Modulatory effects of cadmium om the immune system of small mammals : An in vitro, in vivo and field approach

Summary

Cadmium can be immunotoxic even at relatively low exposure concentrations. By modulating the immune system of environmentally exposed wild small mammals, cadmium may reduce the host resistance to infections (e.g. zoonotic diseases in wild reservoir species). Because the immune status play an important role in preventing and limiting infections transmission, this project assessed the effects of cadmium on the immune responses of small mammals using a sequential approach, from in vitro to in vivo and to field conditions.

In the introductory Chapter 1 the mammalian immune system, the potential effect of environmental pollutants in modulating the immune system of wild small mammals, and cadmium as model metal because its immunotoxic potential are introduced. The immune system is briefly described emphasizing its function in preventing and controlling infections. A short review on cadmium modulatory effects on the immune system of small mammals is presented, including effects in wild rodent species, in laboratory mouse models and in in vitro models. The aim of the study, to gain insight in how cadmium can modulate the immune status of reservoir rodents, is introduced. The sequential study approach is also described, in which by including in vitro, in vivo and field data, allow to stablish causality between exposure to cadmium and effects at cellular level, individual, and even population level. The other five chapters in this thesis are introduced, including four experimental chapters and general discussion.

In Chapter 2 the effects of cadmium were investigated in murine cells from the innate immune system in vitro. In the study, macrophage and mast cell lines were exposed to a range of cadmium concentrations to build dose- response relationships for different toxicological endpoints in resting cells and functional parameters in activated cells. Cadmium depleted cellular glutathione in macrophages and mast cells and had cell-specific immunomodulatory effects. Cadmium was pro-inflammatory in macrophages and suppressive in mast cells. In Chapter 3, the effects of cadmium were studied in mice cells from the adaptive immune system. T helper (Th2) cell and B cell models were exposed to a range of cadmium concentrations to build dose-response relationships for different endpoints, just like as in the previous chapter. Cadmium also led to cell-specific effects, suppressing B cell functions and stimulating T helper (Th2) cells. Cadmium reduced proliferation of activated B cells by reducing DNA/RNA synthesis, while increased IL-4 production in activated T helper (Th2) cells. The suppressive effects of cadmium on B cell proliferation were considered for the following two chapters in vivo. In Chapter 4 laboratory mice were exposed to cadmium in the drinking water for seven weeks and were immunized to sheep red blood cells (SRBC) to evaluate and compare humoral responses between individuals exposed to different cadmium concentrations in vivo. Resulting kidney cadmium concentrations were in the range of environmentally relevant concentrations and higher in female mice. Total blood IgM and IgG immunoglobulin levels were also higher in non-exposed female mice, compared to non-exposed mice. The increasing cadmium concentrations decreased IgM/IgG blood levels, in which the higher IgM/IgG levels found female mice were more susceptible to be decreased by cadmium, compared to males. The lower levels of IgM/IgG in cadmium-exposed mice were correlated with a decrease in the counts of SRBC-specific antibody-producing B cells in spleen, in line with in vitro results in Chapter 3 of inhibition of B cell proliferation by cadmium.

In Chapter 5 environmentally exposed wood mice (Apodemus sylvaticus) were captured from three locations in the Netherlands with known levels of cadmium pollution, to verify the effects observed in vitro and in vivo under laboratory conditions. The animals were immunized to SRBC to evaluate the humoral responses. Kidney cadmium concentrations reflected the different levels of cadmium pollution for each capture location and increased with body mass, as a result of time of exposure (age). Flow cytometry analysis of blood samples showed that cadmium exposure can reduce the counts of B cells of female mice. Humoral responses were measured independently for each capture location, due to natural immune heterogeneity between wild rodent populations. Increasing cadmium concentrations decreased the number of SRBC-specific antibody-producing B cells in wood mice, similar to the observations made in Chapter 4 in laboratory mice, and in line with a decrease of B cell proliferation in vitro in Chapter 3. The immunosuppressive effects of cadmium in the humoral immunity of laboratory mice and wood mice were discussed as a potential mechanism in reducing the host resistance to infections.

Chapter 6 presented a general discussion of the results obtained in this thesis. The discussion went through the results obtained in the different experimental chapters, showing the potential consequences and impacts of the effects of cadmium on the immunity of small mammals at different levels, from sub-cellular, to cellular and individual level. It was possible to establish causality between cadmium exposure and effects at cellular level, providing evidence in how cadmium can modulate and impair small mammal humoral immune responses in vivo, to finally connect those results with effects observed in actual environmentally exposed wood mice living in polluted environments. The immunosuppressive effects of cadmium were discussed in regard of their impacts on host resistance to infections, with emphasis in the risk of increase of zoonotic infections in small mammal reservoir host species.