, respectively 3,4,11,23 . However, the mechanism of keratinocyte activation in psoriasis is not well understood. Chiliveru et al. have found that IFI16 is up-regulated in psoriatic skin lesions and localized to the cytoplasm in a subpopulation of cells. In our study, we confirmed that IFI16 was overexpressed in the epidermis of psoriatic patients, and in vivo evidences suggested that knocking down p204 in imiquimod-treated mice successfully improved epidermal hyperplasia and alleviated skin inflammation, which demonstrated that IFI16 contributes to psoriasis development and may be targeted in psoriasis treatment.
IFI16 belongs to the IFN-inducible PYHIN-200 gene family and acts as a DNA sensor in many cell types, including dendritic cells, myeloid cells, B cells and keratinocytes 9,17,26,27 . IFI16 overexpression has been observed in epidermal lesions of various autoimmune diseases, such as SLE, SSc, and psoriasis 11,22,28,29 . However, no prior studies have addressed the pathogenetic role of IFI16 in these diseases. We found that besides cytosolic dsDNA, the psoriasis-related cytokines IFN-γ , TNF-α , IL-17 and IL-22 also induced the up-regulation of IFI16 in keratinocytes via STAT3 signaling, which is partly consistent with a recent study demonstrating that TNF-α and IL-1β enhance IFI16 expression induced by cytosolic dsDNA in human primary keratinocytes 11,28,29 . In addition, our in vivo data suggested that p204 expression was up-regulated in epidermal keratinocytes in both the early stage of psoriasis and throughout the disease progression.
IFI16 has been shown to promote type I IFN production through the STING-TBK1-IRF3 signaling pathway 30 . Previous studies have reported that after treatment with cytosolic DNA together with TNF-α or IL-1β , IFI16 colocalized with DNA and STING in the cytoplasm and recruited TBK1 in keratinocytes 11 . In addition, IFI16 also has the ability to recruit ASC and forms a functional IFI16-ASC inflammasome, producing the functional IL-1β and IL-18 10,31 . Notably, our in vitro and in vivo data indicated that IFI16 was not involved in cytokine-mediated production of type I IFNs, IL1β , or IL18 in keratinocytes, but was required for the expression of CXCL10 and CCL20, which are also important factors in the immune response of psoriatic lesions. For instance, CXCL10 is detected in both skin lesions and serum obtained from psoriasis patients, and decreasing the expression of CXCL10 successful improved the psoriatic active plaques 32,33 . Moreover, an increase in lesional CCL20 expression is found to incude the recruitment of CCR6 + helper T 17 cells in psoriasis 34 . Given that IFI16 can be up-regulated by T cell-derived cytokines as previously described, a "T cells-IFI16-chemokines-T cells" loop may exist in psoriatic lesion, which leads to the enhancement of local inflammation and contributes to the progression of psoriasis.
The NF-κ B signaling pathway is reported as the upstream of CXCL10 and CCL20 24,25 , and is activated in keratinocytes of psoriatic lesions 35,36 . In agreement with the observation that IFI16 can mediate NF-κ B activation 30 , we found that IFI16 promotes the phosphorylation of the NF-κ B p65 subunit and its translocation into the nucleus both in vitro and in vivo, and that inhibiting the NF-κ B pathway abrogated IFI16-induced up-regulation of CXCL10 and CCL20 expression. Moreover, blocking TBK1 expression in IFI16-overexpressed keratinocytes suppressed the activation of NF-κ B signaling and reduced the expression of CXCL10 and CCL20, confirming that IFI16 modulates these important factors through TBK1-NF-κ B signaling pathway. Therefore, our results indicate that IFI16 plays a critical role in keratinocyte activation in psoriasis.
Accumulated studies have indicated that the subcellular localization of IFI16 is likely to determine its function 37 , and cytoplasmic IFI16 is tended to evoke immune response by sensing cytosolic DNA 38,39 . In psoriatic lesions, cytoplasmic IFI16 is detected in 5-8% of cells, indicating it may reflect immune response in psoriasis 11 . However, exposure of keratinocytes to ultraviolet B light causes the translocation of IFI16 protein from nucleus to cytoplasm, leading to cell apoptosis rather than activation of an immune response 40 . We found that IFI16 was up-regulated in the epidermis of psoriatic lesions and is mainly localized in the nucleus. Therefore, we focused on the biologic functions of IFI16 in the progression of psoriasis, but not its subcellular localization. Reconciling these observations and clarifying the mechanism of immune activation by IFI16 will be the focus of future studies.
In conclusion, we demonstrate that the cytokines-mediated overexpression of IFI16 contributes to the pathogenesis of psoriasis by activating the TBK1-NF-κ B signaling pathway and then inducing the production of CXCL10 and CCL20 in keratinocytes. Our findings consider IFI16 functions as a central hub in the initiation of immune response in psoriasis. Nevertheless, further studies of IFI16 are needed to fully clarify the pathogenic role of IFI16 and evaluate its potential to be a therapeutic target in psoriasis.

Methods
Patients and skin samples. Psoriasis patients with a dermatologist-confirmed diagnosis of chronic plaque psoriasis (12 females and 9 males; age range, 18-59 years; mean age, 32.2 years) and normal volunteers (6 female and 5 male; age range, 21-43 years; mean age, 31.2 years) were recruited for this study. Patient information is shown in Supplementary Table S1. The psoriasis patients required Psoriasis Area Severity Index (PASI) of at least 8 and a typical lesion of at least 1 cm in size that was suitable for biopsy. One 5 mm biopsy was obtained from each normal volunteer. All of the patients enrolled in our study had no other autoimmune or systemic diseases, and the target lesion and surrounding 5 cm area were not treated with any therapeutic measures for at least 2 weeks before the biopsy. Skin samples were incubated with 2.5 mg/ml dispase (Gibco, Grand Island, NY, USA) for 8 h at 4 °C to separate the epidermis from the dermis. All participants provided written, informed consent for their participation. The study protocol was designed and carried out according to the principles of the Declaration of  Mice were treated daily with Aldara IMQ Cream (iNova, Chatswood, Australia) to induce a psoriasis-like mouse model 41 . To detect time-dependent expression of p204 in mice with the IMQ-induced psoriasis-like dermatitis, IMQ was locally applied to the shaved back of mice. Lesions were collected after 0, 1, 2, 3, 4, 5 and 6 days, respectively. To investigate the effect of p204 blockade on psoriasis, the siRNA targeting p204 (Ribobio, Guangzhou, China) for mice was modified by -OMe and -Chol to increase the stability and lipotropy of p204 siRNA molecule in vivo. We used emulsion matrix to blend the siRNA targeting p204 and topically applicated it on the ear skin of mice. For each mouse, the left ear was treated with 2 nmol siRNA mixed with 5 mg emulsion matrix every 48 h. The negative control (NC) siRNA was applied on the right ear in the same way. Both ears were treated with IMQ daily for continuative 6 days. On day 6, skin specimens were obtained from the mice for analysis. The p204 siRNA sequence was as follows: sense, 5′-CCGAAAGAACACAAUCUAUdTdT-3′; antisense, 3′-dTdTGGCUUUCUUGUGUUAGAUA-5′.
Cell lines and reagents. Human primary keratinocytes were obtained from prepuces obtained from healthy individuals who accepted circumcision. Informed consent was obtained from all donors. Keratinocytes grown to 40-60% confluence were stimulated with IL-17A (20 ng/ml), IL22 (20 ng/ml), IFN-γ (10 ng/ml), TNF-α (50 ng/ml), or a mixture of these cytokines (cocktail) for 48 h and then analyzed. Quantitative PCR (qPCR). Total RNA was isolated from cells or tissues using a Total RNA Extraction kit (Anmei Biologicals, Xi'an, China) and used to synthesize cDNA with the PrimeScript RT Reagent kit (Takara Bio, Ohtsu, Japan) according to the manufacturers' protocols. qPCR was performed as described in Supplementary Information online. Expression levels were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primer sequences are showed in Supplementary Table S2. Western blotting. Cells or tissues were lysed with cell lysis solution (DSL, Webster, TX, USA). The total protein concentration in lysates was measured with the bicinchoninic acid assay (Pierce, Rockford, IL, USA). Equal amounts of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes, which were probed with antibodies against IFI16 (Abcam, Cambridge, UK), p204 (Thermo Fisher Scientific, Waltham, MA, USA), p65 (Abcam) and phospho-p65 (Cell Signaling Technology). The information for these antibodies are shown in Supplementary Table S3. Immunohistochemistry. Skin tissues from human donors or mice were fixed in 4% formalin buffered solution and embedded in paraffin. 5 mm tissue sections were staining with hematoxylin and eosin (H&E). Immunohistochemistry was carried out as described in Supplementary information online. Sections were labeled with antibodies against p204 (Thermo Fisher Scientific), IFI16, CXCL10, and CCL20 (all from Abcam). The information for these antibodies are shown in Supplementary Table S3. Immunofluorescence. Immunofluorescence analysis of skin tissue sections and cultured cells were carried out as described in Supplementary information online. Tissue sections or cells were labeled with antibodies against IFI16, p65, and phospho-p65. The information for these antibodies are shown in Supplementary Table S3.
Enzyme-linked immunosorbent assay (ELISA). CXCL10 and CCL20 levels in cell culture media were detected using ELISA kits (Cusabio, Wuhan, China) according to the manufacturer's instructions. Statistical analysis. Each experiment was performed at least three times, and data were analyzed with the unpaired, two-tailed Student's t-test or by one-way analysis of variance using GraphPad Prism v.6.0 (GraphPad, La Jolla, CA, USA). Data were expressed as the mean ± SD. P-values < 0.05 were considered statistically significant.