Co-infection with Pseudomonas aeruginosa and Leishmania donovani affects the antiparasitic immunity of RAW264.7 macrophages via the NOD-like receptor pathway in vitro.

Leishmania infections often involve co-infection. Pseudomonas aeruginosa, a common commensal in the sand fly vector of leishmaniasis, can be co-transmitted alongside Leishmania during blood feeding. However, its role in modulating Leishmania infection remains unclear. This study used an in vitro co-infection model and transcriptome sequencing to investigate the interaction between L. donovani and P. aeruginosa co-infection in murine RAW264.7 macrophages. Co-infection enhances L. donovani invasion of macrophage and simultaneously promotes both pro- and anti-inflammatory responses, as evidenced by significant upregulation of cytokines (IL-4, IL-10, IL-1β and TNF-α), macrophage markers (CD80, CD86, Arg1 and CD206) and MHC class I and II molecules (H2-M3, H2-T24, H2-AB1 and H2-EB1). Transcriptome analysis revealed enrichment of differentially expressed genes in immune-related pathways, particularly the NOD-like receptor pathway. To further explore mechanisms by which co-infection affects anti-Leishmania immunity, hub genes and proteins in the NOD-like receptor pathway were validated via qPCR and Western blot. qPCR results indicated significant upregulation of Nod2 and Ccl2 in co-infected versus Leishmania-infection macrophages at 12 h, with c-Jun and JunB showing increasing trends. Western blot confirmed significant upregulation of p-p38, p-ERK, p-JNK, and RIPK2 proteins in the NOD-like receptor signaling pathway at 12 h. However, the co-infection of heat-killed P. aeruginosa or P. aeruginosa culture supernatant with Leishmania parasites did not significantly affect MHC molecules and the NOD-like receptor pathway in macrophages. These findings highlight the complex immunoregulatory mechanisms underlying co-infection and suggest potential therapeutic targets for leishmaniasis treatment.