Modern Lineages of Mycobacterium tuberculosis Exhibit Lineage-Specific Patterns of Growth and Cytokine Induction in Human Monocyte-Derived Macrophages

Abstract

Background

Strains of Mycobacterium tuberculosis vary in virulence. Strains that have caused outbreaks in the United States and United Kingdom have been shown to subvert the innate immune response as a potential immune evasion mechanism. There is, however, little information available as to whether these patterns of immune subversion are features of individual strains or characteristic of broad clonal lineages of M. tuberculosis.

Methods

Strains from two major modern lineages (lineage 2 [East-Asian] and lineage 4 [Euro-American]) circulating in the Western Cape in South Africa as well as a comparator modern lineage (lineage 3 [CAS/Delhi]) were identified. We assessed two virulence associated characteristics: mycobacterial growth (in liquid broth and monocyte derived macrophages) and early pro-inflammatory cytokine induction.

Results

In liquid culture, Lineage 4 strains grew more rapidly and reached higher plateau levels than other strains (lineage 4 vs. lineage 2 p = 0.0024; lineage 4 vs. lineage 3 p = 0.0005). Lineage 3 strains were characterized by low and early plateau levels, while lineage 2 strains showed an intermediate growth phenotype. In monocyte-derived macrophages, lineage 2 strains grew faster than lineage 3 strains (p<0.01) with lineage 4 strains having an intermediate phenotype. Lineage 2 strains induced the lowest levels of pro-inflammatory TNF and IL-12p40 as compared to other lineages (lineage 2: median TNF 362 pg/ml, IL-12p40 91 pg/ml; lineage 3: median TNF 1818 pg/ml, IL-12p40 123 pg/ml; lineage 4: median TNF 1207 pg/ml, IL-12p40 205 pg/ml;). In contrast, lineage 4 strains induced high levels of IL-12p40 and intermediate level of TNF. Lineage 3 strains induced high levels of TNF and intermediate levels of IL-12p40.

Conclusions

Strains of M. tuberculosis from the three major modern strain lineages possess distinct patterns of growth and cytokine induction. Rapid growth and immune subversion may be key characteristics to the success of these strains in different human populations.

About the Authors:

Rajesh Sarkar

Affiliation: Division of Medical Microbiology, University of Cape Town, Cape Town, South Africa

Laura Lenders

Affiliation: Division of Medical Microbiology, University of Cape Town, Cape Town, South Africa

Katalin A. Wilkinson

Affiliations Clinical Infectious Diseases Research Initiative, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom

Robert J. Wilkinson

Affiliations Clinical Infectious Diseases Research Initiative, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa, Division of Medicine, Imperial College London, London, United Kingdom, MRC National Institute for Medical Research, Mill Hill, London, United Kingdom

Mark P. Nicol

* E-mail: [email protected]

Affiliations Division of Medical Microbiology, University of Cape Town, Cape Town, South Africa, National Health Laboratory Services, Groote Schuur Hospital, Cape Town, South Africa, Clinical Infectious Diseases Research Initiative, Institute of Infectious Diseases and Molecular Medicine, University of Cape Town, Cape Town, South Africa

Introduction

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), infects over 2 billion people world-wide and causes 1.7 million deaths annually [1]. The clinical significance of strain variation in M. tuberculosis remains controversial, partly since virulence cannot be simply defined in relation to defined virulence characteristics as in other bacterial pathogens, such as Clostridium tetani or Staphylococcus aureus. Since modern M. tuberculosis evolves through single nucleotide substitutions, deletion and duplication events, the population structure is strongly clonal [2]. It is therefore feasible that such clonal lineages may evolve specific virulence characteristics [3].

In a robust phylogenetic study based on genomic deletion analysis, M. tuberculosis has been classified into six major lineages. These lineages are highly predominant in specific geographic areas and named according to their geographical distribution: Lineage 1 (also known as Indo-Oceanic lineage), Lineage 2 (also known as East Asian; includes “Beijing”), Lineage 3 (also known as CAS/Delhi), Lineage 4 (also known as Euro-American), Lineage 5 (also known as West African 1) and Lineage 6 (also known West African 2). In the Western Cape region of South Africa the “Lineage 2 (East Asian; includes “Beijing”) and “Lineage 4 (Euro-American; includes LAM3/F11 strains) lineages predominate [4], [5], [6], [7].

Macrophages are the primary site of intracellular replication of M. tuberculosis. In humans, large numbers of alveolar macrophages are not readily available for in vitro study, therefore matured human monocyte derived macrophages (MDM) derived from peripheral blood mononuclear cells (PBMC) are often used as an in vitro model to study M. tuberculosis infection in human cells. Activated macrophages in the presence of T cells and inflammatory cytokines are known to be effective in restricting mycobacterial growth.

Lineage 2 strains are transmitted globally with a high tendency to cause outbreaks and multidrug-resistant tuberculosis infection, suggesting that this lineage may possess particular virulence characteristics [8], [9]. Recent reports indicate that lineage 2 strains are emerging in Western Cape region of South Africa [10], [11]. Lineage 2 strains replicate to high bacillary loads in lungs of infected mice [12]. Strains from this genotype have been shown to replicate more rapidly in the human cell culture model [13], [14], [15]. Several human and animal studies have suggested that the propensity of lineage 2 strains to induce lower levels of protective Th1 cytokines (such as TNF, IL-12 and IFN-γ) may be, in part, responsible for enhanced virulence [16], [17], [18], [19], [20].

Lineage 3 is related to lineage 2 by genome-based phylogeny [7] but is more restricted in geographical location and largely confined to the Indian subcontinent and some regions in East Africa that have experienced a significant Indian migration. This lineage is also common in the United Kingdom amongst Indian populations [21] but is uncommon in Cape Town. Two studies have reported that CAS (central Asian) strains, which belong to lineage 3, grow slowly in liquid broth [19], [22]. Furthermore, a lineage 3 strain (CH), responsible for an outbreak in the United Kingdom, induced less protective IL12p40 and more anti-inflammatory IL-10 than the reference strain H37Rv [22].

Lineage 4 is distributed throughout Europe, America, parts of Africa and the Middle East. This lineage includes Haarlem, Latin American Mediterranean (LAM), X and T families of strains [4], [23]. Strains belonging to the LAM3 family, characterised by the deletion RD761 (RD: region of differentiation) are common in South Africa [2]. The reference strain H37Rv also belongs to lineage 4. H37Rv grows well in the monocyte derived macrophage model [24] and induces similar patterns of cytokines as Harlem and LAM strains in human macrophages [20].

The above experimental evidence suggests that lineages of M. tuberculosis may be characterised by lineage-specific patterns of growth and cytokine induction. The aim of this study was to determine whether strains of M. tuberculosis from the two major circulating lineages in South Africa (lineage 2 and lineage 4) demonstrate such lineage-specific characteristics in vitro models, and whether such patterns are conserved amongst several representatives of these lineages. We also wanted to compare these patterns with those associated with representatives of the lineage 3. We therefore studied in vitro growth rate in liquid broth, intracellular growth rate in monocyte derived macrophages and early pro-inflammatory cytokine induction by strains from each of these different lineages.

Materials and Methods

Ethics statement

The study was conducted with ethical approval from the Faculty of Health Sciences research ethics committee, University of Cape Town (Reference 261/2008).

Selection of M. tuberculosis strains

We selected strains of M. tuberculosis from a previously described collection of isolates from children from the broader Cape Town region of South Africa [6]. Strains from children with tuberculosis are likely to be representative of circulating M. tuberculosis strains in the region, as childhood tuberculosis classically follows exposure to an infectious adult source case. We performed spoligotyping and multiple interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis in order to identify the major strain lineages and strain clusters (Table 1). We selected strains from the major clusters within each of the most common lineages in this strain collection, including three lineage 2 (Beijing) strains (two from the RD181, RD150 sublineage and one from the RD181 sublineage) [25] and three lineage 4 (LAM3/F11) strains. In addition, we selected two lineage 3 (CAS) strains, the previously described CH strain as well as a second lineage 3 strain from the Cape Town paediatric collection. H37Rv was used in all assays as a reference strain.

[Figure omitted. See PDF.]

Table 1. Genotype of selected M. tuberculosis strains.

https://doi.org/10.1371/journal.pone.0043170.t001

Preparation of mycobacterial culture

To ensure a synchronous growth phase amongst all strains, M. tuberculosis was grown at 37°C in a shaking incubator (120 rpm) to mid log phase (0.6 to 0.9 OD) in Middlebrook 7H9 broth (Difco, Detroit, MI, USA) containing 0.2% glycerol, 0.05% Tween 80, and 10% albumin-dextrose-catalase (ADC) growth enrichment (Becton Dickinson, Cockeysville, MD, USA). Multiple vials were stored at −80°C until further use. A new vial of bacilli was thawed before each experimental procedure. The colony forming unit (CFU) concentration of stock vials was calculated by serial dilution and plating in multiple replicates on Middlebrook 7H11 agar (Difco, Detroit, MI, USA) containing 0.5% glycerol and 10% oleic acid-albumin-dextrose-catalase (OADC) growth enrichment (Becton Dickinson, Cockysville, MD, USA).

Axenic growth assays M. tuberculosis growth in liquid 7H9 broth

For growth in axenic media, frozen stock of known CFU concentration were freshly thawed and multiple replicates of 10⁵ CFU/ml of bacilli of M. tuberculosis were inoculated into pre-warmed 7H9 Middlebrook broth supplemented with .2% of glycerol, .05% Tween 80, and 10% ADC growth enrichment and cultured in a shaking incubator (120 rpm) at 37°C for 360 h. To determine growth rate, aliquots were withdrawn at intervals and plated for CFU enumeration by serial dilution and plating in multiple replicates onto 7H11 agar containing 10% OADC.

Monocyte isolation, culture and maturation to monocyte derived macrophages (MDM)

MDM were prepared from healthy donor buffy coats supplied by South African Blood Transfusion Services. Peripheral blood mononuclear cells (PBMC) were separated by centrifugation over Ficol-paque Plus (Pharmacia, Uppsala, Sweden). Cells were washed in RPMI, pooled and counted. Cells were incubated in large tissue culture flask (175 cm²) at 300×10⁶ PBMC per 25 ml RPMI per flask for 2 hours at 37°C in 5% CO₂. This step allows monocytes to adhere to the flask. Non-adherent cells were removed by three washes with 10 ml of pre-warmed RPMI medium (Sigma-Aldrich, Ayrshire, UK). Finally, 10 ml of ice-cold phosphate buffered saline (PBS) was added and the flask incubated at 4°C for 20 minutes. Using a long handled scraper, monocytes were dislodged from the bottom of the flask and pooled in a 50 ml falcon tube for counting. Cells were plated in R10 medium (RPMI containing 10% foetal calf serum (FCS)) at a concentration of 2×10⁵ cells/well in a 96 well tissue culture plate and cultured at 37°C, in 5% CO₂, for 6 days in R10 medium to mature into macrophages.

MDM infection and intracellular growth assay

For the intracellular growth assay, frozen M. tuberculosis stock were freshly thawed and reconstituted at room temperature at the time of infection. Before infection, each stock vial was plated for CFU enumeration to reconfirm the standard concentration of the inoculum. Adherent MDM were co-cultured in duplicate with bacilli at 1∶1 ratio in R10 medium (RPMI+ non heat-inactivated 10% FCS). After 4 h of incubation, extracellular bacteria were removed gently by washing four times with pre-warmed PBS. After 4, 24, 48 or 96 hours infected MDM were subjected to complete lysis by mixing with 100 µl of 0.1% SDS and incubated at room temperature for 12 mins. Lysates were mixed thoroughly for ten times, serially diluted, and plated, in triplicate, on 7H11 agar plates. Plates were incubated for 3–4 weeks at 37°C and CFU enumerated.

Cytokine Analysis

Culture supernatants from control and infected MDM cells were harvested after 48 h and frozen at −80°C. Sterile-filtered culture supernatants were assayed using an enzyme-linked immunosorbent assay (ELISA) kit according to manufacturer's instructions (BD Biosciences Pharmingen, San Diego, CA, USA) to measures levels of tumor necrosis factor (TNF) and IL12p40.

Statistical analysis

Graphpad Prism 5.00 was used for statistical analysis of intracellular growth in monocyte derived macrophages and cytokine assay. The intracellular growth index in MDM infection was compared amongst strains from different groups using the unpaired t test at each time point. Cytokines were analysed using the non-paramatric Mann Whitney test. Axenic growth curve analysis was performed with one-way ANOVA and Bonferroni's post hoc test.

Results

Mycobacterial growth in broth

M. tuberculosis strains belonging to different M. tuberculosis lineages were grown in liquid 7H9 Middlebrook broth to evaluate the growth rate amongst different lineages in axenic media (two independent cultures, each performed in triplicate) (Fig. 1A and 1B). We used C_max and T_max to compare growth of various strains. C_max is defined as the peak point on the bacterial growth curve (maximum number of colony forming units) whilst T_max is the time required to reach C_max. H37Rv grew to a higher C_max than clinical strains (Table 2). Lineage 4 strains showed a higher C_max than clinical strains from other lineages. (Fig. 1C) lineage 2 strains showed an intermediate level of growth whilst lineage 3 strains had the lowest C_max (C_max, H37Rv vs lineage 4/lineage 2/lineage 3 all p<0.0001; lineage 4 vs lineage 2 p = 0.0024; lineage 4 vs lineage 3 p = 0.0005). T_max for H37Rv was 288 h. Lineage 3 strains showed significantly shorter T_max than H37Rv (p<0.05) (Fig. 1D).

[Figure omitted. See PDF.]

Figure 1. Growth of M. tuberculosis strains in liquid 7H9 broth.

Fig. 1 (A) Growth of individual M. tuberculosis strains from different lineages in liquid 7H9 broth. Data represent the mean and range (n = 2) values. Fig. 1 (B) Summarized growth of M. tuberculosis strain lineages. Fig. 1 (C) Comparison of C_max (maximum point on the growth curve) for different strain lineages. Lineage 4 strains showed higher C_max than lineage 2**(p = 0.01) and lineage 3*** strains (p = 0.001). H37Rv showed significantly higher C_max than all clinical strains^# (p = 0.001) Fig. 1 (D) Comparison of T_max (time to reach to C_max) for different strain lineages. H37Rv reached T_max significantly faster than lineage 3 strains* (p = 0.05).

https://doi.org/10.1371/journal.pone.0043170.g001

[Figure omitted. See PDF.]

Table 2. Strain wise comparison of M. tuberculosis growth in axenic media based on Cmax ^# and Tmax ^{# #} values.

https://doi.org/10.1371/journal.pone.0043170.t002

Mycobacterial growth in the monocyte derived macrophage model

We used the MDM infection model to evaluate differences in intracellular growth in different donors (n = 7). We determined initial strain uptake (after 4 h) by expressing the number of CFU at the 4 hour time point from MDM lysates as a percentage of the initial inoculum (Fig. 2). H37Rv showed lower strain uptake by MDM than the clinical strains tested (p<0.001). There were no significant differences in strain uptake between clinical strains.

[Figure omitted. See PDF.]

Figure 2. Strain uptake by monocyte derived macrophages.

Fig. 2 (A) Uptake of M. tuberculosis strains by MDM (4 h CFU calculated as a percentage of initial inoculum). Fig. 2 (B) Summarized uptake of M. tuberculosis strain lineages by MDM. H37Rv showed significantly lower uptake by the than clinical strains^# (p<0.001). Data represent the mean ± SD (n = 7).

https://doi.org/10.1371/journal.pone.0043170.g002

To compare intracellular growth amongst different strain lineages, we determined a growth index, calculated from the log₁₀ of number of CFU at each time point divided by the log₁₀ of number of CFU at the 4 h time point. Growth of all clinical strains was slow for the first 24 hours. Lineage 2 and lineage 4 strains grew faster over the first 48 hours than lineage 3 strains (lineage 3 vs. lineage 4 : p<0.01, lineage 3 vs. lineage 4: p<0.001). lineage 2 strains grew significantly faster than lineage 3 strains (p<0.01) over 96 hours (the mean growth index of lineage 4, lineage 2, lineage 3 and H37Rv was 1.19±.22, 1.25±.17, 1.11±.10 and 1.69±.13 respectively over 96 hours) (Fig. 3). Despite low initial uptake, H37Rv multiplied more rapidly in MDM than clinical strains (H37Rv vs. lineage 2 and lineage 4: p<0.001 at 24 h and 96 h, H37Rv vs. lineage 3 : p<0.001 at all time points). As an alternative approach to compare the growth rate amongst these strains, we have also determined the doubling time of these strains (Doubling time was calculated based on the mean numbers of CFU at 4 hours and 96 hour) (Table 3). The mean doubling time of lineage 2 strains was 23.34±3.0 h, lineage 4 strains 25.10±4.67 h, lineage 3 strains 26.14±2.45 h and H37Rv 17.12±1.61 h (mean ± SD) over the 96 hour time period.

[Figure omitted. See PDF.]

Figure 3. Growth index reflecting the intracellular growth of M. tuberculosis strains in MDM.

Fig. (3A) Growth index of M. tuberculosis strains (calculated by the CFU at each time point divided by the CFU at initial time point). Fig. (3B) Summarized growth index of M. tuberculosis strain lineages. Data represent the mean ± SD (n = 7). Statistical significance is denoted as *p<0.05, **p<0.01, ***p<0.001.

https://doi.org/10.1371/journal.pone.0043170.g003

[Figure omitted. See PDF.]

Table 3. Doubling time⁺ for M. tuberculosis strains from different lineages in human monocyte derived macrophages.

https://doi.org/10.1371/journal.pone.0043170.t003

Cytokine induction

We investigated the release of TNF (n = 8 donors) and IL-12p40 (n = 6 donors) from monocyte-derived macrophages (MDM) during 48 hours of infection with M. tuberculosis strains of various lineages at 48 hours (Fig. 4). Lineage 2 and lineage 3 strains induced lower levels of IL-12p40 when compared to H37Rv (H37Rv: 385±39.89 pg/ml, lineage 2: 91±17.85 89 pg/ml, lineage 3: 123.8±10.1589 pg/ml, median ± S.E, p<0.001) and lineage 4 strains (lineage 4: 205±14.84 pg/ml, median ± S.E, p<0.001) (Fig. 4A). Further, the release of TNF by MDM infected with lineage 2 strains was significantly reduced when compared to H37Rv (H37Rv: 1253±83.52 pg/ml, lineage 2: 362±27.58 pg/ml, median ± S.E, p<0.01) and other lineages (lineage 3: 1818±123 pg/ml, lineage 4: 1207±112 pg/ml, median ± S.E, p<0.001). In contrast to IL-12p40, higher levels of TNF were induced by lineage 3 strains when compared to H37Rv (p<0.01) and lineage 4 strains (p<0.05). Induction of both TNF and IL-12p40 by lineage 4 strains was similar to that induced by H37Rv.

[Figure omitted. See PDF.]

Figure 4. Cytokines detected in culture-supernatant (at 48 h) from MDM infected by different strains of M. tuberculosis.

Fig. 4 (A) IL-12p40 (n = 6 donors). H37Rv and lineage 4 strains induced significantly higher level of IL-12p40 as compared to lineage 3^# and lineage 2^# lineages (p<0.001 for both comparisons) Fig. 4 (B) TNF (n = 8 donors). Lineage 3 strains induced significantly higher levels of TNF as compared to lineage 2^# (p = 0.001), lineage 4* (p<0.05) and H37Rv** (p = 0.01). H37Rv and lineage 4 strains induced higher level of TNF than lineage 2 strain (lineage 4 vs lineage 2 p = 0.001, H37Rv vs lineage 2 p = 0.01). Data represent the median and range.

https://doi.org/10.1371/journal.pone.0043170.g004

Discussion

In this study, we compared lineage-specific in vitro phenotypes of the two prevalent strain lineages from the Western Cape region of South Africa with those of the laboratory strain H37Rv as well as strains from lineage 3. We assessed two virulence-associated characteristics in our study: mycobacterial growth (in liquid broth and MDM) and proinflammatory cytokine induction in MDM. We clearly demonstrated lineage-specific patterns of growth and cytokine induction.

The laboratory-reference strain H37Rv grew most rapidly in liquid culture, suggesting that H37Rv is highly adapted to laboratory media. Interestingly, there were clear lineage-specific differences in growth patterns in axenic media, with lineage 4 strains reaching a higher C_max than the other two lineages and lineage 3 strains peaking early at a lower C_max. Lineage 2 strains had an intermediate phenotype. Our results are in line with previous findings which showed slow growth of lineage 3 (CAS strain) in 7H9 broth [19], [22].

Several previous studies have assessed intracellular growth of M. tuberculosis in human macrophages as a marker of virulence [13], [15], [26], [27]. Individual strains from lineage 2 (Beijing) have been shown to grow more rapidly than comparator strains in in vitro human cell culture models using MDM or monocyte or human macophage cell lines [13], [14], [15]. Zhang et al utilized the monocyte derived macrophage model to study the correlation between the extent of the spread of M. tuberculosis strains in a Los Angeles community setting and the ability of the strains to grow in human macrophages. Multiple isolates of M. tuberculosis strain 210 (a Beijing-family strain [lineage 2] responsible for an outbreak in Los Angeles), grew more rapidly than small cluster or unique cluster strains in human MDM [14]. In contrast Shon et al found no significant difference in growth of a hypervirulent Beijing strain (K-strain) [lineage 2] from Korea compared with H37Rv in murine bone marrow derived macrophages [18].

We found that lineage 2 and lineage 4 strains grew more rapidly than lineage 3 strains during the first 48 hours but that growth declined over the next 48 h. In contrast, H37Rv continued to grow more rapidly than all clinical strains. Initial uptake by MDM was lowest for H37Rv, but similar for all clinical strains. There is evidence of variation in phagocytosis by different strains [28]. For example Schlesinger et al found that phagocytosis of two virulent strains (H37Rv and Erdman) by human monocyte derived macrophages is mediated by the mannose receptor in addition to complement receptor (CR1, CR3 and CR4) whereas the avirulent strain H37Ra uses only complement receptor for phagocytosis [29]. It is unclear whether the differential uptake which we demonstrated is due to differential expression of such pathogen-associated molecular patterns.

There are a number of limitations of this in vitro model. Short term growth in macrophages only partially reflects the more lengthy and complex pathogenesis in humans, we were unable to distinguish bacterial uptake from simple adherence to macrophages and PBMC-derived macrophages differ phenotypically from alveolar macrophages [30]. Rapid growth in macrophages may not necessarily represent a virulence characteristic [18]. Furthermore, due to the limited number of MDM available, we were only able to compare a few strains from each lineage. All of these strains (except CH) were isolated from South African patients, and only represent specific sub-lineages. They do not therefore represent the global diversity of ‘modern’ TB strains.

Early proinflamatory cytokine secretion is a hallmark of M. tuberculosis infection. IL-12 production is essential to induce a protective Th1 response [31]. In humans, mutations in the IL-12p40 and the IL-12 receptor genes result in enhanced susceptibility to mycobacterial infection [32], [33]. TNF is produced by monocytes and macrophages in early infection and plays a key role in protective immunity during tuberculosis infection. In humans, neutralization of TNF using anti-TNF drugs increases the risk of reactivation of latent tuberculosis [34], [35], [36]. Hirsch et al. showed that TNF inhibits the growth of M. tuberculosis within alveolar macrophages [37].

Several studies have reported that heterogeneity exists in the cytokine response induced by various genotype of M. tuberculosis [20], [22]. Modern lineages (lineage 2, lineage 3 and lineage 4) were shown to induce lower levels of proinflamatory cytokines when compared with ancient lineages (lineage 1, lineage 5 and lineage 6) [38]. Lineage 4, a modern M. tuberculosis lineage, consists of a diverse group of M. tuberculosis strains. Haarlem and LAM strains belonging to this lineage induced similar level of TNF, IL-6, IL-10 and GRO-α as H37Rv in human macrophages [20]. Another outbreak strain, CDC1551, which is a representative of lineage 4 (belonging to the X family) induces high levels of pro-inflammatory cytokines in monocyte culture [39], [40]. We studied only modern lineages of M. tuberculosis and were able to demonstrate distinct patterns of cytokine induction by representatives of the three major modern strain lineages. Lineage 4 (LAM3) strains induced similar levels of cytokine to the reference strain H37Rv. Lineage 3 strains have been reported to either induce similar levels of proinflammatory cytokines to lineage 2 strains (low levels) or to induce similar levels to H37Rv (higher levels) [19], [20], [22]. Our findings are in line with those of Newton and colleagues [22] who described high TNF and low IL-12p40 secretion by MDM infected by a lineage 3 strain.

A recent study has demonstrated that ancient Beijing strains (lineage 2) from Brazil (characterized by the absence of an insertion sequence 6110 within the noise transfer function region) induced similar levels of TNF and IL-10 to H37Rv whilst modern MDR Beijing strains (lineage 2) isolated in Russia, induced low TNF and high IL-10 in the THP1 macrophage cell line [41]. In contrast, another study has found that Beijing strains (lineage 2), irrespective of subfamily, showed an immune phenotype of low level of TNF, IL-6, IL-10 and GRO-α [20] production as compared to H37Rv and other genotypes of M. tuberculosis in human macrophages. Low proinflamatory cytokine induction by lineage 2 (Beijing strains) has been repeatedly reported in various human cell culture models including human monocyte-derived macrophages and the THP1 cell line [14], [17], [18], [19], [20], [41]. Our results, with selected modern Beijing strains from South Africa are consistent with other studies showing low levels of proinflamatory cytokine secretion by infected human MDM.

In summary, we have demonstrated that different modern M. tuberculosis lineages exhibit lineage-specific patterns of IL12p40 and TNF induction, with lineage 4 strains inducing high levels of both cytokines, lineage 2 strains low levels of both cytokines and lineage 3 strains high levels of TNF but low levels of IL12p40. This study suggests that modern strain lineages of M. tuberculosis may possess lineage-specific patterns of growth and cytokine induction. These in vitro characteristics may reflect different strategies that M. tuberculosis employs to exploit ecological niches in different human populations.

Acknowledgments

We thank Rachael Jacobson for her assistance with strain genotyping and Kathryn Wood for assistance with ELISA assays.

Author Contributions

Conceived and designed the experiments: MN RW RS. Performed the experiments: RS LL KW. Analyzed the data: RS MN RW. Contributed reagents/materials/analysis tools: MN RW KW. Wrote the paper: RS MN RW.

Citation: Sarkar R, Lenders L, Wilkinson KA, Wilkinson RJ, Nicol MP (2012) Modern Lineages of Mycobacterium tuberculosis Exhibit Lineage-Specific Patterns of Growth and Cytokine Induction in Human Monocyte-Derived Macrophages. PLoS ONE 7(8): e43170. https://doi.org/10.1371/journal.pone.0043170

References

1. WHO Tuberculosis global Factsheet (2010) World Health Organization. Available: http://www.who.int/mediacentre/factsheets/fs104/en/. Accessed 2012 May 15.

2. Gagneux S, Small PM (2007) Global phylogeography of Mycobacterium tuberculosis and implications for tuberculosis product development. The Lancet infectious diseases 7: 328–337.

3. Nicol MP, Wilkinson RJ (2008) The clinical consequences of strain diversity in Mycobacterium tuberculosis. Transactions of the Royal Society of Tropical Medicine and Hygiene 102: 955–965.

4. Gagneux S, DeRiemer K, Van T, Kato-Maeda M, de Jong BC, et al. (2006) Variable host-pathogen compatibility in Mycobacterium tuberculosis. Proceedings of the National Academy of Sciences of the United States of America 103: 2869–2873.

5. Marais BJ, Victor TC, Hesseling AC, Barnard M, Jordaan A, et al. (2006) Beijing and Haarlem genotypes are overrepresented among children with drug-resistant tuberculosis in the Western cape province of South africa. Journal of Clinical Microbiology 44: 3539–3543.

6. Nicol MP, Sola C, February B, Rastogi N, Steyn L, et al. (2005) Distribution of strain families of Mycobacterium tuberculosis causing pulmonary and extrapulmonary disease in hospitalized children in Cape town, South africa. Journal of Clinical Microbiology 43: 5779–5781.

7. Comas I, Chakravartti J, Small PM, Galagan J, Niemann S, et al. (2010) Human T cell epitopes of Mycobacterium tuberculosis are evolutionarily hyperconserved. Nature Genetics 42: 498–503.

8. Bifani PJ, Mathema B, Kurepina NE, Kreiswirth BN (2002) Global dissemination of the Mycobacterium tuberculosis W-Beijing family strains. Trends in Microbiology 10: 45–52.

9. Glynn JR, Whiteley J, Bifani PJ, Kremer K, van Soolingen D (2002) Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis : a systematic review. Emerging infectious diseases 8: 843–849.

10. Cowley D, Govender D, February B, Wolfe M, Steyn L, et al. (2008) Recent and rapid emergence of W-Beijing strains of Mycobacterium tuberculosis in Cape town, South africa. Clinical Infectious Diseases 47: 1252–1259.

11. Van der Spuy GD, Kremer K, Ndabambi SL, Beyers N, Dunbar R, et al. (2009) Changing Mycobacterium tuberculosis population highlights clade-specific pathogenic characteristics. Tuberculosis 89: 120–125.

12. Lopez B, Aguilar D, Orozco H, Burger M, Espitia C, et al. (2003) A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clinical & Experimental Immunology 133: 30–37.

13. Li Q, Whalen CC, Albert JM, Larkin R, Zukowski L, et al. (2002) Differences in rate and variability of intracellular growth of a panel of Mycobacterium tuberculosis clinical isolates within a human monocyte model. Infection and Immunity 70: 6489–6493.

14. Theus SA, Cave MD, Eisenach KD (2005) Intracellular macrophage growth rates and cytokine profiles of Mycobacterium tuberculosis strains with different transmission dynamics. Journal of Infectious Diseases 191: 453–460.

15. Zhang M, Gong J, Yang Z, Samten B, Cave MD, et al. (1999) Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. Journal of Infectious Diseases 179: 1213–1217.

16. Rakotosamimanana N, Raharimanga V, Andriamandimby SF, Soares J-L, Doherty TM, et al. (2010) Variation in Gamma Interferon responses to different infecting strains of Mycobacterium tuberculosis in acid-fast bacillus smear-positive patients and household contacts in Antananarivo, Madagascar. Clinical Vaccine Immunology 17: 1094–1103.

17. Reed MB, Domenech P, Manca C, Su H, Barczak AK, et al. (2004) A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response. Nature 431: 84–87.

18. Sohn H, Lee KS, Kim SY, Shin DM, Shin SJ, et al. (2009) Induction of cell death in human macrophages by a highly virulent Korean isolate of Mycobacterium tuberculosis and the virulent strain H37Rv. Scandinavian journal of immunology 69: 43–50.

19. Tanveer M, Hasan Z, Kanji A, Hussain R, Hasan R (2009) Reduced TNF-alpha and IFN-gamma responses to Central Asian strain 1 and Beijing isolates of Mycobacterium tuberculosis in comparison with H37Rv strain. Transactions of the Royal Society of Tropical Medicine and Hygiene 103: 581–587.

20. Wang C, Peyron P, Mestre O, Kaplan G, van Soolingen D, et al. (2010) Innate immune response to Mycobacterium tuberculosis Beijing and other genotypes. PloS One 5: e13594.

21. Gascoyne-Binzi DM, Barlow REL, Essex A, Gelletlie R, Khan MA, et al. (2002) Predominant VNTR family of strains of Mycobacterium tuberculosis isolated from South Asian patients. The International Journal of Tuberculosis and Lung Disease 6: 492–496.

22. Newton SM, Smith RJ, Wilkinson KA, Nicol MP, Garton NJ, et al. (2006) A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion. Proceedings of the National Academy of Sciences 103: 15594–15598.

23. Brudey K, Driscoll JR, Rigouts L, Prodinger WM, Gori A, et al. (2006) Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. Bio Med Central Microbiology 6: 23.

24. Zhang M, Gong J, Lin Y, Barnes PF (1998) Growth of virulent and avirulent Mycobacterium tuberculosis strains in human macrophages. Infection and immunity 66: 794–799.

25. Tsolaki AG, Gagneux S, Pym AS, Goguet de la Salmoniere Y-OL, Kreiswirth BN, et al. (2005) Genomic Deletions Classify the Beijing/W Strains as a Distinct Genetic Lineage of Mycobacterium tuberculosis. Journal of clinical microbiology 43: 3185–3191.

26. Hoal-van Helden EG, Hon D, Lewis LA, Beyers N, van Helden PD (2001) Mycobacterial growth in human macrophages: variation according to donor, inoculum and bacterial strain. Cell Biology International 25: 71–81.

27. Wong KC, Leong WM, Law HKW, Ip KF, Lam JTH, et al. (2007) Molecular characterization of clinical isolates of Mycobacterium tuberculosis and their association with phenotypic virulence in human macrophages. Clinical Vaccine Immunology 14: 1279–1284.

28. Torrelles JB, Knaup R, Kolareth A, Slepushkina T, Kaufman TM, et al. (2008) Identification of Mycobacterium tuberculosis clinical isolates with altered phagocytosis by human macrophages due to a truncated lipoarabinomannan. Journal of Biological Chemistry 283: 31417–31428.

29. Schlesinger L (1993) Macrophage phagocytosis of virulent but not attenuated strains of Mycobacterium tuberculosis is mediated by mannose receptors in addition to complement receptors. The Journal of Immunology 150: 2920–2930.

30. Gordon S, Taylor PR (2005) Monocyte and macrophage heterogeneity. Nature Reviews Immunology 5: 953–964.

31. Cooper AM, Solache A, Khader SA (2007) Interleukin-12 and tuberculosis: an old story revisited. Current opinion in immunology 19: 441–447.

32. Altare F, Durandy A, Lammas D, Emile JF, Lamhamedi S, et al. (1998) Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency. Science 280: 1432–1435.

33. Altare F, Ensser A, Breiman A, Reichenbach J, Baghdadi JE, et al. (2001) Interleukin-12 receptor beta1 deficiency in a patient with abdominal tuberculosis. The Journal of Infectious Diseases The Journal of infectious diseases 184: 231–236.

34. Keane J, Gershon S, Wise RP, Mirabile-Levens E, Kasznica J, et al. (2001) Tuberculosis associated with Infliximab, A factor α - neutralizing agent. New England Journal of Medicine 345: 1098–1104.

35. Mankia S, Peters J, Kang S, Moore S, Ehrenstein M (2011) Tuberculosis and anti-TNF treatment: experience of a central London hospital. Clinical Rheumatology 30: 399–401.

36. Solovic I, Sester M, Gomez-Reino JJ, Rieder HL, Ehlers S, et al. (2010) The risk of tuberculosis related to tumour necrosis factor antagonist therapies: a TBNET consensus statement. European Respiratory Journal 36: 1185–1206.

37. Hirsch CS, Yoneda T, Averill L, Ellner JJ, Toossi Z (1994) Enhancement of intracellular growth of Mycobacterium tuberculosis in human monocytes by Transforming growth factor-β. Journal of Infectious Diseases 170: 1229–1237.

38. Portevin D, Gagneux S, Comas I, Young D (2011) Human macrophage responses to clinical isolates from the Mycobacterium tuberculosis complex discriminate between ancient and modern lineages. PLoS Pathogens 7: e1001307.

39. Manca C, Reed MB, Freeman S, Mathema B, Kreiswirth B, et al. (2004) Differential monocyte activation underlies strain-specific Mycobacterium tuberculosis pathogenesis. Infection and Immunity 2004/08/24 ed 5511–5514.

40. Manca C, Tsenova L, Barry CE III, Bergtold A, Freeman S, et al. (1999) Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates. Journal of immunology 162: 6740–6746.

41. Lasunskaia E, Ribeiro SCM, Manicheva O, Gomes LL, Suffys PN, et al. (2010) Emerging multidrug resistant Mycobacterium tuberculosis strains of the Beijing genotype circulating in Russia express a pattern of biological properties associated with enhanced virulence. Microbes and Infection 12: 467–475.