Balanced T helper 17/regulatory T cells in gut–lung axis improve chronic obstructive pulmonary disease: The benefits of Polygonatum sibiricum polysaccharide

To the Editor: Chronic obstructive pulmonary disease (COPD), a major global health concern, is marked by progressive airflow limitation and inflammation. While cigarette smoke (CS) is a primary cause, existing treatments remain inadequate.^[1] The gut-lung axis, representing a bidirectional communication pathway, emerges as a promising therapeutic target. Rhizoma Polygonati (Huang Jing), a traditional Chinese medicine, contains bioactive components including polysaccharides, known for multiple bioactivities.^[2] We previously isolated a neutral polysaccharide (PSP-NP) from Polygonatum sibiricum Redoute, revealing its ability to modulate intestinal barrier function and immunity.^[3] This prompts us to investigate whether PSP-NP can improve lung function via the gut-lung axis.

This study used a COPD mouse model to investigate how PSP-NP might improve lung function and its possible connections with intestinal homeostasis and function, thereby providing new evidence supporting the “gut–lung axis” theory. Seventy-five male mice were used in this study (8 weeks age, C57/BL-6J, Sipeifu Biotechnology Co., LTD., Beijing, China) and were divided into 5 groups: the control group, COPD model group, and three PSP-NP-supplied groups with high-dose (200 mg/kg), medium-dose (100 mg/kg), and low-dose (50 mg/kg). All groups, except the control group, were exposed to passive smoking for 10 weeks, along with intranasal administration of lipopolysaccharides (LPS, 2.5 mg/kg) for the initial two weeks to induce a mouse model of COPD (detailed in Supplementary Methods, http://links.lww.com/CM9/C656). The Animal Care and Use Committee of Sichuan Agricultural University reviewed and approved the study (No. SYXK2021-187). One-way analysis of variance were performed for the statistical significance analysis using GraphPad Prism software (version 6.0, San Diego, CA, USA). A P value less than 0.05 was considered statistically significant.

To investigate the potential benefits of PSP-NP on lung diseases, PSP-NP was supplied to COPD mice, which were exposed to passive smoking and received LPS nasal infusion.^[4] PSP-NP administration significantly reversed COPD-induced weight loss [Figure 1A], and blood gas analysis showed decreased PCO₂ and HCO₃^– levels in PSP-NP treated COPD mice indicated improved lung function [Figure 1B]. Besides, histopathological analysis and quantification with hematoxylin and eosin (H&E) staining showed that PSP-NP dose-dependently restored alveolar number and reduced alveolar wall thickening of COPD mice [Figure 1C, Supplementary Figure 1A, and Supplementary Table 1, http://links.lww.com/CM9/C656]. PSP-NP also increased lung content of tight junction proteins zonula occludens-1 (ZO-1) and Occludin, indicating improved epithelial barrier function [Supplementary Figure 1B and C, http://links.lww.com/CM9/C656]. Crucially, PSP-NP suppressed pro-inflammatory cytokines (interleukin [IL]-6, IL-17, IL-21, IL-23, and tumor necrosis factor alpha [TNF-α]) and elevated anti-inflammatory cytokines (IL-2, IL-10, and transforming growth factor beta [TGF-β]) in lung tissue [Supplementary Figure 1D–K, http://links.lww.com/CM9/C656]. These findings underscore the beneficial impact of PSP-NP in enhancing lung function in COPD mice.

Figure 1.

PSP-NP ameliorates chronic obstructive pulmonary disease by balancing the T helper cell 17/regulatory T cells in the gut–lung axis. (A) Quantification shows the body weight changes of the mice in the experiment. *represents the statistical difference between PH and M groups, †represents the statistical difference between PM and M groups, and ‡represents the statistical difference between PL and M groups. (B) The results of blood gas analysis by blood gas analyzer. *stands for the statistical difference compared with control group, and †stands for the statistical difference compared with model group. (C) Representative images of hematoxylin and eosin staining showing the lungs of the mice from the C, M, PL, PM, and PH groups (n = 15), respectively. *indicates alveoli, arrows indicate alveolar walls. Scar bar: 100 μm. (D and E) Western blotting and quantifications show rebalanced ratio of ROR-γt and Foxp3 in the lung (D) and colon (E) of COPD mice supplied with PSP-NP. *stands for the statistical difference compared with control group and †stands for the statistical difference compared with model group. *, †, or ‡: P <0.05; **, ††, or ‡‡: P <0.01; ***, †††, or ‡‡‡: P <0.001. C: Control group; COPD: Chronic obstructive pulmonary disease; M: Chronic obstructive pulmonary disease model group; Foxp3: Forkhead box P3; PL: Low dose of PSP-NP group; PM: Medium dose of PSP-NP group; PH: High dose of PSP-NP group; PSP-NP: Polygonatum sibiricum Polysaccharide - neutral polysaccharide; ROR-γt: Related orphan receptor γt.

We then investigated whether the improvement of pulmonary phenotypes in COPD mice by PSP-NP was related to the improvement of intestinal function. With histological analysis, we observed that PSP-NP dose-dependently repaired jejunal villi fracture/fall-off and colonic gland deficiency, lowering intestinal mucosal injury scores in COPD mice [Supplementary Figure 2A–C, http://links.lww.com/CM9/C656]. Meanwhile, PSP-NP increased intestinal levels of ZO-1 and Occludin, indicating enhanced barrier function [Supplementary Figure 2D and E, http://links.lww.com/CM9/C656]. This finding was corroborated by reduced serum LPS levels [Supplementary Figure 2F, http://links.lww.com/CM9/C656], suggesting a decrease in intestinal permeability. Periodic acid schiff and alcian blue staining showed that PSP-NP restored the number of goblet cells, which are essential for mucosal immunity, in both the jejunum and colon of COPD mice [Supplementary Figure 2G–I, http://links.lww.com/CM9/C656]. Furthermore, PSP-NP also suppressed pro-inflammatory cytokines (IL-6, IL-17, IL-21, IL-23, and TNF-α) and elevated anti-inflammatory cytokines (IL-2, IL-10, and TGF-β) in both the jejunum and colon of COPD mice [Supplementary Figure 2J–Q, http://links.lww.com/CM9/C656], suggesting a bioactive role of PSP-NP in modulating intestinal inflammation.

The observed cytokine shifts mediated by PSP-NP supplement suggested a systemic modulation of immune cell balance, specifically the pro-inflammatory T helper cell 17 (Th17) and anti-inflammatory regulatory T (Treg) cells in COPD mice. Consistent with this, Western blotting showed COPD mice had an increased retinoic acid related orphan receptor γt (ROR-γt, Th17 transcription factor)/forkhead box P3 (Foxp3, Treg transcription factor) ratio in lungs and colon, while treatment with PSP-NP significantly reduced this elevated ratio of ROR-γt/Foxp3 [Figure 1D and E]. In addition, immunohistochemistry confirmed this rebalanced ROR-γt/Foxp3 ratio (Th17/Treg cell ratio) in lungs and colon [Supplementary Figure 3A–H, http://links.lww.com/CM9/C656] of COPD mice with PSP-NP treatment. Importantly, flow cytometry demonstrated that PSP-NP normalized the increased Th17/Treg cell ratio detected in the serum of COPD mice, highlighting a systemic rebalancing of these critical immune regulators across the gut–lung axis [Supplementary Figure 4A–D, http://links.lww.com/CM9/C656].

Given the established link between gut microbiota composition, Th17/Treg cell homeostasis, and microbial metabolites,^[5] we investigated whether specific microbial changes mediated PSP-NP’s effects on Th17/Treg balance in COPD mice. With the adequate sequencing depth confirmed variable region 3 (V3) and variable region 4 (V4) of 16S ribosomal RNA sequencing [Supplementary Figure 5A, http://links.lww.com/CM9/C656], the principal component analysis (PCA) revealed distinct, group-specific clustering [Supplementary Figure 5B, http://links.lww.com/CM9/C656], consistent with significantly reduced microbial richness (Shannon index) and diversity (Simpson index) in COPD mice vs. controls—effects reversed by PSP-NP [Supplementary Figure 5C and D, http://links.lww.com/CM9/C656]. Taxonomic evaluation further detailed these shifts. At the phylum level, COPD mice exhibited a high relative abundance of Firmicutes, Bacteroidetes, and Actinobacteriota, whereas controls and PSP-NP-supplied mice were dominated by Firmicutes, Bacteroidetes, and Verrucomicrobia [Supplementary Figure 5E, http://links.lww.com/CM9/C656]. Class-level analysis showed enrichment of Bacilli and depletion of Bacteroidia in COPD mice, alongside reduced Verrucomicrobiae—all significantly improved by PSP-NP [Supplementary Figure 5F, http://links.lww.com/CM9/C656]. Genus-level profiling identified Lactobacillus and Muribaculaceae as most abundant across groups, followed by Akkermansia in controls, Faecalibaculum in COPD mice, and Dubosiella in PSP-NP-treated mice [Supplementary Figure 5G, http://links.lww.com/CM9/C656]. After further assessing structural differences, partial least squares discrimination analysis (PLS-DA) and hierarchical clustering confirmed distinct compositions per group, though PSP-NP mice clustered closer to controls than COPD mice [Supplementary Figure 6A and B, http://links.lww.com/CM9/C656]. Besides, Venn diagrams confirmed group-unique operational taxonomic units (OTUs) [Supplementary Figure 6C, http://links.lww.com/CM9/C656]. Consequently, linear discriminant analysis Effect size (LEfSe) analysis identified Firmicutes/Lactobacillaceae/Bifidobacteriaceae as COPD-enriched families or genera, vs. Muribaculaceae/Dubosiella/Paludicola enriched by PSP-NP [Supplementary Figure 6D, http://links.lww.com/CM9/C656], demonstrating PSP-NP’s restructuring of gut microbiota in COPD mice.

The microbial changes are always functionally linked to metabolite production. In this work, PSP-NP effectively restored the levels of key gut microbiota-derived short-chain fatty acids (SCFAs; acetic, propionic, butyric, isobutyric, and isovaleric acid), which were significantly reduced in COPD mice [Supplementary Figure 7A–F, http://links.lww.com/CM9/C656]. Critically, correlation analysis revealed negative associations between specific SCFA levels (acetic, propionic, isobutyric, and isovaleric acid) and Th17 cell numbers or the Th17/Treg ratio in colon, liver, and serum [Supplementary Figure 8A, http://links.lww.com/CM9/C656]. Furthermore, the abundance of PSP-NP-enriched genera (Muribaculaceae, Dubosiella, and Paludicola) positively correlated with SCFA levels, whereas COPD-enriched taxa (Firmicutes, Lactobacillaceae, and Bifidobacteriaceae) showed negative correlations [Supplementary Figure 8B, http://links.lww.com/CM9/C656], suggesting SCFAs as a key mechanistic link between PSP-NP-modulated microbiota and immune rebalancing in COPD mice.

To directly test the causal role of the altered microbiota in mediating PSP-NP’s effects, we then performed fecal microbiota transplant (FMT) using microbiota from high-dose PSP-NP-treated COPD mice. Recipient COPD mice exhibited significant improvements mirroring direct PSP-NP treatment: lung histopathology (increased alveolar number), jejunal and colonic histopathology (reduced mucosal injury scores), and enhanced expression of ZO-1/Occludin in lung, jejunum, and colon [Supplementary Figure 9A–I, http://links.lww.com/CM9/C656]. FMT also successfully lowered serum LPS levels and rebalanced the profile of pro- and anti-inflammatory cytokines across lung and gut tissues [Supplementary Figure 9J–Q, http://links.lww.com/CM9/C656]. This compelling evidence confirmed that the beneficial effects of PSP-NP on COPD pathophysiology are primarily mediated through its modulation of gut microbiota and their functional metabolites, and provided evidence revealing how PSP-NP bioactivity is linked to the gut–lung axis.

In this study, PSP-NP treatment increased beneficial gut taxa such as SCFA-producing Muribaculaceae, Dubosiella, and Paludicola in COPD mice. This microbial shift restored SCFA levels, which correlated with rebalanced Th17/Treg responses, reducing systemic and lung inflammation and ameliorating COPD. FMT experiments confirmed that PSP-NP acts through the gut-lung axis by restoring microbiota homeostasis, enhancing SCFAs, and rebalancing Th17/Treg immunity, thereby improving lung and gut barrier integrity. These findings highlight the gut-lung axis as a viable COPD therapeutic target and support PSP-NP as a promising treatment strategy.

Funding

This study was supported by grants from the National Natural Science Foundation of China (No. 82004041), the Key Project of Sichuan Provincial Department of Science and Technology (No. 19ZDYF0734), and the Sichuan Veterinary Medicine and Drug Innovation Group of the China Agricultural Research System (No. SCCXTD-2024-18).

Conflicts of interest

None.

Supplementary Material

cm9-139-472-s001.pdf ^{(5.9MB, pdf)}