Materials and reagents
Hypochlorous acid (HOCl) was used as a gel formulation (0.1% AFC, pH 5.5 ± 0.5) and was supplied as “PR022” by Realm Therapeutics, Inc. (Malvern, PA, USA). PR022 is a proprietary formulation of stabilized hypochlorous acid in a gelling agent and an emollient in development for the treatment of atopic dermatitis. In the Phase 2 clinical study, 2 concentrations of PR022 are being evaluated, 0.05 and 0.1% (NCT03351777). For active sensitization, house dust mite allergen (Dermatophagoides farina, GREER, Lenoir, NC, USA) was used. Poly-L-lysine, laminin, capsaicin, allyl isothiocyanate (AITC), 2-mercaptoethanol and mineral oil were obtained from Sigma (St. Louis, MO, USA). Dispase was purchased from STEMCELL Technologies Inc. (Cambridge, MA, USA). Fura-2-acetoxymethyl ester (Fura-2 AM), phosphate buffered saline (PBS), collagenase, chloroquine, histamine, and SLIGRL-NH2 were ordered from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Fetal bovine serum (FBS), Dulbecco’s modified eagle medium with L-glutamine (DMEM) and RPMI-1640 medium, Ca2+ and Mg2+-free hank’s balanced salt solution (HBSS), penicillin-streptomycin and fetal bovine serum (FBS) were from Mediatech Inc. (Manassas, VA, USA). MEM eagle (EMEM) medium was obtained from Lonza Group Ltd. (Allendale, NJ, USA). For determination of protein content DCs protein assay kit was used (BIO-RAD, Richmond, CA, USA). The murine IgE ELISA (OptEIA) set was ordered from Becton, Dickinson and Company (Franklin Lakes, NJ). Recombinant mouse interleukin (IL-)1β, IL-31, and tumor necrosis factor (TNF)α were purchased from Pepro Tech, Inc. (Rocky Hill, NJ, USA). Enzyme linked immunosorbent assay (ELISA) kits for IL-1β, IL-4, IL-13, thymic stromal lymphopoietin (TSLP), thymus and activation regulated chemokine (TARC), and TNFα were obtained from R&D systems (Minneapolis, MN, USA). Serotonin and tofacitinib were ordered from Tocris Bioscience (Bristol, UK).
NC/Nga (female) mice were ordered from Charles River Japan Laboratories (Tokyo, Japan). The mice arrived at 35 to 40 days of age and were kept in quarantine for at least 1 week. The facility offered a controlled environment (including individually ventilated cages and sentinel animals). The animals were housed at 22 °C with 50% humidity with a 12-h light cycle. The mice were fed with certified pellet diet and received water ad libitum. The study protocol was approved by the Animal Care and Use Committee of the North Carolina State University (IACUC Protocol No. 13–111-B).
Murine model of atopic dermatitis in NC/Nga mice
Prior to first sensitization, NC/Nga mice were clipped on their back. The following day, mice were sensitized with 30 μl of house dust mite allergen (HDM) suspended (10 mg/ml) in mineral oil and applied topically onto the back twice weekly. To accelerate sensitization, mild tape stripping (“Staples” office tape) was performed weekly just before the first HDM sensitization. Tape stripping was terminated as soon as visible lesions had developed. Treatment of HOCl (0.1% in gel, n = 8), tofacitinib (0.5% in lipoderm, n = 8) or vehicle (gel, n = 8) was started on day 15, where the mice showed a mean lesional score of 2.1. The mice were equally distributed according to their clinical score, that each treatment group represents the mean of 2.1. One group of mice (n = 6) was left untreated and served as a base control. The dose selection for tofacitinib was according to a former study in which 0.5% tofacitinib inhibited itch behaviour and inflammation in a mouse model of allergic contact dermatitis, higher doses (when given in acetone) led to irritation of mouse skin, thus it was decided to select a concentration of 0.5% .
During the experimental period, back skin thickness, body weight, clinical scores and scratching behavior were monitored once every week. The clinical score was determined as according to the following system: No symptoms, 0; mild, 1; moderate, 2; severe, 3 and extreme, 4. The mean was calculated from the score for erosions, edema, and erythema as well as skin dryness, as described previously . To monitor scratching behaviour mice were video monitored for 30 min directly after sensitization with HDM once a week. Video monitoring was performed with mouse pairs (belonging to the same treatment group) at the same cage. Only repeated strokes with the hindlimb directed to the area of HDM sensitization were counted as scratching bout. Mice were sacrificed by CO2 inhalation according to AVMA Guidelines for the Euthanasia of Animals and tissue was collected from each group on day 32.
The back skin, blood and dorsal root ganglia (DRG) were obtained from single mice 1 h after last HOCl or tofacitinib application (this means 24 h after last HDM challenge). Samples were processed (or stored) for IgE determination, histology, cytokine determination and functional measurement of intracellular Ca2+ in DRG neurons.
Tissue samples from rostral neck skin were excised, fixed in paraformaldehyde (4% solution), sectioned and stained with haematoxylin-eosin. Edema and cell influx were evaluated semiquantitatively (0, no influx, no edema; 1, mild; 2, moderate; and 3, severe influx, severe edema) in a blinded manner in skin sections of each mouse back skin (n = 8 for treatment groups, n = 6 for untreated control).
Cytokine determination of skin tissues
One part of the rostral neck skin tissue was snap-frozen in liquid nitrogen. Cytokine determination for skin tissues was performed as described previously . IL-1β, − 4, − 13, TARC, TSLP and TNFα were measured by ELISA. Intra-assay variance was < 10%, inter-assay variance was not calculated as each ELISA (30 samples) was done in one setting (= one plate) for the present study.
Determination of total IgE in serum
Blood samples collected from single mice was centrifuged at 3000 g to isolate serum. We used an ELSA according to the manufacturer’s protocol to determine total IgE level present in serum.
Isolation of DRG and calcium imaging on DRG cell culture
Preparation and cultivation of the DRG neurons from the NC/Nga mice was done as described previously . Briefly, DRGs were dissected along the thoracic vertebral column. Single ganglia were digested by means of dispase and collagenase which was dissolved in HBSS for 30 min at 37 °C. A trituration of DRG using fire-polished Pasteur pipettes helped with dissociation. Single cells were centrifuged/washed in DMEM medium containing 10% FBS, resuspended in 50 μl medium and placed onto poly-D-lysine and laminin-coated coverslips. Cells were incubated at 37 °C for 2 h and then flooded with 1 ml of medium and further incubated at 37 °C. Calcium experiments were performed within 24 h of culture.
Changes in intracellular [Ca2+] free concentration in single neurons were measured by digital microscopy connected to equipment for ratiometric recording of single cells as described elsewhere . In brief, coverslips attached dorsal root ganglia cells were loaded with fura-2 (8 μmol/L) in DMEM media and incubated at 37 °C for 30 min. For ratiometric imaging, the cells were transferred to a tempered (37 °C) incubation chamber on the stage of the microscope and constantly perfused with Locke solution. Cells that incorporated fura2 were identified using fluorescence microscopy before starting the ratiometric experiment. Cells were marked with region of interest circles and monitored by sequential dual excitation, 340 and 380 nm. The frequency of image acquisition was 100 ms. DRG neurons were initially exposed to IL-1β (1 μg/ml), TNFα (1 μg/ml), histamine (1 mmol/l), followed by IL-31 (1 μg/ml), chloroquine (10 μmol/l), serotonin (1 mmol/l), AITC (100 μmol/l), capsaicin (1 μmol/l) and KCl (75 mmol/l). However, none of the cells were stimulated with more than 3–4 stimuli in the row and the order was switched randomly from slide to slide always ending with KCl. The cells were superfused with a steady Locke solution flux for at least 180 s. after each stimulus as a recovery for the cells prior to the next stimulus.
All data are displayed as mean (±SD). Statistical significance of the difference was estimated at the 5 and 1% levels of probability. If only the significance of differences between mean values of 2 groups were tested, Student t test was used. For comparisons of more than 2 groups we used a one-way ANOVA followed by Dunnett’s multiple comparison test. Comparisons of proportions (study with dorsal root ganglia) were made by means of the Fisher exact test. The data analysis was performed with Prism 4 (GraphPad Software, San Diego, CA, USA).