Induction of Reactive Intermediates and Autophagy-Related Proteins upon Infection of Macrophages with Rhodococcus equi

Rhodococcus equi (R. equi) is an intracellular macrophage-tropic pathogen with potential for causing fatal pyogranulomatous pneumonia in foals between 1 and 6 months of age. In this study, we sought to determine whether infection of macrophages with R. equi could lead to the induction of autophagy. Murine bone marrow derived macrophages (BMDM) were infected with R. equi for various time intervals and analyzed for upregulation of autophagy proteins and accumulation of autophagosomes relative to uninfected controls. Western blot analysis showed a progressive increase in LC3-II and Beclin1 levels in a time-dependent manner. The functional accumulation of autophagosomes detected with monodansylcadaverine further supported the enhanced induction of autophagy in BMDM infected with R. equi. In addition, infection of BMDM with R. equi induced generation of reactive oxygen species (ROS) in a time-dependent manner. These data are consistent with reports documenting the role of ROS in induction of autophagy and indicate that the infection of macrophages by R. equi elicits innate host defense mechanisms.


Introduction
Rhodococcus equi is an aerobic, Gram-positive, nonspore forming coccobacillus which infects foals within the first few weeks of life and can cause severe pyogranulomatous pneumonia between 1 and 6 months of age [1][2][3]. R. equi persists and replicates intracellularly in murine and equine macrophages [4,5]. Upon infection, R. equi is phagocytosed and resides in an early phagosome compartment, which fails to undergo later maturation stages thereby resulting in an R. equi containing vacuole (RCV). Formation of RCV results in bacterial multiplication and killing of the host macrophage by necrosis with the net effect of R. equi escaping macrophage bactericidal activity [6,7]. A plasmid encoding the virulenceassociated protein antigen (VapA) has been demonstrated to be necessary for virulence and replication of R. equi in macrophages [8][9][10][11][12].
Macrophages are immune cells involved in eliciting primary responses to pathogens, maintenance of tissue homeostasis, as well as coordination of adaptive immune responses, inflammation, resolution of inflammatory states, and tissue repair [13]. Based on distinct functions and physiological roles, macrophages are activated in response to different innate or adaptive immune signals. Various receptors expressed on the surface of macrophages play an important role in influencing and directing immune responses. Activation of macrophages through Fc RI and Toll-like receptor (TLR) signaling is required for phagocytosis of pathogens and can also activate NADPH oxidase (NOX2) to trigger the generation of reactive oxygen species (ROS), such as superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ) which have potential to kill microbes [14,15]. It has been shown by various studies that mitochondria are the main source of ROS for regulation of autophagy [16,17] and that NOX2 2 Scientifica generated ROS is a key regulator of bacterial autophagy [18].
Autophagy is a highly conserved, degradative process which involves the engulfment of a portion of cell cytoplasm and organelles in double membrane vesicles known as autophagosomes which are themselves targeted for lysosomal degradation [19]. In mammals, regulation of autophagy requires several genes termed "autophagy-related genes" (Atg genes) which collectively have diverse functions [20,21]. In addition to cytoplasmic material, xenophagy recognizes intracellular pathogens for targeted lysosomal degradation [19,22]. Xenophagy is an emerging innate defense mechanism against pathogenic agents, including bacteria, viruses, and parasites [22]. Phagocytosis and autophagy act cooperatively as components of the host's innate immune defenses against microbial invasion [18,23]. Autophagy protein Rubicon is shown to be essential for activation of the NADPH oxidase complex, leading to ROS production upon receptor ligation of TLR-2 or upon microbial infection [24]. Rubicon also negatively regulates the maturation step of autophagy as part of a Beclin1-Vps 34-containing autophagy complex [24,25]. To extend these studies in the context of macrophage infection with intracellular bacteria, we investigated induction of autophagy in a longitudinal study by detection of the autophagy proteins Beclin1 and LC3-II and indirectly by monitoring production of ROS upon infection of bone marrow derived primary murine macrophages with R. equi.

2.2.
Bacteria. T194 R. equi strain, a clinical isolate from a pneumonic foal (provided by Dr. Ronald Griffith, Iowa State University), was thawed from −80 ∘ C and streaked on an LB agar plate and was grown for 30 hours in 37 ∘ C. A single colony was inoculated into 20 ml of Brain Heart Infusion (BHI) media and incubated at 37 ∘ C shaker set at 200 rpm to achieve an optical density (O.D) of 0.25. Bacteria were further washed in sterile PBS and resuspended in BHI media.

BMDM Isolation and Cell
Culture. Bone marrow cells were harvested from the femur of C3HeB/FeJ mice according to the IACUC approved protocol. Briefly, the bone marrow cells were flushed out using a 25-gauge needle fixed to 10 ml syringe containing complete cell culture medium (CTCM) with Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 2 mM glutamine, 0.05 M 2-mercaptoethanol, 4.5 g/L glucose, 25 mM HEPES, 100 U/ml penicillin, and 100 g/ml streptomycin. Bone marrow cells were centrifuged at 250 g for 15 min at 4 ∘ C and resuspended in a bone marrow medium containing 30% L-cell conditioned medium, 20% FBS, 50% DMEM, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, and 100 ug/ml streptomycin. Approximately 25 × 10 6 cells were seeded in 15 mm Petri dish and incubated at 37 ∘ C in 5% CO2. On day 2 of culture, the media containing nonadherent cells were replaced with a fresh bone marrow medium. On day 6, the adherent bone marrow derived macrophages were harvested and washed in 1x PBS before resuspension in CTCM.

Confocal Microscopy.
To evaluate the intracellular replication of R. equi, 5 × 10 5 BMDM were allowed to adhere on coverslips in 24-well plates. Cells were infected with R. equi at a multiplicity of infection (moi) of 3 and incubated for 12 h, 24 h, and 48 h at 37 ∘ C. Following infection, cells were fixed with 4% PFA in PBS for 15 min, permeabilized with 0.1% Triton-X 100 for 10 min at room temperature (RT), and blocked with a buffer (10% goat serum, 0.4% bovine serum albumin in 1x PBS) for 30 mins. To label intracellular bacteria, cells were incubated with rabbit polyclonal R. equi (1 : 3000) as primary antibodies for an hour [26]. The coverslips were washed thrice with 1x PBS for 5 min and incubated with goat anti-rabbit Alexa 488 (1 : 3000) as secondary antibodies for 1 h. Coverslips were further washed three times with 1x PBS and mounted on glass slides using ProLong with DAPI (Molecular Probes, Invitrogen). Confocal microscopy was performed either using an Olympus IX-61 microscope equipped with red, green, and blue filter sets with a cooled CCD camera or by an inverted Olympus Fluoview6 1000 (Minneapolis, MN) laser-scanning microscope.

ROS Assay.
Approximately 50,000 cells in complete cell culture medium (CTCM) were seeded per well in a 96-well plate. After infection, cells were incubated with 10 M CM-H 2 DCFDA dye (Invitrogen) in the dark at 37 ∘ C for 30 mins. Cells were washed with Hank's balanced salt solution (HBSS) with calcium and magnesium. Fluorescence was measured using a Fluostar Omega (BMG Biotech) fluorimeter with setup excitation at 485 nm and emission at 540 nm.
2.6. MDC Assay. The MDC (monodansylcadaverine) assay to label autophagosomes has been described previously [27]. After infection, macrophages were incubated with 0.05 mM MDC in a serum-free RPMI medium at 37 ∘ C for 30 mins. Later, macrophages were harvested and lysed in 10 mM Tris-HCl, pH 7.4 containing 1% Triton-X 100. Accumulation of MDC in autophagy vacuoles was measured using a Fluostar Omega plate reader (BMG LABTECH Inc., NC,

Data
Analysis. Data were analyzed by using Prism 4.0 software (GraphPad Prism, San Diego, CA). Results represent mean ± SEM. Statistical analysis was performed with one-way ANOVA followed by Tukey's post hoc test (GraphPad Prism software) in order to compare between treatment groups. Results were considered significantly different if < 0.05.

R. equi Growth in Bone Marrow Derived Macrophages.
First, to investigate the intracellular growth of R. equi, harvested and cultured BMDM were grown on coverslips and were infected with R. equi (moi3). After 2 hrs, extracellular bacteria were killed using gentamycin sulfate (10 ug/ml). Intracellular bacterial infection was allowed for 12 h, 24 h, and 48 h. BMDM were immunostained with anti-R. equi antibody and fluorescence microscopic analysis was performed. In comparison to uninfected BMDM (Figure 1(a)), we observed intracellular R. equi as green fluorescence puncta bodies at 12 h (Figure 1(b)). Moreover, significant increase in R. equi was observed in 24 h and 48 h (Figures 1(c) and 1(d)). Further, higher magnification of intracellular R. equi staining at 24 h revealed that R. equi, in the form of clusters and aggregates, localized in the perinuclear region (Figure 1(e)). This finding verifies the previous study by Hondalus and Mosser [5] showing that intracellular R. equi replicates in clusters.

ROS Increase in Bone Marrow Derived Macrophages
Infected with R. equi. Increase in ROS levels in macrophages in response to bacterial invasion is an important bactericidal mechanism. Therefore, it was important to observe whether R. equi stimulated the increase in ROS levels in BMDM.   (Figure 2). Stimulation of BMDM with 10 ug/ml LPS and 25 ng/ml IFN g for 24 h was considered as positive control [28,29] and showed a significant increase ( < 0.001; app. 1.3fold) in ROS levels. The results of these experiments suggest that R. equi infection elicited the increase in ROS production in the later time points that would result in cell death by necrosis causing failure to sustain ROS at 72 h.

Induction of Autophagy in Bone Marrow Derived Macrophages Infected with R. equi.
Recently, the role of autophagy has been implicated as an important host immune response against microbial infections [22]. In this study we aimed to evaluate the regulation of autophagy by R. equi. BMDM were infected with R. equi (moi3) for 3 h, 6 h, 12 h, 18 h, 24 h, 48 h, and 72 h. Next, we isolated total protein lysates and subjected the lysates to western blotting detection of the autophagy marker, LC3-II a structural component of autophagosomes [30]. In comparison to uninfected control, significant increase in LC3-II levels was observed at 24 h ( < 0.01) and 48 h ( < 0.01  (Figure 3(a)). We further extended this study to observe the formation of autophagy vacuoles. BMDM were infected with R. equi (moi3) for 24 h and 48 h. Later, BMDM were incubated with a fluorescent compound called monodansylcadaverine (MDC) to label autophagy vacuoles. Fluorescence measurement showed a significant increase in accumulation in MDC stained autophagy vacuoles at 24 h ( < 0.001) and 48 h ( < 0.001) infection time points (Figure 3(b)) indicating the induction of autophagy. Stimulation of BMDM with 10 ug/ml LPS and 25 ng/ml IFN g for 24 h was considered as positive control showing a significant increase ( < 0.001; app. 6.5-fold) in autophagy vacuoles.
Next we wanted to clarify if initiation of autophagy has occurred. The whole cell lysates from R. equi infected BMDM at various time points (3 h, 6 h, 12 h, 18 h, 24 h, 48 h, and 72 h) were again subjected to immunoblotting of Beclin1 which is a key regulator in the initiation of autophagy and autophagosome formation through class III PI3K pathway [31]. As shown in Figure 4, we observed the expression levels of Beclin1 at later infection time points, 24 h ( < 0.01) and 48 h ( < 0.01), in comparison to an uninfected control and earlier infection time points (3 h, 6 h, 12 h, and 18 h). Expression level of Beclin1 declined significantly (app. 4.35-fold) at 72 h. Taken together, these results indicate that R. equi infection resulted in autophagy and that the autophagy process is a normal host response and further implicates the importance of class III PI3K pathway in bacterial autophagy.

Discussion
R. equi remains a great challenge to the health of young foals and immunocompromised adult horses. It is also a pathogen of immunocompromised humans [30][31][32]. R. equi is particularly well recognized as a major cause of pneumonia in foals [33]. The incidence of R. equi pneumonia appears to be increasing, possibly because of intensified management of equine breeding farms and climate change. This disease continues to be a major challenge for clinicians and the equine industry in terms of epidemiological pattern and therapeutic control due to its complex host-pathogen interaction [8,33]. The objective of this study was to correlate the induction of LC3-associated phagocytosis with production of ROS in order to gain insight into the macrophage response upon infection with R. equi.
R. equi is an intracellular macrophage-tropic Grampositive coccobacillus which possesses the capability to survive and multiply in murine and equine macrophages [5,34]. Following phagocytosis, R. equi interferes with phagosomal maturation and suppresses acidification of the phagosome to prevent the formation of phagolysosomes [6,35]. In addition to the interference in phagosomal maturation, our data suggest that infection with R. equi induces autophagy, a host response broadly characterized as being protective. Murine BMDM were infected with R. equi at an moi of 3 and the regulation of autophagy was analyzed at specific time points after infection. Detection of autophagy-related proteins, LC3-II and Beclin1, by western blot occurred as early as 3 h with significantly higher amounts of these proteins detected at 24 h and 48 h suggesting that the autophagy machinery could play a role in innate host defense. This study was supported by a progressive accumulation of autophagosomes as detected using a MDC detection assay. Autophagy is a cytoprotective mechanism in which its own cytoplasmic contents are engulfed into autophagosomes and destined for lysosomal degradation. The autophagy mechanism is well studied in various disease states such as cancer, neurodegenerative diseases, aging and immunity, and inflammation [22]. In order to survive and replicate within host cells, some microorganisms have evolved a mechanism to escape degradation by the autophagy machinery [36]. A recently intriguing concept of noncanonical autophagy is characterized by the formation of autophagosomes without the necessity of Atg proteins that are recruited upon a double membrane vacuole containing bacteria [19,37]. LC3-associated phagocytosis involves the formation of a single membrane autophagosome decorated with LC3 within professional phagocytes. This process is independent of the ULK1 complex [38] but requires NADPH oxidase activity with production of superoxide and other reactive intermediates within the vacuole to exert a bactericidal effect [39]. It has been shown that mycobacterial species, including Mycobacterium tuberculosis (M. tuberculosis) and Mycobacterium marinum (M. marinum), survive in macrophages by preventing both phagolysosome formation [40] and recruitment of LC3 to its phagosome [41]. Since R. equi and Mycobacterium spp. belong to the mycolata taxon and R. equi has been shown to inhibit phagolysosomal fusion [6], it is reasonable to speculate that infection with R. equi could induce the same innate host response as infection with M. marinum. Our data are consistent with the notion that induction of autophagy could contribute to the ability of host cells to control infection by intracellular R. equi. A better understanding of this hostpathogen relationship is desirable as there is intense interest in gaining insight into potential therapeutic approaches involving regulation of autophagy.
Depending on the interaction with class III phosphoinositide 3-kinase, Beclin1 plays an important role in localizing autophagy proteins to preautophagosomal structures. In this study, we observed significant levels of Beclin1 at 24 h and 48 h. At this point, it appears that Beclin1 plays an important role in the macrophage's response to infection with R. equi, but further studies are needed to explore the role of Beclin1 in regulating R. equi induced autophagy. It has been shown that M. tuberculosis can escape the phagosome into the cytosol leading to host necrotic cell death via an ESX-1 dependent mechanism. In the study reported here, microscopic necrotic cell death of murine BMDM was observed at 24 h after infection with an moi of 3 R. equi (results not shown), which is consistent with the documented observation that R. equi contains an esx gene cluster encoding ESAT-6 and CFP-10 which are important in virulence and pathogenesis, including necrotic cell death and bacterial release [42,43]. It is unclear what specific role the autophagy machinery plays upon recognition of R. equi in the cytosol and whether it confers a protective effect, possibly delaying host cell necrosis.
Another protective mechanism that appears to be shared by M. tuberculosis and R. equi is mediated by the histone-like protein, lsr2 gene, and the R. equi homologue, which apparently confer resistance against macrophage produced reactive intermediates of oxygen, such as superoxide [44]. However, despite mechanisms of pathogen resistance to ROS, generation of ROS also plays an important mechanistic role in the induction of autophagy by upregulating the NADPH oxidase complex [25]. In this study, when murine BMDM were infected with R. equi at an moi of 3, we observed a time-dependent production of ROS from 18 h until 48 h after infection. It is unclear what the precise mechanism is whereby infection with R. equi leads to enhanced production of ROS and induction of autophagy in macrophages. Although these two events could be independent, recent studies have shown that the RUN domain of Beclin1 interacts with the cysteine-rich Rubicon autophagy protein which, in turn, acts in response to microbial infection as a positive regulator of NADPH oxidase [25]. After microbial infection or ligation of TLR2, Rubicon interacts with the p22phox subunit of the NADPH oxidase complex, accelerating its phagosomal trafficking to induce a burst of ROS and inflammatory cytokines. Rubicon's actions in autophagy and phagocytosis are shown to be functionally and genetically separable while the outcome of both of these functions depends on the environmental stimuli [24,25,38]. However, it is plausible that both autophagy and phagocytosis play important roles in macrophage resistance to infection with R. equi, as Bonilla et al. (2013) showed that autophagy modulates phagocytosis by regulation of scavenger receptors and is required for in vivo eradication of Mycobacterium [45].
The data obtained in the present study provide new insights into the macrophage response upon infection with R. equi, particularly with respect to induction of autophagy and production of reactive intermediates. Similar kinetics in the upregulation of autophagy protein levels of LC3-II and Beclin1 indicated induction of autophagy upon infection of murine BMDM with R. equi. Accumulation of autophagosomes concomitant with an increase in autophagy proteins further supported enhanced induction of autophagy upon infection. ROS generation by murine BMDM is consistent with the necessary signaling role of superoxide in the upregulation of LC3-associated phagocytosis but could also be due to other responses to infection by R. equi. Further studies are needed to characterize the signaling pathways involved in regulation of autophagy in R. equi infected macrophages and whether modulation of autophagy can be used to mitigate R. equi infection in macrophages.

Conflicts of Interest
The authors declare that they have no conflicts of interest.