Hye-Seong Mun

Induction of protective immunity by DNA vaccination with Toxoplasma gondii HSP70, HSP30 and SAG1 genes.

The vaccination with Toxoplasma gondii heat shock protein 70 (T.g.HSP70) gene (a virulent tachyzoite-specific) induced the most prominent reduction in T. gondii loads in various organs of B6 and BALB/c mice at the acute and chronic phases of toxoplasmosis compared with T.g.HSP30 (a bradyzoite-specific) and SAG1 (a tachyzoite-specific) genes. A single gene gun vaccination with 2 microg of T.g.HSP70 gene induced a significant reduction in the number of T. gondii organisms compared with 50 microg of T.g.HSP70 gene vaccination by intramuscular (i.m.) or intraperitoneal (i.p.) injection. The vaccine effects of T.g.HSP70 gene persisted for more than 3 months.

Role of Toxoplasma gondii HSP70 and Toxoplasma gondii HSP30/bag1 in Antibody Formation and Prophylactic Immunity in Mice Experimentally Infected with Toxoplasma gondii

Production of antibodies against Toxoplasma gondii (T. gondii)‐derived stress proteins, T. gondii HSP70 (T.g.HSP70) and T.g.HSP30/bag1, in C57BL/6 and BALB/c mice perorally infected with cysts of the avirulent Fukaya strain of T. gondii was analyzed. Production of anti‐T.g.HSP70 IgG antibodies was transient, whereas production of anti‐T.g.HSP30/bag1 IgG antibodies persisted after infection in both C57BL/6 and BALB/c mice. C57BL/6 mice, a susceptible strain, predominantly produced IgG antibodies specific for T.g.HSP70, whereas BALB/c mice, a resistant strain, predominantly produced IgG antibodies specific for T.g.HSP30/bag1, after T. gondii infection. Immunization with rT.g.HSP30/bag1 enhanced, whereas immunization with rT.g.HSP70 reduced host protective immunity against T. gondii infection with a cyst‐forming avirulent strain, Fukaya, and a virulent strain, RH.

Toxoplasma gondii–derived heat shock protein HSP70 functions as a B cell mitogen

Abstract We have investigated the role of Toxoplasma gondii–derived heat shock protein 70 (TgHSP70) as a B cell mitogen by measuring proliferative responses in vitro. TgHSP70 induced prominent proliferative responses in murine B cells derived not only from T gondii–infected but also from uninfected mice. Nude mice responded to TgHSP70; however, severe combined immunodeficiency, RAG1−/− B6, and μMT mice failed to respond. B220+ spleen cells showed marked proliferation after stimulation with TgHSP70, but neither CD4+ nor CD8+ population responded. This unresponsiveness of CD4+ and CD8+ T cells to TgHSP70 was antigen presenting cells independent. These data indicate that TgHSP70 induced the proliferation of B cells but not T cells. Polymyxin B, a potent inhibitor of lipopolysaccharide (LPS), did not eliminate TgHSP70-induced proliferation. C3H/HeN mice responded well to TgHSP70 stimulation; however, C3H/HeJ mice carrying a point mutation in the Toll-like receptor (TLR) 4 failed to respond. This indicates that TLR4 is required for TgHSP70-induced B cell activation. The involvement of TLR4 in the TgHSP70-induced proliferative responses of spleen cells was also shown by the use of TLR4−/− mice. But TgHSP70-induced, but not LPS-induced, spleen cell proliferation was observed in MyD88−/− mice, indicating that the MyD88 molecule was involved in LPS-induced proliferation but not in TgHSP70-induced proliferation.

Toxoplasma gondii–derived heat shock protein 70 stimulates maturation of murine bone marrow–derived dendritic cells via Toll-like receptor 4

Abstract Toxoplasma gondii –derived heat shock protein 70 (T.g.HSP70) induced maturation of bone marrow–derived dendritic cells (DCs) of wild-type (WT) C57BL/6 mice as evidenced by an increase in surface expression of MHC class I and II molecules and costimulatory molecules such as CD40, CD80, and CD86. Functionally, decreased phagocytic ability and increased alloreactive T cell stimulatory ability were observed in T.g.HSP70-stimulated DCs. These phenotypic and functional changes of T.g.HSP70-stimulated DCs were demonstrated in Toll-like receptor (TLR) 2- and myeloid differentiation factor 88 (MyD88)-deficient but not TLR4-deficient C57BL/6 mice. DCs from WT and TLR2-deficient but not TLR4-deficient mice produced IL-12 after T.g.HSP70 stimulation. T.g.HSP70-stimulated DCs from WT, TLR2-deficient, and MyD88-deficient, but not TLR4-deficient mice expressed IFN-β mRNA. Thus, T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade.

Toll-Like Receptor 4 Mediates Tolerance in Macrophages Stimulated with Toxoplasma gondii-Derived Heat Shock Protein 70

ABSTRACT Peritoneal macrophages (PMs) from toll-like receptor 4 (TLR4)-deficient and wild-type (WT) mice were responsive to recombinant Toxoplasma gondii-derived heat shock protein 70 (rTgHSP70) and natural TgHSP70 (nTgHSP70) in NO release, but those from TLR2-, myeloid differentiation factor 88 (MyD88)-, and interleukin-1R-associated kinase 4 (IRAK4)-deficient mice were not. Polymyxin B did not inhibit PM activation by TgHSP70 and nTgHSP70 from WT and TLR4-deficient mice, while it inhibited PM activation by lipopolysaccharide. Pretreatment of PMs from WT but not from TLR4-deficient mice with rTgHSP70 resulted in suppression of NO release on restimulation with rTgHSP70. Similarly, pretreatment of PMs from WT but not TLR4-deficient mice with nTgHSP70 resulted in suppression of NO release on restimulation with nTgHSP70. Polymyxin B did not inhibit rTgHSP70- and nTgHSP70-induced tolerance of PMs from TLR4-deficient mice. Furthermore, PMs from WT mice increased suppressor of cytokine-signaling-1 (SOCS-1) expression after restimulation with rTgHSP70, while those from TLR4-deficient mice did not. Phosphorylation of JNK and I-κBα occurred in rTgHSP70-induced tolerance of PMs from TLR4-deficient mice, but not in that from WT mice. These data indicated that TgHSP70 signaling mechanisms were mediated by TLR2, MyD88, and IRAK4, but not by TLR4. On the other hand, signaling of TgHSP70-induced tolerance was mediated by TLR4, and the expression of SOCS-1 suppressed the TLR2 signaling pathway.

Toxoplasma gondii Hsp70 as a danger signal in Toxoplasma gondii-infected mice

Abstract Toxoplasma gondii Hsp70, T gondii Hsp30/bag1, and surface antigen 1 messenger RNAs were shown to be useful in analyzing stage conversion of T gondii between bradyzoites and tachyzoites. The high-level expression of T gondii Hsp70 was correlated with mortality in interferon-γ knockout mice infected with T gondii. T gondii Hsp70 inhibited the induction of nitric oxide release by peritoneal macrophages of T gondii-infected mice. These findings identify T gondii Hsp70 as a danger signal during lethal, acute T gondii infection.

Involvement of MyD88 in host defense and the down-regulation of anti-heat shock protein 70 autoantibody formation by MyD88 in Toxoplasma gondii-infected mice

This study investigated the influence of TLR (toll-like receptor)4, TLR2, and MyD88 in Toxoplasma gondii–infected wild-type (WT) mice and TLR4-, TLR2-, and MyD88-deficient mice. Ninety-five percent of MyD88-deficient mice died 10–16 days after intraperitoneal infection with 100 cysts of T. gondii Fukaya strain, whereas 95–100% of TLR4- and TLR2-deficient mice and WT C57BL/6 (B6) mice survived for more than 7 wk after T. gondii infection. The distribution of T. gondii in various organs of TLR4-, TLR2-, and MyD88-deficient mice and WT B6 mice was assessed 2 wk after T. gondii intraperitoneal infection using quantitative competitive polymerase chain reaction. In MyD88-deficient mice, high levels of T. gondii load were observed in the brain, tongue, heart, lungs, spleen, liver, mesenteric lymph node, and kidneys after infection. The T. gondii load was significantly increased in the lungs in both TLR4- and TLR2-deficient mice compared with WT B6 mice. High levels of anti–mouse heat shock protein (mHSP)70 autoantibody and anti-T. gondii HSP70 antibody production were detected in the sera from MyD88-deficient mice.

Anti-HSP70 Autoantibody Formation by B-1 Cells in Toxoplasma gondii-Infected Mice

ABSTRACT Formation of anti-Toxoplasma gondii HSP70 (TgHSP70) antibody cross-reactive to mouse HSP70 (mHSP70) was observed in the sera of BALB/c (a resistant strain) and C57BL/6 (B6; a susceptible strain) mice after peroral infection with T. gondii cysts of the Fukaya strain. The levels of anti-mHSP70 immunoglobulin G (IgG) autoantibody production in B6 mice were higher than those in BALB/c mice. The isotype and subclass of IgG of anti-TgHSP70 monoclonal antibodies cross-reactive to mHSP70 were μ and γ3. Anti-mHSP70 autoantibody in T. gondii-infected BALB/c and B6 mice was shown to be produced by the CD5+subset of B cells (B-1a cells) but not by conventional B cells (B-2 cells). The epitopes recognized by anti-mHSP70 autoantibody were located primarily in the C-terminal fragment of mHSP70.

ROLES OF IFN-γ ON STAGE CONVERSION OF AN OBLIGATE INTRACELLULAR PROTOZOAN PARASITE, Toxoplasma Gondii

IFN-γ downregulates the stage conversion of Toxoplasma gondii (T. gondii), from bradyzoites to tachyzoites, and the expression of heat shock protein 70 (HSP70) of T. gondii ( T.g. HSP70) by tachyzoites. T.g. HSP70 has been shown to downregulate NO release from macrophages and also to induce auto-HSP70 antibody formation and IL-10 secretion by VH11-JH1 B-1 cells, resulting in the suppression of host defense responses to T. gondii infection. A novel category of virulent tachyzoite stage of T. gondii expressing T.g. HSP70 (virulent tachyzoite), which indirectly manifests its pathogenicity by downregulating host defense responses, has been proposed.

Organ Infectivity of Toxoplasma gondii in Interferon-γ Knockout Mice

To determine the influence of interferon (IFN)-γ on the organ infectivity and on the genetic susceptibility of susceptible (C57BL/6) and resistant (BALB/c) strains after peroral infection with cysts of Toxoplasma gondii, IFN-γ knockout (KO) mice in C57BL/6 and BALB/c backgrounds were utilized. The kinetics of the changes in T. gondii abundance were evaluated with a quantitative competitive polymerase chain reaction assay in various organs at different times after peroral infection. In IFN-γ KO mice, a T. gondii-specific gene, SAG1, was detected in all organs examined, and the protozoan proliferated much more actively than in wild-type mice. The abundance of T. gondii was much higher in mesenteric lymph nodes and the heart than in other organs. In contrast, in the nervous system organs and kidneys, only a weakly detectable reaction was observed. Toxoplasma gondii grew at a more rapid rate in the organs of IFN-γ KO C57BL/6 mice than in the organs of IFN-γ KO BALB/c mice during the course of infection. Destruction of the IFN-γ gene showed remarkable effects on the infectivity in both susceptible and resistant mice.