key: cord-0757234-cn7xpyi2
authors: Veras, Flavio P.; Gomes, Giovanni F.; Silva, Bruna M. S.; Almeida, Cicero J. L. R.; Silva, Camila Meirelles S.; Schneider, Ayda H.; Corneo, Emily S.; Bonilha, Caio S.; Batah, Sabrina S.; Martins, Ronaldo; Arruda, Eurico; Fabro, Alexandre T.; Alves-Filho, José C.; Cunha, Thiago M.; Cunha, Fernando Q.
title: Targeting Neutrophils Extracellular Traps (NETs) reduces multiple organ injury in a COVID-19 mouse model
date: 2022-04-27
journal: bioRxiv
DOI: 10.1101/2022.04.27.489676
sha: fa686693144542df5d32ada210f655609eb98646
doc_id: 757234
cord_uid: cn7xpyi2

COVID-19 is characterized by severe acute lung injury, which is associated with neutrophils infiltration and release of neutrophil extracellular traps (NETs). COVID-19 treatment options are scarce. Previous work has shown an increase in NETs release in the lung and plasma of COVID-19 patients suggesting that drugs that prevent NETs formation or release could be potential therapeutic approaches for COVID-19 treatment. Here, we report the efficacy of NET-degrading DNase I treatment in a murine model of COVID-19. DNase I decreased detectable levels of NETs, improved clinical disease, and reduced lung, heart, and kidney injuries in SARS-CoV-2-infected K18-hACE2 mice. Furthermore, our findings indicate a potential deleterious role for NETs lung tissue in vivo and lung epithelial (A549) cells in vitro, which might explain part of the pathophysiology of severe COVID-19. This deleterious effect was diminished by the treatment with DNase I. Together, our results support the role of NETs in COVID-19 immunopathology and highlight NETs disruption pharmacological approaches as a potential strategy to ameliorate COVID-19 clinical outcomes.

Coronavirus diseases 2019 is an infection caused by the severe 4 acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1, 2) and pulmonary-5 related symptoms are one of its hallmarks (3, 4) . Neutrophils have been 6 described as indicators of severity of respiratory symptoms and poor COVID-19 7 prognosis (5-7). Neutrophil extracellular traps (NETs) are one of the most 8 relevant effector mechanisms of neutrophils in inflammatory diseases, playing 9 central role in organs damage (8-10).

We found that exposure of A549 cells to purified NETs significantly increased the 12 percentage of apoptotic cells in comparison with untreated cells. Importantly, the 13 pretreatment of purified NETs with DNase I prevented NETs-induced epithelial 14 Taken together, these results are evidence that DNase I exhibits a protective 16 effect in NETs deleterious functions in lung tissue and could be a potential 17 strategy to block organ injury during 19 DISCUSSION 20

While the number of patients with COVID-19 is growing worldwide, there is no 21 effective treatment for the disease (32, 33). Thus, the understanding of the 22 mechanisms by which the hosts deal with SARS-CoV-2 virus could allow the 23 development of new therapeutic strategies aiming to prevent tissue injuries triggered by the infection. Here, we report that in a COVID-19 mouse model, 1

NETs are released systemically and in higher concentration in the lungs of K18-2 hACE2 mice. Moreover, DNase I treatment reduced multi-organ lesions and 3 improved outcomes associated NETs released. 4

The increase in the number of circulating neutrophils is an indicator of a worse 5 outcome of . In 2004, Brinkmann et al. described, for the first time, 6 that NETs are released by neutrophils, and work as a microbicidal mediator (11). 7

However, following studies demonstrated that NETs have a dual biological role. 8

Besides their microbicidal ability, NETs mediate lesions observed in several 9 inflammatory diseases, including rheumatoid arthritis, lupus, diabetes, and sepsis 10 (8, 10, 35-39). In fact, inhibition of NETs production prevented lung, heart and 11 liver lesions observed in experimental sepsis (38) (39) (40) . 12

There are several stimuli that trigger NETs release, including pathogen-13 associated molecular patterns (PAMPs), damage-associated molecular patterns 14 (DAMPs) and inflammatory mediators, such as cytokines and chemokines (41-15 43) . Our group have previously demonstrated that SARS-CoV-2 can directly 16 infect human neutrophils and is key to trigger NETs production. The first step in 17 neutrophil infection by SARS-CoV-2 is the interaction of the virus with ACE2 and 18 TMPRSS2 expressed in the surface of human neutrophils (8). It is possible to 19 speculate the participation of the cytokines and chemokines released by host 20 cells as activators (44, 45) of NETs production by the neutrophils in the K18-21 hACE2 model; however, this deserves future investigation. 22

The literature describes that NETs can present direct cytotoxic effects on different 23 mammalian cell types, including epithelial and endothelial cells, inducing 24 apoptosis or necrosis (12, 46) . Moreover, NETs could also activate different PRR receptors, such as toll-like receptor (TLR)-4 and 9, which mediate the release of 1 inflammatory mediators; in turn, amplifying the direct effects of NETs (35). In this 2 context, during COVID-19, apoptosis of lung epithelial was previously observed. 3

These events are capable of compromising the lung function, worsening the 4 severity of the disease (3, 4). Considering these findings, in the present study, 5

we observed that DNase I prevented apoptosis in lung tissue from SARS-CoV-2-6 infected mice. In accordance observed in COVID-19 model, isolated NETs from 7 culture of PMA-stimulated human neutrophils induced in vitro apoptosis of 8 epithelial cells with reversal homeostasis in presence of DNase I. Extending this 9

finding to COVID-19 disease, it is possible to suggest that the reduction of viability 10 of the lung cells is a consequence of local production of NETs. In this line, we 11

observed the presence of neutrophils releasing NETs, as well as high 12 concentration of NETs in the lung of SARS-CoV-2-infected mice. Finally, 13 supporting these finds, we observed the presence of neutrophils releasing NETs 14 in the lung tissue of infected mice, detected in the alveolar space. Disease 15 progression was prevented with DNase I treatment in vivo. This observation is 16 tightly correlated with the development of lung injury, suggesting that strategies 17 to reduce NETs levels could have favorable effects on recovering lung function. 18

Together, our findings demonstrate the potentially deleterious role of NETs during 19 COVID-19 and support the use of inhibitors of NETs, such as DNase I as a 20 strategy to ameliorate multi-organ damage during COVID-19.

K18-hACE2 humanized mice (B6.Cg-Tg(K18-ACE2)2Prlmn/J) were obtained 1 from The Jackson Laboratory and were bred in the Centro de Criação de Animais 2 Especiais (Ribeirão Preto Medical School/University of São Paulo). This mouse 3 strain has been previously used as model for SARS-CoV-2-induced disease and 4 it presents signs of diseases, and biochemical and lung pathological changes 5 compatible with the human disease (23). Mice had access to water and food ad 6 libitum. The manipulation of these animals was performed in Biosafety Levels 3 7 (BSL3) facility and the study was approved by Ethics Committee on the Use of 8

Animals of the Ribeirão Preto Medical School, University of São Paulo 9 (#066/2020). 10

Five μm lung, heart and kidney slices were submitted to Hematoxylin and Eosin 2 staining. A total of 10 photomicrographs in 40X magnification per animal were 3 randomly obtained using a microscope ScanScope (Olympus) and Leica. 4

Morphometric analysis was performed in accordance with the protocol 5 established by the American Thoracic Society and European Thoracic Society 6

Lungs were harvested and fixed with PFA 4%. After dehydration and paraffin 18 embedding, 5 μm sections were prepared. The slides were deparaffinized and 19 rehydrated by immersing the through Xylene and 100% Ethanol 90% for 15 20 minutes, each solution. Antigen retrieval was performed with 1.0 mM EDTA, 10 21 mM Trizma-base, pH 9.0 at 95°C for 30 minutes. Later, endogenous peroxidase 

An Official Research Policy Statement of the American