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ORIGINAL ARTICLE
Year : 2023 | Volume
: 41
| Issue : 2 | Page : 94-102
Anti-inflammatory effect of hydrogen sulfide donor sodium-sulfide in an experimental mouse model of contact hypersensitivity
Nermin Ozlen1, Deniz Ercetin2, Melike Sapmaz-Metin2, Ozgur Gunduz1
1 Department of Medical Pharmacology, Faculty of Medicine, Trakya University, Edirne, Turkey
2 Department of Histology and Embryology, Faculty of Medicine, Trakya University, Edirne, Turkey
Correspondence Address:
Assoc. Prof. Ozgur Gunduz
Department of Medical Pharmacology, Faculty of Medicine, Trakya University, Edirne 22030
Turkey
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/ds.DS-D-22-00173
Background: Hydrogen sulfide (H2S), synthesized by most immune cells, has been shown to exert anti-inflammatory and antipruritic effects. The effect of H2S on allergic contact dermatitis (ACD), an inflammatory skin disease that negatively affects the quality of life, is unknown. Objectives: We planned to investigate the antipruritic and anti-inflammatory effects of the H2S donor sodium sulfide (Na2S) in the experimental mouse model of contact hypersensitivity (CHS), which is widely used for ACD research. Methods: CHS was created in Balb/c mice using 1-fluoro-2, 4-dinitrobenzene. Na2S was administered systemically (0.2-2-20 mg/kg/i.p.) and locally (1-3-10 nmol/both ear/i.d.) at 3 h and 25 h after the challenge. Ear thickness and the number of scratches were determined at 24 h and 48 h following the challenge. Ear tissue and serum interferon-gamma, interleukin (IL)-2, IL-4, and IL-5 cytokine levels were evaluated by enzyme-linked immunosorbent assay (ELISA). H and E staining was performed for histopathological studies. CD4+ and CD8+ T cells located in the skin were examined by immunohistochemical staining. Results: Locally (1-3-10 nmol/ear/i.d., P < 0.001, P < 0.0001, P < 0.0001, respectively) and systemically (2–20 mg/kg/i.p., P < 0.01, P < 0.0001, respectively), Na2S administration decreased ear thickness dose dependently. Local (1-3-10 nmol/ear/i.d.) Na2S treatment decreased serum IL-2 levels (P < 0.01, P < 0.05, and P < 0.01, respectively). Na2S administered locally (3–10 nmol/ear/i.d., P < 0.05) and systemically (20 mg/kg/i. p., P < 0.05) decreased the number of CD4+ T lymphocytes. Conclusion: Locally and systemically administered Na2S reduces ear thickness, which is one of the symptoms of CHS, probably by preventing CD4+ T lymphocyte infiltration and proliferation and decreasing IL-2 synthesis.
Keywords: Allergic contact dermatitis, contact hypersensitivity, hydrogen sulfide, inflammation, pruritus
Allergic contact dermatitis (ACD) is a common skin disease, with its prevalence being about 15%–20% and more common in women. Its development against allergens in the work environment leads to loss of workforce, while facial settling causes aesthetic concerns.[1],[2],[3] It is an inflammatory and delayed-type hypersensitivity reaction of the skin, characterized by redness, itching, edema, and thickening that occurs in subsequent contact with the person previously sensitized to the allergen substance.[1],[2],[4]
The number of allergens detected is over 3000. For this reason, avoidance of allergens is the recommended treatment but not always possible.[5] Local corticosteroids, calcineurin inhibitors, and rarely antihistamine drugs are used to treat ACD. However, the undesirable effects of chronic treatment limit the use of these drugs.[4] ACD occurring in humans can be studied using experimental contact hypersensitivity (CHS) models. CHS models are created in 2 steps, sensitization and elicitation (challenge).[6] Although the CHS models are simple in application, they mimic human ACD symptoms quite well.[6] Since it is unknown when the patient will first contact the allergen, it seems more likely to develop agents that are effective in the challenge phase rather than the sensitization phase.[7]
Hydrogen sulfide (H2S) is produced from L-cysteine by the enzymes cystathionine beta-synthase, cystathionine gamma-lyase, and 3-mercaptopyruvate sulfurtransferase.[8],[9] Since it is a highly toxic gaseous compound, there is no direct use for H2S. Instead, H2S donors are used in experimental studies. Various skin diseases such as burns, psoriasis, melanoma, and itching have been treated with H2S donors and essential roles of H2S have been indicated in the pathophysiology of these diseases.[10] Rodrigues et al. showed that locally applied H2S donors reduced the scratching response in a histamine-and 48/80-induced itch model.[9] It has also been demonstrated that all immune cells synthesize endogenous H2S at varying rates and that H2S plays a key role in cytokine synthesis and release.[11] H2S has been shown to affect CD8+ T, CD4+ T, and NK cell survival in a concentration-dependent manner. In terms of survival, CD4+ T cells are much more durable. IL-2 production by activated CD4+ T and CD8+ T cells has been shown to decrease 24 h after H2S administration.[12] In the asthma model induced with ovalbumin, a decrease in T lymphocyte cell count and IL-5 cytokine level was observed as a result of treatment with the H2S donor NAHS.[13]
No research has been conducted to explore the effect of H2S donors on the pathophysiology of ACD. H2S donors can be expected to affect T lymphocyte functions and decrease the synthesis or release of interferon-gamma (IFN gamma), interleukin 2 (IL-2), IL-4, IL-5, and thus may play a role in the treatment of ACD.[12],[13] In this project, we planned to investigate the antipruritic and anti-inflammatory effects of the H2S donor sodium sulfide (Na2S) in the CHS mouse model.
Materials and MethodsAnimals and ethics
In this study, 8–10 weeks old, 80 female BALB/c mice, weighing 20–30g, were purchased in Trakya University Experimental Animals Research Unit. Animals were housed in standard laboratory conditions (12 h light/dark cycles, 50% ±5% humid conditions, and temperature 21 ± 2°C) in an individually ventilated cage system (Tecniplast, Buguggiate, Italy) for the entire duration of the experiment. They were fed a standard laboratory diet and given water ad libitum. Local “Animal Care Ethics Committee” approval (2020.06.01) was obtained for the study. Experimental procedures of this study were conducted according to the national academies press guide for the care and use of laboratory animals.[14]
Reagents and antibodies
1-fluoro-2, 4-dinitrobenzene (DNFB), olive oil, and Na2S were purchased from Sigma Aldrich (MO, USA), whereas acetone was purchased from Merck (Darmstadt, Germany). Primary antibodies used in the immunohistochemical analysis included an anti-CD4 antibody and an anti-CD8 antibody; they were purchased from Santa Cruz Biotechnology (CA, USA).
Animal models of contact hypersensitivity and study design
Eighty BALB/c female mice were randomly divided into two main groups, locally or systemically. Each main group was divided into five subgroups. Systemic treatment groups were determined as control, CHS, CHS + Na2S (0.2 mg/kg/i.p.), CHS + Na2S (2 mg/kg/i.p.), CHS + Na2S (20 mg/kg/i.p.). Local treatment groups were divided into control, CHS, CHS + Na2S (1 nmol/ear/i.d.), CHS + Na2S (3 nmol/ear/i.d.), CHS + Na2S (10 nmol/ear/i.d.).
The experimental CHS model was carried out with minor modifications to the method described by Manresa[6][Figure 1]. First, DNFB was dissolved in acetone/olive oil (4:1 v/v) mixture before use. The abdomen of the mice was shaved with an electric shaver. Sensitization was induced by applying 0.2% 100 microliter DNFB solution to the abdomen on the 1st and 2nd days. On the 7th day, the challenge was done by applying 20 μL of 0.3% DNFB to both ears of the mice. As for the control group, the challenge was performed by application of acetone/olive oil (4:1 v/v) mixture alone.
Figure 1: Effects of local and systemic hydrogen sulfide administrations on ear thickness (a – A and B) and scratching count (b – A and B). In ear thickness: *P < 0.05, **P < 0.0001 versus control group; #P < 0.01, ##P < 0.001, ###P < 0.0001 versus CHS group. Repeated measures two-way ANOVA, post hoc Tukey test. Data are presented as mean ± SD (n = 14–16 ears). In scratching count: &P < 0.05 versus the control group. Repeated measures two-way ANOVA, post hoc Tukey test. Data are presented as mean ± SD (n = 7–8). S: Sensitization, C: Challenge, ♦: Euthanasia, CHS: Contact Hypersensitivity; Na2S: Sodium sulfide, SD: Standard deviation.Na2S, the H2S donor, was administered to the treatment groups at different concentrations, either systemically (0.2-2-20 mg/kg/i.p.) or locally (1-3-10 nmol/both ear/i.d.) at 3 h and 25 h after the challenge. In both local and systemic applications, physiological saline (0.9% NaCl) was used as a vehicle. In systemic applications, the volume was 0.1 ml, whereas a total volume of 10 μL were used for local applications. Ketamine/xylazine anesthesia was applied to the animals previous to local injections. Ear thickness and number of scratching were evaluated 24 h and 48 h after the challenge. After the measurements at 48 h, euthanasia was performed by taking blood from the heart under anesthesia. The collected blood was centrifuged at 2000 rpm for 20 min to obtain serum and stored at −80°C until enzyme-linked immunosorbent assay (ELISA) measurements were made. Both right and left ear tissues were cut immediately. For each group, the right ears from the first four animals and the left ears from the other four animals were stored at −80°C to measure cytokine levels with ELISA. Similarly, opposite ears were fixed in neutral formaldehyde and separated for hematoxylin and eosin (H and E), and toluidine blue (TB) staining.
Measurement of scratching behavior
The number of scratching was assessed at 24 h and 48 h following the challenge. Scratching behaviors were recorded for 1 h with a video camera attached to the room's ceiling. Recordings were made in a soundproof room at the same light intensity. Typically, the mice produced several scratches per second, and such a response is counted as one bout of scratching. The total number of bouts was given for each animal. The same investigator counted the number of bouts by examining the video recording.[15],[16]
Measurement of ear thickness
The same researcher measured the thickness of both ears before and 24 and 48 h after the challenge using a digital caliper (Accud, Istanbul, Turkey) with 0.01 mm resolution sensitivity and ± 0.04 mm accuracy.
Measurement of cytokine levels
The ear tissues were weighed and then added 10 ml of PBS pH 7.4 (Invitrogen, MD, USA) for 1 mg of tissue in safe-lock microcentrifuge tubes homogenized on speed 8 for 10 min with a bullet blender (Next Advance, NY, USA). Samples were centrifuged for 20 min at 14000 rpm at 4°C, and protein supernatants were collected for ELISA. IFN-γ, interleukin 2 (IL-2), IL-4, and IL-5 cytokine concentrations were measured using the sandwich ELISA kit according to the manufacturer's (Andy Gene, Beijing, China) recommendations. The cytokine concentrations were expressed as pg/ml or pg/mg protein. The detection limit of the IFN gamma, IL-2, IL-4, and IL-5 ELISA kits are 1–20 pg/ml, 2–85 pg/ml, 50–1500 pg/ml, and 200–4000 pg/ml, respectively.
Histological analysis
Upper portions of the right ears of each mouse were fixed in 10% neutral formalin, embedded in paraffin, and sectioned into 5 μm slices. Slices were stained with hematoxylin and eosin (H and E) to visualize the morphological structure of the skin and with toluidine blue (TB) for evaluation of mast cells (MCs) number.
H and E stained slides were analyzed using light microscopy (Olympus BX51) at ×40 - ×100 - ×200 - ×400 magnifications. A quantitative histological damage score (0: No alteration; 1: Mild; 2: Moderate; 3: Severe; and 4: Very severe) was performed based on hyperkeratosis, parakeratosis, apoptotic keratinocytes, lymphocyte exocytosis, spongiosis, lymphocytic and neutrophilic infiltrate, follicular infiltration, vasodilatation, perivascular infiltration, and angiogenesis.[17],[18],[19]
The thicknesses of the epidermis and dermis were identified by H and E staining at a magnification of ×100 using an Olympus BX51 light microscope. Epidermis (from stratum corneum to stratum basale) and dermis (from the basement membrane to hypodermis) thickness were measured using AxioVision Rel 4.8 software on the taken micrographs by two blind observers. Two slides were stained per mouse, and five random measurements were examined per slide.
The distribution of metachromatic MCs was assessed at high magnification (×400) from the superficial (subepidermal) and perifollicular dermis. MCs were counted in 10 selected fields per section. Values were expressed as the number of counted cells.
Immunohistochemical analysis
An immunohistochemical examination was performed on the control and lesional tissue samples. Formalin-fixed and paraffin-embedded ear tissues were sectioned at 5 μm. For the immunohistochemical process, the sections were deparaffinized and rehydrated, and heat-mediated antigen retrieval was performed. The sections were then exposed to 3% hydrogen peroxide for 10 min. After overnight incubation with primary antibodies, anti-CD4 antibody (Santa Cruz, MT310, sc-19641, 1:50 dilution); anti-CD8 antibody (Santa Cruz, D-9, sc-7970, 1:100 dilution); and peroxidase-conjugated secondary antibodies were applied. The immunoreactivity was visualized with diaminobenzidine (Vector Laboratories), and counterstaining was performed with hematoxylin. CD4+ and CD8+ cell percentages were obtained at ×400 magnification in 10 random fields subjectively by calculating the ratio of the CD4+ and CD8+ immunolabeled T cells to the average number of cells in a field. Data were expressed as the percentage of cell numbers per section.[20]
Statistical analysis
Comparison of the antipruritic and anti-inflammatory effects of hydrogen sulfide among groups was evaluated using a repeated measure two-way analysis of variance, post hoc Tukey test. Epidermis + dermis thickness and ELISA measurements were assessed using a one-way analysis of variance, followed by the Tukey test. Tissue damage score and immunoreactivity % of CD4+ and CD8+ cells were evaluated using Kruskal–Wallis, post hoc Dunn's multiple comparisons tests.
GraphPad Prism version 6.0c (GraphPad Software Inc., La Jolla, CA, USA) was used to perform the statistics. All data were expressed as mean ± standard deviation P < 0.05 were considered statistically significant.
ResultsEffect of hydrogen sulfide on-ear thickness
A marked increase in ear thickness was observed in both local [[Figure 1]a - A, **P < 0.0001 at both time points] and systemic [[Figure 1]a - B, **P < 0.0001 at both time points] CHS groups compared to their control groups at 24 h and 48 h after the challenge. Local H2S treatment (1-3-10 nmol/ear/i.d.) reduced ear thickness compared to CHS group both at 24 h [[Figure 1]a - A, ###P < 0.0001 all doses], and 48 h [[Figure 1]a - A, ##P = 0.0009, ###P < 0.0001, ###P < 0.0001 respectively]. When given systemically, H2S treatment decreased ear thickness at doses of 0.2 and 20 mg/kg at 24 h [[Figure 1]a - B, #P = 0.045 and ###P < 0.0001, respectively], and at doses of 2 and 20 mg/kg at 48 h [[Figure 1]a - B, #P = 0.0022 and ###P < 0.0001, respectively] compared to CHS group.
Effect of hydrogen sulfide on scratching behavior
A significant increase in the number of scratches was observed in the local CHS group [[Figure 1]b - A, &P = 0.0241 at both time points] compared to their control groups at 24 h and 48 h after the challenge. In the systemic CHS group, augmented the number of ear scratchings was only observed at 24 h [[Figure 1]b - B, &P = 0.0499]. Neither local nor systemic Na2S treatment had any effect on the number of scratches [[Figure 1]b - A and B].
Effect of hydrogen sulfide on cytokine concentrations in skin and serum
Serum IL-2 was apparently enhanced in the local CHS group (*P = 0.0143), whereas did not change in the systemic CHS group [Figure 2]a and [Figure 2]b. Local H2S treatment (1-3-10 nmol/ear/i.d.) reduced serum IL-2 level compared to CHS group [[Figure 2]a, ##P = 0.0081, #P = 0.0187, ##P = 0.0081, respectively]. There was no difference in serum and skin tissue IFN-γ, IL-4 and IL-5 cytokine concentrations following CHS and H2S administrations (Figures are shown in the supplement files [Supplementary Figure 1] and [Supplementary Figure 2].
Figure 2: Effect of local (a) and systemic (b) hydrogen sulfide administration on serum IL-2. *P < 0.05 versus control group; #P < 0.05, ##P < 0.01 versus CHS group. One-way ANOVA, post hoc Tukey test. Data are presented as mean ± SD (n = 6–7). CHS: Contact Hypersensitivity; Na2S: Sodium sulfide, IL-2: Interleukin, SD: Standard deviation.
Effect of hydrogen sulfide on histological changes
The photomicrographs from H and E stained sections of untreated mice (local and systemic controls) showed normal skin with well-defined epidermal and dermal layers. The keratin layer was well-formed and lay just adjacent to the topmost layer of the epidermis [[Figure 3]a – A and F]. In CHS groups, mild to moderate acanthosis and also hyperkeratosis was seen. Apoptosis was identified with pyknotic nuclei among the keratinocytes. The other histological characteristics of affected ear skin were epidermal edema (spongiosis). Mainly neutrophilic and lesser lymphoid infiltrate was localized both in the epidermis and dermis. The papillary dermis showed severely dilated small blood vessels, and vascular proliferation (Figures are shown in the supplement files [Supplementary Figure 3]a, [Supplementary Figure 3]b, [Supplementary Figure 3]c. There was focal and diffuse inflammatory infiltrate in the epidermis and the dermis [[Figure 3]a - B-E and G-J]. Histopathological alterations were also presented by low (×40) and high (×400) magnifications under the light microscopy [Supplementary Figure 4] and [Supplementary Figure 5].
Figure 3: (a) Ear pinna histopathology of control and Na2S-treated mice identified by H and E staining. (A) local control, (B) local CHS, (C) CHS + Na2S (1 nmol/ear/i.d.), (D) CHS + Na2S (3 nmol/ear/i.d.), (E) CHS + Na2S (10 nmol/ear/i.d.), (F) systemic control, (G) systemic CHS, (H) CHS + Na2S (0,2 mg/kg/i.p.) (I) CHS + Na2S (2 mg/kg/i.p.) (J) CHS + Na2S (20 mg/kg/i.p.) ×200. Black-arrow: Apoptotic keratinocyte, arrowhead: Spongiosis, star: Neutrophil, blue-arrow: Lymphocyte, p: parakeratosis. (b-A) Tissue damage score for local treatment groups. (b-B) Tissue damage score for systemic treatment groups. *P < 0.05, **P < 0.01, ***P < 0.001 versus control group. Kruskal–Wallis, post hoc Dunn's multiple comparisons test. Data are presented as mean ± SD. (c-A) Epidermis + dermis thickness for local treatment groups. (c-B) Epidermis + dermis thickness for local treatment groups. *P < 0.01, **P < 0.0001 versus control group; #P < 0.05, ##P < 0.0001 versus CHS group. One-way ANOVA, post hoc Tukey test. Data are presented as mean ± SD. CHS: Contact Hypersensitivity; Na2S: Sodium sulfide, SD: Standard deviation.
Based on histopathological criteria, local injection of 3 and 10 nmol Na2S and systemic injection of 20 mg/kg Na2S improved tissue damage score [[Figure 3]b - A and B]. [Figure 3]c - A and B shows epidermis + dermis thickness (μm) in local and systemic injection groups. DFNB significantly increased in the epidermis + dermis thickness to control groups (**P < 0.0001, *P = 0.0011). Local 3 nmol, 10 nmol/ear/i.d. and systemic 20 mg/kg/i.p. Na2S treatments led to a reduction in epidermis + dermis thickness (##P < 0.0001, ##P < 0.0001 and #P < 0.027, respectively).
The figure of the supplement [Supplementary Figure 6] was shown MCs labeled with TB. Metachromatic cells were counted in the dermis. When comparing MC numbers at 48 h, no difference was observed between the control and CHS groups.
Effect of hydrogen sulfide on T cells infiltration and differentiation in the skin
The immunohistochemical profile revealed atypical lymphocytes which express diffuse CD8+ and focal CD4+. The percentage of CD8+ cells was higher than the CD4+ cell percentage in the DNFB-induced CHS groups [Figure 4]b, [Figure 4]c and [Figure 5]b, [Figure 5]c].
Figure 4: (a) A representative immunohistochemistry staining for the distribution of CD4+ (A-E) and CD8+ (F-J) cells in ear samples from local treatment groups, (A and F) local control, (B and G) local CHS, (C and H) CHS + Na2S (1 nmol/ear/i.d.), (D and I) CHS + Na2S (3 nmol/ear/i.d.), (E and J) CHS + Na2S (10 nmol/ear/i.d.). Star: CD4 + cells, arrow: CD8+ cells. Hematoxylin counterstaining. ×400. (b) The percentage of CD4+ cells. (c) The percentage of CD8+ cells. **P < 0.01, ***P < 0.001, ****P < 0.0001 versus control group. #P < 0.05 versus CHS group. Kruskal–Wallis, post hoc Dunn's multiple comparisons test. Data are presented as mean ± SD. CHS: Contact Hypersensitivity; Na2S: Sodium sulfide, SD: Standard deviation.
Figure 5: (a) A representative immunohistochemistry staining for the distribution of CD4+ (A-E) and CD8+ (F-J) cells in ear samples from (A and F) systemic control, (B and G) systemic CHS, (C and H) CHS + Na2S (0,2 mg/kg/i.p.), (D and I) CHS + Na2S (2 mg/kg/i.p.), (E and J) CHS + Na2S (20 mg/kg/i.p.). Star: CD4+ cells, arrow: CD8+ cells. Hematoxylin counterstaining × 400. (b) The percentage of CD4+ cells, (c) The percentage of CD8+ cells. *P < 0.05, **P < 0.01 versus control group. #P < 0.05 versus CHS group. Kruskal–Wallis, post hoc Dunn's multiple comparisons test. Data are presented as mean ± SD. CHS: Contact hypersensitivity; Na2S: Sodium sulfide, SD: Standard deviation.[Figure 4]a represents CD4+ (A-E) and CD8+ (F-J) immunoreactivity from local treatment groups. Brown cytoplasmic staining was counted for both types of T lymphocytes. CD4+ immunoreactivity was seen in the periphery of focal neutrophilic infiltrates in the epidermis and papillary dermis. CD 8+ T lymphocytes exhibited mainly diffuse distribution in the dermis (G-J). Both CD4+ and CD8+ cell percentage was enhanced in CHS group [****P < 0.0001 and **P = 0.0016, respectively, [Figure 4]b and [Figure 4]c]. Local 3 nmol and 10 nmol/ear/i.d. Na2S treatments decreased CD4+ cell percentage [#P = 0.047 and #P = 0.016, respectively, [Figure 4]c].
[Figure 5]a represents CD4+ (A-E) and CD8+ (F-J) immunoreactivity from systemic treatment groups. The distribution of inflammatory cells was similar to local groups. Both CD4+ and CD8+ cell percentage was enhanced in CHS group [**P = 0.0031 and **P = 0.0065 respectively, [Figure 5]b and [Figure 5]c]. Only Na2S 20 mg/kg/i.p. treatment decreased CD4+ and CD8+ cell percentage [#P = 0.024 and #P = 0.045, respectively, [Figure 5]b and [Figure 5]c].
DiscussionStudies have shown that H2S exerts anti-inflammatory and antipruritic effects.[9],[21] It has also been shown that immune cells synthesize H2S at varying rates.[11] However, the effect of H2S on ACD symptoms is unknown. In our study, we evaluated the effects of local and systemic applications of H2S donor Na2S on CHS.
The parameter usually used as a simple indicator of inflammation in the CHS model is ear thickness due to edema. Similar to previous studies in both local and systemic CHS groups, we determined that the ear thickness increased at 24 h following the challenge, and this increase continued at 48 h due to edema.[22],[23],[24] Local Na2S treatment (1-3-10 nmol/ear/i.d.) reduced ear thickness at all doses, whereas systemic therapy decreased ear thickness at doses of 2 and 20 mg/kg/i.p. Increased capillary permeability plays a major role in the formation of local edema.[25] It is known that activated T lymphocytes and other inflammatory cells increase capillary permeability.[26] Both local and systemic Na2S treatment decreased edema, in addition to the decrease in CD4+ T lymphocyte count at the 48th h; this finding may suggest that CD4+ T lymphocyte-derived cytokines play a role in edema formation. When we evaluated the amounts of IFN-γ, IL-2, IL-4, and IL-5 cytokines in the ear tissue and serum taken at the 48 h following the challenge, we determined a decrease in serum IL-2 level only with local treatment. IL-2 increases capillary permeability and leads to fluid accumulation in the intercellular space.[27] Decreased IL-2 might play a role in preventing edema formation, but our inability to detect the increase in IL-2 in the systemic CHS group was an important shortcoming.
Scratching is an important symptom that reduces the quality of life of ACD patients. Our study determined that local and systemic H2S donor Na2S treatment was ineffective against itching. However, the fact that Na2S was found to be effective on histamine and 48/80-induced scratches and the compatibility of the dose-response graph with our study [[Figure 3]a U-shaped curves] suggest that the effect of Na2S on itching should be re-evaluated with new studies with more animals per group.[9]
When histological findings were examined, a decrease was observed in neutrophilic/lymphocytic infiltration, spongiosis, and apoptosis in the local 3 and 10 nmol/ear/i.d. and systemic 20 mg/kg/i.p. Na2S treatment groups at the 48 h, there was no decrease in the tissue damage score, where all parameters were evaluated together. Studies examining the timing of histological changes have shown that neutrophilic infiltration decreases and lymphocytic infiltration increases starting from the 24 h following the challenge.[28],[29] However, in our study, neutrophilic infiltration was still more intense than lymphocytic infiltration at 48 h in both treatment and CHS groups. This finding suggests that the effect of Na2S is probably on lymphocyte infiltration.
Angiogenesis and enhanced microvascular permeability are hallmarks of many inflammatory diseases such as skin hypersensitivity. Angiogenic blood vessels at the site of inflammation are enlarged and hyperpermeable to maintain the blood flow and meet the increased metabolic demands of the tissue.[30]
When the epidermis and dermis thickness was measured histologically, similar results were obtained in the control and CHS groups as in the previous study.[31] The evaluation of the total thickness of epidermis + dermis, instead of the epidermis or dermis thickness alone, showed that the total thickness increased approximately twice in the CHS groups compared to the control group. The increase was derived from acanthosis and dermal edema due to vascular dilatation and proliferation. In local 3–10 nmol/ear/i.d. and systemic 20 mg/kg/i.p. treatment groups, decreases in total thickness were observed. Except for local 1 nmol/ear/i.d. and systemic 2 mg/kg/i.p. treatment groups, ear thickness measurement, and epidermis + dermis total thickness measurements findings were similar.
The role of MCs in the pathophysiology of CHS is controversial. On the 5th day following the challenge, ear edema increased in genetically MC-deficient mice compared to wild-type mice. It was determined that IL-10 synthesized by MCs suppressed the development of edema.[32] In our study, no difference was found in the number of MCs at 48 h [Supplementary Figure 6]. This observation suggests that the number of MCs does not play a role in the anti-edema effect of Na2S. Studies should be conducted to evaluate the effect of Na2S on IL-10 synthesized from MCs.
When immunohistochemical stainings were examined, no significant T-cell infiltration was observed in the epidermis or dermis in the control groups. The evaluation of lymphocytic infiltration indicated that CD8+ T cell density was higher than CD4+ T cell density in CHS groups. CD8+ T cells were observed to settle in the dermis, while CD4+ T cells were localized around neutrophils in the papillary dermis and epidermis. It is being investigated how the antigen reexposed during the challenge period is presented to effector T lymphocytes. In recent years, it has been shown that presentation occurs in a temporary structure called skin-associated lymphoid tissue (iSALT), consisting of macrophages, dendritic cells, and effector T lymphocytes located around the postcapillary venules.[5],[33] Localisation of CD4+ T cells as local foci resembles the formation of iSALT; this could not be confirmed because macrophage and vessel staining could not be performed in our study. In the treatment with local 3–10 nmol/ear/i.d. and systemic 20 mg/kg/i.p. Na2S doses, decreases in the number of CD4+ T lymphocytes located in foci were detected. This makes us think that Na2S treatment may affect the formation of iSALT. A decrease in CD8+ T lymphocyte count was also detected with systemic administration of 20 mg/kg/i.p. Na2S. This finding suggests that systemic administration may also affect cells located in lymph nodes, apart from local iSALT formation. With systemic administration, higher concentrations of Na2S can reach peripheral lymph nodes. Thus, antigen presentation of dendritic cells to T-lymphocytes may be prevented. In addition, high concentrations of Na2S can prevent the infiltration of effector T-lymphocytes into the skin or impair their survival.[12]
Taken together, local administration of Na2S appears to exert its effect, especially on CD4+ T lymphocytes. In our study, CD4+ T cell subtypes were not determined. Nevertheless, taking into consideration the decrease in serum IL-2 level with local treatment, it can be speculated that the effect is on the Th1 subtype.
An inappropriate finding of our study is that IL-2 levels were not similar in both systemic and local CHS groups. In addition, a significant limitation is that IL-6 and IL-8 cytokine levels, which play a role in angiogenesis and inflammation formation, are not measured.
ConclusionLocal and systemic Na2S application did not reduce itching. However, the fact that Na2S was found to be effective on histamine- and compound 48/80-induced scratches and the compatibility of the dose-response graph with our study suggests that studies with more subjects per group should be conducted. Locally and systemically administered Na2S probably reduces inflammation by reducing effector CD4+ T lymphocyte infiltration and proliferation and suppressing IL-2 synthesis from these cells. Thus, it prevents ear edema, which is one of the symptoms of CHS. Further studies are needed to determine which CD4+ T cell subtypes mediate the effect.
CRediT authorship contribution statement
Nermin OZLEN: designed the study, performed the behavioural test, removed ear tissues, analysed data, ELISA analyses, and manuscript preparation. Deniz ERCETİN: histological and immunohistochemical analysis, manuscript preparation. Melike SAPMAZ-METİN: histological and immunohistochemical analysis: manuscript preparation. Ozgur GUNDUZ: designed the study, performed the behavioural test, removed ear tissues, ELISA analyses, analysed data, and manuscript preparation.
Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
Acknowledgments
We thank Prof. Ahmet ULUGOL for the critical reading of our manuscript and thank Dilşad ERUMİT, Mehmet Zahid ÇETİNKAYA, Buse ÇEVİK, and Kübra DUVAN-AYDEMİR for technical support.
Financial support and sponsorship
This work was supported by Trakya University Research Council (TUBAP-2021/94).
Conflicts of interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
References
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