Unraveling the immune regulatory mechanisms imposed by Anaplasma.

The paper by Zerai Woldehiwet, published in this issue of The Veterinary Journal, reviews mechanisms of immune evasion and immune suppression by the mammalian pathogen, Anaplasma phagocytophilum, which primarily invades and replicates within neutrophils. A. phagocytophilum infection causes a generalized immune suppression in sheep characterized by neutropenia, lymphopenia, and susceptibility to various concurrent bacterial infections resulting from impaired neutrophil and lymphocyte function (Woldehiwet, 2007). The related cattle pathogen, A. marginale, which invades and replicates within erythrocytes, causes anemia, abortion in pregnant heifers, and weight loss, can, during acute infection, reach levels of 109 infected erythrocytes per milliliter of blood (Palmer et al., 1999). Following resolution of acute infection, both pathogens establish persistence, which for A. marginale infected cattle, can be of lifelong duration. Both pathogens evade the immune response through antigenic variation of surface coat proteins, including major surface protein (MSP) 2 and MSP3 in A. marginale, and the related MSP2/P44 in A. phagocytophilum (Palmer et al., 2006). The immunological consequences of this antigenic variation have been documented for A. marginale. During persistent infection, organisms express sequentially variant B cell and T cell epitopes in MSP2 that evade effector IgG2 and CD4+ T cell recognition (French et al., 1999; Brown et al., 2003). In contrast to the generalized immune suppression observed during A. phagocytophilum infection of sheep, infection of cattle with A. marginale does not result in altered numbers of circulating leukocytes, including lymphocytes, and generalized immune suppression has not been reported. However, immunization models using A. marginale MSPs have recently revealed infection-induced regulation of a strong pre-challenge CD4+ T cell response. In the first model, cattle were hyperimmunized by subcutaneous inoculation of gel-purified native MSP2 in Th1 adjuvants and challenged 6 months later with organisms expressing the same major MSP2 variant that was present in the immunogen (Abbott et al., 2005). The predicted protection against infection with organisms expressing this major MSP2 variant was not realized; rather the lack of protection against anemia and rickettsemia was accompanied by a dramatic loss in MSP2-specific CD4+ T cell responses. The robust pre-challenge levels of CD4+ T cell proliferation and IFN-g secreting cells in response to A. marginale, native MSP2, or immunogenic MSP2 peptides in both conserved and hypervariable regions were reduced to background levels just prior to peak rickettsemia. The loss of response to epitopes conserved in all MSP2 variant proteins indicated that the loss of the MSP2-specific response was not due to emergence of new antigenic variants. Importantly, antigen-specific responses were never detected in animals monitored periodically for a year after infection. In contrast, responses to the unrelated Clostridium antigen used to vaccinate the calves prior to the initiation of the experiment remained normal. Although the mechanism of this infection-induced specific immune suppression is not yet known, functional suppressor cells in the peripheral blood of non-responding animals were not detected, the frequencies of circulating CD25+ CD4+ lymphocytes during the course of infection did not vary significantly, and the production of IL-10 and TGF-b by peripheral blood lymphocytes in response to antigen did not increase following infection. These results suggest the sudden loss of MSP2-specific T cell responses in the peripheral blood was not simply due to an increase in regulatory T cells in peripheral blood in response to infection (Abbott et al., 2005). However, the spleens were not evaluated for functional suppressor T cells, which if present, could explain these results, as the spleen is important for removing infected erythrocytes during hemoparasite infections. In a second immunization model, cattle were inoculated intradermally with a DNA construct encoding a T cell epitope (F2-5) and B cell epitopes (the N-terminal repeats) from the conserved MSP1a, boosted once with recombinant protein (Mwangi et al., 2007) and subsequently challenged months later with A. marginale. As observed for MSP2, the strong peripheral blood CD4+ T cell responses measured after immunization and before challenge disappeared just prior to peak rickettsemia (W. C. Brown, unpublished data). The loss of response was again specific for A. marginale, and Clostridium-specific responses remained intact. This experiment confirmed that the infection-induced loss of response was not restricted to the variable MSP2 antigen, but occurred using a different immunization protocol and a conserved protein epitope. Furthermore, for both studies, Anaplasma-specific T cell responses were not detected in peripheral blood lymphocytes from control sham immunized animals following challenge (Abbott et al., 2005; W. C. Brown, unpublished data). This important observation suggests that in spite of high levels of rickettsemia and A. marginale specific IgG antibody titers, antigen-specific T cell responses are difficult to detect in peripheral blood. Examination of splenic lymphocyte responses should be conducted to determine whether Anaplasma-specific lymphocytes (effector cells or regulatory T cells) sequester there. In fact, one study performed by Gale et al. (1996) showed A. marginale-induced lymphocyte proliferation in spleens of persistently infected cattle at time points when peripheral blood responses were not detected. A caveat to these results is that negative control antigens were not included in these assays. Intracellular A. phagocytophilum and A. marginale pose unique problems for vaccine development. In spite of very different intracellular niches, both bacteria achieve persistent infection, which is critical for tick transmission to susceptible individuals. Contributing to the ability to persist are extensive antigenic variation in their immunodominant surface proteins MSP2 (and MSP3 for A. marginale) and evasion the immune response, and modulation of the host immune response. A. phagocytophilum induces a generalized immune suppression attributed to lymphocytopenia, neutropenia and thrombocytopenia, whereas A. marginale infection suppresses a previously elicited A. marginale MSP-specific T cell response and in naive animals infection fails to stimulate consistently detectable peripheral T lymphocyte responses. Unraveling the immune regulatory mechanisms imposed by Anaplasma is an important and exciting area of investigation.