Project 2 (CNR)
Project 2: In vitro models of acute vs. chronic/repeated exposure for assessing nanoparticle-induced inflammation
Fellow: Yang Li (ESR2), 36 months
Tutors: Dr. Diana Boraschi (CNR)
In vitro human cell models of inflammatory reaction were set up during FP6 STREP DIPNA, based on human blood monocytes (professional defence cells), and on differentiated human gut mucosal CaCo-2 carcinoma cells (non professional defence cells). The two models are complementary in assessing the impact of NP on innate/inflammatory immune responses. Particular attention will be devoted to the effects on inflammasome/NALP3 activation and triggering of adjuvant vs. inflammatory effects.
The in vitro kinetic model of inflammation is based on monocytes (isolated from human blood) that will be stimulated so as to mimic the different phases of an inflammatory reaction (initiation, development, and resolution). Cells will be cultured for three days in appropriate conditions (e.g., temperature and oxygen tension reproducing those in inflamed tissue) and exposed sequentially to chemokines (that recruit monocytes to the site of inflammation), LPS or viral components (to simulate the encounter with an infectious agent), IFN-γ (produced upon bacterial infections) or IFN-α (produced upon viral infection) plus TNF-α or other inflammatory cytokines (to mimic the inflammatory environment), IL-10, TGF-β and galectin-3 (to reproduce the late switch-off of inflammation and initiation of tissue repair).
The in vitro model of gut mucosa is based on enterocytes, growing in polarised fashion and establishing tight junctions, derived from in vitro differentiation of the human colon carcinoma cell line CaCo-2. The gut mucosa will be challenged with stressful/infectious agents (to mimic a mucosal infection or injury) and its innate defensive reaction will be assessed. In both models, the possibility that NP could interfere with the development of the reaction, or trigger a reaction by itself, will be examined. While for the monocyte model the exposure to NP cannot take place for more than 3 days, as cells rapidly change in culture, for the mucosa model chronic and cumulative exposures will be possible (up to 3 weeks). The innate immune response of cells will be evaluated in terms of up- and down-regulation of key genes (innate cytokines and receptors, the inflammasome genes NALP3, ASC, Cardinal, caspase-1, IL-1, IL-18) and proteins thereof. In addition, the functional activity of the inflammasome, with cleavage and activation of the inflammatory cytokines IL-1β and IL-18, will be measured. As biological contaminants of NP, in particular LPS, can have a major influence in triggering inflammation, customised procedures for detecting endotoxin contamination of NP batches will be implemented and standardized.
The project will employ one ESR, who will gain skills in cell culture, quantitative gene expression (real-time PCR, microarrays, deep sequencing), protein quantification (MudPIT proteomics, ELISA, cyto-fluorimetry, immunofluorescence), biological functions (proliferation, cytokine production, enzymatic activity). Secondments to partners 3 (chip-based miniaturisation), 4 (assay validation and standardisation), 8 (harmonisation of assays on different tissues), 9 (NP fate during culture), and 10 (standardisation of culture conditions for human primary cells).
In WP1 (task 1.1 and 1.5), WP2 (tasks 2.1 and 2.4), WP3 (task 3.3) and WP4 (task 4.3).