Dietary nanoparticles alter the composition and function of the gut microbiota in mice at dose levels relevant for human exposure.

BACKGROUND: Nanotechnologies provide new opportunities for improving the safety, quality, shelf life, flavor and appearance of foods. The most common nanoparticles (NPs) in human diet are silver metal, mainly present in food packaging and appliances, and silicon and titanium dioxides used as additives. The rapid development and commercialization of consumer products containing these engineered NPs is, however, not well supported by appropriate toxicological studies and risk assessment. Local and systemic toxicity and/or disruption of the gut microbiota (GM) have already been observed after oral administration of NPs in experimental animals, but results are not consistent and doses used were often much higher than the estimated human intakes. In view of the strong evidence linking alterations of the GM to cardiometabolic (CM) diseases, we hypothesized that dietary NPs might disturb this GM-CM axis. MATERIALS AND METHODS: We exposed male C57BL/6JRj mice (n = 13 per dose group) to dietary NPs mixed in food pellets at doses relevant for human exposure: Ag (0, 4, 40 or 400 µg/kg pellet), SiO2 (0, 0.8, 8 and 80 mg/kg pellet) or TiO2 (0, 0.4, 4 or 40 mg/kg pellet). After 24 weeks of exposure, we assessed effects on the GM and CM health (n = 8 per dose group). The reversibility of the effects was examined after 8 additional weeks without NPs exposure (recovery period, n ≤ 5 per dose group). RESULTS: No overt toxicity was recorded. The GM ß-diversity was dose-dependently disrupted by the three NPs, and the bacterial short chain fatty acids (SCFAs) were dose-dependently reduced after the administration of SiO2 and TiO2 NPs. These effects disappeared completely or partly after the recovery period, strengthening the association with dietary NPs. We did not observe atheromatous disease or glucose intolerance after NP exposure. Instead, dose-dependent decreases in the expression of IL-6 in the liver, circulating triglycerides (TG) and urea nitrogen (BUN) were recorded after administration of the NPs. CONCLUSION: We found that long-term oral exposure to dietary NPs at doses relevant for estimated human intakes disrupts the GM composition and function. These modifications did not appear associated with atheromatous or deleterious metabolic outcomes.

Femtosecond pulsed laser microscopy: a new tool to assess the in vitro delivered dose of carbon nanotubes in cell culture experiments.

BACKGROUND: In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs). METHODS AND RESULTS: We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1ß by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1ß responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples. CONCLUSION: The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.

The pulmonary toxicity of carboxylated or aminated multi-walled carbon nanotubes in mice is determined by the prior purification method.

BACKGROUND: Inhalation of multi-walled carbon nanotubes (MWCNTs) poses a potential risk to human health. In order to safeguard workers and consumers, the toxic properties of MWCNTs need to be identified. Functionalization has been shown to either decrease or increase MWCNT-related pulmonary injury, depending on the type of modification. We, therefore, investigated both acute and chronic pulmonary toxicity of a library of MWCNTs derived from a common pristine parent compound (NC7000). METHODS: MWCNTs were thermally or chemically purified and subsequently surface functionalized by carboxylation or amination. To evaluate pulmonary toxicity, male C57BL6 mice were dosed via oropharyngeal aspiration with either 1.6 or 4 mg/kg of each MWCNT type. Mitsui-7 MWCNT was used as a positive control. Necropsy was performed at days 3 and 60 post-exposure to collect bronchoalveolar lavage fluid (BALF) and lungs. RESULTS: At day 3 all MWCNTs increased the number of neutrophils in BALF. Chemical purification had a greater effect on pro-inflammatory cytokines (IL-1ß, IL-6, CXCL1) in BALF, while thermal purification had a greater effect on pro-fibrotic cytokines (CCL2, OPN, TGF-ß1). At day 60, thermally purified, carboxylated MWCNTs had the strongest effect on lymphocyte numbers in BALF. Thermally purified MWCNTs caused the greatest increase in LDH and total protein in BALF. Furthermore, the thermally purified and carboxyl- or amine-functionalized MWCNTs caused the greatest number of granulomatous lesions in the lungs. The physicochemical characteristics mainly associated with increased toxicity of the thermally purified derivatives were decreased surface defects and decreased amorphous content as indicated by Raman spectroscopy. CONCLUSIONS: These data demonstrate that the purification method is an important determinant of lung toxicity induced by carboxyl- and amine-functionalized MWCNTs.

Adverse outcome pathways as a tool for the design of testing strategies to support the safety assessment of emerging advanced materials at the nanoscale.

Toxicity testing and regulation of advanced materials at the nanoscale, i.e. nanosafety, is challenged by the growing number of nanomaterials and their property variants requiring assessment for potential human health impacts. The existing animal-reliant toxicity testing tools are onerous in terms of time and resources and are less and less in line with the international effort to reduce animal experiments. Thus, there is a need for faster, cheaper, sensitive and effective animal alternatives that are supported by mechanistic evidence. More importantly, there is an urgency for developing alternative testing strategies that help justify the strategic prioritization of testing or targeting the most apparent adverse outcomes, selection of specific endpoints and assays and identifying nanomaterials of high concern. The Adverse Outcome Pathway (AOP) framework is a systematic process that uses the available mechanistic information concerning a toxicological response and describes causal or mechanistic linkages between a molecular initiating event, a series of intermediate key events and the adverse outcome. The AOP framework provides pragmatic insights to promote the development of alternative testing strategies. This review will detail a brief overview of the AOP framework and its application to nanotoxicology, tools for developing AOPs and the role of toxicogenomics, and summarize various AOPs of relevance to inhalation toxicity of nanomaterials that are currently under various stages of development. The review also presents a network of AOPs derived from connecting all AOPs, which shows that several adverse outcomes induced by nanomaterials originate from a molecular initiating event that describes the interaction of nanomaterials with lung cells and involve similar intermediate key events. Finally, using the example of an established AOP for lung fibrosis, the review will discuss various in vitro tests available for assessing lung fibrosis and how the information can be used to support a tiered testing strategy for lung fibrosis. The AOPs and AOP network enable deeper understanding of mechanisms involved in inhalation toxicity of nanomaterials and provide a strategy for the development of alternative test methods for hazard and risk assessment of nanomaterials.

Agglomeration of titanium dioxide nanoparticles increases toxicological responses in vitro and in vivo.

BACKGROUND: The terms agglomerates and aggregates are frequently used in the regulatory definition(s) of nanomaterials (NMs) and hence attract attention in view of their potential influence on health effects. However, the influence of nanoparticle (NP) agglomeration and aggregation on toxicity is poorly understood although it is strongly believed that smaller the size of the NPs greater the toxicity. A toxicologically relevant definition of NMs is therefore not yet available, which affects not only the risk assessment process but also hinders the regulation of nano-products. In this study, we assessed the influence of NP agglomeration on their toxicity/biological responses in vitro and in vivo. RESULTS: We tested two TiO2 NPs with different primary sizes (17 and 117 nm) and prepared ad-hoc suspensions composed of small or large agglomerates with similar dispersion medium composition. For in vitro testing, human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic (THP-1) cell lines were exposed to these suspensions for 24 h and endpoints such as cytotoxicity, total glutathione, epithelial barrier integrity, inflammatory mediators and DNA damage were measured. Large agglomerates of 17 nm TiO2 induced stronger responses than small agglomerates for glutathione depletion, IL-8 and IL-1ß increase, and DNA damage in THP-1, while no effect of agglomeration was observed with 117 nm TiO2. In vivo, C57BL/6JRj mice were exposed via oropharyngeal aspiration or oral gavage to TiO2 suspensions and, after 3 days, biological parameters including cytotoxicity, inflammatory cell recruitment, DNA damage and biopersistence were measured. Mainly, we observed that large agglomerates of 117 nm TiO2 induced higher pulmonary responses in aspirated mice and blood DNA damage in gavaged mice compared to small agglomerates. CONCLUSION: Agglomeration of TiO2 NPs influences their toxicity/biological responses and, large agglomerates do not appear less active than small agglomerates. This study provides a deeper insight on the toxicological relevance of NP agglomerates and contributes to the establishment of a toxicologically relevant definition for NMs.

Is aggregated synthetic amorphous silica toxicologically relevant?

BACKGROUND: The regulatory definition(s) of nanomaterials (NMs) frequently uses the term 'agglomerates and aggregates' (AA) despite the paucity of evidence that AA are significantly relevant from a nanotoxicological perspective. This knowledge gap greatly affects the safety assessment and regulation of NMs, such as synthetic amorphous silica (SAS). SAS is used in a large panel of industrial applications. They are primarily produced as nano-sized particles (1-100 nm in diameter) and considered safe as they form large aggregates (> 100 nm) during the production process. So far, it is indeed believed that large aggregates represent a weaker hazard compared to their nano counterpart. Thus, we assessed the impact of SAS aggregation on in vitro cytotoxicity/biological activity to address the toxicological relevance of aggregates of different sizes. RESULTS: We used a precipitated SAS dispersed by different methods, generating 4 ad-hoc suspensions with different aggregate size distributions. Their effect on cell metabolic activity, cell viability, epithelial barrier integrity, total glutathione content and, IL-8 and IL-6 secretion were investigated after 24 h exposure in human bronchial epithelial (HBE), colon epithelial (Caco2) and monocytic cells (THP-1). We observed that the de-aggregated suspension (DE-AGGR), predominantly composed of nano-sized aggregates, induced stronger effects in all the cell lines than the aggregated suspension (AGGR). We then compared DE-AGGR with 2 suspensions fractionated from AGGR: the precipitated fraction (PREC) and the supernatant fraction (SuperN). Very large aggregates in PREC were found to be the least cytotoxic/biologically active compared to other suspensions. SuperN, which contains aggregates larger in size (> 100 nm) than in DE-AGGR but smaller than PREC, exhibited similar activity as DE-AGGR. CONCLUSION: Overall, aggregation resulted in reduced toxicological activity of SAS. However, when comparing aggregates of different sizes, it appeared that aggregates > 100 nm were not necessarily less cytotoxic than their nano-sized counterparts. This study suggests that aggregates of SAS are toxicologically relevant for the definition of NMs.

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-1ß 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.

Mechanisms of lung fibrosis induced by carbon nanotubes: towards an Adverse Outcome Pathway (AOP).

Several experimental studies have shown that carbon nanotubes (CNT) can induce respiratory effects, including lung fibrosis. The cellular and molecular events through which these effects develop are, however, not clearly elucidated. The purpose of the present review was to analyze the key events involved in the lung fibrotic reaction induced by CNT and to assess their relationships. We thus address current knowledge and gaps with a view to draft an Adverse Outcome Pathway (AOP) concerning the fibrotic potential of CNT.As for many inhaled particles, CNT can indirectly activate fibroblasts through the release of pro-inflammatory (IL-1ß) and pro-fibrotic (PDGF and TGF-ß) mediators by inflammatory cells (macrophages and epithelial cells) via the induction of oxidative stress, inflammasome or NF-kB. We also highlight here direct effects of CNT on fibroblasts, which appear as a new mode of toxicity relatively specific for CNT. Direct effects of CNT on fibroblasts include the induction of fibroblast proliferation, differentiation and collagen production via ERK 1/2 or Smad signaling. We also point out the physico-chemical properties of CNT important for their toxicity and the relationship between in vitro and in vivo effects. This knowledge provides evidence to draft an AOP for the fibrogenic activity of CNT, which allows developing simple in vitro models contributing to predict the CNT effects in lung fibrosis, and risk assessment tools for regulatory decision.

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-ß or EGF. MWCNT prolonged ERK 1/2 phosphorylation induced by low concentrations of PDGF or TGF-ß in fibroblasts. This phenomenon and the proliferative activity of MWCNT on fibroblasts was abrogated by the inhibitors of ERK 1/2, PDGF-, TGF-ß- 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.

Does carbonation of steel slag particles reduce their toxicity? An in vitro approach.

Mineral carbonation can stabilize industrial residues and, in the steel industry, may contribute to simultaneously valorize CO2 emissions and slag. We hypothesized that, by restricting the leaching of metals of toxicological concern such as Cr and V, carbonation can suppress the toxicity of these materials. The cytotoxic activity (WST1 assay) of slag dusts collected from a stainless and a Linz-Donawitz (LD) steel plant, before and after carbonation, was examined in J774 macrophages. The release of Cr, V, Fe, Mn and Ni was measured after incubation in artificial lung fluids mimicking the extracellular and phagolysosomal milieu to which particles are confronted after inhalation. LD slag had the higher Fe, Mn and V content, and was more cytotoxic than stainless steel slag. The cytotoxic activity of LD but not of stainless dusts was reduced after carbonation. The cytotoxic activity of the dusts toward J774 macrophages necessitated a direct contact with the cells and was reduced in the presence of inhibitors of phagocytosis (cytochalasin D) or phagolysosome acidification (bafilomycin), pointing to a key role of metallic constituents released in phagolysosomes. This in vitro study supports a limited reduction of the cytotoxic activity of LD, but not of stainless, steel dusts upon carbonation.

Nanometer-long Ge-imogolite nanotubes cause sustained lung inflammation and fibrosis in rats.

BACKGROUND: Ge-imogolites are short aluminogermanate tubular nanomaterials with attractive prospected industrial applications. In view of their nano-scale dimensions and high aspect ratio, they should be examined for their potential to cause respiratory toxicity. Here, we evaluated the respiratory biopersistence and lung toxicity of 2 samples of nanometer-long Ge-imogolites. METHODS: Rats were intra-tracheally instilled with single wall (SW, 70 nm length) or double wall (DW, 62 nm length) Ge-imogolites (0.02-2 mg/rat), as well as with crocidolite and the hard metal particles WC-Co, as positive controls. The biopersistence of Ge-imogolites and their localization in the lung were assessed by ICP-MS, X-ray fluorescence, absorption spectroscopy and computed micro-tomography. Acute inflammation and genotoxicity (micronuclei in isolated type II pneumocytes) was assessed 3 d post-exposure; chronic inflammation and fibrosis after 2 m. RESULTS: Cytotoxic and inflammatory responses were shown in bronchoalveolar lavage 3 d after instillation with Ge-imogolites. Sixty days after exposure, a persistent dose-dependent inflammation was still observed. Total lung collagen, reflected by hydroxyproline lung content, was increased after SW and DW Ge-imogolites. Histology revealed lung fibre reorganization and accumulation in granulomas with epithelioid cells and foamy macrophages and thickening of the alveolar walls. Overall, the inflammatory and fibrotic responses induced by SW and DW Ge-imogolites were more severe (on a mass dose basis) than those induced by crocidolite. A persistent fraction of Ge-imogolites (15% of initial dose) was mostly detected as intact structures in rat lungs 2 m after instillation and was localized in fibrotic alveolar areas. In vivo induction of micronuclei was significantly increased 3 d after SW and DW Ge-imogolite instillation at non-inflammatory doses, indicating the contribution of primary genotoxicity. CONCLUSIONS: We showed that nm-long Ge-imogolites persist in the lung and promote genotoxicity, sustained inflammation and fibrosis, indicating that short high aspect ratio nanomaterials should not be considered as innocuous materials. Our data also suggest that Ge-imogolite structure and external surface determine their toxic activity.

Lung inflammation and thymic atrophy after bleomycin are controlled by the prostaglandin D2 receptor DP1.

Acute lung injury (ALI) can be accompanied by secondary systemic manifestations. In a model of ALI induced by bleomycin (bleo), we examined the response of D prostanoid receptor 1 (DP1)-deficient mice (DP1(-/-)) to better understand these processes. DP1 deficiency aggravated the toxicity of bleo as indicated by enhanced body weight loss, mortality, and lung inflammation including bronchoalveolar permeability and neutrophilia. Thymic atrophy was also observed after bleo and was strongly exacerbated in DP1(-/-) mice. This resulted from the enhanced depletion of immature T lymphocytes in the thymus of DP1(-/-) mice, a phenomenon usually related to increased glucocorticoid release in blood. Serum corticosterone was more elevated in DP1(-/-) mice after bleo than in wild-type (wt) mice. Thymocytes of DP1(-/-) mice were not more sensitive to dexamethasone in vitro, and systemic delivery of dexamethasone or peritoneal inflammation after LPS induced a similar thymic atrophy in wt and DP1(-/-) mice, indicating that pulmonary DP1 was critical to the control of thymic atrophy after bleo. DP1(-/-) mice showed increased lung and/or blood mediators involved in neutrophil recruitment and/or glucocorticoid production/thymic atrophy (osteopontin, leukemia inhibitory factor, and keratinocyte-derived chemokine) after bleo. Finally, local pulmonary DP1 activation or inhibition in wt mice abrogated or amplified thymic atrophy after bleo, respectively. Altogether, our data reveal that ALI can perturb the systemic T-cell pool by inducing thymic atrophy and that both pathological processes are controlled by the pulmonary DP1 receptor. This new pathway represents a potential therapeutic target in ALI.

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.

Azithromycin reduces exaggerated cytokine production by M1 alveolar macrophages in cystic fibrosis.

Macrophages phagocyte pathogenic microorganisms and orchestrate immune responses by producing a variety of inflammatory mediators. The cystic fibrosis (CF) transmembrane conductance regulator chloride channel has been reported to be of pivotal importance for macrophage functions. The exact phenotype and role of macrophages in CF is still unknown. Alveolar and peritoneal macrophages were monitored in CF mice homozygous for the F508 del mutation and in wild-type control animals. Classical (M1) and alternative (M2) macrophage polarization and responses to LPS from Pseudomonas aeruginosa were investigated, and the effect of azithromycin was examined in both cell populations. We show that alveolar macrophage counts were 1.7-fold higher in CF as compared with wild-type mice. The macrophage-related chemokine, chemokine C-C motif ligand (CCL)-2, was found to be at least 10-fold more abundant in the alveolar space of mutant mice. Cell count and CCL-2 protein levels were also increased in the peritoneal cavity of CF mice. Both M1 and M2 macrophage polarization were significantly enhanced in alveolar and peritoneal cells from F508del-CF mice as compared with control animals. LPS-stimulated expression of proinflammatory mediators, such as nitric oxide synthase-2, IL-1beta, and CCL-2, was increased, whereas anti-inflammatory IL-10 expression was decreased in CF macrophages. Azithromycin, added to cell cultures at 1 mg/liter, significantly reduced proinflammatory cytokine expression (IL-1beta, CCL-2, TNF-alpha) in M1-induced CF and wild-type alveolar macrophages. Our findings indicate that CF macrophages are ubiquitously accumulated, and that these cells are polarized toward classical and alternative activation status. Azithromycin down-regulates inflammatory cytokine production by M1-polarized CF alveolar macrophages.

Modulation of cytokine production and silica-induced lung fibrosis by inhibitors of aminopeptidase N and of dipeptidyl peptidase-IV-related proteases.

AIMS: Dipeptidyl peptidase IV (DP IV)-related proteases and aminopeptidase N (APN) are drug targets in various diseases. Here we investigated for the first time the effects of DP-IV-related protease inhibitors and APN inhibitors on chronic inflammatory lung diseases. MAIN METHODS: A murine model of silica (SiO2)-induced lung fibrosis and in vitro cultures of human lung epithelial cells and monocytes have been used and the influence of silica-treatment and inhibitors on inflammation and fibrosis has been measured. KEY FINDINGS: We found increased inflammation and secretion of the chemokines IL-6, MCP-1 and MIP-alpha 2 weeks after SiO2 application, and increased lung fibrosis after 3 months. Treatment with the APN inhibitor actinonin reduced chemokine secretion in the lung and bronchoalveolar lavage fluid, and in cell culture, and decreased the level of fibrosis after 3 months. Treatment with inhibitors of DP-IV-related proteases, or a combination of DP IV inhibitors and APN inhibitors, had no significant effect. We found no obvious side effects of long-term treatment with inhibitors of APN and DP IV. SIGNIFICANCE: Overall, our findings show that actinonin, an inhibitor of aminopeptidase N, might modulate chemokine secretion in the lung and thus attenuate the development of lung fibrosis. Additional targeting of DP-IV-related proteases had no significant effect on these processes.

Markers of macrophage differentiation in experimental silicosis.

Macrophages are characterized by a marked phenotypic heterogeneity depending on their microenvironmental stimulation. Beside classical activation (M1), it has been shown that macrophages could follow a different activation pathway after stimulation with interleukin (IL)-4 or IL-13 (M2). Recently, it has been postulated that those "alternatively activated" macrophages may be critical in the control of fibrogenesis. In an experimental model of silicosis, where pulmonary macrophages play a central role, we addressed the question of whether lung fibrosis development would be associated with alternative macrophage activation. As available markers for alternative macrophage activation, type-1 arginase (Arg-1), Fizz1, Ym1/2, and mannose receptor expression were evaluated at the mRNA and/or protein levels at different stages of the disease. Nitric oxide synthase-2 (NOS-2) expression was also examined to investigate the classical counterpart. We found that the expression of Arg-1, Fizz1, and NOS-2 in adherent bronchoalveolar lavage cells was highly up-regulated 3 days after silica administration but returned to control levels during the fibrotic stage of the disease (60 days). By comparing the early response to silica in C57BL/6 and BALB/c mice, we observed that the amplitude of Arg-1 mRNA up-regulation was not associated with the severity of lung fibrosis. Using a model of manganese dioxide particles (resolutive alveolitis), we showed that this early Arg-1 mRNA was not specific to a fibrogenic lung response. Our data indicate that the modifications of M1/M2 marker expression are limited to the early inflammatory stage of silicosis and that the establishment of a fibrotic process is not necessarily associated with M2 polarization.