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Rodent studies on the effects of engineered nanomaterials (ENM) on the gut microbiome have revealed contradictory results. Our aim was to assess the effects of four well-investigated model ENM using a realistic exposure scenario. Two independent ad libitum feeding studies were performed. In study 1, female mice from the local breeding facility received feed pellets containing 1% CeO₂ or 1% SiO₂ for three weeks. In study 2, both female and male mice were purchased and exposed to 0.2% Ag-PVP or 1% TiO₂ for four weeks. A next generation 16S rDNA sequencing-based approach was applied to assess impacts on the gut microbiome. None of the ENM had an effect on the α- or β-diversity. A decreased relative abundance of the phylum Actinobacteria was observed in SiO₂ exposed mice. In female mice, the relative abundance of the genus Roseburia was increased with Ag exposure. Furthermore, in study 2, a sex-related difference in the β-diversity was observed. A difference in the β-diversity was also shown between the female control mice of the two studies. We did not find major effects on the gut microbiome. This contrast to other studies may be due to variations in the study design. Our investigation underlined the important role of the sex of test animals and their microbiome composition prior to ENM exposure initiation. Hence, standardization of microbiome studies is strongly required to increase comparability. The ENM-specific effects on Actinobacteria and Roseburia, two taxa pivotal for the human gut homeostasis, warrant further research on their relevance for health.

Additional file 1; Fig. S1. Effect of gastrointestinal DEP exposure on blood cell counts. White blood cells (A, E), lymphocytes (B, F), monocytes (C, G), neutrophils (D, H) and red blood cells (I) counts in ApoE−/− (A-D) and C57BL/6 (E-I) mice treated 3x/week with DEP during 3 or 6 months, respectively,. Data were analyzed by a one-way ANOVA followed by a Dunnett’s test (* p ≤ 0.05, ** p ≤ 0.01) and a test for linear trend (indicated p values) (horizontal bars indicate the means, n = 8 for ApoE−/−, n = 10 for C57BL/6). Fig. S2. Effect of gastrointestinal DEP exposure on plasma liver enzymes AST (A, C) and ALT (B, D) were measured in the plasma of ApoE−/− (A, B) and C57BL/6 (C, D) treated 3x/week during 3 or 6 months, respectively, with PBS (CTL), 40, 200 or 1000 ng DEP/gavage. Data were analyzed by a one-way ANOVA followed by a Dunnett’s test and a test for linear trend (n = 8 for ApoE−/−, n = 10 for C57BL/6). Fig. S3. Histological analysis of ileum and colonic mucosa of ApoE−/− mice treated with DEP. Tissue was collected from ApoE−/− mice gavaged with DEP during 3 months. Sections were stained with hematoxylin and eosin. Ileum (A, C, E) and colon (B, D, F) in controls (A, B) or mice treated with 200 ng/gavage (C, D) or 1000 ng/gavage (E, F) 3 times per week. Magnification 100x (bar, 100 μm), inserts 800x. Fig. S4. Histological analysis of ileum and colonic mucosa of C57BL/6 mice treated with DEP. Tissue was collected from C57BL/6 mice gavaged with DEP during 6 months. Sections were stained with hematoxylin and eosin. Ileum (A, D, E, G) and colon (B, D, F, H) in controls (A,B) or mice treated with 40 ng/gavage (C,D), 200 ng/gavage (E,F) or 1000 ng/gavage (G,H) 3 times per week. Magnification 100x (bar, 100 μm), inserts 800x. Fig. S5. Effect of DEP exposure on the relative abundance of phyla in ApoE −/− mice. The relative abundance of bacteria was calculated based on ASVs and taxonomic classification derived from SILVA database. (A) Overview of the relative abundance of bacteria depicted at the phylum level in mice exposed to vehicle control, 200 and 1000 ng DEP/gavage. Fig. S6. Effect of DEP exposure on the relative abundance of family in ApoE −/− mice. The relative abundance of bacteria was calculated based on ASVs and taxonomic classification derived from SILVA database. (A) Overview of the average relative abundance of bacteria depicted at the family level in groups of mice exposed to vehicle control, 200 or 1000 ng DEP/gavage. (B) Relative abundance of selected family was plotted against the dose (Q-values for linear dose effect, n = 8). Fig. S7. Effect of DEP exposure on the relative abundance of genera in ApoE −/− mice. The relative abundance of bacteria was calculated based on ASVs and taxonomic classification derived from SILVA database. Overview of the average relative abundance of bacteria depticted at the genus level in groups of mice exposed to vehicle control, 200 or 1000 ng DEP/gavage. Fig. S8. Effect of DEP exposure on the relative abundance of phyla in C57BL/6 mice. The relative abundance of bacteria was calculated based on ASVs and taxonomic classification derived from SILVA database. (A) Overview of the average relative abundance of bacteria depicted at the phylum level in groups of mice exposed to vehicle control, 40, 200 or 1000 ng DEP/gavage. (B–E) Relative abundance of selected phyla was plotted against the dose (Q-values for linear dose effect, n = 10).Fig. S9. Effect of DEP exposure on the relative abundance of family in C57BL/6 mice. The relative abundance of bacteria was calculated based on ASVs and taxonomic classification derived from SILVA database. (A) Overview of the average relative abundance of bacteria depicted at the family level in groups of mice exposed to vehicle control, 40, 200 or 1000 ng DEP/gavage. (B–C) Relative abundance of selected family was plotted against the dose (Q-values for linear dose effect, n = 10). Fig. S10. Effect of DEP exposure on the relative abundance of genera in C57BL/6 mice. The relative abundance of bacteria at genus level was calculated based on ASVs and taxonomic classification derived from SILVA database. (A) Overview of the average relative abundance of bacteria depicted at the genus level in groups of mice exposed to vehicle control, 40, 200 or 1000 ng DEP/gavage. (B–D) Relative abundance of selected genus was plotted against the dose (Q-values for linear dose effect, n = 10). Fig. S11. Cardiovascular effects of gastrointestinal DEP exposure. Sections of abdominal aorta (H&E staining) from ApoE−/− mice: controls (A) or treated with 200 (B) or 1000 ng/gavage (C), and from C57BL/6 mice: controls (D) or treated with 40 (E), 200 (F) or 1000 ng/gavage (G). Magnification 100x (bar size 100 μm), inserts 800x. Fig. S12. Hydrodynamic mean diameter of SRM2795. The hydrodynamic mean diameter (Z-average) of SRM2795 was measured by DLS on a 5.15 μg/ml suspension in PBS after sonication (concentration of DEP in the suspension used to deliver 1000 ng/gavage). Fig. S13. DEP do not quench SCFA in cecal content. The cecal content of five naive C57Bl/6 mice were pooled, and aliquoted in fractions of about 80 mg to which we added 0, 5, 25 or 125 ng DEP. These samples were carefully mixed, and analyzed for SCFA by LC-MS (p-value for ANOVA; n = 6 technical replicates). The mouse cecal content (200 mg) corresponds on average to 5% of the daily food intake (4000 mg). Thus, the highest spiked amount (125 ng DEP/80 mg cecal content, equivalent to 312.5 ng DEP per cecum) corresponds to a daily intake of 6250 ng DEP, which exceeds the highest gavage dose 1000 ng DEP used in the present study.

Introduction : Lung cancer is the most frequent and the deadliest cancer in the world. In non-small cell lung cancer (NSCLC), which represents 85% of all lung cancers, up to 55% patients relapse following surgery alone or in combination with chemotherapy or radiotherapy. Tumor-infiltrating lymphocytes play a key role in the control of the malignancy and display different phenotypes whilst interacting with cancer cells. Hence, immunoglobulin-A (IgA)-producing cells have been described to have an immunosuppressive function, promoting tumor development and growth in hepatocarcinoma and prostate cancer. Due to the important role of IgA in the lung, we aimed to study the prognostic significance of IgA+ cell infiltration in the epithelial and stromal compartments of resected NSCLCs. [...]