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Evolution of foamy macrophages in the pulmonary granulomas of experimentaltuberculosis models

Neus Caceres a,b,c, Gustavo Tapia b,d, Isabel Ojanguren b,d, Frederic Altare e, Olga Gil a,b,c, Sergio Pinto a,b,c,Cristina Vilaplana a,b,c, Pere-Joan Cardona a,b,c,*

a Unitat de Tuberculosi Experimental, Department of Microbiology, Fundacio Institut per a la Investigacio en Ciencies de la Salut Germans Trias i Pujol,Badalona, Catalonia, Spainb Universitat Autonoma de Barcelona, Badalona, Catalonia, Spainc CIBER Enfermedades Respiratorias, Instituto de Salud Carlos III, Palma de Mallorca, Spaind Pathology Department, Hospital Universitari Germans Trias i Pujol, Badalona, Catalonia, Spaine Inserm U892, CRCNA, Nantes, F-44093, France

a r t i c l e i n f o

Article history:Received 6 July 2008Received in revised form4 October 2008Accepted 12 November 2008

Keywords:Mycobacterium tuberculosisNon-replicating bacilliFoamy macrophagesLipid bodiesTriglyceridesIntracellular lipophilic inclusionsLatent tuberculosis infection

s u m m a r y

The chronic phase of Mycobacterium tuberculosis infection in mouse experimental models is character-ized by the accumulation of foamy macrophages (FM) – which shape the outer ring of the granuloma – inthe alveolar spaces, as detected in paraffin-embedded tissues stained with hematoxylin–eosin. In thisstudy, the use of semi- and ultra-thin sections offers more detailed information about the origin of FMboth in mouse and guinea-pig experimental models. Lipid bodies (LB) are present in macrophages fromthe beginning of infection and accumulate in the chronic phase. LB progress from an early (ELB) to a late(LLB) stage, defined according to their progressive capacity to generate cholesterol crystals, resemblingatherosclerotic lesions. FM arise from massive accumulation of LLB. Electronic microscopy revealsintracellular lipophilic inclusions (ILIs) in those M. tuberculosis bacilli inside FM. It is our hypothesis thatthe accumulation of lipids in M. tuberculosis concomitant to the establishment of the non-replicatingstate prepares the bacilli for future reactivation and for facing future stressful environments.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Mycobacterium tuberculosis infection is characterized by itscapacity to remain for a long time in the host, originating the latenttuberculosis infection. In the murine model there is a long persis-tent chronic phase characterized by the presence of non-replicatingbacilli1,2 and by the accumulation of foamy macrophages (FM) atthe outermost layer of the granuloma.3,4 This accumulation can beobserved by routine hematoxylin–eosin (H&E) staining in 5-mmsections from paraffined blocks. Some FM harbor scarce bacilli,which may reactivate ‘‘in situ’’.3,4 Both the accumulation of FM andthe granulomatous dissemination in mice are directly related to anincrease in the bacillary load.4,5

The aim of this work was to characterize by histopathologicalanalysis the evolution of FM in mouse and guinea-pig experimentalmodels during the course of tuberculosis (TB) infection usingbetter-resolution techniques, such as semi-thin and ultra-thinsections for toluidine blue (TBlue) stains and electronic microscopy(EM), respectively. We chose two different mice strains: the DBA/2,as they accumulate far more FM than other strains4; and the C57BL/6, to allow the relation between the induction of intra-granulomatosis necrosis and the induction of FM.5

Our results demonstrate that the production of FM starts fromthe onset of infection by progressive accumulation of lipid bodies(LB). Both the chemical and physical characteristics of these bodiesvaried with time, with their progressive disruption, loss of TBluestaining and progressive production of large cholesterol crystals ina process that strongly resembles atherosclerotic lesions.6 Once atthe chronic phase of infection, bacilli display intracellular lipophilicinclusions (ILIs). The presence of ILIs in M. tuberculosis was firstdescribed in studies conducted many years ago,7 being linked at thetime to nitrogen-limited cultures8; and it was soon suggested thatthe ILIs concerned could be triglycerides (TAG).9 Studies on ILIs

* Corresponding author. Unitat de Tuberculosi Experimental, Fundacio Institutper a la Investigacio en Ciencies de la Salut Germans Trias i Pujol, Crta de can Rutis/n, Edifici Recerca, 08916 Badalona, Catalonia, Spain. Tel.: þ34 93 497 86 86;fax: þ34 93 497 86 54.

E-mail address: [email protected] (P.-J. Cardona).

Contents lists available at ScienceDirect

Tuberculosis

journal homepage: ht tp: / / int l .e lsevierheal th.com/journals / tube

1472-9792/$ – see front matter � 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.tube.2008.11.001

Tuberculosis 89 (2009) 175–182


evolution in Mycobacterium smegmatis cultures by Nile Red cyto-logical assays showed that TAG were their major componentrelated to stationary-phase cultures.10 ILIs were also identified inacid-fast bacilli obtained from the sputum of TB patients,10 a pres-ence linked to a high expression of M. tuberculosis triacylglycerolsynthase (tgs), as has been recently demonstrated.11

2. Material and methods

2.1. Experimental design

6-week old female spf DBA/2 and C57BL/6 mice (Harlan Iberica,Sant Feliu de Codines, Catalonia, Spain), as well as 10-week oldfemale spf Dunkin-Hartley guinea-pigs (Harlan Iberica, Sant Feliude Codines, Catalonia, Spain) were infected with a M. tuberculosisH37Rv Pasteur strain through aerosol inoculation and induced bya Middlebrook device, which inoculated between 20 and 50 bacilliin the lungs. At different time-points (i.e. at weeks 2, 3, 5, 9 and 17post-infection) mice lungs were extracted immediately after beingeuthanized with a halothane (Zeneca Farma, Pontevedra, Spain)overdose. Guinea-pig samples were extracted at week 18 post-infection following euthanasia with an intraperitoneal (i.p.) injec-tion of ketamine/diazepam (3/1) overdose.

2.2. Histology

Lungs were fixed in buffered formalin and embedded inparaffin; 5-mm sections were stained with H&E and Ziehl Neelsenfor optic microscopy analysis, as described elsewhere.4 Pulmonarygranuloma samples were fixed in glutaraldehyde 0.1 M, post-fixedin osmium tetroxide, dehydrated and embedded in epoxy resin(Epon). Semi-thin sections were stained with TBlue, ultra-thinsections were stained with uranyl citrate and lead citrate, and bothwere examined with a transmission electron microscope (JEOL1010, Tokyo, Japan).

2.3. Animal health

Animals were supervised daily following a protocol that moni-tored weight loss, apparent good health (bristled hair and woundedskin) and behavior (signs of aggressiveness or isolation). Sentinelanimals were used to check for spf conditions within the facility.Tests for 25 known pathogens were all negative. All experimentalproceedings were approved and supervised by the Animal CareCommittee of the ‘‘Germans Trias i Pujol’’ University Hospital inagreement with the European Union Laws for the protection ofexperimental animals.

2.4. Quantification of macrophages and LB from semi-thin slices

Toluidine blue-stained semi-thin slices were analyzed by opticmicroscopy – 1000� – for this purpose. At least two granulomasfrom each time-point (weeks 2, 3, 5 and 9) were observed. Numberof core macrophages and FM were determined. For LB calculation,ten core macrophages were analyzed.

3. Results

3.1. Macrophages accumulated lipid bodies (LB) from the onset ofinfection and changed their morphology as they moved throughoutthe granuloma

Macrophages from the granuloma core accumulated early lipidbodies (ELB) from the beginning of the infection (week 2). Thenumber of ELB inside them increased (Figure 1 and Table 1) andunderwent morphology changes as the infection progressed. These

macrophages emerged in the outer area, inside the alveolar spaces,as the content of their early lipid bodies (ELB) tend to lose the TBluestain positivity, showing a progression towards late lipid bodies(LLB) (Figure 1). At this point, these macrophages had FM charac-teristics, as they were detected by H&E staining (Figure 2). Acid-fastbacilli were detected inside FM using Ziehl Neelsen staining.

3.2. Cholesterol crystals emerged from LLB in the chronic phase ofthe infection

ELB were well defined and homogeneous, but as the infectionbecame chronic, LLB started appearing inside the macrophages,presenting heterogeneity in their size and appearance. This trans-formation developed into the formation of cholesterol crystals,which grew to a large size (Figures 3 and 4).

3.3. Multiple bacilli inside phagosomes were detected at thebeginning of the infection, becoming single and accumulatingintracellular lipophilic inclusions (ILIs) when present inside FM

As macrophages moved out of the granuloma as the infectionprogressed, bacilli could be observed inside their phagosomes. Atthe beginning of infection, phagosomes harbored multiple bacilliand few ELB accumulated in the macrophages (Figures 3 and 4).Once specific immunity was triggered (i.e. at three weeks post-infection) this relationship changed. The accumulation of ELBincreased, as did the proportion of phagosomes containing singlebacilli. While infected macrophages started to be seen at theperiphery of the granuloma, ELB became LLB and thus FM started tobe seen. At this point, ILIs began to be detected inside the bacilli(Figure 5). In the alveolar spaces, some FM destruction took place.In this case, LLB converged and became much bigger, and bacilliimmersed within these coalescent LLB were observed (Figure 5).

3.4. Macrophages filled with ELB and FM were found surroundingintragranulomatous necrosis both in C57BL/6 mice and guinea-pigswith chronic infection

C57BL/6 mice can develop necrosis. Cell clusters containingmacrophages filled with ELB and FM were found among necrotictissue. Fibrin and collagen fibers completed the remaining necroticarea. Blood vessels, polymorphonucleates (PMNs) and also fibro-blasts were found to be spread through this necrotic area, as well asmacrophages and lymphocytes (data not shown). The carefulscreening of necrotic tissue revealed an absence of extracellularbacilli. Bacilli were always intracellular, inside macrophages or FM.

Samples from chronically infected guinea-pigs also showed bothtypes of macrophages in the cellular area surrounding the necrosedcore (Figure 6). As observed in mice, these cells in guinea-pigsshowed a progression from macrophages filled with ELB to FM. Thesecells were also widely found inside the bronchial tree (Figure 6).

4. Discussion

FM may be considered to represent the main cell type respon-sible for the chronicity of M. tuberculosis infection in mice, as theyhave been histologically linked to both the escape of the bacilli –from the granuloma and to new potential focus sites of new gran-uloma formation – and to the increasing granulomatous infiltrationof the lungs.3–5 As it is widely believed,12 a strong specific responseagainst growing bacilli is triggered to stop the acute infection. Oncethe immune response is well established, FM drain the non-repli-cating bacilli following the mechanism usually required forremoving other lesions or toxic products from the lung, replacingthe original parenchyma.13

N. Caceres et al. / Tuberculosis 89 (2009) 175–182176


This study reveals a new aspect in the pathogenesis of M.tuberculosis infection, i.e. the abundance of LB in the macrophagesinside the infected granulomas, from the very beginning of infec-tion. It has recently been demonstrated that LB play a significantrole in the inflammatory response of granulomas.14 LB are mainlygenerated from the phagocytosis of dead cells, in particular their

membranes, which contain large quantities of arachidonic acidprecursors and enzymes related to its processing. The differentmetabolic pathways linked to arachidonic acid may modulate theinflammatory responses against M. tuberculosis either by increasingor decreasing the Th1 response, depending on the induced meta-bolic route.15 The present data clearly suggest the relevance of LBaccumulation inside macrophages and FM, and a prominent role inimmunomodulation.

During the acute phase of infection the number of LB inside coremacrophages considerably increases, as observed between weeks 2and 3 (Table 1), due to the growth of bacilli, which also inducesnecrosis or apoptosis of these macrophages.15 Once specificimmunity has been triggered, infected macrophages are activated,and phagocytosis enhanced. This could explain the increase in thenumber of FM observed between weeks 2 and 3 (Table 1). Growingbacilli are then identified and killed and thus the bacillaryconcentration is controlled.4 At this point, the proportion changesand only a small number of phagosomes, containing single bacilli,are found among abundant LB. In fact, accumulation of LB has beenassociated with the stimulation of phagocytosis.13 In thesecircumstances, there is a regular contact between these twoparticles, thus making feasible the ‘‘kiss and run’’ phenomenon16

Table 1Abundance of ELB related to the presence of core macrophages and FM. The averagevalues were obtained from optic microscopy observation of at least two granulomasfrom each time-point. Toluidine blue-stained semi-thin sections were used for thispurpose.

Infection progression related to macrophage evolution

Time-point in weeks No. LB/coremacrophage

No. coremacrophages(macrophageswith ELB)

No. foamymacrophages(macrophageswith LLB)

Average SD Average SD Average SD

2 12 4 28 8 29 63 48 21 25 7 >150 –5 64 26 29 2 >150 –9 59 18 30 3 >150 –

Figure 1. Pictures from DBA mice granuloma on toluidine blue-stained (TBlue) semi-thin sections. Pictures A and B show an overview of granulomas at weeks 3 and 9 post-infection, respectively. The areas strongly stained with TBlue are located in the center of the granulomas. In the right bottom pictures are detailed with higher magnification(�1000) the accumulation of lipid bodies stained with TBlue. Note that the accumulation of cells stained with TBlue increases with progression of infection. Picture C showsa granuloma at week 9 post-infection showing early lipid bodies (ELB) stained with TBlue inside central macrophages (red arrows) and late lipid bodies (LLB) which do not stainwith TBblue, inside peripheral foamy macrophages (FM) (orange arrow). Pictures D and E show the progressive loss of staining by ELB in macrophages located at the periphery ofthe granuloma (week 9). The classical morphology of the FM can be seen in picture E. The original magnification was 100� for A, 200� for B, 400� for C and 1000� for D and E. Barsrepresent 100 mm for A–C; and 20 mm for the rest.

N. Caceres et al. / Tuberculosis 89 (2009) 175–182 177


favoring both the discharge of the lipid content from the LB to thephagosomes and the production of ILIs inside the bacilli.13

Experiments with M. smegmatis cultures revealed both that ILIsaccumulate in the stationary-phase cultures and that their majorcomponent is TAG.10 More recently, the accumulation of TAG in M.tuberculosis has been linked to stress conditions (low O2 or NOtreatment).17,18 Non-replicating bacilli in latent lesions havegenerally been considered to be in hypoxic condition, as underoxygen depletion in vitro.19 Recently, inhibition of respiration byNO has been found to induce a gene expression pattern that issimilar to that found in a hypoxia-induced non-replicating state.20

This fact is particularly interesting as NO is produced in significantquantities by FM.4

TAG is accumulated in response to an elevated ratio of carbon:nitrogen,21 which is the case inside FM, as it is also for adipocytes,where a prolonged presence of M. tuberculosis bacilli having accu-mulated ILIs has also been reported.22 In this study, a mycobacter-iostatic effect exerted by fatty acids on the bacilli was shown.Different studies in the past have suggested that the mycobacter-icidal action of long-chain fatty acids is due to their detergent-likeaction on the cytoplasmic membrane, and that the determinantfactor for the fatty acid-sensitivity of bacteria is the property of thecell wall, by which fatty acids are adsorbed so that the active site isbrought into contact with the inner membrane.23,24 In this regard,accumulation of TAG in the ILIs could also be a way of neutralizingthe toxic effect of fatty acids.21

Figure 2. Evolution of lung granulomas in the murine experimental model of TB, from the initial infection to the chronic phase (weeks 3–9). Hematoxylin and Eosin stain revealsthe formation of a pre-granuloma characterized by the mixture of different cell types (macrophages, polymorphonucleated cells and lymphocytes) with a non clear spatial structure(pictures A, E). Ziehl Neelsen stain shows growing bacilli inside the macrophages inside this lesion (picture I). Acquisition of specific immunity is revealed by the organization oflymphocytic rings starting on w4 as thin lines that surround infected macrophages (dotted blue line) becoming differentiated structures inside the granuloma (pictures B and F).Ziehl Neelsen stain reveals the presence of growing bacilli inside these macrophages (picture J). In the chronic phase all these patches converge (w9in), and a clear granulomanucleus can be seen where macrophages are the main cell type, and lymphocytes accumulate at the periphery of the granuloma (pictures C and G). At this time-point only fewgrowing intracellular bacilli can be detected in the granuloma center (picture K). Foamy macrophages (FM) accumulate at the outermost ring at the chronic phase (w9out) (picturesD and H). Some of these carry single bacilli (indicated with blue arrows) (picture L). Original magnifications were 100� in pictures A–C; 200� in pictures E–G and D; 400� in pictureH; and 1000� in pictures I–L.



TAG must be accumulated in the cytoplasm as it has never beendetected in the cell wall.25 These lipids can be a major source ofenergy for latent bacilli,26 although it is also possible that they mightalso be accumulated so as to ensure the high fatty acid concentrationrequired to build the complex mycobacterial cell wall for futuregrowth, which requires a high percentage of mycolic acids.25

The recent observation linking the hypervirulent M. tuberculosisBeijing strains with a fast-spreading capacity27 for the accumula-tion of TAG in aerobic cultures and exponential phase cultures invitro (as a consequence of an upward regulation of the DosR reg-ulon28) suggests that lipid storage in M. tuberculosis has a functionbeyond that attributed to it in the traditional view. Whereas theinterpretation applied hitherto has been that ILIs are merely anenergy source for a long-lasting non-replicating period inside anold granuloma.26 It is our hypothesis that the accumulation of lipids

in M. tuberculosis is concomitant to the establishment of the non-replicating state that prepares the bacilli for future reactivation andfor facing future stressful environments.

Macrophages filled with ELB phagocyte the extracellular bacillipresent among the debris from dead cells inside the granuloma,a process that also takes place during the acute phase. However,these bacilli have stopped their growth due to the stressful condi-tions of the new milieu (i.e. low pH and numerous nitrogen andoxygen radicals, together with bactericidal enzymes).29 In thissituation, it is unlikely that the usual low-speed metabolism ofthe bacilli alone is responsible for the non-replicating status duringthe chronic phase. It is more likely that these macrophages havea bacteriostatic effect by producing NO, as demonstrated in mice,20

or reactive nitrogen intermediates (RNIs).4 A lower anti-mycobacterial capacity may also be due to the presence of LB,

Figure 3. Electron microscope pictures from DBA mice granuloma showing the evolution of macrophages at weeks 2 (A), 3 (B), 5 (C), and 9 (D). Note the accumulation of lipidbodies (LB) inside macrophages from the onset of infection and the increase in the number of LB with progression of infection. Black squares show the close relation between bacilliand LB. The original magnification was 3000� in A, 3500� in B, 1500� in C and 4000� in D. Bars represent 2 mm.

Figure 4. Electron microscope details (A–C) and toluidine blue (TBlue) semi-thin section (D) of granuloma macrophages from DBA/2 granulomas showing the evolution of lipidbodies (LB) towards cholesterol crystals. Pictures A–C show LB corresponding to weeks 2, 5 and 9 post-infection. Cholesterol crystals are indicated with red arrows. Growing bacilliare indicated with orange arrows. Picture D shows a large cholesterol crystal among foamy macrophages (FM) at week 9 post-infection. The original magnifications were 4000� inA, 5000� in B and C and 1000� in D. Bars represent 1 mm in A–C, and 20 mm in D.



decreasing previous macrophage activation by limiting the bindingof phagolysosomes.30

FM produce NO,4 do have reduced antigen processing function31

and transiently suppress effector T cells.32 In fact, T cell suppressionby FM has been demonstrated in vivo33 in infected mice; andalveolar macrophages from humans and most mammal speciesinhibit T cell proliferation in vitro.34 In this regard, the suppression

function must not be necessarily linked to the production of NO, asit is demonstrated in guinea-pigs where the function of suppres-sion of T cells by lung macrophages is due to cell-to-cellcontact.33,35 Apoptosis in FM has been found to occur rarely.33,35

Both suppression of T cells surrounding FM and its low rate ofapoptosis can explain why bacilli have more opportunities tosurvive while they remain inside FM.

Figure 5. Electron microscope details of foamy macrophages (FM) from DBA mice in the alveolar spaces at week 9 post-infection. Picture A shows bacilli inside a FM and increasedsize of lipid bodies. Picture B shows broken FM with extensive LB enveloping bacilli. Black arrows indicate bacilli. Original magnification was 2500�. Bar represents 2 mm. Picture onthe right bottom shows a bacillus inside a FM containing ILIs (white arrows). Bar represents 1 mm.

Figure 6. Evolution of foamy macrophages (FM) in guinea-pigs at week 18 post-infection assessed using semi-thin sections and toluidine blue staining (TBlue). Picture A (200�)shows the necrotic tissue in the granuloma core. The white box is magnified in picture B (1000�), and shows macrophages with ELB (black arrow), late lipid bodies (LLB) – which areinside FM – (white arrow) and a transition between both (red arrow). Picture C (400�) shows a bronchus. The white box is amplified in picture D (1000�) which shows FM with latelipid bodies (LLB) (white arrows). Bars represent 100 mm in A and C, and 20 mm in B and D.



Progression of LLB inside these FM observed in the presentstudy resembles what has been described in atherosclerosis.6

Changes in TBlue staining of these FM in semi-thin sections werefirst ascribed to the content of unsaturated fatty acids vs. saturatedfatty acids of LB, because osmium tetroxide post-fixation in elec-tronic microscope samples only binds to unsaturated fatty acids.36

However, analysis of ultra-thin sections in our study did not showa different electrodensity between ELB and LLB. The study of FMinduction in atherosclerosis has provided a mechanistic explana-tion that links this change on the TBlue stain to the accumulation ofcholesterol. In an atherosclerosis experimental model, ELB (i.e.isotropic) and LLB (i.e. anisotropic) were perfectly differentiatedwhen fresh material was evaluated under polarized light37 asa consequence of the lower percentage of TAG in LLB.38 In athero-sclerosis, changes in the nature of LB relate to the different speedmetabolism of their contents: as the metabolism of TAG is fasterthan the metabolism of cholesterol, it seems that the latter tends tocrystallize and transform the material into an anisotropic struc-ture.39 This process is related to the fact that in the atherosclerosisprocess low-density lipoproteins (LDL) become oxidized by freeradicals. This oxidation disrupts the normal system for cholesterolesterification and conversion into LB, leading to the hyper-accu-mulation of both non-esterified and esterified cholesterol.21 Inter-estingly, the extracellular milieu in the tuberculosis granulomahave abundant free radicals29 that can oxidize the lipids comingfrom the cellular debris, thus resembling this process.

The accumulation of FM around the necrotic tissues observed inour studies, has also a parallelism with atherosclerosis, where thisphenomenon is explained by the phagocytosis of LDL in the fibroticmatrix filling the necrotic tissue transuded from the capillaries. Thisphagocytosis induces a proinflammatory response40 that attractsmacrophages and lymphocytes to the necrotic tissue to search forremaining bacilli.

In summary, this study shows the evolution of macrophages toFM, thanks to the accumulation and progressive transformation ofLBs, in a scenario that has a lot of parallelisms with the athero-sclerosis. This context favors the permanence of the intracellularbacilli in a non-replicating state, which would give them someadvantage to face future stressful conditions, and thus to favor theirfuture dissemination and reactivation.

Acknowledgements

FIS 01/3104, Plan Nacional IþDþ I FIS CM06/00123 and GrandChallenge #12.

Funding: None.

Competing interests: None declared.

Ethical approval: Not required.

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