Pseudaminic acid on Campylobacter jejuni flagella modulates dendritic cell IL-10 expression via Siglec-10 receptor: a novel flagellin-host interaction

Abstract

Introduction: Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. At present the identity of host-pathogen interactions that promote successful bacterial colonisation remain ill defined. Herein, we aimed to investigate C. jejuni-mediated effects on dendritic cell (DC) immunity.

Results: We found C. jejuni to be a potent inducer of human and murine DC interleukin 10 (IL-10) in vitro, a cellular event that was MyD88- and p38 MAPK-signalling dependent. Utilizing a series of C. jejuni isogenic mutants we found the major flagellin protein, FlaA, modulated IL-10 expression, an intriguing observation as C. jejuni FlaA is not a TLR5 agonist. Further analysis revealed pseudaminic acid residues on the flagella contributed to DC IL-10 expression. We identified the ability of both viable C. jejuni and purified flagellum to bind to Siglec-10, an immune-modulatory receptor. In vitro infection of Siglec-10 overexpressing cells resulted in increased IL-10 expression in a p38-dependent manner. Detection of Siglec-10 on intestinal CD11c(+) CD103(+) DCs added further credence to the notion that this novel interaction may contribute to immune outcome during human infection.

Conclusions: We propose that unlike the Salmonella Typhimurium flagella-TLR5 driven pro-inflammatory axis, C. jejuni flagella instead promote an anti-inflammatory axis via glycan-Siglec-10 engagement.

Keywords: Campylobacter jejuni; IL-10; Siglec-10; dendritic cells; immune modulation; p38.

Figure 1.

Campylobacter jejuni induces high IL-10 expression in hDC and murine BMDC. Cells were stimulated with either WT C. jejuni 11168H, EPEC E2348/69, Salmonella Typhiumurium SL1344, Clostridium difficile R20291 strains to reach a final MOI of 100, 8 hours post-infection. Cytokine levels were measured 8 hours post-infection. A, Enteropathogen-mediated hDC IL-10, IL-12 and IL-1β production. Bars depict median values from 6 individual donors. B, hDC IL-10/IL-12 ratio. Bars depict median values from 6 individual donors. C, Enteropathogen-mediated BMDC IL-10, IL-12 and IL-1β protein production. Bars show mean values from four independent experiments ± SEM (D) BMDC IL-10/IL-12 ratio. Bars show mean values from four independent experiments ± SEM. Friedman statistical analysis was performed on data sets. *P < .05; **P < .01; ***P < .001. Abbreviations: BMDC, bone-marrow derived dendritic cells; hDC, human dendritic cells; IL, interleukin; WT, wild-type.

Figure 2.

Campylobacter jejuni flagella selectively modulate BMDC IL-10 expression. A, BMDCs were stimulated with C. jejuni 11168H WT, flaA, waaF, kpsM, or pglB isogenic mutants for 24 hours MOI 100. Secreted IL-10, IL-12, TNFα, IL-23 and IL-27 cytokine levels were quantified by ELISA. Bars depict mean values ± SEM from four independent experiments. B, Uptake of 11168H WT and flaA isogenic mutant by BMDCs was quantified 4 hours post-infection (initial infection MOI 100) using the gentamicin protection assay. C, BMDCs were infected with C. jejuni 11168H WT or flaA isogenic mutant (MOI 100) and time-dependent IL-10 and IL-12 p35 subunit mRNA levels were quantified by real-time PCR. Bars depict mean values ± SEM from a minimum of 3 independent experiments. Friedman statistical analysis was performed on data sets. *P < .05; **P < .01; ***P < .001. Abbreviations: BMDC, bone-marrow derived dendritic cells; IL, interleukin; MOI, multiplicity of infection; PCR, polymerase chain reaction; SEM, standard error of the mean; TNFα, tumor necrosis factor α.

Figure 3.

Pseudaminic acid structures on Campylobacter jejuni flagella modulate BMDC and hDC IL-10 expression. A, WT, TRIF^−/−, MyD88^−/−_, and DKO BMDCs were infected with 11168H WT C. jejuni or stimulated with LPS (100 ng/mL) for 24 hours. IL-10 and IL-12 protein levels post-stimulation were measured by ELISA. Bars depict mean levels ± SEM from 3 independent experiments. B, BMDCs were infected for 24 hours with 11168H WT, 11168H flaA, and 11168H rpoN mutants, 81-176 WT and 81-176 flaA mutant, 11168H Cj1324 mutant, and the Cj1324 complemented strain or 11168H Cj1316 mutant (MOI 100). 24 hours post-infection IL-10 protein was measured by ELISA. Bars depict mean values ± SEM from four independent experiments. C, hDCs were infected with 11168H WT, flaA, and Cj1316 mutant strains (MOI 100). Bars depict median levels from 8 individual donors. Friedman statistical analysis was performed on data sets. *P < .05; **P < .01; ***P < .001. Abbreviations: BMDC, bone-marrow derived dendritic cells; DKO, double knockout; ELISA, enzyme-linked immunosorbent assay; hDC, human dendritic cells; IL, interleukin; MOI, multiplicity of infection; SEM, standard error of the mean; WT, wild-type.

Figure 4.

Glycosylated surface structures of Campylobacter jejuni flagella bind to Siglec-10 receptor. A, 11168H WT, flaA, Cj1316, and waaF mutants were assessed for binding to CHO cells overexpressing Siglec-10 receptor at 4°C. Histograms are representative of 3 independent experiments. Control cells alone (dotted line), bacteria/CHO cell co-cultures (solid gray). B, Binding of 11168H WT and Cj1316 mutant to CHO cells is expressed as a bacterial binding index (% cells with bacteria bound × geometric mean fluorescence intensity of the bacteria-positive cells). Bars depict mean values ± SEM from 5 independent experiments. C, The binding capacity of Siglec-10-Fc to purified flagella from WT 11168H, WT 81-176 and 11168H Cj1316 mutant was assessed by ELISA. Bars depict mean values ± SEM from 3 independent experiments performed in duplicate. Friedman statistical analysis was performed on data sets. *P < .05; **P < .01; ***P < .001. Abbreviations: CHO, Chinese hamster ovary; ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MOI, multiplicity of infection; SEM, standard error of the mean; WT, wild-type.

Figure 5.

Siglec-10 receptor overexpression promotes IL-10 expression in response to Campylobacter jejuni flagella. A, Untransduced, GFP control, and Siglec-10 transduced PMA-differentiated THP-1 cells were infected with 11168H WT and flaA isogenic mutant (MOI 100) or LPS (100 ng/mL) for 24 hours. B, Untransduced, GFP control and Siglec-10 transduced RAW264.7 were infected with 11168H WT and Cj1316 isogenic mutant (MOI 100) or purified C. jejuni 11168H LOS (100 ng/mL) for 24 hours. IL-10, IL-12 and TNFα protein levels were assessed by ELISA. Bars depict data from 4 independent experiments ± SEM. Friedman statistical analysis was performed on data sets. *P < .05; **P < .01; ***P < .001. Abbreviations: ELISA, enzyme-linked immunosorbent assay; IL, interleukin; MOI, multiplicity of infection; SEM, standard error of the mean; TNFα, tumor necrosis factor α; WT, wild-type.

Figure 6.

Campylobacter jejuni flagella alter BMDC IL-10 transcription in an NF-κB independent but MAPK-dependent manner. A, BMDCs were transduced with an NF-κB luciferase reporter plasmid on day 4 of differentiation. On day 8 BMDCs were infected with 11168H WT or flaA mutant (MOI of 100) for 6 hours and subsequent luciferase activity quantified. Bars depict values from 3 independent experiments performed in duplicate ± SEM. B, BMDCs were infected with 11168H WT or flaA isogenic mutant (MOI 100) and time-dependent effects on MAPK activation (phosphorylated p38, ERK and JNK) was followed by Western blotting; total ERK was used as a loading control. Representative blots from 3 independent experiments are shown. Densitometric analysis was performed on P-p38 and P-ERK blots using ImageJ software. C, WT, DKO, MyD88^−/− and TRIF^−/− BMDCs were infected with C. jejuni 11168H WT (MOI 100). Time-dependent activation of MAPK pathways (p38, ERK, and JNK) was followed by Western blotting. The presence of phosphorylated MAPKs was indicative of activation; total ERK served as a loading control. D, BMDCs were pre-treated with various MAPK inhibitors for 2 hours and subsequently infected with C. jejuni 11168H WT (MOI 100). 1 μM (black bars; 5 μM PD90859) and 10 μM (gray bars; 50 μM PD90859). IL-10 and IL-12 protein levels were quantified 24 hours post-infection. E, Siglec-10 and GFP control transduced RAW264.7 cells were infected with 11168H WT C. jejuni (MOI of 100) for 24 hours in the presence of the p38 inhibitor SB203580 or DMSO vehicle control. IL-10 levels were analysed by ELISA. Blots are representative of 3 independent experiments. Friedman statistical analysis was performed on data sets. *P < .05; **P < .01; ***P < .001. Abbreviations: BMDC, bone-marrow derived dendritic cells; DKO, double knockout; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; IL, interleukin; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MOI, multiplicity of infection; SEM, standard error of the mean; WT, wild-type.

Figure 7.

Siglec-10 is expressed on human peripheral blood monocyte-derived and gastrointestinal lamina propria dendritic cells. A, Human peripheral blood monocyte-derived DCs were stained with primary mouse monoclonal anti-Siglec-10, 5G6, and PE-conjugated donkey anti-mouse secondary antibody (black line), or secondary antibody alone (solid gray line). The histogram shown is from 1 representative donor of 4. B, Lamina propria cells from colonic biopsy tissue were analysed for expression of Siglec-10, CD103, and CD11c. CD11c⁺ cells were gated on and expression of CD103 and Siglec-10 assessed. FACS plots shown are from 1 representative donor of 3. Abbreviations: DC, dendritic cell; FACS, fluorescence activated cell sorting.