Fungal ligands released by innate immune effectors promote inflammasome activation during Aspergillus fumigatus infection

Abstract

Invasive pulmonary aspergillosis causes substantial mortality in immunocompromised individuals. Recognition of Aspergillus fumigatus by the host immune system leads to activation of the inflammasome, which provides protection against infection. However, regulation of inflammasome activation at the molecular level is poorly understood. Here, we describe two distinct pathways that coordinately control inflammasome activation during A. fumigatus infection. The C-type lectin receptor pathway activates both MAPK and NF-κB signalling, which leads to induction of downstream mediators, such as the transcription factor IRF1, and also primes the inflammasomes. Toll-like receptor signalling through the adaptor molecules MyD88 and TRIF in turn mediates efficient activation of IRF1, which induces IRGB10 expression. IRGB10 targets the fungal cell wall, and the antifungal activity of IRGB10 causes hyphae damage, modifies the A. fumigatus surface and inhibits fungal growth. We also demonstrate that one of the major fungal pathogen-associated molecular patterns, β-glucan, directly triggers inflammasome assembly. Thus, the concerted activation of both Toll-like receptors and C-type lectin receptors is required for IRF1-mediated IRGB10 regulation, which is a key event governing ligand release and inflammasome activation upon A. fumigatus infection.

Figure 1.. Downstream adaptors of TLRs and CLRs are required for inflammasome activation during A. fumigatus infection.

(a) Immunoblot analysis of pro-caspase-1 (p45) and the caspase-1 subunit p20 (p20) of unprimed BMDMs left untreated (medium alone [Med]) or assessed 20 h after infection with live A. fumigatus (A.f) resting conidia (multiplicity of infection [MOI], 20). (b) Release of IL-1β and (c) TNF in unprimed BMDMs left uninfected (Med) or assessed after 20 h with A.f (MOI, 20), n = 4 biologically independent samples, (d) Immunoblot analysis of phospho and total-ERK1/2 (p-ERK, t-ERK) or phospho- and total-IκBα (p-IκBα, t-IκBα) in unprimed WT and Myd88^−/−Trif^−/− BMDMs 0–8 h after infection with live A.f resting conidia (MOI, 20). (e) Immunoblot analysis of NLRP3 in unprimed BMDMs 0–8 h after infection with A.f (MOI, 20). (f) Immunoblot analysis of caspase-1 (as in Fig. 1a), (g) release of IL-1β and (h) TNF in unprimed BMDMs left uninfected (Med) or assessed after 20 h with A.f (MOI, 20), n = 5 biologically independent samples. (i,j,k) Immunoblot analysis of p-, t-ERK and p-, t-IκBα in unprimed WT, Syk^fl/flLysM^Cre, Card9^−/−, and Malt1^−/− BMDMs 0–8 h after infection with live A.f resting conidia (MOI, 20). (b,c,g,h) *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple-comparisons test). Data are representative of (a–k) at least 3 independent experiments (mean ± s.e.m.). The exact P-values are represented in the Supplementary Table 3.

Figure 2.. The SYK-CARD9-MALT1 complex regulates IRF1 expression, whereas the activation of IRF1 is MyD88/TRIF dependent.

(a,b) Immunoblot analysis of IRF1 in unprimed WT, Syk^fl/flLysM^Cre, Card9^−/−, and Malt1^−/− BMDMs 0–8 h after infection with live A.f resting conidia (MOI, 20). (c,d,e) Real-time quantitative RT-PCR analysis of Ifr1, Gbp5 and Irgb10 genes in WT, Card9^−/− and Malt1^−/− BMDMs 0–8 h after infection with A.f presented relative to that of the gene encoding β-actin. (f) Immunoblot analysis of p-, t-IκBα and IRF1 expression in unprimed WT BMDMs and WT BMDMs incubated with NF-κB inhibitor Bay 11-7082. (g) Immunoblot analysis of IRF1 in unprimed WT and Myd88^−/−Trif^−/− BMDMs 0–8 h after infection with live A.f resting conidia (MOI, 20). (h,i,j,k) Real-time quantitative RT-PCR analysis of Ifr1, Gbp2, Gbp5, and Irgb10 genes in WT and Myd88^−/−Trif^−/− BMDMs 0–8 h after infection with A.f presented relative to that of the gene encoding β-actin. (I,m) Immunoblot analysis of IRF1 (top lanes), LaminB (middle lanes, nuclear fraction loading control), and GAPDH (bottom lanes, cytoplasmic fraction loading control) in unprimed (I) nuclear fraction and (m) cytoplasmic fraction of BMDMs 0–8 h after infection with A.f (MOI, 20). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA with Sidak’s multiple-comparisons test), ****(a–m) Data are representative of at least 3 independent experiments (mean ± s.e.m.). The exact P-values are represented in the Supplementary Table 3.

Figure 3.. IRF1 regulates the expression of GBPs and IRGB10 upon A. fumigatus infection.

(a) Immunoblot analysis of caspase-1 (as in Fig. 1a), (b) release of IL-1β, and (c) TNF in unprimed WT and Irf1^−/− BMDMs left uninfected (Med) or assessed after 20 h with A.f (MOI, 20), n = 4 biologically independent samples. (d,e) Immunoblot analysis of caspase-1 in LPS and β-glucan primed WT and Irf1^−/− BMDMs assessed 20 h after infection with live A.f resting conidia (MOI, 20). (f) Immunoblot analysis of IRF1, GBP2, GBP5, and IRGB10 in unprimed WT and Irf1^−/− BMDMs 0–8 h after infection with live A.f resting conidia (MOI, 20). (g,h,i) Real-time quantitative RT-PCR analysis of Gbp2, Gbp5 and Irgb10 genes in WT and Irf1^−/− BMDMs 0–8 h after infection with A.f resting conidia (MOI, 20) presented relative to that of the gene encoding β-actin. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple-comparisons test and Sidak’s multiple-comparisons test), (a–l) Data are representative of at least 2 independent experiments (mean ± s.e.m.). The exact P-values are represented in the Supplementary Table 3.

Figure 4.. IRGB10 and not GBPs contributes to the activation of inflammasomes in response to A. fumigatus.

(a,d) Immunoblot analysis of caspase-1 (as in Fig. 1a), (b,e) release of IL-1β and (c,f) TNF by unprimed WT, (a,b,c) Gbp^chr3−/− and (d,e,f) Irgb10^−/− BMDMs left uninfected (Med) or assessed 20 h with A.f resting conidia (MOI, 20). (b,c) n = 4 biologically independent samples and (e,f) n = 7 biologically independent samples) (g) Immunoblot analysis of p-, t-ERK , p-, t-IκBα, and NLRP3 (as in Fig. 2d,e) in unprimed WT and Irgb10^−/− BMDMs 0–12 h after infection with A.f resting conidia (MOI, 20). (h,i) Real-time quantitative RT-PCR analysis of ll1β and Tnf genes in WT and Irgb10^−/− BMDMs 0–8 h after infection with A.f resting conidia (MOI, 20), presented relative to that of the gene encoding β-actin. (j) Immunoblot analysis of caspase-1 in LPS primed WT and Irgb10^−/− BMDMs assessed 20 h after infection with live A.f resting conidia (MOI, 20). (k) Conidiocidal activity of WT and Irgb10^−/− BMDMs. (I) A.f hyphae inhibition assay of WT and Irgb10−/− BMDMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple-comparisons test and Sidak’s multiple-comparisons test and unpaired t-test two tailed P-value). (a–i) Data are representative of at least 3 independent experiments (mean ± s.e.m.). The exact P-values are represented in the Supplementary Table 3.

Figure 5.. IRGB10 targets intracellular A. fumigatus and IRGB10 peptides inhibit A. fumigatus growth and release fungal ligands which induce inflammasome activation.

(a–c) Immunofluorescence staining of IRGB10 (red) and A.f cell wall (blue) in unprimed primary lung fibroblasts 16 h after infection with A.f. Images were taken by structured illumination microscopy (SIM), (a) A z-plane of a SIM image, (b) Orthogonal-image projection of A.f. (c) Three-dimensional projection of A.f hyphae presented in b. Scale bars 1 μm (a–c). (d) A.f growth in presence of increasing concentrations of IRGB10 peptides 1 and 2 respectively normalized to scramble petides 1 (n = 4 biologically independent samples) and 2 (n = 5 biologically independent samples), (e) A.f growth in increasing concentrations of IRGB10 peptides 3 and scramble peptide 3 (n = 4 biologically independent samples), (f) peptide 4 and 5 normalized to vehicle control (n = 6 biologically independent samples). (g) Conidiocidal (n = 2 biologically independent samples) and (h) hyphae damage activity of IRGB10 peptides 1, 2 and 3 (n = 5 biologically independent samples), (i) Scanning electron microscopy surface analysis of A.f hyphae incubated for 5 h with IRGB10 peptides 1, 2, 3 and vehicle control, (j) Immunoblot analysis of pro-caspase-1 (p45) and the caspase-1 subunit p20 (p20) of unprimed BMDMs left untreated (medium alone [Med]) or assessed after curdlan transfection with Xfect (middle, [Curdlan Xfect]) or Xfect alone (right, [Xfect]). (a-j) Data are representative of at least 2 independent experiments (mean ± s.e.m.).

Figure 6.. IRGB10 provides host protection against infection with A. fumigatus in vivo.

(a) Survival of 7- to 8-week-old wild-type mice (n = 10) and Gbp^chr3−/− mice (n = 12) immunosuppressed with cyclophosphamide and cortisone acetate and infected intranasally with 5.5 × 10⁵ of A.f resting conidia. (b) Survival of 7- to 8-week-old wild-type mice (n = 21) and Irgb10^−/− mice (n = 30) infected intranasally with 5.5 × 10⁵ of A.f resting conidia after immunosuppression with cyclophosphamide and cortisone acetate. (c) Macroscopic pathology of A.f–infected left lobe of lungs collected on day 4 post-infection or uninfected, pictures collected with camera, (d) Hematoxylin-and-eosin and (e) gomori methenamine silver staining and staining on day 4 after infection with A.f. (f) Real-time quantitative PCR analysis of A.f 18S rRNA levels in the lungs of WT and Irgb10^−/− mice on day 4 after infection, WT and Irgb10^−/− mice n = 14. (g) Immunoblot analysis of pro-caspase-1 (Casp-1, p45) and the caspase-1 subunit p10 (Caspl, p10) of lung homogenates after 4 days of infection with A.f resting conidia. (h) Levels of IL-1β and IL6 in lung homogenates after 2 days of infection with A.f resting conidia. **P < 0.01 and ****P < 0.0001, (a,b) log-rank (Mantel-Cox) test and (f) Mann-Whitney test two tailed P-value (h) unpaired t-test two tailed P-value. (a,b) Data are from 2 independents experiments and (c–h) data are representative of at least 1 experiment (mean ± s.e.m.). (d,e) Scale bars 200 μm (10X) and 50 pm (60X). The exact P-values are represented in the Supplementary Table 3.