Figure 1.
(A) HPLC analysis of TFAA hydrolysed C. albicans-chitin (left) compared to commercial crab shell chitin (right). GlcN Glucosamine, Glc Glucose, Man Mannan. (B) Chitin particle size determined by flow cytometry. (C) Chitin size distribution in percentage of analysed chitin extractions, presented as mean ± SEM, n = 6.
Table 1.
Acetylation degree of purified chitin.
Figure 2.
Chitin induced cytokines and synergistic effect on IL-10 secretion.
(A) Cytokine induction in hPBMCs incubated with chitin for 24 h, n = 6, ***p<0.001. (B) Cytokine induction in mBMMφ's incubated with chitin for 24 h, n = 4, *p<0.05. (C) IL-10 and TNF induction after 24 h in mBMMφs incubated with increasing chitin concentrations, n = 4, ***p<0.001 for IL-10, °°°p<0.001 for TNF compared to untreated control. (D) IL-10 and TNF induction in hPBMCs incubated with chitin isolated from different species, n = 3, *p<0.05, **p<0.01. (E) Co-incubation of hPBMCs with either LPS, zymosan, curdlan, Pam3CSK4, flagellin, CpG ODN or C. albicans cell wall proteins (CWPs) and chitin, n = 6, *p<0.05. All data are presented as mean values ± SEM.
Figure 3.
Chitin induced IL-10 secretion depends on mannose receptor, TLR9- and NOD2-signalling.
(A) mBMMφs were incubated with S. c. mannan or laminarin 1 h prior stimulation with chitin, n = 4. (B) mBMMφs from wild type mice (C57BL/6 and 129Sve), dectin-1- and MR –deficient mice were stimulated with chitin, n = 4. (C) mBMMφs from wild type mice (C57BL/6), NOD2-, RICK- and CARD9-deficient mice stimulated with chitin for 24 h, n = 4. (D) mBMMφs from wild type mice (C57BL/6), TLR2-, TLR2/4-, TLR9- and MyD88-deficient mice stimulated with chitin for 24 h, n = 4. All data are presented as mean values ± SEM, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
Figure 4.
Chitin induced IL-10 secretion requires mannose receptor interaction but not uptake.
(A) Chitin was incubated with liposomal transfection reagent DOTAP for 15 min at room-temperature before used to stimulate mBMMφs from wild type, TLR2, TLR9, NOD2, MR and dectin-1-deficient mice for 24 h or (A and B) mBMMφs were treated with CytD for 1 h prior chitin stimulation. Values represent mean ± SEM, n = 4, *p<0.05, **p<0.01.
Figure 5.
Chitin co-localisation with NOD2, TLR9 and MR.
Confocal fluorescence microscopy of mBMMφs from wild type mice stimulated with chitin for 20 min. NOD2 protein was detected with anti-mouse NOD2 antibody, TLR9 with anti-mouse TLR9 antibody, MR with anti-mouse CD206-antibody and chitin was detected using a chitin-binding reporter construct (ChBD-HuCκ). Images show chitin co-localisation with NOD2 (A and C), TLR9 (A and B) and MR (B and C). Images are representative of two independent experiments, scale bars = 10 µm.
Figure 6.
Chitin co-localisation with NOD2 and TLR9 depends on MR.
Fluorescence microscopy of mBMMφs from wild type, TLR2-, NOD2-, MR- and dectin-1-deficient mice stimulated with chitin for 1 h. Nuclei are stained with DAPI (blue) and chitin was detected using a chitin-binding reporter construct (ChBD-HuCκ, green). (A) NOD2 protein was detected with anti-mouse NOD2 antibody (red) (B) TLR9 protein was detected with anti-mouse TLR9 antibody (red) and (C) TLR2 protein was detected with anti-mouse TLR2 antibody (red). White arrows indicate co-localisation of chitin with NOD2 and/or TLR9 (yellow). Chitin did not co-localise with TLR2 in all tested cells (C) and no chitin co-localisation with NOD2 or TLR9 was detectable in MR-deficient macrophages (A and B). Images (A to C) are representative of two independent experiments, scale bars = 15 µm.
Figure 7.
Chitin dampens LPS induced inflammation in vivo.
C57BL/6 mice were injected intraperitoneal with saline, chitin, LPS or chitin and LPS in combination. Infiltrating immune cells and cytokine production were analysed after 4 h (A and D), 24 h (B and E) and 4 days (C and F). Data are presented as mean values ± SEM, n = 5 mice per group, *p<0.05, **p<0.01.
Figure 8.
Reduced cell wall chitin effects late phase cytokine response to C. albicans.
CHIT-1-activity of (A) hPMNs and (B) hMφ's incubated with live C. albicans yeast cells, MOI = 0.4. Values represent means ± SEM, n = 4, *p<0.05, ***p<0.001, ****p<0.0001. (C) Heat-treated yeast cells from C. albicans wild type and chs3Δ mutant were stained for total chitin content with Calcofluor White (CFW) and surface presented chitin with wheat germ agglutinin (WGA) and (D) mean fluorescence intensity (MFI) was analysed. Values are presented as mean ± SEM, n = 30, ****p<0.0001. (E) TEM analysis of heat-treated yeast cells from C. albicans wild type and chs3Δ mutant. Images shown are representative for all analysed yeast cells. (F and G) hPBMCs were incubated with heat-treated C. albicans wild type yeast cells or C. albicans chs3Δ, MOI = 0.4 in the presence or absence of the chitinase inhibitor Bisdionin C. TNF and IL-10 secretion was monitored for a period of 7 days. Values represent means ± SEM, n = 4, *p<0.05, **p<0.01, ****p<0.0001.
Figure 9.
Schematic overview of chitin recognition and involved pathways in negative regulation of inflammation.
Innate recognition of fungal cells by PRRs like Dectin-1 and TLR2 leads to the induction of pro-inflammatory cytokines, such as TNF. The pathogen recognition together with the release of pro-inflammatory cytokines induces the secretion of chitinases (e.g. chitotriosidase) from neutrophils and macrophages. Chitin digestion leads to the generation of small sized chitin particles that are taken up by the mannose receptor and induce IL-10 secretion via the TLR9 and NOD2 pathway. The anti-inflammatory cytokine IL-10 dampens the inflammatory response by down-regulating pro-inflammatory cytokine secretion.