Prevotella intermedia culture supernatant impairs alveolar macrophage function and promotes Mycobacterium avium persistence

Microbiome analyses using 16S ribosomal RNA gene sequencing have revealed a higher prevalence of Prevotella species in patients with nontuberculous mycobacterial pulmonary disease (NTM-PD) than in those with non-NTM-PD bronchiectasis. Prevotella intermedia culture supernatant (P. int. sup.) has been reported to exacerbate pneumonia, but its effect on Mycobacterium avium infection remains unclear. Thus, we manipulated bone marrow cells to differentiate into alveolar macrophage-like cells (AMLCs), infected them with M. avium in the presence of P. int. sup. or control medium, and analyzed their phagocytic and bactericidal activities. Intracellular bacteria were quantified, and antimicrobial gene expression was assessed using real-time reverse transcription PCR and bulk RNA sequencing. In vivo, female C57BL/6J mice (7–8 weeks old) were oropharyngeally inoculated with M. avium with or without P. int. sup. P. int. sup. impaired intracellular killing of M. avium in AMLCs. RNA sequencing revealed downregulation of autophagy, type I interferon, and anti-mycobacterial genes in the P. int. sup.-treated group. Furthermore, flow cytometry analysis revealed that P. int. sup. increases levels of microtubule-associated protein 1 light chain 3 and may inhibit lysosome maturation. In mice, P. int. sup. increased the pulmonary M. avium load and suppressed type I and II interferon expression in the lungs. In this experimental mouse model of NTM-PD, P. int. sup. increased pulmonary M. avium burden, while in mouse derived AMLCs P. int sup. was associated with impaired autophagy‑related and interferon responses and reduced antimicrobial gene expression, supporting a potential role for P. intermedia in NTM-PD progression.