Flower Lose, a Cell Fitness Marker, Predicts COVID-19 Prognosis (2021)
Figure 1

Figure 1. hFwe-Lose biomarker associated with COVID-19 mortality and host comorbidities.

A | A schematic of cell competition process. Our bodies have a natural surveillance system that optimizes tissue fitness. The process of cell competition drives healthy tissues to force suboptimal, yet viable, Loser cells to undergo cell death. Various stressors and insults cause cells to alter their properties and expression of fitness biomarkers. Cellular fitness comparisons lead to the elimination of Loser cells that express hFwe-Lose, a biomarker of reduced fitness. This mechanism is responsible for actively restoring tissue homeostasis and has important implications in response to infections and the development of malignancies. Abbreviations: ROS: reactive oxygen species.

B | hFwe-Lose mRNA expression is more abundant in elderly people. hFwe-Lose mRNA expression was analyzed by RT-qPCR in 86 lung tissue biopsies taken from non-COVID patients with age between 20 and 82 years. Older patients show a significant upregulation of hFwe-Lose expression. A log-Linear regression model demonstrates a positive correlation between age and hFwe-Lose expression (R2= 0.13; slope confidence interval of 95% (CI) = [2.0-12.2]; p-value of the linear regression model < 6⋅10-4).

C | hFwe-Lose expression is elevated in lung tissue biopsies from patients with comorbidities. Box plot illustrates an increased expression of hFwe-Lose in lungs of patients with hypertension (HT; n = 129), obesity (n = 45), chronic obstructive pulmonary disease (COPD n = 51), diabetes (n = 48), cardio-vascular disease (CVD; n = 63) versus disease-free control lungs (n = 42). Patient`s age is depicted in color. Two-sided Student's t-test was performed for each comorbidity (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.

D | hFwe-Lose expression is upregulated in lung tissue of COVID-19 patients. Box plot illustrates an increased expression of hFwe-Lose in lung tissue of patients diagnosed with COVID-19 (n = 11), individuals affected with host comorbidities (n = 216) versus disease-free control lungs (n = 42). Patient's age is depicted in color. Two-sided Student's t-test was performed (compared to disease-free patients), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.

E | SARS-CoV-2-infection manifests histological changes in patients' lungs as demonstrated by H&E staining. 1)For comparison, the normal lung of an elderly individual is shown, containing air-filled empty looking alveolar spaces and thin alveolar septae/gas-exchange membranes. Note that there are almost no inflammatory cells and that capillaries are only merely visible since not congested. 2) Diffuse (proliferative) alveolar damage showing evidence of cell death. An alveolar space filled with desquamated pneumocytes and macrophages, lymphocytes and focal erythrocyte extravasation as well as one multinucleated pneumocyte type II. The still recognizable epithelial lining is detached. Cells and surfactant are lost creating perforations in the alveolar wall, allowing migration of blood cells and fluid to enter inside the alveolar space. The adjacent interstitial space is significantly widened, showing an increase of mononuclear inflammatory cells and extravasation of erythrocytes. On the top and the lower right, dilated and congested capillaries can be seen (Hematoxylin and Eosin (H&E) stain, 200x). Abbreviations: a- Alveolar structure destroyed by lymphocytes, desquamated epithelium, as well as extravasated blood; The alveoli should be empty, but here it is filled with a combination of degenerated cells and fibrin; b- Next alveoli; c- Expanded pulmonary interstitium. The interstitium should be as thin as 10um, but here it is 100um; d- Extravasation of erythrocytes; e- Lymphocytes; f- Congested capillaries and arterioles; g- Multinucleated alveolocyte/pneumocyte type II; h- Detached epithelium; i- A histiocyte with a kidney-shaped nucleus at the bottom of the circle has ingested erythrocytes (left-handed), representing the first step of cellular elimination. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 3) Presence of type II pneumocyte syncytial giant cells in a collapsing alveolar space with detached epithelial lining. The adjacent interstitial space shows analogous changes to 2A1. At the bottom, there is a prominently dilated and congested capillary (H&E, 200x). Abbreviations: a- Alveoli with detached epithelium; b- Next alveoli; c- E Extravasation of erythrocytes; d- Expanded interstitium; e- Lymphocytes; f- Multinucleated pneumocyte type II. 4) Diffuse alveolar damage showing massive extravasation of fibrin (homogeneous eosinophilic material in the center of the alveolar space). The lining pneumocytes are almost all apoptotic/necrotic. The fibrin exudate is intermingled with mononuclear inflammatory cells and cellular debris (H&E, 200x). Abbreviations: a- Alveola; b- Congested capillaries; c- Fibrin; d- Remnants of degenerated / dying epithelium; e- Expanded interstitium; f- Cellular debris consisting of macrophages, detached epithelial cells and lymphocytes; g- Fully degenerated / lacking epithelial coverage within the alveoli. Round inlet with encircled karyopyknotic, karyorrhectic and "ghost-cell" figures indicative of apoptosis. 5) Immunohistochemical stain (IHC) for fibrin showing microthrombi caused by dysfunction of endothelial cells in capillaries of the alveolar membranes/lung interstitium leading to obstruction of the microcirculation (IHC for fibrin, 200x). Abbreviations: a- All small alveolar septal capillaries are filled out with worm-like fibrin thrombi hampering / obstructing circulation; b- Normal interstitium.

F | Healthy lungs show very low cleaved caspase-3 positive cells. IHC staining for cleaved caspase-3 (20x).

G | Expression of cleaved (active) caspase-3 yielding apoptotic and pre-apoptotic cells: IHC for cleaved (active) caspase-3 showing a brown nuclear staining signal in respective cells. 31.5% cells were found to be caspase positive, 11.7% with high caspase-3 positivity and another 19.8% with low. These cells are mainly located in the interstitium, but also in some alveolar epithelial and endothelial compartments. In the lower right, the edge of an alveolar space containing several (pre-)apoptotic pneumocytes is seen, while in the upper left a completely denuded/deepithelialized alveolus with two apoptotic remnants is observable (IHC for cleaved caspase-3, 20x). Abbreviations: a- Apoptotic large mononuclear cells; b-Apoptotic alveolocyte / pneumocyte; i- Amplification of apoptotic alveolocyte / pneumocyte. As shown in the round inlets of 1E2 and 1E4, homogeneous dark nuclear condensation up to a size of 2μm (karyopyknosis), homogeneous pinkish-greyish nuclear condensation, large nuclear inclusion with marginalization of the condensed chromatin, coarse nuclear angulation, nose-like/polar body-like nuclear protrusions, the latter two being karyorrhectic debris, were all morphologically considered evidence of apoptosis. This has been correlated with and was reflected by the results of the cleaved caspase-3 staining on step sections. All cells were counted on the 20X fields for n = 42 disease-free and n = 3 COVID-19 autopsy patients. The expression of hFwe-Lose in COVID-19 patients is significantly higher than in disease-free individuals, regardless of cleaved caspase-3 staining (Cas POS: p < 0.004; Cas NEG: p < 0.006). In COVID-19 patients we observed a significantly higher expression of hFwe-Lose in sections with positive cleaved caspase-3 staining (p < 0.004). Two-sided Student's t-test was performed (compared to disease-free samples and NEG samples, respectively), and p-values are presented on the plot. The central band shows the median, the box indicates the interquartile range, the whiskers extend to the most extreme points within the 1.5-fold distance of the interquartile range above and below the box.