Advertisement

Eosinophils Accelerate Pathogenesis of Psoriasis by Supporting an Inflammatory Milieu that Promotes Neutrophil Infiltration

  • Hee Joo Kim
    Affiliations
    Department of Dermatology, Gachon Gil Medical Center, School of Medicine, Gachon University, Incheon, Korea
    Search for articles by this author
  • Joo Young Roh
    Affiliations
    Department of Dermatology, Gachon Gil Medical Center, School of Medicine, Gachon University, Incheon, Korea
    Search for articles by this author
  • YunJae Jung
    Correspondence
    Correspondence: YunJae Jung, Department of Microbiology, School of Medicine, Gachon University, 155 Gaetbeol-ro, Yeonsu-gu, Incheon 21999, Korea.
    Affiliations
    Department of Microbiology, School of Medicine, Gachon University, Incheon, Korea

    Gachon Advanced Institute for Health Science and Technology, Gachon University, Incheon, Korea
    Search for articles by this author
Open ArchivePublished:March 26, 2018DOI:https://doi.org/10.1016/j.jid.2018.03.1509
      Eosinophils are proinflammatory granulocytes that are involved in the pathogenesis of various inflammatory reactions. However, their roles in psoriasis remain largely unknown. In this study, by examining the inflammatory features of the eosinophilic cell line EoL-1 and an imiquimod-induced murine model of psoriasis, we show that eosinophils provide inflammatory signals that accelerate the pathogenesis of psoriasis. EoL-1 cells constitutively expressed TLR7, which mediates acute and rapidly developing psoriatic inflammation. The activation of TLR7 on EoL-1 cells using R837 resulted in the secretion of inflammatory mediators that support the migration, activation, and survival of neutrophils. The underlying pathologic role of eosinophils in psoriatic inflammation was further substantiated by markedly decreased psoriasiform inflammation in imiquimod-treated ΔdblGATA mice, which have a systemic eosinophil deficiency. While imiquimod-treated wild-type mice showed a significant increase in the eosinophils in their skin, neutrophils remarkably outnumbered the eosinophils in the skin, lymph nodes, and spleen in wild-type mice after imiquimod application. In addition, lesional skin samples from psoriasis patients also showed up-regulated eosinophil cytotoxic granules, accompanied by marked neutrophil infiltration. Based on these collective data, we propose that eosinophils accelerate psoriatic inflammation by supporting inflammatory microenvironments to favor the activation and infiltration of neutrophils.

      Abbreviations:

      CXCL (CXC chemokine ligand), CM (conditioned medium), IMQ (imiquimod), LN (lymph node), WT (wild-type)

      Introduction

      Psoriasis is a chronic inflammatory skin disease characterized by the hyperproliferation and altered differentiation of keratinocytes, accompanied by an increased infiltration of immune cells (
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      Psoriasis.
      ). Although the exact etiology of psoriasis remains unknown, antigenic stimuli induce the activation of innate immune cells, resulting in the production of proinflammatory cytokines in the skin, which leads to the differentiation of Th1 and Th17 cells (
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      Psoriasis.
      ). IL-17 and IL-22 secreted by T cells activate keratinocytes, amplify the production of proinflammatory cytokines and chemokines, and subsequently accelerate the inflammatory features of psoriasis (
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      Psoriasis.
      ). The crucial role of the innate immune system in the initiation and development of psoriasis is emphasized by the efficacy of anti–TNF-α biological agents in psoriasis treatment (
      • Griffiths C.E.
      Immunotherapy for psoriasis: from serendipity to selectivity.
      ).
      The innate immune system detects pathogenic signals through germline-encoded pattern recognition receptors, including TLRs, which are expressed on many types of immune cells, including monocytes, macrophages, dendritic cells, and granulocytes (
      • Netea M.G.
      • van der Graaf C.
      • Van der Meer J.W.
      • Kullberg B.J.
      Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system.
      ). Signaling through TLRs bridges the innate and adaptive immune responses by promoting the development of an inflammatory environment in the skin (
      • Hari A.
      • Flach T.L.
      • Shi Y.
      • Mydlarski P.R.
      Toll-like receptors: role in dermatological disease.
      ). The expression of TLR1 and TLR2 is enhanced on keratinocytes from psoriatic skin (
      • Baker B.S.
      • Ovigne J.M.
      • Powles A.V.
      • Corcoran S.
      • Fry L.
      Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis.
      ), and the activation of TLR9 by self-DNA, complexed with a skin-damage–associated antimicrobial peptide, induces innate cytokine production (
      • Lande R.
      • Gregorio J.
      • Facchinetti V.
      • Chatterjee B.
      • Wang Y.H.
      • Homey B.
      • et al.
      Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.
      ). The TLR7 agonist imiquimod (IMQ) exacerbates psoriasis by enhancing the innate production of cytokines (
      • Gilliet M.
      • Conrad C.
      • Geiges M.
      • Cozzio A.
      • Thurlimann W.
      • Burg G.
      • et al.
      Psoriasis triggered by Toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.
      ) and induces psoriasiform dermatitis in mice (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Accordingly, we have observed increased expression of TLR3, 7, 8, and 9 in the peripheral blood mononuclear cells of a patient with psoriasis, and these findings were associated with activation of the immune system by an intracellular ligand released from damaged tissue (
      • Kim H.J.
      • Kim S.H.
      • Je J.H.
      • Shin D.Y.
      • Kim D.S.
      • Lee M.G.
      Increased expression of Toll-like receptors 3, 7, 8 and 9 in peripheral blood mononuclear cells in patients with psoriasis.
      ).
      Eosinophils are proinflammatory granulocytes implicated in the pathogenesis of allergic disorders and the protective response against helminthic and viral infections (
      • Rothenberg M.E.
      • Hogan S.P.
      The eosinophil.
      ). Mature eosinophils possess cytotoxic granules, including major basic protein, eosinophil-derived neurotoxin, and eosinophil cationic protein (
      • Rothenberg M.E.
      • Hogan S.P.
      The eosinophil.
      ). Eosinophils also release various cytokines, chemokines, and lipid mediators, suggesting that they serve multiple proinflammatory functions (
      • Bochner B.S.
      Novel therapies for eosinophilic disorders.
      ). The TLR-mediated activation of eosinophils can lead to the secretion of several proinflammatory mediators, as demonstrated by the enhanced production of eosinophil-derived neurotoxin and IL-8 from eosinophils stimulated with cognate ligands for TLR7 and TLR9 (
      • Mansson A.
      • Cardell L.O.
      Role of atopic status in Toll-like receptor (TLR)7- and TLR9-mediated activation of human eosinophils.
      ). Although the inflammatory effects of eosinophils are becoming understood, their role in psoriasis has not yet been elucidated. However, a subset of patients with psoriasis showed elevated concentrations of eosinophil cationic protein in serum, and an increased abundance of eosinophils was detected in the lesional skin of patients with rapidly progressive psoriasis (
      • Lundin A.
      • Fredens K.
      • Michaelsson G.
      • Venge P.
      The eosinophil granulocyte in psoriasis.
      ,
      • Michaelsson G.
      • Kraaz W.
      • Gerden B.
      • Hagforsen E.
      • Lundin I.P.
      • Loof L.
      • et al.
      Patients with psoriasis have elevated levels of serum eosinophil cationic protein and increased numbers of EG2 positive eosinophils in the duodenal stroma.
      ). Furthermore, eosinophils influence psoriatic inflammation by regulating cytokine production by plasmacytoid dendritic cells (
      • 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.
      ).
      Here, using an IMQ-induced model of psoriasis, we investigated the role of eosinophils in the pathogenesis of psoriasis. We found that the eosinophilic cell line EoL-1 secreted inflammatory mediators that induced neutrophil activation upon ligation with a TLR7 agonist. Eosinophil-deficient ΔdblGATA mice were protected from psoriatic inflammation and showed a marked decrease of Gr-1+ neutrophils in the skin, axillary lymph nodes (LNs), and spleen compared to wild-type (WT) mice. Gene expression of eosinophil cytotoxic granules was up-regulated in the lesional skin of human psoriasis, and eosinophils were present in the vicinity of infiltrated neutrophils upon microscopic evaluation. Based on these data, we propose that eosinophils accelerate the pathogenesis of psoriasis by modulating local microenvironments to favor inflammatory activation and by promoting the infiltration of neutrophils into lesional psoriatic skin.

      Results

      Expression of TLR7 increased in EoL-1 cells differentiated with butyrate

      The purification of sufficient numbers of eosinophils is difficult because they account for <5% of peripheral blood leukocytes (
      • Rothenberg M.E.
      • Hogan S.P.
      The eosinophil.
      ). A human eosinophilic cell line (EoL-1) represents a useful model for the study of eosinophils and can be differentiated into eosinophilic granule–containing cells by butyrate stimulation (
      • Jung Y.
      Comparative analysis of dibutyric cAMP and butyric acid on the differentiation of human eosinophilic leukemia EoL-1 cells.
      ,
      • Saito H.
      • Hayakawa T.
      • Mita H.
      • Akiyama K.
      • Shida T.
      Effect of butyric acid on induction of differentiation into eosinophil-like cells in human eosinophilic leukemia cells, EoL-1 cell line: possible role of granulocyte-macrophage colony-stimulating factor as an autocrine differentiating factor.
      ,
      • Wong C.K.
      • Ho C.Y.
      • Lam C.W.
      • Zhang J.P.
      • Hjelm N.M.
      Differentiation of a human eosinophilic leukemic cell line, EoL-1: characterization by the expression of cytokine receptors, adhesion molecules, CD95 and eosinophilic cationic protein (ECP).
      ). Eosinophils constitutively express several TLRs, including TLR7 (
      • Nagase H.
      • Okugawa S.
      • Ota Y.
      • Yamaguchi M.
      • Tomizawa H.
      • Matsushima K.
      • et al.
      Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand.
      ), and we observed significantly increased expression of TLR7 in EoL-1 cells compared with that in the HaCaT keratinocyte and HL-60 promyelocytic cell lines (Figures 1a, 1b ). The treatment of EoL-1 cells with butyrate effectively induced the expression of the mature eosinophil marker CCR3 (
      • Rothenberg M.E.
      • Hogan S.P.
      The eosinophil.
      ), and the expression of TLR7 was also significantly up-regulated (Figure 1c). With butyrate treatment, expression of CCR3 and TLR7 peaked at day 2, and the viability of EoL-1 cells decreased in a time-dependent manner (Figure 1d). Therefore, a 2-day treatment with butyrate was used to differentiate EoL-1 cells in further experiments.
      Figure thumbnail gr1
      Figure 1Expression of TLR7 increased in EoL-1 cells differentiated with butyrate. (a) The mRNA expression of TLR7 in HaCaT, HL-60, and EoL-1 cells was analyzed by real-time PCR. Graphs show the mean ± standard error of mean (SEM). P < 0.05 (Mann–Whitney test). (b) The amplified GAPDH and TLR7 products electrophoresed on 2% agarose gel. Representative bands are shown. (c) The mRNA expression of CCR3 and TLR7 in butyrate-treated EoL-1 cells. Graphs show the mean ± SEM. ∗∗P < 0.01, ∗∗∗P < 0.001 (Student t test). (d) Flow cytometric analysis for 7-AAD in butyrate-treated EoL-1 cells. All data are representative of two or more independent experiments. Graphs show the mean ± SEM. ∗∗P < 0.01, ∗∗∗P < 0.001 (Student t test). 7-AAD, 7-amino-actinomycin D.

      TLR7 ligand induced expression of inflammatory cytokines in differentiated EoL-1 cells

      To assess the functions of TLR7 in EoL-1 cells, we evaluated the mRNA expression of cytokines in EoL-1 cells stimulated with a ligand of TLR7 (R837). EoL-1 cells differentiated with butyrate showed increased expression of IL1B, IL23, and IFNG compared with undifferentiated controls, and R837 treatment further enhanced the butyrate-induced expression of cytokines (Figure 2a). Expression of IL6 and TNF, and IL13 in EoL-1 cells was not significantly affected by stimulation with either butyrate or R837 (Figure 2a). R837 exhibited no significant effect on cytokine expression in undifferentiated EoL-1 cells (Figure 2a). To extensively screen the cytokine-producing ability of EoL-1 cells, cytokines in the cell culture supernatants of undifferentiated or butyrate/R837-treated EoL-1 cells were semi-quantitated using a human cytokine array. The secretion of CXC chemokine ligand (CXCL) 12, IL-8, macrophage migration inhibitory factor, and PAI-1, which are involved in neutrophil activation, chemotaxis, and survival (
      • Delano M.J.
      • Kelly-Scumpia K.M.
      • Thayer T.C.
      • Winfield R.D.
      • Scumpia P.O.
      • Cuenca A.G.
      • et al.
      Neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on CXCL12 signaling.
      ,
      • Kunkel S.L.
      • Standiford T.
      • Kasahara K.
      • Strieter R.M.
      Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung.
      ,
      • Roelofs J.J.
      • Teske G.J.
      • Bonta P.I.
      • de Vries C.J.
      • Meijers J.C.
      • Weening J.J.
      • et al.
      Plasminogen activator inhibitor-1 regulates neutrophil influx during acute pyelonephritis.
      ,
      • Santos L.L.
      • Fan H.
      • Hall P.
      • Ngo D.
      • Mackay C.R.
      • Fingerle-Rowson G.
      • et al.
      Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice.
      ), was significantly up-regulated in the butyrate/R837-treated EoL-1 cells (Figure 2b and Supplementary Figure S1 online). The expression of IL-1 receptor antagonist also increased in EoL-1 cells stimulated with butyrate/R837 (Figure 2b and Supplementary Figure S1).
      Figure thumbnail gr2
      Figure 2TLR7 ligand induced the expression of inflammatory cytokines in differentiated EoL-1 cells. (a) mRNA expression of IL1B, IL23, IL6, TNF, IFNG, and IL13 in the undifferentiated control (control), undifferentiated/R837-stimulated EoL-1 cells (control + R837), butyrate-differentiated EoL-1 cells (butyrate day 2), and butyrate-differentiated/R837-stimulated EoL-1 cells (butyrate day 2 + R837) was analyzed by real-time PCR. Graphs show the mean ± standard error of mean. P < 0.05 (Student t test; n = 6–10/group). (b) Thirty-six different cytokines in the culture supernatants of undifferentiated (control) or butyrate/R837-stimulated EoL-1 cells were semi-quantitated using a human cytokine array. Experiments were performed with two independent replicates and representative results are shown. IL-1ra, IL-1 receptor antagonist; MIF, migration inhibitory factor; Pos, positive control.

      Eosinophil deficiency protected mice against IMQ-induced psoriatic inflammation and regulated the expression of cytokines or chemokines implicated in the pathogenesis of psoriasis

      Because the TLR7 ligand R837 induced the production of various inflammatory mediators in EoL-1 cells, we hypothesized that eosinophils play a role in the pathogenesis of psoriasis. To assess this idea, we induced psoriasiform dermatitis in eosinophil lineage–ablated ΔdblGATA mice (
      • Yu C.
      • Cantor A.B.
      • Yang H.
      • Browne C.
      • Wells R.A.
      • Fujiwara Y.
      • et al.
      Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo.
      ) by repeated topical IMQ application and evaluated their macroscopic phenotypic characteristics and expression of inflammatory cytokines. IMQ treatment induced a psoriasiform dermatitis featuring scale, hyperkeratosis, and erythema in both WT and ΔdblGATA mice. However, ΔdblGATA mice developed far less erythema and scaling compared to WT mice (Figures 3a, 3b ), and a microscopic evaluation correspondingly showed less prominent epidermal thickening in IMQ-treated ΔdblGATA mice than in IMQ-treated WT mice (Figures 3b, 3c). Expression of inflammatory mediators in the back skin of IMQ-treated ΔdblGATA mice also supported an attenuation of psoriatic inflammation in ΔdblGATA mice. We analyzed the mRNA expression of several inflammatory cytokines that are important in psoriasis (Il1b, Il6, Tnf, Il22, Il23, and Il17) (
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      Psoriasis.
      ) and chemokines that promote the migration of T cells and leukocytes (Cxcl2 and Ccl5) by quantitative real-time PCR (
      • Lowes M.A.
      • Suarez-Farinas M.
      • Krueger J.G.
      Immunology of psoriasis.
      ,
      • Schall T.J.
      • Bacon K.
      • Toy K.J.
      • Goeddel D.V.
      Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES.
      ). Although IMQ-treated ΔdblGATA mice showed increased expression of cytokines and chemokines compared to the corresponding control mice, the expression of those factors was significantly lower than in IMQ-treated WT mice (Figure 4). Expression of Tnf and Ccl11 (encoding CC chemokine ligand 11, the cognate ligand for CCR3) did not significantly differ between IMQ-treated WT and ΔdblGATA mice (Figure 4). Expression of Il6 and Il23 was lower in ΔdblGATA mice than in WT mice even without IMQ application (Figure 4), suggesting that eosinophils are a cellular source of those inflammatory mediators (
      • Guerra E.S.
      • Lee C.K.
      • Specht C.A.
      • Yadav B.
      • Huang H.
      • Akalin A.
      • et al.
      Central role of IL-23 and IL-17 producing eosinophils as immunomodulatory effector cells in acute pulmonary aspergillosis and allergic asthma.
      ,
      • Melani C.
      • Mattia G.F.
      • Silvani A.
      • Care A.
      • Rivoltini L.
      • Parmiani G.
      • et al.
      Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes.
      ).
      Figure thumbnail gr3
      Figure 3ΔdblGATA mice were protected against psoriasis-like lesions induced by IMQ treatment. (a) Macroscopic phenotypical representation of psoriasis-like skin lesions in BALB/c WT mice and ΔdblGATA mice treated with vehicle or IMQ for 6–8 days. (b) Ear thickness, erythema, and scaling of the back skin measured on the days indicated. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student t test; n = 7–8/group). (c) Hematoxylin and eosin staining of the back skin and ear sections from WT and ΔdblGATA mice treated with vehicle or IMQ for 6–8 days. Scale bar = 50 μm. IMQ, imiquimod; WT, wild-type.
      Figure thumbnail gr4
      Figure 4The absence of eosinophils affected the expression of cytokines or chemokines implicated in the pathogenesis of psoriasis. The mRNA expression of inflammatory cytokines (Il1b, Il6, Tnf, Il22, Il23, and Il17) and chemokines (Cxcl2, Ccl5, and Ccl11) in the back skin of WT and ΔdblGATA mice treated with IMQ or vehicle (con) was analyzed by real-time PCR and expressed as a fold-change normalized to the expression of Gapdh. Data are representative of two or more independent experiments. Graphs show the mean ± standard error of mean. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student t test). IMQ, imiquimod; WT, wild-type.

      Neutrophil infiltration in the skin, axillary LNs, and spleen induced by IMQ treatment was consistently lower in ΔdblGATA mice compared to WT mice

      After 6 to 8 days of consecutive IMQ treatment, we harvested skin, axillary LNs, and spleen, and isolated cells from those tissues. As reported previously (
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ), we found significant spleen enlargement and an increased abundance of splenocytes in IMQ-treated WT mice (data not shown). However, IMQ-treated ΔdblGATA mice showed a marked decrease in the number of total splenocytes compared to their WT counterpart (data not shown), suggesting that the systemic inflammatory activity in response to IMQ stimulation was attenuated in the absence of eosinophils. The compositions of the cell populations isolated from the skin, axillary LNs, and spleen were determined by flow cytometry. Gr-1+ neutrophils were the only cell subset that infiltrated all analyzed tissues following IMQ treatment in both WT and dblGATA mice, and their abundance was significantly lower in IMQ-treated ΔdblGATA mice (Figures 5 and Supplementary Figures S2–S4 online). The abundance of plasmacytoid dendritic cells and macrophages was also markedly lower in the spleen of IMQ-treated ΔdblGATA mice than in WT mice (Figures 5 and Supplementary Figure S4). The abundance of CD4+ T cells increased in skin after IMQ treatment in both WT and ΔdblGATA mice, but no obvious differences were observed between those groups (Figures 5 and Supplementary Figure S2). Although the number of eosinophils was increased in the skin of IMQ-treated WT mice, the abundance of eosinophils in the axillary LNs and spleen was comparable between the control and IMQ-treated groups (Figures 5 and Supplementary Figures S2–S4). Considering that the absolute cell numbers of eosinophils in the skin, LN, and spleen were much lower than those of other cellular subsets (Figure 5), eosinophils could promote the infiltration of other inflammatory cells, particularly neutrophils, upon IMQ stimulation. This idea was further supported by an immunohistochemical analysis of the skin. Compared with the remarkable infiltration of Gr-1+ neutrophils, major basic protein–positive eosinophils showed only minor infiltration after IMQ application in WT mice (Supplementary Figure S5 online). We also observed a marked decrease in the abundance of Gr-1+ neutrophils in the skin of IMQ-treated ΔdblGATA mice (Supplementary Figure S5b).
      Figure thumbnail gr5
      Figure 5The increase in the abundance of neutrophils in the skin, axillary LN, and spleen of IMQ-treated mice was inhibited by the absence of eosinophils. The frequency of CD4+ T cells, pDC, neutrophils, macrophages, and eosinophils in the skin, LN, and spleen of WT and ΔdblGATA mice treated with IMQ or vehicle (con) was analyzed by flow cytometry. Graphs show the mean ± standard error of mean. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Mann–Whitney test for skin and LN, Student t test for spleen). IMQ, imiquimod; LN, lymph node; pDC, plasmacytoid dendritic cell; WT, wild-type.

      Secreted mediators of activated eosinophils promoted the inflammatory features of neutrophils

      To evaluate whether crosstalk occurs between eosinophils and neutrophils, we cultured HL-60 in control media, conditioned media (CM) collected from undifferentiated EoL-1 cells, or CM collected from butyrate-differentiated/R837-stimulated EoL-1 cells for 24 hours. HL-60 cells cultured in CM from butyrate-differentiated/R837-stimulated EoL-1 cells showed significantly augmented proliferation compared with those cultured in control media or CM from undifferentiated EoL-1 cells (Figure 6a). The expression of IL1B, IL6, and TNF also significantly increased in HL-60 cells cultured in CM from butyrate-differentiated/R837-stimulated EoL-1 cells compared to those cultured in CM from undifferentiated EoL-1 cells (Figure 6b). These findings suggested that eosinophils secrete factors that support the proliferation and activation of neutrophils under TLR7-stimulated inflammatory conditions. The patients with psoriasis also showed significant up-regulation of eosinophil cytotoxic granules (PRG2 and eosinophil-derived neurotoxin), accompanied by marked neutrophil infiltration (Figures 6c, 6d). Although expression of CCR3 was not significantly different between the control and psoriasis patients (Figure 6c), histologic analysis found the presence of eosinophils (in 32 of 42 patients) in the vicinity of infiltrated neutrophils (Figure 6d). Taken together, our observations demonstrate that although an increase in eosinophils was not remarkable in psoriasis, activated eosinophils promoted inflammatory activation and infiltration of neutrophils in psoriasis.
      Figure thumbnail gr6
      Figure 6Eosinophilic inflammatory mediators promoted neutrophil proliferation and activation. (a, b) HL-60 cells were stimulated with conditioned media from undifferentiated EoL-1 cells (EoL-1 UCM) or differentiated/R837-stimulated EoL-1 cells (EoL-1 DRCM) for 24 hours. (a) Cell viability was determined with a water-soluble tetrazolium salt assay. (b) Gene expression of IL1B, IL23, IL6, and TNF was analyzed by real-time PCR. (cCCR3 gene expression and eosinophil cytotoxic granules (PRG2, eosinophil-derived neurotoxin [EDN], and eosinophil cationic protein [ECP]) in human psoriatic lesions were analyzed by real-time PCR. (d) Representative photomicrograph of eosinophils (black arrow) and neutrophils (white arrow) in a human psoriatic lesion. Hematoxylin and eosin scale bar = 50 μm. All data are representative of two independent experiments. Graphs show the mean ± standard error of mean. P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (Student t test).

      Discussion

      Eosinophils are inflammatory effector cells that release cytotoxic molecules, lipid mediators, and cytokines, particularly at sites of allergic inflammation (
      • Rothenberg M.E.
      • Hogan S.P.
      The eosinophil.
      ). Although their pathologic role in psoriasis remains unclear, degranulated eosinophils were detected in the skin of certain groups of patients with psoriasis (
      • Lundin A.
      • Fredens K.
      • Michaelsson G.
      • Venge P.
      The eosinophil granulocyte in psoriasis.
      ,
      • Mansur A.T.
      • Goktay F.
      • Yasar S.P.
      Peripheral blood eosinophilia in association with generalized pustular and erythrodermic psoriasis.
      ), and significantly elevated serum levels of the eosinophil marker eosinophil cationic protein have been reported in patients with psoriasis (
      • Michaelsson G.
      • Kraaz W.
      • Gerden B.
      • Hagforsen E.
      • Lundin I.P.
      • Loof L.
      • et al.
      Patients with psoriasis have elevated levels of serum eosinophil cationic protein and increased numbers of EG2 positive eosinophils in the duodenal stroma.
      ). Here, we identified a previously uncharacterized function of eosinophils whereby they accelerate psoriasiform inflammation by stimulating the activity of neutrophils.
      Psoriasis is a chronic inflammatory disorder of the skin in which interactions between T-cell–mediated immunity and epidermal keratinocytes contribute to the maintenance of chronic inflammation (
      • Nestle F.O.
      • Kaplan D.H.
      • Barker J.
      Psoriasis.
      ). However, epidermal hyperplasia and inflammation are not uniform in psoriasis, and an infiltration of neutrophils accompanied by extensive parakeratosis has been observed in highly inflamed parts of psoriatic lesions (
      • 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.
      ). Accumulated neutrophils actively disturb innate immune responses by releasing inflammatory mediators and influence adaptive immunity by promoting antigen presentation to T cells (
      • Wright H.L.
      • Moots R.J.
      • Bucknall R.C.
      • Edwards S.W.
      Neutrophil function in inflammation and inflammatory diseases.
      ). Direct evidence for innate immune responses triggering the pathogenic T-cell cascade has been further supported by the aggravation of psoriasis and induction of a murine model of psoriasis by the TLR7 agonist IMQ (
      • Gilliet M.
      • Conrad C.
      • Geiges M.
      • Cozzio A.
      • Thurlimann W.
      • Burg G.
      • et al.
      Psoriasis triggered by Toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.
      ,
      • van der Fits L.
      • Mourits S.
      • Voerman J.S.
      • Kant M.
      • Boon L.
      • Laman J.D.
      • et al.
      Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
      ). Although diverse types of innate immune cells express TLR7 (
      • Cao W.
      • Bover L.
      • Cho M.
      • Wen X.
      • Hanabuchi S.
      • Bao M.
      • et al.
      Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction.
      ,
      • Gantier M.P.
      • Tong S.
      • Behlke M.A.
      • Xu D.
      • Phipps S.
      • Foster P.S.
      • et al.
      TLR7 is involved in sequence-specific sensing of single-stranded RNAs in human macrophages.
      ,
      • Nagase H.
      • Okugawa S.
      • Ota Y.
      • Yamaguchi M.
      • Tomizawa H.
      • Matsushima K.
      • et al.
      Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand.
      ), human neutrophils lack TLR7 (
      • Tecchio C.
      • Micheletti A.
      • Cassatella M.A.
      Neutrophil-derived cytokines: facts beyond expression.
      ). Here, we observed a significantly higher expression of TLR7 in EoL-1 cells as compared to HaCaT or HL-60 cells (Figure 1). In EoL-1 cells, TLR7 is likely to function as a regulator of secreted inflammatory mediators because the up-regulation of secreted factors, including CXCL12, IL-8, macrophage migration inhibitory factor, and PAI-1, was observed in EoL-1 cells upon the administration of the TLR7 ligand R837 (Figure 2). All of those mediators are important in neutrophil activation (
      • Delano M.J.
      • Kelly-Scumpia K.M.
      • Thayer T.C.
      • Winfield R.D.
      • Scumpia P.O.
      • Cuenca A.G.
      • et al.
      Neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on CXCL12 signaling.
      ,
      • Roelofs J.J.
      • Teske G.J.
      • Bonta P.I.
      • de Vries C.J.
      • Meijers J.C.
      • Weening J.J.
      • et al.
      Plasminogen activator inhibitor-1 regulates neutrophil influx during acute pyelonephritis.
      ,
      • Santos L.L.
      • Fan H.
      • Hall P.
      • Ngo D.
      • Mackay C.R.
      • Fingerle-Rowson G.
      • et al.
      Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice.
      ), and IL-8 is one of the most potent chemoattractants of neutrophils in inflamed tissues (
      • Kunkel S.L.
      • Standiford T.
      • Kasahara K.
      • Strieter R.M.
      Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung.
      ). Therefore, it is plausible that eosinophils contribute to the pathogenesis of psoriasis and that interactions between eosinophils and neutrophils have a role in the inflammatory responses observed in psoriasis. We have substantiated this hypothesis by demonstrating a marked inhibition of inflammatory changes and a significant decrease in neutrophil infiltration in the lesional skin, LN, and spleen of eosinophil-deficient ΔdblGATA mice treated with IMQ (Figure 3, Figure 4, Figure 5).
      Eosinophils have been confirmed as sources of innate IL-23 and IL-17A, which are the signature pathogenic cytokines in psoriasis (
      • Guerra E.S.
      • Lee C.K.
      • Specht C.A.
      • Yadav B.
      • Huang H.
      • Akalin A.
      • et al.
      Central role of IL-23 and IL-17 producing eosinophils as immunomodulatory effector cells in acute pulmonary aspergillosis and allergic asthma.
      ), and the experimental neutralization of IL-23 attenuated the antigen-induced infiltration of neutrophils and eosinophils into the airway (
      • Wakashin H.
      • Hirose K.
      • Maezawa Y.
      • Kagami S.
      • Suto A.
      • Watanabe N.
      • et al.
      IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice.
      ). Eosinophils also constitutively express IL-1β, IL-6, and TNF-α (
      • Cheung P.F.
      • Wong C.K.
      • Lam C.W.
      Molecular mechanisms of cytokine and chemokine release from eosinophils activated by IL-17A, IL-17F, and IL-23: implication for Th17 lymphocytes-mediated allergic inflammation.
      ), and IL-1β released from eosinophils enhances IL-17A expression in activated CD4+ T cells (
      • Esnault S.
      • Kelly E.A.
      • Nettenstrom L.M.
      • Cook E.B.
      • Seroogy C.M.
      • Jarjour N.N.
      Human eosinophils release IL-1β and increase expression of IL-17A in activated CD4+ T lymphocytes.
      ). However, in this study, IMQ-induced psoriatic inflammation was not completely blocked in ΔdblGATA mice, although they showed attenuated inflammatory responses (Figures 3 and 4). In addition, neutrophils were the only cell subset in which abundance was consistently reduced in the skin, LN, and spleen of ΔdblGATA mice compared to that in WT mice after IMQ treatment (Figure 5). Although the abundance of eosinophils in the skin of WT mice slightly increased after the application of IMQ, the numbers of neutrophils in the skin, LN, and spleen of IMQ-treated WT mice were 36 to 59 times higher than eosinophils counted in the same tissues (Figure 5). Neutrophils produce lower amounts of cytokines on a per-cell basis compared to other immune cells, though they constitute the majority of infiltrating cells in inflamed tissues (
      • Tecchio C.
      • Micheletti A.
      • Cassatella M.A.
      Neutrophil-derived cytokines: facts beyond expression.
      ). Therefore, neutrophil-activating signals play an important role in inducing inflammation mediated by neutrophil-derived cytokines (
      • Oishi K.
      • Machida K.
      Some plasma component is essential for IL-6 secretion by neutrophils.
      ,
      • Zimmermann M.
      • Arruda-Silva F.
      • Bianchetto-Aguilera F.
      • Finotti G.
      • Calzetti F.
      • Scapini P.
      • et al.
      IFNalpha enhances the production of IL-6 by human neutrophils activated via TLR8.
      ). Thus, we suggest that eosinophils influence the pathogenesis of psoriasis by modulating microenvironments to favor the activation of neutrophils. This idea was supported by the significantly increased proliferation and expression of inflammatory cytokines in HL-60 cells treated with the CM collected from R837-stimulated EoL-1 cells, and by the up-regulated eosinophil cytotoxic granules in human psoriasis skin (Figure 6). Transcriptomic analyses results of psoriatic lesions also support the pathogenic role of eosinophils, as demonstrated by the inflammatory genetic changes of eosinophils (
      • Swindell W.R.
      • Johnston A.
      • Voorhees J.J.
      • Elder J.T.
      • Gudjonsson J.E.
      Dissecting the psoriasis transcriptome: inflammatory- and cytokine-driven gene expression in lesions from 163 patients.
      ) and up-regulation of an eosinophil activation marker (
      • Suarez-Farinas M.
      • Lowes M.A.
      • Zaba L.C.
      • Krueger J.G.
      Evaluation of the psoriasis transcriptome across different studies by gene set enrichment analysis (GSEA).
      ) in psoriasis skin lesions. Although the IMQ-induced mouse model of psoriasis reproduces several disease features and provides some mechanistic insight into the disease, it is not an exact model of human psoriasis; it is associated with the lack of chronicity or comorbidities of psoriasis, such as arthritis (
      • Flutter B.
      • Nestle F.O.
      TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.
      ). Therefore, we examined human psoriasis samples, and we could observe eosinophils in 32 of the 42 patients with psoriasis in the vicinity of infiltrated neutrophils (Figure 6d), without differences in Th2 phenotypic characteristics (such as eczema or pruritus), between the eosinophil-present and eosinophil-absent groups (odds ratio = 1.596, P = 0.7152). However, considering that expression of CCR3 was not significantly up-regulated in the skin lesions of psoriasis patients compared to the control (Figures 6c), we propose that activated eosinophils accelerate psoriatic inflammation rather than preferentially migrate to the lesional skin. Increased expression of eosinophil cytotoxic granules without a significant difference in CCR3 expression in IMQ-treated WT mice was consistent with this hypothesis (Supplementary Figure S6 online); however, the mechanism underlying the inflammatory response in a murine model might be different from the case for human psoriasis.
      Although eosinophils are generally considered to be proinflammatory cells, they also have physiological functions in the absence of infections or allergic reactions (
      • Jung Y.
      • Rothenberg M.E.
      Roles and regulation of gastrointestinal eosinophils in immunity and disease.
      ). An anti-inflammatory effect of eosinophils associated with down-regulated IFN-α production by plasmacytoid dendritic cells has been reported (
      • Skrzeczynska-Moncznik J.
      • Zabieglo K.
      • Bossowski J.P.
      • Osiecka O.
      • Wlodarczyk A.
      • Kapinska-Mrowiecka M.
      • et al.
      Eosinophils regulate interferon alpha production in plasmacytoid dendritic cells stimulated with components of neutrophil extracellular traps.
      ). EoL-1 cells stimulated with R837 secreted IL-1 receptor antagonist (Figure 2b), which is a member of the IL-1 family that competes with IL-1α and IL-1β for receptor binding and subsequently prevents the development of inflammatory responses (
      • Arend W.P.
      • Malyak M.
      • Guthridge C.J.
      • Gabay C.
      Interleukin-1 receptor antagonist: role in biology.
      ). Considering that the production of IL-1 receptor antagonist is induced in various tissues in response to infection or inflammation (
      • Arend W.P.
      • Gabay C.
      Physiologic role of interleukin-1 receptor antagonist.
      ,
      • Kim H.J.
      • Kim S.H.
      • Park J.
      • Lee M.
      • Kim D.S.
      • Lee M.G.
      Up-regulation of receptor antagonist interleukin-1 family members in psoriasis and their regulation by pro-inflammatory cytokines.
      ), IL-1 receptor antagonist production by stimulated EoL-1 could reflect a physiological effort to counterbalance excessive proinflammatory reactions. However, our observations indicated that the proinflammatory role of eosinophils dominates over their homeostatic function under psoriasis-inducing conditions, and we suggest that TLR7 activation signals are sufficient to drive eosinophil-mediated exacerbation of inflammatory responses.
      Eosinophils interact with various kinds of innate and adaptive immune cells. Interactions between eosinophils and mast cells are a key mechanism in Th2-mediated allergies and parasite infections (
      • Wong C.K.
      • Ng S.S.
      • Lun S.W.
      • Cao J.
      • Lam C.W.
      Signalling mechanisms regulating the activation of human eosinophils by mast-cell-derived chymase: implications for mast cell-eosinophil interaction in allergic inflammation.
      ). Furthermore, human eosinophils differentially affect the generation of Th1 and Th2 chemokines depending on the cytokines present in their microenvironment (
      • Liu L.Y.
      • Bates M.E.
      • Jarjour N.N.
      • Busse W.W.
      • Bertics P.J.
      • Kelly E.A.
      Generation of Th1 and Th2 chemokines by human eosinophils: evidence for a critical role of TNF-alpha.
      ). Eosinophil-derived MBP stimulates neutrophils to produce superoxide and IL-8 (
      • Page S.M.
      • Gleich G.J.
      • Roebuck K.A.
      • Thomas L.L.
      Stimulation of neutrophil interleukin-8 production by eosinophil granule major basic protein.
      ,
      • Shenoy N.G.
      • Gleich G.J.
      • Thomas L.L.
      Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway.
      ), and eosinophils also produce IL-8 (
      • Henkels K.M.
      • Frondorf K.
      • Gonzalez-Mejia M.E.
      • Doseff A.L.
      • Gomez-Cambronero J.
      IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3).
      ,
      • Kita H.
      • Abu-Ghazaleh R.I.
      • Sur S.
      • Gleich G.J.
      Eosinophil major basic protein induces degranulation and IL-8 production by human eosinophils.
      ). Our data demonstrate that eosinophils provide signals that promote an innate immune response, particularly by accelerating the migration and activation of neutrophils. Therefore, studying the inflammatory interactions between eosinophils and neutrophils may provide new insights that could contribute to achieving better control of psoriasis.

      Materials and Methods

      Cell culture

      EoL-1 and HL-60 cells were maintained in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO) and HaCaT cells were maintained in DMEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum in 5% CO2 at 37°C. EoL-1 cells were induced to differentiate by adding 0.5 μM butyrate (Sigma-Aldrich). To examine the effects of a TLR7 ligand on EoL-1 cells, undifferentiated control and butyrate-treated EoL-1 cells were stimulated with 1 μg/mL R837 (Invivogen, San Diego, CA) for 24 hours. The CM was collected from undifferentiated and butyrate-differentiated/R837-stimulated EoL-1 cells, and it was used for stimulating HL-60 cells.

      Mice and treatments

      BALB/c mice (Orientbio, Gapyeng, Korea) and ΔdblGATA mice (Jackson Laboratory, Bar Harbor, ME) were housed in standard laboratory conditions of temperature and humidity at Gachon University. All of the experiments complied with institutional guidelines for animal welfare. At 8–10 weeks old, the mice received a daily topical dose of 62.5 mg IMQ cream (5%) (Aldara; 3M Pharmaceuticals, Maplewood, MN) or vehicle cream (Vaseline; Unilever, Rotterdam, Netherlands) on the shaved back and the right ear for 6–8 consecutive days. Mice were assessed for the severity of the psoriasis-like skin condition. The ear skin thickness was measured in millimeters, and erythema and scaling were scored independently on a 5-point-scale from 0 to 4, as follows: 0 = none; 1 = slight; 2 = moderate; 3 = marked; 4 = very marked.

      Patients

      Forty-two patients (30 male and 12 female patients, mean age 46.26 ± 14.11 years) who were diagnosed with psoriasis based on corroborating clinical and histologic findings were included in the study. The study was approved by the Ethics Committee of the Gachon University Gil Medical Center (GAIRB2017-385) and was conducted according to the Declaration of Helsinki. All participants gave written informed consent. Skin biopsies were taken from an area that was representative of the active disease.

      Statistical analysis

      Data are presented as the mean ± standard error of the mean. When necessary, two-group comparisons were performed using Student t test or Mann–Whitney test. A P value <0.05 was considered statistically significant.
      Detailed methods for the histologic analysis; cell viability assay; PCR analysis; preparation of cell suspensions from the skin, axillary LN, and spleen, followed by flow cytometry; and protein array analysis of the cytokines in the culture supernatant of eosinophils are available in the Supplementary Materials online.

      Conflict of Interest

      The authors state no conflict of interest.

      Acknowledgments

      The authors thank Eun-Hui Lee and Jinsun Jang (Gachon University, Korea) for their technical assistance. This work was supported by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (2016R1D1A1A09916492), and by the Gachon University Gil Medical Center Research Fund (FRD2017-14).

      Supplementary Material

      References

        • Arend W.P.
        • Gabay C.
        Physiologic role of interleukin-1 receptor antagonist.
        Arthritis Res. 2000; 2: 245-248
        • Arend W.P.
        • Malyak M.
        • Guthridge C.J.
        • Gabay C.
        Interleukin-1 receptor antagonist: role in biology.
        Annu Rev Immunol. 1998; 16: 27-55
        • Baker B.S.
        • Ovigne J.M.
        • Powles A.V.
        • Corcoran S.
        • Fry L.
        Normal keratinocytes express Toll-like receptors (TLRs) 1, 2 and 5: modulation of TLR expression in chronic plaque psoriasis.
        Br J Dermatol. 2003; 148: 670-679
        • Bochner B.S.
        Novel therapies for eosinophilic disorders.
        Immunol Allergy Clin N Am. 2015; 35: 577-598
        • Cao W.
        • Bover L.
        • Cho M.
        • Wen X.
        • Hanabuchi S.
        • Bao M.
        • et al.
        Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction.
        J Exp Med. 2009; 206: 1603-1614
        • Cheung P.F.
        • Wong C.K.
        • Lam C.W.
        Molecular mechanisms of cytokine and chemokine release from eosinophils activated by IL-17A, IL-17F, and IL-23: implication for Th17 lymphocytes-mediated allergic inflammation.
        J Immunol. 2008; 180: 5625-5635
        • Delano M.J.
        • Kelly-Scumpia K.M.
        • Thayer T.C.
        • Winfield R.D.
        • Scumpia P.O.
        • Cuenca A.G.
        • et al.
        Neutrophil mobilization from the bone marrow during polymicrobial sepsis is dependent on CXCL12 signaling.
        J Immunol. 2011; 187: 911-918
        • Esnault S.
        • Kelly E.A.
        • Nettenstrom L.M.
        • Cook E.B.
        • Seroogy C.M.
        • Jarjour N.N.
        Human eosinophils release IL-1β and increase expression of IL-17A in activated CD4+ T lymphocytes.
        Clin Exp Allergy. 2012; 42: 1756-1764
        • Flutter B.
        • Nestle F.O.
        TLRs to cytokines: mechanistic insights from the imiquimod mouse model of psoriasis.
        Eur J Immunol. 2013; 43: 3138-3146
        • Gantier M.P.
        • Tong S.
        • Behlke M.A.
        • Xu D.
        • Phipps S.
        • Foster P.S.
        • et al.
        TLR7 is involved in sequence-specific sensing of single-stranded RNAs in human macrophages.
        J Immunol. 2008; 180: 2117-2124
        • Gilliet M.
        • Conrad C.
        • Geiges M.
        • Cozzio A.
        • Thurlimann W.
        • Burg G.
        • et al.
        Psoriasis triggered by Toll-like receptor 7 agonist imiquimod in the presence of dermal plasmacytoid dendritic cell precursors.
        Arch Dermatol. 2004; 140: 1490-1495
        • Griffiths C.E.
        Immunotherapy for psoriasis: from serendipity to selectivity.
        Lancet. 2002; 359: 279-280
        • Guerra E.S.
        • Lee C.K.
        • Specht C.A.
        • Yadav B.
        • Huang H.
        • Akalin A.
        • et al.
        Central role of IL-23 and IL-17 producing eosinophils as immunomodulatory effector cells in acute pulmonary aspergillosis and allergic asthma.
        PLoS Pathog. 2017; 13: e1006175
        • Hari A.
        • Flach T.L.
        • Shi Y.
        • Mydlarski P.R.
        Toll-like receptors: role in dermatological disease.
        Mediators Inflamm. 2010; 2010: 437246
        • Henkels K.M.
        • Frondorf K.
        • Gonzalez-Mejia M.E.
        • Doseff A.L.
        • Gomez-Cambronero J.
        IL-8-induced neutrophil chemotaxis is mediated by Janus kinase 3 (JAK3).
        FEBS Lett. 2011; 585: 159-166
        • Jung Y.
        Comparative analysis of dibutyric cAMP and butyric acid on the differentiation of human eosinophilic leukemia EoL-1 cells.
        Immune Netw. 2015; 15: 313-318
        • Jung Y.
        • Rothenberg M.E.
        Roles and regulation of gastrointestinal eosinophils in immunity and disease.
        J Immunol. 2014; 193: 999-1005
        • Kim H.J.
        • Kim S.H.
        • Je J.H.
        • Shin D.Y.
        • Kim D.S.
        • Lee M.G.
        Increased expression of Toll-like receptors 3, 7, 8 and 9 in peripheral blood mononuclear cells in patients with psoriasis.
        Exp Dermatol. 2016; 25: 485-487
        • Kim H.J.
        • Kim S.H.
        • Park J.
        • Lee M.
        • Kim D.S.
        • Lee M.G.
        Up-regulation of receptor antagonist interleukin-1 family members in psoriasis and their regulation by pro-inflammatory cytokines.
        J Dermatol Sci. 2016; 82: 204-206
        • Kita H.
        • Abu-Ghazaleh R.I.
        • Sur S.
        • Gleich G.J.
        Eosinophil major basic protein induces degranulation and IL-8 production by human eosinophils.
        J Immunol. 1995; 154: 4749-4758
        • Kunkel S.L.
        • Standiford T.
        • Kasahara K.
        • Strieter R.M.
        Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung.
        Exp Lung Res. 1991; 17: 17-23
        • Lande R.
        • Gregorio J.
        • Facchinetti V.
        • Chatterjee B.
        • Wang Y.H.
        • Homey B.
        • et al.
        Plasmacytoid dendritic cells sense self-DNA coupled with antimicrobial peptide.
        Nature. 2007; 449: 564-569
        • Liu L.Y.
        • Bates M.E.
        • Jarjour N.N.
        • Busse W.W.
        • Bertics P.J.
        • Kelly E.A.
        Generation of Th1 and Th2 chemokines by human eosinophils: evidence for a critical role of TNF-alpha.
        J Immunol. 2007; 179: 4840-4848
        • Lowes M.A.
        • Suarez-Farinas M.
        • Krueger J.G.
        Immunology of psoriasis.
        Annu Rev Immunol. 2014; 32: 227-255
        • Lundin A.
        • Fredens K.
        • Michaelsson G.
        • Venge P.
        The eosinophil granulocyte in psoriasis.
        Br J Dermatol. 1990; 122: 181-193
        • Mansson A.
        • Cardell L.O.
        Role of atopic status in Toll-like receptor (TLR)7- and TLR9-mediated activation of human eosinophils.
        J Leukoc Biol. 2009; 85: 719-727
        • Mansur A.T.
        • Goktay F.
        • Yasar S.P.
        Peripheral blood eosinophilia in association with generalized pustular and erythrodermic psoriasis.
        J Eur Acad Dermatol Venereol. 2008; 22: 451-455
        • Melani C.
        • Mattia G.F.
        • Silvani A.
        • Care A.
        • Rivoltini L.
        • Parmiani G.
        • et al.
        Interleukin-6 expression in human neutrophil and eosinophil peripheral blood granulocytes.
        Blood. 1993; 81: 2744-2749
        • Michaelsson G.
        • Kraaz W.
        • Gerden B.
        • Hagforsen E.
        • Lundin I.P.
        • Loof L.
        • et al.
        Patients with psoriasis have elevated levels of serum eosinophil cationic protein and increased numbers of EG2 positive eosinophils in the duodenal stroma.
        Br J Dermatol. 1996; 135: 371-378
        • Nagase H.
        • Okugawa S.
        • Ota Y.
        • Yamaguchi M.
        • Tomizawa H.
        • Matsushima K.
        • et al.
        Expression and function of Toll-like receptors in eosinophils: activation by Toll-like receptor 7 ligand.
        J Immunol. 2003; 171: 3977-3982
        • Nestle F.O.
        • Kaplan D.H.
        • Barker J.
        Psoriasis.
        N Engl J Med. 2009; 361: 496-509
        • Netea M.G.
        • van der Graaf C.
        • Van der Meer J.W.
        • Kullberg B.J.
        Toll-like receptors and the host defense against microbial pathogens: bringing specificity to the innate-immune system.
        J Leukoc Biol. 2004; 75: 749-755
        • Oishi K.
        • Machida K.
        Some plasma component is essential for IL-6 secretion by neutrophils.
        Environ Health Prevent Med. 1997; 2: 89-92
        • Page S.M.
        • Gleich G.J.
        • Roebuck K.A.
        • Thomas L.L.
        Stimulation of neutrophil interleukin-8 production by eosinophil granule major basic protein.
        Am J Respir Cell Mol Biol. 1999; 21: 230-237
        • Roelofs J.J.
        • Teske G.J.
        • Bonta P.I.
        • de Vries C.J.
        • Meijers J.C.
        • Weening J.J.
        • et al.
        Plasminogen activator inhibitor-1 regulates neutrophil influx during acute pyelonephritis.
        Kidney Int. 2009; 75: 52-59
        • Rothenberg M.E.
        • Hogan S.P.
        The eosinophil.
        Annu Rev Immunol. 2006; 24: 147-174
        • Saito H.
        • Hayakawa T.
        • Mita H.
        • Akiyama K.
        • Shida T.
        Effect of butyric acid on induction of differentiation into eosinophil-like cells in human eosinophilic leukemia cells, EoL-1 cell line: possible role of granulocyte-macrophage colony-stimulating factor as an autocrine differentiating factor.
        Int Arch Allergy Immunol. 1993; 100: 240-247
        • Santos L.L.
        • Fan H.
        • Hall P.
        • Ngo D.
        • Mackay C.R.
        • Fingerle-Rowson G.
        • et al.
        Macrophage migration inhibitory factor regulates neutrophil chemotactic responses in inflammatory arthritis in mice.
        Arthritis Rheum. 2011; 63: 960-970
        • Schall T.J.
        • Bacon K.
        • Toy K.J.
        • Goeddel D.V.
        Selective attraction of monocytes and T lymphocytes of the memory phenotype by cytokine RANTES.
        Nature. 1990; 347: 669-671
        • Shenoy N.G.
        • Gleich G.J.
        • Thomas L.L.
        Eosinophil major basic protein stimulates neutrophil superoxide production by a class IA phosphoinositide 3-kinase and protein kinase C-zeta-dependent pathway.
        J Immunol. 2003; 171: 3734-3741
        • 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.
        J Immunol. 2012; 189: 1611-1617
        • Skrzeczynska-Moncznik J.
        • Zabieglo K.
        • Bossowski J.P.
        • Osiecka O.
        • Wlodarczyk A.
        • Kapinska-Mrowiecka M.
        • et al.
        Eosinophils regulate interferon alpha production in plasmacytoid dendritic cells stimulated with components of neutrophil extracellular traps.
        J Interferon Cytokine Res. 2017; 37: 119-128
        • Suarez-Farinas M.
        • Lowes M.A.
        • Zaba L.C.
        • Krueger J.G.
        Evaluation of the psoriasis transcriptome across different studies by gene set enrichment analysis (GSEA).
        PLoS One. 2010; 5: e10247
        • Swindell W.R.
        • Johnston A.
        • Voorhees J.J.
        • Elder J.T.
        • Gudjonsson J.E.
        Dissecting the psoriasis transcriptome: inflammatory- and cytokine-driven gene expression in lesions from 163 patients.
        BMC Genomics. 2013; 14: 527
        • Tecchio C.
        • Micheletti A.
        • Cassatella M.A.
        Neutrophil-derived cytokines: facts beyond expression.
        Front Immunol. 2014; 5: 508
        • 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.
        Exp Dermatol. 2000; 9: 1-10
        • van der Fits L.
        • Mourits S.
        • Voerman J.S.
        • Kant M.
        • Boon L.
        • Laman J.D.
        • et al.
        Imiquimod-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis.
        J Immunol. 2009; 182: 5836-5845
        • Wakashin H.
        • Hirose K.
        • Maezawa Y.
        • Kagami S.
        • Suto A.
        • Watanabe N.
        • et al.
        IL-23 and Th17 cells enhance Th2-cell-mediated eosinophilic airway inflammation in mice.
        Am J Respir Crit Care Med. 2008; 178: 1023-1032
        • Wong C.K.
        • Ho C.Y.
        • Lam C.W.
        • Zhang J.P.
        • Hjelm N.M.
        Differentiation of a human eosinophilic leukemic cell line, EoL-1: characterization by the expression of cytokine receptors, adhesion molecules, CD95 and eosinophilic cationic protein (ECP).
        Immunol Lett. 1999; 68: 317-323
        • Wong C.K.
        • Ng S.S.
        • Lun S.W.
        • Cao J.
        • Lam C.W.
        Signalling mechanisms regulating the activation of human eosinophils by mast-cell-derived chymase: implications for mast cell-eosinophil interaction in allergic inflammation.
        Immunology. 2009; 126: 579-587
        • Wright H.L.
        • Moots R.J.
        • Bucknall R.C.
        • Edwards S.W.
        Neutrophil function in inflammation and inflammatory diseases.
        Rheumatology. 2010; 49: 1618-1631
        • Yu C.
        • Cantor A.B.
        • Yang H.
        • Browne C.
        • Wells R.A.
        • Fujiwara Y.
        • et al.
        Targeted deletion of a high-affinity GATA-binding site in the GATA-1 promoter leads to selective loss of the eosinophil lineage in vivo.
        J Exp Med. 2002; 195: 1387-1395
        • Zimmermann M.
        • Arruda-Silva F.
        • Bianchetto-Aguilera F.
        • Finotti G.
        • Calzetti F.
        • Scapini P.
        • et al.
        IFNalpha enhances the production of IL-6 by human neutrophils activated via TLR8.
        Sci Rep. 2016; 6: 19674