Research ArticleExperimental Studies
Open Access

3-Bromo-4,5-dihydroxybenzaldehyde Attenuates Allergic Contact Dermatitis by Generating CD4+Foxp3+ T cells

SANG-CHUL HAN, JUNG-IL KANG, YOUN KYUNG CHOI, DA HEE YANG, KI JU KIM, HA JEONG BOO, WEON-JONG YOON, HEE-KYOUNG KANG, EUN-SOOK YOO and HYE-JIN BOO
In Vivo January 2025, 39 (1) 201-209; DOI: https://doi.org/10.21873/invivo.13818

Abstract

Background/Aim: Regulatory T cells (Tregs) play a crucial role in inflammatory responses by regulating the activity of various immune cells. M2 macrophages induced by IL-10 and TGF-β exhibit anti-inflammatory functions and induce Treg differentiation. Although the beneficial effects of 3-bromo-4,5-dihydroxybenzaldehyde (BDB) on various diseases have been widely reported, the mechanisms, through which it alleviates allergic contact dermatitis (ACD) via Tregs and macrophages, are not well understood. Therefore, this study aimed to explore whether BDB suppresses ACD and induces Treg generation. Materials and Methods: Mice were sensitized with 1% dinitrochlorobenzene (DNCB), followed by the application of 0.3% DNCB to their ears every 3 days for 31 days. BDB (100 mg/kg) was administered orally once daily throughout the 31 days. Cytokine and transcription factor expression were analyzed via real-time PCR and western blotting, while CD4+Foxp3+ T cell differentiation and T cell proliferation were evaluated using flow cytometry. Results: BDB exhibited therapeutic efficacy in mice with ACD. In this study, the administration of BDB promoted the upregulation of transforming growth factor beta (TGF-β)-dependent CD4+Foxp3+ T cells. BDB elicited T cell hypo-responsiveness and suppressed the expression of cytokines related to the Th1, Th2, and Th17 cell subsets. BDB-M2 macrophages directly mediated the differentiation of CD4+Foxp3+ T cells from CD4+ T cells and concurrently suppressed the proliferation of CD4+ T cells. Conclusion: BDB augments M2 macrophage function and induction of Tregs confers effective protection against ACD in mice. Consequently, BDB may represent a promising therapeutic approach for the treatment of inflammatory skin diseases.

Key Words:

Allergic contact dermatitis (ACD) is an inflammatory dermatological condition frequently observed in children, although it can affect individuals of any age. It is characterized by cutaneous hypersensitivity, manifesting as erythema and severe pruritus (1, 2). Allergens processed by antigen-presenting cells initiate the differentiation of naïve T helper (Th0) cells into Th2 cells during the acute phase of ACD and subsequently induce their differentiation into Th1 cells during the chronic phase, resulting in secretion of various inflammatory cytokines (3, 4). Under homeostatic conditions, the balance between Th1 and Th2 cell interactions sustains the immune response. However, the differentiation of T cells into Th2 cells is upregulated in ACD, leading to elevated levels of inflammatory cytokines and circulating immunoglobulin E (IgE) (5). Additionally, the upregulation of IL-4 suppresses Th1 cell activity, subsequently diminishing cell-mediated immune responses and exacerbating the inflammatory cascade (6).

Upon activation via direct cellular interactions with antigen-presenting cells, CD4+ T cells undergo differentiation into various Th cell subsets in accordance with specific cytokine signaling gradients. Th1 cells differentiate in the presence of IFN-γ or IL-12 and subsequently secrete IFN-γ to orchestrate cell-mediated immunity and inflammatory processes (7, 8). Th2 cells, which differentiate in response to IL-4, produce IL-4 and IL-5 to modulate humoral immune responses and allergic reactions (9). Th17 cells, which differentiate in response to IL-1β or IL-6, produce IL-17 and IL-22 to modulate autoimmune responses (10).

Regulatory T cells (Tregs), which differentiate in response to transforming growth factor beta (TGF-β), express CD25 on their surface, exhibit the transcription factor forkhead box P3 (Foxp3), and secrete IL-10 and TGF-β (11). Additionally, Tregs sustain immune homeostasis by modulating diverse inflammatory responses. In individuals with ACD, Treg population is diminished at these areas of inflammation, contributing to the exacerbation of disease symptoms (12, 13).

Macrophages are integral to the regulation and function of immune response. They execute a defensive function through microbe phagocytosis and are indispensable for the antigen-driven activation of T and B lymphocytes (14, 15). Macrophages undergo polarization into M1 and M2 phenotypes in response to various inflammatory stimuli. M2 macrophages are further categorized into four subtypes— M2a, M2b, M2c, and M2d—based on the cytokines involved in their differentiation (16). Notably, M2c macrophages, induced by IL-10 and TGF-β, demonstrate immuno-suppressive and anti-inflammatory functions, and secrete IL-10 and TGF-β (17).

Recent studies have demonstrated that 3-bromo-4,5-dihydroxybenzaldehyde (BDB), isolated from marine red algae, enhances cardiac function and reduces oxidative stress (18). Additionally, BDB protects human keratinocytes from cytokine-induced stimulation, prevents skin barrier disruption, and exhibits anti-inflammatory properties (19, 20). Although the therapeutic benefits of BDB have been reported across multiple diseases, the mechanisms modulating inflammatory reactions and immune systems mediated by macrophages and Tregs in ACD remain inadequately characterized. Thus, we demonstrated whether BDB reduces allergic reactions and promotes the expansion of Tregs. Additionally, we assessed whether BDB enhances the immunoregulatory functions of M2 macrophages.

Materials and Methods

Experimental animals. BALB/c mice (6 weeks old) were purchased from Samtako Bio (Gyeonggi, Republic of Korea) and maintained under specific pathogen-free conditions in the animal facility of Jeju National University. All animal experiments were approved by the Jeju National University Animal Care and Use Committee.

Assessment of the optimal BDB dosage. To establish the optimal dose of BDB (Sigma-Aldrich, St. Louis, MO, USA), we evaluated the expression of CD4+Foxp3+ T cells following BDB administration in healthy mice. BDB was subsequently administered orally at varying doses (10, 50, and 100 mg/kg) over a period of 14 days. A minimum dose of 50 mg/kg of BDB was necessary to induce CD4+Foxp3+ T cell expression, while a dose of 100 mg/kg resulted in the most pronounced increase in CD4+Foxp3+ T cell levels (data not shown).

Disease models. Experimental ACD was induced in mice through sensitization by administering 300 μl of 1% dinitrochlorobenzene (DNCB; Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan) or the vehicle control to the abdominal region. On Day 7, mice were re-challenged with 100 μl of 0.3% DNCB applied to the ears every three days for a period of up to 31 days. Beginning on Day 0 and continuing through the entirety of the experiment, BDB (100 mg/kg) was administered orally once daily for 31 days. The mice were euthanized on Day 32 (Figure 1A).

Figure 1.
Figure 1.

3-bromo-4,5-dihydroxybenzaldehyde (BDB) treatment suppresses experimental allergic contact dermatitis (ACD). (A) Scheme illustrating the BDB treatment and the establishment of murine disease models. (B) Serum immunoglobulin E levels were quantified using an ELISA assay. (C) Ear thickness was assessed on days 0, 14, 20, 26, and 32. (D) Macroscopic images of the ears and (E) paraffin-embedded ear tissue sections stained with hematoxylin and eosin and toluidine blue (scale bar=0.1 mm). (F) The mRNA expression levels of cytokines and transcription factors in ear tissue were quantified using real-time PCR. (G) Foxp3 expression in ear tissues was evaluated using western blotting (n=5 mice per group). Data are presented as the mean±standard deviation of three independent experiments. *p<0.05; **p<0.01; and ***p<0.001.

Histology and immunohistochemistry (IHC) to detect Foxp3 in tissues. Ear and lymph node (LN) tissues were harvested from mice with ACD, fixed in 10% formalin, and subsequently embedded in paraffin for further analysis. Paraffin-embedded sections of ear tissue were stained with hematoxylin and eosin (H&E) and toluidine blue. Foxp3 expression in LN tissues was analyzed through IHC staining using rabbit anti-Foxp3 antibodies (Novus Biologicals, Cambridge, UK) and a rabbit-specific HRP/DAB IHC detection kit (Abcam, Cambridge, UK), following the manufacturer’s protocol.

Total RNA extraction and real-time PCR. Total RNA was isolated using the TRIzol reagent (Molecular Research Center Inc., Oxford, UK) following the manufacturer’s protocol. Reverse transcription was carried out using a cDNA synthesis kit (MG Med, Seoul, Republic of Korea). Real-time quantitative PCR was conducted using a KAPA SYBR® FAST qPCR kit (Kapa Biosystems, Woburn, MA, USA) and an iQ™5 Multicolor Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) to quantify gene expression levels. The data were analyzed with iQ™5 optical system software, which assessed the amplification of both the target gene and the endogenous control (GAPDH) in the test and reference samples. The data were normalized using GAPDH.

Western blot analysis to detect Foxp3 expression in tissues. Total protein was extracted from tissues using lysis buffer and then transferred to a polyvinylidene difluoride membrane using an iBlot gel transfer device (Invitrogen Corporation, Carlsbad, CA, USA). The membrane was subsequently incubated with rabbit anti-Foxp3 antibody (Novus Biologicals). After washing, the membrane was treated with horseradish peroxidase conjugated anti-rabbit IgG (Cell Signaling Technology Inc., Beverly, MA, USA). Protein bands were visualized using a western blot detection system (Cyanagen Srl, Bologna, Italy) according to the manufacturer’s instructions.

Isolation of CD4+ T cells, CD4+CD25+ T cells, and macrophages. CD4+T cells were purified from the lymphocyte population in the LN using a Dynabeads™ FlowComp™ Mouse CD4 cell kit (Invitrogen), following the protocol provided by the manufacturer.” Cells were initially incubated with CD4 antibody for 15 min, followed by 15 min of incubation in Dynabeads buffer. Bead-bound cells were resuspended in a release buffer for 10 min to facilitate bead detachment.

CD4+CD25+ T cells were purified from the lymphocyte population in the LN using a Dynabeads™ FlowComp™ Mouse CD4+CD25+ Tregs Kit (Invitrogen), following the manufacturer’s instructions. CD4+T cells were initially incubated with CD25 antibody for 15 min, followed by 15 min of incubation in Dynabeads buffer. Bead-bound cells were resuspended in a release buffer for 20 min to facilitate bead detachment.

F4/80+ macrophages were purified from the splenocyte population in the spleen using a MagniSort™ Mouse F4/80 Positive Selection Kit (Invitrogen), following the manufacturer’s instructions. Spleens from each group were processed with 1x RBC lysis buffer (eBioscience, San Diego, CA, USA) to eliminate red blood cells, enabling the preparation of a single-cell suspension. Splenocytes were collected, and the cells were incubated at 37°C for 30 min, after which the culture supernatant containing non-adherent cells was removed. Cells were treated with MagniSort™ positive selection antibody for 15 min, then subsequently incubated in beads buffer for 15 min.

Cell culture and stimulation. CD4+ T cells and macrophages were cultured in RPMI 1640 (Gibco, Uxbridge, UK) with 0.05 mM 2-β-mercaptoethanol (Sigma-Aldrich). For cytokine analysis, CD4+ T cells were stimulated with phorbol 12-myristate-13-acetate (PMA: 20 nM; Sigma-Aldrich) and ionomycin (2 μM; Sigma-Aldrich) for 4 h. For the CD4+Foxp3+ T cell induction and CD4+ T cell proliferation assays, CD4+ T cells were stimulated with anti-CD3 (1 μg/ml; eBioscience) and anti-CD28 (0.5 μg/ml; eBioscience) antibodies, with or without TGF-β (10 ng/ml; PeproTech, Rocky Hill, NJ, USA) for 72 h. To achieve macrophage polarization, the cells were incubated for 48 h with lipopolysaccharide (LPS: 100 ng/ml; Sigma) to induce an M1 phenotype, or with IL-10 and TGF-β (10 ng/ml each; PeproTech) to yield an M2 phenotype.

Co-culture experiments. To evaluate the suppression sensitivity of CD4+ T cells, CD4+CD25+ T cells isolated from wild-type mice were co-cultured at varying ratios with carboxyfluorescein diacetate succinimidyl ester (CFSE; eBioscience)-labeled CD4+ T cells (derived from each experimental group) in the presence of anti-CD3 (1 μg/ml) and anti-CD28 (0.5 μg/ml) with or without TGF-β (10 ng/ml) for 3 days. In the CD4+Foxp3+ T cell induction experiments, CD4+ T cells were co-cultured with M1 or M2 macrophages isolated from each experimental group and stimulated with anti-CD3 antibody (0.25 μg/ml) for 7 days in the presence or absence of anti-IL-10 (10 μg/ml; R&D Systems) and anti-TGF-β (10 μg/ml; R&D Systems)-neutralizing antibodies. For the proliferation assays, CD4+ T cells isolated from healthy mice were labeled with CFSE. These cells were co-cultured with varying ratios of pre-adherent M2 macrophages isolated from different experimental groups and stimulated with anti-CD3 (1 μg/ml) and anti-CD28 (0.5 μg/ml) for 3 days.

Flow cytometric analysis. To evaluate Foxp3 expression, cells were initially permeabilized using the Mouse Foxp3 Buffer Set (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s protocol. The cell suspension was pre-incubated with CD16/CD32 (Invitrogen Corporation) for 20 min, followed by 20 min of staining with anti-CD4-APC or APC Rat IgG2a Embedded Image isotype control (BD Biosciences). The cells were then fixed and permeabilized for 30 min with fixation/permeabilization buffer, and subsequently stained for 20 min with anti-Foxp3-BV421 or BV421 Rat IgG2bEmbedded Image isotype control (BD Biosciences). To assess CD4+CD25+ T cells, the cell suspension was stained with anti-CD4-APC and anti-CD25-BV421 (BD Biosciences) for 20 min each. Data acquisition was performed using a BD LSR Fortessa flow cytometer, and the resulting data were analyzed with FlowJo software.

Statistical analysis. Images were converted into numerical values using Quantity One version 4.2.1 and Image-Pro Plus version 4.5. Student’s t-test and two-way analysis of variance were used to determine the statistical significance of the differences between the induction and BDB groups. The data are expressed as means±standard deviation of three independent experiments. p-Values <0.05 were considered significant.

Results

BDB suppresses the development of experimental ACD. The BDB-treated group demonstrated a significant reduction in IgE levels compared to the induction-only group (mice exposed to DNCB without BDB treatment; Figure 1B). Cutaneous edema was assessed as an indicator of ACD progression, and the impact of BDB on inflammatory cell infiltration was evaluated through H&E and toluidine blue staining of ear tissue sections. The normal and BDB-treated groups exhibited decreased ear thickness, epidermal thickness, and immune cell infiltration compared to the induction group (Figure 1D and E). We also investigated the effect of BDB on the expression of genes and proteins associated with Th cells or Tregs in ear tissues. Compared to the induction group, the normal and BDB-treated groups exhibited significantly reduced IFN-γ, IL-4, IL-13, and IL-17A mRNA levels. Additionally, BDB treatment resulted in increased TGF-β and Foxp3 mRNA levels, as well as elevated Foxp3 protein expression (Figure 1F and G).

Foxp3+ T cells accumulate at inflammation sites. We analyzed the morphologic alterations in the LNs of mice with ACD. The LNs from the induction group were markedly hypertrophic, whereas those from the BDB group exhibited reduced size and lower weight (Figure 2A). We also investigated the potential suppressive effects of BDB on mature CD4+ T cells in an experimental ACD model. Compared to the induction group, BDB-treated mice exhibited decreased mRNA levels of IL-2, IFN-γ, IL-4, IL-13, and IL-17A, along with increased levels of TGF-β, Foxp3, CTLA-4, and granzyme B (Figure 2B). We subsequently evaluated the impact of BDB on Foxp3 expression using IHC staining and western blot analysis of LN sections. Foxp3 expression was significantly increased in the BDB-treated group compared to the induction group (Figure 1C and D).

Figure 2.
Figure 2.

Foxp3+ cells are enriched at inflammatory sites. (A) The lymph nodes (LNs) were imaged and measured to document morphological changes. (B) The mRNA expression levels of cytokines and transcription factors in LN tissues were quantified using real-time PCR. (C) Foxp3 expression in LN tissues was assessed via immunohistochemistry staining (scale bar=0.1 mm). (D) Foxp3 expression in LN tissues was evaluated using western blotting (n=5 mice per group). Data are presented as the mean±standard deviation of three independent experiments. *p<0.05; **p<0.01; and ***p<0.001.

BDB treatment induces the conversion of CD4+ T cells into CD4+Foxp3+ T cells through a TGF-β-dependent mechanism. We investigated the effect of BDB treatment on the regulation of gene expression associated with Th cells and Tregs derived from activated CD4+ T cells. Compared to the induction group, CD4+ T cells from the BDB-treated group exhibited decreased mRNA levels of T-bet, GATA3, and RORγt, along with increased mRNA levels of Foxp3, TGF-β, CTLA4, and GzmB (Figure 3A). Given that TGF-β is known to facilitate T cell differentiations into Tregs (21), we explored the mechanisms, through which BDB and TGF-β influence Treg differentiation. CD4+ T cells in the BDB group exhibited significantly increased TGF-β and Foxp3 mRNA expression after TGF-β stimulation (Figure 3B). The CD4+ T cells treated with TGF-β in the induction and BDB groups exhibited an increase in CD4+Foxp3+ T cells, compared to the untreated ones. Notably, the BDB group demonstrated a significantly higher increase in CD4+Foxp3+ T cells in a TGF-β-dependent manner, compared to the induction group (Figure 3C). However, the CD4+CD25+ Treg populations showed no differences between these two groups (Figure 3D). Furthermore, an analysis of CD4+ T cell proliferation between the induction and BDB groups revealed no significant differences when co-cultured with normal CD4+CD25+ Tregs (suppressor) and CD4+ T cells (responder; isolated from induction or BDB groups). Nevertheless, CD4+ T cells from the BDB group exhibited significantly higher sensitivity to Treg-mediated suppression compared to those from the induction group, particularly in the presence of TGF-β (Figure 3E).

Figure 3.
Figure 3.

3-bromo-4,5-dihydroxybenzaldehyde (BDB) promotes the differentiation of CD4+Foxp3+ T cells from CD4+T cells in a TGF-β-dependent manner. (A) The mRNA expression levels of cytokines and transcription factors in CD4+ T cells were quantified using real-time PCR. (B-D) CD4+ T cells were stimulated with anti-CD3/-CD28 antibodies with or without transforming growth factor beta (TGF-β) for 72 h and analyzed using real-time PCR and flow cytometry. (E) The suppression sensitivity of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+CD25−T cells (responder; in each group) at various ratios was evaluated through incubation with CD4+CD25+ Tregs (suppressors) from normal mice in the presence or absence of TGF-β for 72 h (n=5 mice per group). Data are presented as the mean±standard deviation of three independent experiments. *p<0.05; **p<0.01; and ***p<0.001.

BDB markedly enhances the capacity of M2 macrophages to regulate CD4+Foxp3+ T cell differentiation. We assessed the influence of BDB in modulating IL-10/TGF-β-modified M2 macrophages during the development of Tregs. BDB-M1 macrophages demonstrated a significant reduction in IL-1β, IL-6, and TNF-α mRNA levels compared to induction-M1 macrophages. Additionally, BDB-M2 macrophages exhibited elevated TGF-β mRNA levels relative to induction-M2 macrophages (Figure 4A). Both induction-M2 and BDB-M2 macrophages significantly enhanced Treg differentiation compared to their M1 counterparts, with BDB-M2 macrophages exerting a greater effect on CD4+Foxp3+ T cell generations than induction-M2 macrophages. Conversely, neither induction-M1 nor BDB-M1 macrophages effectively promoted the transformation of CD4+ T cells into CD4+Foxp3+ T cells (Figure 4B). We assessed whether IL-10 or TGF-β mediated the Treg generation by BDB-M2 macrophages. Neutralization studies revealed a significant reduction in Foxp3 expression upon treatment with neutralizing anti-IL-10 or anti-TGF-β antibodies. Furthermore, the anti-TGF-β antibody demonstrated greater efficacy in reducing Foxp3 expression than the anti-IL-10 antibody (Figure 4C). We further examined the effect of polarized macrophages on CD4+ T cell proliferation. In the induction and BDB groups, CD4+ T cell proliferation markedly decreased as the M2 macrophage ratio increased. Notably, at a 5:1 ratio, BDB-M2 macrophages suppressed CD4+ T cell proliferation more strongly than induction-M2 macrophages (Figure 4D).

Figure 4.
Figure 4.

3-bromo-4,5-dihydroxybenzaldehyde (BDB) markedly enhances the capacity of IL-10/transforming growth factor beta (TGF-β)-modified macrophages to promote the differentiation of CD4+Foxp3+ T cells. (A) Cytokine expression was quantified via real-time PCR. (B) CD4+ T cells isolated from normal mice were co-cultured with macrophages from each experimental group and stimulated with anti-CD3 for 7 days. (C) CD4+ T cells from normal mice were co-cultured with macrophages from the BDB group in the presence or absence of anti-IL-10 or anti-TGF-β-neutralizing antibodies for 7 days. (D) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+ T cells isolated from normal mice were co-cultured at various ratios with macrophages from each experimental group and stimulated with anti-CD3 and anti-CD28 antibodies for 72 h (n=5 mice per group). Data are presented as the mean±standard deviation of three independent experiments. *p<0.05; **p<0.01; and ***p<0.001.

Discussion

Recent studies have focused on marine plants as potential sources of bioactive compounds for pharmaceutical and functional food industries. BDB, a compound primarily extracted from marine red algae, such as Polysiphonia morrowii and Polysiphonia urceolata, has garnered particular attention (22). Herein, we investigated how the immuno-modulatory ability of the BDB affects the progression of experimental ACD in mice. IgE, a key activator of mast cells, serves as a critical therapeutic target in the management of inflammatory diseases (23). In ACD, LNs exhibit swelling due to extensive immune cell infiltration. LNs are vital to cell-mediated immunity, as they are central to regulating the functions of mature T and B cells (24, 25). Inflammatory cytokines secreted by activated T cells regulate the persistence and severity of skin inflammation (26). In this study, BDB relieved characteristic ACD symptoms, including skin and LN hypertrophy, elevated serum IgE levels, and heightened inflammatory cytokine levels. It enhanced the production of Treg-related factors in the inflamed area. Furthermore, BDB is believed to modulate immune responses involving Th1, Th2, and Th17 cells. In mice with ACD, BDB increased the number of CD4+Foxp3+ T cells, and its mitigating effects were associated with the elevated expression of CD4+Foxp3+ T cells at the inflamed area. Tregs preserve immune homeostasis and modulate the inflammatory reaction by regulating the immune system (27). The enhancement of the CD4+Foxp3+ T cell population regulated by BDB may reduce CD4+ T cell activation in the inflamed areas, thereby alleviating inflammatory symptoms in mice with ACD. TGF-β, an immunosuppressive cytokine, plays an important role in suppressing inordinate immune responses and facilitates Treg differentiation (28). The immunomodulatory action of BDB facilitates the upregulation of TGF-β secretion and concurrently suppresses the expression of pro-inflammatory cytokines in CD4+ T cells. Herein, BDB did not enhance the CD4+CD25+ population; however, it markedly elevated Treg-associated factor expression in CD4+ T cells and facilitated the TGF-β-dependent differentiation of CD4+Foxp3+ T cells from CD4+ T cells, effectively inhibiting the progression of ACD. M2 macrophages generated by BDB treatment modulate a variety of immune-related factors, indicating the involvement of multiple underlying mechanisms. Macrophages activate T cells through antigen capture, which involves either direct antigen presentation to T cells or the secretion of bioactive substances, including cytokines (29, 30). M2 macrophages mitigate chronic inflammatory diseases (such as rheumatoid arthritis and inflammatory bowel disease) and autoimmune disorders (including systemic lupus erythematosus and multiple sclerosis), while also modulating immune responses by remodeling the extracellular matrix (31-33). IL-10/TGF-β-modified M2 macrophages increased Treg populations in LNs and the inflamed area and promoted CD4+Foxp3+ T cell differentiation from CD4+ T cells (34). Therefore, we hypothesized that BDB treatment would enhance Treg populations and suppress the proliferation of naïve CD4+ T cells through M2 macrophages. BDB-treated M2 macrophages demonstrated elevated TGF-β expression and increased Treg populations. Moreover, two mechanisms were identified for the suppression of CD4+ T cell proliferation by BDB-modified M2 macrophages. First, BDB-treated M2 macrophages restrain CD4+ T cell proliferation by promoting Treg differentiation from CD4+ T cells. Second, they inhibit CD4+ T cell proliferation by increasing TGF-β expression. M2 macrophages are categorized into M2a, M2b, M2c, and M2d subtypes based on specific stimuli (35). Our current research aimed to examine the functions of these M2 macrophage subtypes induced by BDB in ACD animal models. Given that M2a macrophages activated by IL-4 and IL-13 secrete chemokines, such as CCL2 and CCL22, which recruit immune cells to the inflamed area (36), we focused on elucidating the role of BDB-induced M2a macrophages in ACD models and to explore the molecular mechanisms mediating the interaction between M2a macrophages and CD4+ T cells.

Conclusion

In conclusion, BDB treatment mitigated the symptoms of exacerbated ACD by suppressing the inflammatory pathways, modulating CD4+ T cell proliferation, and enhancing the Treg populations through TGF-β-dependent pathways or M2 macrophages. Therefore, BDB could serve as a potential therapeutic option for managing inflammatory skin diseases.

Footnotes

  • Authors’ Contributions

    S.C.H. and H.J.B. conception and design of research; S.C.H., J.I.K., Y.K.C., D.H.Y., K.J.K., H.J.B., and W.J.Y. performed experiments; H.K.K., E.S.Y., and H.J.B analyzed data; S.C.H., H.K.K., E.S.Y., and H.J.B interpreted the results of the experiments; K.J.K. and W.J.Y. prepared the figures; S.C.H. drafted the manuscript; S.C.H. and H.J.B. edited and revised the manuscript. All Authors approved the final version of the manuscript.

  • Funding

    This research was supported by “Regional Innovation Strategy (RIS)” through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (2023RIS-009).

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Received September 5, 2024.
  • Revision received September 30, 2024.
  • Accepted October 14, 2024.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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3-Bromo-4,5-dihydroxybenzaldehyde Attenuates Allergic Contact Dermatitis by Generating CD4+Foxp3+ T cells
SANG-CHUL HAN, JUNG-IL KANG, YOUN KYUNG CHOI, DA HEE YANG, KI JU KIM, HA JEONG BOO, WEON-JONG YOON, HEE-KYOUNG KANG, EUN-SOOK YOO, HYE-JIN BOO
In Vivo Jan 2025, 39 (1) 201-209; DOI: 10.21873/invivo.13818
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