HIF-1α is a key mediator of the lung inflammatory potential of lithium-ion battery particles

BACKGROUND: Li-ion batteries (LIB) are increasingly used worldwide. They are made of low solubility micrometric particles, implying a potential for inhalation toxicity in occupational settings and possibly for consumers. LiCoO2 (LCO), one of the most used cathode material, induces inflammatory and fibrotic lung responses in mice. LCO also stabilizes hypoxia-inducible factor (HIF) -1α, a factor implicated in inflammation, fibrosis and carcinogenicity. Here, we investigated the role of cobalt, nickel and HIF-1α as determinants of toxicity, and evaluated their predictive value for the lung toxicity of LIB particles in in vitro assays. RESULTS: By testing a set of 5 selected LIB particles (LCO, LiNiMnCoO2, LiNiCoAlO2) with different cobalt and nickel contents, we found a positive correlation between their in vivo lung inflammatory activity, and (i) Co and Ni particle content and their bioaccessibility and (ii) the stabilization of HIF-1α in the lung. Inhibition of HIF-1α with chetomin or PX-478 blunted the lung inflammatory response to LCO in mice. In IL-1β deficient mice, HIF-1α was the upstream signal of the inflammatory lung response to LCO. In vitro, the level of HIF-1α stabilization induced by LIB particles in BEAS-2B cells correlated with the intensity of lung inflammation induced by the same particles in vivo. CONCLUSIONS: We conclude that HIF-1α, stabilized in lung cells by released Co and Ni ions, is a mechanism-based biomarker of lung inflammatory responses induced by LIB particles containing Co/Ni. Documenting the Co/Ni content of LIB particles, their bioaccessibility and their capacity to stabilize HIF-1α in vitro can be used to predict the lung inflammatory potential of LIB particles

Mesothelioma response to carbon nanotubes is associated with an early and selective accumulation of immunosuppressive monocytic cells

BACKGROUND: The asbestos-like toxicity of some engineered carbon nanotubes (CNT), notably their capacity to induce mesothelioma, is a serious cause of concern for public health. Here we show that carcinogenic CNT induce an early and sustained immunosuppressive response characterized by the accumulation of monocytic Myeloid Derived Suppressor Cells (M-MDSC) that counteract effective immune surveillance of tumor cells. METHODS: Wistar rats and C57BL/6 mice were intraperitoneally injected with carcinogenic multi-walled Mitsui-7 CNT (CNT-7) or crocidolite asbestos. Peritoneal mesothelioma development and immune cell accumulation were assessed until 12 months. Leukocyte sub-populations were identified by recording expression of CD11b/c and His48 by flow cytometry. The immunosuppressive activity on T lymphocytes of purified peritoneal leukocytes was assessed in a co-culture assay with activated spleen cells. RESULTS: We demonstrate that long and short mesotheliomagenic CNT-7 injected in the peritoneal cavity of rats induced, like asbestos, an early and selective accumulation of monocytic cells (CD11b/c(int) and His48(hi)) which possess the ability to suppress polyclonal activation of T lymphocytes and correspond to M-MDSC. Peritoneal M-MDSC persisted during the development of peritoneal mesothelioma in CNT-7-treated rats but were only transiently recruited after non-carcinogenic CNT (CNT-M, CNT-T) injection. Peritoneal M-MDSC did not accumulate in mice which are resistant to mesothelioma development. CONCLUSIONS: Our data provide new insights into the initial pathogenic events induced by CNT, adding a new component to the adverse outcome pathway leading to mesothelioma development. The specificity of the M-MDSC response after carcinogenic CNT exposure highlights the interest of this response for detecting the ability of new nanomaterials to cause cancer

Vardenafil Restores Pro/Anti Inflammatory Balance in Mouse CF Macrophages

Lung inflammation is a key feature of CF pathology. We have previously shown that, even in basal conditions, CFTRtm1Eur (homozygous F508del-CFTR) mice display an increased number of alveolar and peritoneal macrophages compared to their WT littermates. Promising anti-inflammatory properties have been reported for Vardenafil, a phosphodiesterase type 5 inhibitor (iPDE5). We have recently demonstrated that this iPDE5 reduces LPS-induced inflammatory responses in CF mice (Lubamba et al., 2012). We hypothesized that: 1) the activity of macrophages is altered in CF and 2) macrophages represent target effector cells of the anti-inflammatory effect of vardenafil. Macrophages were isolated and purified from lung homogenates and peritoneal lavages from CF and WT mice. To test the hypothesis that the activity of macrophages is altered in CF, LPS-induced inflammatory responses were first evaluated. Then macrophage differentiation in pro- (M1) and anti-inflammatory (M2) effectors was studied after polarization with LPS and IFN-γ or IL-4 and IL-13, respectively. Pro- (TNF-α, IL-1β and CCL-2) and anti-inflammatory (FIZZ-1 and mYm1) mediators were quantified by ELISA (in culture supernatants) or by quantitative RT-PCR (in cultured macrophages). In each condition, the effect of vardenafil (50µM) was evaluated. CF lung and peritoneal macrophages displayed an exaggerated pro-inflammatory response to LPS and vardenafil treatment reduced it. M1/M2 polarization was altered in CF macrophages. In lung and peritoneal macrophages, M1 response was at least 4-fold larger in CF than in WT cells. In contrast, the M2 response was reduced in CF macrophages. These results confirm a deregulation of the pro/anti-inflammatory balance in CF macrophages. Vardenafil restores the balance by reducing the expression of pro-inflammatory mediators (particularly TNF-α and IL-1β) in lung and peritoneal macrophages. Moreover, vardenafil corrects the overproduction of FIZZ-1 in CF macrophages. Our results show that macrophages play a critical role in inflammatory responses in CF. Differentiation of macrophages from different body compartments is oriented towards a pro-inflammatory profile associated with a decreased anti-inflammatory status. The immunomodulatory effect of vardenafil could thus be beneficial in CF pharmacotherapy

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

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

Beneficial immunomodulatory effect of vardenafil in mouse CF macrophages

Objectives: Mucociliary clearance (MCC), abnormal in CF, is physiologically regulated by epithelial ion transport together with mechanical phenomenon such as cilia movement and cough. Planar Cell Polarity (PCP) is a tightly controlled protein network that drives ciliogenesis and cilia function. Cilia structure and function have been studies in CF. Although the majority of these studies showed no structural abnormality and a normal cilia beat frequency (CBF), it has also been showed that ciliary disorientation, rather than ultrastructural abnormalities or slow CBF, may occur secondary to lung inflammation and result in delayed MCC. We hypothesized that CF HBEs may display abnormalities in PCP network which could further impair coordinated cilia function in the plan of the epithelium. Methods: We profiled PCP genes expression by PCR in HBEs. By quantitative RT-PCR, we determined that expression of CELSR3 and Vangl-1 (Van-Gogh like 1) is down-regulated in CF (F508del/F508del) HBEs as compared to NL cells. In contrast, Fz3, Fz6, Pk1 and Vangl-2 genes expression is upregulated in CF cells. In silico analysis revealed that Vangl-1 and Vangl-2, Pk1, Dvl1, Dvl2, and CELSR3 are putative targets of miR-30 family. Expression of miR-30a, miR-30b, miR-30c, miR-30d and miR-30e is elevated is CF-HBEs. Knock-down of miR-30s expression in CF-HBEs significantly increased expression of CELSR3, Fz6, Vangl-1, Pk1, Dvl 1 and Dvl2 but did not modify expression of Fz3, Vangl-2 and Dvl3. Conclusion: Our results validate several PCP genes as miR-30s target genes and suggest that decreasing miR-30s expression in CF HBEs could modulate cilia function through restoration of PCP genes expression

Dietary silver nanoparticles can disturb the gut microbiota in mice

International audienceBackground: Humans are increasingly exposed via the diet to Ag nanoparticles (NP) used in the food industry. Because of their anti-bacterial activity, ingested Ag NP might disturb the gut microbiota that is essential for local and systemic homeostasis. We explored here the possible impact of dietary Ag NP on the gut microbiota in mice at doses relevant for currently estimated human intake. Methods: Mice were orally exposed to food (pellets) supplemented with increasing doses of Ag NP (0, 46, 460 or 4600 ppb) during 28 d. Body weight, systemic inflammation and gut integrity were investigated to determine overall toxicity, and feces DNA collected from the gut were analyzed by Next Generation Sequencing (NGS) to assess the effect of Ag NP on the bacterial population. Ag NP were characterized alone and in the supplemented pellets by scanning transmission electron microscopy (STEM) and energy dispersive X-ray analysis (EDX). Results: No overall toxicity was recorded in mice exposed to Ag NP. Ag NP disturbed bacterial evenness (alpha-diversity) and populations (beta-diversity) in a dose-dependent manner. Ag NP increased the ratio between Firmicutes (F) and Bacteroidetes (B) phyla. At the family level, Lachnospiraceae and the S24-7 family mainly accounted for the increase in Firmicutes and decrease in Bacteroidetes, respectively. Similar effects were not observed in mice identically exposed to the same batch of Ag NP-supplemented pellets aged during 4 or 8 months and the F/B ratio was less or not modified. Analysis of Ag NP-supplemented pellets showed that freshly prepared pellets released Ag ions faster than aged pellets. STEM-EDX analysis also showed that Ag sulfidation occurred in aged Ag NP-supplemented pellets. Conclusions: Our data indicate that oral exposure to human relevant doses of Ag NP can induce microbial alterations in the gut. The bacterial disturbances recorded after Ag NP are similar to those reported in metabolic and inflammatory diseases, such as obesity. It also highlights that Ag NP aging in food, and more specifically sulfidation, can reduce the effects of Ag NP on the microbiota by limiting the release of toxic Ag ions

Towards predicting the lung fibrogenic activity of nanomaterials: experimental validation of an in vitro fibroblast proliferation assay

Background Carbon nanotubes (CNT) can induce lung inflammation and fibrosis in rodents. Several studies have identified the capacity of CNT to stimulate the proliferation of fibroblasts. We developed and validated experimentally here a simple and rapid in vitro assay to evaluate the capacity of a nanomaterial to exert a direct pro-fibrotic effect on fibroblasts. Methods The activity of several multi-wall (MW)CNT samples (NM400, the crushed form of NM400 named NM400c, NM402 and MWCNTg 2400) and asbestos (crocidolite) was investigated in vitro and in vivo. The proliferative response to MWCNT was assessed on mouse primary lung fibroblasts, human fetal lung fibroblasts (HFL-1), mouse embryonic fibroblasts (BALB-3T3) and mouse lung fibroblasts (MLg) by using different assays (cell counting, WST-1 assay and propidium iodide PI staining) and dispersion media (fetal bovine serum, FBS and bovine serum albumin, BSA). C57BL/6 mice were pharyngeally aspirated with the same materials and lung fibrosis was assessed after 2 months by histopathology, quantification of total collagen lung content and pro-fibrotic cytokines in broncho-alveolar lavage fluid (BALF). Results MWCNT (NM400 and NM402) directly stimulated fibroblast proliferation in vitro in a dose-dependent manner and induced lung fibrosis in vivo. NM400 stimulated the proliferation of all tested fibroblast types, independently of FBS- or BSA- dispersion. Results obtained by WST1 cell activity were confirmed with cell counting and cell cycle (PI staining) assays. Crocidolite also stimulated fibroblast proliferation and induced pulmonary fibrosis, although to a lesser extent than NM400 and NM402. In contrast, shorter CNT (NM400c and MWCNTg 2400) did not induce any fibroblast proliferation or collagen accumulation in vivo, supporting the idea that CNT structure is an important parameter for inducing lung fibrosis. Conclusions In this study, an optimized proliferation assay using BSA as a dispersant, MLg cells as targets and an adaptation of WST-1 as readout was developed. The activity of MWCNT in this test strongly reflects their fibrotic activity in vivo, supporting the predictive value of this in vitro assay in terms of lung fibrosis potential