This cross-sectional study included 113 patients with asthma recruited from the Department of Respiratory Medicine of Jilin University Second Hospital. Asthma was diagnosed in accordance with the 2012 GINA criteria (29). Subjects underwent fractional exhaled nitric oxide (FeNO) tests, pulmonary function tests, and sputum induction. The Asthma Quality of Life Questionnaire (AQLQ) was used to assess patient quality of life, and the 6-item Asthma Control Questionnaire (ACQ6) and Asthma Control Test Questionnaire (ACT) were used to assess asthma control. All examinations were conducted on the same day. Ethical approval was received from the Ethics Committee of the Second Hospital of Jilin University (approval number: 2016-34).

Sputum induction was carried out according to previously described procedures (30, 31). Briefly, participants underwent 15 min of sputum induction with 4.5% hypertonic saline. Dithiothreitol was used to disperse the sputum cells, and the cells were resuspended in 1× phosphate buffered saline, and the total cell count was determined. Cytospins were prepared and stained by May–Grunwald– Giemsa. Cell counts of inflammatory cells (eosinophils, neutrophils, macrophages, epithelial cells, and lymphocytes) were calculated.

According to previously published criteria, the granulocyte cutoff values were 3% for sputum eosinophils (5) and 61% for sputum neutrophils (6). The EA phenotype was defined as sputum eosinophil counts ≥3%; the NA phenotype was defined as neutrophil counts ≥61%; the MA was defined as eosinophil counts ≥3% and neutrophil counts ≥61%; and the PA phenotype was defined as eosinophil counts < 3% and neutrophil counts < 61%.

Quantitative detection of sputum TIPE2 and sputum IL-1β was performed using the commercial human TNFAIP8L2 ELISA Kit (Catalog Number EK6248-2, Signalway Antibody, USA) and Human IL-1β ELISA Kit (CSB-E08053h, CUSABIO, Wuhan, China) according to the manufacturers’ protocols. The levels of other cytokines (IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-22, IL-23, TNF-α, and interferon (IFN)-γ) were measured using the Cytometric Bead Array under the Multi-Analyte Flow Assay Kit (Biolegend, USA) following the manufacturer’s instructions. All incubation steps were performed at room temperature and protected from light. LEGENDplex v8.0 software was used to analyze the data.

The human monocyte leukemia cell line (THP-1) was obtained from the Cell Bank of the Type Culture Collection of the Chinese Academy of Sciences. The cells were cultured in RPMI Medium 1640 basic (1X) supplemented with 10% FBS and 1% penicillin/streptomycin solution (Thermo Fisher Scientific, Inc., USA) under standard conditions (37°C and 5% CO₂ in a humidified incubator). THP-1 cells were cultured with phorbol-12-myristate-13-acetate (PMA, 20 ng/ml; Cat. #M4647, AbMole BioScience) for 48 h to transform into undifferentiated (M0) macrophages. After culture without PMA for 24 h, the M0 macrophages were treated with lipopolysaccharide (LPS, 1 µg/ml; Cat. #L2880, Sigma-Aldrich) for 24 h to induce M1 macrophages.

To overexpress or silence the TIPE2 gene in THP-1, cells were transduced by recombinant lentivirus carrying a TIPE2 over-expression vector (TIPE2-OE) or small hairpin RNA (shRNA) targeting the TIPE2 gene (sh-TIPE2) for 6 h, followed by incubation under standard growth conditions. At 72 h after transfection, overexpression or depletion of TIPE2 was confirmed by qRT-PCR and western blotting.

The production and packaging of the human TIPE2 overexpression vector and the viral vector was conducted by Beijing Mijia Biotech Company. The human TIPE2 sequence was derived from the NCBI database (NM_024575.5) and the TIPE2 shRNA sequence was CCATGACGGCACTTAGCTTTG.

ML385 (32), a small molecule inhibitor of Nrf2, was purchased from TargetMol Company (Catalog Number: T4360, Shanghai, China).

TIPE2 immunofluorescent staining was performed on sputum cytospin smears from asthma patients. Expression of Nrf2 in LPS-induced M1 macrophages was examined by immunofluorescence following treatment with sh-TIPE2, TIPE2-OE or ML385. Briefly, after pretreatment, sputum single cell smears or cell slides were fixed for 30 min or overnight in 4% paraformaldehyde, followed by incubation with primary antibody overnight at 4°C. Cells were then incubated with CY3-conjugated goat Anti-Rabbit IgG (H+L) (E-IR-R321, Elabscience, Wuhan, China) for 60 min at 37°C in the dark. Nuclei were stained with DAPI. Samples were then analyzed using a fluorescence microscope (Olympus, Japan). The primary antibodies included antibodies against TIPE2 (catalog number: 15940-1-AP, 1:100, Proteintech, Wuhan, China) and Nrf2 (Cat. #ab62352, 1:500, Abcam).

Total RNA was isolated using the Cell Total RNA Isolation Kit (FOREGENE, Chengdu, China) according to the manufacturer’s protocols, and RNA was reverse transcribed using the StarScript II First-strand cDNA Synthesis Mix (GenStar, Beijing, China). RT-qPCR was carried out with the CFX96 Touch™ Real-Time qPCR Detection System (BIO-RAD, USA). Results were analyzed using the 2^−ΔΔCt method. GAPDH mRNA was used as an internal control. The primer sequences are provided in Table 1 .

Western blot was performed as previously described (33). Primary antibodies against Nrf2 (cat. #ab62352) and HO-1 (cat. #ab13248) were purchased from Abcam (Cambridge, MA, USA) and used at a dilution 1:1000. The anti-Nrf2 antibody used in our study targets Nrf2 protein with a molecular weight of ~100 kDa. Antibodies against β-actin (1:5000 dilution, Cat. #66009-1-lg, Proteintech, San Ying Biotechnology, China), Tubulin (1:20000, Cat. # 66031-1-Ig, Proteintech) and Lamin B1 (1:2000, Cat. #12987-1-AP, Proteintech) served as internal controls. Appropriate secondary antibodies (Proteintech) were used.

The nuclear and cytoplasmic protein extraction was performed using the nuclear extraction kit from BestBio (Cat. #BB-3115, Shanghai, China) according to manufacturer’s instructions.

Cell culture supernatants were collected for cytokine assay by ELISA (Elabscience, Wuhan, China), performed according to the manufacturer’s instructions. Levels of IL-6, IL-1β and TNF-α were detected.

IBM SPSS Statistics, version 25.0, was used for statistical analysis. Normally distributed data were expressed as the mean ± standard deviation (SD) and a comparison of two groups was performed using Student’s t-test. Comparison of the means among three or more groups was performed using one-way analysis of variance (ANOVA) followed by post-hoc test (least significant difference [LSD] or Tamhane’s T2). Non-normally distributed data were summarized as the median (Q1, Q3) and comparisons between subgroups were performed using Kruskal–Wallis test accompanied by Bonferroni correction or Mann–Whitney U test. For categorical variables, the data were expressed as percentages, and the chi-square test was used for analysis. Spearman correlation coefficients were used to measure the association between clinical parameters of the patients. p<0.05 was considered statistically significant.

This study included 113 patients with asthma and 22 healthy volunteers ( Table 2 ). No significant differences in patient age, sex, body mass index (BMI), percentage of smokers, and smoking index were detected between asthma patients and healthy volunteers (p > 0.05). Compared with the healthy group, the asthma patients had significantly reduced lung function (FEV1/FVC, FEV1% predicted) (p < 0.001). The sputum eosinophil percentage was higher in asthma patients compared with healthy volunteers and the sputum macrophage percentage was lower (p < 0.05).

Sputum cell counts from 102 asthma patients were available, and the patients were classified with asthma inflammatory phenotypes as follows: 48 (47.1%) patients had PA, 31 (30.4%) had EA, 12 (11.8%) had NA, and 11 (10.8%) had MA ( Table 3 ). There were no significant differences in the average age, sex, BMI, and percentage of smokers among the four groups (p > 0.05). Patients with EA had a significantly higher FeNO value and blood eosinophil count compared with NA and PA groups and a significantly lower blood neutrophil count (p < 0.05).

We detected no significant difference in sputum TIPE2 levels between asthma patients and healthy individuals (p > 0.05) ( Table 2 ). However, the sputum TIPE2 level in patients with NA was also significantly lower compared with levels in patients with PA but significantly higher in patients with EA compared with levels in patients with PA (p < 0.05) ( Figure 1A ). We next performed TIPE2 immunofluorescence analysis on sputum inflammatory cells from all asthma phenotypes ( Figure 1B ). TIPE2 expression was lower in sputum neutrophils of all phenotypes compared with sputum eosinophils. Macrophages in NA showed lower levels of TIPE2 staining compared with macrophages in PA, whereas macrophages in EA showed intense expression of TIPE2. All immune cells in NA, including neutrophils, macrophages, and eosinophils, showed lower levels of TIPE2 staining compared with cells in EA ( Figure 1B ). Therefore, sputum TIPE2 expression was down-regulated in neutrophils and NA but was up-regulated in eosinophils and EA. Interestingly, TIPE2 levels in the sputum of patients with MA were significantly higher than those in NA and PA (p < 0.05) but were not significantly different from those in EA (p > 0.05), which may be caused by the different ratio of sputum neutrophils and eosinophils. The expression pattern of TIPE2 in asthma phenotypes may depend on the heterogeneity of airway inflammation.

Sputum TIPE2 was positively correlated with sputum IL-4, IL-5, IL-13, and IL-10 (p < 0.05) ( Figures 2A–D ). Sputum TIPE2 levels were positively correlated with eosinophils and lymphocytes and negatively correlated with macrophages (p < 0.05) ( Figures 2G, I, J ). Sputum TIPE2 was negatively correlated with sputum IL-1β and TNF-α levels (p < 0.05) ( Figures 2E, F ). There was no significant correlation between sputum TIPE2 and sputum neutrophils (p > 0.05) ( Figure 2H ). Sputum TIPE2 was positively correlated with FeNO value and blood eosinophils count (p < 0.05) ( Figures 2K, L ). In addition, there was no significant correlation between sputum TIPE2 and IL-6 (r=-0.144, p=0.290). We also found that sputum TIPE2 expression was similar in patients taking different doses of inhaled corticosteroid (ICS), and there was no significant correlation between sputum TIPE2 and ICS treatment dose (p > 0.05).

To explore the role of TIPE2 in the M1 polarization of macrophages, THP-1 monocytes stimulated with PMA were used as the model of undifferentiated (M0) macrophages. M0 macrophages were then treated with LPS to induce M1 macrophages. M0 and M1 macrophages were initially identified by morphology under the optical microscope ( Figure 3 ). Further analysis by flow cytometry showed a significant increase in CD11b+ cells (7.9% vs. 92%) following PMA stimulation of THP-1 ( Figure 4 ). After LPS continued to stimulate M0, CD80+ cells increased significantly (13.2% vs. 72.9%), while the number of CD11b+ cells exhibited a non-significant change (92% vs. 91%) ( Figure 4 ). These results support the differentiation of THP-1 cells in terms of M0 and M1 macrophages.

M1 macrophages and M1 cytokines (IL-6, IL-1β, and TNF-α) are involved in neutrophilic airway inflammation in asthma (2, 3). Our results showed that TIPE2 mRNA and protein expressions were decreased in M1 macrophages compared with M0 macrophages ( Figures 5A, B ). THP-1 monocytes were then transduced with lentivirus expressing TIPE2 shRNA, and monocytes with TIPE2 shRNA exhibited decreased expression levels of TIPE2 ( Figure 5B ). The expression levels of M1 genes and cytokines in M0 macrophages were measured by RT-qPCR and ELISA following treatment with LPS. Under M1 macrophage–inducing conditions, TIPE2-silenced macrophages exhibited increased expression levels of M1 genes (CD11c and inducible nitric oxide synthase [iNOS] genes) compared with control macrophages ( Figure 5C ). The expressions of IL-1β, IL-6, and TNF-α were also significantly upregulated in TIPE2-silenced M1 macrophages ( Figure 5C ). In addition, compared with control M1 macrophages, M1 macrophages with TIPE2 gene silencing secreted significantly more IL-1β, IL-6, and TNF-α into the supernatant ( Figure 5E ).

Lentivirus was then used to overexpress TIPE2 in macrophages ( Figure 5B ). Compared with the control group, TIPE2 overexpression in macrophages inhibited M1 gene expression and inflammatory cytokine secretion ( Figures 5D, E ). TIPE2 impeded LPS-induced M1 macrophage differentiation and early inflammation.

Activation of the Nrf2/HO-1 pathway restrains the M1 polarization of macrophages and exerts anti-inflammatory effects (26). We next explored whether TIPE2 regulates Nrf2/HO-1 pathway activation. RT-qPCR analysis demonstrated that macrophages with TIPE2 overexpression showed a significant increase in the mRNA expressions of Nrf2 and HO-1 after stimulation with LPS ( Figure 6B ). Relative Nrf2 and HO-1 protein levels were also significantly increased in TIPE2-overexpressing macrophages ( Figure 6D ). In addition, TPE2 overexpression induced significantly increased protein levels of nuclear Nrf2 in M1 macrophages ( Figure 6E ). Immunocytochemistry analysis showed that M1 macrophages with TIPE2 overexpression showed more Nrf2 staining, especially nuclear Nrf2 staining ( Figure 6F ). However, silencing TIPE2 resulted in reduced expression levels of Nrf2, HO-1 and nuclear Nrf2 in LPS-induced M1 macrophages ( Figures 6A, C, E, F ). These data suggested that TIPE2 enhanced activation of the Nrf2/HO-1 pathway in M1 polarization.

ML385 is a specific Nrf2 inhibitor and inhibits activation of the Nrf2 pathway (32). To assess whether TIPE2 inhibits the M1 polarization of macrophages by targeting the Nrf2/HO-1 pathway, we treated macrophages with ML385 (5 nmol) for 48 h to block Nrf2 pathway activation. ML385 treatment decreased the up-regulated expression in HO-1 and Nrf2 mRNA induced by overexpression of TIPE2 in M1 macrophages ( Figure 7A ). ML385 treatment also reduced the increase in Nrf2, and HO-1 protein levels induced by overexpression of TIPE2 ( Figure 7C ). Moreover, M1 macrophages with ML385 treatment exhibited less Nrf2 staining compared with control M1 macrophages, but overexpressing TIPE2 weakened the decrease in Nrf2 expression induced by ML385 treatment in M1 macrophages ( Figure 7B ). Under M1 polarization conditions, TIPE2-overexpressed M1 macrophages had higher expression levels of Nrf2 and HO-1 after ML385 treatment ( Figures 5A, C ). ML385 treatment attenuated the down-regulated expression of M1 genes (CD11c and iNOS genes) induced by TIPE2 overexpression in macrophages following LPS stimulation ( Figure 7D ). Moreover, ML385 treatment significantly diminished the decrease in IL-1β, IL-6, and TNF-α mRNA levels induced by TIPE2 overexpression in M1 macrophages ( Figure 7D ). Upon treatment with ML385, TIPE2-overexpressing M1 macrophages had lower expression levels of CD11c, iNOS, IL-1β, IL-6, and TNF-α compared to control M1 macrophages ( Figure 7D ).

Together, these data indicated that blocking Nrf2 weakened the anti-inflammatory role of TIPE2 in M1 macrophages polarization. Therefore, these findings suggest that TIPE2 inhibits M1 macrophage differentiation and related inflammation by enhancing Nrf2/HO-1 pathway activation.