| Objective:Tuberculosis(TB)is still the public health crisis.In the past 10 years,the global TB situation has been deteriorated due to the emergence of drug-resistance strains,and the number of patients has increased substantially every year.Drug resistance is an important factor for failing to control the disease.According to the epidemiological data,India(27%),China(14%)and the Russian Federation(9%)were the top three countries on a global scale with the largest burden of drug-resistant TB in 2018.The treatment of Mycobacterium tuberculosis(MTB)mainly relies on chemical drugs.However,due to non-standard drug use and bacterial gene mutation,MTB is resistant to a variety of anti-TB drugs,and even appears multidrug-resistance and extensively drug-resistance(XDR)MTB.With the improvement of diagnosis and treatment technology in recent years,it was found that some clinical symptoms and imaging manifestations similar to TB,which was previously identified as MTB,is actually Nontuberculosis Mycobacteria(NTM).However,NTM is naturally resistant to conventional first-line anti-tuberculosis drugs.Therefore,it is urgent to find the fast and effective diagnosis and treatment.From the perspective of mycobacteria,a large number of studies have demonstrated that single nucleotide polymorphism(SNP)mutation was an essential cause of MTB resistance.Can the SNP mutation cause NTM resistance? The lung is the cardinal site of mycobacterial infection,and the macrophages present in the lung and the most critical phagocytic cell after the mycobacteria infection.Macrophages have the functional continuity.The two extremes of this continuity are M1(the classically activated macrophage)and M2(the selectively activated macrophage)types.According to the secretion of cytokines,M2 has been divided into four subtypes: M2 a,M2b,M2 c,and M2 d.Among them,M2 c is also called the inactivated macrophage as a result of high secretion of inhibitory cytokines.Mycobacteria may also change the activated macrophages,resulting in the production of inducible nitric oxide synthase(i NOS),lactic acid and other cytokines.STATs are central factors affecting the polarization of M1 and M2 macrophages.STAT1 is related to the polarization of M1 macrophage,while STAT3 is associated with the activation of M2 macrophage.Both are significant points for the potential regulation of macrophage polarization.Mycobacterial infection plays a key role in the different state and function of macrophage polarization,and the outcome of bacterial infection.The purpose of this study was to understand the mechanism between drug resistance and SNP mutation in clinically isolated mycobacteria and macrophage polarization characteristics.By the construction of the drug-resistance mycobacteria in vitro,it was to analyze the effecting on the macrophage polarized microenvironment providing the theoretical basis on screening the diagnosis and treatment of drug-resistance mycobacteria.Therefore,the following experimental strategies were performed: 1.To isolate and culture of mycobacteria,and to conduct drug susceptibility test and the drug-resistance mechanism of SNP,and to determine the drug resistance status of mycobacteria and the specific mechanism for drug resistance caused by SNP mutation;2.To comprehend the levels of M1/M2 macrophages and the secreted cytokines of clinical samples in the different groups,especially the expression and polarization characteristics of peripheral blood macrophages in patients infected with drug-susceptibility or drug-resistance mycobacteria;3.To construct the drug-resistance mycobacteria by SNP mutation,which was to infect macrophages in vitro,in order to discuss the effect of SNP-mutation drug-resistance mycobacteria on the macrophage polarized microenvironment;4.To investigate the mechanism of the macrophage polarization microenvironment infected with SNP mutation mycobacteria owing to the lack of the anti-inflammatory cytokine,via the small interfering RNA for IL-10(si-IL-10).Methods:1.Proportional drug susceptibility test: It was used for the clinical MTB isolates against isoniazid,rifampicin,ethambutol,streptomycin,kanamycin,para-aminosalicylic acid,levofloxacin and thiophanate.2.Microdilution broth method: It was detected for the clinical Mycobacterium avium isolates with fluoroquinolones and aminoglycosides.And it was also used for induction of drug-resistance BCG;and the successful criterion was the MIC value of the final drug-resistance BCG> the MIC value of drug-susceptibility BCG(8 times).3.Cell culture: It was for the cultivation of Thp1 cells,and M0 macrophages(PMA-Thp1)used in the cell model experiments.4.Flow cytometry: To research FITC-CD11 b,APC-CD86 and PE-CD206 on the surface of the peripheral blood mononuclear cells in clinical samples and for in vitro M0 macrophages infected with BCG.It was the crutial markers for M1/M2 macrophages.5.Flow cytokine microbeads assay: By the macrophage flow cytokine microbeads assay,the antibody on the PE-labeled microbead was combined with the end of the macrophage secreting cytokine,and the APC labeled fluorescent coloring antibody was performed to clarify the secretion of IL-6,TNF-?,IL-1?,IL-12,IL-10,TGF-?1 and arginase-1.It was the necessary indicators of the subtype differentiation as M1/M2 macrophages.6.CCK test: To select different bacteria numbers(MOI = 5,10,20,40)used to measure the macrophage activity resulting in determining appropriate MOI.7.q RT-PCR: To detect the contents of IL-6,TNF-?,IL-1?,IL-12,IL-10 and TGF-?1 in BCG-infecting M0 macrophages.8.Western blot: To perform the expression of STAT1 and STAT3 in the BCG-infecting M0 macrophages.9.Colorimetric method: To identify i NOS and lactic acid in the culture supernatant after BCG-infecting M0 macrophages by the kits.10.Transfection: In M0 macrophages,it was to inquire into the degrees of IL-6,TNF-?,IL-1?,IL-12,IL-10,TGF-?1,i NOS,lactate,STAT1 and STAT3 by si-IL-10.Results:1.Of the studied 132 MTB strains,20(15.15%)strains were multidrugresistance(MDR)-TB,1(0.76%)strain was XDR-TB,and 111 ones were drug-sensitivity.Among them,there were 32(24.24%)isoniazid-resistance,23(17.42%)rifampin-resistance,13(9.85%)ethambutol-resistance,35(26.52%)streptomycin-resistance,2(1.52%)kanamycin-resistance,5(3.79%)para-aminosalicylic acid-resistance,8(6.06%)levofloxacin-resistance strains,and no thiophanate-resistance ones.2.Of the 95 Mycobacterium avium,ofloxacin(89/95,93.68%)and ciprofloxacin(88/95,92.63%)were shown higher drug resistance.Moxifloxacin(18/95,18.95%)has been good activity in the experimental strains.Amikacin(2/95,2.11%)was excellent effect on the clinically isolates,while streptomycin(62/95,65.26%)was weak to the strain.3.The most common SNP of gyr A for fluoroquinolones in the drug-resistance Mycobacterium avium was T ? C(71/95,74.74%),and the amino acid mutation mainly occured in Gly2444Asp(GGT ? GAT)(20/95,21.05%).The main SNP of gyr B was A?C(69/95,72.63%)and its corresponding amino acid substitution was Ile2160Val(ATT ? GTT)(21/95,22.11%).SNP for aminoglycoside antibiotics has been the most commonly found at the G?A position of rps L(73/95,76.84%),and amino acid mutation fundamentally involved in Ala1539985Thr(GCC ? ACC)(19/95,20.00%).4.The amount of M1 macrophages in peripheral blood of patients with TB was significantly lower than that in normal examiners(P = 0.0001).The M1 expression was decreased in the drug-resistance TB group compared to drug-susceptibility TB one(P = 0.0280).The number of M2 macrophages in TB patients was prominently higher than that in normal examiners(P <0.0001).The M2 level in the drug-resistance TB group was overexpressed than that in the drug-susceptibility TB one(P = 0.4950).5.The measurements of peripheral serum secreted cytokines in the different subjects were displayed that IL-6 in TB patients was significantly higher than that in normal examiners(P = 0.0005).IL-10 in TB group was more than that in normal one(P = 0.0624).TGF-?1 in TB one was prominently elevated than that in normal one(P <0.0001),and arginase-1 was remarkably decreased in TB persons than in normal ones(P <0.0001).Depending on the secretion of cytokines,patients with drug-susceptibility TB were dominated by M2 b macrophages,and drug-resistance TB patients were mainly by M2 c macrophages.6.To construct of drug-resistance strain by wild-type and drug-sensitivity BCG.The results exhibited that the MIC value of drug-susceptibility BCG was 0.125?g/ml,and that of drug-resistance BCG was greater than 128?g/ml.The sequencing of isoniazid resistance genes inh A and kat G were shown that inh A had T ? G and kat G had A ? T.Drug-resistance BCG for SNP mutation was successfully induced.7.After drug-susceptibility or drug-resistance BCG infecting M0 macrophages: 1)the expression of M1 cytokines: Compared with the normal control group(M),IL-6 and TNF-? in the drug-resistance group(R)and the drug-sensitive group(S)were increased,but both of them in R group were more downexpressed than that in S group(P = 0.0036,P = 0.0201);IL-1? in group R or S was higher than that in group M(P = 0.0841,P = 0.0468),and there was no difference between group R and S;those results were indicated that BCG infection induced macrophage activation,but the ability of drug-resistance BCG was weaker than that of the drug-sensitive strain.2)the level of M2 cytokines: Compared to M group,IL-10 was significantly increased in R and S group,however it in R one was more overexpressed than that in S one(P = 0.0155);TGF-?1 in R group was distinctly less than that in group S(P = 0.0274);it was suggested that BCG infection can cause the increase of anti-inflammatory cytokines,and the macrophage infected with drug-sensitive BCG can principally express TGF-?1.however,IL-10 was engendered foremost when it was infected with the drug-resistance BCG.3)STAT1 and STAT3: Contrasted to the M group,STAT1 in the S group was significantly risen(P = 0.0043);nevertheless,there was no divergence between group R and M;STAT3 was remarkably more increased in both R and S groups(P = 0.0035,P <0.0001),yet there was no significant difference between the two groups(P = 0.0771);it was made clear that BCG activated macrophages,and drug-resistance BCG infection was given the first place to increase STAT3.8.By si-IL-10,M0 macrophages were infected with drug-sensitive or drug-resistance BCG: 1)IL-6 was more descended in the R' group than in the S' group(P = 0.0004),and it was decline in the R' one more than in the R one(P = 0.0005);it was hinted that the expression of IL-6 was relied on IL-10.2)TGF-?1 decreased to the quite low levels overall in the si-IL-10 group,illustrating that TGF-?1 was dependent on IL-10.3)Under the condition of drug-sensitive BCG(S 'and S groups),STAT1 was influenced in the group S' with si-IL-10 or not;it was clarified that drug-resistance BCG had weaker ability to induce STAT1 contrast with drug-susceptibility one;and STAT3 in the R' one was remarkably less than that in the S' one(P = 0.0018);and it was in the R' one significantly more decreased than that in the R one(P = 0.0041);it was explained that STAT activation is associated with IL-10.4)After si-IL-10,both of i NOS and lactic acid were ascended in the R' group,suggested that IL-10 had a certain inhibitory effect on i NOS and lactic acid during infection by drug-resistance BCG.Conclusions:1.The fluoroquinolones and aminoglycosides resistances to Mycobacterium avium were the similar SNP mechanism to MTB.SNP mutation was an important cause of mycobacterial resistance.2.The expression of M2 macrophages in the peripheral blood of TB patients significantly ascended,and the anti-inflammatory cytokines IL-10 and TGF-?1 increased in patients with TB;M2c macrophage was mainly existence in patients with drug-resistance TB.3.It was successfully constructed SNP-mutation drug-resistance BCG.The ability of macrophages to produce M1 cytokines was reduced,and they were more inclined to differentiate into M2 after drug-resistance BCG infecting for macrophages.4.The decline of pro-inflammatory cytokines,such as IL-6 and TNF-?,attenuated for the removal of drug-resistance bacteria with infected macrophages.5.Drug-resistance BCG activated STAT from macrophage via si-IL-10;while it could strengthen macrophage to differentiate into M2,express IL-6 and produce bactericidal enzymes depending on IL-10. |
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