Deficient macrophage autophagy protects mice from Cerium Oxide nanoparticle-induced lung fibrosis

doi:10.21203/rs.3.rs-41389/v1

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Deficient macrophage autophagy protects mice from Cerium Oxide nanoparticle-induced lung fibrosis

https://doi.org/10.21203/rs.3.rs-41389/v1

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published 01 Feb, 2021

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Background

Cerium (Ce) is a rare earth element, rapidly oxidizing to form CeO₂, and currently used in numerous commercial applications, especially as nanoparticles (NP). The potential health effects of Ce remain uncertain, but literature indicates the development of rare earth pneumoconiosis accompanied with granuloma formation, interstitial fibrosis and inflammation. The exact underlying mechanisms are yet not completely understood, and we propose that autophagy could be an interesting target to study, particularly in macrophages. Therefore, the objective of our study was to investigate the role of macrophagic autophagy after pulmonary exposure to CeO₂ NP in mice. Mice lacking the early autophagy gene Atg5 in their myeloid lineage and their wildtype counterparts were exposed to CeO₂ NP by single oropharyngeal administration and sacrificed up to one month after. At that time, lung remodeling was thoroughly characterized (inflammatory cells infiltration, expression of fibrotic markers such as αSMA, TGFβ1, total and type I and III collagen deposition), as well as macrophage infiltration (quantification and M1/M2 phenotype).

Results:

Such pulmonary exposure to CeO₂ NP induces a progressive and dose-dependent lung fibrosis in the bronchiolar and alveolar walls, together with the activation of autophagy. Blockade of macrophagic autophagy protects from alveolar but not bronchiolar fibrosis, via the modulation of macrophage polarization towards M2 phenotype.

Conclusion:

In conclusion, our findings bring novel insight on the role of macrophagic autophagy in lung fibrogenesis, and add to the current awareness of pulmonary macrophages as potential new therapeutic targets for future anti-fibrotic therapies.

Toxicology

alveolar fibrosis

autophagy

macrophage polarization

nanoparticle

Up to recent years, rare earth elements (REE) received limited attention from environmental and public health researchers. In the last two decades, they have however undergone a fantastic boost in their technological and industrial utilization, which is accompanied by concerns regarding emissions and potential human exposures. One of the main REE currently used in numerous commercial applications is Cerium (Ce), as Ce is very reactive and is a strong oxidizing agent, rapidly oxidizing when in contact with oxygen to form CeO₂. Ce is for example used in flat screen display, alloys, petroleum refining (cracking catalyst), ceramics, glass additives, phosphors, or polishing compounds for glass mirrors, plate glass, television tubes, ophthalmic lenses, precision optics, electronic wafers (1).CeO₂ is also used as additive in cigarettes, and CeO₂ nanoparticles (NP) are predominantly used as diesel fuel additive to increase fuel combustion efficiency (2). Because of these already numerous current and foreseen applications, CeO₂ NP might enter the environment through disposal of consumer and industrial products, and this is accompanied a growing concern for human health.

The potential health effects of Ce remain uncertain, but RE pneumoconiosis accompanied with granuloma and interstitial fibrosis has been reported in workers exposed to asbestos and RE dusts, together with the presence of Ce-containing particle in macrophages from bronchoalveolar lavage (BAL) and in interstitial macrophages (3, 4). Experimental studies conducted in mice or rats have shown that animal exposure to CeO₂ NP lead to the development of pulmonary fibrosis, accompanied by a persistent pulmonary inflammation and the presence of oxidative stress (1, 5–9). The exact underlying mechanisms are yet not completely understood, and we propose that autophagy could be an interesting target to study.

Autophagy is a physiological process mainly responsible for the recycling of damaged cellular organelles and/or macromolecules into simpler forms for cell survival. The resulting degradation products are recycled to maintain nutrient and energy homeostasis (10, 11). Apart from its importance in physiological conditions, numerous studies have suggested that autophagy is regulated under different pathophysiological conditions, in particular in the lung fibrotic process (12, 13). We have recently demonstrated that macrophagic autophagy can be modulated after exposure to NP, which could be of relevance in the context of pulmonary response to Ce exposure (14).

Therefore, the objective of the present study was to investigate the role of macrophagic autophagy after pulmonary exposure to CeO₂ NP in mice. To achieve our aim, mice lacking the early autophagy gene Atg5 in their myeloid lineage and their wildtype counterparts were exposed to CeO₂ NP by single oropharyngeal administration and sacrificed up to one month after. At that time, lung remodeling was thoroughly characterized (inflammatory cells infiltration, expression of fibrotic markers such as αSMA, TGFβ1, total and type I and III collagen deposition), as well as macrophage infiltration (quantification and M1/M2 phenotype).

Characterization of pulmonary remodeling in response to CeO₂ NP

Mice exposure to a single administration of 50 µg CeO₂ NP induced a profound lung fibrotic remodeling characterized by a progressive thickening of bronchiolar and alveolar walls. These modifications appeared gradually, starting from 24 hours post CeO₂ NP administration for bronchiolar modifications, and after 1 week for the alveolar ones (Fig. 1). They were accompanied by a persistent increased total cell count in the BAL (Fig. 1D). Cell differential analysis identified macrophages as the predominant cell population, with a transient significant increase of neutrophil percentage in BAL from CeO₂-exposed animal 24 hours and 1 week after their initial exposure, but which was no more found after 1 month (Additional Fig. 1). As shown in Fig. 1E, CeO₂ NP were internalized in BAL macrophages at all time points.

To further characterize our model, we performed a dose-response experiment and analyzed the lung remodeling achieved in mice 28 days after exposure to one single administration of either 5 or 50 µg CeO₂ NP. As showed in Figs. 2 & 3, a significant thickening of alveolar and bronchiolar walls, together with a significant accumulation of total collagen, collagen I, collagen III, αSMA and TGF-ß in bronchiolar and alveolar area could be observed in mice exposed to 50 µg CeO₂ NP, while these effects where only intermediary in mice exposed to 5 µg CeO₂ NP (except for Collagen III alveolar deposition which was significatively different in mice exposed to 50 µg compared to Saline and 5 µg CeO₂ NP conditions). Quantification of Mac3 expression in lung tissue sections of CeO₂-exposed mice, as an index of total number of macrophages in lung tissue, confirmed the increased total number of macrophages observed in BAL, and demonstrated a predominant alveolar localization of macrophages in CeO₂-exposed animals, both in alveolar spaces as well as infiltrated in the interstitium (Fig. 4). As for BAL macrophages, CeO₂ NP were internalized in macrophages present in the alveolar area (Additional Fig. 1D).

Characterization of autophagy in response to CeO₂ NP exposure

To investigate whether autophagy was induced in our experimental model of lung fibrosis, we utilized GFP-LC3 transgenic mice and exposed them to 50 µg CeO₂ NP. As shown in Fig. 5, we were able to observe the induction of autophagy in CeO₂-exposed GFP-LC3 mice, already 24 h after the initial administration, and up to 28 days. This was observed mainly in epithelial cells in bronchiolar regions, and in macrophages in the alveoli (Fig. 5A-C). These results were confirmed by the observation of increased levels of Atg5 protein, an early marker of autophagy pathway, in similar regions of CeO₂-exposed mice, (Additional Fig. 2, Panel A).

As macrophages are supposedly important players in lung fibrosis (15), and as we recently demonstrated the importance of autophagy in macrophagic response to NP (14), we next focus our work on macrophages. The potential of CeO₂ NP to induce autophagy in macrophages was confirmed by the observed accumulation of GFP-LC3 puncta in peritoneal macrophages obtained from GFP-LC3 mice and exposed to CeO₂ NP in vitro (Fig. 5D), as well as by the induction of Atg5 protein expression in peritoneal macrophages of C57Bl/6 mice exposed to CeO₂ NP in vitro (Fig. 5E). Finally, as autophagy is a highly dynamic process, we addressed the issue of its activation in macrophages exposed to CeO₂ NP. As shown by Fig. 4F, we could observe the colocalization of LC3 staining with that of the lysosomal protein LAMP-1 in CeO₂ NP-exposed macrophages. Overall, these results indicate that pulmonary exposure to CeO₂ NP is followed by the activation of autophagy, especially in macrophages, which are able to phagocyte CeO₂ NP.

Effect of blockade of macrophagic autophagy in response to CeO₂ NP

In order to decipher the specific role of macrophagic autophagy in the development of lung fibrosis, mice lacking Atg5 gene in their myeloid lineage (Atg5^flox/flox LysM^Cre or Atg5^+/−) were exposed to CeO₂ NP. These mice do not present any particular lung phenotype in absence of any exogenous exposure and their macrophages do not express Atg5 in response to CeO₂ NP (Additional Fig. 3). Twenty-eight days after the NP administration, histological analysis of lung tissue sections indicates that Atg5^+/− mice present the same peribronchiolar fibrosis as compared to their wild type (WT) counterparts (Fig. 6, *: p < 0.05). In stark contrast, Atg5^+/− mice were protected against CeO₂ NP-induced alveolar fibrosis; no alveolar wall thickening could be observed in these mice (Fig. 6 *: p < 0.05). This protection against alveolar remodeling was further confirmed by the quantification of total, type I and III collagen deposition, as well as expression of αSMA and TGF-ß: Atg5^+/− mice present the same alveolar phenotype as their wild-type counterpart, whatever their exposure to CeO₂ NP (Fig. 7).

This protection wasn't the result of a differential CeO₂ NP accumulation between WT and Atg5^+/− mice, as X-Ray microfluorescence(micro-XRF) showed that Ce signal could be observed with similar intensity whatever the mice genotype, mainly in the alveolar region, and much less in bronchiolar walls (Additional Fig. 4). Moreover, micro X-ray absorption near edge structure (XANES) spectra of Ce spots detected in CeO₂-exposed mice revealed three different types of modifications that could be observed: 1/ similar to that of the CeO₂ reference (red line in Additional Fig. 4E – Spectrum Type I); 2/ a spectrum presenting a shift of the first peak toward 5727 eV (instead of 5731 eV as in the reference – yellow line – Spectrum Type II), suggesting the reduction of Ce^IV to Ce^III; and 3/ a mixed contribution of Type I and Type II spectra (green line). A similar repartition of the different type of spectra could be observed in all CeO₂-exposed lungs, whatever the genotype (Table 2).

Table 2

Repartition of the different types of XANES spectra in WT and Atg5^+/− samples
	Type I	Type II	Mix Type I + II
WT samples (%)	36.4	45,4	18.2
Atg5^+/− samples (%)	33.3	48.2	18.5

Finally, the protection against alveolar fibrosis was the result of macrophage activity, as treatment of Atg5^+/− mice with clodronate to remove macrophages completely abolished the protection against alveolar fibrosis observed in Atg5^+/− mice, while no modification of bronchiolar fibrosis was observed (Additional Fig. 5 and data not shown).

Characterization of macrophage polarization in response to CeO₂ NP

Blockade of macrophagic autophagy didn't modify the increase in total number of macrophages induced by CeO₂ NP exposure (Fig. 8). However, when assessing the M1 or M2 phenotype of alveolar macrophages as potential contributors to alveolar fibrotic response (15), we observed that WT mice exposed to CeO₂ NP present an M1-like phenotype, with significantly increased expression of CD68, CD80 and iNOS proteins as compared to unexposed mice (Fig. 8C-E and 8I-K, *: p < 0.005), whereas Atg5^+/− mice developed an M2-like phenotype, characterized by increased expression of CD206, CD163 and Arginase1 (Fig. 8F-H and 8L-N,*: p < 0.005). These results were confirmed in vitro; exposure of WT peritoneal macrophages to CeO₂ NP lead to the increased expression of M1 markers, while exposure of macrophages obtained from Atg5^+/− mice lead to an increased expression of M2 markers (Additional Fig. 6).

Finally, this modification of macrophage polarization in Atg5^+/− mice could be responsible of the protective effect against the development of alveolar fibrosis in Atg5^+/− mice as exposure of WT fibroblasts to the supernatant of CeO₂-exposed macrophages induced fibroblast to myofibroblast differentiation, detected by an increased expression of αSMA, Collagen I and III, only when WT macrophages were used, but not macrophages bearing a Atg5^+/− genotype (Fig. 9).

Taken together, our results show the progressive and dose-dependent development of lung fibrosis in mice exposed to CeO₂ NP. They also demonstrate that the blockade of macrophagic autophagy protects from alveolar but not bronchiolar fibrosis, probably via the modulation of macrophage polarization in favor of a M2 phenotype.

The progressive and dose-dependent induction of pulmonary fibrosis that we describe here in response to CeO₂ NP single administration is in accordance with data from literature using other CeO₂ NP (1, 5–9). The same is true for the sustained pulmonary inflammation that we detected and which has been shown to be slow to resolve post exposure (16). We didn't however observe the formation of granuloma as described for example by Park and colleagues in CeO₂-exposed animals (8), but this is probably linked to the more relevant dose of CeO₂ NP that we used in our study; 5–50 µg per mouse versus 3 mg in Park study. Our results also confirm and extend the data obtained by Ma and colleagues, showing that rats exposure to CeO₂ NP lead to the induction of M1 phenotype in BAL macrophages of these animals (17). It must be noted however that 28 days after the initial administration, mRNA expression of Arginase 1 was increased in Park study, thus suggesting a shift toward M2 phenotype that was not observed in our WT animals, although care must be taken as only one single M2 marker was targeted in Park's work.

The development of bronchiolar fibrosis in response to CeO₂ NP was not prevented by the blockade of macrophagic autophagy. Interestingly, this could be paralleled by the very low Ce elemental signal observed by µXRF in bronchiolar regions, as well as the almost lack of macrophages present in these areas, suggesting that CeO₂-induced bronchiolar fibrosis is independent of macrophages. Although we did not investigate further the biological mechanism(s) underlying this bronchiolar fibrosis, the occurrence of epithelial-mesenchymal transition (EMT) could be an explanation. Indeed, Ma and colleagues have recently demonstrated that exposure to CeO₂ NP induces EMT in alveolar type II cells that ultimately plays a role in lung fibrosis (5). Similarly to what we have demonstrated with in vitro exposure of fibroblast to the supernatant of Ce-exposed macrophages, it could be interesting to explore the effect of the secretome from Ce-exposed epithelial cell on fibroblast to myofibroblast differentiation in our system.

The protection against alveolar fibrogenesis observed in Ce-exposed Atg5^+/− mice could have been the result of decreased amounts of Ce present in alveolar regions of Atg5^+/− mice. Indeed, autophagy is known to interplay with macrophage phagocytosis (18–20), and M2 polarization has been shown to enhance Si-NP uptake by macrophages (21). Although we did not strictly quantify the Ce content in WT and Atg5^+/− mice exposed to CeO₂ NP, the similar µXRF signals obtained in both animal genotypes in terms of both intensity and tissue distribution, together with the similar total number of macrophages present in the lungs of WT and Atg5^+/− animals strongly suggest that the protection against alveolar fibrosis observed in Atg5^+/− animals is probably not the result of a decreased amount of CeO₂ NP in these individuals. CeO₂ is highly reactive, and it has been suggested that a change in the Ce³⁺/Ce⁴⁺ ratio may play a significant role in toxicity determination (22), and could thus contribute to lung fibrosis development. In our experiment, while Ce in CeO₂ NP was Ce⁴⁺, Ce in lung tissue was only 33–36% pure Ce⁴⁺, indicating a change in speciation after NP administration. However, both WT and Atg5^+/− mice showed a similar modification of their Ce speciation, with 45–48% as Ce³⁺, and around 18% presenting a mix Ce³⁺/Ce⁴⁺ form. Therefore, the specific protection against alveolar fibrosis observed in Atg5^+/− mice could not be attributed to modifications of Ce speciation.

The modification of macrophage polarization related to their autophagy status could also represent an interesting candidate to explore the underlying mechanism that occurs in Atg5^+/− mice (23). Indeed, macrophages are divided into two distinct sub-populations defined as classically activated pro-inflammatory M1 subtype and alternatively activated M2 subtype responsible for anti-inflammatory, tissue repair and remodeling (Novak and Koh 2013). Both M1 and M2 macrophages have been noted to be involved in the pathogenesis of pulmonary fibrosis, this translating the plasticity of macrophage polarization depending the micro-environment stimuli and signals (25). In our experimental model of lung fibrosis, we demonstrated that macrophages from autophagy-deficient mice tended to polarize into M2a and M2c phenotype whereas their wild-type counterparts exhibiting proficient autophagy skewed towards the M1 subtype. Interestingly, the induction of autophagy by advanced glycation end products or rapamycin triggered macrophage polarization toward M1 phenotype, as well as a sustained inflammation in mice and patients, resulting in delayed wound healing. Moreover, the inhibition of autophagy reduced M1 population but no changes of M2 subtype, which is in accordance with our results (26). However, it must be noted that in mice under high fat diet, there was an increased M1 and decreased M2 polarization in macrophages with defective autophagy, leading to hepatic inflammation and the progression to liver injury (27). This underlines the importance of the environmental context in the overall autophagy effect, which could be a clue to the effects observed in our study, as to understand the underlying mechanism of macrophage specific polarization in absence of autophagy.

Finally, our results suggest that macrophagic autophagy can facilitate fibrosis. Although not in accordance with the initial acceptance that autophagy might be protective in lung fibrosis (11, 13, 28), our data are in line with more recent results from the literature showing an increased number of LC3-II puncta, an index of active autophagy, in fibroblast foci of patients with idiopathic pulmonary fibrosis (IPF), the most common form of pulmonary fibrosis (13, 29). In vitro, these authors also showed that autophagy is induced by TGFß, and that it is necessary for TGFß-induced fibrosis in both non-IPF and IPF fibroblasts (7). Interestingly, such a cell-specific role for autophagy has been recently described in chronic obstructive pulmonary disease (COPD) where autophagy is believed to be protective except in macrophages (10–12, 28). In lung fibrosis, the majority of the studies has focused on lung epithelial cells and/or fibroblasts (28), as the two major cell type involved in fibrogenesis (15). However, the recruitment of inflammatory cells, leading to the activation of effector cells, is often considered as a first trigger of fibrosis (30), and as such, the activation of macrophages occupies a pivotal role in the translation of injury to aberrant repair in lung fibrosis (31, 32). Interestingly, the LC3 puncta observed by Ghavami and colleagues in fibroblast foci of IPF patients are coherent with the presence of macrophages (29).

In conclusion, our finding bring novel insight on the role of macrophagic autophagy in lung fibrogenesis, and add to the current awareness of pulmonary macrophages as potential new therapeutic targets for future anti-fibrotic therapies (33).

Experimental model of lung fibrosis:

Eight to twelve weeks old C57Bl/6 mice received saline or CeO₂ NP (5 or 50 µg, NanoAmor, Houston, TX) via non-surgical oropharyngeal instillation (MicroSprayer® Aerosolizer, PennCentury). Mice were sacrificed 24 h, 1 or 4 weeks later. A broncho-alveolar lavage (BAL) was performed, and the lungs were harvested and collected for further analysis as previously described (34). A subset of mice were treated with Clodronate or liposome as negative control as previously described (35). Myeloid cell specific Atg5 deficient mice (Atg5^fl/fl LysM-Cre^+/− mice further referred as Atg5^+/−) and their littermate wildtype (WT - Atg^fl/fl LysM-Cre^−/−) counterpart were kindly provided by Fatima Clerc (36). GFP-LC3 mice were purchased from Riken, Japan. The experimental protocol received the approval of the French Government (Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation, APAFIS #14914-2018042515599016).

Histological and Immunohistochemistry analyses:

Paraffin embedded lung tissue sections (5 µM) were stained with Hematoxylin and Eosin or Sirius Red for histological observations and total collagen deposition, respectively. Immunohistochemistry experiments were performed using antibodies described in Table 1. At least 10 fields per lung tissue section (magnification 200X) were evaluated for the quantification of histological lesions and immunostainings using ImageJ software as previously described (34). Briefly, for alveolar wall thickening, alveolar images without bronchi were used to build a macro with threshold value for alveolar wall thickness excluding alveolar spaces. For bronchiolar thickening, at least 10 fields with well defined, large and round shaped bronchi were imaged per animal. The thickness of each individual bronchus was measured manually at four different regions in pixels, and the average pixels calculated from these regions was converted into µM for bronchiolar thickening.

Table 1

List of antibodies used in the study
Antibody	Dilution	Reference	Fabricant
Collagen 1	1:100	AB21286	Abcam, Cambridge, UK
Collagen 3	1:1000	AB7778	Abcam, Cambridge, UK
SMA	1:3000	AB5694	Abcam, Cambridge, UK
TGF-ß	1:100	PA5-86215	Thermo-Fisher, France
MAC3	1:50	550292	BD BioScience, France
CD68	1:100	AB125212	Abcam, Cambridge, UK
CD80	1:100	AB64116	Abcam, Cambridge, UK
iNOS	1:100	AB15323	Abcam, Cambridge, UK
CD163	1:250	AB182422	Abcam, Cambridge, UK
CD206	1:500	AB64693	Abcam, Cambridge, UK
Arginase-1	1:500	AB91279	Abcam, Cambridge, UK
LC3	1:1000	PM036	MBL International, MA
LAMP-1	1:50	AF4320	R&D Systems, France

For immunohistological analyses, a representative image of alveolar or bronchiolar regions was opened under ImageJ and zoomed in for 3 colors. The plug-in with color deconvolution was applied on the image followed by choosing of color 1 for DAB stain, color 2 for counter-stain, and color 3 for the white background. The next step was adjusting the threshold for 3 color regions and the specific threshold values for each color were noted and close-all option was employed. The image was then opended and plugin-macro-record and plugin-color deconvolution were applied to the image step-wise. Threshold was adjusted, and the noted values for color 2 and color 1 were set for analyzing the measure for each color.

Lung primary cells culture

Peritoneal macrophages: Primary cultures of peritoneal macrophages were obtained as previously described (37). Briefly, 2 ml of sterile 4% thioglycolate broth (T0157, Sigma-Aldrich, La Verpillère, France) were administered in the peritoneal cavity of C57Bl/6 mice. Seventy-two hours later, peritoneal macrophages were harvested from the peritoneal cavity and cultured in DMEM medium, supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. For immunocytochemistry experiments, cells were seeded in 8-well cell culture chamber slides (LabTek, Nunc, ATGC Biotechnology, France) and exposed for 24 hours to 10 µg/ml CeO₂ NP. After incubation with the primary antibody of interest (Table 1), cells were labelled with secondary antibodies: Alexa Fluor 488 (green) for LC3 and Alexa Fluor 546 (red) for LAMP1. The fluorescence images were captured using Zeiss LSM-510 multitracking laser scanning confocal microscope with a Helium/Neon laser at 543 nm and using AxioVision software (Carl Zeiss).

Lung fibroblasts: Primary fibroblasts cells were isolated from mouse lungs by mechanic dissection and enzymatic digestion (collagenase 4,1% in HBSS). They were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and Glutamax (Gibco, 31966-021) containing 10% foetal bovine serum (Eurobio) and streptomycin penicillin (Life Technologies, E1740384 100 µg/ml).

X-ray microfluorescence experiments:

The localization and speciation of Ce in the lungs were assessed on lung tissue sections embedded in paraffin, using X-ray microfluorescence (micro-XRF) and micro X-ray absorption near edge structure (micro-XANES) as previously described (34). These experiments were performed at the LUCIA beamline of the SOLEIL synchrotron (Orsay, France − (38, 39)). A total of 29 spectra in WT and 22 spectra in Atg5^+/− animals have been recorded.

Statistical analysis:

At least five mice per experimental group were utilized. Taking into account the possibility of non-normal distribution in the mice population, nonparametric tests (Kruskal–Wallis statistical test followed by Dunn’s multiple comparison test) were used (40). Values are expressed as the mean ± SEM. Data were analyzed with GraphPad Prism 6.0 (La Jolla, CA) and STATA v13.0 (College Station, TX). For all statistical tests, p values smaller than 0.05 were considered as significant.

BAL

broncho-alveolar lavage

CeO₂

Cerium Oxide

FBS

Fetal Bovine Serum

µXANES

Micro X-ray absorption near edge structure

µXRF

X-ray microfluorescence

nanoparticles

REE

rare earth elements

Ethical approval and Consent to participate:

The experimental protocol received the approval of the French Government (Ministère de l’Enseignement Supérieur, de la Recherche et de l’Innovation, APAFIS #14914-2018042515599016).

Consent for publication:

Not applicable.

Availability of data and materials:

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests:

No competing interest to declare.

Funding:

This work was supported by a funding of the French "Investissement d'Avenir" program LabEx SERENADE project (B. Annangi recipient of Research Chair).

Author's contributions:

BA contributed to the design of experimental work, acquisition and analysis of data, and wrote the first draft of the manuscript. ZL, JBr, AR and VMS contributed to acquisition of data, DV contributed to acquisition and analysis of data, JBo critically revised the manuscript, and SL designed the work and critically wrote and revised the manuscript.

Acknowledgements:

Authors wish to acknowledge the expertise of Erwan Paineau for the µXRF analyses, and the expert advises of Fatima Clerc on Atg5^+/− mice.

Authors' information: Not applicable.

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AdditionalFigure6.pdf
Additional Figure 6: Characterization of macrophage polarization in vitro iNOS (Panel A), CD68 (Panel B), Arginase 1 (Panel C) or CD206 (Panel D) expression in mice peritoneal macrophages in response to Saline or 10 µg/ml CeO2 NP.
AdditionalFigure5.pdf
Additional Figure 5: Macrophages are responsible of Atg5+/- mice protection against alveolar remodeling Representative lung tissue sections of mice exposed to Saline or CeO2 NP in presence of clodronate, and stained Hematoxylin Eosin. Original magnification x200.
AdditionalFigure4.pdf
Additional Figure 4: X-Ray microfluoresence and XANES spectra of CeO2-exposed mice Representative images of XRF maps of Saline (Panel A and C) and CeO2 NP-exposed (Panel B and D) lungs from WT (Panel A and B) or Atg5+/- (Panel C and D) mice. Original magnification x100. False colors used in correlation XRF maps represent P (green), S (blue) and Ce (red). Panel E: XANES spectra at the Ce edge for Reference (blue line), and representative Ce spots (red, green and yellow lines).
AdditionalFigure3.pdf
Additional Figure 3: Expression of Atg5 peritoneal macrophages of CeO2-exposed mice Expression of Atg5 in peritoneal macrophages from CeO2-exposed in WT and Atg5+/- mice.
AdditionalFigure2.pdf
Additional Figure 2: Expression of Atg5 in lungs Representative lung tissue sections of mice exposed to Saline or 50 µg CeO2 NP and stained, after 28 days, for Atg5. Original magnification x200.
AdditionalFigure1.pdf
Additional Figure 1: Cell differential in CeO2-exposed mice Quantification of cell differential in BAL fluid 24h (Panel A), 1 week (panel B) or 28 days (Panel C) post-exposure to CeO2 NP. Each individual circle represents the value obtained from one animal (empty circle: saline exposure – plain circle: CeO2 NP-exposure). *p<0.05. Representative images of NP internalized by alveolar macrophages (Panel D).

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Deficient macrophage autophagy protects mice from Cerium Oxide nanoparticle-induced lung fibrosis

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Characterization of pulmonary remodeling in response to CeO₂ NP

Characterization of autophagy in response to CeO₂ NP exposure

Effect of blockade of macrophagic autophagy in response to CeO₂ NP

Characterization of macrophage polarization in response to CeO₂ NP

Discussion

Conclusions

Methods

Experimental model of lung fibrosis:

Histological and Immunohistochemistry analyses:

Lung primary cells culture

X-ray microfluorescence experiments:

Statistical analysis:

Abbreviations

Declarations

Ethical approval and Consent to participate:

Funding:

Author's contributions:

Acknowledgements:

References

Supplementary Files

Status:

Journal Publication

Version 1

Deficient macrophage autophagy protects mice from Cerium Oxide nanoparticle-induced lung fibrosis

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Results

Characterization of pulmonary remodeling in response to CeO2 NP

Characterization of autophagy in response to CeO2 NP exposure

Effect of blockade of macrophagic autophagy in response to CeO2 NP

Characterization of macrophage polarization in response to CeO2 NP

Discussion

Conclusions

Methods

Experimental model of lung fibrosis:

Histological and Immunohistochemistry analyses:

Lung primary cells culture

X-ray microfluorescence experiments:

Statistical analysis:

Abbreviations

Declarations

Ethical approval and Consent to participate:

Funding:

Author's contributions:

Acknowledgements:

References

Supplementary Files

Status:

Journal Publication

Version 1

Characterization of pulmonary remodeling in response to CeO₂ NP

Characterization of autophagy in response to CeO₂ NP exposure

Effect of blockade of macrophagic autophagy in response to CeO₂ NP

Characterization of macrophage polarization in response to CeO₂ NP