Inhibition of NF-κB-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival

Abstract

Severe acute respiratory syndrome coronavirus (SARS-CoV) is the etiological agent of a respiratory disease that has a 10% mortality rate. We previously showed that SARS-CoV lacking the E gene (SARS-CoV-ΔE) is attenuated in several animal model systems. Here, we show that absence of the E protein resulted in reduced expression of proinflammatory cytokines, decreased numbers of neutrophils in lung infiltrates, diminished lung pathology, and increased mouse survival, suggesting that lung inflammation contributed to SARS-CoV virulence. Further, infection with SARS-CoV-ΔE resulted in decreased activation of NF-κB compared to levels for the wild-type virus. Most important, treatment with drugs that inhibited NF-κB activation led to a reduction in inflammation and lung pathology in both SARS-CoV-infected cultured cells and mice and significantly increased mouse survival after SARS-CoV infection. These data indicated that activation of the NF-κB signaling pathway represents a major contribution to the inflammation induced after SARS-CoV infection and that NF-κB inhibitors are promising antivirals in infections caused by SARS-CoV and potentially other pathogenic human coronaviruses.

Figures

FIG 1

Lung pathology associated with rSARS-CoV-MA15-ΔE infection in BALB/c mice. Eight-week-old BALB/c mice were infected with 6,000 PFU of rSARS-CoV-MA15-ΔE (ΔE) or wild-type (wt) rSARS-CoV-MA15. After 4 (A) and 6 (B) dpi, mice were sacrificed, and lungs were processed for histopathological examination. Original magnifications were ×4 and ×40 in upper and lower images, respectively, of each panel.

FIG 2

Analysis of lung inflammatory cell infiltrates during rSARS-CoV-MA15-ΔE infection of BALB/c mice. Leukocytes from lungs of 8-week-old BALB/c mice infected with 6,000 PFU of rSARS-CoV-MA15-ΔE or rSARS-CoV-MA15 were isolated at 2, 4, and 6 dpi, and the inflammatory cell populations were identified by flow cytometry as described in Materials and Methods. The total numbers of leukocytes, macrophages and neutrophils and the percentage of macrophages and neutrophils were determined. Error bars represent the means from six mice per group.

FIG 3

Effect of SARS-CoV E protein on clinical disease and proinflammatory response in vivo. Sixteen-week-old BALB/c mice were infected with 100,000 PFU of rSARS-CoV-MA15-ΔE or rSARS-CoV-MA15. (A) Weight loss and survival were evaluated daily in mice infected with rSARS-CoV-MA15-ΔE (ΔE) or rSARS-CoV-MA15 (wt) (10 mice per group). (B) Lung RNAs were extracted at 2 and 4 dpi, and the expression levels of cellular mRNAs corresponding to proinflammatory cytokines (CCL2, CCL5, TNF, CXCL1, CXCL2, CXCL10, and IL-6) and 18S rRNA were measured by RT-qPCR. Numbers indicate the levels of gene expression in rSARS-CoV-MA15-ΔE- or rSARS-CoV-MA15-infected mice compared to mock-infected mice. Error bars represent the means of three mice analyzed for each point.

FIG 4

Expression of proinflammatory cytokines in rSARS-CoV-MA15-ΔE-infected mice. Sixteen-week-old BALB/c mice were infected with 100,000 PFU of rSARS-CoV-MA15-ΔE (ΔE) or rSARS-CoV-MA15 (wt) or were mock infected (mock). Lung proteins were extracted at 2 and 4 dpi, and the accumulation of CCL2 and CXCL10 was measured as described in Materials and Methods. Concentrations of protein are expressed as picograms per milliliter of lung tissue extract. Error bars represent the means of three mice analyzed for each point.

FIG 5

Expression kinetics of proinflammatory cytokines in rSARS-CoV-MA15-ΔE-infected cells. DBT-mACE2 cells were infected at MOIs of 0.5 and 0.05 with rSARS-CoV-MA15-ΔE (ΔE) and rSARS-CoV-MA15 (wt). (A) Cellular RNAs were extracted at 24, 48, and 72 hpi. The expression of the indicated cytokines was determined by RT-qPCR. In each case, the corresponding mRNA expression levels in rSARS-CoV-MA15-ΔE- or rSARS-CoV-MA15-infected cells were plotted as fold change relative to expression levels in uninfected cells. (B) Virus titers in supernatants of rSARS-CoV-MA15-ΔE-infected and rSARS-CoV-MA15-infected DBT-mACE2 cells. Error bars represent standard deviations of the means from three experiments.

FIG 6

Activation of proinflammatory pathways in rSARS-CoV-MA15-ΔE-infected cells. (A) DBT-mACE2 cells were transfected with plasmids expressing firefly and Renilla luciferase, as described in Materials and Methods. Cells were mock infected (mock) or infected with rSARS-CoV-MA15-ΔE (ΔE) or rSARS-CoV-MA15 (wt) at an MOI of 0.05 at 24 h posttransfection. Renilla and firefly luciferase expression levels were quantified 48 h later, and the expression of firefly luciferase was normalized to that of Renilla luciferase. The level of firefly luciferase expression in cells transfected with a plasmid expressing the firefly luciferase under the control of a basal, noninducible promoter (Pr−) was also determined. (B) Cells were infected with rSARS-CoV-MA15-ΔE or rSARS-CoV-MA15 at an MOI of 0.05. At 48 hpi, nuclear extracts were prepared, and the presence of NF-κB was quantified by ELISA as described in Materials and Methods. Error bars represent the means of three independent experiments.

FIG 7

Activation of NF-κB in rSARS-CoV-MA15-ΔE-infected mice. BALB/c mice were infected with 100,000 PFU of rSARS-CoV-MA15-ΔE or rSARS-CoV-MA15. Nuclear lung proteins were extracted at 2 dpi. (A) The accumulation of NF-κB and histone H3 in the nucleus of three infected mice per condition was evaluated by Western blotting. (B) NF-κB and histone H3 amounts were quantified by densitometric analysis. The graph shows the NF-κB/histone H3 ratio in mock-, rSARS-CoV-MA15-ΔE (ΔE)-, and rSARS-CoV-MA15 (wt)-infected mice at 2 dpi. Error bars represent the means of three mice analyzed for each time point.

FIG 8

Effect of NF-κB on proinflammatory cytokine induction after rSARS-CoV-MA15 infection. DBT-mACE2 cells were infected at an MOI of 0.05 with rSARS-CoV-MA15 for 48 h. Mock-infected (mock) or rSARS-CoV-MA15-infected (wt) cells were treated at 43 hpi with the NF-κB inhibitors CAPE, resveratrol, Bay 11–7082, and parthenolide or left untreated (white bars). (A) NF-κB activation was analyzed in cells transfected with luciferase plasmids as described in the legend of Fig. 6. (B) Virus titers in cell culture supernatants were determined by plaque assay in Vero E6 cells. (C) Expression of TNF, CCL2, and CXCL2 and that of 18S rRNA as a control was evaluated by RT-qPCR. The expression levels of the different genes were normalized to the expression of nontreated, mock-infected cells. Error bars represent the means of three independent experiments.

FIG 9

Effect of NF-κB inhibitors in rSARS-CoV-MA15-infected mice. Sixteen-week-old mice were intranasally infected with 100,000 PFU of rSARS-CoV-MA15 or mock infected. At 4 hpi and every 24 h from days 1 to 4, mock-infected and wild-type (wt)-infected mice were intraperitoneally injected with CAPE (wt, +C), parthenolide (wt, +P), both CAPE and parthenolide (wt, +C +P), or vehicle (wt, −). Animals were monitored daily for mortality. Data are representative of three independent experiments, comprising 6 mice per experiment and group (data for treated mock-infected mice are not shown). Differences in survival between nontreated mice and mice treated with CAPE, parthenolide, and CAPE plus parthenolide were statistically significant (P < 0.02).

FIG 10

Effect of NF-κB inhibitors on proinflammatory cytokine induction after rSARS-CoV-MA15 infection in vivo. Mice were infected and treated as described in the legend of Fig. 9. (A) Four days after infection, total RNA from lungs was extracted, and the expression of TNF, CCL2, CXCL2, and 18S rRNA was quantified. The expression levels of the different genes were normalized to levels in nontreated, mock-infected mice. Error bars represent the means of three independent mice per group. (B) Four days after infection, virus titers in lung homogenates were determined. Error bars represent the means of three independent mice per group. Black bars, untreated.

FIG 11

Lung pathology induced after rSARS-CoV-MA15 infection in the presence of NF-κB inhibitors. Mice were mock infected or infected and treated as indicated in the legend of Fig. 9. Four days after infection, lung sections from mock-infected mice (A) and from rSARS-CoV-MA15-infected mice, which were left untreated (B) or treated with CAPE (C), parthenolide (D), or both (E), were evaluated. Three independent mice per group were analyzed. Representative images are shown.