Table 1.
Primer and probe sequences.
Table 2.
Experimental timeline.
Table 3.
Numbers of male (M) and female (F) mice in experimental groups.
Fig 1.
Endothelial Poldip2 is upregulated in human lung after SARS-CoV-2 infection.
Sections of lung tissue were immunostained for CD31 (green), Poldip2 (red) and nuclei (blue). Left: representative images from one control and one infected patient. Right: quantification of Poldip2 staining in CD31-positive areas. Numbers below the bars correspond to individual patients. Bars represent means ± SEM from 4-10 pictures per sample taken at random locations. Data were analyzed using a two-tailed nested Student t-test, n = 3, *P < 0.05.
Fig 2.
Acute infection does not significantly reduce body weights and temperatures.
Male and female human ACE2 transgenic, Poldip2+/+ or Poldip2+/- (hACE2/Poldip2) mice received a single intranasal inoculation of PBS or SARS-CoV-2 on day zero. Body temperatures (A, B) and weights (C, D) were measured every other day. Time courses (A, C) show relative values, calculated by dividing measurements for each mouse by its own initial value (day zero) and multiplied by 100 for weights. The graphs represent means ± SEM of data from n = 8-21 animals. Body temperatures at day 6 (B) and weight changes between day 6 and day zero (D), were analyzed by 2-way ANOVA: ns, not significant. On day zero, average body temperatures were identical between males and females, across all groups (35 ± 0.1°C) and average body weights were higher by a few grams in males (M) than females (F): M 29.4 ± 0.7, F 21.6 ± 0.4 in Poldip2 + /+ and M 25.9 ± 0.5, F 21.4 ± 0.3 in Poldip2 + /- (errors indicate SEM).
Fig 3.
Poldip2 depletion reduces viral load 7 days post-infection.
Male and female hACE2/Poldip2 mice were infected with SARS-CoV-2 by nasal inoculation. Lungs were collected 7 days later for RNA preparation. Viral loads were measured by RT-qPCR using primer pairs and TaqMan probes specific for the viral gene N (A) or subgenomic E (sgE) RNA (B). Bars represent means ± SEM of data from n = 5-7 animals. Groups were compared using two-tailed Mann-Whitney tests: **P < 0.01.
Fig 4.
Poldip2 depletion alleviates lung tissue alterations induced by SARS-CoV-2 infection.
Male and female hACE2/Poldip2 mice were euthanized 7 days after intranasal administration of PBS or SARS-CoV-2. Lungs were processed for histology and paraffin sections were stained with hematoxylin/eosin. (A) SARS-CoV-2 infection increased tissue damage and alveolar wall thickness compared to PBS in Poldip2+/+ (top), but not in Poldip2+/- mice (bottom). (B) Septal thickness was quantified as described in Methods. Bars represent means ± SEM from n = 5-6 mice. Data were analyzed by 2-way ANOVA: ns, not significant, #P = 0.075, *P < 0.05, **P < 0.01.
Fig 5.
Poldip2 depletion reduces neutrophil infiltration in lung after SARS-CoV-2 infection.
Lungs from male and female hACE2/Poldip2 mice were harvested 7 days after nasal PBS or virus instillation and processed for immunofluorescence. Tissue sections were stained to detect the leukocyte common antigen (CD45, red), the neutrophil marker myeloperoxidase (MPO, green) and nuclei (blue), as shown in representative micrographs (left). Cells positive for both CD45 and MPO were counted in 10-13 images per animal (right). Bars represent means ± SEM of data from n = 4-6 mice. Data were analyzed using 2-way ANOVA: ns, not significant; *P < 0.05, **P < 0.01.
Fig 6.
Poldip2 depletion tends to attenuate chemokine expression.
Male and female hACE2/Poldip2 mice were euthanized 7 days after intranasal administration of PBS or SARS-CoV-2. Bronchoalveloar lavage (BAL) fluid was collected before harvesting lungs for RNA extraction as in Figure 3. mRNAs were measured by RT-qPCR in lung tissue (A) and protein ELISAs were carried out in BAL (B). Bars represent means ± SEM of data from n = 4-17 animals. Basal protein levels in +/ + PBS were 40 ± 20 pg/ml (MCP1) and 111 ± 31 pg/ml (CXCL1). Data were analyzed using 2-way ANOVA: ns, not significant; ** P < 0.01; *** P < 0.001.
Fig 7.
Poldip2 depletion variably affects cytokine expression after SARS-CoV-2 infection.
Male and female hACE2/Poldip2 mice received a nasal instillation of PBS or SARS-CoV-2. BAL fluid and lungs were collected after 7 days and processed as in Figure 6 to measure the expression of cytokine mRNAs in lung tissue (A) and proteins in BAL (B). Bars represent means ± SEM of data from n = 5-19 animals. Basal protein levels in +/ + PBS were 1.7 ± 0.1 pg/ml (TNFα), 5.0 ± 0.3 pg/ml (IL-1β), 11.8 ± 0.8 pg/ml (IL-6), 4.2 ± 0.5 pg/ml (IFN-γ). Data were analyzed using 2-way ANOVA: ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.