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Increased Lipocalin-2 Contributes to the Pathogenesis of Psoriasis by Modulating Neutrophil Chemotaxis and Cytokine Secretion

Open ArchivePublished:March 12, 2016DOI:https://doi.org/10.1016/j.jid.2016.03.002
      Psoriasis is characterized by resistance to infections, which is regulated by antimicrobial proteins. Whether antimicrobial proteins play a pathogenic role in psoriasis remains unclear. In this study, we aimed to elucidate the role of lipocalin-2 (Lcn2), an antimicrobial protein, in the pathogenesis of psoriasis. Our results showed that Lcn2 was highly expressed in the lesional skin of psoriatic patients. The neutralization of Lcn2 alleviated epidermal hyperplasia, inflammation, and especially neutrophil infiltration in an imiquimod-induced psoriasis-like murine model. In vitro, Lcn2 stimulated human neutrophils to produce vital proinflammatory mediators, such as IL-6, IL-8, tumor necrosis factor-α, and IL-1α via a specific receptor, 24p3R, on neutrophils, which consequently activated the downstream extracellular signal-regulated kinase-1/2 and p38-mitogen-activated protein kinase signaling pathways. Moreover, Lcn2-induced neutrophil chemotaxis was concentration dependent and mediated by the extracellular signal-regulated kinase-1/2 and p38-mitogen-activated protein kinase signaling pathways in vitro. Furthermore, we demonstrated that both keratinocytes and neutrophils were the sources of Lcn2 in the lesional skin of psoriatic patients. Taken together, these results suggest that Lcn2 is involved in the pathogenesis of psoriasis by modulating neutrophil function, and that it could serve as a potential target for treating psoriasis.

      Abbreviations:

      dHL-60 (differentiated human leukaemia-60), Erk (extracellular signal-regulated kinase), IMQ (imiquimod), Lcn2 (lipocalin-2), MAPK (mitogen-activated protein kinase), rmLcn2 (recombinant Lcn2), TNF (tumor necrosis factor)

      Introduction

      Psoriasis vulgaris is a chronic autoimmune inflammatory skin disease, characterized by the development of erythematous areas covered with thick, silvery scales. It can severely affect the physiological and psychological health of patients (
      • Boehncke W.-H.
      • Schön M.P.
      Psoriasis.
      ). Although the pathogenesis of psoriasis remains controversial, it is generally accepted that the complex interactions between infiltrated immune cells and activated keratinocytes lead to keratinocyte hyperproliferation and the formation of psoriatic lesions (
      • Lowes M.A.
      • Suárez-Fariñas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ). Among those innate effector cells, neutrophils that aggregate in Munro’s microabscesses and scatters in the dermis play a critical pathogenic role by releasing cytokines, enzymes, and neutrophil extracellular traps (
      • Skrzeczynska-Moncznik J.
      • Wlodarczyk A.
      • Zabieglo K.
      • Kapinska-Mrowiecka M.
      • Marewicz E.
      • Dubin A.
      • et al.
      Secretory leukocyte proteinase inhibitor-competent dna deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis.
      ,
      • Terui T.
      • Ozawa M.
      • Tagami H.
      Role of neutrophils in induction of acute inflammation in T-cell-mediated immune dermatosis, psoriasis: a neutrophil-associated inflammation-boosting loop.
      ). Hence, the dysregulation of neutrophil function gives rise to severe psoriatic inflammation. However, the mechanism controlling the activation of neutrophils in psoriasis remains unclear.
      Lipocalin (Lcn)-2, also known as 24p3 and neutrophil gelatinase-associated lipocalin, is a protein stored in the specific granules of human neutrophils (
      • Kjeldsen L.
      • Bainton D.F.
      • Sengeløv H.
      • Borregaard N.
      Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils.
      ). It is regarded as an antimicrobial protein against bacterial infection by sequestrating iron (
      • Flo T.H.
      • Smith K.D.
      • Sato S.
      • Rodriguez D.J.
      • Holmes M.A.
      • Strong R.K.
      • et al.
      Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron.
      ). The other well-established biologic functions of Lcn2 include regulating diverse cellular processes, such as cell growth and migration/invasion (
      • Ding G.
      • Fang J.
      • Tong S.
      • Qu L.
      • Jiang H.
      • Ding Q.
      • et al.
      Over-expression of lipocalin 2 promotes cell migration and invasion through activating ERK signaling to increase SLUG expression in prostate cancer.
      ,
      • Nelson A.M.
      • Zhao W.
      • Gilliland K.L.
      • Zaenglein A.L.
      • Liu W.
      • Thiboutot D.M.
      Neutrophil gelatinase—associated lipocalin mediates 13-cis retinoic acid—induced apoptosis of human sebaceous gland cells.
      ,
      • Wang G.
      • Ma N.
      • Meng L.
      • Wei Y.
      • Gui J.
      Activation of the phosphatidylinositol 3-kinase/Akt pathway is involved in lipocalin-2-promoted human pulmonary artery smooth muscle cell proliferation.
      ). In addition, it can act as a proinflammatory mediator to enhance the production of important cytokines, such as IL-6, IL-8, and CXCL10, resulting in aggravating the diseases (
      • Lin H.
      • Liao C.
      • Lee Y.
      • Hu K.
      • Meng H.
      • Chu S.
      Lipocalin-2-induced cytokine production enhances endometrial carcinoma cell survival and migration.
      ). Recent studies have suggested that Lcn2 is associated with the accumulation of immune cells at the sites of acute or chronic inflammation (
      • Aigner F.
      • Maier H.T.
      • Schwelberger H.G.
      • Wallnöfer E.A.
      • Amberger A.
      • Obrist P.
      • et al.
      Lipocalin-2 regulates the inflammatory response during ischemia and reperfusion of the transplanted heart.
      ,
      • Shashidharamurthy R.
      • MacHiah D.
      • Aitken J.D.
      • Putty K.
      • Srinivasan G.
      • Chassaing B.
      • et al.
      Differential role of lipocalin 2 during immune complex-mediated acute and chronic inflammation in mice.
      ). Hence, the functions of Lcn2 extend beyond those of antimicrobials, and it has also been implicated in the progression of various diseases including infectious disease, cardiac disease, renal injury, tumor metastasis, and chronic inflammatory pain, among others (
      • Barreto R.
      • Elia C.
      • Solà E.
      • Moreira R.
      • Ariza X.
      • Rodríguez E.
      • et al.
      Urinary neutrophil gelatinase-associated lipocalin predicts kidney outcome and death in patients with cirrhosis and bacterial infections.
      ,
      • Nam Y.
      • Kim J.-H.
      • Seo M.
      • Kim J.-H.
      • Jin M.
      • Jeon S.
      • et al.
      Lipocalin-2 protein deficiency ameliorates experimental autoimmune encephalomyelitis: the pathogenic role of lipocalin-2 in the central nervous system and peripheral lymphoid tissues.
      ,
      • Shiratori-Hayashi M.
      • Koga K.
      • Tozaki-Saitoh H.
      • Kohro Y.
      • Toyonaga H.
      • Yamaguchi C.
      • et al.
      STAT3-dependent reactive astrogliosis in the spinal dorsal horn underlies chronic itch.
      ,
      • Yndestad A.
      • Landrø L.
      • Ueland T.
      • Dahl C.P.
      • Flo T.H.
      • Vinge L.E.
      • et al.
      Increased systemic and myocardial expression of neutrophil gelatinase-associated lipocalin in clinical and experimental heart failure.
      ).
      Although the role of Lcn2 in inflammation is appreciated, its involvement in the pathogenesis of psoriasis has not been well elucidated. However, a line of evidence suggests the pathogenic role of Lcn2 in psoriasis. For example, Lcn2 expression can be induced by IL-17A (
      • Ferreira M.C.
      • Whibley N.
      • Mamo A.J.
      • Siebenlist U.
      • Chan Y.R.
      • Gaffen S.L.
      Interleukin-17-induced protein lipocalin 2 is dispensable for immunity to oral candidiasis.
      ), a critical cytokine that bridges the innate and adaptive immune systems. In psoriasis, Lcn2 is markedly increased in the granular layer and hair follicles of psoriatic skin (
      • Seo S.J.
      • Ahn J.-Y.
      • Hong C.-K.
      • Seo E.-Y.
      • Kye K.-C.
      • Lee W.-H.
      • et al.
      Expression of neutrophil gelatinase-associated lipocalin in skin epidermis.
      ). Furthermore, the expression levels of Lcn2 are higher in both lesional skin tissue and serum of psoriatic patients compared with those of normal controls (
      • Ataseven A.
      • Kesli R.
      • Kurtipek G.S.
      • Ozturk P.
      Assessment of lipocalin 2, clusterin, soluble tumor necrosis factor receptor-1, interleukin-6, homocysteine, and uric acid levels in patients with psoriasis.
      ,
      • El-Hadidi H.
      • Samir N.
      • Shaker O.G.
      • Otb S.
      Estimation of tissue and serum lipocalin-2 in psoriasis vulgaris and its relation to metabolic syndrome.
      ,
      • Kamata M.
      • Tada Y.
      • Tatsuta A.
      • Kawashima T.
      • Shibata S.
      • Mitsui H.
      • et al.
      Serum lipocalin-2 levels are increased in patients with psoriasis.
      ,
      • Romaní J.
      • Caixàs A.
      • Ceperuelo-Mallafré V.
      • Carrascosa J.M.
      • Ribera M.
      • Rigla M.
      • et al.
      Circulating levels of lipocalin-2 and retinol-binding protein-4 are increased in psoriatic patients and correlated with baseline PASI.
      ). The fact that the expression of Lcn2 disappears on clearance of psoriatic lesions suggests the association of Lcn2 with the progression of psoriasis (
      • Mallbris L.
      • O’Brien K.P.
      • Hulthén A.
      • Sandstedt B.
      • Cowland J.B.
      • Borregaard N.
      • et al.
      Neutrophil gelatinase-associated lipocalin is a marker for dysregulated keratinocyte differentiation in human skin.
      ).
      In this study, we investigated the role of Lcn2 in the pathogenesis of psoriasis. Our present work shows that psoriasis-involved skin expresses a higher level of Lcn2, which further induces the secretion of important proinflammatory factors and chemotaxis of neutrophils. The neutralization of Lcn2 significantly attenuates disease severity and inflammatory infiltration in vivo. This study not only elucidates the role of Lcn2 in the development of psoriasis, but also identifies its origins, potentiating future targeted therapy.

      Results

      Lcn2 is overexpressed in the lesional skin of psoriatic patients and imiquimod (IMQ)-induced psoriasis-like lesion in mice

      We first examined the expression of Lcn2 in psoriatic skin. Real-time PCR and western blot results showed that the expression levels of Lcn2 mRNA and protein were higher in both the epidermis and the dermis of psoriatic patients than those in normal controls (Figure 1a and b). Furthermore, the immunofluorescence showed that Lcn2 was highly expressed in the thickened epidermis and partly distributed in the inflamed dermis of psoriatic patients, whereas Lcn2 expression was barely detected in normal skin (Figure 1c).
      Figure 1
      Figure 1Lcn2 expression is increased in the lesional skin of psoriatic patients and IMQ-treated mice. (a) Lcn2 expression was evaluated using real-time PCR and (b) western blot in the epidermis and the dermis. The skin samples were treated with 2.5 mg/ml dispase for 8 hours at 4°C. (c) Representative images of immunofluorescence of Lcn2 in normal and psoriatic skin. (d) Lcn2 expression levels in the epidermis and the dermis of IMQ-induced mice were determined by real-time PCR and (e) western blot. Skin samples were from a control (○) or IMQ-induced mice (●). (f) Representative immunohistochemical staining of Lcn2 in the back skin of controls and IMQ-treated mice. Scale bar = 100 μm. Values are presented as mean ± standard deviation. ***P < 0.001. IMQ, imiquimod; Lcn2, lipocalin-2.
      Thereafter, we assessed the Lcn2 expression in IMQ-induced psoriasis-like lesions in mice. As shown in Figure 1d–f, IMQ induced a significant increase in Lcn2 mRNA and protein expression in both the epidermis and dermis as demonstrated by real-time PCR, western blot, and immunohistochemistry.

      Lcn2 monoclonal antibody/recombinant Lcn2 alleviates/exacerbates skin lesions in IMQ-induced psoriasis-like mice

      On the basis of the above findings, we next assessed the role of Lcn2 in the pathogenesis of psoriasis in the IMQ-induced psoriasis model. As shown in Figure 2b and c, neutralization of Lcn2 significantly reduced epidermal thickness and the number of infiltrated inflammatory cells in the dermis, which were paralleled by a reduction in the expression levels of mRNA for critical psoriasis-related cytokines including IL-17 and IL-23, and immune cell markers such as Ly6G (Figure 2d and e). In contrast, recombinant Lcn2 (rmLcn2)-treated mouse skin showed more severe inflammation, thicker epidermis, aggregation and infiltration of more immune cells, and higher expression levels of mRNA for inflammatory mediators (Figure 2b–e). Notably, the expressions of IL-8, IL-6, Ly6G, CD11b, and CXCL2 were all neutrophil associated. Furthermore, the Lcn2-neutralized mice displayed a reduction in the number of neutrophils recruited to lesional skin, whereas the rmLcn2-treated mice had a greater neutrophil infiltration, as demonstrated by increased intensity of immunostaining for Ly6G, a neutrophil marker (Figure 2f). However, the differences in T-cell infiltration (Supplementary Figure S1 online), especially the IL-17A-producing CD4+ T cells (Supplementary Figure S2 online), among the groups were not as remarkable as those in neutrophils. These results suggest that Lcn2 aggravates the inflammation in psoriasis and may be associated with neutrophil function.
      Figure 2
      Figure 2Lcn2 mAb/rmLcn2 alleviates/aggravates epidermal hyperplasia and inflammation in IMQ-induced psoriasis-like mice. (a) Mice were intraperitoneally injected with an anti-Lcn2 mAb, isotype control antibody, recombinant Lcn2 protein, or vehicle, respectively, on the first day and every 48 hours thereafter. (b) Phenotype (upper panel) and hematoxylin and eosin staining (lower panel) of IMQ-treated mice in different groups on day 7. Images are representative of three individual mice per group. (c) Epidermal thickness and infiltrated cells in the dermis. (d) The mRNA expressions of important cytokines and (e) immune cell markers. (f) Neutrophil infiltration was determined by evaluating the intensity of Ly6G staining. Scale bar = 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001. Each experiment was repeated at least 3 times. IMQ, imiquimod; Lcn2, lipocalin-2; mAb, monoclonal antibody.

      Lcn2 induces the gene expression and secretion of critical cytokines/chemokines in neutrophils via the extracellular signal-regulated kinase (Erk)-1/2 and p38-mitogen-activated protein kinase (MAPK) signaling pathways

      To further investigate the regulatory role of Lcn2 in neutrophils, we next identified the receptor for Lcn2 in the peripheral neutrophils from psoriatic patients. Previous studies have identified two receptors of Lcn2, namely megalin and 24p3R (
      • Devireddy L.R.
      • Gazin C.
      • Zhu X.
      • Green M.R.
      A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake.
      ,
      • Hvidberg V.
      • Jacobsen C.
      • Strong R.K.
      • Cowland J.B.
      • Moestrup S.K.
      • Borregaard N.
      The endocytic receptor megalin binds the iron transporting neutrophil-gelatinase-associated lipocalin with high affinity and mediates its cellular uptake.
      ). But our results revealed that 24p3R, not megalin, was expressed to a greater extent in neutrophils from psoriatic patients than in normal neutrophils (Figure 3a). Immunostaining indicated that 24p3R was expressed on the surface of neutrophils (Figure 3b). In addition, 24p3R-expressing neutrophils were identified in the lesional dermis as well (Figure 3c). These findings support the notion that Lcn2 possibly activates neutrophils via 24p3R.
      Figure 3
      Figure 3Lcn2 stimulates the production of critical cytokines by neutrophils. (a) The expression levels of Lcn2 receptors in neutrophils from psoriatic patients and healthy controls. (b) Immunofluorescence analysis of 24p3R on the peripheral neutrophils of psoriatic patients. Scale bar = 10 μm. (c) The colocation of 24p3R and CD15 in the psoriatic dermis. Scale bar = 100 μm. (d) Neutrophils from psoriatic patients were treated with 500 nM of rmLcn2 for the indicated time, followed by real-time PCR analysis of IL-1α, IL-8, IL-6, and TNF-α mRNA expression. (e) Protein levels of IL-1α, IL-8, IL-6, and TNF-α in the supernatant of rmLcn2-stimulated neutrophils. Values are presented as mean ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001. Lcn2, lipocalin-2; TNF-α, tumor necrosis factor-α.
      Because proinflammatory mediators secreted by neutrophils participate in the early immune response and trigger the later inflammatory cascades in psoriasis (
      • Lin A.M.
      • Rubin C.J.
      • Khandpur R.
      • Wang J.Y.
      • Riblett M.
      • Yalavarthi S.
      • et al.
      Mast cells and neutrophils release IL-17 through extracellular trap formation in psoriasis.
      ,
      • Michalak-Stoma A.
      • Pietrzak A.
      • Szepietowski J.C.
      • Zalewska-Janowska A.
      • Paszkowski T.
      • Chodorowska G.
      Cytokine network in psoriasis revisited.
      ), we next evaluated the ability of Lcn2 to induce neutrophils to produce important inflammatory cytokine/chemokines using real-time PCR and ELISA. As shown in Supplementary Figure S3 online, activation of neutrophils with 250 nM, 500 nM, and 1 μM of rmLcn2 resulted in a dose-dependent increase in the expression levels of mRNA for IL-1α, tumor necrosis factor (TNF)-α, IL-6, and IL-8, with a maximum at 500 nM. Moreover, neutrophils from psoriatic patients were found to be more prone to activation compared with that from normal controls (Supplementary Figure S3a and b). The stimulating effect of Lcn2 on neutrophils was time dependent, with a maximum effect after 24 hours of incubation (Figure 3d and e).
      Thereafter, we explored the activated signaling pathways in rmLcn2-stimulated neutrophils. Because the MAPK pathway is primarily activated in antimicrobial peptide-stimulated immune cells (
      • Zheng Y.
      • Niyonsaba F.
      • Ushio H.
      • Nagaoka I.
      • Ikeda S.
      • Okumura K.
      • et al.
      Cathelicidin LL-37 induces the generation of reactive oxygen species and release of human alpha-defensins from neutrophils.
      ,
      • Zheng Y.
      • Niyonsaba F.
      • Ushio H.
      • Ikeda S.
      • Nagaoka I.
      • Okumura K.
      • et al.
      Microbicidal protein psoriasin is a multifunctional modulator of neutrophil activation.
      ), we first determined the protein levels of phosphorylation and dephosphorylation of Erk1⁄2, p38 MAPK, and Jun amino-terminal kinase levels in rmLcn2-activated neutrophils. Our results showed that 500 nM of rmLcn2 markedly induced the phosphorylation of Erk1/2 and p38 MAPK, but not of Jun amino-terminal kinase (not shown), within10 minutes, with a maximum at 30 minutes (Figure 4a). Moreover, immunofluorescence analysis of neutrophils indicated that rmLcn2 induced the translocation of Erk1/2 and p38 MAPK into the nucleus (Figure 4b). To further confirm whether these pathways are required for the Lcn2-mediated production of cytokines, cells were preincubated with specific inhibitors of Erk1/2 or p38 MAPK for 30 minutes. The efficiency of pathway inhibitors was evaluated by western blot (Supplementary Figure S4 online). Incubation with either application of SB203580 (p38 MAPK inhibitor) or PD980599 (Erk1⁄2 inhibitor) almost completely blocked the production of IL-6 and partially suppressed the secretions of IL-1α, TNF-α, and IL-8, as shown by ELISA (Figure 4c).
      Figure 4
      Figure 4The Erk1/2 and p38 MAPK pathways are activated by Lcn2 in neutrophils from psoriatic patients. (a) The phosphorylated p38 MAPK and Erk1/2 levels in neutrophils treated by 500 nM of rmLcn2 were determined by western blot. (b) The nuclear translocation of Erk1/2 and p38 in rmLcn2-stimulated neutrophils. Scale bar = 10 μm. (c) ELISA was employed to assess the inhibiting effects of PD98059 and SB203580 on cytokine secretion from rmLcn2-stimulated neutrophils. (d) Differentiated HL-60 cells were transfected with 24p3R siRNA. (e) The phosphorylated p38 MAPK and Erk1/2 levels and protein levels of cytokines (f) in dHL60 cells stimulated by 500 nM of rmLcn2 for 60 minutes. Similar results were obtained from three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001. dHL-60, differentiated HL-60; Erk, extracellular signal-regulated kinase; Lcn2, lipocalin-2; MAPK, mitogen-activated protein kinase; siRNA, small interfering RNA.
      We next determined whether the phosphorylation of Erk1/2 or p38 MAPK was mediated by activation of 24p3R in neutrophils. Because neutrophils are short-lived and terminally differentiated cells, and constitutively undergo apoptosis, we employed differentiated human leukaemia-60 (dHL-60) cells, a widely used cell line in the study of neutrophil functions. As expected, rmLcn2 treatment enhanced expression levels of the four important cytokines in cells (Supplementary Figure S2c). However, silencing 24p3R abolished the rmLcn2-induced increase in the phosphorylation of Erk1/2 or p38 MAPK in dHL-60 cells (Figure 4d and e). Furthermore, the production of IL-6, IL-8, IL-1α, and TNF-α significantly decreased in 24p3R-silenced dHL-60 cells after rmLcn2 stimulation (Figure 4f).
      Collectively, these data indicate that Lcn2-induced psoriasis-related cytokine/chemokine secretion in neutrophils is via the interaction of Lcn2/24p3R and the activation of the downstream Erk1/2 and p38 MAPK pathways.

      Lcn2 induces neutrophil trafficking in vitro

      In addition to induction of cytokine/chemokine secretion from neutrophils, Lcn2 could independently trigger neutrophil migration. To test this hypothesis, the classical chemokine for neutrophils, N-formylmethionyl-leucyl-phenyl-alanine (10 nM), and rmLcn2 (10 nM, 20 nM, and 50 nM) were added to the lower chamber of a transwell device. As shown in Figure 5a , 20 nM of rmLcn2 was sufficient to attract neutrophils from psoriatic patients in vitro. This neutrophil-attracting effect of rmLcn2 was completely inhibited by an Lcn2 monoclonal antibody (Figure 5a and Supplementary Figure S5a online). To determine whether the activation of Erk1/2 and p38 MAPK pathways was involved in Lcn2-induced chemotaxis, Erk1/2 and p38 MAPK inhibitors were used in the neutrophil trafficking study. As shown in Figure 5b, inhibition of either Erk1/2 or p38 MAPK pathway inhibited rmLcn2-induced migration of neutrophils (Figure 5b and Supplementary Figure S5b). Notably, the optimal incubation time was 45 minutes, and prolonged incubation did not attract additional neutrophils (data not shown). To determine whether rmLcn2-induced neutrophil migration was dose dependent, neutrophil migration was assessed after the incubation of psoriatic neutrophils in a Roswell Park Memorial Institute medium containing rmLcn2 (10 nM and 20 nM). The result demonstrated that rmLcn2 dose dependently induced neutrophil migration in vitro (Figure 5c and Supplementary Figure S5c).
      Figure 5
      Figure 5Lipocalin-2 (Lcn2) induces the migration of human neutrophils in vitro. (a) A transwell experiment was used to determine the migration of neutrophils. A total of 2 ×105 peripheral neutrophils from psoriatic patients were allowed to migrate for 45 minutes toward different concentrations of rmLcn2. N-formylmethionyl-leucyl-phenyl-alanine (10 nM) was a positive control. The chemotaxis effect was determined by crystal violet staining for the lower transwell membrane (left panel) and counting the number of cells in the lower chamber (right panel). (b) Migration of neutrophils from psoriatic patients was assessed after preincubation with PD98059 or SB203580 for 30 minutes. (c) Migration of neutrophils from psoriatic patients was assessed after treatment with various concentrations of recombinant lipocalin-2 (rmLcn2). N = 8. *P < 0.05, **P < 0.01.

      Lcn2 is mainly from activated keratinocytes and infiltrated neutrophils in psoriasis

      To identify the origin of Lcn2 in psoriatic lesions, we assessed Lcn2 expression in HaCaT cells treated with IL-17A, IL-22, TNF-α, and H2O2, all of which are abundant and functional in the psoriatic lesions. Both western blot and ELISA showed that the expression level of Lcn2 in HaCaT cells was significantly upregulated by IL-17A, IL-22, TNF-α, and H2O2 (Figure 6a and b). To localize the expression of Lcn2 in psoriasis, immunostaining was used. In agreement with previous findings that neutrophils secrete antimicrobial proteins in inflammatory conditions, both the dermis and Munro’s microabscesses of psoriatic lesions displayed the intensive staining of Lcn2, mainly localized in CD15+ neutrophils (Figure 6c). Moreover, the peripheral neutrophils from psoriatic patients expressed a higher level of Lcn2 mRNA in comparison with normal controls (Figure 6d and e). These results suggest that both activated keratinocytes in the epidermis and infiltrated neutrophils in the dermis are the main sources of Lcn2 in psoriatic skin.
      Figure 6
      Figure 6Lipocalin-2 (Lcn2) is mainly produced by activated keratinocyte and infiltrated neutrophils in psoriasis. (a, b) Keratinocytes were stimulated with of IL-17A (20 ng/ml), IL-22 (20 ng/ml), TNF-α (50 ng/ml) or H2O2 (800 nM), and expression levels of Lcn2 were evaluated by western blot (a) and ELISA (b). (c) Colocalization of Lcn2 and CD15+ neutrophils in psoriatic dermis and Munro’s macroabscesses was determined by immunofluorescence. Scale bar = 100 μm. (d) The expression levels of Lcn2 mRNA in circulating neutrophils from psoriatic patients and control subjects. ***P < 0.001. (e) Immunofluorescence analysis of Lcn2 in neutrophils from psoriatic patients. Scale bar = 10 μm.

      Discussion

      In this study, we show an increased expression level of Lcn2 in both the lesional skin of psoriatic patients and IMQ-induced psoriasis-like lesions in mice, which is in agreement with previous findings (
      • Seo S.J.
      • Ahn J.-Y.
      • Hong C.-K.
      • Seo E.-Y.
      • Kye K.-C.
      • Lee W.-H.
      • et al.
      Expression of neutrophil gelatinase-associated lipocalin in skin epidermis.
      ). We further demonstrate that Lcn2 is also highly expressed in the peripheral neutrophils of psoriatic patients. In the dermis and Munro’s microabscesses, the majority of Lcn2 is colocalized with infiltrated CD15+ neutrophils. Regarding the origins of Lcn2 in psoriatic lesions, neutrophils are considered as one of the major contributors to Lcn2 in psoriasis. For example,
      • Kjeldsen L.
      • Bainton D.F.
      • Sengeløv H.
      • Borregaard N.
      Identification of neutrophil gelatinase-associated lipocalin as a novel matrix protein of specific granules in human neutrophils.
      have shown that high amounts of Lcn2 protein are released from the prepackaged neutrophil granules in inflammatory conditions. Likewise, inflammatory stimuli, including TNF-α, IFN-γ, and IL-1β, which are abundant in psoriasis, can markedly induce Lcn2 secretion from neutrophils (
      • Jayaraman A.
      • Roberts K.A.
      • Yoon J.
      • Yarmush D.M.
      • Duan X.
      • Lee K.
      • et al.
      Identification of neutrophil gelatinase-associated lipocalin (NGAL) as a discriminatory marker of the hepatocyte-secreted protein response to IL-1beta: a proteomic analysis.
      ,
      • Liu Q.
      • Nilsen-Hamilton M.
      Identification of a new acute phase protein.
      ). Here, our results indicate that Lcn2 is also from activated keratinocytes in psoriatic skin lesions. Although what induce(s) keratinocytes to secrete Lcn2 in psoriasis remains unknown, evidence suggests that epidermal dysfunction could be at least one of the causes. Previous studies have shown that psoriatic patients exhibit a compromised epidermal permeability barrier (
      • Ye L.
      • Lv C.
      • Man G.
      • Song S.
      • Elias P.M.
      • Man M.Q.
      Abnormal epidermal barrier recovery in uninvolved skin supports the notion of an epidermal pathogenesis of psoriasis.
      ), while disruption of the epidermal permeability barrier leads to cytokine release and production (
      • Wood L.C.
      • Elias P.M.
      • Calhoun C.
      • Tsai J.C.
      • Grunfeld C.
      • Feingold K.R.
      Barrier disruption stimulates interleukin-1 alpha expression and release from a pre-formed pool in murine epidermis.
      ). Here we show that inflammatory factors, such as IL-17A, IL-22, TNF-α, and H2O2, stimulate HaCaT cells to produce Lcn2 (Figure 6a), which is supported by previous findings (
      • Roudkenar M.H.
      • Kuwahara Y.
      • Baba T.
      • Roushandeh A.M.
      • Ebishima S.
      • Abe S.
      • et al.
      Oxidative stress induced lipocalin 2 gene expression: addressing its expression under the harmful conditions.
      ,
      • Stallhofer J.
      • Friedrich M.
      • Konrad-Zerna A.
      • Wetzke M.
      • Lohse P.
      • Glas J.
      • et al.
      Lipocalin-2 is a disease activity marker in inflammatory bowel disease regulated by IL-17A, IL-22, and TNF-α and modulated by IL23R genotype status.
      ). Thus, the elevated expression level of Lcn2 in psoriatic epidermis is likely attributed to increased cytokines induced by disruption of the epidermal permeability barrier. In addition to the skin, the expression level of Lcn2 is also elevated in the circulated neutrophils of psoriatic patients, which suggests that it could have a broader spectrum of proinflammatory activity that may not only be limited to the skin but may also have systemic consequences.
      The pathogenesis of psoriasis involves a complex network of immune cells and inflammatory molecules (
      • Lowes M.A.
      • Suárez-Fariñas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ). Although previous studies have shown that Lcn2 is a marker for dysregulated keratinocyte differentiation (
      • Mallbris L.
      • O’Brien K.P.
      • Hulthén A.
      • Sandstedt B.
      • Cowland J.B.
      • Borregaard N.
      • et al.
      Neutrophil gelatinase-associated lipocalin is a marker for dysregulated keratinocyte differentiation in human skin.
      ), additional mechanistic roles for Lcn2 in psoriasis have not been elucidated. But Lcn2 stimulating cytokine production in neutrophils could play a pathogenic role in psoriasis. These cytokines/chemokines, which are secreted by activated neutrophils, can promote the early inflammatory response, link the innate and adaptive immune responses, and amplify psoriatic inflammation (
      • Tecchio C.
      • Micheletti A.
      • Cassatella M.A.
      Neutrophil-derived cytokines: facts beyond expression.
      ). For instance, IL-8, mainly from keratinocytes and infiltrated neutrophils, not only accelerates keratinocyte proliferation but also acts as a critical chemoattractant for neutrophils and T lymphocytes in psoriasis (
      • Pietrzak A.T.
      • Zalewska A.
      • Chodorowska G.
      • Krasowska D.
      • Michalak-Stoma A.
      • Nockowski P.
      • et al.
      Cytokines and anticytokines in psoriasis.
      ). Although the regulatory role of Lcn2 in cytokine expression is well appreciated, the signaling pathway involved in this regulation is largely undetermined. We demonstrate here that Lcn2-induced upregulation of cytokine production in neutrophils occurs via phosphorylation of Erk1/2 and MAPK, which is mediated by the Lcn2 receptor, 24p3R.
      Recent studies have demonstrated that Lcn2 drives neutrophil infiltration in several other diseases (
      • Jha M.K.
      • Jeon S.
      • Jin M.
      • Ock J.
      • Kim J.-H.
      • Lee W.-H.
      • et al.
      The pivotal role played by lipocalin-2 in chronic inflammatory pain.
      ,
      • Wieser V.
      • Tymoszuk P.
      • Adolph T.E.
      • Grander C.
      • Grabherr F.
      • Enrich B.
      • et al.
      Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease.
      ). In this study, we show that rmLcn2 also induces migration of neutrophils from psoriatic patients in vitro. Moreover, the Lcn2-induced chemotaxis is not only concentration dependent but also associated with the activation of the Erk1/2 and p38 MAPK pathways (Figure 5). These migrated neutrophils can contribute to the progression of psoriasis by releasing proinflammatory mediators, enzyme, and antimicrobial peptides, and by forming neutrophil extracellular traps (
      • Skrzeczynska-Moncznik J.
      • Wlodarczyk A.
      • Zabieglo K.
      • Kapinska-Mrowiecka M.
      • Marewicz E.
      • Dubin A.
      • et al.
      Secretory leukocyte proteinase inhibitor-competent dna deposits are potent stimulators of plasmacytoid dendritic cells: implication for psoriasis.
      ,
      • Terui T.
      • Ozawa M.
      • Tagami H.
      Role of neutrophils in induction of acute inflammation in T-cell-mediated immune dermatosis, psoriasis: a neutrophil-associated inflammation-boosting loop.
      ). The role of Lcn2 in the pathogenesis of psoriasis is further supported by our observation that depleting neutrophils with an anti-Ly6G antibody can markedly relieve the inflammation in IMQ-induced mice (Supplementary Figure S6 online). Moreover, neutrophil granule proteases can act as potent IL-36-activating enzymes, leading to cytokine cascade in psoriasis (
      • Henry C.M.
      • Sullivan G.P.
      • Clancy D.M.
      • Afonina I.S.
      • Kulms D.
      • Martin S.J.
      Neutrophil-derived proteases escalate inflammation through activation of IL-36 family cytokines.
      ).
      In conclusion, Lcn2 derived from injured keratinocytes and migrated neutrophils can activate neutrophils to release proinflammatory mediators and induce neutrophil migration, subsequently aggravating inflammation and stimulating keratinocyte proliferation, leading to the formation of psoriasis (Supplementary Figure S7 online). These findings not only reveal the mechanisms by which Lcn2 regulates neutrophil trafficking and cytokine production, but also advance targeted therapy for psoriasis. However, an additional regulator role of Lcn2 in psoriasis remained to be determined.

      Materials and Methods

      Clinical samples

      Skin biopsies and peripheral blood samples were obtained from psoriatic patients (twelve men and eight women, age ranged from 18 to 49 years with a mean of 30 years) who visited our department at Xijing Hospital. Control skin biopsies were obtained from discarded healthy skin from donors (five men and four women, age ranged from 21 to 45 years with a mean of 29.1 years) who were admitted to the Department of Plastic Surgery at Xijing Hospital. Normal control blood samples were collected from age- and sex-matched healthy volunteers working at our department (five men and five women, age ranged from 25 to 42 years with a mean of 28.4 years). This study was performed in accordance with the Declaration of Helsinki Principles and approved by the ethical committee of Fourth Military Medical University. Written informed consent was obtained from each participant. Further details are provided in the Supplementary Materials and Methods online.

      Animal experiments

      Female BALB/c mice aged 8–10 weeks were obtained from the Department of Laboratory Animal Medicine of the Fourth Military Medical University. Briefly, the mice were randomly assigned to five groups and were intraperitoneally injected with an anti-Lcn2 monoclonal antibody (100 μg/mouse, R&D Systems, Minneapolis, MN), rat IgG2a isotype control antibody (100 μg/mouse, R&D Systems), recombinant Lcn2 protein (10 μg/mouse, R&D Systems), or vehicle (Figure 2a). Shaved mouse dorsal skin was treated daily with Aldara (INova Pharmaceuticals, Sydney, Australia) or Vaseline. The animal studies were approved by the institutional review board. Further details are provided in the Supplementary Materials and Methods.

      Preparation of neutrophils from human samples

      The separation of peripheral neutrophils was performed using a magnetic-activated cell sorting method, as previously reported (
      • Brumann M.
      • Matz M.
      • Kusmenkov T.
      • Stegmaier J.
      • Biberthaler P.
      • Kanz K.
      • et al.
      Impact of STAT/SOCS mRNA expression levels after major injury.
      ); the details are described in the Supplementary Materials and Methods. The purity of these selected cells was more than 90%, as confirmed by a flow cytometer.

      HL-60 cell culture and differentiation

      The HL-60 cell line was cultured in the Roswell Park Memorial Institute-1640 medium supplemented with 12% fetal bovine serum (Gibco-Invitrogen, Carlsbad, CA), penicillin (100 U/ml), streptomycin (25 μg/ml), and l-glutamine (1 mmol/l). To be differentiated, HL-60 cells were cultured in a medium containing DMSO (1.3%) for 5 consecutive days as previously reported (
      • Millius A.
      • Weiner O.D.
      Chemotaxis in neutrophil-like HL-60 cells.
      ).

      Neutrophil and dHL-60 cell stimulation

      Peripheral neutrophils or dHL-60 cells were treated with rmLcn2 (R&D Systems). Erk1/2 inhibitor (PD98059, Cell Signaling Technology, Beverly, MA) and p38 MAPK inhibitor (SB203580, Beyotime, Beijing, China) were used for the inhibition experiments. Further details are provided in the Supplementary Materials and Methods.

      Small interfering RNA knockdown of 24p3R

      dHL-60 cells were incubated with 24p3R small interfering RNA (5 μmol/l), as described previously (
      • Millius A.
      • Weiner O.D.
      Chemotaxis in neutrophil-like HL-60 cells.
      ). According to the manufacturer’s instructions, dHL-60 cells were transfected with small interfering RNAs using Lipofectamine 3000 (Invitrogen, Carlsbad, CA), and cultured in a medium supplemented with 1.3% DMSO to maintain differentiation of the HL-60 cells. Further details are provided in the Supplementary Materials and Methods.

      Chemotaxis assay

      Chemotaxis assays were performed using a 24-well transwell chamber with 3-μm pores (Costar, Bodenheim, Germany). The effect of rmLcn2 on neutrophil trafficking was determined by assessing the number of migrated cells in the lower chamber following crystal violet staining of the lower transwell membrane. The detailed protocols are described in the Supplementary Materials and Methods.

      Methods for histopathology, tissue immunohistochemistry and immunofluorescence, cell immunofluorescence, cell culture, real-time PCR, western blot, and ELISA

      Additional materials and methods are detailed in the Supplementary Materials and Methods. The sequences of primers used in the real time PCR were listed in Supplementary Table S1 online.

      Statistical analysis

      Data analysis was performed using the GraphPad Prism version 6.0 software (GraphPad Software, San Diego, CA). Statistically significant differences between two groups were determined by two-tailed unpaired t-tests. P-values of less than 0.05 were considered statistically significant. Data are expressed as mean ± standard error of the mean.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      The authors would like to acknowledge Dr Maoqiang Man (Department of Dermatology, University of California, San Francisco) for critical review of the manuscript. This work was supported by grants from the National Natural Science Foundation of China (81430073, 81301355).

      Supplementary Material

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