Synergistic Effects Of NAC And Anti-TB Antibiotics

Tuberculosis(TB)is an infectious disease caused by M.tb and still has high morbility and mortality in developing countries.With the improvement of people's living standard in our country,the incidence of type 2 diabetes mellitus(T2DM)increases year by year.T2DM patients have become susceptible to TB because of their redox imbalance and immunocompromise which are caused by metabolic disorders.Conventional anti-tuberculosis therapies for standard chemotherapy require long-term administration of multiple antibiotics,which last from six months to nine months,to the patients who have liver function lesion or bear other diseases such as diabetes,the side-effects to liver of antibiotics and drug interactions bring great challenges to the treatment of TB.N-acetylcysteine(NAC),a synthesis precursor of intracellular glutathione(GSH),has been shown to have anti-oxidant effects by improving GSH levels in immune cells,regulates the release of proinflammatory cytokines and anti-inflammatory cytokines,thereby increasing the immune cells'ability to fight Mycobacterium tuberculosis(M.tb)infection.Therefore NAC combined with low-dose first-line anti-TB drugs provide a new idea for the treatment of TB.Most of the current models for the study of TB drugs in vitro are single immune cells,cell lines or animal models,which can not completely simulate the complicated mechanism of human diseases.The establishment of an in vitro granuloma model can systematically study the host immune response mechanism against M.tb.40 ml Peripheral blood samples were collected from 8 healthy individuals and 5 T2DM patients.Peripheral blood mononuclear cells(PBMCs)were isolated by density gradient centrifugation and plasma was kept.PBMCs were infected with virulent M.tb strain Erdman at a multiplicity of infection(MOI)of 0.1:1.6×10~5 cells and 500?l RPMI medium containing human AB serum were added to each well of a 24-well cell culture plate.The minimum inhibitory concentration(MIC)of a single antibiotic isoniazid(INH),rifampcin(RIF)and ethambutol(EMB)with or without 10 mM of NAC,the blank control group was without any conditions,the granulomas formed after cultured several days.The levels of IL-6,IFN-?,TNF-?,IL-12 and IL-10 in plasma from healthy individuals and individuals of T2DM and supernatants from granulomas at 15th day post infection were detected by enzyme-linked immunosorbent assay(ELISA).The granulomas were lysed by repeated freeze-thaw method.The lysates was coated on 7H11 agar solid medium,and the viability of Erdman bacteria in granulomas was calculated by CFU assay,the GSH levels were measured in the rest of the lysates by spectrophotometry.The morphology of different groups of granulomas were identified by hematoxylin and eosin(H.E.)staining on 15th day timepoint.Fluorescent microscope quantified the phagosom-lysosome fusion formed by red fluorescent probe green fluroscent protein labeled bacteria.To explore the relation between different treatment groups of granulomas on the release of cytokines,intracellular glutathione(GSH)levels,the size of granulomas,the number of phagosome-lysosome fusion and the survival of Erdman bacteria in granulomas,and to compare the cytokines and GSH levels in plasma from healthy individuals and individuals with T2DM,and whether there is a relavance of this differences to the ability fight M.tb in vitro granulomas from these 2 groups.The main findings are summarized as follows:1.The levels of TNF-?,IL-6 and IFN-?in plasma of patients with T2DM were significantly lower than those of healthy subjects(P<0.05);IL-12 also decreased to some extent,and the differences were not significant.There was a positive correlation between glutathione(GSH)levels in erythrocytes and a negative correlation with free radical scavenging ability.2.Antibiotic treatment group,both in patients with T2DM or healthy control group,have significant clearance of M.tb in granulomas,a complete clearance was possible in the healthy human population but still a small amount of bacteria residue in the T2DM group when treated with both NAC and antibiotics,but significantly decreased compared to the corresponding group treated with antibiotics alone.3.Granuloma of M.tb scavenging capacity and IL-6,IL-12,IFN-?release levels,NAC promotes their release to some extent.According to the results of H.E.staining,NAC has a positive effect on the size and structure maintaining of granulomas,and the group which contains NAC has a relatively large and strong granuloma structure.We speculate that the mechanism of granuloma elimination of M.tb is due to the upregulation of IL-12 and IL-6 to activate and promote the phago-lysosomal fusion.Lysosome red fluorescent probe method further confirmed this mechanism.There was more Erdman bacteria in acidified compartment than in non-acidified compartment in the NAC cohort group than the antibiotic alone group.Interestingly,TNF-?,a cytokine that plays an important role in the maintenance of the structure of granulomas,is somewhat reduced after treated with NAC,most likely due to the granulomas existed,the need for TNF-?is no longer that strong,the concentration is always maintained at a certain amount,because too much TNF-?can also trigger oxidative stress reaction which is not conductive to the clearance of M.tb.In summary,Granuloma is a good model to study anti-TB infection in the immune system in vitro;NAC plays an important role in the clearance of M.tb and the regulation of immune function,which can be used as an effective adjuvant drug for the treatment of TB;NAC and first-line anti-TB drugs have synergistic effect on the immune response of granuloma-resistant M.tb infection.