{"paper_id":"8ba8e829-6813-47e4-830b-41cd5145bfb4","body_text":"Telocytes (TCs) are a type of mesenchymal (stromal) cells recently discovered by\nPopescu et al 1 . TCs are characterized by a small cellular body and extremely long, thin\ncellular prolongations known as telopodes (Tps), which contain alternating dilated\nsegments (podom) and thin segments (podomer). In recent years, TCs present in\nvarious organs and tissues, including the heart, lungs, spleen, gallbladder, skin,\nand female reproductive system, have been studied extensively 2 – 15 . TCs form extensive intercellular connections with neighboring cells, which\nforms the structural basis for multiple biological functions 16 . In addition, TCs exhibit extensive crosstalk and influence the activity of\nvarious adjacent cells using different biological substances contained within its\nextracellular vesicles. TCs also form neurological connections. Previous studies\nhave shown the relationship between TCs and uterine autonomic nerves 17 .\nEndometriosis (EMs) is a common gynecological disease characterized by the\nimplantation of vascularized endometrial tissue outside the uterine cavity. The\nprimary symptoms include chronic pelvic pain, dysmenorrhea, and dyspareunia 18 . EMs is a major threat to women of reproductive age, as it frequently leads\nto low fertility or infertility. Several theories have been proposed to explain the\npathogenesis of EMs, including Sampson’s theory of menstrual retrograde 19  and Mayer’s theory of coelomic metaplasia 20 . However, none of the theories fully explain the mechanisms underlying the\nformation and development of EMs. Recently, an increasing number of studies have\nfocused on the immune-related pathogenesis of EMs, especially the complex role of\npelvic macrophages and their downstream crosstalk with target endometrial cells 21 – 29 .\nPelvic macrophages are the primary defense mediators of the local immune system 30 . The activation of macrophages involves M1 and M2 differentiation,\naccompanied by different cellular functions under peritoneal immune environments,\nwhich are favorable or unfavorable for the onset of EMs. Generally, the successful\nimplantation of ectopic lesions in the abdominal or pelvic cavity requires various\nfavorable conditions facilitated by M2 macrophages, including immune tolerance,\nneovascularization for ectopic lesions 31 , 32 , a panel of inflammatory factors 33 , and the weakening of self-clearance (phagocytosis) potential 34 , 35 . The RAW264.7 cell line was established using cells from a tumor induced by\nthe Abelson murine leukemia virus. These cells are commonly used as substitutes to\ninvestigate the functions macrophages. Hence, most  in vitro \nexperiments on macrophages use RAW264.7 cells. Among the key pathways involved in\nregulating cellular responses, nuclear factor kappa B (NF-κB) activation is the\ncentral signaling coordination hub associated with macrophage differentiation,\napoptosis, and response to harmful extracellular stimuli 36 – 40 .\nPreviously, we reported that TCs can activate peritoneal macrophages (pMACs) through\ndirect cell-to-cell interactions and paracrine effects, and thereby play a\nsignificant role in the immunoregulation of pMACs 41 . However, there is limited knowledge regarding the crosstalk mechanisms\nexisting between TCs and macrophages. Herein, we investigated the hypothesis that\nTCs induce the differentiation of specific types of macrophages, alter their immune\nstatus and cellular functions, and influence the outcomes of retrograded endometrium\ndebris through certain pathways. In the current study, the effects of TCs on\nmacrophage-related functions, including macrophage proliferation, phagocytosis, and\napoptosis, and the potential involvement of NF-κB signaling were investigated\n in vitro  and  in vivo . These are expected to\nserve as therapeutic targets in EMs.\n\nAdult female BALB/c mice (8- 10-week old, 20–25 g) used in this study were\npurchased from the Experimental Animal Center of Soochow University. All mice\nwere maintained under specific pathogen-free conditions and were provided access\nto standard feed and water in the animal facilities. All animal experiments were\nperformed in compliance with the Guide for Laboratory Animals established by\nSoochow University.\nTCs were isolated according to a method described earlier 41 . The mice were sacrificed by injecting phenobarbital sodium (50 mg/kg;\nFuyang Pharmaceutical Factory, Fuyang, Anhui, China) to obtain the uterine\ntissue. The tissue was washed three times with phosphate-buffered saline (PBS)\nsupplemented with 100 U/mL penicillin and 0.1 mg/mL streptomycin (Sigma-Aldrich,\nSt. Louis, MO, USA). The tissue was then cut into smaller sections and digested\nwith 0.1% type II collagenase (Sigma-Aldrich, St. Louis, MO, USA), placed in a\nshaking incubator at 37°C, and gently dissociated mechanically using a pipette\nevery 15 min, followed by termination of the digestion reaction by the addition\nof fresh and complete medium after 90 min. The mixture was filtered using 100 µm\nand 40 µm mesh filters, followed by centrifugation (302 ×  g , 10\nmin) and re-suspension in DMEM/F12 (Hyclone, Logan, UT, USA) supplemented with\n10% fetal bovine serum (Gibco Life Technologies, Grand Island, NY, USA) and\nseeding in a 10 cm dish (Corning, Glendale, AZ, USA) at 37°C in a humidified\nincubator with 5% CO 2 . After the monolayer attachment of TCs to the\nplate, the complete medium was replaced every 48 h. For the double\nimmunofluorescence staining assay, cells with a specific CD34/vimentin\ndouble-positive immunophenotype were confirmed to be TCs and were used for the\nsubsequent studies. After 48 h of incubation, the medium was discarded and\nreplaced with serum-free DMEM/F12, and the cells were cultured for an additional\n24 h. The supernatant was collected and subsequently referred to as\nTC-conditioned medium (TCM).\nFresh cells were seeded at a suitable density on microscope slides, washed three\ntimes with PBS, fixed with 4% paraformaldehyde for 20 min, and permeabilized by\ntreating with 0.5% Triton X-100 for another 10 min. The cells were blocked by\ntreating with 3% bovine serum albumin (BBI, Shanghai, China) for 1 h. Rat\nanti-vimentin (1:100; Cell Signaling Technologies, Danvers, MA, USA) and rabbit\nanti-CD34 (1:200; Abcam, Cambridge, UK) antibodies were used. The cells were\ntreated with the antibodies overnight at 4°C. After washing three times with\nPBS, the cells were treated with donkey anti-rabbit IgG (H+L) Alexa Fluor 488\n(1:1000; Abcam, Cambridge, UK) or goat anti-mouse IgG (H+L) Alexa Fluor 568\n(1:1000; Abcam, Cambridge, UK) at 37°C for 1 h, followed by treatment with\n4’,6-diamidino-2-phenylindole (DAPI; Cayman Chemical, Ann Arbor, MI, USA). The\nslides were fixed in an antifade medium (1:1000; Beyotime, Shanghai, China) and\nimaged using an inverted fluorescent microscope (Nikon, Tokyo, Japan).\nRAW264.7 cells were purchased from the Bena Culture Collection (Suzhou, Jiangsu,\nChina) and maintained in a cell incubator with 5% CO 2  at 37°C. This\nwas followed by culturing in DMEM/F12 supplemented with 10% fetal bovine serum.\nWhen the cultured cells reached 70%–80% confluence, they were trypsinized and\nsubcultured. In a series of experiments on cell proliferation and apoptosis,\ndexamethasone (DXM) was added at an optimal concentration of 500 ug/mL to the\nculture medium to induce apoptosis, and the cells were referred to as\nDXM-pretreated RAW264.7 cells.\nRAW264.7 cells were seeded in a 6-well plate (1 × 106 cells/well). After 48 h of\nco-culture with TCM or DMEM, the energy metabolism status of RAW264.7 cells was\ndetermined by mitochondrial labeling. The cells were incubated with pre-warmed\nMitoTracker Green (Beyotime, Shanghai, China) working solution at 100 nmol/L for\n15 min in dark. Fluorescence intensity was measured using a fluorescence\nmicroscope (450–490 nm excitation light, 520 nm barrier filter). At least ten\nimages were acquired for each group, and the mean fluorescence intensity (MFI)\nwas semi-quantitatively analyzed using ImageJ (version 1.8.0, Media Cybernetics,\nSilver Spring, Bethesda, MD, USA).\nRAW264.7 cells were seeded in a 96-well plate (5 × 10 5  cells/well).\nThe uptake of neutral red (NR; Sigma Chemical Co, St. Louis, MO, USA) was\ninduced to demonstrate the phagocytosis of macrophages, as described earlier 42 . RAW264.7 cells were co-cultured with DMEM or TCM for 48 h. The medium\nwas then discarded and the cells were washed two times with PBS. Subsequently,\nthe RAW264.7 cells were incubated with NR dye solution (0.1%, diluted in HBSS)\nfor 1 h at 37 °c to facilitate the uptake of the dye by the cells. The plate was\ncarefully cleaned with PBS, and the NR dye solution was extracted from the cells\nusing a lysis solution (composed of 50% ethanol, 1% acetic acid, and 49% water).\nAbsorbance was measured at 540 nm using a microplate reader (Multiscan MK3;\nThermo Labsystems, Waltham, MA, USA). Each sample was analyzed in triplicates\nunder the same conditions. The absorbance value represents the ability of\nmacrophages to engulf the NR dye solution (a higher absorbance value corresponds\nto greater potential for phagocytosis).\nTo measure the expression levels of Fas and FasL (components of the death\nreceptor pathway), the M1 macrophage markers inducible nitric oxide synthase\n(iNOS), tumor necrosis factor alpha (TNF-α), and macrophage-inducible C-type\nlectin (Mincle), and the M2 macrophage marker arginase 1 (Arg1), the cells were\ncollected after 48 h of co-culture with TCM or DMEM, and total RNA was extracted\nusing TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the\nmanufacturer’s instructions. To elaborate, 1 µg of total RNA was added to the\nPrimeScript™ RT Master Mix (TaKaRa, Kyoto, Japan) for reverse transcription to\nobtain cDNA. Then, 1 µL of cDNA was added to TB Green®PreMix Ex TAQ™ (Tli RNAseH\nPlus) (TaKaRa, Kyoto, Japan) with volume adjustment to 20 μL, and quantitative\nreal-time polymerase chain reaction was performed on an ABI QuantStudio3\nDetection System (Applied Biosystems, Carlsbad, CA, USA). The 2 −ΔΔCt \nmethod was used to determine the relative expression in all samples. Each marker\nwas assayed in triplicate under the same conditions. In addition, the\nhousekeeping gene GAPDH was used as a reference gene to homogenize the\nexpression in individual samples. The primer sequences are listed in  Table 1 .\nList of qRT-PCR Primers.\nTo assess M1/M2 differentiation after 24 h and 48 h of culture in DMEM or TCM in\nvitro, the cells were treated with 0.25% EDTA-trypsin, washed with PBS, and\nresuspended in PBS. Subsequently, the cells were stained with phycoerythrin\n(PE)-labeled anti-F4/80 (RAW264.7 cells do not need to be identified as\nmacrophages; however, pMACs need to be applied to assess the purity of\nextraction). Pacific Blue™ anti- mouse CD86 and APC anti-mouse CD206 antibodies\n(eBiosciences, San Diego, CA, USA) were used to detect M1 and M2\ndifferentiation, respectively, with the cells incubated at room temperature for\n30 min. This was followed by flow cytometry analysis using an FACS Calibur\nSystem (BD Biosciences, San Diego, CA, USA).\nThe proliferation of DXM-pretreated RAW264.7 cells was monitored using a Cell\nCounting Kit-8 (CCK8; Dojindo, Kumamoto Prefecture, Kyushu, Japan) according to\nthe manufacturer’s instructions. The cells were seeded in 96-well plates (1 ×\n10 4  cells/well) and treated with DMEM or TCM. After 24, 48, and\n72 h, 10 µL of the CCK8 reagent was added to each well. After 3–4 h of\nincubation, the cell activity was measured using a microplate reader at an\nabsorbance of 450 nm (Multiscan MK3; Thermo Labsystems, Waltham, MA, USA).\nDXM-pretreated RAW264.7 cells were seeded in a 6-well plate (1 × 106 cells/well)\nand incubated at 37°C for 3–4 h to allow the cells to adhere to walls of the\nwells. The unattached cells were washed with PBS, and the medium was replaced\nwith DMEM or TCM. After 48 h of in vitro treatment with DMEM or TCM, a JC-1 kit\n(BD Biosciences, Lake Franklin, NJ, USA) was used to detect changes in the ΔΨm\nvalue of macrophages, as previously described 43 . Both groups of cells were subjected to trypsin digestion and collected\nin a flow tube. The original medium was discarded, and the cells were washed\ntwice with PBS. In each flow tube, the JC-1 working solution (0.5 mL) was added\nand mixed. After incubating for 15 min at 37°C, the working solution was\ndiscarded and the cells were washed twice with 1× buffer solution. Lastly, the\nbuffer solution (200 μL) was added to each flow tube to measure the ΔΨm value\nusing flow cytometry. The non-apoptotic cells were stained red when JC-1 entered\nand aggregated in the mitochondria. In contrast, apoptotic cells appeared green\ndue to JC-1 accumulation in the cytosol. The ratio between green and red\nfluorescence intensities indicated changes in the ΔΨm value.\nAfter 48 h of treatment with DMEM or TCM in vitro, the rate of apoptosis in\nDXM-pretreated RAW264.7 cells was assessed using Annexin V-FITC/7-AAD double\nstaining. Cells from both groups were washed twice with cold PBS, following\nwhich a suspension of 1 × 10 6  cells/mL was prepared using 1× binding\nbuffer. The cell suspension (100 μL) was added to the Falcon test tube along\nwith 5 µL of FITC-conjugated Annexin V (Annexin V-FITC) and 5 µL of 7-AAD (BD\nBiosciences, Lake Franklin, NJ, USA) and incubated in dark for 15 min at room\ntemperature (20∼25°C). The Annexin V-FITC/7-AAD-stained cells were readily\ndetectable using flow cytometry (BD Biosciences, Lake Franklin, NJ, USA), and\nFlowJo software (FlowJo LLC, Ashland, OR, USA) was used to analyze the obtained\ndata. Annexin+/7-AAD+ cells are considered to exhibit late apoptosis/secondary\nnecrosis, whereas Annexin+/7-AAD− cells are considered to exhibit early apoptosis 44 .\nAfter 24 h and 48 h of DMEM or TCM treatment  in vitro , RAW264.7\ncells from both groups were collected to identify the differentiation pathways\ninvolved, and DXM-pretreated RAW264.7 cells were studied to analyze the\napoptosis pathways. Total proteins were extracted by treating with RIPA lysis\nbuffer (Beyotime, Shanghai, China) containing a protease inhibitor cocktail\n(1:100; BBI, Shanghai, China) on ice, and mitochondrial proteins were extracted\nusing a Cell Mitochondria Isolation Kit (Beyotime, Shanghai, China), following\nwhich the samples were treated using a bicinchoninic acid reagent kit (Sangon\nBiotech, Shanghai, China). Subsequently, the sample (20 μg) was separated using\n10% SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane\n(Millipore, Billerica, MA, USA), which was followed by immunoblotting with the\ncorresponding antibodies. The primary antibodies included antibodies against\ncleaved caspase-8, Bax, Bcl-xl, Bcl-2, cleaved caspase-3, cleaved caspase-9,\niNOS, Arg1, NF-κb, p-NF-κb, β-actin, β-tublin, VDAC1, and cytochrome c (1:1000;\nall from Cell Signaling Technologies, Danvers, MA, USA). This was followed by\nlabelling with the corresponding rabbit anti-mouse or goat anti-rabbit\nHRP-conjugated secondary antibodies (1:5000; Absin Bioscience Inc., Shanghai,\nChina) for 1  h. The proteins were detected using an enhanced chemiluminescence\nkit (Absin Bioscience Inc., Shanghai, China) according to the manufacturer’s\ninstructions, followed by imaging using a gel imaging system (Tianneng Company,\nShanghai, China).\nTo further explore the in vivo effects of TCM on macrophages, animal experiments\nwere performed. A mouse model of EMs was established as described previously 45 , 46 . The donor mice were sacrificed, and the uterine tissues were divided\ninto two parts, cut into fragments of 1 mm, and resuspended in 1 mL PBS. Each\nrecipient mouse was administered an equal quantity of both endometrial and\nmyometrial tissue homogenates (equivalent to one uterine horn) via\nintraperitoneal injection (i.p.) using an 18-gauge needle. On day 15, the\nrecipient mice were dissected, and the abdominal cavity was observed. The design\nof the mouse experiments is illustrated in  Fig 1A .\nDesign of the mouse experiment. (A) Establishment of a mouse model of\nendometriosis (EMs) by intraperitoneal (i.p.) injection of endometrial\nand myometrial tissue fragments. On day 15, the ectopic tissue blocks\nwere processed for hematoxylin and eosin (HE) staining and\nimmunofluorescence (IF) staining to confirm the successful development\nof the EMs model. (B) Mice with EMs were divided into two groups: TCM\nand DMEM treatment via i.p. injection. On day 15, to determine M1/M2\ndifferentiation, the ectopic tissue segment was collected for IF\nanalysis, and peritoneal macrophages were collected for flow cytometry\nanalysis. TCM: TC-conditioned medium.\nMacroscopic observation of the ectopic lesion revealed a fresh mass with a\nreddish appearance and cystic texture. For microscopic observation, the ectopic\ntissue was removed, fixed with 4% paraformaldehyde for 24 h, embedded in\nparaffin, cut into slices of 3–5 μm, stained with hematoxylin and eosin (HE),\nand observed under a light microscope. The sections were subjected to vimentin\n(1:500; Cell Signaling Technologies, Danvers, MA, USA) and E-cadherin (1:200;\nR&D Systems, Minneapolis, MN, USA) immunofluorescence staining for\nidentification of the endometrial stromal cells (ESCs) and confirmation of the\nsuccessful development of the EMs model 47 , 48 . The specific operation procedure is referred to as cell\nimmunofluorescence, as mentioned above.\nTo observe the  in vivo  differentiation of tissue and pMACs, the\nEMs model was treated with TCM. Starting from the day of the successful\nestablishment of the mouse model, the mice in the experimental group were\nintraperitoneally injected with TCM (1 mL) administered repeatedly on days +5,\n+7, +9, +11, and +13. The mice in the control group were subjected to identical\nprocedures, with DMEM (1 mL) used instead of TCM. A schematic diagram is shown\nin  Fig. 1B .\nThe mice from both groups were dissected on day +15 to observe the  in\nvivo  differentiation of macrophages. First, to observe the\ndifferentiation of tissue macrophages within EMs lesions, immunofluorescence\ndouble-staining was performed using antibodies specific for iNOS (1:500; Cell\nSignaling Technologies, Danvers, MA, USA) (showing green fluorescence for M1)\nand CD206 (1:500; Santa, Dallas, TX, USA) (showing red fluorescence for M2) to\ntreat formalin-fixed paraffin-embedded EMs sections. The MFI ratio of iNOS and\nDAPI represents the proportion of M1 cells, whereas the MFI ratio of CD206 and\nDAPI represents the proportion of M2 cells 48 , 49 . Second, the pMACs were collected from the abdominal cavity of mice from\nboth groups, and CD86 (M1) and CD206 (M2) were analyzed using flow cytometry, as\ndescribed above.\nData are expressed as mean ± standard deviation (SD) and analyzed using GraphPad\nPrism 8.0 (GraphPad Software, San Diego, CA, USA). mRNA samples were prepared\nusing at least two independent experimental procedures. A Student’s t-test was\nused to compare two independent samples, and the results were presented as *\n P  < 0.05, **  P  < 0.01, ***\n P  < 0.001, and ****  P  < 0.0001.\nStatistical significance was set at  P  < 0.05.\n\nThe characteristic structure and immunophenotype of uterine TCs can be clearly\nidentified after 3–4 days of primary cell culture. TCs are typical mesenchymal\ncells with multiple intercellular connections and fusiform morphology. The\ncharacteristic Tps is composed of alternating thin (podomer) and thick (podom)\nsegments ( Fig. 2A ). TCs\nshow a specific CD34-positive (green) with vimentin-positive (red)\nimmunophenotype along the cellular body and the entire length of Tps, which\noverlap to yield a yellow color in the merged images ( Fig. 2B–D ). Observation of the\ncharacteristic morphology and specific immunophenotype confirmed the successful\nisolation of TCs.\nPrimary telocytes (TCs) with typical morphology and immunophenotype. (A)\nRepresentative morphology of TCs under a light microscope. TCs are\ntypical mesenchymal cells with a characteristic oval cellular body and\nlong extensions named telopodes (Tps), composed of alternating thin\n(podomer) and thick (podom) segments. Scale bar = 10 μm. (B) Labeling\nfor CD34 (green). Scale bar = 50 μm. (C) Labeling for Vimentin (red).\nScale bar = 50 μm. (D) In the merged image, both immunofluorescence\nsignals overlap with each other to form a yellow color along the\ncellular body and the entire length of Tps, with clearly visible\nstructure and the Tps, podomer, and podom indicated. Nuclei were\ncounterstained with DAPI (blue) to confirm the immunophenotype of TCs\n(CD34-positive, vimentin-positive, or c-kit-negative) (images showing\nc-kit-negative staining have not been provided).\nAs shown in  Fig. 3A , the\nRAW264.7 cells treated with TCM exhibited stronger phagocytosis than the cells\ntreated with DMEM after 48 h of co-culture ( P  < 0.05). This\nindicated enhanced cellular function.\nThe phagocytic potential and energy metabolism status of the\nmitochondria. (A) After 48 h, phagocytosis was significantly enhanced in\nTCM-treated RAW264.7 cells compared to that in DMEM-treated cells.\n(* P  < 0.05, Student’s  t  test.\nError bars: SD). (B) Semi-quantitative analysis revealed significantly\nhigher mean fluorescence intensity in the mitochondria in the TCM group\ncompared to that in the DMEM group (****  P  < 0.0001,\nStudent’s t test. Error bars: SD). (C) Fluorescence microscopic\nobservation of MitoTracker Green staining of DMEM-treated RAW264.7\ncells. (D) Fluorescence microscopic observation of MitoTracker Green\nstaining of TCM-treated RAW264.7 cells.\nThe energy metabolism status of RAW264.7 cells from both groups was determined\nusing mitochondrial labeling and semi-quantitative MFI analysis. As shown in\n Fig. 3B–D , the\nmitochondrial MFI value in the TCM group was significantly higher than that in\nthe DMEM group ( P  < 0.0001). Therefore, TCM treatment\nenhanced cell metabolism and subsequent cell proliferation.\nAfter DMEM or TCM treatment for 48 h, the differentiation of RAW264.7 cells was\nassessed. As shown in  Fig.\n4A–C , in the TCM group, flow cytometry revealed a significantly\nhigher proportion of Pacific Blue™-CD86-positive cells ( P  <\n0.05), which is a specific marker for M1 differentiation. In contrast, the\nnumber of CD206 (M2 macrophage marker)-positive cells was lower than that in the\nDMEM group ( P  < 0.05). Meanwhile, as shown in  Fig. 4D–G , qPCR analysis\nfurther confirmed M1 differentiation, as evidenced by the significantly higher\nsecretion of M1-type markers (iNOS, TNF-α, and Mincle) in the TCM group than in\nthe DMEM group ( P  < 0.01,  P  < 0.0001).\nIn contrast, the expression of the M2-type marker (Arg-1) was lower in the TCM\ngroup ( P  < 0.01). These results suggest that TCM treatment\ninduces the differentiation of RAW264.7 cells into the M1 subtype rather than\nthe M2 subtype.\nDifferentiation of TCM-treated and non-treated RAW264.7 cells. (A) Flow\ncytometry analysis of differentiation in RAW264.7 cells. M1 macrophages\nwere marked with the Pacific Blue™-CD86 antibody, whereas M2 macrophages\nwere marked with the APC-CD206 antibody. (B) The percentage of\nCD86-positive macrophages (M1) among TCM-treated RAW264.7 cells was\nhigher than that among DMEM-treated RAW264.7 cells. (* P \n< 0.05, Student’s t test. Error bars: SD). (C) The percentage of\nCD206-positive macrophages (M2) among TCM-treated RAW264.7 cells was\nlower than that among DMEM-treated RAW264.7 cells. (* P \n< 0.05, Student’s t test. Error bars: SD). (D-G) mRNA expression\nlevels of the M1 macrophage markers iNOS (D), TNF-α (E), and Mincle (F),\nand the M2 macrophage marker Arg1 (G). The relative mRNA expression was\ndetermined by normalizing the mRNA expression levels to that of\n GAPDH  (**  P  < 0.01, ****\n P  < 0.0001, Student’s t test, Error bars:\nSD).\nThe activity of DXM-pretreated RAW264.7 cells cultured in DMEM or TCM was\nevaluated at 24 h, 48 h, and 72 h. As shown in  Fig. 5A , the total number of viable\nRAW264.7 cells in the TCM group was significantly higher than that in the DMEM\ngroup during the entire experimental period ( P  < 0.05,\n P  < 0.01). Furthermore, with time, the reduction in\nRAW264.7 activity in the TCM group was significantly slower than that in the\nDMEM group. Therefore, TCM-treated cells exhibited stronger tolerance to\nDXM-induced apoptosis, with greater proliferation potential.\nProliferation and apoptosis in TCM-treated and DMEM-treated\nDXM-pretreated RAW264.7 cells. (A) Among cells that underwent DXM\ntreatment for different durations, TCM-treated RAW264.7 cells exhibited\ngreater proliferation than DMEM-treated RAW264.7 cells.\n(* P  < 0.05, **  P  < 0.01,\nStudent’s t test. Error bars: SD). (B) Flow cytometry analysis for\ndetermination of mitochondrial membrane potential (ΔΨm) based on JC-1\nfluorescence in TCM-treated and DMEM-treated DXM-pretreated RAW264.7\ncells after 48 h. The green/red fluorescence intensity indicates the\nvalue of ΔΨm. (C) TCM can significantly reduce the loss of ΔΨm than that\ninduced upon DMEM treatment. (* P  < 0.05, Student’s\n t  test. Error bars: SD). (D) Flow cytometry\nanalysis of apoptosis in TCM-treated and DMEM-treated DXM-pretreated\nRAW264.7 cells after 48 h. FITC-Annexin V/PerCp-7-AAD double staining\nwas performed to quantitatively analyze the percentage of apoptotic\ncells. (E) The percentage of apoptotic cells among TCM-treated RAW264.7\ncells was considerably lower than that among DMEM-treated RAW264.7 cells\n(** P  < 0.01, Student’s t test. Error bars:\nSD).\nΔΨm was measured in DXM-pretreated RAW264.7 cells using JC-1 fluorescence. As\nshown in  Fig. 5B ,  C , the green/red\nfluorescence ratio in the DMEM group was significantly higher than that in the\nTCM group ( P  < 0.05). The results indicate that TCM can\nreduce the loss of ΔΨm in DXM-pretreated RAW264.7 cells, and can also prevent or\nreverse DXM-induced apoptosis through the mitochondrial pathway.\nThe apoptosis of DXM-pretreated RAW264.7 cells was analyzed after 48 h of\nco-culture with DMEM or TCM. The results of Annexin V/7-AAD double staining flow\ncytometry are shown in  Fig.\n5D ,  E . The\nnumber of DXM-pretreated RAW264.7 cells during early and late apoptosis was\nsignificantly lower in the TCM group than in the DMEM group ( P \n< 0.01). Therefore, TCM treatment could reverse or inhibit DXM-induced\napoptosis and facilitate the survival of RAW264.7 cells through an apoptotic\nchallenge.\nTo explore the mechanisms underlying the differentiation of RAW264.7 cells, the\nexpression of the associated proteins was measured in both groups. As shown in\n Fig. 6A–C , the\nlevels of iNOS and p-NF-κB in TCM-treated RAW264.7 cells were higher than those\nin DMEM-treated cells. In contrast, Arg1 protein expression was suppressed\nvisibly in TCM-treated RAW264.7 cells. The results suggested that the M1/M2\nratio among RAW264.7 cells increased after TCM treatment, and the activation of\nthe NF-κB pathway played a significant role in this.\nExpression of proteins involved in the differentiation of RAW264.7 cells.\nβ-actin is a reference protein. (A) TCM increased the protein expression\nof the M1 marker iNOS. (B) TCM decreased the expression of the M2 marker\nArg1. (C) The expression levels of p-NF-κb increased in TCM-treated\nRAW264.7 cells. TCM: TC-conditioned medium\nAs shown in  Fig. 7A , the\nlevels of the pro-apoptotic proteins Bax, cleaved caspase-3, and cleaved\ncaspase-9 decreased significantly, whereas the levels of the anti-apoptotic\nprotein Bcl-xl and Bcl-2 increased significantly in the TCM group, which\nindicates that the Bax/Bcl-2(or Bcl-xl) ratio has decreased in the TCM group. As\nshown in  Fig. 7B ,\ncompared to that in the DMEM group, the cytoplasmic Cyt c levels were lower in\nthe TCM group, whereas the Cyt C more present in the mitochondria, which\nsuggested that the release of cytochrome C from mitochondria to cytoplasm was\ndown-regulated in the TCM group. Along with the higher expression levels of\np-NF-κB observed in the TCM group ( Fig. 7C ), the results suggested that TCM\nresisted or reversed DXM-induced apoptosis in RAW264.7 cells by inhibiting\nmitochondria-based apoptosis via the activation of the NF-κB-mediated\nBax/Bcl-caspase-9-caspase-3 signaling pathway. Nevertheless, since no\nsignificant difference was observed in the expression of caspase-8 protein and\nFAS/FASL genes (Supplemental Fig. S1 and S2), the death receptor pathway was not\nconsidered among the biological functions of TCM.\nExpression of proteins involved in apoptosis in DXM-pretreated RAW264.7\ncells. β-actin, β-tubin are cytoplasmic reference protein, VDAC1 is a\nmitochondrial reference proteins. (A) Western blotting results revealed\nthe significant reduction in the levels of the pro-apoptotic proteins\nBax, cleaved caspase-3, and cleaved caspase-9 in the TCM group as well\nas the significant increase in the levels of the anti-apoptotic protein\nBcl-xl and Bcl-2 in the TCM group, compared to the corresponding levels\nin the DMEM group. (B) The DMEM group showed higher levels of\ncytoplasmic Cyt C, whereas the TCM group showed higher levels of\nmitochondrial Cyt C. In other words, after TCM treatment, the transport\nof Cyt C from the mitochondria to the cytoplasm was suppressed, and\napoptosis was inhibited. (C) p-NF-κb expression increased in TCM-treated\nDXM-pretreated RAW264.7 cells. TCM: TC-conditioned medium\nThe intraperitoneal injection of uterine fragments for EMs induction is widely\npracticed. As shown in  Fig.\n8A–C ,  a \nmouse model of EMs was successfully established in this study, as evidenced by\nthe formation of a solid cystic ectopic lesion with abundant neovascularization\nin the peritoneum. HE staining of the ectopic lesions revealed a typical uterine\nstructure, which was abundant in the endometrial glands and epithelial cells\n( Fig. 8D–F ).\nImmunofluorescence analysis revealed the presence of vimentin-positive (red) and\nE-cadherin (green)-positive structures ( Fig. 8G–J ). This is consistent with the\nimmunofluorescence characteristics of uterine tissues reported in previous\nstudies, which confirmed the successful establishment of the EMs model.\nA successfully constructed endometriosis (EMs) mice model. (A–C)\nMacroscopic observation of ectopic EMs lesions in the peritoneum of a\nmouse, with round, cystic, solid appearance and abundance of surrounding\nblood vessels. (D–F) Hematoxylin and eosin (HE) staining of ectopic EMs\nlesions. As indicated by the arrows, prominent glandular structures\n(solid arrow) were observed in the lesion, along with columnar\nepithelial cells (dotted arrow). (G–J) Immunofluorescence analysis of\nEMs lesions. The lesions stained positive for E-cadherin (green),\nvimentin (red), and DAPI (blue) in the nucleus. E-cadherin and vimentin\nare immunofluorescence markers for endometrial glandular epithelial\ncells and endometrial stromal cells, respectively.\nFirst, the differentiation of tissue macrophages in EMs lesions was observed\nusing immunofluorescence staining. The results showed that M2 macrophages were\ndominant in untreated EMs lesions( P  < 0.01), as indicated by\npositive CD206 red staining ( Fig. 9B ). In contrast, after TCM treatment, M1 macrophages were\ndominant within EMs lesions( P  < 0.001), as indicated by\npositive iNOS green staining ( Fig. 9C ). Meanwhile, pMAC differentiation in the EMs model was\nanalyzed using flow cytometry ( Fig. 10 ), and the number of peritoneal\nM1 macrophages in the TCM group was significantly higher than that in the DMEM\ngroup ( P  < 0.0001). In contrast, the number of peritoneal M2\nmacrophages in TCM group decreased ( P  < 0.0001). These\nresults indicated that, in the EMs model, compared to cells treated with DMEM,\neither tissue or pMACs differentiate to attain the M1 phenotype rather than the\nM2 phenotype after  in vivo  TCM treatment.\nImmunofluorescence in tissue macrophages in ectopic endometriosis (EMs)\nlesions. (A) The nucleus was labeled with DAPI (blue). CD206 labeling\nindicates the presence of M2 macrophages (red), whereas iNOS labeling\nindicates the presence of M1 macrophages (green). (B) Semi-quantitative\nanalysis revealed the dominance of M2 macrophages in untreated EMs\nlesions. (** P  < 0.01, Student’s t test. Error bars:\nSD). (C) Semi-quantitative analysis revealed the dominance of M1\nmacrophages in TCM treated EMs lesions. (*** P  <\n0.001, Student’s t test. Error bars: SD). TCM: TC-conditioned\nmedium.\nFlow cytometry analysis of peritoneal macrophages in the endometriosis\nmodel. (A) M1 macrophages were marked with the Pacific Blue™-CD86\nantibody, and M2 macrophages were marked with the APC-CD206 antibody.\n(B) The percentage of CD86-positive cells (M1) in the TCM group was\ngreater than that in DMEM group. (**** P  < 0.0001,\nStudent’s t test. Error bars: SD). (C) The percentage of CD206-positive\ncells (M2) was lower in the TCM group than that in DMEM group\n(**** P  < 0.0001, Student’s t test. Error bars:\nSD). TCM: TC-conditioned medium.\n\nSince the first report on TCs by Popescu et al. 50 , the research on cardiovascular, respiratory, digestive, urinary, and female\nreproductive systems has increased. The slender TPs provide the structural basis for\nTCs to form homocellular and heterocellular contacts with various types of adjacent\ncells in 3D patterns within interstitial tissues, and thereby transfer specific\nbiological information, either by direct cell-to-cell contact or via extracellular\nvesicles and secretomes of nanometer dimensions, such as exosomes, which in turn\ninfluences or affects cellular function and behavior 16 . Therefore, TCs are considered to be central signaling coordination hubs in\ntissues and are known to play important roles in stem cell maintenance, tissue\nrepair and regeneration, immune surveillance, and vascular hemostasis 51 . Previously, we have reported the  in vitro  immunoregulatory\nroles of TCs in a series of studies. TCs can activate and maintain the immune\nresponse of pMACs through paracrine signaling and direct intercellular junctions.\nTherefore, TCs are considered to play a role in the onset of EMs 41 , 52 . However, macrophages influence the progression of EMs at multiple molecular\nlevels; among them, differentiation and inadequate phagocytosis are essential steps\nthat lead to the successful implantation of EMs lesions. Here, we investigated the\ndifferentiation of TCM-treated macrophages, related functional alterations, and\nmechanisms underlying the pathways. We found that by inhibiting mitochondria-based\napoptosis via the activation of NF-κB-mediated Bax/Bcl-caspase9-caspase3 signaling,\nTCs induce M1 differentiation and enhance phagocytosis. This might exert a negative\nor inhibitory effect on EMs development.\nEMs is a refractory disease in women of reproductive age. Retrograde menstruation and\nimmunodeficiency are typical mechanisms implicated in the etiology of EMs 21 – 26 , 28 , 34 , 35 . pMACs are the first line of immunocytes to react to the implantation of\nectopic endometrial debris in the abdominal or pelvic cavity, and therefore, play an\nimportant role in the onset of EMs 46 , 53 , 54 . During the entire process, pelvic macrophages tend to be polarized to the M1\nsubtype at the early stage of EMs; these cells primarily play a pro-inflammatory\nrole by recognizing, eliminating, or clearing endogenous ectopic endometrial\ncellular debris, and thereby prevent the development of EMs. While infiltrating\nmacrophages undergo alternative activation (primarily M2 differentiation) in the\nlater stage of EMs, and are characterized by immune tolerance, ineffective immune\nclearance, and poor or impaired phagocytic ability to remove viable retrograde\nendometrial cells within the pelvic cavity, M2 macrophages can produce related\ninflammatory factors that are essential for angiogenesis, tissue remodeling, and\nimplantation, and thereby enhance the growth of ectopic endometrial tissue 55 , 56 .\nThis study showed that TCs can induce M1 differentiation in macrophages in both cell\nculture and in the EMs model, which is characterized by enhanced proliferation and\nphagocytosis and suppressed apoptosis. The differentiation of M1 macrophages\nenhances their pro-inflammatory potential, boosts chemotaxis to sites of invasion\nunder the guidance of inflammatory factors, and strengthens recognition, removal,\ndegradation, and engulfment of the retrograded endometrial debris. Therefore,\nenhanced immune surveillance by M1 macrophages reduces or inhibits the probability\nof EMs development and/or progression. Meanwhile, apoptosis inhibition also\nstrengthens the ability of macrophages to eliminate the retrograde endometrial\ncellular and tissue debris. Conversely, TC-induced transformation from the M2 to the\nM1 subtype also blocks the neovascularization functions of macrophages, which are\nindispensable for the successful implantation of retrograde endometrial tissues 31 , 32 .\nNF-κB is a key responder to immune and inflammatory stimuli and regulator of cell\nproliferation, apoptosis, adhesion, invasion, and angiogenesis in multiple cell types 57 , 58 . These cellular processes are associated with the development of EMs as well\nas other diseases 59 , 60 . Increased NF-κB p65 translocation induces M1 differentiation in macrophages,\nand NF-κB blockade suppresses M1 differentiation and subsequent iNOS production 39 . Macrophage phagocytosis can be enhanced by upregulating NF-κB signaling 61 , 62 . In addition, the activation of NF-κB inhibits both mitochondria-based and\nnon-mitochondria-based macrophage apoptosis 63 , 64 , which may be related to further amplification of the inflammation cascade.\nThese findings were confirmed in the current study. As shown in  Fig. 11 , NF-κB usually binds to specific\ninhibitors (IκB) in the cytoplasm to form the NF-κB complex, which is present in an\ninactive state. When cells are stimulated with TCM, NF-κB and IκB are separated,\nfollowing which NF-κB is translocated to the nucleus and activated. This further\nsuppresses mitochondria-based apoptosis via the inhibition of\nBax/Bcl-caspase9-caspase3 signaling in RAW264.7 cells. Meanwhile, the increase in\nthe proportion of M1 macrophages and enhancement of phagocytosis counteract the\nfunctions of M2 macrophages, which are characterized by the secretion of angiogenic\nfactors, which helps establish a favorable environment for the growth of EMs\nlesions. To the contrary, the enhancement of the activity of TCM-treated macrophages\ncan effectively clear the retrograde endometrial debris, which eventually suppresses\nthe implantation and development of EMs. The findings from this study provide deep\ninsights into the role of uterine TCs in the immunomodulatory functions of\nmacrophages. Additionally, a novel EMs target was identified, which can be explored\nin future research on EMs pathogenesis and immunological treatment.\nTCM induce M1 differentiation of macrophages through the NF-κB pathway, with\nenhanced phagocytosis of retrograded endometrial debris, which helped\nsuppress the onset of endometriosis.\n\nIn summary, although the specific mechanism underlying the action of TCs on\nmacrophages has not been completely elucidated, it is now known that TCs are\ninvolved in the mitochondrial pathway, apoptosis, phagocytosis, and differentiation\nof macrophages. This suggests that TCs may also be potential participants in the\ninitiation of inflammation, and the findings may help develop a novel treatment\nstrategy for obstetrical and gynecological diseases such as EMs.\n\nClick here for additional data file.\nSupplemental Material, sj-docx-1-cll-10.1177_09636897211002762 for Telocytes\nEnhances M1 Differentiation and Phagocytosis While Inhibits\nMitochondria-Mediated Apoptosis Via Activation of NF-κB in Macrophages by\nYue-Lin Huang, Fei-Lei Zhang, Xue-Ling Tang and Xiao-Jun Yang in Cell\nTransplantation\nClick here for additional data file.\nSupplemental Material, sj-docx-2-cll-10.1177_09636897211002762 for Telocytes\nEnhances M1 Differentiation and Phagocytosis While Inhibits\nMitochondria-Mediated Apoptosis Via Activation of NF-κB in Macrophages by\nYue-Lin Huang, Fei-Lei Zhang, Xue-Ling Tang and Xiao-Jun Yang in Cell\nTransplantation","source_license":"CC0","license_restricted":false}