Dectin-2-dependent host defense in mice infected with serotype 3 Streptococcus pneumoniae.

BACKGROUND: Streptococcus pneumoniae, a major causative bacterial pathogen of community-acquired pneumonia, possesses a thick polysaccharide capsule. Host defense against this bacterium is mediated by activation of innate immune cells that sense bacterial components. Recently, C-type lectin receptors (CLRs) have garnered much attention in elucidating the recognition mechanism of pathogen-derived polysaccharides. METHODS: In the present study, we first compared the clinical course and neutrophil accumulation in the lungs of Dectin-2 knock-out (KO) and wild type (WT) mice. Mice were infected intratracheally with a serotype 3 strain of S. pneumoniae, and S. pneumoniae bacterial engulfment by neutrophils and inflammatory cytokine and anti-pneumococcal polysaccharide-specific IgG levels were evaluated in bronchoalveolar lavage fluid (BALF). We also examined the effect of Dectin-2 deficiency on interleukin (IL)-12 production by bone marrow-derived dendritic cells (BM-DCs) stimulated with the bacterial components. RESULTS: S. pneumonia-infected Dectin-2KO mice had a shorter survival time, larger bacterial burden and lower interferon gamma (IFN-γ) production in the lungs than WT mice. Although neutrophilic infiltration in the lungs was equivalent between Dectin-2KO mice and WT mice, S. pneumonia engulfment by neutrophils was attenuated in Dectin-2KO mice compared to WT mice. The anti-pneumococcal polysaccharide-specific IgG and IgG3 levels in BALF were lower in Dectin-2KO mice than in WT mice. When BM-DCs were stimulated with S. pneumoniae culture supernatant or its Concanavalin A (ConA)-bound fraction, IL-12 production was abrogated in Dectin-2KO mice compared to WT mice. CONCLUSIONS: We demonstrated that Dectin-2 is intimately involved in the host defense against infection with a serotype 3 strain of S. pneumoniae. Dectin-2-dependent IL-12 production may contribute to IFN-γ synthesis and subsequent production of serotype-specific anti-capsular polysaccharide IgG after S. pneumoniae infection, which may promote S. pneumoniae bacterial opsonization for engulfment.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation.

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Toll-like receptor 9-dependent activation of bone marrow-derived dendritic cells by URA5 DNA from Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Recently, we reported that Toll-like receptor 9 (TLR9) is involved in host defense against C. neoformans: specifically, it detects the pathogen's DNA. In the present study, we aimed to elucidate the mechanisms underlying TLR9-mediated activation of innate immune responses by using the URA5 gene, which encodes a virulent component of this fungal pathogen. A PCR-amplified 345-bp URA5 gene fragment induced interleukin-12 p40 (IL-12p40) production by bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. Similar activity was detected in the 5' 129-bp DNA fragment of URA5 and in a synthesized oligodeoxynucleotide (ODN) with the same sequence. Shorter ODN fragments, which contained GTCGGT or GACGAT but had only 24 or 21 bases, induced IL-12p40 production and CD40 expression by BM-DCs, but this activity vanished when the CG sequence was replaced by GC or when a phosphorothioate modification was introduced. IL-12p40 production caused by active ODN was strikingly enhanced by treatment with DOTAP, a cationic lipid that increases the uptake of DNA by BM-DCs, though DOTAP failed to induce IL-12p40 production by inactive ODN and did not affect the activity of an ODN-containing canonical CpG motif. There was no apparent difference in intracellular trafficking between active and inactive ODNs. Finally, an extremely high dose of inactive ODN suppressed IL-12p40 production by BM-DCs that had been stimulated with active ODN. These results suggest that the C. neoformans URA5 gene activates BM-DCs through a TLR9-mediated signaling pathway, using a mechanism possibly independent of the canonical CpG motif.

TARM1 contributes to development of arthritis by activating dendritic cells through recognition of collagens.

TARM1 is a member of the leukocyte immunoglobulin-like receptor family and stimulates macrophages and neutrophils in vitro by associating with FcRγ. However, the function of this molecule in the regulation of the immune system is unclear. Here, we show that Tarm1 expression is elevated in the joints of rheumatoid arthritis mouse models, and the development of collagen-induced arthritis (CIA) is suppressed in Tarm1-/- mice. T cell priming against type 2 collagen is suppressed in Tarm1-/- mice and antigen-presenting ability of GM-CSF-induced dendritic cells (GM-DCs) from Tarm1-/- mouse bone marrow cells is impaired. We show that type 2 collagen is a functional ligand for TARM1 on GM-DCs and promotes DC maturation. Furthermore, soluble TARM1-Fc and TARM1-Flag inhibit DC maturation and administration of TARM1-Fc blocks the progression of CIA in mice. These results indicate that TARM1 is an important stimulating factor of dendritic cell maturation and could be a good target for the treatment of autoimmune diseases.

Involvement of Gr-1 dull+ cells in the production of TNF-α and IL-17 and exacerbated systemic inflammatory response caused by lipopolysaccharide.

Systemic inflammatory response syndrome (SIRS) is a life-threatening disease. Recent reports have demonstrated that the immunoregulatory cells that express Gr-1, a granulocyte surface antigen, play a critical role in various pathological conditions. In the present study, we have established a mouse model of SIRS and addressed the possible contribution of Gr-1+ cells in this model. C57BL/6 mice were injected intraperitoneally with anti-Gr-1 mAb or control IgG 1 day before administration of lipopolysaccharide (LPS). All of the mice that received anti-Gr-1 mAb and LPS died early as a result of hypothermia and severe emaciation, whereas mice treated with control IgG and LPS survived the observation period. In mice treated with anti-Gr-1 mAb and LPS, acute inflammatory changes with alveolar hemorrhage were observed in the lung and proximal convoluted tubule necrosis was observed in the kidney. Serum TNF-α and IL-17A levels were markedly increased in anti-Gr-1 mAb-pretreated mice compared with those in control IgG-treated mice at 1 and 3 h after LPS administration, respectively. Flow cytometric analysis revealed an increase in TNF-α and IL-17A expression in Gr-1 dull+ cells in the peripheral blood mononuclear cells. Neutralization of TNF-α by a specific mAb almost completely reversed the clinical course and inhibited the increased production of IL-17A. In addition, IL-17A KO mice were less susceptible to the lethality in this model. Thus, we established a mouse model of severe SIRS and suggested that Gr-1 dull+ cells may play a critical role in the development of this pathological condition.

Dectin-2-dependent NKT cell activation and serotype-specific antibody production in mice immunized with pneumococcal polysaccharide vaccine.

Although thymus-independent type 2 antigens generally do not undergo Ig class switching from IgM to IgG, pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific IgG. How this happens remains unclear, however. In the present study, PPV immunization induced production of IgG as well as IgM specific for a serotype 3-pneumococcal polysaccharide in the sera of wild-type (WT) mice, but this phenomenon was significantly reduced in Dectin-2 knockout (KO) mice. Immunization with PPV caused IL-12p40 production in WT mice, but this response was significantly reduced in Dectin-2KO mice. Likewise, immunization with PPV activated natural killer T (NKT) cells in WT mice but not in Dectin-2KO mice. Furthermore, administration of α-galactosylceramide, recombinant (r)IL-12 or rIFN-γ improved the reduced IgG levels in Dectin-2KO mice, and treatment with neutralizing anti-IFN-γ mAb resulted in the reduction of IgG synthesis in PPV-immunized WT mice. Transfer of spleen cells from PPV-immunized WT mice conferred protection against pneumococcal infection on recipient mice, whereas this effect was cancelled when the transferred spleen cells were harvested from PPV-immunized Dectin-2KO mice. These results suggest that the detection of PPV antigens via Dectin-2 triggers IL-12 production, which induces IFN-γ synthesis by NKT cells and subsequently the production of serotype-specific IgG.

Low-dose interferon-α treatment improves survival and inflammatory responses in a mouse model of fulminant acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is accompanied by severe lung inflammation induced by various diseases. Despite the severity of symptoms, therapeutic strategies for this pathologic condition are still poorly developed. Interferon (IFN)-α is well known as an antiviral cytokine and low-dose IFN-α has been reported to show antiinflammatory effects. Therefore, we investigated how this cytokine affected ARDS in a mouse model. C57BL/6 mice received sequential intratracheal administration of α-galactosylceramide (α-GalCer) and lipopolysaccharide (LPS), which resulted in the development of fulminant ARDS. These mice were then treated intranasally with IFN-α and their survival, lung weight, pathological findings, and cytokine production were evaluated. Administration of low-dose IFN-α prolonged survival of fulminant ARDS mice, but higher doses of IFN-α did not. Histological analysis showed that low-dose IFN-α treatment improved findings of diffuse alveolar damage in fulminant ARDS mice, which was associated with reduction in the wet/dry (W/D) lung weight ratio. Furthermore, IFN-γ production in the lungs was significantly reduced in IFN-α-treated mice, compared with control mice, but tumor necrosis factor (TNF)-α production was almost equivalent for both groups. Low-dose IFN-α shows antiinflammatory and therapeutic effects in a mouse model of fulminant ARDS, and reduced production of IFN-γ in the lung may be involved in the beneficial effect of this treatment.