Apoptotic bodies inhibit inflammation by PDL1–PD1‐mediated macrophage metabolic reprogramming

Abstract Apoptosis triggers immunoregulation to prevent and suppress inflammation and autoimmunity. However, the mechanism by which apoptotic cells modulate immune responses remains largely elusive. In the context of allogeneic mesenchymal stem cells (MSCs) transplantation, long‐term immunoregulation is observed in the host despite the short survive of the injected MSCs. In this study, utilizing a mouse model of acute lung injury (ALI), we demonstrate that apoptotic bodies (ABs) released by transplanted human umbilical cord MSCs (UC‐MSCs) convert the macrophages from a pro‐inflammatory to an anti‐inflammatory state, thereby ameliorating the disease. Mechanistically, we identify the expression of programmed cell death 1 ligand 1 (PDL1) on the membrane of UC‐MSCs‐derived ABs, which interacts with programmed cell death protein 1 (PD1) on host macrophages. This interaction leads to the reprogramming of macrophage metabolism, shifting from glycolysis to mitochondrial oxidative phosphorylation via the Erk‐dependent pathway in ALI. Importantly, we have reproduced the PDL1–PD1 effects of ABs on metabolic switch using alveolar macrophages from patients with ALI, suggesting the potential clinical implications of developing therapeutic strategies for the patients.

the formation of plasma membrane blebs or apoptotic bodies (ABs). 11,12Our previous studies have shown that ABs released by MSCs can be phagocytosed by liver macrophages, resulting in an anti-inflammatory effect. 13,14Nevertheless, the detailed mechanisms underlying the functional impact of ABs derived from MSCs on macrophages, particularly in the context of inflammation, remain largely unknown.
7][18] Moreover, macrophages are widely recognized as the primary 'professional' phagocytes responsible for the clearance of apoptotic cells. 19During inflammation, macrophages undergo metabolic reprogramming, shifting their core metabolism from oxidative phosphorylation (OXPHOS) to glycolysis, upon activation by lipopolysaccharide (LPS), a Gram-negative bacterial product. 20,21This metabolic shift in macrophages leads to increased secretion of pro-inflammatory cytokines. 20,22,23Notably, the metabolic processes that induce glycolytic activation in pro-inflammatory macrophages, triggered by the Toll-like receptor 4 agonist LPS, are downregulated in anti-inflammatory macrophages. 24,25Building upon our previous findings demonstrating the anti-inflammation effects of macrophages following the phagocytosis of ABs derived from MSCs, we hypothesized that ABs from MSCs might regulate the metabolism of macrophages.
The programmed cell death 1 ligand 1-programmed cell death protein 1 (PDL1-PD1) immune checkpoint pathway has been recognized for its role in preventing excessive tissue damage during inflammatory states. 26PDL1 is expressed on various cell types in the body, including MSCs and its expression increases in the presence of inflammation. 26,27In addition, exosomal PDL1 binds to PD1 on T cells, acting as an immunosuppressant that promotes wound healing. 280][31] However, the expression of PDL1 on ABs and the effects of PDL1-PD1 signalling on macrophage metabolism during inflammation remain unknown.
In this study, we aimed to investigate the anti-inflammatory effect of ABs derived from umbilical cord MSCs (UC-MSCs) in an ALI model and the metabolic regulation of ABs in macrophages.We found that UC-MSCs underwent apoptosis following intratracheal transplantation in ALI mice.The therapeutic effect of UC-MSCs on ALI was found to rely on the presence of ABs, which induced a transition of macrophages from a pro-inflammatory state to an antiinflammatory state.Furthermore, UC-MSCs-derived ABs expressed PDL1, and ABs drove the metabolic reprogramming of macrophage by inhibiting glycolysis through the PDL1-PD1 signalling.This metabolic shift promoted the anti-inflammatory properties of macrophages and mitigated the severity of ALI.Our findings reveal that metabolic reprogramming of macrophages by MSC-derived ABs to switch macrophages into anti-inflammatory state holds potent therapeutic potential in inflammatory diseases.Female DBA/1 mice (6-8 weeks old) for inflammatory arthritis model were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd.The animals were maintained under pathogen-free conditions with constant temperature, humidity, and 12:12-h light-dark cycle, and with food and water easily accessible.All animal experiments were performed following the guidelines set by the Animal Care Committee of the Fourth Military Medical University, Xi'an, China.

| Animal models of ALI
ALI model was induced in 8-10 weeks old C57BL/6J mice by intratracheal administration of LPS (Sigma, 5 mg/kg in 50 μL saline).Control mice received 50 μL saline intratracheally.After 4 h, ABs or PKH67 labelled ABs, or ABs + anti-PD1 were intratracheal administered (10 μg ABs in 50 μL saline per mouse).The severity of lung tissue damage and inflammatory cell infiltration in ALI mice was individually scored using a previously described scoring system. 32The total score per mouse was obtained by adding up the scores (ranging from 0 to 4) for four assessment items.Cytokine concentrations were measured on cell-free bronchoalveolar lavage fluid (BALF) using the enzymelinked immunosorbent assay kits (Meimian, Jiangsu, China) following the manufacturer's instructions.

| Isolation and characterization of Abs
UC-MSCs were isolated from umbilical cord of healthy puerperal women as previously described. 33Cells were cultured in basal culture medium containing α-MEM medium (Gibco), 10% FBS (Sijiqing, Zhejiang, China), 2 mM L-glutamine (Invitrogen Life Technology, Carlsbad, California), 100 U/mL penicillin and 100 U/mL streptomycin (Invitrogen).According to the corresponding experiments, UC-MSCs were pre-labelled with PKH67 or treated with staurosporine (STS) at a concentration of 0.2 μM to produce ABs.UC-MSCs were used at Passages 4-8 in the following study.
ABs were isolated from the culture supernatant of UC-MSCs undergoing induced apoptotic using gradient centrifugation, following a previously established method. 13Briefly, UC-MSCs were treated with 0.5 μM STS for 12-16 h to induce apoptosis.The culture supernatant was subjected to additional centrifuged at 800 Â g for 10 min to remove large debris.The supernatant was additional centrifugation at 16,000 Â g for 30 min at 4 C, and the resulting pellet was washed twice with PBS and resuspended in PBS for further use.The samples were observed using a scanning electron microscope (S4800, Hitachi, Japan).Moreover, the size distribution of ABs was measured by nanoparticle tracking analysis (NTA; Ribobio Ltd., Guangzhou, China).
The ABs marker caspase3 was analysed by western blot.ABs were also stained with Annexin-5 (Roche, Basel, Switzerland), and observed using laser scanning confocal microscope (FV1000, Olympus, Japan).

| Primary bone marrow-derived macrophages culture
Primary bone marrow-derived macrophages (BMDMs) were isolated from the femurs and tibiae bone marrow of CIA or C57BL/6J mice.
The cells were cultured in RPMI-1640 medium with 50 ng/mL recombinant macrophage colony-stimulating factor (PeproTech, Rocky Hill, New Jersey) for a period of 5-7 days.Then, naive macrophages were collected and stimulated with 200 ng/mL LPS (Sigma-Aldrich, St. Louis., Missouri) to generate pro-inflammatory macrophages for 24 h.

| Human alveolar macrophages culture
Primary alveolar macrophages were isolated from the lungs of mice or patients (Tangdu Hospital, Xian, China).This was achieved by bronchoalveolar lavage with five washes of normal saline at room temperature.Cells were pelleted and seeded in plates with complete RPMI-1640 medium.Alternatively, the cells were stained with antibodies for flow cytometric analysis (Cytoflex, Beckman Coulter, California).The collection of human alveolar macrophages was performed with the approval and oversight of the Tangdu Hospital ethics committee (202103-121).Written informed consent was obtained from all patients involved.

| Flow cytometry
To assess the transition of macrophages from pro-inflammatory to anti-inflammatory states upon treatment with various agents, CD64 and CD80 (Biolegend, San Diego, California) were chosen as markers of pro-inflammatory macrophages, and CD206 (Biolegend, San Diego, California) as a marker of the anti-inflammatory macrophages.The markers were measured using flow cytometric analysis (Cytoflex, Beckman Coulter, California).

| Immunofluorescence staining
Cells were fixed in 4% paraformaldehyde-PBS for 15 min.After washing with PBS[AUTHOR: Please define (2-NBDG, BCA, qRT-PCR, FCCP, PBS, IgG, TNF-α, IL, KEGG), the cells were permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature.Then cells were blocked in 10% normal goat serum and incubated with antibody overnight at 4 C.After washed in PBS again, cells were incubated with Alexa Fluor 488-labelled secondary antibody for 1 h at room temperature.In addition, 1 mg/mL of 4,6-diamino-2-phenyl indole (DAPI) in PBS were used to stain the nuclei.Fluorescence images were captured with a laser scanning confocal microscope (FV1000, Olympus, Japan).
For in vivo studies, the lungs were harvested from sacrificed mice and fixed overnight with 4% paraformaldehyde, then cryoprotected with 30% sucrose and embedded in the optimal cutting temperature compound.The frozen sections were cut at 10 μm thick (CM1950, Leica, Solms, Germany) and stained with primary antibodies anti-F4/80 and anti-Ly6G (abcam, Cambridge, UK) overnight at 4 C.The sections were then stained by Alexa Fluor 568 goat anti-rabbit IgG for 30 min at room temperature at concentrations of 1:300 and counterstained with DAPI.
For intracellular mitochondria staining, the Mito-Tracker Deep Red (Thermo Fisher Scientific, Massachusetts) was used.Cells were cultured in confocal small dish and supplemented with 100 nM Mito Tracker Deep Red at 37 C for 30 min, followed by incubation with Hoechst for 5 min.Then Fluorescence images were obtained by confocal fluorescence microscope (FV1000, Olympus, Japan).

| Statistical analyses
All experiments were repeated at least three times, and data are presented as mean ± SD.Statistical analysis was performed by Student's t-test (two-tailed) and one-way analysis of variance, with GraphPad Prism 8.0 (GraphPad Software, USA).The difference between groups was considered statistically significant for *p < 0.05, very significant for **p < 0.01, the most significant for ***p < 0.001.Graph analysis was performed using GraphPad Prism 8.0 (GraphPad Software, USA).

| ABs released by UC-MSCs prevent LPSinduced inflammatory lung injury
Since the administration of human UC-MSCs in mice prevented LPS-induced ALI, 10 we intratracheally injected human UC-MSCs pre-labelled with PKH67 to the ALI model in mice.Human UC-MSCs were mainly found in lung tissue after 24 h transplantation (Figure S1A) and then disappeared after 48 h transplantation as observed by confocal microscope in vivo (Figure S1B).To determine whether the UC-MSCs underwent apoptosis in the lung tissue, we performed TdT-mediated dUTP Nick-End Labeling (TUNEL) staining 24 h after transplantation.The UC-MSCs, pre-labelled with PKH67 (green), were examined for the presence of TUNEL-positive cells (red), indicating apoptotic UC-MSCs in the injected lung tissue (Figure S1C).
Next, UC-MSCs were pre-treated with STS to induce apoptosis and ABs were isolated from apoptotic UC-MSCs. 13The morphology and size distribution of the ABs were characterized by scanning electron microscopy and NTA (Figure 1A, B).The ABs typically displayed a round shape (Figure 1A) and composed a homogenous population with sizes ranging from 600 to 1200 nm in diameter (Figure 1B).
Then, we determined ABs by positive ANXA5 (annexin A5) staining (Figure 1C).Western blot analysis showed that the isolated ABs expressed the specific marker caspase 3 (Figure 1D).To evaluate the therapeutic effects of UC-MSCs and ABs on ALI, lung inflammation was assessed by haematoxylin and eosin staining of lung tissue section.The numbers of total cells, neutrophils, and macrophages, as well as the levels of total protein, TNF-α, and IL-6 in BALF were measured.Both UC-MSCs and ABs derived from US-MSCs significantly suppressed lung inflammation in the LPS-induced ALI model (Figures 1E-K and S1D).To investigate the role of ABs in mediating the therapeutic effects of UC-MSCs, the release of ABs was inhibited using sertraline, and the therapeutic effect of UC-MSCs was examined (Figure S1E).We showed that inhibition of ABs release compromised the alleviation of lung inflammation by UC-MSCs (Figure S1F-I), suggesting that the released ABs play a crucial role in the therapeutic effects.To further explore which type of immune cells recognized ABs in inflammatory lung tissue, F4/80 positive macrophages (Red) and Ly6G positive neutrophiles (Red) were stained after administration of ABs pre-labelled with PKH67 (Green).
The results showed that F4/80 positive macrophages were responsible for phagocytosing the ABs (Figure 1L).The data indicated that ABs released from apoptotic UC-MSCs exert therapeutic effects on ALI by suppressing lung inflammation, primarily through their recognition and uptake by macrophages.

| ABs inhibit pro-inflammation of macrophages through PDL1-PD1 pathway
Recent study showed that exosomal PDL1 regulated the inflammatory response to maintain proper immune homoeostasis in wound healing. 28We next sought to unveil whether PDL1 was also expressed by ABs, which are derived from plasma membrane outward blebbing when cells are in the late stage of apoptosis. 35We found that PDL1, but not PD1 was expressed on the plasma membrane of human UC-MSCs (Figure 2A).Moreover, we labelled the surface molecules with biotin and used neutravidin ultralink resin beads to capture biotinylated surface proteins.Western blot analysis was then applied to show expression level of captured proteins by anti-PDL1 antibodies.
The results showed the expression of PDL1 both on the cell surface of UC-MSCs and on the membrane of ABs (Figure 2B).On the other hand, the expression of PD1 on alveolar macrophage in mice was increased after stimulation with LPS from E. coli O111:B4 (Figure 2C).Moreover, we detected the PD1 expressions in interstitial macrophages and monocyte-derived macrophages of ALI mice.The results showed that the expression of PD1 in macrophage population in the inflammatory lung was also increased (Figure S2A,B).Macrophages were reported to be polarized to pro-inflammatory state and to release pro-inflammatory cytokines by LPS, but reversed to antiinflammatory state by MSCs in ALI. 36To know whether ABs regulate the polarization state of macrophages through PDL1-PD1 axis, we stimulated bone marrow-derived macrophages (BMDMs) with LPS and studied the effect of ABs on BMDMs.We showed that ABs decreased the expression of pro-inflammatory makers (CD80 and CD64) and production of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α in BMDMs stimulated by LPS (Figure 2F-J

| ABs contribute to the metabolic switch of macrophages through PDL1-PD1
To gain insights into potential mechanisms through which ABs alter macrophage polarization, we performed global RNA sequencing (RNAseq) on Raw264.7 cells with treatment of LPS, LPS + ABs, or LPS + ABs + anti-PD1 compared with untreated cells.The RNA-seq analysis showed that the anti-PD1 antibody significantly reversed the suppression of pro-inflammatory cytokine and chemokine transcripts in LPS + ABs-treated group (Figure S4J).As the RNA-seq analysis showed that major metabolic pathways were affected by ABs, seahorse extracellular flux analysis, which measures OCR and ECAR, was curtailed the increase in OXPHOS and ATP production induced by ABs in LPS-treated BMDM (Figure 4H-L).Previous study has demonstrated that there is intrinsic connection between structural organization and respiratory status of mitochondria. 37We thus conducted confocal fluorescence imaging studies to assess how ABs altered macrophage mitochondria morphology by overexpressing the fluorophore RFP (red fluorescent protein) specifically targeting to mitochondria.However, no significant changes in mitochondrial networks were observed in BMDMs with supplementation of LPS, LPS + ABs, or LPS + ABs + anti-PD1 (Figure S5).

| ABs reprogramme metabolic pathways of macrophages through Erk
Since above data showed that ABs contributed to the metabolic switch of macrophages through PDL1-PD1 axis, the further detailed mechanism was explored.The Venn diagram shows differentially expressed genes among three pairwise comparisons using principal component analysis (Figure 5A).The RNA-seq analysis showed that ABs downregulated most of the genes involved in the glycolysis, which were upregulated in macrophages after LPS treatment (Figure 5B).In contrast to glycolysis genes, most of the genes involved in tricarboxylic acid (TCA) cycle, a component of mitochondrial respiration, were unchanged or elevated after LPS + ABs treatment compared with LPS alone (Figure 5C).Moreover, RNA-seq results also revealed most genes in fatty acid synthesis and fatty acid oxidation were not changed significantly after LPS + ABs treatment compared with LPS alone (Figure S6A,B).Notably, genes that control the three rate-limiting steps of glycolysis including Hk3, Pgam, and Ldha (Figure 5D) were all upregulated in BMDMs treated with LPS alone, but downregulated by ABs, consistent with the notion that cells upregulated glycolysis to compensate for loss of mitochondrial OXPHOS (Figure 5E-G).However, anti-PD1 antibody increased the expression of Hk3, Pgam, and Ldha in BMDMs treated with LPS and ABs (Figure 5E-G).In addition, the expression levels of Gapdh, Tpl1, and Idh1 were not changed significantly among four groups (Figure S6C-E).These results indicate that ABs switch the glycolysis to OXPHOS in macrophages through PDL1-PD1 interaction, which contributes to the polarization of macrophages from pro-inflammatory to anti-inflammatory state.
PI3K/Akt and MEK/Erk pathways are targets of PD1, and they play important roles in glucose metabolism. 38,39Through GO pathway analysis and gene expression analysis, we found that Erk pathway was one of the most abundant signal transductions but not PI3K/Akt pathway (Figures 5H and S6F).qRT-PCR results confirmed significantly increased gene expression of Erk1 and Erk2, but not Mek expression of BMDMs in LPS and LPS + ABs + anti-PD1 groups compared with control and LPS + ABs groups (Figure 6A-C).Consistently, western blot analysis showed increased expression of pErk1/2 in the same groups (Figure S7A).Both ABs treatment and Erk inhibitor (PD0325901) treatment significantly deceased the expression of pro-inflammatory makers CD80 and CD64 in BMDMs with LPS supplementation (Figures 6D,E and S7B-D).In contrast, activation of the Erk pathway by AR234960 increased the expression of these pro-inflammatory makers in BMDMs with supplementation of LPS and ABs (Figures 6D,E

| ABs promote anti-inflammatory alveolar macrophages from ALI patients by PDL1-PD1 pathway
We also collected the alveolar macrophages of patients with ALI and found that their alveolar macrophages exhibited pro-inflammatory phenotype compared with macrophages from peripheral blood of healthy donors (Figure 7A-E).ABs decreased the expression of CD64 and the production of IL-1β, IL-6, and TNF-α in alveolar macrophages of ALI patients (Figure 7A-E).The anti-inflammation maker CD206 was increased in alveolar macrophages by AB treatment (Figure 7F,G).However, blockade of PDL1-PD1 with anti-PD1 antibody blunted ABs effects (Figure 7A-G).
To further confirm whether ABs regulate the metabolism of alveolar macrophages of ALI patients, seahorse extracellular flux analysis was performed.The results showed that alveolar macrophages from ALI patients exhibited elevated ECAR, indicating increased glycolysis.
However, ABs treatment suppressed the glycolytic activity of alveolar macrophages, leading to a decrease in ECAR.Importantly, when the PDL1-PD1 pathway was blocked with anti-PD1 antibody, the inhibitory effect of ABs on glycolysis was reversed (Figures 7H,I and   S8A,B).Furthermore, ABs treatment induced an elevation in OCR in alveolar macrophages, accompanied by ATP production.However, when the PDL1-PD1 pathway was blocked with anti-PD1 antibody, the increase in OCR and ATP production induced by ABs was reduced (Figures 7J,K and S8C-E).Collectively, these findings indicate that ABs also inhibit pro-inflammatory polarization and glycolytic metabolism of alveolar macrophages through PDL1-PD1 pathway in patients with ALI (Figure 7L).polarization from a pro-inflammatory state to an anti-inflammatory state and reprogrammes the major metabolic pathways of macrophages.Molecular studies showed that PDL1 expressed by ABs made the metabolic switches from glycolysis to OXPHOS of macrophages through the Erk pathway downstream of PDL1-PD1 axis.Importantly, we have confirmed the effects of ABs on metabolic switch of alveolar macrophages from ALI patients, which is dependent on the PDL1-PD1 pathway.
In mammals, impaired clearance of apoptotic cells can lead to exposure of autoantigens, which can trigger autoimmune diseases, such as systemic lupus erythematosus and chronic polyarthritis. 40,41tually, previous research showed that apoptotic cells can exert profound modulatory effects on both the innate and adaptive immune systems. 42MSCs have immunomodulatory properties and undergo apoptosis after in vivo application, which contributes to the therapeutic efficacy. 43,44Our previous study also found that MSCs disappeared gradually in the injection area of MI mice, accompanied by apoptosis. 13According with these results, we found that human UC-MSCs underwent apoptosis after 24 h intratracheally implantation in ALI mice model.Administration of early-stage apoptotic cells has been shown to help alleviating disease severity in inflammation models, probably via elicitation of anti-inflammatory mediators. 45re, we found human UC-MSCs-derived ABs could alleviate inflam- Extracellular vesicles (EVs) from viable cells have been extensively studied in recent years, but our knowledge regarding apoptotic vesicles produced by apoptotic cells is still limited.In vivo, macrophages are responsible for the clearance of dying apoptotic cells, both in normal development/tissue homeostasis 46,47 and at sites of inflammation. 48Recent study reported that metabolites released from apoptotic cells, as tissue messengers, which induce anti-inflammatory effects in macrophages, could alleviate the inflammatory arthritis and lung-graft rejection. 49As to the in vivo fate of ABs, we have found when intratracheally infused, ABs are mainly engulfed by macrophages.EVs derived from viable UC-MSCs, when primed with interferon γ, have been shown to enhance macrophage phagocytosis and more effectively attenuate E. coli-induced lung injury. 50In our study, we have verified that ABs from apoptotic human UC-MSCs downregulate pro-inflammatory macrophages in ALI mice.Intriguingly, ABs from MSCs expressed PDL1, which binds to its receptors PD1 on macrophages induced by LPS in this study.
Through using neutralizing antibody and siRNA approaches to suppress PDL1-PD1 axis, we have verified that ABs switch macrophages from a pro-inflammatory state to an anti-inflammatory state through PDL1-PD1 axis, providing therapeutic benefits in the treatment of ALI and inflammatory arthritis.PDL1-PD1 axis is now gaining great attention as a potential therapeutic target for the treatment of various inflammatory and autoimmune diseases. 51PD1 acts as a metabolic checkpoint in ILC2s, affecting cellular activation and proliferation.Human PD1 agonist ameliorates airway hyper-reactivity and suppresses lung inflammation. 31In our study, the RNA-seq results revealed that the major metabolic pathway was affected by ABs through PDL1-PD1 axis.PD1 has been reported to modulate T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation. 29r results showed that ABs shifted the metabolic pathway from glycolysis to OXPHOS in LPS-treated BMDMs by seahorse analysis.However, little is known about the mechanism of PDL1/PD1 signalling in macrophage metabolism during inflammation.Evidences have pointed out that Erk signalling pathway is a target of PD1/PDL1 and plays important role in promoting the conversion of energy metabolism to glycolysis by affecting the activity of key metabolic regulators. 52,53Previous reports showed that inhibition of Erk can improve the efficacy of anti-PD1 immunotherapy by downregulating PDL1 expression. 54Another study showed PDL1 binding to PD1 promotes M2 macrophage polarization, accompanied by enhanced mitochondrial function and metabolic reprogramming via Erk/Akt/mTOR. 55rthermore, ERK1/2 signalling has been shown to enhance aerobic glycolysis and impair the TCA cycle by promoting the mitochondrial translocation of PGK1, leading to brain tumorigenesis. 56ERK1/2 signalling positively controls the function of pro-inflammatory macrophages by enhancing glycolysis, and its inhibition impairs glucose consumption and lactate production in LPS-activated macrophages. 57 our study, Erk pathway has been confirmed to regulate the expression of three key enzymes in glycolysis, thereby driving the metabolic switch in LPS-treated BMDMs in response to ABs.Recent studies have also showed that macrophages activated by LPS undergo changes toward glycolysis, whereas macrophages activated with IL-10 shift OXPHOS, indicating that cell metabolism can direct macrophage polarization state. 21,24,25,58However, fatty acid synthesis and oxidation were not changed significantly in LPS-stimulated macrophages after ABs treatment in our study.These findings pave the way to the better understanding of PDL1-PD1 function in macrophage metabolism and the other metabolic pathway downstream of PDL1-PD1 signalling.
Notably, we replicated and confirmed this immunoregulatory effects of ABs in alveolar macrophages from ALI patients, which indicates the clinical potential of ABs as a therapeutic approach for lung inflammation.ABs decreased the expression of CD64 and increased the expression of CD206 in alveolar macrophages of ALI patients.

2 |
MATERIALS AND METHODS 2.1 | Animal care 8-to 10-week-old male C57BL/6J mice for ALI model were purchased from Animal Center of the Fourth Military Medical University, China.

2. 10 |
shRNA-mediated knockdown of PD1 and PDL1 in BMDMs and ABs For shRNA knockdown experiments, BMDMs and UC-MSCs were treated with lentivirus, which respectively carried PD1 or PD-L1 shRNAs, according to the manufacturers' protocol (Shanghai Genechem Co., Ltd).Briefly, BMDMs were infected with shPD1 lentivirus at a multiplicity of infection (MOI) of 100 and UC-MSCs were infected with shPD-L1 lentivirus at a MOI of 10.After 48 h of shRNA induction, the cells were analysed for expression levels of PD1 and PD-L1 and used for subsequent experiments.
). Functionally, inhibition of PDL1 expression in UC-MSCs by shRNA downregulated the expression of PDL1 on ABs (Figure 2D,E).shPDL1-ABs treatment increased the expression of pro-inflammatory makers and cytokines in LPS-stimulated BMDMs (Figure 2F-J).Additionally, when the PDL1 is blocked by the anti-PDL1 antibody, the effects of ABs on macrophage polarization are abolished, as indicated by the expression levels of the genes related to pro-inflammatory (IL-1β, IL-6, TNF-α, and NOS2) and anti-inflammatory (Arg1 and IL-10) features of F I G U R E 1 Apoptotic bodies released by umbilical cord mesenchymal stem cells (UC-MSCs) prevent lipopolysaccharide (LPS)-induced inflammatory lung injury.(A) Morphology of UC-MSCs-derived antibodies (ABs) shown by scanning electron microscope.Scale bar, 1 μm.(B) Size distribution of ABs measured by nanoparticle tracking analysis.(C) Confocal microscopy images of ABs stained with ANXA5/annexin V showing phosphatidylserine exposed on the surface.Scale bar, 10 μm.(D) Western blots of the apoptotic marker protein caspase 3 and cleaved-caspase 3 for UC-MSCs-derived ABs.(E) haematoxylin and eosin staining of lung tissue sections (left) and quantification of the severity of lung tissue damage and inflammatory cell infiltration (right) showed UC-MSCs and ABs derived from US-MSCs both suppressed lung inflammation of LPSinduced acute lung injury (ALI; n = 5).Scale bar, 100 μm.(F) Total protein in bronchoalveolar lavage fluid (BALF) was measured by BCA assay (n = 5).(G and H) ELISA for TNF-α (G) and IL-6 (H) in BALF (n = 5).(I-K) Cell infiltration of lung was measured in BALF by flow cytometric analysis, total cells (I), macrophages (J), and neutrophils (K) (n = 5).(L) Immunofluorescence staining of ABs (Green) and F4/80 or Ly6G (Red) in the lungs after intratracheal administration of ABs.Scale bars, 50 and 15 μm.*p < 0.05; **p < 0.01; ***p < 0.001.Error bars are mean ± SD.

F
I G U R E 2 Antibodies (ABs) inhibit pro-inflammation of macrophages through programmed cell death 1 ligand 1-programmed cell death protein 1 (PDL1-PD1) pathway.(A) The expressions of PD1 and PDL1 were assessed by western blot analysis.Left lane, total protein, middle lane, membrane protein, right lane, cytoplasm protein.PDL1 was mainly expressed on plasma membrane of human umbilical cord mesenchymal stem cells (UC-MSCs).(B) Cell surface biotinylation assay of PDL1 on ABs.Right two lanes, western blot for PDL1 following neutravidin pull down from UC-MSCs and ABs.Left two lanes, input, not biotinylated cells.(C) The expression of PD1 on the alveolar macrophage of mice was increased after stimulated by lipopolysaccharide (LPS).(D) PDL1 expression of UC was detected by qRT-PCR analysis (n = 3).(E) Western blot analysis (top) and quantitative analysis of PDL1 protein expression (bottom) showed that PDL1 expression of ABs was decreased after transfected with shPDL1 (n = 3).(F,G) The expression of pro-inflammation surface markers CD80 and CD64 of bone marrow-derived macrophages (BMDMs) in different groups were analysed by flow cytometry (n = 3).Representative flow cytometry histogram of macrophages (top).Quantifications of CD80 and CD64 were presented with mean fluorescence intensity (MFI) (bottom).(H-J) Enzyme-linked immunosorbent assay (ELISA) for IL-1β (H), IL-6 (I), and TNF-α (J) in supernatant of BMDMs in different groups.(K) PD1 expression of BMDMs was detected by qRT-PCR analysis (n = 3).(L) Western blot analysis (top) and quantitative analysis of PD1 protein expression (bottom) showed that PD1 expression of BMDMs was decreased after transfected with shPD1.(M,N) The expressions of CD80 and CD64 of shPD1-BMDMs in different groups were analysed by flow cytometry (n = 3).Representative flow cytometry histogram of shPD1 macrophages (top).Quantifications of CD80 and CD64 were presented with MFI (bottom).(O-Q) ELISA for IL-1β (O), IL-6 (P), and TNF-α (Q) in supernatant of shPD1-BMDMs (n = 3).ns, not significant.*p < 0.05; **p < 0.01; ***p < 0.001.Error bars are mean ± SD. macrophages (Figure S2C-H).On the other hand, ABs were unable to inhibit the expression of pro-inflammatory makers and cytokines production in LPS-stimulated BMDMs with PD1 knockdown (Figure 2K-Q).These data suggest that ABs inhibit pro-inflammatory polarization and cytokines production of macrophages through the interaction between PDL1 on ABs and PD1 on macrophages.F I G U R E 3 Legend on next page.

3. 3 |
Blockade of PDL1-PD1 attenuates the therapeutic effect of ABs on ALIWe next aimed at deciphering whether the therapeutic effect of ABs on ALI depends on PDL1-PD1 axis.First, we detected the expression of PD1 in alveolar macrophages, interstitial macrophages, and monocyte-derived macrophages of ALI mice treated with ABs and anti-PD1 antibody.The results indicated that ABs and anti-PD1 antibody treatment could reduce the expression of PD1 in macrophage population in inflammatory lung (FigureS3A-C).After blockage of PDL1-PD1 interaction with anti-PD1 antibody, the therapeutic effect of ABs on ALI mice was attenuated (Figure3A-D).The alveolar macrophages showed increased expression of pro-inflammatory makers CD80 and CD64 in LPS-induced ALI mice (Figure3E,F).However, blockage of PDL1-PD1 axis by anti-PD1 antibody reversed the effect of ABs on decreasing the expression of these markers in alveolar macrophages from ALI mice (Figure3E,F).Consistently, we found that blockage of PDL1-PD1 by anti-PD1 antibody also attenuated the therapeutic effect of ABs in type-II collagen-induced inflammatory arthritis.This was evidenced by increased lymphocyte infiltration, elevated expression of pro-inflammatory cytokines (IL-1β and TNF-α) in articular cartilage, and higher levels of pro-inflammatory cytokines in the serum (FigureS3D-H).Based on the above observations, we next studied whether blocking PDL1-PD1 pathway switched the polarization state of macrophages.In vitro experiments were conducted using BMDMs treated with LPS and ABs in the presence or absence of an anti-PD1 antibody.The results showed significantly increased expression of anti-inflammatory marker CD206 and deceased expression of pro-inflammatory makers CD80 and CD64 in BMDMs with supplementation of LPS and ABs compared with LPS alone (Figure 3G-I).However, when the PDL1-PD1 axis is blocked by the anti-PD1 antibody, the effects of ABs on macrophage polarization are abolished, as indicated by the expression levels of CD206, CD80, and CD64 (Figure 3G-I).Anti-PD1 antibody treatment also increased the expression of pro-inflammatory factors (IL-1β, IL-6, and TNF-α) and decreased the expression of the anti-inflammatory factor IL-10 in BMDMs treated with LPS and ABs (Figure 3J-M).Consistently, blocking the PDL1-PD1 axis by the anti-PD1 antibody promotes pro-inflammatory polarization in Raw264.7 cells stimulated with LPS and ABs, as evidenced by decreased expression of CD206 and increased expression of CD86 and MHC II (Figure S4A-C).Moreover, qRT-PCR analysis showed that blocking PD1 in BMDMs treated with LPS and ABs leads to significant increases in the expressions of pro-inflammatory cytokine genes, most notably Il-1β, Il-6, Nos2, and TNF-α, while the expressions of anti-inflammatory marker genes of Arg1 and Mrc1 were decreased (Figure S4D-I).
performed on BMDMs and BMDMs with supplementation of LPS, LPS + ABs or LPS + ABs + anti-PD1.The results showed that ABs suppressed the ECAR of BMDMs with supplementation of LPS, while LPS treatment elevated ECAR, consistent with inhibition of glycolysis (Figure 4A-D).However, the blockade of PDL1-PD1 pathway by anti-PD1 antibody partially reversed the glycolysis inhibition in BMDMs treated with LPS and ABs.To validate the ECAR findings, we assessed BMDMs glucose uptake by flow cytometry using a fluorescence tagged, non-metabolizable glucose analogue, 2-NBDdeoxyglucose, and found a decrease in glucose uptake in BMDMs treated with LPS and ABs compared with those treated with LPS alone.The anti-PD1 antibody partially reversed this decrease in glucose uptake (Figure 4E,F).Lactate production by BMDMs was also reduced when supplemented with LPS and ABs, and this reduction was partially reversed by the anti-PD1 antibody (Figure 4G).Concomitant with suppression of ECAR, ABs treatment induced elevation in OCR of BMDMs with LPS administration, indicating a metabolic switch to OXPHOS.The blockade of the PDL1-PD1 pathway partially F I G U R E 3 Blockade of programmed cell death 1 ligand 1-programmed cell death protein 1 (PDL1-PD1) attenuates the therapeutic effect of antibodies (ABs) on acute lung injury (ALI).(A) Haematoxylin and eosin (H&E) staining of lung tissue sections (left) and quantification of the severity of lung tissue damage and inflammatory cell infiltration (right) showed the therapeutic effect of ABs on ALI mice was attenuated after blockage of PDL1-PD1 interaction with anti-PD1 antibody (n = 5).Scale bar, 100 μm.(B) Total protein in bronchoalveolar lavage fluid (BALF) was measured by BCA assay (n = 5).(C,D) enzyme-linked immunosorbent assay (ELISA) for IL-6 (C) and TNF-α (D) in BAL fluid (n = 5).(E) Representative flow cytometry histogram and quantification of CD80 expression in alveolar macrophages from control and acute lung injury (ALI) mice (n = 3).(F) Representative flow cytometric analysis and quantification of CD64 expression in alveolar macrophages from control and ALI mice (n = 3).(G) Representative flow cytometry histogram and quantification of anti-inflammatory marker CD206 expression in bone marrow-derived macrophages (BMDMs) and BMDMs treated with lipopolysaccharide (LPS), ABs or ABs + anti-PD1 (n = 3).(H,I) Representative flow cytometry histograms and quantification of pro-inflammatory markers CD80 (H) and CD64 (I) expressions in BMDMs and BMDMs treated with LPS, ABs, or ABs + anti-PD1 (n = 3).(J-M) ELISA for IL-1β (J), IL-6 (K), IL-10 (L), and TNF-α (M) in supernatant of BMDMs and BMDMs treated with LPS, ABs, or ABs + anti-PD1 (n = 3).ns, not significant.*p < 0.05; **p < 0.01; ***p < 0.001.Error bars are mean ± SD.

F
I G U R E 7 Antibodies (ABs) promote anti-Inflammatory alveolar macrophages from acute lung injury (ALI) patients by programmed cell death 1 ligand 1-programmed cell death protein 1 (PDL1-PD1) pathway.(A-C) Enzyme-linked immunosorbent assay (ELISA) for IL-1β (A), IL-6 (B), and TNF-α (C) in supernatant of control and alveolar macrophages from ALI patients (n = 4).(D-E) Representative flow cytometry histogram (D) and quantification of CD64 expression (E) in control and alveolar macrophages ALI patients (n = 3).(F,G) Representative flow cytometry histogram (F) and quantification of CD206 expression (G) in control and alveolar macrophages from ALI patients (n = 3).(H) Line graph showing extra-cellular acidification rate (ECAR) from seahorse analysis assessed in alveolar macrophages from control and alveolar macrophages from ALI patients (n = 3).(I) Glycolysis in ECAR assay.(J) Line graph showing oxygen consumption rate (OCR) from seahorse analysis assessed in alveolar macrophages from control and alveolar macrophages from ALI patients (n = 3).(K) Basal respiration in OCR assay.(L) Graphic Summary shows the mechanisms of PDL1 on ABs reprogrammed the metabolic process of macrophages by switching the glycolysis to mitochondrial oxidative phosphorylation through Erk dependent pathway.ns, not significant.*p < 0.05; **p < 0.01; ***p < 0.001.Error bars are mean ± SD.
and 7B-D).The Erk inhibitor PD0325901 also decreased the levels of pro-inflammatory factors IL-1β, IL-6, and TNF-α, while increasing the anti-inflammatory factor IL-10 in BMDMs with LPS supplementation (Figure 6F-I).In line with the enhanced pro-inflammatory state after activation of Erk pathway of ABs treated BMDMs, Erk activator AR234960 increased expression of the pro-inflammatory factors IL-1β, IL-6, and TNF-α but decreased immunoregulatory factor IL-10 in BMDMs treated with LPS and ABs (Figure 6F-I).Genes that control the three rate-limiting steps of glycolysis including Hk3, Pgam, and Ldha were all downregulated by ABs and PD0325901 in BMDMs treated with LPS (Figure 6J-L).However, activation of the Erk pathway by AR234960 increased the expression of these genes accordingly (Figure 6J-L).To further confirm, the role of the Erk pathway in shifting the metabolism of BMDMs with LPS treatment, seahorse analysis was performed.The results showed that both ABs and PD0325901 shifted ECAR to OXPHOS in BMDMs supplemented with LPS, indicating a shift from glycolysis to OXPHOS (Figures 6M-P and S7E-I).Intriguingly, the activation of Erk pathway by AR234960 increased ECAR and decreased OXPHOS in BMDMs treated with LPS and ABs (Figures 6M-P and S7E-I).These results suggested that ABs reprogramme major metabolic pathways of macrophages via inhibiting Erk pathway downstream of PDL1-PD1 signalling.
mation of lung tissue, as demonstrated by the decreased inflammatory cell infiltration and decreased expression of inflammatory factors including TNF-α and IL-6 in BALF.Inhibition of ABs production from MSCs impaired the therapeutic effect of UC-MSCs, suggesting the treatment of ALI by human UC-MSCs relies on the presence of ABs.
Moreover, the blockade of the PDL1-PD1 axis attenuated the effects of ABs, suggesting the involvement of this pathway in mediating the immunomodulatory actions of ABs.Metabolic analysis showed that ABs inhibited the pro-inflammatory polarization and glycolytic metabolism of alveolar macrophages through PDL1-PD1 pathway.Considering the above-mentioned properties of ABs and its low immunogenicity, our study expands the possibility of applying ABs to the clinical treatment for inflammation such as ALI.Future clinical applications of ABs could involve administration methods like aerosol inhalation or alveolar lavage, expanding the potential for their use in treating inflammation.In sum, we reveal a key role of ABs in controlling metabolic reprogramming of macrophages via PDL1-PD1 pathway.This metabolic control of ABs can direct the polarization state of macrophages and provide potential therapeutic effect of ALI and other inflammatory diseases.Therapeutic targeting of PDL1-PD1 pathway in macrophages therefore could be beneficial for treatment or prevention of inflammatory diseases associated with ABs.