The RIPK3 Scaffold Regulates Lung Inflammation During Pseudomonas Aeruginosa Pneumonia

Area of Science:

• Immunology
• Molecular Biology
• Pathology

Background:

• Receptor-interacting protein kinase 3 (RIPK3) has dual roles: kinase activity in necroptosis and scaffold function in cytokine production.
• The specific role of RIPK3's kinase versus scaffold function in Pseudomonas aeruginosa pneumonia pathogenesis is unclear.

Purpose of the Study:

• To investigate whether RIPK3's kinase or scaffold domains mediate pathology during Pseudomonas aeruginosa infection.
• To explore RIPK3's role in Pseudomonas-induced inflammation and cell death in vitro and in vivo.

Main Methods:

• Mice with RIPK3 gene deletions or mutations were infected with Pseudomonas aeruginosa to assess survival and inflammation.
• Murine immune cells and human airway epithelial cells were studied in vitro for Pseudomonas-induced cytokine production and cell death.
• RIPK3 binding interactions were blocked using the viral inhibitor M45, and RIPK3 was inhibited using siRNA.

Main Results:

• Deletion of RIPK3 reduced inflammation and mortality in a mouse model of Pseudomonas pneumonia.
• RIPK3 kinase inactivation did not affect mortality or inflammation, indicating the kinase domain is not essential.
• RIPK3's scaffold domain, specifically its RIP homotypic interaction motif (RHIM), was required for driving cytokine production and inflammation, independent of kinase activity.
• Blocking RIPK3 RHIM interactions with M45 reduced in vitro inflammation, and siRNA knockdown decreased inflammation in human cells.

Conclusions:

• The RIPK3 scaffold domain, not its kinase activity, drives detrimental pulmonary inflammation and mortality in Pseudomonas aeruginosa pneumonia.
• This inflammatory response relies on the RIPK3 RHIM, distinct from kinase-mediated necroptosis.
• Inhibiting RIPK3 RHIM signaling presents a potential therapeutic strategy to mitigate lung inflammation during bacterial infections.