## 4.2.10. Immunotoxicity

Author: Nico van den Brink

Review: Manuel E. Ortiz-Santaliestra

Learning objectives:

You should be able to:

- Understand the complexity of potential effects of chemicals on the immune system
- Explain the difference between innate and acquired parts of the immune system
- Explain the most important modes of toxicity that may affect immune cells

Key words: Immune toxicology, pathogens, innate and adaptive immune system, lyme disease

Introduction

The immune system of organisms is very complex with different cells and other components interacting with each other. The immune system has the function to protect the organism from pathogens and infections. It consists of an innate part, which is active from infancy and an acquired part which is adaptive to exposure to pathogens. The immune system may include different components depending on the species (Figure 1).

Figure 1. Simplified diagram of the evolution of the immune system indicating some preserved key immunological functions (adapted from Galloway and Handy, 2003).

The main organs involved in the immune system of mammals are spleen, thymus, bone marrow and lymph nodes. In birds, besides all of the above, there is also the bursa of Fabricius. These organs all play specific roles in the immune defence, e.g. the spleen synthesises antibodies and plays an important role in the dynamics of monocytes; the thymus is the organ where T-cells develop while in bone marrow lymphoid cells are produced, which are transported to other tissues for further development. The bursa of Fabricius is specific for birds and is essential for B-cell development. Blood is an important tissue to be considered because of its role in transporting cells. The innate system generally provides the first response to infections and pathogens, however it is not very specific. It consists of several cell types with different functions like macrophages, neutrophils and mast cells. Macrophages and neutrophils may act against pathogens by phagocytosis (engulfing in cellular lysosomes and destruction of the pathogen). Neutrophils are relatively short lived, act fast and can produce a respiratory burst to destroy the pathogen/microbe. This involves a rapid production of Reactive Oxygen Species (ROS) which may destroy the pathogens. Macrophages generally have a longer live span, react slower but more prolonged and may attack via production of nitric oxide and less via ROS. Macrophages produce cytokines to communicate with other members of the immune system, especially cell types of the acquired system. Other members of the innate immune system are mast cells which can excrete e.g. histamine on detection of antigens. Cells of the acquired, or adaptive immune system mount more specific responses for the immune insult, and are therefore generally more effective. Lymphocytes are the cells of the adaptive immune system which can be classified in B-lymphocytes and T-lymphocytes. B-lymphocytes produce antibodies which can serve as cell surface antigen-receptors, essential in the recognition of e.g. microbes. B-lymphocytes facilitate humoral (extracellular) immune responses against extracellular microbes (in the respiratory gastrointestinal tract and in the blood/lymph circulation). Upon recognition of an antigen, B-lymphocytes produce species antibodies which bind to the specific antigen. This on the one hand may decrease the infectivity of pathogens (e.g. microbes, viruses) directly, but also mark them for recognition by phagocytic cells. T-lymphocytes are active against intracellular pathogens and microbes. Once inside cells, pathogens are out of reach of the B-lymphocytes. T-lymphocytes may activate macrophages or neutrophils to destroy phagocytosed pathogens or even destroy infected cells. Both B- and T-lymphocytes are capable of producing an extreme diversity of clones, specific for antigen recognition. Communication between the different immune cells occurs by the production of e.g. cytokines, including interleukins (ILs), chemokines, interferons (IFs), and also Tumour Necrosis Factors (TNFs). Cytokines and TNFs are related to specific responses in the immune system, for instance IL6 is involved in activating B-cells to produce immunoglobulins, while TNF-α is involved in the early onset of inflammation, therefore one of the cytokines inducing acute immune responses. Inflammation is a generic response to pathogens mounted by cells of the innate part of the immune system. It generally results in increased temperature and swelling of the affected tissue, caused by the infiltration of the tissue by leukocytes and other cells of the innate system. A proper acute inflammatory response is not only essential as a first defence but will also facilitate the activation of the adaptive immune system. Communication between immune cells, via cytokines, not only directs cells to the place of infection but also activates for instance cells of the acquired immune system. This is a very short and non-exhaustive description of the immune system, for more details on the functioning of the immune system see for instance Abbas et al. (2018).

Chemicals may affect the immune system in different ways. Exposure to lead for instance may result in immune suppression in waterfowl and raptors (Fairbrother et al. 2004, Vallverdú-Coll et al., 2019). Decreasing spleen weights, lower numbers of white blood cells and reduced ability to mount a humoral response against a specific antigen (e.g. sheep red blood cells), indicated a lower potential of exposed birds to mount proper immune responses upon infection. Exposure to mercury resulted in decreased proliferation of B-cells in zebra finches (Taeniopygia guttata), affecting the acquired part of the immune system (Lewis et al., 2013). However, augmentation of the immune system upon exposure to e.g. cadmium has also been reported in for instance small mammals, indicating an enhancement of the immune response (Demenesku et al., 2014). Both immune suppression as well as immune enhancement may have negative impacts on the organisms involved; the former may decrease the ability of the organism to deal with pathogens or other infections, while immune enhancement may increase the energy demands of the organism and it may also result in for instance hypersensitivity or even auto-immunity in organisms.

Chemicals may affect immune cells via toxicity to mechanisms that are not specific to the immune system. Since many different cell types are involved in the immune system, the sensitivity to these modes of toxicity may vary considerably among cells and among chemicals. This would imply that as a whole, the immune system may inherently include cells that are sensitive to different chemicals, and as such may be quite sensitive to a range of toxicants. For instance induction of apoptosis, programmed cell death, is essential to clear the activated cells involved in an immune response after the infection is minimised and the system is returning to a state of homeostasis (see Figure 2). Chemicals may induce apoptosis, and thus interfere with the kinetics of adaptive immune responses, potentially reducing the longevity of cells.

Figure 2. Development of an adaptive immune response, with the different cell types involved. Adapted from https://www.memorangapp.com/flashcards/170860/Immunology+Exam+1/.

Toxic effects on mechanisms specific to the immune system may be related to its functioning. Since the production of ROS and nitric oxides are effector pathways along which neutrophils and macrophages of the innate systems combat pathogens (via a high production of reactive oxygen species, i.e. oxidative burst, to attack pathogens), impacts on the oxidative status of these cells may not only result in general toxicity, potentially affecting a range of cell types, but it may also affect the responsiveness of the (innate) immune system particularly. For instance, cadmium has a high affinity to bind to glutathione (GSH), a prominent anti-oxidant in cells, and has shown to affect acute immune responses in thymus and spleens of mice (Pathak and Khandelwal, 2007) via this mechanism. A decrease of GSH by binding of chemicals (like cadmium) may modulate macrophages towards a pro-inflammatory response by changes in the redox status of the cells involved, changing not only their activities against pathogens but potentially also their production and release of cytokines (Dong et al., 1998).

GSH is also involved in the modulation of the acquired immune system by affecting so-called antigen-presenting cells (APCs, e.g. dendritic cells). APCs capture microbial antigens that enter the body, transport these to specific immune-active tissues (e.g. lymph nodes) and present them to naive T-lymphocytes, inducing a proper immune response, so-called T-helper cells. T-helper cells include subsets, e.g. T-helper 1 cells (Th1-cells) and T-helper 2 cells (Th2-cells). Th1 responses are important in the defence against intracellular infections, by activation of macrophages to ingest microbes. Th2-responses may be initiated by infections by organisms too large to be phagocytosed, and mediated by e.g. allergens. As mentioned, GSH depletion may result in changes in cytokine production by APC (Dong et al., 1998), generally affecting the release of Th1-response promoting cytokines. Exposure to chemicals interfering with GSH kinetics may therefore result in a dis-balance between Th1 and Th2 responses and as such affect the responsiveness of the immune system. Cadmium and other metals have a high affinity to bind to GSH and may therefore reduce Th1 responses, while in contrast, GSH promoting chemicals may reduce the organisms’ ability to initiate Th2-responses (Pathak & Khandelwal, 2008).

The overview on potential effects that chemicals may have on the immune system as presented here is not exhaustive at all. This is even more complicated because effects may be contextual, meaning that chemicals may have different impacts depending on the situation an organism is in. For instance, the magnitude of immunotoxic effects may be dependent on the general condition of the organism, and hence some infected animals may show effects from chemical exposure while others may not. Impacts may also differ between types of infection (e.g. Th1 versus Th2 responsive infections). This, together with the complex and dynamic composition of the immune system, limits the development of general dose response relationships and hazard predictions for chemicals. Furthermore, most of the research on effects of chemicals on the immune system is focussed on humans, based on studies on rats and mice. Little is known on differences among species, especially in non-mammalian species which may have completely differentially structured immune systems. Some studies on wildlife have shown effects of trace metals on small mammals (Tersago et al., 2004, Rogival et al., 2006, Tête et al., 2015) and of lead on birds (Vallverdú-Coll et al., 2015). However, specific modes of action are still to be resolved under field conditions. Research on immuno-toxicity in wildlife however is essential not only from a conservational point of view (to protect the organisms and species involved) but also from the perspective of human health. Wildlife plays an important role in the kinetics of zoonotic diseases, for instance small mammals are the prime reservoir for Borrelia spirochetes, the causative pathogens of Lyme-disease while migrating waterfowl are indicated to drive the spread of e.g. avian influenza. The role of wildlife in the kinetics of the environmental spread of zoonotic diseases is therefore eminent, which may seriously be affected by chemical induced alterations of their immune system.

References and further reading

Abbas, A.K., Lichtman, A.H., Pillai, S. (2018). Cellular and Molecular Immunology. 9th Edition. Elsevier, Philadelphia, USA. ISBN: 978-0-323-52324-0

Demenesku, J., Mirkov, I., Ninkov, M., Popov Aleksandrov, A., Zolotarevski, L., Kataranovski, D., Kataranovski, M. (2014). Acute cadmium administration to rats exerts both immunosuppressive and proinflammatory effects in spleen. Toxicology 326, 96-108.

Dong, W., Simeonova, P.P., Gallucci, R., Matheson, J., Flood, L., Wang, S., Hubbs, A., Luster, M.I. (1998). Toxic metals stimulate inflammatory cytokines in hepatocytes through oxidative stress mechanisms. Toxicology and Applied Pharmacology 151, 359-366.

Fairbrother, A., Smits, J., Grasman, K.A. (2004). Avian immunotoxicology. Journal of Toxicology and Environmental Health, Part B 7, 105-137.

Galloway, T., Handy, R. (2003). Immunotoxicity of organophosphorous pesticides. Ecotoxicology 12, 345-363.

Lewis, C.A., Cristol, D.A., Swaddle, J.P., Varian-Ramos, C.W., Zwollo, P. (2013). Decreased immune response in Zebra Finches exposed to sublethal doses of mercury. Archives of Environmental Contamination & Toxicology 64, 327–336.

Pathak, N., Khandelwal, S. (2007). Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice. Toxicology Letters 169, 95-108.

Pathak, N., Khandelwal, S. (2008). Impact of cadmium in T lymphocyte subsets and cytokine expression: differential regulation by oxidative stress and apoptosis. Biometals 21, 179-187.

Rogival, D., Scheirs, J., De Coen, W., Verhagen, R., Blust, R. (2006). Metal blood levels and hematological characteristics in wood mice (Apodemus sylvaticus L.) along a metal pollution gradient. Environmental Toxicology & Chemistry 25, 149-157.

Tersago, K., De Coen, W., Scheirs, J., Vermeulen, K., Blust, R., Van Bockstaele, D., Verhagen, R. (2004). Immunotoxicology in wood mice along a heavy metal pollution gradient. Environmental Pollution 132, 385-394.

Tête, N., Afonso, E., Bouguerra, G., Scheifler, R. (2015). Blood parameters as biomarkers of cadmium and lead exposure and effects in wild wood mice (Apodemus sylvaticus) living along a pollution gradient. Chemosphere 138, 940-946.

Vallverdú-Coll, N., López-Antia, A., Martinez-Haro, M., Ortiz-Santaliestra, M.E., Mateo, R. (2015). Altered immune response in mallard ducklings exposed to lead through maternal transfer in the wild. Environmental Pollution 205, 350-356.

Vallverdú-Coll, N., Mateo, R., Mougeot, F., Ortiz-Santaliestra, M.E. (2019). Immunotoxic effects of lead on birds. Science of the Total Environment 689, 505-515.