7 research outputs found

    Silencing CRACC expression on Kupffer cells alleviates the liver injury induced by Poly I:C/D-GalN.

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    <p><b>A</b>-<b>C</b>, Mice were treated with nanoparticle encapsulated FAM conjunct siRNA by intravenous injection; and 3h later FAM<sup>+</sup> cells among Kupffer cells were analyzed by flow cytometry (<b>A</b>); and the nanoparticle encapsulated FAM conjunct siRNA (green) endocytosed by Kupffer cells (red, stained with APC-F4/80 mAb) were tracked by con-focal microscopy both in isolated Kupffer cells (<b>B</b>, Bar=50µm, 63× objective) and in site of frozen tissue sections(<b>C</b>, Bar=50µm, 40× objective). Kupffer cells were identified as F4/80<sup>+</sup> cells. <b>D</b>, Mice were pre-treated with nanoparticle encapsulated siNeg or siCRACC for 6h, and then treated with Poly I:C/D-GalN in each group. The CRACC expression on Kupffer cells was analyzed by flow cytometry at 12h and 18h time points post the Poly I:C/D-GalN treatment. The Data are from 6-8 mice per group. <b>E</b>-<b>F</b>, The mice were pre-treated with nanoparticle encapsulated siNeg or siCRACC for 6h, and then treated with Poly I:C/D-GalN in each group. The serum ALT was tested (<b>E</b>) and the H&E-Staining analyses were performed at 18h time point post Poly I:C/D-GalN (<b>F</b>). The Data are from 6-8 mice per group. **P < 0.01.</p

    Poly I:C treatment up regulates the expression of CRACC on NK cells.

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    <p>CRACC expression on NK cells (solid line) and NKT cells (long dashed line) from mice treated with Poly I:C for 18h was analyzed by flow cytometry. NK cells were identified as NK1.1 <sup>+</sup> CD3<sup>-</sup> cells and NKT cells were identified as NK1.1 <sup>+</sup> CD3<sup>+</sup> cells. <b>A</b>, Flow Cytometry of the CRACC expression on hepatic NK cells and hepatic NKT cells. <b>B</b>, <i>Upper </i><i>panel</i>: percent of CRACC<sup>+</sup> cells among NK cells from liver, spleen, lung, peripheral blood (PBL), mesenteric lymph nodes (MLN), and bone marrow (BM); <i>Lower </i><i>panel</i>: cells counts of NK cells from indicated organs. <b>C</b>, MFI analyses of SLAM family (2B4, Ly108, Ly9, CD84, CRACC) expression on hepatic NK cells from mice treated with Poly I:C for 18h by flow cytometry. Data are from 5-8 mice per group. **P<0.01; ***P < 0.001.</p

    CRACC-CRACC interaction between NK cells and resident Kupffer cells.

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    <p>Poly I:C stimulation (<b>A</b>) elevated the CARCC expression on NK cells and resident Kupffer cells (<b>B</b>). CRACC-CRACC interaction (<b>B</b>) together with the NKG2D-Rae-1 interaction (<b>C</b>) between NK cells and resident Kupffer cells promote IFN-γ and TNF-α production (<b>D</b>, <b>E</b>), which contributes to the Poly I:C/D-GalN induced liver injury.</p

    Poly I:C/D-GalN induced liver injury correlates to the increased CRACC mRNA expression of liver.

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    <p><b>A</b>, Mice were treated with Poly I:C (1.5 µg per mouse, i.v.) together with D-GalN (10 mg per mouse, i.p.); and then euthanized at 2h, 4h, 6h, 8h, 12h, 18h, 24h, 48h time points post Poly I:C/D-GalN treatment. The expression of CRACC mRNA was assayed by quantitative PCR (upper panel); and the serum ALT was tested (lower panel). <b>B</b>, Mice were treated with saline alone, Poly I:C alone, D-GalN alone or Poly I:C together with D-GalN for 12h; and then the CRACC expression on Kupffer cells and hepatic NK cells was analyzed by flow cytometry. Kupffer cells were identified as F4/80<sup>+</sup> cells and NK cells were identified as NK1.1 <sup>+</sup> CD3<sup>-</sup> cells. Data are from 6-9 mice per group.</p

    CRACC-CRACC Interaction between Kupffer and NK Cells Contributes to Poly I:C/D-GalN Induced Hepatitis

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    <div><p>CD2-like receptor activating cytotoxic cells (CRACC) is known as a critical activating receptor of natural killer (NK) cells. We have previously reported that NK cells contribute to Poly I:C/D-galactosamine (D-GalN)-induced fulminant hepatitis. Since natural killer group 2, member D (NKG2D) is considered critical but not the only activating receptor for NK cells, we investigated the role of CRACC in this model. We found that CRACC was abundant on hepatic NK cells but with low expression levels on Kupffer cells under normal conditions. Expression of CRACC on NK cells and Kupffer cells was remarkably upregulated after poly I:C injection. Hepatic CRACC mRNA levels were also upregulated in Poly I:C/D-GalN-treated mice, and correlated positively with the serum alanine aminotransferase (ALT) levels. CRACC expression on Kupffer cells was specifically silenced by nano-particle encapsulated siRNA <i>in vivo</i>, which significantly reduced Poly I:C/D-GalN-induced liver injury. In co-culture experiments, it was further verified that silencing CRACC expression or blockade of CRACC activation by mAb reduced the production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α. Collectively, our findings suggest that CRACC-CRACC interaction between NK cells and resident Kupffer cells contributes to Poly I:C/D-GalN-induced fulminant hepatitis.</p> </div

    CRACC-CRACC interaction between macrophages and NK cells enhances the cytokine secretion in vitro.

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    <p><b>A</b>, Ana-1 cells pre-transfected with liposome encapsulated siNeg or siCRACC was subsequently stimulated with Poly I:C. The expression of CRACC mRNA was assayed by quantitative PCR at 3h time point post the Poly I:C stimulation. <b>B</b>, <b>C</b> & <b>D</b>, Ana-1 cells pre-transfected with nanoparticle encapsulated siNeg or siCRACC was cultured in absence or presence of NK cells, which were freshly isolated from mice pre-treated by Poly I:C. Then Ana-1 cells cultured with or without NK cells were stimulated with Poly I:C; and D-GalN was made as control. CRACC expression on Ana-1 cells was analyzed by flow cytometry (<b>B</b>) and the cytokine (TNF-α and IFN-γ) in the culture supernatant was assayed by ELISA (<b>C</b>, <b>D</b>) at 24h time point post the Poly I:C or D-GalN stimulation; Ana-1 cells were identified as F4/80<sup>+</sup> cells. <b>E</b>, Ana-1 cells were cocultured with freshly isolated NK cells from mice pre-treated by Poly I:C; and Ana-1 cells alone or NK cells alone were made as control. The Ana-1-NK coculture was stimulated by Poly I:C in the presence of isotype control Ab (Rat IgG), CRACC mAb (anti-CRACC), or NKG2D mAb (anti-NKG2D), respectively. The IFN-γ in culture supernatant was assayed by ELISA at 24h time point post the Poly I:C stimulation. *P < 0.05; **P<0.01; ***P < 0.001.</p

    Poly I:C injection elevates the expression of CRACC on Kupffer cells.

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    <p>CRACC expression on alveolar Mϕ, peritoneal Mϕ and Kupffer cells from mice treated with Poly I:C for 18h was analyzed by flow cytometry. Alveolar Mϕ, peritoneal Mϕ and Kupffer cells were identified as F4/80<sup>+</sup> cells. <b>A</b>, Flow Cytometry of the CRACC expression on alveolar Mϕ, peritoneal Mϕ and Kupffer cells. <b>B</b>, Percent of CRACC<sup>+</sup> cells among alveolar Mϕ, peritoneal Mϕ and Kupffer cells. <b>C</b>, Percent of SLAM family (Ly108, Ly9 and CD84) positive cells among Kupffer cells from mice treated with Poly I:C for 18h. Data are from 6-9 mice per group. *P < 0.05; **P<0.01; ***P < 0.001.</p
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