Invariant natural killer T cells recognize a fungal glycosphingolipid that can induce airway hyperreactivity

Abstract

Aspergillus fumigatus is a saprophytic fungus that is ubiquitous in the environment and is commonly associated with allergic sensitization and severe asthma in humans. Although A. fumigatus is recognized by multiple microbial pattern-recognition receptors, we found that an A. fumigatus-derived glycosphingolipid, asperamide B, directly activates invariant natural killer T (iNKT) cells in vitro in a CD1d-restricted, MyD88-independent and dectin-1-independent fashion. Moreover, asperamide B, when loaded onto CD1d, directly stained, and was sufficient to activate, human and mouse iNKT cells. In vivo, asperamide B rapidly induced airway hyperreactivity, which is a cardinal feature of asthma, by activating pulmonary iNKT cells in an interleukin-33 (IL-33)-ST2-dependent fashion. Asperamide B is thus the first fungal glycolipid found to directly activate iNKT cells. These results extend the range of microorganisms that can be directly detected by iNKT cells to the kingdom of fungi and may explain how A. fumigatus can induce severe chronic respiratory diseases in humans.

Figure 1. An A. fumigatus extract rapidly induces AHR independent of adaptive immunity

(a-e). Meaurement of airway hyperreactivity (AHR) and differenatial cell count of bronchoalveolar lavage (BAL) after treatment with three 100 μg intranasal doses of an A. fumigatus extract. (mean ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 Two-Way ANOVA, Bonferoni post-test). (a) AHR of wild-type BALB/c mice. Pooled data of three experiments are shown (saline n= 11, A.f. n = 13). (b) AHR and (c) BAL count of mice deficient for MHC class II (H2.Ab1-Ea^−/−) and C57BL/6 mice as control. Pooled data of two experiments are shown (WT n = 5, MHCII^−/− Saline n = 7, A.f. n = 8). (d) AHR and (e) BAL count of Il4^−/−Il13^−/− double knockout mice and BALB/c as control. Pooled data of three experiments are shown. (n = 9). (f) Quantification of mRNA expression by QPCR in the lungs of A. fumigatus treated mice. Fold induction of A. fumigatus treated over saline treated mice is shown (mean ± s.d., *** p < 0.001, Two-way ANOVA, Bonferoni post-test) One representative experiment of five is shown.

Figure 2. AHR induced by A. fumigatus extract is dependent on iNKT cells

(a) Measurement of AHR and (b) BAL cell count in CD1d1^−/− and BALB/c treated with three intranasal 100 μg doses of A. fumigatus extract. Pooled data from two experiments are shown (WT n = 7, CD1d1^−/− n = 6), (mean ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001, Two-Way ANOVA, Bonferoni post-test). (c-f)(c) BAL or (d) lung cells from saline or A. fumigatus treated mice were analyzed by FACS for iNKT cells. PBS57 (α-GalCer analogue) loaded CD1d tetramer and TCRβ staining identify iNKT cells with unloaded tetramer as a control. Number shows % of CD45⁺ lymphocytes falling within the iNKT gate. One representative mouse of three is shown. The absolute number of iNKT in the (e) BAL or (f) lung is shown (mean ± s.e.m., * p < 0.05, *** p < 0.001, student's t-test). (g) AHR of Tcra.J18^−/− mice that received 10⁶ PBS57-CD1d tetramer sorted iNKT cells from the spleen of wild-type mice treated with intranasal A. fumigatus extract. One representative experiment of three is shown (n=4, *** p < 0.001 Two-Way ANOVA, Bonferoni post-test).

Figure 3. AHR induced with A. fumigatus extract is dependent on MyD88 but not Ticam1

(a-h). Meaurement of airway hyperreactivity (AHR) and differenatial cell count of bronchoalveolar lavage (BAL) after treatment with the indicated number of 100 μg intranasal doses of an A. fumigatus extract. (mean ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 Two-Way ANOVA, Bonferoni post-test). (a) AHR and (b) BAL count of Myd88^−/− and BALB/c mice administered three doses. Pooled data from three experiments is shown (WT n=9, Myd88^−/− saline n=8, A.f. n=10). (c) AHR and (d) BAL count of Myd88^−/− and BALB/c mice administered one dose. Representative data from one of two experiments are shown (n=4). (e) AHR and (f) BAL count of mice deficient for Ticam1 (Lps2/Lps2) and C57BL/6 mice as control. Pooled data from two experiments is shown (WT n = 7, Lps2 n = 6). (g) AHR and (h) BAL count of BALB/c mice that were administered an antibody to Dectin-1 or isotype control (500 μg/mouse), and intranasal A. fumigatus extract (n=5). (i) Measurement of serum IL-12 by ELISA after treatment with 100 mg Laminarin. An antibody to Dectin-1 or isotype control (500 μg/mouse) was administered 12 h prior (*** p < 0.001, student's t-test).

Figure 4. Purified and synthetic asperamide B activate mouse and human iNKT cells

(a) Structure of isolated Aspergillus fumigatus lipid, asperamide B, with a molecular weight of 738. (b) Asperamide B peak area in a liquid chromatography-mass spectometry chromatogram is shown for A. fumigatus and A. niger (*** p < 0.001, student's t-test). (c-f) Measurement of IL-4 at 48 hours in coculture supernatants of 5 × 10⁴ iNKT and 10⁴ BMDC treated with the indicated lipids (mean ± s.d.). (c) Cocultures treated with DMSO (D), purified asperamide B (pAsp-B), or PBS57 (α-GalCer analogue). (d) Cocultures treated with DMSO (D), synthetic asperamide B (sAsp-B), and PBS57. (c-d) * p < 0.05, ** p < 0.01, One-Way ANOVA, Dunnett's Multiple Comparison Test to DMSO Control. (e) Cocultures with BMDC from BALB/c, CD1d1^−/−, Myd88^−/− or Ticam1^−/− treated with the indicated lipids (** p < 0.01, *** p < 0.001, Two-Way ANOVA, Bonferoni post-test). (f) Cocultures were incubated with an antibody specific for Dectin-1 (2 μg ml⁻¹) and treated with vehicle, A. fumigatus extract, sAsp-B, or PBS57. (g) Measurement of IL-12 by ELISA at 24 hours in BMDC cultures treated with laminarin (500 μg ml⁻¹) with or without anti-dectin-1 mAb (2 μg ml⁻¹), (** p < 0.01, student's t-test). (h) Measurement of IL-12 by ELISA at 24 hours in BMDC cultures treated with A. fumigatus extract or laminarin at the indicated concentrations. (i-j) Measurement of (i) IL-4 and (j) IL-13 by ELISA at 48 hours in cocultures of primary human iNKT cell lines and APCs treated with DMSO, pAspB, sAspB, or PBS57. (* p< 0.05, *** p < 0.001, student's t-test compared to DMSO control) (a–j) mean ± s.d.

Figure 5. Asperamide B loaded CD1d stains and directly activates iNKT cells

(a) FACS plots showing spleen cells stained for CD3 and CD1d tetramers loaded with vehicle (left panel), sAsp-B (middle panel) or PBS57 (right panel). (b-c) FACS plots showing CD4 and Vβ usage by (b) sAsp-B tetramer⁺ cells and (c) PBS57 tetramer⁺ cells. (d) Cytokine production from iNKT cells cultured on plates coated with CD1d monomers loaded with sAsp-B (300 μg ml⁻¹) or no lipid. (e) iNKT stimulated with plate bound CD1d loaded with sAsp-B plus 5 μg ml⁻¹CD28 antibody. (d-e) * p < 0.05, ** p < 0.01, student's t-test. (f) Measurement of AHR and (g) BAL cell count in BALB/c treated with a single intranasal dose of vehicle (n = 9), 5 μg of pAsp-B (n = 8), or 1 μg of PBS57 (n = 9). Pooled data from three experiments are shown. (h) Measurement of IL-4 and IL-13 secreting by iNKT cells in mice treated with vehicle, pAsp-B, or PBS57. (f-h) mean ± s.e.m., ns p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001, Two-Way ANOVA, Bonferoni post-test (i-j) Measurement of AHR in (i)CD1d1^−/− or (j)Il4^−/−Il13^−/− mice treated with 5 μg of pAsp-B or vehicle. BALB/c control is the same in i and j, (WT n ≥ 19, CD1d1^−/− n ≥ 7, Il4^−/−Il13^−/− n ≥ 6). Pooled data from four experiments are shown.

Figure 6. Asperamide B-induced AHR requires MyD88 and ST2

(a-b) Measurement of AHR after treatment with a single intranasal dose of 5 μg of pAsp-B or vehicle (mean ± s.e.m., ** p < 0.01, *** p < 0.001, Two-Way ANOVA, Bonferoni post-test). (a) AHR measured in Myd88^−/− and BALB/c control mice (WT n = 4, Myd88^−/− n = 5) treated with pAsp-B. Representative data from one of two experiments are shown. (b) AHR measured in Il1rl1^−/− (ST2) and BALB/c control mice (WT n = 7, Il1rl1^−/− n = 6). Pooled data of two experiments are shown. (c) IL-33 mRNA measured by QPCR from mice treated with pAsp-B. Fold induction of IL-33 mRNA compared to the zero time-point is shown. One representative experiment of two is shown (mean ± s.e.m., *** p < 0.001 One-Way ANOVA, Tukey's multiple comparison post-test). (d) The number of IL-33⁺ lung macrophages following treatment with vehicle, 5 μg of pAsp-B, or 1 μg of PBS57. Alveolar macrophages were defined as F4/80⁺CD11c⁺ and interstitial macrophages were defined as F4/80⁺CD11c⁻. One representative experiment of two is shown (mean ± s.e.m., * p < 0.05, ** p < 0.01, Two-Way ANOVA, Bonferoni post-test). (e) IL-33 mRNA was measured by QPCR after 24 hours from cocultures of iNKT cells with alveolar macrophages incubated with DMSO, pAsp-B, or PBS57 (mean ± s.d., *** p < 0.001, student's t-test).