Delphine Goven

Bilirubin decreases nos2 expression via inhibition of NAD(P)H oxidase : implications for protection against endotoxic shock in rats

We investigated a possible beneficial role for bilirubin, one of the products of heme degradation by the cytoprotective enzyme heme oxygenase‐1 in counteracting Escherichia coli endotoxin‐mediated toxicity. Homozygous jaundice Gunn rats, which display high plasma bilirubin levels due to deficiency of glucuronyl transferase activity, and Sprague‐Dawley rats subjected to sustained exogenous bilirubin administration were more resistant to endotoxin (LPS)‐induced hypotension and death compared with nonhyperbilirubinemic rats. LPS‐stimulated production of nitric oxide (NO) was significantly decreased in hyperbilirubinemic rats compared with normal animals; this effect was associated with reduction of inducible NO synthase (NOS2) expression in renal, myocardial, and aortic tissues. Furthermore, NOS2 protein expression and activity were reduced in murine macrophages stimulated with LPS and preincubated with bilirubin at concentrations similar to that found in the serum of hyperbilirubinemic animals. This effect was secondary to inhibition of NAD(P)H oxidase since 1) inhibition of NAD(P)H oxidase attenuated NOS2 induction by LPS, 2) bilirubin decreased NAD(P)H oxidase activity in vivo and in vitro, and 3) down‐regulation of NOS2 by bilirubin was reversed by addition of NAD(P)H. These findings indicate that bilirubin can act as an effective agent to reduce mortality and counteract hypotension elicited by endotoxin through mechanisms involving a decreased NOS2 induction secondary to inhibition of NAD(P)H oxidase.

Nuclear Factor Erythroid 2-Related Factor 2 Nuclear Translocation Induces Myofibroblastic Dedifferentiation in Idiopathic Pulmonary Fibrosis

AIMS Oxidants have been implicated in the pathophysiology of idiopathic pulmonary fibrosis (IPF), especially in myofibroblastic differentiation. We aimed at testing the hypothesis that nuclear factor erythroid 2-related factor 2 (Nrf2), the main regulator of endogenous antioxidant enzymes, is involved in fibrogenesis via myofibroblastic differentiation. Fibroblasts were cultured from the lungs of eight controls and eight IPF patients. Oxidants-antioxidants balance, nuclear Nrf2 expression, and fibroblast phenotype (α-smooth muscle actin and collagen I expression, proliferation, migration, and contraction) were studied under basal conditions and after Nrf2 knockdown or activation by Nrf2 or Keap1 siRNA transfection. The effects of sulforaphane (SFN), an Nrf2 activator, on the fibroblast phenotype were tested under basal and pro-fibrosis conditions (transforming growth factor β [TGF-β]). RESULTS Decreased Nrf2 expression was associated with a myofibroblast phenotype in IPF compared with control fibroblasts. Nrf2 knockdown induced oxidative stress and myofibroblastic differentiation in control fibroblasts. Conversely, Nrf2 activation increased antioxidant defences and myofibroblastic dedifferentation in IPF fibroblasts. SFN treatment decreased oxidants, and induced Nrf2 expression, antioxidants, and myofibroblastic dedifferentiation in IPF fibroblasts. SFN inhibited TGF-β profibrotic deleterious effects in IPF and control fibroblasts and restored antioxidant defences. Nrf2 knockdown abolished SFN antifibrosis effects, suggesting that they were Nrf2 mediated. INNOVATION AND CONCLUSION Our findings confirm that decreased nuclear Nrf2 plays a role in myofibroblastic differentiation and that SFN induces human pulmonary fibroblast dedifferentiation in vitro via Nrf2 activation. Thus, Nrf2 could be a novel therapeutic target in IPF.

A Role for Dendritic Cells in Bleomycin-induced Pulmonary Fibrosis in Mice?

RATIONALE Lung dendritic cells (DCs) have been shown to accumulate in human fibrotic lung disease, but little is known concerning a role for DCs in the pathogenesis of fibrotic lung. OBJECTIVES To characterize lung DCs in an in vivo model of bleomycin-induced pulmonary fibrosis in mice. METHODS We characterized the kinetics and activation of pulmonary DCs during the course of bleomycin-induced lung injury by flow cytometry on lung single-cell suspensions. We also characterized the lymphocytes accumulating in bleomycin lung and the chemokines susceptible to favor the recruitment of immune cells. MEASUREMENTS AND MAIN RESULTS We show, for the first time, that increased numbers of CD11c(+)/major histocompatibility complex class II(+) DCs, including CD11b(hi) monocyte-derived inflammatory DCs, infiltrate the lung of treated animals during the fibrotic phase of the response to bleomycin. These DCs are mature DCs expressing CD40, CD86, and CD83. They are associated with increased numbers of recently activated memory T cells expressing CD44, CD40L, and CD28, suggesting that fully mature DCs and Ag-experienced T cells can drive an efficient effector immune response within bleomycin lung. Most importantly, when DCs are inactivated with VAG539, a recently described new immunomodulator, VAG539 treatment attenuates the hallmarks of bleomycin lung injury. CONCLUSIONS These findings identify lung DCs as key proinflammatory cells potentially able to sustain pulmonary inflammation and fibrosis in the bleomycin model.

Dysregulation of elastin expression by fibroblasts in pulmonary emphysema: role of cellular retinoic acid binding protein 2

Background: All-trans retinoic acid (ATRA) stimulates elastin synthesis by lung fibroblasts and induces alveolar regeneration in animal models of pulmonary emphysema. However, ATRA treatment has had disappointing results in human emphysema. It was hypothesised that a defect in the ATRA signalling pathway contributes to the defect of alveolar repair in the human emphysematous lung. Methods: Fibroblasts were cultured from the lung of 10 control subjects and eight patients with emphysema. Elastin and retinoic acid receptor (RAR)-β mRNAs were measured in those cells in the presence of incremental concentrations of ATRA. RARs, retinoic X receptors (RXRs) and cellular retinoic acid binding protein (CRABP) 1 and 2 mRNAs were measured as well as CRABP2 protein content. The effect of CRABP2 silencing on elastin and RAR-β expression in response to ATRA was measured in MRC5 lung fibroblasts. Results: ATRA at 10−9 M and 10−8 M increased median elastin mRNA expression by 182% and 126% in control but not in emphysema fibroblasts. RAR-β mRNA expression was induced by ATRA in control as well as emphysema fibroblasts. RARs, RXRs and CRABP1 mRNAs were similarly expressed in control and emphysema fibroblasts while CRABP2 mRNA and protein were lower in emphysema fibroblasts. CRABP2 silencing abrogated the induction of elastin but not RAR-β expression by ATRA in MRC5 fibroblasts. Conclusion: Pulmonary emphysema fibroblasts fail to express elastin under ATRA stimulation. CRABP2, which is necessary for elastin induction by ATRA in MRC-5 cells, is expressed at low levels in emphysema fibroblasts. This alteration in the retinoic acid signalling pathway in lung fibroblasts may contribute to the defect of alveolar repair in human pulmonary emphysema. These results are the first demonstration of the involvement of CRABP2 in elastin expression.

Role of nitric oxide synthases in elastase-induced emphysema

Nitric oxide (NO) in combination with superoxide produces peroxynitrites and induces protein nitration, which participates in a number of chronic degenerative diseases. NO is produced at high levels in the human emphysematous lung, but its role in this disease is unknown. The aim of this study was to determine whether the NO synthases contribute to the development of elastase-induced emphysema in mice. nNOS, iNOS, and eNOS were quantified and immunolocalized in the lung after a tracheal instillation of elastase in mice. To determine whether eNOS or iNOS had a role in the development of emphysema, mice bearing a germline deletion of the eNOS and iNOS genes and mice treated with a pharmacological iNOS inhibitor were exposed to elastase. Protein nitration was determined by immunofluorescence, protein oxidation was determined by ELISA. Inflammation and MMP activity were quantified by cell counts, RT–PCR and zymography in bronchoalveolar lavage fluid. Cell proliferation was determined by Ki67 immunostaining. Emphysema was quantified morphometrically. iNOS and eNOS were diffusely upregulated in the lung of elastase-treated mice and a 12-fold increase in the number of 3-nitrotyrosine-expressing cells was observed. Over 80% of these cells were alveolar type 2 cells. In elastase-instilled mice, iNOS inactivation reduced protein nitration and increased protein oxidation but had no effect on inflammation, MMP activity, cell proliferation or the subsequent development of emphysema. eNOS inactivation had no effect. In conclusion, in the elastase-injured lung, iNOS mediates protein nitration in alveolar type 2 cells and alleviates oxidative injury. Neither eNOS nor iNOS are required for the development of elastase-induced emphysema.