Tuberculosis 94 (2014) 207e218

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IMMUNOLOGICAL ASPECTS

Differentiation of human mononuclear phagocytes increases their innate response to Mycobacterium tuberculosis infection Diana Castaño a, c, Luis F. García a, c, Mauricio Rojas a, b, c, * a Grupo de Inmunología Celular e Inmunogenética, Instituto de Investigaciones Médicas, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia b Unidad de Citometría de Flujo, Sede de Investigación Universitaria, Universidad de Antioquia UdeA, Calle 70 No. 52-21, Medellín, Colombia c Centro Colombiano de Investigación en Tuberculosis, Medellín, Colombia

a r t i c l e i n f o

s u m m a r y

Article history: Received 15 July 2013 Received in revised form 20 December 2013 Accepted 8 January 2014

The heterogeneity of mononuclear phagocytes, partially explained by cell differentiation, influences the activation of innate responses. It has been reported that Mycobacterium tuberculosis inhibits monocyte differentiation into either dendritic cells or macrophages. To evaluate whether the activation of effector mechanisms against M. tuberculosis differ between less and more differentiated mononuclear phagocytes, we compared monocytes differentiated in vitro for 24 h (MON24) and 120 h (MDM120) infected with M. tuberculosis H37Rv, H37Ra and the clinical isolate UT127 at different multiplicity of infection. MDM120 phagocytosed more M. tuberculosis, inhibited mycobacterial growth and did not die in response to the infection, compared with MON24. In contrast, MON24 become Annexin V and Propidium iodide positive after 36 h of M. tuberculosis infection. Although, there were striking differences between MON24 and MDM120, there were also some differences in the response to the mycobacterial strains used. Finally, in MDM120 infected with M. tuberculosis H37Rv, a lower percentage of mycobacterial phagosomes accumulated transferrin and a higher percentage co-localized with cathelicidin than in MON24. These results demonstrate that innate responses induced by M. tuberculosis depends upon the stage of differentiation of mononuclear phagocytes and support that terminally differentiated cells are more efficient anti-mycobacterial effectors than the less differentiated ones. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Mycobacterium tuberculosis Monocyte Macrophage Mononuclear phagocyte Differentiation Phagocyte heterogeneity

1. Introduction Tuberculosis (TB) is still one of the main causes of morbidity and mortality worldwide, due to a single infectious agent [1,2]. Mycobacterium tuberculosis infects and survives inside macrophages altering different cellular processes; such as inhibiting phagosomeelysosome fusion [3,4], allowing bacilli to remain in compartments with characteristics of early and recycling endosomes, avoiding the activation of bactericidal mechanisms [5,6] and favoring the access to the nutrients and oligoelements needed for its metabolism [7,8]. M. tuberculosis is also capable to eventually induces the death of its host cell [9].

* Corresponding author. Sede de Investigación Universitaria, Universidad de Antioquia, Carrera 53 No. 61-30, Laboratorio 420, Medellín, Colombia. Tel./fax: þ57 4 219 6463. E-mail addresses: [email protected] (D. Castaño), [email protected] (L. F. García), [email protected] (M. Rojas). 1472-9792/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tube.2014.01.001

Besides being the principal reservoirs of M. tuberculosis, macrophages are also considered the first line of defense against this pathogen, as they are the cells responsible for containing mycobacterial replication [10,11]. The ability of mononuclear phagocytes (monocytes and macrophages) to activate innate mechanisms against M. tuberculosis depends on the interaction of several factors, such as the genetic background of the host, bacterial virulence and inoculum size [11e13]. Also, there is evidence that the stage of differentiation of the mononuclear phagocytes may affect the activation of an efficient innate response [14e16]. The differentiation of monocytes into macrophages is characterized by changes in the activation of transcription factors [17] and gene expression [18], that results in changes in the expression of adhesion molecules [19], membrane receptors [20], cytokine production and morphology [21,22]. These phenotypic changes occurring during differentiation should be reflected in the effector responses of the mononuclear phagocytes against pathogens. Our group has previously reported that monocytes differentiated in the presence of M. tuberculosis have alterations in their differentiation process [23]. However, it still remains unclear how the stage of

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differentiation of mononuclear phagocytes influences their effector capacities against M. tuberculosis and how it may affect the course of infection. At the early stages of M. tuberculosis infection, resident alveolar macrophages are considered the main effector cells against mycobacteria. In the murine model it has been demonstrated that, besides macrophages, there is a reservoir of immature monocytes in the lung parenchyma [26]; these cells, like the circulating monocytes [24], can be recruited to the site of infection and become infected with M. tuberculosis before differentiate into macrophages. It has been reported that in murine pulmonary TB, monocytes (CD11bþ/mid/CD11c-) and undifferentiated macrophages (with low levels of F4/80, CD86, MHC class II and MAC-3 expression) are present in the lungs until 21 days post-infection [24,25]. Thus, it is possible that some monocytes enter into the granulomatous lesions at later time points of the infection, and that some of them may have not completed their differentiation. In the zebra fish model, the circulating monocytes that enter to the site of infection rapidly become part of the granuloma [26,27]. In humans, macrophages obtained from bronchoalveolar lavages of TB patients exhibit characteristics of immature cells [28]. In addition, TB patients have increased numbers of circulating CD14þCD16þþ monocytes [29] with characteristics of cells at a lower stage of differentiation. Several studies have contradictory results regarding the capacity of human mononuclear phagocytes to contain mycobacterial replication. Whereas some authors reported that less differentiated mononuclear phagocytes (2 h of culture in vitro) are more permissive for M. tuberculosis growth than those with 3 or 7 days of in vitro differentiation [30]; others have found comparable mycobacterial growth in phagocytes of 2 and 7 days of differentiation [31]. Our group reported a higher replication of M. tuberculosis in the U937 promonocytes than in the macrophages derived from this cell line [15], suggesting that the stage of differentiation of the mononuclear phagocytes plays a critical role in the control of the infection by M. tuberculosis. Vogt and Nathan have reported differences in the antimycobacterial activity of monocyte-derived macrophages (MDM) with a variety of approaches to the in vitro differentiation, such as the source or serum, percentage of oxygen and presence of cytokines and colony stimulating factors (GM-CSF, TNF-a, IFN-g). Actually it was observed a higher mycobacterial replication in undifferentiated (0 and 3 days) than differentiated cells (7, 14, 21 and 28 days) [32]. The present study aimed to determine whether phagocyte responses against M. tuberculosis vary with the host cells stage of differentiation. For this purpose, we compared in vitro the activation of innate mechanisms and the anti-M. tuberculosis effector capabilities of human monocytes differentiated for 24 h (MON24) and 120 h (MDM120). The results show that MDM120 have a higher expression of phagocytic receptors CD11b, CD11c, CD16, CD18, CD64, CD44 and CD206; MDM120 bind and internalized more latex beads and M. tuberculosis H37Rv, H37Ra and the clinical isolate UT127, and more effectively control replication of M. tuberculosis than MON24. Control of M. tuberculosis replication by MDM120 was neither associated with the level of cytokines (IL-12p70, TNF-a, IL-1b, IL-10 and IL-8), H2O2 production, nor with the percentage of acid phagosomes containing M. tuberculosis. However, it was associated with a lower percentage of mycobacterial phagosomes that accumulated transferrin and a higher percentage that co-localized with cathelicidin, as well as a less proportion of cell death. These results demonstrate that differentiated human mononuclear phagocytes have more efficient anti-mycobacterial machinery than the less differentiated cells.

2. Materials and methods 2.1. Culture of mononuclear phagocytes and differentiation Peripheral blood mononuclear cells (PBMC) were isolated from defibrinated blood from healthy individuals by centrifugation on Histopaque-1077 (Sigma Aldrich, St. Louis, MO). PBMCs containing 2.5  105 CD14þ cells/ml in RPMI-1640 (Gibco-BRL, Gran Island, NY) supplemented with 0.5% heat inactivated pooled human serum (PHS), were enriched by adherence to plastic plates (Corning Incorporated Life Science, Lowell, MA). Wells were extensively washed to remove non-adherent cells. Adherent cells were cultured in RPMI-1640 supplemented with 10% heat inactivated PHS for 24 h (MON24) and 120 h to allow differentiation into monocyte-derived macrophages (MDM120). At baseline differentiation, more than 90% of the cells were CD14þ (clone RMO52, Immunotech, Beckman Coulter, Miami, FL). Adherent cells numbers were determined after scraping or lysing to count cells and nuclei, respectively, as described [33]. There were not significant changes in the number of adherent cells during 120 h of culture. 2.2. Expression of phagocytic receptors MON24 and MDM120 monolayers were washed with PBS plus 1% BSA (bovine serum albumin, SigmaeAldrich) and 0.1% NaN3 (SigmaeAldrich), and blocked with PBS plus 2% PHS. Cells were independently stained with anti-CD11b (-PE, clone VIM12, Invitrogen), anti-CD11c (-PE, clone B-ly6, BD-Pharmingen), anti-CD16 (-PE, clone 3G8, BD-Pharmingen), anti-CD18 (-FITC, clone L130, BDPharmingen), anti-CD44 (-FITC, clone L178, BD-Pharmingen), antiCD64 (-PE, clone 10.1, BD-Pharmingen), and anti-CD206 (-RPE, clone 19.2, BD-Pharmingen) or their respective isotype controls for 30 min. Cells were then washed, fixed with 2% PFA for 20 min and scraped with a rubber policeman. Ten thousand cells were acquired in a BD FACS Canto II (Becton Dickinson Biosciences. San Diego, CA). The percentage of stained cells and the mean fluorescence intensity (MFI) were estimated using FlowJo 7.6.1 software (Tree Star, Inc. Ashland, OR). 2.3. Culture of M. tuberculosis and labeling with FDA and CFSE M. tuberculosis H37Rv and H37Ra, obtained from the Instituto Nacional de Salud (Bogotá, Colombia), and UT127 (LAM family), a drug-sensitive clinical isolate from a Colombian HIV negative TB patient (characterized and followed in a 2e3 years cohort study) [34], were grown in Middlebrook 7H9 liquid media (Becton Dickinson, Cockeysville, MD) supplemented with OADC (Oleic Acid Albumin Dextrose Catalase complex, Becton Dickinson). Mycobacteria were harvested after 3 weeks, extensibility washed with PBS (phosphate-buffered saline, Gibco-BRL) and labeled or not with 250 ng/ml FDA (fluorescein diacetate, Invitrogen, Eugene, OR) or 200 mM CFSE (5-(and-6)-carboxyfluoresceinsuccinimidyl ester, Invitrogen) for 60 min at 37  C. The CFSE-labeled mycobacteria were killed with 1.4% PFA (paraformaldehyde, Fisher Scientific, Pittsburgh, PA) for 18 h at room temperature. Labeled and nonlabeled mycobacterias were slightly sonicated to disrupt clumps at 204,8 Watts (Sonics Vibra Cell, model CV33. Newtown, CT). Mycobacterial suspension was centrifuged for 5 min at 200  g and aliquots from the supernatants were frozen at 70  C in a solution containing 20% glycerol. The mycobacterial concentration was calculated by spectrophotometry at 600 nm and verified by counting the number of colony-forming units (CFUs) [11]. The percentage and intensity of FDA and CFSE staining were measured by flow cytometry (FACSort flow cytometer, Becton Dickinson). For

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some experiments, M. tuberculosis H37Rv was opsonized with noninactivated pooled human serum [35].

2.8. Phagosome acidification, transferrin incorporation and indirect immunofluorescence

2.4. In vitro infection with M. tuberculosis

MON24 and MDM120 were detached from culture plates with 0.05% trypsin-EDTA (ethylenediaminetetracetic acid, Gibco-BRL) and plated onto 12 mm of diameter sterile glass cover slides (VWR, West Chester, PA). Adherent cells were infected with live FDA-labeled M. tuberculosis or dead CFSE-labeled M. tuberculosis for 2 h. Extracellular bacteria were washed out; then, cells were treated with 1 mM LysoTracker for 4 h at 37  C, 5% CO2 [36]. Mononuclear phagocytes were washed, fixed with 4% PFA and preserved on glass slides with FluorSave (Calbiochem-Merck). The expression of Rab5, Rab7, CD63, CaMKII (Calcium/Calmodulin-depend protein kinase II) and cathelicidin by MON24 and MDM120 was determined by indirect immunofluorescence. Cells were infected with FDA- and CFSE-labeled M. tuberculosis H37Rv for different times. Thereafter, the cells were washed with PBS, blocked with 2% inactivated PHS and fixed with 4% PFA. Permeabilization was done with 0.1% Triton X-100 and primary antibodies to EEA-1 (early endosome antigen 1) Rab5, CaMKII, Rab7 (Cell Signaling Technology, Danvers, MA), CD63 (Abcam Inc, Cambridge, MA) and cathelicidin (Santa Cruz Biotechnology, Dallas, TX) were added at 1:50 for 1 h. Then, cells were washed and incubated with the respective secondary F(ab)2 anti-rabbit or anti-mouse antibodies labeled with Alexa Fluor 594 (Invitrogen) for 1 h. Cells were washed and preserved as explained above. In some experiments, cells were treated or not with LPS as a positive control for cathelicidin expression. For transferrin incorporation, cells infected with FDA- or CFSElabeled M. tuberculosis for 12 h were incubated in RPMI-1640 without serum for 3 h and treated with 20 mg/ml Alexa Fluor 594-transferrin (Invitrogen) for 1 h. Phagocytes were washed and incubated for 1 h more in RPMI-1640 plus 10% PHS without transferrin; cells were fixed and preserved as explained above. All cells in this set of experiments were stained with Hoescht 33258 for 15 min and were analyzed by epifluorescence and confocal microscopy.

MON24 and MDM120 were infected with FDA- or CFSE-labeled M. tuberculosis H37Rv or not labeled M. tuberculosis H37Rv, H37Ra and UT127 for different time periods at multiplicity of infection (MOI) according to the experimental conditions (for details see the figure legends). Plates were centrifuged for 5 min at 900  g and incubated at 37  C, 5% CO2. After 2 h of infection, monolayers were washed to remove the extracellular non-attached bacteria. At this time, phagocytosis was evaluated or the cells were cultured in RPMI-1640 plus 10% PHS for different times to determine the mycobacterial replication, the cytokines concentrations in culture supernatants and the phagosomal maturation. For evaluation of mononuclear phagocytes death, the bacilli were removed after 12 h of infection. 2.5. Phagocytosis assay MON24 and MDM120 were mixed with fluorescent latex beads (2 mm carboxylate-modified microspheres yellow-green fluorescent, FluoSpheres, Invitrogen) or infected with M. tuberculosis as described at a MOI of 5:1 [29]. Briefly, after 1 h of incubation with beads, cells were extensively washed, fixed with 2% PFA, scraped and immediately acquired in a FACSort flow cytometer (Becton Dickinson). The percentage of cells and the mean fluorescence intensity were estimated using the Cell Quest software (Version 3.3. Becton Dickinson). For bacteria, cells were incubated for 2 h, extensively washed, lysed and serial dilution of each lysate were plated on Middlebrook 7H10 (Becton Dickinson) supplemented with OADC at 37  C. The CFUs were counted at 3 weeks. 2.6. Measurement of M. tuberculosis replication MON24 and MDM120 were infected with non-labeled M. tuberculosis H37Rv, H37Ra and UT127 for 2, 72, 96 and 120 h. At these time periods, the culture plates were centrifuged at 1000  g for 5 min and the supernatants were removed and stored at 70  C. The cells were lysed with 0.1% Triton X-100 (Sigma Aldrich) to release the intracellular mycobacteria. Three serial dilutions of each lysate were plated on Middlebrook 7H10 (Becton Dickinson) supplemented with OADC at 37  C. The CFUs were counted at 3 weeks; the results of 72, 96 and 120 h of infection were normalized and presented as the replication index (RI). RI ¼ number of CFUs at 72, 96 or 120 h of infection/number of CFUs at 2 h of phagocytosis. These RI was used due to the differences observed in the amount of M. tuberculosis bound/internalized between MON24 and MDM120 at 2 h of phagocytosis (0 h of infection). The CFUs at 2 h were considered as the unit of replication. 2.7. Measurement of cytokine production The accumulation of IL-12p70, TNF-a, IL-10, IL-1b and IL-8 was determined in the supernatants of cultures infected with nonlabeled M. tuberculosis H37Rv, H37Ra and UT127 for 2, 72 and 96 h by Cytometric Bead Array, according to manufacturer instructions (Human Inflammatory Cytokine Kit, BD-Pharmingen) by flow cytometry. IL-18 production was determined in supernatants of M. tuberculosis H37Rv infected cultures by ELISA (Medicals & Biological Laboratories Ltd., Naka-ku Nagoya, Japan), according to manufacturer instructions.

2.9. Epifluorescence and confocal microscopy Sequential images from the same focal plane were collected in an inverted epifluorescence and confocal microscopy (IX70 Olympus), using the softwares Cell^M (Olympus, Tokyo, Japan) and Fluoview FV1000 version 3.0 (Olympus). Images were collected with 60 magnification and 1.45 of numerical aperture objective. The colocalization was determined using the “Unbiased Counting” method [37]. The colocalization of M. tuberculosis and the endosomal markers was evaluated by fluorescence distribution and intensity, using the Pearson correlation coefficients between green and red fluorescence. Additionally, the colocalization was corroborated by deconvolution using the software Image Scope Pro (Media Cybernetics, Bethesda, MD). The colocalization of mycobacterial phagosomes in each experimental point was done by counting more than 30 phagosomes, in 20 cells per individual from 10 different fields. Slight changes in brightness and contrast were performed in images to display purposes. For statistical analysis of phagosomes, the Pearson correlation coefficients were transformed to Z values [38] to compare the groups using type II analysis of variance (ANOVA). 2.10. Determination of cell death MON24 and MDM120 were infected with non-labeled M. tuberculosis H37Rv, H37Ra and UT127 for 36 h. Cell death was determined directly on culture plates by simultaneous staining

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with 4 mg/ml AO (acridine orange, Calbiochem-Merck, Darmstadt, Germany) and 4 mg/ml PI (propidium iodide, Becton DickinsonPharmingen) in PBS for 5 min at room temperature in the dark [39]. The percentage of cells with nuclear condensation and fragmentation were visualized with AO and cells with membrane damage by PI counter staining, using an epifluorescence microscope (Eclipse TS-100. Nikon Corporation. Tokyo, Japan) [39]. In parallel experiments, cell death was determined in situ by TACSÔ Annexin V and PI according to manufacturer instructions (Trevigen Inc., Gaithersburg MD). 2.11. Determination of poly-caspases For the evaluation of Poly-caspases (Red FLICA Poly Caspases Assay Kit, ImmunoChemistry Technologies, Bloomington, MN), MON24 and MDM120 were infected as described above and incubated with SR-VAD-FMK sulforhodamine B FLICA, that binds to active caspases 1, 3, 4, 5, 6, 7, 8 and 9, for 1 h at 37  C, 5% CO2. Cells were washed twice with PBS, fixed with 2% PFA, scraped and immediately analyzed using a flow cytometer. 2.12. Statistical analyses Comparisons between paired samples were done by Wilcoxon signed-rank test. The comparisons that include three or more groups and two factor analyses were done by type II ANOVA. The fluorescence distributions between two experimental points were compared by KolmogoroveSmirnov test. A p  0.05 was considered statistically significant. For all analyses, GraphPad Prism 5 (GraphPad Software, Inc, La Jolla, CA) and Statistics Plus 4 software (Statpoint Technologies, Inc, Warrenton, VA) were used. 3. Results 3.1. MDM120 had a superior phagocytic capacity of M. tuberculosis than MON24 The in vitro ability of mononuclear phagocytes to bind or internalize fluorescent latex beads was compared between MON24 and MDM120 by flow cytometry (Supplementary Figure 1). MON24 and MDM120 exhibited the same percentage of cells associated with latex beads, 80% at the 5:1 ratio (Supplementary Figure 1A). However, MDM120 showed greater number of latex beads per cell compared to MON24 (p < 0.001; Supplementary Figure 1B). The binding/internalization of M. tuberculosis by phagocytes was determined by counting the number of CFUs at 2 h post-infection. MDM120 showed a greater number of CFUs of M. tuberculosis H37Rv (p ¼ 0.03),

H37Ra (p ¼ 0.01) and UT127 (p ¼ 0.01) compared to MON24 (Figure 1). The differences between phagocytes were also observed using serum-opsonized M. tuberculosis H37Rv (Data not shown). The number of H37Ra CFUs, albeit higher in MDM120, was very low compared with the CFUs observed with H37Rv and UT127. In addition, there were a significant elevated number of CFUs of M. tuberculosis H37Rv in MON24 compared with H37Ra and UT127 (p  0.005). These results support that macrophages bind and internalize more latex beads and M. tuberculosis per cell than less differentiated cells and that H37Ra is poorly recognized/engulfed by macrophages compared with H37Rv and UT127. 3.2. MDM120 had a higher expression of phagocytic receptors than MON24 Previously, we have reported the expression of the differentiation markers HLA class II, CD14, CD16, CD36, CD40, CD68 and CD86 in MON24 and MDM120 [29]. In addition, the higher numbers of bound/internalized M. tuberculosis by MDM120 prompted us to compare the expression of phagocytic receptors per cell (Figure 2). There were higher levels of FcgR CD16 (p ¼ 0.03) and CD64 (p ¼ 0.01), adhesion molecule CD44 (p ¼ 0.01), mannose receptor CD206 (p ¼ 0.01) and complement receptors CD11b (p ¼ 0.01), CD11c (p ¼ 0.01) and CD18 (p ¼ 0.01) on MDM120 than on MON24. These results suggest that the increased membrane expression of opsonic and non-opsonic phagocytic receptors by MDM120 may explain their higher capacity to bind and internalize more M. tuberculosis bacilli compared to less differentiated cells. 3.3. MDM120 controlled the replication of M. tuberculosis more effectively than MON24 The ability of MON24 and MDM120 to control the intracellular mycobacterial replication was also evaluated. CFUs were determined at 2 h, 72 h, 96 h, and 120 h post-infection and data were normalized based on the replication index (Figure 3). In MON24, there was a time dependent increase of the replication index of M. tuberculosis H37Rv and UT127, that were higher compared with MDM120 (Figure 3). Similar results were found with opsonized M. tuberculosis H37Rv (Data not shown). MON24 controlled the replication of M. tuberculosis H37Ra at all times evaluated (Figure 3); however, there was a time-dependent decrease of the replication index of M. tuberculosis H37Ra in MDM120 (Figure 3). These results indicate that MDM120 control more efficiently the M. tuberculosis replication than less differentiated cells, and that these cells exert a bactericidal effect on

Figure 1. Phagocytosis of M. tuberculosis in MON24 and MDM120. MON24 and MDM120 were infected with M. tuberculosis H37Rv, H37Ra and UT127 for 2 h at a MOI of 5:1; thereafter, monolayers were washed, the cells lysed and the number of colony forming units (CFU) of bound/internalized bacilli was determined. Wilcoxon test, n ¼ 5e6.

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Figure 2. Expression of phagocytic receptors on MON24 and MDM120. Mean fluorescence intensity (MFI) of MON24 and MDM120 positive to CD11b, CD11c, CD16, CD18, CD44, CD64 and CD206. The expression of these receptors was determined by flow cytometry. Wilcoxon test, n ¼ 6.

H37Ra, while exhibit a partial bacteriostatic effect on H37Rv and UT127. 3.4. Production of IL-12p70, TNF-a, IL-10, IL-1b, IL-8 and IL-18 between MON24 and MDM120 in response to mycobacterial infection The production of IL-12p70, TNF-a, IL-10, IL-1b, IL-8 and IL18 by MON24 and MDM120 was compared at 2, 72 and 96 h of infection with live M. tuberculosis H37Rv, H37Ra and UT127. The levels of IL12p70 were very low at the evaluated time points and there were not differences between more and less differentiated phagocytes (data not shown). MON24 showed a trend to produce larger amounts of TNF-a and IL-10 in response to M. tuberculosis H37Rv and UT127 compared to MDM120, reaching statistical significance only at 2 (H37Rv) and 72 h (UT127) for TNFa and at 72 h (UT127) for IL-10 (p  0.01. Figure 4A and B). There were similar levels of IL-1b and IL-8 in supernatants of infected MON24 and MDM120 at 2 and 72 h (Figure 4C and D). However, MDM120 produced significant larger amount of IL-1b at 96 h of H37Rv infection compared with MON24, but lower with

UT127 infection (p  0.05; Figure 4C). Similar to IL-1b production, there was more IL-18 production in response to M. tuberculosis H37Rv at 96 h by MDM120 than MON24 (p  0.01 Data not shown). Importantly, there were lower levels of TNF-a, IL-10, IL-1b and IL-8 in response to M tuberculosis H37Ra, compared with H37Rv and UT127 (Figure 4), without differences between MON24 and MDM120. Although, some differences were found in the cytokine production by MON24 and MDM120 in response to mycobacterial infection; it was not possible to find a pattern of cytokine production by infected MDM120, which can be related with the mycobacterial control observed with these phagocytes. 3.5. MDM120 had more acidic compartments than MON24, however there were similar percentage of acidic phagosomes containing M. tuberculosis between them The experiments described above showed that phagocytosis and M. tuberculosis replication were different between MON24 and MDM120, irrespectively of the strain used to infect the cells; therefore, the next set of experiments were done only with

Figure 3. M. tuberculosis replication in MON24 and MDM120. Replication index (RI) at 72, 96 and 120 h of infection with M. tuberculosis H37Rv, H37Ra and UT127 at a MOI of 5:1 in MON24 and MDM120. The amount of bacilli recovered after 2 h of phagocytosis was considered as the unit and the zero time of replication. Data are shown as the mean  standard deviation (SD). Type II ANOVA, n ¼ 5e6.

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Figure 4. Cytokines production by MON24 and MDM120 infected with M. tuberculosis. The levels of (A) TNF-a, (B) IL-10, (C) IL-1b and (D) IL-8 was evaluated in MON24 and MDM120 culture supernatants after 2 h, 72 h and 96 h of infection with M. tuberculosis H37Rv, H37Ra and UT127, at a MOI of 5:1. The cytokines were measured by Cytometric Bead Array. The data are shown as the mean  standard deviation (SD). Closed triangles (:) correspond to MON24 and closed circles (C) to MDM120. Type II ANOVA, n ¼ 6. *p  0.01.

M. tuberculosis H37Rv. To determine whether the differences in mycobacterial control between MON24 and MDM120 may be related to modifications in the endosomal-phagosome network [7], the acid compartments in non-infected cells were stained with the acidotropic probe “LysoTracker”. MON24 incorporated less amount of the probe than MDM120 (p ¼ 0.04; Figure 5A), the later showing a greater number of acidic compartments per cell (Figure 5B). Whereas most MON24 had 0-4 acid compartments (p < 0.001), MDM120 showed a greater percentage of cells with more than 4 acidic vesicles (p < 0.0001; Figure 5C). However, when MON24 and MDM120 were exposed to live FDA- or dead CFSE-M. tuberculosis H37Rv and stained with LysoTracker, there was a lower number of phagosomes containing live M. tuberculosis that colocalizate with LysoTracker in both MDM120 and MON24 (Figure 5D); whereas a higher colocalization was present in phagosomes containing dead M. tuberculosis, irrespective of the time of cell differentiation (p < 0.0001; Figure 5E). This finding suggest that despite the higher amount of lysosomes in MDM120, the percentage of acid phagosomes containing M. tuberculosis is not higher compared to MON24.

3.6. MDM120 showed lower percentages of transferrin positive phagosomes containing live M. tuberculosis Since no differences were observed in the percentages of acidic mycobacterial phagosomes between MON24 and MDM120, the incorporation of transferrin was evaluated as a marker of immature phagosomes(40). In MON24 there was an evident accumulation of transferrin within almost all phagosomes containing live FDAM. tuberculosis H37Rv, whereas in MDM120, despite the increased cytoplasmic transferrin signal, there was a smaller number of mycobacterial phagosomes that accumulate this protein (Figure 6A). There were higher Pearson correlations coefficients in phagosomes containing live M. tuberculosis with transferrin in MON24 than in MDM120 (p < 0.0001) and the phagosomes containing dead mycobacteria (Figure 6B). Additionally, the expression of Rab5, the EEA-1, CD63 and the CaMKII was assayed in phagosomes containing live and dead M. tuberculosis H37Rv at 2, 6, 12, 24 and 48 h after exposure. The cytoplasmic staining of these markers showed a typical endosomal pattern; however, there was not a clear accumulation of these

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Figure 5. Determination of acid compartments and mycobacterial phagosomes that co-localize with LysoTracker in MON24 and MDM120. (A) LysoTracker incorporation by MON24 and MDM120 was determined by flow cytometry. Wilcoxon test, n ¼ 4. (B) Representative pictures (60) of LysoTracker (red) incorporation by MON24 and MDM120. The chromatin stained with Hoescht 33258 is observed in blue. More than 600 cells evaluated from 4 different healthy individuals. (C) Number of LysoTracker positive compartments in MON24 and MDM120. The figure shows the mean  standard deviation (SD) from more than 900 cells evaluated from 4 different healthy individuals. Type II ANOVA. **p  0.001 and ***p  0.0001. (DeE) The phagosomes containing M. tuberculosis (green) that co-localized with LysoTracker (red) were evaluated in MON24 and MDM120 infected with M. tuberculosis H37Rv at a MOI of 2:1 for 6 h. (D) Representative pictures of MDMs containing live FDA-M. tuberculosis that no-co-localized with LysoTracker, and the positive colocalization with dead CFSE-M. tuberculosis (60). The chromatin stained with Hoescht 33258 is observed in blue. (E) Pearson’s correlation coefficients of evaluated phagosomes. The results are representative from more than 850 phagosomes evaluated from 4 different healthy individuals. The mean is shown in a gray line; type II ANOVA, after transformation of the correlation coefficients to Z values.

molecules in the mycobacterial phagosomes that allowed the comparison between MON24 and MDM120 (Data not shown).

3.7. There were higher percentages of phagosomes containing M. tuberculosis that accumulates cathelicidin in MDM120 To further characterize the phagosomal compartment, the expression of the mature form of the anti-mycobacterial peptide cathelicidin was evaluated in MON 24 h and MDM120 h. Nontreated and LPS-treated MON24, exhibited smaller signal intensity compared with MDM120 (p  0.0001; Figure 7A and B). To determine whether there are differences in the amount of phagosomes containing mycobacteria that co-localize with cathelicidin, MON24 and MDM120 were exposed to live FDA- or dead CFSEM. tuberculosis H37Rv and stained with the anti-cathelicidin. There was a negligible signal of cathelicidin in the phagosomes containing live M. tuberculosis in MON24, whereas in the MDM120 there

was a higher accumulation of this peptide within the mycobacterial phagosomes (Figure 7C); as showed by a higher Pearson correlation coefficients in phagosomes containing live M. tuberculosis with cathelicidin in MDM120 than in MON24 (p < 0.0001; Figure 7D). Moreover, there was more colocalization of phagosomes containing dead M. tuberculosis with cathelicidin in MDM120 than in MON24 (p < 0.0001; Figure 7D).

3.8. MDM120 produced less H2O2 in response to M. tuberculosis infection than MON24 To assess another anti-microbial mechanism in mononuclear phagocytes, the production of H2O2 was determined in MON24 and MDM120 after mycobacterial infection. Although there was a higher basal signal of cationic rhodamine123 in MDM120 compared with MON24 (p ¼ 0.0012; Supplementary Figure 2), in infected cells the rhodamine levels were always lower in MDM120

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Figure 6. Determination of mycobacterial phagosomes that accumulate transferrin in MON24 and MDM120. The phagosomes containing M. tuberculosis (green) that colocalized with transferrin (red) were evaluated in MON24 and MDM120 infected with M. tuberculosis H37Rv at a MOI of 2:1 for 12 h. (A) Representative pictures of MON24 and MDM120 infected with live FDA-labeled M. tuberculosis and treated with Alexa Fluor 594-transferrin (60). The chromatin stained with Hoescht 33258 is observed in blue. (B) Pearson’s correlation coefficients of evaluated phagosomes. The data are representative from more than 650 phagosomes evaluated from 4 different healthy individuals. The mean is shown in a gray line. Type II ANOVA, after transformation of the correlation coefficients to Z values.

compared with MON24, irrespective of the strain of M. tuberculosis used or the addition of PMA (Supplementary Figure 2). 3.9. MDM120 were more resistant to M. tuberculosis-induced cell death than MON24 To determine whether cell death of mononuclear phagocytes in response to M. tuberculosis infection is related with their stage of differentiation, MON24 and MDM120 were infected with M. tuberculosis for 36 h, at a MOI of 15:1 (data not shown) and 30:1, and the cell death was evaluated with AO plus PI or Annexin V plus PI counterstaining (Table 1). Preliminary experiments showed that M. tuberculosis H37Rv infection at 1:1, 5:1 and 10:1 MOIs did not significantly induced cell death, therefore higher MOIs were used for these experiments. In situ staining with AO and PI, showed that most of the MDM120 remained viable after M. tuberculosis H37Rv infection, while 20% of the MON24 were PI-positive (p < 0.001; Table 1). Although, a smaller percentage of MDM120 infected with M tuberculosis H37Ra and UT127 remained viable compared with H37Rv infection, there were more PI-positive cells in infected MON24 (38.8% and 46.8%, respectively. p < 0.001; Table 1). In all experiments, UT127 infection induced a higher percentage of PIpositive cells than the other M. tuberculosis strains (Table 1). Cell death was also evaluated in situ by Annexin V and PI staining. MON24 exhibited a higher percentage of Annexin VþPIþ cells compared with MDM120 (p < 0.001; Table 1), irrespective of the M. tuberculosis strain used to infect them. Finally, to determine whether caspases activation may be associated with the cell death induced by mycobacterial infection, the percentage of MON24 and MDM120 positive to active polycaspases was determined. Indeed, there were higher percentages of poly-caspases positive in MON24 after H37Rv (18.3%  11), H37Ra (20.5%  4.1) and UT127 (22.0  3.1) infection compared to the MDM120 (4.0%  0.6, 7.1%  1.6 and 11.6%  3.4, respectively). These results confirm that MON24 are more prone to die in response to M. tuberculosis infection than MDM120 and this type of cell death involved caspase activity, phosphatidylserine exposure and membrane damage.

4. Discussion The differentiation of mononuclear phagocytes from monocytes into macrophages is a determining factor of the phagocyte heterogeneity and may affect the activation of their innate immune responses. The results from this study demonstrate that the antimycobacterial response of monocyte-derived macrophages (MDM120) differ markedly from the responses of less differentiated cells (MON24). The MDM120 phagocytosed more M. tuberculosis, contained the mycobacterial replication for longer time periods, had lower percentage of phagosomes containing live mycobacteria that accumulated transferrin, had a higher proportion of phagosomes that accumulate cathelicidin than MON24 and did not die in response to infection. Antimycobacterial activity of monocyte-derived macrophages (MDM) differ according the approaches to the in vitro differentiation; macrophages differentiated with GM-CSF, TNF-a and IFN-g could survive the infection and limit the replication of M. tuberculosis [32]. In addition, GM-CSF and Mâ^’ CSF, growth factors traditionally used to differentiate monocytes into dendritic cells or macrophages, were found to alone promote a M1 and M2 like phenotypes, respectively [41]. Therefore, to avoid the interference of the polarization of the mononuclear phagocytes activation in the results presented here, it was used a differentiation approach without any grown factor or cytokine supplementation. Phagocytosis of M. tuberculosis involves opsonic and nonopsonic receptors [42e46] and it has been reported that human macrophages express more receptors that mediate the internalization of M. tuberculosis than circulating monocytes, such as mannose and complement receptors [47e50]. Thus the increased uptake of M. tuberculosis by phagocytes differentiated for 120 h, may be explained by the higher expression of the opsonic (CD11b, CD11c, CD18, CD16 and CD64) and non-opsonic (CD44 and CD206) receptors detected on those cells. The lower amount of M. tuberculosis H37Ra binding/internalization by MDM120 compared with H37Rv was previously reported [46]; and it may be explained by differences in bacterial cell wall components, such as different forms of lipoarabinomannan (LAM) [51], that could affect the bacilli recognition [52]. The decreased phagocytosis observed

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215

Figure 7. Determination of cathelicidin expression and the mycobacterial phagosomes that co-localized with this anti-microbial peptide in MON24 and MDM120. (A) Representative pictures (60) of mature cathelicidin (red) staining in MON 24 and MDM120. The chromatin stained with Hoescht 33258 is observed in blue. (B) The fluorescence intensity of mature cathelicidin was measured in mononuclear phagocytes by confocal microscopy, at a basal stage and after LPS treatment. The data are representative from more than more than 600 cells evaluated from 4 different healthy individuals. Type II ANOVA, after transformation of the correlation coefficients to Z values. (CeD) The phagosomes containing M. tuberculosis (green) that co-localize with mature cathelicidin (red) was evaluated in MON 24 and MDM120 infected with live FDA-or dead CFSE-M. tuberculosis H37Rv at a MOI of 2:1 for 6 h. (C) Representative pictures of MON24 containing live FDA-M. tuberculosis that no-co-localized with cathelicidin, and the positive colocalization in MDM120 (60). The chromatin stained with Hoescht 33258 is observed in blue. (D) Pearson’s correlation coefficients of evaluated phagosomes. The results are representative from more than 850 phagosomes evaluated from 4 different healthy individuals. Type II ANOVA, after transformation of the correlation coefficients to Z values.

with UT127 may be also explain by differences in cell wall components, since it was reported that some clinical isolates contain truncated and more branched forms of mannose-capped LAM (ManLAM) compared with Erdman strain, that allowed to low recognition by mannose receptor and decrease phagocytosis by primary human macrophages [53]. Despite the increased mycobacterial uptake by MDM120, these cells more efficiently controlled the bacterial replication, exerting a bactericidal response for the avirulent M. tuberculosis H37Ra and a bacteriostatic response for virulent M. tuberculosis H37Rv and the clinical isolate UT127. This containment did not seem to be dependent on the mycobacterial load, the production of cytokines (IL-12p70, TNF-a, IL-10, IL-1b, IL-8), the reactive oxygen species (ROS) release and the induction of cell death, since MON24, which showed replication of M. tuberculosis at 72 h post-infection, uptaked less mycobacterias, exhibited a higher production of

H2O2, similar levels of cytokine production, and higher percentage of AVþPIþ cells in response to the infection with H37Rv, H37Ra and UT127, compared with MDM120. It has been reported that the mononuclear phagocytes change their cytokine production according to the stage of differentiation [54,55]. However, there was not a contrasting profile of cytokines production in response to mycobacterial infection between MON24 and MDM120. The increased levels of TNFa, IL-10, IL-1b and IL-8 observed with M. tuberculosis H37Rv and UT127 infection compared with H37Ra, may be explained by the lack of replication of H37Ra and hence induced lower cytokine production. After phagocytosis, phagosomes containing M. tuberculosis mature into acidic and hydrolytic compartments by fusing with lysosomes. Our results confirm previous reports showing an increased number of lysosomes in monocyte-derived macrophages

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Table 1 Evaluation of cell death in MON24 and MDM120 in response to M. tuberculosis infection. MON24*

MDM120*

Vz AO/PI

y

AV/PIy

* y z x {

NC/AVþz x

NI H37Rv H37Ray UT127 NIx H37Rv H37Ra UT127

96.7 78.4 54.7 41.3 97.4 79.9 58.7 28.2

       

0.8 7.5{ 9.0{ 2.7{ 1.7 8.1{ 9.4{ 11.2{

0.7 1.6 6.5 11.9 0.5 1.0 5.2 13.2

       

0.3 2.2 1.3 5.3{ 0.4 0.9 1.5 5.6{

AVþPIþz z

ND ND ND ND 2.0  1.3 18.4  7.9{ 33.5  9.5 50.9  8.1{

PIþz 2.6 20.0 38.8 46.8 0.1 0.7 2.5 7.7

Vz        

0.7 7.9{ 9.5{ 9.2{ 0.2 0.7 2.7 1.8

98.1 97.6 87.6 84.2 98.0 96.5 85.3 83.8

NC/AVþz        

1.2 3.2{ 2.7{ 3.6{ 0.8 1.3{ 5.5{ 2.3{

0.6 0.7 1.3 1.5 0.5 0.8 2.8 1.2

       

0.9 1.2 0.8 1.1{ 0.2 0.6 1.6 1.1{

AVþPIþz

PIþz

ND ND ND ND 1.0 2.6 10.4 13.6

1.3 1.7 10.8 14.3 0.5 0.1 1.4 1.3

   

0.7 1.2{ 4.3 2.8{

       

1.7 3.4{ 2.5{ 4.0k 0.1 0.1 1.3 0.6

MON24 and MDM120 were infected with M. tuberculosis H37Rv, H37Ra and UT127 at 30:1 MOI. Mean  Standard Deviation (SD). AO/PI: Acridine orange plus Propidium iodide. AV/PI: Annexin V plus Propidium iodide. V: Viable. NC/AVþ: Nuclear condensation or Annexin Vþ, AVþPIþ: Annexin Vþ and Propidium iodideþ, PI: Propidium iodideþ. NI: Non-infected cells. .p Values 0.001 by type II ANOVA, Bonferroni post-test. These data are representative from more than 23,000 cells evaluated from 5 to 6 different healthy individuals.

and THP-1 macrophages, than in recently isolated monocytes [56]. However, the higher number of compartments did not correlate with a higher percentage of acidic mycobacterial phagosomes, suggesting that the blockade of the signaling pathways responsible for phagosome acidification by M. tuberculosis is equally efficient in MON24 and MDM120. Even though there were not differences in the percentage of acidic phagosomes, MDM120 controlled the infection more efficiently than MON24. The low accumulation of transferrin in live mycobacteria phagosomes observed in the MDM120, suggests that these phagosomes have less access to early and recycling endosomes [40]. On the contrary in MON24, more than 80% of the phagosomes containing live mycobacteria co-localized with transferrin. Decreased replication of M. tuberculosis has been associated with a reduced communication of phagosomes with the cellular recycling and biosynthetic pathways [40], resulting in a lower access of the mycobacteria to nutrients and oligoelements such as iron, which are important for its metabolism and replication [57e 59]. The finding that a smaller percentage of mycobacterial phagosomes of infected MDM120 accumulates transferrin, may partially explain their capacity to control mycobacterial replication for a longer time. However, it is also possible that other innate effector mechanisms, such as the antimicrobial peptides [60,61], participate in the anti-mycobacterial activity of the MDM120. Indeed, MDM120 showed a high basal expression of the antimicrobial peptide cathelicidin (LL-37). Cathelicidin is known to be induced for and required for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3)-mediated antimicrobial activity against intracellular M. tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis [60,62e64]. Thus, it may be possible that the human serum used in our culture media, reported to have w100 pM of 1,25(OH)2D3 [64] plus the basal levels of ROS detected in MDM120 [65], may explain the high levels of cathelicidin observed in these cells. Cathelicidin may be targeted directly to maturation-arrested mycobacterial phagosomes, where it contribute to kill the bacteria [63]. This signal seems to be important in the early stages of phagosome maturation and requires calcium influx [64]. Therefore, it is reasonable to suggest that the production of cathelicidin participated in the mycobacterial control observed by MDM120. Cathelicidin also have pleiotropic roles in the regulation of inflammatory and immune responses, such induction of autophagy [64], which is known to be involved in the control of M. tuberculosis [66]. It was shown that 1,25(OH)2D3 induces autophagy and colocalization of mycobacterial phagosomes with autophagosomes [64]. Although we observed colocalization of M. tuberculosis and cathelicidin, we cannot rule out an indirect effect of cathelicidin in

the mycobacterial control, such as induction of autophagy. Hence, future studies are required to determine whether there is a higher autophagy induction in MDM120 compared to MON24. Other important components of the innate immune response are the reactive oxygen species (ROS). Although, reactive oxygen intermediates are unlikely to be involved in the direct killing of M. tuberculosis [67], patients with granulomatous disease are susceptible to TB and bacillus Calmette-Guérinerelated complications [68,69]. In addition, ROS have been associated with the regulation of several intracellular signaling in the context of mycobacterial infection [65]. The high basal levels of H2O2 observed in MDM120 may be controlling the expression of different proteins, such cathelicidin [65]. Regarding the induction of cell death by M. tuberculosis, our group and others have extensively demonstrated that the infection with M. tuberculosis induces different types of cell death in monocytes and macrophages [70e73]. Herein, the results show that MON24 infected with M. tuberculosis exhibited alterations associated with apoptosis (nuclear condensation, phosphatidylserine translocation to the outer cell membrane and activation of caspases) and necrosis (cytosolic membrane damage); while the MDM120 showed smaller percentage of dead cells in response to the mycobacterial infection. These findings are in agreement with previous reports that demonstrated that human tissue macrophages are more resistant to apoptosis than monocytes [56,74], and others reports in which differentiated mononuclear phagocytes e namely human alveolar macrophages e did not die in response to low inocula of M. tuberculosis H37Rv [75], but did it with the nonvirulent strain H37Ra infection [70]. According to our finding, the cell death observed here could correspond to the previously described atypical cell death by Kornfeld et al. [75e77]. When M. tuberculosis intracellular load exceeded w20 bacilli per cell, mouse macrophages exhibited apoptotic characteristics and progressed rapidly to necrosis. This is an atypical cell death via lysosomal lipases. Similar to our results, it has been reported that virulent and attenuated mycobacterial strains induced atypical cell death, being higher with the virulent M. tuberculosis Erdman [76]. It has been demonstrated that most heavily loaded (20e40 bacilli) monocytic cells were nonviable in the lung of infected C57BL/6 mice, similar to those observed after high multiplicity challenge in vitro with M. tuberculosis [77]. Thus, it is still necessary to further study the mechanisms involved in the cell death observed in our mononuclear phagocytes. Altogether, our results show that there are clear differences in the anti-mycobacterial responses of mononuclear phagocytes depending on their stage of differentiation. Macrophages can be divided in two groups according the origin, tissues-resident

D. Castaño et al. / Tuberculosis 94 (2014) 207e218

macrophages that are established before birth, that are self renewal and participate mainly in the restoration of homeostasis; and monocytes that under inflammatory conditions are recruited and differentiated in situ into macrophages (MDM), that transiently contribute to the local immune responses [78,79]. Previous report from our group shows that the infection of monocytes with M. tuberculosis alters the in vitro differentiation into macrophages and their capability to activate T cells [23]. Therefore, we hypothesize that during TB, recently recruited monocytes to the site of infection, may become infected before completely differentiated into macrophages, leading to a decreased capacity to contain the mycobacterial replication and, therefore, would favor the establishment and dissemination of the infection. This possibility may be further complicated if CD16þ monocytes are preferentially recruited to the site of infection compared with CD16- monocytes. Previously, we reported that TB patients have increased percentages of circulating CD16þþ monocytes, that produced more TNF-a and were highly susceptible to cell death (with membrane damage) in response to mycobacterial infection [29]. Although the presence of less differentiated cells at the lung lesions is difficult to demonstrate in humans, tracking of monocytes and studies in biopsies may provide evidence about the role of monocytes at the tissue affected by TB. Acknowledgments The authors thank the volunteers that participated in this study. We appreciate the technical support and advices for microscope experiments of Sergio Grinstein (University of Toronto, SickKids Hospital. Toronto, Canada), Patricia Cardona (Grupo de Neurosciencias, Universidad de Antioquia. Medellín, Colombia) and Jean Paul Delgado (Grupo de Genética de Poblaciones, Universidad de Antioquia, Medellín, Colombia). We thank Diana M. Marín (Grupo de Epidemiología, Universidad de Antioquia. Medellín, Colombia) for her help in the statistical analyses of mycobacterial phagosomes. We are grateful with Andrés Arias (Grupo de Inmunodeficiencias, Universidad de Antioquia. Medellín, Colombia) for his advices to perform DHR 123 experiments. Ethical approval: Study and informed consent approved by the Ethics Committee of the Instituto de Investigaciones Médicas of the Facultad de Medicina, Universidad de Antioquia. Medellín, Colombia. Funding: This study was supported by COLCIENCIAS (Bogotá, Colombia) grants:RC431-2004 and 111540520270; and Programa de Sostenibilidad 2013e2014, Universidad de Antioquia. Diana Castaño was a recipient of a doctoral scholarship from COLCIENCIAS. Competing interests:

None declared.

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