TgMAPK1 is a Toxoplasma gondii MAP kinase that hijacks host MKK3 signals to regulate virulence and interferon-γ-mediated nitric oxide production.

The parasite Toxoplasma gondii controls tissue-specific nitric oxide (NO), thereby augmenting virulence and immunopathology through poorly-understood mechanisms. We now identify TgMAPK1, a Toxoplasma mitogen-activated protein kinase (MAPK), as a virulence factor regulating tissue-specific parasite burden by manipulating host interferon (IFN)-γ-mediated inducible nitric oxide synthase (iNOS). Toxoplasma with reduced TgMAPK1 expression (TgMAPK1(lo)) demonstrated that TgMAPK1 facilitates IFN-γ-driven p38 MAPK activation, reducing IFN-γ-generated NO in an MKK3-dependent manner, blunting IFN-γ-mediated parasite control. TgMAPK1(lo) infection in wild type mice produced ≥ten-fold lower parasite burden versus control parasites with normal TgMAPK1 expression (TgMAPK1(con)). Reduced parasite burdens persisted in IFN-γ KO mice, but equalized in normally iNOS-replete organs from iNOS KO mice. Parasite MAPKs are far less studied than other parasite kinases, but deserve additional attention as targets for immunotherapy and drug discovery.

Antigen-specific immunity and cross-priming by epithelial ovarian carcinoma-induced CD11b(+)Gr-1(+) cells.

Both innate and adaptive immune systems are considered important for cancer prevention, immunosurveillance, and control of cancer progression. It is known that, although both systems initially eliminate emerging tumor cells efficiently, tumors eventually escape immune attack by a variety of mechanisms, including differentiation and recruitment of immunosuppressive CD11b(+)Gr-1(+) myeloid suppressor cells into the tumor microenvironment. However, we show that CD11b(+)Gr-1(+) cells found in ascites of epithelial ovarian cancer-bearing mice at advanced stages of disease are immunostimulatory rather than being immunosuppressive. These cells consist of a homogenous population of cells that morphologically resemble neutrophils. Moreover, like dendritic cells, immunostimulatory CD11b(+)Gr-1(+) cells can strongly cross-prime, augmenting the proliferation of functional CTLs via signaling through the expression of costimulatory molecule CD80. Adoptive transfer of these immunostimulatory CD11b(+)Gr-1(+) cells from ascites of ovarian cancer-bearing mice results in the significant regression of s.c. tumors even without being pulsed with exogenous tumor Ag prior to adoptive transfer. We now show for the first time that adaptive immune responses against cancer can be augmented by these cancer-induced granulocyte-like immunostimulatory myeloid (CD11b(+)Gr-1(+)) cells, thereby mediating highly effective antitumor immunity in an adoptive transfer model of immunity.

B7-H1-dependent sex-related differences in tumor immunity and immunotherapy responses.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are immunopathogenic in cancers by impeding tumor-specific immunity. B7-homologue 1 (B7-H1) (CD274) is a cosignaling molecule with pleiotropic effects, including hindering antitumor immunity. In this study, we demonstrate sex-dependent, B7-H1-dependent differences in tumor immunity and response to immunotherapy in a hormone-independent cancer, murine B16 melanoma. Antitumor immunity was better in B7-H1(-/-) females versus males as a result of reduced regulatory T cell function in the B7-H1(-/-) females, and clinical response following B7-H1 blockade as tumor immunotherapy was significantly better in wild-type females than in males, owing to greater B7-H1 blockade-mediated reduction of Treg function in females. Wild-type female Tregs expressed significantly lower B7-H1 versus males but were insensitive to estrogen in vitro. Female B7-H1(-/-) Tregs were exquisitely sensitive to estrogen-mediated functional reduction in vitro, suggesting that B7-H1 effects occur before terminal Treg differentiation. Immune differences were independent of known B7-H1 ligands. Sex-dependent immune differences are seldom considered in designing immune therapy or interpreting immunotherapy treatment results. Our data demonstrate that sex is an important variable in tumor immunopathogenesis and immunotherapy responses through differential Treg function and B7-H1 signaling.

The Hsp90 cochaperone p23 is essential for perinatal survival.

The functions of molecular chaperones have been extensively investigated biochemically in vitro and genetically in bacteria and yeast. We have embarked on a functional genomic analysis of the Hsp90 chaperone machine in the mouse by disrupting the p23 gene using a gene trap approach. p23 is an Hsp90 cochaperone that is thought to stabilize Hsp90-substrate complexes and, independently, to act as the cytosolic prostaglandin E2 synthase. Gene deletions in budding and fission yeasts and knock-down experiments with the worm have not revealed any clear in vivo requirements for p23. We find that p23 is not essential for overall prenatal development and morphogenesis of the mouse, which parallels the observation that it is dispensable for proliferation in yeast. In contrast, p23 is absolutely necessary for perinatal survival. Apart from an incompletely formed skin barrier, the lungs of p23 null embryos display underdeveloped airspaces and substantially reduced expression of surfactant genes. Correlating with the known function of glucocorticoids in promoting lung maturation and the role of p23 in the assembly of a hormone-responsive glucocorticoid receptor-Hsp90 complex, p23 null fibroblast cells have a defective glucocorticoid response. Thus, p23 contributes a nonredundant, temporally restricted, and tissue-specific function during mouse development.

Coupling hippocampal neurogenesis to brain pH through proneurogenic small molecules that regulate proton sensing G protein-coupled receptors.

Acidosis, a critical aspect of central nervous system (CNS) pathophysiology and a metabolic corollary of the hypoxic stem cell niche, could be an expedient trigger for hippocampal neurogenesis and brain repair. We recently tracked the function of our isoxazole stem cell-modulator small molecules (Isx) through a chemical biology-target discovery strategy to GPR68, a proton (pH) sensing G protein-coupled receptor with no known function in brain. Isx and GPR68 coregulated neuronal target genes such as Bex1 (brain-enriched X-linked protein-1) in hippocampal neural progenitors (HCN cells), which further amplified GPR68 signaling by producing metabolic acid in response to Isx. To evaluate this proneurogenic small molecule/proton signaling circuit in vivo, we explored GPR68 and BEX1 expression in brain and probed brain function with Isx. We localized proton-sensing GPR68 to radial processes of hippocampal type 1 neural stem cells (NSCs) and, conversely, localized BEX1 to neurons. At the transcriptome level, Isx demonstrated unrivaled proneurogenic activity in primary hippocampal NSC cultures. In vivo, Isx pharmacologically targeted type 1 NSCs, promoting neurogenesis in young mice, depleting the progenitor pool without adversely affecting hippocampal learning and memory function. After traumatic brain injury, cerebral cortical astrocytes abundantly expressed GPR68, suggesting an additional role for proton-GPCR signaling in reactive astrogliosis. Thus, probing a novel proneurogenic synthetic small molecule's mechanism-of-action, candidate target, and pharmacological activity, we identified a new GPR68 regulatory pathway for integrating neural stem and astroglial cell functions with brain pH.

Mitigating age-related immune dysfunction heightens the efficacy of tumor immunotherapy in aged mice.

Although cancer tends to affect the elderly, most preclinical studies are carried out in young subjects. In this study, we developed a melanoma-specific cancer immunotherapy that shows efficacy in aged but not young hosts by mitigating age-specific tumor-associated immune dysfunction. Both young and aged CD4(+)CD25(hi) regulatory T cells (Treg) exhibited equivalent in vitro T-cell suppression and tumor-associated augmentation in numbers. However, denileukin diftitox (DT)-mediated Treg depletion improved tumor-specific immunity and was clinically effective only in young mice. DT-mediated Treg depletion significantly increased myeloid-derived suppressor cell (MDSC) numbers in aged but not young mice, and MDSC depletion improved tumor-specific immunity and reduced tumor growth in aged mice. Combining Treg depletion with anti-Gr-1 antibody was immunologically and clinically more efficacious than anti-Gr-1 antibody alone in aged B16-bearing mice, similar to Treg depletion alone in young mice. In contrast, DT increased MDSCs in young and aged mice following MC-38 tumor challenge, although effects were greater in aged mice. Anti-Gr-1 boosted DT effects in young but not aged mice. Aged antitumor immune effector cells are therefore competent to combat tumor when underlying tumor-associated immune dysfunction is appropriately mitigated, but this dysfunction varies with tumor, thus also varying responses to immunotherapy. By tailoring immunotherapy to account for age-related tumor-associated immune dysfunctions, cancer immunotherapy for aged patients with specific tumors can be remarkably improved.

Parasite mitogen-activated protein kinases as drug discovery targets to treat human protozoan pathogens.

Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known.

A simple method to detect Toxoplasma gondii-specific cytotoxic T cells in vivo.

Cytotoxic T cells (CTLs) are an important component of adaptive immunity. The study of antigen-specific CTLs in vivo is desirable yet difficult. Identification of the class I-restricted peptide used by CTLs for target recognition is often required for detailed studies, but is generally not known for most antigens. Toxoplasma gondii is a medically important, obligate intracellular parasite and is often used as a model for studies of parasite immunology. No class I-restricted peptides for CTLs are known. We show here a new and convenient method to detect T. gondii-specific CTLs in vivo. We engineered T. gondii tachyzoites to express the model antigen ovalbumin, for which many useful reagents and transgenic mice are available. Using ovalbumin-transgenic T. gondii tachyzoites, antigen-specific CTLs were detected in vivo, and at much earlier time points post-infection than previously reported. This new method has several additional advantages over current methods to detect T. gondii-specific CTLs.