Treatment with the senolytics dasatinib/quercetin reduces SARS-CoV-2-related mortality in mice

Abstract

The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool.

Keywords: COVID-19; SARS-CoV-2; cellular senescence; senolytics; survival.

FIGURE 1

Experimental scheme and impact of SARS‐CoV‐2 in K18‐hACE2 mice. (a) Experimental design for the animal study. Ten‐month‐old K18‐hACE2 mice (males) were divided into three groups, treated with either D/Q, SARS‐CoV‐2 or vehicle (V), and monitored as indicated in Methods. Dpi: Days post‐infection. Negative numbers indicate days pre‐infection (preventive therapy). (b) Percentage survival of mice. p Value was determined by Gehan–Breslow–Wilcoxon test (*p = 0.0398). (c) Viral RNA levels in lungs analyzed by RT–qPCR. p Values were determined by one‐way ANOVA, Fisher's LSD test (**p = 0.0015; ***p ≤ 0.0005; ****p < 0.0001). (d) Weight change (±SEM) relative to day 0 was monitored (N of each group as indicated in [a]). The dashed line indicates the start of SARS‐CoV‐2‐induced deaths, and thus changing the N for each group. p Values were determined by two‐way ANOVA (uncorrected Fisher's LSD; mixed‐effects analysis) test (*p = 0.0412; **p = 0.0054). Data are combined from two independent experiments.

FIGURE 2

Senolytics reduce the onset of SARS‐CoV‐2‐related symptoms. The analyzed symptoms (excluding weight loss) consisted of dyspnea, lethargy and staggering, eye closure, piloerection, and hunched posture. Mock‐infected mice did not exhibit any symptoms throughout the experiment. (a) The number of cumulative symptoms, exhibiting at least one of the previously described, are represented for each group. (b) The individual symptoms are considered as positive when a mouse showed it at any day during the period 1–11 dpi. p Values were determined using the chi‐square test of independence. *p ≤ 0.05; **p ≤ 0.005. Data are combined from two independent experiments.

FIGURE 3

Cytokine and chemokine levels in serum and lungs. (a) Multiplex platform was used to measure the serum levels of the indicated proteins in each experimental group (n as indicated in the graphs). Data are combined from two independent experiments. (b, c) mRNA expression of cytokines and chemokines associated with severe SARS‐CoV‐2 disease (b) or associated with Th2 cytokines and monocyte/macrophage function (c) in lungs of the indicated mouse groups. p Values were determined by one‐way ANOVA, Fisher's LSD test. *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005; ****p ≤ 0.0001. Representative data from one out of two independent experiments.

FIGURE 4

Lung injury. (a) Hematoxylin and eosin staining of representative lung sections from K18‐hACE2 mice. Left: Mouse lung section from the mock‐infected group. Small isolated lymphocyte accumulations were detected in 2 of 4 mice (not shown here; see Figure 5a). Middle: SARS‐CoV‐2‐infected mouse lung, euthanized before the scheduled end of the experiment (<11 dpi). Five of eight mice showed multifocal foci of inflammation (circles). Right: SARS‐CoV‐2‐infected mouse lung, sacrificed at the end of the experiment (11 dpi). The photo shows a 20% lung consolidation due to coalescence of numerous well‐defined foci (arrows). An average of 6–7 foci per lung were detected. Scale bars: 2000 μm. (b) Quantification of areas of dense inflammation foci (SARS‐CoV‐2‐treated mice) and lymphocyte accumulation (mock group), using ImageJ/FIJI software. Below is the numerical data of the area means per group of mice. p Values were determined by one‐way ANOVA, Fisher's LSD test. **p ≤ 0.005; ***p ≤ 0.0005. Data from one out of two independent experiments.

FIGURE 5

Immunohistochemistry (IHC) images of senescence markers in lung tissue from SARS‐CoV‐2 infected K18‐hACE2 mice. (a) Representative IHC of p21^CIP1‐positive cells in the lungs of the indicated groups. Top row: Staining in isolated areas of lymphocyte accumulation (mock group), and in dense inflammation foci (SARS‐CoV‐2‐infected mice). Bottom row: Staining in the lung tissue (excluding inflammation foci), indicated as parenchyma. Arrows indicate p21^CIP1‐positive cells in mock samples. Scale bars: 100 μm (top row) and 50 μm (bottom row). (b) Quantification of p21^CIP1‐positive cells in whole lung sections (left graphic), only in inflammation foci (middle) and parenchyma (right graphic), using ImageJ/FIJI software. In the mock‐infected group, two of four animals displayed a few minor inflammation foci, as shown in the IHC. In the SARS‐CoV‐2 group, four of seven animals showed dense inflammation foci. Bottom numbers: Numerical data of the number of cells analyzed. p Values determined by one‐way ANOVA, Fisher's LSD test. *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005. (c) Representative IHC of p19^ARF‐positive cells in dense inflammation foci in lungs of the indicated mice groups. Arrows indicate p19^ARF‐positive cell in mock sample. Scale bars: 100 μm (20 μm in the magnified square). (d) Quantification of p19^ARF‐positive cells in dense inflammation foci of lung samples from individually analyzed mice, as described in b. p value determined by one‐way ANOVA, Fisher's LSD test. *p ≤ 0.05. Data from one out of two independent experiments.

FIGURE 6

Scheme of the therapeutic challenge with senolytics and effect upon SARS‐CoV‐2 infection in K18‐hACE2 transgenic mice. (a) Experimental design for the therapeutic study. Mice (50% male/female) with a mean age of 12 months, were infected with SARS‐Cov‐2, and then treated with either D/Q or vehicle (V), and monitored as indicated in Methods. Dpi: Days post‐infection. (b) Percentage survival of mice. (c) Viral RNA levels in lungs analyzed by RT–qPCR. p Values were determined by one‐way ANOVA, Fisher's LSD test (*p = 0.0462; **p = 0.0027). (d) Weight change (±SEM) relative to day 0 was monitored (N of each group as indicated in [a]). The dashed line indicates the start of SARS‐CoV‐2‐induced deaths, and thus changing the N for each group. p Values were determined by two‐way ANOVA (uncorrected Fisher's LSD; mixed‐effects analysis) test (*p = 0.0439).