Gua Sha, a press-stroke treatment of the skin, boosts the immune response to intradermal vaccination

Materials

Ovalbumin (OVA) and Freund’s incomplete adjuvant (FIA) were purchased from Sigma-Aldrich (Shanghai, China). Pentobarbital sodium was obtained from Merck, and Tween 20 from Sangon Biotech Co., Ltd (Shanghai, China). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (γ-chain specific), IgG1 (γ1-chain specific), and IgG2a (γ2a-chain specific) were purchased from Southern Biotech (Birmingham, USA). A chromogen, 3,3^′,5,5^′-tetramethylbenzidine (TMB), and the substrate buffer were purchased from Beyotime B.V. (Shanghai, China). Skim milk powder (Yili) was obtained from the local supermarket. All other chemicals were of analytical grade, and all solutions were prepared with Milli-Q water.

Animals

Female BALB/c mice (H-2^d), 6–8 weeks old at the start of the experiments, were purchased from the Experimental Animal Center of Nanjing Medical School (Nanjing, China) and maintained under standardized pathogen-free conditions in the animal facility of the State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University. All mice were housed in standard cages, with free access to food and water. The animals were maintained at a constant temperature (20–21°C) and humidity (55% ± 5%), with a 12-h light/dark cycle. All animals were handled in accordance with the Declaration of Helsinki, 1975 (as revised in 2008) concerning animal rights, and the protocols were approved by the Institutional Animal Care and Use Committee of Model Animal Research Center of Nanjing University (Ref No. 20130015). Efforts were made to minimize the amount of animals used and ameliorate animal suffering.

Gua Sha treatment on experimental mice

Mice were anesthetized by intraperitoneal injection of 100 mg/kg pentobarbital. Gua Sha was performed on the side of the mouse’s back by using a smooth-edged instrument made of bull-horn (Figs. 1A and 1D), after the hair was shaved with a clipper a day prior to the experiment. This material was chosen instead of plastic to avoid accumulation of static electricity during operation. The shaved skin area was wiped with 70% ethanol and left to dry. Then, press-stroke treatment (called “scrape” henceforth) was applied 20 or 40 times in a unidirectional manner, with an angle of ∼90°between the instrument and the mouse’s back. The force of scrape was optimized and standardized at ∼3 N, so that blemishes would appear between 20 and 40 scrapes, while the inner organs would not be damaged. Twenty scrapes took ∼30 s, and a skin area of ∼3 cm² was treated. Lubricant oil was not included in the experiments, as the friction was not harmful and the effect of lubricant oil on the skin tissue and vaccine delivery would need to be excluded. No abrasion-induced open wound was visible during and after the treatment, and the skin remained intact.

Histological imaging of the treated skin

Mice were euthanized by cervical dislocation at the indicated time points after Gua Sha treatment. For histopathological examination, samples of treated/untreated skin tissue were obtained, fixed in Bouin’s buffer, embedded in paraffin, and sectioned. The sections stained with Masson’s trichrome method were analyzed using optical microscopy (Nikon TE2000-U; Nikon, Tokyo, Japan) (Goldner, 1938).

Flow cytometry analysis of the treated skin

At indicated time points after Gua Sha treatment, the mice were euthanized by cervical dislocation. Approximately 3-cm² skin tissue from the treatment site was excised, chopped to small pieces, and digested in 4 mg/ml type I collagenase in Hank’s balanced salt solution (HBSS; Sigma-Aldrich) at 37°C for 2 h. Single cell suspensions were prepared using a 70-μm cell strainer (BD Bioscience, Shanghai) at the concentration of 1 × 10⁷/ml in phosphate-buffered saline (PBS) containing 1% BSA. Cells were stained using APC-conjugated hamster anti-mouse CD11c, PE-conjugated rat anti-mouse F4/80 (BioLegend), or their corresponding isotype controls. After incubation on ice for 30 min, the cells were washed twice with PBS containing 1% BSA and re-suspended for flow cytometry analysis using BD FACS Calibur. Data were analyzed using FlowJo software (Tree Star).

Cytokine profile analysis

Mice were euthanized by cervical dislocation at indicated time points after Gua Sha treatment. Approximately 100 mg of skin tissue from the treatment site was excised and rinsed in cold PBS (at 1 mg/μl), and then homogenized on ice using Lysing Matrix D tubes and Fast Prep-24 homogenizer (MP Biomedicals, Santa Ana, CA). Five groups of skin samples were obtained as follows: (1) untreated skin from naïve mice; (2) treated skin samples taken 1 h after 40 scrapes; (3) untreated skin samples from treated mice 1 h after 40 scrapes; (4) treated skin samples taken 2 h after 40 scrapes; (5) untreated skin samples from treated mice 2 h after 40 scrapes. Supernatants were collected after centrifugation at 14,000 rpm for 20 min at 4°C. Cytokine levels, including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-13, and IL-23 of the lysates were quantified using enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s instructions (4A Biotech Co. Ltd., Beijing, China). Blood samples, 4–5 drops each time per mouse, were collected from the retro-orbital venous sinus of the mice 0.5 and 1 h after 40 scrapes. Blood samples of the untreated mice were collected with the same procedure. Cell-free serum was obtained in collecting tubes with coagulation agent and separation gel (Gongdong, Taizhou, China) by centrifugation after clot formation. TNF-α, IL-1β, and IL-6 levels in the sera were also determined and compared to those of the untreated mice. Serum nitric oxide level was determined using an assay kit (Jiancheng Bioengineering Institute, Nanjing, China) based on the Griess reaction method, following the manufacturer’s instructions (Guevara et al., 1998).

Immunization and serum antibody assays

Mice were intradermally (i.d.) immunized with 5 μg of OVA under anesthesia three times on day 1, 21, and 42 at a site between the back and thigh 10 min after 20 or 40 scrapes, and euthanized on day 56. Groups of OVA alone and OVA with FIA delivered i.d. to naïve mice were included as the negative and positive controls, respectively. Blood samples were drawn from the tail vein one day before each immunization or from the retro-orbital venous sinus during euthanasia under systemic anesthesia. Cell-free sera were obtained as mentioned above and stored at −80°C.

OVA-specific serum IgG, IgG1, and IgG2a titers were determined by ELISA as described previously (Ding et al., 2009). Briefly, ELISA plates (Costar 9018; Elisa, Shanghai, China) were coated with OVA at 4°C overnight. Two-fold serial dilutions of the serum samples were applied, and the antibodies were detected by HRP-conjugated goat anti-mouse IgG, IgG1, or IgG2a by using TMB as the substrate. Antibody titers are expressed as the calculated sample dilution times corresponding to the half of the maximum absorbance at 450 nm of a complete sigmoid absorbance-log dilution curve. If the samples were not diluted in the optimal range, additional measurements with more diluted or concentrated samples were performed to complete the S-shaped curve. Mice whose serum samples did not reach the half-saturated absorbance value at the lowest (ten-fold) dilution were considered non-responders at that time point and the titers were arbitrarily considered 10.

Statistical analysis

Antibody titers were logarithmically transformed for better normality. Analysis was performed as indicated. Statistical analysis was carried out using Graphpad (Prism, San Diego, CA, USA) and a p value of < 0.05 was considered significant.

Skin scrapes lead to blood congestion, blood vessel expansion, and infiltration of immune active cells locally

Treated skin samples were observed with the naked eye as well as with Masson’s staining in order to study the effect of scrapes on the skin. The skin of the naïve mouse after hair removal looked white with a pinkish background. From the dermal side, it was milky white with hardly any capillaries visible (Figs. 1D and 1G). Scrapes were applied 20 or 40 times in a unidirectional manner on the mouse’s back. When observed 30 min after treatment, the skin became darker from the stratum corneum side, with a few blood vessels distinguishable from the dermal side after 20 scrapes (Figs. 1E and 1H). After 40 scrapes, petechiae appeared on the stratum corneum side, and subcutaneous microvascular blood extravasation and bruises could be observed from the dermal side (Figs. 1F and 1I). The vessels in the subcutaneous tissue expanded considerably, with some of them being located closer to the stratum corneum, as shown in the images of Masson’s staining of the Gua Sha-treated skin sections (Fig. 2). The increased diameter of the vessels indicated an enhanced blood and lymphatic flow, allowing more rapid substance exchange with the interstitial fluid. Red blood cells, probably together with other cell types and contents, dispersed through the ruptured peripheral blood vessels into the dermis and subcutaneous fat tissues, followed by accumulation for hours.

In the Gua Sha-treated skin tissue, the ratios of CD11c⁺ cells, including DCs and activated T lymphocytes, and F4/80 macrophages were found to be increased, as observed by flow cytometry analysis (Fig. 3). In the untreated mouse skin, CD11c⁺ cells accounted for ∼4% of the total cell population. This proportion increased to ∼9%, 12%, and 14% at 15, 30, and 60 min after treatment, respectively (Figs. 3A and 3B). A similar trend was observed in case of macrophages present in the treated skin tissue; the proportion of macrophages increased with time after Gua Sha treatment, from 16.5% to ∼20% (Figs. 3C and 3D), probably because of the infiltration from the expanded or ruptured blood vessels.

Skin scrapes cause variations in the local and systemic levels of cytokines and nitric oxide

As Gua Sha treatment enhances microcirculation, more rapid substance exchange occurs among the blood, lymphatic fluid, interstitial fluid, and the infiltrated immune active cells. This changes the microenvironment of the treated skin tissue. The levels of the representative cytokine groups present locally in the treated skin tissue and systemically in the untreated skin area were determined and compared with those of naïve mice (Fig. 4). Local concentrations of most pro-inflammatory cytokines examined, including TNF-α, IL-6, IL-12p70, and IL-23, increased moderately; but a significant increase was observed after the treatment, except IL-1β. Among them, TNF-α level increased in both treated and untreated skin tissues, while IL-6, IL-12p70, and IL-23 levels in the untreated skin area of the treated mice remained constant. The immunosuppressive cytokine IL-10 was present at lower levels in the treated skin tissue 1 h and 2 h after treatment, and in the untreated skin area 2 h after treatment, compared to that in the untreated mice, indicating an overall up-regulation of immune reactivity (Fig. 4D). The levels of anti-inflammatory cytokines IL-4, IL-5, and IL-13 in the skin tissues of treated and untreated mice were also examined, but no remarkable differences were detected (Fig. S1). In the serum samples of the treated mice, the levels of TNF-α, IL-1β, and IL-6 increased significantly, compared to those from untreated mice (Figs. 5A–5C). Consistent with the anatomical and histological observations, nitric oxide content, which leads to vasodilation and increased blood flow, was significantly up-regulated (Fig. 5D). In addition to increasing the infiltration of immune active cells such as neutrophils, monocytes, and macrophages from the blood, nitric oxide enhances immune defense by acting as a free radical with oxidative pressure and is toxic to bacteria and intracellular parasites.

Skin scrapes boost the immune response to intradermal OVA vaccination

Because the levels of some pro-inflammatory cytokines were up-regulated locally, it was hypothesized that the Gua Sha treatment would enhance adaptive immunity against intradermal pathogens. An in vivo vaccination study was performed to examine its effect on the immune defense of the skin. Intradermal injection, instead of dermal application, was chosen for vaccine administration to ensure exact vaccine dosage. The OVA-specific isotype antibody titers of the serum samples from three different time points were determined (Fig. 6). The OVA-specific IgG titers of the Gua Sha-treated groups did not differ significantly from those of the untreated group after prime (Fig. 6A). Interestingly while as expected, the systemic humoral response to OVA was augmented, as evidenced by the IgG titers after the first and second booster doses (Figs. 6B and 6C) and IgG1 titer after second booster dose (Fig. 6D). 20 and 40 scrapes of the skin induced about two and three fold higher IgG titers, respectively after the second booster dose. In the untreated group, IgG2a induction was not very pronounced because half of the animals had titers below the detection limit. However remarkably, mice who received Gua Sha treatment before vaccination had elevated IgG2a titers, without any non-responders (Fig. 6E). The IgG1/IgG2a ratios of the treated groups after the second booster dose were lower than those of the untreated group (Fig. 6F), indicating that Gua Sha treatment of the skin prior to intradermal vaccination may lead to a more Th1-biased immune response.