Matcha alleviates sneezing response in a murine model of allergic rhinitis

Abstract

Matcha, a traditional Japanese tea, has been implicated in various health effects. Herein, we investigated the impact of Matcha on allergic rhinitis by employing an allergen-induced nasal inflammation model in mice. Allergen-induced immediate nasal response, represented by the evocation of sneezing in ovalbumin-immunized mice, and subsequent nasal hyperresponsiveness, assessed by enhanced histamine-triggered sneezes, were significantly suppressed by continuous intragastric administration of hot water-extracted Matcha (250 mg/kg) with its residue. However, accumulation of inflammatory cells, total and allergen-specific IgE production, and intestinal microbiota diversity were unaffected by Matcha. Histamine- and substance P-triggered sneezes, accompanied by c-Fos expression in relevant neurons, tended to be suppressed by Matcha, whereas anti-dinitrophenyl IgE-evoked passive cutaneous anaphylaxis was unaffected. T cell receptor-stimulated CD4⁺ T cell proliferation was slightly enhanced by ethanol-extracted Matcha. Accordingly, RNA-seq analysis demonstrated weak but significant upregulation of iron uptake- and proliferation-related genes in CD4⁺ T cells. Importantly, Matcha treatment nearly abolished histamine-induced c-Fos expression in the ventral spinal trigeminal nucleus caudalis (Sp5C), reducing it to basal levels, suggesting direct suppression of neural mechanisms underlying the sneezing reflex. Matcha potentially alleviates allergic rhinitis symptoms independent of IgE/mast cell- or T-cell-mediated cascades but through a direct down-regulation of the sneezing reflex.

Introduction

Matcha, a finely ground powder made from specially grown and processed green tea leaves, has been traditionally consumed in East Asia, especially Japan, for centuries. Recently, it has gained global popularity not only for its unique flavor but also for its potential health benefits^1,2. Matcha contains various bioactive compounds, including catechins, polyphenols, and the amino acid L-theanine. These compounds have been implicated in numerous health effects, including improved cardiovascular health, enhanced cognitive function, and anti-inflammatory properties^1,2.

Green tea and its components have been shown to alleviate allergic diseases. The reduction of nasal symptoms in patients with Japanese cedar pollinosis by green tea administration was demonstrated in a series of clinical trials^3,4. Consistently, several green tea components, such as epigallocatechin gallate, gallic acid, and pyrogallol, have demonstrated suppressive effects in murine allergic rhinitis models^5–7. However, some of those compounds are hardly extracted and ingested during the standard green tea consumption process. The bioactive compounds can be consumed more efficiently from Matcha because both the extracts and the remaining residue are customarily ingested. However, the influence of Matcha on allergic rhinitis has not been investigated.

Herein, we examined the impact of Matcha on a murine model of allergic rhinitis. Allergen-induced immediate nasal response (INR) and nasal hyperresponsiveness (NHR) in immunized mice were significantly suppressed by Matcha administration without affecting serum IgE response. The mechanisms were further explored by employing a passive cutaneous anaphylaxis (PCA) model, intestinal microbiota diversity assessment, and proliferation and RNA-seq analysis of CD4⁺ T cells. Finally, Matcha was likely to alleviate allergic rhinitis symptoms by directly downregulating the sneezing reflex.

Results

Effect of Matcha on allergen-induced nasal inflammation

We first investigated the effect of Matcha in a murine model of allergic rhinitis. As shown in Fig. 1A, BALB/c mice were immunized with OVA via intraperitoneal injection once a week for four weeks. Two weeks after the final immunization, mice received intranasal phosphate-buffered saline (PBS) or OVA solution once daily for five consecutive days. Matcha extract, prepared by boiling in 65 °C distilled water, was administered intragastrically at 250 mg/kg, three times/week at 1–2-day intervals for five weeks starting from the first immunization. Additionally, Matcha was given 30 min before each intranasal OVA challenge. Neither allergen-immunization nor Matcha treatment affected general health, including weekly body weight changes (Supplementary Fig. 1).

Fig. 1. Effect of Matcha extract on mouse model of allergic rhinitis.

A Schematic of experiments using allergen-immunized mice. BALB/c mice immunized with intraperitoneal (i.p.) administration of ovalbumin (OVA) + alum were challenged by intranasal (i.n.) injection of OVA with intragastric (i.g.) treatment of Matcha. B Immediate nasal response (INR) was assessed immediately after the fourth challenge with PBS or OVA with (+Matcha) or without Matcha (n = 16–21). C Nasal hyperresponsiveness (NHR) was evaluated 6 h after the final OVA challenge (n = 16-22). D Accumulation of eosinophils, neutrophils, lymphocytes, and macrophages in the nasal lavage fluid (NALF) was assessed immediately following the NHR evaluation (n = 4–10). E Total and anti-OVA IgE in the sera, including non-immunized mice (Intact), were measured at day 37 (n = 4–12). Matcha extract (250 mg/kg) was administered to mice by intragastric injection 3 times/week during the immunization period and daily during the challenge period. Data are expressed as the mean ± standard error of the mean from four separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with OVA-challenged mice by one-way analysis of variance with Dunnett’s multiple comparison test, except for (D), which used Kruskal–Wallis test with Dunn’s multiple comparison test.

Repeated intranasal OVA injection significantly induced INR, assessed by counting sneezes immediately after the fourth OVA challenge (Fig. 1B). This model allowed us to assess NHR, characterized by an exaggerated sneezing response to non-specific stimuli⁸. Consistently, NHR, indicated as an increase in histamine-induced sneezing following the final OVA challenge, was significantly evoked (Fig. 1C). Analysis of nasal lavage fluid (NALF) collected immediately after NHR assessment revealed marked infiltration of eosinophils and a non-significant increase in neutrophils, with minor migration of lymphocytes and macrophages into the nasal mucosa upon OVA challenge (Fig. 1D). Matcha treatment significantly attenuated allergen-induced INR and NHR but not affected inflammatory cell infiltration. Total and allergen-specific serum IgE levels, measured at the time of the third OVA injection, were markedly increased in OVA-immunized mice but were not further influenced by subsequent intranasal allergen injection or Matcha administration (Fig. 1E).

Effect of Matcha on intestinal microbiota

High-throughput 16S rDNA sequencing of intestinal microbiota was performed on total genomic DNA extracted from fecal samples at both the beginning and end of the experiment. The rarefaction curves confirmed the appropriateness of the available sample size and the amount of sequencing data included (Supplementary Fig. 2). The PCoA plots (Supplementary Fig. 3), UPGMA clustering trees (Supplementary Fig. 4) indicated that the population of intestinal microbiota was altered by OVA immunization. No significant differences in the Shannon (Fig. 2A) and Chao1 (Fig. 2B) indexes were observed before and after immunization, PBS and OVA challenge, or with and without Matcha treatment. However, given the limited sample size (n = 3 per group) and high variability, these comparisons are underpowered and should be interpreted with caution; both indexes nonetheless exhibited a non‑significant downward trend in OVA-challenged mice regardless of Matcha treatment (Fig. 2A, B).

Fig. 2. Effect of Matcha extract on intestinal microbiota in a murine model of allergic rhinitis.

High-throughput 16S rDNA sequencing was performed on fecal samples from phosphate-buffered saline (PBS)-challenged mice (PA and PB), ovalbumin (OVA)-challenged mice (OA and OB), and Matcha extract-administered and OVA-challenged mice (MA and MB) at the beginning (PA, OA, and MA) and end (PB, OB, and MB) of the experiments. The difference in intestinal microbiota diversity and strain abundances among groups (n = 3, respectively) were compared by (A, B) boxplots of between-group diversity comparison using (A) Shannon index and B Chao1 index, C stacked bar plot of species distribution, and D distribution heatmap representing the relative abundance of the corresponding species in each group. Box plots indicate significantly different abundance strains (E) between the start and end of the experiments in PBS-challenged mice and F between PBS- and OVA-challenged mice. p values calculated by Student’s t-test are indicated.

The top 30 gut microbiota abundance (Fig. 2C) and clustering (Fig. 2D) further indicated OVA immunization-mediated alteration of intestinal bacteria strain distribution. The abundance of Candidatus_Arthromitus, Clostridia_vadinBB60_group, and Lactobacillus was significantly decreased, whereas that of Muribaculaceae and Ruminococcus was increased after the immunization and challenge process, as evaluated in PBS-challenged mice (Fig. 2E).

Not only immunization but also allergen challenge affected the intestinal microbiota. Thus, the abundances of Erysipelatoclostridium and Lactobacillus were significantly higher in mice challenged with OVA compared to those challenged with PBS (Fig. 2F). Lefse analysis and generating cladograms further revealed the difference in bacterial strains between both groups at taxonomic levels (Supplementary Figs. 5 and 6). However, no significant difference in the abundances of bacterial strains was observed in OVA-challenged mice with and without Matcha treatment.

Effect of Matcha on PCA response

Since Matcha displayed a suppressive effect on allergen-induced INR without affecting IgE production, we next examined the effect on IgE/mast cell-dependent response using the PCA model as described previously⁹. Mice were passively immunized by intradermal injection of anti-DNA-IgE into the ear, followed by intravenous administration of dinitrophenyl (DNP)-conjugated bovine serum albumin (BSA) together with Evans blue dye. Prior to allergen challenge, mice received Matcha treatment three times/week at 1–2 day intervals for two consecutive weeks (Fig. 3A). PCA response, assessed by the amount of Evans blue extravasation, was significantly evoked in anti-DNP-IgE immunized ears following DNP-IgE injection. Matcha treatment did not affect the PCA response (Fig. 3B).

Fig. 3. Effect of Matcha extract on mouse model of passive cutaneous anaphylaxis response.

A Schematic of the experimental procedure. Mice were intradermally (i.d.) immunized with anti-dinitrophenyl (DNP) IgE antibody (IgE) or phosphate-buffered saline (-) in the ear. Matcha was intragastrically (i.g.) administered (Matcha) or lest unadministered (Control) 3 times/week at 1–2-day intervals for 2 consecutive weeks. One hour after the last administration, mice were challenged by intravenous (i.v.) injection of DNP-bovine serum albumin mixed with Evans blue dye. B Thirty minutes later, extravasated Evans blue in the ear was extracted and quantified. Data are expressed as the mean ± standard error of the mean for 5–6 animals from two separate experiments. *p < 0.05, ***p < 0.001, compared with IgE-sensitized mice by Student’s t-test.

Effect of Matcha on T cell response

We previously demonstrated that CD4⁺ T cells are responsible for developing INR and NHR. Thus, in addition to the alleviation of allergen- and histamine-induced sneezing responses in allergen-immunized and challenged mice by CD4⁺ cell depletion, NHR was also evoked in non-immunized mice by adoptive transfer of in vitro-differentiated allergen-specific Th2 cells, as well as Th1 and Th17 cells⁸. Therefore, the effect of Matcha extract on T-cell response was evaluated in vitro. Naïve CD4⁺ T cells isolated from DO11.10/RAG2^−/− mouse splenocytes proliferated in response to the OVA (323-339) peptide stimulation. Matcha extract slightly but dose-dependently enhanced the proliferative response (Fig. 4A). We then examined the effect of Matcha extract on stimulation-induced T-cell gene expression by RNA-Seq analysis. Besides the altered expression of more than 9000 genes induced by the stimulation, the expression of only 12 genes was slightly but significantly affected by Matcha extract (Fig. 4B and Table 1). Among them, significant upregulation of Ftl1 and Gfi1 by Matcha was consistent with their profiles for up-taking iron¹⁰, which is richly contained in Matcha, and for accelerating T cell proliferation¹¹ that we observed in Fig. 4A, respectively. Besides a slight upregulation of Ccl5, Matcha extract did not affect gene expression in CD4⁺ T cells, particularly related to immunological and inflammatory responses (Table 1). Given the essential contribution of CD4⁺ T cells, it is unlikely that Matcha’s reduction of allergen‑induced INR and NHR reflects suppression of T‑cell-mediated responses.

Fig. 4. Effect of Matcha extract on T cell response.

A Naïve CD4⁺ T cells from DO11.10/RAG2^−/−were cultured with ovalbumin (323–339) peptide plus irradiated splenocytes in the presence and absence of indicated concentrations of Matcha extract for 4–5 days. The relative proliferative response, as the percent of control without matcha extract, was determined. Data are expressed as the mean ± standard error of the mean of the quadruplicate cultures representative of at least 2 independent experiments. B The expanded CD4⁺ T cells were re-stimulated with anti-CD3 and anti-CD28 antibodies for 6 h in the presence or absence of 25 μg/ml Matcha extract. The volcano plot obtained from RNA-seq analysis data displays the log2 fold change (x-axis) versus the −log10 adjusted p value (q values, y-axis) for each gene. Significantly upregulated and downregulated genes by Matcha extract treatment (q value < 0.05 represented by the horizontal dashed line) are highlighted in red and green, respectively. The vertical dashed lines indicate the log2 fold change thresholds (±1). Genes that do not meet the significance criteria are shown in gray.

Table 1.

Effect of Matcha extract on T cell gene expression^a

Gene	Fold change	q value
Inafm1	1.8	0.025
Ccl5	1.7	0.00000035
Ftl1	1.6	0.00043
Phf1	1.5	0.011
Cd14	1.4	0.03
Gfi1	1.4	0.00074
Rnasek	1.4	0.013
Grina	1.4	0.00043
Prr13	1.4	0.011
Srxn1	1.3	0.03
Lypla2	1.2	0.029
Esf1	0.7	0.011

^aCD4⁺ T cells were re-stimulated with anti-CD3 and anti-CD28 antibodies for 6 h in the presence or absence of 25 μg/ml Matcha extract. Differentially expressed genes in the presence and absence of Matcha extract, showing less than 0.05 false discovery rate (FDR)-adjusted p values (q values) with fold change values compared to Matcha-untreated samples are listed.

Matcha directly alleviates the sneezing reflex

Since IgE/mast cell- and T cell-mediated cascades were unlikely to account for Matcha’s effect on allergen-induced INR and NHR, the impact of Matcha on chemical mediator-induced sneezes was examined. Intranasal administration of histamine or substance P elicited a substantial number of sneezes in unprimed mice. Both responses tended to be attenuated by the Matcha treatment, but this did not reach statistical significance (Fig. 5A, B).

Fig. 5. Effect of Matcha extract on histamine- and substance P-induced sneezing reaction.

Number of sneezes recorded for 5 min following intranasal administration of (A) histamine or (B) substance P in BALB/c mice. c-Fos expression in the ventral spinal trigeminal nucleus caudalis (Sp5C) 45 min after histamine injection, assessed by immunohistochemistry (C) and quantified as relative luminous intensity (arbitrary units, AU) (D). Matcha extract was administered (+) or not (−) by intragastric injection 30 min before stimulation. Data are expressed as the mean ± standard error of the mean from two separate experiments (A, n = 5-6; B, n = 10-12; C, n = 3; D, n = 3). p values evaluated by Student’s t-test (A, B) and by one-way analysis of variance with Dunnett’s multiple comparison test (C, D) are shown.

To further assess whether Matcha directly modulates neural pathways triggering sneezing, we examined c-Fos expression in the ventral spinal trigeminal nucleus caudalis (Sp5C) using immunohistochemistry. Histamine stimulation markedly increased c-Fos expression in this region (Fig. 5C, D), as confirmed by the capsaicin-positive control (Supplementary Fig. 7). Notably, Matcha treatment nearly abolished histamine-induced c-Fos expression, reducing it to basal levels. These findings suggest that Matcha’s inhibitory effect on INR and NHR in immunized mice is mediated, at least in part, by direct suppression of neural mechanisms underlying the sneezing reflex.

Discussion

Our present study demonstrated that Matcha potentially alleviates nasal symptoms in allergic rhinitis by directly affecting the sneezing reflex. This interpretation is consistent with a non-significant trend toward alleviation of histamine-induced c-Fos expression in the ventral spinal trigeminal nucleus caudalis (Sp5C), a key relay of nasal sensory input^12,13, by Matcha, suggesting direct attenuation of trigeminal pathway activation. Among various health-promoting properties of Matcha, several effects related to neuronal activity are implicated¹. Notably, Matcha suppressed stress responses, which are closely related to the autonomic nervous system activity, in animal experiments and clinical studies¹⁴. Besides an essential character as a neuronal reflex initiated by sensory nerve stimulation, the sneeze is affected by autonomic nervous system activity¹⁵. Although identifying the component in Matcha directly affecting the sneezing reflex is needed, the crosstalk between the sensory and autonomic nervous systems may be crucially involved in the mechanisms underlying the down-modulation of the sneezing response by Matcha. Together with the trends toward attenuation of histamine- and substance P-evoked sneezing, the Sp5C c-Fos reduction, albeit based on a small sample size, is consistent with the primary action on neural circuits rather than upstream immune effectors.

Several components involved in green tea leaves displayed alleviating effects on animal models of allergic rhinitis. Fu et al. demonstrated that oral administration of epigallocatechin gallate to immunized mice suppressed allergen-induced nasal symptoms and cytokine production, along with serum IgE and histamine level reduction⁵. The downregulation of nasal symptoms, inflammatory cell infiltration, and cytokine production in allergen-immunized and -challenged mice was achieved by gallic acid administration⁶. We identified pyrogallol from Awa-tea leaves, which exhibited suppressive effects on nasal symptoms in the toluene-2,4-diisocyanate-induced rat rhinitis model. Mechanistically, pyrogallol suppressed Th2 cytokine production by a rat mast cell line, RBL-2H3, partly due to downregulating the nuclear translocation of nuclear factor of activated T cells⁷. However, the amount of epigallocatechin gallate in the Matcha extract administered in this study could not reach the effective dose demonstrated by Fu et al. Although the amounts of gallic acid and pyrogallol involved in the Matcha extract were unknown, we observed that serum IgE production and related T cell gene expression remained unchanged. Therefore, it is likely that these components and Th2 cytokine-related mechanisms do not contribute to the effects of Matcha extract identified in this study. Consistent with this notion, Matcha did not alter IgE/mast cell–dependent PCA responses, while it tended to suppress Sp5C c-Fos, providing a mechanistic bridge between the behavioral phenotype and the absence of systemic immunomodulation.

Despite the limited sample size and high variability, allergen immunization and challenge were associated with a non-significant trend toward shifts in intestinal microbiota composition. Particularly, the lower abundance of Lactobacillus in immunized mice following challenge with OVA than PBS was consistent with previous findings reported by Chen et al.¹⁶. The increase in Ruminococcus demonstrated by the OVA challenge in the report of Chen et al. was only observed as a consequence of allergen immunization, but not that of the OVA challenge in this study. They further displayed the reduction of Bacteroides and Prevotella in OVA-challenged mice, whereas we identified OVA challenge-induced decrease in Erysipelatoclostridium in this study. Among them, several Lactobacillus strains, such as Lactobacillus paracasei KW3110, Lactobacillus acidophilus L-55 and L-92, Lactobacillus casei Shirota, and Lactobacillus plantarum YIT 0132 have been reported to improve symptoms of patients with allergic rhinitis^17–21. The alleviation of allergen-induced IgE response was induced by other Lactobacillus strains in a mouse model²². Therefore, decreased Lactobacillus abundance following OVA immunization and challenge probably affected the symptoms and inflammatory responses observed in the allergic rhinitis model we employed in this study. Although further studies with a larger sample size are warranted, the lack of influence of Matcha on the abundance of bacterial strains argues against the alteration of intestinal microbiota as a primary mechanism of Matcha-mediated alleviation of allergic rhinitis symptoms.

In conclusion, Matcha has the potential to alleviate the nasal symptoms of allergic rhinitis patients, probably through downregulating the neuronal reaction that induces sneezing. Several components, such as L-theanine, arginine, caffeine, and epigallocatechin gallate, have been implicated in Matcha’s stress-reducing effects²³. To further validate the present findings on the health benefits of Matcha, confirmatory studies with larger, adequately powered cohorts are warranted; in parallel, future studies should clarify the specific components responsible for its neurocentric action and elucidate the underlying mechanisms.

Methods

Allergen-induced nasal inflammation

All animal experiments were approved by the Animal Use and Care Committees (A24-37) of Hiroshima University. As shown in Fig. 1A, six- to eight-week-old female BALB/c mice (Jackson Laboratory Japan, Inc., Yokohama, Japan) were weekly (day 0, 7, 14, and 21) immunized four times with intraperitoneal injection of 20 μg OVA (Sigma Aldrich, St. Louis, MO, USA) emulsified in 1 mg aluminum hydroxide (AlumVax Hydroxide vaccine adjuvant; OZ Bioscience, San Diego, CA, USA). Two weeks after the last immunization (day 35), PBS or OVA solution (30 mg/ml in PBS) was intranasally injected into the mice without anesthesia. The same injection was repeated for five consecutive days (days 35–39). Body weight was measured 1 or 2 times/week throughout the process. On day 37, about 50 μl peripheral blood was recovered, and serum levels of total and OVA-specific IgE were measured by enzyme-linked immunosorbent assay (ELISA) using a horse radish peroxidase-conjugated anti-mouse IgE antibody (Serotech, Oxford, UK) as described previously with slight modifications²⁴. The allergen-induced INR was evaluated by counting the number of sneezes for 5 min immediately after the fourth intranasal injection (day 38). To evaluate NHR, six hours after the last OVA injection (day 39), the sneeze number was counted for 5 min immediately following the intranasal injection of 10 μl histamine (100 mM in PBS; Nacalai tesque, Kyoto, Japan). Subsequently, NALF was obtained by introducing 150 μL PBS in the nose immediately after euthanasia. Following the leukocyte number assessment in NALF using a hemocytometer, eosinophils, neutrophils, lymphocytes, and macrophages were classified based on morphologic criteria on a cytocentrifuged preparation upon staining with Diff-Quick (Sysmex Corporation, Kobe, Japan) as described previously²⁵.

PCA response

Seven- to eight-week-old C57BL/6 mice were passively immunized by intradermal injection of 20 μl anti-dinitrophenyl (DNP) IgE antibody (1.5 ng/μl, Sigma Aldrich) in the right ear. For the control, PBS was injected in the left ear. After 24 h, DNP-BSA (2.5 mg/kg, Thermo Fisher Scientific, Waltham, MA, USA) and Evans blue dye (50 mg/kg, Sigma Aldrich) in 200 μl PBS were injected intravenously. Thirty minutes later, the ear (6 mm diameter) was dissected immediately after euthanasia, desiccated in a constant temperature oven, and weighed. The Evans blue in the ear tissue was extracted in formamide (FUJIFILM Wako Pure Chemical Inc., Osaka, Japan) for 48 h and quantified spectrophotometrically at a wavelength of 610 nm. The amount of extravasated Evans blue per dried tissue weight was calculated.

Immunohistochemical detection of histamine-induced c-Fos expression

BALB/c mice were anesthetized with a medetomidine–midazolam–butorphanol cocktail. A histamine solution (300 mM), saline, or capsaicin solution (0.3 mM; positive control) was applied to the left nasal mucosa. After 45 min, mice were decapitated, and the brainstem was rapidly removed and fixed in 4% paraformaldehyde (PFA) in 0.01 M PBS (pH 7.4) for 24–48 h at 4 °C. Tissue was sectioned coronally at 50μm using a vibrating blade microtome (Neo-Linear Slicer NLS-MT, DOSAKA EM, Kyoto, Japan) and stored in cryoprotectant at −20 °C until immunohistochemistry.

Sections were processed simultaneously across all experimental groups to minimize variability. All steps were performed at room temperature on a rocker unless otherwise noted. Sections were blocked in 10% normal goat serum (NGS; Vector Laboratories, Newark, CA, USA, #S-1000) for 90 min, then incubated with guinea pig monoclonal anti-c-Fos antibody (1:2000; Synaptic Systems, Goettingen, Germany, #226308) for 60–72 h at 4 °C. After three rinses with PBS containing 0.05% Tween20 (PBST), sections were incubated with Alexa Fluor647-conjugated goat anti-guinea pig IgG (1:1000; Abcam, Cambridge, UK, #ab150187) for 2 h, followed by DAPI (1:1000; KPL, New Delhi, India, #5930-0005) for 10 min. Sections were mounted on lysine-coated slides, air-dried, and coverslipped with anti-fade medium (Vector Laboratories, #H-5000).

Sections containing the area postrema (~7.8 mm caudal to bregma, reported by Paxinos and Franklin²⁶) were analyzed, as neurons in the spinal trigeminal nucleus caudalis (Sp5C) at this level receive nasal sensory input^12,13. Images were captured using a Zeiss LSM-700 confocal microscope (Oberkochen, Germany) with 405 nm (DAPI) and 639 nm (c-Fos) lasers under a 10× objective. Scans were acquired at 1024 × 1024 pixels, with optical slices of 1–2μm and Z-stacks up to 10 slices. Target regions (340 × 340 μm²) were identified based on capsaicin-challenged controls, which showed strong c-Fos expression in the ventral Sp5C ipsilateral to stimulation. Imaging parameters and Z-stack settings were kept constant across groups to ensure comparability. Each c-Fos-staining in the target region was manually outlined using ZEN software (Carl Zeiss, Jena, Germany), and the area and brightness for each were obtained. Luminous intensity in each c-Fos-staining was calculated by area × brightness, and the sum of these values across the target region was represented as the relative expression levels of c-Fos in Sp5C for each animal.

Matcha sample extraction and treatment

Matcha green tea powder from standard matcha-grade leaves harvested in May at the Kyoto Uji area was provided by Kyoeiseicha Co., Ltd. (Osaka, Japan). Table 2 lists the nutrients contained in Matcha analyzed by the manufacturer. For in vivo treatments, Matcha extracted in 65 °C distilled water (25 mg/ml) for 120 min was administered to mice (250 mg/kg) with its residue by intragastric injection in each treatment. In the allergen-induced nasal inflammation model, mice were treated with Matcha 3 times/week at 1–2-day intervals for 5 consecutive weeks from the start of allergen immunization. The same treatment was performed 30 min before each intranasal OVA injection. In some experiments, the sneezing response triggered by intranasal injection of 10 μl histamine (300 mM in PBS) or substance P (1 mM in PBS; FUJIFILM Wako Pure Chemical Inc.) in BALB/c mice was assessed 30 min after the Matcha treatment. In PCA response experiments, mice were pretreated with Matcha 3 times/week at 1–2-day intervals for 2 consecutive weeks. The last Matcha treatment was performed 1 h before DNP-BSA administration. For immunohistochemical analysis, mice were treated with Matcha 30 min before being anesthetized. For in vitro experiments, to better reflect post-digestive availability, Matcha components were extracted following the Folin-Ciocalteu method²⁷ with modifications. Briefly, Matcha powder was suspended in EtOH, sonicated for 1 min, and centrifuged at 2500 rpm for 5 min. After recovering the supernatant, including hydrophilic and lipophilic constituents, the same extraction process was repeated three times on the remaining residue. All the supernatants were mixed and dried up. The recovery rate of the extract from the original Matcha powder was 16.4% (w/w). The extract was resolved in dimethyl sulfoxide (DMSO) at 25 mg/ml and stored at −80 °C until usage.

Table 2.

Nutrient components in Matcha^a

Component		(mg/kg)
Water		39
Proteins		324
Lipids		65
Carbohydrates		465
Sugars		153
Dietary fiber		312
Ash		77
Sodium		0.03
Caffeine		30
Phylloquinone		0.029
Vitamin C		0.67
Theanine		19
Polyphenol		94
Epicatechin		2.4
Epicatechin gallate		5.8
Epigallocatechin		11
Epigallocatechin gallate		41
Catechin		0.019
Catechin gallate		0.046
Gallocatechin		0.052
Gallocatechin gallate		0.043
Chlorophyll (total)		10
	Chlorophyll-a	6.8
	Chlorophyll-b	3.3
Carotenoids (total)		1.25
	Lutein	0.63

^aThe component analysis was performed at Japan Food Research Laboratories (Tokyo, Japan) and Food Analysis Technology Center (Mie, Japan).

High-throughput sequencing for 16S rDNA of intestinal microbiota

In the allergen-induced nasal inflammation model, total genomic DNA was extracted from fecal samples at the beginning and end of the experiments. The sequencing library was constructed using the MetaVX Library Preparation Kit (GENEWIZ, Inc., South Plainfield, NJ, USA). Briefly, 20–50 ng of DNA was used to generate amplicons covering the V3 and V4 hypervariable regions of the bacterial 16S rRNA gene. The forward and reverse primers contained the sequences “CCTACGGRRBGCASCAGKVRVGAAT” and “GGACTACNVGGGTWTCTAATCC”, respectively. Next-generation sequencing was conducted on an Illumina Miseq/Novaseq Platform (Illumina, San Diego, CA, USA). Automated cluster generation and 250/300 paired-end sequencing with dual reads were performed according to the manufacturer’s instructions. Sequence data associated with this project have been deposited in the NCBI Sequence Read Archive database under BioProject ID PRJDB37765 and will be released upon publication.

Metagenomic data analysis

Double-end sequencing reads were filtered using DADA2 software (Illumina) for denoising and chimera removal, yielding amplicon sequence variants (ASVs). The Silva 138 database served as the 16S rRNA reference. A taxonomic analysis of representative ASV sequences was performed using the Ribosomal Database Program Classifier (v2.2) with the Bayesian algorithm, determining community composition at various taxonomic levels for each sample. Upon confirming the appropriateness of the available sample size and the amount of sequencing data of ASVs by rarefaction curves, random sequence sampling was performed to ensure evenness, and the Shannon and Chao1 alpha diversity indices were calculated. Principal Coordinates Analysis (PCoA) was performed and plotted, and the Unweighted Pair Group Method with Arithmetic Mean (UPGMA) clustering method was used to cluster the samples based on Bray-Curtis distance matrices. Differential analysis of species composition between groups was performed based on differential abundance between groups. These analyses were conducted using R (v3.3.1). Linear Discriminant Analysis (LDA) Effect Size (Lefse) analysis was performed to identify differentially abundant taxa across multiple groups, and cladograms were generated to illustrate the hierarchical relationships and evolutionary context of these differences.

T cell activation

Naïve CD4⁺ T cells were isolated from DO11.10/RAG2^−/− mouse splenocytes by magnetic cell sorting with an EasySep Mouse CD4 T Cell Isolation Kit (Veritas, Santa Clara, CA, USA), as described previously⁹. The CD4⁺ T cells (10⁵/ml) with >98% purity determined by flow cytometry were cultured in the presence or absence of Matcha extract with synthetic OVA (323-339) plus 30 Gy γ-ray-irradiated splenocytes (2 × 10⁵/ml) from BALB/c mice in CTS OpTmizer T Cell Expansion SFM medium (Thermo Fisher Scientific) with recombinant interleukin (IL)-2 (10 U/mL, PeproTech, Inc., Cranbury, NJ, USA) and 10% fetal calf serum. After 4–5 days, the proliferative response was analyzed using CellTiter 96 AQueous One Solution Cell Proliferation Assay reagent (Promega, Madison, WI, USA) according to the manufacturer’s protocols. Data are presented as optical density values measured at 490 nm (OD490), from which the values of background wells were subtracted. In some experiments, the expanded CD4⁺ T cells were employed for the RNA-seq analysis.

RNA-seq analysis

The expanded CD4⁺ T cells were re-stimulated by the cross-linkage of 5 min pretreated 1 μg/ml anti-CD3 and anti-CD28 antibodies (GioGems, Inc.,Westlake Village, CA, USA) with 10 μg/ml rabbit anti-Hamster IgG (H + L) for 6 h in the presence or absence of 25 μg/ml Matcha extract. Total RNA was extracted using an RNeasy mini kit (Qiagen, Hilden, Germany). Following the assessment of the quality, including the concentration, 28S/18S, RNA integrity number, or RNA quality number through Agilent 2100 and Fragment Analyzer and Qsep-400, RNA was converted into libraries using a SureSelect Strand-Specific RNA Library Preparation kit (Agilent Technologies, Santa Clara, CA, USA). Transcriptome analysis was performed by BGI Genomics (Shenzhen, China). The data were processed using the Dr. Tom platform provided by BGI Genomics to identify differentially expressed genes. The resulting data were deposited in the NCBI Sequence Read Archive database under BioProject ID PRJDB37765 and will be released upon publication.

Statistics

Experimental data are presented as means ± standard error of the mean or box plots representing medians, quartiles, minimums, and maximums. Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA) with Student’s t-test, one-way analysis of variance with Dunnett’s multiple comparison test, whereas datasets failing these assumptions were analyzed by the Kruskal-Wallis test with Dunn’s multiple comparison test, with outliers that violated the homogeneity-of-variance assumption excluded. Statistical significance was set at p < 0.05. To evaluate the possibility of false positives, false discovery rate (FDR)-adjusted p values (q values) were calculated for RNA-seq data comparison.

Author contributions

S.O.: Conducting experiments and drafting the manuscript. N.U.: Conducted experiments. K.M.: Funding acquisition, conducting experiments, analyzing the data, data curation, and drafting the manuscript. U.E.: Funding acquisition, conducting experiments. N.Y.: Funding acquisition, conducting experiments, data curation. N.H.: Review editing. A.M.: Review editing, methodology. R.H.: Conducting experiments, analyzing the data. N.M.: Conducting experiments, analyzing the data. F.H.: Review editing. M.G.: Analyzing the data, data curation. S.N.: Funding acquisition, methodology, review editing. N.K.: Funding acquisition, methodology, review editing. M.G.: Review editing. S.S.: Methodology, conducting experiments, analyzing the data, data curation, and drafting the manuscript. O.K.: Responsible for the conceptualization and design of the research project, funding acquisition, supervision, conducting experiments, collecting and analyzing the data, validation, and review editing.

Data availability

High-throughput 16S rDNA analysis and RNA-seq analysis data have been deposited in the NCBI Sequence Read Archive database under BioProject ID PRJDB37765 and will be made publicly available upon publication. The custom R code used for the metagenomic analysis has also been deposited on GitHub (https://github.com/maribetg/Matcha-alleviates-sneezing-response). All the immunohistochemistry images in Fig. 5 are available on Figshare at 10.6084/m9.figshare.30370537. Other data generated or analyzed during this study are included in this article and its supplementary information files.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41538-026-00777-9.