Anti-ManLAM antibodies circulating in Multi Drug Resistant (MDR) and Drug-sensitive tuberculosis (TB) patients exert differential effect on fagocytosis and costimulatory potential of Dendritic Cells

SCHIERLOH P; COLMAN E; LANDONI V.I; LABORDE E; BASILE JI; BALBOA L; SABIO Y GARCIA C; ROMERO M; GEFFNER L; BARRERA L; CASTAGNINO J; SASIAIN MC

Buenos Aires Argentina

Congreso; First French-Argentine Immunology Congress.; 2010

Mannosilated lipoarabinomannan (ManLAM) is an abundant, complex and bioactive lipoglycan present in cell wall of pathogenic slow-growing mycobacteria (1,2). This molecule exerts its immunomodulatory effect through recognition by innate immune receptors expressed on host cells (3,4). Anti-ManLAM antibodies (¥áManLAM Abs) have been detected in serum from tuberculosis patients (TB) but its role in physiopathology remains poorly studied (5-8). In order to address the role of ¥áManLAM Abs on dendritic cell (DC) function and factors/mechanisms involved, ¥áManLAM Abs were characterized in serum samples from 20 MDR and 20 Drug-sensitive (DS) TB patients and 17 healthy donors (N), thereafter, well characterized serum subgroups were employed to perform bioassays on monocytes derived DC from normal blood donors. Immunochemical characterization of circulating aManLAM Abs  For ¥áManLAM Abs quantification in sera, we developed an in-house indirect ELISA using purified ManLAM (Colorado State Univ.) as capture antigen (5,6). An optimal 1/1000 serum dilution were established in preliminary experiments. Polivalent goat antihuman IgG/A/M Ab conjugated with HRP (Sigma) was used as 2¨¬ reagent. We confirm the presence of circulating aManLAM Abs in 85% of TB sera (34/40). No significant differences were observed among mean titres of MDR (N=20) and DS-TB (N=20). In order to analyze chemical nature (lipidic vs glycosidic) of ¥áManLAM epitopes, high titer sera (MDR N=6; DS N=6) were diluted 1/1000 and pre-incubated with excess of total lipidic extract of Mtb (Tlip, Colorado State Univ.) or yeast derived mannan (Sigma) before ¥áManLAM ELISA assay (8). Competition ELISA showed that both kinds of ManLAM epitopes were recognized by most sera without clear association to any TB group. Next, to analyze cell wall epitope-display, we developed a whole Bacteria ELISA method by using intact M.tuberculosis (Mtb) 6006 local strain (ANLIS-Malbran) as antigen. We establish an optimal 1/500 serum dilution and optimal sensitivity/specificity range for this method. All ¥áManLAM high titer sera were above cut-off value (100% positivity) and whole bacteria binding correlates well with ¥áManLAM titre (p<0.01). Furthermore, pre-incubation with excess of ManLAM partially inhibit whole bacteria binding in all samples studied (12/12; whole Bacteria competition ELISA). Finally, we wanted to address the isotype class of ¥áManLAM Abs that¡¯s bind to the surface of Mtb cells. To do this, we performed a bacteria based flow cytometry assay by incubating Mtb 6006 local strain with 1/500 serum dilution pre-incubated or not with excess of ManLAM. FITC-conjugated goat antihuman Igk, IgG, IgA, IgM and IgE were used as 2¨¬ reagents. As figure 1 show, isotype composition of ¥áManLAM Ab was variable among samples with no association to any TB group. Interestingly IgG and IgM ¥áManLAM Abs were inversely correlated between individuals (r=-0.39; p<0.05).