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The study aims to discover the immunological basis of the constitution of Tanshi (Flegm-Dampness)-a type of sub-series in traditional Chinese medicine (TCM)-using unique RNA sequencing (SCRNA-SEQ) of mononuclear cells of peripheral blood (PBMC). TCM classifies individuals in nine constitutions, with tanshi linked to metabolic disorders such as diabetes and characterized by symptoms such as heaviness, abdominal glow and oily skin. By analyzing the PBMCs of Tanshi and non-taunsthi individuals, research aims to fill TCM constitutional theory with modern molecular immunology, providing a scientific basis for the concept of TCM preventive medicine.

Thirteen adult volunteers have been recruited, including four with the constitution of Tanshi (scores ≥40 via an investigation at 8 questions) and nine non-taunsthi witnesses. The PBMCs were isolated from their blood and the SCRNA-SEQ was carried out using the 10x genomic platform, generating high-resolution transcriptomic data for 51,196 cells. Bioinformatics analyzes, including cell grouping via UMAP, have identified 15 subtypes of immune cells, such as B cells, T cells, monocytes and dendritic cells. Comparative analysis has revealed significant differences in the composition of immune cells between groups.

The main results include a significant reduction in T cells (MASit) associated with mucous membranes in Tanshi individuals, confirmed by sorting cells activated by fluorescence (facs) in an independent validation cohort of 10 additional participants. Masit cells, essential for innate immunity, have been quantified using multicolored flow cytometry targeting CD3, TCR Vα7.2 and CD161 markers. At the same time, the Tanshi subjects had an increased proportion of conventional CD14 + monocytes and a trend towards a decrease in non -conventional CD16 + monocytes, with facs showing a significant reduction in the CD16 + cellular ratio compared to CD14 + cells.

Transcriptomical analyzes have identified differently expressed genes (DEG) associated with increased inflammatory paths in Tanshi PBMCs. Genetic ontology (GO) has revealed a positive regulation of TNFα-NFκB signaling, components of the Jak-Stat route (for example, IL-1β, SOCS3) and interferon responses (for example, IRF1, IFI44L). GSEA has also highlighted the activation of “Hallmark_TNFA_SIGNALING via NFKB” and “Interferon response” Set of genes in conventional monocytes and Masit cells. In addition, genes related to hypoxia and apoptosis have been enriched, potentially linked to sleep apnea – a common comorbidity in the constitution of Tanshi which induces intermittent hypoxia.

The study validates the constitutional theory of TCM by molecular evidence, demonstrating that Tanshi individuals have a chronic inflammatory state of low grade and an immune deregulation. The reduction of Masit cells, previously associated with the severe results of COVID-19, is aligned with clinical observations of the potential sensitivity of Tanshi subjects to infectious diseases. The increase in conventional monocytes can contribute to metabolic inflammation, supporting the association between the constitution of Tanshi and metabolic syndrome.

The high resolution of SCRNA-SEQ profile allows the characterization of the specific changes to the cellular type, overcoming the limits of the lock of BRRA RNA by resolving heterogeneity in the PBMC populations. Facs validation ensures the reliability of the results of the SCRNA-SEQ, in particular for the quantification of Masit cells, which requires trigger strategies in several stages to exclude contamination of T γδ cells. The integration of TCM diagnosis with modern OMS technologies establishes a framework to translate traditional concepts into exploitable biomedical information.

Beyond the constitution of Tanshi, this approach is promising to study other constitutions of the MTC and monitor the therapeutic responses to TCM interventions. For example, the SCRNA-SEQ of PBMCs before and after plant-based treatment could elucidate how TCM module the immune ways. In addition, the combination of SCRNA-SEQ with metabolomics, microbiomic and neurophysiological data can disentangle the holistic mechanisms underlying the regulating theories of TCM of TCM of TCM, such as the interaction between the autonomic nervous system and the immunity highlighted in research on acupuncture.

In summary, this study provides a molecular snapshot of the immunological landscape of the constitution of Tanshi, connecting the preventive philosophy of TCM to the concrete biological paths. By demonstrating the usefulness of SCRNA-SEQ in the decoding of TCM constitutional theory, research opens up new paths for personalized medicine, where immune profiling could shed light on early intervention strategies for health risks specific to the Constitution. As such, it fills ancient wisdom with contemporary science, offering a basic base to advance TCM to the era of precision medicine.