A Novel TLR4-Binding Domain of Peroxiredoxin From Entamoeba histolytica Triggers NLRP3 Inflammasome Activation in Macrophages

Abstract

Macrophages promote early host responses to infection by releasing pro-inflammatory cytokines, and they are crucial to combat amoebiasis, a disease affecting millions of people worldwide. Macrophages elicit pro-inflammatory responses following direct cell/cell interaction of Entamoeba histolytica, inducing NLRP3 inflammasome activation with high-output IL-1β/IL-18 secretion. Here, we found that trophozoites could upregulate peroxiredoxins (Prx) expression and abundantly secrete Prxs when encountering host cells. The C-terminal of Prx was identified as the key functional domain in promoting NLRP3 inflammasome activation, and a recombinant C-terminal domain could act directly on macrophage. The Prxs derived from E. histolytica triggered toll-like receptor 4-dependent activation of NLRP3 inflammasome in a cell/cell contact-independent manner. Through genetic, immunoblotting or pharmacological inhibition methods, NLRP3 inflammasome activation was induced through caspase-1-dependent canonical pathway. Our data suggest that E. histolytica Prxs had stable and durable cell/cell contact-independent effects on macrophages following abundantly secretion during invasion, and the C-terminal of Prx was responsible for activating NLRP3 inflammasome in macrophages. This new alternative pathway may represent a potential novel therapeutic approach for amoebiasis, a global threat to millions.

Keywords: Entamoeba histolytica; NLRP3 inflammasome; TLR4-binding domain; macrophage; peroxiredoxin.

Figure 1

Priming of NLRP3 inflammasome in ALA tissue and Secretion of Prx in E histolytica. (A) Construction of mice ALA model. Male C57BL/6 mice were inoculated with 1 × 10⁶ trophozoites of the E histolytica SAW755CR strain/mouse. Five days after treatment, the ALA tissue was obtained. Sections from normal or ALA tissue were observed by HE and PAS staining. Bars: 100 μm. Black arrows: trophozoites. Experiments were repeated twice. (B) Immunohistochemical staining analysis of ALA. Sections of ALA tissue were incubated with anti-F4/80 antibody, mAb 4G6, or anti-IL-1β antibody to detect distribution of macrophages, Prx of E histolytica, or IL-1β in ALA tissue, respectively. Brown areas were considered positive. Bars: 100μm. (C) Expression of NLRP3 inflammasome-related genes in ALA tissue. Male C57BL/6 mice were inoculated with 10⁶ trophozoites of the E histolytica SAW755CR strain. Five days after infection, the expression levels of related genes in ALA tissue were measured through qPCR, and shown as 2-ΔΔCt of the target gene relative to β-actin, normalized with corresponding values in normal mice. In sham group, n = 6; in ALA group, n = 8. Experiments were repeated twice. (D) The location of Prx in trophozoites co-incubated with CHO-K1 or RAW264.7 cells. Trophozoites during the logarithmic growth phase were harvested and co-incubated with CHO-K1 or RAW264.7 cells. An hour after incubation, an E histolytica 4G6 monoclonal antibody (mAb) was used to measure the expression level and localization of Prx through immunofluorescence assay and laser confocal microscopy. White arrows: engulfed cells; red arrows: trophozoites membrane. Bars, 25 μm (the left column) or 7.5 μm (right three columns). (E) Quantitative analysis of Prx fluorescence intensity of E histolytica. Cells were observed with the same laser intensity by laser confocal microscopy. The fluorescence intensity of Prx was analyzed using ImageJ 1.52a. In all groups, n = 4. Experiments were repeated three times. (F) Prx secretion from E histolytica trophozoites was detected by western blotting. After incubated with CHO-K1 or RAW264.7 cells in serum-free cell medium under anaerobic condition, cell supernatant was collected. Cold acetone precipitation was conducted to extract protein from culture supernatant. Afterwards, protein from the same volume of supernatant was analyzed by western blotting. Experiments were repeated twice. CHO, CHO-K1 cells; RAW, RAW264.7 cells; SN, supernatant; Lys, lysate. Statistical analysis was conducted by Student’s t-test, and data are presented as mean ± standard error of mean (SEM). *p < 0.05, **p < 0.01, ns, not significant.

Figure 2

Priming of NLRP3 inflammasome in macrophages by native Prx. (A) Gene expression levels detected by qPCR. RAW264.7 cells were treated with 5 μg/mL of native Prx, while LPS (1 μg/ml) and Tris-HCl (pH 8.0) was used as the control. Six hours after the treatment, the expression levels of related genes were measured by qPCR, and shown as 2-ΔΔCt of the target gene relative to β-actin, normalized with corresponding values. In all groups, n = 3. Experiments were repeated three times. (B) Western blot analysis of NLRP3 inflammasome-related proteins. RAW264.7 cells were pretreated with TAK242 (1 μM) for 1 h. Next, the cells were treated with either 5 μg/mL of native Prx or Tris-HCl (pH 8.0). Six hours after the treatment, the cells were collected, and the cell lysis was analyzed by western blotting. In all groups, n = 3. Experiments were repeated three times. Statistical analysis was conducted by Student’s t-test, and data are expressed as mean ± SEM. *p < 0.05, **p < 0.01.

Figure 3

Eh-rPrx-Activation of NLRP3 Inflammasome in RAW264.7 Cells. (A) Female C57BL/6 mice were treated by intraperitoneal injection of Eh-rPrx (100 μg/mouse); Tris-HCl (pH 8.0) was used as the negative control. Twelve hours after the treatment, PEC (containing macrophages) were collected, and the expression levels of related genes were measured by qPCR and shown as 2-ΔΔCt of the target gene relative to β-actin, normalized with corresponding values in the negative control. In control group, n = 6; in treatment group, n = 11. Experiments were repeated twice. (B) Gene expression levels detected by qPCR. RAW264.7 cells were treated with 5 μg/mL of Eh-rPrx, while LPS (1 μg/ml) and Tris-HCl (pH 8.0) was used as the control. Six hours after the treatment, the expression levels of related genes were measured by qPCR, and shown as 2-ΔΔCt of the target gene relative to β-actin, normalized with corresponding values. In all groups, n = 3. Experiments were repeated three times. (C) Western blot analysis of NLRP3 inflammasome-related proteins. RAW264.7 cells were pretreated with TAK242 (1 μM) for 1 h or oATP (500 μM) for 2 h. Next, the cells were treated with either 5 μg/mL of Eh-rPrx, 1 μg/mL of LPS, or Tris-HCl (pH 8.0). For the positive control, ATP (4 mM) was added at the last 2 h of incubation. Six hours after the treatment, the cells were collected, and the cell lysis was analyzed by western blotting. In all groups, n = 3. Experiments were repeated three times. (D) Release of IL-1β and IL-18 in the supernatant of Eh-rPrx-treated cells. RAW264.7 cells were pretreated with TAK242 (1 μM) for 1 h or oATP (500 μM) for 2 h. Next, cells were treated with either 5 μg/mL of Eh-rPrx, 1 μg/mL of LPS, or Tris-HCl (pH 8.0). For the positive control, ATP (4 mM) was added at the last 2 h of incubation. Cell culture supernatant was collected after 12 and 24 h of treatment, and analyzed by enzyme-linked immunosorbent assay. In all groups, n=3. Experiments were repeated three times. Statistical analysis was conducted by Student’s t-test, and data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

Figure 4

Mechanism of Eh-rPrx recognition via TLR4 receptor. (A) Eh-rPrx recognition through TLR4 receptor was confirmed via TLR4 siRNA. RAW264.7 cells were treated with control siRNA (50 nM) and TLR4 siRNA (50 nM) for 24 h. Next, the supernatant was removed, and the cells were treated with Eh-rPrx (5 μg/mL) for 6 h. Expression levels of NLRP3-related genes and proteins were detected by qPCR and w estern blotting, respectively. In all groups, n = 3. Experiments were repeated three times. (B) Activation of NLRP3 Inflammasome independently of Prx’s reductase activity. Eh-rPrx functioned independently of antioxidant activity. For detection of NLRP3-related gene expression levels, RAW264.7 cells were pretreated with TLR4 inhibitor TAK242 (1 μM) for 1 h, prior to 5 μg/mL of Eh-rPrx or mutant Eh-rPrx. LPS (1 μg/mL) and Tris-HCl (pH 8.0) were used as the positive and negative controls, respectively. Six hours after the treatment, the expression levels of related genes were measured by qPCR and shown as 2-ΔΔCt of the target gene relative to β-actin, normalized with corresponding values in the negative control. In all groups, n = 3. Experiments were repeated three times. (C) The C-terminal of Eh-Prx was identified as the functional domain responsible for activating NRLP3 inflammasome. Three fragments of Eh-Prx were constructed; fragment-1 to fragment-3 each contained 100 amino acids at the N-terminal, middle segment, and C-terminal. Following incubation of the fragments and RAW264.7 cells for 6 h, the expression levels of related genes were measured by qPCR and shown as 2-ΔΔCt of the target gene relative to β-actin, normalized with corresponding values in the negative control. In all groups, n = 3. Experiments were repeated three times. Statistical analysis was conducted by Student’s t-test, and data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

Figure 5

Inhibition of TLR4 receptor blocked apoptosis and mitochondrial oxidative damage induced by Prx. (A) Following pretreatment with TAK242 (1 μM) for 1 h, RAW264.7 cells were incubated with Eh-rPrx (5 μg/ml) for 24 h, then suspended in 500 μL of 1× binding buffer (Sigma-Aldrich, APOAF) at a concentration of 10⁶ cells/ml. Following incubation at room temperature for 10 min, 5 μL of Annexin V FITC conjugate (Sigma-Aldrich, APOAF) and 10 μL of propidium iodide solution (Sigma-Aldrich, APOAF) were added to each suspension. Fluorescence of the cells was immediately determined by using a flow cytometer. In all groups, n = 3. Experiments were repeated three times. (B) Quantitative analysis of mitochondrial ROS MFI in RAW264.7 cells treated with rPrx. RAW264.7 cells were pretreated with TAK242 (1 μM) for 1 h or oATP (500 μM) for 2 h. Next, the cells were treated with 5 μg/mL of Eh-rPrx or Tris-HCl (pH 8.0) as the negative control. After 24 h or 36 h of treatment, the live cells were stained with MitoSOX (5 μM) for 10 min, and then with CFSE (2 μM) for counterstaining. The cells were immediately observed and quantified through high-content screening analysis. In all groups, n = 3. Experiments were repeated three times. (C) The fluorescence images of RAW264.7 cells after 36 h of rPrx treatment. Red fluorescence, MitoSOX™ Red; green fluorescence, CFSE; scale bar, 100 μm. In all groups, n = 3. Experiments were repeated three times. Statistical analysis was conducted by Student’s t-test, and data are expressed as mean ± SEM. *p < 0.05, **p < 0.01, ns, not significant.