key: cord-1021982-lt938wg6
authors: Wei, Jinhuan; Shang, Rui; Wang, Jiaqi; Zhu, Shengze; Yin, JianQiang; Chen, Ying; Zhao, Yayu; Chen, Gang
title: ACE2 overexpressing mesenchymal stem cells alleviates COVID-19 lung injury by inhibiting pyroptosis
date: 2022-03-10
journal: iScience
DOI: 10.1016/j.isci.2022.104046
sha: 7d89a663f97275f813a4ed5ad2c4f9f22a53e4f9
doc_id: 1021982
cord_uid: lt938wg6

Mesenchymal stem cells (MSCs) have shown some efficacy in the COVID-19 treatment. We proposed that exogenous supplementation of ACE2 via MSCs (ACE2-MSCs) might have better therapeutic effects. We constructed SARS-CoV-2 spike glycoprotein stably transfected AT-II and Beas-2B cells, and used SARS-CoV-2 spike pseudovirus to infect hACE2 transgenic mice. The results showed that spike glycoprotein transfection triggers apoptotic bodies’s release and membrane pores’s formation in pyroptosis. Inflammatory factors and pyroptosis factors were highly upregulated by spike glycoprotein transfection. SARS-CoV-2 spike pseudovirus worsened lung injury and increased the main factors of cytokine storm and pyroptosis. Compared to using MSCs or rh-ACE2 alone, the administration of ACE2-MSCs could much more significantly reduce these factors and alleviate lung injury in vivo and in vitro, which might be due to the increased activities of secretory ACE2. Our proposal is a promising therapeutic solution for preclinical or clinical research.

COVID-19 patients than using MSCs alone. have better therapeutic effects than MSCs alone to alleviate COVID-19-induced lung 13 injury by inhibiting pyroptosis.

scanning electron microscopy (SEM) and found that in both spike protein-transfected 1 AT-II and Beas-2B cells, apoptotic bodies were released ( Figure 1F and 1G), which is 2 consistent with previous reports that SARS-CoV-2 disrupts nuclear integrity and 3 causes tissue damage (Ackermann et al., 2020; Hekman et al., 2020). 4 Surprisingly, SEM results also showed that some AT-II-S and Beas-2B-S cells with 5 pores were characteristic markers of pyroptosis, while there were no pores in the 6 control group cells ( Figure 1H and 1I). These results are consistent with the finding 7 that COVID-19 induces pyroptosis, which leads to the appearance of many pores on 13 in both AT-II cells and Beas-2B cells 14 Although some researchers have proposed targeting inflammasomes and pyroptosis 15 for COVID-19 patient treatment (Freeman and Swartz, 2020; Yap et al., 2020), few 16 efforts have been put into clinical trials. To confirm the profile of the inflammatory 17 response and pyroptosis, we performed qRT-PCR and checked the transcript levels of 18 some main characteristics. The results demonstrated that in AT-II cells and Beas-2B 19 cells stably transfected with the SARS-CoV-2 spike protein, most of the tested genes 20 were upregulated compared with their untransfected cells. In AT-II-S cells, the Figure 2C ). TNF-α, IL-8, ASC and AIM2 were significantly upregulated in Beas-2B-S 10 cells compared with Beas-2B cells ( Figure 2D ), but we did not detect any dramatic 11 alterations in these genes in the LPS treatment groups ( Figure S2D ). Although IL-1β, 12 IL-18, GSDMD, NLRP3, NLRC4, and NLRP6 were also increased by LPS treatment 13 in Beas-2B-S cells compared with Beas-2B cells, their change folds within a narrower 14 range than that in the non-LPS treatment group ( Figure 2D ). We also found that LPS 15 significantly increased the transcript expression levels of IL-6 and NLRP1 in 16 Beas-2B-S cells ( Figure 2D ), but no detectable change was observed in Beas-2B cells 17 compared with their relative control group ( Figure S2B ). These results suggested that 18 LPS treatment could aggravate the inflammatory response and pyroptosis in 19 SARS-CoV-2 spike protein-transfected AT-II and Beas-2B cells compared with their 20 corresponding untransfected cells. They also indicated that the two different types of 21 lung cells have different reactions to spike protein transfection and LPS stimulation. To examine the therapeutic effects of ACE2-MSCs on COVID-19, we overexpressed 4 ACE2 in hUC-MSCs (ACE2-MSCs) and used the empty vector as a control 5 (GFP-MSCs) ( Figure 3A ). ACE2 activities in cell lysates and culture medium were 6 measured, and the results indicated that ACE2 activities were increased with culture 7 time in ACE2-MSCs ( Figure 3B and Figure 3C ), which suggested that hUC-MSCs 8 successfully overexpressed ACE2 and would be effective for subsequent research. We 9 treated AT-II, AT-II-S, Beas-2B, and Beas-2B-S cells with LPS for 24 hours and then 10 cocultured them with ACE2-MSCs or GFP-MSCs via a Transwell coculture system. 11 The transcript levels of the main factors of inflammation and pyroptosis were 12 examined by qRT-PCR, and the results showed that ACE2-MSCs very obviously 13 inhibited all these factors in LPS-treated AT-II-S cells compared with the GFP-MSCs 14 treatment group ( Figure 3D) . Surprisingly, in LPS-treated AT-II cells, some genes, 15 such as IL-6, IL-8, and NLRP6, were not changed, while other pyroptosis main factors, 16 such as GSDMD, Casp1, Casp4, ASC, AIM2, NLRP1, and NLRC4, were obviously and pyroptosis induced by spike protein transfection and LPS treatment compared 1 with the administration of GFP-MSCs. In vitro, we proved that the spike protein of SARS-CoV-2 transfection somehow 6 mirrored the phenomena (pyroptosis, apoptosis and inflammation) caused by 7 SARS-CoV-2 virus infection and that the administration of ACE2-MSCs suppressed 8 the main factors of pyroptosis and inflammation. Next, we aimed to confirm whether 9 ACE2-MSCs are much more effective in COVID-19 treatment than MSCs alone in 10 vivo. Considering the biosafety and operability to construct a SARS-CoV-2 11 virus-infected mouse model, we preferred to use the SARS-CoV-2 pseudovirus for 12 infection. Pseudoviruses expressing the SARS-CoV-2 spike protein that could 13 simulate the real virus to infect target cells without replication have been used widely Here, we performed intratracheal injection of SARS-CoV-2 pseudovirus in hACE2 19 transgenic mice to minor COVID-19. The experimental strategy is shown in Figure   20 4A. Unfortunately, if we only intratracheally injected the pseudovirus for 24 hours, 21 J o u r n a l P r e -p r o o f we could not observe detectable histopathological changes ( Figure S3 ). However, 1 based on LPS-induced ALI, intratracheal injection of SARS-CoV-2 pseudovirus 2 deteriorated lung injury ( Figure 4B ). The results of gross pathology and 3 histopathology of lungs from two times saline injection (sham group), LPS injection 4 plus saline (control group) and LPS injection plus SARS-CoV-2 pseudovirus injection 5 showed that LPS treatment induced the lung injury, and the pseudovirus made the 6 lung injury worse: much more edema, inflammatory cell exudation, hemorrhage, 14 The mRNA levels of pyroptosis main factors were then determined. Notably, in 15 addition to IL-1β, another downstream marker of pyroptosis, IL-18, its transcript was increased to different extents ( Figure 4E ), suggesting that pyroptosis was stirred up by 1 SARS-CoV-2 pseudovirus infection. Taken together, these results implied that our 2 COVID-19-infected mouse model was valuable for subsequent studies. To ensure that tail intravenous injection of MSCs homed to the lung tissues to exert 6 their functions, we traced the distribution of MSCs from 12 hours to 84 hours after 7 injection. The results demonstrated that in the LPS-treated group at every tested time 8 point, the number of MSCs was greater than that in the control group, and at the peak 9 time of accumulation (24 hours), the difference was obvious ( Figure 5A and 5B). 10 Considering the accumulation and actual action of MSCs on lung tissues, we treated 11 lung injury with MSCs for 36 hours and sampled lung tissues for the following 12 experiments ( Figure 5C ). Figure 5D shows the histopathology of PBS-, GFP-MSCs- 13 or ACE2-MSCs-treated injured lung tissues. Compared with the PBS-treated group, 14 the GFP-MSCs group had significantly reduced lung injury, but the ACE2-MSCs of ACE2-MSCs made the damaged lung tissue recover to near normal ( Figure 5D ). 19 Histopathological examination by hematoxylin-eosin staining showed that 20 inflammatory cell exudation, hemorrhage, and hyaline membrane were barely 21 J o u r n a l P r e -p r o o f detected in the ACE2-MSCs treatment group ( Figure 5D ). Statistical analysis of the 1 histopathological scores found that edema, inflammatory cell exudation, hemorrhage, 2 and hyaline membrane were all decreased by the administration of GFP-MSCs and 3 ACE2-MSCs to different degrees ( Figure 5E ). Again, ACE2-MSCs functioned much 4 more effectively than MSCs alone to treat COVID-19-induced lung injuries. Besides, 5 we compared the therapeutics of soluble ACE2 and the therapeutics of ACE2-MSCs. 6 Even though compared with the control group, human ACE-2 (rh-ACE2) protein 7 could attenuate the lung injury, its therapeutic effects were weaker than that of To explore the mechanisms underlying the therapeutic effect, we tested the IL-1β were significantly repressed ( Figure 5F ), which indicated that the therapeutic effects of ACE2-MSCs were much stronger than the therapeutic effects of rh-ACE2.

In summary, our study proved that the administration of MSCs could alleviate 2 COVID-19-induced lung injury and repress cytokine storms and pyroptosis at the 3 molecular level. In addition, ACE2-MSCs function more effectively than GFP-MSCs 4 or rh-ACE2. Beas-2B, and transfected the cells with SARS-CoV-2 spike protein containing 20 plasmid DNA, which showed apoptosis, necrosis and pyroptosis; in vivo, hACE2 transgenic mice were treated with LPS first and infected with SARS-CoV-2 1 pseudovirus, which demonstrated severe lung injuries, cytokine storm and pyroptosis.

The application of MSCs significantly reduced the transcript levels of the main factors 3 of the inflammatory response, cytokine storm and pyroptosis, especially ACE2-MSCs, 4 which were much more effective than MSCs alone. 5 We tried to use different doses of SARS-CoV-2 spike RBD recombinant protein to 6 infect AT-II and Beas-2B cells for several days but did not yield obvious results. pyroptosis phenomena yet, here, in both AT-II and Beas-2B cells we found that 2 besides the cell growth was repressed, apoptosis, and necrosis, more obviously, Pyroptosis is well known to be activated by inflammasome components (NLRP3, 19 active Casp-1) and then executed by gasdermin family members. GSDMD is the most which indicated that by recombinant SARS-CoV-2 spike protein and pseudovirus 16 dominantly triggered the classical pyroptosis pathway but also induced the 17 noncanonical pathway. 18 There are many preclinical and clinical trials targeting NLRP3 and GSDMD to treat 20 we also found that these two factors were highly increased. However, we should also 21 take notice of AIM2 and NLRC4 because their transcript expression was much higher upregulated by spike protein transfection in AT-II and Beas-2B cells ( 8 and Casp1 than GFP-MSCs. Therefore, based on the in vivo and in vitro results, we 9 speculate that ACE2-MSCs are much more effective in treating COVID-19 patients 10 than GFP-MSCs or rh-ACE2 alone. However, the molecular mechanisms underlying 11 this process are poorly understood and will be the focus of upcoming studies. 12 In conclusion, in COVID-19-infected cells or animals, MSCs modified with the ACE2 13 gene or even other vital gene(s) will be a promising approach for the treatment of 14 COVID-19 patients. In addition, targeting core members of the cytokine storm or 15 pyroptosis in therapy provides some new strategies for developing new treatments for 16 COVID-19 patients. ACE2-MSCs modulate cellular and signaling networks in response to 10 microenvironmental cues in COVID-19 is also necessary. On the clinical side, 11 regarding optimal therapeutic doses and optimal route(s) of administration of 12 ACE2-MSCs should be the prime concern. Recently, WHO warned that with the rapid 13 transmission and spread of Omicron in global area, the high infection rate might 14 precipitate the emergence of new variant strain(s); people may never be able to 15 vanquish the SARS-CoV-2 virus, and it will eventually become part of the ecosystem. Declaration of interests 15 The authors declare no competing interests. respectively, compared with that in their corresponding cells. The relative expression 8 of target genes was normalized to 18S rRNA. The results are presented as the 9 mean ± SD. Student's t-test was used to compare the differences between groups, 10 followed by Bonferroni's test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 11 versus the control group.

(C) qRT-PCR results showed that the inflammatory factors TNFα, IL-6, IL-8, IL-1β, 13 and IL-18 and pyroptosis members GSDMD, Casp1, Casp4, AIM2, NLRP3, NLRC4 14 and NLRP6 were upregulated in LPS-treated AT-II-S cells compared with LPS-treated 15 AT-II cells. The relative expression of target genes was normalized to 18S rRNA. The 16 results are presented as the mean ± SD. Student's t-test was used to compare the 17 differences between groups, followed by Bonferroni's test. *P < 0.05, **P < 0.01, 18 ***P < 0.001, ****P < 0.0001 versus the control group. results are presented as the mean ± SD. Student's t-test was used to compare the 4 differences between groups, followed by Bonferroni's test. *P < 0.05, **P < 0.01, 5 ***P < 0.001, ****P < 0.0001 versus the control group. 

(B and C) ACE2 activity in cell lysates and cell culture medium was measured by an 12 ACE2 activity assay kit, respectively. The results are presented as the mean ± SD. 13 Student's t-test was used to compare the differences between groups, followed by 14 Bonferroni's test. **P < 0.01, ***P < 0.001, versus control group. to 18S rRNA. The results are presented as the mean ± SD. Student's t-test was used to 3 compare the differences between groups, followed by Bonferroni's test. *P < 0.05, 4 **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the control group. ## P < 0.01, 5 ### P < 0.001, #### P < 0.0001 versus the control group. normalized to 18S rRNA. The results are presented as the mean ± SD. Student's t-test 12 was used to compare the differences between groups, followed by Bonferroni's test.

*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the control group. 14 # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 versus the control group. hyaline membrane. The results are presented as the mean ± SD. One-way ANOVA 8 was used to compare the differences between groups, followed by Bonferroni's test.

N=5 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the NS 24 rRNA. The results are presented as the mean ± SD. T-test or one-way ANOVA 15 was used to compare the differences between groups, followed by Bonferroni's test. 16 N=3 or 4 per group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus the 17 NS 24 h + NS 24 h group. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001 versus the 18 LPS 24 h + NS 24 h group. 19 (E) qRT-PCR detected the main pyroptosis factor mRNAs: GSDMD, Casp1, Casp4, normalized to 18S rRNA. The results are presented as the mean ± SD. One-way 1 ANOVA was used to compare the differences between groups, followed by 2 Bonferroni's test. N=3 or 4 per group, *P < 0.05, **P < 0.01, ***P < 0.001, 3 ****P < 0.0001 versus the NS 24 h + NS 24 h group. # P < 0.05, ## P < 0.01, 4 ### P < 0.001, #### P < 0.0001 versus the LPS 24 h + NS 24 h group. injection. The results are presented as the mean ± SD. Student's t-test was used to 12 compare the differences between groups, followed by Bonferroni's test. N=6 per 13 group, **P < 0.01, ***P < 0.001 verse control group. Further information and requests for resources and reagents should be directed to and 4 will be fulfilled by the lead contact, Gang Chen (chengang6626@ntu.edu.cn).

This study did not generate new unique reagents.

Data and code availability 8 Data reported in this paper will be shared by the lead contact upon request. This paper 9 does not report original code. Any additional information required to reanalyze the 10 data reported in this paper is available from the lead contact upon request. Cell culture 13 AT-II cells (BFN6080397) and Beas-2B cells (BFN608009328, RRID: CVCL_UR57) 14 were purchased from BFB BluefBio. hUC-MSCs were donated by 15 Genesis Stem-cell Co., Ltd. Cells were grown in complete medium containing 90% 16 basic medium, 10% FBS, and 1% PSS. The basic media for AT-II and Beas-2B cells 17 were pyruvic acid-free DMEM and RPMI 1640, respectively. hUC-MSCs culture reader.

Coculture system 10 AT-II and AT-II-S coculture systems were plated at 2*10 5 cells/well; Beas-2B and 11 Beas-2B-S cells were plated at 3*10 5 cells/well, all in 6-well plates. Twelve hours 12 later, all the cells were treated with LPS (2 μg/ml). ACE2-MSCs or GFP-MSCs

(2*10 5 cells/well) were plated in a 0.45 μm Transwell membrane in a 6-well plate.

14 After 24 hours, AT-II/-S or Beas-2B/-S culture medium with LPS was replaced by 15 Opti-MEM, and ACE2-MSCs or GFP-MSCs cultured in transwell membrane were 16 put on the uperside. Cells were cocultured for 24 hours, and then the cells were 17 collected for the next experiment. 7 We administered SARS-CoV-2 pseudovirus (OBiO Scientific Service, H7657) 5*10 5 8 TU to every mouse by intratracheal injection to simulate a COVID-19-infected model. Then, the cells were dehydrated using graded ethanol followed by isoamyl acetate 

Statistical analysis 15 All data are expressed as the mean ± SD, as indicated in the figure legends. Student's 16 t-test (two groups), one-way ANOVA or two-way ANOVA was used to compare the highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors 28 a high capacity to mediate membrane fusion. Cell. Res. 30, 343-355. 29 10.1038/s41422-020-0305-x. 

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