Overexpression of Bcl-2 in hepatocytes protects against injury but does not attenuate fibrosis in a mouse model of chronic cholestatic liver disease.

International audienceThe role of hepatocyte apoptosis in the physiopathology of obstructive cholestasis is still controversial. Although some data have strongly suggested that hepatocellular cholestatic injury is due to Fas-mediated hepatocyte apoptosis, some others concluded that necrosis, rather than apoptosis, represents the main type of hepatocyte death in chronic cholestasis. Moreover, it has also been suggested that the reduced liver injury observed in the absence of Fas receptor after bile duct ligation was not due to lower hepatocyte apoptosis but to the indirect role of this receptor in non-hepatocytic cells such as cholangiocytes and inflammatory cells. The aim of this work was therefore to determine whether a protection against cell death limited to hepatocytes could be sufficient to reduce liver injury and delay cholestatic fibrosis. With this purpose, we performed bile duct ligation in transgenic mice overexpressing Bcl-2 in hepatocytes and in wild-type littermates. We found that, compared with necrosis, apoptosis was negligible in this model. Our results also showed that hepatocyte Bcl-2 expression protected hepatocytes against liver injury only in the early steps of the disease. This protection was correlated with reduced mitochondrial dysfunction and lipid peroxidation. However, in contrast to Fas receptor-deficient lpr mice, fibrosis progression was not hampered and liver inflammatory response was not reduced by Bcl-2 overexpression. These results therefore comfort the hypothesis that Fas-mediated apoptotic hepatocyte pathway is not a significant contributing factor to the clinical features observed in cholestasis. Moreover, in the absence of a blunted inflammatory response in transgenic mice, Bcl-2 protection against hepatocyte mitochondrial dysfunction and lipid peroxidation was not sufficient to block fibrosis progression

Increased susceptibility to liver fibrosis with age is correlated with an altered inflammatory response.

International audienceIt has been suggested that increasing age is correlated with an acceleration of the progression of liver fibrosis induced by various agents, such as hepatitis C virus or chronic alcohol consumption. However, the cellular and molecular changes underlying this predisposition are not entirely understood. In the context of an aging population, it becomes challenging to decipher the mechanisms responsible for this higher susceptibility of older individuals to this acquired liver disorder. To address this issue, we induced liver fibrosis by carbon tetrachloride (CCl(4)) chronic administration to 8-week- and 15-month-old mice. We confirmed that susceptibility to fibrosis development increased with age and showed that aging did not affect fibrosis resolution capacity. We then focused on the impairment of hepatocyte proliferation, oxidative stress, and inflammation as potential mechanisms accelerating the development of fibrosis in the elderly. We detected no inhibition of hepatocyte proliferation after CCl(4) injury in 15-month-old mice, whereas it was inhibited after a partial hepatectomy. Finally, we observed that, in a context in which liver oxidative stress was not differentially increased in both experimental groups, there was a higher recruitment of inflammatory cells, including mostly macrophages and lymphocytes, oriented toward a T helper 2 (T(H)2) response in older mice. Our data show that in conditions of equivalent levels of oxidative stress and maintained hepatocyte proliferative capacity, an increased inflammatory reaction mainly composed of CD4(+) lymphocytes and macrophages expressing T(H)2 cytokines is the main factor involved in the higher susceptibility to fibrosis with increasing age

Slow CCL2-dependent translocation of biopersistent particles from muscle to brain.

BACKGROUND: Long-term biodistribution of nanomaterials used in medicine is largely unknown. This is the case for alum, the most widely used vaccine adjuvant, which is a nanocrystalline compound spontaneously forming micron/submicron-sized agglomerates. Although generally well tolerated, alum is occasionally detected within monocyte-lineage cells long after immunization in presumably susceptible individuals with systemic/neurologic manifestations or autoimmune (inflammatory) syndrome induced by adjuvants (ASIA). METHODS: On the grounds of preliminary investigations in 252 patients with alum-associated ASIA showing both a selective increase of circulating CCL2, the major monocyte chemoattractant, and a variation in the CCL2 gene, we designed mouse experiments to assess biodistribution of vaccine-derived aluminum and of alum-particle fluorescent surrogates injected in muscle. Aluminum was detected in tissues by Morin stain and particle induced X-ray emission) (PIXE) Both 500 nm fluorescent latex beads and vaccine alum agglomerates-sized nanohybrids (Al-Rho) were used. RESULTS: Intramuscular injection of alum-containing vaccine was associated with the appearance of aluminum deposits in distant organs, such as spleen and brain where they were still detected one year after injection. Both fluorescent materials injected into muscle translocated to draining lymph nodes (DLNs) and thereafter were detected associated with phagocytes in blood and spleen. Particles linearly accumulated in the brain up to the six-month endpoint; they were first found in perivascular CD11b+ cells and then in microglia and other neural cells. DLN ablation dramatically reduced the biodistribution. Cerebral translocation was not observed after direct intravenous injection, but significantly increased in mice with chronically altered blood-brain-barrier. Loss/gain-of-function experiments consistently implicated CCL2 in systemic diffusion of Al-Rho particles captured by monocyte-lineage cells and in their subsequent neurodelivery. Stereotactic particle injection pointed out brain retention as a factor of progressive particle accumulation. CONCLUSION: Nanomaterials can be transported by monocyte-lineage cells to DLNs, blood and spleen, and, similarly to HIV, may use CCL2-dependent mechanisms to penetrate the brain. This occurs at a very low rate in normal conditions explaining good overall tolerance of alum despite its strong neurotoxic potential. However, continuously escalating doses of this poorly biodegradable adjuvant in the population may become insidiously unsafe, especially in the case of overimmunization or immature/altered blood brain barrier or high constitutive CCL-2 production.European Community's Seventh Framework Programme in the project ENDOSTEM "Activation of vasculatur

Immunosuppressive properties of lung fibroblasts.

<p>(a) Proliferation of phytohemagglutinin (PHA)-stimulated PBMC estimated ³H thymidine incorporation after 5-day exposure to supernatants collected from 24-h cultured MSC, DF and lung fibroblasts from C-NS, C-S and COPD subjects. (b) Proliferation of CD3/CD28 activated PBMC estimated by CFSE staining 4 days after exposure to supernatants collected from 24-h cultured lung fibroblasts. (a, b)NS: non significant, * p<0.05. MSC, mesenchymal stem cells, DF, dermal fibroblasts; C-NS: non-smokers controls; C-S: smoker controls; COPD: smokers with COPD. Results are expressed as means ± SEM of at least n = 3 independent experiments made with the 25 patient fibroblasts.</p

<i>In vitro</i> plasticity of lung fibroblasts.

<p>(a)Rate of lung fibroblast conversion into myofibroblasts after 3, 4 and 5 days exposure to TGF-β1. * p<0.05 compared to untreated cells. # p<0.05 compared to non- smoker (C-NS) and smoker controls (C-S) exposed to TGF-β1 for 4 or 5 days. (b) Oil-red O staining of, MSC, DF and lung fibroblasts from C-NS, C-S and COPD subjects12 days after exposure to adipogenic differentiation medium. (c)Alcian blue staining of MSC, DF and lung fibroblasts at day 24 following chondrogenesis initiation. (d,e)<b>(D, E)</b> Comparative real-time PCR analysis of mRNA expression of genes encoding for (d) adipogenic peroxysome proliferative activated receptor gamma 2 (PPAR-γ 2; early), lipoprotein lipase (LPL; late) and (e)chondrogenic Sox-9 marker. (f) Alizarin red staining of cells following 17 days of osteoblastic induction and (g)real-time PCR analysis of mRNA expression of osteoblastic alkaline phosphatase (ALP, early) and osteocalcin (OC, late) markers. (d,e,g) Results represent means ± SEM of at least 3 independent experiments performed with donor fibroblasts. NS: non significant; * p<0.05, ** p<0.01. MSC, mesenchymal stem cells; DF, dermal fibroblast; C-NS, non-smokers controls; C-S, smoker controls; COPD, smokers with COPD.</p

Lung Fibroblasts Share Mesenchymal Stem Cell Features Which Are Altered in Chronic Obstructive Pulmonary Disease via the Overactivation of the Hedgehog Signaling Pathway

<div><p>Background</p><p>Alteration of functional regenerative properties of parenchymal lung fibroblasts is widely proposed as a pathogenic mechanism for chronic obstructive pulmonary disease (COPD). However, what these functions are and how they are impaired in COPD remain poorly understood. Apart from the role of fibroblasts in producing extracellular matrix, recent studies in organs different from the lung suggest that such cells might contribute to repair processes by acting like mesenchymal stem cells. In addition, several reports sustain that the Hedgehog pathway is altered in COPD patients thus aggravating the disease. Nevertheless, whether this pathway is dysregulated in COPD fibroblasts remains unknown.</p><p>Objectives and Methods</p><p>We investigated the stem cell features and the expression of Hedgehog components in human lung fibroblasts isolated from histologically-normal parenchymal tissue from 25 patients—8 non-smokers/non-COPD, 8 smokers-non COPD and 9 smokers with COPD—who were undergoing surgery for lung tumor resection.</p><p>Results</p><p>We found that lung fibroblasts resemble mesenchymal stem cells in terms of cell surface marker expression, differentiation ability and immunosuppressive potential and that these properties were altered in lung fibroblasts from smokers and even more in COPD patients. Furthermore, we showed that some of these phenotypic changes can be explained by an over activation of the Hedgehog signaling in smoker and COPD fibroblasts.</p><p>Conclusions</p><p>Our study reveals that lung fibroblasts possess mesenchymal stem cell-features which are impaired in COPD via the contribution of an abnormal Hedgehog signaling. These processes should constitute a novel pathomechanism accounting for disease occurrence and progression.</p></div

Subject characteristics.

<p>Data are expressed as median and minimum and maximum quartiles (in parentheses) $ p<0.05 COPD patients vs. non smokers and smokers</p><p>*p<0.05 COPD patients vs. smokers.</p><p>** p< 0.01 COPD patients vs. non-smokers.</p><p>VC, vital capacity; FEV₁, forced expiratory volume in 1 sec; COPD, chronic obstructive pulmonary disease; GOLD, Global Initiative for Chronic Obstructive Lung Disease classification</p><p>Subject characteristics.</p

Cell-surface expression of MSC-associated markers.

<p>(a) Representative flow cytometry of CD34, CD117, CD45, CD133, CD15, CD166, VEGF-R, HLA-DR, CD44, CD105 and HLA-I cell-surface markers in mesenchymal stem cells, dermal fibroblasts and lung fibroblasts from non-smoker controls (C-NS), smoker controls (C-S) and COPD smokers (COPD). The dotted black line represents control immunoglobulin and the red refers to the specific marker. (b)Flow cytometry of CD90, CD13 and STRO-1. The dotted black line represents control immunoglobulins; the red line refers to the specific marker in non-smoker control fibroblasts (C-NS) and the blue represents smoker control (C-S) or COPD lung fibroblasts. (c) Histograms representing mean fluorescence variations for CD90, CD13 and STRO-1 between lung fibroblasts from the 3 groups (C-NS: n = 8, C-S: n = 8 and COPD: n = 9). NS, non significant, * p<0.05.</p

Primer sequences used for qPCR assays.

<p>Primer sequences used for qPCR assays.</p

Protection against Hepatocyte Mitochondrial Dysfunction Delays Fibrosis Progression in Mice

Accumulating evidence indicates that oxidative stress is involved in the physiopathology of liver fibrogenesis. However, amid the global context of hepatic oxidative stress, the specific role of hepatocyte mitochondrial dysfunction in the fibrogenic process is still unknown. The aim of this study was to determine whether a targeted protection of hepatocytes against mitochondrial dysfunction could modulate fibrosis progression. We induced liver fibrogenesis by chronic carbon tetrachloride treatment (3 or 6 weeks of biweekly injections) in transgenic mice expressing Bcl-2 in their hepatocytes or in normal control mice. Analyses of mitochondrial DNA, respiratory chain complexes, and lipid peroxidation showed that Bcl-2 transgenic animals were protected against mitochondrial dysfunction and oxidative stress resulting from carbon tetrachloride injury. Picrosirius red staining, α-smooth muscle actin immunohistochemistry, and real-time PCR for transforming growth factor-β and collagen α-I revealed that Bcl-2 transgenic mice presented reduced fibrosis at early stages of fibrogenesis. However, at later stages increased nonmitochondrial/nonhepatocytic oxidative stress eventually overcame the capacity of Bcl-2 overexpression to prevent the fibrotic process. In conclusion, we demonstrate for the first time that specific protection against hepatocyte mitochondrial dysfunction plays a preventive role in early stages of fibrogenesis, delaying its onset. However, with the persistence of the aggression, this protection is no longer sufficient to impede fibrosis progression