To investigate the role of the Toll-like receptor (TLR) family in host defense against Toxoplasma gondii, we infected TLR2-, TLR4- and MyD88-deficient mice with the avirulent cyst-forming Fukaya strain of T. gondii. All TLR2- and MyD88-deficient mice died within 8 days, whereas all TLR4-deficient and wild-type mice survived after i.p. infection with a high dose of T. gondii. Peritoneal macrophages from T. gondii-infected TLR2- and MyD88-deficient mice did not produce any detectable levels of NO. T. gondii loads in the brain tissues of TLR2- and MyD88-deficient mice were higher than in those of TLR4-deficient and wild-type mice. Furthermore, high levels of IFN-gamma and IL-12 were produced in peritoneal exudate cells (PEC) of TLR4-deficient and wild-type mice after infection, but low levels of cytokines were produced in PEC of TLR2- and MyD88-deficient mice. On the other hand, high levels of IL-4 and IL-10 were produced in PEC of TLR2- and MyD88-deficient mice after infection, but low levels of cytokines were produced in PEC of TLR4-deficient and wild-type mice. The most remarkable histological changes with infiltration of inflammatory cells were observed in lungs of TLR2-deficient mice infected with T. gondii, where severe interstitial pneumonia occurred and abundant T. gondii were found.

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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 -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-␤ mRNA. Thus, T.g.HSP70 stimulates murine DC maturation via TLR4 through the MyD88-independent signal transduction cascade.

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 and ␥3. 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.Toxoplasma gondii, an obligate intracellular parasitic protozoan, causes a life-threatening toxoplasmosis in fetuses or in immunocompromised humans and mice, and the mechanisms of antigen presentation of T. gondii-infected cells have been analyzed (2,45,46,48). We have previously demonstrated a potential role of human heat shock cognate protein 70 (hHSC70) in antigen processing and presentation of T. gondiiinfected cells to CD4 ϩ cytotoxic T lymphocytes in humans (44) and have reported anti-T. gondii HSP70 (TgHSP70) antibody formation in T. gondii-infected BALB/c and B6 mice (23).Members of the HSP family have been shown to have important functions as (i) intracellular detergents for aggregated and denatured molecules formed as a result of exposure of cells to physical stressors and (ii) molecular chaperones in peptide and protein transport between cell organelles (14,35,41). Of the HSP family members, HSP70 has been shown to be a major immunodominant antigen in bacterial and parasite infections as well as the preferred target of the humoral and cell-mediated immune responses to infection (7,28,49). The nucleic acid sequence of HSP70 is known to be highly homologous among different species. Indeed, the sequence of TgHSP70 is 68.1 and 74.1% homologous to those of mouse HSP70 (mHSP70) and hHSC70, respectively (6, 8, 47, 50); thus, it is possible that the high homology of HSP70 between T. gondii and hosts could induce autoreactive immune responses in T. gondii-infected hosts. Autoantibody induction against host components has been reported in infection with other parasites such as Trypanosoma cruzi (16), Plasmodium falciparum (21), Schistosoma mansoni (15), and Onchocerca volvulus (27). In particular, Mattei et al. demonstrated that sera from humans infected with malaria recognized the human HSP70, indicating that autoantibodies directed against host HSP70 could be induced by the homologous parasite protein (21). In contrast, humans infected with Echinococcus granulosus and Leishmania braziliensis did not induce autoimmune reactivity against homologous hHSP70, although specific antibodies reactive with parasite HSP70 were detected (4, 34).No such anti-host HSP70 autoantibody was induced in dogs during viscero-cutaneous leishmaniasis either ...