Lipoteichoic acid of Streptococcus gordonii as a negative regulator of human dendritic cell activation.

Streptococcus gordonii, an opportunistic Gram-positive bacterium, causes an infective endocarditis that could be fatal to human health. Dendritic cells (DCs) are known to be involved in disease progression and immune responses in S. gordonii infection. Since lipoteichoic acid (LTA) is a representative virulence factor of S. gordonii, we here investigated its role in the activation of human DCs stimulated with LTA-deficient (ΔltaS) S. gordonii or S. gordonii LTA. DCs were differentiated from human blood-derived monocytes in the presence of GM-CSF and IL-4 for 6 days. DCs treated with heat-killed ΔltaS S. gordonii (ΔltaS HKSG) showed relatively higher binding and phagocytic activities than those treated with heat-killed wild-type S. gordonii (wild-type HKSG). Furthermore, ΔltaS HKSG was superior to wild-type HKSG in inducing phenotypic maturation markers including CD80, CD83, CD86, PD-L1, and PD-L2, antigen-presenting molecule MHC class II, and proinflammatory cytokines such as TNF-α and IL-6. Concomitantly, DCs treated with the ΔltaS HKSG induced better T cell activities, including proliferation and activation marker (CD25) expression, than those treated with the wild-type. LTA, but not lipoproteins, isolated from S. gordonii weakly activated TLR2 and barely affected the expression of phenotypic maturation markers or cytokines in DCs. Collectively, these results demonstrated that LTA is not a major immuno-stimulating agent of S. gordonii but rather it interferes with bacteria-induced DC maturation, suggesting its potential role in immune evasion.

Gamma-irradiation-killed Streptococcus pneumoniae potently induces the expression of IL-6 and IL-8 in human bronchial epithelial cells.

Streptococcus pneumoniae is a major respiratory pathogen that can cause pneumonia, meningitis, and otitis media. Although capsular polysaccharide-based vaccines are commercially available, there is a need for broad-spectrum, serotype-independent, and cost-effective vaccines. Recently, an intranasal vaccine formulated with gamma-irradiated nonencapsulated S. pneumoniae whole cells has been developed and its immunogenicity is under investigation. Since innate immunity influences the subsequent adaptive immunity, in the present study, we investigated the immunostimulatory activity of gamma-irradiated S. pneumoniae (r-SP) in the human bronchial epithelial cell-line, BEAS-2B, by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP). r-SP potently induced interleukin (IL)-6 and IL-8 at both mRNA and protein levels in a dose- and time-dependent manner, whereas h-SP and f-SP poorly induced them. Of note, the mRNA levels of IL-6 and IL-8 were approximately two-fold higher when cells were stimulated with 3 × 107 CFU/ml of r-SP for 3 h, while the protein levels of IL-6 and IL-8 were approximately five-fold higher after stimulation with 3 × 107 CFU/ml of r-SP for 24 h. Furthermore, r-SP exhibited potent activation of Toll-like receptor 2 compared with h-SP or f-SP. The expression of IL-6 and IL-8 induced by r-SP was mediated through the activation of mitogen-activated protein kinases. Remarkably, when r-SP was further treated with heat or formalin, there was a decrease in the aforementioned activities. Taken together, we suggest that r-SP stimulates the human respiratory epithelial cells to produce the cytokines IL-6 and IL-8, which might influence the induction of adaptive immune responses.

Gamma-irradiation of Streptococcus pneumoniae for the use as an immunogenic whole cell vaccine.

Streptococcus pneumoniae is a major respiratory pathogen that causes millions of deaths worldwide. Although subunit vaccines formulated with the capsular polysaccharides or their protein conjugates are currently-available, low-cost vaccines with wide serotype coverage still remain to be developed, especially for developing countries. Recently, gamma- irradiation has been considered as an effective inactivation method to prepare S. pneumoniae vaccine candidate. In this study, we investigated the immunogenicity and protective immunity of gamma-irradiated S. pneumoniae (r-SP), by comparing with heat-inactivated S. pneumoniae (h-SP) and formalin-inactivated S. pneumoniae (f-SP), both of which were made by traditional inactivation methods. Intranasal immunization of C57BL/6 mice with r-SP in combination with cholera toxin as an adjuvant enhanced S. pneumoniaespecific antibodies on the airway mucosal surface and in sera more potently than that with h-SP or f-SP under the same conditions. In addition, sera from mice immunized with r-SP potently induced opsonophagocytic killing activity more effectively than those of h-SP or f-SP, implying that r-SP could induce protective antibodies. Above all, immunization with r-SP effectively protected mice against S. pneumoniae infection. Collectively, these results suggest that gamma- irradiation is an effective method for the development of a killed whole cell pneumococcal vaccine that elicits robust mucosal and systemic immune responses.

Staphylococcal LTA antagonizes the B cell-mitogenic potential of LPS.

Lipoteichoic acid (LTA) of Gram-positive bacteria is regarded as the counterpart biomolecule of lipopolysaccharide (LPS) of Gram-negative bacteria because of their structural and immunological similarities. Although LPS induces a strong polyclonal expansion of B cells, little is known about the effect of LTA on B cell proliferation. In the present study, we prepared LTAs from Gram-positive bacteria and examined their effect on splenic B cell proliferation. Unlike LPS, LTA did not induce B cell proliferation. Instead, Staphylococcus aureus LTA (Sa.LTA) appeared to inhibit LPS-induced B cell proliferation in vitro, ex vivo, and in vivo models. Such effect was observed neither in splenocytes from Toll-like receptor 2 (TLR2)-deficient mice nor in the purified splenic B cells. Furthermore, decreased ERK phosphorylation appeared to be responsible for this phenomenon. Collectively, our results support that Sa.LTA inhibited LPS-induced B cell proliferation through the decrease of ERK phosphorylation via TLR2 signaling pathway.

Serine-Rich Repeat Adhesins Contribute to Streptococcus gordonii-Induced Maturation of Human Dendritic Cells.

Dendritic cells (DCs) play a pivotal role in the induction of immunity by recognition, capture, process, and presentation of antigens from infectious microbes. Streptococcus gordonii is able to cause life-threatening systemic diseases such as infective endocarditis. Serine-rich repeat (SRR) glycoproteins of S. gordonii are sialic acid-binding adhesins mediating the bacterial adherence to the host and the development of infective endocarditis. Thus, the SRR adhesins are potentially involved in the bacterial adherence to DCs and the maturation and activation of DCs required for the induction of immunity to S. gordonii. Here, we investigated the phenotypic and functional changes of human monocyte-derived DCs treated with wild-type S. gordonii or the SRR adhesin-deficient mutant. The mutant poorly bound to DCs and only weakly increased the expression of CD83, CD86, MHC class II, and PD-L1 on DCs compared with the wild-type. In addition, the mutant induced lower levels of TNF-α, IL-6, and IL-12 than the wild-type in DCs. When DCs sensitized with the mutant were co-cultured with autologous T cells, they induced weaker proliferation and activation of T cells than DCs stimulated with the wild-type. Blockade of SRR adhesin with 3'-sialyllactose markedly reduced S. gordonii binding and internalization, causing attenuation of the bacterial immunostimulatory potency in DC maturation. Collectively, our results suggest that SRR adhesins of S. gordonii are important for maturation and activation of DCs.

Wall teichoic acid is an essential component of Staphylococcus aureus for the induction of human dendritic cell maturation.

Staphylococcus aureus is a Gram-positive pathogen that can cause chronic skin inflammation, pneumonia, and septic shock. The immunomodulatory functions of wall teichoic acid (WTA), a glycopolymer abundantly expressed on the Gram-positive bacterial cell wall, are poorly understood. Here, we investigated the role of WTA in the phenotypic and functional activation of human monocyte-derived dendritic cells (DCs) treated with ethanol-killed S. aureus. WTA-deficient S. aureus mutant (ΔtagO) exhibited attenuated binding and internalization to DCs compared to the wild-type. ΔtagO induced lower expression of maturation markers on and cytokines in DCs than the wild-type S. aureus. Furthermore, autologous human peripheral blood mononuclear cells cocultured with ΔtagO-treated DCs exhibited a marked reduction in T cell proliferative activity, the expression of activation markers, and the production of cytokines compared to the wild-type S. aureus-stimulated DCs. Collectively, these results suggest that WTA is an important cell wall component of S. aureus for the induction of DC maturation and activation.