TLR2-/- Mice Display Decreased Severity of Giardiasis via Enhanced Proinflammatory Cytokines Production Dependent on AKT Signal Pathway

Abstract

Giardia infection is one of the most common causes of waterborne diarrheal disease in a wide array of mammalian hosts, including humans globally. Although numerous studies have indicated that adaptive immune responses are important for Giardia defense, however, whether the host innate immune system such as TLRs recognizes Giardia remains poorly understood. TLR2 plays a crucial role in pathogen recognition, innate immunity activation, and the eventual pathogen elimination. In this study, we investigated the role of TLR2 as a non-protective inflammatory response on controlling the severity of giardiasis. RT-PCR analysis suggested that TLR2 expression was increased in vitro. We demonstrated that Giardia lamblia-induced cytokines expression by the activation of p38 and ERK pathways via TLR2. Interestingly, the expression of IL-12 p40, TNF-α, and IL-6, but not IFN-γ, was enhanced in TLR2-blocked and TLR2^-/- mouse macrophages exposed to G. lamblia trophozoites compared with wild-type (WT) mouse macrophages. Further analysis demonstrated that G. lamblia trophozoites reduced cytokines secretion by activating AKT pathway in WT mouse macrophages. Immunohistochemical staining in G. lamblia cysts infected TLR2^-/- and WT mice showed that TLR2 was highly expressed in duodenum in infected WT mice. Also, infected TLR2^-/- and AKT-blocked mice showed an increased production of IL-12 p40 and IFN-γ compared with infected WT mice at the early stage during infection. Interestingly, infected TLR2^-/- and AKT-blocked mice displayed a decreased parasite burden, an increased weight gain rate, and short parasite persistence. Histological morphometry showed shortened villus length, hyperplastic crypt and decreased ratio of villus height/crypt depth in infected WT mice compared with in infected TLR2^-/- and AKT-blocked mice. Together, our results suggested that TLR2 deficiency leads to alleviation of giardiasis and reduction of parasite burden through the promotion of proinflammatory cytokines production. For the first time, our results demonstrated that TLR2 played a negative role in host defense against Giardia.

Keywords: AKT; Giardia; TLR2; TLR2−/−; cytokines; giardiasis.

Figure 1

Giardia lamblia trophozoites induced cytokines secretion in a TLR2-independent way. RT-PCR analysis of relative mRNA level for TLR2 in total RNA isolated from 2 × 10⁶ mouse peritoneal macrophages incubated in medium alone, with 1 × 10⁶ G. lamblia trophozoites, Pam₃CSK₄ (10 µg/ml), respectively. The mRNA level was normalized to β-actin (A). TLR2^−/− and wild-type (WT) mouse peritoneal macrophages were stimulated with 1 × 10⁶ G. lamblia trophozoites or Pam₃CSK₄ (10 µg/ml). After 18 h of incubation, the levels of TNF-α, IFN-γ, IL-6, and IL-12 p40 in cell culture supernatant were detected by ELISA (B). Data are expressed as the mean ± SD from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, stimulated cells versus those cultured in medium alone.

Figure 2

Giardia lamblia trophozoites induced the phosphorylation of p38 and ERK via TLR2. 2 × 10⁶ wild-type (WT) mouse peritoneal macrophages were stimulated with 1 × 10⁶ G. lamblia trophozoites for different times (0–240 min), cell lysates were used for Western blot analysis to measure the levels of phosphorylation of p38 and ERK (A). WT, TLR2^−/−, and TLR2-blocked mouse peritoneal macrophages were stimulated with G. lamblia trophozoites or Pam3CSK4 for 30 min (B). Densitometric analysis of Western blot in panels (A,B), respectively (C,D). Data are expressed as the mean ± SD from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, stimulated cells versus those cultured in medium alone.

Figure 3

Giardia lamblia trophozoites-mediated cytokine expression was blocked by the inhibition of p38 and ERK. 2 × 10⁶ wild-type mouse peritoneal macrophages were pretreated for 60 min with inhibitors of p38 (SB203580; 30 µM) or ERK (PD98059; 40 µM) before stimulation with G. lamblia trophozoites. After incubation, the secretion levels of TNF-α, IFN-γ, IL-6, and IL-12 p40 in cell supernatant were measured by ELISA (A–D). The mean ± SD levels of G. lamblia trophozoites-induced TNF-α, IFN-γ, IL-6, and IL-12 p40 were set to 100%, and the relative loss of cytokine in the presence of each inhibitor was calculated and expressed as the percentage of control (% ctrl). Data were expressed as the mean ± SD from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, cells treated with G. lamblia trophozoites and the inhibitor versus cells stimulated with G. lamblia trophozoites alone.

Figure 4

Giardia lamblia trophozoites enhanced cytokine expression by inhibition of AKT signal pathway via TLR2. 2 × 10⁶ wild-type (WT) mouse peritoneal macrophages were stimulated with 1 × 10⁶ G. lamblia trophozoites for different times (0–240 min), cell lysates were used for Western blot analysis to measure the levels of phosphorylation of AKT (A). WT, TLR2-blocked, and TLR2^−/− mouse peritoneal macrophages were stimulated with G. lamblia trophozoites for 30 min (B). Densitometric analysis of Western blot in panels (A,B), respectively (C,D). 2 × 10⁶ WT mouse peritoneal macrophages were transfected with small-interfering RNA (siRNA)-control or siRNA AKT (E). Densitometric analysis of Western blot in panel (E) (F). 2 × 10⁶ WT mouse peritoneal macrophages were transfected with siRNA AKT or pretreated for 60 min with AKT inhibitor (AKT inhibitor IV) before stimulation with G. lamblia trophozoites. After incubation, the secretion levels of TNF-α, IFN-γ, IL-6, and IL-12 p40 in cell supernatant were measured by ELISA (G–J). The mean ± SD levels of G. lamblia trophozoites-induced TNF-α, IFN-γ, IL-6, and IL-12 p40 were set to 100%, and the relative loss of cytokine in the presence of AKT inhibitor or siRNA AKT was calculated and expressed as the percentage of control (% ctrl). Data were expressed as the mean ± SD from three separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001, cells treated with G. lamblia trophozoites and the inhibitor versus cells stimulated with G. lamblia trophozoites alone.

Figure 5

Giardia lamblia trophozoites induced changes of subcellular localization of NF-κB/rel subunits. Confocal microscopy [(A), oil-immersion objective] and Western blot analysis (B,C) were used to measure the effects of G. lamblia trophozoites on the translocation of NF-κB from the cytoplasm to the nucleus in both wild-type (WT) and TLR2^−/− mouse peritoneal macrophages. Scale bars: 20 µm (A). 2 × 10⁶ WT mouse peritoneal macrophages were stimulated with 1 × 10⁶ G. lamblia trophozoites for different times (0–120 min), cell lysates were used for Western blot analysis. The phosphorylation of p65, IκBα, and total IκBα levels in the cytoplasmic of WT mouse peritoneal macrophages was detected (B). WT and TLR2^−/− mouse peritoneal macrophages were stimulated with G. lamblia trophozoites for 60 min. The phosphorylation of p65 and IκBα in the cytoplasmic was detected by Western blot (C). Data were acquired by three independent experiments.

Figure 6

Challenge with Giardia lamblia WB cysts in wild-type (WT) mice was longer persistence and caused weight loss in compared with TLR2^−/− mice. TLR2^−/−, AKT-blocked, and WT mice were infected on day 0 with WB strain of G. lamblia. Mice were euthanized on the indicated days post-infection, and parasite loads in the small intestine were calculated as described in the Section “Materials and Methods” (A). Growth curves measuring the percentage of change in weight beginning on the day of infection with G. lamblia WB cysts (n = 10 mice per group) or PBS control (n = 10 mice per group) (B). Data were acquired by three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, mice challenge with G. lamblia WB cysts versus mice challenge with PBS.

Figure 7

Infected TLR2^−/− and AKT-blocked mice challenged with Giardia lamblia WB cysts showed lighter lesions intestinal mucosa damage and crypt hyperplasia. H&E-stained duodenum sections from a representative PBS-challenged mouse at 9 dpi. The green box represents villus height. The green line represents crypt depth (A). H&E-stained duodenum sections from a representative Giardia-challenged wild-type (WT) mouse at 9 dpi. Black arrows indicate G. lamblia (B). H&E-stained duodenum sections from a representative Giardia-challenged TLR2^−/− mouse at 9 dpi (C). H&E-stained duodenum sections from a representative Giardia-challenged AKT-blocked mouse at 9 dpi. Black arrows indicate G. lamblia (D). H&E-stained duodenum sections from a representative AKT-blocked control mouse at 9 dpi (E). Villus and crypt architectural changes following G. lamblia infection in WT and TLR2^−/− mice at 9 dpi (n = 10 mice per group) (F–H). (A–C) Original magnification: ×20. *p < 0.05, **p < 0.01, ***p < 0.001, mice challenge with G. lamblia WB cysts versus mice challenge with PBS.

Figure 8

Wild-type (WT) mice challenged with Giardia lamblia WB cysts showed activation of TLR2 in intestinal duodenum. Immunohistochemical analysis of duodenum sections from a representative PBS-challenged mouse at 9 dpi with monoclonal TLR2 antibody (A). Immunohistochemical analysis of duodenum sections from a representative Giardia-challenged WT mouse at 9 dpi with a monoclonal TLR2 antibody (B). Immunohistochemical analysis of duodenum sections from a representative Giardia-challenged TLR2^−/− mouse at 9 dpi with a monoclonal TLR2 antibody (C). The degree of expression of TLR2 in the small intestine was measured as described in the Section “Materials and Methods” (n = 10 mice per group) (D). Original magnification: ×20. *p < 0.05, **p < 0.01, ***p < 0.001, mice challenge with G. lamblia WB cysts versus mice challenge with PBS.

Figure 9

Cytokines production in the small intestine of wild-type (WT), AKT-blocked, and TLR2^−/− mice infected with Giardia lamblia. WT, AKT-blocked, and TLR2^−/− mice were infected with G. lamblia and euthanized at 5, 9, and 17 dpi. The proportions of CD4⁺ (A) and CD8⁺ (B) cells in mesenteric lymph nodes were detected during the course of infection. Production of IL-12 (C), IFN-γ (D), TNF-α (E), IL-4 (F), IL-6 (G), and IL-10 (H) in small intestine were measured using ELISA. *p < 0.05, **p < 0.01, ***p < 0.001, mice challenge with G. lamblia WB cysts versus mice challenge with PBS. Data presented are means and SEM for 10 mice per time point.