Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.
Tuberkulose (TB) holder stadig en stor trussel mod sundheden for mennesker verden over, og der er et behov for omkostningseffektiv, men pålidelige modeller til at hjælpe os med at forstå de mekanismer, sygdom og fremme opdagelser nye behandlingsmuligheder. In vitro cellekulturer af monolag eller co-kulturer mangler den tredimensionale (3D) miljø og vævsreaktioner. Heri beskriver vi en innovativ in vitro model af en human lungevæv, som lover at være et effektivt værktøj til at studere de komplekse begivenheder, der opstår under infektion med Mycobacterium tuberculosis (M. tuberculosis). 3D vævsmodellen består af vævsspecifikke epitelceller og fibroblaster, som er dyrket i en matrix af collagen på toppen af en porøs membran. Ved lufteksponering, epitelcellerne stratificere og udskiller slim ved apikale side. Ved at indføre humane primære makrofager inficeret med M. tuberkulose til vævet tilstandl, har vi vist, at immunceller vandre ind i inficeret væv og danne tidlige stadier af TB granulom. Disse strukturer rekapitulere særligt træk ved human tuberkulose, den granulom, som adskiller sig fundamentalt eller ej almindeligvis observeret i vid udstrækning anvendes eksperimentelle dyremodeller. Denne organotypisk kultur metode gør det muligt for 3D-visualisering og robust kvantitativ analyse, der giver afgørende oplysninger om rumlige og tidslige funktioner i værtscellelinjer-patogen interaktioner. Tilsammen lungevævet model giver en fysiologisk relevant væv mikro-miljø for undersøgelser af tuberkulose. Således lungevævet model har potentielle konsekvenser for både grundlæggende mekanistiske og anvendte studier. Vigtigere er det, model tillader tilføjelse eller manipulation af individuelle celletyper, som derved udvider dets anvendelse til modellering en vifte af smitsomme sygdomme, der påvirker lungerne.
Hos mennesker, reaktioner på infektion, vævsinflammation, cellulær rekruttering, vævsombygning og regulering af væv homeostase er komplekse begivenheder, der involverer forskellige celletyper. Derfor er disse processer bedst undersøgte i lokale vævsmiljø. Tidligere har det hovedsagelig været muligt ved hjælp af eksperimentelle dyremodeller. Men den udbredte forsøgsdyr holde mange grænser, da de ofte reagere på patogener på en anden måde end mennesker, og også vise et andet sygdomsforløb 1. Et menneske in vitro lungevæv model holder mulighederne for at studere specifikke immunresponser i human lunge.
Human tuberkuloseinfektion (TB) er hovedsagelig en sygdom, der påvirker lungerne. Mycobacterium tuberculosis (M. tuberculosis), det kausative agens for tuberkulose, når lungen via aerosoldråber, der transporteres til det alveolære rum, hvor bakterierne er opslugt af pulmonal dendritic celler og alveolære makrofager som en del af svaret på infektionen 2,3 medfødte immunforsvar. Fagocytose af patogenet fører til opdeling af fejlen inden for en fagosomet og ideelt resulterer i neutralisering og drab af patogenet ved phagocyt. Op til 50% af personer udsat for M. tuberkulose menes at være i stand til at fjerne infektionen gennem immunrespons medfødte 4. Andre resultater af infektion er efter det adaptive immunsystem på et senere tidspunkt, latent infektion eller i værste tilfælde kronisk aktiv sygdom 5.
Tidligere har der ikke været in vitro væv modeller til studier af menneskelig TB. Encellede kulturer af humane makrofager eller andre celler fra perifert blod er ofte blevet anvendt 6,7. Ulempen ved denne fremgangsmåde er, at de ikke kan afspejle dynamikken i forskellige celletyper, der sammen i en lungevæv udsat for M. tuberkulose </em>. Således er der et behov for en in vitro model for at kunne udføre funktionelle og mekanistiske undersøgelser på TB. Den cellebaserede in vitro humant lungevæv model beskrives her blev oprindeligt etableret af vores gruppe til undersøgelser af dendritiske celle funktioner 8. Vi har tilpasset denne metode til undersøgelse af TB.
Den menneskelige lungevæv model præsenteres her består af vævsspecifikke epitelceller og fibroblaster 8. Disse celler dyrkes i en matrix af collagen på toppen af en porøs membran i en transwell indsatsen og danne strukturer ligner normalt lungevæv human (figur 1). Når de udsættes for luft cellerne begynder at udskille slim på den apikale side 8. Ved at implantere humane primære makrofager inficeret med M. tuberkulose til modellen, har vi observeret, hvordan de immunceller migrerer i vævet og danne tidlige stadier af TB granulomer 9. Dette er den første humant væv model descrIBED for TB og det udgør et lovende redskab til at studere medfødte immunrespons til tuberkulose og andre sygdomme i lungerne. Til dato har vi kun anvendes monocytter og makrofager som immunceller i modellen, men graden af kompleksitet kan forøges ved medtagelse af yderligere relevante celletyper.
Figur 1. Skematisk oversigt over lungevævet model. (A) Modellen består af humane lunge-specifikke epitelceller, M. tuberculosis-inficerede primære makrofager og røde farvestof mærkede monocytter podet på collagen indlejret fibroblaster fremstillet på en transwell filter. Eksponering af vævet model til luft initierer produktionen af ekstra-cellulære matrixproteiner, slim sekretion og lagdeling af epitel. 3D-væv-modellen således udviklet er et nyttigt redskab til at studere M. tuberkuloseinfektion i et miljø, Closely ligner en menneskelig lunge. (B) Repræsentative mikroskopiske billeder af de forskellige trin i udarbejdelsen af vævet model. (C) Komplet struktur af lungen model vævssnit. Scale -. 100 um Klik her for at se en større version af dette tal.
The ability to recruit and form organized cell clusters at the site of infection is the hallmark of human TB 11. These dynamic structures known as tubercle granulomas primarily consist of immune cells (macrophages, monocytes, T-cells and B-cells) and multi-nucleated giant cells surrounding M. tuberculosis. The role of the granuloma has long been considered to wall off the infection, preventing local spread of bacteria. However, more recent studies show that granuloma formation is critical for early bacterial survival, growth and dissemination 12. A strategy of new studies is to identify molecules or pathways that could efficiently be targeted to inhibit the cellular migration in granuloma formation and/or TB dissemination.
A caveat for novel studies on TB is the lack of models that recapitulate human TB. The most widely used experimental animals do not form true granuloma upon M. tuberculosis infection, and are therefore not appropriate choices for studies of TB 13-16. Non-human primates have the closest resemblance to human TB 17, but are not the preferred choice owing to high operational costs and ethical issues. Human TB is a complex immunological process and is difficult to model in vitro. Cell cultures of monolayers or co-cultures lack the 3D environment and tissue responses. Therefore, we have developed an innovative lung tissue model based on human primary immune cells and human lung-specific cell lines 8,9. The model displays characteristic features of human lung tissue, including epithelia with evenly integrated macrophages, formation of extracellular matrix, stratified epithelia and mucus secretion 9.
The 3D human lung tissue model has several benefits over the in vitro single or co-cultures seeded on tissue culture plates or transwell inserts. First, the human lung-specific cells (fibroblasts and epithelial cells) are not commonly included in the in vitro single or co-cultures. Second, the immune cells and lung-specific cells are embedded in a 3D physiological context (collagen rich extra-cellular matrix products). The response of cells to a stimulus/infection and the migratory behaviour of cells, for instance formation of a granuloma, differ significantly between a 2D and 3D environment. Furthermore, the described method enables the 3D visualization and robust 3D quantitative analysis that provides pivotal information on spatial distribution and intricate cellular interactions.
Experimental infection in the model tissue with M. tuberculosis resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma (Figure 2 and 3). We have recently demonstrated that mutant strains defective in the ability to secrete the virulence factor ESAT-6 or Mycobacterium bovis BCG that lacks ESAT-6 did not induce the clustering of monocytes (no early granuloma), in contrast to the virulent M. tuberculosis 9. These data are consistent with the observations made from Mycobacterium marinum-infected zebrafish embryos, whose transparency allows for elegant live imaging of granuloma formation 12. As there is no gold-standard model for TB, we took advantage of the surgically resected tissue biopsies from TB patients for validation of the method 9. Our in vitro tissue model shares several characteristics with the lung and lymph node biopsies from TB patients, including the aggregation of macrophages in granuloma, the presence of both intra- and extracellular bacteria 18 and induction of necrosis 11.
Although the described model has physiological relevance to human TB and has several advantages over other in vitro models, it has some limitations. For instance, out of more than 20 collagen proteins identified in humans, only type I is included to the model to mimic the extra-cellular matrix. However, type I collagen is a complex mixture of extra-cellular matrix products and is the most abundant collagen in the human body. Further, we have demonstrated the presence of collagen IV and several extra-cellular matrix proteins such as tropoelastin, vimentin and laminin, which are produced by the epithelial cells and fibroblasts in the tissue model, indicating the synthesis of new collagen 8. Presently, the lung tissue model only has monocytes and macrophages, besides lung-specific cells. It lacks neutrophils and lymphocytes that are also known to be present in the granuloma. Remarkably the model is not limited to the introduction of additional immune cells and is of interest to explore how they contribute to the complex cellular interactions in human TB. Implantation of primary alveolar macrophages, skin-specific cells and lung carcinoma cells has already been tested in the model. Since our objective was to use a model that closely resembles human TB, introduction of mouse cells have not been attempted.
In summary, the lung tissue model has implications for both basic mechanistic and applied studies. Potential applications of the lung model include the study of innate immunity, investigating mechanistic aspects of host defences such as phagosomal maturation, autophagy, production of cytokines, chemokines and anti-microbial peptides, and functional characterization of individual cell types. Strikingly, the in vitro tissue model allows manipulation of one or more cells types and provides a relevant tissue micro-environment, not only for studies on TB, but for a variety of infectious and non-infectious diseases that affect the lungs.
The authors have nothing to disclose.
The authors acknowledge the Microscopy core facility at the Faculty of Health Sciences, Linköping University for providing access to advanced imaging systems; Karl-Eric Magnusson (Emeritus Scientist) at the Dept. of Clinical and Experimental Medicine, Linköping University for providing access to Imaris 3D/4D image processing software (Bitplane, Switzerland); and S. Braian for his help with the lung model cartoon. This work was supported by funds from the Swedish Research Council (Alternatives to animal research, 2012-1951) and Swedish Research Council (2012-3349) to M.L. and Swedish Foundation for Strategic Research to S.B. S.B. receive grants from the Karolinska Institutet, Swedish Research Council, the Swedish International Development Cooperation Agency (Sida) and the Swedish Civil Contingencies Agency (MSB), and the Swedish Heart and Lung Foundation (HLF). M.S. received grants from the Karolinska Institutet and Stockholm County Council.
Cell culture inserts | BD Falcon | 353092 | |
6-well culture plates | BD Falcon | 353046 | |
MRC-5 cells, lung fibroblasts | ATCC#CCL-171 | ||
16HBE cells, lung epithelial cells | Gift from Dr. Dieter Gruenert, Mt. Zion Cancer Center, University of California, San Fransisco, USA | ||
5 x Dulbecco’s modified Eagle’s medium (5 x DMEM) | Gibco | 12800-082 | Made from powder but add 5 times less water. Adjust pH to 7.3 and filter it using a 0.2 µm filter. |
Dulbecco’s modified Eagle’s medium with glucose (DMEM) 1x | Gibco | 41965-039 | |
Minimum Essential Medium (MEM) 1x with Earle’s salts | Sigma | M4655 | |
Non-Essential Amino Acids Solution, 100x | Life Technologies | 11140-035 | |
L-glutamine 200 mM (100x) | Gibco | 25030-024 | |
Sodium Pyruvate | Life Technologies | 11360-039 | |
NaHCO3 (71.2 mg/ml) | Prepared in house | ||
Heat inactivated Fetal Bovine Serum (FBS) | Gibco | 10270-106 | Heat inactivated for 30 min, 56 °C |
Gentamicin (50 mg/ml) | Gibco | 15750-060 | |
Hepes buffer solution 1M | Gibco | 15630-056 | |
Penicillin Streptomycin (Pen Strep) | Gibco | 15140-122 | |
Lymphoprep | Axis-Shield | 7801 | |
Ultrapure 0.5 M EDTA | Gibco | 15575 | |
Bovine Collagen PA treated (500 ml) | Organogenesis | 200-055 | |
Pure col purified Bovine Collagen solution (100 ml) | Advanced biomatrix | 5005-B | |
Extracellular matrix protein, Fibronectin (1 mg) | BD | 354008 | |
Primary human monocytes/macrophages | Isolated from human whole blood or buffy coats. | ||
PKH26 Red fluorescent cell linker | Sigma | MINI26 | |
Mycobacterium tuberculosis H37Rv expressing green fluorescent protein | M. tuberculosis H37Rv wild type was transformed with the pFPV2 plasmid constitutively expressing GFP. | ||
Middlebrook 7H9 medium | Difco | 271310 | |
BBL Middlebrook ADC Enrichment | BBL | 211887 | |
Tween-80 | |||
Glycerol | |||
Kanamycin B sulfate (20 µg/ml) | Sigma | B5264 | |
Prolong Gold anti=-fade reagent with DAPI | Invitrogen | P36935 | |
Trypsin -EDTA | |||
Bovine serum albumin | |||
Paraformaldehyde | |||
DAPI | |||
LSM700 Confocal microscope | Zeiss | ||
iMaris Scientific 3D/4D image processing software, version 7.6.8 | Bitplane AG |