Mycobacterium Tuberculosis Lipids Effects On Human Peripheral Mononuclear Cells

Background and Objective: Pulmonary tuberculosis(TB) is one of the oldest infectious diseases, as far as we know. Mycobacterium tuberculosis(M.tb) is the critical factor for sickness. Therefore, it is one of the important discussion issues for every government around the world to find out useful drugs or vaccines to against M.tb. From now on, the job has not yet conclusion. Although bacillus Calmette-Guérin( BCG) has somewhat protection, particularly in children TB such as Miliary TB and caseous pneumonic TB, it is still unreliable in protective effects on adults to against M.tb. Today, many studies focus on getting the components or self-secreting proteins from M.tb as antigens to induce different immune cell responses, in order to further know relative pathogenesis. The cells of M.tb contain abundant lipid components. They just like a lay of thick capsule to protect M.tb from bad environment and themselves also mediate in both innate and adaptive immune responses, which imply maybe they play an potential target in researching progress of anti-M.tb drugs and vaccinum. Firstly, we investigated comparation of M.tb Whole Cell Lysate effect on proliferation of human peripheral blood mononucelear cell(PBMC)with human AB type serum or fetal bovine serum. Secondly, we selected some antigens of M.tb to culture with PBMC for assaying their proliferation and activation, so as to signify whether lipid antigens of M.tb have specific immune responses in host against M.tb infection or not.Methods:(1) PBMC from healthy adults were stimulated by WCL or WCL with recombinant human interleukin 2( rh IL-2) or phytohemagglutinin(PHA) or none as control for four groups. Each of the group was cultured with RPMI-1640 medium containing 10% AB serum or 10% FBS for six days respectively. The proliferation of lymphocytes were carefully investigated under the inverted microscope and Flow Cytometry(FCM).(2) By means of FCM, we detected respectively CD11+c, CD14+and CD1+a cell proportion of non adherent cells, which divided from PBMC, prior to and after treatment with interleukin(IL)-4 and GM-CSF. We also detected CD83 cell proportion after induced im DCs cultured with antigens of M.tb that we screened. These included total lipid(TLIP), Acetone-Soluble Lipids(ASLIP), Purified Sulfolipid(PSLI P), Purified Lipoarabinomannan(LAM), Lipomannan(LM). The obtained results were compared with same specimens adding Whole Cell Lysate(WC L), Culture Filtrate Proteins(CFP), lipopolysaccharide(LPS), M.tb heat-resistant antigen(M.tb- HAg), and negative(Blank) respectively.(3) We relied on FCM to detect proliferation, interferon(IFN)-γ Tumor Necrosis Factor(TNF)-α and IL-4 of specific T cells of healthy donors, to M.tb lipid antigens reaction respectively. PBMCs were stimulated using autologous immature dendritic cells(im DCs) prior to culture with antigens. IFN-γ/ IL-4/ TNF-α concentrations of secreting T cells(CD4+, CD8+, γδT cells) were measured by enzyme-linked immunosorbent assay(ELISA) or FCM. Proliferation assay of T cell subsets( NKT, CD4+, CD8+, γδT cells) also banked on FCM with labeled 5,6- carboxyfluorescein diacetate,succinimidyl ester(CFSE).Results: Our results showed that(1) The proliferation of lymphocytes stimulated by WCL in medium with 10% AB serum is better than that in medium with 10% FBS(P < 0.05). The similar result was obtained when PBMCs were stimulated by WCL and rh IL-2(P < 0.05).(2) After non-adherent cells induced by IL-4 and GM-CSF, we found CD14+ cell proportion declined(P < 0.05), while CD1a+ cell proportion increased(P < 0.01) when compared with originals.(3) Immature DCs, added all lipid antigens that we screened, cloud express up CD83(mature marker) and meanwhile produced interleukin IL-12(P<0.05).(4) Proliferation of NKT and CD8+ T cell proportion significantly increased in all lipid antigens compared with Blank group(P < 0.05).(5) The TNF-α concentration of supernatant fluids from culture medium at 3rd day showed increased in group PSLIP, but was decreased in group LM, when compared with Blank group(P < 0.05).(6) At the end of 9th day, TNF-α-secreted cell proportion of proliferative CD8+ T and γδT cells in group LM sharply decreased, but rest lipid groups increase(P < 0.05).(7) IL-4- secreted cell proportion of non proliferative CD4+ T cells was increased in group-ASLIP, LAM and LM compared with negative one(P < 0.05).(8) But IFN-γ- secreted cell proportion of proliferative CD4+ T, γδT and CD8+ T cells were higher in all lipid antigen groups than negative one(P < 0.05).Conclusion:(1) When human PBMCs were stimulated with M.tb WCL, the proliferation of lymphocytes are better in the culture medium supplemented with human AB serum than in that with FBS serum.(2) our results demonstrated that all lipid antigens we screened play an important role in effects on the T cell response of host against M.tb infection. Lipid antigens cloud be potential vaccine and therapy target of host against to M.tb.