# Mycobacterium avium Infection of Multinucleated Giant Cells Reveals Association of Bacterial Survival to Autophagy and Cholesterol Utilization

Authors: Jayanthi J. Joseph; Amy Leestemaker-Palmer; Soheila Kazemi; Lia Danelishvili; Luiz E. Bermudez
Journal: Cellular Microbiology
 (2023)

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## Abstract

Mycobacterium avium subsp. hominissuis (M. avium) is an opportunistic environmental pathogen that typically infects patients with existing lung conditions such as cystic fibrosis or COPD. Pulmonary M. avium infection generates peribronchial granulomas that contain infected macrophages and multinucleated giant cells (MGCs). While granuloma formation with MGCs is a common feature of mycobacterial infection, the role of MGCs within the granulomas as well as in the host-pathogen interaction is poorly understood. To shed light on the role of MGCs, we established a novel in vitro model utilizing THP-1 cells stimulated with a combination of IFN-γ and TNF-α. In this study, we show that MGCs can take up M. avium, which replicates intracellularly before leaving the cell. Bacteria that escape the MGC exhibit a highly invasive phenotype, which warrants further evaluation. Characterization of MGCs with transmission electron microscopy revealed an accumulation of cytoplasmic lipid droplets, autophagic activity, and multiple nuclei. Autophagy markers are upregulated in both uninfected and infected MGCs early in infection, measured by RT-qPCR analysis of Beclin-1 and LC3. Inhibition of autophagy with siRNA significantly reduced M. avium survival significantly in THP-1 macrophages. Depletion of host cholesterol and sphingomyelin in MGCs also resulted in decreased survival of M. avium. These processes potentially contribute to the formation of a supportive intracellular environment for the pathogen. Collectively, our results suggest that M. avium is adapted to replicate in MGCs and utilize them as a springboard for local spread.


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## Body

## 1. Introduction

Mycobacterium avium subsp. hominissuis (M. avium) is an environmental opportunistic pathogen that causes chronic, granulomatous pulmonary infections in patients with underlying lung pathology or immunosuppression [1]. In recent years, the number of M. avium infections has been increasing worldwide [2]. Treatment of pulmonary infections involves multiple antibiotics for a minimum of 12 months and is only effective in 50-60% of patients [1, 3]. Airway infections are characterized by the formation of pulmonary lesions, nodules, and both nonnecrotic and necrotic peribronchiolar granulomas [4, 5]. Upon infection, M. avium traverses the bronchial epithelium and is phagocytosed by macrophages and circulating monocytes. To sustain a chronic infection, mycobacteria must evade the immune system and find supportive niches to foster bacterial growth and dissemination. One strategy is to survive intracellularly, which M. avium accomplishes by inhibiting phagosome acidification, which promotes bacterial survival and replication [6, 7]. There is growing evidence that mycobacteria hijack the host’s granulomatous response for their benefit. Traditionally, granulomas were described as protective structures formed by the host immune system to “wall off” pathogenic bacteria and curb dissemination. However, over the past decade, research has shown that mycobacteria can survive and persist in granulomas [8]. Studies on Mycobacterium marinum infection in zebrafish demonstrated that granulomas facilitate cell-to-cell spread, increase bacterial loads, and contribute to dissemination [9, 10]. Additionally, disruption of granulomas in zebrafish can promote host survival as shown in studies performed by Cronan and colleagues, suggesting that granulomatous inflammation is propathogen [11].Langhans multinucleated giant cells (MGCs) are a characteristic feature of granulomas formed in response to pathogens, rather than foreign materials [12]. MGCs in mycobacterial granulomas contain high bacterial loads in vivo, shown by histological examination of granuloma tissues from rabbits, mice, and humans [13–17]. Multinucleated giant cells are formed by the fusion of monocytes and are characterized by multiple nuclei, elevated cholesterol metabolism, and upregulation of iNOS [15, 18]. The interactions between mycobacteria and multinucleated giant cells have not been fully characterized. Previous studies have shown that M. avium-induced MGCs are able to take up bacteria [15, 16]. Understanding the role of MGCs in mycobacterial pathogenesis may shed light into the balance of host-pathogen interactions during chronic infection. We developed an in vitro model of MGCs to help understand the contribution of MGCs in mycobacterial pathogenesis and the balance of the host-pathogen interaction during chronic infection of M. avium.Lipids are a well-known source of nutrition for mycobacteria in the host [19]. Host lipid metabolism shifts during mycobacterial infections, and the inhibition of lipid accumulation in mycobacterium-infected macrophages reduces bacterial survival [20, 21]. Host lipids utilized by mycobacteria include triacylglycerols, cholesterol, and sphingomyelin. Cholesterol is required for persistence of Mycobacterium tuberculosis (M. tb) in the lungs of chronically infected mice and Mycobacterium avium subsp. paratuberculosis in human macrophages [22, 23]. In humans, cholesterol accumulates in M. tb granulomas, accompanied by an increase in host lipid metabolism and providing a nutrient-rich environment for mycobacteria [24]. M. avium possesses genes involved in cholesterol metabolism, including several mce (mammalian cell entry) genes, which are associated with cholesterol uptake and utilization in mammalian hosts [23, 25]. Sphingolipids are important components of eukaryotic cell membranes, and several intracellular pathogens have evolved mechanisms to utilize this class of lipids for growth [26]. Several intracellular pathogens manipulate host sphingolipid metabolism, including M. tb, P. aeruginosa, and L. pneumophila [26]. M. tuberculosis possesses several genes involved in cholesterol metabolism, a neutral sphingomyelinase SpmT (Rv0888), and alkaline ceramidase (Rv0669c) [27, 28]. Although M. avium lacks homologs for either of these genes, it possesses several uncharacterized hypothetical genes that may have similar activity [23, 25]. For example, Pseudomonas aeruginosa uses host sphingomyelin and ceramide as a nutrient by destroying them with a host-mimicking sphingomyelin synthase and ceramidase [29, 30]. M. tb utilizes host sphingomyelin by expressing the SpmT (rv0888) gene to support intracellular replication in macrophages [27]. While M. avium lacks the SpmT gene, it possesses other genes involved in lipid uptake and degradation, which may play a similar role [25].In this study, we developed anin vitro MGC model and characterized the cellular morphology, which included multiple nuclei, accumulation of cytoplasmic lipid droplets, and the presence of autophagy. We determined the uptake and survival of M. avium in MGCs and found that M. avium is well adapted at intracellular replication in MGCs. We evaluated the MGC-M. avium interaction and identified possible explanations for the contribution of increased host autophagy and cytosolic lipid droplets toward bacterial survival.


## 2. Materials and Methods

### 2.1. Bacterial Culture

Mycobacterium avium subsp. hominissuis strains 104 (MAH 104), A5 (MAH A5), 101 (MAH 101), and 109 (MAH 109) were originally isolated from the blood of AIDS patients [31]. MAH 101 is the standard M. avium strain for susceptibility testing, and MAH 109 has been used extensively in preclinical studies [32]. MAH A5 exhibits prolific biofilm formation and eDNA export [33]. MAH 104 and MAH A5 were grown on Middlebrook 7H10 agar supplemented with 10% w/v oleic acid, albumin, dextrose, and catalase (OADC, Hardy Diagnostics) for 7-10 days at 37°C.


### 2.2. Tissue Culture and Formation of Multinucleated Giant Cells

THP-1 (TIB-202) human monocytes were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products) at 37°C with 5% CO2. THP-1 cells were seeded in 48-well plates at a density of 3.5×105 cells per well and differentiated with 50 ng/ml of Phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich) for 24 hours, followed by 24 hours in RPMI 1640 supplemented with 10% FBS without PMA, prior to use in experiments. Differentiated macrophages were treated with IFN-γ (100 ng/ml) (R&D Systems) and TNF-α (25 ng/ml) (VWR). The cytokines were used in combination to promote multinucleated giant cell (MGC) formation. Cytokines in RPMI 1640 supplemented with 10% FBS were added every 48 hours for 6 days after initial PMA treatment triggered differentiation. The fusion index was calculated using the following formula: No.ofnucleiinMGCs/totalNo.ofnuclei×100%.


### 2.3. Flow Cytometry

For CD40 surface marker analysis, THP-1 cells were seeded in a 6-well plate (1×106 cells/ml) and differentiated with 50 ng/ml PMA for 24 h. MGCs were formed as described above. Cells were detached from the plate using 0.25% EDTA-free trypsin (Thermo Fisher Scientific) in 1X phosphate-buffered saline (PBS) for 5 min at 37°C, followed by gentle pipetting. Cell suspensions were washed with PBS and either left unstained or stained with fluorochrome-conjugated antibodies: CD40 FITC (5C3) (eBioscience) or mouse IgG1 kappa isotype control (P3.6.2.8.1) (eBiosciene) FITC, for 1 hour at 4°C. After staining, cells were fixed with 2% paraformaldehyde (PFA) for 10 min at 37°C to prevent bacterial contamination of the machine, washed, and analyzed immediately. Gating was performed to exclude dead cells. Flow cytometry was performed using an Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using Accuri C6 software, FlowJo, and GraphPad Prism 9.


### 2.4. Mycobacterial Infection and Survival in MGCs

MAH bacteria were added to multinucleated giant cells at an MOI of 10, synchronized by centrifugation for 10 min at150×g, and allowed to infect for 1 hour. Inoculums for these assays were formed in 1X HBSS supplemented with 0.05% Tween-20 (Sigma-Aldrich). Extracellular bacteria were removed by two wash steps with 1X HBBS followed by treatment with gentamicin sulfate (Sigma-Aldrich) for 1 hour (100 μg/ml) and an additional wash step with 1X HBSS to remove the antibiotic and dead extracellular bacteria. Cells were lysed at appropriate time points with 0.1% Triton-X for 10 min, and resulting lysates were diluted and plated. Colony forming units (CFUs) were enumerated at 1, 24, 48, and 72 hours postinfection (h.p.i.). Potential extracellular bacteria escaping from cells were quantified at each time point via spot plating. Briefly, 5 μl of media removed from infected wells was plated on 7H10 agar. Colonies were counted after 7 days of incubation at 37°C.


### 2.5. Collection of MGC-Passaged Bacteria

MGCs, formed as described previously, were infected with wild-type MAH A5 for 1 h at an MOI of 10. The infection was carried out as described previously. Fresh media were replaced every 24 h. Supernatants were collected at 72 h.p.i, and immediately kept on ice. Cell debris was removed by centrifugation for 5 min,150×g at 4°C. The supernatant containing bacteria was pelleted by centrifugation for 15 min, 2000×g at 4°C. The supernatant was removed, and the pellet resuspended in 2 ml of HBSS and stored at 4°C for up to 7 days before use in experiments.


### 2.6. qPCR of Autophagy-Associated Genes during MGC Infection

THP-1 and MGC RNA were isolated with the RNeasy mini kit (Qiagen), followed by treatment with DNase I recombinant (Roche Diagnostics) for 2 hours at 37°C to remove contaminating genomic DNA. The DNase was inactivated with Turbo DNase-inactivation reagent (Turbo DNA-free kit, Thermo Fisher Scientific) for 2 min at 37°C. Inactivation reagent was removed via centrifugation for 1 min at10,000×g, and RNA transferred to new collection tubes. RNA samples were stored at -4°C for future processing. cDNA was transcribed from host RNA using the iScript cDNA synthesis kit (Bio-Rad). The quality of cDNA was tested by PCR with Gold 360 Master Mix using the manufacturer’s specifications (Thermo Fisher Scientific). The RT-qPCR reaction was performed using iQ SYBR Green Supermix (Bio-Rad) and an iCycler (CFX Connect Real-Time Systems, Bio-Rad) as previously described [34]. All gene expression data are presented as relative expression to beta-actin. Primers were designed in Primer3 using sequences from GenBank (National Center for Biotechnology Information). Primer sequences are as follows: beclin-1, 5′-AGCTGCCGTTATACTGTTCTG-3′ (forward) and 5′-ACTGCCTCCTGTGTCTTCAATCTT-3′ (reverse); LC3-II, 5′-GATGTCCGACTTATTCGAGAGC-3′ (forward) and 5′-TTGAGCTGTAAGCGCCTTCTA-3′ (reverse); and beta-actin: 5′-CATGTACGTTGCTATCCAGGC-3′ (forward) and 5′-CTCCTTAATGTCACGCACGAT-3′ (reverse) [35].


### 2.7. Fluorescent and Transmission Electronic Microscopy of MGCs

Multinucleated giant cells were generated with cytokines in chamber slides (Falcon, VWR) and visualized by fluorescence microscopy. The cells were fixed in 2% PFA for 15 min, washed twice with 1X HBSS, and permeabilized for 10 min with 0.1% Triton-X before staining with 1μg/ml Hoechst 34580 (Invitrogen) for 15 min in the dark. Hoechst was removed, and samples were washed twice with 1X HBSS and stained with 0.165 μM Alexa Fluor 488 phalloidin (Thermo Fisher Scientific) for 15 min in the dark, followed by two wash steps with 1X HBSS. Slides were allowed to dry before affixing glass coverslips with Cytoseal (Thermo Fisher Scientific). Slides were imaged with a Leica DM4000B fluorescent microscope (Leica Microsystems) and QICAM Fast 1394 camera (QImaging) at a magnification of 1000x under oil immersion. Images were acquired and processed with QCapture Pro 7 software. For TEM, cells were detached from 6-well plates by treatment with 5 mM EDTA for 30 min, washed in 1X HBSS (HBSS, Cellgro), and suspended in fixative buffer with 2.5% glutaraldehyde, 1% formaldehyde, and 0.1 M sodium cacodylate for 24 hours prior to submission to the electron microscopy facility for processing. Samples were sectioned, dehydrated, and visualized by a FEIT Titan 80-200 TEM/STEM microscope at Oregon State University Electron Microscopy Facility.


### 2.8. siRNA Targeting of Autophagy Genes during Mycobacterial Infection

To examine the role of ATG5 and Beclin-1 in activation of autophagy, we inhibited the functionality of selected targets in THP-1 macrophages using siRNA technology in accordance with the manufacturer’s recommendations (Santa Cruz Biotechnology, Inc). THP-1 cells were differentiated and seeded at 80% confluence in 6-well plates and, prior to infection, transfected with ATG5 and Beclin siRNAs. Briefly, siRNAs were diluted in RPMI without serum at a final concentration of 25 nM and 3μl of Continuum™ transfection reagent (Gemini). Macrophage monolayers were washed once with siRNA transfection medium and replenished with new transfection medium containing target-specific siRNA transfection reagent mixture. Cells were incubated at 37°C in the presence of 0.5% CO2. Untreated and control siRNA- (nontargeting sequences) transfected cells served as negative controls. After 48 h, monolayers were infected for different time points and lysed and bacterial CFUs were recorded on Middlebrook 7H10 agar plates. Before infection, the ATG5, Beclin, and β-actin protein levels from control and experimental wells were analyzed by semiquantitative western blotting. Lysed macrophages were resolved by electrophoresis on 12.5% SDS-PAGE gels, transferred to nitrocellulose membranes, and blocked overnight with 5% Bovine Serum Albumin (BSA). After incubation with primary antibodies at a dilution of 1 : 200 for 2 h, membranes were washed three times with PBS and then probed with corresponding IRDye secondary antibody (Li-Cor Biosciences, Inc) at a dilution of 1 : 5000 for 1 h. Proteins were visualized using Odyssey Imager (Li-Cor).


### 2.9. Statistical Analysis

All described experiments were repeated at least three times, and data shown are representative of the biological replicates. Comparisons between two groups were analyzed in Microsoft Excel using the two-tailed Studentt-test. Results with P values below 0.05 were considered significant.


## 2.1. Bacterial Culture

Mycobacterium avium subsp. hominissuis strains 104 (MAH 104), A5 (MAH A5), 101 (MAH 101), and 109 (MAH 109) were originally isolated from the blood of AIDS patients [31]. MAH 101 is the standard M. avium strain for susceptibility testing, and MAH 109 has been used extensively in preclinical studies [32]. MAH A5 exhibits prolific biofilm formation and eDNA export [33]. MAH 104 and MAH A5 were grown on Middlebrook 7H10 agar supplemented with 10% w/v oleic acid, albumin, dextrose, and catalase (OADC, Hardy Diagnostics) for 7-10 days at 37°C.


## 2.2. Tissue Culture and Formation of Multinucleated Giant Cells

THP-1 (TIB-202) human monocytes were obtained from the American Type Culture Collection (ATCC) and maintained in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gemini Bio-Products) at 37°C with 5% CO2. THP-1 cells were seeded in 48-well plates at a density of 3.5×105 cells per well and differentiated with 50 ng/ml of Phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich) for 24 hours, followed by 24 hours in RPMI 1640 supplemented with 10% FBS without PMA, prior to use in experiments. Differentiated macrophages were treated with IFN-γ (100 ng/ml) (R&D Systems) and TNF-α (25 ng/ml) (VWR). The cytokines were used in combination to promote multinucleated giant cell (MGC) formation. Cytokines in RPMI 1640 supplemented with 10% FBS were added every 48 hours for 6 days after initial PMA treatment triggered differentiation. The fusion index was calculated using the following formula: No.ofnucleiinMGCs/totalNo.ofnuclei×100%.


## 2.3. Flow Cytometry

For CD40 surface marker analysis, THP-1 cells were seeded in a 6-well plate (1×106 cells/ml) and differentiated with 50 ng/ml PMA for 24 h. MGCs were formed as described above. Cells were detached from the plate using 0.25% EDTA-free trypsin (Thermo Fisher Scientific) in 1X phosphate-buffered saline (PBS) for 5 min at 37°C, followed by gentle pipetting. Cell suspensions were washed with PBS and either left unstained or stained with fluorochrome-conjugated antibodies: CD40 FITC (5C3) (eBioscience) or mouse IgG1 kappa isotype control (P3.6.2.8.1) (eBiosciene) FITC, for 1 hour at 4°C. After staining, cells were fixed with 2% paraformaldehyde (PFA) for 10 min at 37°C to prevent bacterial contamination of the machine, washed, and analyzed immediately. Gating was performed to exclude dead cells. Flow cytometry was performed using an Accuri C6 flow cytometer (BD Biosciences). Data were analyzed using Accuri C6 software, FlowJo, and GraphPad Prism 9.


## 2.4. Mycobacterial Infection and Survival in MGCs

MAH bacteria were added to multinucleated giant cells at an MOI of 10, synchronized by centrifugation for 10 min at150×g, and allowed to infect for 1 hour. Inoculums for these assays were formed in 1X HBSS supplemented with 0.05% Tween-20 (Sigma-Aldrich). Extracellular bacteria were removed by two wash steps with 1X HBBS followed by treatment with gentamicin sulfate (Sigma-Aldrich) for 1 hour (100 μg/ml) and an additional wash step with 1X HBSS to remove the antibiotic and dead extracellular bacteria. Cells were lysed at appropriate time points with 0.1% Triton-X for 10 min, and resulting lysates were diluted and plated. Colony forming units (CFUs) were enumerated at 1, 24, 48, and 72 hours postinfection (h.p.i.). Potential extracellular bacteria escaping from cells were quantified at each time point via spot plating. Briefly, 5 μl of media removed from infected wells was plated on 7H10 agar. Colonies were counted after 7 days of incubation at 37°C.


## 2.5. Collection of MGC-Passaged Bacteria

MGCs, formed as described previously, were infected with wild-type MAH A5 for 1 h at an MOI of 10. The infection was carried out as described previously. Fresh media were replaced every 24 h. Supernatants were collected at 72 h.p.i, and immediately kept on ice. Cell debris was removed by centrifugation for 5 min,150×g at 4°C. The supernatant containing bacteria was pelleted by centrifugation for 15 min, 2000×g at 4°C. The supernatant was removed, and the pellet resuspended in 2 ml of HBSS and stored at 4°C for up to 7 days before use in experiments.


## 2.6. qPCR of Autophagy-Associated Genes during MGC Infection

THP-1 and MGC RNA were isolated with the RNeasy mini kit (Qiagen), followed by treatment with DNase I recombinant (Roche Diagnostics) for 2 hours at 37°C to remove contaminating genomic DNA. The DNase was inactivated with Turbo DNase-inactivation reagent (Turbo DNA-free kit, Thermo Fisher Scientific) for 2 min at 37°C. Inactivation reagent was removed via centrifugation for 1 min at10,000×g, and RNA transferred to new collection tubes. RNA samples were stored at -4°C for future processing. cDNA was transcribed from host RNA using the iScript cDNA synthesis kit (Bio-Rad). The quality of cDNA was tested by PCR with Gold 360 Master Mix using the manufacturer’s specifications (Thermo Fisher Scientific). The RT-qPCR reaction was performed using iQ SYBR Green Supermix (Bio-Rad) and an iCycler (CFX Connect Real-Time Systems, Bio-Rad) as previously described [34]. All gene expression data are presented as relative expression to beta-actin. Primers were designed in Primer3 using sequences from GenBank (National Center for Biotechnology Information). Primer sequences are as follows: beclin-1, 5′-AGCTGCCGTTATACTGTTCTG-3′ (forward) and 5′-ACTGCCTCCTGTGTCTTCAATCTT-3′ (reverse); LC3-II, 5′-GATGTCCGACTTATTCGAGAGC-3′ (forward) and 5′-TTGAGCTGTAAGCGCCTTCTA-3′ (reverse); and beta-actin: 5′-CATGTACGTTGCTATCCAGGC-3′ (forward) and 5′-CTCCTTAATGTCACGCACGAT-3′ (reverse) [35].


## 2.7. Fluorescent and Transmission Electronic Microscopy of MGCs

Multinucleated giant cells were generated with cytokines in chamber slides (Falcon, VWR) and visualized by fluorescence microscopy. The cells were fixed in 2% PFA for 15 min, washed twice with 1X HBSS, and permeabilized for 10 min with 0.1% Triton-X before staining with 1μg/ml Hoechst 34580 (Invitrogen) for 15 min in the dark. Hoechst was removed, and samples were washed twice with 1X HBSS and stained with 0.165 μM Alexa Fluor 488 phalloidin (Thermo Fisher Scientific) for 15 min in the dark, followed by two wash steps with 1X HBSS. Slides were allowed to dry before affixing glass coverslips with Cytoseal (Thermo Fisher Scientific). Slides were imaged with a Leica DM4000B fluorescent microscope (Leica Microsystems) and QICAM Fast 1394 camera (QImaging) at a magnification of 1000x under oil immersion. Images were acquired and processed with QCapture Pro 7 software. For TEM, cells were detached from 6-well plates by treatment with 5 mM EDTA for 30 min, washed in 1X HBSS (HBSS, Cellgro), and suspended in fixative buffer with 2.5% glutaraldehyde, 1% formaldehyde, and 0.1 M sodium cacodylate for 24 hours prior to submission to the electron microscopy facility for processing. Samples were sectioned, dehydrated, and visualized by a FEIT Titan 80-200 TEM/STEM microscope at Oregon State University Electron Microscopy Facility.


## 2.8. siRNA Targeting of Autophagy Genes during Mycobacterial Infection

To examine the role of ATG5 and Beclin-1 in activation of autophagy, we inhibited the functionality of selected targets in THP-1 macrophages using siRNA technology in accordance with the manufacturer’s recommendations (Santa Cruz Biotechnology, Inc). THP-1 cells were differentiated and seeded at 80% confluence in 6-well plates and, prior to infection, transfected with ATG5 and Beclin siRNAs. Briefly, siRNAs were diluted in RPMI without serum at a final concentration of 25 nM and 3μl of Continuum™ transfection reagent (Gemini). Macrophage monolayers were washed once with siRNA transfection medium and replenished with new transfection medium containing target-specific siRNA transfection reagent mixture. Cells were incubated at 37°C in the presence of 0.5% CO2. Untreated and control siRNA- (nontargeting sequences) transfected cells served as negative controls. After 48 h, monolayers were infected for different time points and lysed and bacterial CFUs were recorded on Middlebrook 7H10 agar plates. Before infection, the ATG5, Beclin, and β-actin protein levels from control and experimental wells were analyzed by semiquantitative western blotting. Lysed macrophages were resolved by electrophoresis on 12.5% SDS-PAGE gels, transferred to nitrocellulose membranes, and blocked overnight with 5% Bovine Serum Albumin (BSA). After incubation with primary antibodies at a dilution of 1 : 200 for 2 h, membranes were washed three times with PBS and then probed with corresponding IRDye secondary antibody (Li-Cor Biosciences, Inc) at a dilution of 1 : 5000 for 1 h. Proteins were visualized using Odyssey Imager (Li-Cor).


## 2.9. Statistical Analysis

All described experiments were repeated at least three times, and data shown are representative of the biological replicates. Comparisons between two groups were analyzed in Microsoft Excel using the two-tailed Studentt-test. Results with P values below 0.05 were considered significant.


## 3. Results

### 3.1. IFN-γ and TNF-α Induce Multinucleated Giant Cell Formation In Vitro

To triggerin vitro MGC formation, THP-1 cells were differentiated with Phorbol 12-myristate 13-acetate (PMA) for 24 hours, followed by stimulation with a combination of TNF-α and IFN-ɣ for 6 days. After differentiation with PMA, THP-1 cells do not proliferate and become adherent. TNF-α and IFN-ɣ were selected because they have been shown to drive granuloma formation and contribute to MGC formation in vivo [36–39]. A fusion index of 56.5% was observed when cells were treated with 25 ng/ml TNF-α and 100 ng/ml IFN-ɣ over a 6-day period (Figure 1(a)). The resulting mixed population of MGCs and activated THP-1 macrophages was utilized for further experiments. MGCs formed with this protocol have 2-4 nuclei (Figures 1(b)–1(d)). CD40 is a marker of MGCs associated with mycobacterial infection and is involved in MGC formation [40, 41]. MGCs exhibited a significant shift in mean fluorescent intensity (MFI) of FITC-CD40, indicating that 98% of MGCs are CD40 positive (Figure 1(e)). THP-1 cells exhibited a small shift in fluorescent intensity, indicating a low percentage of CD40-positive cells (Figure 1(f)). MGCs have an MFI of FITC-CD40 that is significantly higher than THP-1 macrophages, MGCs stained with a FITC-isotype control, and unstained MGCs (Figure 1(g)). These results indicate that MGCs retain the CD40 marker and are biologically similar to MGCs observed in vivo.Figure 1
IFN-γ and TNF-α induce formation of multinucleated giant cells in vitro. THP-1 cells were fluorescently labeled after cytokine treatment to determine MGC formation via microscopy at 1000x magnification. The expression of CD40, a marker associated with classical macrophage activation and Langhans giant cells, was measured via flow cytometry. (a) The macrophage fusion index determined the percentage of multinucleated cell having at least 3 nuclei in 10 fields of view. (b) Hoechst stain of MGC nuclei (white arrow). (c) Alexa Fluor Phalloidin 488 stain of actin cytoskeleton (white arrow). (d) Merged image of MGC (white arrow). (e) FITC-CD40 levels in MGCs (black: unstained, blue: isotype control, and red: CD40+). (f) FITC-CD40 levels in PMA-differentiated THP-1 cells (black: unstained, red: CD40+). (g) Mean fluorescent intensity of FITC-CD40 (black: unstained, blue: isotype control, and red: CD40+). All groups were compared to MGC CD40+. Data are representative of 3 independent experiments. Statistical comparisons: ∗P<0.01, ∗∗P<0.001, and ∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)


### 3.2. Intracellular Survival and Growth ofM. avium in Multinucleated Giant Cells

The intracellular stages of mycobacteria in MGCs have not been fully described. Survival assays were performed to characterize the uptake and intracellular growth ofM. avium in the in vitro MGC model and compared to THP-1 macrophages. M. avium A5 infected MGCs at a similar rate to THP-1 macrophages (Figure 2(a)). Once cells were infected, bacterial survival was determined by CFU counts in either MGCs or THP-1 macrophages over 3 and 6 days, respectively, and fold change relative to 1 h.p.i calculated. The time points selected for THP-1 survival assays are standard practice. The time points for MGC survival assays were selected from day 5 of stimulation up to the point where bacterial growth was observed at 3 d.p.i. Additionally, by 6 d.p.i, the MGCs would have been on the plate for 12 days, which would reduce the number of viable cells. At 4 d.p.i for THP-1 and 48 h.p.i for MGC, fold change of bacterial growth was 4 (Figures 2(b) and 2(c)). In THP-1 macrophages, M. avium intracellular growth exhibited a fold change of 11 at 6 d.p.i, whereas M. avium intracellular growth in MGCs exhibited a fold change of 7 at 3 d.p.i (Figures 2(b) and 2(c)). During the survival assay, CFU/milliliter of extracellular bacteria that exited MGCs was enumerated (Figure 2(d)). The number of bacteria leaving MGCs was significantly greater at 72 h.p.i. compared to other time points. These data indicate that M. avium A5 can enter MGCs with the same efficacy as THP-1 macrophages. Additionally, M. avium A5 survives in the MGC environment and shows a similar growth trend to THP-1 macrophages. Our data support the idea of a favorable niche existing inside the MGC environment that M. avium A5 can utilize to survive and eventually leave to infect other cells nearby.Figure 2
Infection and survival ofM. avium A5. (a) Percent uptake of M. avium A5 in THP-1 macrophages and MGCs after 1 hour of infection. (b) Fold change relative to 1 h.p.i (dashed line) of M. avium growth over 6 days in THP-1 macrophages. (c) Fold change relative to 1 h.p.i (dashed line) of M. avium growth over 3 days in MGCs. (d) Extracellular bacterial CFUs recovered after infection of MGCs. Data shown are representative of results from three independent biological replicates. Statistical comparisons: ∗P<0.05; ∗∗P<0.005; ∗∗∗P<0.0005.
(a)(b)(c)(d)


### 3.3. Characterization ofM. avium Phenotype upon Leaving Multinucleated Giant Cells

To determine if the phenotype of MGC-passagedM. avium A5 is different than WT M. avium A5, uptake and survival assays were carried out in THP-1 macrophages. To passage M. avium A5, MGCs were infected with WT M. avium A5 for 1 h, and at 72 h.p.i, bacteria that exited the MGCs (MGC-passaged A5) were collected in cell culture media and kept on ice or at 4°C while being pelleted and washed. THP-1 cells were infected for 1 hour with MGC-passaged bacteria or M. avium A5 from a 7H10 streak plate. Uptake of M. avium A5 was evaluated by inhibiting macropinocytosis and receptor-mediated phagocytosis. Wortmannin inhibits the macropinocytosis pathway by preventing complete closure of the cytoplasmic ruffles. Blocking complement receptor 3 (CR3) with a CD11b antibody inhibits complement receptor-mediated phagocytosis. MGC-passaged A5 exhibited a percent uptake approximately 3 times greater than M. avium A5 grown on a plate, under normal infection conditions (Figure 3(a)). Blocking the CR3 receptor with an anti-CD11b antibody for 1 hour prior to infection led to a 52% reduction in uptake for both bacterial phenotypes (Figure 3(b)). Inhibition of macropinocytosis with wortmannin for 2 h prior to infection led to a 33% reduction in uptake for both bacterial phenotypes (Figure 3(b)). Inhibiting uptake pathways reduced the uptake of both phenotypes but did not completely block them, showing that M. avium of either phenotype can circumvent the effect of the inhibitors and entering the macrophage via an alternate route. This suggests that MGC-passaged bacteria can enter THP-1 cells via alternate routes. Survival in THP-1 macrophages over 6 days with MGC-passaged A5 is comparable to A5 from the plate; thus, once bacteria invade macrophages, there was no added advantage to MGC-passaged bacteria (Figure 3(c)). These data suggest that the MGC environment increases the invasiveness of M. avium A5 but that they retain their ability to enter the next host cell in multiple ways.Figure 3
Uptake and survival of MGC-passagedM. avium A5 in THP-1 cells. (a) Percent uptake of M. avium A5 inoculums in THP-1 cells after 1 h of infection. (b) Percent reduction of uptake by THP-1 cells treated with either wortmannin for 2 h or a CD11b antibody for 1 h, prior to infection. (c) Fold change relative to 1 h.p.i (dashed line) of bacterial survival by plate or MGC-passaged M. avium in THP-1 macrophages over 6 days. “Plate” refers to M. avium A5 grown on a 7H10 plate, and “MGC-passaged” refers to M. avium A5 that was collected from infected-MGCs at 72 h.p.i. Data are representative of three independent experiments. Statistical comparisons: ∗P<0.01, ∗∗P<0.001, and ∗∗∗P<0.0001.
(a)(b)(c)


### 3.4. Electron Microscopy Observations ofM. avium A5 in MGCs

To increase our understanding of the behavior of bacteria inside MGCs, transmission electron microscopy (TEM) was used. MGCs were infected withM. avium over 72 h then processed for TEM. THP-1 macrophages that are not treated with cytokines or infected with bacteria show normal cellular structures including a single nucleus and cell size. After cytokine exposure, MGCs were formed and autophagosomes were visible in the cytosol, characterized by double membranes (Figure 4(a)). In MGCs infected with M. avium A5 for 72 h, bacteria can be seen inside autophagosomes (Figure 4(b)), and bacterium-containing vacuoles can be seen interacting with autophagosomes (Figures 4(c) and 4(d)). Comparing multiple cells, numerous autophagic vacuoles exist within MGCs, and once these cells are infected with M. avium A5, the number of autophagosomes increased substantially.Figure 4
Autophagic activity in MGCs. (a) Autophagosomes containing cellular components in uninfected MGCs. (b) Autophagosomes containingM. avium 72 h.p.i. of MGCs. (c) Vacuoles containing M. avium interacting with an autophagosome at 72 h.p.i. (d) Same as (c). Black arrows: bacteria; orange arrows: autophagosomes.
(a)(b)(c)(d)The formation of MGCs causes several cellular changes, and one of the changes observed was the accumulation of lipid droplets in the cytosol (Figure5(a)). At 1 h.p.i, bacteria were present the cytosol near lipid droplets (Figure 5(b)) as well as in autophagosomes (Figures 5(c) and 5(e)). Intracellular lipid inclusions were present in intracellular bacteria at several time points (Figures 5(b)–5(e)). Vacuoles with multiple bacteria were observed at 72 h.p.i. (Figure 5(d)). After 24 h, bacteria started to leave MGCs and were observed near the plasma membrane (Figure 5(f)).Figure 5
Characteristics of intracellularM. avium in MGCs. (a) Uninfected MGC with three nuclei surrounded by lipid droplets. (b) Cytosolic M. avium inside MGCs at 1 h.p.i. (c) M. avium bacterium in a vacuole at 1 h.p.i in MGCs. (d) Vacuole containing multiple M. avium 72 h.p.i. inside MGCs. (e) Intracellular M. avium in autophagosome in MGC at 1 h.p.i. (f) 24 h.p.i., extracellular M. avium from an MGC. Blue arrows: host lipid droplets, black arrows: bacteria, green arrows: intrabacterial lipid inclusions, white arrows: nuclei, and orange arrows: autophagosomes.
(a)(b)(c)(d)(e)(f)


### 3.5.In Vitro Multinucleated Giant Cells Exhibit Increased Autophagic Activity Compared to Macrophages

To assess autophagic activity in MGCs, we quantified mRNA levels of LC3 and Beclin-1 via real-time PCR. Beclin-1 controls phagophore formation, and LC3 is involved in phagophore elongation [42]. Beclin-1 gene levels after 4 h indicate that MGCs have naturally greater expression compared to THP-1 macrophages, and once infected, MGCs exhibited even greater gene expression of Beclin-1 (Figure 6(a)). The trend continued through 24 h.p.i, except there was no difference between infected and uninfected MGC gene expression. Later in the infection, there was no difference in Beclin-1 gene expression between experimental groups. LC3 expression by MGCs at 4 h showed significant increase compared to LC3 expression by THP-1 macrophages (Figure 6(b)). After 24 h, LC3 gene expression by uninfected MGCs increases, and expression by infected MGCs decreased. This could be due to suppression of autophagic flux by M. avium, which has been demonstrated in M. abscessus [43]. After 72 h, LC3 gene expression was undetectable for all experimental groups. The reduction in autophagic marker expression by 72 h.p.i could be due to the absence of cytokine stimulation after the start of infection. For infected MGCs, the reduction could be due to bacterial exit from the cell. Collectively, these data support our earlier observation that infected MGCs have a greater number of autophagosomes than uninfected MGCs at early time points.Figure 6
Gene expression of autophagic markers is elevated in MGCs. Either MGCs or THP-1 cells were infected with WTM. avium A5 for 4 h. (a) Beclin-1 transcripts were measured by RT-qPCR at indicated times. mRNA levels are presented as relative expression to actin. (b) LC3 transcripts were measured at indicated time points. mRNA levels are represented relative to actin expression. Data are representative of two independent experiments. Statistical comparisons: ∗P<0.01; ∗∗P<0.001; ∗∗∗P<0.0001.
(a)(b)


### 3.6. Silencing of Beclin and ATG5 Reduces Intracellular Survival ofM. avium in THP-1 Macrophages

Since there is a clear tendency of MGCs cells to express autophagy uponM. avium infection, which is similar to what is observed at later time points in macrophages, we questioned if autophagy was an important occurrence for survival in host cells. Macrophages, the preferred cell type of M. avium and precursor of MGCs, were selected for this experiment. To assess the importance of autophagy during M. avium infection in THP-1 macrophages, siRNA technology was used to knock down expression of Beclin-1 and ATG5 host genes. THP-1 macrophages treated with siRNA targeting either Beclin, ATG5, empty vector, or no vector showed reduced but not absent protein levels inside of cells, confirmed by western blot (Figure 7(a)). These THP-1 macrophages were then infected with multiple strains of M. avium including 101, 104, and 109. Our results demonstrate that inhibition of ATG5 or Beclin, markers of autophagy, decreased the ability for bacteria to survive in macrophages over 4 days (Figure 7(b)). These data suggest that M. avium uses autophagy for intracellular survival in resting macrophages. It is possible that the autophagic activity in MGCs aids bacterial survival by a similar mechanism.Figure 7
siRNA targeting of autophagy markers decreases protein levels. THP-1 cells were depleted of either ATG5 or Beclin-1 48 h after siRNA targeting. (a) Western blot band intensities were quantified usingβ-actin as a loading control by Odyssey imager software (Li-Cor). (b) Survival of MAH strains 109, 101, and 104 at 48 h.p.i. in THP-1 macrophages transfected with ATG5 or Beclin-1 siRNA. CFU/milliliter of data is representative of three experiments. Statistical comparisons: ∗P<0.01; ∗∗P<0.001; ∗∗∗P<0.0001.
(a)(b)


### 3.7. Intracellular Cholesterol Transport andM. avium A5 Survival in MGCs

M. avium is clearly utilizing host lipids in MGCs, as shown by the presence of intrabacterial lipid inclusions (Figures 4(b), 5(b), and 5(c)). To determine whether M. avium A5 utilizes MGC-derived cholesterol as a source of nutrients for intracellular growth, uptake and survival assays were carried out in MGCs treated with (3β)-3-[2-(diethylamino) ethoxy] androst-5-en-17-one hydrochloride (U18666A), an intracellular cholesterol transport inhibitor. The final concentration was chosen based on previously published studies in mouse macrophages and human fibroblasts [44, 45]. To rule out direct bacterial inhibition, a growth curve of M. avium A5 in 7H9 broth supplemented with 3 μg/ml of U18666A was conducted (Figure 8(a)). U18666A had no effect on the growth of M. avium A5 over 6 days (Figure 8(a)). Since U18666A did not affect bacterial growth, we then used the inhibitor on MGCs infected with M. avium A5. U18666A was added 1 hour after infection of MGCs and replenished daily over 72 h. MGCs were lysed, and CFUs were counted at 1, 24, and 72 h.p.i. In MGCs that were infected with M. avium A5, treatment with U18666A did not affect whether bacteria were taken up by the cells (Figure 8(b)). M. avium A5 intracellular growth over 72 h was significantly hindered by U18666A compared to without inhibition, with a reduction of about 25% (Figure 8(b)). This indicates that M. avium can utilize alternate sources of energy if cholesterol is unavailable in host cell during early stages of infection. However, M. avium is unable to overcome the lack of cholesterol later in infection and intracellular growth plateaus. Since the inhibition of intracellular cholesterol transport with U18666A reduces but does not prevent intracellular growth of M. avium, we propose that cholesterol is not essential for intracellular growth.Figure 8
Effects of altering lipid cellular components during mycobacterial infection. (a) Growth curve ofM. avium A5 in 7H9 supplemented with either DMSO, U18666A (3 μg/ml), or D609 (100 μM). D609 was added at specified time points after inoculation of growth media. (b) Survival of M. avium in MGCs in the presence of intracellular cholesterol transport inhibitor U18666A (3 μg/ml) or in media only. (c) Survival of M. avium in MGCs in the presence of sphingomyelin synthase inhibitor D609 (100 μM) or in media only. (d) D609 was added to MGCs at 24 or 48 h.p.i.; then, CFUs were determined over 72 h. (e) Extracellular CFUs from (d) supernatants (SN) from infected MGCs were plated at each time point. Statistically significant treatments are compared to untreated. Data shown are from 3 independent experiments. Statistical comparisons: ∗P<0.01; ∗∗P<0.001.
(a)(b)(c)(d)(e)


### 3.8. Effect of Sphingomyelin Synthase Inhibition onM. avium A5 Survival in MGCs

Since sphingomyelin has been shown to be advantageous for TB in macrophages, we determined whetherM. avium A5 utilizes sphingomyelin in MGCs to aid intracellular growth [27]. MGCs were either treated with tricyclodecan-9-yl-xanthogenate (D609) or untreated. D609 is an inhibitor of sphingomyelin synthase that reduces intracellular diacylglycerol and sphingomyelin and increases ceramide levels [46–48]. D609 was added to infected MGCs 1 hour postinfection and was replenished daily over the course of three days (Figure 8(c)). The concentration of 100 μM was chosen based on previously established inhibitory concentrations in human lung fibroblast and monocyte cell lines [46, 47]. 100 μM of D609 had no effect on the growth of M. avium A5 in 7H9 broth after 4 days of growth (Figure 8(a)). To account for the effects of D609 on bacterial growth at day 6, all in vitro experimental time points were determined by day 3. MGCs were infected then lysed, and CFUs were counted at 1, 24, and 72 h.p.i. In MGCs that were treated with 100 μM of D609, uptake of M. avium A5 was not affected as D609 was added after infection. M. avium A5 intracellular growth was significantly hindered with treatment only at 72 h.p.i, with a reduction of about 30% (Figure 8(c)). This indicates that during early stages of infection, M. avium can utilize alternate sources of energy if sphingomyelin is unavailable in the host cell.To determine the effect of sphingomyelin synthase inhibition after the infection had been more established, MGC survival assays were conducted in which D609 was added to the cells at either 24 h.p.i or 48 h.p.i (Figure8(d)). When D609 was added to M. avium A5-infected MGCS at 24 h.p.i, there was a reduction in bacterial survival at 48 h.p.i. However, M. avium A5 was able to overcome the effect of the inhibitor and resumed growth, which is shown by the increase in CFUs from 48 to 72 h.p.i. When D609 was added to M. avium A5-infected MGCS at 48 h.p.i, there was a reduction in bacterial survival at 72 h.p.i. The number of extracellular bacteria leaving the MGCs was counted for untreated, 24 h D609, and 48 h D609 groups (Figure 8(e)). In both the untreated and 24 h D609 groups, the number of extracellular bacteria increases between 48 and 72 h.p.i; however, the 48 h D609 group shows no increase during this period. Our results show that M. avium A5 can overcome sphingomyelin depletion when it occurs at 24 h.p.i, but not 48 h.p.i., and suggest M. avium dependency on sphingomyelin at later stages of infection.


## 3.1. IFN-γ and TNF-α Induce Multinucleated Giant Cell Formation In Vitro

To triggerin vitro MGC formation, THP-1 cells were differentiated with Phorbol 12-myristate 13-acetate (PMA) for 24 hours, followed by stimulation with a combination of TNF-α and IFN-ɣ for 6 days. After differentiation with PMA, THP-1 cells do not proliferate and become adherent. TNF-α and IFN-ɣ were selected because they have been shown to drive granuloma formation and contribute to MGC formation in vivo [36–39]. A fusion index of 56.5% was observed when cells were treated with 25 ng/ml TNF-α and 100 ng/ml IFN-ɣ over a 6-day period (Figure 1(a)). The resulting mixed population of MGCs and activated THP-1 macrophages was utilized for further experiments. MGCs formed with this protocol have 2-4 nuclei (Figures 1(b)–1(d)). CD40 is a marker of MGCs associated with mycobacterial infection and is involved in MGC formation [40, 41]. MGCs exhibited a significant shift in mean fluorescent intensity (MFI) of FITC-CD40, indicating that 98% of MGCs are CD40 positive (Figure 1(e)). THP-1 cells exhibited a small shift in fluorescent intensity, indicating a low percentage of CD40-positive cells (Figure 1(f)). MGCs have an MFI of FITC-CD40 that is significantly higher than THP-1 macrophages, MGCs stained with a FITC-isotype control, and unstained MGCs (Figure 1(g)). These results indicate that MGCs retain the CD40 marker and are biologically similar to MGCs observed in vivo.Figure 1
IFN-γ and TNF-α induce formation of multinucleated giant cells in vitro. THP-1 cells were fluorescently labeled after cytokine treatment to determine MGC formation via microscopy at 1000x magnification. The expression of CD40, a marker associated with classical macrophage activation and Langhans giant cells, was measured via flow cytometry. (a) The macrophage fusion index determined the percentage of multinucleated cell having at least 3 nuclei in 10 fields of view. (b) Hoechst stain of MGC nuclei (white arrow). (c) Alexa Fluor Phalloidin 488 stain of actin cytoskeleton (white arrow). (d) Merged image of MGC (white arrow). (e) FITC-CD40 levels in MGCs (black: unstained, blue: isotype control, and red: CD40+). (f) FITC-CD40 levels in PMA-differentiated THP-1 cells (black: unstained, red: CD40+). (g) Mean fluorescent intensity of FITC-CD40 (black: unstained, blue: isotype control, and red: CD40+). All groups were compared to MGC CD40+. Data are representative of 3 independent experiments. Statistical comparisons: ∗P<0.01, ∗∗P<0.001, and ∗∗∗P<0.0001.
(a)(b)(c)(d)(e)(f)(g)


## 3.2. Intracellular Survival and Growth ofM. avium in Multinucleated Giant Cells

The intracellular stages of mycobacteria in MGCs have not been fully described. Survival assays were performed to characterize the uptake and intracellular growth ofM. avium in the in vitro MGC model and compared to THP-1 macrophages. M. avium A5 infected MGCs at a similar rate to THP-1 macrophages (Figure 2(a)). Once cells were infected, bacterial survival was determined by CFU counts in either MGCs or THP-1 macrophages over 3 and 6 days, respectively, and fold change relative to 1 h.p.i calculated. The time points selected for THP-1 survival assays are standard practice. The time points for MGC survival assays were selected from day 5 of stimulation up to the point where bacterial growth was observed at 3 d.p.i. Additionally, by 6 d.p.i, the MGCs would have been on the plate for 12 days, which would reduce the number of viable cells. At 4 d.p.i for THP-1 and 48 h.p.i for MGC, fold change of bacterial growth was 4 (Figures 2(b) and 2(c)). In THP-1 macrophages, M. avium intracellular growth exhibited a fold change of 11 at 6 d.p.i, whereas M. avium intracellular growth in MGCs exhibited a fold change of 7 at 3 d.p.i (Figures 2(b) and 2(c)). During the survival assay, CFU/milliliter of extracellular bacteria that exited MGCs was enumerated (Figure 2(d)). The number of bacteria leaving MGCs was significantly greater at 72 h.p.i. compared to other time points. These data indicate that M. avium A5 can enter MGCs with the same efficacy as THP-1 macrophages. Additionally, M. avium A5 survives in the MGC environment and shows a similar growth trend to THP-1 macrophages. Our data support the idea of a favorable niche existing inside the MGC environment that M. avium A5 can utilize to survive and eventually leave to infect other cells nearby.Figure 2
Infection and survival ofM. avium A5. (a) Percent uptake of M. avium A5 in THP-1 macrophages and MGCs after 1 hour of infection. (b) Fold change relative to 1 h.p.i (dashed line) of M. avium growth over 6 days in THP-1 macrophages. (c) Fold change relative to 1 h.p.i (dashed line) of M. avium growth over 3 days in MGCs. (d) Extracellular bacterial CFUs recovered after infection of MGCs. Data shown are representative of results from three independent biological replicates. Statistical comparisons: ∗P<0.05; ∗∗P<0.005; ∗∗∗P<0.0005.
(a)(b)(c)(d)


## 3.3. Characterization ofM. avium Phenotype upon Leaving Multinucleated Giant Cells

To determine if the phenotype of MGC-passagedM. avium A5 is different than WT M. avium A5, uptake and survival assays were carried out in THP-1 macrophages. To passage M. avium A5, MGCs were infected with WT M. avium A5 for 1 h, and at 72 h.p.i, bacteria that exited the MGCs (MGC-passaged A5) were collected in cell culture media and kept on ice or at 4°C while being pelleted and washed. THP-1 cells were infected for 1 hour with MGC-passaged bacteria or M. avium A5 from a 7H10 streak plate. Uptake of M. avium A5 was evaluated by inhibiting macropinocytosis and receptor-mediated phagocytosis. Wortmannin inhibits the macropinocytosis pathway by preventing complete closure of the cytoplasmic ruffles. Blocking complement receptor 3 (CR3) with a CD11b antibody inhibits complement receptor-mediated phagocytosis. MGC-passaged A5 exhibited a percent uptake approximately 3 times greater than M. avium A5 grown on a plate, under normal infection conditions (Figure 3(a)). Blocking the CR3 receptor with an anti-CD11b antibody for 1 hour prior to infection led to a 52% reduction in uptake for both bacterial phenotypes (Figure 3(b)). Inhibition of macropinocytosis with wortmannin for 2 h prior to infection led to a 33% reduction in uptake for both bacterial phenotypes (Figure 3(b)). Inhibiting uptake pathways reduced the uptake of both phenotypes but did not completely block them, showing that M. avium of either phenotype can circumvent the effect of the inhibitors and entering the macrophage via an alternate route. This suggests that MGC-passaged bacteria can enter THP-1 cells via alternate routes. Survival in THP-1 macrophages over 6 days with MGC-passaged A5 is comparable to A5 from the plate; thus, once bacteria invade macrophages, there was no added advantage to MGC-passaged bacteria (Figure 3(c)). These data suggest that the MGC environment increases the invasiveness of M. avium A5 but that they retain their ability to enter the next host cell in multiple ways.Figure 3
Uptake and survival of MGC-passagedM. avium A5 in THP-1 cells. (a) Percent uptake of M. avium A5 inoculums in THP-1 cells after 1 h of infection. (b) Percent reduction of uptake by THP-1 cells treated with either wortmannin for 2 h or a CD11b antibody for 1 h, prior to infection. (c) Fold change relative to 1 h.p.i (dashed line) of bacterial survival by plate or MGC-passaged M. avium in THP-1 macrophages over 6 days. “Plate” refers to M. avium A5 grown on a 7H10 plate, and “MGC-passaged” refers to M. avium A5 that was collected from infected-MGCs at 72 h.p.i. Data are representative of three independent experiments. Statistical comparisons: ∗P<0.01, ∗∗P<0.001, and ∗∗∗P<0.0001.
(a)(b)(c)


## 3.4. Electron Microscopy Observations ofM. avium A5 in MGCs

To increase our understanding of the behavior of bacteria inside MGCs, transmission electron microscopy (TEM) was used. MGCs were infected withM. avium over 72 h then processed for TEM. THP-1 macrophages that are not treated with cytokines or infected with bacteria show normal cellular structures including a single nucleus and cell size. After cytokine exposure, MGCs were formed and autophagosomes were visible in the cytosol, characterized by double membranes (Figure 4(a)). In MGCs infected with M. avium A5 for 72 h, bacteria can be seen inside autophagosomes (Figure 4(b)), and bacterium-containing vacuoles can be seen interacting with autophagosomes (Figures 4(c) and 4(d)). Comparing multiple cells, numerous autophagic vacuoles exist within MGCs, and once these cells are infected with M. avium A5, the number of autophagosomes increased substantially.Figure 4
Autophagic activity in MGCs. (a) Autophagosomes containing cellular components in uninfected MGCs. (b) Autophagosomes containingM. avium 72 h.p.i. of MGCs. (c) Vacuoles containing M. avium interacting with an autophagosome at 72 h.p.i. (d) Same as (c). Black arrows: bacteria; orange arrows: autophagosomes.
(a)(b)(c)(d)The formation of MGCs causes several cellular changes, and one of the changes observed was the accumulation of lipid droplets in the cytosol (Figure5(a)). At 1 h.p.i, bacteria were present the cytosol near lipid droplets (Figure 5(b)) as well as in autophagosomes (Figures 5(c) and 5(e)). Intracellular lipid inclusions were present in intracellular bacteria at several time points (Figures 5(b)–5(e)). Vacuoles with multiple bacteria were observed at 72 h.p.i. (Figure 5(d)). After 24 h, bacteria started to leave MGCs and were observed near the plasma membrane (Figure 5(f)).Figure 5
Characteristics of intracellularM. avium in MGCs. (a) Uninfected MGC with three nuclei surrounded by lipid droplets. (b) Cytosolic M. avium inside MGCs at 1 h.p.i. (c) M. avium bacterium in a vacuole at 1 h.p.i in MGCs. (d) Vacuole containing multiple M. avium 72 h.p.i. inside MGCs. (e) Intracellular M. avium in autophagosome in MGC at 1 h.p.i. (f) 24 h.p.i., extracellular M. avium from an MGC. Blue arrows: host lipid droplets, black arrows: bacteria, green arrows: intrabacterial lipid inclusions, white arrows: nuclei, and orange arrows: autophagosomes.
(a)(b)(c)(d)(e)(f)


## 3.5.In Vitro Multinucleated Giant Cells Exhibit Increased Autophagic Activity Compared to Macrophages

To assess autophagic activity in MGCs, we quantified mRNA levels of LC3 and Beclin-1 via real-time PCR. Beclin-1 controls phagophore formation, and LC3 is involved in phagophore elongation [42]. Beclin-1 gene levels after 4 h indicate that MGCs have naturally greater expression compared to THP-1 macrophages, and once infected, MGCs exhibited even greater gene expression of Beclin-1 (Figure 6(a)). The trend continued through 24 h.p.i, except there was no difference between infected and uninfected MGC gene expression. Later in the infection, there was no difference in Beclin-1 gene expression between experimental groups. LC3 expression by MGCs at 4 h showed significant increase compared to LC3 expression by THP-1 macrophages (Figure 6(b)). After 24 h, LC3 gene expression by uninfected MGCs increases, and expression by infected MGCs decreased. This could be due to suppression of autophagic flux by M. avium, which has been demonstrated in M. abscessus [43]. After 72 h, LC3 gene expression was undetectable for all experimental groups. The reduction in autophagic marker expression by 72 h.p.i could be due to the absence of cytokine stimulation after the start of infection. For infected MGCs, the reduction could be due to bacterial exit from the cell. Collectively, these data support our earlier observation that infected MGCs have a greater number of autophagosomes than uninfected MGCs at early time points.Figure 6
Gene expression of autophagic markers is elevated in MGCs. Either MGCs or THP-1 cells were infected with WTM. avium A5 for 4 h. (a) Beclin-1 transcripts were measured by RT-qPCR at indicated times. mRNA levels are presented as relative expression to actin. (b) LC3 transcripts were measured at indicated time points. mRNA levels are represented relative to actin expression. Data are representative of two independent experiments. Statistical comparisons: ∗P<0.01; ∗∗P<0.001; ∗∗∗P<0.0001.
(a)(b)


## 3.6. Silencing of Beclin and ATG5 Reduces Intracellular Survival ofM. avium in THP-1 Macrophages

Since there is a clear tendency of MGCs cells to express autophagy uponM. avium infection, which is similar to what is observed at later time points in macrophages, we questioned if autophagy was an important occurrence for survival in host cells. Macrophages, the preferred cell type of M. avium and precursor of MGCs, were selected for this experiment. To assess the importance of autophagy during M. avium infection in THP-1 macrophages, siRNA technology was used to knock down expression of Beclin-1 and ATG5 host genes. THP-1 macrophages treated with siRNA targeting either Beclin, ATG5, empty vector, or no vector showed reduced but not absent protein levels inside of cells, confirmed by western blot (Figure 7(a)). These THP-1 macrophages were then infected with multiple strains of M. avium including 101, 104, and 109. Our results demonstrate that inhibition of ATG5 or Beclin, markers of autophagy, decreased the ability for bacteria to survive in macrophages over 4 days (Figure 7(b)). These data suggest that M. avium uses autophagy for intracellular survival in resting macrophages. It is possible that the autophagic activity in MGCs aids bacterial survival by a similar mechanism.Figure 7
siRNA targeting of autophagy markers decreases protein levels. THP-1 cells were depleted of either ATG5 or Beclin-1 48 h after siRNA targeting. (a) Western blot band intensities were quantified usingβ-actin as a loading control by Odyssey imager software (Li-Cor). (b) Survival of MAH strains 109, 101, and 104 at 48 h.p.i. in THP-1 macrophages transfected with ATG5 or Beclin-1 siRNA. CFU/milliliter of data is representative of three experiments. Statistical comparisons: ∗P<0.01; ∗∗P<0.001; ∗∗∗P<0.0001.
(a)(b)


## 3.7. Intracellular Cholesterol Transport andM. avium A5 Survival in MGCs

M. avium is clearly utilizing host lipids in MGCs, as shown by the presence of intrabacterial lipid inclusions (Figures 4(b), 5(b), and 5(c)). To determine whether M. avium A5 utilizes MGC-derived cholesterol as a source of nutrients for intracellular growth, uptake and survival assays were carried out in MGCs treated with (3β)-3-[2-(diethylamino) ethoxy] androst-5-en-17-one hydrochloride (U18666A), an intracellular cholesterol transport inhibitor. The final concentration was chosen based on previously published studies in mouse macrophages and human fibroblasts [44, 45]. To rule out direct bacterial inhibition, a growth curve of M. avium A5 in 7H9 broth supplemented with 3 μg/ml of U18666A was conducted (Figure 8(a)). U18666A had no effect on the growth of M. avium A5 over 6 days (Figure 8(a)). Since U18666A did not affect bacterial growth, we then used the inhibitor on MGCs infected with M. avium A5. U18666A was added 1 hour after infection of MGCs and replenished daily over 72 h. MGCs were lysed, and CFUs were counted at 1, 24, and 72 h.p.i. In MGCs that were infected with M. avium A5, treatment with U18666A did not affect whether bacteria were taken up by the cells (Figure 8(b)). M. avium A5 intracellular growth over 72 h was significantly hindered by U18666A compared to without inhibition, with a reduction of about 25% (Figure 8(b)). This indicates that M. avium can utilize alternate sources of energy if cholesterol is unavailable in host cell during early stages of infection. However, M. avium is unable to overcome the lack of cholesterol later in infection and intracellular growth plateaus. Since the inhibition of intracellular cholesterol transport with U18666A reduces but does not prevent intracellular growth of M. avium, we propose that cholesterol is not essential for intracellular growth.Figure 8
Effects of altering lipid cellular components during mycobacterial infection. (a) Growth curve ofM. avium A5 in 7H9 supplemented with either DMSO, U18666A (3 μg/ml), or D609 (100 μM). D609 was added at specified time points after inoculation of growth media. (b) Survival of M. avium in MGCs in the presence of intracellular cholesterol transport inhibitor U18666A (3 μg/ml) or in media only. (c) Survival of M. avium in MGCs in the presence of sphingomyelin synthase inhibitor D609 (100 μM) or in media only. (d) D609 was added to MGCs at 24 or 48 h.p.i.; then, CFUs were determined over 72 h. (e) Extracellular CFUs from (d) supernatants (SN) from infected MGCs were plated at each time point. Statistically significant treatments are compared to untreated. Data shown are from 3 independent experiments. Statistical comparisons: ∗P<0.01; ∗∗P<0.001.
(a)(b)(c)(d)(e)


## 3.8. Effect of Sphingomyelin Synthase Inhibition onM. avium A5 Survival in MGCs

Since sphingomyelin has been shown to be advantageous for TB in macrophages, we determined whetherM. avium A5 utilizes sphingomyelin in MGCs to aid intracellular growth [27]. MGCs were either treated with tricyclodecan-9-yl-xanthogenate (D609) or untreated. D609 is an inhibitor of sphingomyelin synthase that reduces intracellular diacylglycerol and sphingomyelin and increases ceramide levels [46–48]. D609 was added to infected MGCs 1 hour postinfection and was replenished daily over the course of three days (Figure 8(c)). The concentration of 100 μM was chosen based on previously established inhibitory concentrations in human lung fibroblast and monocyte cell lines [46, 47]. 100 μM of D609 had no effect on the growth of M. avium A5 in 7H9 broth after 4 days of growth (Figure 8(a)). To account for the effects of D609 on bacterial growth at day 6, all in vitro experimental time points were determined by day 3. MGCs were infected then lysed, and CFUs were counted at 1, 24, and 72 h.p.i. In MGCs that were treated with 100 μM of D609, uptake of M. avium A5 was not affected as D609 was added after infection. M. avium A5 intracellular growth was significantly hindered with treatment only at 72 h.p.i, with a reduction of about 30% (Figure 8(c)). This indicates that during early stages of infection, M. avium can utilize alternate sources of energy if sphingomyelin is unavailable in the host cell.To determine the effect of sphingomyelin synthase inhibition after the infection had been more established, MGC survival assays were conducted in which D609 was added to the cells at either 24 h.p.i or 48 h.p.i (Figure8(d)). When D609 was added to M. avium A5-infected MGCS at 24 h.p.i, there was a reduction in bacterial survival at 48 h.p.i. However, M. avium A5 was able to overcome the effect of the inhibitor and resumed growth, which is shown by the increase in CFUs from 48 to 72 h.p.i. When D609 was added to M. avium A5-infected MGCS at 48 h.p.i, there was a reduction in bacterial survival at 72 h.p.i. The number of extracellular bacteria leaving the MGCs was counted for untreated, 24 h D609, and 48 h D609 groups (Figure 8(e)). In both the untreated and 24 h D609 groups, the number of extracellular bacteria increases between 48 and 72 h.p.i; however, the 48 h D609 group shows no increase during this period. Our results show that M. avium A5 can overcome sphingomyelin depletion when it occurs at 24 h.p.i, but not 48 h.p.i., and suggest M. avium dependency on sphingomyelin at later stages of infection.


## 4. Discussion

Multinucleated giant cells play a significant role in the host-pathogen interaction during the granulomatous response to mycobacteria. Past studies have usedin vitro multinucleated giant cell models that utilize mycobacteria or mycobacterial components and a combination of primary lymphocytes and macrophages to trigger the formation of cellular aggregates with few MGCs [49–51]. The MGC model described in this study focuses on a single cell type, avoiding the use of bacterial components and complexities of cell-aggregate models (formed with several cell types) to detail characteristics of MGCs in the absence of confounding bacterial factors. The absence of bacterial components allows this model to have broader applications beyond mycobacterial studies. In addition, the model utilizes an immortalized cell line, rather than primary cells; this makes it affordable and reproducible. Foreign body multinucleated giant cell models induce macrophage fusion with IL-4 stimulation; however, these MGCs are typically found near medical implants and biomaterials and are significantly different than MGCs found in mycobacterial granulomas [52, 53]. In addition, MGCs formed in this model have the CD40 surface marker, which is present during acute tuberculosis infection and is required for MGC formation in tuberculosis [40, 41].MGCs are a common feature of mycobacterial granulomasin vivo and typically contain large bacterial loads [14–17]. It is not clear whether MGCs play a host-protective role or act in favor of the pathogen. In previous studies, MGCs have been shown to exhibit increased phagocytic activity compared to macrophages [15, 54, 55]. Recent research by Gharun and colleagues supports the idea that MGCs are permissive to mycobacterial replication [15]. In this study, we demonstrated that MGCs formed by the fusion of macrophages after IFN-γ and TNF-α stimulation can phagocytose M. avium and allow intracellular replication. Additionally, M. avium that leaves the MGC after 3 days exhibits a highly invasive phenotype. Previous work by Early and colleagues have shown that macrophage-passaged M. avium exhibits a more invasive phenotype than plate-grown M. avium [56]. Their work shows that macrophage-passaged M. avium enters uninfected macrophages primarily via macropinocytosis rather than complement receptor-mediated phagocytosis, which is the typical pathway [56]. Our work shows that blocking macropinocytosis significantly reduces the uptake of macrophages by MGC-passaged M. avium. These findings also indicate that the MGC environment increases the invasiveness of M. avium and that MGC-passaged bacteria can enter macrophages in multiple ways. Previous studies by Clay and colleagues, in the M. marinum zebrafish model, demonstrated that infected macrophages control the bacterial burden, migrate into tissues, and recruit additional cells to form granulomas in early stages of infection [57]. Additionally, granulomas themselves facilitate cell-to-cell spread of bacteria and increase bacterial burden [9, 10]. Our finding that M. avium exits MGCs and exhibits a highly invasive phenotype suggests that the intracellular environment of MGCs may enhance and contribute to the bacterial replicative niche and drive cell-to-cell spread.After infection,M. avium survives in a cytoplasmic vacuole of macrophages and prevents the acidification and vacuole fusion with lysosomes [58]. The lack of Rab7 marker on M. avium late phagosomes influences the phagolysosome fusion [59]. Furthermore, intracellular M. avium is resistant to ROS and nitric oxide and capable of subverting macrophage killing mechanisms [60]. Presumably, M. avium uses similar strategies for survival in MGCs.In some cases, autophagy is used as a mechanism to kill intracellular pathogens, such asSalmonella enterica serovar Typhimurium and M. tuberculosis [61, 62]. However, some bacterial species have strategies to avoid autophagic clearance, while others utilize autophagy to fuel intracellular growth or to drive their escape from the host cell [63, 64]. For instance, Francisella tularensis, an intracellular pathogen that infects macrophages, exploits autophagy to amass nutrients for intracellular growth [65]. M. tuberculosis suppresses macrophage autophagy through the inhibition of production of reactive oxygen species (ROS) [62]. Mycobacterium marinum, a model organism for M. tuberculosis, employs host autophagic machinery for cell-to-cell transmission of bacteria and is able to prevent maturation to autophagolysosomes [66]. Our work demonstrates that the inhibition of autophagy using RNAi in THP-1 cells leads to a significant reduction in intracellular survival of M. avium. Altogether, data collected on the increased autophagy in MGCs, the presence of M. avium in autophagosomes, and the permissiveness for bacterial growth suggest that M. avium utilizes autophagy to fuel intracellular growth in both macrophages and MGCs.When we inhibited intracellular cholesterol transport in MGCs,M. avium survival was significantly reduced at 72 h.p.i. These data indicate that cholesterol is not vital for intracellular survival and that M. avium is able to overcome the lack of cholesterol and replicate in MGCs. Work from Knight et al.’s group has indicated that accumulation of lipids in M. tuberculosis-infected macrophages is not triggered by the bacteria but by the host [67]. These findings agree with our observation that M. avium makes use of the cholesterol only because it is available; however, it does not affect bacterial survival if cholesterol is limited or absent. Depletion of host sphingomyelin with D609 during M. avium infection at different time points temporarily reduced bacterial intracellular growth in MGCs. M. avium was able to resume intracellular growth 24 hours after each D609 treatment, indicating that it may use host lipids as an energy source if they are present but can utilize alternative sources of energy if host lipids are not available. Improving our understanding of the role of host lipids in intracellular replication is an important avenue of future research. Characterization of MGC lipid droplets by lipidomics would provide further information about the MGC model and might point to specific lipids utilized by intracellular bacteria.M. avium that leaves the MGCs readily enters neighboring macrophages. Previous work by Early and colleagues has shown that macrophage-passaged M. avium exhibit higher uptake and induces apoptosis in macrophages as a mechanism of cell exit [56]. Apoptosis was not observed during visual inspection of macrophages infected with MGC-passaged M. avium. To follow up, an experiment is to measure apoptosis with an Annexin V assay is necessary to confirm this observation. The study published by Early et al. shows that macrophage-passaged M. avium enters uninfected macrophages primarily via macropinocytosis rather than complement receptor-mediated phagocytosis, which is the typical pathway [56]. Our work demonstrates that blocking macropinocytosis or complement receptor-mediated phagocytosis significantly reduces the uptake of macrophages by MGC-passaged M. avium, suggesting that this phenotype uses multiple routes of entry. The precise escape mechanism of M. avium from MGCs remains unclear. M. marinum utilizes autophagy to escape from host macrophages in a nonlytic mechanism [68]. M. avium may utilize a similar autophagy-mediated pathway of cell exit. Together, these findings demonstrate that exposure to the MGC environment increases the uptake of M. avium, by naïve macrophages which may help minimize the time spent in the extracellular space and maximize transmission. Further work is needed to describe the precise mechanism utilized by M. avium to exit MGCs.Previous studies by Clay and colleagues, in theM. marinum zebrafish model, demonstrated that infected macrophages control the bacterial burden, migrate into tissues, and recruit additional cells to form granulomas in early stages of infection [57]. Additionally, granulomas themselves facilitate cell-to-cell spread of bacteria and increase bacterial burden [9, 10]. Our finding that M. avium exits MGCs and exhibits higher uptake suggests that the intracellular environment of multinucleated giant cells may enhance and contribute to the bacterial replicative niche and drive cell-to-cell spread. Characterizing the cell exit mechanism utilized by MGC-passaged M. avium is an important future direction. Probing the involvement of autophagy on bacterial cell exit would be a good starting point.Using thein vitro model established in this study, we characterized some of the interactions between MGCs and M. avium. Bacterial survival assays in MGCs confirm a favorable intracellular environment promoting M. avium growth and escape. We also demonstrated that autophagy is elevated in MGCs, and its inhibition reduces M. avium survival in phagocytic cells. M. avium also appears to utilize host cholesterol and sphingomyelin to fuel intracellular growth. Further study is needed to define the role of lipids versus autophagy in nutrient acquisition by intracellular M. avium. Our data indicate that while M. avium is well adapted to replicate in MGCs, the host environment stimulates the uptake of bacteria and efficient escape from phagocytic cells likely for local spread.



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*Source: 5064371-2023-02-11.xml*