Probiotic microbes induce different resonses from bovine bronchial alveolar lavage cells in vitro

Objective Probiotics are fed to improve enteric health, and they may also affect respiratory immunity through their exposure to the upper respiratory tract upon ingestion. However, their effect on the respiratory system is not known. Our aim was to determine how probiotics affect functions and markers of bronchoalveolar lung lavage cells (BAL) isolated from lungs of calves at slaughter. Results Treatments consisted of ten probiotic species and one control treatment. Probiotics and BAL were incubated 1:1 for 2 h at 37° C and 5% CO2. The cell surface markers measured included CD14, CD205, and CD18, and E. coli bioparticles were used to measure phagocytosis and oxidative burst. Differences were considered significant at P ≤ 0.05 and were noted for percent cells fluorescing and mean fluorescence intensity for CD14 and CD205. Additionally, oxidative burst was different as measured by both percentage of cells fluorescing and mean fluorescence intensity, and phagocytosis differed among species as measured by mean fluorescence intensity. Overall, probiotic species differed in their ability to suppress or increase leukocyte function showing that probiotic bacteria differentially modulate BAL. Cell counts of C. a L. L. In contrast, our data show only suppressed fluorescence of phagocytic activity by Bifidobacterium animalis (BB-12) and Bacillus subtillus (EB-15) compared to controls, and no differences were evident in the number of cells that were phagocytizing. These microbes also decreased the percentage of CD14 expressing cells, demonstrating the importance of the CD14 molecule in phagocytosis of the E. coli bioparticles. We did see enhanced number of cells with oxidative burst by Propionibacterium freudenreichii (PF-24) compared to controls and to 4 other probiotic microbes. Oxidative burst fluorescence was not different from controls for any treatment, but differences among the treatments that had enhanced fluorescence E. faecium , CH-212) and on with suppressed were evident. There are differences in the approaches used in these 2 studies. their study vivo mice. Our work contrast used harvested BAL tested their responses ex vivo . CD14 changes in PF-24 were also reflected by enhanced number cells burst, similarly the CD14 M-74 appears that many

coli bioparticles were used to measure phagocytosis and oxidative burst. Differences were considered significant at P ≤ 0.05 and were noted for percent cells fluorescing and mean fluorescence intensity for CD14 and CD205. Additionally, oxidative burst was different as measured by both percentage of cells fluorescing and mean fluorescence intensity, and phagocytosis differed among species as measured by mean fluorescence intensity.
Overall, probiotic species differed in their ability to suppress or increase leukocyte function showing that probiotic bacteria differentially modulate BAL.

Background
Probiotics have been investigated for many health benefits, however little research has investigated their effects on the respiratory immune cells (1). Lungs were previously believed to be a sterile environment, but that has recently been revealed to not be the case in the upper respiratory tract (2). Others have shown a communication between the gut and respiratory immune mechanisms (3,4,5). Because most applications of probiotics are by an oral route, we hypothesized that some of the probiotic may be having an impact on the respiratory system as well as the intended enteric system (5,6). As an initial step, our aim was to determine how bronchial alveolar lavage cells (BAL) responded 3 to 8 potential probiotic microbes and two synbiotics designed for livestock.

Methods
Lungs were recovered from 5 beef calves after slaughter and placed on ice for transport to the laboratory approximately 10 min away. The lungs were lavaged with 100 mL of warm (37º C) HBSS (Gibco, ThermoFischer). A minimum of 50 mL of lavage fluid was obtained with 2 washes. The lavage fluid was filtered over sterile gauze into a 50 mL sterile tube.
The tubes were centrifuged at 1800 x g for 15 minutes at 4°C. Supernatants were discarded, and BAL were resuspended in 10 mL of cold sterile HBSS, then centrifuged a second time. Supernatants were discarded, and cells were resuspended in RPMI + glutamine at 10 6 cells/mL. The synbiotics (probiotics plus prebiotics) that were used for stimulation were US (a 3-strain lactobacillus probiotic, USDA-ARS), and Probios® synbiotic

Results
Cell counts determined that BAL were > 60% macrophages. Preliminary data were used to determine that a 1:1 ratio of the probiotic to leukocyte was appropriate for a good response. Differences were declared at P ≤ 0.05. Mean fluorescence of phagocytosis of E.
coli bioparticles was less for BB-12 and EB-15 than CNT, but percentage (%) of cells was reflected in reduced oxidative burst. It appears that there are many facets of the BAL interaction with various probiotic microbes that show the variation in whether their interaction will be favorable. BB-12 benefits for upper respiratory infections in humans were dependent on timing (17). Method of delivery and duration of supplementation have been cited as reasons for difference in the effectiveness of probiotic supplements on upper respiratory symptoms, some showed benefit in rate while others showed a reduction in duration or severity but not on incidence.
8 Because we used a static system, in vitro, we would not expect large shifts in cell population percentages such as in our phagocytosis data where little change was evident in the percentage of cells, but the mean expression showed some substantial differences.
It is possible that effects in vivo may be more dramatic because of the increased chance to affect the cell population development.
Other benefits attributed to probiotics are increased expression of mucin genes and mucin secretion in intestines (18), and antimicrobial peptide producing cells, whether that is true for respiratory mucosal surfaces is not known. Additionally, many probiotics have mechanical actions that are antagonistic to pathogens (19).

Declarations
Ethics approval: no humans were used in these studies. Because the lungs were collected at the abbatoir, no animal use approval was required.

Consent for publication: not applicable.
Availability of data and materials: All data generated or analysed during this study are included in this published article.

Competing interests: CCM and SDE have no competing interests, KAB is employed by
Chr. Hansen, Inc. which provided the probiotics used in this study.