Thymic stromal lymphopoietin (TSLP) has multifaceted immunological functions ranging from maintenance of tolerance to induction of disease. Two human transcript variants of TSLP are described: a long form (variant 1; lfTSLP) consisting of four exons and an alternative, short form (variant 2; sfTSLP) that lacks two exons compared with variant 1. SfTSLP has not been described at the protein level or functionally studied. Here, we demonstrate that the human sfTSLP is the predominant form of TSLP, constitutively expressed at the mRNA and protein level in keratinocytes of oral mucosa and skin and in salivary glands, is released in saliva, and is not regulated in the same manner as the long form. Compared with lfTSLP, sfTSLP exhibits a markedly stronger antibacterial activity. Synthetic sfTSLP did not activate signal transducer and activator of transcription 5 (STAT5) signaling in CD1c(+) dendritic cells nor interfered with STAT5 activation by lfTSLP. SfTSLP may, therefore, act as an antimicrobial peptide in the oral cavity and on the skin to create a defense barrier that aids in the control of both commensal and pathogenic microbes. The results show that the two translational products of the TSLP gene have a different expression and different biological properties, and emphasize the importance of analyzing the two TSLP isoforms separately.

BackgroundCarriage of and infection with Streptococcus pneumoniae is known to predominantly induce T helper 17 (Th17) responses in humans, but the types of Th cells showing reactivity towards commensal streptococci with low pathogenic potential, such as the oral commensals S. mitis and S. salivarius, remain uncharacterized.MethodsMemory CD4+ T helper (Th) cell subsets were isolated from healthy human blood donors according to differential expression of chemokine receptors, expanded in vitro using polyclonal stimuli and characterized for reactivity against different streptococcal strains.ResultsTh cells responding to S. mitis, S. salivarius and S. pneumoniae were predominantly in a CCR6+CXCR3+ subset and produced IFN-γ, and in a CCR6+CCR4+ subset and produced IL-17 and IL-22. Frequencies of S. pneumoniae-reactive Th cells were higher than frequencies of S. mitis- and S. salivarius-specific Th cells. S. mitis and S. pneumoniae isogenic capsule knock-out mutants and a S. mitis mutant expressing the serotype 4 capsule of S. pneumoniae showed no different Th cell responses as compared to wild type strains. S. mitis-specific Th17 cells showed cross-reactivity with S. pneumoniae.ConclusionsAs Th17 cells partly control clearance of S. pneumoniae, cross-reactive Th17 cells that may be induced by commensal bacterial species may influence the immune response, independent of capsule expression.

Streptococcus mitis (S. mitis) is a pioneer commensal bacterial species colonizing many of the surfaces of the oral cavity in healthy individuals. Yet, not much information is available regarding its interaction with the host. We used examination of its transcriptional regulation in oral keratinocytes to elucidate some of its potential roles in the oral cavity. Transcription factor analysis of oral keratinocytes predicted S. mitis-mediated activation of aryl hydrocarbon receptor (AhR). Activation and functionality of AhR was confirmed through nuclear translocation determined by immunofluorescence microscopy and real-time polymerase chain reaction with reverse transcription analysis of CYP1A1, the hallmark gene for AhR activation. Addition of Streptococcus mutans or Streptococcus gordonii did not induce CYP1A1 transcription in the keratinocyte cultures. Introduction of an AhR-specific inhibitor revealed that S. mitis-mediated transcription of CXCL2 and CXCL8 was regulated by AhR. Elevated levels of prostaglandin E2 (enzyme-linked immunosorbent assay) in supernatants from S. mitis-treated oral epithelial cells were also attenuated by inhibition of AhR activity. The observed AhR-regulated activities point to a contribution of S. mitis in the regulation of inflammatory responses and thereby to wound healing in the oral cavity. The concept that the oral commensal microbiota can induce AhR activation is important, also in view of the role that AhR has in modulation of T-cell differentiation and as an anti-inflammatory factor in macrophages.

Streptococcus mitis colonizes all niches of the human oral cavity from early infancy and throughout life. Monocytes patrol blood vessels, lymphoid and non-lymphoid tissues and migrate into infected tissue where they participate in the inflammatory cascade and immune regulation. Here, we studied the effect of S. mitis on monocytes. Transcriptome analysis of monocytes exposed to S. mitis (SmMo) revealed increased transcription of chemotactic factors (CCL2, CCL3, CCL20, CXCL1, CXCL2) and cytokines (IL1A, IL1B, IL6, IL23, IL36G, TNF), indicating that S. mitis may trigger recruitment of leucocytes and initiate inflammation. Increased transcription in SmMo of IL1B, IL6 and IL23 indicated that S. mitis may participate in the induction of Th17 responses and agreed with our earlier findings of S. mitis-mediated memory Th17 reactivity. Furthermore, S. mitis inhibited tetanus toxoid-specific CD4 T cell proliferation. This can be due to the increased secretion of IL-10 and expression of PD-L1 that was observed in SmMo. PGE2 can modulate IL-10 and PD-L1 expression, concomitant with that of CCR7, IL-12 and IL-23 that also were changed. This, along with increased SmMo transcription of PTGS2 (COX2) and PTGER4 (EP4), pointed to a role of PGE2. Measurement of PGE2 secretion by SmMo showed indeed a marked increase, and chemical inhibition of PGE2 production lowered the PD-L1 expression on SmMo. In conclusion, our findings show that S. mitis may trigger immune modulation by recruiting immune cells to the site of infection, while at the same time dampening the severity of the response through expression of IL-10, PGE2 and PD-L1.

Streptococcus mitis is a colonizer of the oral cavity and the nasopharynx, and is closely related to Streptococcus pneumoniae. Both species occur in encapsulated and unencapsulated forms, but in S. mitis the role of the capsule in host interactions is mostly unknown. Therefore, the aim of this study was to examine how capsule expression in S. mitis can modulate interactions with the host with relevance for colonization. The S. mitis type strain, as well as two mutants of the type strain, an isogenic capsule deletion mutant, and a capsule switch mutant expressing the serotype 4 capsule of S. pneumoniae TIGR4, were used. Wild-type and capsule deletion strains of S. pneumoniae TIGR4 were included for comparison. We found that capsule production in S. mitis reduced adhesion to oral and lung epithelial cells. Further, exposure of oral epithelial cells to encapsulated S. mitis resulted in higher interleukin-6 and CXCL-8 transcription levels relative to the unencapsulated mutant. Capsule expression in S. mitis increased the sensitivity to human neutrophil peptide 1-3 but reduced the sensitivity to human β-defensin-3 and cathelicidin. This was in contrast with S. pneumoniae in which capsule expression has been generally associated with increased sensitivity to human antimicrobial peptides (AMPs). Collectively, these findings indicate that capsule expression in S. mitis is important in modulating interactions with epithelial cells, and is associated with increased or reduced susceptibility to AMPs depending on the nature of the AMP.