The Defect in Autophagy Induction by Clinical Isolates of Mycobacterium Tuberculosis Is Correlated with Poor Tuberculosis Outcomes
The Defect in Autophagy Induction by Clinical Isolates of Mycobacterium Tuberculosis Is Correlated with Poor Tuberculosis Outcomes
Furong Li 0 1 2
Bo Gao 0 1 2
Wei Xu 0 1 2
Ling Chen 0 1 2
Sidong Xiong 0 1 2
0 1 Institute for Immunobiology, Department of Immunology, Shanghai Medical College of Fudan University , Shanghai 200032 , P.R. China , 2 Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University , Suzhou 215006 , P.R. China , 3 Department of Respiratory Medicine, Affiliated Hospital of Zunyi Medical College , Zunyi 563000 , P.R. China
1 Funding: This work was supported by grants from the National Science & Technology Key Projects during the Twelfth Five-Year Plan Period of China (2013ZX10003007), Major State Basic Research Development Program of China ( 2013CB530501, 2013CB531502), the National Natural Science Foundation of China (31470839, 81072428) , Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Jiangsu Provincial Innovative Research Team, and the Program for Changjiang Scholars and Innovative Research Team
2 Editor: Giovanni Delogu, The Catholic University of the Sacred Heart , Rome, ITALY
Tuberculosis (TB) represents a major global health problem. The prognosis of clinically active tuberculosis depends on the complex interactions between Mycobacterium tuberculosis (Mtb) and its host. In recent years, autophagy receives particular attention for its role in host defense against intracellular pathogens, including Mtb. In present study, we aim to investigate the relationship of autophagy induction by clinical isolates of Mtb with the clinical outcomes in patients with TB.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
in University of Ministry of Education of China
(PCSIRT-IRT1075). The funders had no role in study
design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
These data indicated that the defect in autophagy induction by Mtb isolates increased the
risk of poor clinical outcomes in TB patients, and detection of clinical isolates-induced
autophagosome formation might help evaluate the TB outcomes.
Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), is a major public
health problem worldwide with up to 10 million new cases each year, leading to 1.5 million
deaths annually. China occupies second place, behind India, among the top five high-burden
countries for the last decade [
]. The application of BCG vaccine and anti-TB antibiotics has
been effective in preventing and controlling TB. However, the high rates of latent tuberculosis
infection (LTBI), emergence of drug-resistant Mtb and HIV co-infection, etc., have made the
control and treatment of TB become difficult in recent years [
]. Novel and effective
therapeutic strategies against TB are therefore urgently needed. Accumulating evidence indicates
that the interaction between Mtb and the host is of great importance in determining the
outcome of TB [
]. Of those strategies targeting Mtb-host interaction, autophagy receives
particular attention in recent years [
Autophagy is an evolutionarily conserved process in which organelles and proteins are
sequestered into a double-membrane-bound autophagosome, and delivered to the lysosome
for degradation. Recent reports reveal that autophagy is involved in diverse pathophysiological
processes, including cell survival, aging, neurodegeneration, cancer and the clearance of
intracellular pathogen [
]. Accumulating evidence indicates that autophagy may function as a
crucial anti-TB strategy of the host, although these data are mainly obtained from the
investigation on BCG or standard H37Rv strain. On the other hand, it is also suggested that through the
intimate and persistent interaction with its human host, Mtb may have evolved strategies to
counter the antibacterial effect of autophagy [
9, 10, 12, 16–18
]. The pathogenesis of TB has
been thought to be mainly related to host factors in earlier investigations, however, it appears
clear now that bacterial factors also play crucial roles . Reports indicate that clinical isolates
of Mtb display different characteristics from those of BCG or H37Rv [
]. Different clinical
isolates of Mtb are also found to induce different host immune responses [
]. It is therefore
of interest to further investigate the role of autophagy in TB pathogenesis using clinical isolates
In this study, we collected 185 clinical isolates of Mtb from Zunyi, one of the
highest-incidence-rate areas with TB in China [
], and investigated the effect of these clinical isolates on
autophagosome formation in macrophages, and its associated clinical significance. Our data
showed that most clinical isolates of Mtb were able to induce autophagosome formation in
macrophages, however different clinical isolates of Mtb differed in their ability to induce
autophagosome formation. Of importance, it was found that the extent of clinical isolates of
Mtbinduced autophagy was negatively correlated with the clinical outcomes in patients with TB.
Materials and Methods
A total of 185 Mtb isolates were obtained from clinical patients, from 2011 to 2014 in Zunyi,
Guizhou Province, one of the highest-incidence-rate areas with TB in China. Of these samples,
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177 specimens were from sputum, 6 from urine, 1 from the cerebrospinal fluid, and 1 from the
scrotum. Mtb isolates were grown in 7H11 medium (Difco BD, NY) supplemented with 0.05%
Tween 80 and 10% oleic albumin dextrose catalase enrichment (Difco, Detroit, MI), and the
identification of all these isolates were performed according to the TB diagnosis bacteriology
test criteria of the China Antituberculosis Association. Single-cell suspensions of mycobacteria
at a concentration of 107 CFU/ml were prepared and used to infect cells.
Following verbal and written consent, socio-demographic, clinical, radiographic and laboratory
data were obtained from patients' medical record, and the data were analyzed anonymously.
The Ethics Committee of Fudan University and Affiliated Hospital of Zunyi Medical College
specifically approved this study, and this work was also performed in compliance with the
Helsinki Declaration. The radiographic extent of disease was categorized to be "minimal",
"moderately advanced", or "far advanced" according to the classification of the National Tuberculosis
and Respiratory Disease Association [
]. The TB treatment outcomes were defined by WHO
criteria as "favourable" (cured and treatment completed) and "unfavourable" (defaulted, failed
and died) [
]. Retreatment cases were those having history of previous TB treatment of more
than one month.
Macrophage stimulation with mycobacterial strains
The murine macrophage cell line RAW264.7 (ATCC number: TIB-71) was maintained at 37°C
in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS (HyClone, Logan,
UT, USA) and antibiotics in a 5% CO2 atmosphere. The human monocyte cell line THP-1
(ATCC number: TIB-202) was cultured in RPMI1640 (Invitrogen) supplemented with 10%
FBS (HyClone). Prior to Mtb infection, THP-1 cells were treated with 50 ng/ml Phorbol
12-myristate 13-acetate (PMA) for 24 hours to allow differentiation into macrophages. BMDM
of > 95% purity were obtained from BALB/c as described previously [
stimulation with Mtb strains was performed according to previous reports [
macrophages were infected with clinical isolates of Mtb or H37Rv at multiplicity of infection (MOI)
of 10:1. Four hours after infection, macrophages were washed twice with prewarmed
serumfree RPMI1640 or DMEM to remove unbound bacilli, and were further cultured in
serumsupplemented RPMI1640 or DMEM for another 4 hours. No toxicity was observed in
Western blot analysis
Western blot was performed as described previously [
]. Antibody against LC3 was obtained
from Sigma (St. Louis, Mo, USA), and anti-GAPDH was from CWBio (Beijing, China).
THP-1 cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 10 min,
permeabilized with 0.25% Triton X-100 in PBS for 10 min. Cells were then incubated
sequentially with rabbit anti-LC3 antibody and tetramethyl rhodamine isothiocyanate
(TRITC)-conjugated goat anti-rabbit IgG (red), followed by the staining with 4',6-diamidino-2-phenylindole
(DAPI) to visualize the nuclei (blue). Fluorescence images were acquired with a confocal
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Categorical variables were compared using pearson chi-square test, and the difference in
continuous variables was analyzed by one-way analysis of variance (One-way ANOVA). Variables
with a p-value < 0.2 in univariate logistic regression analyses were further subjected to
multivariate logistic regression analysis to identify independent variables that evaluated the role of
autophagy in the pathogenesis of TB. A p-value <0.05 was considered to be statistically
significant. Data were entered and analyzed using a statistical software package (SPSS18.0).
Clinical isolates of Mtb were able to induce autophagosome formation in
To investigate the effect of Mtb on autophagy induction, we infected human THP-1
macrophages with two clinical isolates of Mtb or H37Rv for 4 hr, followed by detecting the
autophagosome formation using western blot. Results showed that, similar to H37Rv, two clinical
isolates of Mtb (M1, M2) were able to significantly increase the level of LC3
(microtubule-associated protein light chain 3)-II, a hallmark of autophagy induction (Fig 1A). As the the increase
in LC3II level could mean that there is an increase in autophagy induction or there is an
inhibition of flux, we examined the effect of Bafilomycin A1 (Baf A1) treatment on Mtbs-induced
autophagy. It was found that Baf A1 treatment could further increase the LC3II level in
Mtbsinfected THP1 cells (Fig 1A). We have also investigated the effect of Mtbs infection on the
expression level of p62, which is incorporated into autophagosomes and degraded along with
other substrates by lysosomal hydrolyses. Our results showed that infection with clinical
isolates (M1, M2) or H37Rv led to the downregulation of p62 significantly in THP1 cells at 4
hours postinfection (Fig 1B). Together, these data indicated that these Mtbs-induced autophagy
was functional rather than blocking autophagical flux at 4 hours postinfection. We also
examined the effect of clinical isolates of Mtb on autophagy induction in mouse macrophage
RAW264.7 cells and mouse bone marrow-derived macrophages (BMDM). Similar to the
observation in THP-1 cells, infection with clinical isolates (M1, M2) or H37Rv led to the
increase in LC3II level while downregulated p62 expression in RAW264.7 cells (Fig 1C) and
BMDM cells (Fig 1D). We further determined the effect of clinical Mtb isolate on
autophagosome formation using the immunofluorescence technique. The confocal result showed that
clinical Mtb isolates (M1, M2) or H37Rv stimulated the formation of LC3 punctuate in THP-1
cells significantly (Fig 1E).
Different clinical isolates of Mtb induced autophagosome formation in
macrophages to different extent
Above data showed that two clinical isolates of Mtb were able to induce autophagosome
formation in macrophages, we therefore further collected more clinical isolates of Mtb to a total of
185 during the period from 2011 to 2014, and examined the effect of these clinical isolates of
Mtb on autophagosome formation in THP-1 macrophages using western blot. It was found
that most of these clinical isolates of Mtb could induce autophagosome formation, however,
the autophagy-inducing ability appeared to vary greatly among different isolates (Fig 2A). To
better define the clinical isolates of Mtb-induced autophagosome formation, the extent of
autophagosome formation was graded on LC3-II/GAPDH ratio into four classes: absent (0–
0.25), weak (0.26–0.50), moderate (0.51–0.75), and strong (>0.75) (Fig 2B).
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Fig 1. Clinical isolates of Mtb could induce autophagy in macrophages. (A) THP-1 were infected with H37Rv and two clinical isolates of Mtb (M1 and
M2) (MOI = 10) for 4 hr in the absence or presence of Baf A1. Cells were harvested and subjected to western blot analysis using anti-LC3. The expression of
GAPDH was used as a loading control. The immunoblots were scanned and subjected to densitometric analysis. LC3-II/GAPDH ratio was calculated, and
the mean value of at least five samples from three independent experiments was shown at the bottom of each lane. (B) THP-1 were infected with clinical Mtb
isolates (M1, M2) or H37Rv (MOI = 10) for 4 hr. Cells were harvested and subjected to western blot analysis using antibodies against p62 and GAPDH.
Protein was quantitified by densitometry. P62/GAPDH ratio was calculated, and the mean value of at least five samples from three independent experiments
was shown at the bottom of each lane. (C and D) RAW264.7 and BMDMs macrophages were treated as in B. Cells were harvested and subjected to western
blot analysis using antibodies against p62 and GAPDH. Protein was quantitified by densitometry. LC3-II/GAPDH or p62/GAPDH ratio was calculated,
respectively, and the mean value of at least five samples from three independent experiments was shown at the bottom of each lane. (E) THP-1
macrophages were infected with a clinical isolates of Mtb (MOI = 10) for 4 h. Cells were then incubated sequentially with anti-LC3B antibody and TRITC goat
anti-rabbit IgG (red), followed by the staining with DAPI to visualize the nuclei (blue), the right panel was the quantification of LC3 punctuate per cell. The data
shown represent mean ± SE from three independent experiments.
Characteristics of patients harbouring clinical isolates of Mtb with
different autophagy-inducing ability
As above data demonstrated that clinical isolates of Mtb differed in their ability to induce
autophagosome formation in THP-1 macrophages and autophagy were reported to be crucial
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Fig 2. Different clinical isolates of Mtb induced autophagy in THP-1 cells to a different extent. (A) THP-1 cells were infected with clinical isolates of Mtb
for 4 hr. Cells were harvested and subjected to western blot analysis using anti-LC3. GAPDH was used as a loading control. Representative immunoblots
(sample 1–7, 13–19, 40–50, 73–80, 112–122, 155–165) were shown. The immunoblots were scanned and subjected to densitometric analysis. LC3-II/
GAPDH ratio was calculated, and the mean value of at least five samples from three independent experiments was shown at the bottom of each lane. (B) The
extent of autophagosome formation was graded on LC3-II/GAPDH ratio into four classes: absent (0–0.25), weak (0.26–0.50), moderate (0.51–0.75), and
for Mtb clearance, we would like to know the relationship between the extent of Mtb-induced
autophagosome formation and the clinical outcomes of TB patients. The clinicopathological
comparisons among patients harbouring clinical isolates of Mtb with different
autophagyinducing ability were shown in Table 1. It was found that patients infected by Mtb with poor
autophagy-inducing ability displayed more severe radiographic extent of disease (p<0.001)
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Abbreviation: PTB, pulmonary tuberculosis; EPTB, extra-pulmonary tuberculosis; ESR, erythrocyte sedimentation rate; CO2CP, Carbon Dioxide
bOne way ANOVA.
and were more likely to have unfavorable treatment outcomes (p<0.001). We also observed
that re-treatment TB cases were more likely to harbour isolates with poor autophagy-inducing
ability (p<0.001). There was no significant association of the extent of Mtb-induced autophagy
with some socio-demographic characteristics (including gender, age, tobacco smoking and
alcohol consumption), the coexistence of pulmonary and extra pulmonary tuberculosis (PTB
+EPTB), and some laboratory tests [such as hemoglobin, leukocyte count, erythrocyte
sedimentation rate (ESR), and carbon dioxide combining power (CO2CP)].
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The defect in autophagy induction by Mtb increased the risk of far
advanced radiographic disease and unfavorable treatment outcomes in
Results from Table 1 showed that there was a higher portion of patients with far-advanced
radiographic disease in the poor autophagy-inducing group as compared with the strong
autophagy-inducing group. We further determined the association between them by logistic
regression analysis. The univariate analysis showed that the defect in Mtb isolates-induced
autophagosome formation was positively correlated with the far-advanced radiographic disease
[odd ration (OR), 4.574; 95% confidence interval (CI) 1.93–10.86; p = 0.001]. When controlling
for age, tobacco consumption and ESR, the poor autophagy-inducing ability by Mtb remained
as a significant risk factor for the far-advanced radiographic disease [adjusted OR (aOR),
4.710; 95% CI 1.93–11.50; p = 0.001]. Besides, over 50 years of age was also revealed to be
associated with the far-advanced radiographic disease (aOR, 2.915; 95% CI 1.12–7.58; p = 0.028) in
the final regression model. Details are showed in Table 2.
We further investigated the correlation between the extents of Mtb-induced autophagosome
formation with the treatment outcome of TB patient. Result from the univariate analysis
showed that the defect in Mtb-induced autophagosome formation was positively correlated
with the unfavorable treatment outcomes of TB patients (OR, 7.881; 95% CI 2.27–27.43;
p = 0.001). In further multivariate analysis adjusted for age, tobacco smoking and leucocyte
count, the poor autophagy-inducing ability by Mtb remained as a significant risk factor for
unfavorable outcomes in TB patients (aOR, 8.310; 95% CI 2.22–28.97; p = 0.001). Of the
covariates included in the final model, over 50 years of age (aOR, 4.274; 95% CI 1.32–13.86;
p = 0.015) and PTB+EPTB (aOR, 2.504; 95% CI 1.18–5.33; p = 0.031) were also revealed as
the risk factors for unfavorable treatment outcomes in TB patients. Details are presented in
Together, these data indicated that the defect in autophagy induction by Mtb posed as an
independent risk factor for poor clinical outcomes in patients with TB.
The current study revealed that most of clinical isolates of Mtb was able to induce
autophagosome formation in macrophages. The results also revealed that clinical isolates of Mtb differed
significantly in their ability to induce autophagosome formation. Furthermore, our data
revealed that the defect in autophagy induction by clinical isolates was positively correlated
with the poor clinical outcomes in TB patients.
Autophagy is revealed to play a crucial role in host defense against Mtb by both in vitro and
in vivo investigation [
9, 12, 16
]. Results from a genome-wide analysis of the host intracellular
network also indicate that autophagy is implicated in the regulation of Mtb survival [
Autophagy may contribute to the elimination of Mtb through the fusion of Mtb-containing
autophagosomes with lysosomes, leading to the xenophagic degradation of Mtb, and/or the
enhancement of antigen presentation, and the consequent activation of adaptive immunity. On
the other hand, through long battles with the host, Mtb may have developed various strategies
to evade the autophagy-mediated antibacterial activities [
]. In present investigation, we
focus on the effect of clinical isolates of Mtb on autophagy induction and its possible
association with clinical outcomes in TB patients.
We found that although most of clinical isolates of Mtb could induce autophagy in
macrophages, the autophagy-inducing ability varied significantly among these clinical isolates.
Reports indicate that the genotype of Mtb may have an important role in its behaviors [
], we thus would like to know whether the autophagy-inducing ability of these clinical
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isolates was related to their genetic background. We examined the genotypes of these Mtb
isolates by the mycobacterial interspersed repetitive unit-variable number of tandem repeats
(MIRU-VNTR) technique, and determined the possible association between the genotype and
autophagy induction by cluster analysis using BioNumerics software. Our results revealed that
there was no significant correlation between the clinical isolates-induced autophagosome
formation and their genotypes (data not shown). We further investigated the association between
the extent of autophagy induction by clinical isolates of Mtb with various clinical variables,
including the socio-demographic characters, radiographic findings, laboratory tests and
treatment outcomes, etc. Our results showed that that the defect in autophagy induction by clinical
isolates was an independent risk factor for far-advanced radiographic disease and unfavorable
treatment outcomes in TB patients. Additionally, our data also identified the Mtb-induced
autophagosome formation was an independent risk factor for re-treatment TB cases (OR,
8.754; 95% CI 3.64–21.08; p<0.001) (S1 Table). Collectively, these data indicated that the
Abbreviation: OR, odd ratio; aOR, adjusted OR; CI, confidence interval
*For the regression analysis of radiographic findings, the radiographic extent of disease was categorized into two group: Minimal to moderate-advanced
group and Far-advanced group.
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autophagy-inducing ability by clinical isolates of Mtb might play a crucial role in determining
the clinical outcomes in TB patients.
Given the importance of clinical isolates of Mtb-induced autophagy in TB outcomes, an
interesting question was raised. Why different clinical isolates of Mtb possessed different ability
in inducing autophagosome formation. This work was now undertaken in our laboratory.
Reports indicated that H37Rv infection could suppress the autophagy flux in macrophages [
], however, which could be rescued by Mtb-specific T cells . We think that, besides the
manipulation of autophagy induction, clinical isolates of Mtb may have developed other
strategies for their survival. It is of interest in further investigation to determine whether clinical
isolates of Mtb could impair autophagy flux, and the possible involvement of host factors, such as
Mtb-specific T cells, in this process.
In conclusion, this study revealed that clinical isolates of Mtb differed in their ability to
induce autophagy, which was closely correlated with clinical outcomes in TB patients,
indicating the control of autophagy induction might be an important strategy manipulated by the
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bacteria to evade host immune responses. To our knowledge, this is the first report
investigating the association between the autophagy induction and TB outcomes using clinical isolates.
These data may help better understand the role of autophagy in the pathogenesis of
tuberculosis, and provide important information for the better control of TB infection.
S1 Table. Univariate and multivariate logistic regression analysis of retreatment TB cases.
S2 Table. Raw data of patients’ characteristics.
Conceived and designed the experiments: FL BG SX. Performed the experiments: FL. Analyzed
the data: FL BG. Contributed reagents/materials/analysis tools: BG LC WX SX. Wrote the
paper: BG SX.
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1. Global Tuberculosis Report 2014 . World Health Organization 2014 .
2. Zumla A , George A , Sharma V , Herbert N , Baroness MOI . WHO's 2013 global report on tuberculosis: successes, threats, and opportunities . Lancet . 2013 ; 382 : 1765 - 1767 . doi: 10 .1016/S0140- 6736 ( 13 ) 62078 - 4 PMID: 24269294
3. Rubin EJ . Troubles with tuberculosis prevention . N Engl J Med . 2014 ; 370 : 375 - 376 . doi: 10 .1056/ NEJMe1312301 PMID: 24450896
4. Miotto P , Cirillo DM , Migliori GB . Drug resistance in Mycobacterium tuberculosis: molecular mechanisms challenging fluoroquinolones and pyrazinamide effectiveness . Chest . 2015 ; 147 : 1135 - 1143 . doi: 10 .1378/chest.14-1286 PMID: 25846529
5. Guidelines on the Management of Latent Tuberculosis Infection . World Health Organization, Geneva, Switzerland. World Health Organization, 2015 .
6. Harding CV , Ramachandra L , Wick MJ . Interaction of bacteria with antigen presenting cells: influences on antigen presentation and antibacterial immunity . Curr Opin Immunol . 2003 ; 15 : 112 - 119 . PMID: 12495742
7. Bruns H , Stenger S . New insights into the interaction of Mycobacterium tuberculosis and human macrophages . Future Microbiol . 2014 ; 9 : 327 - 341 . doi: 10 .2217/fmb.13.164 PMID: 24762307
8. Wang J , Li BX , Ge PP , Li J , Wang Q , Gao GF , et al. Mycobacterium tuberculosis suppresses innate immunity by coopting the host ubiquitin system . Nat Immunol . 2015 ; 16 : 237 - 245 . doi: 10 .1038/ni.3096 PMID: 25642820
9. Gutierrez MG , Master SS , Singh SB , Taylor GA , Colombo MI , Deretic V . Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages . Cell . 2004 ; 119 : 753 - 766 . PMID: 15607973
10. Songane M , Kleinnijenhuis J , Netea MG , van Crevel R. The role of autophagy in host defence against Mycobacterium tuberculosis infection . Tuberculosis (Edinb) . 2012 ; 92 : 388 - 396 .
11. Deretic V , Saitoh T , Akira S. Autophagy in infection, inflammation and immunity . Nat Rev Immunol . 2013 ; 13 : 722 - 737 . doi: 10 .1038/nri3532 PMID: 24064518
12. Bento CF , Empadinhas N , Mendes V. Autophagy in the Fight Against Tuberculosis . Dna Cell Biol . 2015 ; 34 : 228 - 242 . doi: 10 .1089/dna. 2014 .2745 PMID: 25607549
13. Levine B , Kroemer G. Autophagy in the pathogenesis of disease . Cell . 2008 ; 132 : 27 - 42 . doi: 10 .1016/ j.cell. 2007 . 12 .018 PMID: 18191218
14. Choi AM , Ryter SW , Levine B. Autophagy in human health and disease . N Engl J Med . 2013 ; 368 : 1845 - 1846 .
15. Murrow L , Debnath J . Autophagy as a stress-response and quality-control mechanism: implications for cell injury and human disease . Annu Rev Pathol . 2013 ; 8 : 105 - 137 . doi: 10 .1146/annurev-pathol020712-163918 PMID: 23072311
16. Kim JJ , Lee HM , Shin DM , Kim W , Yuk JM , Jin HS , et al. Host cell autophagy activated by antibiotics is required for their effective antimycobacterial drug action . Cell Host Microbe . 2012 ; 11 : 457 - 468 . doi: 10 .1016/j.chom. 2012 . 03 .008 PMID: 22607799
17. Petruccioli E , Romagnoli A , Corazzari M , Coccia EM , Butera O , Delogu G , et al. Specific T cells restore the autophagic flux inhibited by Mycobacterium tuberculosis in human primary macrophages . J Infect Dis . 2012 ; 205 : 1425 - 1435 . doi: 10 .1093/infdis/jis226 PMID: 22457295
18. Romagnoli A , Etna MP , Giacomini E , Pardini M , Remoli ME , Corazzari M , et al. ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells . Autophagy . 2012 ; 8 : 1357 - 1370 . doi: 10 .4161/auto.20881 PMID: 22885411
19. Sassetti CM , Boyd DH , Rubin EJ . Comprehensive identification of conditionally essential genes in mycobacteria . Proc Natl Acad Sci U S A . 2001 ; 98 : 12712 - 12717 . PMID: 11606763
20. Vrba-Pech A , Fol M , Krawczyk M , Kowalewicz-Kulbat M , Kwiatkowska S. Clinical Mycobacterium tuberculosis isolates from the population of Lodz, Poland stimulated macrophages to the lower production of IL-12 and NO when compared to the virulent H37Rv strain . Tuberculosis (Edinb) . 2014 ; 94 : 383 - 388 .
21. Romero MM , Balboa L , Basile JI , Lopez B , Ritacco V , de la Barrera SS , et al. Clinical isolates of Mycobacterium tuberculosis differ in their ability to induce respiratory burst and apoptosis in neutrophils as a possible mechanism of immune escape . Clin Dev Immunol . 2012 ; 2012 :152546. doi: 10 .1155/ 2012 / 152546 PMID: 22778761
22. Krishnan N , Robertson BD , Thwaites G . Pathways of IL-1beta secretion by macrophages infected with clinical Mycobacterium tuberculosis strains . Tuberculosis (Edinb) . 2013 ; 93 : 538 - 547 .
23. Chen L , Li N , Liu Z , Liu M , Lv B , Wang J , et al. Genetic diversity and drug susceptibility of Mycobacterium tuberculosis isolates from Zunyi, one of the highest-incidence-rate areas in China . J Clin Microbiol . 2012 ; 50 : 1043 - 1047 . doi: 10 .1128/JCM.06095-11 PMID: 22205809
24. Falk AJ , O'Connor B , Pratt PC . Classification of pulmonary tuberculosis . 12th ed. New York: National Tuberculosis and Respiratory disease Association; 1969 .
25. World Health Organization. Treatment of tuberculosis: guidelines for national programmes , 4th ed., Geneva , Switzerland. 2009 .
26. Zhang W , Xu W , Xiong S. Blockade of Notch1 signaling alleviates murine lupus via blunting macrophage activation and M2b polarization . J Immunol . 2010 ; 184 : 6465 - 6478 . doi: 10 .4049/jimmunol. 0904016 PMID: 20427764
27. Li S , Yue Y , Xu W , Xiong S. MicroRNA-146a represses mycobacteria-induced inflammatory response and facilitates bacterial replication via targeting IRAK-1 and TRAF-6 . PLoS One . 2013 ; 8:e81438 . doi: 10.1371/journal.pone.0081438 PMID: 24358114
28. Gao B , Wang Y , Xu W , Li S , Li Q , Xiong S. Inhibition of histone deacetylase activity suppresses IFNgamma induction of tripartite motif 22 via CHIP-mediated proteasomal degradation of IRF-1 . J Immunol . 2013 ; 191 : 464 - 471 . doi: 10 .4049/jimmunol.1203533 PMID: 23729439
29. Kumar D , Nath L , Kamal MA , Varshney A , Jain A , Singh S , et al. Genome-wide analysis of the host intracellular network that regulates survival of Mycobacterium tuberculosis . Cell . 2010 ; 140 : 731 - 743 . doi: 10 .1016/j.cell. 2010 . 02 .012 PMID: 20211141
30. Chakraborty P , Kulkarni S , Rajan R , Sainis K. Drug resistant clinical isolates of Mycobacterium tuberculosis from different genotypes exhibit differential host responses in THP-1 cells . PLoS One . 2013 ; 8: e62966 . doi: 10.1371/journal.pone.0062966 PMID: 23667550
31. Srilohasin P , Chaiprasert A , Tokunaga K , Nishida N , Prammananan T , Smittipat N , et al. Genetic diversity and dynamic distribution of Mycobacterium tuberculosis isolates causing pulmonary and extrapulmonary tuberculosis in Thailand . J Clin Microbiol . 2014 ; 52 : 4267 - 4274 . doi: 10 .1128/JCM.01467-14 PMID: 25297330