Inactivated Rothia nasimurium promotes a persistent antiviral immune status in porcine alveolar macrophages
Globalization has increased in the incidence of infectious diseases in livestock, further aggravated by the reduction of antibiotic consumption. To minimise the resulting economic consequences to the meat production industry, as well as the higher risk of zoonotic events, immunostimulants have risen as a potential strategy to enhance animal resilience to diseases. The capability of bacterial-based immunostimulants to modulate innate immune cells functionality makes them cost-effective candidates to be used in livestock, whether as vaccine adjuvants, as antimicrobials, or as preventive immunostimulators inducing long-term innate immune memory. Here we characterized in vitro the immunostimulatory properties of a strain of Rothia nasimurium isolated from warthog fecal microbiota. Stimulation with heat-inactivated bacteria induced cytokine production by porcine immune cells, and a robust innate immune transcriptomic signature in porcine alveolar macrophages. Interestingly, the bacteria induced inflammasome activation and IL1b production, thus confirming its proinflammatory properties, and suggesting its potential as vaccine adjuvant. Importantly, this immunostimulatory status functionally resulted in an antimicrobial state, hampering the replication levels of two major porcine viral pathogens: the porcine reproductive and respiratory syndrome virus (PRRSV) and the African swine fever virus (ASFV). Moreover, alveolar macrophages showed an enhanced cytokine response upon ASFV infection several days after heat-inactivated bacteria stimulation, suggesting the induction of an innate immune memory phenotype. This nonspecific response resulted in a significant reduction of ASFV replication kinetics, demonstrating the capacity of the bacteria to induce a more resistant state in macrophages against a highly lethal virus infection. Altogether, these results demonstrate the immunostimulatory capability of heat-inactivated Rothia nasimurium in porcine macrophages, showing potential to enhance animal resilience to diseases through the modulation of innate immune cells responsiveness to infections. Overall design: Four lots of PAMs were seeded at 5x10^6 cells/well in a 24-well flat-bottom plate with RPMI supplemented with 10% FCS, 1% of P/S, 1% of L-glutamine, and 0.5% of nystatin, and left overnight at 37ºC and 5% CO2. Cells were then stimulated for 6 or 24 hours with LPS (tlrl-eblps InvivoGen) at 10 ug/ml, Pam3CKS (InvivoGen tlrl-pm2s-1) at 10 ug/ml, or heat-inactivated Rothia nasimurium (HI-Ro) at MOI 50. Non-stimulated cells were used as a reference.