Metformin Promotes Acquisition of Immune Tolerance in Allergen-Specific Immunotherapy

Yu-mi Park; Ji An Kim; Hana Kim; Jong-seo Yoon; Hyun Hee Kim; Yoon Hong Chun

doi:10.4168/aair.2026.18.2.254

. 2026 Mar 3;18(2):254–270. doi: 10.4168/aair.2026.18.2.254

Metformin Promotes Acquisition of Immune Tolerance in Allergen-Specific Immunotherapy

Yu-mi Park ¹, Ji An Kim ¹, Hana Kim ¹, Jong-seo Yoon ², Hyun Hee Kim ³, Yoon Hong Chun ^1,^✉

PMCID: PMC13047430 PMID: 41914532

Abstract

Purpose

Subcutaneous immunotherapy (SCIT) is a safe, effective immunotherapy method. However, it has several limitations, most notably the prolonged build-up phase. Metformin regulates Th17/regulatory T-cell (Treg) homeostasis by increasing Tregs and anti-inflammatory cytokines. To overcome the limitation of the long build-up phase of SCIT, we propose combining SCIT with metformin.

Methods

Sensitized BALB/c mice received intraperitoneal metformin (100 mg/kg or 300 mg/kg) for 9 days, except for the negative, positive, and SCIT groups. The SCIT, SCIT combined with 100 mg/kg metformin (SCIT-Met(100)), and SCIT combined with 300 mg/kg metformin (SCIT-Met(300)) groups received 3 subcutaneous injections of house dust mite (HDM) extract at 2-day intervals for immunotherapy. All groups except the negative control were given intranasal HDM extract for 5 days. Nasal symptoms, ear swelling, eosinophil count in nasopharyngeal wash-out lavage, antibody levels, and nasal mucosa histopathology were analyzed.

Results

All immunotherapy groups exhibited reduced nasal symptoms, ear swelling, eosinophil counts in nasopharyngeal lavage, eosinophils, mast cells, and goblet cells in the nasal mucosa compared to the positive and metformin-injected (Met) groups. HDM-specific immunoglobulin G1 levels increased in all immunotherapy groups. The SCIT-Met groups induced more Treg than the Met(100), Met(300) and SCIT groups. Interleukin (IL)-4, IL-5 and IL-13 mRNA levels were lower in the Met groups and SCIT-Met(300) group than in the SCIT and SCIT-Met(100) groups. Foxp3 mRNA level was significantly higher in the Met groups and SCIT-Met groups than in the SCIT group.

Conclusions

Overall, the SCIT-Met(300) group showed relatively favorable outcomes compared with the other groups. Combining immunotherapy with metformin may alleviate allergy symptoms and enhance immune tolerance in a murine model of allergic rhinitis. Further studies are needed to confirm these findings.

Keywords: Allergens, immunotherapy, metformin, eosinophils, mast cells, goblet cells, mites, rhinitis

INTRODUCTION

Allergic disease treatment primarily aims to alleviate symptoms, with allergen-specific immunotherapy (AIT) being the only curative option. Subcutaneous immunotherapy (SCIT) is one of the most proven, safe, effective immunotherapy methods.¹

SCIT induces immune tolerance, improves allergic symptoms, and modifies disease progression.² However, SCIT has several limitations, including a prolonged build-up phase to reach the target antigen concentration, frequent injections, the need for long-term maintenance, and potential side effects such as anaphylaxis following immunotherapy injections.¹ Immunotherapy prevents new allergic sensitizations¹ and helps stop allergic rhinitis from progressing to asthma.³ Early immunotherapy in children is more effective in preventing asthma.⁴ Rush or cluster applications shorten the build-up phase that induces allergic immune responses, and are used in adults.⁵ This method is safe, and improves adherence to immunotherapy.⁶ Rush and cluster build-up schedules have also been attempted in pediatric patients and shown to be effective; however, their safety has not yet been completely evaluated.⁷ Because these accelerated approaches may increase the risk of systemic reactions,⁸ they are not yet widely applicable across all age groups. Shortening treatment duration, such as rush or cluster application in adults, could improve accessibility and facilitate early immunotherapy initiation.

Metformin exhibits hypoglycemic, anti-inflammatory antioxidant properties. It enhances therapeutic efficacy when used in combination therapy for cancers, liver diseases, inflammatory bowel diseases, kidney diseases, and neurologic disorders.⁹ Metformin activates AMP-activated protein kinase (AMPK), promoting regulatory T-cells (Tregs) proliferation, increasing Foxp3 expression and suppressing Th17 cells. By regulating Th17/Treg cell homeostasis, metformin modulates inflammatory cytokines and improves inflammatory diseases.¹⁰,¹¹,¹²,¹³,¹⁴ In asthma mouse models, AMPK activation via metformin improved outcomes, reduced exacerbations,¹⁵,¹⁶,¹⁷ decreased airway inflammation, and increased Treg cell infiltration in airways.¹⁸ Additionally, metformin reduces eosinophilic inflammation and airway remodeling, regardless of obesity.¹⁹

Therefore, it is hypothesized that combining metformin with SCIT enhances Treg production, improving the effectiveness of immunotherapy.²⁰ Using an allergic rhinitis mouse model,²¹,²² different metformin dosages were tested to evaluate their impact on accelerating immune response induction among the conventional SCIT and metformin combined SCIT groups. This combination may promote immune tolerance during AIT.

MATERIALS AND METHODS

Study design

Six-week-old female BALB/c mice (OrientBio, Seongnam, Korea) were acclimated for 1 week under controlled conditions of 20°C ± 2°C and 40%–60% humidity. After stabilization, the mice were randomly divided into the following groups: the negative control (NC), positive control (PC), house dust mite (HDM)-SCIT, SCIT combined with 100 mg/kg metformin intraperitoneal injection [SCIT-Met(100)], SCIT combined with 300 mg/kg metformin intraperitoneal injection (SCIT-Met(300)), 100 mg/kg metformin intraperitoneal injection [Met(100)], and 300 mg/kg metformin intraperitoneal injection [Met(300)]. Each group consisted of 6 mice (Table).

Table. Treatment protocol for each group of mice (n = 6).

Variable	i.p. sensation	Metformin injection (i.p.)	SCIT treatment	Challenge
NC	5 µg HDM/alum, × 3	PBS, × 3	PBS, × 3	PBS
PC	5 µg HDM/alum, × 3	PBS, × 3	PBS, × 3	25 µg HDM, × 5
Met(100)	5 µg HDM/alum, × 3	100 mg/kg metformin, × 9	PBS, × 3	25 µg HDM, × 5
Met(300)	5 µg HDM/alum, × 3	300 mg/kg metformin, × 9	PBS, × 3	25 µg HDM, × 5
HDM-SCIT	5 µg HDM/alum, × 3	PBS, × 3	250 µg HDM, × 3	25 µg HDM, × 5
SCIT-Met(100)	5 µg HDM/alum, × 3	100 mg/kg metformin, × 9	250 µg HDM, × 3	25 µg HDM, × 5
SCIT-Met(300)	5 µg HDM/alum, × 3	300 mg/kg metformin, × 9	250 µg HDM, × 3	25 µg HDM, × 5

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i.p., intraperitoneal; SCIT, subcutaneous immunotherapy; NC, negative control; HDM, house dust mite; alum, aluminum hydroxide; PBS, phosphate-buffered saline; PC, positive control; Met(100), 100 mg/kg metformin; Met(300), 300 mg/kg metformin; SCIT-Met(100), subcutaneous immunotherapy combined with 100 mg/kg metformin; SCIT-Met(300), subcutaneous immunotherapy combined with 300 mg/kg metformin.

Mice were sensitized with 5 µg of HDM antigen (Dermatophagoides pteronyssinus; GREER Laboratories, Lenoir, NC, USA) and 20 µL of Inject Alum (ThermoFisher Scientific, Rockford, IL, USA) as an adjuvant, mixed in 0.1 mL of phosphate-buffered saline, via intraperitoneal injection on days 0, 7, and 14 (Supplementary Fig. S1). The HDM-SCIT, SCIT-Met(100), and SCIT-Met(300) groups received 3 subcutaneous injections of 250 µg of HDM antigen on days 28, 30, and 32. The Met(100), Met(300), SCIT-Met(100), and SCIT-Met(300) groups received intraperitoneal metformin (100 or 300 mg/kg) from days 26 to 34.¹²,¹⁵ To monitor the potential side effects of metformin, such as hypoglycemia, body weight was measured every 2 days for a total of 9 days after metformin administration. To induce allergic rhinitis symptoms, all groups except NC received intranasal instillation of 25 µg of HDM antigen on days 42, 43, 44, 45, and 46. Twenty-four hours after the final intranasal challenge, the mice were sacrificed under inhalation anesthesia with isoflurane (1.5%–2%). Ear swelling test was performed on day 45, and rhinitis clinical scores²³,²⁴ were recorded on day 46. Twenty-four hours after the final intranasal administration of HDM antigen, nasopharyngeal lavage fluid, nasal mucosal tissue, and serum samples were collected. Eosinophil counts in the nasopharyngeal lavage fluid were analyzed. Nasal mucosal tissues were prepared as paraffin blocks to examine eosinophils, goblet cells, and mast cells. HDM-specific antibodies were measured in the serum. Splenocytes were cultured with 100 µg/mL HDM antigen for 72 hours, harvested, and analyzed by flow cytometry to detect CD4⁺CD25⁺Foxp3⁺ Treg and CD4⁺ interferon (IFN)-γ⁺ cells, and by quantitative reverse transcription polymerase chain reaction to analyze the expression levels of interleukin (IL)-4, IL-5, IL-13, IL-17, Foxp3, transforming growth factor (TGF)-β, and IFN-γ mRNA. All animal experiments were conducted in accordance with the guidelines and approval of the Animal Experimentation Ethics Committee of Incheon St. Mary’s Hospital (CIMH-2024-002).

Statistical analyses

GraphPad prism software 9.1.0 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analyses. All experimental values are expressed as the mean ± standard deviation. To test differences between the experimental groups, Tukey’s multiple comparison test and one-way analysis of variance were applied. P < 0.05 was considered statistically significant.

Further experimental details are provided in the online repository (Supplementary Data S1).

RESULTS

Ear swelling test results and rhinitis clinical scores

The number of sneezes and rubbing was counted after the final HDM antigen stimulation in the nasal cavity on day 46. The SCIT (24.50 ± 4.32, P < 0.05), SCIT-Met(100) (17.33 ± 4.97, P < 0.01), and SCIT-Met(300) (15.83 ± 5.81, P < 0.01) groups showed significantly lower number of sneezes than the PC (33.33 ± 8.60) group (Fig. 1A). The number of nose rubbing was low in the SCIT, SCIT-Met(100), and SCIT-Met(300) groups compared to that in the PC group; however, the difference was not statistically significant [PC (43.00 ± 16.63) vs. SCIT (32.33 ± 4.93), SCIT-Met(100) (30.67 ± 8.62) and SCIT-Met(300) (32.17 ± 5.19)] (Fig. 1B). However, no significant differences were observed in the number of sneezes and rubbing behaviors between the Met(100) and Met(300) groups. Sneezes and rubbing counts in these groups were similar to those of the PC group.

Fig. 1 — AIT, allergen-specific immunotherapy; SCIT, conventional subcutaneous immunotherapy with house dust mite; SCIT-Met(100), conventional subcutaneous immunotherapy combined with 100 mg/kg metformin intraperitoneal injection; SCIT-Met(300), conventional subcutaneous immunotherapy combined with 300 mg/kg metformin intraperitoneal injection; NC, negative control; PC, positive control; Met(100), 100 mg/kg metformin intraperitoneal injection; Met(300), 300 mg/kg metformin intraperitoneal injection.

^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

For the skin swelling response test performed on day 45, after intradermal injection of HDM antigen in the ear, the SCIT (0.06 ± 0.02 mm, P < 0.01), SCIT-Met(100) (0.06 ± 0.02 mm, P < 0.01), SCIT-Met(300) (0.05 ± 0.01 mm, P < 0.001) groups had significantly less swelling than the PC (0.12 ± 0.03 mm) group. The Met(100) and Met(300) groups showed no reduction in skin swelling compared to the PC group (Fig. 1C).

Immune cell findings in nasopharyngeal lavage and nasal mucous tissues

The number of eosinophils in nasopharyngeal lavage significantly increased in the PC group compared to that in the NC group (NC: 4.67 ± 1.21 vs. PC: 23.00 ± 6.10, P < 0.0001). Eosinophil counts were significantly lower in the SCIT (15.17 ± 2.14, P < 0.05), SCIT-Met(100) (13.67 ± 4.63, P < 0.05), and SCIT-Met(300) (11.83 ± 3.92, P < 0.01) groups than in the PC group (Fig. 2A).

Fig. 2 — H&E, hematoxylin and eosin; AIT, allergen-specific immunotherapy; PC, positive control; PAS, periodic acid-Schiff; NC, negative control; Met(100), 100 mg/kg metformin intraperitoneal injection; Met(300), 300 mg/kg metformin intraperitoneal injection; SCIT, conventional subcutaneous immunotherapy with house dust mite; SCIT-Met(100), conventional subcutaneous immunotherapy combined with 100 mg/kg metformin intraperitoneal injection; SCIT-Met(300), conventional subcutaneous immunotherapy combined with 300 mg/kg metformin intraperitoneal injection.

^*P < 0.05; ^**P < 0.01; ^***P < 0.001; ^****P < 0.0001.

Eosinophil infiltration in the nasal mucous tissues confirmed by hematoxylin and eosin (H&E) staining was significantly greater in the PC group than in the NC group (NC: 30.83 ± 3.19 vs. PC: 59.83 ± 5.88, P < 0.0001). SCIT (41.17 ± 3.06, P < 0.0001), SCIT-Met(100) (41.33 ± 5.28, P < 0.001), SCIT-Met(300) (40.83 ± 1.60, P < 0.0001) and Met(300) (52.00 ± 2.83, P <0.05) groups showed significantly lower infiltration than in the PC group (Fig. 2B). The number of goblet cells, determined by Periodic Acid-Schiff (PAS) staining, was significantly higher in the PC group than in the NC group (NC: 37.50 ± 4.93 vs. PC: 61.50 ± 5.17, P < 0.0001). The number of goblet cells were significantly lower in the SCIT (44.33 ± 4.41, P < 0.0001), SCIT-Met(100) (42.83 ± 3.25, P < 0.0001), and SCIT-Met(300) (44.17 ± 2.56, P < 0.0001) groups than in the PC group (Fig. 2C). The number of mast cells, observed after toluidine blue staining, was significantly higher in the PC group than in the NC group (NC: 23.83 ± 4.67 vs. PC: 39.83 ± 4.45, P < 0.0001), whereas mast cells in the SCIT (29.50 ± 3.73, P < 0.01), SCIT-Met(100) (27.33 ± 4.32, P < 0.001), and SCIT-Met(300) (26.00 ± 2.10, P < 0.0001) groups were significantly reduced compared to those in the PC group (Fig. 2D).

Serum HDM-specific antibodies

Serum total immunoglobulin (Ig) E levels were significantly higher in the PC group than in the NC group (11,220.00 ± 6,960.00 ng/mL vs. 4,105.00 ± 3,543.00 ng/mL, P < 0.05). Although the SCIT (23,578.00 ± 3,432.00 ng/mL, P < 0.01) and SCIT-Met(100) (21,721.00 ± 1,768.00 ng/mL, P < 0.01) groups showed a more significant increase in serum total IgE levels compared to the PC group, the SCIT-Met(300) group showed a marginal increase in total IgE compared to the PC group (Fig. 3A).

Fig. 3 — Ig, immunoglobulin; HDM, house dust mite; SCIT, conventional subcutaneous immunotherapy with house dust mite; SCIT-Met(100), conventional subcutaneous immunotherapy combined with 100 mg/kg metformin intraperitoneal injection; SCIT-Met(300), conventional subcutaneous immunotherapy combined with 300 mg/kg metformin intraperitoneal injection; NC, negative control; PC, positive control; Met(100), 100 mg/kg metformin intraperitoneal injection; Met(300), 300 mg/kg metformin intraperitoneal injection.

^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

HDM-specific IgE level was significantly higher in the PC group than in NC group (1,230.00 ± 437.40 ng/mL vs. 358.6 ± 252.10 ng/mL, P < 0.01), whereas no difference was observed in HDM-specific IgE levels between the PC and the SCIT/SCIT-Met(100)/SCIT-Met(300) groups. HDM-specific IgE levels were significantly lower in the Met(100) (964.1 ± 393.60 ng/mL, P < 0.01) and Met(300) (297.1 ± 33.90 ng/mL, P < 0.001) groups than in the PC group (Fig. 3B).

The HDM-specific IgG1 level was significantly higher in the Met(300) (40,176.00 ± 35,470.00 ng/mL, P < 0.05), SCIT (116,929.00 ± 9,679.00 ng/mL, P < 0.01), SCIT-Met(100) (149,029.00 ±140,536.00 ng/mL, P < 0.05), and SCIT-Met(300) (119,255.00 ± 46,484.00 ng/mL, P < 0.001) groups than in the PC group (20,472.00 ± 10,628.00 ng/mL) (Fig. 3C).

The HDM-specific IgG2a level was significantly higher in the SCIT (784.30 ± 296.20 ng/mL, P < 0.05) group than in the PC group (453.10 ± 161.20 ng/mL). Although SCIT-Met(100) (900.40 ± 473.1 ng/mL) and SCIT-Met(300) (980.5 ± 889.2 ng/mL) showed an increase in HDM-specific IgG2a levels compared to the PC group, the difference was not significant (Fig. 3D).

Flow cytometric analysis

Splenic mononuclear cells were sorted based on the expression levels of Foxp3 and CD25. CD4⁺CD25⁺Foxp3⁺ T cells accounted for 6.18% (minimum, maximum: 5.59, 7.42), 5.67% (3.27, 9.62), 6.15% (4.59, 7.60), 6.44% (5.57, 7.20), 7.11% (6.04, 8.42), 7.88% (7.37, 8.46), and 8.38% (7.47, 9.59) of total splenic mononuclear cells in the NC, PC, Met(100), Met(300), SCIT, SCIT-Met(100), and SCIT-Met(300) groups, respectively. The percentage of CD4⁺CD25⁺Foxp3⁺ T cells in the SCIT-Met(300) group was significantly higher than in the PC (P < 0.05), SCIT (P < 0.05) and Met groups (P < 0.01) groups. The percentage of CD4⁺CD25⁺Foxp3⁺ T cells in SCIT-Met(100) group showed significantly higher than that in the Met(100) (P < 0.01) and Met(300) (P < 0.01) groups (Fig. 4B).

Fig. 4 — IFN, interferon; SCIT-Met(300), conventional subcutaneous immunotherapy combined with 300 mg/kg metformin intraperitoneal injection; PC, positive control; Met, metformin intraperitoneal injection; SCIT, conventional subcutaneous immunotherapy with house dust mite; SCIT-Met(100), conventional subcutaneous immunotherapy combined with 100 mg/kg metformin intraperitoneal injection; NC, negative control; Met(100), 100 mg/kg metformin intraperitoneal injection; Met(300), 300 mg/kg metformin intraperitoneal injection.

^*P < 0.05; ^**P < 0.01; ^***P < 0.001.

Splenic mononuclear cells expressing CD4 and IFN-γ were sorted. CD4⁺IFN-γ T cells accounted for 2.22% (minimum, maximum: 1.17, 2.87), 2.63% (1.62, 3.36), 2.24% (1.18, 3.76), 3.17% (2.45, 4.11), 2.00% (1.51, 2.96), 3.36% (1.75, 4.63), and 4.16% (3.04, 5.31) of the total splenic mononuclear cells in the NC, PC, Met(100), Met(300), SCIT, SCIT-Met(100), and SCIT-Met(300), respectively. The percentage of CD4+ IFN-γ T cells was significantly higher in the SCIT-Met(300) group than in the PC (P < 0.01), Met(100) (P < 0.01) and Met(300) (P < 0.05) groups. The percentage of these cells increased more significantly in the SCIT-Met(100) (P < 0.05) and SCIT-Met(300) (P < 0.001) groups compared to the SCIT group (Fig. 4D).

Analysis of mRNA expression on splenocytes

After stimulation with HDM antigen in splenocyte culture for 72 hours, total mRNA was extracted to analyze gene expression. The mRNA expression levels of IL-4, IL-5, and IL-13 cytokines, primarily secreted by Th2 lymphocytes, were compared among the PC, Met(100), Met(300), SCIT, SCIT-Met(100), and SCIT-Met(300) groups.

The SCIT-Met(300) and Met groups showed significantly lower IL-4 mRNA levels than the SCIT and SCIT-Met(100) groups (Fig. 5A). The SCIT-Met(300) group and Met groups showed significantly lower IL-5 mRNA levels than the SCIT group (Fig. 5B). The IL-13 mRNA level was highest in the SCIT group. The IL-13 mRNA levels were more markedly reduced in the Met and the SCIT-Met(300) groups than in the SCIT and SCIT-Met(100) groups (Fig. 5C). The IL-17 mRNA level was lowest in the SCIT-Met(300) group, showing a significant reduction compared to the SCIT-Met(100), SCIT-Met(300), the SCIT, and SCIT-Met(100) groups (Fig. 5D). The IFN-γ mRNA level was highest in the SCIT group. A significant reduction in IFN-γ mRNA level was observed in the SCIT-Met(300) and Met(300) groups than in the SCIT group (Fig. 5G).

The Foxp3 mRNA level was significantly higher in the Met groups, SCIT-Met(100) group and SCIT-Met(300) group than in the SCIT group. Moreover, the SCIT-Met(300) group showed a significant increase in Foxp3 mRNA levels compared to both the Met(100) and SCIT-Met(100) groups (Fig. 5E). The TGF-β mRNA level significantly increased in the SCIT-Met(300) group compared to the Met(100) and SCIT groups (Fig. 5F).

DISCUSSION

This study investigated the effect of metformin on inducing immune tolerance when combined with conventional SCIT in an HDM-induced allergic rhinitis mouse model. Metformin combined with SCIT promoted Treg cell production more than SCIT and metformin alone. Foxp3 mRNA levels increased, Th2 (IL-4, IL-5, IL-13) and IL-17 cytokine levels decreased, and these changes were more pronounced with higher doses of metformin.

AIT increases Treg cells and suppressor cytokines (TGF-β and IL-10). This alters allergen-specific T cell and B cell responses, reducing allergic inflammation. Ultimately, it decreases IgE levels while increasing IgG1 and IgG4, promoting immune tolerance.¹,²⁰,²⁵,²⁶,²⁷ Among these, Treg cells are key in initiating and maintaining immune responses. Rapid Treg induction during SCIT build-up phase may promptly suppress allergen-specific Th2-Th1 cell responses.²,²⁷,²⁸ In addition, the early formation of IgG1 and IgG2a can accelerate protective immune responses, leading to earlier symptom relief and ultimately contributing to the shortening of both build-up phase and overall immunotherapy duration.

Metformin promotes Treg cell production by activating the AMPK pathway and inducing IL-10 production,¹¹,¹²,¹⁵ thereby suppressing allergic inflammation.¹⁶,¹⁹ It was hypothesized that metformin amplifies SCIT effects and suppresses Th17 cells, enhancing SCIT efficacy and allergic rhinitis symptom control.²⁹,³⁰ Additionally, metformin influences mast cell regulation, crucial in allergic disease.³¹ Therefore, it was hypothesized that combining metformin with SCIT could induce an early immune response, enhance immunotherapy effectiveness and help manage allergic rhinitis symptoms.

Treg cells were more effectively induced in SCIT-Met groups than the SCIT group and metformin only groups. Furthermore, the SCIT-Met(300) group showed a more significant increase in Treg cells than the other groups. Combining metformin with SCIT enhances immunotherapy induction, with higher doses being more effective. Foxp3 mRNA expression, a transcription factor that regulates Treg cell differentiation, was higher in SCIT-Met groups and Met groups than that in the SCIT group, and increased with higher metformin doses. This supports metformin’s role in increasing Treg cell production and suggests a potential synergistic effect between SCIT and high-dose metformin rather than a simple additive effect. Additionally, TGF-β mRNA levels, which play a role in promoting Treg cell induction, reduces Th2 cytokines, and enhances IL-10 secretion during the early phase of SCIT,²⁶,²⁷ increased in the high-dose metformin combination group [SCIT-Met(300) group]. This indicates using metformin combined with SCIT could facilitate early immune response induction. However, the TGF-β mRNA levels were also significantly increased in the NC and PC groups. TGF-β is a cytokine secreted by various cells and plays diverse roles depending on the cell type that produces it.³² Eosinophils can secrete TGF-β,³³ contributing to airway remodeling in asthma.³⁴,³⁵ Therefore, the TGF-β mRNA level may be elevated in the NC and PC groups. Immunotherapy likely altered cell distribution in allergic inflammation in all SCIT groups, leading to a gradual increase in TGF-β expression from Treg cells. This may explain the observed mRNA levels in the experimental results. Consequently, TGF-β mRNA levels were lower in all SCIT groups than those in the PC group. However, the SCIT-Met(300) group had significantly higher TGF-β mRNA than SCIT group. This aligned with increased Treg cells, linking TGF-β mRNA elevation to Treg induction.

Total IgE levels were higher in both the SCIT and SCIT-Met groups than those in the PC group. This aligns with the role of regulatory B cells in maintaining immune tolerance during SCIT. Regulatory B cells produce IL-10 and TGF-β, promoting an increase in IgG4 and a decrease in IgE levels.¹ These immunological changes typically occur in the later phase of SCIT. During the early phase, total and allergen-specific IgE levels often rise and begin to decline after approximately 6 months.²⁰,³⁶,³⁷,³⁸,³⁹,⁴⁰ This transient IgE elevation can lead to neosensitization to new allergens.³⁷ Nevertheless, therapeutic effects of SCIT emerge as IgG4—an IgE-blocking antibody—increases early and inhibits allergen–IgE binding.³⁶,³⁷,⁴¹,⁴² In the present study, HDM-specific IgE levels were significantly elevated in the SCIT and SCIT-Met groups compared with the NC, which reflects a typical early-phase response. The expected reduction in HDM-specific IgE, observed in later SCIT stages, could not be detected due to the relatively short observation period of the mouse model. In mice, IgG1 functions similarly to human IgG4,⁴³,⁴⁴ and its levels increased significantly in both SCIT and SCIT-Met groups, confirming successful induction of allergen-specific immunity. The decreased total and HDM-specific IgE levels in the Met groups may be attributed to metformin’s anti-inflammatory effects, as metformin alone did not cause additional sensitization. However, the absence of a significant increase in HDM-specific IgG1 or IgG2a suggests that, although metformin exhibits anti-inflammatory activity, it does not sufficiently induce blocking antibodies required for long-term immunological tolerance unless continuously administered. Therefore, while metformin may act as an adjuvant to accelerate the build-up phase of SCIT, its contribution to long-term maintenance of immune tolerance appears to be limited.

Th2 cytokines (IL-4, IL-5, and IL-13) typically decrease during immunotherapy. Higher metformin doses led to greater reduction of Th2 cytokine mRNA levels induced that in SCIT-Met groups than the SCIT group. The expression of IL-4, IL-5, IL-13, and IL-17 mRNA was lower in the SCIT-Met groups and Met groups than that in the SCIT group. When combined with SCIT, higher doses of metformin resulted in a greater reduction of Th2 cytokines. However, the PC group showed no increase in Th2 cytokine mRNA levels compared to the NC group. Contrastingly, IL-4, IL-5, and IL-13 mRNA levels were significantly higher in the SCIT and the SCIT-Met(100) groups than those in the other groups. This study assessed the mRNA levels expressed in splenocytes, not cytokines in nasal mucosa (target site). Increased mRNA levels for a given gene are expected to correspond to an increase in associated protein levels; however, this is not always the case. Although mRNA expression and protein production can sometimes align, various regulatory processes—such as translation, post-translational modifications, and proteasomal pathways that promote protein degradation—collectively influence protein levels. Therefore, mRNA expression may not necessarily directly correspond to protein abundance. Increased mRNA levels can result in low protein expression, and conversely, protein expression may be elevated despite low mRNA levels.⁴⁵ Therefore, mRNA levels alone may not provide an accurate reflection of the current protein expression status of a gene. Therefore, in this study, mRNA levels may not accurately reflect immune response changes to SCIT and metformin, requiring caution when interpreting the results. In the metformin-only groups, H&E staining revealed a reduction in eosinophil counts, and mRNA levels of IL-4, IL-5, and IL-13 were comparable to or even lower than those observed in the SCIT groups—except for the SCIT-Met(300) group—confirming that metformin alone exerts an anti-inflammatory effect capable of suppressing type 2 cytokine responses. Moreover, although Foxp3 and TGF-β mRNA levels in the metformin-only group appeared comparable to those in the SCIT-Met groups, flow cytometric analysis did not reveal a significant increase in Treg cell populations. Thus, consistent with previous reports, our findings confirm that metformin alone can attenuate allergic inflammation; however, its effect was insufficient to elicit the robust immunological changes required to fully support the therapeutic efficacy of SCIT. Taken together, these findings suggest that combining metformin with SCIT can enhance the therapeutic efficacy of allergen immunotherapy, with the effect being most pronounced in the SCIT-Met(300) group. This observation supports the potential of metformin as a promising adjuvant strategy to improve SCIT outcomes.

Metformin promotes the phosphorylation of AMPK in IFN-γ-producing CD4⁺ T cells, increasing AMPK activity and inhibiting IFN-γ production. Additionally, it suppresses granulocyte-macrophage colony-stimulating factor production, inhibiting CD4⁺IFN-γ⁺ T cells differentiation.⁴⁶ However, SCIT shifts the immune response by decreasing Th2 and increasing Th1 immune responses upon allergen exposure.²,²⁷,²⁸ Therefore, during immunotherapy, Th2 cytokines decrease, whereas CD4⁺IFN-γ⁺ T cells and IFN-γ levels increase. Flow cytometry confirmed an increase in CD4⁺IFN-γ⁺ T cells in splenic mononuclear cells. These results can be interpreted as an outcome of SCIT; however, the proportion of CD4⁺IFN-γ⁺ T cells significantly increased as the administered dose of metformin combined with SCIT increased, suggesting that metformin contributes to this effect. Contrary to the previously mentioned role of metformin in reducing CD4⁺IFN-γ⁺ T cell generation, metformin can enhance Th1 cell activation and IFN-γ production.⁴⁷,⁴⁸ The effect of metformin on Th1 cells and IFN-γ production varies depending on experimental conditions and the immune environment. Therefore, in this study, metformin combined with SCIT likely triggered an antagonistic regulation mechanism, though not fully understood, leading to decreased Th2 cytokine mRNA levels (IL-4, IL-5, and IL-13) in splenocyte cultures, ultimately contributing to the increased CD4⁺IFN-γ⁺ T cells. In the SCIT-Met groups, CD4⁺IFN-γ⁺ T cells increased but IFN-γ⁺ mRNA levels significantly decreased with increasing metformin dose. This suggests that metformin increased AMPK expression and activity, but inhibited IFN-γ+ production in CD4⁺IFN-γ⁺ T cells. IFN-γ mRNA levels were measured in splenocytes, making its correlation with IFN-γ protein expression unclear.⁴⁴ Moreover, in the shift from a Th2 to a Th1 immune response, IL-4 decrease is more crucial than the IFN-γ increase.⁴⁹ Therefore, as Th2 cytokine levels decreased, IFN-γ changes likely had minimal impact on SCIT outcomes.

Metformin has hypoglycemic effects; however, rather than directly lowering glucose levels, it inhibits hepatic gluconeogenesis and improves insulin resistance. It is a well-established, safe medication,⁵⁰ without considerable side effects in combination treatments. However, to observe the safety and possible side effects of the metformin administration, body weight and activity were monitored throughout the experiment. No changes in body weight or activity were observed during and after metformin administration. Ideally, hypoglycemia in mice should be assessed via tail vein blood sampling. The lack of direct measurements should be considered when interpreting the safety and metabolic effects of metformin in this study.

This study provides preliminary data on the immune response to metformin. However, data on the optimal dose, metformin administration schedule to enhance immune responses, and human safety remain insufficient. Therefore, further studies are needed to obtain additional data on dosing and safety profiles. In addition, the lack of direct blood glucose measurements should be considered a limitation when interpreting the safety and metabolic effects of metformin in this study, as potential hypoglycemic events could not be precisely evaluated. Also, measuring only mRNA levels in splenocytes did not fully reflect actual gene expression. Therefore, further research should include western blot analysis to assess protein expression and supplement findings.

Combining metformin with SCIT reduced allergic symptoms and supported immune tolerance. Higher metformin doses more effectively reduced allergic inflammation and facilitated early immune tolerance. Metformin-SCIT combination demonstrated comparable superior results to conventional SCIT, enhancing immune response induction more than SCIT monotherapy. Therefore, combining metformin with SCIT may enhance immunological tolerance; however, further studies are required to evaluate potential clinical benefits such as treatment compliance or modification of the build-up phase.

ACKNOWLEDGMENTS

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (RS-2022-NR073135) and a Grant of Translational R&D Project through Institute for Bio-Medical convergence, Incheon St. Mary’s Hospital, The Catholic University of Korea.

Footnotes

Disclosure: There are no financial or other issues that might lead to conflict of interest.

SUPPLEMENTARY MATERIALS

Supplementary Data S1

Methods

aair-18-254-s001.doc^{(36.5KB, doc)}

Supplementary Fig. S1

Schedules for immunotherapy in the HDM-induced murine allergic rhinitis model. HDM sensitization was induced by 3 i.p. injections of 5 µg of HDM extract absorbed to alum on days 0, 7, and 14, followed by 3 SCIT treatments with 250 µg of HDM extract per mouse on days 28, 30, and 32. In the experimental groups, metformin was administered at a dose of 100 or 300 mg/kg, respectively, by 9 i.p. injections (from days 26 to 34). i.n. challenge with 25 µg of HDM extract was performed daily from days 42 to 46. Clinical symptoms were checked immediately after the last i.n. challenge. Twenty-four hours later, the mice were sacrificed and serum, nasopharyngeal lavage, tissues, and splenocytes were collected for further analysis.

aair-18-254-s002.ppt^{(515.5KB, ppt)}

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Data S1

Methods

aair-18-254-s001.doc^{(36.5KB, doc)}

Supplementary Fig. S1

aair-18-254-s002.ppt^{(515.5KB, ppt)}

[B1] 1.Alvaro-Lozano M, Akdis CA, Akdis M, Alviani C, Angier E, Arasi S, et al. EAACI allergen immunotherapy user’s guide. Pediatr Allergy Immunol. 2020;31 (Suppl 25):1–101. doi: 10.1111/pai.13189. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.Shamji MH, Sharif H, Layhadi JA, Zhu R, Kishore U, Renz H. Diverse immune mechanisms of allergen immunotherapy for allergic rhinitis with and without asthma. J Allergy Clin Immunol. 2022;149:791–801. doi: 10.1016/j.jaci.2022.01.016. [DOI] [PubMed] [Google Scholar]

[B3] 3.Schmitt J, Schwarz K, Stadler E, Wüstenberg EG. Allergy immunotherapy for allergic rhinitis effectively prevents asthma: results from a large retrospective cohort study. J Allergy Clin Immunol. 2015;136:1511–1516. doi: 10.1016/j.jaci.2015.07.038. [DOI] [PubMed] [Google Scholar]

[B4] 4.Hamelmann E, Hammerby E, Scharling KS, Pedersen M, Okkels A, Schmitt J. Quantifying the benefits of early sublingual allergen immunotherapy tablet initiation in children. Allergy. 2024;79:1018–1027. doi: 10.1111/all.15985. [DOI] [PubMed] [Google Scholar]

[B5] 5.Lee JH, Choi JH, Jeong KB, Lee SJ, Lee MK, Lee WY, et al. Safety and utility of rush immunotherapy with aqueous allergen extracts for treatment of respiratory allergies. J Korean Med Sci. 2021;36:e18. doi: 10.3346/jkms.2021.36.e18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Caruso C, Bramé B, Bagnasco D, Cocconcelli A, Ortolani V, Pravettoni V, et al. Adherence to allergen subcutaneous immunotherapy is increased by a shortened build-up phase: a retrospective study. BioMed Res Int. 2020;2020:7328469. doi: 10.1155/2020/7328469. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Gomes Dos Reis Pimentel RA, Oliveira G, Ferreira Chaves Loureiro E Lemos CS. Accelerated subcutaneous immunotherapy in pediatric population - systematic review. Pulmonology. 2018;24:182–193. doi: 10.1016/j.rppnen.2017.07.009. [DOI] [PubMed] [Google Scholar]

[B8] 8.Hajirawala M, Hardeman A, Huntwork MP, Carlson JC. A short, safe rush-induction protocol for aeroallergen immunotherapy. Ann Allergy Asthma Immunol. 2023;131:661–662. doi: 10.1016/j.anai.2023.07.016. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Hasanvand A. The role of AMPK-dependent pathways in cellular and molecular mechanisms of metformin: a new perspective for treatment and prevention of diseases. Inflammopharmacology. 2022;30:775–788. doi: 10.1007/s10787-022-00980-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Liu X, Sun Z, Wang H. Metformin alleviates experimental colitis in mice by up-regulating TGF-β signaling. Biotech Histochem. 2021;96:146–152. doi: 10.1080/10520295.2020.1776896. [DOI] [PubMed] [Google Scholar]

[B11] 11.Nath N, Khan M, Paintlia MK, Singh I, Hoda MN, Giri S. Metformin attenuated the autoimmune disease of the central nervous system in animal models of multiple sclerosis. J Immunol. 2009;182:8005–8014. doi: 10.4049/jimmunol.0803563. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Sun Y, Tian T, Gao J, Liu X, Hou H, Cao R, et al. Metformin ameliorates the development of experimental autoimmune encephalomyelitis by regulating T helper 17 and regulatory T cells in mice. J Neuroimmunol. 2016;292:58–67. doi: 10.1016/j.jneuroim.2016.01.014. [DOI] [PubMed] [Google Scholar]

[B13] 13.Kim JW, Kim SM, Park JS, Hwang SH, Choi J, Jung KA, et al. Metformin improves salivary gland inflammation and hypofunction in murine Sjögren’s syndrome. Arthritis Res Ther. 2019;21:136. doi: 10.1186/s13075-019-1904-0. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Duan W, Ding Y, Yu X, Ma D, Yang B, Li Y, et al. Metformin mitigates autoimmune insulitis by inhibiting Th1 and Th17 responses while promoting Treg production. Am J Transl Res. 2019;11:2393–2402. [PMC free article] [PubMed] [Google Scholar]

[B15] 15.Ma W, Jin Q, Guo H, Han X, Xu L, Lu S, et al. Metformin ameliorates inflammation and airway remodeling of experimental allergic asthma in mice by restoring AMPKα activity. Front Pharmacol. 2022;13:780148. doi: 10.3389/fphar.2022.780148. [DOI] [PMC free article] [PubMed] [Google Scholar]

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[B17] 17.Li CY, Erickson SR, Wu CH. Metformin use and asthma outcomes among patients with concurrent asthma and diabetes. Respirology. 2016;21:1210–1218. doi: 10.1111/resp.12818. [DOI] [PubMed] [Google Scholar]

[B18] 18.Guo Y, Shi J, Wang Q, Hong L, Chen M, Liu S, et al. Metformin alleviates allergic airway inflammation and increases Treg cells in obese asthma. J Cell Mol Med. 2021;25:2279–2284. doi: 10.1111/jcmm.16269. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B19] 19.Medeiros ML, Oliveira AL, Mello GC, Antunes E. Metformin counteracts the deleterious effects of methylglyoxal on ovalbumin-induced airway eosinophilic inflammation and remodeling. Int J Mol Sci. 2023;24:9549. doi: 10.3390/ijms24119549. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20.Gonzalez M, Doña I, Palomares F, Campo P, Rodriguez MJ, Rondon C, et al. Dermatophagoides pteronyssinus immunotherapy changes the T-regulatory cell activity. Sci Rep. 2017;7:11949. doi: 10.1038/s41598-017-12261-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Kim SM, Kim JA, Kim HS, Yoon JS, Kim HH, Chun YH. Development of a house dust mite-induced mouse model for allergen-specific immunotherapy in allergic rhinitis. Allergy Asthma Respir Dis. 2022;10:195. [Google Scholar]

[B22] 22.Hesse L, Petersen AH, Nawijn MC. Methods for experimental allergen immunotherapy: subcutaneous and sublingual desensitization in mouse models of allergic asthma. Methods Mol Biol. 2021;2223:295–335. doi: 10.1007/978-1-0716-1001-5_20. [DOI] [PubMed] [Google Scholar]

[B23] 23.Saito H, Howie K, Wattie J, Denburg A, Ellis R, Inman MD, et al. Allergen-induced murine upper airway inflammation: local and systemic changes in murine experimental allergic rhinitis. Immunology. 2001;104:226–234. doi: 10.1046/j.0019-2805.2001.01253.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B24] 24.Brimnes J, Kildsgaard J, Jacobi H, Lund K. Sublingual immunotherapy reduces allergic symptoms in a mouse model of rhinitis. Clin Exp Allergy. 2007;37:488–497. doi: 10.1111/j.1365-2222.2006.02624.x. [DOI] [PubMed] [Google Scholar]

[B25] 25.Drazdauskaitė G, Layhadi JA, Shamji MH. Mechanisms of allergen immunotherapy in allergic rhinitis. Curr Allergy Asthma Rep. 2021;21:2. doi: 10.1007/s11882-020-00977-7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B26] 26.Akdis CA, Akdis M. Mechanisms of allergen-specific immunotherapy and immune tolerance to allergens. World Allergy Organ J. 2015;8:17. doi: 10.1186/s40413-015-0063-2. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B27] 27.O’Hehir RE, Gardner LM, de Leon MP, Hales BJ, Biondo M, Douglass JA, et al. House dust mite sublingual immunotherapy: the role for transforming growth factor-beta and functional regulatory T cells. Am J Respir Crit Care Med. 2009;180:936–947. doi: 10.1164/rccm.200905-0686OC. [DOI] [PubMed] [Google Scholar]

[B28] 28.Pawankar R, Ra C. Heterogeneity of mast cells and T cells in the nasal mucosa. J Allergy Clin Immunol. 1996;98:S248–S262. doi: 10.1016/s0091-6749(96)70073-8. [DOI] [PubMed] [Google Scholar]

[B29] 29.Amin K, Issa SM, Ali KM, Aziz MI, Hama Amieen HM, Bystrom J, et al. Evidence for eosinophil and IL-17 mediated inflammation in allergic rhinitis. Clin Mol Allergy. 2020;18:6. doi: 10.1186/s12948-020-00117-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Metformin Promotes Acquisition of Immune Tolerance in Allergen-Specific Immunotherapy

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