Modulation of lung CD11b+ dendritic cells by acupuncture alleviates Th2 airway inflammation in allergic asthma

Abstract

Background: Allergic asthma features Th2-mediated airway inflammation, with dendritic cells (DCs) playing a crucial role. Acupuncture shows promise in modulating immune responses, yet its mechanisms in allergic asthma are not well understood. This study explores how acupuncture alleviates Th2 airway inflammation by modulating lung CD11b⁺ DCs.

Methods: Female BALB/c mice were assigned to control and asthma model groups, with subsets receiving acupuncture at Dazhui (GV14), Fengmen (BL12), and Feishu (BL13). We evaluated airway hyperresponsiveness (AHR), immune cell infiltration, histological changes, Th2 cytokine production, and serum IgE levels. The effects of acupuncture serum on T cell activation and bone marrow-derived dendritic cells (BMDCs) were assessed. The role of CD11b⁺ DCs was analyzed using flow cytometry, cytokine assays, and adoptive transfer experiments. Epithelial-derived alarmins and type 2 innate lymphoid cells (ILC2s) were also examined.

Results: Acupuncture significantly reduced AHR, immune cell infiltration, goblet cell hyperplasia, and serum IgE levels in HDM-induced allergic asthma. It also suppressed Th2 cytokine production and differentiation. While acupuncture serum did not directly affect T cell activation, it modulated BMDC activity. Adoptive transfer of acupuncture-treated lung DCs into asthmatic mice reduced Th2 cell recruitment and ameliorated airway inflammation. Acupuncture reduced the population of lung CD11b⁺ DCs and downregulated the expression of activation markers such as CD86 and OX40L, along with Th2-promoting chemokines CCL17 and CCL22. Furthermore, it influenced CD11b⁺ DC migration by modulating CCR7, CCL2, and CCL8 expression. Acupuncture suppressed epithelial-derived alarmins IL-25, IL-33, and TSLP, attenuating ILC2 accumulation and activation, which indirectly affected CD11b⁺ DCs and Th2 responses.

Conclusions: Acupuncture alleviates Th2 airway inflammation in allergic asthma by modulating lung CD11b⁺ DC activities. These findings provide new insights into acupuncture-based therapeutic strategies for asthma.

Keywords: Acupuncture; Airway epithelium; Alarmin cytokines; Allergic asthma; Dendritic cells; Immunoregulation; Th2 cell; Type 2 innate lymphoid cells.

Fig. 1

Acupuncture alleviates airway inflammation and hyperresponsiveness in an HDM-induced allergic asthma model. A Schematic representation of the experimental timeline for HDM/PBS sensitization and challenge, with acupuncture treatment administered at Dazhui (GV14), Fengmen (BL12), and Feishu (BL13) on designated days. B Illustration of acupoint locations used in the study. GV14 is located in the depression below the spinous process of the C7 vertebra, BL12 is positioned in the foveola laterally between the T2 and T3 vertebrae, and BL13 is found in the foveola laterally between the T3 and T4 vertebrae. C Airway resistance measured in response to increasing doses of methacholine (normalized to baseline). D Dynamic lung compliance assessed in response to increasing methacholine doses (normalized to baseline). E Total and differential counts of inflammatory cells (lymphocytes, macrophages, eosinophils) in BALF. F ELISA analysis of serum IgE levels, including (G) HDM-specific IgE. H Representative H&E-stained lung sections depicting inflammatory changes across different groups. Scale bar = 200 μm. I Quantification of airway inflammation scores based on H&E staining. J PAS staining of lung sections to evaluate goblet cell hyperplasia. Upper scale bar = 200 μm; lower scale bar = 50 μm. K Quantification of the mean goblet cell index from PAS staining. L Representative images of immunohistochemical staining for Muc5ac in lung tissues, highlighting mucus overproduction. Scale bar = 100 μm. M Semi‑quantitative assessment of Muc5ac protein expression in lung tissues. N–O Relative mRNA expression levels of Muc5ac (N) and Muc5b (O) gene in lung tissues. NC: normal control, AS: asthma model, AA: asthma + acupuncture, NA: normal + acupuncture. Data are presented as means ± SEM; n = 5–6 mice/group; *P < 0.05, **P < 0.01 as indicated. For C and D, ##P < 0.05, ##P < 0.01 compared with NC; *P < 0.05, **P < 0.01 compared with AS

Fig. 2

Acupuncture reduces Th2 cytokine production and Th2 cell differentiation in an HDM-induced asthma model. A–C ELISA analysis of Th2 cytokines IL-4 (A), IL-5 (B), and IL-13 (C) in BALF. D Relative mRNA expression levels of Gata3, Il4, Il5, and Il13 in lung tissues. E Flow cytometry analysis of IL-4⁺CD4⁺ T cells (Th2 cells) in lung tissues. F Quantification of the percentage of Th2 cells in lung tissues. G Percentage of IL-17 A⁺CD4⁺ T cells (Th17 cells) determined by flow cytometry. H Cytokine concentrations of IL-4, IL-5, and IL-13 in the supernatants of MLN cell cultures restimulated with HDM in vitro. Data are shown as means ± SEM; n = 5–6 mice/group; *P < 0.05, **P < 0.01 as indicated

Fig. 3

Acupuncture serum does not directly affect T cells but modulates BMDC activity. A Flow cytometry analysis of T cell proliferation in response to anti-CD3/CD28 antibodies (αCD3-CD28) stimulation with cyclosporine A (CsA) or varying acupuncture-treated asthma (AA)-Serum concentrations. CFSE dilution reflects the percentage of proliferating cells. B Quantification of proliferative cells, showing that AA serum did not significantly suppress T cell proliferation compared to controls. C Flow cytometry plots illustrating CD25 and CD69 expression in treated T cells, and D quantification of CD25⁺CD69⁺ activated T cells. E, F Relative mRNA expression levels of Il4 and Gata3 in T cells under Th2-polarizing conditions, measured by qRT-PCR. G Flow cytometry histograms illustrating MHC II expression in LPS-stimulated BMDCs with normal control (NC)-Serum or AA-Serum, and H corresponding relative MFI quantification. I Flow cytometry histograms illustrating CD86 expression in LPS-stimulated BMDCs, and J corresponding relative MFI quantification. K–M Levels of IL-1β, TNF-α, and IL-6 in culture supernatants of LPS-stimulated BMDCs treated with AA-serum, assessed by ELISA (n = 5/group). N Flow cytometry histograms of dextran uptake in immature BMDCs (imBMDCs) treated with dexamethasone (Dex), NC-serum, or AA-serum, assessing endocytosis. O Quantification of relative endocytosis. Data are presented as means ± SEM, representing three experiments for flow cytometry analyses; ns, not significant; *P < 0.05, **P < 0.01 as indicated

Fig. 4

Acupuncture-treated lung DCs alleviates HDM-induced allergic airway inflammation. A Mice were sensitization with NC-DCs, AS-DCs, AA-DCs, or NA-DCs by intravenous (i.v.) injection, followed by five consecutive intranasal (i.n.) administrations of HDM for challenge. B Airway hyperresponsiveness to methacholine at incremental doses was analyzed 24 h after the final challenge. C, D Total and differential cell counts (lymphocytes, macrophages, and eosinophils) in BALF. E, F Serum HDM-specific IgE Levels (E) and Th2 cytokine concentrations (IL-4, IL-5, and IL-13) (F) in BALF were examined using ELISA assays. G Flow cytometry plots illustrating the percentage of CD4⁺IL-4⁺ Th2 cells in lung tissues, and H quantification of Th2 cells. Data are presented as means ± SEM; n = 5–6 mice/group; *P < 0.05, **P < 0.01 as indicated

Fig. 5

Acupuncture treatment reduces both the population and immune activation of lung CD11b⁺ DCs in allergic asthma. A Gating strategy for identifying total DCs from CD45⁺ live cells, along with the analysis of total DC numbers and frequencies. B Representative flow cytometry plots showing lung CD11b⁺CD103⁻ and CD11b⁻CD103⁺ DC subsets (gated on live CD45⁺CD11c⁺MHC II⁺ cells), and C quantitative analysis of CD11b⁺ and CD103⁺ DC subsets. D t-distributed stochastic neighbor embedding (t-SNE) plot illustrating the distribution of total lung DCs based on flow cytometry analysis. Cells are colored according to the four identified clusters. E Pie chart representation of DC subset composition in lungs. F Ratio of CD11b⁺ DCs to CD103⁺ DCs in the lung tissues. G Flow cytometry analysis of CD86 expression on total DCs, with representative histograms and MFI quantification. H Flow cytometry analysis of CD86 expression specifically on CD11b⁺ DCs, including representative histograms and MFI quantification. I, J Flow cytometry overlay histogram and MFI analysis of OX40L expression on total DCs (I) and CD11b⁺ DCs (J). Data are presented as means ± SEM; n = 5 mice/group; ns, not significant; *P < 0.05, **P < 0.01 as indicated

Fig. 6

Acupuncture modulates lung CD11b⁺ DC migration and suppresses pro-inflammatory chemokine and cytokine expression. A Representative histograms depicting CCR7 expression on lung CD11b⁺ DCs. B MFI quantification of CCR7 expression in lung CD11b⁺ DCs. C–F Relative mRNA levels of Ccl2, Ccl8, Ccl17, and Ccl22 in lung tissues, measured by RT-qPCR. G–H ELISA quantification of CCL17 and CCL22 in BALF. I IL-6 production by lung DCs following HDM restimulation in vitro at 6 h and 24 h. Data are presented as means ± SEM; n = 5–6 mice/group; ns, not significant; *P < 0.05, **P < 0.01 as indicated

Fig. 7

Acupuncture reduces ILC2 accumulation and suppresses epithelial-derived alarmins in the lung. A Representative flow cytometry plots identifying ILC2s in the lung, gated as Lineage⁻CD45⁺KLRG1⁺CD90.2⁺ cells. B Quantification of ILC2 percentage and absolute cell number in lung tissues. C–E Relative mRNA expression levels and protein concentrations of epithelial-derived alarmins IL-25 (C), IL-33 (D) and TSLP (E) in the lung. Data are presented as means ± SEM; n = 5 mice/group; *P < 0.05, **P < 0.01 as indicated