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.
Highlights
Giardia duodenalis, known as Giardia lamblia and Giardia intestinalis, is an opportunist protozoan parasite that predominantly parasitizes the duodenum of humans, as well as of numerous domestic and wild animals, causing giardiasis
We further found that TLR9 was significantly activated in mouse macrophages with G. duodenalis stimulation, an effect that involved proinflammatory cytokine production mediated by the TLR9–p38/ERK signaling pathways
The results showed that PMφs incubated with G. duodenalis exhibited significantly enhanced transcription levels of TLR9 within 12 h compared to the control PMφs, peaking at 8 h and decreasing (Figure 1A)
Summary
Known as Giardia lamblia and Giardia intestinalis, is an opportunist protozoan parasite that predominantly parasitizes the duodenum of humans, as well as of numerous domestic and wild animals, causing giardiasis. Giardiasis has been included in the World Health Organization’s neglected disease initiative since 2004 (Savioli et al, 2006), owing to its severe impact on children, which includes severe malnutrition, physical retardation, and poor cognitive function (Berkman et al, 2002). Giardiasis has been reported to the Centers for Disease Control and Prevention (CDC) of the United States since 1992 and became a nationally notifiable disease in 2002 (Coffey et al, 2021). According to the CDC, because of poverty, poor drinking water quality, and limited treatment options, nearly 33% of the population in developing countries is afflicted with giardiasis, as is 2% of the adult population in the developed world (Kunz et al, 2017). Giardia infection has clearly become a non-negligible problem, attracting widespread research attention from an increasing number of scientists
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