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Role of Nano-Titanium Dioxide Immunotoxicity in Infectious Disease paThology

Final Report Summary - TITOXPATH (Role of Nano-Titanium Dioxide Immunotoxicity in Infectious Disease paThology)

The rise in use of nanotechnology has significantly increased risks of human exposure and interaction of nanoparticles with the immune system, potentially jeopardizing host responses to infection. Titanium dioxide nanoparticle (nano-TiO2) has been increasingly used in cosmetics, building materials, medications and food, resulting in estimated environmental concentration of 0.025 µg/mL. Nanoparticles can be phagocytized and can induce cytokine and antibody production, as well as interfere with phagocyte granule exocytosis. Neutrophils have been shown to manifest significant changes in phagocytosis, oxidative burst, degranulation, and extracellular trap release following exposure to nanoparticles. Notwithstanding these observations, nano metallic-oxide particles are currently deemed safe, but regulators acknowledge that standardized toxicity assays can often miss the effects of chronic exposures or potential toxicity to specific organ systems.

Exposure to nano-TiO2 has been found to interfere with neutrophil function and modify gene expression, but its contribution to infectious disease pathology is currently unclear. There is a critical need to define the functional immunological consequences of chronic nanoparticle exposure. Lack of this knowledge could potentially confound efforts to prevent, diagnose, and treat infectious diseases in people whose immune system responses may have been affected by exposure to nanomaterials. The objective of the proposed studies was to define genetic and innate immune responses of neutrophils to exposure to nano-TiO2 as well as functional consequences in host responses to disease causing pathogens using a fish model. The rationale for the research approach was that insights into nano-TiO2 dependent changes in host immune response to pathogens will allow better assessment of risks associated with chronic exposures to metallic oxide nanoparticles.

The public health relevance of the performed research is reflected in the fact that nano-technology products are rapidly accumulating in the environment and their potential for causing adverse health effects is growing proportionately. The project results provided us with better understanding of the contribution of environmentally-relevant nanoparticle doses to modulation of infectious disease pathology, but further experiments are needed in order to increase our mechanistic understanding of the biological activity of nano-metallic oxides and assess their potential for chronic toxicity, as well as to allow us to evaluate the safety of metallic oxide nanoparticles. Integration of principal scientist to the EU scientific community network was significantly enhanced with CIG support to retain and expand collaborations and knowledge transfer from the U.S. to Germany.

During this project, we have completed the embryonic zebrafish exposure to nano-TiO2 particles and quantum dots and determined that there is no direct toxicity of embryos exposed to those two nanoparticles but, there is observed interaction between innate immune system and nanoparticles. We then focused on clarifying the mechanisms that may be causing different dynamics of pathogenesis in fish exposed to nanoparticles and infection. The analysis of molecular data has also been complemented with in silico analysis of regulation of signaling pathways involved in organismal responses to nanoparticles both alone and in presence of infectious disease agents. We used multiple molecular regulatory network information to identify 6 microRNA targets for future studies.

We have also exposed the neutrophils to effects of nanoplastic particles (polyvinyl and polycarbonate) to verify that similar patterns of immunomodulation occur with other types of nanoparticles. Our second major objective was to determine if exposure to nanoparticles can change the outcome of infection and we have determined that there is difference in infection progression depending on the bacterium species used in the challenge studies, as well that there are molecular mechanisms based on microRNA regulated pathways involved in several major responses to toxicants and infection: oxidative stress, DNA damage and repair and inflammation regulation. We have developed regulatory gene network models and identified 6 potential targets for further studies as biomarkers of nanoparticle toxicity, and we are now focusing on clarifying the mechanisms that may be causing different dynamics of pathogenesis in fish exposed to nanoparticles and infection.

Outreach and dissemination activities took form in laboratory open days, where general public was invited to visit on total of four occasions, however, only a limited number of visitors was attending, mostly students of LMU veterinary faculty. The regional television (Bayerishe Rundfunk) has prepared and broadcasted a short video featuring our laboratory in relation to possible fish welfare aspects.

The international collaboration and dissemination was on the other hand increasingly successful. As a project activity, we have hosted exchange scientists from Egypt, Thailand, Uganda, Serbia and also extended visits to hold information events and workshops in Serbia, Thailand and South Africa in years 3 and 4 of the project.

The training of young scientists and expanding collaborative networks was also very successful. One habilitant and two doctoral students who were partially involved with the project activities have received their respective degrees (habilitation and doctoral degrees) from LMU in January 2016. Two female doctoral students participated in the project from 2014-2016, and one doctoral student started his participation in 2016 (expected to complete in 2019).Four undergraduate/veterinary students have been involved in project activities as student aids and received training in basic research methodology and science concepts.

The principal scientist has successfully established collaborations with State (Ministry of Ecology and Ministry of Agriculture research institutes), National (Fridrich Loeffer Institute, Max Planck Institute), and several International institutions: Austria (U of Vet Med, Vienna), Canada (UPEI Atlantic Veterinary College), Croatia (Rudjer Boskovic Institute), Egypt – Mansoura University; Greece, U of Thessaly; Hungary – University of Veterinary Medicine Budapest; Norway – Norwegian Veterinary Institute; Romania (The University of Agricultural Sciences and Veterinary Medicine of Bucharest), Serbia (Veterinary and Agriculture Faculties of Belgrade University, Biology Faculty of Nis University), South Africa (University of Pretoria), Thailand (Chulalongkorn University Bangkok, and Maejo University, Chiang Mai), and U.S.A (Iowa State University). Prof. Palić collaborated on peer reviewed manuscripts and proposal submissions with the above institutions, as well as several other institutions since CIG grant award. The CIG allowed funding for several visits of Prof. Palić to exchange visits with colleagues in the E.U. and worldwide, and also for corresponding visits to LMU.

Project website is integral part of the LMU Fish Disease Chair web pages system. The specific project activities (publications, presentations etc) will be published on the web site as they become available ( so that information about project activities and result dissemination is publicly available. Further activities of the project were also disseminated through social media pages through World Aquatic Veterinary Medical Association portals, through Regional TV broadcasting, as well as news items in regional online and printed newspaper editions.

Highlights of integration are authorship (co-author or senior/corresponding author) on 4 research (two published and two in preparation) and two review articles (in preparation) with colleagues from the E.U. and submission of 5 proposals in collaboration with E.U. colleagues (two to H2020 as consortium of several countries, two to DFG/German Science Foundation as cross-border collaboration of Germany and Austria, and one to LEAP-AGRI as consortium of E.U. and African partners).

Highlights of research are characterization of nanoparticles, determination of interaction between innate immune system and nanoparticles, and observation of difference in disease pathogenesis followed by development and delineation of possible regulatory networks involving microRNA.

Further contact information:

Prof. Dr Dušan Palić, Chair of Fish Diseases and Fisheries Biology
Faculty of Veterinary Medicine
Ludwig-Maximilians-Universität München