Corynebacterium pseudodiphtheriticum 090104 was cultured in trypticase soy broth and harvested by centrifugation at 3,000 × g for 10 min, washed three times with sterile 0.01 M phosphate buffer saline (PBS, pH 7.2), and resuspended in sterile PBS. Non-viable C. pseudodiphtheriticum 090104 was obtained as described previously (Tomosada et al., 2013). Briefly, bacteria were killed by tyndallization in a water bath at 80°C for 30 min and the lack of bacterial growth was confirmed by plating on to soy broth agar plates.

In order to evaluate the susceptibility to antimicrobials of C. pseudodiphtheriticum 090104, the broth microdilution method was followed as recommended by the CLSI for infrequently isolated or fastidious bacteria (M45-A, CLSI-2006). The antimicrobial agents were penicillin (PEN), cefotaxime (CTX), ceftriaxone (CRO), meropenem (MER), vancomycin (VAN), gentamicin (GEN), ciprofloxacin (CIP), erythromicin (ERY), tetracycline (TET). MIC results were interpreted following CLSI guidelines (M45-A, CLSI-2006). Evaluation of bacterial translocation in infant mice was monitored by determining the presence of C. pseudodiphtheriticum lung, blood and spleen samples.

Female 3-week-old BALB/c mice were obtained from the closed colony kept at CERELA (San Miguel de Tucumán, Argentina) or Tohoku University (Sendai, Japan). They were housed in plastic cages at room temperature. Mice were housed individually during the experiments. Viable and non-viable C. pseudodiphtheriticum 090104 were nasally administered to mice for 5 consecutive days at a dose of 10⁸ cells/mouse/day in 50 μl of PBS. All groups were fed a conventional balanced diet ad libitum. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Guidelines for Animal Experimentation of CERELA and all efforts were made to minimize suffering. The Institutional Animal Welfare Committee of CERELA reviewed and approved the protocols used in this study.

Administration of the viral pathogen molecular pattern poly(I:C) was performed on day 6, after a 5 days treatment with viable or non-viable C. pseudodiphtheriticum 090104. Mice were lightly anesthetized and 100 μl of PBS, containing 250 μg poly(I:C) (equivalent to 10 mg/kg body weight), was administered dropwise, via the nares (Tomosada et al., 2013). Control animals received 100 μl of PBS. Mice received three doses of poly(I:C) or PBS with 24 h rest period between each administration.

Human RSV strain A2 was grown in Vero cells as described by Murawski et al. (2009). Briefly, Vero cells were infected with RSV at a multiplicity of infection (MOI) of 1 in 5 ml of Dulbecco's modified Eagle's medium (DMEM). Cells were infected for 2.5 h at 37°C and 5% CO₂. After infection, 7 ml of DMEM with 10% FBS (Sigma, Tokyo, Japan), 0.1% penicillin–streptomycin (Pen/Strep) (Sigma, Tokyo, Japan), and 0.001% ciprofloxacin (Bayer) were added to the flask and further incubated. When extensive syncytia formed, cells were scraped from the flask and sonicated three times, 5 s per pulse, at 25 W on ice. Cell lysates were centrifuge at 700 × g for 10 min at 4°C. Cell-free virus stocks were stored in 30% sucrose at −80°C. Uninfected cells were treated identically to generate a virus- and cell-free supernatant control. Mice were slightly anesthetized and intranasally challenged with 2.4 × 10⁶ PFU RSV strain A2 or an equivalent volume of Vero cell lysate on day 6 after viable or non-viable C. pseudodiphtheriticum 090104 treatment.

Intact lung tissue was removed and stored in 30% sucrose for plaque assays. RSV immune-plaque assay was done as previously explained (Chiba et al., 2013). Briefly, lungs were homogenized using a pellet pestle and centrifuged at 2,600 × g for 10 min at 4°C. Twenty-four-well tissue culture plates were seeded with 1.5 × 10⁵ Vero cells/well in DMEM (10% FBS, 0.1% Pen/Strep, and 0.001% ciprofloxacin) and virus plaques assays were performed in triplicate. Plates were incubated at 37°C and 5% CO₂ for 2.5 h. After incubation, supernatant was removed, and 1 ml of fresh DMEM medium supplemented with 10% FBS, 0.1% Pen/Strep, and 0.001% ciprofloxacin was overlaid on monolayers. When extensive syncytia developed, the overlay was removed and monolayers were fixed with ice-cold acetone:methanol (60:40). Primary RSV anti-F (clones131-2A; Chemicon) and anti-G (Mouse monoclonal [8C5 (9B6)] to RSV glycoprotein, Abcam) antibodies were added to wells for 2 h, followed by secondary horseradish peroxidase anti-mouse immunoglobulin antibody (Anti-mouse IgG, HRP-linked Antibody #7076, Cell signaling Technology) for 1 h. Plates washed twice with PBS containing 0.5% Tween 20 (Sigma) after each antibody incubation step. Individual plaques were developed using a DAB substrate kit (ab64238, Abcam) following manufacture's instructions. Results for immune-plaque assay were expressed as log₁₀ PFU/g of lung.

Streptococcus pneumoniae serotype 6B (ANLIS, Argentina) was obtained from the respiratory tract of a patient from Hospital del Niño Jesús, Tucumán, Argentina. Pneumococci were grown on blood agar for 18 h. Colonies were suspended in Todd Hewitt broth (Oxoid), incubated overnight at 37°C, harvested and washed with sterile PBS. Cell density was adjusted to 4 × 10⁷ CFU/ml. Challenge with pneumococci was performed 5 days after the last administration of poly(I:C) or RSV challenge. Viable or non-viable C. pseudodiphtheriticum-treated as well as control infant mice were challenged intranasally with the pathogen by dripping 25 μl of inoculums containing 10³ CFU (log phase) in PBS into each nostril.

Treated and control mice were sacrificed 2 days after S. pneumoniae infection. Lungs were excised, weighed and homogenized in sterile peptone water. Homogenates were diluted appropriately, plated in duplicate on blood agar and incubated for 18 h at 37°C. Streptococcus pneumoniae was identified by standard techniques and the results were expressed as log of CFU/g of lung. Bacteremia was monitored in blood samples obtained by cardiac puncture which were plated on blood agar. Results were reported as negative or positive hemocultures.

BAL samples were obtained as described previously (Villena et al., 2005) by performing lavages of lungs with sterile PBS. After centrifugation, cell-free supernatants were kept at −70°C. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, IFN-β, IFN-α, interleukin (IL)-6, and IL-10 concentrations in serum and BAL were measured by enzyme-linked immunosorbent assay (ELISA) following the manufacturer's recommendations (R&D Systems, MN, USA) (Salva et al., 2011).

Single lung cells were prepared using the previously described method (Villena et al., 2012). Briefly, mice were anesthetized and lungs were removed, finely minced and incubated for 90 min with 300 U of collagenase (Yakult Honsha Co., Tokyo, Japan) in 15 ml of RPMI 1640 medium (Sigma, Tokyo, Japan). After removal of debris, erythrocytes were depleted by hypotonic lysis. The cells were washed with RPMI medium supplemented with 0.1% Pen/Strep and suspended in a medium supplemented with 10% heat-inactivated fetal calf serum (FCS). Cells were counted using Trypan Blue and adjusted to 5 × 10⁶ cells/ml.

Single lung cells from mice were prepared as previously described (Villena et al., 2012; Zelaya et al., 2014, 2015). Lungs were removed, finely minced and incubated for 90 min with 300 U of collagenase (Yakult Honsha Co., Tokyo, Japan) in 15 ml of RPMI 1640 medium (Sigma, Tokyo, Japan). To dissociate the tissue into single cells, collagenase-treated minced lungs were gently tapped into a plastic dish. After removal of debris, erythrocytes were depleted by hypotonic lysis. The cells were washed with RPMI medium supplemented with 100 U/ml of penicillin and 100 mg/ml of streptomycin and then resuspended in a medium supplemented with 10% heat-inactivated fetal calf serum (FCS). Cells were counted using Trypan Blue exclusion and then resuspended at 5 × 10⁶ cells/ml.

Lung cell suspensions were pre-incubated with anti-mouse CD32/CD16 monoclonal antibody (Fc block) for 15 min at 4°C. Cells were incubated in the antibody mixes for 30 min at 4°C and washed with FACS buffer. Then, cells were stained with fluorochrome-conjugated antibodies against CD3, CD4, CD8, CD11c, CD11b, CD103, MHC-II, IFN-γ, IL-10, sialic acid-binding immunoglobulin-like lectin F (SiglecF) (BD Bioscience), IFN-β, and CD45 (eBioscience). Cells were then acquired on a BD FACSCalibur™ flow cytometer (BD Biosciences) and data were analyzed with FlowJo software (TreeStar). The total number of cells in each population was determined by multiplying the percentages of subsets within a series of marker negative or positive gates by the total cell number determined for each tissue (Villena et al., 2012; Zelaya et al., 2014, 2015).

To measure increased permeability of the bronchoalveolar–capillarity barrier, we quantified albumin and protein content in cell-free BAL. Furthermore, lactate dehydrogenase (LDH) activity was quantified as an indicator of general cytotoxicity (Villena et al., 2012; Zelaya et al., 2014, 2015). Lung wet:dry weight ratio was determined as described before. Briefly, mice were euthanized and exsanguinated; lungs were removed, weighed (wet weight), dried at 55°C for 7 days, and weighed again (dry weight). The wet:dry weight ratio is a measure of intrapulmonary fluid accumulation. Histopathological examination was also performed in order to evaluate tissue damage during respiratory superinfection. Lungs were aseptically removed, fixed in 4% formalin and embedded in histowax (Leica Microsystems). Histopathological assessment was performed on five-micron tissue sections stained with hematoxylin-eosin.

Experiments were performed in triplicate and results were expressed as mean ± standard deviation (SD). Five to six animals were used in each replicate per experimental group. Normal distributed data were tested by 2-way ANOVA was used. Tukey's test (for pairwise comparisons of the means) or the Fisher's least significant difference (LSD) test (for multi-comparison) were used to evaluate the differences between the groups. Differences were considered significant at p < 0.05.

In order to determine whether viable or non-viable C. pseudodiphtheriticum could modulate mucosal immune responses in the respiratory tract, we quantified key cytokines in broncho-alveolar lavages (BAL) and serum samples as well as immune cell populations in lungs. Both treatments elicited an increase in the levels of the proinflammatory cytokines TNF-α and IL-6 in serum and BAL (Figure 1). Furthermore, C. pseudodiphtheriticum induced a significant increase in the production of IFN-γ and a slight but still significant enhancement of IFN-β, while no effect was observed in IFN-α levels. The immunoregulatory cytokine IL-10 was also detected at higher concentrations in viable or non-viable C. pseudodiphtheriticum-treated infant mice than in control mice without bacterial stimulation (Figure 1). In general, the effect of the live bacterium was stronger than the elicited by non-viable C. pseudodiphtheriticum. In addition, the changes in cytokine levels were more pronounced in BAL (Figure 1A) than in serum samples (Figure 1B).

Flow cytometry indicated that there were no significant differences between the experimental groups when the numbers of lung antigen presenting cells including dendritic cells (CD11c⁺CD103-CD11b^high and CD11c⁺CD103⁺CD11b^low cells) and alveolar macrophages (CD45⁺CD11c⁺SiglecF⁺ cells) were compared (Figure 2). However, activated lung antigen presenting cells differed in mice nasally treated with C. pseudodiphtheriticum (Figure 2). For instance, CD11c⁺CD103⁺MHCII⁺ and CD11c⁺CD11b^highMHCII⁺ populations were significantly greater (p < 0.05) in viable or non-viable C. pseudodiphtheriticum-treated infant mice than in control mice. In both cases, live bacteria had a stronger influence than non-viable C. pseudodiphtheriticum in antigen presenting cells activation. No differences were observed between the groups when total counts of lung CD3⁺CD4⁺ and CD3⁺CD8⁺ T cells were compared (Figure 3). Nevertheless, C. pseudodiphtheriticum differentially modulated specific IFN-γ producing populations of CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes. Viable bacteria significantly induced higher numbers of IFN-γ specific CD3⁺CD4⁺ T cells, whereas heat-killed C. pseudodiphtheriticum increased IFN-γ specific CD3⁺CD8⁺ cells (Figure 3). A slight but not significant increase of lung CD3⁺CD4⁺IL-10⁺ T cells was observed in C. pseudodiphtheriticum-treated mice.

After treatment with viable or non-viable C. pseudodiphtheriticum, mice were nasally challenged for three consecutive days with poly(I:C). In order to evaluate the extent of lung injury induced by TLR3-triggered inflammation (Figure 4), we determined total protein and albumin concentrations, and LDH activity in BAL 2 days after challenge. In addition, we also determined the magnitude of edema by calculating the lung wet:dry ratio. Administration of poly(I:C) resulted in water retention due to the inflammatory response in the lungs in comparison with unchallenged mice. Viable C. pseudodiphtheriticum somehow prevented lungs from retaining fluids as it is shown in Figure 4 reaching levels close to the unchallenged lung wet:dry ratios. Other lung injury parameters, such as protein content, albumin and LDH activity were also significantly (p < 0.05) reduced when mice were previously treated with viable or non-viable C. pseudodiphtheriticum, being the treatment with live bacteria more effective than heat-killed bacteria.

As we have described previously (Tomosada et al., 2013), nasal challenge with poly(I:C) increased the levels of proinflammatory cytokines and IL-10 (Figure 5), and immune cells (Figure 6) in the respiratory tract when compared to basal levels. The nasal pretreatments of mice with viable or non-viable C. pseudodiphtheriticum differentially modulated the profiles of cytokines measured in BAL. IFN-γ was the most influenced cytokine, showing a significant increase in mice treated with live bacteria in comparison to untreated control mice challenged with poly(I:C), whereas non-viable bacteria slightly increased the levels of IFN-γ (Figure 5). To a lesser extent, TNF-α, IL-6, IFN-β, and IL-10 concentrations in BAL were also higher in viable C. pseudodiphtheriticum-treated mice. In contrast, only TNF-α and IL-6 were modulated by non-viable bacteria.

The numbers of lung CD11c⁺CD103⁺MHCII⁺ and CD11c⁺CD11b^highMHCII⁺ dendritic cells were significantly greater (p < 0.05) in viable or non-viable C. pseudodiphtheriticum-treated infant mice than in control mice (Figure 6). In addition, higher numbers of CD45⁺SiglecF⁺IFN-β⁺ alveolar macrophages and CD3⁺CD4⁺IL-10⁺ T cells were observed when mice were previously treated with viable C. pseudodiphtheriticum in comparison to control mice (Figure 6). In contrast, non-viable bacteria did not produce significant changes in the numbers of these immune cells populations. CD3⁺CD4⁺IFNγ⁺ lymphocyte counts increased in mice which received viable or non-viable C. pseudodiphtheriticum before poly(I:C) challenge, being this effect stronger with the viable bacteria (Figure 6).

Nasal administration of viable C. pseudodiphtheriticum improved health state of infant mice infected with RSV as reflected by the increase in body weight during the studied period (Figure 7). Mice treated with non-viable bacteria did not show the improvement of body weight. Virus load was significantly lower in mice treated with viable C. pseudodiphtheriticum when compared to those receiving heat-killed bacteria or controls (Figure 7). The markers of lung tissue damage in RSV-infected mice showed that the viral infection induced a significant cellular damage and alveolar-capillary barrier alterations (Figure 7). Both, BAL LDH and albumin concentrations were significantly lower in infant mice previously treated with viable C. pseudodiphtheriticum than in RSV-challenged controls or mice receiving heat-killed bacteria (Figure 7).

Finally, we addressed whether the nasal treatments with viable and non-viable C. pseudodiphtheriticum where able to increase the resistance of infant mice to secondary pneumococcal pneumonia. For that purpose, mice were nasally primed with viable or non-viable bacteria, infected with RSV, and 5 days after virus infection, they were challenged with S. pneumoniae. Pneumococcal colonization and bacteremia were evaluated on day 2 post-pneumococcal challenge. In addition, RSV titers as well as lung tissue damage were studied before (day 0) and after (day 2) infection with S. pneumoniae. RSV was detected in lungs of infected infant mice before and after pneumococcal infection (Figure 8). In addition, pneumococci were detected in lungs and blood of control infant mice (Figure 8). Viable C. pseudodiphtheriticum significantly reduced RSV titers as well as lung bacterial cell counts and prevented the dissemination of S. pneumoniae into the blood (Figure 8). No protective effect was observed for non-viable C. pseudodiphtheriticum. When lung injury was studied it was observed that the secondary pneumococcal pneumonia induced a significant increase of the BAL biochemical parameters that evaluate cellular damage and alveolar-capillary barrier alterations (Figure 8). Viable C. pseudodiphtheriticum significantly reduced pulmonary damage as demonstrated by the lower LDH and albumin content in BAL when compared to controls and non-viable bacteria-treated mice (Figure 8). Moreover, histological examination of lung of infected infant mice revealed severe inflammatory cell recruitment around alveoli and blood vessels, focal hemorrhage and a significant reduction of gas exchange spaces (Figure 8). Lung histology analysis of viable C. pseudodiphtheriticum-tretaed mice showed a significant reduction in the alterations of gas exchange spaces, hemorrhage and inflammatory cells infiltration (Figure 8) while mice treated with non-viable bacteria were not different from control animals (data not shown).

The authors 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.