Oral StreptococciS. anginosusandS. mitisinduce distinct morphological, inflammatory, and metabolic signatures in macrophages

Abstract Oral streptococci are the pioneer colonizer and structural architect of the complex oral biofilm. Disruption of this architectural framework causes oral dysbiosis associated with various clinical conditions, including dental caries, gingivitis, and oral cancer. Among the genus Streptococcus , S. anginosus is associated with esophageal, gastric, and pharyngeal cancer tissues, while S. mitis is correlated with oral cancer. However, no study has investigated mechanistic links between these Streptococcus species and cancer-related inflammatory responses. To explore the underlying involvement of S. anginosus and S. mitis in inflammation-associated cancer development, we investigated the innate immune response elicited by S. anginosus versus S. mitis using the RAW264.7 macrophage cell line. Compared to untreated or S. mitis infected macrophages, S. anginosus infected macrophages exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1β, NOS2, and COX2, accompanied by enhanced NF-κB activation. Mitostress analysis revealed an increased extracellular acidification rate in macrophages infected with S. anginosus compared to S. mitis. Further, macrophages infected with S. anginosus for 6h displayed upregulated aconitate decarboxylase, which catalyzes itaconate production. In contrast, no significant alterations were observed in succinate dehydrogenase that converts succinate to fumarate. At 24h, S. anginosus induced significant shifts in succinate and itaconate, emphasizing a unique macrophage metabolic profile, and an augmented inflammatory response in response to S. anginosus. This study underscores the capacity of S. anginosus to elicit a robust proinflammatory response in macrophages and opens new avenues of secretory immune metabolites in response to oral streptococci. Importance The surge in head and neck cancer cases among individuals devoid of typical risk factors such as HPV infection, tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiome’s influence on physiology, the oral microbiome, notably oral streptococci, a gatekeeper of systemic health, has been underappreciated in mucosal immunopathogenesis. S. anginosus, a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to S. anginosus compared to the early colonizer S. mitis as an initial ride towards understanding the impact of distinct oral Streptococcus species on the host immune response in the progression of OSCC.