Study provides a molecular snapshot of Tanshi constitution's immunological landscape

The study aims to uncover the immunological basis of Tanshi (phlegm-dampness) constitution—a subhealth type in traditional Chinese medicine (TCM)—using single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs). TCM categorizes individuals into nine constitutions, with Tanshi linked to metabolic disorders like diabetes and characterized by symptoms such as heaviness, abdominal flabbiness, and oily skin. By analyzing PBMCs from Tanshi and non-Tanshi individuals, the research seeks to bridge TCM constitutional theory with modern molecular immunology, providing a scientific foundation for TCM's preventive medicine concept.

Thirteen adult volunteers were recruited, including four with Tanshi constitution (scored ≥40 via an 8-questionnaire survey) and nine non-Tanshi controls. PBMCs were isolated from their blood, and scRNA-seq was performed using the 10x Genomics platform, generating high-resolution transcriptomic data for 51,196 cells. Bioinformatics analyses, including cell clustering via UMAP, identified 15 immune cell subtypes, such as B cells, T cells, monocytes, and dendritic cells. Comparative analysis revealed significant differences in immune cell composition between groups.

Key findings include a notable reduction in mucosal-associated invariant T (MAIT) cells in Tanshi individuals, confirmed by fluorescence-activated cell sorting (FACS) in an independent validation cohort of 10 additional participants. MAIT cells, critical for innate immunity, were quantified using multi-color flow cytometry targeting CD3, TCR Vα7.2, and CD161 markers. Concurrently, Tanshi subjects exhibited an increased proportion of classical CD14+ monocytes and a trend toward decreased non-classical CD16+ monocytes, with FACS showing a significant reduction in CD16+ cell ratio relative to CD14+ cells.

Transcriptomic analyses identified differentially expressed genes (DEGs) associated with heightened inflammatory pathways in Tanshi PBMCs. Gene ontology (GO) enrichment revealed upregulation of TNFα-NFκB signaling, JAK-STAT pathway components (e.g., IL-1β, SOCS3), and interferon responses (e.g., IRF1, IFI44L). GSEA further highlighted activation of "HALLMARK_TNFA_SIGNALING VIA NFKB" and "INTERFERON RESPONSE" gene sets in classical monocytes and MAIT cells. Additionally, genes related to hypoxia and apoptosis were enriched, potentially linked to sleep apnea—a common comorbidity in Tanshi constitution that induces intermittent hypoxia.

The study validates TCM's constitutional theory through molecular evidence, demonstrating that Tanshi individuals exhibit a chronic low-grade inflammatory state and immune dysregulation. The reduction in MAIT cells, previously associated with severe COVID-19 outcomes, aligns with clinical observations of Tanshi subjects' potential susceptibility to infectious diseases. The increase in classical monocytes may contribute to metabolic inflammation, supporting the association between Tanshi constitution and metabolic syndrome.

ScRNA-seq's high-resolution profiling enables the characterization of cell-type-specific changes, overcoming limitations of bulk RNA sequencing by resolving heterogeneity in PBMC populations. FACS validation ensures the reliability of scRNA-seq findings, particularly for MAIT cell quantification, which requires multi-step gating strategies to exclude γδ T cell contamination. The integration of TCM diagnosis with modern omics technologies establishes a framework for translating traditional concepts into actionable biomedical insights.

Beyond Tanshi constitution, this approach holds promise for studying other TCM constitutions and monitoring therapeutic responses to TCM interventions. For instance, scRNA-seq of PBMCs before and after herbal treatment could elucidate how TCM modulates immune pathways. Moreover, combining scRNA-seq with metabolomics, microbiomics, and neurophysiological data may unravel the holistic mechanisms underlying TCM's "mind-body" regulatory theories, such as the interaction between the autonomic nervous system and immunity highlighted in acupuncture research.

In summary, this study provides a molecular snapshot of Tanshi constitution's immunological landscape, linking TCM's preventive philosophy to concrete biological pathways. By demonstrating scRNA-seq's utility in decoding TCM constitutional theory, the research opens new avenues for personalized medicine, where immune profiling could inform early intervention strategies for constitution-specific health risks. As such, it bridges ancient wisdom with contemporary science, offering a data-driven foundation for advancing TCM in the precision medicine era.