BCG-Infected Dendritic Cells Induce TNF-alpha Dependent Cell Cluster Formation that Promotes Bacterial Dissemination through an In Vitro Model of the Blood Brain Barrier

Abstract

Central nervous system tuberculosis (CNSTB) is the most severe manifestation of extrapulmonary tuberculosis infection, but the mechanism of how mycobacteria cross the blood-brain barrier (BBB) is not well understood. Here, we report a novel murine in vitro BBB model combining primary brain endothelial cells, Mycobacterium bovis Bacillus Calmette-Guérin (BCG)-infected dendritic cells (DCs), peripheral blood mononuclear cells (PBMCs), and bacterial antigen-specific CD4⁺ T cells. We show that mycobacterial infection limits DC mobility and also induces cellular cluster formation that has a similar composition to pulmonary mycobacterial granulomas. Within the clusters, infection from DCs disseminate to the recruited monocytes, promoting bacterial expansion. Mycobacterium-induced in vitro granulomas have been described previously, but this report shows for the first time that they can form on brain endothelial cell monolayers. Cellular cluster formation leads to cluster associated damage (CAD) of the endothelial cell monolayer defined by mitochondrial stress, disorganization of the tight junction proteins ZO-1 and claudin-5, upregulation of the adhesion molecules VCAM-1 and ICAM-1, and increased transmigration of bacteria-infected cells across the BBB. TNF-α inhibition reduces cluster formation on brain endothelial cells and mitigates CAD. These data describe a novel model of bacterial dissemination across the blood-brain barrier (BBB) shedding light on a mechanism that might contribute to CNSTB infection and facilitate treatments.

1. Introduction

One of the leading causes of death among infectious diseases is tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb). These bacteria primarily infect the lung but can disseminate to other parts of the body including the central nervous system (CNS), leading to tuberculous meningitis (1). CNSTB is a devastating disease which causes serious neurological damage and severe disability with a high mortality rate (2–4). It has been proposed that mycobacterial lesion formation on the vessels of the CNS facilitates the dissemination (5), however, the mechanism of how mycobacteria traverse the BBB to access the brain parenchyma is still unknown.

There are three biological barriers of the CNS, which could serve as an entry route for mycobacteria from blood vessels including the meninges, the choroid plexus, and the blood-brain barrier (6). A study using a human in vitro blood-brain barrier (BBB) model showed that mycobacteria are capable of invading endothelial cells and traverse the endothelial monolayer without a cellular carrier (7). On the other hand, previous studies have described mycobacteria crossing the cellular layers within carrier cells (4) in a lung in vitro system (8) and in zebrafish models (9, 10), providing a basis for the Trojan horse theory. Mycobacteria are intracellular microorganisms, and in the body, they reside mainly in macrophages and dendritic cells (11). Previous studies have shown that CD11c^high DCs are the sole emigrating cell type from primary granulomas carrying live mycobacteria that result in the dissemination and formation of new granulomas through capturing of antigen specific T-cells (12). Mycobacterium carrying DCs promote granuloma formation (12) that can be facilitated by their limited migration (11–13).

In vitro granuloma models offer a unique opportunity to elucidate the mechanisms of early granuloma formation and analyzing host-mycobacteria interactions in a controlled environment (14). Several groups have established in vitro granuloma models using human peripheral blood mononuclear cells (PBMCs) infected with Mtb or the attenuated strain Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Pasteur. With these models, multiple aspects of the granuloma pathogenesis were studied: (i) secretion of cytokines promoting granuloma formation such as TNF-α (15, 16), (ii) dormancy (16), (iii) formation of multinucleated giant cells (17), (iv) differences between active and latent TB infections (18), and (v) 3D environment to study host-pathogen interactions (19). These models are suitable to analyze intercellular interactions within an artificial granuloma, but lack the possibility to study Mtb dissemination in situ or across a vessel wall. To address this, we developed a novel combination model of an in vitro granuloma and BBB co-culture system (20). This model allows us to study the interaction of Mycobacterium-infected DCs, Mycobacterium antigen-specific P25 T cells, PBMCs, and the BBB. Here, we show for the first time that in vitro granulomas form on the surface of brain endothelial cells when BCG-infected DCs are co-cultured with PBMCs and P25 T cells. These in vitro cell clusters are complex in cellular composition, resemble in vivo lung granuloma morphology, and facilitate in situ bacterial dissemination. We also confirmed that DCs are restricted in migration after infection (12, 13) which we show to be a possible cause of altered intracellular actin redistribution. The in vitro cell aggregates interfere with BBB and brain endothelial cell functions. The in vitro granuloma cell cluster formation leads to reactive oxygen stress of endothelial cells, cluster associated damage (CAD), and increased transmigration of infected cells across the endothelial cell (EC) monolayer. Treatment with the TNF-α blocker MP6-XT22 decreased cluster formation, adherence to brain endothelial cells, and diminished the CAD effect. Our data contribute to the understanding of mycobacterial dissemination across brain endothelial monolayers giving insight for developing therapeutic targets to inhibit TB meningitis.

2. Materials and Methods

2.1. Experimental animals and ethics statement

All experimental procedures were performed in accordance with the guidelines of the University of Wisconsin Institutional Animal Care and Use Committee using approved protocols. Wild type C57BL/6J, CD11c-eYFP (B6.Cg-Tg(Itgax-Venus)1Mnz/J) (21), and CX3CR1-GFP (B6.129P-Cx3cr1tm1Litt/J) mice were purchased from The Jackson laboratory (Bar Harbor, ME, USA) and housed in micro-isolator cages at the University of Wisconsin pathogen-free facility Animal Care Unit (Madison, WI, USA). The P25 mice express a transgenic T-cell antigen receptor recognizing Mycobacterium tuberculosis (Mtb) Antigen 85b^(250−254) epitope restricted to MHC class II IAb (11) were a gift of Drs. Rothfuchs and Sher (NIH, Bethesda, MD, USA). The mT/mG tdTomato-expressing P25 T-cells were isolated from the F1 generation of a cross between homozygous tdTomato and P25 mice. The LifeAct-RFP mice (22) were a gift from Dr. Jordan Jacobelli (Department of Biomedical Research, National Jewish Health; Department of Immunology and Microbiology, University of Colorado School of Medicine). LifeAct-RFP mice were generated to study filamentous actin dynamics in living cells that express the 17-amino-acid LifeAct peptide driven by a chicken actin promoter and CMV enhancer. These are transgenic global reporter mice that express LifeAct fused to mRFPruby on F-actin. LifeAct-RFP positive mice were crossed with CD11c-eYFP mice generating CD11c-eYFP⁺LifeAct-RFP⁺ mice from which primary cells were isolated.

2.2. Bacterial strains and culture

The Mycobacterium bovis Bacillus Calmette-Guérin (BCG) Pasteur strain was provided by Dr. Glen Fennelly (Albert Einstein College, Bronx, NY). BCG was electroporated with tdTomato (pTEC27), or E2-Crimson (pTEC19) plasmids which were generated by Dr. Lalita Ramakrishnan (Addgene plasmid #30182 and #30178, respectively, Cambridge, MA, USA) (23). Strains were stored as frozen aliquots at −80 °C. All BCG was grown at 37 °C in Middlebrook 7H9 media (BD, Franklin Lakes, NJ, USA) supplemented with 10 % oleic acid-albumin-dextrose-catalase (OADC) enrichment (BD, Franklin Lakes, NJ, USA) and 0.05% Tween 80 (Sigma-Aldrich, St. Louis, MO, USA) in the presence of Hygromycin B Gold (100 μg/mL; InvivoGen, San Diego, CA, USA) in a shaking incubator at 37 °C.

2.3. Bone marrow derived dendritic cell differentiation

Dendritic cells were generated from the bone marrow of adult wild type C57BL/6J or CD11c-eYFP or CD11c-eYFP⁺LifeAct-RFP⁺ mice as described previously (24–27). Briefly, bone marrow was extracted from the femurs of adult wild type or CD11c-eYFP mice, cell suspensions were treated with ammonium chloride potassium-containing lysis buffer (Stemcell Technologies, Vancouver, Canada) to lyse the erythrocytes, washed, then plated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin/streptomycin, 1 % HEPES, 1 % GlutaMAX (Thermo Fisher, Waltham, MA, USA), 1 % Non-essential amino acids (Thermo Fisher, Fitchburg, WI, USA), 1 % Essential amino acids (Thermo Fisher, Fitchburg, WI, USA), 1 % Na-pyruvate (Thermo Fisher, Fitchburg, WI, USA), 50 μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 20 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF, Peprotech, Rocky Hill, NJ, USA). Cells were seeded onto 100 mm non-adherent Petri dishes (Falcon, Corning, Corning, NY, USA), and maintained at 37 °C in a humidified incubator with 5% CO₂. Cells were fed on day 3 and non-adherent and loosely adherent BMDC precursors were passaged on day 6 using the same medium containing 10 ng/ml GM-CSF. Surface expression of CD11b, CD11c, and MHC II were confirmed by flow cytometry. Cultures were infected with BCG on day 8 for 1–4 hours with MOI 1:1 in 2% FBS-RPMI in a 48-well non-adherent plate. Cells were washed after infection and used for experiments. Infection was confirmed with both flow cytometry and fluorescent microscopy.

2.4. In vitro dendritic cell migration and Morphokinetic Assay

3D collagen chemotaxis assays were performed with μ-Slide Chemotaxis^3D following the manufacturers protocol (Ibidi, Fitchburg, Wi, USA). The μ-Slide Chemotaxis^3D device is composed of three observation areas each of which are flanked on both sides by a reservoir such that once the observation area is seeded and the collagen has solidified, a concentration gradient develops through the addition of media plus cellular stimuli to one reservoir and media without stimuli to the other which serves as a no-chemokine control for each experiment. Infected or uninfected CD11c-eYFP or CD11c-eYFP⁺LifeAct-RFP⁺ bone marrow-derived DCs at a concentration of 3×10⁶ cells/ml were suspended in 5 mg/ml of type I collagen gel (Ibidi, Fitchburg, Wi, USA) at a final concentration of 1.5 mg/ml supplemented with 10x DMEM (Sigma-Aldrich, St. Louis, MO, USA), 1x DMEM (Sigma-Aldrich, St. Louis, MO, USA), NaOH in ultrapure H₂O, 7.5 % NaHCO₃ (Sigma-Aldrich St. Louis, MO, USA), and ultrapure H₂O on ice. Immediately after suspension, the cell-collagen mixture was injected into the observation area of the μ-Slide and placed into an incubator for 30 minutes to allow for gelation. Once fibrils are visible, 200 ng/ml of recombinant mouse CCL2/JE/MCP-1 (PeproTech, Rocky Hill, NJ, USA) chemokine in RPMI 1640 (Corning, Corning, NY, USA) was added to one reservoir and only RPMI 1640 to the other as a no chemokine control. For measuring distance and velocity, the slide was placed in a stage-top Tokai Hit Bio-chamber at 37°C and 5% CO₂ and pictures were taken every 2 minutes for 3 hours on a Leica SP8 3X STED Super-resolution/confocal microscope at 10X magnification. For morphodynamic analysis, CD11c-eYFP⁺LifeAct-RFP⁺ DCs were used in the same setup with pictures of BCG-infected and uninfected cells taken at 63X every 30 seconds for 10 minutes. Single cell protrusion outputs and actin localization were analyzed using the ImageJ open source plugin Automated Detection and Analysis of Protrusions (ADAPT) version 1.185. Observation of assembly and evaluation of the timing of in vitro granuloma formation was performed using a 2-well culture insert micro-dish (Ibidi, Fitchburg, WI, USA). Endothelial cells (ECs) were grown on the ibiTreat surface and the model was assembled on the top of the confluent EC monolayer using 7 × 10⁴ tdTomato BCG-infected DC co-cultured with 7 × 10⁴ P25 PBMCs. Before the assembly, PBMC cultures were labeled with anti-CD4-Alexa 647 conjugated antibody. Cell imaging was performed using confocal live cell imaging (Leica SP8, Leica Microsystems, Wetzlar, Germany). Stable temperature and CO₂ levels were maintained by a stage top incubator (Tokai Hit, Fujinomiya, Japan). Cell aggregate formation was followed 1–4 hours after assembly while pictures were taken every 2 minutes for 3 hours. Picture analysis was performed using ImageJ (National Institute of Health open source software).

2.5. In vitro mouse blood-brain barrier model

Primary mouse brain ECs were isolated from adult wild type C57BL/6J mice, according to previously described methods (28–30). Harvested cells were plated on plastic surfaces coated with collagen type IV and fibronectin (100 µg/ml each, Sigma-Aldrich, St. Louis, MO, USA). Cell culture medium consisted of DMEM/F12, FBS (15 %, Corning, Corning, NY, USA), heparin (100 µg/ml, Sigma-Aldrich, St. Louis, MO, USA), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml, Sigma-Aldrich, St. Louis, MO, USA), basic fibroblast growth factor (1 ng/ml; Sigma-Aldrich, St. Louis, MO, USA) and gentamycin (50 µg/ml, Sigma-Aldrich, St. Louis, MO, USA). Cells were cultured for four days in the presence of 4 µg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA) to eliminate contaminating cell types (31). Primary mouse brain ECs were seeded on Transwell, polycarbonate membrane (3 µm pore size, Corning) and co-cultured with glial cells (28–30). After two days of co-culture, 550 nM hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA) was added to the medium to facilitate intercellular junction formation (32, 33). Primary cultures of mouse glial cells were obtained from one- or two-day-old C57BL/6J wild type mice as described previously (29, 30).

2.6. Evaluation of barrier integrity

Transendothelial electrical resistance (TEER) was measured by the EVOM2 voltohmmeter (World Precision Instruments, Sarasota, FL, USA) using STX-2 electrodes. Resistance was calculated according to the surface of the Transwell inserts (Ω × cm²) and TEER of cell-free inserts (70 Ω) was subtracted from these values. TEER values were confirmed prior to all experiments (150 Ω × cm²), and a low permeability value was validated with showing 3.36 × /10⁻⁶ cm/s permeability coefficient (P_e) for sodium fluorescein and 0.45 × /10⁻⁶ cm/s for Evans-blue labeled albumin.

2.7. Peripheral blood mononuclear cell (PBMC) isolation

Primary blood lymphocytes were isolated from 8–12 weeks old C57BL/6J wild-type, P25, CD11c-eYFP reporter, or CX3CR1-GFP reporter mice using the Ficoll gradient method (GE Healthcare, Chicago, IL, USA). Blood from anesthetized mice was collected into 10 mL phosphate buffered saline (PBS) with 20 U/ml heparin (Sigma-Aldrich, St. Louis, MO, USA) and layered to the top of 7 ml Ficoll gradient in a 50 ml centrifuge tube. The gradient was centrifuged at 740 g for 30 minutes at 20 °C with the brake off. The upper layer of the gradient containing blood plasma and platelets was discarded and the mononuclear cell layer was collected and processed for in vitro granuloma studies.

2.8. In vitro granuloma model assembly

The in vitro co-culture BBB model was established as described previously (20). For each experiment performed using the Transwell inserts, 10⁵ uninfected or infected DCs were cultured on the top of brain endothelial cells with or without the addition of 10⁵ P25 PBMCs. All groups received 20 % liver granuloma supernatant to enhance in vitro granuloma formation (20). The cell culture medium consisted of DMEM/F12, 10 % FBS (Corning, Corning, NY, USA), heparin (100 µg/ml, Sigma-Aldrich, St. Louis, MO, USA), insulin (5 µg/ml), transferrin (5 µg/ml), sodium selenite (5 ng/ml, Sigma-Aldrich, St. Louis, MO, USA) and gentamycin (10 µg/ml, Sigma-Aldrich, St. Louis, MO, USA). When TNF-α neutralization was tested, neutralizing antibody MP6-XT22 (34) was added at 50 ng/ml (BioLegend, San Diego, CA, USA).

2.9. Immunohistochemistry

The cellular composition, morphology, and functional changes of the in vitro aggregates were characterized by immunohistochemical staining. The in vitro granuloma models were fixed one or two days after the assembly with 2 % paraformaldehyde (PFA, Electron Microscopy Sciences, Hatfield, PA, USA) in PBS for 1 h and cells were stored in PBS until staining. To define cellular composition of the in vitro aggregates, cultures were stained with antibodies for CD4 conjugated to PE (clone RM4–5, 1:200, 553048, BD), CD11b conjugated to PE (clone M1/70, 1:200, 553311, BD) or APC (clone M1/70, 1:200, eBioscience, Santa Clara, CA, USA, 17–0112-82) and B220 conjugated to PE (clone RA3–6B2, 1:200, 553090, BD, Franklin Lakes, NJ, USA). Non-conjugated primary antibodies against integral membrane tight junction marker Claudin-5 (rabbit-anti-Cl5, 1:400, SAB4502981, Sigma-Aldrich, St. Louis, MO, USA ), junctional associated protein Zonula Occludens-1 (rabbit-anti-ZO-1, 1:400, 61–7300, Thermo Fisher, Fitchburg, WI, USA), vascular cell adhesion molecule-1 (rat-anti-VCAM-1, CD106, 1:100, 14–1061-82, Thermo Fisher, Fitchburg, WI, USA), and biotinylated intercellular adhesion molecule-1 (ICAM-1, CD54, 1:200, BAF796, R&D Systems, Minneapolis, MN, USA) were used to test barrier characteristics. Secondary antibodies goat-anti-rabbit Alexa 568 (1:400, A-11008, Thermo Fisher, Fitchburg, WI, USA), goat-anti-rat Alexa 568 (1:400, A-11077, Thermo Fisher, Fitchburg, WI, USA) and streptavidin-Alexa 647 (1:200, 405237, Biolegend, San Diego, CA, USA) were used to detect stainings. All samples were permeabilized with 0.1 % Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) in PBS detergent for 10 min, 4 °C, and blocked in 1 % bovine serum albumin-PBS for 3 h, room temperature. Conjugated antibodies were incubated for 1 h at room temperature with the nucleus dye. Cells were washed three times with PBS between each step and incubations were always performed in blocking buffer. Samples were mounted on slides using the Fluoromount G mounting medium (Southern Biotech, Birmingham, AL, USA). Images were obtained using the Olympus FV1200 IX83 confocal microscope (Olympus Corporation, Shinjuku, Japan) and analyzed with ImageJ (National Institute of Health open source software).

2.10. Cell cluster quantification and in vitro granuloma formation

Cell cluster formation was analyzed with two methods to determine cluster size and cell number threshold. During the first 24 hours of incubation, floating and attached immune cell cluster formation was observed using the 2-well culture insert micro-dish (Ibidi) with phase contrast microscopy. Before the addition of infected and uninfected DCs and PBMCs, primary brain ECs were grown to confluency. Pictures were taken after 30 min of assembly at 4 sides of the micro dish and cluster number was quantified with the ImageJ software. Cell clusters were defined as cellular aggregates consisting of at least 5 cells per cluster. This threshold was kept uniform during the analysis.

In subsequent experiments co-culture of cells were kept together using cell culture Transwell inserts. After 24 and 48 hours, supernatants were removed and the barrier forming primary brain ECs with attached cells and cell clusters were fixed and stained using immunohistochemistry. Images were taken of entire inserts, where aggregates consisting of at least 5 cells were counted manually.

2.11. Mitotracker staining

Mitochondrial network was visualized using the Mitotracker Orange dye (Thermo Fisher) according to the manufacturer’s protocol. Briefly primary brain ECs were grown on collagen type IV coated round cover slips (VWR, Radnor, PA, USA) until confluency, and the in vitro granuloma model was established using 3 × 10⁵ uninfected or BCG infected DCs with or without 3 × 10⁵ P25 PBMCs. After one day of co-culture, supernates were removed, cultures were washed once with PBS, and cells were incubated with 0.1 µM of Mitotracker Orange (Thermo Fisher) in serum free DMEM for 40 min at 37 °C. After staining, cells were washed with PBS and fixed with 2 % PFA in PBS for 1 h. Samples were mounted and analyzed with confocal microscopy.

2.12. Transmigration assays

To count CD11c-eYFP cells that transmigrated the membrane and remained adherent underneath the cell culture inserts, we used Z-stack fluorescent confocal imaging. Here, only CD11c-eYFP that carried bacteria and associated with cell clusters were counted.

Migration of CD11c-eYFP cells across the EC monolayer was studied by cytofluorometry of the cells in the bottom wells. 10⁴ Cell Trace Violet (CTV) labeled wild type DCs were added to the bottom well as a buffer cell population. CTV labeling was performed according to the protocol of the manufacturer (Invitrogen, Life Technologies). Absolute cell numbers were determined with the AccuCheck flow cytometry counting beads (Life Technologies). Data was collected using a BD LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo v. 8.7 (Flowjo LLC, Ashland, OR, USA).

2.13. Flow cytometry

Cells were incubated with cell viability Ghost dye (1:100, Tonbo Biosciences, San Diego, CA, USA) in PBS for 10 min at 4 °C, then fixed for 40 min with 2 % PFA-PBS at room temperature. Specification of anti CD4, CD8, B220, CD11c, CD11b antibodies are listed in the immunohistochemistry section. Data was collected using an LSRII flow cytometer and analyzed with FlowJo v. 8.7.

2.14. Statistical analyses

For statistical analysis GraphPad Prism 5.0 software (GraphPad Software, San Diego, CA, USA) was used. Results are given as mean ± the standard error of the mean (S.E.M). Multiple comparisons were made using one-way and two-way ANOVA with Bonferroni-tests. Two-tailed unpaired t-test analysis was used to compare measures made between two groups. P values < 0.05 were being considered statistically significant. All types of experiments were repeated at least two to three times using independent cell isolations for all types of primary cells with n >= 3.

3. Results

3.1. DC mobility is reduced after BCG infection.

The migration of infected DCs and dissemination of BCG from primary granulomas by infected DCs can result in antigen-specific T-cell arrest and the subsequent formation of secondary and tertiary granulomas (12). To study the effect of mycobacterial infection on the migratory capacity and morphokinetics of DCs, we infected bone marrow-derived DCs from the CD11c-eYFP reporter mice with BCG-E2-Crimson. Infected and uninfected DC migration was analyzed using Ibidi μ-slide chemotaxis chambers with media supplemented with 200ng/μl CCL2 on one side and only media on the other. CCL2 contributes to the recruitment of immune cells to the site of infection and is necessary for DC migration into the CNS in experimental autoimmune encephalomyelitis (35–37). A mixture of infected (CD11c-eYFP⁺BCG-E2-Crimson⁺) and uninfected (CD11c-eYFP⁺BCG-E2-Crimson⁻) DCs were placed in a 3D collagen suspension and loaded into the migration chamber where a picture was taken every 2 minutes for 3 hours to create a video where single cell migration kinetics were tracked using the manual tracking plugin from ImageJ. Tracking data from a total of 52 uninfected and 93 infected cells from 3 separate experiments showed a reduction in track length by infected cells compared to uninfected cells (Fig. 1A) that correlated with a reduction in the accumulated distance by infected DCs (Fig. 1B). Additionally, the migration velocity of uninfected DCs was roughly four times greater than that of BCG-infected DCs within the 3-hour time frame (Fig. 1C). This data show that the migration kinetics of DCs during infection with BCG is diminished both in velocity and in distance traveled.

FIGURE 1.

Migration analysis using live cell imaging of BCG-infected and uninfected DCs. Using Ibidi 𝜇-slide chamber infected and uninfected DCs were imaged every 2 minutes for 3 hours. (A) Representative cell tracks from the Manual Cell Tracker ImageJ plugin of uninfected (left) and infected (right) DCs. (B) Quantification of the accumulated velocity and (C) distance of infected and uninfected DCs from three different experiments. Unpaired t-test, n=52 (uninfected) and n=93 (infected), ****, p<0.0001, data is shown as mean +/− SEM

3.2. BCG infection reduces DC polarity and leading-edge migratory actin distribution.

As we have shown, BCG has the ability to restrict DC migration during infection (Fig. 1). Next, we wanted to determine the effect of BCG infection on DC polarity and actin distribution to find a possible cause for the reduction of migration. To study actin localization and distribution in regard to DC cell polarity during BCG infection, DCs from CD11c-eYFP⁺LifeAct-RFP⁺ mice were used to visualize the actin distribution and kinetics in combination with live-cell imaging. DCs were infected with E2-Crimson BCG for 1 hour at a multiplicity of infection (MOI) of 1, then suspended in a 3D collagen matrix and injected into the μ-slide migration chamber. Pictures were taken of 10 infected and 11 uninfected cells at the single cell level every 30 seconds for 10 minutes to create a video for analysis. The ImageJ plugin ADAPT was used to measure actin distribution and kinetics within each cell. Our data show that infected DCs have a round morphology with reduced continued polarization in any specific direction, whereas uninfected cells expressed polarization usually in a single direction (Fig. 2A). For cells to polarize and subsequently migrate toward a chemotactic signal, they first must be able to protrude in one direction (leading edge) of the cell while simultaneously retracting the other end of the cell (38). We measured the protrusion and retraction of the cell membrane and found that infected cells displayed a tendency to either protrude or retract (Fig. 2A bottom row, cell outline all green or red), while uninfected cells were able to accomplish both actions simultaneously and more often (Fig. 2A top row, cell outline green and red). Analysis showed that in infected DCs, about 60% of the time the DC cell membrane either protruded or retracted while uninfected DCs experienced this movement only around 5% of the time (Fig. 2B). Additionally, uninfected DCs were able to simultaneously protrude and retract about 75% of the time in contrast to infected cells which was observed less than 20% of the time (Fig. 2C). Next, we studied the actin distribution and expression by measuring the mean fluorescence intensity (MFI) at the center of the cell (arbitrary units=0) to the outer boundary (arbitrary units =1) (Fig. 2E). We found that actin distribution was visibly expressed at the center of the infected DCs through the outer boundary whereas uninfected cells had most of the actin localized to the outer boundary (Fig. 2D). Compared to uninfected DCs, MFI analysis confirmed our observations that infection increased actin expression toward the center of the cell and through to the outer cell boundary (Fig. 2F). Our data show that infected DCs exhibit different morphokinetic and actin distribution than uninfected DCs, which may play a role in the infection-mediated reduction of cell migration.

FIGURE 2.

BCG-infection reduces DC polarization and actin localization to a leading edge during infection. (A) Automated detection and analysis of protrusions (ADAPT) analysis of representative time-lapse images showing protrusion (green) and retraction (red) events of CD11c-eYFP⁺LifeAct-RFP⁺ dendritic cells uninfected (top) and infected (bottom). (B) Quantification of the time cells have spent with 70% of the cell body either protruding or retracting and (C) 40–50% of the cell body either protruding or retracting. (D) Representative images of actin localized within uninfected (top) and infected (bottom) DCs. (E) Quantification of actin localization by measuring the mean fluorescent intensity (MFI) from the center of the cell (0) to the cell’ edge (1) in arbitrary units. 63x. Experiments were repeated three times, n=10 (infected) and n=11 (uninfected). Analysis was performed using t-test, *, p<0.05; ***, p<0.001, data is shown as mean +/− SEM

3.3. BCG-infected dendritic cells facilitate cellular cluster formation and promote attachment of cell clusters to the surface of brain endothelial monolayers in vitro.

In vitro granuloma models are useful to understand the formation of granulomas and define cellular interactions in mycobacterial infections (14, 39). By combining BCG-infected or uninfected CD11c-eYFP reporter DCs with or without PBMCs from mycobacterial antigen-specific P25 TCR transgenic mice and the primary mouse brain endothelial cell BBB model, we were able to visualize and characterize early cellular interactions of infected DCs with other immune cell types on brain endothelial cells (Fig. 3A). We detected cellular cluster formation as early as 30 minutes when infected DCs were used (Fig. 3B). However, after 1-hour, small clusters began to form more frequently in the presence of P25 PBMCs with infected DCs (Fig. 3C). To observe the dynamics of cluster formation, live cell imaging was performed between 1 and 4 hours after the assembly (Supplementary video 1). We found that when BCG-infected DCs and PBMCs from P25 TCR transgenic mice are co-cultured on brain endothelial monolayers, cluster formation is dynamic. Single cells come together to form smaller clusters which then form larger aggregates (Supplemental video 1). This data show for the first time that BCG-infected DCs induce rapid cellular cluster formation on brain endothelial cell monolayers.

FIGURE 3.

In vitro granuloma formation of Mycobacterium-infected dendritic cells is facilitated by P25 Mycobacterium antigen-specific CD4⁺ T-cell containing peripheral blood mononuclear cells, which promote attachment of cellular aggregates to the surface of brain endothelial monolayers. (A) Scheme of the in vitro system used to model granuloma formation on brain microvessels. Primary mouse brain endothelial cells were co-cultured with primary mouse astrocytes to create the in vitro blood-brain barrier model on a Transwell insert system. To induce in vitro granuloma formation primary bone marrow derived dendritic cells from CD11c-eYFP reporter mice were infected with Mycobacterium bovis bacillus Calmette-Guérin (BCG) and co-cultured with primary mouse peripheral blood mononuclear cells (PBMC) isolated from P25 Mycobacterium antigen-specific CD4⁺ T-cell transgenic mice. Liver granuloma supernate was also added to the system to facilitate cell aggregate formation. (B) Comparison of aggregate formation induced by infected and uninfected DCs in the presence or absence of P25 PBMCs on primary brain endothelial cells at different time points was followed with confocal microscopy. Cell cluster formations are outlined with yellow. (C) Quantification of cluster formation induced by infected vs. uninfected DCs in the presence or absence of P25 PBMCs on primary brain endothelial cells at different time points. Cell clusters were quantified by analyzing phase contrast images from three different experiments, 4 field of view / time point, n=2–3 / group / experiment. Two-way Anova with Bonferroni post-test, **, p<0.01; ***, p<0.001, data is shown as mean +/− SEM.

3.4. In vitro cluster formation by BCG-infected CD11c-eYFP cells and P25 PBMCs facilitates transmigration of infected CD11c-eYFP cells across brain endothelial monolayers.

Next, we wanted to understand if the formation of these clusters on the in vitro BBB affects the migration of infected cells across the barrier. To answer this question, we quantified the amount of BCG-infected DC migration across the brain EC monolayer. 3D rendering of fluorescent confocal images organized in Z-stacks of clusters from BCG-infected DCs co-cultured with PBMCs show CD11c-eYFP⁺ and CD11c-eYFP⁻ cells traversing the EC monolayer carrying bacteria (Fig. 4A and 4C). Analyzing cellular migration of BCG-infected DCs and infected DCs with PBMCs for 24 and 48 hours using cytofluorimetry showed that infected DCs were able to migrate across the monolayer (Fig. 4B). However, BCG-infected DC migration across the EC monolayer significantly increased in the presence of PBMCs: ~8 fold after 24 hours and ~3 fold after 48 hours (Fig. 4B). Confocal microscopic analysis of the inserts showed that some infected DCs remained adherent to the bottom side of the membrane (Fig. 4C i-iii). This action was amplified in the presence of PBMCs when compared to groups without PBMCs (Fig. 4D). Together, these results indicate that cluster formation facilitates the dissemination of infected CD11c-eYFP cells across the brain EC monolayer.

FIGURE 4.

In vitro granuloma formation facilitates transmigration of infected CD11c-eYFP cells across brain endothelial monolayers. (A) Representative picture of a cell cluster formed by BCG-infected DCs and P25 PBMCs attached to the brain endothelial monolayers (i) and illustrated representation (ii). 3D rendering of cluster (iii). Cross section view showing cells attached to the lower side of the Transwell insert (iv) and an illustrated representation (v). (B) Fold change of infected CD11c-eYFP positive cells in the presence and absence of P25 PBMCs that migrated across the brain endothelial monolayer quantified by flow cytometry. Analysis was done from three separate experiments, n=3 technical parallels / experiment. Absolute number of cells was quantified by counting beads. Unpaired t-test, *, p<0.05, error bars mean +/− SEM. (C) Bone marrow derived dendritic cells from wild type C57/bl6 animals were infected with BCG and co-cultured with peripheral blood mononuclear cells isolated from CD11c-eYFP reporter mice on the in vitro blood-brain barrier model for up to 48 hours. Representative picture of a peripheral blood mononuclear derived CD11c-eYFP cell (green) transmigrating and carrying BCG (white) across the brain endothelial monolayer (i). Cross-section view of the confocal microscopy Z-stack picture shows the transmigrating cell (ii). Illustration clarifying the interpretation of the cross section view of the transmigration: yellow dashed line: Transwell insert porous membrane, green: CD11c-eYFP cell, white: E2-Crimson BCG bacteria, white dashed lines: top and bottom of the Z-stacks (iii). (D) Quantification of BCG-infected CD11c-eYFP positive cells that migrated across the brain endothelial monolayer and adhere to the lower side of the insert. Cells were counted manually after taking individual Z-stacks of each aggregate. Analysis was done from two different experiments, n=3 insert / experiment; at least 10 aggregates/insert were counted. Unpaired t-test, *, p<0.05, data is shown as mean +/− SEM.

3.5. Cellular composition of in vitro clusters on brain endothelial cells mimics in vivo granuloma structures and facilitates in situ bacteria dissemination to CD11b⁺, PBMC-derived CD11c⁺ and CX3CR1⁺ cells.

To determine the cellular composition and distribution of cells within the clusters formed on the surface of brain endothelial monolayers, BCG-infected CD11c-eYFP DCs and PBMCs were co-cultured for 24 and 48 hours and analyzed using confocal fluorescent microscopy (Fig. 5A). Eighty cell clusters were analyzed to determine cell composition and distribution throughout the cluster. Our data show that CD11b⁺ cells were the most abundant cell type making up on average 56% of the cells present in the clusters (Fig. 5A and 5B). CD11c-eYFP DCs and CD4⁺ T cells make up 23% and 15% of the cellular aggregates, respectively (Fig. 5B). DCs were found throughout the cluster and in contact with CD4⁺ cells (Fig. 5A). The number of B220⁺ cells present in these clusters was below 4% (Fig. 5B). These results point to similarities in cellular formation and localization between clusters in our in vitro model and granulomas of pulmonary TB infections (40).

FIGURE 5.

Brain endothelial cell-adherent clusters contain different cell types mimicking in vivo granuloma structures. (A) Representative images showing the composition of cell clusters formed by BCG-infected dendritic cells and PBMCs attached to brain endothelial monolayers after 24 and 48 hours of co-culture which were stained with CD4, CD11b and B220 markers. (B) Quantification of cell types from images. A total number of 80 clusters were counted for the BCG infected groups from two different experiments, n=3.

3.6. In situ dissemination of BCG occurs with CD11c⁺ and CX3CR1⁺ cells on the brain endothelial cell monolayer

Upon analysis of the clusters, CD11c-eYFP negative cells were harboring BCG E2-Crimson despite only infecting CD11-eYFP⁺ DCs (Fig. 5A). Pulmonary granulomas provide a niche that can be conducive for the localized dissemination of bacteria from one cell to another. To fully understand if localized dissemination was occurring within cell aggregates on the BBB, PBMCs from CD11c-eYFP and fractalkine CX3CR1-GFP reporter mice were isolated and co-cultured with BCG-infected DCs from C57BL/6J wild-type mice on brain endothelial monolayers. BCG bacilli were found in both CD11c-eYFP (Fig. 6A) and CX3CR1-GFP cells (Fig. 6C), indicating that both DCs and macrophages contribute to in situ dissemination of BCG within clusters on brain endothelial cell monolayers (Fig. 6B and 6D). These results support local in situ dissemination of mycobacteria within cellular clusters of DCs and PBMCs on the in vitro BBB.

FIGURE 6.

In situ dissemination of bacteria within CD11c-eYFP and CX3CR1-GFP cells on brain endothelial monolayers. Bone marrow derived dendritic cells from wild type C57/bl6 animals were infected with BCG and were co-cultured with peripheral blood mononuclear cells isolated from CD11c-eYFP reporter mice (A) and CX3CR1-GFP reporter mice (C) on the Transwell blood-brain barrier model for up to 48 hours. Confocal microscopy pictures show PBMC derived BCG infected CD11c-eYFP (A) and BCG infected and uninfected CX3CR1-GFP (C) cells in the culture nearby or inside cell aggregates (yellow arrows) indicating in situ dissemination in the system to PBMC derived CD11c⁺ and CD11b⁺ cells. (B) and (D) show the percentage of infected cells of PBMC-derived CD11c-eYFP origin (B) or CX3CR1-GFP origin (D) of all reporter cells within a single cell culture insert (n=3).

3.7. Clusters induce inflammatory characteristics of the brain endothelial cell monolayer.

Mtb-induced pulmonary granulomas, although protective against TB infection, can also considerably contribute to disease pathology by inducing damage to the surrounding tissue (41, 42). Our next aim was to understand the damaging effects of cell clusters on the brain endothelial cell monolayer. To study this, inserts were co-cultured with uninfected DCs, BCG-infected DCs, and BCG-infected DCs with PBMCs for 24 and 48 hours then fluorescently labeled to identify any cluster-mediated inflammation on the brain endothelial cell monolayer.

We examined the effects of cluster formation on barrier integrity by analyzing the morphology of two essential inter-endothelial junctional proteins supporting physiological BBB function. Fluorescently labeled tight junction protein Claudin-5 (Cl-5) and tight junction-associated protein ZO-1 were analyzed for morphological changes using fluorescent confocal microscopy (Fig. 7A). In the absence of infection, both ZO-1 and Cl-5 expression remained intact and localized to the cell borders, while infection led to a more perturbed Cl-5 expression showing small, discontinuous gaps in the staining (Fig. 7A, white arrows) and holes (Fig. 7A, asterisk) on the monolayer, while the ZO-1 staining was mildly affected. This morphology was more noticeable in groups containing PBMCs with infected DCs suggesting cluster formation likely plays a role in regulating barrier integrity.

FIGURE 7.

Brain endothelial cell tight junction organization and adhesion molecule expression during cluster formation. (A) Zonula occludens-1 (ZO-1) and claudin-5 junctional stainings of brain endothelial cells 24 hours after the model assembly. Groups of uninfected dendritic cells, BCG infected dendritic cells and infected dendritic cells with P25 PBMCs were co-cultured on the in vitro blood-brain barrier system. White arrows point at endothelial junctional perturbations and discontinuity; star indicates major junctional rearrangements. (B) and (D) are representative images of adhesion molecule stainings for VCAM-1 and ICAM-1 of the system. Adhesion molecule VCAM-1 (C) and ICAM-1 (E) expression intensity of brain endothelial cells was quantified by confocal microscopy. Pictures were taken with the same microscope settings at sites without cell aggregates. Analysis was done from two different experiments, n=3, using 5 pictures / group / experiment. One-way Anova with Bonferroni post-test, *, p<0.05; ****, p<0.0001, data is shown as mean +/− SEM

Another critical characteristic of inflammation is the expression of adhesion molecules. Endothelial cells facilitate leukocyte transmigration to sites of inflammation through increasing adhesion molecule expression, specifically ICAM-1 and VCAM-1, ligands for LFA-1 and VLA-4, respectively (43). Fluorescent antibody labeling revealed a slight increase in both VCAM-1 (Fig. 7B) and ICAM-1 (Fig. 7D) expression on brain endothelial cell monolayers with infected DCs only. However, there was a substantial increase in the expression of both adhesion molecules in the presence of PBMCs, which was validated using image analysis and quantifying mean gray value staining intensity of the VCAM-1 and ICAM-1 labeling (Fig. 7C and 7E). This data supports that Mycobacterium infections lead to brain endothelial cell inflammation and possible barrier function loss.

3.8. BCG induces mitochondrial stress in CD11c-eYFP and brain endothelial cells while clusters cause damage to the brain endothelial monolayer.

To further understand the cellular mechanism of BBB damage as a result of Mycobacterium infection, we visualized cluster-induced cellular stress. Mitochondria play many roles in the cellular process, including contribution to endothelial dysfunction and vascular disease (44). To understand mitochondrial stress in the presence of BCG-induced cluster formation, we stained inserts with Mitotracker dye and evaluated mitochondrial network organization. BCG-infected DCs have disorganized mitochondrial network morphology with more punctate mitochondrial staining both in the cytoplasm of infected DCs (arrow heads) and the underlying endothelial cell monolayer (asterisk), an effect that was exacerbated by the addition of PBMCs (Fig. 8A). DCs and the underlying ECs of uninfected samples expressed an evenly distributed and continuous network of mitochondria suggesting BCG-infected DC-induced cluster formation on the EC monolayer causes cellular stress in both infected cells and in the underlying ECs as well.

FIGURE 8.

Mitochondrial stress and cluster associated damage on CD11c-eYFP and brain endothelial cells. (A) Mitotracker staining of the in vitro co-cultures for all conditions. Asterisks indicate mitochondrial network disassembly of brain endothelial cells. White arrows point at mitochondrial disassembly of dendritic cells, lymphocytes and monocytes. (B) Representative images of 24- and 48-hour clusters and cluster associated damage (CAD) outlined in yellow on the brain endothelial cell monolayer. (C) CAD area was measured on 9 and 13 separate clusters from 2 inserts after 24 and 48 hours, respectively. Analysis was done from two different experiments. Two-way Anova with Sidak’s multiple comparison test, **, p<0.01, data is shown as mean +/− SEM

Next, to assess barrier damage directly associated with the clusters over time, BCG-infected CD11c-eYFP DCs were co-cultured with wild type PBMCs and purified P25 transgenic CD4⁺ T cells in the in vitro BBB system for 24 and 48 hours. P25 T cells were stained with CellTrace Far Red before assembly. After 24 and 48 hours, inserts were fluorescently labeled for ICAM-1 to identify areas of damage in the endothelial cell monolayers. Areas that lacked ICAM-1 expression and had direct contact with the cell clusters were termed cluster associated damage (CAD). Nine and 13 clusters from 24- and 48-hour time points were selected in an unbiased manner, and the endothelial monolayer directly below was examined. CADs were observed at clusters formed both after 24 and 48 hours (Fig. 8B). Using ImageJ, a region of interest (ROI) was drawn around the clusters. Our analysis indicates that clusters are similar in size after 24 and 48 hours (Fig. 8C). Similarly, to measure the CAD area, the area of each cluster was subtracted from the ROI around all areas of damage/decreased ICAM-1 expression which showed an increase in CAD area after 48 hours when compared to 24 hours (Fig. 8C). CAD appeared to be the result of prolonged cluster formation on the endothelial cell monolayer rather than the size of the clusters, since CAD area grew independently of cluster size (Fig. 8C). The collection of this data indicates that clusters are capable of inducing increased cellular stress in brain endothelial cells and cause an increase in cluster associated damage which indicates a decrease in barrier function and integrity.

3.9. TNF-α is necessary for BCG-infected DC-induced cluster formation on brain endothelial cell monolayers.

Cytokines play an important role in protection against Mtb. TNF-α is among the most potent cytokines affording this protection as TNF-α-deficient mice are quickly overcome by the infection and lack granuloma formation (45). As TNF-α is required for pulmonary granuloma formation, we tested if TNF-α affects cluster formation on brain endothelial cells.

TNF-α neutralizing antibody MP6-XT22 is frequently used to block the effects of TNF-α during Mtb infection in mouse studies (34, 45). To understand the mechanism of BCG-infected DC-induced cluster formation on brain endothelial cells, BCG-infected CD11c-eYFP DCs, WT isolated PBMCs, and purified P25 CD4⁺ T cells stained with CellTrace Far Red dye were co-cultured in the presence and absence of MP6-XT22 for 24 and 48 hours, then analyzed using IHC and confocal imaging. Confocal fluorescent microscopy analysis of the brain endothelial cell monolayers showed a pronounced absence of clusters in the presence of TNF-α neutralizing antibody at both time points (Fig. 9A). Multi-area mosaic photos of 4–6 inserts over 24 and 48 hours were stitched and tightly compact clusters consisting of ≥5 cells were counted revealing that on average there were 150 clusters per brain endothelial monolayer in the absence of TNF-α neutralizing antibody after 24 hours with a modest decrease to slightly over 100 clusters after 48 hours (Fig. 9B). Neutralization of TNF-α decreased cluster formation to under 50 per insert over 24 and 48 hours which was a significant decrease compared to inserts containing TNF-α (Fig. 7B). The absence of clusters in the presence of TNF-α neutralizing antibody MP6-XT22 reveals that cluster formation on brain endothelial cells is a TNF-α dependent process.

FIGURE 9.

Cluster formation over 24 and 48 hours in the presence and absence of TNF-a. (A) Representative images of inserts showing BCG-infected DC-induced cluster formation in the presence and absence of MP6-XT22. Neutralization of TNF-a reduces cluster formation over 24 and 48 hours. (B) Quantification of cluster formation. 4 entire inserts were imaged from each group and clusters containing >=5 cells aggregated tightly together were counted. Experiments were repeated twice. Two-way ANOVA with Tukey’s multiple comparison posttest, **, p<0.01; ****, p<0.0001, data is shown as mean +/− SEM

Discussion

Of the various manifestations of the tuberculosis infections, CNSTB is the least understood in part due to the lack of appropriate in vivo or in vitro models. First observations regarding CNSTB were described by pathologists Rich and McCordock in 1933 during autopsies of Mtb-infected individuals (5). It was proposed that Mtb bacilli travel systemically through the blood and reach the vasculature of the CNS, where it can traverse meningeal vessels. In the CNS, inflammatory lesions (Rich focus) are formed and damage the underlying vasculature, leading to a rupture and spread of bacilli into the perivascular space (5). Recent findings of Brilha et al. showed that tight-junction expression decreases in an in vitro BBB model, leading to reduced barrier integrity in the presence of Mtb-infected monocyte-conditioned medium (46). In another cell culture BBB system, Jain et al. published that Mtb can invade and transverse the human in vitro BBB monolayer through actin rearrangement of the brain endothelial cells (7, 46).

Here, we present an in vitro BBB-Mycobacterium induced granuloma co-culture model, which enables the study of the early interactions between Mycobacterium-infected CD11c-eYFP DCs, CX3CR1-GFP macrophages, PBMCs, and Mycobacterium-specific CD4⁺ T cells to understand how these effects can contribute to the dissemination of Mtb into the CNS. While it has been proposed that extracellular bacilli travel to the CNS through systemic circulation and traverse the BBB (7), we found that Mtb-carrying DCs populate the brain parenchyma and provide a carrier for BCG (4), suggesting intracellular Mycobacterium drives to disseminate across the BBB. This “Trojan horse” theory has been implicated as the transport mechanism of other diseases involving neuroinvasion such as West Nile Virus and Cryptococcus neoformans (47, 48). In a mouse model of TB infection, inflammatory DCs can be seen carrying bacilli away from BCG-induced liver granulomas, leading to the dissemination of infection and formation of multiple secondary and tertiary granulomas as infected DCs encounter localized Mtb-specific CD4 T cells (12).

Human dendritic cells that disseminate Mtb infection have been shown to express reduced levels of cell surface integrins, and the ability to migrate toward a chemokine gradient (13). We have demonstrated that BCG-infected DCs showed a severely impaired migratory capacity that was correlated with two distinct actin profiles associated with infected and uninfected cells. Cells migrate through protruding and retracting forces mediated by actin polymerization at the front (or leading edge) of the cells and depolymerization at the rear of the cell usually resulting in the polarization of the cell (38, 49–51). During infection with BCG, infected DCs were less likely to protrude and retract simultaneously, making it rare to find BCG-infected DCs that could polarize in manner that was observed in migrating uninfected DC. As actin expression in infected DCs was increased toward the center and maintained throughout the cell, this was rarely observed in uninfected migrating DCs that mostly expressed actin toward the edges. Multiple studies have identified the effect of Mycobacterium on actin polymerization within multiple cell types (7, 52, 53). Bacterial surface protein heparin-binding haemagglutinin (HBHA) binds to G-actin and alters actin dynamics (54) and the Mycobacterium tuberculosis-secreted tyrosine phosphatase (MptpA) has been shown to affect macrophage phagocytosis through inhibition of actin-mediated processes (55). DCs encounter pathogens, process antigens, and travel to the lymph nodes to prime CD4 T cells and elicit the adaptive immune response (11, 56). Data from our lab supports that BCG may at least delay this process as BCG-infected DCs showed impaired migration toward CCL21 in a CCR7 dependent manner and CD11c⁺BCG⁺ liver granuloma-derived DCs had a reduction in CCR7 expression (12), a receptor that is used for trafficking to the lymph nodes. While it is apparent that Mycobacterium infection impairs DC migration, it seems to have an opposite effect on an infected DC’s ability to recruit PBMCs and Mtb-specific T cells to the site of infection. Although infection limits DC mobility, it increases the ability of DCs to initiate local granuloma-like aggregates on the brain endothelial cells. These aggregates diminish the integrity of the barrier and promote bacterial dissemination into the brain.

Lung granuloma formation is the hallmark immune response to pulmonary Mtb infection in vivo (57–59). During early lung granuloma formation bacteria is engulfed by alveolar macrophages that secrete proinflammatory cytokines leading to the recruitment of monocytes, DCs, T cells, B cells and neutrophils (41, 58, 60, 61). Typical pulmonary granulomas mainly consist of macrophages located at the center, while T-cells accumulating and distribute around the periphery (58, 62). As early as 30 minutes into culture assembly, BCG-infected DCs induce cluster formation on the brain endothelial monolayer that increased in quantity and size in the presence of Mycobacterium-specific CD4 T cells and PBMCs. The multi-cellular clusters morphologically resembled pulmonary TB granulomas as a tight cluster of cells containing BCG rods with CD11b⁺ macrophages (56%) and CD11c⁺ DCs (23%) while CD4⁺ T cells (15%) were found consistently toward the edges in contact with other cells.

Furthermore, in situ bacterial dissemination from CD11b⁺/CD11c⁺ cells to CD11c-eYFP or CX3CR1-GFP cells was observed in the present model, further drawing similarities to pulmonary lesions. Likewise, similar to granuloma formation of pulmonary Mtb infections, cluster formation on the brain endothelial cell monolayer is also dependent on the presence of TNF-α. TNF-α-dependent clusters on the brain endothelial monolayer induce inflammation and impairment of the basic function of the BBB. Clusters increased brain endothelial oxidative stress shown by mitochondrial network disassembly (44), reduction in tight junction protein Claudin-5 integrity, and increased expression of adhesion molecules VCAM-1 and ICAM-1. Likewise, examination of brain endothelial cells directly in contact with clusters revealed substantial cluster associated damage to the barrier that increased over time conceivably due to sustained pro-inflammatory cytokine secretion as cluster size did not significantly change the damaged area. Lung granulomas are well known to contribute to the pathology of pulmonary Mtb infections through the sustained secretion of proinflammatory factors causing damage to the surrounding tissue (5, 63, 64). The nature of our in vitro co-culture systems permits a more in-depth investigation into the effects BCG-infected DC-induced cluster formation has on the BBB. Bacterial meningitis has been shown to compromise tight junction integrity, which was also seen in an in vitro model of CNSTB using Mtb-infected monocyte-conditioned medium (46). ICAM-1 and VCAM-1 expression is elevated on endothelial cells in the presence of inflammatory stimuli to increase adherence and enhance leukocyte transmigration to the inflamed tissue (65), which correspond with our findings and demonstrating BCG-infected DC-induced clusters promote an inflammatory phenotype on the brain endothelial monolayer. The inflammatory characteristics demonstrated here present a potentially devastating situation. Clusters comprised of Mtb niche-creating macrophages and DCs promote bacterial growth and localized dissemination directly in the vicinity of barrier integrity loss resulting in a possible opportunity for DC-mediated dissemination of Mtb across the barrier.

DCs play a key role in tuberculosis infection through Mtb antigen presentation in the lymph nodes that induce an Mtb-specific immune response (66). Continuous antigen sampling by DCs control disease progression (67). Inflammatory DCs are less capable of engulfing and killing Mtb compared to macrophages, although both cell types provide a niche that fosters the survival and replication of Mtb. Our studies show that cell clusters forming on the BBB contribute to the dissemination of mycobacteria across the barrier by inducing damage to the endothelial layer.

Overall, these findings suggest that Mycobacterium employs a host-mediated mechanism for dissemination into the CNS. Infected DCs are arrested at the BBB and induce cellular cluster formation. The resulting clusters, as increasing numbers of PBMCs arrive, will release pro-inflammatory cytokines, thus increasing the concentration gradient at a single inflammatory focus. Sustained secretion of these factors results in inter-endothelial junction protein ZO-1 and Cl-5 dysregulation, upregulation of ICAM-1 and VCAM-1 expression, cellular stress, as well cluster associated damage leading to the loss of barrier integrity. Over time, clusters foster bacterial growth and simultaneously promote barrier damage, thus providing an opportunity for the dissemination of Mycobacterium-harboring immune cells spreading infection across the BBB and into the CNS. DC-induced local lesions on vessels may represent a general mechanism for mycobacterial dissemination.

Key points:

1. BCG-infected DCs form cellular clusters with PBMCs promoting local dissemination

2. Clusters on brain endothelial cells induce inflammation and barrier integrity loss

3. TNF-α inhibition reduces cluster formation and mitigates endothelial damage

Abbreviations

BBB

blood-brain barrier

BCG

Mycobacterium bovis bacillus Calmette-Guérin

BMDC

bone marrow-derived dendritic cell

CAD

cluster associated damage

CNSTB

central nervous system tuberculosis

DC

dendritic cell

ICAM-1

intercellular adhesion molecule-1

PBMC

peripheral blood mononuclear cell

VCAM-1

vascular cell adhesion molecule-1

ZO-1

zonula occludens-1