Myeloid-specific Notch1 knockout mice (C57BL/6J background) were generated by targeted gene deletion, and the knockout mice (Notch1^cre+) and wild-type control mice (Notch1^flox+) were produced by Cyagen Biotechnology (Suzhou, China) and referred to as Cre and Flox mice respectively. Male mice (4–6 weeks old, 14–18 g) were selected for the experiments. The experimental mice were maintained under specific pathogen-free (SPF) conditions, with temperature regulated at 22 $\pm$ 1 °C and relative humidity maintained at 60%. All animal care and experimental protocols were approved by the Animal Ethics Committee of Wuhan University (Wuhan, China, Project No. WP20240088).

The ALI models were established following previously described methods [13]. Notch1^flox mice were divided into three experimental groups (n = 5 per group): the control group (saline), the lipopolysaccharide (LPS) group (LPS), and the LPS+Mvc group (LPS + Maraviroc). Specifically, mice were anesthetized with sodium pentobarbital (1%, 70 mg/kg, i.p.) and then administered LPS via tracheal injection. Notch1^cre+ mice were randomly assigned to two groups (n = 5 per group): the saline control group and the LPS group. For the ALI model, mice were given 3 mg/kg LPS (S1732, Beyotime, Shanghai, China) via intratracheal injection. A dose of 10 µg/g Maraviroc (CCR5 antagonist, HY-13004, MedChemExpress, Monmouth County, NJ, USA) was administered 30 minutes prior to LPS induction. After 24 hours, all mice were humanely euthanized by CO₂ asphyxiation (20% chamber volume per minute) to minimize suffering, followed by cervical dislocation as a secondary confirmatory method. Peripheral blood was collected via retro-orbital venous plexus puncture. The thoracic cavity was rapidly accessed through a midline sternotomy, and bilateral lung tissues were resected en bloc. The harvested specimens were used for subsequent histopathological evaluation, real-time quantitative reverse transcription PCR (qRT-PCR), and western blotting analysis.

Post-fixation, lung tissue specimens underwent sequential ethanol dehydration followed by paraffin embedding to generate histological sections. After the Hematoxylin and eosin (G1120, Solarbio, Beijing, China) staining of lung tissue sections, the slides were observed under an optical microscope (Leica, Wetzlar, Germany) for histopathological examination and quantitative lung injury scoring [14]. For each tissue slide, five different areas were selected and examined at high magnification (200$\times$).

Fresh lung tissue was carefully blotted with absorbent paper to remove surface moisture and blood, and its wet weight was recorded. Then, the samples were incubated in a 60 °C constant-temperature incubator for 72 hours. Following complete desiccation, the tissue mass was recorded. The gravimetric ratio was determined to assess pulmonary edema severity.

500 µL of chilled phosphate-buffered saline (PBS) was injected through intratracheal instillation and gently aspirated to obtain BALF. This lavage procedure was iterated three times to maximize fluid recovery. The pooled BALF was subjected to centrifugation and the supernatant was aliquoted for protein quantification. The remaining cellular fraction was washed twice with PBS before resuspension for quantitative analysis.

Immortalized bone marrow-derived macrophages (iBMDMs) were purchased from Cyagen Bioscience Inc., Guangzhou, China. We validated all cell lines by Short Tandem Repeat (STR) profiling. All cells were confirmed to be mycoplasma-free by testing with a PCR-based mycoplasma detection kit (C0301S, Beyotime, Shanghai, China), ensuring the absence of contamination. The iBMDM cell line was maintained in high-glucose DMEM containing 10% FBS (Gibco, Carlsbad, CA, USA) under standard culture conditions (37 °C, 5% CO₂). For experimental treatments, cells were pre-incubated with MK-0752 ($\gamma$-secretase inhibitor, HY-10974; MedChemexpress, Monmouth County, NJ, USA) for 24 hours prior to LPS (1 µg/mL) challenge.

After protein extraction, samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for molecular separation. Subsequently, the resolved proteins were electrophoretically transferred onto polyvinylidene fluoride (PVDF) membranes, which were then incubated with 5% non-fat dry milk for 60 minutes at ambient temperature to prevent non-specific binding. Primary antibodies were applied according to the experimental requirements and incubated overnight at 4 °C: CCR5 (BS77962, 1:1000, Bioworld Biotechnology, Nanjing, China), NICD detection using Cleaved Notch1 (V1744) antibody (4147S, 1:1000, Cell Signaling Technology, Danvers, MA, USA), GAPDH (GB15004, 1:6000, Servicebio Biotech, Wuhan, China). After washing with Tris-Buffered Saline with Tween 20 (TBST), the membrane was incubated with the secondary antibody, Goat Anti-Rabbit IgG (H+L) HRP (A0208, 1:1000, Beyotime, Shanghai, China) at room temperature for 1 hour, followed by washing with TBST. Protein bands were visualized using Enhanced Chemiluminescence (ECL) (A10016, Abmart, Shanghai, China) detection. The quantification of blots was performed by using ImageJ software (version 1.8.0, NIH, Bethesda, MD, USA).

Total RNA isolation from pulmonary tissues and cultured cells was carried out with Trizol reagent (15596026, Thermo Fisher Scientific, Waltham, MA, USA). RNA integrity and concentration were assessed spectrophotometrically using a Nanodrop One instrument (Thermo Fisher Scientific). Subsequently, cDNA was synthesized by reverse transcription. Quantitative real-time PCR analysis was conducted with 2$\times$Universal Blue SYBR Green qPCR Master Mix (G3326, Servicebio Biotech, Wuhan, China) in triplicate technical replicates per sample. Relative mRNA expression levels were determined through the comparative $\mathrm{\Delta }$$\mathrm{\Delta }$Ct method. Primer sequences are listed in Table 1.

Fresh lung tissue was prepared for flow cytometry analysis. 1$\times$ red blood cell lysis buffer was added into the cell suspension, after a standing time of 3 minutes, the mixture was then centrifuged. The cells were then resuspended in 100 µL PBS and blocked with 2 µL of anti-mouse CD16/CD32 (65080-1-Ig, 0.5 mg/mL, Proteintech Group, Wuhan, China) antibody at 4 °C for 10 minutes. The cells were stained with fluorescently conjugated antibodies: APC-A750 anti-mouse CD45 antibody (E-AB-F1136, 5 µL/mL, Elabscience, Wuhan, China) and PE-A anti-mouse CD64 antibody (E-AB-F1186, 5 µL/mL, Elabscience, Wuhan, China) for 60 minutes in the dark at 4 °C. After washing, the samples were analyzed by flow cytometer (Beckman Coulter, Brea, CA, USA).

Enzyme Following the manufacturer’s protocols, the ELISA kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to measure the concentrations of interleukin (IL)-6 (H007-1), IL-1$\beta$ (H002-1), and tumour necrosis factor-alpha (TNF-$\alpha$) (H052-1) in mouse serum.

Lung tissue sections embedded in paraffin were subjected to deparaffinization and rehydration processes. Antigen retrieval was achieved through microwave-mediated heating. Primary antibody incubation was performed overnight at 4 °C using anti-CCR5 (1:50 dilution, BS77962, Bioworld Biotechnology, Nanjing, China) and anti-F4/80 (1:200 dilution, ab6640; Abcam, Cambridge, UK). Following three washes with PBS, samples were exposed to species-specific secondary antibodies: AF488-labeled Goat Anti-Rabbit IgG (H+L) (1:300 dilution, A0423, Beyotime, Shanghai, China), Cy3-labeled Goat Anti-Rat IgG (H+L) (1:300 dilution, A0507, Beyotime, Shanghai, China) for 1 hour at 37 °C under light-protected conditions. Nuclei were counterstained with 4^′,6-diamidino-2-phenylindole (DAPI, C1005, Beyotime, Shanghai, China) for 10 minutes. Fluorescence imaging was conducted using an inverted fluorescence microscope (Olympus, Tokyo, Japan).

iBMDM cells were cultured in a 37 °C incubator until reaching 80% confluence. A sterile pipette tip was used to create a wound in the cell, and the cells were treated with 100 nM MK-0752 either in the presence or absence of LPS. The relative change in the scratch area over 12 hours was measured using ImageJ software to assess the effect of MK-0752 on LPS-induced cell migration. Images were captured at 0 hours, 6 hours, and 12 hours. The extent of cell migration was represented as the relative change in the scratch area compared to the 0-hour time point.

iBMDM cells were seeded into the Transwell system and were divided into four groups: Control, LPS (1 µg/mL), LPS+CCL5 (10 ng/mL, P6780, Beyotime, Shanghai, China), and LPS+CCL5+MK-0752 (100 nM). A complete culture medium (600 µL) was added to the lower chamber, and 200 µL serum-free cell suspension with or without treatment was added to the upper chamber. LPS and MK-0752 were added to the upper chamber, while CCL5 was added to the lower chamber. After incubation at 37 ℃ for 12 hours, the unmigrated cells in the upper chamber were removed. The rest was stained with 0.1% crystal violet (C0121, Beyotime, Shanghai, China) for 20 minutes.

All data were analyzed and processed using GraphPad Prism (version 10.0, GraphPad Software, LLC, San Diego, CA, USA) and were presented as the mean $\pm$ SD. Differences between the two groups were assessed using an unpaired t-test. Brown-Forsythe test was used to analyze whether the data complied with the homogeneity of variance. The Shapiro-Wilk test is used to verify the normal distribution. One-way ANOVA was used to compare groups before Tukey’s post hoc test. The p 0.05 was considered statistically significant.

To investigate the pathological mechanisms, initial analyses were conducted to evaluate pulmonary tissue alterations along with NICD and CCR5 expression levels in Notch1^flox+ mice subjected to ALI. LPS stimulation destroys lung tissue architecture, which includes alveolar wall thickening, infiltration of red blood cells, and inflammatory cells in the alveoli and interstitium (Fig. 1A). Statistical evaluation demonstrated markedly elevated expression levels of CCR5 and NICD proteins in lung tissue homogenates obtained from LPS-treated animals relative to untreated control groups (p 0.05, Fig. 1B). The experimental data indicate that LPS-triggered ALI activates the Notch pathway, and promotes protein expression of NICD, concomitant with enhanced CCR5 protein activity in pulmonary tissues.

Fig. 1.

Lung tissue pathology and protein expression in ALI mice. (A) hematoxylin and eosin (HE) staining of lung tissue sections and quantification of lung injury, n = 5. The magnification of the objective lens was 10$\times$ (Scale bar = 200 µm) and 40$\times$ (Scale bar = 50 µm), with an eyepiece magnification of 10$\times$. (B) Western blot analysis of CCR5 and NICD levels in lung tissue and quantitative analysis, n = 3. *p 0.05, ***p 0.001. ALI, acute lung injury; NICD, Notch intracellular domain; LPS, lipopolysaccharide.

To investigate the therapeutic potential of CCR5 inhibition, mice were administered Maraviroc prior to LPS-induced ALI. Histopathological evaluation of pulmonary tissues demonstrated that Maraviroc pretreatment attenuated LPS-mediated lung injury, as evidenced by reduced alveolar septal thickening and inflammatory cell infiltration (Fig. 2A), accompanied by significantly lower histopathological scores (Fig. 2B, p 0.05). Quantitative assessments revealed that Maraviroc administration significantly decreased pulmonary edema (wet-to-dry weight ratio), BALF cellularity, and total protein concentration compared to vehicle-treated controls (Fig. 2C–E, p 0.05). Furthermore, pre-administration of Maraviroc markedly suppressed pulmonary expression of pro-inflammatory cytokines, including TNF-$\alpha$, IL-6, and IL-1$\beta$, at the transcriptional level. Concurrently lessened their serum levels (Fig. 2F–K, p 0.05). The successful establishment of the LPS-induced ALI model was confirmed by characteristic pathological features, including severe pulmonary damage, a significant increase in protein content in BALF, and elevated levels of proinflammatory cytokines in lung tissue. Inhibition of CCR5 alleviated lung tissue damage in ALI mice and reduced the expression of inflammatory cytokines.

Fig. 2.

CCR5 inhibition attenuated pulmonary damage in ALI. (A,B) Representative hematoxylin and eosin (H&E) lung sections and corresponding histopathological scoring n = 5; The magnification of the objective lens was 10$\times$ (Scale bar = 200 µm) and 40$\times$ (Scale bar = 50 µm). (C) Quantification of pulmonary edema through W/D weight ratio analysis n = 5. (D,E) Total cellularity and protein concentration in BALF n = 5. (F–H) qRT-PCR analysis of TNF-$\alpha$, IL-6, and IL-1$\beta$ mRNA levels in lung tissue, n = 5. (I–K) Enzyme-Linked Immunosorbent Assay (ELISA) detection of TNF-$\alpha$, IL-6, and IL-1$\beta$ levels in serum, n = 3. *p 0.05, **p 0.01, ***p 0.001. W/D, wet/dry; BALF, bronchoalveolar lavage fluid; qRT-PCR, real-time quantitative reverse transcription PCR; LPS+Mvc, LPS + Maraviroc.

CCR5, as a receptor for inflammatory cell chemoattractants, is upregulated during the acute phase of ALI, potentially exacerbating secondary alveolar damage. It is therefore speculated that Notch may regulate CCR5 expression through ligand recognition, driving the accumulation of inflammatory cells and contributing to the pathological process.

To verify this hypothesis, we used macrophage Notch1 knockout mice to examine the effects of Notch1 deficiency on CCR5 activity in ALI. Analysis of BALF from ALI mice showed that both the total cell counts and total protein level in Notch1^cre+ mice were significantly lower than those of Notch1^flox+ mice (p 0.05) (Fig. 3A,B). Flow cytometry analysis indicated that the percentage of CD45+CD64+ macrophages in lung tissue was comparable between Notch1^cre+ and Notch1^flox+ mice under baseline conditions. However, following LPS induction, the number of CD45+CD64+ cells in Notch1^cre+ mice was significantly reduced compared to the Notch1^flox+ control group (p 0.05, Fig. 3C). Regarding CCR5 expression, no baseline differences were observed between Notch1^flox+ and Notch1^cre+ mice. Under physiological conditions, pulmonary NICD expression was markedly reduced in Notch1^cre+ (p 0.05, Fig. 3D). In the LPS-induced ALI model, both NICD and CCR5 protein levels in the lung tissue of Notch1^cre+ and Notch1^flox+ mice were increased, but the increase in Notch1^cre+ mice was lower than in Notch1^flox+ mice (p 0.05, Fig. 3E). In different conditions, the transcriptional level of CCR5 in murine pulmonary tissue was correlated with its corresponding protein expression (Fig. 3F).

Fig. 3.

Notch1-mediated CCR5 anti-inflammatory effect. (A,B) Total cell count and protein content in BALF, n = 5. (C) Flow cytometric analysis of lung tissue from mice and representative gating plots; Bar graphs represent the percentage of CD45+ and CD64+ macrophages of total cells. (D) Western blot analysis assessing NICD and CCR5 expression in lung tissue under normal conditions, n = 3. (E) Western blot analysis assessing NICD and CCR5 expression in lung tissue of wild-type and Notch1 knockout mice with LPS treatment, n = 3. (F) qRT-PCR detection of CCR5 mRNA levels in lung tissue of LPS-induced ALI mice, n = 5. (G) Immunofluorescence detection of CCR5 expression (green) in lung tissue macrophages (red). F4/80, red; CCR5, green; DAPI, blue; white box in each image indicates magnified views in the inset; magnification 40$\times$, scale bar = 100 µm. The scale bar in the large image is 10 µm. (H,I) Quantification of F4/80 and CCR5 fluorescence intensity. (J) Percentage of F4/80 and CCR5 double-positive cells among F4/80-positive cells, n = 5. *p 0.05, **p 0.01, ***p 0.001, ns indicates no statistical significance. DAPI, 4^′,6-diamidino-2-phenylindole.

Immunofluorescence was used to detect CCR5 expression in alveolar macrophages, with anti-F4/80 (red) and anti-CCR5 (green) antibodies marking the macrophages to analyze CCR5 expression in macrophages of lung tissue. We found that under physiological conditions, the proportion of F4/80 and CCR5 double-positive cells in the lung tissue was low, with no significant difference between Notch1^flox+ and Notch1^cre+ mice (p $>$ 0.05) (Fig. 3G). After LPS stimulation, F4/80 positivity increased in the lung tissues of both Notch1^flox+ and Notch1^cre+ mice, with higher fluorescence intensity observed in Notch1^flox+ mice (p 0.05, Fig. 3H). In contrast to Notch1^flox+ mice, where CCR5 showed high expression after LPS stimulation, the expression of green fluorescent CCR5 was lower in the Notch1^cre+ mice (p 0.05, Fig. 3I). Furthermore, cells double-positive for F4/80 and CCR5 were scarcely observed in Notch1^cre+ mice (p 0.05, Fig. 3J).

The results indicate that although CCR5 expression in the lung tissue of Notch1^cre+ mice increased after LPS stimulation, it was lower than that in Notch1^flox+ mice. Additionally, the rare presence of F4/80 and CCR5 double-positive cells in Notch1^cre+ mice suggests that Notch1 deficiency in alveolar macrophages impedes the expression of CCR5 on their cell membrane during LPS-induced ALI.

To elucidate the regulatory role of the Notch signaling pathway on CCR5 expression under in vitro conditions, we employed MK-0752, a specific $\gamma$-secretase inhibitor, to suppress intracellular Notch signaling activity in iBMDM cells derived from murine sources. Following pharmacological inhibition, the protein expression levels of CCR5 were quantitatively assessed in the treated iBMDM cellular model.

As illustrated in Fig. 4A, LPS stimulation markedly enhanced CCR5 fluorescence intensity on the cellular membrane, a phenomenon that was substantially inhibited by MK-0752 administration. Consistent with these findings, western blot analysis demonstrated a concomitant increase in both CCR5 and NICD protein levels upon LPS challenge, which were prominently downregulated in iBMDM cells pretreated with MK-0752 prior to LPS exposure (p 0.05) (Fig. 4B,C). Furthermore, quantitative PCR analysis revealed that pharmacological blockade of $\gamma$-secretase activity significantly suppressed LPS-induced transcriptional upregulation of IL-6, IL-1$\beta$, and TNF-$\alpha$ (p 0.05), as evidenced in Fig. 4D–F. These findings indicate that the intracellular Notch signaling in macrophages is closely linked to CCR5 expression and the transcription of pro-inflammatory cytokines, which was dependent on the activity of $\gamma$-secretase.

Fig. 4.

Inhibition of the Notch pathway reduces CCR5 transcription and pro-inflammatory cytokine expression in LPS-induced iBMDM cells. (A) Immunofluorescence was used to detect CCR5 expression (green) on iBMDM cells, with DAPI staining (blue) for nuclei. The fluorescence intensity was quantified. Magnification: 40$\times$, scale bar = 50 µm. (B,C) Western blot analysis was used to assess NICD and CCR5 protein levels in iBMDM cells under different treatments, with quantitative analysis of the protein expression. (D–F) qRT-PCR was used to detect the mRNA levels of TNF-$\alpha$, IL-6, and IL-1$\beta$ in LPS-induced iBMDM cells, n = 3. *p 0.05, **p 0.01, ***p 0.001. iBMDM, immortalized bone marrow-derived macrophages.

The activation of Notch receptors on macrophages, upon binding with ligands from neighboring cells, triggers the release of chemokines that orchestrate the recruitment and migration of immune cells to the lungs, thereby exacerbating lung injury [15]. CCR5, as a chemokine receptor, plays an important role in regulating macrophage migration [16, 17]. Therefore, we investigated whether Notch signaling affects macrophage migration through CCR5 expression. Since primary macrophages in vitro are adherent and difficult to track for migration efficiency, this part of the experiment utilized LPS-induced iBMDM cell migration for analysis.

The wound healing assay showed that under MK-0752-mediated $\gamma$-secretase inhibition, LPS-induced migration of iBMDM cells was also suppressed (Fig. 5A,B). We further used the Transwell assay to verify whether this migratory inhibition occurs through the CCR5 pathway. After LPS stimulation, the migration of iBMDM cells treated with CCL5 significantly increased. However, under $\gamma$-secretase inhibition by MK-0752, the ability of CCL5 to recruit cells was notably reduced (Fig. 5C,D). These experimental findings provide additional evidence that Notch signaling activation in macrophages potentially mediates the CCL5-induced macrophage aggregation by modulating CCR5 activity.

Fig. 5.

Inhibition of the Notch pathway reduces the migration efficiency of iBMDM cells. (A) Migration of iBMDM cells under Notch inhibition was assessed using the wound healing assay. (B) Quantification of the migration rate of iBMDM cells at 6 and 12 hours. LPS group VS LPS+MK-0752 group **p 0.01. (C) Migration of iBMDM cells was observed through crystal violet staining. (D) Quantitative analysis of the number of migrated iBMDM cells in each group, n = 3, magnification: 10$\times$, scale bar = 100 µm. *p 0.05, ***p 0.001, ****p 0.0001.