Specific pathogen-free female BALB/c mice were obtained from Jiangsu Qinglongshan Biotechnology Co., Ltd. All animals were group-housed under controlled conditions with a 12-hour light/dark cycle, maintained at 22 ± 2 °C with 40−60% relative humidity. All experimental procedures were approved by the Institutional Animal Care and Ethics Committee of Bengbu Medical University.

Nine-week-old female mice were randomly assigned to the following groups: control (PBS-treated), OVA, TNF-α antibody blockade (TNF-Ab), and isotype control (OVA + Iso Ab). Mice in the OVA group received intraperitoneal injections of 100 μg OVA (Sigma-Aldrich, USA) and 2 mg Al(OH)₃ adjuvant (Thermo Fisher Scientific, USA) dissolved in 200 μL PBS on days 0, 3, and 5. The control group received an equal volume of PBS only. On days 10, 12, and 14, airway challenge was performed via intranasal instillation of 50 μg OVA in the OVA group. The control group received an equal volume of PBS only. In the TNF-Ab group, a TNF-α neutralizing antibody (26405-1-AP, Proteintech, China) was administered intranasally at a dose of 300 μg/kg 12 hours before each airway challenge. The isotype control group (OVA + Iso Ab) received an equivalent amount of isotype control antibody following the same protocol. All assessments were conducted 24 hours after the final challenge. Endpoints were assessed 24 hours post-final challenge.

Lung tissues were fixed in 4% paraformaldehyde (PFA), dehydrated through an ethanol gradient, cleared in xylene, and embedded in paraffin. Sections (5 μm) were floated on a 45 °C water bath and dried. For H&E staining, sections were deparaffinized, rehydrated, stained with hematoxylin (3 min) and eosin Y (2 min), differentiated in acid ethanol, stained blue in ammonia water, dehydrated, and mounted. Samples were examined under light microscopy.

Airway resistance (RL) was measured using an invasive pulmonary function system. Mice were anesthetized with amobarbital sodium (40 mg/kg), intubated, and ventilated (150 breaths/min, 10 ml/kg tidal volume). Aerosolized acetylcholine chloride (0–30 mg/mL) (MCE, USA) was administered, and airway hyperresponsiveness was assessed by measuring changes in the respiratory system resistance (Rrs).

The left lung was rinsed, blotted, and weighed (W), dried at 65 °C for 72 hours, then reweighed (D). The wet/dry weight ratio (W/D) was calculated as W/D.

The concentrations of IL-4 (Cusabio, China), IL-5 (Cusabio, China), IL-1β (Cusabio, China), and TNF-α (Invitrogen, USA) were quantified using commercial ELISA kits according to the manufacturer’s protocols. Optical density (OD) was measured at 450 nm using a microplate reader.

Mice were humanely killed, and bronchoalveolar lavage was performed using ice-cold PBS. BALF was centrifuged, red blood cells were lysed, and cells were resuspended in RPMI-1640 with 10% FBS. AMs were purified by adherence at 37 °C for 2 hours, and Siglec-F⁺ (Invitrogen, USA)/CD11c⁺ (Invitrogen, USA) cell purity (>95%) was confirmed by flow cytometry ( Supplementary Figure S1 ).

sCD36 in BALF: BALF was centrifuged at 300 × g for 10 min to remove cells and debris. The resulting supernatant was collected and subjected to ELISA (Cusabio, China) for detection.

sCD36 in cell culture supernatant: AMs isolated from BALF were seeded in 6-well plates. After 48 h of siRNA transfection, the culture medium was collected, centrifuged at 300 × g for 5 min to remove cellular components, and analyzed by ELISA.

BALF was obtained from mice. After centrifugation, the cell precipitates were resuspended in PBS. Wright’s staining was used to produce and stain cell smears. Lastly, a 400× magnification microscope was used to count the eosinophils.

After sufficient AEC or Jurkat cells were harvested, they were centrifuged and the supernatant was discarded. The cell pellet was resuspended in PBS, and camptothecin (Beyotime, China) was added to a final concentration of 10 µM. The cells were then incubated at 37 °C for 24 h. After induction, the cells were washed with PBS and resuspended again. Pre-warmed Cell-Tracker Green CMFDA (Maokang Biotech, China) was added to 5 µM, and the suspension was incubated at 37 °C in a CO₂ incubator in the dark for 24 h. Finally, the cells were washed with PBS to obtain the apoptotic population ( Supplementary Figure S2 ).

The phagocytosis of apoptotic cells by AMs was examined by plating AMs (isolated from the BALF) into 24-well plates at a concentration of 1×10⁵ cells per well. After adherence, cells were incubated with Cell-Tracker Deep Red (Maokang Biotech, China) live cell marker at 37 °C in an atmosphere of 5% CO₂ for 40 minutes. Subsequently, 3×10⁵ apoptotic AEC/Jurkat cells labeled with Cell-Tracker Green CMFDA (Maokang Biotech, China) were added to each well and co-cultured for 6 hours. Cells were then blown into flow cytometry tubes, washed twice with PBS containing 1% FBS, centrifuged at 100 × g for 5 minutes, and fixed with 300 μL 4% PFA. The phagocytosis of apoptotic cells by AMs was then analyzed using a Cytek DxP Athena flow cytometer. Gating strategy: AMs were first gated based on FSC/SSC characteristics, followed by exclusion of doublets using FSC versus FSCW. Then, AMs were identified using a Cell-Tracker Deep Red single-stained sample to set the gate in the APC channel. The phagocytic capacity of this gated population was finally assessed in the FITC channel by measuring the percentage of cells that had ingested CMFDA-labeled apoptotic cells. Corresponding representative gating plots are provided in Supplementary Figure S3 .

To evaluate the expression of CD36, MERTK, and ADAM17 on the surface of AMs, cells were incubated with anti-CD36 (Invitrogen, USA), anti-MERTK (Invitrogen, USA), and anti-ADAM17 (Bioss, China) primary antibodies, and isotype-matched control antibodies on ice for 30 minutes. After staining, cells were fixed with 1% PFA and analyzed by flow cytometry. Data are expressed as mean fluorescence intensity (MFI).

Total RNA from AMs was extracted and converted into cDNA using a reverse transcription kit. qRT-PCR was conducted with Roche’s LightCycler 480 and a quantitative kit, following the manufacturer’s instructions. All the primers used are shown in Supplementary Table S1 .

A total of 5×10⁵ AMs were seeded in 6-well plates and cultured until they reached approximately 50% confluence. Adam17-targeted small interfering RNA (siRNA) or non-targeting control siRNA (Sangon Biotech, China) was transfected into the cells using Lipofectamine 2000 transfection reagent (Invitrogen, USA), following the manufacturer’s instructions. The medium containing low serum was replaced 6 hours post-transfection, and subsequent experiments were performed after an additional 42 hours of incubation. All the target sequences were shown in Supplementary Table S2 .

Membrane proteins were extracted using a commercial membrane protein extraction kit (Proteintech, China), determining protein concentration via a BCA assay, and separating equal protein amounts on SDS-PAGE gels before transferring them to PVDF membranes. The membranes were blocked with 5% skim milk for 2 hours at room temperature, then incubated overnight with primary antibodies at 4 °C, followed by a 1-hour incubation with HRP-labeled secondary antibodies. Protein bands were detected using the Gel Doc XR system and analyzed with Image Lab software. The antibodies used in this study are as follows: anti-ADAM17 (# bs-4236R, Bioss), Na⁺/K⁺-ATPase (#CY5159, Abways).

Data are presented as the mean ± SD and were analyzed using GraphPad Prism 9. For comparisons involving multiple groups, one-way (or two-way) analysis of variance (ANOVA) followed by Tukey’s post hoc test was used. Comparisons between two groups were performed using Student’s t-test. A P-value < 0.05 was considered to be statistically significant.

We developed an OVA-induced asthmatic mouse model to examine the phagocytic function of AMs in asthma ( Figure 1A ). we found that, compared to control mice, OVA-induced mice displayed significant airway inflammation, increased airway resistance, and increased total cell counts, eosinophil numbers, and pro-inflammatory cytokine levels (IL-1β, IL-4, and IL-5) in the BALF ( Figures 1B–I ), indicating that the murine OVA-induced model used in this study represented the type 2-high endotype. Our flow cytometry data revealed that AMs from OVA-induced mice displayed significantly impaired phagocytosis of both apoptotic AEC and apoptotic thymocytes (Jurkat cells) compared to control AMs ( Figures 1J, K ). Together, these results indicated that the phagocytic capacity of AMs was markedly reduced in OVA-induced mice.

Macrophage phagocytosis is mediated by receptors that directly or indirectly bind phosphatidylserine (PS). Thus, we assessed the expression of CD36, a direct PS-binding receptor, in AMs. Our flow cytometry data showed that CD36 surface expression levels were significantly downregulated on the AMs of OVA-induced mice compared to control mice ( Figures 2A–D ). In contrast, no significant difference in the expression of MERTK, an indirect PS-binding receptor, was observed between AMs isolated from control and OVA-induced mice. Next, we investigated the functional role of CD36 in phagocytosis by treating AMs with a CD36-blocking antibody. Blockade of CD36 led to significantly impaired phagocytosis by AMs from both control and OVA-induced mice ( Figure 2E ), confirming the essential role of CD36 in phagocytosis. Together, these findings suggested that the impaired phagocytic capacity of AMs in OVA-induced mice may be primarily attributed to reduced CD36 expression.

To investigate the reduced CD36 expression on AMs in OVA-induced mice, we measured soluble CD36 (sCD36) in BALF. The sCD36 levels were significantly higher in the OVA group than in controls ( Figure 3A ), suggesting CD36 cleavage and release into body fluids. ADAM17, a membrane-bound metalloprotease, modulates the expression of phagocytic receptors on the cell surface, thereby regulating cellular phagocytic capacity (14). Given ADAM17’s enzymatic activity in cleaving cell surface receptors, we assessed its expression on the surface of AMs. Our flow cytometry data revealed that there was a marked upregulation in the expression of ADAM17 on the surface of AMs from OVA-induced mice compared to control mice ( Figure 3B ).

To investigate the regulatory role of ADAM17 in AMs, we knocked down ADAM17 in AMs using small interfering RNA, and the knockdown efficiency is shown in Figures 3C–E . The expression of ADAM17 in AMs was reduced by approximately 70% following transfection with the specific siRNA. To further clarify the effect of ADAM17 on phagocytosis-related molecules, we examined the expression of CD36 on the surface of AMs. As shown in Figure 3F , ADAM17 knockdown significantly increased surface CD36 expression on AMs. Furthermore, the concentration of sCD36 in the culture supernatant of this group was markedly reduced compared to the control group ( Figure 3G ). The elevated sCD36 levels in BALF ( Figure 3A ) reflected the total CD36 shedding in the airway microenvironment in vivo. These experiments demonstrate that ADAM17 can mediate the proteolytic cleavage of CD36. The effects of ADAM17 on phagocytosis were next examined in AMs transfected with ADAM17-siRNA. We found that ADAM17 knockdown markedly enhanced the phagocytic capacity of AMs compared to scrambled siRNA controls ( Figure 3H ), suggesting that ADAM17 acted as a negative regulator of phagocytosis. These results indicated that ADAM17 inhibited phagocytosis by AMs through downregulation of CD36 surface expression.

Asthma, a chronic inflammatory airway disease, is associated with the release of multiple inflammatory mediators during its progression. To investigate the role of TNF-α, a known regulator of macrophage phagocytic function (15)in AMs-mediated phagocytosis, we first analyzed TNF-α levels in the BALF of different treatment groups of mice. Flow cytometry analysis revealed a significant increase in TNF-α levels in OVA-induced mice compared to wild-type controls ( Figure 4A ). Next, the impact of TNF-α on phagocytosis was determined by treating AMs from wild-type mice with TNF-α at concentrations ranging from 10 to 500 pg/ml for 24 hours. We found that TNF-α treatment markedly suppressed the phagocytic capacity of AMs, with the most significant inhibition observed at 200 pg/mL ( Figures 4B, C ). Flow cytometry analysis revealed that treatment with 200 pg/ml TNF-α significantly upregulated ADAM17 surface expression levels ( Figure 4D ), while downregulating CD36 expression levels ( Figure 4E ) and the level of sCD36 in the supernatant of AMs was upregulated ( Figure 4F ) compared to untreated controls. The effects of TNF-α on AMs-mediated phagocytosis were next examined in vivo by isolating AMs from OVA-induced mice following TNF-α blockade and quantifying their ADAM17 and CD36 surface expression levels, as well as the level of sCD36 in BALF. Compared to the isotype-matched control antibody, TNF-α blockade significantly downregulated ADAM17 and upregulated CD36 expression levels in the OVA+TNF-α Ab group ( Figures 4G, H ), along with a reduction in the level of sCD36 in BALF ( Figure 4J ). The functional consequences of TNF-α blockade were next determined by quantifying the phagocytic capacity of AMs from OVA-induced mice. Flow cytometry analysis revealed that TNF-α blockade significantly enhanced the phagocytic capacity of AMs in the OVA+TNF-α Ab group compared to the isotype-matched control antibody ( Figure 4I ). Next, we sought to elucidate the mechanism by which TNF-α regulates AMs-mediated phagocytosis by knocking down ADAM17 expression in AMs in vitro. Then, we observed the effects of TNF-α on the expression of CD36 receptors on the surface of AMs, the content of sCD36 in the cell supernatant, and the phagocytic function of AMs.The results revealed that CD36 expression levels were significantly upregulated ( Figure 4K ), the content of sCD36 decreased ( Figure 4L ), and the phagocytic capacity was markedly enhanced ( Figure 4M ) in the TNF-α+ADAM17-siRNA group compared to the TNF-α+si-NC group. These results demonstrated that knockdown of ADAM17 in AMs attenuated the TNF-α-induced downregulation of CD36 expression and suppression of phagocytic capacity. Collectively, our findings indicated that TNF-α upregulated ADAM17 to reduce CD36 surface expression, thereby impairing phagocytosis by AMs.