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

Abstract

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.

Fig. 1

Effect of Dectin-2 deficiency on S. pneumoniae infection. WT mice and Dectin-2KO mice were infected with S. pneumoniae. a The number of live mice was recorded daily. Closed circles, WT mice (n = 6); open squares, Dectin-2KO mice (n = 6). b Bacterial load in the lungs was examined on day 3 post-infection. Each symbol shows each mouse, and the bars indicate the mean ± SD. Similar results were obtained in three independent experiments. *p < 0.05

Fig. 2

Equivalent level of neutrophil infiltration in lungs between WT and Dectin-2KO mice. WT mice and Dectin-2KO mice were infected with S. pneumoniae. a The lung section obtained 12 h post-infection was stained with hematoxylin-eosin (H-E), and then observed using light microscopy. Original magnifications: ×100 and × 1000. b Cells in BALF at 12 h and 24 h post-infection were stained with Diff-Quick, and the composition of neutrophils was then quantified. Each group consists of five to seven mice. Similar results were obtained in three independent experiments. NS, not significantly different

Fig. 3

Effect of Dectin-2 deficiency on the engulfment of pneumococcus by neutrophils. WT mice and Dectin-2KO mice were infected with S. pneumoniae. The phagocytic rate (a) and phagocytic index (b) of neutrophils in BALF were calculated 12 h and 24 h after infection. The number of neutrophils was estimated by multiplying the total cell count by its proportion identified in morphological analysis. Each group consists of five to seven mice. Similar results were obtained in three independent experiments. *, p < 0.05; NS, not significantly different

Fig. 4

Effect of Dectin-2 deficiency on cytokine production in the lungs after pneumococcal infection. WT mice and Dectin-2KO mice were infected with S. pneumoniae. Cytokine concentrations in BALF were measured 12 h post-infection. Each group consists of five to seven mice. Similar results were obtained in three independent experiments. *, p < 0.05; NS, not significantly different

Fig. 5

Cellular source of IFN-γ production in the lungs after pneumococcal infection. WT mice were infected with S. pneumoniae. The lung leukocytes prepared at 12 h post-infection were stained with various mAbs to identify populations of myeloid cells and lymphocytes, and intracellular IFN-γ expression was analyzed in each population using flow cytometry. Cut-off lines were determined on the histograms stained with isotype-matched IgG. Ex Mφ, exudate macrophages; Alv Mφ, alveolar macrophages; Neu, neutrophils; IgG, isotype-matched IgG

Fig. 6

Reduced production of serotype-specific Ab against pneumococcal capsular polysaccharides in Dectin2KO mice. Anti-PPS3 IgG (a), IgG3 (b) and IgM (c) concentrations in BALF before infection or 24 h post-infection were measured as OD450 values at × 1, ×3 and × 9 dilution, respectively. Each group consists of six to seven mice. Similar results were obtained in three independent experiments. *, p < 0.05, compared with Dectin-2KO mice 24 h post-infection. Open circles, WT mice 24 h post-infection; closed circles, Dectin-2KO mice 24 h after infection; open triangles, WT mice before infection; closed triangles, Dectin-2KO mice before infection

Fig. 7

Effect of Dectin-2 deficiency on IL-12p40 production by BD-DCs upon stimulation with S. pneumoniae. BM-DCs from WT mice (open columns) or Dectin-2KO mice (solid columns) were cultured with live S. pneumoniae (a); lysates from S. pneumoniae or sham-operated PBS (Sham) (b); S. pneumoniae culture supernatant or Todd-Hewitt broth (THB) (c); or LPS (1 μg/ml), CpG (1 μg/ml) or mannan (3 mg/ml) for 24 h. IL-12p40 concentrations in the culture supernatants were measured using ELISA. Each column shows the mean ± SD of triplicate culture. Similar results were obtained in three independent experiments. MOI, multiplicity of infection. *, p < 0.05; NS, not significantly different

Fig. 8

Dectin-2-dependent IL-12p40 production by BM-DCs in the ConA-bound fraction of S. pneumoniae culture supernatant. a BM-DCs derived from WT mice were stimulated with the ConA-Sepharose4B-treated or sham-treated supernatant from S. pneumoniae or mannan for 24 h. b BM-DCs from WT mice (open columns) or Dectin-2KO mice (solid columns) were cultured with the ConA-Sepharose4B-bound fraction of S. pneumoniae culture supernatants, LPS (1 μg/ml) or mannan (3 mg/ml) for 24 h. The IL-12p40 concentration in the culture supernatants was measured by ELISA. Each column shows the mean ± SD of triplicate culture. Similar results were obtained in three independent experiments. Sup-sham, sham-treated supernatants; Sup-tx, ConA-Sepharose4B-treated supernatants; Mannan-sham, sham-treated mannan; Mannan-tx, ConA-Sepharose4B-treated mannan; ConA-bound Fr, CpnA-Sepharose4B-bound fraction. * p < 0.05