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.

Keywords: ERK; Giardia duodenalis; TLR9; cytokines; p38.

FIGURE 1

Giardia duodenalis trophozoites induced cytokine production in a TLR9-dependent manner. (A) RT-qPCR analysis of the relative fold change in the levels of TLR9 mRNA extracted from 3 × 10⁶ mouse peritoneal macrophages stimulated with 1 × 10⁶ Giardia lamblia virus (GLV)–containing Giardia trophozoites for various periods (0, 2, 4, 6, 8, and 12 h). (B) RT-qPCR analysis of the relative fold change in the levels of TLR9 mRNA extracted from 3 × 10⁶ mouse peritoneal macrophages stimulated with gDNA (3 μg, extracted from 1 × 10⁶ GLV-free Giardia trophozoites), 1 × 10⁶ GLV-free Giardia trophozoites, 1 × 10⁶ GLV-containing Giardia trophozoites, or CpG ODN 1668 (5 μM/mL). The TLR9 mRNA level was normalized to that of β-actin. (C) The secretion levels of IL-6, TNF-α, and IL-12 p40 in cell culture supernatants were measured by ELISA. Data are expressed as means ± SD from three separate experiments. ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Giardia duodenalis trophozoites activated the p38 and ERK/MAPK signaling pathways via TLR9. (A) A total of 3 × 10⁶ wild-type (WT) mouse peritoneal macrophages were stimulated with 1 × 10⁶ G. duodenalis trophozoites for various periods (0–6 h) following which the phosphorylation levels of p38 and ERK were analyzed by Western blot. (B) The secretion levels of IL-6, TNF-α, and IL-12 p40 in cell culture supernatants were measured by ELISA. (C) Macrophages treated or not with small-interfering RNA (siRNA) targeting TLR9 (siTLR9) were stimulated with Giardia lamblia virus (GLV)–free or GLV-containing Giardia trophozoites for 3 h. (D) A total of 3 × 10⁶ WT macrophages pretreated or not with siTLR9 were incubated for 3 h with 1 × 10⁶ GLV-free Giardia trophozoites or 1 × 10⁶ GLV-containing Giardia trophozoites, following which TLR9 expression levels were analyzed by Western blot. Relative protein expression was quantified by densitometric analysis using β-actin as an internal reference. Data are expressed as means ± SD from three separate experiments. ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Giardia duodenalis trophozoites–induced cytokine production was disrupted by p38 and ERK inhibitor treatment. (A) A total of 3 × 10⁶ wild-type (WT) mouse peritoneal macrophages were pretreated for 30 min with the p38 inhibitor SB203580 (30 μM) or the ERK inhibitor PD98059 (40 μM) before stimulation with 1 × 10⁶ Giardia lamblia virus (GLV)–free Giardia trophozoites or 1 × 10⁶ GLV-containing Giardia trophozoites. The phosphorylation levels of p38 and ERK were subsequently analyzed by Western blot. Relative protein expression was quantified by densitometric analysis using β-actin as an internal reference. (B) The production of IL-6, TNF-α, and IL-12 p40 in cell supernatants was measured by ELISA. Data are expressed as means ± SD from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Giardia duodenalis trophozoites induced the phosphorylation of AKT via TLR9. (A) A total of 3 × 10⁶ wild-type (WT) mouse peritoneal macrophages were stimulated with 1 × 10⁶ G. duodenalis trophozoites for various periods (0–6 h) after which the phosphorylation level of AKT was analyzed by Western blot. (B) A total of 3 × 10⁶ macrophages treated or not with siRNA targeting TLR9 were stimulated with 1 × 10⁶ Giardia lamblia virus (GLV)–free Giardia trophozoites or 1 × 10⁶ GLV-containing Giardia trophozoites for 3 h. Data are expressed as means ± SD from three separate experiments. ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Giardia duodenalis trophozoites suppressed cytokine production by activating the AKT signaling pathway. (A) A total of 3 × 10⁶ wild-type (WT) mouse peritoneal macrophages were pretreated for 30 min with the AKT inhibitor MK-2206 2HCl (5 μM) before stimulation with Giardia lamblia virus (GLV)–free or GLV-containing Giardia trophozoites. The AKT phosphorylation level was subsequently analyzed by Western blot. Relative protein expression was quantified by densitometric analysis using β-actin as an internal reference. (B) The secretion levels of IL-6, TNF-α, and IL-12 p40 in cell supernatants were measured by ELISA. Data are expressed as means ± SD from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Giardia duodenalis trophozoites activated the NF-κB signaling pathway by inducing NF-κB p65 nuclear translocation. (A) Laser confocal microscopic images showing the facilitation of the nuclear translocation of NF-κB p65 by G. duodenalis in wild-type (WT) mouse macrophages treated or not with small-interfering RNA (siRNA) targeting TLR9 (siTLR9). (B) A total of 3 × 10⁶ WT macrophages were stimulated with 1 × 10⁶ G. duodenalis trophozoites for various periods (0–120 min) after which the phosphorylation levels of NF-κB p65 and IκBα were analyzed by Western blot. (C) A total of 3 × 10⁶ WT macrophages treated or not with siTLR9 were stimulated with 1 × 10⁶ Giardia lamblia virus (GLV)–free Giardia trophozoites or 1 × 10⁶ GLV-containing Giardia trophozoites for 60 min. Relative protein expression was quantified by densitometric analysis using β-actin as an internal reference. Data are expressed as means ± SD from three separate experiments. ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001.