Giardia duodenalis Induces Proinflammatory Cytokine Production in Mouse Macrophages via TLR9-Mediated p38 and ERK Signaling Pathways

Abstract

Giardia duodenalis, also known as Giardia lamblia or Giardia intestinalis, is an important opportunistic, pathogenic, zoonotic, protozoan parasite that infects the small intestines of humans and animals, causing giardiasis. Several studies have demonstrated that innate immunity-associated Toll-like receptors (TLRs) are critical for the elimination of G. duodenalis; however, whether TLR9 has a role in innate immune responses against Giardia infection remains unknown. In the present study, various methods, including reverse transcriptase–quantitative polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, immunofluorescence, inhibitor assays, and small-interfering RNA interference, were utilized to probe the role of TLR9 in mouse macrophage-mediated defenses against G. lamblia virus (GLV)–free or GLV-containing Giardia trophozoites. The results revealed that in G. duodenalis–stimulated mouse macrophages, the secretion of proinflammatory cytokines, including interleukin 6 (IL-6), tumor necrosis factor α (TNF-α), and IL-12 p40, was enhanced, concomitant with the significant activation of TLR9, whereas silencing TLR9 attenuated the host inflammatory response. Notably, the presence of GLV exacerbated the secretion of host proinflammatory cytokines. Moreover, G. duodenalis stimulation activated multiple signaling pathways, including the nuclear factor κB p65 (NF-κB p65), p38, ERK, and AKT pathways, the latter three in a TLR9-dependent manner. Additionally, inhibiting the p38 or ERK pathway downregulated the G. duodenalis–induced inflammatory response, whereas AKT inhibition aggravated this process. Taken together, these results indicated that G. duodenalis may induce the secretion of proinflammatory cytokines by activating the p38 and ERK signaling pathways in a TLR9-dependent manner in mouse macrophages. Our in vitro findings on the mechanism underlying the TLR9-mediated host inflammatory response may help establish the foundation for an in-depth investigation of the role of TLR9 in the pathogenicity of G. duodenalis.

Giardia lamblia regulates the production of proinflammatory cytokines through activating the NOD2–Rip2–ROS signaling pathway in mouse macrophages

“…38 The production of TNF-a, IL-6 and IL-12p40 induced by G. lamblia was significantly influenced by ERK and p38 signals via TLR2 or TLR9. 12,15 Our data indicated that the NOD2-Rip2 signaling pathway regulated the production of these cytokines via the NF-jB and ERK signaling pathways, suggesting ERK signaling may play a key role in regulating cytokine secretion in PMs challenged with G. lamblia.…”

“…20 NOD2-Rip2 signaling pathway regulated the NF-jB, p38, c-Jun N-terminal kinase and ERK signals during porcine reproductive and respiratory syndrome virus infection, 39 suggesting that signaling pathways controlled by NOD2 vary in different pathogen infections. In addition, both TLR9 15 and NOD2 regulated the activation of ERK signaling in macrophages challenged with G. lamblia. However, TLR9 regulated the activation of the p38 signaling pathway, but not the NF-jB p65 pathway, 15 while NOD2 induced the activation of the NF-jB p65 pathway, but not the p38 signaling pathway.…”

“…In addition, both TLR9 15 and NOD2 regulated the activation of ERK signaling in macrophages challenged with G. lamblia. However, TLR9 regulated the activation of the p38 signaling pathway, but not the NF-jB p65 pathway, 15 while NOD2 induced the activation of the NF-jB p65 pathway, but not the p38 signaling pathway. During ischemia/reperfusion injury, TLR9 regulated the NF-jB p65 and ERK signaling pathways, while NOD2 controlled the NF-jB p65 signaling pathway, but not the ERK pathway, in the liver, 40,41 suggesting that the signaling pathways controlled by NOD2 or TLR9 were different for the same injury or stimulation.…”

“…12 G. lamblia triggered the TLR9-p38/ERK signaling pathway to induce proinflammatory cytokine production in mouse macrophages. 15 Besides, NLR family pyrin domaincontaining 3 inflammasome signal was involved in G. lamblia infection. 16 Nucleotide oligomerization domain (NOD) 1 and NOD2 are NLRs that are capable of initiating innate immune responses as well as appropriate adaptive immune responses during pathogen infections.…”

“…G . lamblia triggered the TLR9–p38/ERK signaling pathway to induce proinflammatory cytokine production in mouse macrophages 15 . Besides, NLR family pyrin domain‐containing 3 inflammasome signal was involved in G. lamblia infection 16 …”