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. 2019 Aug 5;216(8):1733-1748.
doi: 10.1084/jem.20182227. Epub 2019 Jun 12.

CXCR4 Regulates Plasmodium Development in Mouse and Human Hepatocytes

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Free PMC article

CXCR4 Regulates Plasmodium Development in Mouse and Human Hepatocytes

Hironori Bando et al. J Exp Med. .
Free PMC article

Abstract

The liver stage of the etiological agent of malaria, Plasmodium, is obligatory for successful infection of its various mammalian hosts. Differentiation of the rod-shaped sporozoites of Plasmodium into spherical exoerythrocytic forms (EEFs) via bulbous expansion is essential for parasite development in the liver. However, little is known about the host factors regulating the morphological transformation of Plasmodium sporozoites in this organ. Here, we show that sporozoite differentiation into EEFs in the liver involves protein kinase C ζ-mediated NF-κB activation, which robustly induces the expression of C-X-C chemokine receptor type 4 (CXCR4) in hepatocytes and subsequently elevates intracellular Ca2+ levels, thereby triggering sporozoite transformation into EEFs. Blocking CXCR4 expression by genetic or pharmacological intervention profoundly inhibited the liver-stage development of the Plasmodium berghei rodent malaria parasite and the human Plasmodium falciparum parasite. Collectively, our experiments show that CXCR4 is a key host factor for Plasmodium development in the liver, and CXCR4 warrants further investigation for malaria prophylaxis.

Figures

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Figure 1.
Figure 1.
The HGF–MET–PKCζ–NF-κB signaling pathway is required for liver-stage P. berghei development. (A) Images illustrating sporozoite differentiation into EEFs in Huh7 cells. Rod-shaped sporozoites initially acquire a bulbous shape and subsequently become spherical. Bars, 5 µm. (B–D) Effect of addition of anti-HGF (20 µg/ml) antibody to the culture medium on P. berghei sporozoite development in Huh7 cells. (B) EEFs were counted using IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (C) Luciferase intensity 48 h after sporozoite infection. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (D) Parasite shapes were assessed by IFA at the times after sporozoite infection indicated at the graphs. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (E) WT, PKCζ-KO, or MET-KO Huh7 cells were treated with 50 ng/ml HGF for 1 min. Expression of the genes indicated to the right was detected in the cell lysates by Western blotting. Numbers (in kilodaltons) to the left indicate the position of size markers. Data are representative of three independent experiments. (F and G) WT or MET-KO Huh7 cells were infected with P. berghei sporozoites. (F) Parasite shapes were assessed by IFA at the times after sporozoite infection indicated at the graphs. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (G) Luciferase intensity 48 h after sporozoite infection. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. ***, P < 0.001 (Student’s t test). (H and I) WT or PKCζ-KO Huh7 cells were infected with P. berghei sporozoites. (H) Luciferase intensity 48 h after sporozoite infection. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (I) Parasite shapes were assessed by IFA at the times after sporozoite infection indicated at the graphs. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. ***, P < 0.001 (Student’s t test). (J–L) WT mice (n = 15) or PKCζ-KO mice (n = 15) were infected with P. berghei sporozoites. (J) The progression of infection 24 or 48 h after sporozoite infection was measured using an in vivo imaging system. Data are representative of three independent experiments. (K) The survival rate was analyzed. Data are cumulative of three independent experiments. P < 0.0001 (log-rank test). (L) Blood–brain barrier integrity was measured by Evans Blue (EB) perfusion. Uninfected mice (n = 15) were used as controls. Data are cumulative of three independent experiments. Indicated values are means (n = 15) of ± SD. Each point represents the value of each mouse. **, P < 0.01 (Student’s t test). (M) IFA image of WT or PKCζ-KO mice liver at 15 h after sporozoite infection. Bars, 5 µm. Images are three representatives out of five independent sections. (N) Effect of the addition of IKKβ inhibitor PF184 (500 nM) or ERK inhibitor FR180204 (10 µM) to the culture medium on P. berghei sporozoite development in Huh7 cells. Luciferase intensity 48 h after sporozoite infection is shown. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (O) Model of host signaling pathway for transformation of P. berghei sporozoite. N.S., not significant; hpi, hours post-infection.
Figure 2.
Figure 2.
CXCR4 is necessary and sufficient for liver-stage P. berghei development. (A) Selection of PKCζ-dependent genes in 2.5 × 105 WT or PKCζ-KO Huh7 cells treated with HGF (50 ng/ml) or mosquito debris or infected with 5 × 105 P. berghei sporozoites for 6 h. Among 26 genes shared with both conditions (fully described in Fig. S2 A and the legend), the top 10 genes with indicated names are shown. Among the 10 genes, the expression of 7 genes could be assessed using commercially available antibodies (“Antibody availability”). Among these 7 genes, we confirmed that Huh7 cells with the genome editing of 4 genes showed reduction of the corresponding proteins (“Knock-down confirmation”; Fig. S2 B). The Huh7 cells with successful reduction of the indicated proteins were further tested for EEF formation assay in Fig. 2 B. Expression values are scaled up to the rows and range from −4 to +4. Data are from one experiment. (B) WT, PKCζ-KO, CXCR4-KD, SULF2-KD, ZNF426-KD, or CD160-KD Huh7 cells were infected with P. berghei sporozoites. Luciferase intensity was measured at 48 h after sporozoite infection. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (C) WT or PKCζ-KO Huh7 cells were treated or not with 50 ng/ml HGF for 24 h. The expression in the cell lysates of the genes indicated to the right was assessed by Western blotting. Numbers (in kilodaltons) to the left indicate the position of size markers. Data are representative of three independent experiments. (D) Huh7 cells were treated or not with PF184 (500 nM) or FR180204 (10 µM) for 4 h, as indicated. Also, cells were treated or not with 50 ng/ml HGF for 6 h and infected or not with sporozoites or treated with mosquito debris for 6 h, as indicated. The graphs report the quantitative RT-PCR analysis of CXCR4 mRNA levels. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (E) WT or CXCR4-KD Huh7 cells were infected with P. berghei sporozoites. Parasite shapes were assessed by IFA at 24 h after sporozoite infection. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (F) WT, PKCζ-KO, or CXCR4-KD Huh7 cells were infected with P. berghei sporozoites. EEFs at 48 h after sporozoite infection were counted using IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated (Student’s t test). (G and H) WT, CXCR4-KD, or PKCζ-KO Huh7 cells that were transfected with an empty vector (Empty) or with a vector expressing CXCR4 (+CXCR4) were infected with P. berghei sporozoites. (G) Luciferase intensity at 48 h after sporozoite infection. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. ***, P < 0.001 (Student’s t test). (H) Parasite shapes at 24 h after sporozoite infection were assessed by IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (I and J) Mice untreated or treated with the CXCR4 inhibitor AMD3100 (10 mg/kg) were infected with P. berghei sporozoites. (I) The progression of infection 24 h after sporozoite challenge was measured using an in vivo imaging system. Data are representative of three independent experiments. (J) Comparison of survival rate. Data are cumulative of three independent experiments. P = 0.021 (log-rank test). (K) IFA image of DMSO- or AMD3100-treated mice liver 15 h after sporozoite infection. Bars, 5 µm. Images are three representatives out of five independent sections. N.S., not significant; hpi, hours post-infection.
Figure 3.
Figure 3.
CXCR4-mediated intracellular Ca2+ increase in hepatocytes triggers P. berghei sporozoite differentiation into EEFs. (A and B) WT, PKCζ-KO, or CXCR4-KD Huh7 cells were stained with Rhod-4 (red) and DAPI (blue) and then treated or not with 50 ng/ml HGF for 1 h. (A) Representative images of the cells. Bars, 5 µm. Data are representative of three independent experiments. (B) Analysis of calcium flux in indicated cells. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (C–E) Effect of the addition of Ca2+ (1 mM) to the culture medium on host cell–free P. berghei sporozoite development. (C) Parasite shapes at 0 or 24 h after sporozoite infection were assessed by IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. ***, P < 0.001 (Student’s t test). (D) RT-PCR analysis of S-type rRNA or 18s rRNA level in sporozoites. Data are representative of three independent experiments. (E) Expression of the genes indicated to the right was detected in the parasite lysates by Western blotting. Data are representative of three independent experiments. (F) WT, PKCζ-KO, CXCR4-KD, anti-HGF (20 µg/ml), or AMD3100 (1 µM) pretreated Huh7 cells were treated with Rhod-4 and then infected with P. berghei sporozoites for 1 h. Representative IFA images of the sporozoites in cells stained with YFP (green), DAPI (blue), and Rhod-4 (red). Bars, 5 µm. Data are representative of three independent experiments. (G–J) WT, PKCζ-KO, CXCR4-KD, anti-HGF (20 µg/ml), or AMD3100 (1 µM) pretreated Huh7 cells were treated or not with ionomycin (1 µg/ml) for 15 min, and cells were washed. Then cells were infected with P. berghei sporozoites. (G) Luciferase intensity at 48 h after sporozoite infection. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. ***, P < 0.001 (Student’s t test). (H) Parasite shapes 24 h after sporozoite infection were assessed by IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. *, P < 0.05; **, P < 0.01 (Student’s t test). (I) Images of IFA of sporozoite in cells stained with YFP (green), UIS4 (red), and DAPI (blue). Bars, 5 µm. Data are representative of three independent experiments. (J) Images of IFA of sporozoite in cells stained with anti-UIS3 (green), anti-UIS4 (red), and DAPI (blue). Bars, 5 µm. Data are representative of three independent experiments. N.S., not significant; hpi, hours post-infection.
Figure 4.
Figure 4.
CXCR4 is required for liver P. falciparum development. (A) Images illustrating P. falciparum sporozoite differentiation into EEFs in HC-04 cells. Rod-shaped sporozoites initially acquire a bulbous shape and subsequently become spherical. Bars, 5 µm. (B–E) WT, PKCζ-KO, or CXCR4-KD HC-04 cells were infected with P. falciparum sporozoites. (B and C) Parasite shapes 24 h after sporozoite infection were assessed by IFA. Bars, 5 µm. (B) Data are representative of three independent experiments. (C) Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. *, P < 0.05; **, P < 0.01 (Student’s t test). (D and E) EEFs at 7 d after sporozoite infection were counted using IFA. Bars, 10 µm. (D) Data are representative of three independent experiments. (E) Indicated values are means (n = 3) of ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (F) HC-04 cells were uninfected or infected with P. falciparum sporozoites. Localization of p65/RelA at 2 h after sporozoite infection was assessed by IFA. Bars, 5 µm. Data are representative of three independent experiments. (G) HC-04 cells were treated or not with PF184 (500 nM) for 4 h, as indicated, and then infected or not with P. falciparum sporozoites or treated with mosquito debris for 6 h, as indicated. The graphs report the quantitative RT-PCR analysis of CXCR4 mRNA levels. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (H and I) Effect of AMD3100 (1 µM) on P. falciparum development in HC-04 cells. (H) EEFs at 7 d after sporozoite infection were counted using IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (I) Parasite shapes 24 h after sporozoite infection were assessed by IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (J) Effect of AMD3100 (1 µM) on P. falciparum development in human primary hepatocytes. EEFs at 7 d after sporozoite infection were counted using IFA. Indicated values are means (n = 3) ± SEM. Each point represents the mean of one experiment. Three experiments were repeated. **, P < 0.01 (Student’s t test). (K) Scheme of host signaling pathway for liver stage P. berghei or P. falciparum transformation. N.S., not significant; dpi, days post-infection; hpi, hours post-infection.

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