Transient Induction of Salivary SIgA by Intranasal Hinokitiol in Aged Mice

Hideki Yoshimatsu; Ryuhei Kanda; Mirai Hide; Masahiro Inoue; Hiroki Kishida; Yojiro Maeda; Daoyi Liu; Toshiro Yamamoto; Keita Kano

doi:10.20944/preprints202604.2156.v1

Submitted:

29 April 2026

Posted:

30 April 2026

You are already at the latest version

Abstract

This study aimed to determine whether intranasal hinokitiol modulates short-term sal-ivary secretory IgA (SIgA) secretion dynamics and IgA antibody-forming cell (AFC) ac-tivity in the submandibular glands of aged mice, a model of age-associated mucosal immune decline. Aged BALB/c mice received intranasal hinokitiol (50 μg) once weekly for 4 weeks. Saliva was collected on days 0, 7, 14, and 21 at baseline, 0.5 h, 1.5 h, 3 h, and 6 h after each administration. SIgA levels were measured using an enzyme-linked im-munosorbent assay. On Day 21, IgA AFCs were enumerated using an enzyme-linked immunosorbent spot assay, and their viability and proliferative activity were assessed using the MTT assay. Salivary SIgA rose transiently after each dose, peaking at 1.5 h and returning to baseline by 6 h. By Day 21, baseline SIgA secretion was significantly higher than at Day 0, indicating a cumulative effect. IgA AFCs were unchanged in number, but viability and proliferation increased at 0.5 and 1.5 h, coinciding with SIgA peaks. Flow cytometry revealed significant expansion of B220⁺CD38⁺ memory B-cells; B220⁺CD138⁺ plasma cells were unaffected. Intranasal hinokitiol transiently enhances salivary SIgA secretion in aged mice, likely through short-term modulation of salivary gland immune activity. This non-invasive approach may aid mucosal defense in aging populations.

Keywords:

hinokitiol

;

intranasal administration

;

secretory IgA (SIgA)

;

mucosal immunity

;

submandibular gland

Subject:

Biology and Life Sciences - Life Sciences

1. Introduction

The oral cavity, as the entry point to the gastrointestinal tract, is essential for food intake and swallowing. The oral mucosa, which forms a boundary with the external environment, is constantly exposed to numerous antigens and allergens and hosts approximately 400 to 700 microbial species [1]. This environment positions the oral mucosa as both a sentinel and a mediator, eliminating pathogenic threats while maintaining tolerance to dietary antigens and commensal microorganisms [2]. To sustain this balance, the oral mucosa functions through a specialized “mucosal immune system” distinct from systemic immunity that maintains immune homeostasis at this critical interface [2].

A central feature of oral mucosal immunity is the secretion of secretory immunoglobulin A (SIgA) in saliva and the presence of serum-derived immunoglobulin G (IgG) in gingival crevicular fluid [3]. The coexistence of mucosal and systemic immunoglobulins highlights the oral cavity’s dual role in immune defense. In humans, about 99% of SIgA is produced by mucosal tissues and exocrine glands [4]. These antibodies protect through neutralization and agglutination of microbes and foreign particles, playing a key role in oral immune surveillance and pathogen exclusion.

For decades, efforts have focused on developing strategies to induce antigen-specific SIgA responses in saliva, aiming to prevent common oral infections such as dental caries and periodontal disease [5,6,7]. However, these approaches have not produced clinically applicable methods, and the regulation of salivary SIgA secretion remains incompletely understood.

Hinokitiol (HNK), a natural tropolone derivative first isolated by Japanese researchers in the early 20th century, has attracted attention due to its broad-spectrum antimicrobial activity against bacteria and fungi [8,9,10]. It is now widely used as a quasi-drug ingredient in oral hygiene and personal care products such as toothpastes, mouthwashes, and hair tonics. Beyond its antimicrobial properties, we previously showed that HNK induces apoptosis and inhibits proliferation in human oral squamous cell carcinoma cells [11,12,13], underscoring its therapeutic potential.

The present study, therefore, investigated whether intranasal administration of HNK alters short-term SIgA secretion dynamics in aged mice. Rather than focusing on antigen-specific adaptive immune induction, we aimed to determine whether HNK modulates the functional activity of IgA-producing cells and salivary secretion responses. Because aging is associated with reduced SIgA output and impaired mucosal defense, understanding transient regulatory mechanisms of SIgA secretion may provide insight into novel non-invasive strategies to support oral mucosal immunity in older individuals.

2. Materials and Methods

2.1. Animals

Female BALB/c mice, aged 48 weeks, were used for immunization. The animals were housed in groups of five per cage within horizontal laminar flow cabinets under specific pathogen-free conditions at Osaka Dental University. Sterilized food and water were provided ad libitum. The experimental groups were categorized based on the timing of sample collection relative to HNK administration: immediately before administration (−0 h), and at 0.5, 1.5, 3, and 6 h post-administration. A total of five groups were established. All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Osaka Dental University and were approved by the Institutional Animal Care and Use Committee of Osaka Dental University (Approval Nos. 23-02013).

2.2. Reagent

HNK (Tokyo Chemical Industry Co., Tokyo, Japan) was dissolved in dimethyl sulfoxide (DMSO; Kishida Chemical Co., Osaka, Japan) to prepare a 100 mg/mL stock solution.

2.3. Nasal Immunization and Sample Collection Schedule

A total of 50 μg of HNK (prepared by diluting 0.5 μL of a 100 mg/mL HNK stock solution) was adjusted to a final volume of 6 μL with phosphate-buffered saline (PBS) and administered intranasally once per week for four consecutive weeks (3 μL per nostril per dose). All mice were immunized nasally under intraperitoneal anesthesia with hydrochloric acid medetomidine (0.3 mg/kg), midazolam (4 mg/kg), and butorphanol tartrate (5 mg/kg). Saliva samples were collected using procedures modified from previous studies [14]. On each administration day, saliva was collected at five timepoints: immediately before HNK administration (−0 h) and at 0.5, 1.5, 3, and 6 h post-administration (Figure 1). On day 21, after saliva collection, mice were euthanized, and their submandibular glands (SMGs) were harvested. Mononuclear cells were then isolated from the SMGs as previously described [15]. Briefly, excised SMGs were enzymatically digested with 0.5 mg/mL collagenase type IV (Sigma-Aldrich Japan, Tokyo, Japan), and the resulting cell suspension was subjected to density gradient centrifugation with Percoll (GE Healthcare Japan, Tokyo, Japan) to obtain mononuclear cells.

2.4. Measurement of SIgA and IgA Antibody-Forming Cells (AFCs)

Saliva samples were collected on days 0, 7, 14, and 21 and analyzed for SIgA concentrations using a Mouse IgA ELISA Quantitation Kit (Bethyl Laboratories, Montgomery, TX) according to the manufacturer’s instructions. Absorbance at 450 nm was measured using a microplate reader (SpectraMax M5; Molecular Devices Japan, Tokyo, Japan), and SIgA concentrations were calculated using SoftMax Pro software, version 6 (Molecular Devices Japan). On day 21, IgA AFCs in the SMGs were quantified by an enzyme-linked immunospot (ELISPOT) assay using 96-well polyvinylidene fluoride plates (Mouse IgA ELISpot Basic Kit, 3865-2A; Mabtech, Cincinnati, OH) following the manufacturer’s protocol. Spots corresponding to IgA-secreting cells were visualized using 3-amino-9-ethylcarbazole (AEC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and counted under a stereomicroscope (Stemi 305; Carl Zeiss Microscopy Co., Tokyo, Japan).

2.5. Relative Metabolic Activity of IgA AFCs

On day 21, mononuclear cells isolated from the SMGs at each time point were incubated with biotin-conjugated rat anti-mouse IgA antibody (clone 407003; BioLegend, San Diego, CA). After washing, the cells were labeled with streptavidin-conjugated magnetic beads and positively selected using the MojoSort™ Magnetic Cell Separation System (BioLegend) to isolate IgA AFCs. Purified IgA AFCs (2 × 10⁵ cells per well) were seeded into 96-well microplates (BD Biosciences, Franklin Lakes, NJ), followed by the addition of 10 μL of CytoSelect™ MTT Cell Proliferation Assay Reagent (CELL BIOLABS Inc., San Diego, CA). The plates were incubated for 4 h at 37 °C in a CO₂ incubator. After incubation, 100 μL of Detergent Solution was added to each well, and the plates were kept at room temperature in the dark for 2 h. Absorbance was measured at 540 nm using a microplate reader (SpectraMax M5; Molecular Devices Japan), and cell viability and proliferative activity were quantified using SoftMax Pro software, version 6 (Molecular Devices Japan).

2.6. Flow Cytometric Analysis for B220⁺CD38⁺ Memory B-Cell Populations

Mononuclear cells were isolated from the SMGs of mice 3 h after the final administration of HNK or vehicle control. Vehicle control solutions contained the same final concentration of DMSO diluted in PBS without HNK.

The cells were then stained with Brilliant Violet 421-conjugated anti-mouse B220 and APC-conjugated anti-mouse CD38 monoclonal antibodies (BioLegend). Flow cytometric analysis was conducted using a FACSVerse flow cytometer equipped with FlowJo software (BD Biosciences).

2.7. Statistical Analysis

The data are presented as means ± standard error from three independent experiments. The statistical significance of the differences between means was calculated by Student’s t-test using GraphPad Prism (version 7; GraphPad Software, San Diego, CA). A p-value < 0.05 was considered significant, with * p < 0.05.

3. Results

3.1. Temporal Changes in Salivary SIgA Antibody Levels Following HNK Administration

To examine the time-dependent kinetics of salivary SIgA antibody (SIgA Ab) induction and resolution following HNK administration, saliva samples were collected at −0 h (before administration), 3 h, and 6 h post-administration on days 0, 7, 14, and 21. Initially, we attempted to monitor SIgA Ab levels at additional timepoints (12 and 24 h post-administration), but no significant differences were observed between these later timepoints and the baseline (−0 h) levels on the same day (data not shown). Therefore, the analysis focused on the −0-h, 3-h, and 6-h timepoints.

As shown in Figure 2, on days 0, 7, 14, and 21, SIgA Ab levels peaked at 3 h post-HNK administration and returned to baseline levels by 6 h. Notably, on day 21, the 3 h post-administration level was significantly higher than baseline (−0 h), indicating a robust immune response after repeated HNK exposure.

A cumulative effect of repeated HNK administration was observed: salivary SIgA Ab levels at each corresponding time point (−0, 3, and 6 h) progressively increased across days 0 to 21. A significant difference was found between SIgA Ab levels at 3 h on day 0 and those at 3 h on day 21 (Figure 2), indicating enhanced mucosal immune responsiveness during the immunization schedule.

3.2. Rapid Kinetics of Salivary SIgA Antibody Secretion Following HNK Administration

Based on the results described in Figure 2, we investigated the short-term kinetics of salivary SIgA Ab secretion to determine the time required to reach peak levels after HNK administration. Specifically, SIgA Ab concentrations were measured at 0.5 and 1.5 h post-administration on days 0, 7, 14, and 21.

As shown in Figure 2, on all treatment days, the highest SIgA Ab levels occurred 1.5 h post-HNK administration, consistently exceeding those at 3 h post-administration, as shown in Figure 2. On day 21, SIgA Ab concentrations at 0.5 and 1.5 h post-administration were significantly higher than those at baseline (−0 h), indicating a rapid and robust mucosal immune response.

These results confirm that salivary SIgA Ab secretion peaked at 1.5 h post-administration and subsequently declined to baseline.

Consistent with the trends in Figure 2, SIgA Ab levels progressively increased with successive immunizations. Specifically, on day 21, SIgA Ab levels at 0.5 and 1.5 h post-HNK administration were significantly higher than the corresponding values on day 0 (Figure 2), indicating a cumulative enhancement of mucosal IgA responses with repeated HNK exposure.

3.3. Quantification of IgA AFCs in SMGs on the Final Day of HNK Administration

To assess whether HNK administration influenced the number of IgA AFCs in the SMGs, we quantified IgA AFCs at different timepoints on day 21, the final day of administration. Measurements were taken at −0 h (before administration), and at 0.5, 1.5, 3, and 6 h post-administration.

As shown in Figure 3, no significant differences were observed in the number of IgA AFCs at any time points compared with the pre-administration baseline. These findings indicate that short-term changes in salivary SIgA antibody levels after HNK exposure are not associated with changes in the number of AFCs in the SMGs.

3.4. Evaluation of the Viability and Proliferative Capacity of IgA AFCs in SMGs on the Final Day of HNK Administration

To assess the functional status of IgA AFCs, we evaluated their viability and proliferative capacity at different timepoints on day 21, the final day of HNK administration.

The viability and proliferative capacity of IgA AFCs before HNK administration (−0 h) were set as the baseline (100%), and subsequent values were compared to this reference (Figure 4). No significant changes were observed at 3 and 6 h post-administration, whereas significant increases occurred at 0.5 and 1.5 h post-administration. The highest viability and proliferative activity were noted at 0.5 h post-HNK administration.

3.5. Induction of B220⁺CD38⁺ Memory B-Cells in SMGs

The frequency of B220⁺CD38⁺ memory B-cells in the SMGs of mice that received intranasal vaccination with or without HNK was evaluated using flow cytometry. Intranasal immunization with HNK significantly increased the proportion of B220⁺CD38⁺ memory B-cells in the SMGs compared to mice immunized without HNK (Figure 5).

4. Discussion

The most notable finding of this study is the rapid and transient increase in salivary SIgA levels occurring within 0.5–1.5 hours after intranasal HNK administration. Such kinetics are inconsistent with de novo adaptive immune induction, which typically requires several days. Instead, these findings indicate that HNK primarily influences short-term regulation of SIgA secretion dynamics.

One possible explanation is the involvement of a neuro-immune reflex pathway linking nasal mucosal stimulation to parasympathetic regulation of the salivary glands. Sensory stimulation of the nasal mucosa can modulate salivary gland secretion through trigeminal pathways and the superior salivatory nucleus [27,28]. Given the rapid onset and transient nature of the response observed here, HNK may function as a local mucosal stimulant rather than an immunogenic antigen.

HNK exhibits antimicrobial activity against various bacteria and fungi, including periodontal pathogens such as Porphyromonas gingivalis [16] and opportunistic organisms such as Candida albicans [9]. However, while the antimicrobial properties of HNK are well characterized, its influence on host mucosal immune regulation remains poorly understood. Only limited studies have addressed its immunobiological effects, such as suppression of inflammatory cytokine production in macrophages and modulation of lymphocyte proliferation [18,19]. The present findings, therefore, extend previous knowledge by demonstrating that HNK can acutely modulate mucosal immune secretion without inducing classical adaptive immune activation.

Dose-response observations from preliminary experiments further support a functional rather than proliferative mechanism. A lower dose (5 µg) induced a similar kinetic pattern with reduced magnitude, whereas a higher dose (100 µg) markedly suppressed SIgA secretion and reduced both the number and viability of IgA AFCs (data not shown). These findings suggest that excessive HNK or its solvent (DMSO) may impair mucosal immune cell function, and that 50 µg represents an optimal stimulatory dose under the present conditions.

Importantly, the number of IgA AFCs in the SMGs did not change following HNK administration. This indicates that the transient elevation in SIgA secretion is unlikely to result from expansion of IgA-producing cells. Instead, the short-term increase in relative metabolic activity of IgA AFCs suggests temporary functional modulation of pre-existing cells or alterations in secretory processes within the glandular tissue [29]. The temporal association between increased AFC metabolic activity at 0.5 h and peak SIgA secretion at 1.5 h further supports this interpretation.

Although repeated HNK administration was associated with a gradual increase in baseline SIgA secretion measured before dosing, each response remained short-lived and returned to baseline within hours. Therefore, the present data do not support the classification of HNK as a classical mucosal adjuvant but rather suggest a modulatory effect on secretory function.

Flow cytometric analysis demonstrated an increased proportion of B220⁺CD38⁺ memory B cells in the SMGs following repeated HNK administration. While this suggests that HNK exposure may influence the local immune environment, the relationship between these cellular changes and the rapid SIgA secretion observed remains unclear. Further mechanistic studies are needed to determine whether neural, epithelial, or immune signaling pathways are primarily responsible for the observed effects, including possible interactions between neural reflex pathways and local immune cells [30].

These findings may have particular relevance in the context of aging. Elderly individuals commonly exhibit reduced salivary flow rates and diminished SIgA secretion, which are associated with increased susceptibility to mucosal infections and reduced quality of life [21,22]. Because the present study was conducted in aged mice, the observed responsiveness to HNK suggests that mucosal secretory mechanisms remain modifiable even in immunosenescent conditions.

Several limitations should be considered. First, the SIgA measured in this study was total SIgA and not antigen-specific. Therefore, the observed increase likely reflects a transient enhancement of nonspecific mucosal defense rather than induction of adaptive antigen-specific immunity. Such nonspecific SIgA secretion may contribute to first-line barrier protection through immune exclusion of microorganisms at mucosal surfaces. Second, neural pathways were not directly tested, and the proposed neuro-immune mechanism remains hypothetical. Third, because HNK was dissolved in DMSO, minor mucosal effects of the vehicle cannot be fully excluded despite the inclusion of vehicle controls [31]. Finally, extrapolation of these findings to other age groups or infectious conditions requires caution [32].

In conclusion, intranasal HNK administration in aged mice induces a rapid and transient increase in salivary SIgA secretion without expanding IgA-producing cell numbers. These findings point to short-term regulatory effects on salivary gland secretion rather than classical adaptive immune induction and provide a basis for further investigation into neural and local regulatory mechanisms of mucosal immunity.

5. Conclusions

In summary, intranasal HNK induces a rapid and transient increase in salivary SIgA secretion without increasing IgA AFC numbers, suggesting modulation of secretion dynamics rather than adaptive immune induction. These findings highlight a potential neuro-immune regulatory mechanism influencing salivary gland immunity in aging.

Author Contributions

Conceptualization, H.Y. and R.K.; methodology, K.K.; software, K.K.; validation, H.Y., R.K., T.Y. and K.K.; formal analysis, H.Y.; investigation, H.Y.; resources, R.K.; data curation, R.K.; writing—original draft preparation, H.Y.; writing—review and editing, M.H., M.I., H.K., Y.M. T.Y. and D.L.; visualization, H.Y.; supervision, H.Y.; project administration, R.K.; funding acquisition, H.Y. All The authors have read and agreed to the published version of the manuscript.

Funding

This project was partially supported by JSPS KAKENHI (grant number 23K16240), Japan.

Institutional Review Board Statement

All animal procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Osaka Dental University and were approved by the Institutional Animal Care and Use Committee of Osaka Dental University (Approval Nos. 23-02013; date of approval: ).

Informed Consent Statement

Not applicable.

Data Availability Statement

We encourage all authors of articles published in MDPI journals to share their research data. In this section, please provide details regarding where data supporting reported results can be found, including links to publicly archived datasets analyzed or generated during the study. Where no new data were created, or where data is unavailable due to privacy or ethical restrictions, a statement is still required. Suggested Data Availability Statements are available in the section “MDPI Research Data Policies” at https://www.mdpi.com/ethics.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:

HNK	Hinokitiol
SIgA	Secretory immunoglobulin A
AFC	Antibody-forming cell
DC	Dendritic cell
SMG	Submandibular gland
DMSO	Dimethyl sulfoxide
PBS	Phosphate-buffered saline
ELISA	Enzyme-linked immunosorbent assay
ELISPOT	Enzyme-linked immunospot
NALT	Nasal-associated lymphoid tissue

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Figure 1. Schedule of intranasal HNK administration and saliva collection. Female mice (48 weeks old) received intranasal administration of HNK (50 μg) once per week for a total of four times. On each administration day, saliva was collected at the following time points: immediately before administration (−0), and at 0.5, 1.5, 3, and 6 hours post-administration.

Figure 2. Salivary IgA antibody levels before and after intranasal HNK administration (up to 6 hours). Female mice (48-51 weeks old, n = 5) received intranasal administration of HNK (50 μg). Saliva samples were collected at three time points: immediately before administration (−0), and at at 0.5 hours (0.5) 1.5 hours (1.5) 3 hours (3) and 6 hours (6) post-administration. Salivary IgA antibody levels were measured using an ELISA.

Figure 3. Comparison of IgA antibody-forming cell (AFC) numbers in the submandibular gland (SMG) before and after the final intranasal HNK administration (Day 21). On the final day of HNK administration (Day 21), submandibular glands (SMGs) were collected from mice at the following time points: immediately before administration (−0), and at 0.5, 1.5, 3, and 6 hours post-administration. The number of IgA antibody-forming cells (AFCs) in the SMGs was quantified using the ELISPOT assay.

Figure 4. Viability and proliferative capacity of SMG IgA AFCs before and after the final intranasal HNK administration (Day 21). On the final day of HNK administration (Day 21), submandibular glands (SMGs) were harvested from mice at the following time points: immediately before administration (−0), and at 0.5, 1.5, 3, and 6 hours post-administration. IgA AFCs were isolated from SMGs using magnetic bead separation. Cell viability and proliferative activity at each time point were assessed using the MTT assay. The viability and proliferation of IgA AFCs before HNK administration were set as 100%.

Figure 5. Mononuclear cells were isolated from the submandibular glands (SMGs) of mice one week after the final administration of Hinokitiol or vehicle control. The cells were stained with BV421-conjugated anti-mouse B220 and PE-conjugated anti-mouse CD38 monoclonal antibodies. Representative flow cytometry plots show the gating strategy used to identify B220⁺CD38⁺ memory B cells. The percentages of each population are indicated. Data are representative of three independent experiments.

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Transient Induction of Salivary SIgA by Intranasal Hinokitiol in Aged Mice

Abstract

Keywords:

Subject:

1. Introduction