key: cord-0299692-h6giz8f9
authors: Tojo, K.; Natsuhiro, Y.; Tamada, N.; Mihara, T.; Abe, M.; Goto, T.
title: Early Alveolar Epithelial Cell Necrosis is a Potential Driver of ARDS with COVID-19
date: 2022-01-24
journal: nan
DOI: 10.1101/2022.01.23.22269723
sha: 016b340fcaedf32e57de3a26d2e69a2d729901af
doc_id: 299692
cord_uid: h6giz8f9

Rationale: Acute respiratory distress syndrome (ARDS) with COVID-19 is aggravated by hyperinflammatory responses even after passing the peak of viral load. However, the underlying mechanisms remain unclear. Objectives : Here, we assess whether alveolar epithelial cell necrosis and subsequent releases of damage associated molecular patterns (DAMPs) at an early disease stage aggravate ARDS with COVID-19 Methods: In patients with COVID-19 with and without ARDS and healthy adults, serum levels of the following were quantified: an epithelial total cell death marker, cytokeratin18-M65; an epithelial apoptosis marker, CK18-M30; HMGB-1; and alveolar epithelial and endothelial injury markers, sRAGE, angiopoietin-2, and surfactant protein-D. Molecular mechanisms of alveolar epithelial cell death and effects of HMGB-1 neutralization on alveolar tissue injury were assessed using a mouse model of COVID-19-induced ARDS. Measurements and main results: The levels of CK18-M65, CK18-M30, and alveolar tissue injury markers were elevated in early stages of ARDS. The median M30/M65 ratio, an epithelial apoptosis indicator, was 31.50% in patients with ARDS, a value significantly lower than that of non-ARDS patients or healthy subjects. Serum levels of HMGB-1, one of DAMPs released from necrotic cells, were also significantly elevated in ARDS versus non-ARDS patients. In a COVID-19-induced ARDS mouse model, alveolar epithelial cell necrosis involved two forms of programmed necrosis, necroptosis and pyroptosis. Finally, neutralization of HMGB-1 attenuated alveolar tissue injury in the mouse model. Conclusions: Necrosis, including necroptosis and pyroptosis, seems to be the primary form of alveolar epithelial cell death, and subsequent release of DAMPs is a potential driver of COVID-19-induced ARDS.

Acute respiratory distress syndrome (ARDS) with COVID-19 is aggravated by hyperinflammatory responses even after passing the peak of viral load. However, the underlying mechanisms remain unclear.

Here, we assess whether alveolar epithelial cell necrosis and subsequent releases of damage associated molecular patterns (DAMPs) at an early disease stage aggravate ARDS with COVID-19

In patients with COVID-19 with and without ARDS and healthy adults, serum levels of the following were quantified: an epithelial total cell death marker, cytokeratin18-M65; an epithelial apoptosis marker, CK18-M30; HMGB-1; and alveolar epithelial and endothelial injury markers, sRAGE, angiopoietin-2, and surfactant protein-D. Molecular mechanisms of alveolar epithelial cell death and effects of HMGB-1 neutralization on alveolar tissue injury were assessed using a mouse model of COVID-19-induced ARDS.

. CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint 2

The levels of CK18-M65, CK18-M30, and alveolar tissue injury markers were elevated in early stages of ARDS. The median M30/M65 ratio, an epithelial apoptosis indicator, was 31.50% in patients with ARDS, a value significantly lower than that of non-ARDS patients or healthy subjects. Serum levels of HMGB-1, one of DAMPs released from necrotic cells, were also significantly elevated in ARDS versus non-ARDS patients. In a COVID-19-induced ARDS mouse model, alveolar epithelial cell necrosis involved two forms of programmed necrosis, necroptosis and pyroptosis. Finally, neutralization of HMGB-1 attenuated alveolar tissue injury in the mouse model.

Necrosis, including necroptosis and pyroptosis, seems to be the primary form of alveolar epithelial cell death, and subsequent release of DAMPs is a potential driver of COVID-19-induced ARDS. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022.

Infection with a novel strain of coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causes coronavirus disease 2019 (COVID- 19) pneumonia. In the most severe cases, the disease progresses to acute respiratory distress syndrome (ARDS), which is associated with severe alveolar tissue injury (1, 2) .

Interestingly, the disease severity is exacerbated by hyperinflammatory responses even after passing the peak of viral load (3, 4) . However, the mechanisms that underly disease aggravation remain unclear. We and others previously reported that alveolar epithelial injury at a very early disease stage is a hallmark of COVID-19-induced ARDS(5, 6), suggesting that alveolar epithelial injury may be a trigger of subsequent disease progression. Therefore, elucidating the detailed mechanisms by which alveolar epithelial injury occurs in COVID-19 may reveal a therapeutic target that prevents disease aggravation.

In ARDS, alveolar epithelial cells undergo cell death (7, 8 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01. 23.22269723 doi: medRxiv preprint shown to be involved in alveolar epithelial cell death in several animal ARDS models(10-13). In contrast to apoptosis, which does not elicit inflammation, necrosis causes the release of damage-associated molecular patterns (DAMPs) such as high mobility group box (HMGB)-1 from dead cells(14, 15). Therefore, it is possible that alveolar necrosis during early disease stages, and the subsequent release of DAMPs, may drive disease progression in COVID-19-associated ARDS (16, 17) .

Here, we assess whether alveolar epithelial cell necrosis and subsequent release of DAMPs aggravate COVID-19-associated ARDS. To determine alveolar epithelial cell death patterns in COVID-19 patients with or without ARDS, we analyzed serum levels of full-length (CK18-M65 antigen) and caspase-cleaved (CK18-M30 antigen) cytokeratin 18, which are epithelial total cell death and epithelial apoptosis markers respectively, in addition to the other several alveolar epithelial and endothelial injury markers. Moreover, we investigated the detailed mechanisms of alveolar epithelial cell death using the animal model mimicking COVID-19-induced ARDS. Finally, we evaluated whether blockade of HMGB-1, one of DAMPs released from necrotic cells, can attenuate alveolar tissue injury in the animal model. Some of the preliminary results of the study were published previously(5).

. CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint 5

Detailed methods are provided in online supplementary materials (Text E1).

This single-center observational study included adult patients with available serum samples who were admitted to Yokohama City University Hospital with COVID-19

from January 2020 to January 2021. ARDS was diagnosed based on the Berlin definition (18) . The study protocol was approved by the Yokohama City University Hospital institutional review board (B200700100). The requirement for informed consent was waived due to the observational nature of the study.

Clinical data were retrospectively collected from each patient's medical chart.

Using enzyme-linked immunosorbent assay (ELISA) kits, serum levels of the following were measured: soluble receptors for advanced glycation end products is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint and HMGB-1. Concentrations of the above markers at admission (first or second hospital day) of ARDS, non-ARDS, and control individuals were compared. Temporal changes in marker levels in patients with ARDS throughout the first 8 days of admission were assessed.

All animal experimental protocols were approved by Yokohama City University Animal Research Committee. Male specific-pathogen-free C57/BL6J mice aged 8-10 weeks were used. Mice were euthanized 24 h after intratracheal instillation, and tissue and bronchoalveolar lavage fluid (BALF) samples were collected as previously described.

Twelve animals were randomly allocated to one of the following three groups (n = is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint

To evaluate effects of anti-HMGB-1 neutralizing antibodies in severe COVID-19 animal model, six animals were randomly placed in either anti-HMGB-1 antibody or isotype control groups (n = 3 for each). The severe COVID-19 animal model was established as described above. Then, 100 μg anti-HMGB-1 neutralizing or isotype control antibodies dissolved in 100 μL PBS were intravenously administered via the tail vein 4 h after intratracheal instillation.

BALF leukocyte and protein levels were quantified as described previously.

Concentrations of sRAGE, ANG-2, CK18-M30, CK18-M65, and HMGB-1 were measured by ELISAs. Cytokines and chemokines were analyzed using a semiquantitative multiplex cytokine assay.

Mixed lineage kinase domain-like (MLKL), phospho-MLKL, gasdermin D (GSDMD), and cleaved-GSDMD levels in lung tissues were analyzed by immunoblotting.

. CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint

Lung tissue sections were stained with hematoxylin and eosin to assess histopathology. Lung tissue sections were stained with anti-phospho-MLKL and anti-GSDMD n-terminal antibodies.

Statistical analyses were performed using Prism 9 (GraphPad Software, La Jolla, CA). Values of P < 0.05 were considered significant. Data from clinical studies were presented as medians and interquartile ranges (IQRs), and analyzed as non-parametric data. Log-transformed animal experiment data are presented as means ± standard deviation (SD), and analyzed as parametric data. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint

Forty-eight (18 non-ARDS and 30 ARDS) among a total of 84 patients hospitalized with COVID-19 during the study period and 18 healthy volunteers matched as closely as possible for age and sex, were included in analyses. Characteristics of patients with COVID-19 are presented in Table 1 . Patients with ARDS had higher acute physiology and chronic health evaluation-II (APACHE-II) scores, white blood cell counts, C-reactive protein (CRP) levels, D-dimer levels, and lower ratios of partial pressure of arterial oxygen to fraction of inspired oxygen (P/F ratios) and lymphocyte counts than patients without ARDS. Eight patients with ARDS (26.7%) died. Among patients with ARDS, five developed acute kidney injury, with only a small increase in total bilirubin concentration was observed in several patients. Thus, organ dysfunction in most patients was primarily limited to the lungs.

We evaluated the circulating levels of three alveolar tissue injury markers: an alveolar epithelial injury marker (sRAGE) (19, 20) and an endothelial injury marker (ANG-2) (21, 22) , along with an alveolar permeability indicator (SP-D) (23, 24) . All the alveolar tissue injury markers levels after admission were significantly higher in patients with ARDS versus healthy controls ( Fig. 1A-C) . However, only serum sRAGE . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint and SP-F levels of patients with and without ARDS significantly differed ( Fig. 1A -C).

In patients with ARDS, sRAGE levels were significantly elevated immediately after admission, and gradually decreased thereafter (Fig. 1D, G) . On the other hand, ANG-2 and SP-F levels peaked later ( Fig. 1E-G) . Collectively, these results agree with prior work that demonstrated that severe alveolar epithelial cell injury at a very early disease stage is a hallmark of COVID-19-induced ARDS(5, 6).

Next, levels of epithelial cell death markers were evaluated to elucidate the dominant form of alveolar epithelial cell death at early disease stage of COVID-19induced ARDS. Serum levels of CK18-M65 and -M30 antigens were measured to distinguish alveolar necrosis from apoptosis. CK18 is exclusively expressed in epithelial cells, and is released upon cell death. The M65 antigen is an indicator of both epithelial cell necrosis and apoptosis. In contrast, the M30 antigen produced after caspase cleavage of CK18 is an indicator apoptotic epithelial cell death (25) . Although CK18 is expressed in all kinds of epithelial cells, it thought to be derived from alveolar epithelial cells in this cohort because the organ damage was limited almost exclusively to the . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint lungs. Levels of both M65 and M30 at admission was positively correlated with disease severity in patients with COVID-19 ( Fig. 2A, B) (Fig. 2C) , indicating that epithelial cell death in COVID-19 ARDS is mainly due to necrosis. Additionally, levels of HMGB-1, a kind of DAMPs released from necrotic cells, were significantly elevated in ARDS patients versus non-ARDS patients and healthy controls (Fig. 2D ). The analysis of correlations among these biomarkers demonstrated that HMGB-1 levels were most strongly correlated with M65 levels, suggesting that epithelial cell death contributes to HMGB-1 release to circulation (Fig. E1 in the online data supplement).

To elucidate mechanisms of alveolar epithelial cell death in COVID-19-induced ARDS, a COVID-19 mouse model was established. Previously, it was reported that innate immune responses to components of SARS-CoV-2 are principal drivers of inflammation and alveolar tissue injury in COVID-19 (26) (27) (28) . As expected, a . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. Further, levels of several chemokines and cytokines previously reported to be elevated in COVID-19 patients (29, 30) were significantly increased in the BALF of animal models of severe COVID-19 versus controls (Fig. 3F) .

Both M30 and M65 levels were increased in the BALF of COVID-19 models versus controls (Fig. 4A, B) , indicating that both necrosis and apoptosis are involved in alveolar epithelial cell death. M30/M65 ratio, an indicator of apoptosis fraction relative to total epithelial cell death, decreased as lung injury increased in severity, as was observed in COVID-19 patients (Fig. 4C) . Additionally, HMGB-1 levels were . CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint 13 significantly elevated in severe COVID-19 animal model versus the other two groups (Fig.4D) . Taken together, these results demonstrated that animal models of severe COVID-19 exhibit the same pattern of alveolar epithelial cell death as COVID-19 patients with ARDS.

Next, we evaluated whether some forms of programmed necrosis are involved in alveolar epithelial cell death animal models of COVID-19. Necroptosis and pyroptosis are two types of programmed necrosis that are induced by inflammation (31) . In lung tissues of animal models of severe COVID-19, levels of phospho-MLKL and cleaved GSDMD, executioners of necroptosis(32, 33) and pyroptosis (34) , respectively, were significantly elevated versus the other two groups (Fig. 4E, F, Fig. E3 in the online data supplement)). Moreover, immunohistochemical analysis demonstrated that both phospho-MLKL and GSDMD are localized within alveolar walls (Fig. 4G) .

Collectively, these results indicate that necroptosis and pyroptosis contribute to alveolar epithelial cell death in COVID-19-induced ARDS.

. CC-BY-NC 4.0 International license It is made available under a perpetuity.

is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. Alveolar tissue injury in severe COVID-19 is aggravated after passing the peak viral load (3, 4) . Therefore, hyperinflammatory responses only against SARS-CoV-2 per se cannot fully explain mechanisms that underly disease progression. Recently, we published findings suggesting that alveolar epithelial injury at a very early disease stage may trigger subsequent COVID-19 progression(5). The present study indicates that initial alveolar epithelial necrosis, and the subsequent release of DAMPs may exacerbate alveolar tissue injury, which can progress even after viral loads have peaked.

Previously, it was shown that both necrosis and apoptosis are involved in alveolar epithelial cell injury in ARDS(8). Because apoptosis can be easily assessed by TUNEL staining or caspase detection, the contribution of alveolar epithelial apoptosis in ARDS has been extensively studied(7). However, we recently demonstrated that necrosis is the is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint dominant form of alveolar epithelial cell death in LPS-induced ARDS by quantification of CK18-M30 and M65 in addition to cell labeling techniques(9). In particular, quantification of CK18-M30 and M65 levels using commercially available ELISA kit can be applied for evaluation of epithelial apoptosis and necrosis in clinical setting. In fact, patterns of epithelial cell death such as sepsis (35, 36) and graft rejection after lung transplantation (37) have been analyzed previously. This is the first study to suggest that necrosis is the primary form of alveolar epithelial cell death in human ARDS, similar to LPS-induced animal models of ARDS. Further studies will be needed to identify alveolar patterns of epithelial cell death in ARDS that is induced by disease etiologies other than COVID-19.

Necrosis was previously thought to cause accidental cell death. However, it has been demonstrated that some forms of necrosis, which are called as programmed necrosis, are regulated via molecular pathways (38) . Several animal studies have demonstrated that programmed necrosis is involved in alveolar epithelial cell death in ARDS(10-13). Moreover, studies have suggested that SARS-CoV-2 activates intracellular necroptosis and pyroptosis pathways (31, 39, 40) , and it has been reported that the circulating level of receptor-interacting protein kinase 3, a kinase required for necroptosis, is elevated in critically ill patients with COVID-19 (41) . In line with is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint findings of these studies, our animal experiments suggest that necroptosis and pyroptosis are involved in alveolar epithelial cell death in COVID-19 ARDS.

The release of DAMPs to extracellular spaces is a characteristic of necrosis that distinguishes it from apoptosis (42) . Several previous studies have also reported that circulating levels of DAMPs, such as HMGB-1 (43, 44) is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. Our data suggest that plasma M30/M65 ratio (an indicator of apoptosis in relation to total levels of epithelial cell death) is a potential marker of COVID-19 severity. Our findings agreed with a previous study that showed M30/M65 ratios of hospitalized COVID-19 patients were lower than those of non-hospitalized patients (48) .

Additionally, some studies have indicated that subtypes of COVID-19 respond to treatments differently (49, 50) . M30/M65 ratio may serve as a marker for selecting patients likely to benefit from anti-DAMPs treatment.

This study has some limitations. First, only patients admitted to a single center were included in the analysis due to the limited availability of clinical samples. Further is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. 

In conclusion, our data indicate that necrosis, including necroptosis and pyroptosis, is the primary form of alveolar epithelial cell death in COVID-19-induced ARDS.

DAMPs released from necrotic alveolar epithelial cells are potential drivers of progressive alveolar tissue damage in COVID-19; therefore, they are promising targets for preventing the aggravation of ARDS in patients with COVID-19. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint

The copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint

A systematic review of pathological findings in COVID-19: a pathophysiological timeline and possible mechanisms of disease progression

Increased Angiotensin-Converting Enzyme 2 and Loss of Alveolar Type II Cells in COVID-19-related Acute Respiratory Distress Syndrome

Treating COVID-19: are we missing out the window of opportunity?

DAMPs and NETs in Sepsis

The role of death domain proteins in host response upon SARS-CoV-2 infection: modulation of programmed cell death and translational applications

Role of DAMPs in respiratory virus-induced acute respiratory distress syndrome-with a preliminary reference to SARS-CoV-2 pneumonia

Acute Respiratory Distress Syndrome: The Berlin Definition

Receptor for Advanced Glycation End-Products Is a Marker of Type I Cell Injury in Acute Lung Injury

Plasma receptor for advanced glycation end products and clinical outcomes in acute lung injury

Angiopoietin-2, permeability oedema, occurrence and severity of

ALI/ARDS in septic and non-septic critically ill patients

Plasma angiopoietin-2 in clinical acute lung injury

Plasma surfactant protein levels and clinical outcomes in patients with acute lung injury

Prognostic value of surfactant proteins A and D in patients with acute lung injury*

Differentiation between Cell Death Modes Using Measurements of Different Soluble Forms of Extracellular Cytokeratin 18

SARS-CoV-2 spike protein interacts with and activates TLR41

TLR2 senses the SARS-CoV-2 envelope protein to produce inflammatory cytokines

RIG-I triggers a signaling-abortive anti-SARS-CoV-2 defense in human lung cells

Cytokine elevation in severe and critical COVID-19: a rapid systematic review, meta-analysis, and comparison with other inflammatory syndromes

Characterization of the Inflammatory Response to Severe COVID-19 Illness

Synergism of TNF-α and IFN-γ Triggers Inflammatory Cell Death, Tissue Damage, and Mortality in SARS-CoV-2 Infection and Cytokine Shock Syndromes

Activation of the pseudokinase MLKL unleashes the four-helix bundle domain to induce membrane localization and necroptotic cell death

Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion

Cell death serum biomarkers are early predictors for survival in severe septic patients with hepatic dysfunction

Serum Levels of Caspase-Cleaved Cytokeratin-18 and Mortality Are Associated in Severe Septic Patients: Pilot Study

Circulating Cell Death Biomarkers May Predict Survival in Human Lung Transplantation

Programmed necrosis in inflammation: Toward identification of the effector molecules

SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation

Hamster organotypic modeling of SARS-CoV-2 lung and brainstem infection

Serum levels of receptor-interacting protein kinase-3 in patients with COVID-19

DAMP-sensing receptors in sterile inflammation and inflammatory diseases

HMGB1 as a potential biomarker and therapeutic target for severe COVID-19

High Mobility Group Box 1 and Interleukin 6 at Intensive Care Unit Admission as Biomarkers in Critically Ill COVID-19 Patients

Circulating mitochondrial DNA is an early indicator of severe illness and mortality from COVID-19

Cytokine Signature Induced by SARS-CoV-2 Spike Protein in a Mouse Model

The SARS-CoV-2 spike protein subunit S1 induces COVID-19-like acute lung injury in Κ18-hACE2 transgenic mice and barrier dysfunction in human endothelial cells

Cytokeratin 18 cell death assays as biomarkers for quantification of apoptosis and necrosis in COVID-19: a prospective, observational study

Identifying clinical and biochemical phenotypes in acute respiratory distress syndrome secondary to coronavirus disease-2019

Latent Class Analysis Reveals COVID-19-related Acute Respiratory Distress Syndrome Subgroups with Differential Responses to Corticosteroids

We would like to thank Ms. Yuki Yuba (Department of Anesthesiology and Critical Care Medicine) for technical assistance. We also thank Department of Emergency Medicine (Prof. Ichiro Takeuchi), Department of Microbiology (Prof. Akihide Ryo), and Yokohama City University Center for Novel and Exploratory Clinical Trials for

It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted January 24, 2022. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted January 24, 2022. shown. Values are presented as means ± standard error. *p<0.05, **p<0.01, ***p<0.0001.. CC-BY-NC 4.0 International license It is made available under a perpetuity.is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprintThe copyright holder for this this version posted January 24, 2022. ; https://doi.org/10.1101/2022.01.23.22269723 doi: medRxiv preprint