Tumor Necrosis Factor Alpha Modulates the Dynamics of the Plasminogen-Mediated Early Interaction between Bifidobacterium animalis subsp. lactis and Human Enterocytes

HT29 cell culture conditions.HT29 cells were grown in Dulbecco's modified Eagle's minimal essential medium with 4.5 g/liter glucose (DMEM; PAA Laboratories) as reported by O'Hara et al. (22). For adhesion and intracellular invasion assays, 2.5 × 10⁵ HT29 cells per well were seeded in 24-well tissue culture plates (TPP) and 12-mm-diameter glass coverslips and grown to confluent monolayers. For the transmigration assay, 1 × 10⁵ cells were layered on 3-μm-pore size transwell inserts (Falcon; Becton Dickinson) in 24-well tissue culture plates and allowed to grow to confluent and fully differentiated monolayers. The tightness of the cell layers was verified as described by Attali et al. (2). Twenty-four hours before each assay, the cell medium was replaced with interaction medium (IM) (DMEM, 25 mM HEPES, 1 g/liter glucose [Gibco], 1% fetal calf serum [FCS]); when necessary, 2 ng/ml human recombinant TNF-α (Thermo Scientific) was added to induce a proinflammatory response (22).

Role of human Plg in the interaction process of B. animalis subsp. lactis BI07 and HT29 cells.To assess whether the HT29 cell endogenous Plg contributed to the early interaction between B. animalis subsp. lactis BI07 and HT29 cells, the impact of the enterocyte pretreatment with polyclonal goat anti-human Plg IgG (Kordia) (5 μg/ml) on B. animalis subsp. lactis BI07 adhesion was determined. B. animalis subsp. lactis BI07 adhesion to HT29 monolayers was measured by quantitative PCR (qPCR) as reported by Candela et al. (9). For each experimental condition, six independent replica experiments were performed. According to our data, the anti-human Plg pretreatment resulted in a slight, but not significant (P > 0.05), decrease of the bifidobacterial adhesion to the enterocyte surface (−16%). These data indicated that the endogenously produced Plg of HT29 cells exerted only a minimal contribution to the B. animalis subsp. lactis BI07-HT29 cell adhesion process. Consequently, in order to investigate the role of human Plg in the B. animalis subsp. lactis BI07-enterocyte interaction process, bacterial cells were preincubated with human Plg (Sigma) as reported by Attali et al. (2) and then subjected to a conventional HT29 cell adhesion assay (9). Thirty-minute preincubation at 37°C with 100 μg/ml human Plg significantly enhanced B. animalis subsp. lactis BI07 adhesion to HT29 cells (+225%; P < 0.001) (Table 1). The incubation of Plg-pretreated B. animalis subsp. lactis BI07 cells with 5 μg/ml anti-human Plg IgG resulted in the complete abolition of the Plg contribution to the bifidobacterial adhesion to the HT29 cell surface. These data demonstrated that human Plg captured on the B. animalis subsp. lactis BI07 cell surface significantly enhanced bacterial adhesion to the host enterocytes. As suggested by Pancholi et al. (24), the bacterium-bound Plg can act as a molecular bridge between bacterial and enterocyte receptors, enhancing bacterial adhesion to the host epithelium. Incubation of Plg-pretreated B. animalis subsp. lactis BI07 cells with 1 μg/ml uPA (Sigma) decreased the HT29 cell adhesion by ∼50% with respect to the value obtained in the absence of uPA (P < 0.001) (Table 1). The addition of 8 U/ml of the plasmin inhibitor aprotinin (Sigma) (2) was effective in the complete recovery of the Plg-dependent enterocyte adhesion of B. animalis subsp. lactis BI07 cells preincubated with Plg plus uPA (Table 1), proving that the acquisition of a cell surface plasmin activity in the presence of uPA was sufficient to dampen the contribution of Plg to the B. animalis subsp. lactis BI07 adhesion to the enterocyte surface.

Next, we investigated HT29 cell internalization and transmigration of untreated, pretreated with Plg or with Plg and uPA B. animalis subsp. lactis BI07 bacteria. Enterocyte internalization was analyzed by double immunofluorescence microscopy, which enables a differential staining of intracellular and adherent bacterial cells, as reported by Bergmann et al. (4). For each experimental condition, three independent adhesion experiments were carried out. Antibodies against B. animalis subsp. lactis BI07 were generated in BALB/c mice according to standard protocols. Microscopic analysis of the whole HT29 cell layer revealed no bacterial internalization by the HT29 enterocyte cell line in any of the tested conditions (Fig. 1A). For comparison and as a control, the internalization of Salmonella enterica serovar Typhimurium by HT29 cells was determined using a gentamicin protection assay (13). S. Typhimurium, provided by A. Essig (Department of Medical Microbiology, University of Ulm, Germany), was cultured as reported by Candela et al. (9). In accordance with data reported by Hess et al. (13), we determined a HT29 cell internalization value of (9.32 ± 1.58) × 10⁴ CFU of S. Typhimurium after 1 h of incubation with 1 × 10⁸ CFU of this microorganism. In order to analyze the B. animalis subsp. lactis BI07 transmigration through HT29 monolayers, a transwell system-based transmigration assay was performed as reported by Attali et al. (2). Untreated, Plg-pretreated, and Plg- and uPA-pretreated B. animalis subsp. lactis BI07 cells showed no transmigration through the HT29 monolayers (data not shown). In a control experiment, the transmigration of S. Typhimurium bacteria across HT29 monolayers was determined. In accordance with the data reported by Hess et al. (13), (7.84 ± 1.03) × 10⁵ bacteria penetrated from the apical compartment to the basal compartment of HT29 cells after 1 h of coincubation with 2 × 10⁷ CFU. These last data proved that in B. animalis subsp. lactis BI07, the acquisition of a surface-associated plasmin activity did not result in enterocyte internalization or bacterial transmigration through enterocyte monolayers, supporting the strict commensal nature of this health-promoting species.

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Fig 1

Intracellular invasion assays of B. animalis subsp. lactis BI07 in untreated (A) and TNF-α-pretreated (B) HT29 cells. HT29 cell monolayers grown to confluence were incubated with 5 × 10⁷ CFU of B. animalis subsp. lactis BI07 untreated (BI07), pretreated with Plg (+Plg) and pretreated with Plg and uPA (+Plg+uPA). Intracellular and adherent bacteria were differentiated by double immunofluorescence microscopy. Adherent B. animalis subsp. lactis BI07 bacteria (indicated by arrows) were stained with mouse antibifidobacterial antiserum followed by a secondary Alexa Fluor 488-conjugated anti-mouse antibody (green). After HT29 cell permeabilization, internalized bacteria were stained with mouse antibifidobacterial antiserum and a secondary Alexa Fluor 568-conjugated anti-mouse antibody (red). HT29 cells are visualized in the corresponding phase-contrast images shown in the lower panel (magnification, ×100). For each experimental condition, the assay was repeated three times. Results of a representative experiment are shown.

Even if the modality of interaction between B. animalis subsp. lactis and the components of the host Plg system resembles the one described for several pathogens (16, 25, 29, 30, 32, 36), our data demonstrated that the consequences of this interaction with respect to host colonization are different. In fact, for both B. animalis subsp. lactis and pathogenic bacteria, cell-bound Plg enhances bacterial adhesion to the host epithelium (2, 24), but in the presence of host PAs, the phenotype of interaction with the host becomes totally different. Different from the case for pathogens, in B. animalis subsp. lactis, the acquisition of surface-associated plasmin activity does not result in pericellular invasion but rather supports bacterial migration to the luminal compartment. Thus, common to symbionts and pathogens (1, 15), the bacterial capacity to intervene with the host Plg system cannot be viewed as a determinant of pathogenicity by itself. Instead, it could be regarded as an ancestral mechanism of bacterium-host interaction evolved prior to the radiation of pathogens from commensals (6, 21). Acting in concert with other virulence attributes (2, 21), the capacity to intervene with the host Plg system appears to be a fundamental prerequisite for host invasion only for pathogens (37).

Impact of TNF-α on the Plg-mediated early interaction between B. animalis subsp. lactis BI07 and HT29 cells.To mimic an inflammatory status, HT29 cell layers were preincubated with the proinflammatory cytokine TNF-α, and their early interaction with Plg- or Plg- and uPA-pretreated and untreated B. animalis subsp. lactis BI07 cells was investigated by qPCR and immunomicroscopic analysis as reported above. Interestingly, we detected only a minor increase of B. animalis subsp. lactis BI07 adhesion in TNF-α-stimulated HT29 monolayers after Plg pretreatment (Table 1). Moreover, after the TNF-α preincubation of HT29 layers, Plg-pretreated and Plg- and uPA-pretreated B. animalis subsp. lactis BI07 cells exhibited comparable adhesion values (Table 1). The addition of aprotinin effectively restored the Plg-dependent B. animalis subsp. lactis BI07 adhesion to TNF-α-stimulated HT29 layers for both Plg- and Plg- and uPA-pretreated B. animalis subsp. lactis BI07 cells (Table 1). Analogous to the data obtained with noninflamed HT29 cells, no bacterial internalization (Fig. 1B) or transmigration (data not shown) was detected when TNF-α-stimulated HT29 cells were incubated with Plg-pretreated, Plg- and uPA-pretreated, and untreated B. animalis subsp. lactis BI07 cells. Taken together, these data indicated that, by enhancing the enterocyte capacity to activate Plg, a TNF-α-mediated inflammatory response can modulate the dynamics of the Plg-mediated early interaction between B. animalis subsp. lactis and human enterocytes, lowering bacterial adhesion to the enterocyte surface and shifting migration to the luminal compartment. Confirming this hypothesis, in a conventional plasmin activity assay (2, 7), we demonstrated that the TNF-α pretreatment increased by 2-fold the HT29 cell capacity to activate Plg (P < 0.001).

Our data suggest that B. animalis subsp. lactis BI07 shows a different phenotype of interaction with the host Plg system depending on the inflammatory status of the host GIT epithelium. While Plg enhances B. animalis subsp. lactis adhesion in a noninflamed GIT epithelium, inflammation would result in the activation of bacterium-bound Plg and mediate the migration of the microorganisms to the luminal compartment. We hypothesize that this particular dynamic of interaction between B. animalis subsp. lactis and the components of the host Plg system could be of some relevance in the bifidobacterial microecology in the human GIT. In noninflamed regions of the human GIT, which represent the suitable ecological niche for B. animalis subsp. lactis, the microorganism can take advantage of the host Plg for the adhesion to the mucosal surface. On the other hand, in inflamed tissue sites, B. animalis subsp. lactis would acquire a surface-associated plasmin activity that, facilitating the bacterial migration to the luminal compartment, could function as a bacterial escape mechanism to circumvent the host inflammatory response. Unable to face inflammation (34), B. animalis subsp. lactis may thus utilize the host Plg system to sense and escape intestinal inflammation, abandoning inflamed gastrointestinal sites in favor of noninflamed ones. Our in vitro model of interaction could explain, at least in part, the observed decrease of the relative abundance of Bifidobacterium in inflammatory bowel disease (IBD) subjects (33, 39).