Bartonella henselae Persistence within Mesenchymal Stromal Cells Enhances Endothelial Cell Activation and Infectibility That Amplifies the Angiogenic Process
ABSTRACT
Some bacterial pathogens can manipulate the angiogenic response, suppressing or inducing it for their own ends. In humans, Bartonella henselae is associated with cat-scratch disease and vasculoproliferative disorders such as bacillary angiomatosis and bacillary peliosis. Although endothelial cells (ECs) support the pathogenesis of B. henselae, the mechanisms by which B. henselae induces EC activation are not completely clear, as well as the possible contributions of other cells recruited at the site of infection. Mesenchymal stromal cells (MSCs) are endowed with angiogenic potential and play a dual role in infections, exerting antimicrobial properties but also acting as a shelter for pathogens. Here, we delved into the role of MSCs as a reservoir of B. henselae and modulator of EC functions. B. henselae readily infected MSCs and survived in perinuclearly bound vacuoles for up to 8 days. Infection enhanced MSC proliferation and the expression of epidermal growth factor receptor (EGFR), Toll-like receptor 2 (TLR2), and nucleotide-binding oligomerization domain-containing protein 1 (NOD1), proteins that are involved in bacterial internalization and cytokine production. Secretome analysis revealed that infected MSCs secreted higher levels of the proangiogenic factors vascular endothelial growth factor (VEGF), fibroblast growth factor 7 (FGF-7), matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), interleukin 6 (IL-6), platelet-derived growth factor D (PDGF-D), chemokine ligand 5 (CCL5), and C-X-C motif chemokine ligand 8 (CXCL8). Supernatants from B. henselae-infected MSCs increased the susceptibility of ECs to B. henselae infection and enhanced EC proliferation, invasion, and reorganization in tube-like structures. Altogether, these results indicate MSCs as a still underestimated niche for persistent B. henselae infection and reveal MSC-EC cross talk that may contribute to exacerbate bacterium-induced angiogenesis and granuloma formation.
INTRODUCTION
Endemic among domestic cats, Bartonella henselae is a fastidious Gram-negative bacterium that, in humans, can cause subclinical intraerythrocytic bacteremia, mainly transmitted by cat fleas. In immunocompetent individuals, B. henselae infection can also lead to cat-scratch disease (CSD), characterized by lymphadenopathy with suppurative granulomas. Atypical clinical presentations of CSD, ranging from prolonged fever of unknown origin to hepatosplenic, ocular, and neurological manifestations, have also been reported (1).
Individuals who are unable to mount an immune response against B. henselae tend to develop a tumor-like vascular proliferative response in the skin and/or internal organs, which can lead to bacillary angiomatosis or bacillary peliosis (2). After infection, B. henselae survives and stimulates the migration and the production of proangiogenic factors by human endothelial cells (ECs) (2–5). In addition, other cell types, such as monocytes and macrophages (6), recruited to the vasoproliferative lesions stimulate EC proliferation in a paracrine manner through the production of vascular endothelial growth factor (VEGF) and C-X-C motif chemokine ligand 8 (CXCL8) (7). Mononuclear phagocytes, CD34+ progenitor cells, and ECs can also function as a reservoir from which B. henselae periodically enters the bloodstream and disseminates within the host (8). Despite the clinical implications of protracted Bartonella infections, the underlying mechanism of intracellular B. henselae persistence is poorly understood, and the existence of different reservoirs remains to be determined.
Mesenchymal stromal cells (MSCs) are multipotent adult stem cells that are present in various tissues, including the bone marrow (BM) and the adipose tissue, and that have recently received much attention due to their regenerative potential and immunomodulatory properties (9). MSCs actively participate in angiogenesis through several mechanisms, including paracrine cytokines and exosomes and cell contact interactions with ECs (10, 11). Diverse and multitasking roles of MSCs during bacterial infection have recently emerged (12, 13). MSCs can sense pathogens and mount an appropriate cytokine/chemokine response through the activation of Toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and the scavenger receptors MARCO and SR-B1 (12). Moreover, MSCs express epidermal growth factor receptor (EGFR) (a member of the ErbB receptor tyrosine kinase family), which has been shown to enhance their proliferation and the release of angiogenic factors (14). However, despite the emerging role of MSCs in infectious diseases, the mechanisms regulating the interplay between MSCs and bacteria are yet to be defined. Recent evidence suggests that MSCs can have a double-edged sword effect by playing a role in clearing infection but also promoting persistent bacterial infection. MSCs exert antimicrobial effects by secreting antimicrobial peptides and expressing indoleamine 2,3-dioxygenase, and MSC administration reduces the pathogen burden in animal models of antimicrobial sepsis (12). However, MSCs can also serve as a niche in which Mycobacterium tuberculosis can survive and persist during antimicrobial therapy. Indeed, viable M. tuberculosis was recovered from MSCs infiltrating tuberculosis granulomas in humans and in a tuberculosis mouse model (15, 16). It is likely that other chronic bacterial pathogens may exploit MSCs to favor their survival in the host, and we hypothesized that B. henselae infects MSCs and that infected MSCs contribute to angiogenesis via interactions with ECs, which represent one of Bartonella preferential targets.
Here, we show that B. henselae can invade and survive within human MSCs, and we demonstrate that TLR2, NOD-containing protein 1 (NOD1), and EGFR are implicated in bacterial recognition and cytokine production. Moreover, we provide evidence for MSC-EC cross talk involved in bacteria intracellular survival and activation of a proangiogenic program.
RESULTS
B. henselae invades and persists in MSCs.
To characterize the interaction of MSCs with B. henselae, adipose tissue-derived MSCs were infected at a multiplicity of infection (MOI) of 100 for 1, 2, 3, 4, or 8 days and then treated with gentamicin to kill all residual extracellular bacteria. Subsequently, the number of viable intracellular bacteria was measured by CFU assay. The number of B. henselae organisms invading MSCs increased progressively over a 3-day period, and the number of CFU in MSCs remained unchanged up to 8 days (P < 0.05) (Fig. 1A). At day 8 postinfection (pi), the vast majority of MSCs contained B. henselae, as demonstrated by the strong cytoplasmic reactivity of an anti-B. henselae monoclonal antibody (MAb) (Fig. 1B, upper and middle). The presence of internalized bacteria was confirmed by immunofluorescence (Fig. 1B, lower). To assess B. henselae intracellular survival after the initial infection and gentamicin treatment, MSCs were cultured in medium without gentamicin for an additional 4 days. The number of viable intracellular bacteria recovered, which remained stable during the first 96 h, was significantly lower at day 8, compared to day 4 (Fig. 1C). The ability of B. henselae to invade MSCs was further assessed by comparing its infection efficiency in MSCs versus human umbilical vein ECs (HUVECs), a known target of B. henselae infection. The number of intracellular bacteria recovered from MSCs after 24 h of infection was significantly higher than that recovered from HUVECs (Fig. 1D).
FIG 1
Next, we followed MSC infection by fluorescence microscopy. At day 1 pi, B. henselae stained with 4′,6-diamidino-2-phenylindole (DAPI) (cyan) remained mainly anchored to the MSC membrane, with only a few bacteria present in the cytoplasm (Fig. 2A, upper right, arrowhead). From day 2 pi onward, the number of internalized bacteria increased, and most B. henselae organisms were enclosed in perinuclear vesicles (Fig. 2A, lower left and middle, thin arrows). After 8 days pi, aggregates of bacteria colocalized with F-actin in globular structures called invasomes, which were first described in Bartonella-infected ECs (Fig. 2A, lower right, large arrow, and Fig. 2B), as attested by three-dimensional immunofluorescence analysis.
FIG 2
Altogether, these findings indicate that B. henselae is internalized by MSCs even more efficiently than by HUVECs, where it can persist for a long time.
B. henselae infection enhances MSC proliferation.
We next asked whether B. henselae infection would affect MSC survival. B. henselae infection did not induce cell death in MSCs, as demonstrated by similar amounts of annexin V-positive cells found in uninfected versus infected MSCs (Fig. 3A). This finding was further supported by the unaltered Bcl-2 (antiapoptotic)/Bax (apoptotic) expression ratio observed in these cells (Fig. 3B). We then assessed the effect of infection on the proliferation rate of MSCs. Infected MSCs grew significantly faster than their uninfected counterparts. Conversely, heat-inactivated B. henselae failed to enhance MSC proliferation (Fig. 3C).
FIG 3
Roles of TLR2, EGFR, and NOD1 in MSC infection with B. henselae.
TLRs and NODs play key roles in bacterial detection, and their cooperation becomes relevant in the context of infections. Interactions between cell surface TLR2 and intracellular surveillance NOD1/2 are of relevance in the recognition of pathogens and in the induction of the inflammatory response (17). However, a number of cell surface receptors, such as EGFR, that signal through pathways not related to TLRs and NODs are also used by pathogens, and an interaction between TLRs and EGFR has been demonstrated (18, 19).
Therefore, we assessed the expression of these receptors in response to B. henselae infection. Interestingly, B. henselae infection led to a >6-fold increase in TLR2 expression at both the mRNA and protein levels, while TLR4 expression remained basically unchanged (Fig. 4A and B). Furthermore, real time--PCR analysis showed significant upregulation of NOD1 mRNA at days 2 and 4 pi (Fig. 4A). NOD2 gene expression was not detected in uninfected or infected MSCs. Lastly, B. henselae infection significantly increased EGFR mRNA and phosphorylation levels (Fig. 4A and C, respectively). Specifically, we detected increased phosphorylation as early as 30 min pi, which remained above basal levels up to 120 min pi (Fig. 4C).
FIG 4
The involvement of these receptors in the production of CXCL8, a cytokine shown to be triggered by Bartonella in different cell types (20), was evaluated. Bartonella infection of MSCs enhanced their ability to produce CXCL8, which was neutralized by incubation with an anti-TLR2 neutralizing antibody (Fig. 4D, upper). Similarly, treatment with the EGFR inhibitor gefitinib or with the selective receptor-interacting serine/threonine-protein kinase 2 (RIPK2 or RIP2) inhibitor GSK583 significantly reduced the release of CXCL8 in B. henselae-infected MSCs (Fig. 4D, lower), suggesting that the EGFR/NOD pathway may play a role in CXCL8 transcriptional regulation. Finally, to address the role of bacterium-activated EGFR in Bartonella entry, we treated MSCs with the EGFR inhibitor gefitinib and a neutralizing anti-EGFR antibody and detected bacterial internalization reduced by about 70% and 50%, respectively, compared to untreated cells (Fig. 4E).
B. henselae-infected MSCs promote angiogenesis and infection of ECs.
Since MSCs regulate vascular remodeling and angiogenesis (21), we assessed the proangiogenic activity of conditioned medium (CM) from B. henselae-infected MSCs. To this end, CM from uninfected or B. henselae-infected MSC cultures were tested in a scratch wound healing assay using HUVECs. CM from B. henselae-infected MSCs (CM-MSC B. henselae) induced more rapid repair of the HUVEC monolayer (Fig. 5A). In addition, the CM-MSC B. henselae was 9-fold more powerful than CM from uninfected MSCs (CM-MSC control [CTRL]) in an aspheroid-based sprouting assay, which faithfully recapitulates the proliferation, invasion, and reorganization in tube-like structure of ECs (Fig. 5B). In keeping with the proangiogenic activity of MSCs, the CM-MSC CTRL induced the formation of radial sprouts, similar to what is induced by spheroid stimulation with 30 ng/ml of VEGF-A (Fig. 5B, right). Importantly, CM-MSC B. henselae but not CM-MSC CTRL accelerated the morphogenesis of HUVECs seeded on Cultrex extracellular matrix, as judged by the number of closed structures formed at 18 h pi (Fig. 5C).
FIG 5
Although ECs and MSCs can exhibit cross talk through soluble mediators (22), there are no data on the effects of MSCs on the susceptibility of ECs to bacterial infection. Therefore, we assessed the extent of Bartonella internalization at day 1 pi in HUVECs pretreated with CM-MSC CTRL or CM-MSC B. henselae. While there were no differences in the yield of bacteria between control HUVECs and HUVECs pretreated with CM-MSC CTRL, significantly more bacteria were detected in HUVECs pretreated with CM-MSC B. henselae (Fig. 5D). After 1 day of culture, we did not observe a significant increase in the proliferation of infected HUVECs pretreated with CM-MSC B. henselae, compared with those pretreated with CM-MSC CTRL or directly infected. The values (mean ± standard error of the mean [SEM]) for the number of cells harvested/number of cells seeded were 1.23 ± 0.2 (unconditioned medium), 1.063 ± 0.06 (B. henselae infected), 1.125 ± 0.1 (CM-MSC CTRL), and 1.25 ± 0.05 (CM-MSC B. henselae). In accordance with our observation, EC proliferation during B. henselae infection has been shown after 3 or 4 days of incubation (7, 23). Our results indicate that the treatment with CM-MSC B. henselae makes HUVECs more infectible and the increase in intracellular CFU does not depend on HUVEC proliferation.
Angiogenic expression profile of B. henselae-infected MSCs.
Finally, we assessed the impact of B. henselae infection on the ability of MSCs to modulate the expression of proinflammatory and proangiogenic molecules. For this purpose, we probed an antibody angiogenesis array with CM from uninfected and 4-day-infected MSCs. Among the 55 proteins of the assay, 27 were detected in CM of both uninfected and infected MSCs. Densitometric analysis showed the upregulation of fibroblast growth factor 7 (FGF-7), CXCL8, matrix metallopeptidase 9 (MMP-9), placental growth factor (PIGF), serpin E1, thrombospondin 1 (TSP-1), urokinase-type plasminogen activator (uPA), and VEGF in B. henselae-infected MSC CM, compared to that from uninfected MSCs (Fig. 6A and B). Intriguingly, activin A was the only growth factor downregulated in B. henselae-infected MSCs (Fig. 6A and B). Of note, the elevated expression of monocyte chemoattractant protein-1 (MCP-1), pentraxin 3 (PTX3), and tissue inhibitor of metalloproteinases 1 (TIMP-1) was not modulated by infection (Fig. 6A and B). The quantification by enzyme-linked immunosorbent assay (ELISA) of the increased production of CXCL8 and VEGF in the supernatants of MSCs infected for 2, 4, and 7 days was in good agreement with the array data (Fig. 6C). Finally, the expression of other molecular factors that are known for their angiogenic activity but were not included in our array, such as interleukin 6 (IL-6), chemokine ligand 5 (CCL5), and platelet-derived growth factor D (PDGF-D), was also induced following B. henselae infection (Fig. 6C).
FIG 6
DISCUSSION
Bartonella species exploit several mechanisms to hide inside erythrocytes and ECs to evade immune responses and persist in both animal reservoirs and human hosts. Numerous findings indicate that the blood-stage phase is preceded by the infection of cellular niches that periodically release bacteria able to invade erythrocytes. ECs were the first cell type considered a primary niche, because they support B. henselae replication and reside in proximity to the bloodstream (2, 24). However, later studies identified additional B. henselae persistence sites, including hematopoietic progenitor cells and dendritic cells (8, 25).
Here, we show that, once inside, B. henselae resides in MSCs without proliferating for several days. During this time, B. henselae localizes in numerous perinuclear membrane-bound vacuoles, as previously shown in HUVECs and Mono Mac cells (26, 27), or, at late time points of infection, as aggregated bacteria enclosed in F-actin-rich cell membrane protrusions identified as invasome structures (28).
MSCs sense microorganisms through the expression of various pattern recognition receptors, including TLRs and NLRs. The engagement of such receptors modulates MSC functions and their abilities to secrete cytokines (29). Our studies revealed that TLR2, NOD1, and EGFR are involved in the recognition of and responses to B. henselae by MSCs. Upon infection with B. henselae, MSCs secret large amounts of CXCL8, which is curbed by incubation with an anti-TLR2 antibody. A central role of TLR2 signaling during B. henselae infection is consistent with previous findings indicating that B. henselae, despite being Gram negative, preferentially activates TLR2 (25). In infected cells, NOD1 and NOD2 recognize bacterial peptidoglycan derivatives released into the cytosol and, upon ligand association with the adaptor protein RIPK2, trigger proinflammatory signaling (30). In our experimental system, inhibition of RIPK2 with the highly RIPK2-specific compound GSK583 (31) decreased CXCL8 release, indicating that NOD1 activation and signaling through RIPK2 during MSC infection is, in part, responsible for inducing the inflammatory response to B. henselae infection. Consistent with our results, NOD1 mediates CXCL8 induction after recognition of Helicobacter pylori, Escherichia coli (32, 33), and Chlamydia pneumoniae (34). Importantly, gefitinib, an inhibitor of the EGFR tyrosine kinase domain that is used to treat various forms of cancer, can hamper B. henselae-mediated induction of CXCL8, suggesting a role of EGFR in this pathway. Gefitinib also exerts an off-target inhibitory activity on the expression of RIPK2 (35); therefore, the inhibition of CXCL8 secretion may be due to blockage of NOD/RIPK2 signaling alongside that of EGFR. In support of this hypothesis, EGFR-NOD cooperation was recently involved in cytokine production in dengue virus-infected monocytes (36). Moreover, a growing body of literature highlights the importance of EGFR/ErbB in several bacterial and viral inflammatory responses (18, 37) and in pathogenic angiogenesis (38). In addition to stimulation of EGFR tyrosine phosphorylation, B. henselae enhanced EGFR mRNA expression, suggesting that this upregulation could serve as a positive feedback system. A functional role of EGFR signaling in the immune response against B. henselae is further supported by the observation that treatment of MSCs with the kinase inhibitor gefitinib or an anti-EGFR antibody significantly decreases B. henselae internalization. In this regard, EGFR was recently shown to act as a cofactor in mediating pathogen internalization in host cells (e.g., hepatitis B virus, hepatitis C virus, Chlamydia, and Candida) (18). Our finding indicates an important role of EGFR activation in B. henselae invasion; however, because these EGFR inhibitors do not completely abrogate B. henselae uptake by MSCs, it is likely that receptors other than EGFR may play a role in B. henselae infection. Moreover, it remains to be investigated whether EGFR activation is due to the direct interaction of B. henselae with the EGFR extracellular domain or its transactivation by EGFR ligands (i.e., epidermal growth factor [EGF], heparin-binding EGF-like growth factor [HBEGF], transforming growth factor α [TGF-α], betacellulin [BTC], amphiregulin [AREG], epiregulin [EREG], and epithelial mitogen [EPGN]), as shown for H. pylori and Neisseria spp. (39, 40). EGFR signaling pathways exert antiapoptotic activity in Pseudomonas- and Helicobacter-infected cells (41, 42), suggesting that EGFR activation by B. henselae promotes the survival and proliferation of infected MSCs.
These effects may also be explained at least in part by the robust release of cytokine/growth factors caused by B. henselae infection. In addition to CXCL8, angiogenic factors upregulated in infected MSCs include FGF-7, MMP-9, PIGF, serpin E1, TSP-1, uPA, IL-6, CCL5, and VEGF, leading to the induction of a proangiogenic phenotype in ECs as well as increased susceptibility of ECs to infection. Data reporting a role of MSCs in facilitating the infection of other cell types are sparse and concern mainly phagocytic cells. MSCs were shown to enhance bacterial uptake and clearance by polymorphonuclear leukocytes (43) and to mediate the reactivation of HIV in monocytic cells (44). A secretome rich in inflammatory angiogenic cytokines and matrix-remodeling factors was previously described in B. henselae-infected myeloid angiogenic cells (MACs). Similar to our observation with MSCs, CM from MACs increased angiogenic sprouting (45). In the past, infected ECs have been shown to upregulate the expression of VEGF and CXCL8, which lead directly to host cell proliferation and potentiate angiogenesis (23, 46); in parallel, B. henselae triggers the release of proinflammatory chemokines, which recruit monocytes/macrophages in the vasoproliferative lesions, and the production of angiogenic factors by phagocytic cells upon infection plays a central role in mediating angiogenesis (7, 20, 45). Since MSCs localize in contact with ECs at sites of infection/inflammation (22, 47), we propose that infected MSCs may support this angiogenic loop.
A role for MSCs can be envisioned in different scenarios of B. henselae infections. For instance, MSCs are recruited in tuberculosis around the lymph node granulomas to establish persistent infection and likely to suppress T cell responses (48). Moreover, MSCs are found in oral pyogenic granuloma tissues (49). Granulomatous lymphadenitis is the pathological hallmark of CSD, whereby MSCs could also be recruited in B. henselae granulomas to contribute to the immunopathogenesis. MSCs reside in the BM, interacting with other cellular components. We previously showed the colocalization of dendritic cells and MSCs in human BM (50). The role of MSC-EC cross talk has been characterized in the maintenance of the hematopoietic stem cell niche and in infection-induced emergency myelopoiesis (51, 52). Interestingly MSCs were shown to regulate proliferation and erythroid differentiation of CD34+ stem cells (53). Because B. henselae can infect CD34+ BM progenitor cells, BM has been proposed as one of the potential niches. In this regard, multifocal BM involvement was shown in CSD (54, 55), and a contribution of B. henselae to ineffective erythropoiesis was suggested (56). B. henselae-infected MSCs, releasing soluble molecules, can recruit and activate ECs, which in turn collaborate with MSCs in fine regulation of the hematopoietic stem cell niche.
In conclusion, this study provides novel insights into the role of MSCs serving as a reservoir during B. henselae infection and identifies TLR2, NOD1, and EGFR as the receptors involved in the recognition of B. henselae. Infection of MSCs triggers a potent proangiogenic program, which activates and enhances EC susceptibility to bacterial infection. A better understanding of the involvement of MSCs in B. henselae-induced angiogenesis may allow the development of targeted therapeutic strategies for the treatment of vascular proliferative disorders.
MATERIALS AND METHODS
Cell cultures.
Human MSCs were isolated from adipose tissues as described previously (50). Human adipose tissue samples were collected by lipoaspiration from healthy donors after written consent was obtained, in compliance with the Declaration of Helsinki and with approval by the local ethics committee (Comitato Etico Interaziendale A.O.U. Città della Salute e della Scienza di Torino, A.O. Ordine Mauriziano, ASL TO1, approval number 0009806). Subsequently, MSCs were analyzed by flow cytometry to verify that their phenotype was positive for CD73, CD90, and CD105 and negative for CD11b, CD34, and CD45.
HUVECs were isolated from umbilical cords of healthy informed volunteers in compliance with the Declaration of Helsinki. HUVECs were used at early passages (passages 1 to 4) and grown on culture plates coated with porcine gelatin in M199 medium (Gibco Life Technologies, Thermo Fisher Scientific) supplemented with 20% heat-inactivated fetal calf serum (FCS) (Gibco Life Technologies), EC growth factor (ECGF) (10 μg/ml), and porcine heparin (100 μg/ml) (Sigma-Aldrich) or in complete EC growth basal medium 2 (EBM2) (Lonza Group Ltd., Basel, Switzerland).
Bacterial cultures.
B. henselae Houston I strain (ATCC 49882; ATCC, Manassas, VA, USA) was grown on Columbia 5% sheep blood agar plates (bioMérieux, Lyon, France) at 37°C for 10 days under anaerobic conditions (i.e., candle jar). Bacteria were harvested under a laminar flow hood by gently scraping colonies off the agar surface. They were then suspended in Microbank cryopreservative solution and stored at −80°C in 1-ml aliquots. For biological assays, frozen bacteria were incubated in Schneider’s insect medium (Sigma-Aldrich) supplemented with 10% FCS, as described by Riess et al. (57), at 37°C in 5% CO2 for 6 days. Spectrophotometry was performed to evaluate bacterial growth (optical density at 600 nm [OD600] of 0.6, corresponding to 1 × 108 bacteria/ml) and confirmed by plating serial dilutions on Columbia 5% sheep blood agar plates. Bacteria, washed three times with 1× phosphate-buffered saline (PBS), were then added to cell cultures. Where indicated, B. henselae organisms were killed by heating the thawed bacteria at 56°C for 30 min.
Preparation of CM.
MSCs, cultured in 12-well plates at a density of 0.5 × 105 cells/well in RPMI 1640 medium with 10% FCS without antibiotics, were left untreated or infected for 96 h with B. henselae. Cells were then extensively washed to remove extracellular bacteria, and fresh RPMI 1640 medium was replaced for 72 h. CM was collected, centrifuged at 4,000 rpm for 10 min, filtered, aliquoted, and stored at −20°C.
Infection assay.
B. henselae invasion of MSCs was assessed by gentamicin protection assay (GPA). Briefly, 12,500 MSCs/cm2 were seeded for 24 h in RPMI 1640 medium supplemented with 10% FCS. To compare MSCs with HUVECs, infection was carried out with 60,000 cells/well seeded in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FCS or complete EBM2 (Lonza Group Ltd.), respectively. The next day, cells were washed twice and cultured in RPMI 1640 medium supplemented with 10% FCS without antibiotics. B. henselae (MOI of 100) was added to the cells, which were immediately centrifuged at 1,200 × g for 5 min to allow the association of bacteria with the cellular surface and incubated for 1, 2, 3, 4, or 8 days. At the end of the infection period, gentamicin sulfate (Sigma-Aldrich) (100 μg/ml) was added to the medium for 2 h to kill all extracellular bacteria. This assay was performed in triplicate, and control wells were left uninfected. Cells were then washed extensively, lysed by the addition of 200 μl of distilled water for 5 min, and sonicated for 1.30 min. Lysates were serially diluted and plated on Columbia blood agar plates, and CFU were counted after 1 week of incubation. To determine intracellular survival after 96 h of infection, extracellular bacteria were killed by gentamicin treatment for 2 h. Cells were further incubated in normal medium for the remaining time of the indicated infection period. When indicated, cells were pretreated for 6 h with the specific inhibitors gefitinib (10 μM) and GSK583 (1 μM) (both from MedChemExpress, Monmouth Junction, NJ, USA) or with a specific antibody against EGFR (mouse IgG1, clone LA1) or its corresponding isotype control antibody (both from EMD Millipore Corp., Burlington, MA, USA) at 10 μg/ml. The GPA was performed as described above after 1 or 2 days. In some experiments, HUVECs were cultured in the presence of CM from untreated and infected MSCs. Briefly cells seeded at 60,000 cells/well were pretreated overnight with the indicated CM and then infected with B. henselae (MOI of 100) for 24 h. Cells were harvested and counted directly with an hemacytometer. Proliferation is reported as an index calculated as the number of cells harvested/number of cells seeded. In parallel, a GPA was performed.
Staining procedures.
MSCs (1 × 104) were seeded on glass coverslips and infected with B. henselae at an MOI of 100. For immunohistochemical staining, cells were fixed in methanol, saturated with 0.1% bovine serum albumin (BSA) in PBS, and incubated for 1 h with an anti-B. henselae MAb (1:50 dilution, mouse IgG2b, clone H2A10; Abcam, Cambridge, UK). The H2A10 clone reacts with a 43-kDa epitope present only in B. henselae strains and not in other B. henselae species (58). After washing, an anti-mouse IgG biotinylated antibody was added for 30 min, and the slides were then stained with horseradish peroxidase (HRP)-conjugated streptavidin or with the chromogen 3,3′-diaminobenzidine (DAB) (Thermo Fisher Scientific). For immunofluorescence analysis, the slides were incubated with anti-B. henselae MAb, followed by goat anti-mouse IgG-Alexa Fluor 594 (A21023, 1:500 dilution; Thermo Fisher Scientific). Nuclei were counterstained with DAPI (Thermo Fisher Scientific). To follow bacterial infection, MSCs were seeded at 0.25 × 104 on glass coverslips, infected with B. henselae (MOI of 100), and incubated for 1, 2, 3, 4, or 8 days. At the end of the infection period, cells were fixed with 4% paraformaldehyde (PFA) for 10 min, washed with PBS, and permeabilized with 0.25% saponin in PBS. Samples were then saturated with blocking solution (PBS with 5% normal goat serum and 2% BSA) for 1 h at room temperature. After washes in PBS, samples were incubated with wheat germ agglutinin (WGA)-Alexa Fluor 594 or -Alexa Fluor 488 conjugate, Alexa Fluor 594-phalloidin (A12381) (1 h), and DAPI (5 min) (all from Thermo Fisher Scientific) to stain cell membranes, actin, and nuclei/bacteria, respectively. Cells were analyzed under a Zeiss Observer Z1 epifluorescence microscope equipped with a Plan-Apochromat 100×/1.4 numerical aperture oil objective and ApoTome2 imaging system for optical sectioning. Z-stack images were elaborated through AxioVision 3D and extended focus modules.
Immunoblotting.
Total cell lysates from cells that were untreated or treated for 30, 60, or 120 min with B. henselae (MOI of 100) or with 50 ng/ml EGF (R&D Systems, Minneapolis, MN, USA) for 15 min were prepared in cold lysis buffer (1% Triton X-100 and 1% Nonidet P-40 in PBS [pH 7.4]) containing a cocktail of protease and phosphatase inhibitors (Sigma-Aldrich). Samples (10 to 20 μg) were analyzed by 10% SDS-PAGE under denaturing conditions, followed by Western blotting using antibodies against EGFR (clone A10, sc-373746) and phospho-Y1068-EGFR (sc-377547) and HRP-conjugated secondary antibodies (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Chemiluminescence signals (Clarity Western enhanced chemiluminescence [ECL] substrate; Bio-Rad Laboratories) were acquired by the ChemiDoc imaging system (Bio-Rad Laboratories).
Real-time PCR.
Total MSC RNA isolated with the Qiagen RNeasy minikit was treated with DNase I (Qiagen, Hilden, Germany) and retrotranscribed into cDNA by the iScript cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Gene-specific primers were as follows: TLR-2: sense, 5′-CTCATTGTGCCCATTGCTCTT-3′; antisense, 5′-TCCAGTGCTTCAACCCACAAC-3′; TLR-4: sense, 5′-GGCCATTGCTGCCAACAT-3′; antisense, 5′-CAACAATCACCTTTCGGCTTTT-3′; Bax: sense, 5′-AGAGGATGATTGCCGCCGT-3′; antisense, 5′-CAACCACCCTGGTCTTGGATC-3′; Bcl-2: sense, 5′-TGCACCTGACGCCCTTCAC-3′; antisense, 5′-AGACAGCCAGGAGAAATCAAACAG-3′; hypoxanthine phosphoribosyltransferase (HPRT): sense, 5′-TGACCTTGATTTATTTTGCATACC-3′; antisense, 5′-CGCTTTCCATGTGTGAGGTGATG-3′; ribosomal protein L13a (RPL13A): sense, 5′-CATAGGAAGCTGGGAGCAAG-3′; antisense, 5′-GCCCTCCAATCAGTCTTCTG-3′. For EGFR, NOD1, and NOD2, validated primers from Bio-Rad Laboratories were used (unique assay identifiers qHsaCID0007564, qHsaCED0005079, and qHsaCED0056944, respectively). For quantitative real-time PCR, the iQTM SYBR green supermix (Bio-Rad Laboratories) was used according to the manufacturer’s instructions. Reactions were run in duplicate on a CFX96 real-time system and analyzed by CFX Maestro software (Bio-Rad Laboratories). Gene expression was normalized to the HPRT or RPL13A mRNA content.
MTT assay.
MSC viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). Cells were seeded at a density of 2 × 103 cells/well in 96-well plates. After 24 h of incubation in RPMI 1640 medium with 10% FCS without antibiotics, cells were infected with B. henselae (MOI of 100). The medium was changed after 4 days to wash out all extracellular bacteria. When indicated, cells were treated with heat-killed B. henselae. Cells were then incubated for 3 h with 20 μl MTT (final concentration, 0,5 mg/ml). Formazan crystals were solubilized for 10 min in 100 μl dimethyl sulfoxide (DMSO), and the OD570 was measured using a microplate reader (Victor3; PerkinElmer, MA, USA). To determine the contribution of bacteria in MTT reduction to overall values for infected MSCs, bacterial suspensions with the same concentrations per milliliter as those recovered from cells were assessed in parallel, and the values obtained were subtracted from results from infected cells.
Annexin V assay.
MSCs that were untreated or infected for 96 h with B. henselae were stained with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) (Sigma-Aldrich) according to the manufacturer’s instructions. Samples were analyzed by FACSCalibur (Becton, Dickinson), and results were quantified using FlowLogic (Miltenyi Biotec, Bergisch Gladbach, Germany).
Flow cytometry.
MSCs were collected at the indicated times after infection, preincubated for 30 min at 4°C in 1× PBS supplemented with 2% goat serum and 0.2% sodium azide, and washed twice with 1% BSA. Successively, cells were incubated for 30 min at 4°C with anti-human TLR-2 FITC (mouse IgG2a) and anti-human TLR-4-phycoerythrin (mouse IgG2a) or the respective isotype controls (all from BioLegend, San Diego, CA, USA). Flow cytometry analysis was performed using FACSCalibur and FlowLogic as described above.
Cytokine measurements.
MSCs seeded in 24‐well plates were infected with an MOI of 100 B. henselae for the indicated times. For some experiments, cells were pretreated with the pharmacological inhibitors gefitinib and GSK583 or the neutralizing antibody anti‐TLR2 (anti‐human TLR2 IgA, clone B4H2) or the human IgA2 isotype control (both purchased from InvivoGen, San Diego, CA, USA). Cell-free supernatants were then harvested to measure human VEGF-A, CXCL8, IL-6, and CCL5 production by ELISA (R&D Systems). To quantify human PDGF-D, a specific kit from Elabscience (Wuhan, Hubei, China) was employed.
Angiogenesis array.
The human angiogenesis array (Proteome Profiler antibody array; R&D Systems) was used to assess the expression of 55 angiogenesis-related proteins in MSCs that were uninfected or infected with B. henselae for 96 h. The array membranes were probed with pooled supernatants derived from three independent experiments according to the manufacturer’s instructions. Chemiluminescence signals were acquired with the ChemiDoc imaging system (Bio-Rad Laboratories).
The signal intensity of each antigen-specific antibody spot was quantified using Fiji-ImageJ (NIH) software. For comparison of the relative expression of proteins in uninfected versus infected cells, the mean pixel density of the pair of duplicate spots for each protein, after subtraction of the mean pixel density of the negative-control spots of the respective array, was normalized to the mean pixel density of the positive-control spots. Heat-map analysis using the normalized data was performed with GraphPad Prism v8.0 software.
Sprouting assay.
Sprouting of HUVEC spheroids was assessed as described previously (59). Briefly, spheroids were prepared in 20% methylcellulose medium, embedded in a fibrin gel, and stimulated with recombinant human VEGF-A165 (30 ng/ml; R&D Systems) or with different concentrations of CM from uninfected or infected MSCs. The number of radially growing cell sprouts was counted after 24 h using an Axiovert 200M microscope equipped with an LD A-Plan 20×/0.30 Ph1 objective (Carl Zeiss) and was expressed as the relative increase over untreated spheroids.
Motility assay.
The HUVEC motility assay was based on scratch wounding of a confluent monolayer. Briefly, HUVECs (1 × 105) were seeded onto 0.1% collagen type I (BD Biosciences, Italy)-coated six-well plates in complete medium until a confluent monolayer was formed. The cell monolayers were scratched using a pipette tip, washed with 1× PBS to remove the undetached cells, and treated with MSC CM. After 24 h, cells were photographed under an Axiovert 200M microscope (Carl Zeiss) equipped with an LD A-Plan 20×/0.30 Ph1 objective. The healed area was quantified through computerized analysis by subtracting the wound area at 24 h from the initial area.
Tube formation assay.
EC vessel formation was assessed by the tube morphogenesis assay in a three-dimensional collagen matrix. To this end, HUVECs were seeded onto a Cultrex reduced growth factor basement membrane matrix (Trevigen)-coated μ-slide angiogenesis chamber (Ibidi, Martinsried, Germany) at a density of 4.0 × 104 cells/cm2 in the absence or presence of CM from untreated or infected MSCs. After 48 h, cells were photographed using an Axiovert 200M microscope, and the number of meshes per field was counted.
Statistical analysis.
Statistical significance was determined by the nonparametric Student's t test and one-way analysis of variance followed by Tukey’s multiple-comparison test. Results were analyzed by GraphPad Prism v8.0 software.
Data availability. All data and materials are available upon request.
ACKNOWLEDGMENTS
We thank William Vermi, Department of Molecular and Translational Medicine, University of Brescia, for immunohistochemical analysis of the MSC-B. henselae interaction.
This work was supported by funds from the Compagnia di San Paolo, Fondazione Ricerca Molinette, and Associazione Italiana Ricerca sul Cancro project IG15811 (2015 to 2017), project IG20776 (2017), and project IG17276. E. Grillo was also supported by a FUV Fellowship.
We declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
S. Scutera, S. Mitola, S. Sozzani, and T. Musso participated in the design of the study. S. Scutera, R. Sparti, V. Salvi, E. Grillo, G. Piersigilli, M. Bugatti, D. Alotto, and T. Schioppa participated in data acquisition and analysis. T. Musso, S. Mitola, and S. Scutera wrote the manuscript. S. Sozzani participated in data interpretation and manuscript revision.
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Information & Contributors
Information
Published In
Infection and Immunity
Volume 89 • Number 8 • 15 July 2021
Editor: Victor J. Torres, New York University School of Medicine
PubMed: 34031126
Copyright
© 2021 American Society for Microbiology. All Rights Reserved.
History
Received: 6 May 2021
Accepted: 7 May 2021
Accepted manuscript posted online: 21 May 2021
Published online: 15 July 2021
Keywords
Contributors
Editor
Victor J. Torres
Editor
New York University School of Medicine
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