LACC1 attenuates LPS-induced acute lung injury by inhibiting inflammatory response in alveolar macrophage through LPAR3/PI3K/AKT/mTOR signaling pathway

Abstract

Objective

Laccase domain containing 1 (LACC1) is an enzyme abundantly expressed in inflammatory macrophages that regulate diverse inflammatory diseases. This study aimed to investigate the role of LACC1 in lipopolysaccharide (LPS)-induced acute lung injury (ALI) and to elucidate its underlying regulatory mechanisms.

Methods

Single-cell RNA sequencing of bronchoalveolar lavage fluid (BALF) from patients with ALI was performed to characterize LACC1 expression patterns in pulmonary macrophages. Then, LACC1 conditional knockout mouse model was established to investigate the in vivo function of LACC1 during LPS-induced lung injury. ln vitro function of LACC1 was further explored using LACC1-silenced THP-1 cells. RNA sequencing was conducted to profile gene expression changes in LACC1-knockdown THP-1 cells, followed by GO and KEGG enrichment analysis. Key downstream molecules and signaling pathways modulated by LACC1 were validated by Western blot and qRT-PCR.

Results

Single-cell RNA sequencing analysis based on BALF of ALI patients revealed that LACC1 expression was markedly elevated in pulmonary macrophages. Conditional knockout of LACC1 in macrophages exacerbated LPS-induced pulmonary inflammation. In vitro, LACC1 knockdown in THP-1 cells markedly exacerbated the inflammatory response after LPS treatment. RNA sequencing identified LPAR3 as a key downstream target, and enrichment analysis indicated significant involvement of phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) signaling pathway followed by LACC1 knockdown. Western blot analysis confirmed that LACC1 knockdown reduced LPAR3 expression and inhibited PI3K/AKT/mTOR pathway activation.

Conclusions

LACC1 might exert a protective effect against LPS-induced lung injury by suppressing inflammatory responses via modulation of LPAR3/PI3K/AKT/mTOR signaling pathway.

Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s40364-026-00967-3.

Introduction

Acute lung injury (ALI) is a common and heterogeneous clinical syndrome that arises from various causes such as pneumonia, aspiration, trauma, and sepsis, and is associated with high mortality [1, 2]. Pathologically, ALI is marked by abnormal increased pulmonary vascular permeability, breakdown of the alveolar–capillary barrier, and subsequent pulmonary edema, followed by overwhelming inflammatory responses [3]. This process involves extensive damage and apoptosis of alveolar epithelial cells, excessive release of pro-inflammatory cytokines, and impaired integrity of the pulmonary endothelial barrier [4]. Despite recent advances in the understanding of pathogenesis and supportive treatment of ALI, its incidence and fatality rate remains distressingly high, typically between 40% and 60% [2]. Moreover, ALI has a substantial impact on global public health and represents a major cause of death in severe pneumonia, including cases linked to SARS-CoV-2 infection [5, 6]. Therefore, elucidating the immunopathogenesis of ALI and developing targeted therapeutic interventions to mitigate ALI progression represent critical priorities, which are pivotal for reducing the global health burden of ALI.

In the innate immune system, alveolar macrophages (AMs) play a critical role in the pathogenesis of ALI, acting as the key immune sentinels protecting the lungs from invading pathogens [7]. Residing within the airway lumen and adjacent to alveolar epithelial cells, AMs are essential for maintaining pulmonary homeostasis and immune competence [8]. As central regulators of innate defense system, the activation of AMs is instrumental throughout all phases of the inflammatory response. Based on their functional roles in host defense, macrophages are broadly classified into classically activated (M1) and alternatively activated (M2) phenotypes [9]. During the initial phase of ALI, resting (M0) AMs polarize towards pro-inflammatory M1 phenotype, initiating the release of cytokines such as TNF-α, IL-6, and iNOS, thereby driving the progression of ALI and ultimately leading to tissue destruction. During the resolution phase, AMs undergo a phenotypic shift towards anti-inflammatory M2 phenotype, releasing anti-inflammatory mediators like IL-10 to facilitate lung tissue repair and resolution of injury [10, 11]. Given the central role of AMs in the pathogenesis of lung injury, targeting AM function presents a promising approach for reestablishing immune homeostasis in ALI.

Laccase domain-containing 1 (LACC1), also known as C13orf31 or FAMIN, has been closely linked with various human immune-mediated and inflammatory disorders [12]. Genetic variation of LACC1 has been linked to susceptibility to Crohn’s disease (CD), leprosy, and systemic juvenile idiopathic arthritis (JIA) [13–15]. LACC1 encodes a multifunctional enzyme highly expressed in inflammatory macrophages, involved in key metabolic pathways such as endogenous isocyanic acid synthesis and purine nucleotide metabolism [16, 17]. Studies have shown that treatment of mouse bone marrow-derived macrophages (BMDMs) with LPS significantly upregulates LACC1 expression [18]. Furthermore, the absence of LACC1 led to an accelerated onset of arthritis and significantly worse arthritis and inflammation in mice [18]. Notably, LACC1 has been proposed to serve as an intermediary linking inducible nitric oxide synthase (NOS2) with polyamine immunometabolism during the arginine metabolism pathway [17]. These observations strongly suggested that LACC1 was involved in a series of autoimmune and inflammatory diseases and played a significant regulatory role in macrophage immunometabolic functions. However, the biological role of LACC1 in inflammatory conditions such as ALI remains largely unexplored.

Materials and methods

Experimental animals

SPF-level 8-week-old male C57BL/6 mice purchased from GemPharmatech (Nanjing, China) were used for animal experiments. Before experiments, these mice were housed under a 12 h light and dark cycle and had free access to maintenance food and water for at least one week. All animal experiment procedures were according to the experiment protocols approved by the Animal Review Committee of Zhongshan Hospital, Fudan University.

Establishment of LACC1^flox/flox Lyz2-Cre⁺ mice models

LACC1^flox/flox mice and lysozyme 2 (Lyz2)-Cre⁺ mice were generated using CRISPR-Cas9 system by GemPharmatech (Nanjing, China). To obtain alveolar macrophage-specific LACC1-deficient mice, Lyz2-Cre⁺ mice were crossed with LACC1^flox/flox mice, yielding Lyz2-Cre⁺-LACC1^flox/flox offspring (designated as LACC1 cKO). Littermate LACC1^flox/flox mice (designated as LACC1 WT) served as controls in experiments.

Murine model of LPS-induced ALI

Mouse model of ALI was established by intratracheal instillation of LPS. Mice were randomly divided into control and ALI group, and then anesthetized using 3%-5% isoflurane. After disinfection of midline neck skin, an incision was made to sufficiently expose the trachea. Thereafter, 100 µL LPS solution (0111:B4, L2630, Sigma, USA) was slowly administered intratracheally at a dose of 1 mg/kg. Mice in the control group received an equal volume of intratracheal instillation of sterile PBS. Then, mice undergoing LPS instillation were sacrificed through intraperitoneal injection 1% pentobarbital (150 mg/kg) after 24 h, 48 h and 72 h and lung tissues were collected for subsequent assays.

Acquisition of bronchoalveolar lavage fluid (BALF)

Bronchoalveolar lavage fluid (BALF) samples were collected from patients under sterile conditions using fiberoptic bronchoscopy. The recovered BALF was immediately filtered through a 70-µm cell strainer to remove mucus and debris, followed by centrifugation at 300 × g for 10 min at 4 °C. The cell pellet was washed twice with ice-cold phosphate-buffered saline (PBS) containing 0.04% bovine serum albumin (BSA) to preserve cell viability. Cell viability and concentration were assessed using trypan blue exclusion and an automated cell counter, ensuring a viability rate above 85%. All procedures involving human samples were conducted under protocols approved by the Ethics Committee of Zhongshan Hospital, Fudan University, and informed consent was obtained from all participants (B2023-074R).

Single-cell RNA sequencing (scRNA-seq) and library preparation

Single-cell encapsulation, barcoding, and library construction were performed by SeekGene (XunYin Biotechnology, Beijing, China) using SeekOne^® high-throughput single-cell platform. SeekOne^® DD (digital droplet) single-cell chemistry was applied for cell partitioning and molecular barcoding, per the provider’s standard operating procedures. Single-cell suspensions were loaded onto the SeekOne device to generate uniquely barcoded single-cell partitions. Reverse transcription and second-strand synthesis were carried out according to SeekOne^® reagent kit protocols to produce full-length cDNA. cDNA amplification was performed using kit-specified cycles, and amplified cDNA was purified using magnetic bead–based cleanup. Library fragment size distributions and concentrations were evaluated on an Agilent 2100 Bioanalyzer and quantified with a Qubit fluorometer.

Final libraries were pooled and sequenced on an Illumina NovaSeq 6000 system, targeting an average sequencing depth of 30,000–50,000 reads per cell. Demultiplexing, read alignment to the human reference genome (GRCh38), and generation of gene expression matrices were performed using SeekGene’s analysis pipeline and quality-control filters prior to downstream bioinformatic processing.

RNA sequencing and data analysis

Total RNA was isolated from alveolar macrophages obtained from murine bronchoalveolar lavage fluid (BALF) using TRIzol reagent (Invitrogen, USA). RNA concentration and integrity were assessed with NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, USA). RNA-seq library construction and sequencing were performed by Sangon Biotech (Shanghai, China). Qualified libraries were sequenced on the Illumina NovaSeq 6000 platform to generate 150 bp paired-end reads. Raw reads were processed using FastQC and Trimmomatic for quality control and adapter removal. Differentially expressed genes (DEGs) were identified using DESeq2 (p value < 0.05, |log₂FoldChange| ≥ 2). Functional enrichment of DEGs was conducted via clusterProfiler for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.

Isolation of murine alveolar macrophages

Alveolar macrophages were isolated from freshly collected mice BALF. BALF samples were first filtered through a sterile 70-µm cell strainer to remove mucus and debris, followed by centrifugation at 1000 rpm for 10 min at 4 °C. The resulting cell pellet was resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. The cell suspension was then seeded into 6-well culture plates and incubated at 37 °C with 5% CO₂ for 2 h to allow macrophage adherence. After incubation, non-adherent cells, including alveolar lymphocytes and epithelial cells, were gently removed by washing the wells three times with pre-warmed PBS. The adherent cell population, representing enriched alveolar macrophages, was then maintained in fresh complete RPMI-1640 medium for downstream experiments. Cell purity was assessed by morphological evaluation under light microscopy. Then alveolar macrophages were used for subsequent assays, including RNA extraction, Western Blot, or RNA sequencing.

Cell culture

The human monocytic cell line THP-1 was cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, USA) and maintained at 37 °C in a humidified incubator with 5% CO₂. For differentiation into macrophage cells, THP-1 monocytes were seeded at a density of 1 × 10⁶ cells/mL and treated with 100 nM phorbol 12-myristate 13-acetate (PMA) (Biosharp, Shanghai, China) for 48 h. After stimulation, the medium was replaced with fresh medium, and cells were allowed to rest for an additional 24 h to recover and stabilize before subsequent experiments. THP-1 cells were routinely tested for mycoplasma contamination and were confirmed to be negative.

Lentiviral transduction and validation of LACC1 knockdown

To suppress LACC1 expression, lentivirus encoding LACC1-targeting shRNA and negative control vectors (NC) were obtained from Genechem (Shanghai, China). THP-1 cells, cultured in suspension, were seeded at a density of 1 × 10⁶ cells/mL and infected with lentivirus. To enhance infection efficiency, the virus–cell mixture was subjected to centrifugation at 1,000 × g for 60 min at room temperature, followed by incubation for 6 h. The medium was then replaced with fresh complete RPMI-1640 medium supplemented with 10% FBS, and cells were cultured for an additional 48 h before collection. Knockdown efficiency was confirmed at both the mRNA and protein levels by quantitative real-time PCR (qRT-PCR) and Western blot analysis, respectively. All experiments were performed using three independent biological replicates per group.

Hematoxylin-eosin (H&E) staining

Mouse lung tissues were collected and immediately fixed in 4% paraformaldehyde (pH 7.4) at room temperature for 24 h. After fixation, tissues were dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Paraffin-embedded lungs were sectioned at a thickness of 4 μm using a rotary microtome. The sections were firstly deparaffinized in xylene, and then rehydrated through decreasing concentrations of ethanol, and stained sequentially with hematoxylin and eosin solutions. After staining, slides were washed, dehydrated, cleared, and mounted with neutral resin. Tissue morphology was observed and imaged under a light microscope (Leica Microsystems, Germany).

Quantitative real-time pcr (qRT-PCR)

Total RNA was isolated from THP-1 cells and mouse lung tissues using TRIzol reagent (Thermo Fisher Scientific, USA). cDNA synthesis was carried out using the SuperRT III All-in-One RT Mix with gDNA Remover (Yeasen, Shanghai, China). Quantitative PCR was performed with the Hieff^® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) on a CFX96™ Real-Time PCR Detection System (Bio-Rad, USA) according to the standard conditions: 95 °C for 5 min, followed by 45 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s. Primer pairs were synthesized by Sangon Biotech (Shanghai, China), and sequences were provided in Table 1. Relative gene expression levels were quantified using the 2⁻ΔΔCt method. All reactions were performed in triplicate from three independent biological replicates.

Enzyme-linked Immunosorbent assay (ELISA)

IL-6 and TNF-α concentrations in cell culture supernatants and murine BALFs were quantified by ELISA kits according to the instructions of manufacturer (Abclonal, Wuhan, China).

Western blot analysis

Total protein was extracted from THP-1 cells and mouse lung tissues using RIPA lysis buffer (Yeasen, Shanghai, China) supplemented with 1% phosphatase inhibitor cocktail and protease inhibitor cocktail (Yeasen, Shanghai, China) on ice. Protein concentrations were determined using BCA Protein Assay Kit (Epizyme, Shanghai, China). Total protein (40 µg) were separated on 8–12% SDS–PAGE gels and transferred to PVDF membranes (Millipore, USA). Then membranes were blocked by non-fat powdered milk and subsequently incubated with primary antibodies overnight at 4 °C, followed by HRP-conjugated anti-mouse or anti-rabbit IgG secondary antibodies for 1 h at room temperature. The following antibodies were used: LACC1 (1:1000, sc-376231, Santa Cruz); β-actin (1:1000, 30101ES10, Yeasen); GAPDH (1:1000, 60004, Proteintech); LPAR3 (1:1000, 19509-1-AP, Proteintech); PI3K(1:1000, AF6241, Affinity); P-PI3K(1:1000, AF3241, Affinity); AKT(1:1000, ET1609-51, HUABIO); P-AKT(1:1000, 66444-1-lg, Proteintech); mTOR (1:1000, ET1608-5, HUABIO); P-mTOR (1:1000, AF3309, Affinity); Vinculin (1:1000, 26520-1-AP, Proteintech). Protein bands were visualized using enhanced chemiluminescence detection system (Tanon, Shanghai, China), and signal intensities were quantified with ImageJ software.

Statistical analysis

Quantitative data was presented as the mean ± standard deviation (SD). Statistical analysis was conducted using GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were performed with an unpaired Student’s t-test, multiple group comparisons were analyzed by one-way ANOVA, and experiments involving two independent variables were assessed using two-way ANOVA. A p-value < 0.05 was considered indicative of statistical significance.

Results

Bronchoalveolar immune landscapes in patients with acute lung injury

To profile the cellular landscape of the pulmonary microenvironment during acute lung injury, we collected BALF samples from patients with lung injury and performed single-cell RNA sequencing on BALF cells from three healthy controls and three patients with lung injury.

Based on classic marker genes, clustering analysis revealed 9 major distinct clustering of major immune and epithelial cell populations composed of B cells, T cells, epithelial cells, macrophages, neutrophils, dendritic cells (DCs), monocytes, NK cells and mast cells (Fig. 1A). Heatmap displayed the scaled expression of DEGs across clusters, indicating differential expression patterns corresponding to specific cell types (Fig. 1B). The results showed that macrophages, monocytes and neutrophils all exhibited high expression of inflammatory markers including MRC1, MSR1, CXCL8, etc., further supporting their critical roles in ALI pathogenesis. Then we quantified the relative abundance of each cell type for all samples, and the cell type proportion results showed BALFs of patients with lung injury exhibited a marked decrease of macrophages, whereas monocytes and neutrophils exhibited a significant expansion, consistent with the well-established immunological dynamics during the early stage of ALI (Fig. 1C). The dot plot displayed the expression pattern of DEGs in distinct cell types. Notably, we observed that LACC1, a gene of interest, was specifically identified and highly expressed in immune cell populations including AMs, DCs and monocytes, suggesting its potential involvement during ALI (Fig. 1D). Given the established role of AMs as the major effector populations in orchestrating early inflammatory responses in ALI [19], our subsequent analysis focused on elucidating the specific role of LACC1 in AMs. Together, these findings demonstrated significant cellular heterogeneity in BALF during ALI and LACC1 might play a significant role in ALI pathogenesis.

Fig. 1.

Bronchoalveolar immune landscapes in patients with acute lung injury. (A) UMAP plot of major distinct cell clusters from human BALF samples. (B) Heatmap of DEGs across the identified cell clusters. (C) Bar plot of distinct cell type proportions in BALF samples of ALI and healthy control groups. (D) Dot plot of DEGs expression levels across the identified cell clusters

LACC1 was upregulated in AMs of patients with acute lung injury

To further dissect the expression pattern of LACC1, we subclustered and annotated macrophages and monocytes into 10 distinct subpopulations and UMAP feature plots further confirmed the spatially restricted expression of LACC1 within macrophage clusters and monocyte clusters (Fig. 2A, C). Differential gene expression heatmap analysis demonstrated that LACC1 was preferentially enriched in macrophage and monocyte populations (Fig. 2B). Dot plot profiling across major cell clusters indicated that LACC1 expression was largely restricted to macrophages, DCs and monocytes, with minimal expression detected in epithelial cells, neutrophils, NK cells, or T cells and further sub-clustering analysis elucidated LACC1 was mainly distributed among MARCKS⁺ macrophages, CD16⁺ monocytes, and CD14⁺ CD16⁺ monocytes, cellular populations that collectively represent the most dynamically responsive components of the mononuclear phagocyte system during acute inflammatory injury. (Fig. 2D, E). Consistently, violin plots indicated significantly higher LACC1 expression in macrophage subtypes, particularly in MARCKS⁺ macrophages (Fig. 2F). Collectively, these findings suggested that LACC1 was markedly upregulated in AM subsets within BALF microenvironment of acute lung injury patients, suggesting its potential involvement in regulating pulmonary inflammatory responses.

Fig. 2.

LACC1 was upregulated in alveolar macrophages of patients with acute lung injury. (A) UMAP plot of macrophage and monocyte subclusters from human BALF samples. (B) Heatmap of DEGs across macrophage and monocyte populations. (C) UMAP plot of the spatial distribution and expression intensity of LACC1. (D) Dot plot of LACC1 expression levels across major cell clusters. (E) Dot plot of LACC1 expression levels across macrophage and monocyte subclusters. (F) Violin plot of LACC1 expression levels across macrophage and monocyte subclusters

LACC1 was upregulated in LPS-induced inflammatory AMs

To further validate the elevated expression of LACC1 in alveolar macrophages, we established LPS-induced acute lung injury mouse model. AMs from mice BALFs were purified for transcriptomic analysis. A total of 2933 genes were identified as DEGs (log₂|FoldChange| > 2, p value < 0.05) and created as an overall volcano plot. Among these DEGs, LACC1 expression was significantly elevated with a log₂|FoldChange| of 4.609116 in the LPS group, as displayed in the volcano plot (Fig. 3A) and heatmap (Fig. 3B). Compared with control group, mice exhibited pronounced alveolar wall thickening, inflammatory cell infiltration, and hemorrhage after LPS treatment, all of which progressively aggravated over time (Fig. 3C). Consistently, the lung injury score, lung wet/dry ratio, total cell counts, as well as protein leakage in BALF were remarkably increased in a time-dependent manner following LPS exposure (Fig. 3D-G). The production of proinflammatory cytokines, including TNF-α and IL-6, was also elevated in the BALF of LPS-challenged mice over time (Fig. 3H-K). Western blot analysis further confirmed a time-dependent increase in LACC1 protein expression in lung tissues after LPS stimulation (Fig. 3L, M), consistent with elevated LACC1 mRNA expression (Fig. 3N). Similarly, LACC1 was significantly increased at protein and mRNA levels in mice primary AMs at 48 h (Fig. 3O-Q). Together, these findings demonstrated that LACC1 expression was robustly upregulated in lung tissue and AMs during LPS-induced acute lung injury, displaying a time-dependent increase that suggested a significant regulatory role in the inflammatory response.

Fig. 3.

LACC1 was upregulated in LPS-induced inflammatory alveolar macrophages. (A) Volcano plot depicting the distribution of DEGs in alveolar macrophages isolated from control and LPS-treated mice. DEGs were screened using a threshold of |log2 FoldChange| >2 and p value < 0.05. (B) Heatmap of DEGs in alveolar macrophages isolated from control and LPS-treated mice. (C) Representative H&E images of lung sections from mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). Scale bar, 100 μm. (D, E) Lung injury score and lung wet/dry weight ratio of mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). (F, G) Quantification of total cell counts and protein concentrations in BALF from mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). (H, J) Relative mRNA expression levels of proinflammatory cytokines IL-6 and TNF-α in lung tissues from mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). (I, K) Relative expression levels of proinflammatory cytokines IL-6 and TNF-α in BALF from mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). (L) Representative Western blot images of LACC1 protein levels in lung tissues from mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). (M) Quantification of relative LACC1 protein expression levels in lung tissues from mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). (N) Relative mRNA expression level of LACC1 in lung tissues from mice undergoing LPS stimulation for 12 h, 24 h and 48 h (n = 3). (O) Representative Western blot images of LACC1 protein levels in isolated primary alveolar macrophages from mice undergoing LPS stimulation for 48 h (n = 3). (P) Quantification of relative LACC1 protein expression levels in isolated primary alveolar macrophages from mice undergoing LPS stimulation for 48 h (n = 3). (Q) Relative mRNA expression level of LACC1 in in isolated primary alveolar macrophages from mice undergoing LPS stimulation for 48 h (n = 3). Data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; determined by one-way ANOVA with Tukey’s post hoc test

LACC1 deficiency in alveolar macrophages exacerbates LPS-induced acute lung injury

We next generated conditional LACC1 cKO mice by crossing LACC1 floxed mice with Lyz2-Cre mice to investigate the functional role of LACC1 in AMs during acute lung injury, and the disruption of LACC1 in macrophages was verified through qRT-PCR (Fig. S1). LACC1 WT and cKO mice were intratracheally instilled with LPS, and lung injury was assessed 48 h post-treatment. H&E analysis revealed that compared with LACC1 WT mice, LACC1 cKO mice developed more severe pulmonary injury after LPS exposure, characterized by extensive alveolar wall thickening, interstitial edema, and dense inflammatory cell infiltration (Fig. 4A). Besides, LACC1 deficiency in AMs significantly increased cell counts and protein leakage in BALF (Figs. 4B, C), as well as lung injury scores and wet/dry weight ratios (Figs. 4D, E), indicating aggravated pulmonary damage and edema. Moreover, the loss of LACC1 in AMs led to a marked elevation of proinflammatory cytokines including TNF-α and IL-6 compared with LACC1 WT group following LPS treatment (Figs. 4F-I). These findings suggested that LACC1 deficiency in AMs exacerbated LPS-induced lung injury, highlighting the significant role of LACC1 in maintaining pulmonary homeostasis during acute inflammatory responses.

Fig. 4.

LACC1 deficiency in alveolar macrophages exacerbated LPS-induced acute lung injury. (A) Representative H&E images of lung sections from LACC1 WT and LACC1 cKO mice under control or LPS-stimulated conditions (n = 3). Scale bar, 100 μm. (B, C) Quantification of total cell counts and protein concentrations in BALF from LACC1 WT and LACC1 cKO mice after LPS treatment (n = 3). (D, E) Lung injury score and lung wet/dry weight ratio of LACC1 WT and LACC1 cKO mice after LPS treatment (n = 3). (F, G) Relative mRNA expression levels of proinflammatory cytokines IL-6 and TNF-α in lung tissues from LACC1 WT and LACC1 cKO mice after LPS treatment (n = 3). (H, I) Relative expression levels of proinflammatory cytokines IL-6 and TNF-α in BALF from LACC1 WT and LACC1 cKO mice after LPS treatment (n = 3). Data were presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; determined by two-way ANOVA with Tukey’s post hoc test

LACC1 knockdown enhanced LPS-induced inflammatory responses in THP-1 cells

To assess the significance of LACC1 in inflammatory responses, we first analyzed the expression of LACC1 in THP-1 cells following LPS stimulation. LACC1 expression was significantly upregulated after 24 h of LPS treatment, indicating a strong induction of LACC1 under inflammatory conditions (Fig. 5A). Besides, pro-inflammatory cytokines including IL-6 and TNF-α levels were markedly elevated in the LPS-treated group compared to control (Fig. 5B-D). Next, we established LACC1 knockdown THP-1 cells through LACC1 lentivirus shRNAs. Compared to the control group (sh-NC), sh-LACC1-2 cells showed a marked reduction of approximately 70–80% in LACC1 expression at mRNA and protein levels and therefore it was selected for subsequent experiments (Fig. 5E-G). To explore the effect of LACC1 knockdown on inflammatory response in macrophages, THP-1 cells were induced to differentiate into macrophages and then exposed to LPS for 24 h. The loss of LACC1 significantly increased the levels of pro-inflammatory cytokines after LPS stimulation, further supporting the role of LACC1 in regulating inflammatory response in response to LPS (Fig. 5H-J).

Fig. 5.

LACC1 knockdown enhanced LPS-induced inflammatory responses in THP-1 cells. (A) Relative mRNA expression level of LACC1 in THP-1 cells treated with LPS for 24 h (n = 3). (B, C) Relative expression levels of proinflammatory cytokines IL-6 and TNF-α in THP-1 cells treated with LPS for 24 h (n = 3). (D) Relative mRNA expression levels of IL-6 and TNF-α in THP-1 cells treated with LPS for 24 h (n = 3). (E) Relative mRNA expression level of LACC1 in THP-1 cells transfected with LACC1 lentiviral shRNAs (n = 3). (F) Representative Western blot images of LACC1 protein levels in THP-1 cells transfected with LACC1 lentiviral shRNAs (n = 3). (G) Quantification of relative LACC1 protein expression levels in THP-1 cells transfected with LACC1 lentiviral shRNAs (n = 3). (H, I) Relative expression levels of proinflammatory cytokines IL-6 and TNF-α in LACC1 knockdown THP-1 cells treated with LPS for 24 h (n = 3). (J) Relative mRNA expression levels of IL-6 and TNF-α in LACC1 knockdown THP-1 cells treated with LPS for 24 h (n = 3). Data were presented as mean ± SD; **p < 0.01, ***p < 0.001, ****p < 0.0001; determined by unpaired two-tailed Student’s t-tests and one-way ANOVA followed by Tukey’s post hoc test

LACC1 deficiency facilitated inflammatory response through LPAR3/PI3K/AKT/mTOR signaling pathway in THP-1 cells

To further explore the molecular mechanisms by which LACC1 regulated inflammatory response, we performed RNA sequencing on THP-1 cells after LACC1 knockdown and comprehensively integrated differential expression magnitude, pathway enrichment relevance, and protein-protein interaction (PPI) network analysis. Volcano plot analysis identified 66 DEGs in the sh-LACC1 group relative to controls. Notably, LPAR3 exhibited a marked reduction upon LACC1 silencing, with a log₂|FoldChange| of -2.40491 (Fig. 6A). Hierarchical clustering also demonstrated distinct transcriptional signatures between sh-LACC1 and sh-NC cells (Fig. 6B). PPI network analysis identified LPAR3 as a central node gene closely associated with multiple inflammatory and signaling molecules including PI3K/AKT signaling pathway, chemokine signaling pathway (Fig. 6C). GO enrichment analysis indicated the most significantly affected pathways following LACC1 knockdown were primarily associated with cell cycle regulation, DNA replication, and chromosome segregation (Fig. 6D). KEGG pathway analysis further identified significant enrichment in PI3K-AKT, TNF, and NF-κB signaling pathways, implicating these pathways in LACC1-mediated immune regulation (Fig. 6E). We selected four representative hub genes identified from our RNA-seq screen and found the mRNA expression levels of four candidates were significantly and robustly downregulated following LACC1 knockdown in THP-1 cells, which was consistent with our transcriptomic profiling data (Figure S2).

Fig. 6.

LACC1 deficiency facilitated inflammatory response through LPAR3/PI3K/AKT/mTOR signaling pathway in THP-1 cells. (A) Volcano plot depicting the distribution of DEGs between sh-LACC1 and sh-NC THP-1 cells. (B) Heatmap showing hierarchical clustering of DEGs between sh-LACC1 and sh-NC THP-1 cells. (C) PPI network constructed from the identified DEGs. (D) GO enrichment analysis of DEGs classified by biological process (BP), cellular component (CC), and molecular function (MF). (E) KEGG pathway enrichment analysis of the top significantly enriched pathways. (F) Representative Western blot images of LPAR3 protein levels in LPS-treated THP-1 cells (n = 3). (G) Quantification of relative LPAR3 protein expression levels in LPS-treated THP-1 cells (n = 3). (H) Representative Western blot images of LPAR3, mTOR, p-mTOR, AKT, p-AKT, PI3K, p-PI3K protein levels in shNC and shLACC1 THP-1 cells with or without LPS stimulation for 24 h (n = 3). (I)-(K) Quantification of relative LPAR3, mTOR, p-mTOR, AKT, p-AKT, PI3K, p-PI3K protein expression in shNC and shLACC1 THP-1 cells with or without LPS stimulation for 24 h (n = 3). Data were presented as mean ± SD; **p < 0.01, ***p < 0.001, ****p < 0.0001; determined by unpaired two-tailed Student’s t-tests

Fig. 7.

LACC1 modulates inflammatory response in alveolar macrophage through LPAR3/PI3K/AKT/mTOR signaling pathway. In ALI, LACC1 mediates its effect through the activation of LPAR3, which triggers the PI3K/AKT signaling cascade, leading to the subsequent phosphorylation and activation of mTOR, thereby controlling the macrophage’s overall inflammatory phenotype within the alveolar macrophages

Notably, LPAR3 exhibited a pronounced downregulation following LACC1 knockdown and emerged as a key hub gene within the PPI network, displaying strong connectivity with PI3K/AKT-related signaling pathway. Furthermore, KEGG pathway enrichment analysis identified PI3K/AKT/mTOR signaling as one of the most significantly altered pathways after LACC1 silencing. Based on its significant differential expression, central network topology, and established biological relevance to PI3K/AKT signaling, LPAR3 was prioritized as a candidate downstream effector for mechanistic validation to determine whether LACC1-mediated inflammatory regulation is functionally linked to modulation of the PI3K/AKT/mTOR axis. LPAR3, a member of the G protein-coupled receptor family, also known as LPA3 or EDG7, could bind endogenous lysophosphatidic acids (LPAs) and have been implicated in diverse biological processes, including cell proliferation, migration, survival, and inflammatory regulation [20]. Previous studies have further demonstrated that LPAR3-mediated signaling can activate the PI3K/AKT/mTOR axis in multiple pathological contexts, including cardiac remodeling, tumor progression, and inflammatory injury [20–23]. Given the established role of LPAR3 in PI3K/AKT/mTOR activation, we next investigated the downstream pathway. To determine whether LACC1 was involved in the regulation of LPAR3, we assessed the expression of LPAR3 in LPS-stimulated THP-1 cells. LPS treatment induced the elevation of LPAR3 expression at both mRNA and protein levels, indicating a physiological compensatory response of macrophages under acute inflammatory stress (Fig. 6F-H), which was markedly suppressed following LACC1 knockdown. Furthermore, LPS exposure led to an increase in the phosphorylation levels of PI3K, AKT, and mTOR. However, the phosphorylation levels of PI3K, AKT, and mTOR were significantly reduced after LACC1 knockdown (Fig. 6H-L). Collectively, these results demonstrated that LACC1 deficiency could downregulate the expression of LPAR3 and prevent the activation of the protective PI3K/AKT/mTOR signaling pathway, thereby contributing to the modulation of macrophage inflammatory responses (Fig. 7).

To further evaluate whether this LACC1-regulated LPAR3 expression subsequently governs downstream cascade dynamics under inflammatory challenge, we systematically quantified the phosphorylation-to-total ratios of the PI3K/AKT/mTOR axis across the four experimental groups. In sh-NC cells, LPS stimulation triggered a profound hyper-phosphorylation and activation of the downstream cascade, as evidenced by a sharp increase in the ratios of P-PI3K/PI3K, P-AKT/AKT, and P-mTOR/mTOR (Fig. 6H–K, referencing image_2c6607.png). Strikingly, LACC1 silencing thoroughly disrupted this protective signaling response; in sh-LACC1 cells, the phosphorylation levels of PI3K, AKT, and mTOR plummeted to a deep baseline deficit and entirely lost their capacity to be activated or induced upon LPS challenge (Fig. 6H–K, referencing image_2c6607.png). Collectively, these comprehensive four-group results demonstrated that LACC1 deficiency fundamentally downregulates LPAR3 expression, thereby paralyzing the compensatory activation of the protective PI3K/AKT/mTOR signaling pathway and contributing to the hyper-inflammatory breakdown of macrophage homeostasis during acute injury (Fig. 7).

Discussion

Our study first identified LACC1 as a crucial regulator that restrains macrophage-driven inflammation during acute lung injury. By integrating single-cell RNA sequencing data, in vivo conditional knockout models, and in vitro mechanistic assays, we demonstrated that the loss of LACC1 aggravated lung injury through inhibiting LPAR3-dependent PI3K/AKT/mTOR signaling pathway.

Single-cell transcriptomic profiling of BALF samples provided an integrated view of pulmonary immune landscape and specific expression pattern of LACC1 during acute lung injury. Although LACC1 displayed the highest average expression within DC clusters, it concurrently exhibited substantial and heterogeneous expression across macrophage and monocyte populations. Crucially, considering the significance of AMs as critical components driving early inflammation in ALI, sub-clustering analysis was then performed and results demonstrated that LACC1 exhibited marked heterogeneity and was strongly enriched in MARCKS⁺ macrophages, CD14⁺ CD16⁺ monocytes, key components of the mononuclear phagocyte system that mediate innate immune activation and inflammatory responses [24]. Consequently, these findings suggested that while DC clusters showed relatively high baseline LACC1 expression, the pronounced and functionally relevant enrichment of LACC1 within macrophage and monocyte subsets represented the critical cellular targets mediating LACC1-driven immunometabolic regulation during ALI.

To clarify the functional role of LACC1, we constructed LPS-induced mouse model of ALI, followed by isolation and RNA sequencing of alveolar macrophages. Consistently, LACC1 expression was elevated in both murine lung tissue and primary alveolar macrophages. More importantly, conditional knockout of LACC1 in macrophages aggravated lung injury, as evidenced by more severe lung damage and higher levels of pro-inflammatory cytokines, indicating that LACC1 might exert a protective role in maintaining pulmonary immune homeostasis. In vitro experiments further indicated LACC1 knockdown in THP-1 derived macrophages enhanced LPS-induced expression of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. Mechanistically, RNA sequencing and pathway analysis uncovered that LACC1 could regulate LPAR3/PI3K/AKT/mTOR signaling pathway, which was known to play a key role in inflammatory regulation. Previous studies have highlighted that activation of PI3K/AKT signaling could attenuate inflammatory response and block the release of proinflammatory cytokines [25–27], consistent with our observations that diminished PI3K/AKT/mTOR expression under LACC1 deficiency was accompanied by enhanced inflammatory responses. Therefore, our findings suggested that LACC1 might exert its protective effect by enhancing LPAR3-mediated activation of PI3K/AKT/mTOR pathway, thereby suppressing excessive inflammatory responses in macrophages.

Most functional studies focused on the role of LACC1 in macrophages due to its predominant expression in myeloid cells [28]. Our above findings aligned with previous studies showing that LACC1 acted as a crucial molecule in several immunometabolic pathways including NOS2-linked polyamine metabolism, mitochondrial respiration, fatty acid oxidation, and autophagy, which was indispensable for maintaining macrophage homeostasis in chronic inflammatory disorders [12, 16, 17, 29]. Our work revealed that LACC1 modulated macrophage inflammatory response by sustaining LPAR3 expression and downstream PI3K/AKT/mTOR signaling, which was known to inhibit inflammatory response. Collectively, these data extended the functions of LACC1 as an essential molecule protecting against acute lung injury by maintaining LPAR3/PI3K/AKT/mTOR activity to suppress inflammation.

Our study had several limitations. Firstly, despite the observed association between LACC1 and LPAR3, precise molecular mechanism by which LACC1 regulated LPAR3 expression is not fully defined and requires further investigation. What’s more, our study primarily relied on LPS-induced ALI mouse models, which could not fully represent the heterogeneity of clinical ALI. Further validation in additional ALI models is required to reinforce the role of LACC1 in ALI. Finally, whether pharmacological activation of LACC1 or restoration of LPAR3/PI3K/AKT/mTOR signaling can achieve therapeutic benefit remains to be tested in further studies. More studies are necessary for exploring metabolic nodes within the LACC1-LPAR3 axis and evaluating their potential for macrophage-centered interventions in human ALI.

Taken together, our study demonstrated that LACC1 acted as a regulatory molecule protecting against acute lung injury by modulating inflammatory response through the LPAR3/PI3K/AKT pathway. These results not only broaden the understanding of LACC1’s biological function beyond chronic inflammatory diseases but also provide a potential therapeutic target for macrophage-centered interventions in acute pulmonary inflammation. Further studies are warranted to explore whether pharmacological activation of LACC1 or its downstream signaling can ameliorate inflammatory lung injury in clinical settings.

Conclusions

LACC1 was markedly upregulated in alveolar macrophages derived from BALFs of patients with ALI, as well as in experimental models of LPS-induced lung injury. Macrophage-specific deletion of LACC1 significantly exacerbated pulmonary inflammation and aggravated lung injury following LPS challenge. Mechanistically, LACC1 deficiency enhanced LPS-induced inflammatory responses in THP-1 and was accompanied by suppression of the LPAR3/PI3K/AKT/mTOR signaling pathway. Collectively, these findings suggested that LACC1 might play a protective role in ALI and represent a promising therapeutic target for intervention in ALI.

Supplementary Information

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Author contributions

Yiran He, Yi Huang, and Linli Wang contributed equally to this work. Yiran He, Yi Huang, and Linli Wang conceived and designed the study, performed the experiments, analyzed the data, and drafted the manuscript. Shengyu Hao, Xu Wang, Mingchen Han, and Kunlin Zhou assisted with experiments, data acquisition, and statistical analysis. Lizhen Xuan and Minjie Ju supervised the study, provided critical intellectual input, secured funding, and revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Funding

This research was supported by the Innovation Fund of Zhongshan Hospital, Fudan University (Grant No.2023-2ZSCX12), the Shanghai Public Health Talent Development Program for Outstanding Discipline Leaders (Grant No. GWVI-11.2-XD36), the Shanghai Science and Technology Commission (Grant No. 20S31905300) and the Chinese Medical Association Medical Education Research Project (Grant No. 2025B54).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Decleration

Ethics approval

All procedures involving human samples adhered to the principles of the Declaration of Helsinki (1964) and were proved by the Ethics Committee of Zhongshan Hospital, Fudan University (B2023-074R). All animal protocols were reviewed and approved by the Animal Review Committee of Zhongshan Hospital, Fudan University.

Competing interests

The authors declare no competing interests.