Sialoadhesin promotes rapid proinflammatory and type I IFN responses to a sialylated pathogen, Campylobacter jejuni

Abstract

Sialoadhesin (Sn) is a macrophage (Mφ)-restricted receptor that recognizes sialylated ligands on host cells and pathogens. Although Sn is thought to be important in cellular interactions of Mφs with cells of the immune system, the functional consequences of pathogen engagement by Sn are unclear. As a model system, we have investigated the role of Sn in Mφ interactions with heat-killed Campylobacter jejuni expressing a GD1a-like, sialylated glycan. Compared to Sn-expressing bone marrow-derived macrophages (BMDM) from wild-type mice, BMDM from mice either deficient in Sn or expressing a non-glycan-binding form of Sn showed greatly reduced phagocytosis of sialylated C. jejuni. This was accompanied by a strong reduction in MyD88-dependent secretion of TNF-α, IL-6, IL-12, and IL-10. In vivo studies demonstrated that functional Sn was required for rapid TNF-α and IFN-β responses to i.v.-injected sialylated C. jejuni. Bacteria were captured within minutes after i.v. injection and were associated with Mφs in both liver and spleen. In the spleen, IFN-β-reactive cells were localized to Sn⁺ Mφs and other cells in the red pulp and marginal zone. Together, these studies demonstrate that Sn plays a key role in capturing sialylated pathogens and promoting rapid proinflammatory cytokine and type I IFN responses.

FIGURE 1.

Binding and phagocytosis of GB11 C. jejuni by WT and Sn^−/− BMDM in vitro. (A and B) IFN-α–stimulated BMDM were incubated with CFSE-labeled heat-killed sialylated (sia) or non-sialylated GB11 C. jejuni and analyzed by flow cytometry. To quench the fluorescence of extracellular bound but not phagocytosed bacteria, trypan blue (TB) was added for 15 min and washed out before the analysis. Opsonized E. coli were used as a positive control. One representative experiment (A) and data pooled from three independent experiments (B) are shown. Bars represent the average ± SD. Two-way ANOVA tests were used to assess differences and interactions between groups. *p < 0.01. (C) Live cell video microscopy images showing binding and engulfment of C. jejuni by WT and Sn^−/− BMDM. In the upper panels, the first image shows initial contact between the WT Mϕs (red) and C. jejuni (green). Subsequent images show C. jejuni being engulfed by the Mϕs. In the later images, colocalization of C. jejuni with LysoTracker Red DND-99 can be seen (yellow) indicating phagolysosomal fusion. The lower panel shows Sn^−/− Mϕ binding to two C. jejuni bacteria. The bacteria remain associated with the Mϕ for 10 min and then dissociate. The time points at which images were taken are displayed; images are representative from two independent experiments. Scale bars, 10 μm.

FIGURE 2.

In vitro cytokine production by BMDM in response to C. jejuni. (A) Heat-killed sialylated (sia) or non-sialylated GB11 C. jejuni were either untreated or opsonized (ops) with mouse anti-C. jejuni antiserum and then incubated with BMDM from WT and Sn^−/− mice for 90 min. After removal of unbound bacteria, BMDM were cultured for a further 24 h. Concentrations of TNF-α, IL-6, IL-12, and IL-10 in cell culture supernatants were measured by ELISA. Bars show the average of three independent experiments (three replicates) ± SD. Media from unstimulated cells were analyzed as a negative control. Heat-killed Salmonella were used as a positive control. (B) Sn expression on IFN-α–stimulated WT, Sn^−/−, and MyD88^−/− BMDM. (C) IFN-α–stimulated WT, Sn^−/−, and MyD88^−/− BMDM were incubated with CFSE-labeled heat-killed sialylated C. jejuni and binding and phagocytosis analyzed by flow cytometry. To quench the fluorescence of extracellular bound but not phagocytosed bacteria, trypan blue (TB) was added for 15 min and washed out before the analysis. (D) BMDM from WT, Sn^−/−, and MyD88^−/− mice were incubated for 90 min with heat-killed sialylated C. jejuni, and after removal of unbound bacteria, cells were cultured for a further 24 h. Concentrations of TNF-α and IL-6 in cell culture supernatants were measured by ELISA. Bars show the average of two independent experiments with three replicates ± SD. Media from unstimulated cells were analyzed as a negative control. Two-way ANOVA tests were used to assess differences and interactions between groups. *p < 0.01. US, Unstimulated cells.

FIGURE 3.

In vivo cytokine responses to C. jejuni in Sn^−/− mice. Groups of WT and Sn^−/− mice were injected i.v. with 10⁸ heat-killed sialylated (+sia) or non-sialylated (−sia) C. jejuni or PBS (control) and cytokine responses assessed after 1 h by (A) ELISA measurements of serum cytokine concentrations or (B) changes in mRNA levels in spleen (top panels) or liver (bottom panels) by real-time quantitative PCR. Data are presented as means ± SD and are shown for one representative experiment out of two, with five to six mice per group injected with C. jejuni and three mice per group injected with PBS as a control. *p < 0.01 (Mann–Whitney U test).

FIGURE 4.

In vivo cytokine responses to C. jejuni in Sn^W2QR97A mice. Groups of WT and Sn^W2QR97A mice were injected i.v. with 10⁸ heat-killed sialylated C. jejuni and cytokine responses measured at 1 h and 2 h by (A) ELISA measurements of serum cytokine concentrations or (B) changes in mRNA levels in spleen by real-time quantitative PCR. Data are presented as means ± SD and are shown for one experiment with three mice per group. *p < 0.05 (Mann–Whitney U test).

FIGURE 5.

Localization of C. jejuni in spleen and liver after i.v. injection. Groups of WT and Sn^W2QR97A mice were injected i.v. with 10⁸ PKH67-labeled heat-killed sialylated C. jejuni. Spleens and livers were collected at 5 min or 20 min after the injection. (A) Representative images of spleen sections 5 min after injection are shown after staining with Abs to F4/80, Sn, CD68, and B220. Red pulp (RP) and white pulp (WP) areas are indicated. Scale bars, 100 μm. (B) Representative images of liver sections are shown after staining with anti-Sn Abs and DAPI. Scale bars, 100 μm. Data shown are representative of one experiment of two performed, each with four mice per group.

FIGURE 6.

Quantification of C. jejuni in spleen and liver after i.v. injection. Groups of WT and Sn^W2QR97A mice were injected i.v. with 10⁸ PKH67-labeled heat-killed sialylated C. jejuni, and tissues were collected at 5 min or 20 min after the injection. (A) Spleen, average area (μm²) of C. jejuni per field of view. (B) Liver, average area (μm²) of C. jejuni per field of view. At least 20 images per sample were analyzed for each of the 5-min and 20-min time points. Data are shown are averages from two experiments with four mice per group. Data are presented as means ± SD. *p < 0.01 (paired t test).

FIGURE 7.

Sn-dependent IFN-β production in spleen. WT and Sn^W2QR97A mice were injected i.v. with 10⁸ sialylated C. jejuni, 100 μg poly(I:C), or PBS as a control. Spleens were collected after 1 h and cryostat sections stained with Abs to Sn, F4/80, and IFN-β and analyzed by confocal microscopy. Scale bars, 50 μm. Data are shown for one representative experiment of two with five to six mice per experimental group and three PBS control mice per group.

FIGURE 8.

Localization of IFN-β–associated cells in the red pulp and marginal zone of WT spleen. A WT mouse was injected with sialylated C. jejuni and the spleen collected after 1 h. Cryostat sections were stained with Abs to Sn, F4/80, and IFN-β. White boxes show areas selected for higher-power images of red pulp (RP) (middle panels) and marginal zone (MZ) (bottom panels). White arrows point to examples of IFN-β–associated, non-Mϕ cells. Scale bars, 50 μm.