Maternal Fine Particulate Matter Exposure Impairs Inguinal White Adipose Tissue Plasticity in Middle-Aged Male Mouse Offspring

Abstract

The rising prevalence of metabolic diseases represents a global health challenge, among which metabolically unhealthy normal-weight individuals constitute a largely ignored subgroup. Fine particulate matter (PM_2.5), which contains substantial nanoscale particulate matter, is a recognized extrinsic environmental trigger of metabolic disorders in both obese and nonobese situations, whereas the loss of plasticity in inguinal white adipose tissue (iWAT) is a critical intrinsic pathological feature of metabolic diseases. However, the long-term metabolic effects of maternal PM_2.5 exposure on nonobese offspring, particularly in iWAT plasticity, and underlying cellular mechanisms remain poorly understood. Here, we revealed that maternal PM_2.5 exposure induced insulin resistance in middle-aged male mouse offspring and identified iWAT as a susceptible adipose depot with impaired plasticity, which is characterized by adipocyte hypertrophy, inflammation, fibrosis, and metabolic dysfunction. Using single-cell RNA sequencing on iWAT from middle-aged male mouse offspring, we found that maternal PM_2.5 exposure altered the fate decisions of adipose-derived stem cells from adipogenesis to fibrosis through increasing CD142⁺ adipogenesis-regulatory cell expansion and inducing fibrogenesis in DPP4⁺ adipose stem cells. Mechanistically, maternal PM_2.5 exposure induced IgG production from plasma cells, which promoted fibrogenesis in DPP4⁺ adipose stem cells by activating macrophages. This process was further exacerbated by monocyte- and macrophage-mediated inflammation. Finally, maternal PM_2.5 exposure induced endothelial cell heterogeneity shifts and dysfunction, facilitating immune cell recruitment and naïve B cell differentiation into plasma cells, ultimately initiating IgG-triggered plasticity impairment. This study provided insights into the adverse effects of maternal exposure to environmental pollution on the metabolic health of offspring at single-cell resolution.

Introduction

Over the past decades, the rising prevalence of metabolic diseases has emerged as a global health burden. While obesity-related metabolic diseases have been well studied, the metabolically unhealthy normal weight (MUHNW) phenotype, which affects ∼20% of the normal-weight population, has been largely overlooked. Air pollution contributes to 7 million deaths annually, representing one of the predominant threats to global health. Fine particulate matter (PM_2.5), a key component of air pollution containing substantial nanoscale particulate matter, has been demonstrated to contribute to metabolic diseases in both obese and normal weight individuals in our previous studies. − Growing evidence has indicated that adult metabolic diseases may originate from early-life environmental influences on fetal programming, − a concept known as the Developmental Origins of Health and Diseases (DOHaD). Previous studies have linked maternal exposure to nanoparticles, such as nano-titanium dioxide and polystyrene nanoplastics, to various adverse effects on offspring health. − Given the persistent severity of air pollution over recent decades, identifying the metabolic consequences of maternal exposure to PM_2.5 with nanoparticles on offspring is essential for understanding the high prevalence of the MUHNW phenotype and advancing our knowledge of its developmental etiology.

Adipose tissue is an extraordinarily flexible and heterogeneous organ that exhibits remarkable adaptability at metabolic, cellular, and structural levels upon sensing internal and external stimuli, a property termed adipose tissue plasticity, which is critical for maintaining metabolic homeostasis. , Adipose tissue is divided into white (WAT), brown (BAT), and beige adipose tissue. WAT, comprising visceral (predominantly epididymal, eWAT) and subcutaneous (primarily inguinal, iWAT) depots, primarily serves as the major site for lipid storage and mobilization, whereas BAT and beige adipose tissue are specialized for thermogenesis. Despite consensus on the role of eWAT in MUHNW, little attention has been given to the functional importance of iWAT. In fact, evidence has been established for a strong association between low iWAT mass and the MUHNW phenotype in humans, , implicating impaired iWAT plasticity in its pathogenesis. Notably, iWAT fibrosis was both a symptom and a contributor to its plasticity loss and has been noted in obesity, ,,, whereas it was never explored in the setting of PM_2.5 exposure. Based on the DOHaD theory, it is clinically imperative to investigate the ignored issue of iWAT plasticity in maternal ambient PM_2.5-exposed offspring, and further mechanistic exploration of iWAT fibrosis is warranted for the development of effective intervention strategies.

iWAT fibrosis involves a large number of nonadipocyte cells, collectively termed the stromal-vascular fraction (SVF), , which contains highly heterogeneous populations of adipose-derived stem cells (ADSCs), endothelial cells (ECs), immune cells, and other cell types. Current understanding of iWAT fibrosis is partial and one-sided due to existing studies predominantly focusing on the pro-fibrogenic changes in either whole tissue or individual cell types, ,,− overlooking the cellular heterogeneity and multicellular crosstalk within adipose tissue. Fortunately, the emergence of single-cell RNA sequencing (scRNA-seq) provides an unparalleled tool for dissecting the landscape of complex tissues at single-cell resolution, , promoting a comprehensive understanding of intercellular interactions in iWAT of offspring from PM_2.5-exposed dams. Immunoglobulin G (IgG), the most abundant immunoglobulin in circulation, is secreted by plasma cells and serves as vital for adaptive immunity. A recent discovery identified IgG as the trigger for WAT fibrosis and metabolic dysfunction during aging. However, whether IgG contributes to maternal PM_2.5 exposure-induced iWAT fibrosis in offspring and the underlying cellular and molecular mechanisms has yet to be elucidated.

In this study, we investigated the long-term adverse metabolic consequences of maternal PM_2.5 exposure on mouse offspring, systematically characterized the pathogenic impairment of iWAT plasticity in middle-aged male mouse offspring through scRNA-seq, and explored the underlying mechanisms of iWAT fibrosis under in vivo and in vitro conditions. This study established the theoretical and experimental foundations for elucidating the etiology of metabolic disorders of MUHNW and the potential pathogenic mechanisms of fetal-derived metabolic diseases induced by maternal ambient PM_2.5 exposure.

Results

PM_2.5 Concentration and Components

To investigate the long-term metabolic consequences of maternal PM_2.5 exposure on mouse offspring, pregnant mice were exposed to PM_2.5 during gestation until gestational day (GD) 18 using a whole-body inhalation exposure system. First, we monitored the average daily concentration of PM_2.5 in the chambers. During the exposure period, the mean daily PM_2.5 concentration in the PM_2.5 chamber was 182.50 ± 22.83 μg/m³ versus 13.79 ± 2.77 μg/m³ in the filtered air (FA) chamber (Figure S1A). Then, we characterized PM_2.5 collected during the exposure period. SEM characterization showed that filter-collected PM_2.5 existed as spherical particle aggregates with irregular surfaces, which were composed of numerous nano-sized particles (Figure S1B). As for the component analysis, NO₃ ^– (13.13 ± 1.63 μg/m³), SO₄ ^2– (5.89 ± 0.54 μg/m³), and NH₄ ⁺ (5.44 ± 0.52 μg/m³) were the main water-soluble inorganic ions attached to PM_2.5 (Figure S1C). Moreover, the most abundant trace metal and nonmetal elements in PM_2.5 were Fe (0.55 ± 0.04 μg/m³) and S (1.99 ± 0.17 μg/m³), respectively (Figure S1D,E).

iWAT Was the Susceptible Adipose Depot in Response to Maternal PM_2.5 Exposure

On GD 18, pregnant mice were moved from the PM_2.5 chamber and housed in the FA environment until delivery, and both male and female pups were raised until 12 months old (middle-aged) under the FA conditions (Figure A). The mean birth weights of newborn pups per litter showed no significant difference after maternal PM_2.5 exposure (Figure S2A). However, the body weight of maternal PM_2.5-exposed male offspring decreased in both adulthood and middle age (Figure B). Consistent with this, male offspring from PM_2.5-exposed dams exhibited significantly reduced fat mass during growth (Figure C), whereas lean mass remained comparable between groups (Figure S2B). At middle age, maternal PM_2.5 exposure exerted insulin resistance in male offspring (Figure D,E), despite inducing no significant effect on either glucose tolerance (Figure S1C,D) or energy metabolism (e.g., VO₂ consumption, VCO₂ production, respiratory exchange ratio, and heat production) (Figure S2E–H). In addition, no significant alterations of these metabolic indices were observed in female offspring (Figure S3). These results indicated that maternal PM_2.5 exposure induced insulin resistance in nonobese middle-aged male offspring but not in females. Based on these sex-specific outcomes, subsequent studies were conducted to elucidate the metabolic implications of maternal PM_2.5 exposure, specifically in middle-aged male offspring.

1.

Maternal PM_2.5 exposure induced insulin resistance and iWAT metabolic dysfunction in middle-aged male offspring. (A) Schematic experimental design. (B) Body weights of male offspring at adulthood and middle age. n = 8–13 per group. (C) Fat mass growth curve of male offspring. n = 8–13 per group. (D and E) ITT and area under the curve of middle-aged male offspring. n = 5 per group. (F and G) H&E staining and quantification of adipocyte size in iWAT, eWAT, and BAT of middle-aged male offspring. Scale bars: 100 μm. n = 7 per group. (H) qRT-PCR analysis of adipokine gene expression in iWAT of middle-aged male offspring. n = 7 per group. (I and J) Western blotting and quantification analysis for AKT phosphorylation levels in iWAT, eWAT, and BAT of middle-aged male offspring. n = 4 per group. All data were expressed as means ± SEM. Data were compared using Student’s t-test (B, E, G, H, and J) and two-way repeated-measures ANOVA followed by Bonferroni’s post-hoc test (C and D). *P < 0.05 and **P < 0.01.

Adipose tissue serves as a critical metabolic organ. Thus, we collected WAT (iWAT and eWAT) and BAT and found that maternal PM_2.5 exposure did not alter the mass of these adipose tissue depots in middle-aged male offspring (Figure S4A). Notably, histological analysis revealed hypertrophy, a significant increase in average adipocyte size in iWAT of middle-aged male offspring from PM_2.5-exposed dams, whereas eWAT and BAT showed no significant morphological alterations (Figure F,G). Consistently, the mRNA expression of insulin resistance-promoting adipokines (Rbp4 and Retn) was significantly increased in iWAT of middle-aged male offspring from PM_2.5-exposed dams (Figure H), while insulin-sensitizing adipokines (Adipoq and Lep) showed a compensatory increase in BAT (Figure S4B). No significant alterations in these adipokines were detected in eWAT (Figure S4C). Moreover, maternal PM_2.5 exposure specifically inhibited AKT phosphorylation in iWAT of middle-aged male offspring but not in eWAT or BAT (Figure I,J), suggesting depot-specific susceptibility to developmental programming by PM_2.5 exposure. Consequently, we detected the mRNA expression of glucose- and lipid-metabolism-related enzymes and found that maternal PM_2.5 exposure perturbed glucose and lipid metabolic homeostasis in the iWAT of middle-aged male offspring (Figure S4D). These results demonstrated that maternal PM_2.5 exposure impaired insulin signaling and induced metabolic dysfunction in the iWAT of middle-aged male offspring, subsequently contributing to systemic insulin resistance.

Maternal PM_2.5 Exposure Induced a Fibrotic Phenotypic Switch in iWAT of Middle-Aged Male Offspring

Fibrosis is a pathological hallmark of metabolic dysfunction in adipose tissue. To determine whether maternal PM_2.5 exposure induces iWAT fibrosis in middle-aged male offspring, we performed Masson’s trichrome staining and Sirius red staining. Both histological analyses exhibited significant collagen deposition in the iWAT of middle-aged male offspring from PM_2.5-exposed dams (Figure A–C). Consistent with this, maternal PM_2.5 exposure significantly increased COL1A1 deposition (Figure A,D) and upregulated mRNA expression of the main fibrillary collagen genes in iWAT of middle-aged male offspring, including Col1a1, Col3a1, and Col6a1 (Figure E). TGF-β is a well-established master regulator of fibrosis by mediating epithelial-mesenchymal transition (EMT). Supportively, maternal PM_2.5 exposure significantly upregulated the TGF-β protein expression in iWAT of middle-aged male offspring (Figure F,G). Furthermore, we observed significantly reduced E-cadherin protein expression in iWAT of middle-aged male offspring from PM_2.5-exposed dams, while vimentin protein expression remained unchanged (Figure F,G), suggesting partial EMT activation. However, the aforementioned fibrosis-related alterations in iWAT were not detected in either PM_2.5-exposed pregnant mice or male mice (Figures S5 and S6), indicating that this fibrotic phenotypic switch of iWAT was specific to maternal PM_2.5-exposed male offspring. These results suggested that maternal PM_2.5 exposure specifically induced a phenotypic switch to fibrosis in iWAT of middle-aged male offspring.

2.

Maternal PM_2.5 exposure induced iWAT fibrosis in middle-aged male offspring. (A) Masson’s trichrome staining, Sirius red staining, and immunohistochemical staining of iWAT from middle-aged male offspring. Scale bars: 100 μm. (B–D) Quantification of fibrotic area in Masson’s trichrome staining, Sirius red staining, and immunohistochemical staining in iWAT of middle-aged male offspring. n = 6 per group. (E) qRT-PCR analysis of collagen-related gene expression in iWAT from middle-aged male offspring. n = 7 per group. (F and G) Western blotting and quantification analysis for TGF-β, vimentin, and E-cadherin protein levels in iWAT of middle-aged male offspring. n = 4 per group. All data were expressed as means ± SEM. Data were compared using Student’s t-test. *P < 0.05 and **P < 0.01.

scRNA-Seq Delineated the Cellular Landscape of iWAT in Middle-Aged Male Offspring

To clarify the cellular mechanism underlying maternal PM_2.5 exposure-induced phenotypic switch and metabolic dysfunction in iWAT, we performed scRNA-seq on iWAT isolated from middle-aged male offspring of PM_2.5-exposed dams and defined iWAT cellular heterogeneity and its dynamic changes. Following stringent quality control, a total of 42,896 single-cell transcriptomes were obtained (24,012 for the male/FA group; 18,884 for the male/PM_2.5 group). Eleven major cell types were identified based on the expression of canonical markers in mouse adipose tissue, including ADSCs, mononuclear phagocytes (MPs), T cells, ECs, epithelial cells, B cells, mast cells, mural cells, adipocytes, group 2 innate lymphoid cells (ILC2), and neutrophils (Figures A,B and S7). iWAT from maternal PM_2.5-exposed middle-aged male offspring exhibited higher proportions of epithelial cells and B cells, but lower abundances of adipocytes and ILC2 compared to maternal FA-exposed controls (Figure A,C). Notably, while EC proportions were similar between groups, maternal PM_2.5 exposure profoundly rewired the ECs’ transcriptional profiles in iWAT of middle-aged male offspring (Figure D). These results suggested that maternal PM_2.5 exposure-induced pathogenic remodeling of iWAT in middle-aged male offspring was associated with cell type-specific reprogramming.

3.

A scRNA-seq atlas of iWAT from middle-aged male offspring. (A) t-SNE visualization of 42,896 cells from iWAT of middle-aged male offspring (left). The bar plots illustrate the distribution of cell numbers and percent contribution for each cluster in Male/FA and Male/PM_2.5 groups (right). n = 3 mice per group. (B) Individual gene t-SNE plots of marker genes for ADSCs, ECs, MPs, and B cells. (C) t-SNE visualization of each cluster in Male/FA and Male/PM_2.5 groups. (D) Distribution of DEGs (left) and number of DEGs (right) for each cluster in middle-aged male offspring from the Male/PM_2.5 group.

Maternal PM_2.5 Exposure Altered the Fate Decision of ADSCs from Adipogenesis to Fibrosis

Based on the dual adipogenic and fibrogenic potential of ADSCs, we focused on the ADSCs and identified three subpopulations (ADSC_1, ADSC_2, and ADSC_3) with distinct gene signatures through unsupervised transcriptomic clustering (Figure A,B). ADSC_1, defined by Col4a1 and Icam1 expression (Figure B,C), was enriched for adipogenic markers, including Aoc3, Cd36, Fabp4, Pdgfra, and Pparg (Figure S8A,B). Consistent with this, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that ADSC_1-specific expression genes were markedly enriched in the PI3K-Akt signaling pathway and PPAR signaling pathway (Figure B). Assessment of adipogenic score further demonstrated that ADSC_1 exhibited higher adipogenic potential than ADSC_2 and ADSC_3 (Figure D), suggesting its identity as “committed preadipocytes.” ADSC_2 expressed Clec11a and F3 (encoding CD142) (Figure B,C), genes marking “adipogenesis-regulatory cells” (Aregs) that functionally inhibit adipogenesis in fat depots. ADSC_3, marked by Dpp4 and Pi16 expression (Figure B,C), highly expressed stem cell markers (e.g., Cd34 and Ly6a) (Figure S8C,D), and exhibited a significantly higher stem cell score (Figure E). Therefore, ADSC_3 was classified as “adipose stem cells,” which can adopt either adipogenic or profibrogenic fates depending on the adipose tissue microenvironment cues. Next, we predicted ADSCs’ differentiation trajectories. CytoTRACE analyses revealed that ADSC_3 had the highest differentiation potential, consistent with its stem cell characteristics (Figure F). Monocle2 analyses inferred that ADSC_3 had two differentiation trajectories: transitioning to ADSC_2 or ADSC_1 (Figure G). These results suggested that ADSC_3 adipose stem cells were the source of ADSC_1 committed preadipocytes and ADSC_2 Aregs.

4.

Maternal PM_2.5 exposure induced fibrogenic activation of ADSC_3 and inhibited adipogenesis by increasing the expansion of ADSC_2 in iWAT of middle-aged male offspring. (A) t-SNE visualization of three ADSC subpopulations. (B) Heatmap of the most highly DEGs across three ADSC subpopulations and the KEGG pathways significantly enriched among these DEGs specific to one of the three subpopulations. (C) Individual gene t-SNE plots of representative marker genes. (D and E) Adipogenic and stem cell scores in three ADSC subpopulations. (F) Cell differentiation potential of three ADSC subpopulations predicted by using CytoTRACE. (G) Pseudotemporal cell ordering of three ADSC subpopulations along differentiation trajectories by using Monocle2. Pseudotime is depicted from dark to light yellow (left). ADSC subpopulations were overlaid on the pseudotime trajectory map (right). (H and I) t-SNE visualization and proportions of three ADSC subpopulations in iWAT of middle-aged male offspring from Male/FA and Male/PM_2.5 groups. Data were expressed as means ± SEM, n = 3 mice per group. (J and K) Adipogenic score in ADSC, ADSC_1, ADSC_2, and ADSC_3 between middle-aged male offspring from Male/FA and Male/PM_2.5 groups. (L) GO terms enriched with downregulated DEGs in ADSC_1 from middle-aged male offspring in Male/PM_2.5 groups. (M) GO terms enriched with upregulated DEGs in ADSC_3 from middle-aged male offspring in Male/PM_2.5 groups. (N) Dot plot of differential ligand–receptor pairs mediating intercellular communication from ADSC_2 to both ADSC_1 and ADSC_3. Circle sizes indicate P values, and colors represent mean expression. Data were compared using Student’s t-test (I), Kruskal–Wallis test (D and E), and Mann–Whitney test (J and K). **P < 0.01 and ***P < 0.001.

Next, we examined the effects of maternal PM_2.5 exposure on these three subpopulations, aiming to identify which specific subpopulation contributed to fibrosis. Maternal PM_2.5 exposure significantly expanded the ADSC_2 population in the iWAT of middle-aged male offspring (Figure H,I). Given ADSC_2-mediated adipogenesis suppression, we scored the adipogenic capacity of ADSCs and demonstrated that maternal PM_2.5 exposure markedly inhibited adipogenic differentiation in ADSCs, especially in ADSC_1 (Figure J,K). Consistently, downregulated genes in ADSC_1 from middle-aged male offspring in the Male/PM_2.5 group were highly enriched in gene ontology (GO) terms associated with angiogenesis and fat cell differentiation, as well as the PPAR signaling pathway in KEGG analysis (Figures L and S8E). It is noteworthy that ADSC_3 in middle-aged male offspring from PM_2.5-exposed dams exhibited increased expression of extracellular matrix (ECM) and fibrosis-related genes (Figure M). Next, to explore the potential mechanisms underlying ADSC_2-mediated anti-adipogenesis, we performed cell–cell interaction analysis. The differential interaction pairs, which were significantly enriched in the Male/PM_2.5 group but not in the Male/FA group, were summarized in Figure N. Notably, several reported adipogenesis-inhibitory interactions were identified, including PDGFA_PDGFRA, RSPO2_LGR5, and TGF-β signaling. These results demonstrated that maternal PM_2.5 exposure induced a pro-fibrotic phenotypic switch in ADSC_3, while simultaneously inhibiting adipogenesis by increasing the expansion of ADSC_2.

Pro-fibrotic Phenotypic Switch of ADSC_3 Was Induced by Plasma Cell-Derived IgG through Macrophage Activation under Maternal PM_2.5 Exposure

IgG has recently been reported to drive adipose tissue fibrosis and metabolic decline during aging. To explore the mechanism of ADSC_3 fibrogenesis in iWAT from maternal PM_2.5-exposed middle-aged male offspring, we examined and detected significant IgG accumulation in the iWAT of middle-aged male offspring from PM_2.5-exposed dams by immunohistochemistry staining (Figure A) and western blotting (Figure B,C). Furthermore, maternal PM_2.5 exposure also strikingly increased IgG1 (the predominant IgG subtype in adipose tissue) levels in the iWAT of middle-aged male offspring (Figure B,C).

5.

Plasma cell-derived IgG promoted ADSC_3 fibrogenesis by activating macrophages. (A) Immunohistochemical staining and quantification of IgG in iWAT of middle-aged male offspring. Scale bars: 100 μm. n = 6 per group. (B and C) Western blotting and quantification analysis for protein levels of IgG and IgG1 heavy chains in iWAT of middle-aged male offspring. n = 4 per group. (D) t-SNE visualization of distinct subtypes of B cells, including naïve B cells, memory B cells, GCB cells, and plasma cells. (E) Individual gene t-SNE plots of marker genes for naïve B cells, memory B cells, GCB cells, and plasma cells. (F and G) t-SNE visualization and proportions of B cell subtypes in iWAT of middle-aged male offspring from Male/FA and Male/PM_2.5 groups. n = 3 mice per group. (H) qRT-PCR analysis of plasma cell marker gene expression in iWAT of middle-aged male offspring. n = 5–6 per group. (I) Dot plot showing expression of immunoglobulin-coding genes across distinct B cell subtypes from Male/FA and Male/PM_2.5 groups. Circle sizes indicate the percentage expressed, and colors represent the normalized average expression. (J) Schematic diagram of the structures for immunoglobulins. Upregulated immunoglobulins and their encoding genes are labeled in red. (K) qRT-PCR analysis of fibrosis-related gene expression in 3T3-L1 preadipocytes treated with CM collected from vehicle- or IgG-treated BMDMs. n = 4 per group. (L) Dot plot of ligand–receptor pairs mediating intercellular communication from macrophages to ADSC_3 in iWAT of middle-aged male offspring derived from PM_2.5-exposed dams. Circle sizes indicate P values, and colors represent mean expression. (M) qRT-PCR analysis of the indicated gene expression in BMDMs treated with vehicle or different concentrations of IgG (50 and 100 μg/mL). n = 4 per group. All data were expressed as means ± SEM. Data were compared using Student’s t-test (A, C, G, and H) and one-way ANOVA followed by Tukey’s post-hoc test (K and M). *P < 0.05, **P < 0.01, and ***P < 0.001.

Next, we further asked how maternal PM_2.5 exposure caused IgG accumulation in the iWAT of middle-aged male offspring. IgG is derived from the following sources: neonatal Fc receptor (FcRn)-mediated IgG recycling and de novo IgG production by plasma cells. , As shown in Figure S9, Fcgrt (encoding FcRn) mRNA expression levels showed no significant change in the iWAT of middle-aged male offspring from PM_2.5-exposed dams, excluding the recycling pathway. Regarding the de novo origin from the plasma cell that belongs to B cells, we first classified the B-cell subtypes based on the canonical marker expression, including plasma cells, naïve B cells, memory B cells, and germinal center B (GCB) cells (Figure D,E). Middle-aged male offspring from PM_2.5-exposed dams showed a significant expansion in plasma cells and a marked reduction in naïve B cells within iWAT (Figure F,G). Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of plasma cell markers confirmed maternal PM_2.5 exposure-induced plasma cell accumulation (Figure H). Additionally, we observed elevated expression of genes encoding the heavy chain of multiple IgG subtypes (e.g., IgG1, IgG2b, IgG2c, and IgG3), predominantly in plasma cells in iWAT of middle-aged male offspring from PM_2.5-exposed dams (Figure I,J). These results indicated that maternal PM_2.5 exposure triggered plasma cell expansion in the iWAT of middle-aged male offspring, resulting in enhanced IgG production and deposition.

To delineate the potential mechanisms through which IgG exerts its pro-fibrotic effects on ADSC_3, we treated 3T3-L1 preadipocytes with IgG. However, no significant alterations were detected in the expression of fibrosis-related genes (Figure S10), excluding the direct effect of IgG on the pro-fibrotic phenotype switch and leading us to explore the indirect possibility mediated by other cellular types. Consequently, to determine which cellular population mediated the pro-fibrotic effects of IgG on ADSC_3, we compared gene expression values of various IgG receptors across all clusters. MPs exhibited the highest expression levels (Figure S11A,B). MPs were further divided into six subtypes representing macrophages, monocytes, cDC1, cDC2, mature DCs, and proliferating MPs based on the marker gene expression profile (Figure S11C,D). Maternal PM_2.5 exposure significantly decreased the proportion of macrophages, while increasing that of monocytes in iWAT of middle-aged male offspring (Figure S11E,F). Among these subtypes, IgG receptors were predominantly expressed in macrophages (Figure S11G), suggesting that macrophages may serve as a potential medium for IgG-mediated iWAT fibrosis. As expected, conditioned media (CM) collected from IgG-treated bone marrow-derived macrophages (BMDMs) significantly upregulated the mRNA expression of genes related to fibrosis (e.g., Col1a1, Col3a1, and Col6a1) in 3T3-L1 preadipocytes (Figure K). Next, to further delineate the mechanism by which macrophages mediate IgG-driven pro-fibrotic effects on ADSC_3, we analyzed potential ligand–receptor interaction pairs between macrophages and ADSC_3 (Figure L). qRT-PCR analysis of IgG-treated BMDMs confirmed the significant increase in the mRNA expression of six ligands: Ccl2, Cxcl10, Igf1, Pdgfc, Tgfb1, and Tnf (Figures M and S12), suggesting that these genes might be involved in the fibrogenic activation of ADSC_3 mediated by IgG-activated macrophages.

Maternal PM_2.5 Exposure Stimulated Infiltration of Immune Cells into iWAT via EC and Differentiation from Naïve B Cells to Plasma Cells

ECs serve as a critical interface between the circulation and adipose tissue microenvironment to orchestrate immune cell trafficking. Building upon the finding that ECs exhibited the highest number of differentially expressed genes (DEGs) in iWAT of middle-aged male offspring from PM_2.5-exposed dams (Figure D), we classified ECs into four subtypes based on established marker genes, including venous ECs (VECs), lymphatic ECs (LECs), capillary ECs (CapECs), and arterial ECs (AECs) (Figure A,B). Notably, maternal PM_2.5 exposure induced shifts in EC heterogeneity, dramatically increasing the proportion of VECs and LECs while reducing the number of CapECs and AECs (Figure C,D). Based on these alterations in CapECs and AECs, we assessed hypoxia scores in EC subpopulations and found significantly elevated hypoxia scores in VECs, CapECs, and AECs (Figure E). KEGG enrichment analysis revealed that VECs, LECs, and AECs in iWAT of middle-aged male offspring from PM_2.5-exposed dams shared significant upregulation of the AGE-RAGE signaling pathway and antigen processing and presentation (Figure F), implicating activated stress and immune response. VECs, in particular, also showed activation of pro-inflammatory signaling pathways (e.g., TNF signaling pathway, IL-17 signaling pathway, and cell adhesion molecules) (Figure F). In contrast, pathways critical for maintaining vascular barrier function and endothelial metabolic homeostasis (e.g., PPAR signaling pathway, Rap1 signaling pathway, adherens junction, and focal adhesion) were significantly downregulated in VECs, CapECs, and AECs (Figure F). CapECs also exhibited inhibition of the Wnt signaling pathway and Hippo signaling pathway, indicating impaired proliferation and angiogenesis (Figure F). Furthermore, maternal PM_2.5 exposure led to dysregulated mRNA expression of genes associated with proliferation and maturation, leukocyte migration, and injury-induced activation in distinct EC subtypes (Figure G). These results suggested that maternal PM_2.5 exposure induced EC heterogeneity shifts accompanied by EC dysfunction.

6.

ECs enhanced naïve B cell, plasma cell, and monocyte infiltration into iWAT and differentiation from naïve B cells to plasma cells. (A) t-SNE visualization of distinct EC subtypes, including VECs, LECs, CapECs, and AECs. (B) Individual gene t-SNE plots of marker genes for VECs, LECs, CapECs, and AECs. (C and D) t-SNE visualization and proportions of EC subtypes in iWAT of middle-aged male offspring from Male/FA and Male/PM_2.5 groups. Data were expressed as means ± SEM, n = 3 mice per group. (E) Hypoxia score in VECs, LECs, CapECs, and AECs between middle-aged male offspring from Male/FA and Male/PM_2.5 groups. (F) KEGG enrichment analysis of DEGs in VECs, LECs, CapECs, and AECs from middle-aged male offspring in Male/PM_2.5 groups. (G) Heatmap showing fold-change expression of indicated genes across distinct EC subtypes. (H) Dot plot of differential ligand–receptor pairs mediating intercellular communication from four EC subtypes to monocytes, naïve B cells, and plasma cells. Circle sizes indicate P value, and colors represent mean expression. Data were compared using Student’s t-test (D) and Mann–Whitney test (E and G). *P < 0.05, **P < 0.01, and ***P < 0.001.

To examine the mechanistic role of ECs in mediating maternal PM_2.5 exposure-enhanced plasma cell and monocyte infiltration in iWAT of middle-aged male offspring, we performed a cell–cell interaction analysis between four EC subtypes and three immune cell types (naïve B cell, plasma cell, and monocyte). The differential interaction pairs, which were significantly enriched in the Male/PM_2.5 group but not in the Male/FA group, were summarized in Figure H. We found that all four EC subtypes recruited monocytes through the secretion of distinct chemokines, including CCL2_CCR2 between VECs and monocytes; CCL7_CCR5, CX3CL1_CX3CR1, and CXCL12_CXCR4 between LECs and monocytes; CSF1_CSF1R between CapECs and monocytes; and CCL7_CCR2 between AECs and monocytes (Figure H). Furthermore, maternal PM_2.5 exposure augmented the adhesion of VECs and AECs to monocytes through THY_ADGRE5 (Figure H). Regarding B cells, CXCL13_CXCR5 and TF_TFRC were presented between VECs and naïve B cells in iWAT from PM_2.5-exposed dams (Figure H). CXCL13_CXCR5 is known to mediate B-cell migration, while TF_TFRC participates in naïve B cell to plasma cell differentiation. Furthermore, CXCL12 from LECs showed a strong interaction with naïve B cells and plasma cells through CXCR4 (Figure H). This CXCL12_CXCR4 was reported to have chemoattractant activity on B cells. These results suggested that ECs promoted the recruitment of monocytes, naïve B cells, and plasma cells and facilitated naïve B cell differentiation into plasma cells in iWAT of middle-aged male offspring from PM_2.5-exposed dams.

Adipose Tissue Macrophage-Mediated Inflammation Additionally Contributed to a Pro-fibrotic Phenotypic Switch of ADSC_3

Given the established heterogeneity of adipose tissue macrophages, we sought to identify which macrophage subpopulation specifically mediated IgG-induced pro-fibrotic switching in ADSC_3. To address this, we performed unsupervised clustering of gene expression profiles, classifying macrophages into six subpopulations (Mac_1–6) (Figure A), each exhibiting unique gene expression signatures (Figure B). According to previously established classification markers for adipose tissue macrophages, , Mac_1 (expressing Lyve1, Timd4, and Cd163) and Mac_4 (expressing Ear2 and Fcrls) were identified as perivascular macrophages (PVMS) and non-perivascular macrophages (NPVMs), respectively (Figure C). Mac_2, characterized by Adrb2 expression (Figure C), was designated as “sympathetic neuron-associated macrophages (SAMs).” Mac_3, a previously uncharacterized macrophage subpopulation, showed a high expression of antigen processing and presentation-related genes (Figure B). Mac_5 was classified as “inflammatory macrophages (IMs)” because of its high expression of inflammatory cytokines, such as Ccl4, Ccl3, and Il1b (Figure B). Mac_6, which highly expressed Lpl, Trem2, and Cd9 (Figure C), was known as “lipid-associated macrophages (LAMs).” In iWAT of middle-aged male offspring from PM_2.5-exposed dams, a substantial decrease in the abundance of Mac_3 was observed, concomitant with a marked increase in Mac_4 and monocyte populations (Figure D,E).

7.

Monocyte- and macrophage-induced inflammation contributed to the pro-fibrotic phenotypic switch of ADSC_3 in iWAT of middle-aged male offspring from PM_2.5-exposed dams. (A) t-SNE visualization of monocytes and six macrophage subpopulations. (B) Heatmap of the most highly DEGs across six macrophage subpopulations and the KEGG pathways significantly enriched among these DEGs specific to one of the three subpopulations. (C) Violin plots of selected gene expression levels. (D and E) t-SNE visualization and proportions of monocytes and six macrophage subpopulations in iWAT of middle-aged male offspring from the Male/FA and Male/PM_2.5 groups. Data were expressed as means ± SEM, n = 3 mice per group. (F) RNA velocity analysis of six macrophage subpopulations and monocytes. Velocity fields were projected on the t-SNE plot. (G) Dot plot of the indicated gene expression in six macrophage subpopulations from Male/FA and Male/PM_2.5 groups. Circle sizes indicate the percentage expressed, and colors represent the normalized average expression. (H) Proinflammatory score in six macrophage subpopulations and monocytes between middle-aged male offspring from Male/FA and Male/PM_2.5 groups. (I) qRT-PCR analysis of inflammation-related gene expression in iWAT from middle-aged male offspring, n = 5 per group. (J and K) Western blotting and quantification analysis for IL-6 and IL-1β protein levels in iWAT of middle-aged male offspring. Data were expressed as means ± SEM, n = 4 per group. (L and M) Spearman’s correlation between the protein expression of IL-6 and IL-1β and quantification of positive area in Masson’s trichrome staining, Sirius red staining, and immunohistochemical staining in iWAT of middle-aged male offspring, respectively. Data were compared using Student’s t-test (E, I, and K) and Mann–Whitney test (H). *P < 0.05, **P < 0.01, and ***P < 0.001.

To delineate the dynamic reorganization of these macrophage subpopulations induced by maternal PM_2.5 exposure, we performed RNA velocity analysis to predict the cellular differentiation trajectories (Figure F). The trajectory modeling revealed that Mac_3 exhibited high differentiation capacity, capable of transitioning into Mac_1, Mac_2, Mac_4, or Mac_6 through divergent trajectories. A subset of Mac_3 cells followed a sequential differentiation route, first transitioning to Mac_6 and further to Mac_4. Furthermore, monocytes can transit to Mac_5. These results indicated that maternal PM_2.5 exposure drove a Mac_3-to-Mac_4 phenotypic shift in the iWAT of middle-aged male offspring. Next, we analyzed the expression profiles of the six aforementioned ligands and found that maternal PM_2.5 exposure specifically upregulated the mRNA expression of Cxcl10 and Tnf in Mac_5, concomitantly enhancing the mRNA expression of Tgfb1 in Mac_6 (Figure G). Furthermore, maternal PM_2.5 exposure increased the mRNA expression of Ccl2 and Pdgfc in multiple macrophage subpopulations, but downregulated the mRNA expression of Igf1 (Figure G). These results implied that the mechanism underlying IgG-activated macrophage subpopulation-induced ADSC_3 fibrogenesis possibly involved the genes Cxcl10, Tnf, Tgfb1, Ccl2, and Pdgfc.

Previous studies have established an association between IgG accumulation and adipose tissue inflammation. ,, Indeed, we observed upregulated mRNA expression of pro-inflammatory genes in IgG-treated BMDMs, such as Ccl2, Cxcl10, and Tnf (Figure M). Therefore, we further characterized the inflammatory response in macrophage subpopulations and monocytes. Monocytes and most macrophage subpopulations (e.g., Mac_2, Mac_4, Mac_5, and Mac_6) exhibited a significant increase in pro-inflammatory scores (Figure H). Consistent with this, KEGG enrichment analysis showed that maternal PM_2.5 exposure specifically activated pro-inflammatory signaling pathways in Mac_4, Mac_5, and monocytes (Figure S13). Based on these results, the inflammatory response was validated in iWAT of middle-aged male offspring. Maternal PM_2.5 exposure significantly upregulated the mRNA expression of Ccl2, Cxcl10, and Tnf (Figure I). Western blotting analysis further confirmed significantly elevated protein expression of IL-6 and IL-1β in the iWAT of middle-aged male offspring from PM_2.5-exposed dams (Figure J,K). Furthermore, the mRNA expression of these inflammatory cytokines exhibited a strong positive correlation with the mRNA levels of the main fibrillary collagen genes (Figure S14), while their protein expression was positively correlated with the extent of collagen deposition (Figure L,M). These results demonstrated that IgG-activated macrophage subpopulations (particularly Mac_4, Mac_5, and Mac_6) and monocytes promoted inflammation in iWAT, contributing to ADSC_3 fibrogenesis in middle-aged male offspring from PM_2.5-exposed dams.

Discussion

The current study delineated the adverse impacts of maternal exposure to environmental pollutants on the metabolic health of mouse offspring at single-cell resolution. The main findings of this study are as follows: (i) maternal PM_2.5 exposure induced insulin resistance in nonobese, middle-aged male mouse offspring, with iWAT identified as the susceptible adipose depot that exhibited impaired iWAT plasticity; (ii) maternal PM_2.5 exposure altered the fate decisions of ADSCs from adipogenesis to fibrosis; (iii) maternal PM_2.5 exposure led to IgG production by accumulated plasma cells, promoting a pro-fibrotic phenotypic switch in the ADSC_3 subpopulation through macrophage activation; (iv) maternal PM_2.5 exposure recruited immune cells into iWAT, followed by plasma cell differentiation due to EC heterogeneity shifts and dysfunction; (v) monocyte- and macrophage-mediated inflammation further contributed to the pro-fibrotic phenotypic switch of ADSC_3.

Maternal PM_2.5 Exposure, Metabolic Disorder, and Impaired iWAT Plasticity

Emerging studies have established adverse maternal factors during pregnancy as significant risk determinants for developing metabolic diseases in offspring during adulthood. − In this study, we demonstrated that maternal PM_2.5 exposure induced insulin resistance in middle-aged male offspring but not female offspring, highlighting the long-term metabolic consequences of maternal PM_2.5 exposure on male offspring. Although a great number of studies have focused on PM_2.5 and metabolic disorders, very limited toxicological studies have demonstrated obesity and β-cell dysfunction in maternal PM_2.5-exposed adult offspring , and only one epidemiological study reported an association between prenatal and perinatal PM_2.5 exposure and raised hemoglobin A1c (HbA1c) levels in children aged 4–6 years, suggesting maternal PM_2.5 exposure as a potential diabetes risk. Furthermore, given the pivotal role of sex hormones in mediating sex-specific disparities in health and disease, − we hypothesize that androgens may exacerbate metabolic susceptibility in male offspring, whereas estrogens may confer protection in female offspring. Elucidating the functional role of these hormones will be the focus of our further investigation. Focusing on the long-term impacts of maternal PM_2.5 exposure, our findings filled the critical knowledge gap of the large population with metabolic dysfunction, underscoring the necessity and urgency of further investigation into the underlying mechanisms.

Adipose tissue is a highly flexible and heterogeneous organ. Its loss of plasticity, as characterized by fibrosis, drives insulin resistance and other obesity-related pathological consequences. In this study, we identified iWAT as the susceptible adipose depot in response to maternal PM_2.5 exposure, showing significantly impaired plasticity, characterized by adipocyte hypertrophy, inflammation, fibrotic phenotypic switch, and metabolic dysfunction. Furthermore, we identified three ADSC subpopulations in iWAT of middle-aged male offspring using scRNA-seq, including ADSC_1 committed preadipocytes, ADSC_2 Aregs, and ADSC_3 adipose stem cells. Given the fibrosis-versus-adipogenesis fate axis in ADSCs, an important finding of this study was that maternal PM_2.5 exposure altered the fate decision of ADSCs from adipogenesis to fibrosis.

On the one hand, maternal PM_2.5 exposure inhibited adipogenesis by increasing the expansion of ADSC_2, explaining the observed adipocyte hypertrophy linked with the decline in adipose tissue plasticity and systemic metabolic disorder. TGF-β and PDGFA are known to inhibit adipogenesis by directly suppressing the PPARγ-C/EBPα complex. , Moreover, Dong et al. demonstrated that CD142⁺ cells, corresponding to our ADSC_2, suppress adipogenesis via RSPO2 paracrine signaling. Moreover, they revealed that elevated circulating RSPO2 levels are significantly associated with metabolic disorders, such as insulin resistance and impaired glucose homeostasis, in both mice and male obese individuals. Based on the cell–cell interaction analysis, this ADSC_2-mediated adipogenesis suppression in maternal PM_2.5-exposed male offspring may be achieved by cytokines, including TGF-β, PDGFA, and RSPO2.

On the other hand, maternal PM_2.5 exposure induced a pro-fibrotic phenotypic switch of ADSC_3, leading to iWAT fibrosis in middle-aged male offspring. Similarly, Hepler et al. identified a LY6C⁺PDGFRβ⁺ADSC subpopulation with a pro-fibrogenic/pro-inflammatory phenotype in eWAT of adult mice, and they named it fibro-inflammatory progenitors (FIPs). ADSC_3 in iWAT exhibited transcriptomic similarity to FIPs from eWAT, implying that their pro-fibrotic activity was conserved across distinct fat depots. Collectively, our results highlighted the crucial role of ADSC-fate decisions in maternal PM_2.5 exposure-induced fibrosis phenotypic switch of iWAT in middle-aged male offspring. Notably, regulating ADSC-fate decisions to favor adipogenesis may represent a potential therapeutic strategy for ameliorating the pathogenic remodeling of iWAT and combating metabolic diseases.

Plasma Cell-Derived IgG, Macrophages, and the Fibrotic Phenotypic Switch of iWAT

Next, we explored how maternal PM_2.5 exposure induced a pro-fibrotic phenotypic switch of iWAT in middle-aged male offspring. Here, we noticed significant IgG accumulation and enhanced expression of multiple IgG subtypes in iWAT of middle-aged male offspring from PM_2.5-exposed dams. IgG levels, which could accumulate in aged or obese mice, ,, were maintained by two biological processes: FcRn-mediated IgG recycling and de novo IgG production by plasma cells. , Notably, we observed increased plasma cells, but not FcRn levels, in iWAT of middle-aged male offspring, indicating that maternal PM_2.5 exposure-induced IgG deposition was mediated through de novo production by plasma cells rather than FcRn-mediated recycling.

We further asked how IgG exerts its pro-fibrotic effects on ADSC_3. Since no direct pro-fibrotic effect of IgG on 3T3-L1 preadipocytes was detected, we explored the indirect way under the illumination of scRNA-seq data. First, macrophages highly expressed IgG receptors, providing a material basis for IgG to act on macrophages. Next, we treated BMDMs with IgG in vitro and collected CM. This CM significantly upregulated the mRNA expression of fibrosis-related genes in 3T3-L1 preadipocytes, confirming the role of macrophages in mediating IgG-induced fibrogenesis in 3T3-L1 preadipocytes. Consistently, Yu et al. recently demonstrated that IgG drove WAT fibrosis and metabolic decline during aging, while inhibiting IgG accumulation restored WAT integrity and metabolic health in aged mice. Collectively, our results demonstrated that plasma cell-derived IgG unexpectedly mediated the pro-fibrotic phenotypic switch of ADSC_3 by activating macrophages, implying that blocking IgG accumulation could be a potential therapeutic strategy for fetal-originated metabolic diseases induced by maternal PM_2.5 exposure.

To further delineate the mechanism by which macrophages mediated the IgG-driven pro-fibrotic phenotypic switch of ADSC_3, we analyzed potential ligand–receptor interaction pairs between macrophages and ADSC_3 and found that macrophage-derived TGF-β1 and PDGFC exhibited strong interactions with ADSC_3 by activating TGF-β receptors and PDGFRα, respectively. Furthermore, in vitro experiments demonstrated that IgG treatment markedly upregulated the mRNA expression of Tgfb1 and Pdgfc in BMDMs. Consistently, previous studies demonstrated that IgG stimulated TGF-β expression in macrophages to induce WAT fibrosis during aging, and activation of PDGFRα contributed to promoting progenitor fibrogenesis and ultimately driving WAT fibrosis. Thus, the TGF-β1 and PDGFC secreted by IgG-activated macrophages promoted a pro-fibrotic phenotypic switch of ADSC_3 in middle-aged male offspring from PM_2.5-exposed dams.

ECs, Inflammation, and iWAT Fibrosis

In this study, we found increased immune cell infiltration (e.g., plasma cells and monocytes) in the iWAT of maternal PM_2.5-exposed male offspring. As a critical interface between the circulation and adipose tissue microenvironment, ECs orchestrate B cell and monocyte trafficking through well-known B cell chemoattractants (e.g., CXCL12 and CXCL13), and canonical monocyte chemokines (e.g., CCL2 and CCL7). ,,, In this study, VECs and LECs exhibited maternal PM_2.5 exposure-specific interactions with naïve B cells and plasma cells through CXCL12_CXCR4 and CXCL13_CXCR5, promoting their recruitment. Furthermore, maternal PM_2.5 exposure promoted monocyte infiltration by EC-derived canonical monocyte chemokines and concurrently activated inflammatory responses in both monocytes and macrophages. This inflammatory response was further confirmed in both the iWAT of maternal PM_2.5-exposed male offspring and IgG-treated BMDMs, as demonstrated by the significantly increased expression of pro-inflammatory cytokines. Thus, our results suggested that ECs mediated maternal PM_2.5 exposure-induced immune cell infiltration, which subsequently promoted inflammation in the iWAT of middle-aged male offspring.

Given the increased infiltration but decreased proportion of naïve B cells in iWAT of maternal PM_2.5-exposed male offspring, we speculated that maternal PM_2.5 exposure promoted naïve B cells into plasma cell differentiation. The TF_TFRC interaction was demonstrated to play a critical role in B cell differentiation. Indeed, in this study, cell–cell interaction analysis revealed that TF from VECs showed a significant interaction with naïve B cells through TFRC after maternal PM_2.5 exposure, suggesting that maternal PM_2.5 exposure promoted naïve B cells into plasma cell differentiation. Thus, this enhanced circulatory infiltration and increased naïve B cell differentiation synergistically drove plasma cell accumulation in the iWAT of middle-aged male offspring from PM_2.5-exposed dams, establishing the cellular basis for IgG production and deposition.

Adipose tissue inflammation and fibrosis constitute a reciprocally reinforcing bidirectional process, wherein inflammatory cytokines (e.g., TNF-α) stimulate collagen synthesis and ECM deposition, while fibrotic remodeling conversely exacerbates the inflammatory response, ultimately leading to a decline in adipose tissue plasticity. ,, In this study, the mRNA expression of Tnf was markedly upregulated in both the iWAT of maternal PM_2.5-exposed male offspring and IgG-treated BMDMs. Notably, cell–cell interaction analysis revealed that macrophage-derived TNF-α exhibited a strong interaction with ADSC_3 via TNFR2, suggesting its potential role in promoting ADSC_3 fibrogenesis. Thus, these results suggested that maternal PM_2.5 exposure-induced inflammation promoted iWAT fibrosis in middle-aged male offspring through alternative pathways, leading to a decline in iWAT plasticity and subsequently promoting metabolic disorders.

However, this study has several limitations. First, we demonstrated the adverse metabolic impacts of maternal PM_2.5 exposure on offspring, whereas the intergenerational transmission mechanisms of such metabolic dysfunction remain unclear. Current studies provide two mechanisms: (i) direct particle translocation across the placental barrier into fetal circulation, , and (ii) indirect mechanisms mediated by PM_2.5-induced epigenetic modifications , and gut microbiota dysbiosis. − This warrants further investigation. Second, our results revealed pronounced sexual dimorphism in the developmental programming of insulin resistance by maternal PM_2.5 exposure, with male offspring being more susceptible. Notably, previous studies have consistently reported sex-dependent impacts of maternal PM_2.5 exposure on metabolic health in offspring. ,, Therefore, the mechanistic basis for these sexually dimorphic effects warrants further investigation, which has critical implications for the identification and targeted intervention of vulnerable populations. Third, although we have systematically characterized the single-cell landscape of iWAT in middle-aged male offspring and delineated maternal PM_2.5 exposure-induced multicellular alterations with their interactive networks, the precise mechanistic contributions of these cell type-specific alterations in offspring insulin resistance pathogenesis require systematic experimental validation in the future.

Conclusions

In summary, our study demonstrated that maternal exposure to PM_2.5 with nanoparticles triggered a fibrosis phenotypic switch of iWAT by altering cell-fate decisions in ADSCs from adipogenesis to fibrosis, which was mediated by expanded ADSC_2 populations and an IgG-driven pro-fibrotic phenotypic switch of ADSC_3. This impaired iWAT plasticity subsequently exerted systemic insulin resistance in nonobese, middle-aged male mouse offspring. From the perspective of maternal exposure to environmental factors, this study not only provided etiological evidence for the MUHNW but also advanced the mechanistic understanding of fetal-originated metabolic diseases.

Methods

Animals

All animal experiments were performed following the protocols evaluated and approved by the Institutional Animal Care and Use Committee of Zhejiang Chinese Medical University (ZCMU; Ethics Approval Number: IACUC-202409–23). The C57BL/6N mouse line was acquired from Charles River Laboratories. Mice were maintained under a 12 h light–dark cycle (from 06:00 to 18:00), with unrestricted access to water and food. Mice were fed a standard chow diet (Jiangsu Synergy Pharmaceutical Bioengineering Co., Ltd., China), which is suitable for gestation periods, breeding, lactation, and maintenance.

Experimental Strategy for Maternal PM_2.5 Exposure

Following a 1-week acclimation, 8-week-old virgin female mice were mated with male mice at 20:00 in a 2:1 ratio and checked for a vaginal plug (a positive sign of mating) the next morning. Once the vaginal plug was observed (GD 0), female mice were randomized to either the FA group or the concentrated PM_2.5 exposure group (PM_2.5). The exposure protocol was 12 h per day (from 08:00 to 20:00) for 19 successive days (GD 0–18), utilizing a whole-body inhalation exposure system located on the ZCMU campus.

The exposure system has two temperature-controlled chambers (FA chamber and one PM_2.5 chamber) holding cages, in which mice were housed and allowed to access food and water freely. The principles and design criteria were described in detail previously. , Briefly, the exposure system drew ambient air directly from the atmosphere and delivered it to the FA and PM_2.5 chambers. The FA chamber had a HEPA filter at the inlet valve to filter airborne particles, while the PM_2.5 chamber was equipped with a cyclone inlet to eliminate particles larger than 2.5 μm and a series of virtual impactors for the PM_2.5 concentration. Subsequently, clean air and concentrated PM_2.5 were continuously pumped to the FA and PM_2.5 chambers, respectively, and were evenly distributed within the chambers. During the non-exposure period (from 20:00 to 08:00 the next day), both groups of mice were housed in the FA chamber under the same conditions. After a 19-day exposure, some of the pregnant mice (GD 18) were euthanized and necropsied to collect iWAT for further analysis, while the other pregnant mice were removed from the FA and PM_2.5 chambers and housed individually in clean air until delivery (∼GD 19) to avoid offspring being exposed to PM_2.5. Notice that only female mice were subjected to PM_2.5 exposure during gestation throughout the experiment, while male mice used for mating and offspring were housed in clean air.

At birth (postnatal day 0, PND 0), the pups were weighed in whole litters and then indiscriminately culled to 6–8 per litter. The remaining pups were reared with their dams in a clean air environment. At PND 21, all pups were weaned and caged according to sex. Then, all weaned offspring were fed a standard chow diet and weighed weekly. At PND 56 (adult) and PND 365 (12 months old, middle-aged), the offspring from each litter were euthanized and necropsied to collect serum and organs (i.e., iWAT, eWAT, and BAT) for further analysis.

For the PM_2.5-exposed male mice model, 8-week-old male mice were randomly exposed to either FA or the concentrated PM_2.5 using the same exposure system for 12 h per day (from 08:00 to 20:00), 6 days per week, over 8 weeks. iWAT of male mice was collected post-euthanasia at the end of the exposure period.

PM_2.5 Concentration Analysis and Characterization

The PM_2.5 concentration in the PM_2.5 chamber was monitored in real-time by using an aerosol monitor model (pDR-1500, Thermo Scientific, USA). In addition, to obtain more accurate data on PM_2.5 concentration, PM_2.5 samples from FA and PM_2.5 chambers were captured on Teflon membranes (R2PJ037, Pall Corporation, USA) and quartz membranes (1851–047, GE Healthcare, UK). The membranes were weighed before and after sampling to calculate weight gain using a microbalance (Excellence Plus XP, Mettler-Toledo, Switzerland) in a room with controlled temperature and humidity. The formula for calculating PM_2.5 concentration is as follows: PM_2.5 concentration (μg/m³) = weight gain of the membrane (μg)/[sampling time (min) × sampling flow rate (L/min) × 0.001]. The sampling flow rate was determined using an air flow calibrator (Gilibrator 2, Sensidyne, USA). According to the exposure protocol (12 h per day), the average daily PM_2.5 concentration (including the non-exposed time) inhaled by mice was normalized using the following equation: average daily PM_2.5 concentration (μg/m³) = [PM_2.5 concentration in the PM_2.5 chamber (μg/m³) × 12 (hour) + PM_2.5 concentration in the FA chamber (μg/m³) × 12 (hour)]/24 (hour).

For PM_2.5 characterization, as in our previous study, water-soluble inorganic ions and the elemental composition of PM_2.5 were detected by an ion chromatograph (ICS6000, Thermo Scientific, USA) and a wavelength-dispersive X-ray fluorescence spectrometer (S8 Tiger, Bruker, Germany), respectively. The scanning electron microscope (SEM; S-4800 N, HITACHI Inc., Japan) was used to assess the morphology of PM_2.5.

Whole-Body Energy Metabolism Analysis

Heat production, O₂ consumption, CO₂ production, and respiratory exchange ratio of middle-aged offspring were determined using the Columbus Instruments Laboratory Animal Monitoring System (CLAMS, Columbus, USA) over 48 h.

Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)

Middle-aged offspring were fasted (12 h for GTT; 4.5 h for ITT) and then injected i.p. with glucose (2 mg/g body weight) or insulin (0.5 U/kg body weight), respectively. Blood glucose levels were monitored at 0 min pre-injection and at 30, 60, 90, and 120 min post-injection with a Precision Neo Blood Glucose Meter (Abbott Diabetes Care Inc., USA).

Body Composition Analysis

The benchtop time-domain NMR (TD-NMR) analyzer (minispec LF50, Bruker, Germany) was used to measure the lean and fat mass of offspring at 1, 2, 4, 8, and 12 months of age.

Histology and Immunohistochemistry

Adipose tissues collected from middle-aged male offspring were fixed in 4% PFA, paraffin-embedded, sectioned into 5 μm slices, and finally stained with hematoxylin and eosin according to the standard protocol. Images were captured using a microscope (DM4B, Leica Microsystems, Germany). Adipocyte sizes were quantified by using ImageJ.

For Masson’s trichrome staining, adipose tissue paraffin sections (5 μm) were deparaffinized and rehydrated. After incubation with Bouin’s solution (BP0140, Biosci Biotechnology Co., Ltd., China) overnight, these sections were stained with hematoxylin solution, differentiated in hydrochloric acid alcohol, reblued in lithium carbonate solution, and stained with ponceau acid fuchsin solution (rinsing with water after each step of processing). Subsequently, sections were incubated in a phosphomolybdate acid solution and stained with aniline blue solution. After alcohol dehydration and xylene clearing, sections were coverslipped using neutral balsam. Images were captured by using a microscope. The positive area in Masson’s trichrome staining was quantified using Image-Pro Plus.

For Sirius Red staining, after deparaffinization and rehydration, adipose tissue paraffin sections (5 μm) were immersed in 0.1% Sirius Red at room temperature. After differentiation and dehydration in alcohol, sections were cleared in xylene and coverslipped using neutral balsam. Images were captured using a microscope. The positive area in Sirius Red staining was quantified by using Image-Pro Plus.

For immunohistochemistry, after deparaffinization and rehydration, adipose tissue paraffin sections (5 μm) underwent heat-induced antigen retrieval. Then, sections were blocked (3% bovine serum albumin solution) and incubated overnight at 4 °C with rabbit anti-COL1A1 (1:500; A22090, ABclonal, China) and rabbit anti-IgG (1:6400; 46540, Abcam, UK). After washing, sections were incubated with HRP-linked secondary antibodies at room temperature, stained with diaminobenzidine (DAB), redyed with hematoxylin, and coverslipped using neutral balsam. Images were captured using a microscope. Quantitative analysis was performed by using Image-Pro Plus.

scRNA-Seq

The fresh iWAT from middle-aged male offspring was immediately preserved on ice in an sCelLive Tissue Preservation Solution (Singleron, China). After washing with HBSS, iWAT was minced and digested for 15 min at 37 °C in sCelLive Tissue Dissociation Solution (Singleron, China) using the Singleron PythoN Tissue Dissociation System. The resulting suspension was filtered through a 40-μm strainer, treated with red blood cell lysis buffer (Singleron, China) (room temperature, 5–8 min), and centrifuged (300 g, 4 °C, 5 min). The pellet was resuspended in PBS (HyClone, USA), and the cell viability was assessed using trypan blue staining.

Using the Singleron Matrix Single Cell Processing System, single-cell suspensions (2 × 10⁵ cells/mL) were loaded onto a microwell chip. After collecting the barcoding beads, the captured mRNA was reverse transcribed into cDNA and amplified by PCR. The amplified cDNA was then fragmented, ligated with adapters, and then used to construct libraries with the GEXSCOPE Single Cell RNA Library Kits (Singleron, China) per manufacturer’s instructions. Individual libraries were pooled at 4 nM and sequenced on an Illumina Novaseq 6000 (Illumina, USA) platform with 150-bp paired-end reads.

scRNA-Seq Data Analysis

Raw reads were processed using CeleScope (https://github.com/singleron-RD/CeleScope) to filter low-quality reads and then trimmed using Cutadapt v1.17. Following cell barcode and unique molecular identifier (UMI) extraction, reads were aligned to the GRCm38 (Ensembl version 92 annotation) with STAR v2.6.1a, achieving 83.26% mapping efficiency. Gene expression matrices were subsequently generated using featureCounts v2.0.1 for downstream analysis.

Quality control, dimensionality reduction, and clustering were executed utilizing Seurat 3.0. Cells were filtered according to three criteria: gene counts <200, top 2% gene and UMI counts, and mitochondrial content >10%. After filtering, 42896 cells were remained (average 2406.632 genes and 7527.484 UMIs per cell) for the downstream analyses. For dimensionality reduction and clustering, gene expression was normalized and scaled using the NormalizeData and ScaleData functions in Seurat v3.1.2, and the top 2000 variable genes were selected with the FindVariableFeatures function for PCA analysis. Cell clusters were identified using the top 20 principal components with FindClusters and projected in a two-dimensional space using the t-distributed stochastic neighbor embedding (t-SNE) method.

DEGs were identified using the Seurat FindMarkers function (Wilcox test) with thresholds set at >10% expression in a cluster and an average log (fold change) > 0.25. Cell types were annotated based on canonical markers from DEGs using the SynEcoSys database. Doublets, defined as cells coexpressing markers for multiple cell types, were manually removed. For high-resolution subclustering, ADSCs and macrophages were extracted and reclustered at resolutions of 0.3 and 0.2, respectively. Cell-type marker expression was visualized through heatmaps and feature plots using the DoHeatmap and FeaturePlot functions in Seurat v3.1.2, respectively.

Gene enrichment analysis (e.g., GO and KEGG) of DEGs was conducted using the “clusterProfiler” R package 3.16.1. GO terms and pathways with adjusted P values <0.05 were deemed significantly enriched.

The cell differentiation trajectory was inferred with Monocle2, ordering cells by pseudotime based on highly variable genes (HVGs). DDRTree was used to perform FindVariableFeatures and dimensionality reduction. Subsequently, the trajectory was visualized by the plot_cell_trajectory function. Differentiation potential was further predicted with CytoTRACE. RNA velocity analysis was analyzed in Python (velocyto v0.2.3; scVelo v0.17.17) under default parameters and visualized on t-SNE plots.

CellPhoneDB v2.1.0 was applied to identify receptor–ligand interactions. The statistical significance of interactions was assessed by randomly permuting all cell cluster labels 1000 times to derive a null distribution for average expression levels. Interactions with an average log expression >0.1 and P-value <0.05 were considered significant.

Gene set scoring was conducted per cell using the R package UCell v1.1.061, which employs a Mann–Whitney U test on ranked gene expression.

Cell Culture

3T3-L1 cells (CL-173, ATCC, USA) were maintained in high-glucose DMEM (11995040, Gibco, USA) containing 10% donor bovine serum (10371029, Gibco, USA) and 1% penicillin/streptomycin (P/S; abs9244, Absin, China). In the IgG-treated assay, experimental protocols and IgG treatment concentrations were referenced from previous studies. 3T3-L1 preadipocytes in 24-well plates were treated with IgG (I5381, Sigma, USA) at different concentrations (0, 50, and 100 μg/mL) for 24 h and then harvested for further examination. In the CM-treated assay, 3T3-L1 cells were treated with CM from BMDMs in 24-well plates for 24 h and then harvested for further examination.

As previously described, BMDMs were flushed from male mice femurs and tibias using α-MEM (C12571500CP, Gibco, USA) supplemented with 2% fetal bovine serum (FBS; 10099–141, Gibco, USA) and 1% P/S, then incubated with red blood cell lysis buffer (abs9101, Absin, China) to eliminate erythrocytes. The remaining cells were maintained in α-MEM supplemented with 10% FBS, 1% P/S, 1% GlutaMAX (35050–061, Gibco, USA), 1% NEAA (11140–050, Gibco, USA), and 10 ng/mL M-CSF (315–02, Pepro Tech, USA). In the IgG-treated assay, experimental protocols and IgG treatment concentrations were referenced to previous studies. After culturing in 24-well plates for 4 days, IgG was added to BMEMs at varying concentrations (0, 50, and 100 μg/mL) for 2 days. Two days later, CMs were collected to treat 3T3-L1 cells, and the cells were harvested for further examination.

RNA Isolation and qRT-PCR

RNA was isolated from adipose tissues and cells using RNAiso Plus (9108, TaKaRa, Japan), quantified with a spectrophotometer (NanoDrop 2000, Thermo Scientific, USA), and then reverse-transcribed into cDNA with Prime Script RT Master Mix (6210, TaKaRa, Japan). Subsequently, qRT-PCR was performed using PowerUP SYBR Green Master Mix (A25742, Applied Biosystems, USA) on a QuantStudio 7 Flex instrument (Applied Biosystems, USA). Relative gene expression was normalized to 36B4 or β-actin reference gene and analyzed via the 2^{−ΔΔ Ct} method, with primers listed in Table S1.

Western Blotting

Protein was extracted from adipose tissues using RIPA lysis buffer (AR0102, Boster, China) containing PMSF (ST506, Beyotime, China) and a phosphatase inhibitor cocktail (4906837001, Roche, Switzerland). Protein concentration was measured with a BCA protein assay kit (P0010S, Beyotime, China). Equal protein amounts (10–20 μg) per sample were loaded, separated by using SDS-PAGE, and transferred onto PVDF membranes. Subsequently, the membranes were blocked with 5% milk and incubated with rabbit anti-pAKT (Ser473) (1:1000; 9271, CST, USA), rabbit anti-AKT (1:1000; 9272, CST, USA), rabbit anti-TGFβ (1:1000; 3711, CST, USA), rabbit anti-Vimentin (1:1000; 5741, CST, USA), rabbit anti-E-Cadherin (1:1000; 3195, CST, USA), rabbit anti-IgG (1:2000; 46540, Abcam, UK), mouse anti-IL-6 (1:500; sc-32296, Santa Cruz, USA), rabbit anti-IL-1β (1:2000; 16806–1-AP, ProteinTech Group, China), and rabbit anti-HSP90 (1:2000; 13171–1-AP, ProteinTech Group, China) overnight at 4 °C. After washing, the membranes were incubated with HRP-linked secondary antibodies (1:5000; SA00001–1 and SA00001–2, ProteinTech Group, China) at room temperature and then visualized using a Clarity Western chemiluminescence (ECL) substrate (1705061, Bio-Rad, USA) and an ECL Super Kit (RM02867, ABclonal, China) on a ChemiDoc Imaging System (12003153, Bio-Rad, USA). IgG1 was detected directly using a secondary antibody (HRP-conjugated Goat anti-Mouse IgG1; 1:2000; AS006, ABclonal, China). Quantitative analysis was performed using ImageJ.

Statistical Analysis

All data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism version 8.0. Comparisons between the two groups used an unpaired Student’s t-test (two-tailed); comparisons among the three groups used one-way ANOVA with Tukey’s test; data involving two variables or repeated measures were analyzed by two-way ANOVA or two-way repeated-measures ANOVA, respectively, both followed by Bonferroni’s test. A P value < 0.05 was considered significant. The statistical parameters and n-values are shown in the figure legends.

scRNA-seq data generated in this study have been deposited at GEO with the accession number GSE307673 and are publicly available as of the date of publication.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsnano.5c18921.

• PM_2.5 concentration and components; effects of maternal PM_2.5 exposure on birth weight, lean mass, glucose tolerance, and energy metabolism in male offspring; effects of maternal PM_2.5 exposure on body composition, energy metabolism, and glucose homeostasis in female offspring; effects of maternal PM_2.5 exposure on adipose tissues in male offspring; effects of PM_2.5 exposure on iWAT fibrosis in pregnant mice; effects of PM_2.5 exposure on iWAT fibrosis in male mice; individual gene t-SNE plots of marker genes for T cells, mural cells, ILC2, epithelial cells, neutrophils, adipocytes, and mast cells; effects of maternal PM_2.5 exposure on ADSCs in iWAT from middle-aged male offspring; qRT-PCR analysis of Fcgrt gene expression in iWAT from middle-aged male offspring; qRT-PCR analysis of fibrosis-related gene expression in 3T3-L1 preadipocytes treated with vehicle or different concentrations of IgG (50 and 100 μg/mL); effects of maternal PM_2.5 exposure on MPs in iWAT from middle-aged male offspring; qRT-PCR analysis of indicated gene expression in BMDMs treated with vehicle or different concentrations of IgG (50 and 100 μg/mL); KEGG enrichment analysis of DEGs in Mac_4, Mac_5, and monocytes from middle-aged male offspring in Male/PM_2.5 groups; correlation between fibrosis and inflammation in iWAT from middle-aged male offspring (PDF)

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R.J.H. and R.L. contributed equally. C.Q.L. conceptualized and designed the study. R.L. and C.Q.L. provided scientific advice and resources. R.J.H. and R.L. performed animal experiments. R.J.H. performed cell experiments. R.J.H., R.L., L.M.W., S.D.L., H.S.C., L.Z., and L.Q. were involved in the collection, preparation, interpretation, validation, and critical review of the data. R.J.H. created the figures and Supporting Information and drafted the manuscript. R.L., Q.H.S., and C.Q.L. edited and reviewed the manuscript text. R.L. and C.Q.L. obtained funding and supervised research.

The authors declare no competing financial interest.