key: cord-0917758-5exojpih authors: Wang, Yizhuo; Liu, Shuyun; Li, Lan; Li, Ling; Zhou, Xueli; Wan, Meihua; Lou, Peng; Zhao, Meng; Lv, Ke; Yuan, Yujia; Chen, Younan; Lu, Yanrong; Cheng, Jingqiu; Liu, Jingping title: Peritoneal M2 macrophage-derived extracellular vesicles as natural multi-target nanotherapeutics to attenuate cytokine storm after severe infections date: 2022-03-14 journal: bioRxiv DOI: 10.1101/2022.03.13.484180 sha: 1f4df79f33ed07af0b23b389dc44529d8a1837db doc_id: 917758 cord_uid: 5exojpih Cytokine storm is a primary cause for multiple organ damage and death after severe infections, such as SARS-CoV-2. However, current single cytokine-targeted strategies display limited therapeutic efficacy. Here, we report that peritoneal M2 macrophages-derived extracellular vesicles (M2-EVs) are multi-target nanotherapeutics to resolve cytokine storm. In detail, primary peritoneal M2 macrophages exhibited superior anti-inflammatory potential than immobilized cell lines. Systemically administrated M2-EVs entered major organs and were taken up by phagocytes (e.g., macrophages). M2-EVs treatment effectively reduced excessive cytokine (e.g., TNF-α and IL-6) release in vitro and in vivo, thereby attenuated oxidative stress and multiple organ (lung, liver, spleen and kidney) damage in endotoxin-induced cytokine storm. Moreover, M2-EVs simultaneously inhibited multiple key proinflammatory pathways (e.g., NF-κB, JAK-STAT and p38 MAPK) by regulating complex miRNA-gene and gene-gene networks, and this effect was collectively mediated by many functional cargos (miRNAs and proteins) in EVs. In addition to the direct anti-inflammatory role, human peritoneal M2-EVs expressed angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2 spike protein, and thus could serve as nanodecoys to prevent SARS-CoV-2 pseudovirus infection in vitro. As cell-derived nanomaterials, the therapeutic index of M2-EVs can be further improved by genetic/chemical modification or loading with specific drugs. This study highlights that peritoneal M2-EVs are promising multifunctional nanotherapeutics to attenuate infectious diseases-related cytokine storm. Infectious diseases, caused by various types of pathogens (e.g., bacteria, viruses, fungi 48 and parasites), have been a huge public health problem worldwide, which can affect 49 ~10% of all patients per year according to the World Health Organization report [1] . 50 Although mild infections may be eliminated by the host immune system, severe 51 infections frequently trigger immune disorders and hyper-inflammatory status, which 52 is also known as cytokine storm [2, 3] . Cytokine storm, featured by excessive release 53 of proinflammatory cytokines (e.g., IL-6 and TNF-α) and chemokines (e.g., MCP-1 and 54 CCL5), has been recognized as one of the leading causes for development of multiple 55 organ damage and failure after severe infections [4, 5] . For example, the recent COVID-56 19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-57 2) is highly accompanied with elevated levels of cytokines (e.g., TNF-α and IL-6, ~2-58 5 fold vs. normal healthy), which are mainly secreted by disordered immune cells (e.g., 59 macrophages) in patients, and this effect is primarily responsible for the following acute 60 respiratory distress syndrome and high mortality (up to ~9.6%) [6, 7] . Therefore, 61 therapeutics that can effectively resolve cytokine storm are urgently required in clinic. 62 Some therapies aimed to counter or neutralize a single cytokine (e.g., IL-6 or TNF-α 63 blockers) had been developed and shown certain beneficial effects in pre-clinical 64 models [8, 9] , but they showed controversial results in clinical trials of COVID-19 distribution of EVs was analyzed using a Nanoparticle Tracking Analyzer (NTA, 155 Particle Metrix, Meerbusch, Germany) as described before [21] . Western blotting was 156 performed to detect the positive markers (anti-TSG101, anti-Alix, anti-HSP70) and 157 negative marker (GM130) of EVs. Green PCR mix (Vazyme Biotech). Primers used in this study are listed in Table S1 . 242 The data were analyzed using Bio-Rad CFX Manager software, and the relative changes 243 of mRNA were calculated by delta-delta Ct method with GAPDH as the internal 244 reference gene. Flow cytometric analysis (FCA) 246 The FCA samples of mouse spleen were prepared as previously reported [19] . In 247 brief, mouse spleen tissues were ground in PBS and then passed through a 70 μm filter. Spleen samples were then incubated in red blood cell lysis buffer (R1010, Solarbio, Beijing, China) for 20 min to lyse red blood cell, followed by re-suspending the spleen England Biolabs) was used to produce small RNA libraries. cDNAs were prepared 304 using adaptor-specific primers, and DNA fragments (∼140-160 bp) were recovered. The library quality was evaluated using DNA High Sensitivity Chips on an Agilent 306 Bioanalyzer 2100 system. Sequencing of libraries was conducted on an Illumina 307 Novaseq 6000 system and 50 bp single-end reads were generated. All identical 308 sequences with sizes of 18 to 32 nt were counted and removed from the initial data set. EVs were taken up by PMφ (Fig. 2E) . The mechanisms of EV uptake by recipient cells macrophages) in vivo [39] . Consistently, we observed the colocalization of Cy7-labeled 433 M2-EVs with the CD68 + macrophages in tissue sections, such as lung, liver and spleen 434 (Fig. 3E) . However, no detectable signal was observed in dye control (Cy7 alone) 435 groups ( Fig. 3A-E) . These results suggest that large amounts of M2-EVs can arrive at (decreased by ~78% for TNF-α, decreased by ~65% for IL-6) and tissue cytokines 449 (decreased by ~50-70% for TNF-α, decreased by ~60-80% for IL-6) levels of multiple 450 organs (liver, lung and kidney) compared to LPS groups ( Fig. 4B-C) . Moreover, the 451 expression of vital chemokines (e.g., ICAM-1) was suppressed by M2-EVs treatment 452 in lung, spleen and liver tissues (Fig. 4F, G) . Similarly, we also observed a slightly 453 higher anti-inflammatory efficacy in M2-EVs groups compared to MSC-EVs groups at 454 same concentrations (Fig. 4B, C, F, G) . (FCA) method (Fig. S4, Fig. 2D) . Indeed, the population of proinflammatory M1 462 macrophages (F4/80 + /CD11c + ) increased markedly in spleen at the early stage (4 h) 463 after LPS challenge, while its population was reduced by M2-EVs treatment (Fig. 4D -464 E). MSC-EVs treatment led to a slight reduction in proinflammatory macrophages 465 (F4/80 + /CD11c + ) in spleen (Fig. 4D, E) . However, there was no significant difference inflammatory cell infiltrations in multiple organs (lung, liver and spleen) (Fig. 5A ). Lung edema and thickened alveolar walls, liver necrosis, and dilated spleen sinus with 479 a disorganized white pulp with loss in boundary definitions and barely distinct follicular 480 structure, were also observed in mice with LPS challenge (Fig. 5A) . The levels of 481 kidney injury molecule-1 (KIM-1, a marker of renal damage) was elevated in kidneys 482 from LPS group (Fig. S5A-B) . In contrast, M2-EVs treatment suppressed these lesion 483 formations in multiple organs, and reduced the overall tissue injury scores of lung, liver 484 and spleen in mice with LPS priming (Fig. 5B, S5B) . Meanwhile, MSC-EVs also 485 exhibited similar effects on tissue protection in vivo. 486 Excessive cytokines can induce cell death by triggering oxygen reactive species in genes expression between groups (Fig. 6A) . Compared with control group, 3008 507 upregulated genes and 2211 downregulated genes were observed in LPS/IFN-γ group. After M2-EVs treatment, 543 upregulated genes and 732 downregulated genes were 509 found compared to LPS/IFN-γ group (Fig. 6B) . Heatmap analysis showed that many 674-5p, miR-24-3p, miR-21a-5p, miR-23b-3p, miR-146a-5p, miR-378a-5p and let-7b-529 5p were shown (Fig. 7A ). Next, we performed miRNA-gene network analysis to 530 explore the possible interactions between these miRNAs carried by EVs and the 531 intracellular genes regulated by M2-EVs (Fig. 7B) . Again, the results indicated that Interestingly, the expression of ACE2 receptor has been found on the surface of 573 primary alveolar macrophages [52] . Based on these findings, we speculated that hM2- EVs may also express ACE2 and thus are capable of neutralizing SARS-CoV-2 virus. Our results showed that large amounts (~25%) of human peritoneal macrophages 576 (hPMφ) could be collected from peritoneal dialysate of PD patients (Fig. S7A ). These 577 cells were further polarized towards M2 phenotype in vitro, as indicated by elevated 578 expression of Mrc1 and Arg1 (Fig. S7B-C) . We isolated EVs from hPMφ and human 579 MSCs, and the resulting hM2-EVs and hMSC-EVs were bi-layer lipid membrane 580 vesicles with average sizes of ~150 nm and ~170 nm, respectively ( Fig. 8A-B) . Both EVs expressed markers such as Alix and TSG101 (Fig. 8C) . We found that hM2-PMφ 582 and hM2-EVs highly expressed ACE2, while a much weaker ACE2 expression was 583 found in hMSC and hMSC-EVs, which was consistent with previous report that hMSCs cells. In addition, we found that ACE2 + hM2-EVs could also reduce cytokine release in 599 mPMφ with LPS challenge (Fig. S7D) , which was similar to mouse M2-EVs. Taken The authors declare that they have no known competing financial interests or personal 631 relationships that could have appeared to influence the work reported in this paper. Burden of endemic health-care-associated infection in developing countries: 642 systematic review and meta-analysis Association between cytokine levels, sepsis severity and 645 clinical outcomes in sepsis: a quantitative systematic review protocol The 'cytokine storm': molecular mechanisms and therapeutic 648 prospects The cytokine storm in COVID-650 19: An overview of the involvement of the chemokine/chemokine-receptor system Clinical features of patients infected 655 with 2019 novel coronavirus in Origin and physiological roles of inflammation Different Profiles of Antibodies and Cytokines Were Found Between Severe and 660 Moderate COVID-19 Patients Current concepts in the diagnosis and management of cytokine release syndrome The use of anti-inflammatory drugs in the treatment of people with severe 669 coronavirus disease 2019 (COVID-19): The Perspectives of clinical immunologists from China Amelioration of systemic inflammation via the display of two different decoy protein receptors 676 on extracellular vesicles Macrophage activation syndrome in adults: 679 recent advances in pathophysiology, diagnosis and treatment Macrophage-derived 682 extracellular vesicles: diverse mediators of pathology and therapeutics in multiple diseases Chemerin aggravates DSS colitis by suppressing M2 macrophage polarization Extracellular vesicle-based therapeutics for 687 the regeneration of chronic wounds: current knowledge and future perspectives From M2b Macrophages Attenuate DSS-Induced Colitis Over-expression of miR-223 induces M2 macrophage 692 through glycolysis alteration and attenuates LPS-induced sepsis mouse model, the cell-based 693 therapy in sepsis Peritoneal M2 macrophage transplantation as a potential cell therapy for enhancing renal 696 repair in acute kidney injury Therapeutic potential of regulatory macrophages generated from peritoneal 699 dialysate in adriamycin nephropathy Mesenchymal Stem Cell-Derived Extracellular Vesicles Attenuate 702 Mitochondrial Damage and Inflammation by Stabilizing Mitochondrial DNA miRNet 2.0: network-based visual analytics for 705 miRNA functional analysis and systems biology Therapeutic effects of 708 serum extracellular vesicles in liver fibrosis Extracellular vesicles containing ACE2 efficiently prevent 711 infection by SARS-CoV-2 Spike protein-containing virus A Reservoir of Mature Cavity Macrophages that Can Rapidly Invade 714 Visceral Organs to Affect Tissue Repair Targeting RAW 264.7 macrophages (M1 type Withaferin-A decorated mannosylated liposomes induces repolarization via downregulation of 717 NF-kappaB and controlled elevation of STAT-3 Macrophage polarization and 719 function with emphasis on the evolving roles of coordinated regulation of cellular signaling 720 pathways Exotoxins and endotoxins: Inducers of inflammatory cytokines Molecular mechanisms of innate memory and tolerance to LPS Regulation of the Immune Response by TGF-beta: From 726 Extracellular Vesicles Protect 729 the Neonatal Lung from Hyperoxic Injury through the Epigenetic and Transcriptomic 730 Reprogramming of Myeloid Cells Routes and mechanisms of extracellular vesicle 732 uptake Delivery Vehicles Cellular 736 internalization of exosomes occurs through phagocytosis MSC-exosome: A novel cell-free therapy for 738 cutaneous regeneration Therapeutic Features and Updated Clinical Trials of Mesenchymal Stem Cell (MSC)-Derived Exosomes Immunopathogenesis and treatment of cytokine storm in COVID-19 The mechanism of 745 superantigen-mediated toxic shock: not a simple Th1 cytokine storm Macrophage-dependent clearance of systemically 749 administered B16BL6-derived exosomes from the blood circulation in mice Laboratory models of sepsis and septic shock Cytokine Storms: Understanding COVID-19 Acute kidney 756 injury from sepsis: current concepts, epidemiology, pathophysiology, prevention and treatment Injury in Non-human Primate Models of Viral Infections Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic 762 acute kidney injury by disrupting TFAM-mediated mtDNA maintenance Vitamin D3 decreases TNF-alpha-induced inflammation in lung epithelial cells through a 766 reduction in mitochondrial fission and mitophagy Proinflammatory Cytokine Signaling by Targeting STAT3 Inhibition of miR-378a-3p by 774 Inflammation Enhances IL-33 Levels: A Novel Mechanism of Alarmin Modulation in 775 Trials of anti-tumour necrosis factor therapy for COVID-19 are urgently needed Nature's second pandemic progress report Millisecond dynamic of SARS-CoV-2 spike and its 782 interaction with ACE2 receptor and small extracellular vesicles Engineering Extracellular Vesicles Enriched with Palmitoylated 786 ACE2 as COVID-19 Therapy Distinct uptake, amplification, 789 and release of SARS-CoV-2 by M1 and M2 alveolar macrophages Mesenchymal Stem Cells Improves the Outcome of Patients with 795 COVID-19 Pneumonia Ventilator-Free in Patients With Moderate or Severe Acute Respiratory Distress Syndrome and 803 COVID-19: The CoDEX Randomized Clinical Trial Updates on clinical trials evaluating the regenerative potential of 805 allogenic mesenchymal stem cells in COVID-19