Negative Regulation of Schistosoma japonicum Egg-Induced Liver Fibrosis by Natural Killer Cells

The role of natural killer (NK) cells in infection-induced liver fibrosis remains obscure. In this study, we elucidated the effect of NK cells on Schistosoma japonicum (S. japonicum) egg-induced liver fibrosis. Liver fibrosis was induced by infecting C57BL/6 mice with 18–20 cercariae of S. japonicum. Anti-ASGM1 antibody was used to deplete NK cells. Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly I∶C) was used to enhance the activation of NK cells. Results showed that NK cells were accumulated and activated after S. japonicum infection, as evidenced by the elevation of CD69 expression and IFN-γ production. Depletion of NK cells markedly enhanced S. japonicum egg-induced liver fibrosis. Administration of poly I∶C further activated NK cells to produce IFN-γ and attenuated S. japonicum egg-induced liver fibrosis. The observed protective effect of poly I∶C on liver fibrosis was diminished through depletion of NK cells. Disruption of IFN-γ gene enhanced liver fibrosis and partially abolished the suppression of liver fibrosis by poly I∶C. Moreover, expression of retinoic acid early inducible 1 (RAE 1), the NKG2D ligand, was detectable at high levels on activated hepatic stellate cells derived from S. japonicum-infected mice, which made them more susceptible to hepatic NK cell killing. In conclusion, our findings suggest that the activated NK cells in the liver after S. japonicum infection negatively regulate egg-induced liver fibrosis via producing IFN-γ, and killing activated stellate cells

Evaluation of the C-Terminal Fragment of Entamoeba histolytica Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess.

Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. E. histolytica invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that E. histolytica possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of E. histolytica trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after in vitro stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses

Evaluation of a Major Surface Antigen of Babesia microti Merozoites as a Vaccine Candidate against Babesia Infection

Babesia species are tick-borne intraerythrocytic protozoa that cause babesiosis in humans worldwide. No vaccine has yet proven effective against Babesia infection. Surface antigens of merozoites are involved in the invasion of erythrocytes by Babesia. Surface antigens may be presented by both babesial sporozoites and merozoites and provide a general target for antibody-mediated inhibition of erythrocyte invasion. Here we evaluated a major surface antigen of B. microti merozoites, BMSA, as a potential vaccine to prevent babesiosis. Our data indicated that bmsa is transcribed during different phases, including ring form, amoeboid form, and merozoites, and that its expression is significantly increased in mature merozoites. The protein was found to be located in the membrane of B. microti and in the cytoplasm of infected erythrocytes. The immune response induced by BMSA had a significant inhibitory effect on parasite invasion of the host erythrocytes (83.3% inhibition of invasion) and parasite growth in vivo. The levels of parasitemia significantly decreased after BMSA vaccination when mice were infected with babesia parasite. Importantly, protective immunity was significantly related to the upregulation of the Th17 cytokine interleukin-17, the Th1 cytokine interleukin-12p70 and the Th2 cytokines, such as interleukin-4, -6, and -10. Ingenuity Pathway Analysis indicated that interleukin-17 facilitated the secretion of Th2 cytokines, such as interleukin-10, -4, and -6, thereby inducing a predominately Th2 protective immune response and promoting the expression a high level of special IgG1 against Babesia infection. Further, an anti-BMSA monoclonal antibody successfully protected NOD/SCID mice from a challenge with B. microti. Taken together, our results indicated that BMSA induces a protective immune response against Babesia infection and may serve as a potential vaccine

Reactivity of sera from hamsters immunized with C-Igl and sham-immunized hamsters to C-Igl, rIgl1, and rIgl2 in ELISA (A), Western immunoblot (B) and dot blot analysis (C).

<p>(A) Mean value of ELISA results from serial dilutions of sera from C-Igl-immunized hamsters reacted with C-Igl (●), rIgl-1 (■), or rIgl-2 (▲) and sera from sham-immunized hamsters reacted with C-Igl (<b>▽</b>), rIgl-1 (◇), or rIgl-2 (○). Error bars represent the standard errors of the means calculated from each individuals from three independent experiments.*<i>P</i> < 0.001. (B) Western immunoblot analysis of the reactivity of pooled sera from immunized hamsters to <i>E</i>. <i>histolytica</i> trophozoites. Crude antigen from the HM-1: IMSS strain was subjected to SDS-PAGE under reduced conditions. The strip in lane 1 was stained with CBB. Strips were treated with 1:400 diluted sera from C-Igl-immunized hamsters (lane 2) and sham-immunized hamsters (lane 3). Biostep Prestained Protein Marker (Tanon, China) was used and numbers to the left indicate the molecular masses of the marker. (C) Various concentrations (0.01, 0.1, and 1 μg) of rIgl-1 and rIgl-2 were spotted on nitrocellulose membranes. One strip was stained with Coomassie brilliant blue (CBB). Other strips were treated with different dilutions of pooled sera. HRP-conjugated goat antibody to hamster IgG was used as a secondary antibody.</p

Evaluation of the C-Terminal Fragment of <i>Entamoeba histolytica</i> Gal/GalNAc Lectin Intermediate Subunit as a Vaccine Candidate against Amebic Liver Abscess

<div><p>Background</p><p><i>Entamoeba histolytica</i> is an intestinal protozoan parasite that causes amoebiasis, including amebic dysentery and liver abscesses. <i>E</i>. <i>histolytica</i> invades host tissues by adhering onto cells and phagocytosing them depending on the adaptation and expression of pathogenic factors, including Gal/GalNAc lectin. We have previously reported that <i>E</i>. <i>histolytica</i> possesses multiple CXXC sequence motifs, with the intermediate subunit of Gal/GalNAc lectin (i.e., Igl) as a key factor affecting the amoeba's pathogenicity. The present work showed the effect of immunization with recombinant Igl on amebic liver abscess formation and the corresponding immunological properties.</p><p>Methodology/Principal Findings</p><p>A prokaryotic expression system was used to prepare the full-length Igl and the N-terminal, middle, and C-terminal fragments (C-Igl) of Igl. Vaccine efficacy was assessed by challenging hamsters with an intrahepatic injection of <i>E</i>. <i>histolytica</i> trophozoites. Hamsters intramuscularly immunized with full-length Igl and C-Igl were found to be 92% and 96% immune to liver abscess formation, respectively. Immune-response evaluation revealed that C-Igl can generate significant humoral immune responses, with high levels of antibodies in sera from immunized hamsters inhibiting 80% of trophozoites adherence to mammalian cells and inducing 80% more complement-mediated lysis of trophozoites compared with the control. C-Igl was further assessed for its cellular response by cytokine-gene qPCR analysis. The productions of IL-4 (8.4-fold) and IL-10 (2-fold) in the spleen cells of immunized hamsters were enhanced after <i>in vitro</i> stimulation. IL-4 expression was also supported by increased programmed cell death 1 ligand 1 gene.</p><p>Conclusions/Significance</p><p>Immunobiochemical characterization strongly suggests the potential of recombinant Igl, especially the C-terminal fragment, as a vaccine candidate against amoebiasis. Moreover, protection through Th2-cell participation enabled effective humoral immunity against amebic liver abscesses.</p></div

NKG2D/RAE1 interaction is involved in the cytotoxicity of NK cells against activated HSCs.

<p>(A, B) HSCs were isolated from <i>S. japonicum</i>-infected mice or uninfected mice. Expression of α-SMA (A) and various NKG2D ligands (B) on HSCs was detected by quantitative PCR. Values were normalized to β-actin in the same samples and expressed as fold change in comparison with the samples from uninfected controls (0 wk). Data were shown as mean ± SEM from three independent experiments. (C, D) NK cells isolated from <i>S. japonicum</i>-infected mice were used as effector cells. (C) HSCs isolated from normal mice (quiescent HSCs) or 8-week <i>S. japonicum</i>-infected mice (activated HSCs) were used as target cells. (D) HSCs isolated from 8-week <i>S. japonicum</i>-infected mice (activated HSCs) were used as target cells. A dose of 10 µg/mL anti-NKG2D (blocking) mAb was added in the coculture for the blockade. T/E, target-effector ratio.</p

Disruption of the IFN-γ gene accelerates hepatic fibrosis and partially abolish poly I∶C-mediated suppression.

<p>(A–C) Wild-type and IFN-γ−/− mice were infected with 18–20 cercariae of <i>S. japonicum</i>, and injected with poly I∶C (0.5 µg/g, i.p.) or saline from week 5 to week 10 post-infection. All mice were euthanized at week 10 post-infection. (A–B) Liver tissues were subjected to stain for quantifying collage (A) and α-SMA positive areas (B). Data were shown as mean ± SEM from 7–9 mice per each group. *, <i>P</i><0.05 versus corresponding saline-treated wild-type mice. #, <i>P</i><0.05 versus corresponding poly I∶C-treated wild-type mice. (C) Intrahepatic IL-4, IL-5, and IL-13 mRNA expression levels were measured by quantitative PCR. Results were expressed as fold amplification over normal, uninfected liver following normalization with β-actin. Data were expressed as mean ± SEM (n≥7 for each group).</p

Analysis of hepatic NK cell activation during <i>S. japonicum</i> infection.

<p>C57BL/6 mice were percutaneously infected with 18–20 cercariae of <i>S. japonicum</i>. (A–C) After various time periods post-infection, hepatic MNCs were prepared for flow cytomery analysis. The relative proportion (A) and absolute number (B) of CD3-NK1.1+ cells were shown. (C) Expression of CD69 on hepatic NK cells from uninfected (thin lines) or infected mice (bold lines) during infection (right panel). For the kinetic study, the percentage of CD69+ NK cells among total NK cells was calculated and represented (left panel). Data were shown as mean ± SEM (n = 5 for each group). *, <i>P</i><0.05 versus corresponding 0 week groups. (D) Liver MNCs isolated from uninfected or <i>S. japonicum</i>-infected mice, and then were analyzed by flow cytometry for intracellular IFN-γ of NK cells (CD3-NK1.1+) and NKT (CD3+NK1.1+) cells. (E) Hepatic MNCs were isolated from control IgG or anti-ASGM1 pretreated mice at week 6 post-infection. After culture for 48 hours, the supernatants were collected for IFN-γ measurement by ELISA. Data were presented as mean ± SEM; *, <i>P</i><0.05 compared with corresponding control IgG treated group.</p

Injection of poly I∶C further activates NK cells in <i>S. japonicum</i> infected mice.

<p><i>S. japonicum</i>-infected mice were injected intraperitoneally with poly I∶C (0.5 µg/g) since week 5 post-infection. (A–D) At various time points, hepatic MNCs were isolated and subjected for flow cytomery analysis. A representative FACS analysis of NK1.1 and CD3 was shown in (A). The percentage of NK cells among hepatic MNCs and the total number of NK cells are summarized in (B) and (C), respectively. The percentage of CD69+ NK cells among total NK cells was calculated and represented in (D). Data were presented as mean ± SEM (n = 5 for each group). *, <i>P</i><0.05 versus corresponding saline-treated group. (E) Mice were sacrificed at week 6 post-infection, and IFN-γ expression by hepatic NK cells (CD3-NK1.1+) was examined by flow cytometry. (F) Hepatic MNCs from control IgG or anti-ASGM1 pretreated mice (denoted as “MNCs” and “MNCs without NK cells”, respectively) were incubated with poly I∶C (100 µg/ml) or saline for 48 hours. IFN-γ secretion in the supernatant was measured by ELISA. Data were presented as mean ± SEM; *, <i>P</i><0.05.</p