Cell wall N-glycan of Candida albicans ameliorates early hyper- and late hypo-immunoreactivity in sepsis

Abstract

Severe infection often causes a septic cytokine storm followed by immune exhaustion/paralysis. Not surprisingly, many pathogens are equipped with various anti-inflammatory mechanisms. Such mechanisms might be leveraged clinically to control septic cytokine storms. Here we show that N-glycan from pathogenic C. albicans ameliorates mouse sepsis through immunosuppressive cytokine IL-10. In a sepsis model using lipopolysaccharide (LPS), injection of the N-glycan upregulated serum IL-10, and suppressed pro-inflammatory IL-1β, TNF-α and IFN-γ. The N-glycan also improved the survival of mice challenged by LPS. Analyses of structurally defined N-glycans from several yeast strains revealed that the mannose core is key to the upregulation of IL-10. Knocking out the C-type lectin Dectin-2 abrogated the N-glycan-mediated IL-10 augmentation. Furthermore, C. albicans N-glycan ameliorated immune exhaustion/immune paralysis after acute inflammation. Our results suggest a strategy where the immunosuppressive mechanism of one pathogen can be applied to attenuate a severe inflammation/cytokine storm caused by another pathogen.

Fig. 1. N-glycan of C. albicans up-regulates IL-10 production and improves mouse septic responses with LPS.

a, b Serum cytokines after i.v. injection with a low dose of LPS (15 μg/20 g mouse weight) and J-1012 N-glycan (400 μg N-glycan/20 g mouse weight) in WT (a) or TLR4KO mice (b). c, d Serum IL-10 after i.v. injection with J-1012 N-glycan purified after β-elimination (c) or J-1012 N-glycan treated by lyticase in WT mice (d). e Effects of deficient of Dectin-1 on serum IL-10. f Effects of anti-IL-10 on serum cytokines. g Serum IL-10 after i.p. injection with a lethal dose (100 μg/20 g mouse weight) of LPS and J-1012 N-glycan. h, i Survival rates of mice after i.p. injection with J-1012 N-glycan and the high dose of LPS in the absence (h) or the presence (i) of mAbs, as indicated. These experiments were repeated twice, and compiled results are shown. a–g Data are expressed as the mean ± SD (n = 3) (a, c–g), (n = 6) (b) and are representative of at least two independent experiments. Statistical analyses were performed between LPS and LPS + J-1012 (a, g), LPS + J-1012 and LPS + J-1012 with β-elimination (c), LPS + J-1012 and LPS + J-1012 with lyticase (d), LPS + J-1012 + R.IgG and LPS + J-1012 + αIL-10 (f). p value at the peak point of cytokine production was determined by unpaired two-tailed Student’s t-test. N.S., not significant (p > 0.05). h, i p value was determined by the Wilcoxon test.

Fig. 2. α-mannans of N-glycan of C. albicans are involved in augmentation of IL-10 through Dectin-2.

a Schematic structures of N-glycans and mannose core (black circle; α1,6-mannose (in main chain), blue circle; β-mannose, green circle; α1,3-mannose, light blue circle; α-mannose (linked to the phosphate group), red circle; α1,6-mannose, yellow circle; α1,2-mannose, light brown rectangle; Mannose₁₀ core). b–f Analyses of serum cytokines after i.v. injection with LPS and J-1012 N-glycan treated with α-mannosidase (b), S. cerevisiae (mnn1/mnn4) (c), C. lusitaniae (d), C. parapsilosis and C. stellatoidea (e), and S. cerevisiae (mnn2) (f) N-glycan as in Fig. 1a. g Serum IL-10 of 2 h after i.v. injection with LPS and J-1012 N-glycan in mice pre-treated by clodronate liposome. h–k Cytokine production by rpMϕ after stimulation by LPS for 24 h on plates coated with intact (h, k), NaIO₄ treated (i) or α-mannosidase treated (j) J-1012 N-glycan. Effects of signaling inhibitors on IL-10 production by rpMϕ (k). l IL-10 production of SIGNR1KO rpMϕ in response to plate-coated J-1012 N-glycan (left panel) and serum IL-10 in SIGNR1KO mice as in Fig. 1a (right panels). m Serum IL-10 in Dectin-2KO mice upon stimulation with LPS in the presence of J-1012 N-glycan as in Fig. 1a. n Survival of WT and Dectin-2KO mice as in Fig. 1h. The experiment was repeated twice, and compiled results are shown. o Serum IL-10 in Dectin-2KO mice after injection with N-glycan from S. cerevisiae X2180-1A-5 (mnn2). b–m, o Data are expressed as the mean ± SD (n = 3) (b–l), (n = 4) (m) and (n = 4) (o), and are representative of at least two independent experiments. In (b), (c), (l right panel) and (o), statistical analyses were performed between LPS + J-1012 and LPS + J-1012 with α-mannosidase, LPS + J-1012 and LPS + mnn1/4, WT + J-1012 and SIGNR1KO + J-1012, and WT + mnn2 + LPS and Dectin-2KO + mnn2 + LPS, respectively. b, d, l (right panel), m, o, p value at the peak point of cytokine production was determined by unpaired two-tailed Student’s t-test. g–l p value was determined by unpaired two-tailed Student’s t-test. N.S., not significant (p > 0.05). n p value was determined by the Wilcoxon test.

Fig. 3. Augmentation of IL-10 production by mannose core in vitro.

a–e Cytokine production by rpMϕ from WT(a–c) and Dectin-2KO (d, e) mice after stimulation with Man₉Core-BSA, Man₃Core-BSA, Man₅₁-BSA (bound more than 51 mannose monomers), and J-1012 N-glycan, immobilized on plates as in Fig. 2h. Schematic structures of Man₉Core-BSA and Man₃Core-BSA are depicted. f IL-10 production by BMDCs from WT and Dectin-2KO mice in response to Man₉Core-BSA. g IL-10 production by BMDCs from WT mice upon stimulation with Man₉Core-BSA, Man₃Core-BSA, Man₅₁-BSA, J-1012 N-glycan, and J-1012 N-glycan treated with α-mannosidase. h Schematic structure of (Man₉CoreCys)₅-biotin. εAHX, ε-amino-n-hexanoic acid. i Cytokine production by BMDCs from WT and Dectin-2KO mice upon stimulation with (Man₉CoreCys)₅-biotin bound to an avidin-coated plate. These experiments were repeated at least twice, and representative results are shown. Data are expressed as the mean ± SD. p value was determined by unpaired two-tailed Student’s t-test. N.S., not significant (p > 0.05).

Fig. 4. Amelioration of hypo-immunoreactivity by J-1012 N-glycan.

a Recovery of delayed-type hypersensitivity in mouse footpad by J-1012 N-glycan. b, c Analyses of cytokine production by splenocytes from mice after 2 weeks of sepsis induction with i.v injection of OVA, J-1012 N-glycan and LPS without (b) or with anti-IL-10 mAb (c). The splenocytes were restimulated as described for 4 days. d Expression of CD62L and PD-1 of transferred DO11.10 T cells (CD4⁺KJ1-26⁺) (Supplementary Fig. 10) from mice after 2 weeks of sepsis induction with OVA, LPS and J-1012 N-glycan. The experiment was repeated twice, and compiled results are shown (right panels) (n = 6). a–c Data are expressed as the mean ± SD and are representative of at least two independent experiments (n = 3). p value was determined by unpaired two-tailed Student’s t-test. N.S., not significant (p > 0.05). R.IgG, control rat IgG. SRBC sheep red blood cells.