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. 

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