Edited by: Attila Mócsai, Semmelweis University, Hungary
Reviewed by: Wolfgang Bäumer, Freie Universität Berlin, Germany; Krisztina Futosi, Semmelweis University, Hungary
This article was submitted to Autoimmune and Autoinflammatory Disorders, a section of the journal Frontiers in Immunology
†These authors have contributed equally to this work
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
Protease-activated receptors (PARs) constitute a family of G protein-coupled receptors activated by proteolytic cleavage of their extracellular N-termini. PARs may be activated by various proteases generated by exogenous (e.g., bacteria, mites, plants, and allergens) or endogenous sources (plasma coagulation proteases and proteases from epithelium, endothelium, fibroblasts, or immune cells) (
By activation of PAR2 in the skin, proteases such as house dust mite (HDM) allergens, bacterial proteases, kallikreins, matriptase, trypsin-4, or prostasin may contribute to important biological processes including epidermal barrier homeostasis, innate and adaptive immunity, leukocyte recruitment, pigmentation, fibrosis, pruritus, and pain (
AD is one of the most common chronic inflammatory skin diseases. It is characterized by skin changes such as erythema, edema, and lichenification, in addition to the hallmark symptom of pruritus (itch) (
It is generally accepted that exogenous agents such as
To test this hypothesis, we utilized mice that overexpress PAR2 in keratinocytes (PAR2OE) and stimulated them with house dust mite proteases. Our
PAR2 (a.k.a. F2RL1) and Par2 (a.k.a. F2rl1) refer to human and mouse protease-activated receptor-2 protein, respectively.
PAR2 overexpressing mice were generated by inserting a cassette consisting of the mouse Par2 coding sequence followed by an internal ribosomal entry site and the lacZ reporter gene at the start codon of the grainyhead-like-3 (
Eight-week-old mice were shaved with a clipper and a shaver, and 100 mg of Biostir AD (extract of HDM; Biostir Inc., Kobe, Japan) were applied onto the nape of neck on the next day. From then on, we applied the HDM extract twice per week for 6 weeks. Before each application of the HDM extract, re-grown hair was shaved, 150 μl of 4% sodium dodecyl sulfate solution were applied for barrier disruption, and mice were air-dried for 2–3 h before HDM application. Mice were euthanized at 14 weeks of age and multiple 4 μm sections from treated skin were obtained for histological analyses.
Slides were incubated with primary antibodies for 1 h at room temperature following rinsing with PBS. Antigen retrieval was performed for 10 min in TEG buffer. Slides were washed in 50 mM NH4Cl in PBS for 30 min and blocked by 1% BSA, 0.2% gelatine, 0.05% Saponin in PBS at room temperature for 10 min, three times. Primary antibody was diluted in 0.1% BSA, 0.3% Triton X-100 in PBS, overnight at 4°C. Antibodies against involucrin, loricrin, and filaggrin were purchased from Covance (Denver, PA). Rabbit polyclonal antibody PAR2 (H-99; sc-5597) was provided by Santa Cruz Biotechnology (Dallas, TX). Slides were rinsed three times for 10 min in PBS containing 0.1% BSA, 0.2% gelatine, and 0.05% saponin at room temperature and the secondary biotinylated antibody (goat-anti rabbit; Vector Labs, Burlingame, CA) was diluted in 0.1% BSA, 0.3% Triton X-100 in PBS. Elite Standard Vectastain ABC kit and DAB kit (both Vector Labs) were finally applied according to the manufacturer's instructions. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Dako, Glostrup, Denmark).
For hematoxylin and eosin (HE)-staining, paraffin-embedded sections of 4 μm were used. Microscopic analyses were performed using an Axioskop2 (Zeiss, Oberkochen, Germany) microscope and the Axiovision software Rel4.7 (Zeiss).
Five-micrometer sections of frozen samples were fixed with methanol, followed by inactivation of endogenous peroxidase with 0.3% H2O2, blocking of endogenous biotin with Biotin-Blocking System (DAKO) and unspecific binding with 5% rabbit serum. The samples were incubated with the respective primary antibody, followed by incubation with the relevant horseradish peroxidase-labeled secondary antibody (Vector). Antibodies against CD11b (M1/70), CD3 (DaA3), CD4 (RM4-5), CD8 (Ssa1) were from Immunotools, Friesoythe, Germany; Gr1 (RB6–8C5) from BD, Heidelberg, Germany; ET-1 from Bachem, Torrance, CA; PGP9.5 from Abcam, Cambridge, MA. After incubation with streptavidin-peroxidase (Vector) and AEC+-Solution (Dako), samples were finally counterstained with hematoxylin (Dako).
20 μm sections were incubated with a fluorescein isothiocyanate-conjugated rat anti-mouse NGF antibody (M-20; Santa Cruz).
LacZ expression was detected by incubating the tissue at 30°C overnight in 0.1% X-gal, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 1 mM magnesium chloride 0.002% NP-40, 0.01% sodium deoxycholate, PBS, pH 7.0. Finally, serum samples were used to determine total IgE by ELISA (eBioscience, San Diego, CA).
Skin samples were minced to <0.5 mm3 fragments, rinsed three times in 0.1 mol/l cacodylate buffer, and pre-fixed in half-strength Karnovsky's fixative, followed by postfixation in reduced 1% osmium tetroxide (OsO4) containing 1.5% ferrocyanide or in 0.2% ruthenium tetroxide (RuO4). Selected samples were immersed for 2 h in absolute pyridine for visualization of the cornified lipid envelope, followed by OsO4 postfixation, as described previously. The combination of osmium (OsO4) and ruthenium tetroxide (RuO4) postfixation protocols with pyridine pretreatment allowed us to assess the CE scaffold in relation to the extracellular lamellar bilayer system, as described previously. After staining with 2% aqueous uranyl acetate and embedding in Epon epoxy, ultrathin sections (600Å) were assessed using a Zeiss 10A electron microscope, operated at 60 kV.
The number of SC layers was counted at ×3.5 to ×10 magnification. CE thickness was quantitated with an image analyzer, attached to the electron microscope camera, in the lowest SC layer (first SC layer above the SG–SC junction) vs. outermost SC layer by an unbiased observer who did 30 measurements taken from five images at ×125 magnification. The length of corneodesmosomes was measured between the first and second SC layer above the SG–SC junction at ×25 and expressed as corneodesmosome length/total CE length.
Skin fragments prepared as described above were immersed in 4% lanthanum nitrate in 0.05 mol/l Tris buffer (pH 7.4) containing 2% glutaraldehyde and 1% paraformaldehyde for 1 h at room temperature. After lanthanum perfusion, the samples were washed and processed for electron microscopy, as described above.
We assessed LB and the extent of LB secretion to determine whether Par2 knock-in interferes with secretion of LB contents. LB numbers were determined in granular cells two to three layers below the SG–SC junction by counting LBs at ×16 magnification using a calibrated grid. To assess the LB secretory system, the following criteria were assessed: (i) amount of accumulated lipid material at the SG–SC junction; (ii) presence of “entombed” LB within the corneocyte cytosol; and (iii) extent of extracellular delivery vs. corneocyte retention of a lipid hydrolase (acid lipase), which is concentrated in LB and normally secreted and segregated in toto within the SC interstices. For quantification of LB secretion, areas of secretion at the SG–SC junction were measured and correlated with the length of the bottom surface of the first SC layer on 10 random images at 16K magnification. Finally, on RuO4 postfixed tissue, the maturation and supramolecular organization of extracellular lamellar bilayer quantities were determined.
Skin biopsies and DRGs were homogenized in liquid nitrogen using a Mikro-Dismembrator U (Braun Biotech, San Diego, CA) and RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA). Samples from skin biopsies were tested with primers for murine PAR2, NGF, ETAR, and TSLPR. One microgram of RNA were reversed transcribed using SuperScript II (Invitrogen, Carlsbad, CA). Primers PAR2: forward, 5′-CCACGTCCGGGGATGCGAAG-3′; reverse, 5′-GTTGCGTCCCGGTGCAAGGT-3′; NGF: forward, 5′-TGATCGGCGTACAGGCAGA-3′; reverse, 5′-GAGGGCTGTGTCAAGGGAAT-3′; TSLPR: forward, 5′-CATCCGCGGGTGACCCCT-3′; reverse, 5′-TCCAGGGAAGGAGCCGCTGG-3′; ETAR: forward, 5′-GCTGGTTCCCTCTTCACTTAAGC-3′; reverse 5′-TCATGGTTGCCAGGTTAATGC-3′; GAPDH: forward, 5′-GCCTTCTCCATGGTGGTGAA-3′; reverse, 5′-GCACAGTCAAGGCCGAGAAT-3′. Twenty-five nanograms of cDNA were amplified per reaction, either in the presence of SYBR green master mix, or in the presence of TaqMan® universal master mix (Applied Biosystems, Foster City, CA). Gene-specific PCR products were measured by means of an ABI PRISM® 7000 Sequence Detection Systems (Applied Biosystems; stage 1, 50°C for 2 min, stage 2, 95°C for 10 min and stage 3, 95°C for 15 s, 60°C for 1 min, repeated 40 times). Gene expressions were related to the housekeeping gene and are presented as relative units of expression.
The fur on the rostral back was shaved and mice were habituated to the Plexiglas recording arena 1 week prior to testing. On the experiment day, animals were placed in an arena and videotaped for 30 min to assess spontaneous scratching. Following the recording, animals were tested with id injection of 10 μl of one of the following: vehicle (isotonic saline), histamine (Sigma-Aldrich, St. Louis, MO, 35 μg in saline), the PAR2/MrgprC11 agonist SLIGRL-NH2 (Quality Controlled Biochemicals, Hopkinton, MA, and GenScript, Piscataway, NJ; 35 μg in saline), or serotonin (Sigma, St. Louis, MO; 3 μg in saline). Intradermal (id) microinjections were made. Immediately following the id microinjection, mice were placed in the arena and videotaped for 30 min from above. Scratching elicited by each pruritogen subsided by the end of the 30-min recording period. Investigators left the room during videotaping. Videotapes were reviewed by investigators blinded to the treatment, and the number of scratch bouts was counted. A scratch bout was defined as one or more rapid back-and-forth hind paw motion(s) directed toward and contacting the injection site, and ending with licking or biting of the toes and/or placement of the hind paw on the floor. Hind paw movements directed away from the injection site (e.g., ear-scratching) and grooming movements were not counted. One-way ANOVA followed by the Bonferroni post-test or unpaired
Alloknesis was assessed as follows. At 5-min intervals, von Frey stimuli (bending force: 0.7 mN) were applied on the border of the lesional skin at 5 randomly selected sites. In pilot experiments we determined that application of von Frey stimuli within the lesional skin was ineffective. The presence or absence of a positive response, i.e., a hindlimb scratch bout directed to the site of mechanical stimulation, was noted for each stimulus before the next one was given. The alloknesis score was the total number of positive responses elicited by the three stimuli, i.e., 0, 1, 2, 3, 4, or 5. In one set of experiments, we tested the effect of the μ-opiate antagonist naltrexone on scratching and alloknesis. Naltrexone (1 mg/kg s.c., Dupont; Garden, NY) or saline was administered. In addition to this, we also performed subcutaneous injections with lidocaine (1%).
The animal was euthanized under sodium pentobarbital anesthesia, and upper- to mid-cervical DRGs were acutely dissected and enzymatically digested at 37°C for 10 min in HBSS (Invitrogen, Carlsbad, CA) containing 20 U/ml papain (Worthington Biochemical, Lakewood, NJ) and 6.7 mg/ml L-cysteine (Sigma), followed by 10 min at 37°C in HBSS containing 3 mg/ml collagenase (Worthington Biochemical). The ganglia were then mechanically triturated using fire-polished glass pipettes. DRG cells were pelleted; suspended in MEM with Earle's balanced salt solution (Gibco, Life Technologies, Carlsbad, CA) containing 100 U/ml penicillin, 100 μg/ml streptomycin (Gibco), 1× vitamin (Gibco), and 10% horse serum (Quad Five, Ryegate, MT); plated on poly-d-lysine-coated glass coverslips; and cultured for 16–24 h.
DRG cells were incubated in Ringer's solution (pH 7.4, 140 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, and 4.54 mM NaOH) with 10 μM Fura-2 AM and 0.05% of Pluronic F-127 (Invitrogen). Coverslips were mounted on a custom-made aluminum perfusion block and viewed through an inverted microscope (Nikon TS100, Technical Instruments, Burlingame, CA). Fluorescence was excited by UV light at 340 and 380 nm alternately, and emitted light was collected via a CoolSNAP camera attached to a Lambda LS lamp and a Lambda optical filter changer (Sutter Instrument, Novato, CA). Ratiometric measurements were made using Simple PCI software (Hamamatsu, Sewickley, PA) every 3 s.
Solutions were delivered by a solenoid-controlled eight-channel perfusion system (ValveLink, AutoMate Scientific, San Francisco, CA). One of the following agents was delivered for 30 s: histamine (100 μM), 5-HT (100 μM), and the PAR2/MrgprC11 agonist SLIGRL-NH2 (100 μM). Potassium chloride (144 mM) was always delivered at the end of each experiment. Ratios were normalized to baseline. Cells were judged to be sensitive if the ratio value increased by >10% of the resting level following chemical application. Only cells responsive to high potassium were included for analysis. Unpaired
We utilized
Par2 overexpression in the epidermis results in spontaneous eczema formation and intense pruritus.
Although PAR2OE mice were born without any overt abnormalities, they started to develop eczematous skin lesions spontaneously after several weeks of life. This ultimately evolved to severe dermatitis with weight loss that necessitated euthanasia (
Using an arbitrary skin lesion score for dermatitis severity that evaluated “erythema/hemorrhage,” “edema,” “excoriation/erosion,” and “scaling/dryness” [maximum three points each (
The skin phenotype was accompanied by severe scratching behavior of the PAR2OE mice, with an ~3-fold increase in scratching bouts (
Since exposure to house dust mites (HDM) is associated with flare-ups of the skin disease in AD patients, and HDM allergens are known activators of PAR2, we investigated the effect of topical treatment with HDM extract (
Application of house dust mites triggers eczematous skin lesions and pruritus in PAR2OE mice.
For a deeper understanding of the pathophysiological processes in our PAR2OE mice, we first examined alterations of keratinocyte differentiation markers by immunohistochemistry (IHC), focusing on filaggrin, involucrin, and loricrin. IHC staining for these proteins revealed less signal in PAR2OE lesional skin compared to either WT skin or non-lesional PAR2OE skin (
Structural impairment of barrier function in PAR2OE mice.
To functionally assess the epithelial barrier, we used the water-soluble tracer colloidal lanthanum for perfusion and visualization by transmission electron microscopy (TEM). Colloidal lanthanum is normally excluded from both the corneocyte cytosol and the extracellular matrix, and its perfusion stops in the stratum granulosum (SG). As expected, WT littermates showed this pattern of colloidal lanthanum staining. Interestingly, colloidal lanthanum was observed intercellularly in the stratum corneum (SC) of both lesional and non-lesional skin from PAR2OE mice (
Detailed morphological analyses of the epidermis of lesional PAR2OE skin by TEM revealed an abundance of abnormalities, thus we focus here on alterations in non-lesional PAR2OE skin (
Morphological analyses of subcellular compartments and components by electron microscopy in lesional and non-lesional skin of PAR2OE mice, and WT mice.
To further characterize our mouse model, we next assessed the infiltration of inflammatory cells in PAR2OE and littermate mouse skin with and without HDM treatment (as in
The eczematous skin lesions in HDM-treated PAR2OE mice display immunological characteristics of atopic dermatitis. All samples derive from 14-week-old PAR2OE and WT mice, treated with HDM or vehicle for the last 6 weeks.
Due to the significant scratching behavior in PAR2OE mice (
Evidence for increased epidermo-neuronal communication, and Par2 upregulation on dorsal root ganglion neurons. All samples derive from 14-week-old PAR2OE and WT mice, treated with HDM or vehicle for the last 6 weeks.
To address the functional consequences of increased nerve fiber density and increased Par2 expression on DRG neurons, we injected the Par2/MrgprC11 agonist peptide (SLIGRL) as well as two independent pruritogens, histamine and serotonin (5-HT). Despite ubiquitous Par2 overexpression in DRG neurons of PAR2OE mice, injection of SLIGRL only resulted in significantly increased scratching bouts (hyperknesis) in lesional PAR2OE (lesPAR2OE) relative to WT littermates (
Analysis of sensitization of itch signaling pathways in PAR2OE mice.
PAR2OE mice exhibited a significant increase in touch-evoked scratching (alloknesis score ~2) compared to WT animals, which exhibited no alloknesis (alloknesis score = 0).
To further investigate the basis of scratching behavior, we used calcium imaging to investigate pruritogen-evoked responses in DRG cells from PAR2OE and WT littermates. Mean calcium responses following stimulation with the pruritogens histamine, SLIGRL, and 5-HT are shown in
The cellular circuits that link skin epithelium, immune cells and the skin nervous system in AD are very poorly understood. Here, we present
Our findings of spontaneous development of AD-like skin disease in PAR2OE mice fit well with what is currently known about barrier dysfunction in AD pathogenesis. SPINK5 knockout mice (Spink5−/−) have been used to investigate the lack of one key serine protease inhibitor LEKTI, which models the human disease Netherton syndrome. Loss of LEKTI leads to hyperactivity of epidermal proteases, followed by stratum corneum detachment, resulting in enhanced allergen absorption and formation of acanthosis, papillomatosis, parakeratosis, and influx of immune cells (
KLK5 activity is deregulated upon loss of LEKTI. KLK5 can activate PAR2, and PAR2 contributes to TSLP overexpression in LEKTI deficient mouse skin (
PAR2 activation has been shown to induce TSLP in keratinocytes, which is considered a key trigger in the initiation and maintenance of AD and the “atopic march” (
With regard to skin barrier function, WT skin and non-lesional PAR2OE skin revealed no significant differences in filaggrin, involucrin, and loricrin levels, but lesional PAR2OE skin showed a remarkable decrease in the level of all three epidermal proteins. Interestingly, even non-lesional skin of PAR2OE mice revealed a loss of the epidermal calcium gradient and a leaky epidermal water barrier. By morphological analysis of non-lesional PAR2OE skin, we found a regular but thinner cornified envelope, increased lipid secretion, and abnormal SC extracellular lamellar bilayers in comparison to WT mice. The response to barrier damage includes secretion of preformed lamellar bodies, followed by increased lipid synthesis, further production/secretion of new lamellar bodies, and organization of secreted and processed lipids into mature lamellar membrane structures, thereby restoring barrier function (
Analyses of the inflammatory infiltrate in HDM-treated PAR2OE mice revealed an increase of CD4+ helper T cells, but not of CD8+ effector T cells. Mast cells and eosinophilic granulocytes were also significantly elevated in HDM-treated PAR2OE mice. These effects were absent in HDM-treated littermates, vehicle-treated PAR2OE, and WT mice. Increased total IgE levels were observable, although not all HDM-treated PAR2OE mice displayed an identical increase in IgE levels. The same immune cell subpopulations were described to be elevated in
Due to the severe scratching phenotype in PAR2OE mice especially after HDM treatment, we investigated possible links between altered keratinocyte signaling and sensory dorsal root ganglion (DRG) neurons. In skin lesions of HDM-treated PAR2OE mice, we observed increased nerve fiber density and an increased expression of epidermal ET-1, which we recently described as a potent inducer of histamine-independent pruritus in chronic itch (
In summary, we present a body of evidence using dermatological, behavioral, neuroscientific, and immunological approaches indicating that increased epidermal Par2 activity is sufficient to drive many features of human AD in a mouse model. Additionally, increased epidermal Par2 activity facilitates skin sensitization to exposure to HDM extract and pruritogens, another feature of human AD. The remarkable effects of HDM extract in this model may be due to direct activation of keratinocyte PAR2 by PAR2-activating proteases known to be present in HDM extracts and/or to other effects of HDM enabled and enhanced by the PAR2-driven barrier defect. This complex project did not investigate protease content and level changes in the skin mediated by Par2 knock-in, in different ages of the mice, different eczema stages, and different topical treatments initially and over several weeks (e.g., by HDM, SDS, or petrolatum). While sustained barrier defects (regardless of cause) stimulate pro-inflammatory immune cascades (
Our results suggest that Par2 signaling in keratinocytes triggers epidermal responses that are sufficient to trigger neuronal sensory and inflammatory responses in our AD model. Studies of global Par2 deficiency suggests that Par2 can play a necessary role in related models. PAR2OE mice displayed striking parallels to human AD: (i) no overt skin disease at birth, (ii) slowly crescendo-type development of spontaneous skin lesions, in association with significant pruritus, (iii) aggravation of the skin disease including pruritus upon topical exposure to HDM, (iv) an initially intact skin barrier that becomes dysfunctional over time and seems to precede the skin lesions, (v) dermal infiltration of characteristic immune cells and increased IgE production, (vi) involvement of different pruritogenic pathways resulting in direct and indirect activation of skin-innervating DRG cells, (vii) secondary upregulation of neuronal Par2 on DRG neurons during chronification of the AD lesions in PAR2OE, and (viii) increased sensitivity to various pruritogens. These and other data raise the question of whether the keratinocyte-protease-PAR2 system may mediate barrier defects, sensory signaling and neuro-immune communication in human AD, and whether PAR2 antagonists and/or selective protease inhibitors may be a novel approach for its treatment. This initial description of pathophysiological processes in PAR2OE mice warrants further in-depth analysis of the different mechanisms involved (e.g., on triggers of the skin barrier defect and its time course, on more details of the infiltrating immune cells, their activation, mediators, and regulation, etc.) to better understand its fidelity as a model for human AD and its potential utility for evaluation of candidate therapeutic approaches.
The datasets generated for this study are available on request to the corresponding author.
The animal study was reviewed and approved by UCSF and UCD-Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for Care and Use of Laboratory Animals.
TB, AI, SR, and MSt designed the research. TB, AI, CK, FC, MSu, and JB performed experiments for
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank Jutta Schulz, Ronald Manlapaz, and Wendy Cedron for excellent technical assistance. Parts of this work have been presented at a national and a European conference on skin research. These poster abstracts were published as Buhl et al., J Invest Dermatol 2014, 134, S1, and Buhl et al., Exp Dermatol 2015, 24, E1.
atopic dermatitis
cornified envelope
dorsal root ganglion
house dust mite
healthy skin
immunohistochemistry
interferon
interleukin
lamellar body
lesional skin of PAR2OE
protease-activated receptor 2
PAR2 overexpression
stratum corneum
stratum granulosum
transmission electron microscopy
T helper cell type 1
T helper cell type 2
T helper cell type 17
T helper cell type 22
tumor necrosis factor
thymic stromal lymphopoietin.