Linden E. Craig

The CD40/CD40 Ligand Interaction Is Required for Resistance to Toxoplasmic Encephalitis

ABSTRACT Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-γ), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-γ when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-γ or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L−/− mice infected withT. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-γ but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L−/− mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-γ, to activate macrophages in vitro to control replication ofT. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.

Identification of a Role for NF-κB2 in the Regulation of Apoptosis and in Maintenance of T Cell-Mediated Immunity to Toxoplasma gondii

The NF-κB family of transcription factors are involved in the regulation of innate and adaptive immune functions associated with resistance to infection. To assess the role of NF-κB2 in the regulation of cell-mediated immunity, mice deficient in the NF-κB2 gene (NF-κB2−/−) were challenged with the intracellular parasite Toxoplasma gondii. Resistance to this opportunistic pathogen is dependent on the production of IL-12, which is required for the development of innate NK cell and adaptive T cell responses dominated by the production of IFN-γ necessary to control replication of this parasite. Although wild-type controls were resistant to T. gondii, NF-κB2−/− mice developed severe toxoplasmic encephalitis and succumbed to disease between 3 and 10 wk following infection. However, NF-κB2 was not required for the ability of macrophages to produce IL-12 or to inhibit parasite replication and during the acute stage of infection, NF-κB2−/− mice had no defect in their ability to produce IL-12 or IFN-γ and infection-induced NK cell responses appeared normal. In contrast, during the chronic phase of the infection, susceptibility of NF-κB2−/− mice to toxoplasmic encephalitis was associated with a reduced capacity of their splenocytes to produce IFN-γ associated with a loss of CD4+ and CD8+ T cells. This loss of T cells correlated with increased levels of apoptosis and with elevated expression of the pro-apoptotic molecule Fas by T cells from infected NF-κB2−/− mice. Together, these results suggest a role for NF-κB2 in the regulation of lymphocyte apoptosis and a unique role for this transcription factor in maintenance of T cell responses required for long-term resistance to T. gondii.

MATERNAL ANTIBODIES AGAINST PLASMODIUM SPP. IN AFRICAN BLACK-FOOTED PENGUIN (SPHENISCUS DEMERSUS) CHICKS

Anti-Plasmodium spp. antibody titers of mating pairs of adult, captive-reared, African black-footed penguins (Spheniscus demersus) and their chicks were determined using the enzyme-linked-immunosorbent assay (ELISA). Two Plasmodium falciparum antigens were used for the ELISA: R32tet32 (sporozoite antigen), and crude red blood cell extract (CRBCE). Eighteen chicks were bled weekly for ten weeks starting with their day of hatching. The yolk sacs of two penguin eggs were biopsed for ELISA-detectable maternal antibodies (MAB). None of the 28 adult penguins were parasitemic by Giemsa-stained thin blood smear; however, all had anti-Plasmodium spp. immunoglobulins reacting with P. falciparum antigens. All 18 newly hatched chicks had anti-Plasmodium spp. MAB while housed in a mosquito-free environment. The level of MAB in the newly hatched chicks was correlated significantly (P < 0.001) with antibody level detected in their female parents (R32tet32: r = 0.87, CRBCE: r = 0.89). No correlation was found between antibody titers of the newly hatched chicks and their male parents. The level of maternal-fetal antibodies was regressed significantly (P < 0.001) over the 10-week period. Penguin chicks over 10 weeks of age had no anti-Plasmodium spp. MAB. Egg-yolk samples had significantly (P < 0.03) higher MAB titers than female parents that laid these eggs.

Blockade of costimulation prevents infection-induced immunopathology in interleukin-10-deficient mice.

ABSTRACT Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected with Toxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-γ) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice with T. gondii resulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice with T. gondii did not affect serum levels of IFN-γ or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-γ and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-γ but not IL-12. Further reduction of IFN-γ production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.

Contribution of Interleukin-12 (IL-12) and the CD28/B7 and CD40/CD40 Ligand Pathways to the Development of a Pathological T-Cell Response in IL-10-Deficient Mice

ABSTRACT The ability of interleukin-10 (IL-10) to suppress accessory cell functions required for optimal T-cell activation makes it an important inhibitor of cell-mediated immunity. Thus, after infection with the protozoan parasite Toxoplasma gondii, IL-10 knockout (KO) mice develop a CD4+-T-cell-dependent shock-like reaction with high levels of IL-12 and gamma interferon (IFN-γ) in serum, leading to death of mice during the acute phase of infection. Previous studies from this laboratory have shown that simultaneous blockade of CD28 and CD40 can prevent this lethal reaction by inhibiting the production of IFN-γ. However, the blockade of costimulation did not affect systemic levels of IL-12. To better understand the relationship between IL-12 and the CD28 and CD40 pathways in mediating immune hyperactivity, antagonists of these factors were used to determine their effects on the development of a pathological T-cell response in IL-10 KO mice. Blockade of IL-12 or the CD28/B7 interaction alone did not affect survival; however, the combined blockade of both pathways resulted in decreased production of IFN-γ and the survival of IL-10 KO mice. To assess the role of the two ligands for CD28, B7.1 and B7.2, IL-10 KO mice were treated with αIL-12 plus αB7.1 or αB7.2 or the combination of all three antibodies. These studies revealed that blockade of both B7 molecules is required for decreased production of IFN-γ and survival of infected IL-10 KO mice, suggesting that B7.1 and B7.2 can contribute to the lethal shock-like reaction in IL-10 KO mice. In contrast, neutralization of IL-12 and blockade of the CD40/CD40 ligand (CD40L) interaction in vivo did not alter the production of IFN-γ and only resulted in a small delay in time to death of mice. Together, these data suggest that the CD28/B7 interaction has a central role in the development of a pathological T-cell response in IL-10 KO mice, which is distinct from the role of the CD40/CD40L and IL-12 pathways.