CD5L Inhibits Allergic Airway Inflammation by Lysophosphatidylcholine-Induced ILC2 Apoptosis

Abstract

Aims:

Group 2 innate lymphoid cells (ILC2s) play key roles in allergic asthma development. We have previously discovered that CD5 antigen-like protein (CD5L) can inhibit allergic airway inflammation. In this study, we investigate the effect of CD5L on ILC2s and the underlying mechanism.

Results:

Our findings demonstrated that CD5L suppresses allergic airway inflammation by inhibiting ILC2s. CD5L inhibited NF-κB, MAPK, and PI3K-AKT pathways in ILC2s, thus reducing interleukin (IL)-5 and IL-13 production. CD5L increased the level of lysophosphatidylcholine (lysoPC) in ILC2s through the transforming growth factor beta (TGF-β) signaling pathway. The elevated lysoPC further induced reactive oxygen species (ROS) production in ILC2s, and the increased ROS fed back to increase the level of lysoPC. The accumulated ROS induced ILC2 apoptosis. The scavenger receptor CD36 mediated the inhibitory effect of CD5L on ILC2s and allergic airway inflammation. Finally, CD5L was shown to be potential therapeutic for allergic asthma.

Innovation:

This study is the first to demonstrate that CD5L suppresses allergic airway inflammation by inhibiting ILC2 responses. It is the initial discovery that CD5L promotes ILC2 apoptosis, whereas CD5L was previously recognized as an apoptosis inhibitor. The regulation of TGF-β signaling pathway on lysoPC is demonstrated for the first time.

Conclusion:

This study demonstrated that CD5L inhibits ILC2 activation and induces ILC2 apoptosis, thereby suppressing allergic airway inflammation. CD5L can serve as a novel therapeutic strategy for allergic asthma. Antioxid. Redox Signal. 44, 393–409.

Keywords

asthma CD5 antigen-like protein group 2 innate lymphoid cells lipid metabolism lysophosphatidylcholine oxidative stress

Introduction

Allergic asthma is a chronic inflammatory airway disease. The main manifestations of asthma are airway hyperresponsiveness (AHR), chronic inflammation, and reversible airflow obstruction. Type 2 cytokines mediate asthma symptoms by promoting eosinophil recruitment, mucus overproduction, AHR, and immunoglobulin E production (Lambrecht et al., 2019). In allergic asthma, type 2 cytokines are mainly produced by type 2 T helper (Th2) cells and ILC2s. Unlike Th2 cells, ILC2s lack antigen receptors and respond rapidly to the cytokines interleukin (IL)-33, IL-25, and thymic stromal lymphopoietin (TSLP), which are primarily produced by epithelial cells. Hence, ILC2s are indicators of early-stage initiation of type 2 immunity (Halim et al., 2016). Therefore, regulation of ILC2 homeostasis is an important therapeutic strategy to allergic asthma.

Many studies have demonstrated that lipid metabolism is significantly altered in asthma. Abnormal lipid metabolism, especially lipid mediator synthesis, is closely related to the development of asthma (Leuti et al., 2020; Samuchiwal and Boyce, 2018). As for ILC2s, fatty acids (FAs) serve as the major source of energy production (Wilhelm et al., 2016). However, some lipid mediators play roles in the negative regulation of ILC2s. For instance, prostaglandin E2 suppresses ILC2 activation by decreasing the expression of GATA binding protein 3 (GATA3) and ST2 (Zhou et al., 2018). Prostaglandin I2 can also inhibit type 2 cytokine production in ILC2s by activating the AC/cAMP/PKA pathway (Zhou et al., 2016). Therefore, lipid metabolism is tightly linked to ILC2 proliferation and activation as well as allergic asthma development, although the underlying mechanism remains to be fully elucidated.

CD5L, a member of the scavenger receptor cysteine-rich superfamily, is produced primarily by mature macrophages (Martinez et al., 2014). CD5L has been reported to participate in many diseases, including cancer (LaFargue et al., 2023; Li et al., 2011; Lu et al., 2024; Sanchez-Moral et al., 2023), infectious diseases (Martinez et al., 2014; Oliveira et al., 2024; Stalenhoef et al., 2018), and atherosclerosis (Arai et al., 2005). Recently, CD5L has been demonstrated to be effective in the treatment of sepsis (Oliveira et al., 2024). Multiple studies have also indicated that CD5L can regulate lipid metabolism (Iwamura et al., 2012; Sanjurjo et al., 2015; Wang et al., 2015). For instance, CD5L could transform Th17 cells from pathogenic to nonpathogenic cells by modulating the intracellular lipidome (Wang et al., 2015). Our previous results indicated that CD5L can increase the number of CD11c^high alveolar macrophages and attenuate allergic airway inflammation (Weng et al., 2022). However, it remains unclear whether CD5L inhibits allergic asthma by acting on ILC2s.

In this study, we demonstrated that CD5L inhibited allergic airway inflammation by inhibiting ILC2 responses. CD5L inhibited NF-κB, MAPK, and PI3K-AKT pathways in ILC2s, thus decreasing the production of IL-5 and IL-13. CD5L treatment increased the level of lysoPC through the TGF-β signaling pathway. The upregulated lysoPC induced oxidative stress in ILC2s, thereby inducing ILC2 apoptosis. The inhibitory effect of CD5L on ILC2s and allergic airway inflammation was mediated by scavenger receptor CD36. In addition, CD5L exhibited therapeutic potential for allergic asthma.

Innovation

ILC2s play a vital role in asthma. For the first time, this study reveals that CD5L restrains allergic airway inflammation by suppressing ILC2 responses. CD5L inhibits ILC2 activation and promotes ILC2 apoptosis; the inductive effect of CD5L on apoptosis is first discovered. The regulation of TGF-β signaling pathway on lysoPC is demonstrated for the first time. This study further demonstrates that CD5L has therapeutic potential for the treatment of allergic airway inflammation (Fig. 8).

Results

CD5L inhibits ILC2s and restrains allergic airway inflammation

Our previous study had indicated that CD5L can inhibit allergic airway inflammation (Weng et al., 2022). In this study, we used an inflammation mouse model using rmIL-33 combination with rmCD5L or PBS to further evaluate the effect of CD5L on ILC2s in vivo (Fig. 1A). As expected, rmCD5L treatment significantly decreased ILC2 number as well as the production of IL-5 and IL-13 (Fig. 1B–E). In addition, the number of eosinophils was also decreased by rmCD5L treatment (Fig. 1F). Meanwhile, rmCD5L treatment significantly attenuated inflammatory cell infiltration and mucus secretion (Fig. 1G,H). Altogether, these data indicated that CD5L can decrease the number and activation of ILC2s and inhibit airway inflammation in vivo.

FIG. 1.

CD5L inhibits ILC2s and restrains allergic airway inflammation. (A) Establishment of IL-33-induced allergic asthma and treated with rmCD5L using C57BL/6 mice. (B) Percentage and number of ILC2s in the indicated lungs. (C–E) Percentage and number of IL-5⁺ILC2s (C), IL-13⁺ILC2s (D), and IL-5⁺IL-13⁺ILC2s (E). (F) Percentage and number of eosinophils in the indicated lungs. (G) H&E and PAS staining of the indicated lung tissue sections. Scale bar: 100 µm. (H) The inflammation and PAS score of lungs. (I) Representative histogram of the expression of IL-5 and IL-13 in ILC2s detected by flow cytometry. (J) MFI of IL-5 and IL-13 in ILC2s. (K) Flow cytometric analysis of ILC2 apoptosis, and representative plots were shown. (L) Percentage of AnnexinV⁺PI⁻ILC2s (left), AnnexinV⁺PI⁺ILC2s (median), and AnnexinV⁺ILC2s (right). (M) IL-5 (left) and IL-13 (right) levels in the supernatant. For A–H, n = 4–6, per group. For I–M, n = 3, per group. The data are representative of three independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM. CD5L, CD5 antigen-like protein; ILC2s, group 2 innate lymphoid cells; IL, interleukin; H&E, hematoxylin and eosin; PAS, periodic acid–Schiff; MFI, mean fluorescence intensity.

We next examined whether CD5L can directly inhibit lung ILC2 responses. Lung ILC2s were sorted by flow cytometry as lineage⁻ST2⁺CD127⁺CD90.2⁺and cultured in the presence or absence of rmCD5L for 3 days. We found that the production of IL-5 and IL-13 by ILC2s was significantly decreased by rmCD5L treatment (Fig. 1I,J). CD5L is also known as apoptosis inhibitor expressed by macrophages; therefore, we detected the effect of CD5L on ILC2 apoptosis. Interestingly, rmCD5L treatment promoted the apoptosis of ILC2s as shown by Annexin-V/PI staining (Fig. 1K,L). The secretion of IL-5 and IL-13 in supernatant was also decreased by rmCD5L treatment (Fig. 1M). These results indicated that CD5L directly decreases the production of IL-5 and IL-13 in ILC2s and promotes ILC2 apoptosis in vitro.

CD5L ameliorates ILC2-dependent airway inflammation

To exclude the affection of adaptive immunity, we further determined the effect of CD5L on asthma using Rag2^−/− mice that lack any mature B and T cells (Fig. 2A). Treatment with rmCD5L decreased the number of ILC2s and production of IL-5 and IL-13 in ILC2s (Fig. 2B–E). Although the percentage of IL-5⁺IL-13⁺ILC2s was comparable between IL-33- and IL-33+CD5L-treated mice, the absolute numbers were decreased by rmCD5L treatment (Fig. 2E). We also detected eosinophils in lungs and found that the numbers were decreased by rmCD5L treatment (Fig. 2F). Hematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining showed that the inflammatory cell infiltration and mucus secretion were both decreased by rmCD5L treatment (Fig. 2G,H). These results indicated that CD5L can inhibit ILC2s and IL-33-driven airway inflammation in the absence of adaptive immunity.

FIG. 2.

CD5L ameliorates ILC2-dependent airway inflammation. (A) Rag2^−/− mice were used to establish IL-33-induced allergic asthma and treated with rmCD5L. (B) Percentage and number of ILC2s in the indicated lungs. Lung lymphocytes were fractionated by Percoll. (C–E) Percentage and number of IL-5⁺ILC2s (C), IL-13⁺ILC2s (D), and IL-5⁺IL-13⁺ILC2s (E) in the indicated lungs. Lung lymphocytes were fractionated by Percoll. (F) Percentage and number of eosinophils in the indicated lungs. (G) H&E and PAS staining of the indicated lung tissue sections. Scale bar: 100 µm. (H) The inflammation and PAS score of lungs. For all experiments, n = 4–5 per group. The data are representative of three independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001. Data are represented as mean ± SEM.

CD5L suppresses the activation of ILC2s

GATA3 is the master regulator for transcription of the Il5 and Il13 genes (Klein Wolterink et al., 2013; Thio et al., 2018; Yagi et al., 2014); given the results that rmCD5L treatment decreased the production of IL-5 and IL-13 in ILC2s (Fig. 1I,J), we detected the expression of GATA3 in ILC2s by flow cytometry. As expected, the expression of GATA3 was decreased after rmCD5L treatment (Fig. 3A,B). Previous studies have shown that NF-κB (Guo et al., 2012; Kabata et al., 2018; Wang et al., 2021), PI3K/AKT (Bartemes and Kita, 2021; Helou et al., 2022; Sakano et al., 2024), MAPK (He et al., 2022; Lei et al., 2018; Petrova et al., 2020; Suzuki et al., 2015; Yamamoto et al., 2018), and AMPK pathways can regulate the stability of GATA3 (Wang et al., 2021) and are principal regulators of ILC2 functions. Therefore, we analyzed the effects of CD5L on these pathways in ILC2s by flow cytometry. The results showed that rmCD5L treatment decreased the expression of phosphorylation of p65 (p-p65), phosphorylation of p38 (p-p38), phosphorylation of ERK (p-ERK), and phosphorylation of AKT (p-AKT) (Fig. 3C–J), whereas the expression of phosphorylation of AMPKα (p-AMPKα) was not affected (Fig. 3K,L). These results indicated that CD5L can regulate NF-κB, MAPK, and PI3K-AKT pathways in ILC2s, thus inhibiting the production of IL-5 and IL-13.

FIG. 3.

CD5L suppresses the activation of ILC2s. (A) Representative histogram of the expression of GATA3 in ILC2s detected by flow cytometry. (B) MFI of GATA3 in ILC2s. (C) Representative histogram of the expression of p-p65 in ILC2s detected by flow cytometry. (D) MFI of p-p65 in ILC2s. (E) Representative histogram of the expression of p-p38 in ILC2s detected by flow cytometry. (F) MFI of p-p38 in ILC2s. (G) Representative histogram of the expression of p-ERK in ILC2s detected by flow cytometry. (H) MFI of p-ERK in ILC2s. (I) Representative histogram of the expression of p-AKT in ILC2s detected by flow cytometry. (J) MFI of p-AKT in ILC2s. (K) Representative histogram of the expression of p-AMPKα in ILC2s detected by flow cytometry. (L) MFI of p-AMPKα in ILC2s. For all experiments, n = 3, per group. The data are representative of three independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01. Data are represented as mean ± SEM. GATA3, GATA binding protein 3; p-p65, phosphorylation of p65; p-p38, phosphorylation of p38; p-ERK, phosphorylation of ERK; p-AKT, phosphorylation of AKT; p-AMPKα, phosphorylation of AMPKα.

Upregulated lysoPC mediates CD5L-induced ILC2 apoptosis

Metabolites are central to the development and function of cells. To investigate the mechanism by which CD5L induces ILC2 apoptosis, we isolated pulmonary ILC2s and treated them with or without rmCD5L for 3 days. Subsequently, we performed metabolomics analysis to quantify the metabolite landscape. Principal component analysis (PCA) based on all metabolites revealed a clear separation of PBS-treated ILC2s from CD5L-treated ILC2s (Fig. 4A). An total of 1033 upregulated and 641 downregulated metabolites were induced by rmCD5L treatment and were enriched in lipids and lipid-like molecules (35.59%) (Fig. 4B,C). Further analysis of lipids and lipid-like molecules showed a significant increase in lysoPC levels after rmCD5L treatment, including lysoPC (22:5(4Z,7Z,10Z,13Z,16Z)) and lysoPC (20:3(5Z,8Z,11Z)) (Fig. 4D,E). As lysoPC is primarily derived from the turnover of phosphatidylcholine by phospholipase A2 (PLA2), we detected the activity of PLA2 in ILC2s. The results showed that rmCD5L treatment increased the activity of cytosolic PLA2 (cPLA2) (Fig. 4F), whereas the activities of secreted PLA2 and calcium-independent PLA2 were low in ILC2s and showed no significant difference after rmCD5L treatment (Fig. 4G,H). These results indicated that CD5L can increase the activity of cPLA2, thus increasing the level of lysoPC.

FIG. 4.

Upregulated lysoPC mediates CD5L-induced ILC2 apoptosis. (A) PCA of LC-MS/MS metabolomics data of ILC2s. (B) Comparison of total differentially regulated metabolites. (C) Total differentially regulated metabolites. (D) Heat map diagrams of top 10 upregulated and downregulated lipids and lipid-like molecules. (E) LysoPC levels detected by metabolomics. (F) Activity of cPLA2 in ILC2s. (G) Activity of sPLA2 in ILC2s. (H) Activity of iPLA2 in ILC2s. (I) ILC2 apoptosis detected by flow cytometry and representative plots were shown. (J) Percentage of AnnexinV⁺PI⁻ILC2s (left), AnnexinV⁺PI⁺ILC2s (median), and AnnexinV⁺ILC2s (right). (K) Flow cytometric analysis of ILC2 apoptosis. Representative plots were shown. (L) Percentage of AnnexinV⁺PI⁻ILC2s (left), AnnexinV⁺PI⁺ILC2s (median), and AnnexinV⁺ILC2s (right). For all experiments, n = 3, per group. The data are representative of three independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM. lysoPC, lysophosphatidylcholine; PCA, principal component analysis; LC-MS/MS, liquid chromatography -tandem mass spectrometry; cPLA2, cytosolic phospholipase A2; sPLA2, secreted PLA2; iPLA2, calcium-independent PLA2.

To investigate the role of lysoPC in CD5L-induced ILC2 apoptosis, we treated cultured ILC2s with lysoPC and found that lysoPC significantly increased the apoptosis of ILC2s (Fig. 4I,J). Furthermore, we treated the cells with arachidonyl trifluoromethyl ketone (AACOCF3), an inhibitor of PLA2, and found that AACOCF3 partially reversed the induction of ILC2 apoptosis by CD5L (Fig. 4K,L). Taken together, these results demonstrated that CD5L upregulates lysoPC levels, thereby inducing ILC2 apoptosis.

CD5L induces oxidative stress in ILC2s via upregulating lysoPC through the TGF-β signaling pathway

To further explore the mechanism of CD5L-mediated increased ILC2 apoptosis, RNA sequencing (RNA-Seq) was performed on PBS- and CD5L-treated ILC2s. PCA revealed that rmCD5L treatment resulted in distinct transcriptomic states (Fig. 5A). A total of 665 genes were differentially expressed, including 432 upregulated genes and 233 downregulated genes (Fig. 5B). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that these differentially expressed genes were enriched in biological processes, including TGF-β signaling pathway (Fig. 5C). Consistent with the KEGG results, the phosphorylation of Smad2, a key marker of TGF-β pathway activation, was strongly induced by CD5L (Fig. 5D,E). These results showed that rmCD5L treatment activates TGF-β signaling pathway in ILC2s.

FIG. 5.

CD5L induces oxidative stress in ILC2s via upregulating lysoPC through the TGF-β signaling pathway. (A) PCA of RNA-Seq data of ILC2s. (B) Comparison of differentially expressed genes. (C) KEGG pathway enrichment of differentially expressed genes. (D) Representative histogram of the expression of p-Smad2 in ILC2s detected by flow cytometry. (E) MFI of p-Smad2 in ILC2s. (F) The concentration of lysoPC in the indicated ILC2 culture supernatant (left) and the indicated ILC2 lysates (right). (G) Activity of cPLA2 in ILC2s. (H) The concentration of lysoPC in the indicated ILC2 culture supernatant (left) and the indicated ILC2 lysates (right). (I) Activity of cPLA2 in ILC2s. (J) Heat map diagrams of oxidative stress-related genes. (K) MFI of the indicated ROS⁺ILC2s. (L) Flow cytometric analysis of ILC2 apoptosis and representative plots were shown. (M) Percentage of AnnexinV⁺PI⁻ILC2s (left), AnnexinV⁺PI⁺ILC2s (median), and AnnexinV⁺ILC2s (right). (N) MFI of the indicated ROS⁺ILC2s. (O) MFI of the indicated ROS⁺ILC2s. (P) Flow cytometric analysis of ILC2 apoptosis and representative plots were shown. (Q) Percentage of AnnexinV⁺PI⁻ILC2s (left), AnnexinV⁺PI⁺ILC2s (median), and AnnexinV⁺ILC2s (right). (R) The concentration of lysoPC in the indicated ILC2 culture supernatant (left) and the indicated ILC2 lysates (right). (S) The concentration of lysoPC in the indicated ILC2 lysates (left) and the indicated ILC2 culture supernatant (right). (T) MFI of the indicated ROS⁺ILC2s. For all experiments, n = 3, per group. The data are representative of three independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM. TGF-β, transforming growth factor beta; RNA-Seq, RNA sequencing; KEGG, Kyoto Encyclopedia of Genes and Genomes; p-Smad2, phosphorylation of Smad2.

To investigate the role of TGF-β signaling pathway on the increased lysoPC by CD5L, we first treated cultured lung ILC2s with rmTGF-β and detected the level of lysoPC and the activity of cPLA2. We found that rmTGF-β treatment increased the level of lysoPC and the activity of cPLA2 (Fig. 5F,G). In addition, we further determined the role of TGF-β signaling pathway by the addition of LY2157299, a TGF-β receptor type I (TGF-βRI) kinase inhibitor. The results showed that LY2157299 pretreatment partially reversed the increased effects of rmCD5L on lysoPC levels and cPLA2 activity (Fig. 5H,I). Taken together, these results indicated that CD5L increases lysoPC levels in ILC2s through the TGF-β signaling pathway.

Previous studies have shown that elevated lysoPC can induce oxidative stress (Colles and Chisolm, 2000; Hsu et al., 2011; Sueajai et al., 2022; Yasunari et al., 2001). Consistent with this, our RNA-Seq results indicated altered expression of oxidative stress-related genes following rmCD5L treatment (Fig. 5J). To confirm RNA-seq results, we examined intracellular reactive oxygen species (ROS) production in ILC2s by flow cytometry. The results showed that rmCD5L treatment significantly increased ROS levels in ILC2s (Fig. 5K). To determine whether CD5L exerted its inhibitory effect on ILC2s by inducing oxidative stress, we pretreated ILC2s with ROS scavenger N-acetyl-L-cysteine (NAC). The results indicated that ROS scavenging partially reversed the induction of ILC2 apoptosis by rmCD5L treatment (Fig. 5L,M). These observations collectively demonstrated that CD5L induces ILC2 apoptosis by inducing oxidative stress.

To determine the function of lysoPC in CD5L-induced oxidative stress in ILC2s, we first treated cultured lung ILC2s with lysoPC and detected ROS levels. LysoPC significantly increased the level of ROS in ILC2s (Fig. 5N). We then treated lung ILC2s with AACOCF3 and found that ablation of lysoPC with AACOCF3 reduced the elevated level of ROS induced by rmCD5L treatment (Fig. 5O). Consistent with these findings, NAC pretreatment partially reversed the induction of ILC2 apoptosis by lysoPC (Fig. 5P,Q). Taken together, these results showed that CD5L induces oxidative stress in ILC2s via upregulating lysoPC.

Previous studies have shown that the elevated ROS can increase the level of lysoPC (Aoto et al., 2009; Nandi et al., 2017; Yasuda et al., 1999). Therefore, we hypothesized that the increased ROS by rmCD5L treatment may also regulate lysoPC levels in ILC2s. We pretreated ILC2s with NAC and detected the level of lysoPC in ILC2s. The results showed that ROS scavenging partially reversed the inductive effects of CD5L on the lysoPC levels (Fig. 5R). These results collectively demonstrated that CD5L-induced elevation of lysoPC and ROS may mutually feedback regulate their levels. Furthermore, we conducted a time course study to detect the induction of lysoPC and ROS by rmCD5L treatment. The results showed that after rmCD5L treatment, the level of lysoPC showed a significant increase at 3 h, whereas the level of ROS did not increase until 4 h in ILC2s (Fig. 5S,T), indicating that lysoPC accumulation preceded the increase in ROS after rmCD5L treatment in ILC2s.

CD5L inhibits ILC2s through scavenger receptor CD36

CD5L colocalizes with scavenger receptor CD36 on the surface of adipocytes (Kurokawa et al., 2010). The functions of CD5L are mainly mediated by its regulation of macrophages, hepatocytes, and other cells via CD36 (Kurokawa et al., 2010; Nemoto et al., 2023; Sanjurjo et al., 2015). Importantly, ILC2s can also express CD36 (Fali et al., 2021). To investigate whether CD36 serves as the receptor for CD5L acting on ILC2s, we first analyzed the effect of CD5L on CD36 expression in ILC2s. We found that CD5L-treated ILC2s had higher CD36 expression both in vivo and in vitro, including the percentage and mean fluorescence intensity (MFI) of CD36⁺ILC2s (Fig. 6A,B). To investigate the role of CD36 in the inhibitory effects of CD5L on airway inflammation and ILC2s, we first evaluated the effects of CD5L using Cd36^−/−mice (Fig. 6C). As expected, rmCD5L treatment decreased eosinophil numbers, ILC2 numbers, the production of IL-5 and IL-13 in ILC2s, inflammatory cell infiltration, and mucus secretion in IL-33-treated wild-type (WT) mice (Fig. 6D–K). However, in Cd36^−/−mice, the number of eosinophils and ILC2s, the activation of ILC2s as well as the inflammatory cell infiltration and mucus secretion showed no significant difference between IL-33-treated mice and IL-33+CD5L-treated mice (Fig. 6D–K). Furthermore, we sorted Cd36^−/−ILC2s from lung of Cd36^−/−mice and treated with rmCD5L in vitro. The results showed that the inhibition of IL-5 and IL-13 production and induction of ILC2 apoptosis by rmCD5L treatment were diminished in Cd36^−/−ILC2s (Fig. 6L–O). Besides, we treated cultured lung ILC2s from WT mice with the irreversible CD36 inhibitor sulfosuccinimidyl oleate (SSO), and the results showed that SSO treatment partially reversed the inhibitory effects of rmCD5L on the production of IL-5 and IL-13 as well as the induction of ILC2 apoptosis (Supplementary Fig. S1A-C). Taken together, these results suggested that CD5L suppresses ILC2 responses through CD36.

FIG. 6.

CD5L inhibits ILC2s through scavenger receptor CD36. (A) Percentage and MFI of CD36⁺ILC2s in the indicated lungs. (B) Percentage and MFI of CD36⁺ILC2s cultured in vitro. (C) Establishment of IL-33-induced allergic asthma and treated with rmCD5L using Cd36^−/−mice. (D) Percentage and number of eosinophils in the indicated lungs. (E) Percentage and number of ILC2s in the indicated lungs. (F–H) Percentage and number of IL-5⁺ILC2s (F), IL-13⁺ILC2s (G), and IL-5⁺IL-13⁺ILC2s (H). (I) H&E staining of the indicated lung tissue sections. Scale bar: 100 µm. (J) PAS staining of the indicated lung tissue sections. Scale bar: 100 µm. (K) The inflammation and PAS score of lungs. (L) MFI of IL-5 and IL-13 in ILC2s detected by flow cytometry. (M) Flow cytometric analysis of ILC2 apoptosis and representative plots were shown. (N) Percentage of AnnexinV⁺PI⁻ILC2s (left), AnnexinV⁺PI⁺ILC2s (median), and AnnexinV⁺ILC2s (right). (O) IL-5 (left) and IL-13 (right) levels in the supernatant. (P) MFI of GATA3, p-p65, p-p38, p-ERK, p-AKT, and p-AMPKα in ILC2s. (Q) MFI of p-Smad2 in ILC2s. (R) The concentration of lysoPC in the indicated ILC2 lysates (left) and the indicated ILC2 culture supernatant (right). For A and C–K, n = 4–5 per group. For B and L–R, n = 3, per group. The data are representative of three independent experiments. ns: nonsignificant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM.

To further determine the role of CD36, we detected the expression of GATA3, p-p65, p-p38, p-ERK, p-AKT, and p-AMPKα in Cd36^−/−ILC2s. The results showed that, in Cd36^−/−ILC2s, the expression of GATA3, p-p65, p-p38, p-ERK, p-AKT, and p-AMPKα was comparable between PBS- and CD5L-treated groups (Fig. 6P). These results indicated that the inhibitory effect of CD5L on NF-κB, MAPK, and PI3K-AKT pathways in ILC2s is dependent on CD36. Furthermore, we detected the phosphorylation of Smad2 in Cd36^−/−ILC2s. The results showed that, in contrast to ILC2s from WT mice, CD5L cannot increase the phosphorylation of Smad2 in Cd36^−/−ILC2s (Fig. 6Q), which indicated that the activation of the TGF-β signaling pathway by CD5L in ILC2s is dependent on CD36. Meanwhile, unlike ILC2s from WT mice, rmCD5L treatment did not affect the level of lysoPC in cells and supernatant of Cd36^−/−ILC2s (Fig. 6R). These results collectively demonstrated that the inhibitory effect of CD5L on ILC2s is mediated by CD36.

CD36 is a scavenger receptor involved in lipid metabolism such as fatty acid translocation and uptake (Chen et al., 2022; Pepino et al., 2014). Since CD5L increased the expression of CD36 in ILC2s both in vivo and in vitro, we investigated the effect of rmCD5L on fatty acid uptake. ILC2s were stained with BODIPY FL C16 to measure exogenous fatty acid uptake. We found that rmCD5L treatment led to an increase in fatty acid uptake (Supplementary Fig. S2A,B). Excess free fatty acids (FFAs) produce harmful bioactive lipids and disrupt mitochondrial membrane integrity. This phenomenon is known as lipotoxicity. Cells counteract this potential damage by formatting lipid droplets (LDs) to sequester FFAs (Chitraju et al., 2017). We detected LDs using BODIPY 493/503 and found that rmCD5L decreased the formation of LDs (Supplementary Fig. S2C,D). The above results showed that rmCD5L increased fatty acid uptake in ILC2s and decreased LD formation. Therefore, we hypothesized that rmCD5L could induce lipotoxicity, thereby inhibiting production of IL-5 and IL-13 and increasing ILC2 apoptosis.

CD5L is a potential therapeutic for allergic asthma

Our previous study has showed that rmCD5L treatment can inhibit allergic airway inflammation induced by house dust mite (HDM), a clinically relevant allergy (Weng et al., 2022). Here, we further evaluate the effects of CD5L on ILC2s in HDM-induced allergic asthma (Fig. 7A). The number of ILC2s in lungs was significantly reduced by rmCD5L treatment (Fig. 7B). Meanwhile, the activation of ILC2s was also inhibited by rmCD5L as demonstrated by the decreased number of IL-5⁺ILC2s (Fig. 7C), IL-13⁺ILC2s (Fig. 7D), and IL-5⁺IL-13⁺ILC2s (Fig. 7E).

FIG. 7.

CD5L is a potential therapeutic for allergic asthma. (A) Establishment of HDM-induced allergic asthma and treated with rmCD5L using BALB/c mice. (B) Percentage and number of ILC2s in the indicated lungs. (C–E) Percentage and number of IL-5⁺ILC2s (E), IL-13⁺ILC2s (D), and IL-5⁺IL-13⁺ILC2s (E). (F) Establishment of therapeutic models using C57BL/6 mice. (G) Percentage and number of eosinophils in the indicated lungs. (H) Percentage and number of ILC2s in the indicated lungs. (I–K) Percentage and number of IL-5⁺ILC2s (I), IL-13⁺ILC2s (J), and IL-5⁺IL-13⁺ILC2s (K). (L) H&E and PAS staining of the indicated lung tissue sections. Scale bar: 100 µm. (M) The inflammation and PAS score of lungs. (N) Establishment of humanized mice model using Rag2^−/−Il2rg^−/−mice. (O) Number of ILC2s in the indicated lungs. (P) Number of eosinophils in the indicated lungs. (Q) H&E and PAS staining of lung tissue sections. Scale bar: 100 µm. (R) The inflammation and PAS score of lungs. For all experiments, n = 4–5, per group. The data are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are represented as mean ± SEM. HDM, house dust mite.

To investigate the therapeutic effect of CD5L for allergic asthma, we used an inflammation mouse model using rmIL-33 and administered rmCD5L for three consecutive days after the last treatment of rmIL-33 (Fig. 7F). The effects of CD5L on airway inflammation and ILC2 responses were detected. The results showed that rmCD5L treatment significantly decreased the number of ILC2s and eosinophils in lungs as well as the production of IL-5 and IL-13 in ILC2s (Fig. 7G,K). Meanwhile, the inflammatory cell infiltration and mucus secretion were both decreased by rmCD5L treatment (Fig. 7L,M). These results demonstrated that CD5L exerts therapeutic effects against IL-33-induced airway inflammation.

Pushing for more translational potential, we further evaluated the effect of CD5L in humanized mice. We isolated ILC2s from human peripheral blood mononuclear cells (PBMCs) of healthy donors and adoptively transferred to Rag2^−/−Il2rg^−/− mice that lack T cells, B cells, NK cells, and ILCs. Subsequently, the humanized mice were employed an inflammation model using rhIL-33 and treated with rhCD5L (Fig. 7N). Strikingly, the number of ILC2s in lungs was significantly reduced in rhCD5L-treated mice (Fig. 7O). Meanwhile, rhCD5L treatment significantly decreased the number of eosinophils in lungs (Fig. 7P). Histological analysis revealed fewer inflammatory cells and reduced mucus secretion in rhCD5L-treated mice (Fig. 7Q,R). Altogether, these results suggested that CD5L has the potential to inhibit airway inflammation in patients.

Discussion

In this study, we demonstrated for the first time the therapeutic potential of CD5L for allergic asthma by acting on ILC2s. CD5L inhibited NF-κB, MAPK, and PI3K-AKT pathways in ILC2s, thus decreasing IL-5 and IL-13 production. CD5L increased the level of lysoPC through the TGF-β signaling pathway. The elevated lysoPC induced oxidative stress and promoted ILC2 apoptosis. CD5L inhibited ILC2s and allergic airway inflammation through the scavenger receptor CD36. Finally, we demonstrated that CD5L can potentially serve as a viable therapeutic option to treat allergic asthma.

Many studies suggest the important role of ILC2s in allergic asthma. Furthermore, ILC2s are considered as a promising target for asthma treatment. Previous studies have reported that serum CD5L levels were significantly lower in asthmatic children (Gao et al., 2020). In addition, serum CD5L levels were negatively correlated with disease progression, which increased following treatment (Gao, 2021). Our previous work has demonstrated the inhibitory effect of CD5L on allergic airway inflammation in HDM-induced allergic asthma mice through CD11c^high macrophages (Weng et al., 2022). In this study, we demonstrated that CD5L inhibits ILC2s, including apoptosis induction and decreased IL-5 and IL-13 levels, thereby restraining allergic airway inflammation in IL-33-induced allergic asthma model. CD5L was originally discovered as a macrophage apoptosis inhibitor, but our study indicated that CD5L can promote ILC2 apoptosis. This discrepancy may be caused by the different cells. CD5L has also been reported to inhibit B lymphocyte proliferation in combination with TGF-β1 (Yusa et al., 1999). This is the initial discovery of the inductive effect on apoptosis of CD5L in ILC2s.

Metabolites are biochemical intermediates and end products of metabolic pathways. Metabolic reprogramming caused by abnormal metabolites displays a profound effect on the immune response and disease progression. Lipid metabolism plays an important regulatory role in a variety of diseases, including asthma. Previous studies have demonstrated altered lipid levels in the airway epithelium and sputum of asthma patients, correlating with disease stratification (Brandsma et al., 2023; Johnson et al., 2022). This suggests that lipids are closely related to the pathological process of asthma. In addition, studies have also shown that some lipid components and lipid metabolism regulators can influence the onset and progression of asthma, such as the commonly used leukotriene regulator Zafirlukast (Gusach et al., 2019; Hjelmsø et al., 2020; Pein et al., 2018). CD5L plays an important regulatory role in various diseases by regulating lipid metabolism in cells (Iwamura et al., 2012; Sanjurjo et al., 2015; Wang et al., 2015). However, its role in ILC2s has not been described. Our metabolomics results showed that CD5L significantly alters the lipid metabolism of ILC2s.

LysoPC and its synthetic analogs are bioactive lysolipids that display diverse biological and cytotoxic effects that are highly dependent upon cell type, degree of cell maturation, and exposure time (Zhang et al., 2023). It has been observed that the level of lysoPC (15:0) was different in the lung tissue and plasma of mice with asthma (Yu et al., 2017), indicating a potential involvement for lysoPC in asthma. However, the role of lysoPC in asthma has not been described. Our metabolomics results showed increased levels of lysoPC (22:5) and lysoPC (20:3) in ILC2s following rmCD5L treatment, indicating the regulatory capacity of CD5L on lysoPC. Further experiments showed that lysoPC treatment induced ILC2 apoptosis. Conversely, the absence of lysoPC partially reversed the inductive effects of CD5L on ILC2 apoptosis. Therefore, we demonstrated that the ILC2 apoptosis induced by CD5L was dependent on lysoPC.

However, the regulatory mechanism of intracellular lysoPC content remains to be fully elucidated. Our RNA-seq results indicated that the TGF-β signaling pathway was altered by rmCD5L treatment. A previous study demonstrated that CD5L can inhibit B cell proliferation, which requires pretreatment of cells with TGF-β1 (Yusa et al., 1999). Our results showed that rmTGF-β treatment increased the level of lysoPC and activity of cPLA2, whereas inhibiting TGF-β signaling pathway reduced the increase in lysoPC level and cPLA2 activity by CD5L. Current studies suggest both positive and negative effects of TGF-β on ILC2s. Rigas et al. have reported that TGF-β suppressed IL-5 and IL-13 production of ILC2s (Rigas et al., 2017), and Ogasawara et al. have also reported the inhibitory effect on IL-4, IL-5, and IL-13 production of human ILC2s (Ogasawara et al., 2018). However, Denney et al. showed that TGF-β enhanced ILC2 migration (Denney et al., 2015). Our results supported an inhibitory effect of TGF-β on ILC2s. In addition, in this study, we revealed the regulation of lysoPC by the TGF-β signaling pathway for the first time.

ILC2s exhibit context-dependent metabolism that primarily relies on FA metabolism (Karagiannis et al., 2020; Wilhelm et al., 2016). However, FFA accumulation can induce lipotoxicity and affect cell behaviors via multiple processes. These include activation of signaling cascades and death receptors, endoplasmic reticulum stress, modification of mitochondrial function, and oxidative stress (Marra and Svegliati-Baroni, 2018). LDs prevent lipotoxicity of externally acquired FFAs. The important role of LDs in ILC2s has been revealed (Karagiannis et al., 2020). Our results showed that rmCD5L treatment increases the uptake of FFAs of ILC2s, but inhibits the formation of LDs. Thus, these results further revealed the inhibitory mechanism of CD5L on ILC2 responses.

Overproduction of H₂O₂ and other ROS induces oxidative stress. It can lead to cellular damage, which in turn leads to a disorder in organ system. Therefore, it is crucial for normal cells to maintain ROS homeostasis and overcome the potential toxicity of ROS. In PM2.5-induced pulmonary inflammation, the upregulation of the Cd5l gene expression and oxidative stress have been observed (Wang et al., 2019). However, the interrelationships between CD5L and oxidative stress have not been revealed. In this study, we demonstrated that CD5L induces oxidative stress in ILC2s. Lipids play an important role in oxidative stress (Astudillo et al., 2023; Pokharel et al., 2023). Some FAs accumulation can induce oxidative stress (Palomer et al., 2018; Xu et al., 2020). Meanwhile, the excessive ROS induces lipid peroxidation, leading to irreversible cell damage and death. It has been reported that lysoPC can induce oxidative stress in vascular smooth muscle cells (Yoon et al., 2012). In this study, we showed that lysoPC treatment increases the level of ROS in ILC2s. Meanwhile, the deletion of ROS partially reversed the induction of ILC2 apoptosis by lysoPC. Therefore, our study revealed that CD5L induced oxidative stress by upregulating lysoPC level, thereby inducing ILC2 apoptosis. However, the deeper mechanisms need to be further explored. Oxidative stress-induced lipid accumulation is mediated by LD homeostasis, which sequesters vulnerable unsaturated triglycerides into LDs to prevent further peroxidation (Zhang et al., 2024). Our results suggested that CD5L treatment reduced LDs in ILC2s that may further exacerbate oxidative stress-induced lipid metabolism disorder and cell damage.

In summary, our findings demonstrated that CD5L suppressed allergic airway inflammation by inhibiting ILC2 responses. These results highlighted the therapeutic potential of CD5L for allergic asthma.

FIG. 8.

A working model of CD5L regulates ILC2s and inhibits allergic airway inflammation.

Materials and Methods

Mice

WT C57BL/6 and BALB/c mice were purchased from the Animal Experimental Center of Chongqing Medical University. Rag2-deficient (Rag2^−/−) mice and Cd36^−/−mice were purchased from Cyagen Biosciences. Rag2/Il2rg double knockout (Rag2^−/−Il2rg^−/−) mice were obtained from the Children’s Hospital of Chongqing Medical University. For all experiments, female mice aged 6–8 weeks, weight 16–18 g, were used. Mice were housed in specific pathogen-free conditions. Mouse experimental procedures were performed in accordance with the Guide for the Care and use of Laboratory animals and approved by the Institutional Animal Care and Use Committee at Chongqing Medical University (Approval No. IACUC-CQMU-2023-0034).

Models of Mouse Allergic Airway Inflammation

For IL-33-induced airway inflammation model, each mouse was intranasally treated with 0.5 μg rmIL-33 (Biolegend) for 3 consecutive days; rmCD5L (0.67 μg/mouse, Biolegend) or PBS was intranasally administered during rmIL-33 treatment. After 24 h, mice were sacrificed. For HDM-induced airway inflammation model, mice were intranasally sensitized with 1 μg HDM (Greer Laboratories) on day 0. From days 7 to 11, the mice were subsequently challenged with 10 μg HDM intranasally. The mice were administered with rmCD5L (0.67 μg/mouse) or PBS intranasally on days 7, 9, and 11. Mice were sacrificed and analyzed at day 14. For therapeutic models, rmCD5L (0.67 μg/mouse, Biolegend) or PBS was intranasally administered thrice after the inflammation models were established. After 24 h, mice were sacrificed.

Acquisition of lung single cell suspension

Mouse lungs were obtained, minced, and digested with 1 mg/mL type IV collagenase (Solarbio) and 5 U/mL DNase I (Beyotime Biotechnology) in complete RPMI-1640 medium for 40 min. Following this, crude tissues were passed through 70 µm cell strainers and then washed with RPMI-1640 to recover cells. For some experiments, the obtained cells were suspended in 40% Percoll (Cytiva) and added to 80% Percoll. Then, the gradient was centrifuged at 800 g for 20 min to obtain lymphocytes.

Flow Cytometry Analysis

For mouse surface markers, the obtained cells were counted and incubated with anti-CD16/32 (Biolegend) for 20 min at 4°C to avoid nonspecific immunofluorescent staining. Then cells were stained with appropriate antibodies for 30 min at 4°C. For mouse intracellular cytokine staining, cells were suspended and stimulated in complete RPMI-1640 medium containing 2 µL/mL cell stimulation Cocktail (plus protein transport inhibitors) (eBioscience) for 5 h at 37°C, 5% CO₂. After stimulation, the cells were stained with anti-CD16/32 and surface antibodies. Subsequently, the cells were permeabilized and fixed with Foxp3/Transcription Factor Staining Buffer Set (eBioscience). Then, cells were incubated with anti-CD16/32 and then stained with cytokine antibodies for 30 min at 4°C. For human surface protein staining, the obtained PBMCs were counted and incubated with mouse plasma for 20 min at 4°C to avoid false-positive staining. Then, the cells were incubated with specific antibodies for 30 min at 4°C. All stained cells were acquired on a BD FACSCanto plus (BD Biosciences, Franklin, NJ, USA). Mouse ILC2s were gated as Lineage⁻ST2⁺CD127⁺CD90.2⁺, mouse eosinophils were gated as CD45⁺CD11c⁻SiglecF⁺, and human ILC2s were gated as CD45⁺Lineage⁻CD127⁺CRTH2⁺. The data were analyzed by FlowJo software.

Neutral lipid staining and detection of FFAs uptake

For flow cytometry analysis, cultured ILC2s were stained with 100 ng/mL neutral lipid dye 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY 493/503) (Thermo Fisher Scientific) or 25 ng/mL fluorescently labeled long-chain FA palmitate 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Hexadecanoic Acid (BODIPY FL C16) (Thermo Fisher Scientific) for 30 min at 37°C and analyzed by flow cytometry. For Confocal microscopy detection, cells were incubated with 100 ng/mL BODIPY 493/503 or 25 ng/mL BODIPY FL C16 for 30 min at 37°C, then cells were stained with 4',6-Diamidino-2-phenylindole (DAPI) for 10 min at room temperature. The images were obtained using a Leica DMI8 confocal microscope.

Mouse ILC2s sorting and in vitro culture

Murine ILC2s (Lineage⁻ST2⁺CD127⁺CD90.2⁺) were sorted on FACSCanto II (BD Biosciences, Franklin, NJ, USA), plated in 96-well plate (10,000 per well). The cells were cultured in complete RPMI-1640 medium containing rmIL-2 (10 ng/mL, Biolegend), rmIL-7 (10 ng/mL, Biolegend), rmIL-33 (10 ng/mL, Biolegend) plus rmTSLP (10 ng/mL, R&D). For co-treatments, different combinations of 10 ng/mL rmCD5L with or without LysoPC (50 µg/mL) were added into culture medium. For pretreatment, SSO (100 µM, Cayman Chemical), AACOCF3 (10 µM, Cayman Chemical), NAC (100 µM, Sigma-Aldrich), and LY2157299 (10 µM, MedChemExpress, MCE) were supplied into culture medium 1 h before rmCD5L treatment. For rmTGF-β treatment, 200 ng/mL rmTGF-β (Biolegend) were supplied into culture medium. After 3 days, ILC2s were stained with H₂DCFDA (MCE) to detect ROS production. The apoptosis was detected using FITC Annexin V Apoptosis Detection Kit with PI (Biolegend). The production of IL-5 and IL-13 was detected by enzyme-linked immunosorbent assay (ELISA, Biolegend) using supernatant, and the level of lysoPC was also detected by ELISA (Cloud-Clone Corp) using supernatant and cell lysates. The activity of PLA2 was detected by ELISA (Cayman Chemical), following the manufacturer’s instructions.

Human ILC2s isolation and humanized mice

All human studies were conducted with informed consent and approved by Biomedical Ethics Committee at Chongqing Medical University (Approval No.2024032). All participants provided written informed consent. Human fresh blood was diluted with equal volume of balanced salt solution, added to centrifuge tube prefilled with Ficoll-Paque media (Cytiva). Then, the gradient was centrifuged at 400 g for 30 min to collect PBMCs. Human ILC2s were sorted as CD45⁺Lineage⁻CD127⁺CRTH2⁺ by flow cytometry and cultured with rhIL-2 (20 ng/mL, Peprotech) and rhIL-7 (20 ng/mL, Peprotech) for 3 days. Subsequently, the cells were adoptively transferred to Rag2^−/−Il2rg^−/− mice through the tail vein (5 × 10⁴ cells per mouse). After 24 h, each mouse was treated with 0.5 µg rhIL-33 (Peprotech) for 3 consecutive days; rhCD5L (0.67 µg/mouse, MCE) or PBS was intranasally administered during rhIL-33 treatment. On day 4, mice were sacrificed and analyzed.

RNA-Seq

Murine ILC2s were sorted and treated (see above). After that, the cultured ILC2s were collected in TRIzol^TM Reagent. The library was constructed, and samples were sequenced on an Illumina NovaSeq^TM 6000 (LC Sciences, USA). The transcriptomics data have been deposited in the Sequence Read Archive database under the accession code: PRJNA1313446.

LC-MS/MS metabolomics

Murine ILC2s were sorted and treated (see above). For metabolites analysis, cells were washed and resuspended in extraction solution to extract metabolites. LC-MS/MS analyses were performed using an UHPLC system (Vanquish, Thermo Fisher Scientific). The mass spectrometry data have been deposited in the MetaboLights database under the accession code: MTBLS12937.

Histological analysis

Lungs were harvested and fixed immediately with 4% paraformaldehyde overnight. The lung tissue was embedded in paraffin, cut into 4 µm sections, and stained with H&E and PAS according to standard protocols.

The inflammatory degree was measured as previous described (Weng et al., 2022). The PAS scores were quantified according to a modified five-point scoring system (grades 0–4) based on the percentage of goblet cells in the epithelium: grade 0 (no goblet cells), grade 1 (<25%), grade 2 (25%–50%), grade 3 (50%–75%), and grade 4 (>75%).

Statistical Analysis

The data in the article are representative of three independent experiments. All data were analyzed with Prism Software (GraphPad). For comparisons between two groups, unpaired, two-tailed Student’s t tests were applied. For multigroup comparisons, we used one-way analysis of variance (ANOVA) or two-way ANOVA with Tukey’s multiple-comparison test. p Values <0.05 were considered statistically significant.

Electronic laboratory notebook was not used.

Authors’ Contributions

Y.W. and T.C. equally designed, performed experiments, analyzed results, and wrote the article. Y.Q., M.L., S.Y., X.Y., and Q.K. contributed to perform experiments and analysis of results. X.S. and Y.J. contributed to analysis of omics data. M.Z., X.Z., and Y.Y. contributed to interpretation of data and reviewed the article. W.X. supervised, designed the experiments, interpreted the data, and critically reviewed the article. All authors read and approved the final article.

Footnotes

Acknowledgments

The authors thank Prof. Xiaodong Zhao from Children’s Hospital of Chongqing Medical University for providing the Rag2^−/−Il2rg^−/−mice. The authors are grateful to the Flow Cytometry and Informatic and Data Analysis Core facilities from Ministry of Education Key Laboratory of Development and Disorders at Children’s Hospital of Chongqing Medical University.

Author Disclosure Statement

The authors declare that they have no competing interests.

Funding Information

This work was supported by National Natural Science Foundation of China (grant no. 82270030, grant no. 82302011), Natural Science Foundation of Chongqing (grant no. CSTB2023NSCQ-MSX0704), and Chongqing Yuzhong District Science and Technology Bureau (grant no. 20210125).

Data Availability

The data supporting the findings of this study are available within the article or from the corresponding author on request.

Supplemental Material

Abbreviations

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