Trypanosoma brucei Secreted Aromatic Ketoacids Activate the Nrf2/HO-1 Pathway and Suppress Pro-inflammatory Responses in Primary Murine Glia and Macrophages.

African trypanosomes, such as Trypanosoma brucei (T. brucei), are protozoan parasites of the mammalian vasculature and central nervous system that are best known for causing fatal human sleeping sickness. As exclusively extracellular parasites, trypanosomes are subject to constant challenge from host immune defenses but they have developed very effective strategies to evade and modulate these responses to maintain an infection while simultaneously prolonging host survival. Here we investigate host parasite interactions, especially within the CNS context, which are not well-understood. We demonstrate that T. brucei strongly upregulates the stress response protein, Heme Oxygenase 1 (HO-1), in primary murine glia and macrophages in vitro. Furthermore, using a novel AHADHinT. brucei cell line, we demonstrate that specific aromatic ketoacids secreted by bloodstream forms of T. brucei are potent drivers of HO-1 expression and are capable of inhibiting pro-IL1ß induction in both glia and macrophages. Additionally, we found that these ketoacids significantly reduced IL-6 and TNFα production by glia, but not macrophages. Finally, we present data to support Nrf2 activation as the mechanism of action by which these ketoacids upregulate HO-1 expression and mediate their anti-inflammatory activity. This study therefore reports a novel immune evasion mechanism, whereby T. brucei secretes amino-acid derived metabolites for the purpose of suppressing both the host CNS and peripheral immune response, potentially via induction of the Nrf2/HO-1 pathway.

Ethyl Pyruvate Induces Tolerogenic Dendritic Cells.

Dendritic cells (DC) are professional antigen presenting cells that have a key role in shaping the immune response. Tolerogenic DC (tolDC) have immuno-regulatory properties and they are a promising prospective therapy for multiple sclerosis and other autoimmune diseases. Ethyl pyruvate (EP) is a redox analog of dimethyl fumarate (Tecfidera), a drug for multiple sclerosis treatment. We have recently shown that EP ameliorates experimental autoimmune encephalomyelitis, a multiple sclerosis murine model. Here, we expanded our study to its tolerogenic effects on DC. Phenotypic analysis has shown that DC obtained from mice or humans reduce expression of molecules required for T cell activation such as CD86, CD83, and HLA-DR under the influence of EP, while CD11c expression and viability of DC are not affected. Furthermore, EP-treated DC restrain proliferation and modulate cytokine production of allogeneic lymphocytes. These results demonstrate that EP has the ability to direct DC toward tolDC.

Implications of antibodies to pyruvylated glucose in healthy populations for mycobacterioses and other infectious diseases.

Members of the Mycobacterium avium-Mycobacterium intracellulare (MAI) complex are typeable because each serovar is characterized by its own specific antigenic glycolipid. By means of an enzyme-linked immunosorbent assay, we studied serum specimens obtained from 148 healthy college students for antibodies to these glycopeptidolipids. Ninety-two (61.5%) of the serum specimens were positive to the specific glycolipid antigen from MAI serovar 8. In a study of a pediatric population, antibodies appeared to develop during adolescence. Individuals with overt mycobacterial disease had a significantly lower incidence (tuberculosis patients, 34.5%; leprosy patients, 25%). We found a lower incidence of positive results in a survey of 96 Japanese serum specimens (29.1%), but the results from a survey of sera obtained from Bombay, India, indicated a large degree of reactivity (55.5%). Antibodies to other MAI serovars (serovars 2, 4, and 11) were not found, except antibodies to MAI serovar 21 were seen in the same individuals with antibodies to serovar 8. The dominant epitope of the MAI serovar 8-specific glycopeptidolipid is a terminal pyruvylated 3-O-methylglucose residue [4,6-(1'-carboxyethylidene)-3-O-methyl-alpha-D-glucopyranosyl] unit, whereas that of the MAI serovar 21 has the same terminal pyruvylated glucose devoid of the 3-methoxy group. Thus the antibodies appear specific for the pyruvylated glucose. It is unclear whether the high prevalence of antibodies to these epitopes reflects a high incidence of subclinical colonization or infection with certain MAI serovars or whether they are acquired through contact with some other related antigen source.

Characterization of phosphonoglycosphingolipids containing pyruvate: localization in Aplysia nerve bundles.

A phosphonoglycosphingolipid, designated as FGL-IIb, was first identified in nerve fibers of Aplysia kurodai by two-dimensional TLC (Abe, S. et al. (1986) Biomed. Res. 7, 47-51), and its chemical structure has been determined to be 3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNac alpha 1----3(Fuc alpha 1----2)(2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide (Araki, S. et al., submitted). Cryostat and paraffin sections of the nervous tissue and skin of Aplysia were examined immunohistochemically with antiserum against FGL-IIb. With this antiserum, only nerve bundles were stained distinctly: nerve cells in ganglia and in subcutaneous and muscular tissues and other cell elements were not stained. From histochemical findings in cryostat sections pretreated with chloroform-methanol (2 : 1, v/v) and from the results of Western blot analysis of the nervous tissue, the staining was concluded to be due to glycolipid antigens. The antiserum reacted with FGL-IIb and other phosphonoglycosphingolipids named FGL-I, FGL-IIa, FGL-V, and F-9 on TLC plates. This reactivity of FGL-IIb was abolished by mild acid-methanol treatment, and the lost reactivity was recovered by alkaline hydrolysis. These findings suggest that the free carboxyl group of the pyruvic acid of FGL-IIb is essential for the immunological reaction and that all the glycolipids listed above have the same epitope as that of FGL-IIb. Immunohistochemical findings indicated that these glycolipids including FGL-IIb are localized specifically in nerve bundles of Aplysia.

Locomotion of human lymphoblasts towards glucose and other sugars. Effects of pH, temperature and oxygen.

The effect of basic physical and chemical changes in the environment on the locomotion of human lymphoblasts was studied. The lymphoblasts were obtained by culture of human blood lymphocytes either with mitogens or in mixed lymphocyte culture. Lymphoblasts required glucose for optimal locomotion, and, if placed in glucose free media, they migrated towards glucose sources. A filter checkerboard assay showed chemokinesis and was suggestive of chemotaxis to glucose. Among other sugars tested only mannose enhanced locomotion, and products of glycolysis such as lactate and pyruvate inhibited locomotion. Lymphoblast locomotion was inhibited by azide, fluoride and 2-deoxyglucose but the effect of 2-deoxyglucose could be reversed by the addition of glucose. Lymphoblasts migrated faster at 40 degrees C than at 37 degrees C and their pH optimum was between 7.1 and 7.4. They migrated well in anaerobic conditions.

Immunochemical determination of the configuration of a haptenic substituent.

Quantitative inhibition of specific immune precipitation, a rapid microanalytical technique requiring no expensive equipment, was used to determine the stereoconfiguration of pyruvyl groups attached as acetals to two hydroxyls of nonreducing lateral end groups of the capsular polysaccharides of Klebsiella serotypes K11 and K21. The R and S isomers of 4,6-O-pyruvyl-D-alpha-methylgalactoside were used as inhibitors with appropriate polysaccharide antigens and antisera to the two serotypes. The R isomer was a potent inhibitor of precipitation in both antisera, showing that the pyruvylgalactosyl residues in the polysaccharides of both K11 and K21 are in the R form, in which the methyl group of pyruvyl is equatorial to the plane of the acetal ring.