A polysaccharide virulence factor from Aspergillus fumigatus elicits anti-inflammatory effects through induction of Interleukin-1 receptor antagonist

Abstract

The galactosaminogalactan (GAG) is a cell wall component of Aspergillus fumigatus that has potent anti-inflammatory effects in mice. However, the mechanisms responsible for the anti-inflammatory property of GAG remain to be elucidated. In the present study we used in vitro PBMC stimulation assays to demonstrate, that GAG inhibits proinflammatory T-helper (Th)1 and Th17 cytokine production in human PBMCs by inducing Interleukin-1 receptor antagonist (IL-1Ra), a potent anti-inflammatory cytokine that blocks IL-1 signalling. GAG cannot suppress human T-helper cytokine production in the presence of neutralizing antibodies against IL-1Ra. In a mouse model of invasive aspergillosis, GAG induces IL-1Ra in vivo, and the increased susceptibility to invasive aspergillosis in the presence of GAG in wild type mice is not observed in mice deficient for IL-1Ra. Additionally, we demonstrate that the capacity of GAG to induce IL-1Ra could also be used for treatment of inflammatory diseases, as GAG was able to reduce severity of an experimental model of allergic aspergillosis, and in a murine DSS-induced colitis model. In the setting of invasive aspergillosis, GAG has a significant immunomodulatory function by inducing IL-1Ra and notably IL-1Ra knockout mice are completely protected to invasive pulmonary aspergillosis. This opens new treatment strategies that target IL-1Ra in the setting of acute invasive fungal infection. However, the observation that GAG can also protect mice from allergy and colitis makes GAG or a derivative structure of GAG a potential treatment compound for IL-1 driven inflammatory diseases.

Figure 1. GAG inhibits Aspergillus-induced human T-helper cell cytokine production.

(A) TNFα, IL-6, IL-8 and IL-10 concentrations in culture supernatants of human PBMCs stimulated for 24 hours with 10 µg/ml GAG and IFN-γ, IL-17, IL-5 and IL-9 concentrations after 7 days of stimulation. (B) TNFα, IL-6 and IL-10 concentrations in culture supernatants of human PBMCs (n = 6 donors) stimulated for 24 hours with heat inactivated A. fumigatus conidia (1×10⁷/ml) in the presence or absence of 10 µg/ml GAG. (C,D) IL-17, IL-22 and IFN-γ concentrations in culture supernatants of human PBMCs stimulated for 7 days with heat inactivated A. fumigatus conidia (1×10⁷/ml) (n = 10 donors for IL-17 and IL-22, n = 6 donors for IFN-γ) (c), IL-1β/IL-23 (50/100 ng/ml) (n = 14 donors) or IL-12/IL-18 (50/100 ng/ml) (n = 10 donors) in the presence or absence of GAG (10 µg/ml). Data are represented as mean +/− SEM.

Figure 2. GAG induces interleukin 1 receptor antagonist.

(A) IL-1 bioactivity measured as IL-2 production by NOB-1 cells stimulated with 50 ng/ml IL-1β in the presence of culture supernatant of unstimulated PBMCs (medium) or culture supernatants of PBMCs that were exposed to 10 µg/ml GAG for 24 hours (GAG conditioned medium) (n = 6 donors). (B) IL-1Ra, IL-1β and IL-1α concentrations in culture supernatants of human PBMCs stimulated with for 24 hours with 10 µg/ml GAG. Data are represented as mean +/− SEM.

Figure 3. Suppression of IL-17 and IL-22 by GAG is dependent on IL-1Ra.

(A) IL-17, IL-22 and IFN-γ concentrations in culture supernatants of PBMCs stimulated for 7 days with heat inactivated A. fumigatus conidia 1×10⁷/ml, IL-1β/IL-23 (50/100 ng/ml) or IL-12/IL-18 (50/100 ng/ml) in the presence or absence of recombinant human IL-1Ra (10 ng/ml). Data are represented as mean +/− SEM. (B) Inhibition of IL-1β/IL-23 (50/100 ng/ml) induced IL-17 and IL-22 by GAG (10 µg/ml) in human PBMCs in the presence of isotype control (10 µg/ml) or anti-IL-1Ra (10 µg/ml). The IL-17 and IL-22 production by IL-1β/IL-23 in absence of GAG was set at 100% and mean percentage changes relative to the control are represented +/− SEM.

Figure 4. GAG induces IL-1Ra in vivo and IL-1Ra increases susceptibility to aspergillosis.

BALB/c and Il1ra^−/− mice were intranasally infected with Aspergillus conidia and treated with GAG (250 μg/kg intranasally) the day of infection, and 1, 2 and 3 days post-infection. (A) Il1ra mRNA expression in lung homogenates of mice with invasive aspergillosis, (B) Il1ra mRNA expression in purified cells from lungs of naive mice pre-stimulated with Aspergillus conidia or LPS for 1 hour, and exposed to different GAG concentrations for an additional 18 hours. (C) Survival, (D) fungal growth (CFU/lung, mean +/−SEM), (E) protein levels of IL-1Ra, (F) Mpo expression in lung homogenates, and (G) BAL morphometry [% polymorphonuclear (PMNs) cells and lung histology (PAS stained sections, bars indicate 20× magnification) of Aspergillus-infected mice with or without GAG treatment. Assays were done a day after the last GAG treatment. (H) BAL morphometry [% PMNs or eosinophils (Eo)] and lung histology (PAS stained sections, bars indicate 20× magnification), and (I) expression of Th transcription factors and cytokines in total cells from the draining lymph nodes in mice with ABPA and treated with or without GAG. Naïve means uninfected mice, and none means untreated mice and/or unstimulated cells. (J) IL-5, IL-13 and IL-17 concentrations in culture supernatants of PBMCs pre-incubated 1 h either with medium or GAG (10 μg/ml). After washing, the cells were stimulated for 7 days with heat inactivated 1×10⁷/ml A. fumigatus conidia (n = 4 donors). Data are represented as mean +/− SEM. *, p<0.05; **, p<0.01.

Figure 5. GAG protects mice from experimental DSS-induced colitis.

(A) Body weight losses, (B) stool and histological score, (C) histology of colonic sections and (D) cytokine concentrations present in total colonic cells a day after the 7-day of DSS rest in C57BL/6 and p47^phox−/− (CGD) mice with or without GAG treatment. *P<0.05, GAG treated vs. untreated mice.