Macrophage migration inhibitory factor of zoonotic Giardia duodenalis triggers TLR4-MAPK/AKT/NLRP3 pathways to regulate inflammatory cytokines production in mouse macrophages
Description
Giardia duodenalis is an important zoonotic protozoan that has a significant negative impact on public health worldwide. G. duodenalis activates Toll-like receptors (TLRs) and NOD-like receptors (NLRs)-dependent inflammation, but which parasite component activates these immune pathways remain to be further explored. G. duodenalis macrophage migration inhibitory factor (GdMIF) has close similarity with mammalian MIF. However, the immunomodulatory mechanisms of GdMIF remain poorly understood. Here, we found that G. duodenalis could secrete the GdMIF protein, and demonstrated that recombinant GdMIF (rGdMIF) acts as a potent immunostimulatory molecule in mouse peritoneal macrophages (PMϕs). rGdMIF stimulation significantly upregulated TLR2 and TLR4 expression and triggered robust secretion of cytokines (IL-6, IL-10, IL-12, TNF-α, and IL-1β). Mechanistically, rGdMIF activated the MAPK (p38, ERK), AKT, and NF-κB signaling pathways. TLR4 deficiency (TLR4-/-) markedly attenuated rGdMIF-induced IL-6, IL-10, IL-12, TNF-α and IL-1β secretion, and significantly reduced phosphorylation of p38, ERK, and AKT, whereas TLR2-/- had no effect. Subsequently, pretreatment of PMϕs with specific inhibitors of p38, ERK further suppressed, while AKT inhibition slightly enhanced rGdMIF-induced cytokines secretion. Besides, rGdMIF also potently activated the NLRP3 inflammasome, evidenced by increased NLRP3 expression, Caspase-1 cleavage (p20), mature IL-1β (p17) production, IL-1β secretion, LDH release, and cytoplasmic NLRP3 aggregation. The NLRP3 activation was abolished in NLRP3-/- and TLR4-/- PMϕs, but still observed in TLR2-/- PMϕs. These findings demonstrated that GdMIF activated TLR4-dependent MAPK and AKT signaling to regulate IL-6, IL-10, IL-12, TNF-α and IL-1β secretion, and drived TLR4-mediated NLRP3 inflammasome activation for IL-1β maturation and release. GdMIF is a novel G. duodenalis-derived pathogen-associated molecular pattern (PAMP) recognized by TLR4. This study provides new insights into the molecular mechanisms of G. duodenalis-host innate immune interactions.