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Polymorphisms in Autophagy Genes and Susceptibility to Tuberculosis

  • Mario Songane ,

    Contributed equally to this work with: Mario Songane, Johanneke Kleinnijenhuis

    Affiliations Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

  • Johanneke Kleinnijenhuis ,

    Contributed equally to this work with: Mario Songane, Johanneke Kleinnijenhuis

    Affiliations Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

  • Bachti Alisjahbana,

    Affiliations Department of Internal Medicine, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia, Health Research Unit, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

  • Edhyana Sahiratmadja,

    Affiliations Health Research Unit, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia, Department of Biochemistry, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

  • Ida Parwati,

    Affiliation Department of Clinical Pathology, Faculty of Medicine, Universitas Padjadjaran, Bandung, Indonesia

  • Marije Oosting,

    Affiliations Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

  • Theo S. Plantinga,

    Affiliations Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

  • Leo A. B. Joosten,

    Affiliations Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

  • Mihai G. Netea,

    Affiliations Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

  • Tom H. M. Ottenhoff,

    Affiliation Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands

  • Esther van de Vosse,

    Affiliation Department of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands

  • Reinout van Crevel

    r.vancrevel@aig.umcn.nl

    Affiliations Department of Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, Nijmegen Institute for Infection, Inflammation and Immunity (N4i), Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Polymorphisms in Autophagy Genes and Susceptibility to Tuberculosis

  • Mario Songane, 
  • Johanneke Kleinnijenhuis, 
  • Bachti Alisjahbana, 
  • Edhyana Sahiratmadja, 
  • Ida Parwati, 
  • Marije Oosting, 
  • Theo S. Plantinga, 
  • Leo A. B. Joosten, 
  • Mihai G. Netea, 
  • Tom H. M. Ottenhoff
PLOS
x

Abstract

Recent data suggest that autophagy is important for intracellular killing of Mycobacterium tuberculosis, and polymorphisms in the autophagy gene IRGM have been linked with susceptibility to tuberculosis (TB) among African-Americans, and with TB caused by particular M. tuberculosis genotypes in Ghana. We compared 22 polymorphisms of 14 autophagy genes between 1022 Indonesian TB patients and 952 matched controls, and between patients infected with different M. tuberculosis genotypes, as determined by spoligotyping. The same autophagy polymorphisms were studied in correlation with ex-vivo production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 in healthy volunteers. No association was found between TB and polymorphisms in the genes ATG10, ATG16L2, ATG2B, ATG5, ATG9B, IRGM, LAMP1, LAMP3, P2RX7, WIPI1, MTOR and ATG4C. Associations were found between polymorphisms in LAMP1 (p = 0.02) and MTOR (p = 0.02) and infection with the successful M. tuberculosis Beijing genotype. The polymorphisms examined were not associated with M. tuberculosis induced cytokines, except for a polymorphism in ATG10, which was linked with IL-8 production (p = 0.04). All associations found lost statistical significance after correction for multiple testing. This first examination of a broad set of polymorphisms in autophagy genes fails to show a clear association with TB, with M. tuberculosis Beijing genotype infection or with ex-vivo pro-inflammatory cytokine production.

Introduction

Mycobacterium tuberculosis (M. tuberculosis), the main cause of tuberculosis (TB) worldwide, is an intracellular pathogen that primarily infects macrophages [1], [2]. This pathogen resides and multiplies within a host-derived phagosome where it persists through interference with phagosome-lysosome biogenesis [3], [4]. Recent studies suggest that autophagy, a homeostatic process involved in nutrient regeneration and immune responses, is involved in intracellular killing of M. tuberculosis [3], [5], [6], and that physiological or pharmacological induction of this process in vitro (i.e.: with ATP, IFN-γ, vitamin D3) promotes fusion of phagosomes containing M. tuberculosis with lysosomes and subsequent killing of the pathogen in autophagic characteristic double-membrane autolysosomes [1], [3], [7]. In addition, intracellular survival of M. tuberculosis was shown to depend on its ability to escape or inhibit autophagy [5], [8], and a study by Kumar et al. found that genes that regulate intracellular survival of M. tuberculosis, regardless of its genotype, are in the autophagy pathway itself or in pathways that affect autophagy [9].

Susceptibility to TB is partly genetically determined and variations in genes involved in the autophagic pathway may affect the host response to M. tuberculosis infection. Indeed, mice deficient in autophagy and autophagy related genes were found to be more susceptible to infection with M. tuberculosis [10], [11] and human mononuclear cells with certain polymorphisms in autophagy related genes displayed an impaired ability to control M. tuberculosis growth [12], [13], thus suggesting that polymorphisms in autophagy and autophagy related genes may be associated with TB. This appears to be the case as various polymorphisms in one autophagy gene IRGM, a downstream effector of IFN-γ, have been associated with increased protection against M. tuberculosis infection in African-American [14] and Chinese individuals [15] and infection by particular M. tuberculosis genotypes in Ghana [16]. In addition, polymorphisms in a number of genes which affect autophagy, such as P2RX7, have also been associated with TB [17], [18]. However, to our knowledge, besides IRGM no other gene of the autophagy pathway itself has been examined in TB patients. We have therefore examined a selection of autophagy genes in a large cohort of TB patients and healthy controls in Indonesia. Since susceptibility to TB may depend on the interplay between host and mycobacterial genotype [2], [9], [19], we also grouped patients’ M. tuberculosis isolates into W-Beijing genotype strains, which account for one-third of all M. tuberculosis infections in Indonesia [20], [21], and non-W-Beijing genotypes. Furthermore, in a Caucasian cohort that was genotyped for the 22 SNPs, we measured cytokine production in peripheral blood mononuclear cells (PBMCs) stimulated with M. tuberculosis.

Materials and Methods

Subject Recruitment

We previously recruited consecutive TB patients diagnosed in two outpatient clinics and two hospitals in Jakarta and Bandung (Indonesia) from January 2001 to December 2006, for a series of genetic studies examining host susceptibility to TB [19], [22], [23].

Diagnosis of pulmonary TB (PTB) was done according to World Health Organization criteria by clinical presentation and chest radiograph examination, followed by confirmation with microscopic detection of acid-fast bacilli in Ziehl-Neelsen-stained sputum smears and positive culture of M. tuberculosis on 3% Ogawa medium. For M. tuberculosis genotype analysis, mycobacterial DNA was extracted by bringing 2 loops of bacterial mass from an M. tuberculosis culture in saline solution and subsequently heating it at 95°C for 5 min. M. tuberculosis genotype was determined by using a commercially available Spoligotyping kit (Isogen Bioscience, Maarssen, The Netherlands) as previously described [20]. M. tuberculosis Beijing genotype was defined as a spoligo-pattern showing hybridization to at least 3 of the 9 spacers 35–43 and absence of hybridization to spacers 1–34. Spoligotyping was done at the Hasan Sadikin Hospital, Bandung, Indonesia. In addition, for quality control purposes, spoligotyping of 10% of the isolates and of all isolates lacking hybridization were also done at Gelre Hospital, Apeldoorn, The Netherlands.

We excluded from the genetic studies patients with a confirmed diagnosis of extra-pulmonary TB (n = 93), diabetes mellitus (fasting blood glucose >126 mg/dL) (n = 139) and HIV-positive subjects (n = 10). Standard regimen for treatment of TB consisted of isoniazid, rifampin, pyrazinamide, and ethambutol (2HRZE/4H3R3) was administered free of charge to all patients according to the Indonesian National TB program.

During the above mentioned period we also recruited 1000 randomly selected age and gender matched, but genetically unrelated control subjects from the same, mostly poor and densily populated areas where TB is abundant. All control individuals were subjected to the same physical examination, blood tests and chest radiography as the TB patients. A total of 952 control subjects were enrolled in the study after excluding individuals with symptoms or chest X-ray abnormalities suggesting active TB or a history of TB.

A structured questionnaire was used for patients and control subjects to record clinical information, age, gender, self and parental ethnicity, socio-economic status and concurrent medical history.

Ethics Statement

All individuals recruited signed a written informed consent. The study protocol was reviewed and approved by the local institutional review boards of the medical faculty of university of Indonesia, the Eijkman institute for molecular biology in Jakarta in Indonesia and the Medical Ethical Committee Arnhem-Nijmegen in The Netherlands.

Genotyping

Using NCBI SNP database we selected SNPs in autophagy genes previously associated with TB (P2RX7- rs2393799 [17]), other diseases (ATG16L1- rs2241880 [24], [25], ATG5- rs2245214 [26] IRGM rs72553867 [27] rs4958847 [28] or with a minor allele frequency of at least 5% (Table 1).

Blood samples were obtained by venapuncture. Genomic DNA was isolated from EDTA blood of patients, controls and a cohort of healthy volunteers using standard methods, and 5 ng of DNA was used for genotyping. Multiplex assays were designed using Mass ARRAY Designer Software (Sequenom) and genotypes were determined using Sequenom MALDI-TOF MS according to manufacturer’s instructions (Sequenom Inc., San Diego, CA, USA). Briefly, the SNP region was amplified by a locus-specific PCR reaction. After amplification a single base extension from a primer adjacent to the SNP was performed to introduce mass differences between alleles. This was followed by salt removal and product spotting onto a target chip with 384 patches containing matrix. MALDI-TOF MS was then used to detect mass differences and genotypes were assigned real-time using Typer 4 software (Sequenom Inc. San Diego, CA, USA). As quality control, 5% of samples were genotyped in duplicate and each 384-well plate also contained at least 8 positive and 8 negative controls, no inconsistencies were observed. DNA samples of which half or more of the SNPs failed (N = 90) were excluded from analyses. Variants with call-rates below 90% were also excluded from further analyses (n = 0).

For quality control purposes the genotype of at least two samples for each homozygous genotype were confirmed by sequencing using Sanger method with Big Dye Terminator version 3 (Applied Biosystems). After the cycle sequence reaction, the samples were purified by ethanol precipitation and analysed on a 3730 Sequence Analyzer (Applied Biosystems).

Previously polymorphisms in various genes were genotyped on a two-stage genome-wide association study (GWAS) using Illumina’s GoldenGate Assay according to manufacturer instructions, aiming to discover genes relevant in pulmonary TB susceptibility in the same Indonesian cohort involved in the current study [29]. Among the SNPs studied, five were in autophagy genes and were included in our data analysis (Table 1). The overlap of study subjects between the current study and the GWAS is shown in Figure 1.

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Figure 1. The overlap of study subjects between the current study (n = 1974) and the previous genome-wide association study (GWAS, n = 1139) [29].

https://doi.org/10.1371/journal.pone.0041618.g001

Cytokine Production by M. tuberculosis Stimulated PBMC

Cells isolated from healthy Caucasian volunteers bearing various genotypes were examined for cytokine production induced by sonicated M. tuberculosis H37Rv (n = 67). These individuals were aged 23–73 years, 77% was male and none had a known TB contact. All gave written informed consent, and the study was approved by the Ethical Committee of the Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Blood samples were obtained by venapuncture. The mononuclear cell fraction was isolated from blood by density centrifugation of blood, diluted 1∶1 in pyrogen-free saline over Ficoll-Paque (Pharmacia Biotech, PA, USA). Cells were washed twice in saline and resuspended in culture medium (RPMI, Invitrogen, CA, USA) supplemented with gentamicin 10 µg/ml, L-glutamine 10 mM, and pyruvate 10 mM. Cells were counted in a Coulter counter (Coulter Electronics) and the number was adjusted to 5×106 cells/ml. A total of 5×105 mononuclear cells in a 100 µl volume of RPMI was added to round-bottom 96-wells plates (Greiner) with or without sonicated M. tuberculosis H37Rv (final concentration: 1 µg/ml). After 24 hours, 48 hours (both without serum) or 7 days of incubation (in the presence of 10% serum), supernatants were stored at −20°C. Cytokine concentrations were assessed in the supernatants using enzyme-linked immunosorbent assay (ELISA).

Cytokine measurements of TNF, IL-1β, IL-6, IL-8 (after 24 hours incubation); IFN-γ (after 48 hours incubation), and IL-17 (7 days incubation) were performed in the supernatants using commercial ELISAs from R&D Systems, MN, USA (TNF, IL-1β, IL-8, and IL-17) or Sanquin, Amsterdam, The Netherlands (IL-6 and IFN-γ).

Statistical Analysis

All data collected from the questionnaires and genotyping were analysed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). The Hardy-Weinberg equilibrium (HWE) was checked for each SNP using the program HWE Version 1.10 (Rockefeller University, New York). The program Conting was used to calculate the χ2 and the associated values for a contingency table. Patient data were stratified for the M. tuberculosis genotype with which they were infected; Beijing or non-Beijing strains and the χ2 was calculated with SPSS. Differences in cytokine production were analyzed using the Wilcoxon signed rank test. All statistical analyses were 2-sided, and P<0.05 was considered to be statistically significant.

The available number of study subjects allowed us to observe a 5% allele difference between patients and controls for the SNP in IRGM (rs4958847), based on previously reported allele distribution in the general population, a power (β) of 0.80 and a significance level (α) of 0.05.

Results

Study Subjects

A total number of 1022 confirmed pulmonary TB patients and 952 age- and gender matched community controls were included in the data analysis. As shown in Table 2, 78% of patients and control subjects were Javanese (a population group with relatively low genetic variance in Indonesia [30]) with similar age, gender distribution, and likelihood of having a BCG scar. Furthermore, both groups also had a similar socioeconomic status (not shown) and previous analysis in this cohort [29] showed that population stratification was minimal.

Association between Polymorphisms in Autophagy Genes and Susceptibility to TB

Polymorphisms rs11235604 (in ATG16L2), rs77228473 and rs77833427 (in ATG2A), rs74719094 (in ATG2B), rs72553867 (in IRGM), rs10493328 and rs10493329 (in ATG4C) were rare in the study subjects. With the exception of the SNP rs3759601 in ATG2B (HWE: 2p  = 0.034), all polymorphisms were in Hardy–Weinberg equilibrium in the healthy controls. The distribution of the alleles for all polymorphisms analyzed in the current study is presented in Table 3. After Chi-square testing we did not detect significant associations between any genetic polymorphism and susceptibility to TB. This was also the case when the largest group (Javanese) was analysed separately (data not shown).

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Table 3. Distribution of polymorphism allele frequencies in cases and controls.

https://doi.org/10.1371/journal.pone.0041618.t003

Association between Polymorphisms in Autophagy Genes and M. tuberculosis Genotype

To examine a possible association between host and mycobacterial genotype, autophagy gene polymorphisms were compared between patients infected with M. tuberculosis Beijing genotype and other M. tuberculosis genotypes. One hundred and sixty-one patients (33%) were infected with M. tuberculosis Beijing genotype strains, 322 with a non-Beijing strain, while no strain information was available for the remainder (n = 540). Patients infected with M. tuberculosis Beijing and non-Beijing strains were not significantly different in terms of age, sex, or history of previous tuberculosis treatment. The distribution of the alleles for all polymorphisms among patients infected with M. tuberculosis Beijing and non-Beijing strains is shown in Table 4. The polymorphism in LAMP1 (rs9577229) showed an association with TB caused by M. tuberculosis Beijing strains, when the TC was combined with the low prevalent TT genotype (p = 0.02). The same was true for the polymorphism in MTOR (rs6701524); when combining the AG with the low prevalent GG genotype, MTOR was significantly associated with infection with M. tuberculosis Beijing strains (p = 0.02). However, both associations lost statistical significance after correction for multiple testing.

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Table 4. Polymorphism in autophagy genes according to M. tuberculosis genotypes strain.

https://doi.org/10.1371/journal.pone.0041618.t004

Polymorphisms in Autophagy Genes and M. tuberculosis Induced Cytokine Production

Association between host genotype and M. tuberculosis induced cytokine production by PBMC was examined in healthy Caucasian individuals. Table 5 shows the difference in M. tuberculosis induced production of TNF, IL-1β, IL-6, IL-8, IFN-γ and IL-17 by PBMC isolated from individuals stratified for different genotype of autophagy related genes. Six of these polymorphisms showed no polymorphic distribution in the Caucasian individuals and could therefore not be analysed. With the exception of ATG10 (rs1864183), for which a significant difference was found in IL-8 production between individuals with an AA and GG genotype (p = 0.04), no associations were observed between the investigated cytokines and the autophagy related polymorphisms. Figure 2 presents scatter plots of TNF, IFN-γ, and IL-17 stratified for genotypes of both investigated polymorphisms in IRGM which was previously linked with susceptibility to TB.

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Figure 2. Scatter plots of TNF, IFN-γ, and IL-17 produced after stimulation of PBMC with M. tuberculosis stratified for genotypes of both investigated polymorphisms in IRGM.

https://doi.org/10.1371/journal.pone.0041618.g002

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Table 5. Variation in cytokine level associated with polymorphisms in autophagy genes.

https://doi.org/10.1371/journal.pone.0041618.t005

Discussion

In-vitro data strongly support a role for autophagy in control of M. tuberculosis, and a study involving 2010 patients with pulmonary TB and 2346 control subjects from Ghana has previously reported an association between a polymorphism in the autophagy gene IRGM and TB [16]. To further explore a role of autophagy in TB we examined polymorphisms in a number of autophagy genes in TB patients and matched controls from Indonesia. Among almost 2000 subjects, no association was found between TB and 22 SNPs in 14 different autophagy and autophagy related genes, including IRGM and P2RX7 which were previously associated with TB. When TB patients were stratified according to M. tuberculosis genotype, associations were observed between SNPs in LAMP1, MTOR and infection with M. tuberculosis Beijing genotype, but statistical significance was lost after correction for multiple testing. No significant correlation was found between M. tuberculosis induced cytokine production and genotype of autophagy related genes in a separate cohort of healthy Caucasian volunteers.

IRGM, a downstream effector protein of IFN-γ, induces autophagy and subsequent generation of large autolysosomal organelles as a mechanism for the elimination of intracellular M. tuberculosis [31]. In a cohort of 2010 Ghanaian patients and 2346 controls a polymorphism (rs9637876) in IRGM was associated with decreased susceptibility to TB caused by M. tuberculosis Euro-American (EUAM) lineage, although not for M. tuberculosis East-African-Indian (EAI), Beijing, Delhi, M. africanum and M. bovis lineages [16]. In a study in the US, a polymorphism in IRGM (rs10065172) was more common in 370 African-American TB patients compared to controls, but not in 177 Caucasian patients compared to 110 Caucasian controls [14]. We did not find an association between TB, which in Indonesia is mainly caused by the M. tuberculosis Beijing lineage, and two different polymorphisms in IRGM.

P2RX7 is an autophagy related gene. It encodes for the P2X7 receptor, a plasma membrane receptor which mediates ATP-induced autophagy and subsequent intracellular killing of M. tuberculosis upon upregulation in mature macrophages [32], [33], [34]. P2RX7 displays a high genetic heterogeneity [12], and a polymorphism with a C allele at position -762 in the P2RX7 promoter region was found to have a protective effect against TB in over 300 TB patients and 160 ethnically matched controls subjects from The Gambia [18]. However, no association was found between the same polymorphism and TB in our cohort of Indonesian subjects. It is noteworthy that the protective effect of this polymorphism in Gambian subjects was weak and that it did not correlate with altered receptor expression or activity, suggesting the effect of this SNP might be influenced by other host and pathogen factors [18]. In addition, the relative importance of the role of P2X7 receptor in the control of M. tuberculosis growth is still debated since mice deficient for P2X7 receptor displayed a similar ability to control pulmonary M. tuberculosis infection compared to wild-type mice [35]. Unfortunately, studies on the effect of P2RX7 polymorphisms in susceptibility to pulmonary TB in humans have not yet been done either in vivo or in other ethnic groups.

Polymorphisms in various genes have been associated with TB, but only polymorphisms in VDR [36], [37], [38], NRAMP1 [39], [40] and MBL [41], [42] were found to be associated with TB in different geographic regions and ethnic groups. However, the effect of SNPs in these genes varies among racial groups. SNPs in NRAMP1 were associated with an increased risk of PTB in Gambians [39] but were found to have a protective effect in Cambodians [40], polymorphisms in MBL were associated with protection against TB in South Africans [41] but in South Indians increased susceptibility to this disease [42], while SNPs in VDR were found to increase susceptibility to PTB in three African countries [36] but to have no effect in Cambodians [40]. As suggested by Fernando et al, these contrasting findings between different ethnic groups may be due to differences in allele frequencies [17]. In addition, the phylogeography of mycobacteria implies that M. tuberculosis lineages have become differentially adapted to genetic variations among racial groups [2].

The development of TB is the result of a complex interaction between the host and pathogen influenced by environmental factors [43]. After stratification according to M. tuberculosis genotype, we found a suggestive association between TB caused by M. tuberculosis Beijing genotype and a polymorphism in LAMP1, similar to what we have previously shown for polymorphisms in SLC11A/NRAMP1 [19]. However, nine major M. tuberculosis genotypes have been previously identified in Indonesia [20] and some polymorphisms analysed here may be associated with TB caused by other genotypes not identified in this study.

LAMP1 and LAMP2 are two major protein components of late endosome and lysosome membranes, thought to form a protective barrier against degradation by hydrolytic enzymes [44], [45]. Mice lacking Lamp2 display impaired autophagy and lysosome biogenesis, while deletion of both Lamp1 and Lamp2 is embryonically lethal [44]. However, the contribution of these two lysosomal membrane proteins to phagosomal maturation and killing of intracellular pathogens still needs to be clarified.

Our group recently showed that inhibition of autophagy (genetically or with either siRNA or 3MA) increased IL-1β production [46], [47], [48]. However, with the exception of a SNP in ATG10 and IL-8, no differences in cytokine production were observed in M. tuberculosis stimulated PBMCs of healthy volunteers stratified for genotype of autophagy related genes.

Our paper has several limitations. First and most importantly, no tuberculin skin testing was performed in the control population. However, exposure to tuberculosis must be common in this group, as the majority of controls lived in households of tuberculosis patients, who mostly had a productive cough (98%) for a median of 3 months before first presentation at the TB clinic [49]. Second, as we powered our study on an expected 5% difference in allele frequency between the groups, we cannot exclude possible associations amongst SNPs with a lower frequency.

This is the first paper to investigate the relation of different SNPs in a broad set 14 autophagy genes with susceptibility to TB, as well as with the infecting M. tuberculosis genotype and ex-vivo cytokine production. These data further supports the belief that susceptibility to TB has a polygenic nature and polymorphisms in more than one gene may be required to render individuals more or less susceptible to develop active disease.

Author Contributions

Conceived and designed the experiments: MS JK MN TO EV RC. Performed the experiments: MS JK BA ES IP MO TP. Analyzed the data: MS JK MN TO EV RC. Wrote the paper: MS JK LJ MN TO EV RC.

References

  1. 1. Yuk JM, Shin DM, Lee HM, Yang CS, Jin HS, et al. (2009) Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Cell Host Microbe 6: 231–243.
  2. 2. Gagneux S, Small PM (2007) Global phylogeography of Mycobacterium tuberculosis and implications for tuberculosis product development. Lancet Infect Dis 7: 328–337.
  3. 3. Gutierrez MG, Master SS, Singh SB, Taylor GA, Colombo MI, et al. (2004) Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119: 753–766.
  4. 4. Goren MB, D’Arcy HP, Young MR, Armstrong JA (1976) Prevention of phagosome-lysosome fusion in cultured macrophages by sulfatides of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 73: 2510–2514.
  5. 5. Deretic V, Levine B (2009) Autophagy, immunity, and microbial adaptations. Cell Host Microbe 5: 527–549.
  6. 6. Xie Z, Klionsky DJ (2007) Autophagosome formation: core machinery and adaptations. Nat Cell Biol 9: 1102–1109.
  7. 7. Biswas D, Qureshi O, Lee WY, Croudace J, Mura M, et al. (2008) ATP-induced autophagy is associated with rapid killing of intracellular mycobacteria within human monocytes/macrophages. BMC Immunol 9: 35.
  8. 8. Vergne I, Chua J, Singh SB, Deretic V (2004) Cell biology of mycobacterium tuberculosis phagosome. Annu Rev Cell Dev Biol 20: 367–394.
  9. 9. Kumar D, Nath L, Kamal MA, Varshney A, Jain A, et al. (2010) Genome-wide analysis of the host intracellular network that regulates survival of Mycobacterium tuberculosis. Cell 140: 731–743.
  10. 10. MacMicking JD, Taylor GA, McKinney JD (2003) Immune Control of Tuberculosis by IFN-Inducible LRG-47. Science 302: 654–659.
  11. 11. Divangahi M, Mostowy S, Coulombe Fo, Kozak R, Guillot Lc, et al. (2008) NOD2-Deficient Mice Have Impaired Resistance to Mycobacterium tuberculosis Infection through Defective Innate and Adaptive Immunity. J Immunol 181: 7157–7165.
  12. 12. Fernando SL, Saunders BM, Sluyter R, Skarratt KK, Wiley JS, et al. (2005) Gene Dosage Determines the Negative Effects of Polymorphic Alleles of the P2X7 Receptor on Adenosine Triphosphate Mediated Killing of Mycobacteria by Human Macrophages. J Infect Dis 192: 149–155.
  13. 13. Ferwerda G, Girardin SE, Kullberg BJ, Le BL, de Jong DJ, et al. (2005) NOD2 and toll-like receptors are nonredundant recognition systems of Mycobacterium tuberculosis. PLoS Pathog 1: 279–285.
  14. 14. King KY, Lew JD, Ha NP, Lin JS, Ma X, et al. (2011) Polymorphic allele of human IRGM1 is associated with susceptibility to tuberculosis in African Americans. PLoS One 6: e16317.
  15. 15. Che N, Li S, Gao T, Zhang Z, Han Y, et al. (2010) Identification of a novel IRGM promoter single nucleotide polymorphism associated with tuberculosis. Clin Chim Acta 411: 1645–1649.
  16. 16. Intemann CD, Thye T, Niemann S, Browne EN, Amanua CM, et al. (2009) Autophagy gene variant IRGM -261T contributes to protection from tuberculosis caused by Mycobacterium tuberculosis but not by M. africanum strains. PLoS Pathog 5: e1000577.
  17. 17. Fernando SL, Saunders BM, Sluyter R, Skarratt KK, Goldberg H, et al. (2007) A Polymorphism in the P2X7 Gene Increases Susceptibility to Extrapulmonary Tuberculosis. Am J Resp Crit Care Med 175: 360–366.
  18. 18. Li CM, Campbell SJ, Kumararatne DS, Bellamy R, Ruwende C, et al. (2002) Association of a polymorphism in the P2X7 gene with tuberculosis in a Gambian population. J Infect Dis 186: 1458–1462.
  19. 19. van Crevel R, Parwati I, Sahiratmadja E, Marzuki S, Ottenhoff THM, et al. (2009) Infection with Mycobacterium tuberculosis Beijing Genotype Strains Is Associated with Polymorphisms in SLC11A1/NRAMP1 in Indonesian Patients with Tuberculosis. J Infect Dis 200: 1671–1674.
  20. 20. Parwati I, van Crevel R, Sudiro M, Alisjahbana B, Pakasi T, et al. (2008) Mycobacterium tuberculosis Population Structures Differ Significantly on Two Indonesian Islands. J Clin Microbiol 46: 3639–3645.
  21. 21. van Crevel R, Nelwan RH, de LW, Veeraragu Y, van der Zanden AG, et al. (2001) Mycobacterium tuberculosis Beijing genotype strains associated with febrile response to treatment. Emerg Infect Dis 7: 880–883.
  22. 22. Davila S, Hibberd ML, Hari DR, Wong HE, Sahiratmadja E, et al. (2008) Genetic association and expression studies indicate a role of toll-like receptor 8 in pulmonary tuberculosis. PLoS Genet 4: e1000218.
  23. 23. Sahiratmadja E, Baak-Pablo R, de Visser AW, Alisjahbana B, Adnan I, et al. (2007) Association of polymorphisms in IL-12/IFN-gamma pathway genes with susceptibility to pulmonary tuberculosis in Indonesia. Tuberculosis (Edinb ) 87: 303–311.
  24. 24. Cheng JF, Ning YJ, Zhang W, Lu ZH, Lin L (2010) T300A polymorphism of ATG16L1 and susceptibility to inflammatory bowel diseases: a meta-analysis. World J Gastroenterol 16: 1258–1266.
  25. 25. Lacher M, Schroepf S, Ballauff A, Lohse P, von Schweinitz D, et al. (2009) Autophagy 16-like 1 rs2241880 G allele is associated with Crohn’s disease in German children. Acta Paediatr 98: 1835–1840.
  26. 26. Gateva V, Sandling JK, Hom G, Taylor KE, Chung SA, et al. (2009) A large-scale replication study identifies TNIP1, PRDM1, JAZF1, UHRF1BP1 and IL10 as risk loci for systemic lupus erythematosus. Nat Genet 41: 1228–1233.
  27. 27. Moon CM, Shin DJ, Kim SW, Son NH, Park A, et al. (2012) Associations between genetic variants in the IRGM gene and inflammatory bowel diseases in the korean population. Inflamm Bowel Dis.
  28. 28. Sehgal R, Berg A, Polinski JI, Hegarty JP, Lin Z, et al. (2012) Mutations in IRGM are associated with more frequent need for surgery in patients with ileocolonic Crohn’s disease. Dis Colon Rectum 55: 115–121.
  29. 29. Png E, Alisjahbana B, Sahiratmadja E, Marzuki S, Nelwan R, et al. (2012) A genome wide association study of pulmonary tuberculosis susceptibility in Indonesians. BMC Med Genet 13: 5.
  30. 30. Shepard EM, Chow RA, Suafo’a E, Addison D, Perez-Miranda AM, et al. (2005) Autosomal STR variation in five Austronesian populations. Hum Biol 77: 825–851.
  31. 31. Singh SB, Davis AS, Taylor GA, Deretic V (2006) Human IRGM Induces Autophagy to Eliminate Intracellular Mycobacteria. Science 313: 1438–1441.
  32. 32. Di VF (1995) The P2Z purinoceptor: an intriguing role in immunity, inflammation and cell death. Immunol today 16: 524–528.
  33. 33. Lammas DA, Stober C, Harvey CJ, Kendrick N, Panchalingam S, et al. (1997) ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors. Immunity 7: 433–444.
  34. 34. Stober CB, Lammas DA, Li CM, Kumararatne DS, Lightman SL, et al. (2001) ATP-Mediated Killing of Mycobacterium bovis Bacille Calmette-Guerin Within Human Macrophages Is Calcium Dependent and Associated with the Acidification of Mycobacteria-Containing Phagosomes. J Immunol 166: 6276–6286.
  35. 35. Myers AJ, Eilertson B, Fulton SA, Flynn JL, Canaday DH (2005) The Purinergic P2X7 Receptor Is Not Required for Control of Pulmonary Mycobacterium tuberculosis Infection. Infect Immun 73: 3192–3195.
  36. 36. Bornman L, Campbell SJ, Fielding K, Bah B, Sillah J, et al. (2004) Vitamin D Receptor Polymorphisms and Susceptibility to Tuberculosis in West Africa: A Case-Control and Family Study. J Infect Dis 190: 1631–1641.
  37. 37. Martineau AR, Timms PM, Bothamley GH, Hanifa Y, Islam K, et al. (2011) High-dose vitamin D(3) during intensive-phase antimicrobial treatment of pulmonary tuberculosis: a double-blind randomised controlled trial. Lancet 377: 242–250.
  38. 38. Roth DE, Soto G, Arenas F, Bautista CT, Ortiz J, et al. (2004) Association between Vitamin D Receptor Gene Polymorphisms and Response to Treatment of Pulmonary Tuberculosis. J Infect Dis 190: 920–927.
  39. 39. Bellamy R, Ruwende C, Corrah T, McAdam KPWJ, Whittle HC, et al. (1998) Variations in the NRAMP1 Gene and Susceptibility to Tuberculosis in West Africans. N Eng J Med 338: 640–644.
  40. 40. Delgado JC, Baena A, Thim S, Goldfeld AE (2002) Ethnic-Specific Genetic Associations with Pulmonary Tuberculosis. J Infect Dis 186: 1463–1468.
  41. 41. Hoal-Van Helden EG, Epstein J, Victor TC, Hon D, Lewis LA, et al. (1999) Mannose-binding protein B allele confers protection against tuberculous meningitis. Pediatr Res 45: 459–464.
  42. 42. Selvaraj P, Narayanan PR, Reetha AM (1999) Association of functional mutant homozygotes of the mannose binding protein gene with susceptibility to pulmonary tuberculosis in India. Tuber Lung Dis 79: 221–227.
  43. 43. Bellamy R (2003) Susceptibility to mycobacterial infections: the importance of host genetics. Genes Immun 4: 4–11.
  44. 44. Eskelinen EL (2006) Roles of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol Aspects Med 27: 495–502.
  45. 45. Binker MG, Cosen-Binker LI, Terebiznik MR, Mallo GV, McCaw SE, et al. (2007) Arrested maturation of Neisseria-containing phagosomes in the absence of the lysosome-associated membrane proteins, LAMP-1 and LAMP-2. Cell Microbiol 9: 2153–2166.
  46. 46. Crisan TO, Plantinga TS, van de Veerdonk FL, Farcas MF, Stoffels M, et al. (2011) Inflammasome-independent modulation of cytokine response by autophagy in human cells. PLoS One 6: e18666.
  47. 47. Kleinnijenhuis J, Oosting M, Plantinga TS, van der Meer JW, Joosten LA, et al. (2011) Autophagy modulates the Mycobacterium tuberculosis-induced cytokine response. Immunology 134: 341–348.
  48. 48. Plantinga TS, Crisan TO, Oosting M, van dV, de Jong DJ, et al. (2011) Crohn’s disease-associated ATG16L1 polymorphism modulates pro-inflammatory cytokine responses selectively upon activation of NOD2. Gut 60: 1229–1235.
  49. 49. Alisjahbana B, van CR, Sahiratmadja E, den HM, Maya A, et al. (2006) Diabetes mellitus is strongly associated with tuberculosis in Indonesia. Int J Tuberc Lung Dis 10: 696–700.