The commensal Streptococcus salivarius K12 downregulates the innate immune responses of human epithelial cells and promotes host-microbe homeostasis

Abstract

Streptococcus salivarius is an early colonizer of human oral and nasopharyngeal epithelia, and strain K12 has reported probiotic effects. An emerging paradigm indicates that commensal bacteria downregulate immune responses through the action on NF-kappaB signaling pathways, but additional mechanisms underlying probiotic actions are not well understood. Our objective here was to identify host genes specifically targeted by K12 by comparing their responses with responses elicited by pathogens and to determine if S. salivarius modulates epithelial cell immune responses. RNA was extracted from human bronchial epithelial cells (16HBE14O- cells) cocultured with K12 or bacterial pathogens. cDNA was hybridized to a human 21K oligonucleotide-based array. Data were analyzed using ArrayPipe, InnateDB, PANTHER, and oPOSSUM. Interleukin 8 (IL-8) and growth-regulated oncogene alpha (Groalpha) secretion were determined by enzyme-linked immunosorbent assay. It was demonstrated that S. salivarius K12 specifically altered the expression of 565 host genes, particularly those involved in multiple innate defense pathways, general epithelial cell function and homeostasis, cytoskeletal remodeling, cell development and migration, and signaling pathways. It inhibited baseline IL-8 secretion and IL-8 responses to LL-37, Pseudomonas aeruginosa, and flagellin in epithelial cells and attenuated Groalpha secretion in response to flagellin. Immunosuppression was coincident with the inhibition of activation of the NF-kappaB pathway. Thus, the commensal and probiotic behaviors of S. salivarius K12 are proposed to be due to the organism (i) eliciting no proinflammatory response, (ii) stimulating an anti-inflammatory response, and (iii) modulating genes associated with adhesion to the epithelial layer and homeostasis. S. salivarius K12 might thereby ensure that it is tolerated by the host and maintained on the epithelial surface while actively protecting the host from inflammation and apoptosis induced by pathogens.

Figures

FIG. 1.

Venn diagram illustrating the overlap of differentially expressed genes across the four conditions studied.

FIG. 2.

Impact of S. salivarius on interferon signaling pathways. Underlined genes were upregulated by S. salivarius K12 alone. IFNA/B, alpha/beta interferon; IFNG, gamma interferon; IFNAR, alpha interferon receptor; IFNGR, gamma interferon receptor; ISRE, interferon stimulated response element; GAS, gamma interferon activated site; GEF, guanine nucleoside exchange factor; CRKL, CRK-like protein.

FIG. 3.

Streptococcus salivarius K12 downregulates IL-8 release from human bronchial epithelial cells (16HBE14O-) in response to LL-37, Pseudomonas aeruginosa, endotoxin, and flagellin. IL-8 release from 16HBE14O- cells was monitored after incubation with LL-37 (A and B), with P. aeruginosa (D), and with flagellin (E) in the presence (black bars) or absence (gray bars) of S. salivarius (MOI, 50). LL-37 was incubated with cells for either 24 h (A) or 48 h (B). Growth properties of S. salivarius on 16HBE14O- cells in the presence of LL-37 (25 and 40 μg/ml) was monitored (C). P. aeruginosa (MOI, 50) was incubated with cells for 6 h. S. salivarius was incubated for 2 h before flagellin (0.5 and 1 μg/ml) was added and further incubated for 5 h (7 h of total incubation). The data are the result of a minimum of three biological repeats and two technical repeats. A two-tail-distribution, unpaired Student t test was performed (**, P < 0.01; *, P < 0.05). OD600, optical density at 600 nm.

FIG. 4.

Streptococcus salivarius downregulates IL-8 secretion from primary normal human bronchial epithelial cells and primary keratinocytes. IL-8 release from pnHBE cells (A) or primary keratinocytes (B) was monitored after incubation with P. aeruginosa PAK, the PAK fliC-negative mutant, or flagellin (1 μg/ml) in the presence (black bars) or absence (gray bars) of S. salivarius K12. Conditions were as follows: S. salivarius was added to the cells at the same time as P. aeruginosa (MOI, 50:1) or flagellin, and the cells were incubated for 6 h. **, P < 0.01.

FIG. 5.

Streptococcus salivarius K12 downregulates Groα release from human bronchial epithelial (16HBE14O-) cells in response to flagellin by inhibiting the NF-κB signaling pathway. Groα release from 16HBE14O- cells was monitored after they were incubated with flagellin (1 μg/ml) and compared with the level in the control. 16HBE14O- cells were incubated with S. salivarius (MOI, 50) (black bars), with the NF-κB inhibitor Bay 11-7085 (40 μM) (hatched bars), or nothing (gray bars). Conditions were as follows. S. salivarius was added to the epithelial cells 2 h prior to the addition of flagellin (0.5 and 1 μg/ml) and incubated for 24 h (26 h of total incubation). The NF-κB inhibitor Bay 11-7085 (40 μM) was added to the epithelial cells 30 min prior to the addition of flagellin, and then the cells were incubated for 24 h. **, P < 0.01; *, P < 0.05.

FIG. 6.

S. salivarius inhibits NF-κB P65 subunit translocation into the nucleus. Western blotting of P65 NF-κB subunits was performed with nuclear extracts of 16HBE14O- cells following their incubation with 1 μg/ml flagellin or S. salivarius (MOI, 50:1) or with S. salivarius (MOI, 50:1) plus 1 μg/ml flagellin. Reaction with antibody against histone H2AX was used as a loading control. Results are representative of three independent experiments.