Resolution of bleomycin-induced murine pulmonary fibrosis via a splenic lymphocyte subpopulation

Abstract Background Idiopathic pulmonary fibrosis (IPF) is a progressive disease with high mortality, and the pathogenesis of the disease is still incompletely understood. Although lymphocytes, especially CD4+CD25+FoxP3+ regulatory T cells (Tregs), have been implicated in the development of IPF, contradictory results have been reported regarding the contribution of Tregs to fibrosis both in animals and humans. The aim of this study was to investigate whether a specific T cell subset has therapeutic potential in inhibiting bleomycin (BLM)-induced murine pulmonary fibrosis. Methods C57BL/6 mice received BLM (100 mg/kg body weight) with osmotic pumps (day 0), and pulmonary fibrosis was induced. Then, splenocytes or Tregs were adoptively transferred via the tail vein. The lungs were removed and subjected to histological and biochemical examinations to study the effects of these cells on pulmonary fibrosis, and blood samples were collected by cardiac punctures to measure relevant cytokines by enzyme-linked immunosorbent assay. Tregs isolated from an interleukin (IL)-10 knock-out mice were used to assess the effect of this mediator. To determine the roles of the spleen in this model, spleen vessels were carefully cauterized and the spleen was removed either on day 0 or 14 after BLM challenge. Results Splenocytes significantly ameliorated BLM-induced pulmonary fibrosis when they were administered on day 14. This effect was abrogated by depleting Tregs with an anti-CD25 monoclonal antibody. Adoptive transfer of Tregs on day 14 after a BLM challenge significantly attenuated pulmonary fibrosis, and this was accompanied by decreased production of fibroblast growth factor (FGF) 9-positive cells bearing the morphology of alveolar epithelial cells. In addition, BLM-induced plasma IL-10 expression reverted to basal levels after adoptive transfer of Tregs. Moreover, BLM-induced fibrocyte chemoattractant chemokine (CC motif) ligand-2 production was significantly ameliorated by Treg adoptive transfer in lung homogenates, accompanied by reduced accumulation of bone-marrow derived fibrocytes. Genetic ablation of IL-10 abrogated the ameliorating effect of Tregs on pulmonary fibrosis. Finally, splenectomy on day 0 after a BLM challenge significantly ameliorated lung fibrosis, whereas splenectomy on day 14 had no effect. Conclusions These findings warrant further investigations to develop a cell-based therapy using Tregs for treating IPF

Circulating AIM as an indicator of liver damage and hepatocellular carcinoma in humans.

BackgroundHepatocellular carcinoma (HCC), the fifth most common cancer type and the third highest cause of cancer death worldwide, develops in different types of liver injuries, and is mostly associated with cirrhosis. However, non-alcoholic fatty liver disease often causes HCC with less fibrosis, and the number of patients with this disease is rapidly increasing. The high mortality rate and the pathological complexity of liver diseases and HCC require blood biomarkers that accurately reflect the state of liver damage and presence of HCC.Methods and findingsHere we demonstrate that a circulating protein, apoptosis inhibitor of macrophage (AIM) may meet this requirement. A large-scale analysis of healthy individuals across a wide age range revealed a mean blood AIM of 4.99 ± 1.8 µg/ml in men and 6.06 ± 2.1 µg/ml in women. AIM levels were significantly augmented in the younger generation (20s-40s), particularly in women. Interestingly, AIM levels were markedly higher in patients with advanced liver damage, regardless of disease type, and correlated significantly with multiple parameters representing liver function. In mice, AIM levels increased in response to carbon tetrachloride, confirming that the high AIM observed in humans is the result of liver damage. In addition, carbon tetrachloride caused comparable states of liver damage in AIM-deficient and wild-type mice, indicating no influence of AIM levels on liver injury progression. Intriguingly, certain combinations of AIM indexes normalized to liver marker score significantly distinguished HCC patients from non-HCC patients and thus could be applicable for HCC diagnosis.ConclusionAIM potently reveals both liver damage and HCC. Thus, our results may provide the basis for novel diagnostic strategies for this widespread and fatal disease

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

<div><p>Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (<em>SGMS1</em> and <em>2</em>) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.</p> </div

Relationship between the SGMS genes and HCV infection.

<p>(<b>A, B</b>) The correlation between SGMS1/2 and liver HCV-RNA of HCV infected humanized chimeric mice (n = 7). (<b>C</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on HCV in HCV-infected cells. (<b>D</b>) The effect of silencing HCV genome RNA with siRNA (siE-R7: 1 nM) on the expression of SGMS1/2 mRNA measured by RTD-PCR. (<b>E</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM each) measured by RTD-PCR. (<b>F</b>) The effect of silencing SGMS1/2 mRNA with siRNA (3 nM) on HCV replication in FLR 3-1. In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01.</p

Effects of NA808 treatment on sphingomyelin (SM) and ceramide (total and individual molecular species).

<p>(<b>A, B</b>) Relative ratio of total ceramide (<b>A</b>) and SM (<b>B</b>) in uninfected mice (white, n = 4), HCV genotype 1a-infected mice (black, n = 5), and HCV-infected mice treated with NA808 for 14 days (dark gray, 5 mg/kg, n = 4; light gray, 10 mg/kg, n = 3). *<i>p</i><0.05 and **<i>p</i><0.01 compared with HCV-infected mice. (<b>C</b>) SM levels (bars) and HCV RNA levels (black arrowhead) in the livers of mice treated for 14 days with NA808 (5 or 10 mg/kg/day) and untreated chimeric mice. (<b>D, E</b>) Relative intensities of individual ceramide molecular species (<b>D</b>) and individual SM molecular species (<b>E</b>) in uninfected mice (white, n = 3), HCV-infected mice (black, n = 3), and HCV-infected mice treated with NA808 for 14 days (dark gray, 5 mg/kg, n = 2; light gray, 10 mg/kg, n = 1). In all cases, error bars indicate SDs.</p

HCV alters sphingolipid metabolism.

<p>(<b>A, B</b>) Time-course studies of humanized chimeric mice inoculated with human serum samples positive for HCV genotype 1a (<b>A</b>) or 2a (<b>B</b>). (<b>C</b>) mRNA expression of <i>SGMS1</i> and <i>SGMS2</i> in uninfected (white, n = 5) and HCV genotype 1a-infected (black, n = 7) chimeric mice. (<b>D, E</b>) Effects of HCV infection on hepatocyte SM and ceramide levels in humanized chimeric mice. Relative intensity of total ceramide (<b>D</b>) and total shingomyelin (SM) (<b>E</b>) in uninfected mouse hepatocytes (white bar, n = 4), HCV genotype 1a-infected mouse hepatocytes (black bar, n = 5), and HCV genotype 2a-infected mouse hepatocytes (dark gray bar, n = 3). (<b>F</b>) Mass spectrum of SM in Bligh & Dyer extracts of a human hepatocyte cell line (HuH-7 K4). (<b>G, H</b>) Effects of HCV infection on hepatocyte SM and ceramide levels in humanized chimeric mice. Relative intensity of individual ceramide molecular species (<b>G</b>) and individual SM molecular species (<b>H</b>) in uninfected mouse hepatocytes (white bar, n = 3), HCV genotype 1a-infected mouse hepatocytes (black bar, n = 3), and HCV genotype 2a-infected mouse hepatocytes (dark gray bar, n = 3). In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01 compared with uninfected hepatocytes.</p

Inhibition of sphingolipid biosynthesis with hepatotropic serine palmitoyltransferase (SPT) inhibitor NA808 exerts anti-HCV effect.

<p>(<b>A</b>) Serum HCV-RNA levels in response to treatment with NA808 (blue, 5 mg/kg/day, purple, 10 mg/kg/day, n = 6 each), or PegIFN-α (pink, 30 µg/kg twice weekly, n = 4). (<b>B</b>) Effect of NA808 (5 mg/kg/day) on serum HCV-RNA levels. A star indicates that HCV-RNA was not detected. (<b>C</b>) Levels of liver HCV-RNA (black) and HCV core protein (gray) after the 14-day treatment. *<i>p</i><0.05 and **<i>p</i><0.01 compared with no treatment. (<b>D</b>) Histological analysis using immunofluorescent labeling of HCV core protein (green) and fluorescent staining of nuclei (blue). (<b>E</b>) Serum HCV-RNA levels in response to no treatment (pink, n = 3) or NA808 treatment (blue, 5 mg/kg/day, n = 4). (<b>F</b>) Liver HCV-RNA levels in genotype 2a-infected mice after the 14-day treatment. *<i>p</i><0.05 and **<i>p</i><0.01 compared with no treatment. In all cases, error bars indicate SDs.</p

Specific sphingomyelin molecular species upregulated by HCV promote HCV replication on the detergent-resistant membrane fraction.

<p>(<b>A</b>) Comparison of the relative amounts of SM, as measured by MS analysis, in whole cells and the DRM fraction of mock-infected (HuH-7 K4 cells) (white, n = 6; whole cells, n = 3; DRM fraction) and HCV (JFH-1)-infected cells (JFH/K4 cells) (black, n = 6; whole cells, n = 3; DRM fraction). (<b>B</b>) Composition ratio of SM molecular species in whole cells and DRM fraction of HCV-infected cells. (<b>C</b>) Relative intensities of each SM molecular species in the DRM fraction of mock-infected cells (white, n = 2) and HCV-producing cells without (black, n = 2) or with NA808 treatment (gray, n = 2). (<b>D</b>) <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002860#s2" target="_blank">Results</a> of the ELISA SM binding assay (n = 3 each). (<b>E</b>) Average activation kinetics of each SM molecular species on HCR6 (genotype 1b) RdRp (n = 3 each). (<b>F</b>) Scheme of HCV-RNA replicase assay using digitonin-permeabilized cells. (<b>G, H</b>) Effect of each SM molecular species on HCV-RNA in digitonin-permeabilized replicon cells treated without (G) or with 10 nM NA808 (H) (n = 3 each). In all cases, error bars indicate SDs. *<i>p</i><0.05 and **<i>p</i><0.01.</p