The antimicrobial peptide hBD2 promotes itch through Toll-like receptor 4 signaling in mice

To the Editor

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 psoriatic patients also suffer from chronic itch, which significantly impairs quality of life ¹. Although recent exciting studies have identified a positive correlation between the intensity of psoriatic itch and the expression levels of nerve growth factor (NGF), neuropeptides, and many cytokines ², 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 ³. Besides potent antimicrobial activity, hBD2 has diversified roles in regulating adaptive immunity, wound healing, and male fertility ⁴. Interestingly, hBD2 also promotes inflammation by recruiting multiple types of immune cells through interacting with both C-C chemokine receptor type 2 (CCR2) and 6 (CCR6) in vitro ^5,6. 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-B). In addition, mBD4, 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.

Fig 1.

TRPV1 is the downstream mediator of hBD2-induced itch. A, Time course of the scratching response induced by vehicle (circle), hBD2 at 2 (square) and 5 (triangle) μg/50 μl recorded for 30 min after intradermal injection. B, Bar charts illustrate dose-dependent scratching response produced by intradermal injections of hBD2. n=7 for vehicle, n=6 for 2 μg and n=9 for 5 μg hBD2. *p˂0.05, ****p˂0.0001, ANOVA; C-E, hBD2-induced scratching was reduced in Trpv1^-/- (C, n=5) but not Trpa1^-/-(D, n=4) or mast cell-deficient sash mice (E, n=5). n=6 for wild-type mice in all groups. ****p˂0.0001, n.s, not significant, Student's t test; F, Representative traces showing [Ca²⁺]_i responses in individual DRG neurons freshly isolated from wild-type mice in the presence of 10 μM hBD2. 100nM capsaicin and 100 mM KCl were used as positive controls. Each colored line represents an individual cell; G-H, Representative I-V curves illustrate that 10 μM hBD2 did not activate HEK293 cells expressing mTRPV1 (G) or hTRPA1 (H) which were activated by 100 nM capsaicin or 100 μM AITC.

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 non-histaminergic itch ⁷. Furthermore, the TRPV1-expressing sensory fibers mediate skin inflammation through facilitating the function of dermal immune cells in a mouse model of psoriasis ⁸. To investigate if TRPA1 and/or TRPV1 are also downstream mediators of hBD2-induced itch, we tested if genetic ablation of TRPA1 or TRPV1 function affects hBD2-induced scratching. Strikingly, the number of the hBD2-induced scratching bouts was markedly reduced in the Trpv1^-/- but not the Trpa1^-/- mice when compared with wild-type mice (Fig 1, C-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 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-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).

Since 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 if mast cells are involved in hBD2-elicited itch by measuring hBD2-induced scratching in mast cell-deficient Kit^W-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 anti-histamines in more than 80% patients ^1,2.

Since hBD2 receptors CCR2 and CCR6 are abundantly expressed by skin-resident cells ^5,6, 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-B), suggesting that neither CCR2 nor CCR6 mediates hBD2-induced itch. Since TLR4 mediates mBD2-induced activation of dendritic cells and mBD2 and hBD2 share structural and functional similarities ⁹, 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 if 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 LysM^cre; Tlr4^f/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 LysM^cre; Tlr4^f/f mice was substantially reduced when compared with their wild-type littermates (Fig 2, D). To assess if 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 LysM^cre; Tlr4^f/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 E. coli or S. enterica did not evoke significant scratching. Since 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 LPS signaling pathway is associated with the pain sensation while the stimulation of TLR4 by hBD2 primarily generate 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.

Fig 2.

TLR4 but not CCR2 or CCR6 mediates hBD2-induced itch. A-D, hBD2-induced acute itch was severely attenuated in Tlr4^-/- mice (C, n=7) and LysM-cre; Tlr4^f/f conditional knockout mice (D, n=6) but not ccr2^-/- (A, n=6) or ccr6^-/- (B, n=6) mice, compared with their control groups. **p˂0.01, n.s, not significant, Student's t test; E-H, Representative traces showing hBD2-evoked [Ca²⁺]_i response in skin-resident cells freshly isolated from ear preparations of wild-type (E), Tlr4^-/- (F), Tlr4^CKO (G) mice and in acutely dissociated DRG neurons from the Tlr4^-/- (H) mice (n=5 independent repeats). 100nM capsaicin, 100 mM KCl and 1 μM ionomycin were used as positive controls in relevant experiments. Each colored line represents an individual cell.

In conclusion, here we first report that hBD2, which is markedly up-regulated in differentiated keratinocytes of psoriatic patients, 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.

Methods

Animals

C57BL/6J, ccr2^-/- ccr6^-/- Tlr4^-/-, LysA^cre, Tlr4^f/f, and mast cell-deficient Kit^W-sh “sash” mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA). Trpvl^+/+ and congenic Trpv1^−/− mice on the C57BL/6J background were obtained from Jackson Laboratory (Bar Harbor, ME, USA). The Trpa1^+/+ and congenic Trpa1^−/− mice on the C57BL/6J background were described previously ^E1. 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 Tlr4^f/fmice and wild-type littermates were used as the control in the behavior testing. All mice were housed under a 12 h 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 cell culture and transfection

HEK293T cells were grown as a monolayer maintained in DMEM (Life Technologies, Carlsbad, CA, USA), supplemented with 10% FBS (Life Technologies, Carlsbad, CA, USA), 100 units mL⁻¹ penicillin, and 100 μg·mL⁻¹ streptomycin in a humidified incubator at 37°C with 5% CO₂. The cells were transiently transfected with cDNAs for mouse TRPV1 (mTRPV1) or human TRPA1 (hTRPA1) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Following transfection, the cells were maintained in DMEM at 37°C for 24 hours before use.

Fresh isolation of mouse skin-resident cells

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^E2. Samples were mashed through 70 μrn cell strainers and washed with DMEM media supplemented 10% FBS (Life Technologies, Carlsbad, CA, USA), 100 units/ml penicillin, and 100 μg·mL⁻¹ streptomycin. Single-cell suspensions were used for subsequent Ca²⁺ imaging assays.

Isolation and short-term culture of mouse DRG neurons

Mouse spinal columns were removed and placed in ice-cold HBSS; neurons were acutely dissociated and maintained as described ^E3,4. 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 1-cysteine and 20 U papain (Worthington, Lakewood, NJ, USA), and incubated at 37°C for 10 min. The suspension of DRG was centrifuged, the supernatant was removed, 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 min. After digestion, neurons were pelleted, suspended in neurobasal medium containing 2% B-27 supplement, 1% L-glutamine, l00 U·mL⁻¹ penicillin plus 100 μg·mL⁻¹ streptomycin, and 50 ng·mL⁻¹ nerve growth factor, plated on a 12 mm coverslip coated with poly-L-lysine (10 μg·mL⁻¹) and cultured under a humidified atmosphere of 5% CO₂/95% air at 37°C for 18–24 hr before use.

Live-cell Ca²⁺ imaging on freshly isolated skin-resident cells and DRG neurons

Fura-2-based ratiometric measurement of [Ca²⁺]_i was performed as described previously ^E5. Freshly isolated skin-resident cells and cultured DRG neurons were loaded with 4 μM Fura-2 AM (Life Technologies, Carlsbad, CA, USA) in culture medium at 37°C for 60 min. Cells were then washed three times and incubated in HBSS at room temperature for 30 min 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, USA). Fura-2 ratios (F340/F380) reflecting changes in intracellular Ca²⁺ upon stimulation were recorded. Values were obtained from 100–250 cells in time-lapse images from each coverslip. Threshold of activation was defined as 3 standard deviations above the average (∼ 20% above the baseline).

Whole-cell patch-clamp recordings

Whole-cell patch-clamp recordings were performed using an multiclamp 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) at room temperature (22–24 °C) on the stage of an inverted phase-contrast microscope equipped with a filter set for green fluorescence protein visualization (Nikon Instruments Inc., Melville, NY, USA). Pipettes pulled from borosilicate glass (BF 150-86-10; Sutter Instrument Company, Novato, CA, USA) with a Sutter P-97 pipette puller had resistances of 2–4 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⁻¹. 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/1 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 using or Clampex 10 (Molecular Devices, Sunnyvale, CA, USA). Currents were filtered at 2 kHz and digitized at 10 kHz.

Acute itch behavior

Mice were shaved on the nape of the neck two days before assay. On the day of experiment, mice were acclimated for 1 hr 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 min without any person in the recording room. After the recording, the videotapes were played back and the number of scratching bouts towards the injection site was counted by an investigator blinded to the treatment.

Supplementary Material

Fig E1 Both hBD3 and mBD4 produce scratching responses when injected intradermally. A, intradermal injections of hBD3 (5 μg/50 ul) elicited a scratching response in wild-type mice. n=7 for each group. *p˂0.05, Student's t test; B, intradermal injection of mBD4 (5 μg/50 ul) induced a scratching response in wild-type mice. n=6 for each group. **p˂0.01, Student's t test.

Fig E2 hBD2-treated skin superfusates evoked a robust [Ca²⁺]_i response in wild-typet 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).

Fig E3 TLR4 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 ∼45% CD45⁺; B, Macrophages were defined as I-A^b-lo/- F4/80⁺ CD11b⁺ CD11c^-, dendritic cells were defined as I-A^b-hi F4/80^+/- CD11b^+/- CD11c⁺, mast cells were defined as c-Kit⁺ FcERIa/IgE⁺, eosinophils were defined as Siglec-F⁺, neutrophils were defined as CD11b⁺ Ly6-G⁺ I-A^b- F4/80^-; C, Bar chart showing the percentages of cells found in each of the specified gates. Data are representative of three independent experiments.

Fig E4 hBD2 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 percentage of human skin-resident myeloid derived cells responded to hBD2, hBD2 plus LPS-RS. ****p<0.0001, Student's t test.

Fig E5 Injection of LPS, a TLR4 ligand, evoked acute pain but not itch sensation. A-B, Intradermal injections of LPS (5 μg and 50 μg) from E. coli and S. enterica into wild-type mice didn't caused significantly 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˂0.001, ****p˂0.0001, n.s, not significant, ANOVA.

Abbreviations

AMPs

antimicrobial peptides

[Ca²⁺]_i

intracellular Ca²⁺

CCR2

C-C chemokine receptor type 2

CCR6

C-C chemokine receptor type 6

hBD2

human beta-defensin 2

hBD3

human beta-defensin 3

mBD2

murine beta-defensin 2

mBD4

murine beta-defensin 4

NGF

nerve growth factor

TRPA1

transient receptor potential cation channel subfamily A member 1

TRPV1

transient receptor potential cation channel subfamily V member 1