Nocardia, a rare but life-threatening pathogen, can invade multiple tissues and organs, such as lungs, brain, skin and soft tissue. In this study, we determined whether nitric oxide (NO) contributes to the severity of experimental pulmonary nocardiosis. BALB/c mice with or without aminoguanidine (AG) treatment were infected with N. farcinica through intranasal or intraperitoneal routes. Over experimental period, weight and mortality were monitored, and lung tissues were collected for NO production, cytokines detection, histopathological analysis, and bacterial load assessment. Next, alveolar MH-S macrophages were treated with various inhibitors to explore the impacts of NO, MAPK, and NF-κB against N. farcinica infection. AG treatment improved weight loss, lowered pulmonary bacterial load, and attenuated inflammatory response in infected mice. Similar effects were observed in alveolar MH-S macrophages. And all AG-treated mice survived infection. Furthermore, we suggest that NO is induced by N. farcinica through MAPK JNK and NF-κB signaling. Our study demonstrates the causative role of inducible NO on the severity of N. farcinica infection.

Nocardia, an easily missed but potentially fatal opportunistic pathogen, can lead to serious infections like lung and brain abscesses. Intranasal inoculation (IN) is the traditional approach for constructing a Nocardia-induced pneumonia mice model, while it usually only results in limited local bacterial infection in the lungs. To comprehensively assess infection dynamics across distinct pulmonary inoculation routes in mice models, this study compared the pathogenicity of three different Nocardia farcinica pneumonia models established via IN, intratracheal aerosolization (ITA), and intratracheal instillation (ITI). C57BL/6J mice were infected with N. farcinica through IN, ITA and ITI with comparative analyses of bacterial distribution in lungs, survival rate, weight, bacterial load, inflammatory cytokines, histopathological characteristics and transcriptome differences. The findings suggest that ITA N. farcinica infections caused severer clinical symptoms, higher mortality, pulmonary bacterial load, levels of inflammatory cytokines in bronchoalveolar lavage fluid, and more significant histopathological damage to lungs than IN and ITI. Furthermore, ITA resulted in better lung bacterial distribution and delivery efficiency than ITI and IN. Transcriptome analysis of lungs from N. farcinica infected mice via IN, ITA and ITI revealed significant differential gene expression, whereas ITA route resulted in a larger fold change. ITA provides a more consistent and severe model of N. farcinica pneumonia in mice than IN and ITI, which can make the bacteria more evenly distributed in the lungs, leading to more severe pathological damage and higher mortality rates. In conclusion, ITA is an optimal route for developing animal models of N. farcinica pneumonia infections.