Large Peritoneal Macrophages Promote the Resolution of Inflammation in Injured Endometrium

The human endometrium undergoes a monthly process of remodeling, shedding, and regeneration, commonly referred to as menstruation [ 1]. This multicellular, bi-layered tissue Guangfeng Zhao [email protected] Sunan Shen [email protected] Yayi Hou [email protected] 1 The State Key Laboratory of Pharmaceutical Biotechnology, Division of Immunology, Medical School, Nanjing University, Nanjing 210093, China 2 Department of Obstetrics and Gynecology, Nanjing Drum Tower Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210093, China 3 Jiangsu Key Laboratory of Molecular Medicine, Nanjing 210093, China 4 Jiangsu International Laboratory of Immunity and Metabolism, The Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou 221004, China

Macrophages play a significant role in the repair of endometrial injuries. While large peritoneal macrophages (LPMs) have been reported to migrate to injured organs and repair tissues within the peritoneal cavity, their involvement in the repair of injured endometrium remains unclear. In this study, we utilize a mouse model of endometrial injury that does not involve laparotomy, a procedure that typically results in a substantial loss of LPMs. Strikingly, we find that LPMs reach the endometrium within 6 h post-modeling. By depleting or supplementing LPMs, our results reveal that these cells are capable of engulfing dead cells in the endometrium and resolving inflammation. Additionally, we observe that the migra - tion efficiency of LPMs is enhanced with increased levels of 17β-estradiol (E2) in mice. In vitro assays further confirm that E2 accelerates the migration of LPMs towards apoptotic endometrial stromal cells. Overall, our findings demonstrate that LPMs rapidly migrate into injured endometrium in relation to E2 levels and facilitate the process of tissue repair.

Large peritoneal macrophages · Endometrium · Endometrial injury · Endometrial repair · 17β-estradiol Received: 21 March 2025 / Revised: 23 May 2025 / Accepted: 17 June 2025 / Published online: 2 August 2025 © The Author(s) 2025 Large Peritoneal Macrophages Promote the Resolution of Inflammation in Injured Endometrium Jingman Li1 · Lijie Yin1 · Jiali Wang1 · Yuchen Pan4 · Chen Peng1 · Yue Dong1 · Sunan Shen1,3 · Yayi Hou1,3 · Guangfeng Zhao2 1 3 Inflammation (2025) 48:4428–4442 the early phases of pathologic endometrial injury [ 11–13], leading to the infiltration of neutrophils and monocytes/ macrophages [ 14]. These cells carry out vital functions, including the clearance of cellular debris and the resolution of inflammation [ 15, 16]. Among them, macrophages are deemed the most crucial cells contributing to the repair and regeneration of the endometrium [17]. Current research sug- gests that the macrophages involved in endometrial repair originate from two distinct sources: in situ proliferation of resident macrophages and macrophages derived from mono- cytes [18]. However, the potential roles of other sources of macrophages in endometrial repair remain unexplored. In recent years, an increasing number of studies have concentrated on the role of a specific group of resident macrophages found in the mammalian peritoneal cavity, known as large peritoneal macrophages (LPMs) [19]. LPMs are not a homogeneous population but exhibit heterogene - ity in terms of their origin, phenotype, and function. The ontogeny of LPMs involves both embryonic-derived resi - dent macrophages and monocyte-derived macrophages that can replenish the LPM pool under certain conditions [ 20, 21]. This heterogeneity and ontogeny are crucial for under- standing the diverse roles LPMs play in health and disease. LPMs constitute the primary population of peritoneal mac - rophages, with their main function being the phagocytosis of apoptotic cells [ 22]. Research has indicated that LPMs possess the ability to rapidly migrate to the site of injury and facilitate tissue repair in mouse models of liver injury [23] and damage to the intestinal serosal layer [24]. Further- more, when the peritoneal cavity wall is injured, clusters of LPMs physically seal the wound, promoting swift recovery in mouse model [ 25]. Given that the uterus is also a vital organ situated within the peritoneal cavity, it remains to be further explored whether LPMs similarly migrate to the endometrium to exert their effects. In this study, we employed our previously established mouse model of endometrial injury, which obviates the need for peritoneal cavity incision [ 11]. This model was initially designed to minimize the risk of systemic infec - tions associated with open surgical procedures and to more closely mimic the conditions of human endometrial trauma. Notably, within this model, we observed the presence of LPMs in the damaged uterine tissue. These LPMs appeared earlier than monocyte—derived macrophages and nearly simultaneously with neutrophils. We utilized flow cytom - etry assays to demonstrate the capacity of LPMs to migrate from the peritoneal cavity to the injured endometrium. Fur- thermore, through experiments involving the depletion and supplementation of LPMs, we elucidated their pivotal role in clearing dead cells and resolving inflammation at the site of injury. Additionally, our findings revealed that estradiol (E2) facilitates the migration of LPMs both in vivo and in vitro. These results collectively highlight the crucial func - tion of LPMs in endometrial repair and provide evidence that administering E2 following uterine cavity surgery can expedite the migration of LPMs, thereby enhancing their reparative capabilities.

Animals and Experimental Protocol Female BALB/c mice (8–10 weeks old) and female C57BL/6 mice (8–10 weeks old) were brought from Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. (Taizhou, China) and were housed in a pathogen-free condi- tion in a 12-h light and dark cycle. All procedures involved in mice were approved by the institutional guidelines for animal care and used based the Animal Care Committee at Nanjing University and followed all the ARRIVE guide - lines [26]. Endometrial Injury Time-Course Experiment Balb/c female mice (n = 4–6) were anesthetized with isoflu- rane and fixed in the supine position. The probe of electric scratching tool was inserted into the uterus of mice through the vaginal opening. The switch was pressed, vibrated for 8 s, and then paused for 8 s. This process was repeated twice more. The probe was slowly pulled out and the mouse was placed under a warm lamp for recovery from anesthe - sia. Samples were harvested at 0, 3, 6, 12, 24 or 48 h after modeling. CLL Eliminate LPMs Experiment Balb/c female mice ( n = 4–6) were injected intraperitone - ally with 100 μL PBS/con-CLL (7 mg/mL, FormuMax, F70101-A)/CLL (7 mg/mL, FormuMax, F70101C-A) 24 h in advance. Next, endometrium of mice was injured using the electric scratching tool for 12 h. Intrauterine Supplementation of LPMs Vaginal secretions from female BALB/c mice (8–10 weeks old) were scraped using a sterile disposable 200 μL suction tip, suspended in PBS, and placed under a light microscope to observe cell morphology. The mouse model was created at estrus based on the cell morphology in vaginal secretion smear of mice. Balb/c female mice (n = 3–5) were anesthe- tized with isoflurane and fixed in the supine position. They were then disinfected and hair from their abdominal sur - faces were removed. The abdominal skin and muscles were 1 3 4429 Inflammation (2025) 48:4428–4442 then cut. One side of the uterine horn was found. A syringe needle (30 G) was inserted and the lining of the uterus was scratched 50 times. 10 µL of saline solution contained LPMs (8 × 104) or not was then injected into the uterine cav- ity. Both ends of the uterine horn were held with forceps for 5 min to ensure that liquid is fully absorbed by the tissue. The muscle layer and skin were sutured. The mouse was put under a warm lamp for recovery from anesthesia. Samples were harvested after 12 h. Isolation of Primary Mouse Cells Large Peritoneal Macrophage (LPMs) LPMs were obtained from female Balb/c mice (8–10 weeks old). The mice need to be fasted for 8 h and sacrificed by cer- vical vertebra dislocation. Then inject cold DMEM medium 5 mL into peritoneal cavity of mice and gently massage them for about 2 min. Subsequently, collect the peritoneal fluid and 300 × g centrifugate for 5 min to obtain cells. Lyse erythrocytes if necessary. Inoculate the peritoneal fluid cells into cell culture plates with warm DMEM medium supple - mented with 10% FBS for 4 h. Then abandon the superna - tant and wash cells attached to the bottom with warm PBS buffer solution to removal suspension cells. The remaining cells are LPMS for subsequent experiments. Endometrial Stromal Cells (ESCs) Uterine tissues from mice were cut into pieces and digested with trypsin with 0.1% EDTA for 10 min at 37 °C in a 5% CO2-humidified atmosphere. After termination of trypsin digestion, the cells were digested in HBSS medium contain- ing 0.8 mg/mL collagenase type I for 2 h at 37 °C in a 5% CO2-humidified atmosphere. Then digestive fluid contained tissue residue were fractionated with 40-μm cell strainers. 300 × g centrifugate the filtrate for 5 min and the cell precip- itates were cultured in cell culture dishes with DMEM/F12 medium containing 10% FBS, 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a 5% CO2-humidified atmo- sphere. After 2–3 days of culture, the cells that could con - tinuously proliferate were ESCs. Primary ESCs from P2/P3 were used for subsequent experiments. Cells Treatment and Experiments in vitro ESCs Apoptosis Inducing Method ESCs cultured to passage 2 or 3 were seeded in 24-well plates (1 × 104 cells/well) and treated with 20% ethanol for 1, 5 and 25 min or not. 20% ethanol system was absolute ethanol and DMEM/F12 medium containing 10% FBS (1:4). The subsequent experiments used 5 min as treatment condition. Phagocytosis Assay of LPMs on ESCs ESCs cultured to passage 2 or 3 were seeded in 24-well plates (1 × 104 cells/well) and treated with carboxyfluores - cein succinimidyl amino ester (CFSE, Invitrogen, catalog # 65–0850-85) for 10 min at 37 °C in a 5% CO 2-humidified atmosphere. Then the cells labeled by CFSE were induced apoptosis and cocultured with LPMs (1:3) isolated from mice for 12 h. E2 and Fulvestrant Treatment of LPMs The LPMs isolated from mice were seeded in 24-well plates (5 × 104 cells/well) and treated by fulvestrant (100 nM) or not for 1 h. Next, the LPMs treated by E2 (1, 10 or 100 nM) or not for 24 h. The fulvestrant and E2 was purchased from MedChemExpress (MCE) (catalog # HY-13636 and HY-B0141). LPMs Migration Model and Crystal Violet Staining LPM was inoculated into the transwells (1 × 104 cells/well) and pre-treated by fulvestrant or E2 or not for 24 h. The tran- swells within LPMs were cocultured with apoptotic ESCs in 24 well plates for 6 h. Then the transwells were fixed with 4% paraformaldehyde in phosphate buffer for 15 min at room temperature. After washed twice with phosphate buf - fer, the transwells were fixed with 1% crystal violet solution (biosharp, catalog # BL802A) in phosphate buffer for 15 min. They were washed with phosphate buffer until there was no residual crystal violet solution. Cells on the side of the transwells were gently wiped off with a cotton swab, leaving only the bottom cells. Finally, they were viewed and photographed under a Nikon Eclipse Ti-U microscope equipped with a digital camera (DS-Ri1, Nikon). Flow Cytometry Assay All cells were filtered through a 70 μm cell strainer and then washed with PBS to generate single-cell suspensions. An Fc-receptor blocker (CD16/32, eBioscience, catalog # 14–0161-82) was used to reduce non-specific antibody binding. Single cell suspensions were labeled with flow cytometry antibodies (manufacturer and catalog number are showed in the Supplementary material Table S1) and detected by BD FACS Calibur or Beckman Coulter Cyto - flex S. Data analysis was performed by FlowJo software. 1 3 4430 Inflammation (2025) 48:4428–4442 Statistical Analysis All values with a normal distribution presented on the graphs are shown as means ± S.E.M. Unpaired Student’s t-tests was used to analyze statistical significance, and P-values < 0.05 were considered statistically significant. All statistics were performed with GraphPad Prism 8.

LPMs Migrate Into Injured Endometrium To examine the alterations in inflammation-associated immune cells within the injured endometrium, we induced endometrial damage in mice using an electric scratching tool, as previously described [ 11], at various time points: 3, 6, 12, 24, and 48 h prior to sample collection (Fig. 1A). Flow cytometry results showed that, after endometrium was injured, the proportion of neutrophils ( CD11bintLy6G+) in uterus increased to the peak at 6 h and gradually disappeared (Fig. 1B). The increase in monocytes ( CD11b+CCR2+) and uterine resident macrophages ( CD11b+CD102−F4/80int) were slightly later, reach the peak at 12 h (Fig. 1C and D). Remarkably, there were a group of cells characterized as CD11bhigh, CD102+ and F4/80high. Their phenotype dif - fered from that of neutrophils but increased at a similar rate (Fig. 1E). CD102 is the specific marker of cavity resident macrophages [ 27]. Given that the uterus is located in the peritoneal cavity, we supposed that these cells might be peritoneal macrophages. Next, we measured the proportions of LPMs and SPMs in peritoneal cavity at each moment of endometrial injury by flow cytometry. The CD11b + myeloid cells in the peritoneal After removing dead cells and adherent cells, we refer the CD11b+CD102+ cells as LPMs, CD11b+CD102−MHCII+ cells as SPMs, CD11b+CD102−MHCII− cells as new cells, CD11bintLy6G+ cells as neutrophils, CD11b+CCR2+ cells as monocytes, and CD11b+F4/80intCD102− cells as uterine resident macrophages. Immunofluorescence Staining Mouse uterus tissues or primary mouse cells were fixed with 4% paraformaldehyde in phosphate buffer. Paraffin-embed- ded samples were sectioned at 2 μm. Tissue slices or cells on glass slides were incubated with primary antibodies (manu- facturer and catalog number are showed in the Supplemen - tary material Table S2) overnight at 4 °C. After rinsing three times in PBS, the samples were incubated with secondary antibody (manufacturer and catalog number are showed in the Supplementary material Table S2) for 1.5 h at room tem- perature in the dark, and then the nuclei were stained with DAPI (Bioword, China). The slides were visualized using a FV3000 Laser Scanning Confocal Microscope (Olympus). Enzyme-Linked Immunosorbent Assay (ELISA) and Biochemical Detection The levels of IL-1β, IL-6 and TNF-α in mouse serum were detected using the corresponding mouse ELISA kit accord- ing to the manufacturer’s instructions (Biolegend, catalog # 432601, 431301 and 430901). The level of E2 was detected using the corresponding mouse ELISA kit according to the manufacturer’s instructions (Cloud-Clone Corp., catalog # CEA461Ge). Serum E2 levels were determined at Service - bio Biotechnology Co. LTD (Wuhan, Hubei, China). Quantitative real-Time PCR Quantitative real-time PCR was carried out as previously described [14]. The primer sequences we used are showed in the Supplementary material Table S3. RNA Sequencing Assay Mouse LPMs samples were analyzed via RNA sequencing in Hangzhou Lianchuan Biotechnology Co., Ltd. (Hang - zhou, China). Preliminary progression was analyzed after obtaining raw data. Genes differential expression analysis was performed by DESeq2 software between E2 and con - trol groups. The fold value represented the degree of differ- ential expression between E2 and control groups. The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 was considered differentially expressed genes. Fig. 1 LPMs migrate into injured endometrium. A The endometrium of Balb/c female mice (8–10 weeks old) was injured using the elec - tric scratching tool and samples were collected at 3-, 6-, 12-, 24- and 48-h post-injury. B-E The statistical figures of percentages of neutro - phils (CD11bint Ly6G+), monocytes (CD11b+CCR2+), uterine resident macrophages ( CD11b+F4/80intCD102−) and LPMs ( CD11b+F4/80 +CD102+) in uterus determined by flow cytometry assay. F The statis- tical figures of percentages of LPMs (CD11b+CD102+F4/80+) in peri- toneal cavity determined by flow cytometry assay. G Flow cytometry assay data of the F4/80 and GATA6 expressing levels on cells from above gates. Among them, Q2 represented LPMs. H Immunofluores- cence staining images (cross section) of CD102 and GATA6 expres- sions in uterus at 0 h (control) and 6 h after injured. CD102 (red) was located on the cell membrane and GATA6 (green) was located in the nucleus. Bar = 50 μm. I The statistical figures of percentages of LPMs (CD11b+CD102+F4/80+GATA6+) in uterus determined by flow cytom- etry assay. J Immunofluorescence staining images (cross section) of TUNEL in uterus at 0 h (control) and 6 h after injured. Bar = 100 μm. K The level of IL-1β, IL-6 and TNF-α in serum detected by ELISA assay. Bar graphs show the mean ± SEM; unpaired Student’s t test was used to compare the experimental groups. Bar = 50 μm. n = 4–6; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 1 3 4431 Inflammation (2025) 48:4428–4442 1 3 4432 Inflammation (2025) 48:4428–4442 liver, or bone marrow, except for a marked reduction in macrophages in the spleen (Supplementary Fig. 3B). More- over, level of IL-6 in serum was slightly increased, whereas the levels of IL-1β and TNF-α did not change significantly (Supplementary Fig. 3 C). Taken together, these results sug- gested that intraperitoneal injection of CLL could deplete LPMs from the peritoneal cavity of mice without causing too many additional effects. We subsequently depleted LPMs in the peritoneal cav - ity using CLL 24 h prior to experimentally inducing endo - metrial damage in mice with our electric scratching tool, with samples collected 12 h later (Fig. 2A). Flow cytometry

indicated that CLL effectively hindered the migration of LPMs from the peritoneal cavity to the uterus, whereas con-CLL did not exert such an effect (Fig. 2B and C). The absence of LPMs prevented their accumulation in the injured uterus. Furthermore, the lack of LPMs exacerbated both the elevation of inflammatory factors in the serum resulting from uterine injury (Fig. 2D) and the accumula - tion of dead cells in the endometrium (Fig. 2E). Based on these observations, we hypothesized that LPMs may play a crucial role in phagocytosing dead cells, mitigating inflam - mation, and facilitating endometrial repair. LPMs Relieve Inflammation and Phagocytose Dead Cells in Injured Endometrium To validate our hypothesis regarding the function of LPMs in the injured endometrium, we conducted a LPMs trans - plantation experiment. In this study, we accessed the peri - toneal cavity of the mice and induced endometrial damage using a syringe needle. This procedure mirrored the estab - lishment of the mechanical damage IUA mouse model described in our earlier publications [11, 28] (Fig. 3A). Our findings revealed that the introduction of LPMs was capa - ble of abrogating the upregulation of serum inflammatory factors ( IL-1β, IL-6, and TNF-α) triggered by endometrial injury (Fig. 3B). We also noted that LPMs were colocalized with dead cells within the endometrium (Fig. 3C), indicat- ing their capacity to phagocytose these cells in that location. To further substantiate this observation, we conducted an in vitro phagocytosis assay. Given that endometrial stromal cells (ESCs) constitute the primary cell population in the endometrium, we utilized ESCs derived from mouse uteri as the target for phagocytosis. ESCs were seeded in well plates and subsequently treated with ethanol for 5 min to induce apoptosis (Supplementary Fig. 4 A and B). The immuno - fluorescence images obtained in vitro demonstrated that LPMs engaged in trogocytosis, shredding, and endocytos - ing apoptotic ESCs, confirming their ability to phagocytose these cells (Fig. 3D). Collectively, these results established that LPMs can alleviate inflammation and phagocytose dead CD102 (red) was located on the cell membrane and GATA6 (green) was located in the nucleus. They were divided into three groups based on the levels of CD102 and MHCII, including LPMs ( CD102+), SPMs ( CD102−MHCII+) and an undefined subgroup called new cells ( CD102−MHCII−) (Supplementary Fig. 1B). Among them, LPMs had the highest proportion (> 90%). Moreover, LPMs exhibited F4/80high and GATA6+ specifically, which was different from the other two populations (Supplementary Fig. 1 C and Supplementary Fig. 1D). Flow cytometry results showed that, after damage to the endometrium, the proportion of LPMs in peritoneal cavity was decreased at 3 h and came to the valley bottom at 6 h (Fig. 1F). Then the proportion gradually returned to normal. This pattern of change was just corresponded to that of CD11b+CD102+F4/80+ cells in injured endometrium (Fig. 1E). In addition, the proportion of SPMs was increased at 24 h after damage (Supplemen - tary Fig. 1E) and new cells were increased to the peak at 6 h (Supplementary Fig. 1 F). These data indicated that LPMs may be the cell population which migrated from peritoneal cavity into the damaged endometrium. To validate this hypothesis, we conducted a detailed characterization of CD11b+, CD102+, and F4/80+ cells within the injured endometrium and discovered that these cells expressed GATA6, a specific marker for LPMs (Sup - plementary Fig. 1 A, Fig. 1G and H). Our flow cytometry analysis revealed that, subsequent to endometrial injury, the proportion of LPMs in the uterus increased notably at 3 h, reaching a peak at 6 h (Fig. 1I), followed by a gradual decline. Correspondingly, this treatment caused the increase of dead cells number in the endometrium (Fig. 1J) and the upregulation of inflammatory factors ( IL-1β、IL-6 and TNF-α) in serum (Fig. 1K). All the above experiments were carried out on Balb/c mice. A similar phenomenon was also observed in C57BL/6 mice (Supplementary Fig. 2 A, B, C). Overall, these findings indicate that LPMs could transfer from peritoneal cavity to injured endometrium. This process was as rapid as the infiltration of neutrophils, and could be observed in different mouse strains. LPMs Absence Enhances Inflammation and Accumulation of Dead Endometrial Cells Our findings underscored the capacity of LPMs to swiftly migrate to the site of uterine injury. To delve deeper into the roles of LPMs in the injured uterus, we employed a strat - egy to deplete LPMs by administering clodronate liposomes (CLL) intraperitoneally to the mice, with con-CLL served as the control. Notably, a substantial majority of LPMs were effectively eliminated within 48 h post-injection (Supple - mentary Fig. 3 A). Additionally, we observed no significant alterations in the proportions of macrophages in the uterus, 1 3 4433 Inflammation (2025) 48:4428–4442 were numerous large scaly cells, i.e. the keratinized endo - metrial epithelial cells (Supplementary Fig. 4 A). Indeed, the level of E2 was higher in the serum of proestrus mice than that in the serum of estrus mice (Supplementary Fig. 4B). We then divided mice into estrus and proestrus groups by the above characteristics, and damaged their endometrium with our electric scratching tool at 6 h before sample harvesting respectively (Fig. 4A). The results showed that the migra - tion efficiency of LPMs in proestrus mice was significantly higher than that in estrus mice, although their migration to the uterus occurred in both stages of mice (Fig. 4B and C). cells in the injured endometrium, which is crucial for uter - ine repair. Estradiol can Accelerate the Migration of LPMs to the Damaged Endometrium We identified the estrous periods according to the cell mor- phology in mouse vaginal secretion smear of mice. In the vaginal secretions of proestrus mice, there were mainly nucleated, round, large cells, i.e. the normal endometrial epi- thelial cells. In the vaginal secretions of estrus mice, there Fig. 2 LPMs absence enhances inflammation and accumulation of dead endometrial cells. A The schematic diagram of modelling process. Balb/c female mice (8–10 weeks old) were injected intra- peritoneally with 100 μL PBS/con- CLL/CLL 24 h in advance. Next, endometrium of mice was injured using the electric scratching tool for 12 h. B The statistical figures of percentages of LPMs (CD11b+C D102+F4/80+) in peritoneal cavity determined by flow cytometry assay. C The statistical figures of percentages of LPMs (CD11b+CD 102+F4/80.+) in uterus determined by flow cytometry assay. D The level of IL-1β, IL-6 and TNF-α in serum detected by ELISA assay. E Immunofluorescence staining images (cross section) of TUNEL in uterus. Bar graphs show the mean ± SEM; unpaired Student’s t test was used to compare the experimental groups. Bar = 100 μm. n = 4–6; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 1 3 4434 Inflammation (2025) 48:4428–4442 Fig. 3 LPMs relieve inflammation and phagocytose dead cells in injured endometrium. A The schematic diagram of modelling pro - cess. Balb/c female mice (8–10 weeks old) were carried out surgery and intrauterine injected LPMs (8 × 104). Samples were harvested after 12 h. B The level of IL-1β, IL-6 and TNF-α in serum detected by ELISA assay. C Immunofluorescence staining images (cross section) of CD102 (pink), GATA6 (green) and TUNEL (yellow) expressions in uterus from ‘injury + LPMs’ group mice. D Immunofluorescence stain- ing images of LPMs (F4/80 +, red) and ESCs (CFSE labeled, green) isolated in vitro. Bar graphs show the mean ± SEM; unpaired Student’s t test was used to compare the experimental groups. Bar = 50 μm. n = 3–5; *P < 0.05, **P < 0.01 1 3 4435 Inflammation (2025) 48:4428–4442 factors in proestrus mice were significantly lower than those in estrus mice (Fig. 4F). These data suggested that the differ- ent E2 levels may influence the migration of LPMs into the impaired uterus to suppress inflammation. Moreover, at 6 h after endometrium injury, the proportion of LPMs in peritoneal cavity of mice was inversely pro - portional to the level of E2 (Fig. 4D), but directly propor - tional in uterus (Fig. 4E). The serum levels of inflammation Fig. 4 The migration efficiency of LPMs into injured uterus is influ - enced by E2 level. A The schematic diagram of modelling process. Balb/c female mice (8–10 weeks old) were divided into estrus group and proestrus group. The endometrium was injured using the elec - tric scratching tool for 6 h. B The statistical figures of percentages of LPMs (CD11b + CD102 + F4/80 +) in peritoneal cavity determined by flow cytometry assay. C The statistical figures of percentages of LPMs (CD11b + CD102 + F4/80 +) in uterus determined by flow cytometry assay. D Correlation analysis graph of percentage of LPMs in perito - neal cavity and E2 of serum. E Correlation analysis graph of percent- age of LPMs in uterus and E2 of serum. F The level of IL-1β, IL-6 and TNF-α in serum detected by ELISA assay. Bar graphs show the mean ± SEM; unpaired Student’s t test was used to compare the experimental groups. n = 4–8; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 1 3 4436 Inflammation (2025) 48:4428–4442 non-vascular route, subsequently contributing to tissue repair [23]. Subsequent research has further elucidated the migratory behavior and tissue repair capabilities of LPMs in both mouse intestinal [ 24] and peritoneal cavity wall [25] injuries. Furthermore, pericardial macrophages, which, akin to LPMs, belong to the category of cavity—resident macrophages, have been demonstrated to infiltrate damaged cardiac tissue and exert a reparative influence in a mouse model [21]. Notably, GATA6⁺ macrophages have been iden- tified within human pericardial fluid. This finding provides substantial support for the hypothesis that the reparative function observed in the mouse model holds clinical rele - vance in human cardiac diseases [21]. Given that the uterus is also situated within the peritoneal cavity, we are intrigued by the possibility that LPMs may similarly respond to endo- metrial injury. It is known that injury to the peritoneal cavity wall causes LPMs to form adherent aggregates at the wound site, which leads to a significant reduction of LPMs in peritoneal fluid [25]. In addition, peritoneal cavity infection caused by lapa- rotomy can also trigger a large loss of LPMs [19]. Therefore, the laparotomy during the establishment of animal models is bound to interfere with the study of LPMs. Our modeling tool skillfully avoided disruption of the normal peritoneal E2/ER-β Axis is Necessary for the Migration of LPMs to Apoptotic ESCs Above results hinted E2 may drive the migration of LPMs to damaged uterus. To further explore roles of E2 in migration of LPMs, we intraperitoneally injected E2 to estrus mice, and then damaged their endometrium with the electric scratch - ing tool at 6 h before sample harvesting (Fig. 5A). Dimethyl sulfoxide (DMSO) was used as control. As showed in flow cytometry assay results, E2 promoted the accumulation of LPMs in impaired uterus, while there was no difference in the percentage of LPMs in peritoneal cavity between ‘Injury + DMSO’ group and ‘Injury + E2’ group (Fig. 5B and C). In addition, the transcriptome RNA sequencing (RNA-seq)

showed that E2 increased the expression of many genes related to cytoskeleton, adhesion and migration in LPMs (Fig. 5D). Indeed, we found that E2 could elongate the cytoskeleton and increase pseudopodia of LPMs in vitro (Fig. 5E), which were both necessary for cells to migrate. To better simulate the migration of LPMs to the damaged uterus, an in vitro migration assay was designed. ESCs are considered as the main cells in endometrium. ESCs were thus seeded in well plates, and then treated with ethanol for 5 min to induce apoptosis. Correspondingly, LPMs were seeded in the upper transwells for co-culture with apop - totic ESCs. The results showed that E2 indeed promote the migration of LPMs to apoptotic ESCs in a dose-dependent manner (Fig. 5F). Accumulated evidence has revealed that there are three types of estrogen receptors (ERα, ERβ, and GPR30). Impor- tantly, LPMs were detected to express only ER-β (gene named Esr-2) specifically, but not ER-α (gene named Esr-1) and GPR30 (gene named Gpr30) (Fig. 5G). Moreover, E2 (10 nM) significantly up-regulated the expression of ER-β in LPMs (Fig. 5H and I), indicating ER-β in LPMs specifi - cally responded to the stimulation of E2. Estrogen receptor antagonist fulvestrant (Ful) is showed to completely inhibit estrogen-mediated changes in gene transcription. In in vitro migration assay, Ful eliminated the promotion of E2 on the migration of LPMs to apoptotic ESCs (Fig. 5J). Combined with all above results, we concluded that E2/ ER-β axis was necessary for the migration of LPMs to apoptotic ESCs.

Over the past decade, large peritoneal macrophages (LPMs) have garnered increasing attention. As the primary subpop- ulation of macrophages within the mammalian peritoneal cavity, they fulfill a pivotal immune function in maintain - ing homeostatic balance. A seminal study illustrated that LPMs can swiftly migrate to the site of liver injury via a Fig. 5 E2/ER-β axis is necessary for the migration of LPMs to apop - totic ESCs. A The schematic diagram of modelling process. The endo- metrium of Balb/c female mice (8–10 weeks old) which were in estrus period was injured using the electric scratching tool for 6 h. B The sta- tistical figures of percentages of LPMs (CD11b + CD102 + F4/80 +) in peritoneal cavity determined by flow cytometry assay. C The statistical figures of percentages of LPMs ( CD11b + CD102 + F4/80 +) in uterus determined by flow cytometry assay. D RNA-seq analysis of LPMs isolated from mouse peritoneal cavity treated with or without E2 (10 nM) for 24 h. The heat map represented the differential expression of genes, sorted from top to bottom according to fold change (log2). E Immunofluorescence staining images of LPMs isolated from mouse peritoneal cavity in vitro. LPMs were treated by E2 (10 nM) for 24 h. Phalloidin (green) stained the cytoskeleton. Bar = 30 μm. F Crys- tal violet staining images of LPMs which migrated from inside of the transwell to the bottom of that. LPMs in transwell were pre-treated by E2 (1, 10 or 100 nM) or not for 24 h. The transwell within LPMs was cocultured with Ap-ESC for 6 h. Ap-ESC represents the apoptotic ESCs. Bar = 100 μm. The column graph was the statistic of the purple cell number. G The expression level (2∆Ct) of Esr-1, Esr-2 and gpr30 in LPMs (isolated from estrus or proestrus mice) or 3T3-L1 cells detected by qRT-PCR assay. H Relative expression of mRNAs of Esr- 2 in LPMs from those mice detected by qRT-PCR assay. The LPMs isolated from mice were treated by E2 (1, 10 or 100 nM) or not for 24 h. I Immunofluorescence staining images of LPMs isolated from mouse peritoneal cavity in vitro. The LPMs were treated by E2 (10 nM) or not for 24 h. J Crystal violet staining images of LPMs which migrated from inside of the transwell to the bottom of that. LPMs in transwell were pre-treated by fulvestrant (Ful, 100 nM) or not for 1 h and treated by E2 (10 nM) or not for 24 h. The transwell within LPMs was cocultured with Ap-ESC for 6 h. Bar = 100 μm. The column graph was the statistic of the purple cell number. Bar graphs show the mean ± SEM; unpaired Student’s t test was used to compare the experimental groups. n = 3; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 1 3 4437 Inflammation (2025) 48:4428–4442 1 3 4438 Inflammation (2025) 48:4428–4442 accumulation of dead uterine cells. These findings imply that LPMs may contribute to the phagocytosis of dead cells, suppression of inflammation, and ultimately, the promo - tion of endometrial repair. To further scrutinize the impact of LPMs on damaged endometrium, we administered addi - tional LPMs directly into the uterus. Given that intrauterine injection necessitated opening the peritoneal cavity of mice, we adopted the endometrial injury method utilized in the conventional IUA mouse model [ 28]. This method offered the advantage of more severely disrupting the endometrium, thereby accentuating the effects of LPMs. Our results indi - cated that LPMs significantly reduced inflammation levels following uterine injury in mice, and we observed colocal - ization of LPMs with TUNEL-positive cells in the endo - metrium. However, due to technical constraints, it was not feasible to determine whether the TUNEL-positive cells were dead uterine cells or LPMs that gradually disappeared after entering the uterus, potentially due to death and subse- quent removal by other phagocytes. Therefore, we investi - gated the phagocytosis of LPMs on apoptotic ESCs in vitro, hoping to sequentially mimic their possible behavior in the damaged endometrium. In addition, the limitation of this part of the study is that the histological changes of the endo- metrium in the early stage (within 12 h) after injury are not obvious, so we only indirectly reflected the effect of LPMs on endometrium repair through the level of inflammation and the number of dead cells. More indicators of early stage after endometrium injury need to be further explored. After surgical separation of adhesions in patients with IUA, E2 is usually administered as an adjunctive therapy. Although there are several evidences showed E2 promotes the regeneration of endometrium [ 29–31], the influence of E2 on LPMs is unknown. Since the E2 levels in mice are highest during proestrus and lowest during estrus [ 32], we investigated whether there was any difference in migration efficiency of LPMs into impaired uterus between proestrus and estrus in mice. The amount of LPMs migrating in estrus mice tended to be significantly lower than in proestrus mice. This finding suggested that female sex hormones may be able to influence the migration of LPMs. The main hormones that change during the female cycle are E2 and progesterone P4, and their levels alternately rise and fall [ 33]. In human hormone cycle, the early stage of endometrium regeneration is mainly regulated by E2 [ 18]. Mice undergo an estrous cycle similarly to the hormonal cycle of humans, and that can be divided into four stages. Among them, proestrus and estrus are dominated by E2, while metestrus and diestrus are P4-dominant stages [34]. Our experimental results con- firmed that the E2 level in proestrus mice is higher than that in estrus. Considering the high migration efficiency of LPMs in proestrus mice, and there was some evidence that E2 or E2 analogs could promote cell migration [ 35–37]. We only environment, allowing us to study the behavior of LPMs in response to endometrial injury with maximum accuracy. It is worth mentioning that during the experiments, we found that intraperitoneal anesthetics also caused the depletion of LPMs, so all our animal experiments were performed with inhaled anesthetics. In addition, we strictly followed sterile operation when injecting drugs or fluorescent dye intraper - itoneally. All these efforts have been made to ensure that the behavioral patterns of LPMs can be simulated precisely under normal conditions in endometrial damage. It is recently reported that there are two distinct popu - lations of macrophages in the peritoneal cavity—the small peritoneal macrophages (SPMs) and LPMs. SPMs are bone marrow derived with the F4/80 low, CD11b low, CD102 negative and MHCII positive phenotype. LPMs are embryo derived and F4/80 high, CD11b high, and CD102 positive. They selectively express the zinc finger tran - scription factor GATA-binding protein 6 ( GATA6) and are maintained in the peritoneal cavity through self-renewal [27]. Using electric scratching tool to injure the endome - trium of mice for different durations, we found that LPMs could transfer from peritoneal cavity to injured endo - metrium rapidly, with a peak at 6 h. In this process, the migration of LPMs was accompanied by a rapid increase of CD11b+CD102−MHCII−F4/80−GATA6− cells in the peri - toneal cavity. We called them the ‘new cells’, and consid - ered them may be a population of undifferentiated myeloid cells derived from bone marrow. Immediately thereafter, the proportion of SPMs began to rise. Many studies have suggested that SPMs are the differentiation precursors of LPMs, and that new cells may also be the differentiation precursors of SPMs [ 22]. Their amount was increased in order to supplement the amount of LPMs. This was essen - tial to maintain the homeostasis of peritoneal cavity envi - ronment. Our researches suggested the ability of LPMs to quickly migrate into injured mouse uterus. However, due to the limit of experiment condition, we did not verify this phenomenon on humans. More efforts should be made to explore the changes of LPMs in human endometrial repair. We observed that there was a large accumulation of dead cells in the uterus and an inflammatory response in the body after endometrial injury. These occur at approxi - mately the same time as the migration of LPMs into uterus. Therefore, we hypothesized that LPMs may play a role in clearing apoptotic cells and resolving inflammation in the endometrium. We initially employed CLL to deplete LPMs. This approach proved highly effective in eliminating LPMs, and by restricting the intraperitoneal injection of CLL to a 48-h window, we ensured that tissue-resident macrophages in the uterus, liver, and bone marrow remained unaffected. The absence of LPMs notably reduced their presence in the injured uterus, resulting in heightened inflammation and an 1 3 4439 Inflammation (2025) 48:4428–4442 Supplementary Information The online version contains supplementary material available at h t t p s : / / d o i . o r g / 1 0 . 1 0 0 7 / s 1 0 7 5 3 - 0 2 5 - 0 2 3 3 5 - z. Author Contributions Y .H., S.S., J.L., G.Z. acquired and interpreted data, and drafted the manuscript; Y .H., S.S., J.W. acquired data and revised the manuscript; Y .H., G.Z., S.S., J.L., J.W., L.Y ., Y .P., C.P., Y .D. contributed to data interpretation and revision of the manuscript; Y .H., G.Z., J.L. conceived and supervised the work, acquired, and in- terpreted data, and drafted the manuscript. All authors read and ap - proved the final manuscript. Funding This work was supported by the National Key R&D Program of China (2023YFC2308200), National Natural Science Foundation of China (82471663,82071600, 82271653), and the Key Research and Development Program of Jiangsu Province (BE2019706). Data Availability No datasets were generated or analysed during the current study. Declarations Ethics Approval and Consent to Participate The animal study was ap - proved by Nanjing University Animal Care and Use Committee. The study was conducted inaccordance with the local legislation and insti- tutional requirements. Patient Consent for Publication Not applicable. Competing interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and explored the effect of E2 on LPMs in this study, but not P4. We found that the amount of LPMs migrating to the dam - aged uterus in mice was proportional to E2 levels. Further in vitro experiments also confirmed that E2 could promote LPMs migration by regulating the expression of a series of migration-related genes and promoting cytoskeletal elon - gation. Our results also showed that ER-β was specifically expressed in LPMs and its expression level was upregulated by E2 stimulation. There may be a positive feedback mecha- nism to drive the migration of LPMs to the uterus, that is, the higher the E2 level is, the more ER-β is expressed, and the migration of LPMs to the uterus is promoted. The closer to the uterus, the E2 level is further increased, which fur - ther promotes the recruitment of LPMs there. E2 has been widely applied in the treatment of diseases related to endo - metrium injury [ 29–31], our study may provide some new explanations for its therapeutic effects. In conclusion, our study has demonstrated the capacity of LPMs to swiftly migrate into the damaged endometrium of the uterus (Fig. 6). This response occurs rapidly, requiring only a few hours. Once in the uterus, LPMs contribute to tissue repair through phagocytosis of dead cells and sup - pression of inflammation. Additionally, we observed that the migration efficiency of LPMs was greater in mice with higher levels of E2. 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