Pseudorabies virus induces natural killer cell depletion by GSDMD-mediated inflammation and pyroptosis to promote infection and lung injury

Pseudorabies virus (PRV) infection induces a hyperinflammatory response to promote inflammatory injury in multiple tissues. However, the relative mechanism remains elusive. Herein, we first confirmed that PRV infection could trigger pyroptosis to cause lung injury, evidenced by the increased release of pro-inflammatory cytokines and lactate dehydrogenase (LDH) in lung tissue of infected piglets and mice. Subsequently, we observed that PRV-induced lung lesions and mortality were improved in <i>Gsdmd^-/-</i> mice rather than in <i>Ripk3^-/-</i> mice. The number of NK cells was markedly higher in <i>Gsdmd^-/-</i> mice than in WT mice. Based on this finding, refilling NK cells could significantly alleviate the replication of PRV in the lungs of mice and alleviate lung injury caused by PRV infection, whereas depletion of NK cells aggravated PRV infection and led to severe lung lesions. Notably, gasdermin D (GSDMD) deficiency could reduce the production of tumor necrosis factor-α (TNF-α) positive macrophages and the depletion of NK cells induced by PRV infection to alleviate the pathogenicity of PRV, while TNF-α neutralizing antibody could also reduce the PRV-induced NK cell depletion to alleviate the pathogenicity of PRV. Ultimately, we found that necrosulfonamide (NSA) showed a good protective effect on PRV-infected mice and had good anti-PRV activity <i>in vivo</i>. Collectively, our findings illuminate a new regulatory mechanism by which PRV infection induces NK cell depletion to weaken the antiviral ability of the host to promote virus replication and activate GSDMD-mediated pyroptosis in lung tissue, leading to severer lung injury, and NSA is expected to be a candidate agent for the prevention and control of PRV infection.IMPORTANCENecroptosis and pyroptosis are the most common regulated necrotic cell death pathways in various pathogenic infection-induced tissue damage. Herein, we found that gasdermin D (GSDMD)-mediated pyroptosis is a critical cell death pathway in pseudorabies virus (PRV)-induced lung inflammatory injury rather than receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis. PRV infection induces NK cell depletion to weaken the antiviral ability of the host to promote viral replication. GSDMD deficiency can reduce the depletion of NK cells induced by PRV infection by reducing the production of tumor necrosis factor-α (TNF-α)-positive macrophages, thereby attenuating lung tissue lesions in PRV-infected mice. Whereas the use of necrosulfonamide (NSA) showed a good protective effect on PRV-infected mice. This study reveals that PRV infection can excessively activate macrophages to secrete more TNF-α to induce NK cell depletion to facilitate PRV infection and aggravate viral pathogenicity, and clarifies the roles of GSDMD in modulating macrophage activity and NK cell death, and also provides an effective inhibitor for use against PRV infection.