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Mycobacterium tuberculosis replicates within necrotic human macrophages

Mycobacterium tuberculosis replicates within necrotic human macrophages JCB: Report Mycobacterium tuberculosis replicates within necrotic human macrophages 1 1 1 3 2 1 Thomas R. Lerner, * Sophie Borel, * Daniel J. Greenwood, * Urska Repnik, Matthew R.G. Russell, Susanne Herbst, 2 2 3 1 Martin L. Jones, Lucy M. Collinson, Gareth Griffiths, and Maximiliano G. Gutierrez 1 2 Host-Pathogen Interactions in Tuberculosis Laboratory and Electron Microscopy Science Technology Platform, The Francis Crick Institute, London NW1 1AT, England, UK Department of Biosciences, University of Oslo, 0371 Oslo, Norway Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) in- tegrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor–differentiated macrophages more than in granulocyte–macrophage colony-stimulating factor–differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial rep- lication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host. Introduction The intracellular lifestyle of Mycobacterium tuberculosis rep- nity and inflammation (Vogt and Nathan, 2011; Martinez and resents a crucial stage in the pathogenesis of tuberculosis, and Gordon, 2015). Macrophages differentiated in vitro are often successful drug discovery programs have to include in vitro referred to as classical or alternatively activated macrophages, studies using infected host cells (Young et al., 2008; Lechart- or M1 and M2 macrophages, respectively. Several stimuli are ier et al., 2014). Most of the in vitro studies of infection with used to differentiate macrophages in vitro, and in this context, M. tuberculosis rely on infected macrophages and survival anal- granulocyte–macrophage colony-stimulating factor (GM-CSF; ysis by colony-forming units (CFUs), luciferase, or fluorescent considered M1)– and macrophage colony-stimulating factor reporters. The use of primary human macrophages is of clear (M-CSF; considered M2)–differentiated human macrophages advantage because there are some fundamental differences be- are widely used models of macrophage biology (Lacey et al., tween human macrophages and those originating from other 2012; Martinez and Gordon, 2015). GM-CSF macrophages species. Important differences are obvious, such as the lack of a are generally proinflammatory and display enhanced antigen large set of IFN-γ–inducible GTPases that are exclusively pres- presentation, phagocytosis, and microbicidal capacity. How- ent in mouse macrophages (Kim et al., 2012) or the differential ever, M-CSF macrophages display an antiinflammatory cyto- production of nitric oxide when compared with mouse macro- kine profile after stimulation (Lacey et al., 2012; Martinez and phages (Thomas and Mattila, 2014). Gordon, 2014). Although GM-CSF– and M-CSF–differenti- In vivo, macrophages are composed of a very hetero- ated macrophages clearly respond differently to extracellu- geneous population as a result of multiple differentiation and lar stimuli (Lacey et al., 2012), the nature of this differential activation stimuli present in tissues (Martinez and Gordon, response is less clear. 2014). Numerous experimental approaches are used to differ- Adding more complexity to in vitro systems of infection, entiate human macrophages in vitro with the aim of mimicking there are a wide range of methods described in the literature to the heterogeneity present in tissue macrophages during immu- activate macrophages. One of the key cytokines used for the activation of human macrophages is IFN-γ. This cytokine is a key modulator of the phagocytic and mycobactericidal activity *T.R. Lerner, S. Borel, and D.J. Greenwood contributed equally to this paper. of mouse macrophages (Flynn and Chan, 2001). Although stud- Correspondence to Maximiliano G. Gutierrez: [email protected] ies show that genetic errors of IFN-γ immunity result in severe Abbreviations used: CFU, colony-forming unit; CLEM, correlative live-cell and tuberculosis in children (Abel et al., 2014), the role of IFN-γ in electron microscopy; DPSS, diode-pumped solid-state; GM-CSF, granulocyte– macrophage colony-stimulating factor; HyD, hybrid detector; IDO, indoleam- the antimycobacterial activity of human macrophages and its ine 2,3-dioxygenase; LM, light microscopy; M-CSF, macrophage colony- stimulating factor; PBMC, peripheral blood mononuclear cell; PI, propidium iodide; PM, plasma membrane; SBF SEM, serial block face scanning EM; © 2017 Lerner et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https ://creativecommons .org /licenses /by /4 .0 /). TEM, transmission EM. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 3 583–594 https://doi.org/10.1083/jcb.201603040 JCB 583 T H E J O U R N A L O F C E L L B I O L O G Y role in pulmonary tuberculosis in adults is still unclear (Abel et at the single-cell level. A quantitative analysis of the number of al., 2014; Lerner et al., 2015). GFP-positive pixels per macrophage confirmed that M.  tuber- Although in vitro and in vivo studies highlight the im- culosis replicated over time at the single-cell level (Fig.  1  B). portance of host cell death modes during mycobacterial control In parallel, the proportion of M. tuberculosis–infected cells per or dissemination, the mode of host cell death in the pathogen- sample significantly increased between hour 2 and day 7, sug- esis of human tuberculosis is not completely understood. In gesting that the infection was spreading between cells (Fig. 1 C). tuberculosis, apoptosis is generally considered to be a part of Activation of GM-CSF macrophages with IFN-γ had only a lim- a host protective response, whereas necrosis is considered to ited effect on bacterial replication (Fig. 1, A–C). We observed a be a pathway for bacterial dissemination and granuloma cavity similar level of M. tuberculosis replication in M-CSF compared formation (Behar et al., 2010; Ramakrishnan, 2012; Wong and with GM-CSF macrophages by CFUs (Fig.  1  D) and also by Jacobs, 2016). Apoptosis is believed to help with the eradica- microscopy at the single-cell level (Fig.  1, E and F). Interest- tion of M. tuberculosis (Keane et al., 2000; Behar et al., 2010), ingly, we observed that at 7 d after infection in IFN-γ–activated and, unsurprisingly, this pathogen has strategies to inhibit apop- M-CSF macrophages, the mean burden of M.  tuberculosis per tosis (Velmurugan et al., 2007). Apoptosis is protective in part cell was much higher than all of the other conditions (Fig. 1, B because bacteria are internalized via efferocytosis and subse- and E). Furthermore, infection of macrophages with GFP-Mtb quently eliminated (Martin et al., 2012). However, necrosis of ΔRD1 (which lacks the ESX-1 type VII secretion system; Fig. infected cells helps bacterial dissemination. M. tuberculosis in- S2, A–D) revealed that this mutant strain had a reduced bacte- hibits the plasma membrane (PM) repair pathway, resulting in rial burden (Fig. S2, A and D) and a reduced cell-to-cell spread the progression to necrosis and mycobacterial release into the (Fig. S2 B) compared with wild type and lacked the ability to extracellular environment (Divangahi et al., 2009). Neverthe- induce cell death overall (Fig. S2 C), even after 5 d of infection less, whether host cell necrosis directly affects bacterial replica- (Fig. S2 D and Video  1). Bacterial viability was independent tion has not yet been demonstrated. of the time of activation or IFN-γ concentration because pre- In this study, we show that M.  tuberculosis replicates stimulation overnight or treatment after infection with a higher to a similar extent in GM-CSF– and M-CSF–differentiated concentration of IFN-γ rendered similar results (not depicted). macrophages (GM-CSF macrophages and M-CSF macro- In resting GM-CSF macrophages, bacteria were mostly local- phages, respectively). Remarkably, IFN-γ activation enhanced ized in compartments negative for the phagolysosomal marker M.  tuberculosis replication in M-CSF macrophages but not in LAMP-2, as was expected for bacteria residing in an arrested GM-CSF macrophages, and this correlated with an increased early phagosome (Fig. 1, G and H). IFN-γ activation did not sig- susceptibility to necrosis compared with the other macrophage nificantly change this localization (Fig. 1, G and H). In M-CSF populations. Long-term live-cell imaging of infected macro- macrophages, M. tuberculosis was also localized in LAMP-2– phages revealed that at the single-cell level, M.  tuberculosis negative compartments (Fig. 1, I and J), but IFN-γ increased the infection induces loss of PM integrity to replicate in damaged fraction of mycobacteria found in late endocytic compartments cells before entering the extracellular milieu. Collectively, after 48 h of infection (Fig. 1, I and J). Overall, our results are in our data define a differential susceptibility to the necrosis of agreement with a study indicating that in GM-CSF and M-CSF GM-CSF– and M-CSF–differentiated macrophages after in- macrophages, M.  tuberculosis grows at similar rates (Tailleux fection and IFN-γ activation. Importantly, these differences et al., 2003), but our results are also in disagreement with other are critical for the replication of M.  tuberculosis in damaged studies (Douvas et al., 1985; Akagawa et al., 2006; Wong and cells before dissemination. Jacobs, 2013). It is likely that differences in donor samples, my- cobacterial strains, MOI, and also cell differentiation protocols are explanations for discrepancies among studies. However, Results and discussion our data highlight important physiological differences between GM-CSF and M-CSF macrophages during the late stages of M. tuberculosis replicates in both resting M.  tuberculosis infection in vitro. We suggest that the alveo- and IFN-γ–activated GM-CSF– and M-CSF– lar macrophage phenotype conferred by GM-CSF (Akagawa, differentiated human macrophages 2002) makes the macrophages permissive to M.  tuberculosis First, we characterized each macrophage subset by flow cytom- replication, allowing bacterial dissemination after primary in- etry using surface markers of activation. Then, we verified that fection of the lungs. In contrast, in M-CSF macrophages, IFN-γ they showed a mature phenotype (CD86 positive; Fig. S1 A) will promote bacterial replication and, later on, dissemination. and were responsive to IFN-γ by looking at the expression of The interplay of these differentiation factors, their contribution the IFN-γ receptor (Fig. S1 B) and by inducing indoleamine to bacterial replication and dissemination in vivo, and the phys- 2,3-dioxygenase (IDO) and other IFN-γ–inducible proteins iological differences between human and mouse macrophages (Fig. S1 C and not depicted). Next, we analyzed the replica- remain to be investigated. Although we observed variability tion of M. tuberculosis H37Rv expressing EGFP (GFP-Mtb) in across the donors tested in this work, IFN-γ unexpectedly did GM-CSF human primary macrophages by CFUs. We observed not have any major impact on bacterial growth in GM-CSF that on average, bacteria replicated by ∼2.5-fold after 72 h of macrophages in vitro. It is not clear why IFN-γ did not control infection (Fig.  1  A). Beyond the 72-h time point, it was not bacterial replication in our system compared with other stud- possible to determine intracellular bacterial replication using ies. A possible cause is that our study was not performed under CFUs because the dying cells detached, resulting in an under- hypoxic conditions, which were shown to be antimycobacterial estimation of the actual number of intracellular bacteria in the (Vogt and Nathan, 2011). Also, reported differences in nitric sample. Therefore, to analyze longer periods of infection, we oxide production between human and mouse macrophages can used a microscopy-based method, which more closely reflected account for reduced potency in human cells (Thomas and Mat- how the infection progressed over an extended in vitro infection tila, 2014). Our data are consistent with a study showing that in 584 JCB • Volume 216 • NumBer 3 • 2017 Figure 1. Dynamic growth of M. tuberculosis in distinct subpopulations of human primary macrophages. (A) Intracellular replication of GFP-Mtb in resting (blue line) or IFN-γ–activated (red line) GM-CSF– differentiated macrophages, normalized to 2 h and expressed as the percent increase in CFUs over time. (B) Intracellular replication of GFP-Mtb in resting (blue dots) or IFN-γ–activated (red dots) GM-CSF–differentiated macrophages (expressed as the mean num- ber of GFP-positive pixels per cell) at 2 h, 72 h, and 7 d after infection. (C) Quantification of the mean proportion of GFP-Mtb infected cells in resting (blue dots) or activated (red dots) GM-CSF–differentiated macrophages at 2 h, 72 h, and 7 d after infection. (D–F) Same as in A–C, but with M-CSF–differentiated macrophages. (A–F) Data presented are means ± SEM from three (A, B, D, and E) or at least three (C and F) independent experiments. (G) Analysis of the association of GFP-Mtb with LAMP-2 in resting and activated GM-CSF macrophages infected at an MOI of 1 for 72 h. Representative confocal microscopy images of GFP-Mtb and LAMP-2 marker. Nuclei were stained with DAPI. Insets show magnifications of the outlined cells, highlighting the low level of as- sociation of LAMP-2 to bacteria. (H) Quantification of the association of GFP-Mtb with LAMP-2 in resting (blue) or activated (red) GM-CSF macrophages. The number of bacteria analyzed for each condition is displayed as well as the proportions of M. tuberculosis that are considered to be positive for LAMP-2 association (i.e., a mean intensity value of ≥150). (I and J) Same as G and H, but with M-CSF–differentiated macrophages. Bars, 10 µm. human macrophages at 5–10% O , IFN-γ induced the growth detect macrophage extracellular traps in our system. Our data of mycobacteria and the induction of macrophage extracellu- are also in agreement with studies in mouse macrophages show- lar traps (Wong and Jacobs, 2013). Our data are also consistent ing that when a threshold of intracellular bacteria is reached, with early studies showing that IFN-γ enhanced M. tuberculo- IFN-γ had little influence on bacterial growth (Repasy et al., sis growth (Douvas et al., 1985), although we were not able to 2013). Additionally, studies in human patients—particularly mtb replicates within necrotic human macrophages • lerner et al. 585 Figure 2. Differential susceptibility to necrosis in macrophages leads to M. tuberculosis growth. (A) Quantification of the proportion of cells that had a PI-positive (PI+) nucleus in resting (blue dots) or activated (red dots) GM-CSF macrophages. A PI-positive nucleus indicates that the cell membrane has become permeable to the extracellular medium and therefore is likely to be dead. Each data point represents one independent experiment, and the bars show means ± SEM. (B) Same as in A, but with M-CSF macrophages. (C) Representative confocal images of macrophages infected with GFP-Mtb (green) and labeled with PI (red). Macrophages were infected at an MOI of 1 for 2 h. 7 d after infection, macrophages were stained with 25 µg/ml PI in RPMI for 15 min and fixed. PI-positive cells are outlined. (D) Same as in C, but with M-CSF macrophages. Bars, 10 µm. (E) Quantification of GFP-Mtb replication in 586 JCB • Volume 216 • NumBer 3 • 2017 children—have shown that IFN-γ is a key antimycobacterial cy- activation accelerated both PM damage and bacterial cell growth tokine (Abel et al., 2014). It remains to be defined which in vitro (Fig.  2, I and J). We concluded that during in vitro infection, systems with human macrophages better capitulate macrophage loss of PM integrity permits continued M.  tuberculosis repli- biology in vivo. In this context, cytokines such as IFN-γ could cation and that the extent of cell death in the sample correlates be affecting other cell types with potential relevance to the pa- with the total bacterial burden. Thus, IFN-γ enhanced PM dam- thology of human tuberculosis (Lerner et al., 2015). age (without causing significant chromatin decondensation) in M-CSF macrophages, which allowed greater bacterial replica- M. tuberculosis replication continues after tion per macrophage. This is consistent with data showing that the loss of PM permeability fast replicating bacteria induced more necrosis in vivo (Repasy We then focused on understanding the differential effect of et al., 2013, 2015). Alternatively, IFN-γ could regulate macro- IFN-γ in enhancing the replication of M. tuberculosis in M-CSF phage cell death and thereby bacterial replication, as shown for macrophages compared with GM-CSF macrophages. To moni- mouse macrophages (Lee and Kornfeld, 2010). tor the proportion of cells that had lost PM integrity, we stained Necrotic macrophages provide a niche for the cells with propidium iodide (PI) before fixation (Fig. 2, A M. tuberculosis replication and B). We observed relatively low levels of cell death in any We next focused on characterizing the environment where condition up to 72 h after infection, but by day 7 after infection, cell death was apparent to differing extents in the macrophage M.  tuberculosis replicates in human macrophages. Because populations (Fig.  2, A and B). Although there was noticeable M.  tuberculosis can be localized in the cytosol under some variation between donors, activated M-CSF macrophages had conditions (van der Wel et al., 2007), we performed an ul- the largest mean proportion of PI-positive cells, with approx- trastructural analysis of M.  tuberculosis localization using imately double the mean of the other macrophage populations EM during the growth of M.  tuberculosis in GM-CSF and (Fig.  2, A and B). We observed that in PI-positive cells at 7 M-CSF macrophages, as described previously (Fig. 3; Lerner d after infection, the bacterial burden was higher in both GM- et al., 2016). Resting GM-CSF macrophages had an increased CSF (Fig.  2, C and E) and M-CSF (Fig.  2, D and F) macro- proportion of bacteria present in the cytosol compared with phages in both resting and activated conditions. We therefore M-CSF after 48 h (45% and 30%, respectively; Fig. 3 B), and hypothesized that these necrotic cells were providing a niche IFN-γ activation did not affect the ultimate localization of the for M.  tuberculosis replication. Using microscopy with fixed bacteria (Fig. 3 B). It was interesting to note that after 5 h plus samples provided only snapshots in time, which meant that we 2 h of infection, there was already up to 15% of the bacteria could not determine whether the cells were dying because of the present in the cytosol in both GM-CSF and M-CSF macro- high bacterial burden or if the cells became more permissive for phages (Fig. 3 A, subpanels B and F; and Fig. S2 E). To fur- replication after they became necrotic. To investigate these dy- ther characterize at the ultrastructural level the environment namic events in more detail, we performed live-cell imaging of where M. tuberculosis replicated, we followed M. tuberculosis infected cells with the presence of PI in the medium to monitor growth by live-cell imaging as described previously and per- macrophage PM integrity. We observed that when macrophages formed correlative live-cell and electron microscopy (CLEM; became leaky (positive for PI), bacterial replication continued Fig.  4; Russell et al., 2016). As shown by live-cell imaging, in necrotic cells that nevertheless retained their cellular mor- after 56 h of infection, the PM became permeable and bacteria phology (Fig.  2  G and Video  2). Continued M.  tuberculosis started to replicate (Fig.  4  A). At 70  h after infection, cells replication after a loss of PM integrity is not solely caused by were fixed and processed for EM. Although the multistage fix- M. tuberculosis gaining access to and obtaining nutrients from ation protocol affected the morphology of the cell, we imaged the extracellular RPMI medium because the growth rate in leaky after each step to document the changes (Fig. 4 A). By mor- macrophages (Fig. 2 H, red lines) was increased compared with phology, we could confirm that M.  tuberculosis was growing the growth rate in medium alone (Fig. 2 H, green lines). A quan- in a necrotic cell with features of late necrotic cell death that titative analysis of GFP fluorescence intensity at the single-cell include PM damage, swollen and dilated organelles, as well as level suggests that in a proportion of macrophages, detectable autophagic structures (Fig. 4 B). We observed that replicating mycobacterial replication does not begin until after the cells bacteria were in close apposition to host membranes and were have become leaky (Fig. 2, I and J). Bacterial replication was exposed to an environment rich in cellular membranes and observed in both resting and activated GM-CSF macrophages undigested organelles. Collectively, already at 48  h, a large (Fig.  2  I), consistent with the previous data (Fig.  1, A–F). In proportion of the bacilli were found in the cytosol, and then at resting M-CSF macrophages, mycobacterial replication started later stages, mycobacteria were mostly replicating in an early after macrophages became leaky (Fig.  2  J). However, IFN-γ to late necrotic macrophage environment. PI-negative or PI-positive cells in resting (black squares) or activated (red squares) GM-CSF macrophages. Images were analyzed, and the GFP signal per cell was plotted. Data represent the means ± SEM of one representative experiment. (F) Same as in E, but with M-CSF macrophages. (G) Representative snapshots at the indicated time points (Video 2) of resting GM-CSF macrophages infected with GFP-Mtb (green) in the presence of PI. Macrophages were infected at an MOI of 1 for 2 h, and after 24-h infection, the media were replaced with RPMI medium containing 0.4 µg/ml PI. Cells were imaged for at least 7 d. Bar, 20 µm. (H) Comparison of GFP-Mtb growth rates as measured by live-cell imaging. Three conditions were tested: M. tuberculosis in RPMI medium alone (green lines), M. tuberculosis within GM-CSF–differentiated human monocyte-derived macrophages (HMDMs) before the moment the cell becomes PI positive (blue lines), and M. tuberculosis after human monocyte-derived macrophages become PI positive (red lines). Each line shows the growth rate of M. tuberculosis represented by the fold change in GFP signal (RAWIntDen) of the GFP-Mtb signal over time, normalized to the first frame in the video. (I) Quantification of intracellular replication of GFP-Mtb before and after membrane damage in resting or activated GM-CSF and M-CSF macrophages at the single-cell level. Red dashed lines indicate when the cell’s nucleus becomes PI positive. GFP-Mtb signal was monitored in three representative cells from each condition using ImageJ software. (J) Same as in I, but with M-CSF macrophages. mtb replicates within necrotic human macrophages • lerner et al. 587 Figure 3. M. tuberculosis is localized in the cytosol of primary human macrophages. (A) TEM images representing the observed localization of M. tu- berculosis in GM-CSF– and M-CSF–differentiated macrophages at 48 h after infection. “Membrane bound” represents bacteria in a phagosome (with one surrounding host membrane, such as in subpanels A and E, highlighted by white arrowheads), bacteria in phagolysosomes (loose vesicles with one surrounding host membrane, such as in subpanels C and G), or bacteria in autophagosomes (with two or more surrounding host membranes, such as in subpanels D and H); “Cytosol” represents bacteria with no surrounding host membranes (such as in subpanels B and F). Bars, 500 nm. (B) Quantification of GFP-Mtb subcellular localizations in resting (blue-outlined bars) or activated (red-outlined bars) GM-CSF and M-CSF macrophages at 2, 24, and 48 h after infection. Quantification was performed by stereological analysis of TEM images of resin sections of infected macrophages. Data are from one experiment, and the total numbers of cells analyzed in each condition (n) are shown. Inhibition of host cell necrosis limits hibitor (Degterev et al., 2013). Resting and activated GM-CSF M. tuberculosis replication and M-CSF macrophages were infected with GFP-Mtb for 7 d Because M.  tuberculosis most efficiently grows within ne- (Fig.  5  A), and the percent inhibition of M.  tuberculosis rep- crotic cells, we reasoned that we should be able to modulate lication was determined (Fig.  5  B). In parallel, the proportion bacterial replication using chemical inhibitors of necrosis. of PI-positive cells was measured to confirm that the inhibitors We used three inhibitors: IM-54, a potent inhibitor of oxida- were indeed preventing cell death (Fig. 5 C). We also confirmed tive stress–induced necrosis (Dodo et al., 2005); MCC950, a that the inhibitors themselves did not affect M.  tuberculosis specific inhibitor of the NLRP3 inflammasome (Coll et al., growth at the same concentration in vitro (Fig. S3 A). Notably, 2015); and Necrostatin-1s (Nec-1s), a RIP1 kinase (RIPK1) in- all of the drug treatments inhibited to differing extents the mean 588 JCB • Volume 216 • NumBer 3 • 2017 Figure 4. M. tuberculosis replicates in a cellular necrotic environment. (A) Snapshots taken from live-cell imaging of GM-CSF macrophages infected with GFP-Mtb over 4 d; the graph below shows how the growth of GFP-Mtb occurs only after the cell becomes PI positive. (B) SBF SEM and TEM were performed on the same sample as in A. This allowed for the GFP-Mtb signal to be overlaid with the EM images. Subpanels i–iii show magnified regions of the ultra- structure taken from the TEM image. The bottom subpanel shows segmentation of the PM in red using the SBF SEM data (with reference to the adjacent TEM image) with the LM data and TEM image overlay. Black arrows indicate PM damage. M.  tuberculosis replication compared with the DMSO control repair program, thereby inducing necrosis and a release of in each macrophage population analyzed (Fig. 5, A and B). In- bacteria that can then reinfect surrounding cells (Divangahi et terestingly, Nec-1s treatment in resting M-CSF macrophages al., 2009). In addition, our data revealed that M. tuberculosis increased the mean bacterial burden; consistent with this, in this induces PM damage, seemingly to disarm the host cell, and particular condition, Nec-1s actually increased the proportion then replicates in this nutrient-rich environment. Once repli- of PI-positive cells (Fig. 5 C). This suggests that RIPK1 could cation occurs in this protected and rich environment, bacteria be differentially activated in the different macrophage popula- immediately get access to the extracellular media after rein- tions. The lack of effect on cell death and bacterial replication fection. Our data therefore provide evidence suggesting that in M-CSF macrophages was reversed by IFN-γ activation, in- M.  tuberculosis actively avoid the extracellular environment dicating that this cytokine activates in macrophages a signaling that can be detrimental for long periods of time. A study per- pathway via RIPK1 that results in necrosis, as was previously formed with a high burden of bacterial infection (Lee et al., reported in fibroblasts (Thapa et al., 2013). The precise mech- 2006) could mimic some of our results, but our experimen- anism and signaling pathways that regulate this differential tal setting is different and could reflect more the cells that in susceptibility to PM integrity after infection and IFN-γ acti- vivo are infected with fewer bacteria and not the ones that vation between M-CSF and GM-CSF macrophages remains to take up large bacterial aggregates. In our system, we observed be investigated. Importantly, the necrosis inhibitors were more by live-cell imaging that once macrophages internalized big efficient in activated than in resting M-CSF macrophages, cor - clumps of bacteria (either by cell transfer or efferocytosis), relating with the increased replication and enhanced cell death cell death followed rapidly (unpublished data). Collectively, observed (Figs. 1 E and 2 B). Thus, we concluded that inhibi- in this study, we analyzed for the first time the dynamic repli- tion of necrosis resulted in less efficient M. tuberculosis growth. cation of M. tuberculosis in human macrophages by long-term Our results highlight the possibility that bacteria repli- live-cell imaging in different functional populations of macro- cate within cells after PM damage before being released into phages. Our studies provide insights into the dynamic interac- the extracellular medium. If this scenario holds in vivo, extra- tions between human macrophages and M. tuberculosis. Thus, cellular bacteria would originate from two different sources: in addition to the known role of dissemination, leaky cells un- the pool that replicated intracellularly in damaged cells and dergoing early stages of necrosis represent a niche that helps a pool of bacteria that grows extracellularly. Necrotic cells bacteria to grow even before getting freely into the extracel- represent a much better environment for bacterial replica- lular environment. Our studies uncover an important aspect tion, as they are rich in nutrients in a host cell with severely in the biology of tuberculosis, revealing that M.  tuberculosis affected cell-autonomous immunity. Our data are consistent continues to replicate in host cells after PM integrity is lost with the idea that virulent mycobacteria inhibits the cell PM before disseminating to other cells. mtb replicates within necrotic human macrophages • lerner et al. 589 Figure 5. Inhibition of host cell necrosis limits M. tuberculosis replication. (A) Example confocal microscopy images showing GFP-Mtb (green), PI (red), and DAPI (blue) staining of resting or activated GM-CSF or M-CSF macrophages at 7 d after infection in control cells (DMSO only) or cells treated with 10 µM of the shown necrosis inhibitors. Cells outlined in white dotted lines are considered to be PI positive. Bars, 10 µm. (B) Quantification of the effect of treating infected resting (blue) or activated (red) GM-CSF (dots) or M-CSF (squares) with three necrosis inhibitors compared with untreated control macrophages at 7 d after infection with GFP-Mtb. (C) Quantification of the effect of treating infected resting (blue) or activated (red) GM-CSF (dots) or M-CSF (squares) with three necrosis inhibitors on the proportion of cells with a PI-positive (PI+) nucleus compared with untreated control macrophages at 7 d after infection with GFP-Mtb. Data points show the percent reduction for each independent experiment, and the bars show the overall mean. 590 JCB • Volume 216 • NumBer 3 • 2017 Materials and methods min and permeabilized with 0.05% saponin/1% BSA in PBS for 10 min. Coverslips were incubated with the mouse anti–human LAMP-2 Preparation of monocyte-derived macrophages antibody (Hybridoma Bank) at a dilution of 1:100 for 1  h at room Peripheral blood mononuclear cells (PBMCs) from healthy anonymous temperature, followed by the goat anti–mouse Alexa Fluor 546 anti- donors were isolated from leukocyte provided by the National Blood body (Jackson ImmunoResearch Laboratories, Inc.) at a dilution of and Transplant Service, UK. Red blood cells were removed by centrif- 1:800 for 1  h at room temperature. Nuclei were stained with DAPI, ugation on Ficoll-Paque (28-4039-56 AD; GE Healthcare) and red cell and coverslips were mounted with mounting medium (Dakok). lysis buffer (11814389001; Sigma-Aldrich). Recovered PBMCs were Samples were analyzed using a laser-scanning confocal microscope washed to remove platelets. Monocytes were isolated from PBMCs (SP5; Leica Biosystems). using a magnetic cell separation system with anti-CD14 mAb-coated For analysis, RGB images or frames were split into separate microbeads (130-050-201; Miltenyi Biotec). CD14-positive mono- color channels. Bacteria (green channel) were thresholded per single cytes were cultured in complete RPMI 1640 medium (Lonza) sup- cell, and the mean fluorescence intensity of LAMP-2 associated to the plemented with 9.1% heat-inactivated FCS supplemented with either phagosome was measured for each frame by redirecting measurements 50 ng/ml GM-CSF or 50 ng/ml M-CSF (130-093-862/130-093-963; to the channel of interest. Fluorescence intensity values were plotted Miltenyi Biotec) at 37°C under a humidified 5% CO atmosphere for using Prism software (GraphPad Software). The number of GFP-posi- 6 d. Media were replaced at 3 d. On day 6, the cells were washed and tive pixels per cell (or a value called RAWIntDen, which correlates to detached with 0.5 mM EDTA in ice-cold PBS and plated in a 24-well the number of GFP-positive pixels multiplied by 255) was plotted as a plate (containing coverslips for microscopy) or a live-cell imaging dish measure of the intracellular bacterial replication over time. The propor- at a concentration of 2 × 10 cells/well with complete RPMI media (for tion of infected cells per sample was also determined. resting macrophages). Macrophages were either treated overnight or after infection with recombinant human IFN-γ (PHC 4031; Gibco) at a Quantification of bacterial burden with cell death inhibitors concentration of 100 U/ml. Out-of-focus cells were manually cropped from tile scans, and the GFP per cell was quantified with an in-house R script using the RBio- Bacterial culture Formats (Oleś, 2014) and EBImage (Pau et al., 2010) packages (Fig. GFP-Mtb or M.  tuberculosis H37Rv ΔRD1 expressing EGFP (GFP- S3 B). For each image, nuclei were identified by DAPI staining, seg- Mtb ΔRD1) was grown at 37°C in Middlebrook 7H9 medium (Difco mented by watershed, and used as seeds for a Voronoi segmentation Laboratories) containing 0.5% glycerol and 0.05% Tween 80 and was of the field by Euclidian distance. Pixels in the GFP channel above supplemented with 10% oleic acid–albumin–dextrose–catalase supple- a predetermined threshold were counted per segment, and segments ment (OADC; BD) or on Middlebrook 7H10 plates (Difco Laborato- with ≥5 GFP-positive pixels were considered infected cells. A mini- ries) supplemented with 10% OADC. mum of 349 cells were analyzed per condition across three biological replicates, with a mean of 1,450. Infection of human monocyte-derived macrophages Macrophages were seeded onto 24-well culture plates the day be- PM integrity in fixed cells fore the infection. M.  tuberculosis cultures were grown to the midex- Before collecting the coverslips, the macrophages were stained by ponential phase. On the day of infection, the bacteria were pelleted 25 µg/ml PI (Sigma-Aldrich) in RPMI for 15 min at room temperature and washed with PBS and RPMI/10% FCS at 3,000 rpm for 5 min. and fixed in 3% PFA overnight at 4°C.  Images were obtained using An equal volume of sterile glass beads (2.5–3.5 mm) that matched an SP5 confocal microscope with an argon laser emitting dually at the pellet size was added (usually three to four beads) and then vig- 488 nm for excitation of GFP and at 561 nm for PI. To determine the orously shaken for 1 min to break up bacterial clumps. The bacteria replication of bacteria in PI-positive or -negative cells, bacteria (green were washed again with complete RPMI at 1,200 rpm for 5 min. The channel) were thresholded per single cell, and the pixel numbers were supernatant was transferred into a new tube, and the measured OD measured in each positive and negative propidium cell. Values were plot- was then diluted to 0.1 in complete RPMI. Next, cells were infected ted using Prism software. with M. tuberculosis at an MOI of 1 for 2 h and then washed once with PBS and incubated with complete RPMI media containing IFN-γ and/ Live-cell imaging or 10 µM of the necrosis inhibitors IM-54 (Sigma-Aldrich), MCC950 For live-cell imaging, 10 macrophages were seeded on 35-mm (Cayman Chemicals), or Nec-1s (Source Biosciences), where relevant. glass-bottom dishes (MatTek Corporation) and treated overnight with 100 U/ml IFN-γ in RPMI complete media. Cells were washed with PBS CFU determination and infected with M. tuberculosis at an MOI of 1 for 2 h. After infection, For counting bacterial viability, macrophages were washed once with cells were washed with PBS and replaced with RPMI complete media. PBS and lysed with water–Tween 80 (0.05%) for 30 min at room tem- 24  h after infection, the media were replaced with imaging medium: perature. The lysed solution from triplicate wells was taken and seri- complete RPMI medium with 0.4 µg/ml PI. M.  tuberculosis was also ally diluted in PBS in 10-fold steps until 1:1,000 dilution. 25 µl from grown in imaging medium alone by simply adding the infection inocu- each dilution was plated in triplicate onto complete 7H11 agar plates. lum into a glass-bottom dish containing no macrophages. Imaging was Agar plates were incubated for 2–3 wk at 37°C. Triplicate plates were performed with an SP5 laser-scanning confocal microscope equipped averaged, and CFU were calculated and plotted as the mean CFU per with an environment control chamber (EMB LEM). During imaging, a milliliter from triplicate wells. single focal plane was monitored in time (xyt scanning mode) using a 63×/1.4 NA HCX-PLA PO oil objective, a 488-nm argon laser, and a Indirect immunofluorescence 561-nm diode-pumped solid-state (DPSS) laser (scanner frequency 400 Coverslips were washed once with PBS and fixed overnight with 3% Hz; line averaging 2) using photomultiplier detectors and/or hybrid de- PFA (Electron Microscopy Sciences) in PBS, pH 7.4, at 4°C.  After tectors (HyDs) at a scanning resolution of 1,024 × 1,024 pixels. Analysis fixation, PFA was removed and replaced with PBS. Then, cells were was performed using FIJI (ImageJ; National Institutes of Health). For incubated with 50  mM glycin/PBS, pH 7.4 (Sigma-Aldrich), for 10 analysis, RGB images or frames were split into separate color channels. mtb replicates within necrotic human macrophages • lerner et al. 591 Bacteria (green channel) were thresholded per single cell. Pixels were glutaraldehyde and 4% PFA in 0.1 M phosphate buffer. The cells were quantified and plotted using Prism software. Bacteria replication was cal- again imaged before processing for serial block face scanning EM culated in PI-positive and -negative cells for each time point of interest. (SBF SEM) commenced, using a modified National Center for Micros- copy and Imaging Research protocol (Lerner et al., 2016). Coverslips EM were postfixed in 2% osmium tetroxide/1.5% potassium ferrocyanide GM-CSF and M-CSF macrophages were prepared as described in the for 1 h on ice, incubated in 1% wt/vol thiocarbohydrazide for 20 min Preparation of monocyte-derived macrophages section (2 × 10 cells before a second staining with 2% osmium tetroxide, and then incubated seeded per T25 flask) and infected at an MOI 1 for 5 h, with 200 ng/ml overnight in 1% aqueous uranyl acetate at 4°C. Cells were stained with IFN-γ added after infection where appropriate. The protocol for resin Walton’s lead aspartate for 30 min at 60°C and dehydrated through embedding was adapted from Rohde et al. (2012). Samples were fixed an ethanol series on ice, incubated in a 1:1 propylene oxide/Durcu- by adding warm 2% glutaraldehyde in 200 mM Hepes, pH 7.4, directly pan resin mixture, and then embedded in Durcupan resin according to to the cell culture medium at a 1:1 volume ratio. After 5 min, the mix- the manufacturer’s instructions (TAAB Laboratories Equipment Ltd.). ture of the fixative and medium was replaced with 1% glutaraldehyde SBF SEM images were collected using a 3View 2XP system (Gatan in Hepes buffer, and the samples were incubated overnight at 4°C. Cells Inc.) mounted on a Sigma VP scanning electron microscope (ZEISS). were scraped and embedded in 1% low melting point agarose. Agarose Images were collected at 1.8 kV using the high-current setting with a blocks were postfixed with 2% osmium tetroxide solution containing 20-µm aperture at 6 Pa chamber pressure and a 2-µs dwell time. The 1.5% potassium ferricyanide for 1 h on ice and then stained with 0.5% dataset was 33.59 × 33.59 × 0.75 µm in xyz, consisting of 15 serial tannin for 20 min and with 1.5% aqueous uranyl acetate for 1 h. Next, images of 50-nm thickness and a pixel size of 4.1 × 4.1 nm. The total cells were dehydrated at room temperature using a graded ethanol se- volume of the dataset was ∼846 µm . After SBF SEM imaging, the ries (70, 80, 90, 95, and 100%), followed by gradual infiltration with sample was removed, and 70-nm sections were cut on a ultramicrotome Spurr’s resin (Polysciences) over 2 d. Ultrathin sections (∼70 nm) were (UC7; Leica Biosystems). Images were then collected in a transmission cut with an ultramicrotome Ultracut EM UCT ultramicrotome (Leica electron microscope (Tecnai G2 Spirit BioTwin; Thermo Fisher Scien- Microsystems) and contrasted with 0.2% lead citrate. Cells were exam- tific) using a charge-coupled device camera (Orius; Gatan Inc.). The ined with a transmission electron microscope (JEM-1400; JEOL). The SBF SEM dataset was segmented manually using Amira 6.0 (Thermo images were recorded digitally with a TemCam-F216 camera (TVI PS). Fisher Scientific). The PM was manually selected by morphology with Between 24 and 89 infected cells per sample were imaged by system- reference to transmission EM (TEM) images (in which membrane gap atic and random sampling. Cross points of the stereological test grid morphology was more readily apparent, because of the higher struc- over bacteria were counted with regard to the subcellular localization tural resolution). Only larger gaps (greater than ∼100 nm) were seg- of bacteria, and fractions of membrane-bound and cytosolic bacteria mented to account for the reduced resolution in the SBF SEM images were calculated from total counts per sample. The data were plotted as and to prevent false positive assignment of gaps. the proportion of M. tuberculosis in the cytosol versus the proportion in membrane-bound compartments for each condition. CLEM alignment Alignment between light microscopy (LM), SBF SEM, and TEM was CLEM performed using the BigWarp plugin in Fiji. We first mapped the TEM GM-CSF macrophages were prepared as described in the Preparation image onto the final image of the SBF SEM image stack at full resolu- of monocyte-derived macrophages section, and cells were seeded to tion, using landmarks in 2D. The warped and exported TEM image was ∼30–50% confluence onto gridded dishes (MatTek Corporation) in then appended to the SBF SEM stack to form a hybrid EM stack. Note 400 µl RPMI + 10% FCS for a 2-h attachment time at 37°C and 5% that the slightly different slice thickness of the SEM stack and single CO . They were then infected with GFP-Mtb at an MOI of 1 as de- TEM slice can be essentially ignored because the axial diffraction limit scribed in the Infection of human monocyte-derived macrophages sec- of the LM image is much larger than the EM slice thickness. The LM tion and incubated for 24 h. The infection medium was then replaced image stack was then mapped onto the hybrid EM stack (containing with 2 ml RPMI + 10% FCS containing 0.4 µg/ml PI, and then the dish both SBF SEM and TEM data) using landmarks in 3D at corresponding was securely fastened into a custom-made dish holder for the stage positions in the two stacks. of a confocal microscope ready for imaging. The microscope was set up with the argon laser set to 20% and the DPSS laser switched on. A Flow cytometry sequential scan for each channel was set up: (a) argon 488-nm laser (set Isolated monocytes and differentiated macrophages were incubated to 10%) with the HyD in brightR mode; or (b) DPSS 561-nm laser (set with an Fc receptor–blocking antibody (Human BD Fc Block; BD) on to 2%) with the HyD in brightR mode, and simultaneously a brightfield ice for 20 min, followed by staining with anti–CD119-PE (clone GIR- image was obtained using SCAN-BF mode with gain set to 493 V. Ini- 208; eBioscience), anti–CD86-BV421 (clone 2331), anti–CD206-APC tially, a 10× objective was used to determine the grid reference of the (clone 19.2), anti–CD14-AF488 (clone M5E2), or anti–CD16-APC-H7 cell of interest. A 63× oil objective was then used with the following (clone 3G8; BD) for 20 min at room temperature in the dark. Cells imaging conditions: 1,024 × 1,024–pixel resolution; line averaging 2; were washed once in PBS, fixed in 1% PFA, and acquired on a flow zoom 1; and frames every 15 min. At the desired time point, warm cytometer (Fortessa; BD) or a FACS CyAnTM (Dako). Respective double-strength fixative (8% PFA in 0.2 M phosphate buffer, pH 7.4) isotype controls were mouse IgG1 K-PE (clone P3.6.2.8.1; eBiosci- was directly added to the cell culture medium (1:1 ratio) for 15 min ence), mouse IgG1, κ-BV421 (clone X40), mouse IgG1, κ-APC (clone at room temperature. The cell of interest was relocated and imaged. MOPC-21), mouse IgG2a, κ-AF488 (clone G155-178), and mouse The fixative was then removed, and a 1-mm layer of 4% low melting IgG1, κ-APC-H7 (clone MOPC-21; BD). point agarose (Sigma-Aldrich) in 0.1 M phosphate buffer was added to form a protective layer over the cells. This was placed on ice quickly Western blotting to solidify. Next, 4% PFA in 0.1  M phosphate buffer was added for Macrophages were washed once with PBS and lysed in NP-40 15 min at room temperature, and again the cells were imaged. Finally, for 40 min on ice. 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M.C. Inserra, I. Vetter, L.S. Dungan, B.G. Monks, A. Stutz, et al. 2015. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat. Med. 21:248–255. Online supplemental material Degterev, A., J.L.  Maki, and J.  Yuan. 2013. Activity and specificity of Fig. S1 is a characterization of GM-CSF and M-CSF macrophages, necrostatin-1, small-molecule inhibitor of RIP1 kinase. Cell Death Differ. including the expression of specific differentiation markers and the 20. http ://dx .doi .org /10 .1038 /cdd .2012 .133 IFN-γ receptor. Also shown are Western blots of the IFN-γ–induced Divangahi, M., M.  Chen, H.  Gan, D.  Desjardins, T.T.  Hickman, D.M.  Lee, protein IDO. Fig. S2 (A–C) measures the GFP per cell, the propor- S.  Fortune, S.M.  Behar, and H.G.  Remold. 2009. Mycobacterium tuberculosis evades macrophage defenses by inhibiting plasma membrane tion of infected cells, and the proportion of PI-positive cells in rest- repair. Nat. 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Cell Host Microbe. 12:432– lication of GFP-Mtb in activated GM-CSF macrophages (some of 444. http ://dx .doi .org /10 .1016 /j .chom .2012 .09 .007 which are positive for PI). Lacey, D.C., A. Achuthan, A.J. Fleetwood, H. Dinh, J. Roiniotis, G.M. Scholz, M.W.  Chang, S.K.  Beckman, A.D.  Cook, and J.A.  Hamilton. 2012. Defining GM-CSF– and macrophage-CSF–dependent macrophage Acknowledgments responses by in vitro models. J.  Immunol. 188:5752–5765. http ://dx .doi .org /10 .4049 /jimmunol .1103426 We would like to thank the Electron Microscopy Laboratory at the Lechartier, B., J.  Rybniker, A.  Zumla, and S.T.  Cole. 2014. Tuberculosis drug discovery in the post-post-genomic era. EMBO Mol. Med. 6:158–168. Department of Biosciences, University of Oslo. We also thank the Lee, J., and H. Kornfeld. 2010. Interferon-γ regulates the death of M. tuberculo- Host-Pathogen Interactions in Tuberculosis Laboratory and Douglas sis-infected macrophages. J. Cell Death. 3:1–11. Young for useful discussions and comments on the manuscript. Lee, J., H.G. Remold, M.H. Ieong, and H. Kornfeld. 2006. Macrophage apoptosis in response to high intracellular burden of Mycobacterium tuberculosis This work was supported by the Francis Crick Institute (to M.G. Gutierrez), is mediated by a novel caspase-independent pathway. J.  Immunol. 176:4267–4274. http ://dx .doi .org /10 .4049 /jimmunol .176 .7 .4267 which receives its core funding from Cancer Research UK (grant Lerner, T.R., S. Borel, and M.G. Gutierrez. 2015. The innate immune response FC001092), the UK Medical Research Council (grants MC_UP_1202/11 in human tuberculosis. Cell. Microbiol. 17:1277–1285. http ://dx .doi .org and FC001092), and the Wellcome Trust (grant FC001092). /10 .1111 /cmi .12480 Lerner, T.R., C.  de Souza Carvalho-Wodarz, U.  Repnik, M.R.  Russell, The authors declare no competing financial interests. 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Mycobacterium tuberculosis replicates within necrotic human macrophages

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Pubmed Central
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0021-9525
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DOI
10.1083/jcb.201603040
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Abstract

JCB: Report Mycobacterium tuberculosis replicates within necrotic human macrophages 1 1 1 3 2 1 Thomas R. Lerner, * Sophie Borel, * Daniel J. Greenwood, * Urska Repnik, Matthew R.G. Russell, Susanne Herbst, 2 2 3 1 Martin L. Jones, Lucy M. Collinson, Gareth Griffiths, and Maximiliano G. Gutierrez 1 2 Host-Pathogen Interactions in Tuberculosis Laboratory and Electron Microscopy Science Technology Platform, The Francis Crick Institute, London NW1 1AT, England, UK Department of Biosciences, University of Oslo, 0371 Oslo, Norway Mycobacterium tuberculosis modulation of macrophage cell death is a well-documented phenomenon, but its role during bacterial replication is less characterized. In this study, we investigate the impact of plasma membrane (PM) in- tegrity on bacterial replication in different functional populations of human primary macrophages. We discovered that IFN-γ enhanced bacterial replication in macrophage colony-stimulating factor–differentiated macrophages more than in granulocyte–macrophage colony-stimulating factor–differentiated macrophages. We show that permissiveness in the different populations of macrophages to bacterial growth is the result of a differential ability to preserve PM integrity. By combining live-cell imaging, correlative light electron microscopy, and single-cell analysis, we found that after infection, a population of macrophages became necrotic, providing a niche for M. tuberculosis replication before escaping into the extracellular milieu. Thus, in addition to bacterial dissemination, necrotic cells provide first a niche for bacterial rep- lication. Our results are relevant to understanding the environment of M. tuberculosis replication in the host. Introduction The intracellular lifestyle of Mycobacterium tuberculosis rep- nity and inflammation (Vogt and Nathan, 2011; Martinez and resents a crucial stage in the pathogenesis of tuberculosis, and Gordon, 2015). Macrophages differentiated in vitro are often successful drug discovery programs have to include in vitro referred to as classical or alternatively activated macrophages, studies using infected host cells (Young et al., 2008; Lechart- or M1 and M2 macrophages, respectively. Several stimuli are ier et al., 2014). Most of the in vitro studies of infection with used to differentiate macrophages in vitro, and in this context, M. tuberculosis rely on infected macrophages and survival anal- granulocyte–macrophage colony-stimulating factor (GM-CSF; ysis by colony-forming units (CFUs), luciferase, or fluorescent considered M1)– and macrophage colony-stimulating factor reporters. The use of primary human macrophages is of clear (M-CSF; considered M2)–differentiated human macrophages advantage because there are some fundamental differences be- are widely used models of macrophage biology (Lacey et al., tween human macrophages and those originating from other 2012; Martinez and Gordon, 2015). GM-CSF macrophages species. Important differences are obvious, such as the lack of a are generally proinflammatory and display enhanced antigen large set of IFN-γ–inducible GTPases that are exclusively pres- presentation, phagocytosis, and microbicidal capacity. How- ent in mouse macrophages (Kim et al., 2012) or the differential ever, M-CSF macrophages display an antiinflammatory cyto- production of nitric oxide when compared with mouse macro- kine profile after stimulation (Lacey et al., 2012; Martinez and phages (Thomas and Mattila, 2014). Gordon, 2014). Although GM-CSF– and M-CSF–differenti- In vivo, macrophages are composed of a very hetero- ated macrophages clearly respond differently to extracellu- geneous population as a result of multiple differentiation and lar stimuli (Lacey et al., 2012), the nature of this differential activation stimuli present in tissues (Martinez and Gordon, response is less clear. 2014). Numerous experimental approaches are used to differ- Adding more complexity to in vitro systems of infection, entiate human macrophages in vitro with the aim of mimicking there are a wide range of methods described in the literature to the heterogeneity present in tissue macrophages during immu- activate macrophages. One of the key cytokines used for the activation of human macrophages is IFN-γ. This cytokine is a key modulator of the phagocytic and mycobactericidal activity *T.R. Lerner, S. Borel, and D.J. Greenwood contributed equally to this paper. of mouse macrophages (Flynn and Chan, 2001). Although stud- Correspondence to Maximiliano G. Gutierrez: [email protected] ies show that genetic errors of IFN-γ immunity result in severe Abbreviations used: CFU, colony-forming unit; CLEM, correlative live-cell and tuberculosis in children (Abel et al., 2014), the role of IFN-γ in electron microscopy; DPSS, diode-pumped solid-state; GM-CSF, granulocyte– macrophage colony-stimulating factor; HyD, hybrid detector; IDO, indoleam- the antimycobacterial activity of human macrophages and its ine 2,3-dioxygenase; LM, light microscopy; M-CSF, macrophage colony- stimulating factor; PBMC, peripheral blood mononuclear cell; PI, propidium iodide; PM, plasma membrane; SBF SEM, serial block face scanning EM; © 2017 Lerner et al. This article is available under a Creative Commons License (Attribution 4.0 International, as described at https ://creativecommons .org /licenses /by /4 .0 /). TEM, transmission EM. The Rockefeller University Press $30.00 J. Cell Biol. Vol. 216 No. 3 583–594 https://doi.org/10.1083/jcb.201603040 JCB 583 T H E J O U R N A L O F C E L L B I O L O G Y role in pulmonary tuberculosis in adults is still unclear (Abel et at the single-cell level. A quantitative analysis of the number of al., 2014; Lerner et al., 2015). GFP-positive pixels per macrophage confirmed that M.  tuber- Although in vitro and in vivo studies highlight the im- culosis replicated over time at the single-cell level (Fig.  1  B). portance of host cell death modes during mycobacterial control In parallel, the proportion of M. tuberculosis–infected cells per or dissemination, the mode of host cell death in the pathogen- sample significantly increased between hour 2 and day 7, sug- esis of human tuberculosis is not completely understood. In gesting that the infection was spreading between cells (Fig. 1 C). tuberculosis, apoptosis is generally considered to be a part of Activation of GM-CSF macrophages with IFN-γ had only a lim- a host protective response, whereas necrosis is considered to ited effect on bacterial replication (Fig. 1, A–C). We observed a be a pathway for bacterial dissemination and granuloma cavity similar level of M. tuberculosis replication in M-CSF compared formation (Behar et al., 2010; Ramakrishnan, 2012; Wong and with GM-CSF macrophages by CFUs (Fig.  1  D) and also by Jacobs, 2016). Apoptosis is believed to help with the eradica- microscopy at the single-cell level (Fig.  1, E and F). Interest- tion of M. tuberculosis (Keane et al., 2000; Behar et al., 2010), ingly, we observed that at 7 d after infection in IFN-γ–activated and, unsurprisingly, this pathogen has strategies to inhibit apop- M-CSF macrophages, the mean burden of M.  tuberculosis per tosis (Velmurugan et al., 2007). Apoptosis is protective in part cell was much higher than all of the other conditions (Fig. 1, B because bacteria are internalized via efferocytosis and subse- and E). Furthermore, infection of macrophages with GFP-Mtb quently eliminated (Martin et al., 2012). However, necrosis of ΔRD1 (which lacks the ESX-1 type VII secretion system; Fig. infected cells helps bacterial dissemination. M. tuberculosis in- S2, A–D) revealed that this mutant strain had a reduced bacte- hibits the plasma membrane (PM) repair pathway, resulting in rial burden (Fig. S2, A and D) and a reduced cell-to-cell spread the progression to necrosis and mycobacterial release into the (Fig. S2 B) compared with wild type and lacked the ability to extracellular environment (Divangahi et al., 2009). Neverthe- induce cell death overall (Fig. S2 C), even after 5 d of infection less, whether host cell necrosis directly affects bacterial replica- (Fig. S2 D and Video  1). Bacterial viability was independent tion has not yet been demonstrated. of the time of activation or IFN-γ concentration because pre- In this study, we show that M.  tuberculosis replicates stimulation overnight or treatment after infection with a higher to a similar extent in GM-CSF– and M-CSF–differentiated concentration of IFN-γ rendered similar results (not depicted). macrophages (GM-CSF macrophages and M-CSF macro- In resting GM-CSF macrophages, bacteria were mostly local- phages, respectively). Remarkably, IFN-γ activation enhanced ized in compartments negative for the phagolysosomal marker M.  tuberculosis replication in M-CSF macrophages but not in LAMP-2, as was expected for bacteria residing in an arrested GM-CSF macrophages, and this correlated with an increased early phagosome (Fig. 1, G and H). IFN-γ activation did not sig- susceptibility to necrosis compared with the other macrophage nificantly change this localization (Fig. 1, G and H). In M-CSF populations. Long-term live-cell imaging of infected macro- macrophages, M. tuberculosis was also localized in LAMP-2– phages revealed that at the single-cell level, M.  tuberculosis negative compartments (Fig. 1, I and J), but IFN-γ increased the infection induces loss of PM integrity to replicate in damaged fraction of mycobacteria found in late endocytic compartments cells before entering the extracellular milieu. Collectively, after 48 h of infection (Fig. 1, I and J). Overall, our results are in our data define a differential susceptibility to the necrosis of agreement with a study indicating that in GM-CSF and M-CSF GM-CSF– and M-CSF–differentiated macrophages after in- macrophages, M.  tuberculosis grows at similar rates (Tailleux fection and IFN-γ activation. Importantly, these differences et al., 2003), but our results are also in disagreement with other are critical for the replication of M.  tuberculosis in damaged studies (Douvas et al., 1985; Akagawa et al., 2006; Wong and cells before dissemination. Jacobs, 2013). It is likely that differences in donor samples, my- cobacterial strains, MOI, and also cell differentiation protocols are explanations for discrepancies among studies. However, Results and discussion our data highlight important physiological differences between GM-CSF and M-CSF macrophages during the late stages of M. tuberculosis replicates in both resting M.  tuberculosis infection in vitro. We suggest that the alveo- and IFN-γ–activated GM-CSF– and M-CSF– lar macrophage phenotype conferred by GM-CSF (Akagawa, differentiated human macrophages 2002) makes the macrophages permissive to M.  tuberculosis First, we characterized each macrophage subset by flow cytom- replication, allowing bacterial dissemination after primary in- etry using surface markers of activation. Then, we verified that fection of the lungs. In contrast, in M-CSF macrophages, IFN-γ they showed a mature phenotype (CD86 positive; Fig. S1 A) will promote bacterial replication and, later on, dissemination. and were responsive to IFN-γ by looking at the expression of The interplay of these differentiation factors, their contribution the IFN-γ receptor (Fig. S1 B) and by inducing indoleamine to bacterial replication and dissemination in vivo, and the phys- 2,3-dioxygenase (IDO) and other IFN-γ–inducible proteins iological differences between human and mouse macrophages (Fig. S1 C and not depicted). Next, we analyzed the replica- remain to be investigated. Although we observed variability tion of M. tuberculosis H37Rv expressing EGFP (GFP-Mtb) in across the donors tested in this work, IFN-γ unexpectedly did GM-CSF human primary macrophages by CFUs. We observed not have any major impact on bacterial growth in GM-CSF that on average, bacteria replicated by ∼2.5-fold after 72 h of macrophages in vitro. It is not clear why IFN-γ did not control infection (Fig.  1  A). Beyond the 72-h time point, it was not bacterial replication in our system compared with other stud- possible to determine intracellular bacterial replication using ies. A possible cause is that our study was not performed under CFUs because the dying cells detached, resulting in an under- hypoxic conditions, which were shown to be antimycobacterial estimation of the actual number of intracellular bacteria in the (Vogt and Nathan, 2011). Also, reported differences in nitric sample. Therefore, to analyze longer periods of infection, we oxide production between human and mouse macrophages can used a microscopy-based method, which more closely reflected account for reduced potency in human cells (Thomas and Mat- how the infection progressed over an extended in vitro infection tila, 2014). Our data are consistent with a study showing that in 584 JCB • Volume 216 • NumBer 3 • 2017 Figure 1. Dynamic growth of M. tuberculosis in distinct subpopulations of human primary macrophages. (A) Intracellular replication of GFP-Mtb in resting (blue line) or IFN-γ–activated (red line) GM-CSF– differentiated macrophages, normalized to 2 h and expressed as the percent increase in CFUs over time. (B) Intracellular replication of GFP-Mtb in resting (blue dots) or IFN-γ–activated (red dots) GM-CSF–differentiated macrophages (expressed as the mean num- ber of GFP-positive pixels per cell) at 2 h, 72 h, and 7 d after infection. (C) Quantification of the mean proportion of GFP-Mtb infected cells in resting (blue dots) or activated (red dots) GM-CSF–differentiated macrophages at 2 h, 72 h, and 7 d after infection. (D–F) Same as in A–C, but with M-CSF–differentiated macrophages. (A–F) Data presented are means ± SEM from three (A, B, D, and E) or at least three (C and F) independent experiments. (G) Analysis of the association of GFP-Mtb with LAMP-2 in resting and activated GM-CSF macrophages infected at an MOI of 1 for 72 h. Representative confocal microscopy images of GFP-Mtb and LAMP-2 marker. Nuclei were stained with DAPI. Insets show magnifications of the outlined cells, highlighting the low level of as- sociation of LAMP-2 to bacteria. (H) Quantification of the association of GFP-Mtb with LAMP-2 in resting (blue) or activated (red) GM-CSF macrophages. The number of bacteria analyzed for each condition is displayed as well as the proportions of M. tuberculosis that are considered to be positive for LAMP-2 association (i.e., a mean intensity value of ≥150). (I and J) Same as G and H, but with M-CSF–differentiated macrophages. Bars, 10 µm. human macrophages at 5–10% O , IFN-γ induced the growth detect macrophage extracellular traps in our system. Our data of mycobacteria and the induction of macrophage extracellu- are also in agreement with studies in mouse macrophages show- lar traps (Wong and Jacobs, 2013). Our data are also consistent ing that when a threshold of intracellular bacteria is reached, with early studies showing that IFN-γ enhanced M. tuberculo- IFN-γ had little influence on bacterial growth (Repasy et al., sis growth (Douvas et al., 1985), although we were not able to 2013). Additionally, studies in human patients—particularly mtb replicates within necrotic human macrophages • lerner et al. 585 Figure 2. Differential susceptibility to necrosis in macrophages leads to M. tuberculosis growth. (A) Quantification of the proportion of cells that had a PI-positive (PI+) nucleus in resting (blue dots) or activated (red dots) GM-CSF macrophages. A PI-positive nucleus indicates that the cell membrane has become permeable to the extracellular medium and therefore is likely to be dead. Each data point represents one independent experiment, and the bars show means ± SEM. (B) Same as in A, but with M-CSF macrophages. (C) Representative confocal images of macrophages infected with GFP-Mtb (green) and labeled with PI (red). Macrophages were infected at an MOI of 1 for 2 h. 7 d after infection, macrophages were stained with 25 µg/ml PI in RPMI for 15 min and fixed. PI-positive cells are outlined. (D) Same as in C, but with M-CSF macrophages. Bars, 10 µm. (E) Quantification of GFP-Mtb replication in 586 JCB • Volume 216 • NumBer 3 • 2017 children—have shown that IFN-γ is a key antimycobacterial cy- activation accelerated both PM damage and bacterial cell growth tokine (Abel et al., 2014). It remains to be defined which in vitro (Fig.  2, I and J). We concluded that during in vitro infection, systems with human macrophages better capitulate macrophage loss of PM integrity permits continued M.  tuberculosis repli- biology in vivo. In this context, cytokines such as IFN-γ could cation and that the extent of cell death in the sample correlates be affecting other cell types with potential relevance to the pa- with the total bacterial burden. Thus, IFN-γ enhanced PM dam- thology of human tuberculosis (Lerner et al., 2015). age (without causing significant chromatin decondensation) in M-CSF macrophages, which allowed greater bacterial replica- M. tuberculosis replication continues after tion per macrophage. This is consistent with data showing that the loss of PM permeability fast replicating bacteria induced more necrosis in vivo (Repasy We then focused on understanding the differential effect of et al., 2013, 2015). Alternatively, IFN-γ could regulate macro- IFN-γ in enhancing the replication of M. tuberculosis in M-CSF phage cell death and thereby bacterial replication, as shown for macrophages compared with GM-CSF macrophages. To moni- mouse macrophages (Lee and Kornfeld, 2010). tor the proportion of cells that had lost PM integrity, we stained Necrotic macrophages provide a niche for the cells with propidium iodide (PI) before fixation (Fig. 2, A M. tuberculosis replication and B). We observed relatively low levels of cell death in any We next focused on characterizing the environment where condition up to 72 h after infection, but by day 7 after infection, cell death was apparent to differing extents in the macrophage M.  tuberculosis replicates in human macrophages. Because populations (Fig.  2, A and B). Although there was noticeable M.  tuberculosis can be localized in the cytosol under some variation between donors, activated M-CSF macrophages had conditions (van der Wel et al., 2007), we performed an ul- the largest mean proportion of PI-positive cells, with approx- trastructural analysis of M.  tuberculosis localization using imately double the mean of the other macrophage populations EM during the growth of M.  tuberculosis in GM-CSF and (Fig.  2, A and B). We observed that in PI-positive cells at 7 M-CSF macrophages, as described previously (Fig. 3; Lerner d after infection, the bacterial burden was higher in both GM- et al., 2016). Resting GM-CSF macrophages had an increased CSF (Fig.  2, C and E) and M-CSF (Fig.  2, D and F) macro- proportion of bacteria present in the cytosol compared with phages in both resting and activated conditions. We therefore M-CSF after 48 h (45% and 30%, respectively; Fig. 3 B), and hypothesized that these necrotic cells were providing a niche IFN-γ activation did not affect the ultimate localization of the for M.  tuberculosis replication. Using microscopy with fixed bacteria (Fig. 3 B). It was interesting to note that after 5 h plus samples provided only snapshots in time, which meant that we 2 h of infection, there was already up to 15% of the bacteria could not determine whether the cells were dying because of the present in the cytosol in both GM-CSF and M-CSF macro- high bacterial burden or if the cells became more permissive for phages (Fig. 3 A, subpanels B and F; and Fig. S2 E). To fur- replication after they became necrotic. To investigate these dy- ther characterize at the ultrastructural level the environment namic events in more detail, we performed live-cell imaging of where M. tuberculosis replicated, we followed M. tuberculosis infected cells with the presence of PI in the medium to monitor growth by live-cell imaging as described previously and per- macrophage PM integrity. We observed that when macrophages formed correlative live-cell and electron microscopy (CLEM; became leaky (positive for PI), bacterial replication continued Fig.  4; Russell et al., 2016). As shown by live-cell imaging, in necrotic cells that nevertheless retained their cellular mor- after 56 h of infection, the PM became permeable and bacteria phology (Fig.  2  G and Video  2). Continued M.  tuberculosis started to replicate (Fig.  4  A). At 70  h after infection, cells replication after a loss of PM integrity is not solely caused by were fixed and processed for EM. Although the multistage fix- M. tuberculosis gaining access to and obtaining nutrients from ation protocol affected the morphology of the cell, we imaged the extracellular RPMI medium because the growth rate in leaky after each step to document the changes (Fig. 4 A). By mor- macrophages (Fig. 2 H, red lines) was increased compared with phology, we could confirm that M.  tuberculosis was growing the growth rate in medium alone (Fig. 2 H, green lines). A quan- in a necrotic cell with features of late necrotic cell death that titative analysis of GFP fluorescence intensity at the single-cell include PM damage, swollen and dilated organelles, as well as level suggests that in a proportion of macrophages, detectable autophagic structures (Fig. 4 B). We observed that replicating mycobacterial replication does not begin until after the cells bacteria were in close apposition to host membranes and were have become leaky (Fig. 2, I and J). Bacterial replication was exposed to an environment rich in cellular membranes and observed in both resting and activated GM-CSF macrophages undigested organelles. Collectively, already at 48  h, a large (Fig.  2  I), consistent with the previous data (Fig.  1, A–F). In proportion of the bacilli were found in the cytosol, and then at resting M-CSF macrophages, mycobacterial replication started later stages, mycobacteria were mostly replicating in an early after macrophages became leaky (Fig.  2  J). However, IFN-γ to late necrotic macrophage environment. PI-negative or PI-positive cells in resting (black squares) or activated (red squares) GM-CSF macrophages. Images were analyzed, and the GFP signal per cell was plotted. Data represent the means ± SEM of one representative experiment. (F) Same as in E, but with M-CSF macrophages. (G) Representative snapshots at the indicated time points (Video 2) of resting GM-CSF macrophages infected with GFP-Mtb (green) in the presence of PI. Macrophages were infected at an MOI of 1 for 2 h, and after 24-h infection, the media were replaced with RPMI medium containing 0.4 µg/ml PI. Cells were imaged for at least 7 d. Bar, 20 µm. (H) Comparison of GFP-Mtb growth rates as measured by live-cell imaging. Three conditions were tested: M. tuberculosis in RPMI medium alone (green lines), M. tuberculosis within GM-CSF–differentiated human monocyte-derived macrophages (HMDMs) before the moment the cell becomes PI positive (blue lines), and M. tuberculosis after human monocyte-derived macrophages become PI positive (red lines). Each line shows the growth rate of M. tuberculosis represented by the fold change in GFP signal (RAWIntDen) of the GFP-Mtb signal over time, normalized to the first frame in the video. (I) Quantification of intracellular replication of GFP-Mtb before and after membrane damage in resting or activated GM-CSF and M-CSF macrophages at the single-cell level. Red dashed lines indicate when the cell’s nucleus becomes PI positive. GFP-Mtb signal was monitored in three representative cells from each condition using ImageJ software. (J) Same as in I, but with M-CSF macrophages. mtb replicates within necrotic human macrophages • lerner et al. 587 Figure 3. M. tuberculosis is localized in the cytosol of primary human macrophages. (A) TEM images representing the observed localization of M. tu- berculosis in GM-CSF– and M-CSF–differentiated macrophages at 48 h after infection. “Membrane bound” represents bacteria in a phagosome (with one surrounding host membrane, such as in subpanels A and E, highlighted by white arrowheads), bacteria in phagolysosomes (loose vesicles with one surrounding host membrane, such as in subpanels C and G), or bacteria in autophagosomes (with two or more surrounding host membranes, such as in subpanels D and H); “Cytosol” represents bacteria with no surrounding host membranes (such as in subpanels B and F). Bars, 500 nm. (B) Quantification of GFP-Mtb subcellular localizations in resting (blue-outlined bars) or activated (red-outlined bars) GM-CSF and M-CSF macrophages at 2, 24, and 48 h after infection. Quantification was performed by stereological analysis of TEM images of resin sections of infected macrophages. Data are from one experiment, and the total numbers of cells analyzed in each condition (n) are shown. Inhibition of host cell necrosis limits hibitor (Degterev et al., 2013). Resting and activated GM-CSF M. tuberculosis replication and M-CSF macrophages were infected with GFP-Mtb for 7 d Because M.  tuberculosis most efficiently grows within ne- (Fig.  5  A), and the percent inhibition of M.  tuberculosis rep- crotic cells, we reasoned that we should be able to modulate lication was determined (Fig.  5  B). In parallel, the proportion bacterial replication using chemical inhibitors of necrosis. of PI-positive cells was measured to confirm that the inhibitors We used three inhibitors: IM-54, a potent inhibitor of oxida- were indeed preventing cell death (Fig. 5 C). We also confirmed tive stress–induced necrosis (Dodo et al., 2005); MCC950, a that the inhibitors themselves did not affect M.  tuberculosis specific inhibitor of the NLRP3 inflammasome (Coll et al., growth at the same concentration in vitro (Fig. S3 A). Notably, 2015); and Necrostatin-1s (Nec-1s), a RIP1 kinase (RIPK1) in- all of the drug treatments inhibited to differing extents the mean 588 JCB • Volume 216 • NumBer 3 • 2017 Figure 4. M. tuberculosis replicates in a cellular necrotic environment. (A) Snapshots taken from live-cell imaging of GM-CSF macrophages infected with GFP-Mtb over 4 d; the graph below shows how the growth of GFP-Mtb occurs only after the cell becomes PI positive. (B) SBF SEM and TEM were performed on the same sample as in A. This allowed for the GFP-Mtb signal to be overlaid with the EM images. Subpanels i–iii show magnified regions of the ultra- structure taken from the TEM image. The bottom subpanel shows segmentation of the PM in red using the SBF SEM data (with reference to the adjacent TEM image) with the LM data and TEM image overlay. Black arrows indicate PM damage. M.  tuberculosis replication compared with the DMSO control repair program, thereby inducing necrosis and a release of in each macrophage population analyzed (Fig. 5, A and B). In- bacteria that can then reinfect surrounding cells (Divangahi et terestingly, Nec-1s treatment in resting M-CSF macrophages al., 2009). In addition, our data revealed that M. tuberculosis increased the mean bacterial burden; consistent with this, in this induces PM damage, seemingly to disarm the host cell, and particular condition, Nec-1s actually increased the proportion then replicates in this nutrient-rich environment. Once repli- of PI-positive cells (Fig. 5 C). This suggests that RIPK1 could cation occurs in this protected and rich environment, bacteria be differentially activated in the different macrophage popula- immediately get access to the extracellular media after rein- tions. The lack of effect on cell death and bacterial replication fection. Our data therefore provide evidence suggesting that in M-CSF macrophages was reversed by IFN-γ activation, in- M.  tuberculosis actively avoid the extracellular environment dicating that this cytokine activates in macrophages a signaling that can be detrimental for long periods of time. A study per- pathway via RIPK1 that results in necrosis, as was previously formed with a high burden of bacterial infection (Lee et al., reported in fibroblasts (Thapa et al., 2013). The precise mech- 2006) could mimic some of our results, but our experimen- anism and signaling pathways that regulate this differential tal setting is different and could reflect more the cells that in susceptibility to PM integrity after infection and IFN-γ acti- vivo are infected with fewer bacteria and not the ones that vation between M-CSF and GM-CSF macrophages remains to take up large bacterial aggregates. In our system, we observed be investigated. Importantly, the necrosis inhibitors were more by live-cell imaging that once macrophages internalized big efficient in activated than in resting M-CSF macrophages, cor - clumps of bacteria (either by cell transfer or efferocytosis), relating with the increased replication and enhanced cell death cell death followed rapidly (unpublished data). Collectively, observed (Figs. 1 E and 2 B). Thus, we concluded that inhibi- in this study, we analyzed for the first time the dynamic repli- tion of necrosis resulted in less efficient M. tuberculosis growth. cation of M. tuberculosis in human macrophages by long-term Our results highlight the possibility that bacteria repli- live-cell imaging in different functional populations of macro- cate within cells after PM damage before being released into phages. Our studies provide insights into the dynamic interac- the extracellular medium. If this scenario holds in vivo, extra- tions between human macrophages and M. tuberculosis. Thus, cellular bacteria would originate from two different sources: in addition to the known role of dissemination, leaky cells un- the pool that replicated intracellularly in damaged cells and dergoing early stages of necrosis represent a niche that helps a pool of bacteria that grows extracellularly. Necrotic cells bacteria to grow even before getting freely into the extracel- represent a much better environment for bacterial replica- lular environment. Our studies uncover an important aspect tion, as they are rich in nutrients in a host cell with severely in the biology of tuberculosis, revealing that M.  tuberculosis affected cell-autonomous immunity. Our data are consistent continues to replicate in host cells after PM integrity is lost with the idea that virulent mycobacteria inhibits the cell PM before disseminating to other cells. mtb replicates within necrotic human macrophages • lerner et al. 589 Figure 5. Inhibition of host cell necrosis limits M. tuberculosis replication. (A) Example confocal microscopy images showing GFP-Mtb (green), PI (red), and DAPI (blue) staining of resting or activated GM-CSF or M-CSF macrophages at 7 d after infection in control cells (DMSO only) or cells treated with 10 µM of the shown necrosis inhibitors. Cells outlined in white dotted lines are considered to be PI positive. Bars, 10 µm. (B) Quantification of the effect of treating infected resting (blue) or activated (red) GM-CSF (dots) or M-CSF (squares) with three necrosis inhibitors compared with untreated control macrophages at 7 d after infection with GFP-Mtb. (C) Quantification of the effect of treating infected resting (blue) or activated (red) GM-CSF (dots) or M-CSF (squares) with three necrosis inhibitors on the proportion of cells with a PI-positive (PI+) nucleus compared with untreated control macrophages at 7 d after infection with GFP-Mtb. Data points show the percent reduction for each independent experiment, and the bars show the overall mean. 590 JCB • Volume 216 • NumBer 3 • 2017 Materials and methods min and permeabilized with 0.05% saponin/1% BSA in PBS for 10 min. Coverslips were incubated with the mouse anti–human LAMP-2 Preparation of monocyte-derived macrophages antibody (Hybridoma Bank) at a dilution of 1:100 for 1  h at room Peripheral blood mononuclear cells (PBMCs) from healthy anonymous temperature, followed by the goat anti–mouse Alexa Fluor 546 anti- donors were isolated from leukocyte provided by the National Blood body (Jackson ImmunoResearch Laboratories, Inc.) at a dilution of and Transplant Service, UK. Red blood cells were removed by centrif- 1:800 for 1  h at room temperature. Nuclei were stained with DAPI, ugation on Ficoll-Paque (28-4039-56 AD; GE Healthcare) and red cell and coverslips were mounted with mounting medium (Dakok). lysis buffer (11814389001; Sigma-Aldrich). Recovered PBMCs were Samples were analyzed using a laser-scanning confocal microscope washed to remove platelets. Monocytes were isolated from PBMCs (SP5; Leica Biosystems). using a magnetic cell separation system with anti-CD14 mAb-coated For analysis, RGB images or frames were split into separate microbeads (130-050-201; Miltenyi Biotec). CD14-positive mono- color channels. Bacteria (green channel) were thresholded per single cytes were cultured in complete RPMI 1640 medium (Lonza) sup- cell, and the mean fluorescence intensity of LAMP-2 associated to the plemented with 9.1% heat-inactivated FCS supplemented with either phagosome was measured for each frame by redirecting measurements 50 ng/ml GM-CSF or 50 ng/ml M-CSF (130-093-862/130-093-963; to the channel of interest. Fluorescence intensity values were plotted Miltenyi Biotec) at 37°C under a humidified 5% CO atmosphere for using Prism software (GraphPad Software). The number of GFP-posi- 6 d. Media were replaced at 3 d. On day 6, the cells were washed and tive pixels per cell (or a value called RAWIntDen, which correlates to detached with 0.5 mM EDTA in ice-cold PBS and plated in a 24-well the number of GFP-positive pixels multiplied by 255) was plotted as a plate (containing coverslips for microscopy) or a live-cell imaging dish measure of the intracellular bacterial replication over time. The propor- at a concentration of 2 × 10 cells/well with complete RPMI media (for tion of infected cells per sample was also determined. resting macrophages). Macrophages were either treated overnight or after infection with recombinant human IFN-γ (PHC 4031; Gibco) at a Quantification of bacterial burden with cell death inhibitors concentration of 100 U/ml. Out-of-focus cells were manually cropped from tile scans, and the GFP per cell was quantified with an in-house R script using the RBio- Bacterial culture Formats (Oleś, 2014) and EBImage (Pau et al., 2010) packages (Fig. GFP-Mtb or M.  tuberculosis H37Rv ΔRD1 expressing EGFP (GFP- S3 B). For each image, nuclei were identified by DAPI staining, seg- Mtb ΔRD1) was grown at 37°C in Middlebrook 7H9 medium (Difco mented by watershed, and used as seeds for a Voronoi segmentation Laboratories) containing 0.5% glycerol and 0.05% Tween 80 and was of the field by Euclidian distance. Pixels in the GFP channel above supplemented with 10% oleic acid–albumin–dextrose–catalase supple- a predetermined threshold were counted per segment, and segments ment (OADC; BD) or on Middlebrook 7H10 plates (Difco Laborato- with ≥5 GFP-positive pixels were considered infected cells. A mini- ries) supplemented with 10% OADC. mum of 349 cells were analyzed per condition across three biological replicates, with a mean of 1,450. Infection of human monocyte-derived macrophages Macrophages were seeded onto 24-well culture plates the day be- PM integrity in fixed cells fore the infection. M.  tuberculosis cultures were grown to the midex- Before collecting the coverslips, the macrophages were stained by ponential phase. On the day of infection, the bacteria were pelleted 25 µg/ml PI (Sigma-Aldrich) in RPMI for 15 min at room temperature and washed with PBS and RPMI/10% FCS at 3,000 rpm for 5 min. and fixed in 3% PFA overnight at 4°C.  Images were obtained using An equal volume of sterile glass beads (2.5–3.5 mm) that matched an SP5 confocal microscope with an argon laser emitting dually at the pellet size was added (usually three to four beads) and then vig- 488 nm for excitation of GFP and at 561 nm for PI. To determine the orously shaken for 1 min to break up bacterial clumps. The bacteria replication of bacteria in PI-positive or -negative cells, bacteria (green were washed again with complete RPMI at 1,200 rpm for 5 min. The channel) were thresholded per single cell, and the pixel numbers were supernatant was transferred into a new tube, and the measured OD measured in each positive and negative propidium cell. Values were plot- was then diluted to 0.1 in complete RPMI. Next, cells were infected ted using Prism software. with M. tuberculosis at an MOI of 1 for 2 h and then washed once with PBS and incubated with complete RPMI media containing IFN-γ and/ Live-cell imaging or 10 µM of the necrosis inhibitors IM-54 (Sigma-Aldrich), MCC950 For live-cell imaging, 10 macrophages were seeded on 35-mm (Cayman Chemicals), or Nec-1s (Source Biosciences), where relevant. glass-bottom dishes (MatTek Corporation) and treated overnight with 100 U/ml IFN-γ in RPMI complete media. Cells were washed with PBS CFU determination and infected with M. tuberculosis at an MOI of 1 for 2 h. After infection, For counting bacterial viability, macrophages were washed once with cells were washed with PBS and replaced with RPMI complete media. PBS and lysed with water–Tween 80 (0.05%) for 30 min at room tem- 24  h after infection, the media were replaced with imaging medium: perature. The lysed solution from triplicate wells was taken and seri- complete RPMI medium with 0.4 µg/ml PI. M.  tuberculosis was also ally diluted in PBS in 10-fold steps until 1:1,000 dilution. 25 µl from grown in imaging medium alone by simply adding the infection inocu- each dilution was plated in triplicate onto complete 7H11 agar plates. lum into a glass-bottom dish containing no macrophages. Imaging was Agar plates were incubated for 2–3 wk at 37°C. Triplicate plates were performed with an SP5 laser-scanning confocal microscope equipped averaged, and CFU were calculated and plotted as the mean CFU per with an environment control chamber (EMB LEM). During imaging, a milliliter from triplicate wells. single focal plane was monitored in time (xyt scanning mode) using a 63×/1.4 NA HCX-PLA PO oil objective, a 488-nm argon laser, and a Indirect immunofluorescence 561-nm diode-pumped solid-state (DPSS) laser (scanner frequency 400 Coverslips were washed once with PBS and fixed overnight with 3% Hz; line averaging 2) using photomultiplier detectors and/or hybrid de- PFA (Electron Microscopy Sciences) in PBS, pH 7.4, at 4°C.  After tectors (HyDs) at a scanning resolution of 1,024 × 1,024 pixels. Analysis fixation, PFA was removed and replaced with PBS. Then, cells were was performed using FIJI (ImageJ; National Institutes of Health). For incubated with 50  mM glycin/PBS, pH 7.4 (Sigma-Aldrich), for 10 analysis, RGB images or frames were split into separate color channels. mtb replicates within necrotic human macrophages • lerner et al. 591 Bacteria (green channel) were thresholded per single cell. Pixels were glutaraldehyde and 4% PFA in 0.1 M phosphate buffer. The cells were quantified and plotted using Prism software. Bacteria replication was cal- again imaged before processing for serial block face scanning EM culated in PI-positive and -negative cells for each time point of interest. (SBF SEM) commenced, using a modified National Center for Micros- copy and Imaging Research protocol (Lerner et al., 2016). Coverslips EM were postfixed in 2% osmium tetroxide/1.5% potassium ferrocyanide GM-CSF and M-CSF macrophages were prepared as described in the for 1 h on ice, incubated in 1% wt/vol thiocarbohydrazide for 20 min Preparation of monocyte-derived macrophages section (2 × 10 cells before a second staining with 2% osmium tetroxide, and then incubated seeded per T25 flask) and infected at an MOI 1 for 5 h, with 200 ng/ml overnight in 1% aqueous uranyl acetate at 4°C. Cells were stained with IFN-γ added after infection where appropriate. The protocol for resin Walton’s lead aspartate for 30 min at 60°C and dehydrated through embedding was adapted from Rohde et al. (2012). Samples were fixed an ethanol series on ice, incubated in a 1:1 propylene oxide/Durcu- by adding warm 2% glutaraldehyde in 200 mM Hepes, pH 7.4, directly pan resin mixture, and then embedded in Durcupan resin according to to the cell culture medium at a 1:1 volume ratio. After 5 min, the mix- the manufacturer’s instructions (TAAB Laboratories Equipment Ltd.). ture of the fixative and medium was replaced with 1% glutaraldehyde SBF SEM images were collected using a 3View 2XP system (Gatan in Hepes buffer, and the samples were incubated overnight at 4°C. Cells Inc.) mounted on a Sigma VP scanning electron microscope (ZEISS). were scraped and embedded in 1% low melting point agarose. Agarose Images were collected at 1.8 kV using the high-current setting with a blocks were postfixed with 2% osmium tetroxide solution containing 20-µm aperture at 6 Pa chamber pressure and a 2-µs dwell time. The 1.5% potassium ferricyanide for 1 h on ice and then stained with 0.5% dataset was 33.59 × 33.59 × 0.75 µm in xyz, consisting of 15 serial tannin for 20 min and with 1.5% aqueous uranyl acetate for 1 h. Next, images of 50-nm thickness and a pixel size of 4.1 × 4.1 nm. The total cells were dehydrated at room temperature using a graded ethanol se- volume of the dataset was ∼846 µm . After SBF SEM imaging, the ries (70, 80, 90, 95, and 100%), followed by gradual infiltration with sample was removed, and 70-nm sections were cut on a ultramicrotome Spurr’s resin (Polysciences) over 2 d. Ultrathin sections (∼70 nm) were (UC7; Leica Biosystems). Images were then collected in a transmission cut with an ultramicrotome Ultracut EM UCT ultramicrotome (Leica electron microscope (Tecnai G2 Spirit BioTwin; Thermo Fisher Scien- Microsystems) and contrasted with 0.2% lead citrate. Cells were exam- tific) using a charge-coupled device camera (Orius; Gatan Inc.). The ined with a transmission electron microscope (JEM-1400; JEOL). The SBF SEM dataset was segmented manually using Amira 6.0 (Thermo images were recorded digitally with a TemCam-F216 camera (TVI PS). Fisher Scientific). The PM was manually selected by morphology with Between 24 and 89 infected cells per sample were imaged by system- reference to transmission EM (TEM) images (in which membrane gap atic and random sampling. Cross points of the stereological test grid morphology was more readily apparent, because of the higher struc- over bacteria were counted with regard to the subcellular localization tural resolution). Only larger gaps (greater than ∼100 nm) were seg- of bacteria, and fractions of membrane-bound and cytosolic bacteria mented to account for the reduced resolution in the SBF SEM images were calculated from total counts per sample. The data were plotted as and to prevent false positive assignment of gaps. the proportion of M. tuberculosis in the cytosol versus the proportion in membrane-bound compartments for each condition. CLEM alignment Alignment between light microscopy (LM), SBF SEM, and TEM was CLEM performed using the BigWarp plugin in Fiji. We first mapped the TEM GM-CSF macrophages were prepared as described in the Preparation image onto the final image of the SBF SEM image stack at full resolu- of monocyte-derived macrophages section, and cells were seeded to tion, using landmarks in 2D. The warped and exported TEM image was ∼30–50% confluence onto gridded dishes (MatTek Corporation) in then appended to the SBF SEM stack to form a hybrid EM stack. Note 400 µl RPMI + 10% FCS for a 2-h attachment time at 37°C and 5% that the slightly different slice thickness of the SEM stack and single CO . They were then infected with GFP-Mtb at an MOI of 1 as de- TEM slice can be essentially ignored because the axial diffraction limit scribed in the Infection of human monocyte-derived macrophages sec- of the LM image is much larger than the EM slice thickness. The LM tion and incubated for 24 h. The infection medium was then replaced image stack was then mapped onto the hybrid EM stack (containing with 2 ml RPMI + 10% FCS containing 0.4 µg/ml PI, and then the dish both SBF SEM and TEM data) using landmarks in 3D at corresponding was securely fastened into a custom-made dish holder for the stage positions in the two stacks. of a confocal microscope ready for imaging. The microscope was set up with the argon laser set to 20% and the DPSS laser switched on. A Flow cytometry sequential scan for each channel was set up: (a) argon 488-nm laser (set Isolated monocytes and differentiated macrophages were incubated to 10%) with the HyD in brightR mode; or (b) DPSS 561-nm laser (set with an Fc receptor–blocking antibody (Human BD Fc Block; BD) on to 2%) with the HyD in brightR mode, and simultaneously a brightfield ice for 20 min, followed by staining with anti–CD119-PE (clone GIR- image was obtained using SCAN-BF mode with gain set to 493 V. Ini- 208; eBioscience), anti–CD86-BV421 (clone 2331), anti–CD206-APC tially, a 10× objective was used to determine the grid reference of the (clone 19.2), anti–CD14-AF488 (clone M5E2), or anti–CD16-APC-H7 cell of interest. A 63× oil objective was then used with the following (clone 3G8; BD) for 20 min at room temperature in the dark. Cells imaging conditions: 1,024 × 1,024–pixel resolution; line averaging 2; were washed once in PBS, fixed in 1% PFA, and acquired on a flow zoom 1; and frames every 15 min. At the desired time point, warm cytometer (Fortessa; BD) or a FACS CyAnTM (Dako). Respective double-strength fixative (8% PFA in 0.2 M phosphate buffer, pH 7.4) isotype controls were mouse IgG1 K-PE (clone P3.6.2.8.1; eBiosci- was directly added to the cell culture medium (1:1 ratio) for 15 min ence), mouse IgG1, κ-BV421 (clone X40), mouse IgG1, κ-APC (clone at room temperature. The cell of interest was relocated and imaged. MOPC-21), mouse IgG2a, κ-AF488 (clone G155-178), and mouse The fixative was then removed, and a 1-mm layer of 4% low melting IgG1, κ-APC-H7 (clone MOPC-21; BD). point agarose (Sigma-Aldrich) in 0.1 M phosphate buffer was added to form a protective layer over the cells. This was placed on ice quickly Western blotting to solidify. Next, 4% PFA in 0.1  M phosphate buffer was added for Macrophages were washed once with PBS and lysed in NP-40 15 min at room temperature, and again the cells were imaged. Finally, for 40 min on ice. 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Journal

The Journal of Cell Biology – Pubmed Central

Published: Mar 6, 2017

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