A total of forty patients with a diagnosis allergic asthma were enrolled in this study. The clinical study was conducted in accordance with the protocol previously published by our team [18]. Briefly, patients received acupuncture treatment every other day over a 6-week period, comprising a total of 20 treatment sessions. The selected acupoints included GV14, bilateral BL12, and bilateral BL13. Following needle insertion, standardized manual stimulation was consistently applied, consisting of twisting and lifting–thrusting manipulations for 20 seconds every 10 minutes. Each acupuncture session lasted 30 minutes, after which the needles were carefully withdrawn. In the current mechanistic study, peripheral blood samples were collected from a subset of patients assigned to the acupuncture treatment arm both before and after the intervention. Collected blood samples were centrifuged, and the resulting serum was aliquoted and stored at –80 °C until further analysis of inflammatory molecules, including CC10. Asthma control status was assessed using the validated Asthma Control Test (ACT) questionnaire, while peripheral blood leukocyte counts were measured by routine hematological analyses. This clinical study was prospectively registered (ClinicalTrials.gov identifier: NCT01931696, registered on 26 August 2013) and approved by Yueyang Hospital, affiliated with Shanghai University of Traditional Chinese Medicine (approval number: 2013–041). All participants provided written informed consent prior to study initiation.

Female C57BL/6 wild-type (Cc10^WT) mice and CC10 knockout (Cc10^-⁣/-) mice, generated using CRISPR/Cas9 gene-editing technology by the Shanghai Model Organisms Center on a C57BL/6 background, aged 6–8 weeks, were used in this study. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (Approval no. PZSHUTCM2309080009). All animal experiments were performed and reported in accordance with the ARRIVE 2.0 guidelines. Animals were maintained under specific pathogen-free conditions with a controlled 12-hour light-dark cycle, and were provided ad libitum access to standard rodent chow and water.

Mice were randomly assigned to four groups: PBS group (control mice sensitized and challenged with PBS), HDM group (asthmatic mice sensitized and challenged with HDM), HDM+Acu (asthmatic mice treated with acupuncture), and PBS+Acu (control mice treated with acupuncture). A murine model of allergic asthma was established as previously described [13]. Briefly, mice were intranasally sensitized with 10 µg HDM extract (Cat. no. 322781, Greer Laboratories, Lenoir, NC, USA) on days 0 and 7. Subsequently, from day 14 to day 18, mice underwent daily intranasal challenges with 20 µg HDM. Control animals received an equivalent volume of PBS at corresponding time points.

All acupuncture procedures were performed under inhalational anesthesia using 2% isoflurane to minimize animal stress. Beginning immediately after the sensitization phase, acupuncture was applied every other day for two consecutive weeks. Mice in the HDM+Acu and PBS+Acu groups received manual acupuncture at acupoints GV14, bilateral BL12, and bilateral BL13, localized according to murine anatomical analogs of the human World Health Organization (WHO) Standard Acupuncture Point Locations. Disposable stainless-steel needles (0.30 mm in diameter and 13 mm in length; Suzhou Medical Appliance Factory, Suzhou, China) were inserted perpendicularly to a depth of approximately 2.5–3 mm. Immediately upon insertion, needles were subjected to standardized manual manipulation, consisting of bidirectional rotations (360°, 200 rotations per minute) for 30 seconds continuously. This manipulation was repeated every 3 minutes, with a total needle retention time of 30 minutes per session. In contrast, animals in the PBS and HDM groups underwent identical anesthesia and handling without needle insertion. All acupuncture procedures were performed by the same licensed acupuncturist, who was extensively trained in this protocol and blinded to the group assignments.

AHR was assessed 24 hours after the final HDM challenge (day 20) using the FinePointe RC system (Data Sciences International, St. Paul, MN, USA). Mice were anesthetized by intraperitoneal injection of 1% pentobarbital sodium (10 mL/kg, Cat. no. P3761, Sigma-Aldrich, St. Louis, MO, USA). Following surgical tracheotomy, an 18-gauge catheter was inserted into the trachea and connected to a computer-controlled mechanical ventilator set at 140 breaths/min, a tidal volume of 0.2 mL, and a positive end-expiratory pressure (PEEP) of 2 cm H₂O.

After a 5-minute stabilization period with aerosolized PBS, animals were sequentially exposed to increasing concentrations of aerosolized methacholine (MCh; 0, 1.5, 3, 6, and 12 mg/mL; Cat. no. A6625, Sigma-Aldrich, St. Louis, MO, USA) using an integrated ultrasonic nebulizer (Data Sciences International, St. Paul, MN, USA). Airway resistance (R_L) was continuously recorded for 3 minutes following MCh administration at each concentration. R_L values, expressed as cmH₂O$\cdot$s/mL, were used as quantitative indicators of bronchoconstriction and normalized to baseline PBS responses. Dose-response curves were generated for each group, and the areas under the curves (AUC) were calculated as integrative measures of airway responsiveness.

Following AHR assessment, mice were humanely euthanized by intraperitoneal overdose of pentobarbital sodium ($\ge$120 mg/kg), and BALF was collected. A tracheal cannula was inserted, and the lungs were lavaged three times with 1 mL of sterile PBS. Collected BALF samples were centrifuged at 500 $\times$g for 10 minutes at 4 °C, and the resulting cell pellets were resuspended in 100 µL of PBS supplemented with 1% fetal bovine serum (FBS, Cat. no. 35-081-CV, Corning Inc., Corning, NY, USA) for leukocyte differential analysis. Total and differential cell counts, including macrophages, lymphocytes, neutrophils, and eosinophils, were determined using an automatic hematology analyzer (BC-5000 Vet, MINDRAY, Shenzhen, China).

Lung tissues were harvested, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at a thickness of 4 µm. To assess airway inflammation, sections were stained with hematoxylin and eosin (H&E). The following standardized semi-quantitative scoring system, based on the number of cell layers surrounding the bronchi and blood vessels was used to evaluate inflammatory cell infiltration: 0 = no inflammatory cells; 1 = few scattered cells; 2 = 1–2 cell layers; 3 = 3–4 cell layers; and 4 = more than 4 cell layers. Goblet cell metaplasia in the airway epithelia was evaluated using periodic acid-Schiff (PAS) staining. The percentage of PAS-positive goblet cells relative to the total number of epithelial cells was quantified in 5–10 randomly selected airway regions per mouse. All histological evaluations were independently performed by two experienced pathologists blinded to group allocation to minimize observer bias. Microscopic images were captured using a Nikon 80i light microscope (Nikon Corporation, Tokyo, Japan).

Lung tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) at 95 °C for 20 minutes. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide for 10 minutes, followed blocking with 5% normal goat serum to prevent non-specific binding. Sections were subsequently incubated overnight at 4 °C with a rabbit anti-CC10 primary antibody (Cat. no. ab40873, Abcam, Cambridge, UK; diluted 1:1000 in PBS supplemented with 1% BSA). After washing, sections were incubated with HRP-conjugated secondary antibody (Cat. no. sc-2004, Santa Cruz Biotechnology, Dallas, TX, USA) for 30 minutes at room temperature. Immunoreactivity was visualized using DAB substrate (Cat. no. P0203, Beyotime Biotechnology, Shanghai, China) and counterstained with hematoxylin. Images were acquired using a Nikon 80i light microscope (Nikon Corporation, Tokyo, Japan), and quantification of CC10-positive areas was performed using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA).

Following euthanasia, spleens were excised and immediately weighed. The spleen index was determined by calculating the ratio of spleen weight (mg) to body weight (g).

Serum levels of HDM-specific IgE were determined by enzyme-linked immunosorbent assay (ELISA) using a modified protocol based on a commercial kit (Cat. no. 432404, BioLegend, San Diego, CA, USA). Briefly, 96-well plates were coated overnight at 4 °C with a capture antibody, followed by blocking with assay buffer. Diluted serum samples were incubated for 2 hours at room temperature. Subsequently, biotinylated HDM extract (Cat. no. 02.01.88, CITEQ, Quebec City, QC, Canada) was applied for 1 h, followed by incubation with streptavidin-HRP conjugate. Colorimetric development was initiated using TMB substrate, and the reaction was terminated with 1 M H₂SO₄. Absorbance was measured at 450 nm using a microplate reader (Synergy H1, BioTek Instruments, Winooski, VT, USA). HDM-specific IgE levels were expressed as optical density (OD) values.

Levels of interleukin (IL)-4, IL-5, and IL-13 in BALF and mediastinal lymph node (MLN) cell culture supernatants were measured using commercial ELISA kits. Specifically, IL-4 and IL-5 levels were assessed using BioLegend kits (Cat. nos. 431101 and 431201, respectively; BioLegend, San Diego, CA, USA), while IL-13 was quantified using an Invitrogen kit (Cat. no. 88-7137-88, Thermo Fisher Scientific, Waltham, MA, USA). All assays were performed strictly in accordance with the manufacturers’ instructions.

Total RNA was extracted from lung tissues using TRIzol reagent (Cat. no. 15596026, Thermo Fisher Scientific, Waltham, MA, USA) in accordance with the manufacturer’s guidelines. Subsequently, 1 µg of total RNA was reverse-transcribed into complementary DNA (cDNA) using a reverse transcription kit (Cat. no. K1622, Thermo Fisher Scientific, Waltham, MA, USA). qRT-PCR was performed on a LightCycler 96 System (Roche, Switzerland) using SYBR Green PCR Master Mix (Cat. no. QPK-201, TOYOBO, Osaka, Japan). The relative mRNA expression of the target genes, Cc10 and Muc5ac, was normalized to the housekeeping gene Gapdh using the 2^{-Δ⁢Δ⁢Ct} method. Primer sequences are detailed in Supplementary Table 1.

Lung tissues were homogenized in RIPA lysis buffer (Cat. no. P0039, Beyotime Biotechnology, Shanghai, China) supplemented with protease and phosphatase inhibitors (Cat. no. 04693132001, Roche Diagnostics, Mannheim, Germany). Protein concentrations were quantified using a BCA assay, and equal amounts of total protein (20 µg) were separated by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked for 1 hour at room temperature with 5% non-fat milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). Membranes were incubated overnight at 4 °C with primary antibodies against CC10 (1:1000, Cat. no. ab40873, Abcam, Cambridge, UK) and $\beta$-actin (1:2000, Cat. no. 4967, Cell Signaling Technology, Danvers, MA, USA). Following extensive washing, membranes were incubated for 1 hour at room temperature with HRP-conjugated secondary antibodies. Protein bands were visualized using enhanced chemiluminescence reagents (Cat. no. A38555, Thermo Fisher Scientific, Waltham, MA, USA), and densitometric analysis was performed using ImageJ software (version 1.53, National Institutes of Health, Bethesda, MD, USA).

Lung single-cell suspensions were prepared by mechanical dissociation followed by enzymatic digestion in RPMI 1640 medium supplemented with 1 mg/mL collagenase IV and 200 U/mL DNase I at 37 °C for 45 minutes. The digested tissues were filtered through a 70 µm cell strainer, and red blood cells were removed using an RBC lysis buffer. Cells were then stained with fluorochrome-conjugated antibodies against CD45, CD11c, MHC II, CD11b, and CD103 (BioLegend and BD Biosciences). Dead cells were excluded using a fixable viability dye (Cat. no. 65-0865-18, Thermo Fisher Scientific, San Diego, CA, USA). Detailed information on all antibodies used for flow cytometry is provided in Supplementary Table 2. Data were acquired on an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA, USA), and analyzed using FlowJo software (version 10.9, BD Biosciences, Ashland, OR, USA). DCs were identified as live CD45⁺CD11c⁺MHC II⁺ cells, and DC subsets were defined based on differential expression of CD11b and CD103. Dimensionality reduction and subset visualization were performed using t-distributed stochastic neighbor embedding (t-SNE).

All data are presented as mean $\pm$ SEM. Paired Student’s t-tests were used to compare clinical parameters before and after acupuncture treatment in patients. Comparisons among multiple groups were performed using one-way or two-way ANOVA, followed by Tukey’s post hoc tests, as appropriate. Two-way repeated-measures ANOVA was applied to analyze dose–response curves. Statistical significance was set at p 0.05.

To investigate the clinical relevance of CC10 and the effects of acupuncture in patients with allergic asthma, we first measured serum CC10 concentrations, asthma symptom control, and peripheral blood leukocyte counts before and after acupuncture treatment. As shown in Fig. 1A,B, acupuncture significantly increased serum CC10 levels from 16.90 $\pm$ 0.96 ng/mL to 19.99 $\pm$ 1.13 ng/mL (p 0.01). Concurrently, asthma symptom control, assessed by ACT scores, improved markedly following acupuncture intervention (from 17.88 $\pm$ 0.67 to 22.10 $\pm$ 0.52; p 0.01; Fig. 1C). Additionally, total peripheral white blood cell counts were significantly reduced after treatment (6.71 $\pm$ 0.23 $\times$ 10⁹/L to 6.05 $\pm$ 0.22 $\times$10⁹/L; p 0.01; Fig. 1D). Collectively, these results indicate that acupuncture enhances systemic CC10 production and promotes clinical improvement in asthma control in patients with allergic asthma.

Fig. 1.

Acupuncture elevates serum CC10 levels, improves asthma control, and reduces peripheral leukocyte counts in allergic asthma patients. (A,B) Serum CC10 concentrations were significantly elevated post-acupuncture treatment (n = 40). (C) Asthma symptom control, measured by Asthma Control Test (ACT) scores, significantly improved following acupuncture. (D) Total peripheral white blood cell counts significantly decreased after acupuncture treatment. Data are expressed as mean $\pm$ SEM. Statistical analysis was conducted using paired t-tests. CC10, Clara cell 10-kDa protein; SEM , standard error of the mean.

To further elucidate the mechanisms underlying the therapeutic effects of acupuncture, we employed a murine model of HDM-induced allergic asthma (Fig. 2A). As expected, HDM sensitization and subsequent challenge significantly enhanced AHR, evidenced by increased R_L in response to increasing doses of MCh compared with PBS-treated controls (Fig. 2B,C). Notably, acupuncture treatment markedly ameliorated HDM-induced AHR, resulting in a pronounced reduction in R_L toward baseline levels.

Fig. 2.

Acupuncture reduces airway hyperresponsiveness and allergic airway inflammation in an HDM-induced asthma mouse model. (A) Experimental timeline illustrating sensitization and challenge with HDM or PBS, with concurrent acupuncture treatments at GV14, BL12, and BL13 on the indicated days. (B) Airway resistance (R_L) measured in response to increasing doses of methacholine (0–12 mg/mL), normalized to baseline values. (C) Area under the curve (AUC) analysis derived from R_L data in panel (B). (D) Representative hematoxylin and eosin (H&E) staining of lung sections. Scale bars = 100 µm. (E) Quantitative inflammation scores from H&E-stained lung sections. (F) Total leukocyte counts in BALF. (G) Eosinophil counts in BALF. (H) Serum levels of HDM-specific IgE. (I–K) Concentrations of Th2 cytokines IL-4 (I), IL-5 (J), and IL-13 (K) in BALF. Data represent mean $\pm$ SEM. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. Two-way repeated measures ANOVA (Analysis of Variance) was used for dose–response curves in panel (B). *p 0.05, **p 0.01, ***p 0.001, ns, not significant; In panel (B), ## indicates p 0.01 compared with the PBS group, and ** indicates p 0.01 compared with the HDM group. PBS, negative control group; HDM, asthma model group; HDM+Acu, acupuncture-treated asthma group; PBS+Acu, acupuncture-treated control group; BALF, Bronchoalveolar Lavage Fluid; Th2, T helper type 2; IL, interleukin.

Histopathological examination using H&E staining revealed extensive peribronchial and perivascular inflammatory cell infiltration, accompanied by pronounced airway epithelial damage in HDM-challenged mice. Acupuncture treatment markedly alleviated inflammatory cell infiltration and facilitated epithelial repair, thereby preserving airway epithelial integrity (Fig. 2D,E). Consistent with these histological findings, acupuncture significantly decreased both the elevated total leukocyte and eosinophil counts in BALF induced by HDM exposure (Fig. 2F,G). Additionally, serum HDM-specific IgE levels, a hallmark of allergic sensitization, were substantially increased following HDM challenge but were significantly reduced after acupuncture intervention (Fig. 2H).

Further analysis demonstrated that HDM exposure markedly increased the levels of Th2-associated cytokines, including IL-4, IL-5, and IL-13, in BALF. Acupuncture treatment robustly suppressed the production of these cytokines (Fig. 2I–K). In summary, these results demonstrate that acupuncture effectively attenuates airway hyperresponsiveness and suppresses allergic airway inflammation in the murine model of HDM-induced asthma.

Considering the clinically observed elevation in serum CC10 levels following acupuncture treatment in allergic asthma patients, we next investigated whether acupuncture similarly modulates pulmonary CC10 expression in the murine model of HDM-induced asthma. Immunohistochemical staining revealed a marked reduction in CC10 protein expression within airway epithelial cells of HDM-challenged mice compared with PBS-treated control mice. Importantly, acupuncture intervention effectively restored CC10 expression, approaching levels observed in control animals (Fig. 3A). Consistent with these histological findings, qRT-PCR analysis demonstrated a significant downregulation of pulmonary Cc10 mRNA expression in HDM-induced asthmatic mice, which was robustly reversed following acupuncture treatment (Fig. 3B). Moreover, Western blot analysis further confirmed a pronounced decrease in CC10 protein levels in lung homogenates from asthmatic mice, whereas acupuncture effectively normalized CC10 expression (Fig. 3C,D). These findings suggest that HDM-induced asthma suppresses CC10 expression in the lung, and that acupuncture exerts a protective effect, at least in part, by restoring CC10 production within the airway epithelium.

Fig. 3.

Acupuncture restores pulmonary CC10 expression in HDM-induced asthmatic mice. (A) Immunohistochemical staining illustrating CC10 protein expression (arrows) in airway epithelial cells. Scale bars = 50 µm. (B) Relative pulmonary Cc10 mRNA expression (n = 5 per group) quantified by quantitative Real-Time PCR (qRT-PCR). (C) Representative Western blot depicting CC10 protein expression in lung homogenates. (D) Densitometric quantification of CC10 protein normalized to $\beta$-actin from three independent experiments. Data are presented as mean $\pm$ SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post hoc test. *p 0.05, **p 0.01.

To define the indispensable role of CC10 in mediating the anti-asthmatic effects of acupuncture, we employed Cc10 knockout mice (Cc10^-⁣/-) and their wild-type littermates (Cc10^WT) in an HDM-induced allergic asthma model, with or without acupuncture intervention. As shown in Fig. 4A,B, Cc10^-⁣/- mice exhibited significantly aggravated AHR following HDM challenge, as indicated by increased R_L and elevated AUC values compared with HDM-challenged Cc10^WT mice. Although acupuncture robustly attenuated AHR in Cc10^WT mice, this protective effect was substantially blunted in Cc10^-⁣/- mice.

Fig. 4.

CC10 deficiency exacerbates asthma severity and reduces the therapeutic efficacy of acupuncture. (A) Airway resistance (R_L) response curves to escalating methacholine doses (0–12 mg/mL). (B) Corresponding area under the curve (AUC) analysis of R_L data from panel (A). (C) Representative H&E staining of lung sections illustrating airway inflammation. Scale bars = 100 µm. (D) Quantitative inflammation scores derived from H&E-stained sections. (E) Representative periodic acid-Schiff (PAS) staining of lung sections depicting goblet cell metaplasia. Scale bars = 100 µm. (F) Quantification of PAS-positive goblet cells as a percentage of total airway epithelial cells. (G) Relative expression of Muc5ac mRNA in lung tissues measured by qRT-PCR. (H) Spleen index expressed as spleen-to-body weight ratio. Data represent mean $\pm$ SEM. Statistical significance was assessed using two-way ANOVA followed by Tukey’s post hoc test. Two-way repeated measures ANOVA was used for dose–response analysis in panel (A). *p 0.05, **p 0.01, ***p 0.001. ns, not significant.

Histopathological examination further revealed that CC10 deficiency substantially intensified pulmonary inflammation (Fig. 4C,D) as well as airway goblet cell metaplasia, as demonstrated by PAS staining and subsequent quantification (Fig. 4E,F). Correspondingly, pulmonary Muc5ac mRNA expression, indicative of mucus hypersecretion, was significantly increased in asthmatic Cc10^-⁣/- mice compared with Cc10^WT asthmatic mice (Fig. 4G). Notably, the acupuncture-mediated attenuation of inflammation and mucus hypersecretion observed in Cc10^WT mice was markedly diminished or completely abolished in Cc10^-⁣/- mice. Additionally, the spleen index, reflecting systemic inflammatory burden, was significantly elevated in Cc10^-⁣/- asthmatic mice, with acupuncture’s protective effect substantially diminished (Fig. 4H). Taken together, these results underscore the indispensable role of CC10 in mediating the protective effects of acupuncture against airway hyperresponsiveness, inflammation, and mucus hypersecretion.

We next assessed the impact of CC10 deficiency on allergic airway inflammation, with particular focus on Th2 immune responses and the immunomodulatory effects of acupuncture. Analysis of BALF revealed markedly increased total leukocyte counts and eosinophil proportions in asthmatic Cc10^-⁣/- mice compared with their asthmatic Cc10^WT counterparts. Importantly, the inhibitory effect of acupuncture on inflammatory cell infiltration was substantially diminished in the absence of CC10 (Fig. 5A).

Fig. 5.

CC10 deficiency impairs acupuncture-mediated suppression of airway Th2 inflammation. (A) Total leukocyte counts and differential analysis of neutrophils, lymphocytes, eosinophils, and macrophages in BALF. (B) Serum HDM-specific IgE levels. (C) Th2 cytokines (IL-4, IL-5, IL-13) quantified in BALF. (D) Th2 cytokine levels (IL-4, IL-5, IL-13) measured in culture supernatants from mediastinal lymph node (MLN) cells stimulated ex vivo with HDM for 72 hours. Data represent mean $\pm$ SEM. Statistical analyses were performed using two-way ANOVA followed by Tukey’s post hoc test for multiple comparisons. *p 0.05, **p 0.01, ***p 0.001. ns, not significant.

In parallel, serum levels of HDM-specific IgE were significantly elevated in Cc10^-⁣/- asthmatic mice. While acupuncture treatment effectively reduced HDM-specific IgE concentrations in Cc10^WT mice, this therapeutic benefit was largely impaired in Cc10^-⁣/- mice (Fig. 5B). Furthermore, levels of the Th2-associated cytokines IL-4, IL-5, and IL-13 in BALF were profoundly increased in Cc10^-⁣/- asthmatic mice, indicative of exacerbated Th2-driven inflammation. Importantly, acupuncture-mediated suppression of these cytokines was significantly weakened or completely abolished in the absence of CC10 (Fig. 5C). Consistent with these in vivo findings, ex vivo stimulation of MLN cells with HDM extract resulted in enhanced Th2 cytokine production in cells derived from Cc10^-⁣/- mice, and acupuncture treatment exhibited limited capacity to modulate this exaggerated response (Fig. 5D). Together, these results strongly support a pivotal role of CC10 in restraining Th2-mediated inflammation and highlight its indispensability for the full immunomodulatory effects of acupuncture.

Building on our previous findings that acupuncture suppresses airway Th2 inflammation through modulation of pulmonary CD11b⁺ DCs, we further investigated whether CC10 serves as the molecular mediator of these regulatory effects. Flow cytometric analysis was performed using a refined gating strategy to identify total DC populations (CD45⁺CD11c⁺MHCII⁺ live cells) and their subsets (Fig. 6A). Although the overall frequency of pulmonary DCs remained unaffected by either CC10 deficiency or acupuncture treatment (Fig. 6B), distinct phenotypic shifts were observed within specific DC subpopulations.

Fig. 6.

Acupuncture modulates pulmonary CD11b⁺ dendritic cells via CC10. (A) Flow cytometric gating strategy identifying pulmonary DCs. Cells were gated sequentially as total lung cells, single cells, live cells, CD45⁺ cells, and further defined as CD11c⁺MHCII⁺ DCs. (B) Frequency of total pulmonary DCs (CD11c⁺MHCII⁺CD45⁺ live cells). (C) Representative flow cytometry plots showing frequencies of pulmonary CD11b⁺ DCs (CD11b⁺CD103⁻) and CD103⁺ DCs (CD11b⁻CD103⁺), gated on live CD45⁺CD11c⁺MHCII⁺ cells. (D,E) Quantification of frequencies of CD11b⁺ DCs (D) and CD103⁺ DC subsets (E). (F) t-Distributed Stochastic Neighbor Embedding (t-SNE) visualization of pulmonary DC subset distributions, illustrating distinct clusters identified by flow cytometry. Each color indicates a unique DC subset. (G) Pie charts summarizing compositional proportions of pulmonary DC subsets. Data are expressed as mean $\pm$ SEM. Statistical analyses were performed using two-way ANOVA followed by Tukey’s post hoc test. ***p 0.001. ns, not significant.

Notably, the CD11b⁺ DC subset (CD11b⁺CD103^–), a key driver of Th2-type immunity, was significantly expanded in asthmatic Cc10^-⁣/- mice compared to their Cc10^WT counterparts. Acupuncture significantly reduced the frequency of CD11b⁺ DCs in Cc10^WT mice; however, this suppressive effect was markedly diminished in CC10-deficient mice (Fig. 6C,D). In contrast, the CD103⁺ DC subset (CD11b^–CD103⁺), typically associated with Th1 immune responses, exhibited differential patterns (Fig. 6E).

Further dimensionality reduction using t-SNE analysis provided an integrated visualization of pulmonary DC subset distributions. Acupuncture substantially contracted the CD11b⁺ DC clusters (blue) in Cc10^WT mice (Fig. 6F); conversely, this subset rebalancing was notably impaired in Cc10^-⁣/- mice. This observation was further substantiated by pie chart analysis (Fig. 6G), which revealed a dominant expansion of CD11b⁺ DCs in Cc10^-⁣/- mice that remained refractory to acupuncture treatment. Overall, these results indicate that CC10 is indispensable for acupuncture-induced immunoregulation via the modulation of CD11b⁺ DC expansion, thereby providing a clear mechanistic link between acupuncture and airway immune homeostasis.