Aging compromises oral mucosal immunity and is increasingly discussed within the clinical concept of oral frailty, a transitional state between healthy oral function and overt functional decline that is associated with adverse health outcomes. Oral frailty is operationalized using multi-item criteria that include not only tooth loss and impaired mastication/swallowing but also subjective dry mouth, making salivary dysfunction a clinically relevant component of the syndrome (Tanaka et al., 2018, Tanaka et al., 2024). The salivary glands and saliva play central roles in oral host defense: antimicrobial peptides, such as β-defensins (DEFB1/DEFB2) and secretory immunoglobulin A (IgA), contribute to epithelial integrity and microbiota regulation. Although immunosenescence and inflammaging are known to influence mucosal immunity, organ-specific effects within the oral cavity—and their relationship to measurable salivary biomarkers—remain incompletely characterized (Mittelbrunn and Kroemer, 2021, Palmer et al., 2018).

Immune-regulatory receptors, including programmed cell death 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and lymphocyte-activation gene 3 (LAG-3), modulate activation thresholds and functional persistence of lymphocytes and are increasingly viewed as part of age-associated immune remodeling, in which chronic low-grade inflammation and altered antigenic experience reshape mucosal immune balance (Darvin et al., 2018, Li et al., 2019, Mittelbrunn and Kroemer, 2021, Qin et al., 2019). In the present study, we quantified checkpoint-related transcripts in whole salivary gland tissue as exploratory tissue-level immune-regulatory markers in the context of aging. This approach is intended to capture integrated tissue-level immune-regulatory status without implying cell type-specific localization. Salivary glands comprise multiple epithelial and immune cell populations; single-cell transcriptomic atlases highlight marked cellular heterogeneity, underscoring that bulk-tissue measurements reflect integrated tissue-level signals rather than cell type-specific localization (Hauser et al., 2020).

Dietary polyphenols can modulate epithelial host-defense programs, providing a plausible strategy for reinforcing oral barrier function in aging. In gingival epithelial cells, green tea extract and its major catechin EGCG stimulate human β-defensin secretion and protect defensins from proteolytic degradation by periodontal pathogens, supporting a direct mechanistic link between catechins and epithelial antimicrobial defense (Lombardo Bedran et al., 2014). Green tea catechins also enhance gingival epithelial barrier integrity and protect against pathogen-driven disruption, consistent with a broader barrier-supporting effect in the oral mucosa (Lagha et al., 2018). In infection-related contexts, EGCG can induce defensin responses (e.g., DEFB3) via MAPK signaling, further supporting its capacity to modulate antimicrobial peptide pathways (Mou et al., 2020). EGCG has also been reported to counteract drug-associated suppression of epithelial defensin programs in epithelial models, supporting the plausibility of restoring host-defense markers under mucosal stress (Takahashi et al., 2014). Building on this rationale and our previous research on oral biomarkers, we demonstrated that salivary IgA levels reflect intestinal IgA responses during mucosal injury induced by an oral anticancer agent (afatinib), supporting saliva as a practical proxy matrix for monitoring mucosal immune changes (Uemura et al., 2025). We also reported that dietary modulation can enhance oral immune markers in vivo, supporting the feasibility of targeting oral defenses via nutritional interventions (Uemura et al., 2024b).

We hypothesized that aging leads to measurable impairment of salivary gland-associated oral defense markers, reflected by alterations in salivary flow, protein output, Defb1/Defb2 expression, and salivary IgA, and that EGCG ameliorates these changes (Liu et al., 2021, Mittelbrunn and Kroemer, 2021, Palmer et al., 2018). To test this hypothesis, we compared young and aged mice and evaluated whether oral EGCG administration modulates (i) salivary secretion and protein output, (ii) Defb1/Defb2 expression in salivary tissues, and (iii) IgA-related markers. In addition, we quantified checkpoint-related transcripts (Pd-1, Ctla-4, Lag-3) in whole salivary gland tissue as exploratory tissue-level immune-regulatory markers in aging, without attributing changes to specific cell populations (Yun et al., 2010).