Although there is increasing literature on probiotics and the intestinal microbiome, there are few studies on the effects of probiotics on respiratory immunity in cattle. Lima et al. (7) showed the changes in healthy and diseased calves’ upper respiratory tract microbiome from 3 days of age to 35 days of age. Bosch et al. (8) suggested that imbalances of the upper respiratory tract microbiome may lead to invasion by and overgrowth by pathogenic bacteria. Homan et al. (1) determined that the microbiome of cattle on the day of arrival into a feedlot and after 60 days were significantly different. Corbett et al. (9) noted that feeding probiotics did not reduce respiratory susceptibility in cattle. However, Adjei-Fremeh et al. (6) reported that feeding of probiotics induced global gene expression upregulation of genes associated with both innate and adaptive immunity. Cytokines, chemokines, TLRs, and stress-related signaling molecules that are related to the inflammatory response and to the maintenance of homeostasis were predominant. The cattle used in this study had undergone transport stress and movement into the abattoir prior to our samples, thus this could have resulted in cortisol release altering immune profiles and functions.
In this in vitro work we sought to determine whether probiotic microbes could stimulate the immune functions of leukocytes obtained by lung lavage by changes in cell surface markers. We measured CD14 as part of the LPS recognition molecule, CD18 as a marker of cell activation and adhesion, CD205 to determine the role of dendritic cells, and phagocytosis of E. coli bioparticles and the associated oxidative burst to determine phagocytic function. Most literature reports on Lactobacillus strains in disease prevention of pneumococcal infections (3) and Lactobacillus have been used to determine some of the mechanisms that reduce susceptibility in vitro (10). However, Bacillus subtilis delivered intranasally increased TLR expression in tonsils of pigs (11). Monocyte derived DCs were not affected in numbers or maturation by the soluble mediators of Lactobacillus rhamnosus, but their capacity to modulate T cell responses was enhanced (12). Additionally, L. rhamnosus CLR 1505 modulated the TLR3-mediated immune response in the respiratory tract of mice (13). Lehtoranta et al. (14) reviewed some common probiotics’ effectiveness in humans and mice. They concluded the variability in outcomes may be attributed to the strains of probiotic in use, bacterial dose provided, and additives contained within the probiotic products. TLR3 is an important component in the inflammatory response to viral infection, and with the associated pathology. Our data showed an increase in the number of cells expressing the CD205 dendritic cell marker for P. freudenreichii PF-24 compared to other probiotic microbes, but it was not statistically different than the control cells. Lactobacillus animalis LA-51, B. animalis BB-12 and B. amyloliquifaciens ZM-16, and the Probios product showed a decrease in the number of CD205-expressing cells compared to P. freudenreichii PF-24. In contrast, the mean fluorescence of CD205 was greatest for E. faecium M-74 compared to CNT, but Lactobacillus such as LA-51 and US (3 strains of Lactobacillus) both resulted in lower CD205 mean fluorescence than E. faecium M-74 and similar to CNT. In concurrence with Forsythe’s (15) observation that microbes have effects on dendritic cell phenotype and function, our data show that dendritic cells are certainly playing a role in the ability of the leukocytes to modulate immunity. The increase in the % of cells with oxidative burst corresponds with the increase in the % of cells expressing the DC marker. This would be a desirable characteristic of a probiotic affecting the respiratory tract.
The recognition of gram negative bacteria requires the expression of CD14 as part of the LPS recognition molecule which it binds only in the presense of LPS-binding protein. In the current in vitro study, differences among the probiotic microbes were evident in the % of cells expressing CD14 molecules (no differences from CNT), but E. faecium M-74 CD14 fluorescence was reduced compared to all other treatments and this corresponds to the decrease in oxidative burst due to E. faecium M-74 microbe stimulation.
Nasally delivered L. lactis NZ900 improved clearance of S. pneumoniae, possibly by a competitive exclusion mechanism (3) and by enhanced IgA and IgG in BAL fluid in mice. Marranzion et al. (16) demonstrated TNF-α concentration was not altered in BAL compared with serum and intestinal fluid, but IFN-g was increased by 2 or 3 strains of Lactobacillus compared to controls in BAL, both in ex vivo and in vitro experiments.
The oxidative burst of those 2 strains was also greater than controls (16). Cell counts of pathogenic C. albicans in lungs of infected mice showed a reduction with L. casei CRL431 and L. rhamnosus CRL1505 treatments. In contrast, our data show only suppressed fluorescence of phagocytic activity by B. animalis BB-12 and B. subtillus EB-15 compared to controls, and no differences were evident in the number of cells that were phagocytizing. These microbes also decreased the % 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 P. 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 with suppressed fluorescence (E. faecium M-74) were evident. There are numerous differences in the approaches used in these 2 studies including different species, method of probiotic delivery and duration of treatment. Marranzino et al. (16) did much of their study in vivo in mice. Our work in contrast used harvested bovine BAL and tested their responses ex vivo. CD14 changes in P. freudenreichii PF-24 were also reflected by enhanced number of cells with oxidative burst, and similarly the suppression of CD14 fluorescence by E. faecium M-74 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. Bifidobacterium animalis 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.
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 % 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).