Differential activation of programmed cell death in patients with severe SARS-CoV-2 infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes severe lower airway disease and death in a subset of patients. Knowledge on the relative contribution of programmed cell death (PCD) to lung pathology is limited to few human autopsy studies with small sample size/scope, in vitro cell culture, and experimental model systems. In this study, we sought to identify, localize, and quantify activation of apoptosis, ferroptosis, pyroptosis, and necroptosis in FFPE lung tissues from patients that died from severe SARS-CoV-2 infection (n = 28) relative to uninfected controls (n = 13). Immunofluorescence (IF) staining, whole-slide imaging, and Image J software was used to localize and quantify expression of SARS-CoV-2 nucleoprotein and the following PCD protein markers: cleaved Caspase-3, pMLKL, cleaved Gasdermin D, and CD71, respectively. IF showed differential activation of each PCD pathway in infected lungs and dichotomous staining for SARS-CoV-2 nucleoprotein enabling distinction between high (n = 9) vs low viral burden (n = 19). No differences were observed in apoptosis and ferroptosis in SARS-CoV-2 infected lungs relative to uninfected controls. However, both pyroptosis and necroptosis were significantly increased in SARS-CoV-2-infected lungs. Increased pyroptosis was observed in SARS-CoV-2 infected lungs, irrespective of viral burden, suggesting an inflammation-driven mechanism. In contrast, necroptosis exhibited a very strong positive correlation with viral burden (R² = 0.9925), suggesting a direct SARS-CoV-2 mediated effect. These data indicate a possible novel mechanism for viral-mediated necroptosis and a potential role for both lytic programmed cell death pathways, necroptosis and pyroptosis, in mediating infection outcome.

Fig. 1. Patients who died from severe SARS-CoV-2 infection exhibit significant lung pathology with variable viral burden.

A Representative H&E image (100×) from a patient that died from severe SARS-CoV-2 infection. Diffuse alveolar damage is noted throughout. The white arrows indicate type II pneumocyte hyperplasia, yellow arrows indicate loose interstitial fibrosis, blue arrows indicate vascular congestion, black stars highlight intra-alveolar fibrinous exudates, and yellow stars indicate hemorrhage. B Representative fluorescent images show variable staining with antibodies targeting SARS-CoV-2 nucleoprotein (green) in SARS-CoV-2-positive lungs and no signal in control lung tissues (DAPI counterstain). C. Image J was used to quantify raw integrated intensity in control (open circles) versus SARS-CoV-2+ (enclosed circles) lung tissues. The dashed box highlights a dichotomous population of patients with severe COVID, exhibiting either high or low SARS-CoV-2 burden in the lungs at the time of death. T tests were used to determine statistical significance set at a p value ≤ 0.05. Scale bars shown in (A) and (B) indicate 50 μm.

Fig. 2. Apoptosis and ferroptosis are detected but not significantly upregulated in the lungs of patients who died from severe SARS-CoV-2 infection relative to control lung tissue.

A Representative fluorescent images show comparable cleaved caspase 3 (apoptosis marker) and CD71 (ferroptosis marker) in the lungs of patients that died from severe SARS-CoV-2 infection relative to control lung tissues. Scale bars shown indicate 100 μm. B Image J was used to quantify raw integrated intensity for cleaved caspase 3 in control (open circles) versus SARS-CoV-2+ (enclosed circles) lung tissues. C Pearson correlation and linear regression showed no correlation between cleaved caspase 3 and SARS-CoV-2 burden. Similarly, (D) Image J was used to quantify CD71 raw integrated intensity and (E) linear regression showed no correlation between CD71 and viral burden. T tests or one-way ANOVA with Tukey’s post-test were used to determine statistical significance set at a p value ≤ 0.05.

Fig. 3. Pyroptosis is significantly upregulated in the lungs during severe SARS-CoV-2 infection but does not correlate with viral burden.

A Representative fluorescent images show increased cleaved gasdermin D (pyroptosis marker) in the lungs of patients that died from severe SARS-CoV-2 infection relative to control lung tissues. Scale bars shown indicate 50 μm. B Quantification of raw integrated intensity for cleaved gasdermin D shows significantly increased signal in SARS-CoV-2 infected lungs relative to control lung tissues. C Pearson correlation and linear regression show no correlation between cleaved gasdermin D and SARS-CoV-2 burden. T tests or one-way ANOVA with Tukey’s post-test analyses were used to determine statistical significance set at a p value ≤ 0.05.

Fig. 4. Necroptosis is significantly upregulated in the lungs during severe SARS-CoV-2 infection and exhibits a strong positive correlation with viral burden.

A Representative fluorescent images show phosphorylated MLKL (necroptosis marker) in the lungs of patients who died from severe SARS-CoV-2 infection with minimal detection in control lung tissues. Additionally, increased signal was observed in SARS-CoV-2+ lung tissues with high viral burden relative to low viral burden. Scale bars shown indicate 50 μm. B Quantification of raw integrated intensity for pMLKL shows significantly increased signal in SARS-CoV-2-infected lungs with high viral burden relative to samples with low viral burden and control lung tissues. C Pearson correlation and linear regression show a strong positive correlation between pMLKL and SARS-CoV-2 burden. D Image J was used to quantify % co-localization of pMLKL with SARS-CoV-2 nucleoprotein. T-tests or one-way ANOVA with Tukey’s post-test were used to determine statistical significance set at a p value ≤ 0.05.