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Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, Tokyo, JapanInstitute for Environmental and Gender Specific Medicine, Juntendo University Graduate School of Medicine, Chiba, Japan
Correspondence: Kei Ohnuma, Department of Therapy Development and Innovation for Immune Disorders and Cancers, Graduate School of Medicine, Juntendo University, 2-1-1, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Psoriasis is a chronic inflammatory skin disease characterized mainly by epidermal hyperplasia, scaling, and erythema; T helper 17 cells have a role in its pathogenesis. Although IL-26, known as a T helper 17 cytokine, is upregulated in psoriatic skin lesions, its precise role is unclear. We investigated the role of IL-26 in the imiquimod-induced psoriasis-like murine model using human IL-26 transgenic mice. Erythema symptoms induced by daily applications of imiquimod increased dramatically in human IL-26 transgenic mice compared with controls. Vascularization and immune cell infiltration were prominent in skin lesions of human IL-26 transgenic mice. Levels of fibroblast growth factor (FGF) 1, FGF2, and FGF7 were significantly upregulated in the skin lesions of imiquimod-treated human IL-26 transgenic mice and psoriasis patients. In vitro analysis demonstrated that FGF1, FGF2, and FGF7 levels were elevated in human keratinocytes and vascular endothelial cells following IL-26 stimulation. Furthermore, IL-26 acted directly on vascular endothelial cells, promoting proliferation and tube formation, possibly through protein kinase B, extracellular signal–regulated kinase, and NF-κB pathways. Moreover, similar effects of IL-26 were observed in the murine contact hypersensitivity model, indicating that these effects are not restricted to psoriasis. Altogether, our data indicate that IL-26 may be a promising therapeutic target in T cell–mediated skin inflammation, including psoriasis.
). Initiating events cause release of cytokines by keratinocytes, recruiting neutrophils and macrophages to inflammatory sites and activating dendritic cells. Release of cytokines such as IFN-α by dendritic cells induces Th helper (Th) 1 and Th17 T cell differentiation to contribute to the psoriatic events. The proinflammatory cascade continues with additional recruitment of inflammatory cells and cause hyperproliferation of the epidermal layer (
Th17 cells play an important role in the pathogenesis of psoriasis and other inflammatory disorders by producing cytokines promoting keratinocyte proliferation and other psoriatic changes (
). We recently showed that IL-26 receptor is expressed on both human and murine fibroblasts, and IL-26 activates fibroblasts, leading to increased collagen production (
). IL-26 induces monocyte and natural killer cell production of selected cytokines, including TNF-α, upregulates cell surface tumor necrosis factor–related apoptosis-inducing ligand expression and promotes generation of Th17 cells (
Imiquimod 5% cream for the treatment of actinic keratosis: results from a phase III, randomized, double-blind, vehicle-controlled, clinical trial with histology.
). The topical application of IMQ-containing cream to the skin of mice is now widely accepted as a convenient and cost-effective murine model for studying early events of psoriasis (
In the present study, because the gene encoding IL-26 is absent in mice, we investigate the role of IL-26 in the IMQ-induced psoriasis-like murine model using human IL-26 bacterial artificial chromosome transgenic (hIL-26Tg) mice (
). We found that vascularization and immune cell infiltration induced by daily applications of IMQ were enhanced dramatically in hIL-26Tg mice, and associated with increased expression of fibroblast growth factor (FGF) 1, FGF2, and FGF7 in the skin lesions. Moreover, the effect of IL-26 on angiogenesis and inflammation was commonly observed in the murine contact hypersensitivity model. These results strongly suggest that IL-26 may represent a promising therapeutic target for T cell–mediated skin inflammation, including psoriasis and contact hypersensitivity reactions.
Results
IL-26 exacerbates skin inflammation by inducing vascular invasion and immune cell infiltration in the IMQ-induced psoriasis model
Although the IL-26 gene is absent in rodents, IL-20RA and IL-10RB,which are part of the IL-10 family of cytokine receptors, are also expressed in mice, and human IL-26 functions in both human and murine cells (
). To explore the role of IL-26 in the pathology of psoriasis, hIL-26Tg mice were compared with Δ conserved noncoding sequence (CNS)-77 transgenic (Tg) mice (control Tg mice with deleting human IL-26 transcription) utilizing the IMQ-induced psoriasis model. We applied daily 40 mg of IMQ cream on the back skin of each mouse and assessed disease severity every day utilizing the clinical Psoriasis Area and Severity Index (PASI) score. We first confirmed that there was no significant difference in the appearance and the PASI scores between ΔCNS-77 Tg mice and C57BL/6 wild-type mice (Supplementary Figure S1 online), validating the use of ΔCNS-77 Tg mice as a control group compared with hIL-26Tg mice in this study. The back skin of hIL-26Tg mice appeared markedly affected and especially exhibited increased erythema symptoms compared with ΔCNS-77 Tg mice, and all of the PASI scores of hIL-26Tg mice were higher than those of control mice (Figure 1a, 1b). Excessive blood vessel formation and vascular invasion were observed in subcutaneous tissues of hIL-26Tg mice (Figure 1c, 1d). Moreover, CD31-positive blood vessels were markedly increased in the back skin of hIL-26Tg mice (Figure 1e). We next conducted histologic studies of IMQ-induced psoriatic skin. There was increased infiltration of blood cells and blood vessels in the skin of hIL-26Tg mice compared with control mice from day 2 (Figure 1f).
Figure 1Markedly enhanced angiogenesis is observed in psoriatic skin lesions of IMQ-applied hIL-26Tg mice. (a) Phenotypical representation of IMQ-induced skin inflammation in each mice. (b) Time course of Psoriasis Area and Severity Index scores (erythema, thickness, and scaling were scored daily on a scale from 0 to 4, respectively) in each mice. (c, d) Subcutaneous vascular formation of IMQ-treated back skin in each mice (c) was measured by ImageJ software (d). (e, f) Immunofluorescence staining (CD31, green) (e) or HE staining (f) of IMQ-treated back skin from each mice on day 3. Scale bar = 200 μm, 100 μm (right panels in f). n = 8 mice for each group at each time point. (b, d) Mean ± standard error of the mean of each group. ∗P < 0.01. HE, hematoxylin and eosin; hIL-26Tg, human IL-26 transgenic; IMQ, imiquimod.
The infiltrating cell types in the IMQ-induced psoriatic skin of hIL-26Tg mice were then characterized by flow cytometry. The cell type with the most prominent difference between hIL-26Tg mice and ΔCNS-77 Tg mice was neutrophils (Figure 2a). In addition, the number of TCRβ+CD4+ T cells infiltrating the skin lesions of hIL-26Tg mice was significantly increased compared with ΔCNS-77 Tg mice on day 3 and day 4 (Figure 2b). There was no significant difference in the number of TCRβ+CD8+ T cells and γδ T cells between hIL-26Tg mice and control mice (Figure 2c, 2d), and similar results were also seen with macrophages and mast cells (data not shown). To better evaluate the level of neutrophil invasion, we performed immunofluorescence staining for Ly6g. Ly6g-positive cells were markedly increased in the back skin of hIL-26Tg mice (Figure 2e). mRNA expression levels of the neutrophil recruitment chemokines CXCL1 and CXCL2 in the skin were also markedly increased in hIL-26Tg mice compared with ΔCNS-77 Tg mice (Supplementary Figure S2 online). Taken together, these results indicate that IL-26 exaggerates the severity of psoriatic skin inflammation by inducing angiogenesis and immune cell infiltration.
Figure 2IL-26 enhances infiltration of neutrophils and CD4+ T cells in IMQ-induced psoriatic skin lesions. Absolute cell numbers of neutrophils (a), CD4+ T cells (b), CD8+ T cells (c), and γδ T cells (d) in IMQ-treated skin lesions from each mice (n = 5 mice for each group at each time point) were quantified by flow cytometry. Single-suspension cells isolated from the skin lesions were analyzed, and the percentages of CD11b+CD11clow/negaLy6G+F4/80nega (a), TCRβ+TCRγδnegaCD4+CD8nega (b), TCRβ+TCRγδnegaCD4negaCD8+ (c), or TCRβnegaTCRγδ+ (d) were calculated. Mean ± standard error of the mean of each group. ∗P < 0.01. (e) Immunofluorescence staining (Ly6g, green) of IMQ-treated back skin from each mice on day 3. n = 4 mice for each. Scale bar = 200 μm. IMQ, imiquimod.
IL-26 upregulates expression of FGF1, FGF2, and FGF7 in IMQ-induced psoriasis model
To elucidate the molecular mechanism associated with IL-26–dependent angiogenesis, we examined the kinetics of mRNA expression of neoangiogenesis factors. mRNA expression of human IL-26 was markedly increased in the skin of hIL-26Tg mice following daily application of IMQ cream, whereas no expression of human IL-26 was detected in the skin of ΔCNS-77 Tg mice (Figure 3a). Expression of vascular endothelial growth factor (VEGF)-A was gradually increased from day 1, but the difference was hardly observed between hIL-26Tg mice and control mice (Figure 3a). On the other hand, in the skin lesions from hIL-26Tg mice, the mRNA expression levels of VEGF-C, epidermal growth factor, hypoxia-inducible factor1-α, FGF1, FGF2, FGF7, angiopoietin-1, and angiopoietin-2 were significantly increased compared with control mice (Figure 3a). There was no marked difference in expression levels of VEGF-D, TNF-α, and TYMP between hIL-26Tg mice and control mice (Supplementary Figure S2).
Figure 3IL-26 increases levels of FGF1, FGF2, FGF7, and other biomarkers associated with angiogenesis in IMQ-induced psoriasis-like skin inflammation. (a) Kinetics of mRNA expression levels of IL-26 or angiogenesis factors in IMQ-treated skin lesions from each mice (n = 6 mice for each group at each time point). Mean ± standard error of the mean of each group. ∗P < 0.01. (b, d) Immunofluorescence staining of IMQ-treated back skin from each mice on day 3 (b) or human skin samples (d) using anti-CD4 (red) plus IL-26, FGF1, FGF2, or FGF7 (IL-26 and FGFs, green). Scale bar = 200 μm, 100 μm (right panels of CD4/IL-26 in b, lower panels in d). (c) HE staining of human skin samples. Scale bar = 100 μm. For each, n = 4 mice (b), n = 3 (c, d). ANGPT, angiopoietin; EGF, epidermal growth factor; FGF, fibroblast growth factor; HE, hematoxylin and eosin; HIF, hypoxia inducible factor; IMQ, imiquimod; ND, not detectable.
Although VEGF-A is a particularly important angiogenic factor, FGF2 also functions as a main growth factor associated with angiogenesis, and the serum level of FGF2 is increased and significantly correlated with PASI value in psoriasis patients (
Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: an autocrine mechanism contributing to angiogenesis.
). We therefore focused on FGF1, FGF2, and FGF7 and examined their protein expression levels by immunofluorescence staining of the IMQ-induced skin lesions. IL-26–producing cells (shown in green, Supplementary Figure S3 online) were hardly observed in hIL-26Tg and control mice at day 0, whereas these cells were clearly detected in the dermis, particularly under the basement membrane of hIL-26Tg mice (Figure 3b). In addition, the infiltration of CD4-positive cells (shown in red) was prominent in the skin of hIL-26Tg mice as compared with control mice (Figure 3b). Likewise, prior to IMQ application (day 0), protein expression of FGF1, FGF2, and FGF7 was hardly observed both in hIL-26Tg and control mice (Supplementary Figure S3), while expression of FGF1, FGF2, and FGF7 was remarkably increased in hIL-26Tg mice (Figure 3b). In IMQ-treated hIL-26Tg mice, FGF1 was seen in the epidermal tissue, particularly in the upper layer around the corneum, and the expression of FGF2 and FGF7 was observed in the dermis and adipose tissue, under the basement membrane and around blood vessels. These results indicate that IL-26 enhances the expression of FGF1, FGF2, and FGF7 in the IMQ-induced skin. To confirm the expression and localization of IL-26 and FGFs in humans, we conducted immunofluorescence staining of human skin specimens obtained from patients with psoriasis and healthy volunteers. Histologic findings of the skin specimens of psoriasis patients revealed characteristics of psoriatic skin, such as acanthosis, parakeratosis, papillomatosis, and infiltration of inflammatory cells (Figure 3c). Similar to these findings, protein expression of FGF1, FGF2, FGF7, and IL-26 was markedly increased in the skin lesions of psoriasis patients compared with healthy controls (Figure 3d). These findings suggest that IL-26 and FGFs play an important role in the pathophysiology of psoriasis in humans, as well as the IMQ-induced psoriasis model.
IL-26 enhances the production of FGF1, FGF2, and FGF7 from keratinocytes and vascular endothelial cells
We hypothesized that IL-26 induces angiogenesis in psoriatic skin through production of FGFs from the cells constituting the skin tissue. To validate this assumption, we examined FGF production from normal human epidermal keratinocytes (NHEKs) and normal human dermal fibroblasts (NHDFs) by ELISA following IL-26 stimulation. FGF1 and FGF2 production from NHEKs was enhanced by IL-26 stimulation in a dose-dependent manner (Figure 4a, 4b). Meanwhile, constitutive production of FGF2 and FGF7 from NHDFs was not influenced by IL-26 treatment (Figure 4b, 4c). We next investigated whether IL-26 directly affected FGF expression in vascular endothelial cells. IL-26 enhanced FGF2 production from human umbilical vein endothelial cells (HUVECs) (Figure 4b). Although the amount of FGF7 produced from HUVECs was relatively small compared with NHDFs, IL-26 also enhanced FGF7 production from HUVECs (Figure 4c).
Figure 4Stimulation with IL-26 and psoriasis-associated cytokines induces enhanced expression of FGF1, FGF2, and FGF7 in keratinocytes and vascular endothelial cells.(a, b, c) NHEKs, NHDFs, and HUVECs were stimulated with IL-26 for 24 hours. Production of FGF1 (a), FGF2 (b), or FGF7 (c) was evaluated using specific ELISA. The dashed lines indicate the detection limit. NHEKs (d), NHDFs (e), and HUVECs (f) were stimulated with IL-26 alone (10 ng/ml) or in combination with PAC (10 ng/ml each) for 6 hours. mRNA expression levels of FGF1, FGF2, or FGF7 were quantified by quantitative real-time reverse transcriptase PCR. (a–f) Mean ± standard deviation of triplicate samples. ∗P < 0.01. FGF, fibroblast growth factor; HUVEC, human umbilical vein endothelial cells; IMQ, imiquimod; ND, not detectable; NHDF, normal human dermal fibroblast; NHEK, normal human epidermal keratinocyte; PAC, psoriasis-associated cytokines.
Stimulation of inflammatory cytokines and antimicrobial peptides, such as IL-17 and LL-37, enhances angiogenesis in psoriasis through the induction of angiogenic factors from keratinocytes (
). We therefore examined the effect of IL-26 in combination with various psoriasis-associated inflammatory cytokines on expression of FGFs. We examined mRNA expression levels of FGF1 and FGF2 in NHEKs, and FGF2 and FGF7 in NHDFs and HUVECs by quantitative real-time reverse transcriptase PCR. Consistent with our findings regarding protein production, stimulation with IL-26 alone enhanced expression levels of FGF1 and FGF2 in NHEKs (Figure 4d), and FGF2 and FGF7 in HUVECs (Figure 4f), while having no effect on FGF2 and FGF7 levels in NHDFs (Figure 4e). Intriguingly, stimulation with IL-26 in combination with psoriasis-associated cytokines resulted in much greater enhancement in mRNA expression levels of FGF1 and FGF2 in NHEKs and FGF2 and FGF7 in HUVECs (Figure 4d, 4f). Among psoriasis-associated cytokines used in this study, we found that IL-1β in NHEKs and IFN-α in HUVECs were key cytokines for enhancing FGF2 expression in synergy with IL-26 (Figure 4d, 4f). Moreover, addition of IFN-α plus IFN-γ resulted in increased enhancement of FGF7 expression in HUVECs in synergy with IL-26 (Figure 4f). There were no specific factors identified for enhancing FGF1 expression in NHEKs. On the other hand, stimulation with psoriasis-associated cytokines without IL-26 led to elevated expression of FGF2 and FGF7 in NHDFs, but no additional enhancement was observed when cells were stimulated with these cytokines in the presence of IL-26 (Figure 4e). These results indicate that IL-26 acts directly on not only keratinocytes, but also vascular endothelial cells to enhance production of FGFs.
IL-26 acts directly on vascular endothelial cells, resulting in enhanced proliferation and tube formation
We next examined IL-26 effect on vascular endothelial cell function. For this purpose, we assayed for HUVECs proliferation and tube formation following IL-26 stimulation. Surprisingly, IL-26 promoted proliferation and tube formation of HUVECs in a dose-dependent manner, similar to VEGF effect (Figure 5a, 5b). We confirmed that both FGF2 and FGF7 enhanced HUVECs proliferation and tube formation (Supplementary Figure S4 online). From these results, we next conducted knockdown experiments using small interfering RNA (siRNA) against FGF2 and FGF7 in HUVECs to determine whether FGF2 or FGF7 production was involved in HUVECs activation following IL-26 stimulation. Expression level of FGF2 and FGF7 in HUVECs stimulated with IL-26 or vehicle was determined by quantitative real-time reverse transcriptase PCR in the presence of control siRNA or two different sequences of FGF2-siRNA or FGF7-siRNA. FGF2-siRNA treatment clearly reduced FGF2 expression as compared with control siRNA or FGF7-siRNA, which was associated with a significant decrease in IL-26–stimulated proliferation and tube formation of HUVECs (Figure 5c, 5e, 5f). Treatment with FGF7-siRNA markedly reduced FGF7 expression compared with control siRNA or FGF2-siRNA (Figure 5d), which was associated with a partial reduction in IL-26–stimulated HUVECs tube formation, and only a slight effect on HUVECs proliferation (Figure 5e, 5f). Similar results were also obtained with a different siRNA sense 2, as described in Materials and Methods (data not shown). On the other hand, stimulation with VEGF led to enhancement of HUVECs proliferation and tube formation regardless of FGF2 or FGF7-siRNA treatment (Figure 5e, 5f), strongly suggesting that the observed reduction in proliferation or tube formation of FGF2 or FGF7-siRNA–treated HUVECs following IL-26 stimulation was not due to non-specific toxicity of the transfection procedure or off-target effects. Similar results were observed in the experiment utilizing FGF2 or FGF7-neutralizing antibodies (Supplementary Figure S5 online). Given our findings on angiogenesis in the IMQ-induced psoriatic skin of hIL-26Tg mice, we conducted studies to investigate the direct effect of IL-26 on murine blood vessels by evaluating whether IL-26 stimulation induced vessel formation in mouse aortic rings. Aortic rings stimulated with IL-26 strongly produced sprouts in a dose-dependent manner (Figure 5g). Taken together, our data strongly suggest that IL-26 acts directly on vascular endothelial cells, resulting in enhanced FGF2 and FGF7 production and prominent blood vessel formation.
Figure 5IL-26 enhances proliferation and tube formation of HUVECs by inducing FGF2 and FGF7 production. HUVECs were stimulated with IL-26 or VEGF for 48 hours (a) or 9 hours (b). (a) Proliferation was assessed by cell confluence. (b) Tube formation was assessed by cell sprouts formation. Mean ± standard deviation of triplicate samples. ∗P < 0.01. Scale bar = 300 μm. HUVECs were transfected with siRNA and stimulated with IL-26 for 6 hours. mRNA expression levels were quantified by quantitative real-time reverse transcriptase PCR (c, d). Transfected HUVECs were stimulated with IL-26 or VEGF for 48 hours (e) or 9 hours (f), and assessed as described here. Mean ± standard deviation of triplicate samples. ∗P < 0.01. (g) Mouse aorta ring explants were stimulated with IL-26 or VEGF for 10 days. FGF, fibroblast growth factor; HUVEC, human umbilical vein endothelial cell; siRNA, small interfering RNA; VEGF, vascular endothelial growth factor.
PI3K-Akt, Raf-MEK-ERK, and IκB–NF-κB signalings are indispensable for IL-26–mediated activation of vascular endothelial cells
We next examined the signaling events in HUVECs following IL-26 stimulation. Downstream signaling of IL-26 involves the JAK–STAT3 pathway substantially, but other pathways mediated by Akt or ERK1/2 have been also reported (
). We therefore examined phosphorylation of Akt, ERK1/2, p38, c-Jun N-terminal kinase, IκB, and STAT3 in HUVECs following IL-26 stimulation by Western blotting. Stimulation with IL-26 resulted in the prominent phosphorylation of Akt, ERK1/2, p38, and IκB, whereas c-Jun N-terminal kinase and STAT3 phosphorylation was not significantly enhanced in HUVECs following IL-26 stimulation (Figure 6a). Of note, the intensity of Akt, ERK1/2, p38, and IκB phosphorylation following IL-26 stimulation was as strong as FGF2 stimulation (Figure 6a). To identify the signals involved in the activation of HUVECs following IL-26 stimulation, we examined the effect of signal inhibitors. The inhibitor against Akt, mitogen-activated protein kinase/ERK kinase 1/2, and NF-κB markedly inhibited HUVECs proliferation and tube formation following IL-26 stimulation in a dose-dependent manner (Figure 6b, 6c). In addition, to characterize the signals involved in FGF2 or FGF7 production from HUVECs, we performed quantitative real-time reverse transcriptase PCR analysis. The Akt inhibitor and NF-κB inhibitor suppressed expression of both FGF2 and FGF7 in HUVECs following IL-26 stimulation (Figure 6d, 6e). Taken together, these data strongly suggest that stimulation with IL-26 activates various signal pathways in HUVECs, and among them, PI3K-Akt, Raf-MEK-ERK, and IκB–NF-κB signaling events are particularly important for IL-26–mediated angiogenesis.
Figure 6PI3K-Akt, Raf–MEK-ERK, and IκB-NF-κB–mediated signaling are indispensable for HUVECs activation following IL-26 stimulation. (a) HUVECs were stimulated with IL-26 or FGF2. Phosphorylation of each protein was detected by immunoblotting. The same blots were stripped and reprobed with anti-pan protein antibodies. Band intensity of phospho-proteins was normalized to pan proteins, respectively. Mean ± standard error of the mean from three independent experiments. ∗P < 0.01. HUVECs were stimulated with IL-26 for 48 hours (b), 9 hours (c) or 6 hours (d, e) in the presence of vehicle or signal inhibitors. (b) Proliferation was assessed by cell confluence. (c) Tube formation was assessed by cell sprouts formation. (d, e) mRNA expression levels were quantified by quantitative real-time reverse transcriptase PCR. Mean ± standard deviation of triplicate samples. ∗P < 0.01. HUVEC, human umbilical vein endothelial cell; JNK, c-Jun N-terminal kinase; MEK, mitogen activated protein kinase/ERK kinase.
Finally, to determine whether the effect of IL-26 on angiogenesis and inflammation is specific for the IMQ-induced psoriasis model or can be commonly observed in other types of inflammatory models, we evaluated the role of IL-26 in the DNFB-induced contact hypersensitivity model (
). Similar to the IMQ-induced psoriasis model, excessive blood vessel formation and vascular invasion were observed in the ear skin of hIL-26Tg mice with DNFB-induced contact hypersensitivity (Supplementary Figure S6a online). Thickening of the ear skin was also enhanced in hIL-26Tg mice compared with ΔCNS-77 Tg mice (Supplementary Figure S6b). Moreover, IL-26–producing cells and CD31-positive blood vessels were clearly detected in the dermis of hIL-26Tg mice (Supplementary Figure S6c, S6d). Histologic findings showed that infiltration of inflammatory cells was increased in the ear skin of hIL-26Tg mice compared with control mice (Supplementary Figure S6e). These results indicate that IL-26 plays an important and potentially universal role in angiogenesis and inflammation in skin inflammatory lesions, including psoriasis and T cell–mediated contact hypersensitivity reactions.
Discussion
Our present work showed that vascularization and immune cell infiltration induced by daily applications of IMQ were dramatically enhanced in hIL-26Tg mice. We demonstrate that IL-26 acts directly on vascular endothelial cells and enhances proliferation and tube formation, at a level similar to VEGF. Moreover, the effect of IL-26 on angiogenesis and inflammation was commonly observed in the DNFB-induced contact hypersensitivity model, indicating that this angiogenic effect of IL-26 is not restricted to psoriasis but is also seen in T cell–mediated contact hypersensitivity reactions.
Our in vitro assay showed the direct effect of IL-26 on vascular endothelial cells to promote proliferation and tube formation, involving both FGF2 and FGF7. Besides FGF2 and FGF7, recombinant FGF1 enhanced both proliferation and tube formation of HUVECs (Supplementary Figure S4), suggesting that the markedly increased expression of FGF1 in the keratinocytes of IMQ-induced hIL-26Tg mice shown in Figure 3b may also be associated with in vivo blood vessel formation. In addition, because expression levels of various angiogenic factors other than FGFs, such as VEGF-C, angiopoietin-1, and angiopoietin-2, were increased in the IMQ-induced skin lesions of hIL-26Tg mice (Figure 3a), further work will be needed to better characterize the cellular mechanisms involved in in vivo IL-26–induced excessive blood vessel formation in inflammatory lesions. Thickening and scaling of the back skin were also enhanced in hIL-26Tg mice compared with ΔCNS-77 Tg mice (Figure 1b). Enhanced expression of FGF1, FGF2, and FGF7 in the IMQ-induced skin of hIL-26Tg mice was likely associated with the excessive and abnormal proliferation of keratinocytes. Furthermore, increased levels of neutrophils and CD4+ T cells infiltrating the skin lesions may exacerbate inflammatory responses, in turn affecting the thickness and scaling of the skin of hIL-26Tg mice.
Binding of IL-26 to a distinct cell surface receptor consisting of IL-20RA and IL-10RB results in functional activation via STAT3 phosphorylation (
). Our data indicated that IL-26 activated Akt, ERK1/2, p38, and IκB in HUVECs, whereas STAT3 phosphorylation was not clearly enhanced following IL-26 stimulation (Figure 6a). These results strongly suggest that the signals transduced in HUVECs following IL-26 stimulation were not mediated by the well-known receptor IL-20RA/IL-10RB. In fact, quantitative real-time reverse transcriptase PCR analysis did not detect mRNA expression of IL-20RA in HUVECs, while IL-10RB was easily detected (data not shown). IL-26 is an unusual cationic and amphipathic cytokine, closely resembling the structure of antimicrobial peptides (
). Because polycationic proteins bind to various molecules, it is possible that activation of vascular endothelial cells by IL-26 is mediated by a heretofore unknown receptor other than IL-20RA and IL-10RB.
The hIL-26Tg mice carry a 190-kb bacterial artificial chromosome transgene with the human IFNG gene and 90 kb of both upstream and downstream sequences, and distal regulatory elements in addition to human IFNG and IL26 genes are contained in this region (
). While immunofluorescence staining of skin sections of hIL-26Tg mice indicated that IL-26 was produced by CD4-positive cells as well as other cell types, the cells involved may differ in the IMQ-induced psoriasis model from psoriasis patients (Figure 3b, 3d). In addition to Th17 cells, other IL-26–producing cells have been identified recently (
). Further studies are needed to identify the signaling events regulating IL-26 expression in psoriatic patients and psoriasis murine model.
Localization of FGFs differed in human patients compared with the IMQ-induced murine model. In the mouse model, FGF2 was observed in the epidermis, dermis, and adipose tissue, while FGF7 was seen mainly in the dermis and adipose tissue (Figure 3b). Meanwhile, FGF2 and FGF7 expression was found mainly in the epidermal tissue, as well as in the dermis of psoriasis patients (Figure 3d). While the IMQ-induced psoriasis model is a widely accepted murine model for studying early events of psoriasis, it does not adequately replicate the chronic and complex human psoriatic inflammatory condition. Because we examined FGF expression in skin samples obtained from only three untreated psoriasis patients, additional in-depth work with more patient samples is needed to address important issues relating to FGF biology in psoriatic patients.
Invasion of blood vessels in the dermis is a histologic hallmark of psoriatic skin lesions (
). Our current work indicates that IL-26 plays a significant role in angiogenesis and leukocyte recruitment, and control of the excessive angiogenesis in the inflammatory skin lesions, including psoriasis and T cell–mediated contact hypersensitivity reactions by modulating IL-26 is potentially important in the clinical setting.
Materials and Methods
Cell culture and reagents
NHEKs were cultured in Humedia-KGM2 medium (Kurabo, Osaka, Japan). NHDFs were cultured in FGM-2 medium (Lonza, Walkersville, MD). HUVECs were cultured in EGM-2 medium (Lonza). Cells were cultured at 37°C in a humidified 5% CO2 incubator. For cell stimulation, recombinant human IL-1β, IL-6, IL-17A, IL-21, IL-22, IL-23, TNF-α, IFN-α, IFN-γ, and LL-37 (all the mixture was used as psoriasis-associated cytokines) were purchased from BioLegend (San Diego, CA). Recombinant human IL-26 dimer was purchased from R&D Systems (Minneapolis, MN). Commercial inhibitors used in this study are shown in Supplementary Table S1 online. siRNAs against FGF2 and FGF7 were purchased from ThermoFisher Scientific (Waltham, MA) (sequences are shown in Supplementary Table S2 online), and negative control siRNA (oligonucleotide sequences are not disclosed) was purchased from Qiagen (Hilden, Germany).
Antibodies
Commercial antibodies used in this study are shown in Supplementary Table S3 online. Mouse anti-human IL-26 monoclonal antibody (clone 69-10) was developed in our laboratory by subcutaneously immunizing BALB/c mice with recombinant human IL-26 protein (R&D Systems). Biotinylated anti-IL-26 monoclonal antibody was used for immunofluorescence staining.
Mice
hIL-26Tg mice and ΔCNS-77 Tg mice were kindly provided by Thomas Aune’s laboratory (
). The characteristics and the details of these mice are described in Supplementary Materials and Methods online. All mice used in this study were kept under specific pathogen-free facility in micro-isolator cages. Female mice at 8–12 weeks of age were used.
IMQ-induced psoriasis model
Mice received a daily topical dose of 40 mg 5% IMQ cream (Beselna Cream; Mochida Pharmaceutical, Tokyo, Japan) on the shaved back for 5 consecutive days. The severity of inflammation of the back skin was measured by an objective scoring system based on the clinical PASI (
). Subcutaneous vessels were measured by ImageJ software (National Institutes of Health, Bethesda, MD).
Clinical samples
Skin punch biopsies were obtained from three untreated psoriasis patients (two males, one female; aged 72, 55, and 77 years, PASI score, 7.8, 18, and 23.1) and three healthy controls (two males, one female; aged 69, 49, and 38 years) at Juntendo University Urayasu Hospital.
Proliferation assay for HUVECs
HUVECs or siRNA-transfected HUVECs (5 × 103) were incubated in the EGM-2 containing 2% fetal calf serum on 96-well plates (Corning, Tewksbury, MA) for 12 hours at 37°C, and then stimulated with IL-26 or each angiogenic factor in the presence or absence of signal inhibitors and neutralizing antibody. Cell growth was measured as cell confluence using IncuCyte ZOOM (Essen Biosciense, Ann Arbor, MI).
Tube formation assay
HUVECs or siRNA-transfected HUVECs (1 × 106) were incubated in EGM-2 containing 2% fetal calf serum for 12 hours at 37°C. After incubation, cells (1.5 × 103) were seeded on 50 μl of Cultex Basement Membrane Extract (R&D Systems) in a 96-well plate. Seeded cells were stimulated with IL-26 or each angiogenic factor. Tube form length was measured using the MetaMorph image analysis system (Molecular Device, Sunnyvale, CA).
Aortic ring assay
Mouse thoracic aortas and renal arteries were dissected from C57BL/6 mice at 6 weeks of age, and cleared of fat and connective tissues. Mouse aortas were cut into 0.5–1.0-mm-thick rings and embedded in 100 μl of Cultex Basement Membrane Extract containing IL-26 or VEGF. The aorta rings were cultured in 500 μl of EGM-2 containing 2% fetal calf serum for 10 days. Thereafter, images of endothelial sprouts and interconnected capillary tubes were observed using a microscope (Carl Zeiss, Oberkochen, Germany).
Methods for immunofluorescence staining and immunohistochemical staining of skin sections, quantitative real-time reverse transcriptase PCR, ELISA, Western blotting and flow cytometry
Data were analyzed by two-tailed Student t test for two-group comparison or by one-way analysis of variance test with Tukey’s for multiple comparison testing. The assay was performed in triplicate, and data are presented as mean ± standard deviation of triplicate samples of the representative experiment, or mean ± standard error of triplicate samples of independent experiments. Significance was analyzed using MS Excel (Microsoft, Redmond, WA) and values of P < 0.01 were considered significant and are indicated in the corresponding figures and figure legends.
Study approval
Human study protocols were approved by the Ethics Committees at Juntendo University and performed according to the principles set out in the Declaration of Helsinki. Written informed patient consent was obtained from all participants (three psoriasis patients and three healthy volunteers) prior to inclusion in this study. Animal experiments were conducted following protocols approved by the Animal Care and Use Committees at Juntendo University.
Conflicts of Interests
The authors state no conflict of interest.
Acknowledgments
The authors thank Yasushi Suga and Utako Kimura (Department of Dermatology, Juntendo University Urayasu Hospital) for excellent technical assistance in obtaining human skin samples.
This study was supported in part by a grant of the Ministry of Health, Labour and Welfare, Japan (grant numbers 150401-01 and 180101-01 to CM), Japanese Society for the Promotion of Science KAKENHI grant numbers JP16H05345 to CM, JP15H04879 and JP18H02782 to KO, JP17K10008 and 26860760 to RH, Japanese Society for the Promotion of Science Research Fellowships for Young Scientists to RH, and a Grant-in-Aid (S1311011) from the Foundation of Strategic Research Projects in Private Universities from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to KO and CM). This work was also supported by a research grant from the Japan Research Institute of Industrial Science (to RH and TI), a research grant from the joint research program of Juntendo University of Faculty of Health and Sports Science (TI), and a research grant from the Japanese Society of Hematology (RH).
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