Yousof Yakoub

Thickness of multiwalled carbon nanotubes affects their lung toxicity.

Two samples of highly pure multiwalled carbon nanotubes (MWCNTs) similar in hydrophobicity and surface reactivity were prepared with similar length, <5 μm, but markedly different diameter (9.4 vs 70 nm). The samples were compared for their cytotoxic activity, uptake, and ability to induce oxidative stress (ROS production and intracellular GSH depletion) in vitro in murine alveolar macrophages (MH-S). The in vivo toxicity was evaluated by measuring biochemical (LDH activity and total proteins) and cellular responses in bronchoalveolar lavage (BAL) after intratracheal instillation in rats. Both samples were internalized in MH-S cells. However, thin MWCNTs appeared significantly more toxic than the thicker ones, both in vitro and in vivo, when compared on a mass-dose basis. The data reported herein suggest that the nanotube diameter is an important parameter to be considered in the toxicological assessment of CNTs.

Platelet-Derived Growth Factor–Producing CD4+ Foxp3+ Regulatory T Lymphocytes Promote Lung Fibrosis

RATIONALE There is evidence that CD4(+) effector T lymphocytes (T eff) participate in the development of lung fibrosis, but the role of their CD4(+) regulatory T-cell (T reg) counterparts remains to be determined. OBJECTIVES To elucidate the contribution of T reg cells in a mouse model of lung fibrosis induced by silica (SiO(2)) particles. METHODS Lung T reg and T eff cells purified from SiO(2)-treated Foxp3-GFP transgenic mice were cocultured with naive lung fibroblasts or transferred to the lungs of healthy mice. DEREG mice, which express the diphtheria toxin receptor under the control of the foxp3 gene, were used to deplete T reg cells during fibrogenesis. MEASUREMENTS AND MAIN RESULTS CD4(+) Foxp3(+) T reg cells were persistently recruited in the lungs in response to SiO(2). T reg accumulation paralleled the establishment of pulmonary immunosuppression and fibrosis. T reg cells highly expressed platelet-derived growth factor (PDGF)-B via a TGF-β autocrine signaling pathway, directly stimulated fibroblast proliferation in vitro, and increased lung collagen deposition upon transfer in the lung of naive mice. The direct profibrotic effects of T reg cells were abolished by the inhibitor of the PDGF-B/TGF-β signaling pathway, imatinib mesylate. Neutralization of T reg-immunosuppressive activity resulted in enhanced accumulation of T eff cells and IL-4-driven pulmonary fibrogenesis, further demonstrating that T reg cells control T eff cell functions during inflammatory fibrosis. CONCLUSIONS Our study indicates that T reg cells contribute to lung fibrosis by stimulating fibroblasts through the secretion of PDGF-B in noninflammatory conditions and regulate detrimental T eff cell activities during inflammation-related fibrosis.

The alarmin IL-1α is a master cytokine in acute lung inflammation induced by silica micro- and nanoparticles

BackgroundInflammasome-activated IL-1β plays a major role in lung neutrophilic inflammation induced by inhaled silica. However, the exact mechanisms that contribute to the initial production of precursor IL-1β (pro-IL-1β) are still unclear. Here, we assessed the implication of alarmins (IL-1α, IL-33 and HMGB1) in the lung response to silica particles and found that IL-1α is a master cytokine that regulates IL-1β expression.MethodsPro- and mature IL-1β as well as alarmins were assessed by ELISA, Western Blot or qRT-PCR in macrophage cultures and in mouse lung following nano- and micrometric silica exposure. Implication of these immune mediators in the establishment of lung inflammatory responses to silica was investigated in knock-out mice or after antibody blockade by evaluating pulmonary neutrophil counts, CXCR2 expression and degree of histological injury.ResultsWe found that the early release of IL-1α and IL-33, but not HMGB1 in alveolar space preceded the lung expression of pro-IL-1β and neutrophilic inflammation in silica-treated mice. In vitro, the production of pro-IL-1β by alveolar macrophages was significantly induced by recombinant IL-1α but not by IL-33. Neutralization or deletion of IL-1α reduced IL-1β production and neutrophil accumulation after silica in mice. Finally, IL-1α released by J774 macrophages after in vitro exposure to a range of micro- and nanoparticles of silica was correlated with the degree of lung inflammation induced in vivo by these particles.ConclusionsWe demonstrated that in response to silica exposure, IL-1α is rapidly released from pre-existing stocks in alveolar macrophages and promotes subsequent lung inflammation through the stimulation of IL-1β production. Moreover, we demonstrated that in vitro IL-1α release from macrophages can be used to predict the acute inflammogenic activity of silica micro- and nanoparticles.

Lung fibrosis induced by crystalline silica particles is uncoupled from lung inflammation in NMRI mice.

Previous studies in rats have suggested a causal relationship between progressive pulmonary inflammation and lung fibrosis induced by crystalline silica particles. We report here that, in NMRI mice, the lung response to silica particles is accompanied by a mild and non progressive pulmonary inflammation which is dispensable for the development of lung fibrosis. We found that glucocorticoid (dexamethasone) dramatically reduced lung injury, cellular inflammation and pro-inflammatory cytokine expression (TNF-α, IL-1β and KC) but had no significant effect on silica-induced lung fibrosis and expression of the fibrogenic and suppressive cytokines TGF-β and IL-10 in mice. Other anti-inflammatory molecules such as the COX inhibitor piroxicam or the phosphodiesterase 5 inhibitor sildenafil also reduced lung inflammation without modifying collagen, TGF-β or IL-10 lung content. Our findings indicate that the development of lung fibrosis in silica-treated NMRI mice is not driven by inflammatory lung responses and suggest that suppressive cytokines may represent critical fibrotic factors and potential therapeutic targets in silicosis.

IL-1α induces CD11b(low) alveolar macrophage proliferation and maturation during granuloma formation.

Macrophages play a central role in immune and tissue responses of granulomatous lung diseases induced by pathogens and foreign bodies. Circulating monocytes are generally viewed as central precursors of these tissue effector macrophages. Here, we provide evidence that granulomas derive from alveolar macrophages serving as a local reservoir for the expansion of activated phagocytic macrophages. By exploring lung granulomatous responses to silica particles in IL‐1‐deficient mice, we found that the absence of IL‐1α, but not IL‐1β, was associated with reduced CD11bhigh phagocytic macrophage accumulation and fewer granulomas. This defect was associated with impaired alveolar clearance and resulted in the development of pulmonary alveolar proteinosis (PAP). Reconstitution of IL‐1α−/− mice with recombinant IL‐1α restored lung clearance functions and the pulmonary accumulation of CD11bhigh phagocytic macrophages. Mechanistically, IL‐1α induced the proliferation of CD11blow alveolar macrophages and differentiated these cells into CD11bhigh macrophages which perform critical phagocytic functions and organize granuloma. We newly discovered here that IL‐1α triggers lung responses requiring macrophage proliferation and maturation from tissue‐resident macrophages. Copyright

Type I Interferon Signaling Contributes to Chronic Inflammation in a Murine Model of Silicosis

Lung disorders induced by inhaled inorganic particles such as crystalline silica are characterized by chronic inflammation and pulmonary fibrosis. Here, we demonstrate the importance of type I interferon (IFN) in the development of crystalline silica-induced lung inflammation in mice, revealing that viruses and inorganic particles share similar signaling pathways. We found that instillation of silica is followed by the upregulation of IFN-beta and IRF-7 and that granulocytes (GR1(+)) and macrophages/dendritic cells (CD11c(+)) are major producers of type I IFN in response to silica. Two months after silica administration, both IFNAR- and IRF-7-deficient mice produced significantly less pulmonary inflammation and chemokines (KC and CCL2) than competent mice but developed similar lung fibrosis. Our data indicate that type I IFN contributes to the chronic lung inflammation that accompanies silica exposure in mice. Type I IFN is, however, dispensable in the development of silica-induced acute lung inflammation and pulmonary fibrosis.

Uncoupling between inflammatory and fibrotic responses to silica: evidence from MyD88 knockout mice.

The exact implication of innate immunity in granuloma formation and irreversible lung fibrosis remains to be determined. In this study, we examined the lung inflammatory and fibrotic responses to silica in MyD88-knockout (KO) mice. In comparison to wild-type (WT) mice, we found that MyD88-KO animals developed attenuated lung inflammation, neutrophil accumulation and IL-1β release in response to silica. Granuloma formation was also less pronounced in MyD88-KO mice after silica. This limited inflammatory response was not accompanied by a concomitant attenuation of lung collagen accumulation after silica. Histological analyses revealed that while pulmonary fibrosis was localized in granulomas in WT animals, it was diffusely distributed throughout the parenchyma in MyD88-KO mice. Robust collagen accumulation was also observed in mice KO for several other components of innate immunity (IL-1R, IL-1, ASC, NALP3, IL-18R, IL-33R, TRIF, and TLR2-3-4,). We additionally show that pulmonary fibrosis in MyD88-KO mice was associated with the accumulation of pro-fibrotic regulatory T lymphocytes (T regs) and pro-fibrotic cytokine expression (TGF-β, IL-10 and PDGF-B), not with T helper (Th) 17 cell influx. Our findings indicate that the activation of MyD88-related innate immunity is central in the establishment of particle-induced lung inflammatory and granuloma responses. The development of lung fibrosis appears uncoupled from inflammation and may be orchestrated by a T reg-associated pathway.

PET/CT with 18F-FDG– and 18F-FBEM–Labeled Leukocytes for Metabolic Activity and Leukocyte Recruitment Monitoring in a Mouse Model of Pulmonary Fibrosis

Idiopathic pulmonary fibrosis is characterized by a progressive and irreversible respiratory failure. Validated noninvasive methods able to assess disease activity are essential for prognostic purposes as well as for the evaluation of emerging antifibrotic treatments. Methods: C57BL/6 mice were used in a murine model of pulmonary fibrosis induced by an intratracheal instillation of bleomycin (control mice were instilled with a saline solution). At different times after instillation, PET/CT with 18F-FDG– or 18F-4-fluorobenzamido-N-ethylamino-maleimide (18F-FBEM)–labeled leukocytes was performed to assess metabolic activity and leukocyte recruitment, respectively. Results: In bleomycin-treated mice, a higher metabolic activity was measured on 18F-FDG PET/CT scans from day 7 to day 24 after instillation, with a peak of activity measured at day 14. Of note, lung mean standardized uptake values correlated with bleomycin doses, histologic score of fibrosis, lung hydroxyproline content, and weight loss. Moreover, during the inflammatory phase of the model (day 7), but not the fibrotic phase (day 23), bleomycin-treated mice presented with an enhanced leukocyte recruitment as assessed by 18F-FBEM–labeled leukocyte PET/CT. Autoradiographic analysis of lung sections and CD45 immunostaining confirm the higher and early recruitment of leukocytes in bleomycin-treated mice, compared with control mice. Conclusion: 18F-FDG– and 18F-FBEM–labeled leukocyte PET/CT enable monitoring of metabolic activity and leukocyte recruitment in a mouse model of pulmonary fibrosis. Implications for preclinical evaluation of antifibrotic therapy are expected.

CCR2+ monocytic myeloid‐derived suppressor cells (M‐MDSCs) inhibit collagen degradation and promote lung fibrosis by producing transforming growth factor‐β1

Monocytes infiltrating scar tissue are predominantly viewed as progenitor cells. Here, we show that tissue CCR2+ monocytes have specific immunosuppressive and profibrotic functions. CCR2+ monocytic cells are acutely recruited to the lung before the onset of silica‐induced fibrosis in mice. These tissue monocytes are defined as monocytic myeloid‐derived suppressor cells (M‐MDSCs) because they significantly suppress T‐lymphocyte proliferation in vitro. M‐MDSCs collected from silica‐treated mice also express transforming growth factor (TGF)‐β1, which stimulates lung fibroblasts to release tissue inhibitor of metalloproteinase (TIMP)‐1, an inhibitor of metalloproteinase collagenolytic activity. By using LysMCreCCR2loxP/loxP mice, we show that limiting CCR2+ M‐MDSC accumulation reduces the pulmonary contents of TGF‐β1, TIMP‐1 and collagen after silica treatment. M‐MDSCs do not differentiate into lung macrophages, granulocytes or fibrocytes during pulmonary fibrogenesis. Collectively, our data indicate that M‐MDSCs contribute to lung fibrosis by specifically promoting a non‐degrading collagen microenvironment. Copyright

Towards predicting the lung fibrogenic activity of MWCNT: Key role of endocytosis, kinase receptors and ERK 1/2 signaling

Abstract Carbon nanotubes (CNT) have been reported to induce lung inflammation and fibrosis in rodents. We investigated the direct and indirect cellular mechanisms mediating the fibrogenic activity of multi-wall (MW) CNT on fibroblasts. We showed that MWCNT indirectly stimulate lung fibroblast (MLg) differentiation, via epithelial cells and macrophages, whereas no direct effect of MWCNT on fibroblast differentiation or collagen production was detected. MWCNT directly stimulated the proliferation of fibroblasts primed with low concentrations of growth factors, such as PDGF, TGF-β or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-β in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-β- and EGF-receptors. This activity was also reduced by amiloride, an endocytosis inhibitor. Finally, the lung fibrotic response to several MWCNT samples (different in length and diameter) correlated with their in vitro capacity to stimulate the proliferation of fibroblasts and to prolong ERK 1/2 signaling in these cells. Our findings point to a crosstalk between MWCNT, kinase receptors, ERK 1/2 signaling and endocytosis which stimulates the proliferation of fibroblasts. The mechanisms of action identified in this study contribute to predict the fibrogenic potential of MWCNT.