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The antimicrobial peptide human beta-defensin 2 promotes itch through Toll-like receptor 4 signaling in mice

2017; Elsevier BV; Volume: 140; Issue: 3 Linguagem: Inglês

10.1016/j.jaci.2017.03.035

1097-6825

Jing Feng, Jialie Luo, Madison R. Mack, Yang Pu, Feng Zhang, Guan Wang, Xuan Gong, Tao Cai, Zhinan Mei, Brian Kim, Shijin Yin, Hongzhen Hu,

Asthma and respiratory diseases

Chronic skin inflammation is considered the most prominent feature for clinical diagnosis of psoriasis, a long-lasting autoimmune disease characterized by patches of red, itchy, and scaly skin. Besides skin inflammation, up to 84% of patients with psoriasis suffer from chronic itch, which significantly impairs quality of life.1Yosipovitch G. Goon A. Wee J. Chan Y.H. Goh C.L. The prevalence and clinical characteristics of pruritus among patients with extensive psoriasis.Br J Dermatol. 2000; 143: 969-973Crossref PubMed Scopus (346) Google Scholar Although recent exciting studies have identified a positive correlation between the intensity of psoriatic itch and the expression levels of nerve growth factor, neuropeptides, and many cytokines,2Reich A. Szepietowski J.C. Mediators of pruritus in psoriasis.Mediators Inflamm. 2007; 2007: 64727Crossref PubMed Scopus (59) Google Scholar the molecular and cellular mechanisms underlying psoriatic itch are not fully understood. In response to TH1 or TH17 cytokines, excessive antimicrobial peptides (AMPs) are locally released by rapidly differentiating psoriatic keratinocytes. Among them, human beta-defensin 2 (hBD2) is increased by nearly 400-fold in patients with severe psoriasis and serves as a biomarker for psoriasis activity.3Jansen P.A. Rodijk-Olthuis D. Hollox E.J. Kamsteeg M. Tjabringa G.S. de Jongh G.J. et al.β-Defensin-2 protein is a serum biomarker for disease activity in psoriasis and reaches biologically relevant concentrations in lesional skin.PloS One. 2009; 4: e4725Crossref PubMed Scopus (143) Google Scholar Besides potent antimicrobial activity, hBD2 has diversified roles in regulating adaptive immunity, wound healing, and male fertility.4Suarez-Carmona M. Hubert P. Delvenne P. Herfs M. Defensins: "Simple" antimicrobial peptides or broad-spectrum molecules?.Cytokine Growth Factor Rev. 2015; 26: 361-370Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar Interestingly, hBD2 also promotes inflammation by recruiting multiple types of immune cells through interacting with both C-C chemokine receptor type 2 (CCR2) and C-C chemokine receptor type 6 (CCR6) in vitro.5Röhrl J. Yang D. Oppenheim J.J. Hehlgans T. Human β-defensin 2 and 3 and their mouse orthologs induce chemotaxis through interaction with CCR2.J Immunol. 2010; 184: 6688-6694Crossref PubMed Scopus (232) Google Scholar, 6Yang D. Chertov O.B. Bykovskaia S.N. Chen Q. Buffo M.J. Shogan J. et al.β-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.Science. 1999; 286: 525-528Crossref PubMed Scopus (1524) Google Scholar However, the role of hBD2 in itch sensation has not been determined. We therefore investigated whether hBD2 could elicit scratching in wild-type C57BL/6J mice. Strikingly, intradermal injections of hBD2 produced a robust scratching response in wild-type mice in a dose-dependent manner (Fig 1, A and B). In addition, murine beta-defensin 4, the hBD2 ortholog in mouse, and hBD3 could also elicit scratching in mice although the itch intensity varied among different AMPs (see Fig E1 in this article's Online Repository at www.jacionline.org), which provides a proof of concept that both human and mouse AMPs could serve as endogenous pruritogens. Previous studies have demonstrated that sensory transient receptor potential cation channel subfamily V member 1 (TRPV1) and subfamily A member 1 (TRPA1) channels are selectively expressed by a subpopulation of primary afferent nociceptors and serve as molecular integrators for numerous endogenous pruritogens released by skin-resident cells to provoke both histaminergic and nonhistaminergic itch.7Luo J. Feng J. Liu S. Walters E.T. Hu H. Molecular and cellular mechanisms that initiate pain and itch.Cell Mol Life Sci. 2015; 72: 3201-3223Crossref PubMed Scopus (56) Google Scholar Furthermore, the TRPV1-expressing sensory fibers mediate skin inflammation through facilitating the function of dermal immune cells in a mouse model of psoriasis.8Riol-Blanco L. Ordovas-Montanes J. Perro M. Naval E. Thiriot A. Alvarez D. et al.Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation.Nature. 2014; 510: 157-161Crossref PubMed Scopus (331) Google Scholar To investigate whether TRPA1, TRPV1, or both are also downstream mediators of hBD2-induced itch, we tested whether genetic ablation of TRPA1 or TRPV1 function affects hBD2-induced scratching. Strikingly, the number of hBD2-induced scratching bouts was markedly reduced in the Trpv1−/− but not the Trpa1−/− mice when compared with wild-type mice (Fig 1, C and D). One possibility for the involvement of TRPV1 in hBD2-induced itch is that hBD2 might promote excitability of cutaneous pruriceptors through directly activating TRPV1. To test this possibility, we examined the effect of hBD2 on dorsal root ganglion (DRG) neurons isolated from wild-type mice using live-cell Ca2+ imaging. Surprisingly, no intracellular Ca2+ ([Ca2+]i) response was observed when 10 μM hBD2 was applied to wild-type DRG neurons (Fig 1, F). Consistent with the Ca2+ imaging data in DRG neurons, hBD2 did not activate membrane currents in HEK293 cells transfected with either mouse TRPV1 or human TRPA1 DNA construct (Fig 1, G and H). These results suggest that TRPV1 is a key downstream mediator of hBD2-induced itch but hBD2 does not directly activate TRPV1. This conclusion was further supported by the finding that TRPV1 deficiency abolished [Ca2+]i response in DRG neurons elicited by applications of hBD2-treated wild-type skin superfusates (see Fig E2 in this article's Online Repository at www.jacionline.org). Because mast cell–derived histamine is one of the best studied pruritogens, especially in allergic itch, and TRPV1 is the major downstream mediator of histaminergic itch, we asked whether mast cells are involved in hBD2-elicited itch by measuring hBD2-induced scratching in mast cell–deficient KitW-sh "sash" mice. Surprisingly, hBD2 evoked comparable scratching responses between the sash mice and the wild-type mice (Fig 1, E), suggesting that mast cells are dispensable and histamine might not play an essential role in hBD2-induced itch, which is consistent with clinical observations that psoriatic itch is refractory to oral antihistamines in more than 80% of the patients.1Yosipovitch G. Goon A. Wee J. Chan Y.H. Goh C.L. The prevalence and clinical characteristics of pruritus among patients with extensive psoriasis.Br J Dermatol. 2000; 143: 969-973Crossref PubMed Scopus (346) Google Scholar, 2Reich A. Szepietowski J.C. Mediators of pruritus in psoriasis.Mediators Inflamm. 2007; 2007: 64727Crossref PubMed Scopus (59) Google Scholar Because hBD2 receptors CCR2 and CCR6 are abundantly expressed by skin-resident cells,5Röhrl J. Yang D. Oppenheim J.J. Hehlgans T. Human β-defensin 2 and 3 and their mouse orthologs induce chemotaxis through interaction with CCR2.J Immunol. 2010; 184: 6688-6694Crossref PubMed Scopus (232) Google Scholar, 6Yang D. Chertov O.B. Bykovskaia S.N. Chen Q. Buffo M.J. Shogan J. et al.β-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6.Science. 1999; 286: 525-528Crossref PubMed Scopus (1524) Google Scholar we tested hBD2-elicited scratching in both ccr2−/− and ccr6−/− mice. To our surprise, hBD2 induced comparable scratching responses among wild-type, ccr2−/−, and ccr6−/− mice (Fig 2, A and B), suggesting that neither CCR2 nor CCR6 mediates hBD2-induced itch. Because Toll-like receptor 4 (TLR4) mediates murine beta-defensin 2–induced activation of dendritic cells and murine beta-defensin 2 and hBD2 share structural and functional similarities,9Biragyn A. Ruffini P.A. Leifer C.A. Klyushnenkova E. Shakhov A. Chertov O. et al.Toll-like receptor 4-dependent activation of dendritic cells by β-defensin 2.Science. 2002; 298: 1025-1029Crossref PubMed Scopus (815) Google Scholar we tested hBD2-induced scratching in Tlr4−/− mice. Strikingly, the hBD2-induced scratching response was markedly reduced in the Tlr4−/− mice compared with wild-type mice (Fig 2, C), suggesting that hBD2-induced itch requires TLR4. To further test whether TLR4-expressing skin-resident immune cells are involved in the hBD2-induced itch, we conditionally ablated TLR4 expression in the myeloid cell lineage by generating the LysMcre; Tlr4f/f mice as we found that TLR4 was primarily expressed by CD11b+/CD11c− dermal macrophages besides a small percentage of dendritic cells and eosinophils (see Fig E3 in this article's Online Repository at www.jacionline.org). Indeed, the number of hBD2-induced scratching bouts in the LysMcre; Tlr4f/f mice was substantially reduced when compared with their wild-type littermates (Fig 2, D). To assess whether hBD2 directly activates TLR4 in skin-resident cells, we performed live-cell Ca2+ imaging on skin-resident cells freshly isolated from mouse ear skin preparations. Consistent with behavioral testing, 10 μM hBD2 elicited a robust [Ca2+]i response in 4.8% of the skin-resident cells examined (Fig 2, E) and the [Ca2+]i response was completely absent from the skin-resident cells isolated from the Tlr4−/− and LysMcre; Tlr4f/f mice or Tlr4−/− DRG neurons (Fig 2, F-H). Moreover, hBD2 also activated human skin–resident myeloid cells, which was nearly abolished by a selective TLR4 antagonist LPS-RS (see Fig E4 in this article's Online Repository at www.jacionline.org). Surprisingly, the classic TLR4 ligand LPS from either Escherichia coli or Salmonella enterica did not evoke significant scratching. Because it is known that itch is constitutively inhibited by pain, we thus tested the effects of intraplantar injections of hBD2 or LPS on mechanical threshold. Indeed, we found that LPS markedly reduced the mechanical threshold while hBD2 had no effect (see Fig E5 in this article's Online Repository at www.jacionline.org), suggesting that the LPS signaling pathway is associated with the pain sensation while the stimulation of TLR4 by hBD2 primarily generates itch sensation without inducing pain responses. Taken together, these results provide strong evidence that TLR4 expressed by skin-resident immune cells but not DRG neurons mediates hBD2-induced itch in mice. In conclusion, here we first report that hBD2, which is markedly upregulated in differentiated keratinocytes of patients with psoriasis, promotes itch sensation by activating TLR4-expressing cutaneous immune cells in mice. Our findings suggest that hBD2 could act as a potent endogenous pruritogen, which expands the roles of the antimicrobial beta-defensin family and may also provide new therapeutic targets against psoriatic itch. C57BL/6J, ccr2−/−, ccr6−/−, Tlr4−/−, LysMcre, Tlr4f/f, and mast cell–deficient KitW-sh "sash" mice were obtained from Jackson Laboratories (Bar Harbor, Me). Trpv1+/+ and congenic Trpv1−/− mice on the C57BL/6J background were obtained from Jackson Laboratories. The Trpa1+/+ and congenic Trpa1−/− mice on the C57BL/6J background were described previously.E1Cruz-Orengo L. Dhaka A. Heuermann R.J. Young T.J. Montana M.C. Cavanaugh E.J. et al.Cutaneous nociception evoked by 15-delta PGJ2 via activation of ion channel TRPA1.Mol Pain. 2008; 4: 30Crossref PubMed Scopus (117) Google Scholar All transgenic mice were extensively backcrossed to C57BL/6J for 10 or more generations. Conditional knockout TLR4 in the myeloid cell lineage was generated by mating the LysM-cre mice with Tlr4f/f mice and wild-type littermates were used as the control in the behavior testing. All mice were housed under a 12-hour light/dark cycle with food and water provided ad libitum. All behavioral tests were videotaped from a side angle, and behavioral assessments were done by observers blind to the treatments or genotypes of animals. All experiments were performed in accordance with the guidelines of the National Institutes of Health and the International Association for the Study of Pain, and were approved by the Animal Studies Committee at Washington University School of Medicine. HEK293T cells were grown as a monolayer maintained in Dulbecco modified Eagle medium (DMEM) (Life Technologies, Carlsbad, Calif), supplemented with 10% FBS (Life Technologies), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C with 5% CO2. The cells were transiently transfected with cDNAs for mouse TRPV1 or human TRPA1 using Lipofectamine 2000 (Invitrogen, Carlsbad, Calif). Following transfection, the cells were maintained in DMEM at 37°C for 24 hours before use. Fresh mouse ear skin preparations were cut and separated using forceps and digested in 0.25 mg/mL Liberase TL (Roche, Risch-Rotkreuz, Switzerland) in DMEM media for 90 minutes at 37°C as described.E2Broggi A. Cigni C. Zanoni I. Granucci F. Preparation of single-cell suspensions for cytofluorimetric analysis from different mouse skin regions.J Vis Exp. 2016; : e52589PubMed Google Scholar Samples were mashed through 70-μm cell strainers and washed with DMEM media–supplemented 10% FBS (Life Technologies), 100 units/mL penicillin, and 100 μg/mL streptomycin. Single-cell suspensions were used for subsequent Ca2+ imaging assays. Mouse spinal columns were removed and placed in ice-cold HBSS; neurons were acutely dissociated and maintained as described.E3Yin S. Luo J. Qian A. Yu W. Hu H. LE135, a retinoid acid receptor antagonist, produces pain through direct activation of TRP channels.Br J Pharmacol. 2014; 171: 1510-1520Crossref PubMed Scopus (9) Google Scholar, E4Yin S. Luo J. Qian A. Du J. Yang Q. Zhou S. et al.Retinoids activate the irritant receptor TRPV1 and produce sensory hypersensitivity.J Clin Invest. 2013; 123: 3941-3951Crossref PubMed Scopus (48) Google Scholar In brief, laminectomies were performed and bilateral DRG were dissected out. After removal of connective tissues, DRG were transferred to a 1 mL Ca2+/Mg2+-free HBSS containing 2 μL saturated NaHCO3, 0.35 mg l-cysteine, and 20 U papain (Worthington, Lakewood, NJ), and incubated at 37°C for 10 minutes. The suspension of DRG was centrifuged, the supernatant was removed, and 1 mL Ca2+/Mg2+-free HBSS containing 4 mg collagenase type II and 1.25 mg dispase type II (Worthington) was added and incubated at 37°C for 10 minutes. After digestion, neurons were pelleted, suspended in neurobasal medium containing 2% B-27 supplement, 1% l-glutamine, 100 U/mL penicillin plus 100 μg/mL streptomycin, and 50 ng/mL nerve growth factor, plated on a 12-mm coverslip coated with poly-l-lysine (10 μg/mL), and cultured under a humidified atmosphere of 5% CO2/95% air at 37°C for 18 to 24 hours before use. Fura-2–based ratiometric measurement of [Ca2+]i was performed as described previously.E5Liu S. Feng J. Luo J. Yang P. Brett T.J. Hu H. Eact, a small molecule activator of TMEM16A, activates TRPV1 and elicits pain- and itch-related behaviours.Br J Pharmacol. 2016; 173: 1208-1218Crossref PubMed Scopus (22) Google Scholar Freshly isolated skin-resident cells and cultured DRG neurons were loaded with 4 μM Fura-2 AM (Life Technologies) in culture medium at 37°C for 60 minutes. Cells were then washed 3 times and incubated in HBSS at room temperature for 30 minutes before use. Fluorescence at 340 and 380 nm excitation wavelengths was recorded on an inverted Nikon Ti-E microscope equipped with 340, 360, and 380 nm excitation filter wheels using NIS-Elements imaging software (Nikon Instruments Inc, Melville, NY). Fura-2 ratios (F340/F380) reflecting changes in intracellular Ca2+ upon stimulation were recorded. Values were obtained from 100 to 250 cells in time-lapse images from each coverslip. Threshold of activation was defined as 3 SDs above the average (∼20% above the baseline). Whole-cell patch-clamp recordings were performed using a multiclamp 700B amplifier (Molecular Devices, Sunnyvale, Calif) at room temperature (22°C-24°C) on the stage of an inverted phase-contrast microscope equipped with a filter set for green fluorescence protein visualization (Nikon Instruments Inc). Pipettes pulled from borosilicate glass (BF 150-86-10; Sutter Instrument Company, Novato, Calif) with a Sutter P-97 pipette puller had resistances of 2 to 4 MΩ when filled with pipette solution containing 140 mM CsCl, 1 mM MgCl2, 0.5 mM EGTA, and 10 mM HEPES with pH 7.3 and 315 mOsm/L. A Ca2+-free extracellular solution was used for whole-cell recording to avoid Ca2+-dependent desensitization of TRPV1 or TRPA1 containing 140 mM NaCl, 5 mM KCl, 0.5 mM EGTA, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH was adjusted to 7.4 with NaOH, and the osmolarity was adjusted to ≈340 mOsm/L with sucrose). The whole-cell membrane currents were recorded using voltage ramp from −100 to +100 mV for 500 ms at holding potential of 0 mV. Data were acquired and analyzed using Clampex 10.4 and Clampfit 10 (Molecular Devices). Currents were filtered at 2 kHz and digitized at 10 kHz. Mice were shaved on the nape of the neck 2 days before assay. On the day of experiment, mice were acclimated for 1 hour by placing each of them individually in the recording chamber followed by intradermal injection of hBD2 to the nape of the neck (50 μL per site). Immediately after the injection, mice were videotaped for 30 minutes without any person in the recording room. After the recording, the videotapes were played back and the number of scratching bouts toward the injection site was counted by an investigator blinded to the treatment.Fig E2hBD2-treated skin superfusates evoked a robust [Ca2+]i response in wild-type but not Trpv1−/− DRG neurons. A, Representative traces showing that vehicle-treated skin superfusate did not evoke a [Ca2+]i response in the wild-type DRG neurons (n = 5 independent repeats). B, Representative traces showing that 10 μM hBD2-treated skin superfusate evoke a [Ca2+]i response in the wild-type DRG neurons (n = 5 independent repeats). C, The [Ca2+]i response evoked by hBD2-treated skin superfusate was not present in the Trpv1−/− DRG neurons (n = 5 independent repeats).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3TLR4 is expressed primarily by dermal macrophages. A, Representative FACS plots of TLR4-positive cells in the skin and fluorescence minus one (FMO) negative control. TLR4+ cells are approximately 45% CD45+. B, Macrophages were defined as I-Ab−lo/− F4/80+ CD11b+ CD11c−, dendritic cells were defined as I-Ab-hi F4/80+/− CD11b+/− CD11c+, mast cells were defined as c-Kit+ FcERIa/IgE+, eosinophils were defined as Siglec-F+, and neutrophils were defined as CD11b+ Ly6-G+ I-Ab− F4/80−. C, Bar chart showing the percentages of cells found in each of the specified gates. Data are representative of 3 independent experiments. FACS, Fluorescence-activated cell sorting; FSC-H, forward scatter-height.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4hBD2 stimulates human skin–resident myeloid-derived cells via TLR4. A, Representative traces showing that 10 μM hBD2-evoked a [Ca2+]i response in myeloid-derived cells freshly isolated from human forearm skin (n = 5 independent repeats). B, The selective TLR4 antagonist LPS-RS (2 μg/mL) nearly abolished the hBD2-induced [Ca2+]i responses in the human skin–resident myeloid-derived cells (n = 5 independent repeats). C, Bar charts illustrated that the percentage of human skin–resident myeloid-derived cells responded to hBD2, hBD2 plus LPS-RS. ****P < .0001, Student t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5Injection of LPS, a TLR4 ligand, evoked acute pain but not itch sensation. A and B, Intradermal injections of LPS (5 μg and 50 μg) from Escherichia coli and Salmonella enterica into wild-type mice did not cause significant scratching responses compared with vehicle control. n = 6 for each group. n.s., Not significant, ANOVA. C, Time course of changes in Paw withdrawal thresholds in response to von Frey filaments before and at several time points after intraplantar injections of 5 μg hBD2 or LPS from E coli and S enterica. n = 6 for each group. ***P < .001, ****P < .0001, n.s., Not significant, ANOVA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)