Inflammasomes are important sentinels of innate immune defence that are activated in response to diverse stimuli, including pathogen-associated molecular patterns (PAMPs)1. Activation of the inflammasome provides host defence against aspergillosis2,3, which is a major health concern for patients who are immunocompromised. However, the Aspergillus fumigatus PAMPs that are responsible for inflammasome activation are not known. Here we show that the polysaccharide galactosaminogalactan (GAG) of A. fumigatus is a PAMP that activates the NLRP3 inflammasome. The binding of GAG to ribosomal proteins inhibited cellular translation machinery, and thus activated the NLRP3 inflammasome. The galactosamine moiety bound to ribosomal proteins and blocked cellular translation, which triggered activation of the NLRP3 inflammasome. In mice, a GAG-deficient Aspergillus mutant (Δgt4c) did not elicit protective activation of the inflammasome, and this strain exhibited enhanced virulence. Moreover, administration of GAG protected mice from colitis induced by dextran sulfate sodium in an inflammasome-dependent manner. Thus, ribosomes connect the sensing of this fungal PAMP to the activation of an innate immune response.
Specific PAMPs that activate the inflammasome in response to bacterial and viral infection are known1, but the identities of fungal PAMPs remain unclear. Conidia of A. fumigatus do not activate the inflammasome, whereas hyphae of this species are competent for inflammasome activation4. We hypothesized that the component that activates the inflammasome is present only in hyphal A. fumigatus. The fungal cell-wall surface is composed largely of polysaccharides, and this composition changes throughout the fungal life cycle5. GAG is present on hyphae but is completely absent on conidia, and it is considered to be a major virulence factor6,7. Polysaccharides from other microbial pathogens trigger activation of the inflammasome2,8,9. We therefore hypothesized that GAG might be central for inflammasome activation induced by A. fumigatus.
To test this, we generated a strain of A. fumigatus that does not produce GAG. Despite extensive studies on GAG synthesis in A. fumigatus, the GAG synthase has not previously been identified10. We bioinformatically characterized the GAG biosynthetic gene cluster11, and found that GT4C (AFUA_3G07860) is potentially the GAG synthase (Extended Data Fig. 1a). Genes in the cluster containing GT4C were upregulated during A. fumigatus growth (Extended Data Fig. 1b, c). GT4C possesses transmembrane regions and is part of the major facilitator superfamily (Extended Data Fig. 1d). To confirm that GT4C is involved in GAG synthesis, we generated an A. fumigatus strain that lacks GT4C (Δgt4c) (Extended Data Fig. 1e, f). GAG is required for A. fumigatus adherence7. ∆gt4c mycelium lost their adherence capability (Fig. 1a, b), and introduction of purified GAG from wild-type A. fumigatus partially restored its adhesion (Fig. 1c), which suggests that the GT4C gene is involved in adherence and the synthesis of GAG. In addition, scanning electron microscopy showed that wild-type fungus was decorated with fibrillar material in the extracellular matrix, which is characteristic of GAG being present6,7, and the fibrils adhered to both fungal cells and abiotic surfaces (arrows in Fig. 1d). These structures were completely absent in the ∆gt4c strain6,7 (Fig. 1d), and immunostaining with an anti-GAG monoclonal antibody confirmed the absence of GAG (Fig. 1e).
GAG is a large polysaccharide polymer that is composed of α1,4-linked galactose and α1,4-linked N-acetylgalactosamine or galactosamine (GalN) residues randomly distributed6 (Fig. 1f). The ∆gt4c-mutant cell wall completely lacked the GalN residue, whereas the parental strain contained GalN (Fig. 1g–i). Together, these data confirm that GT4C is essential for GAG synthesis and that the GT4C protein is the putative GAG synthase for A. fumigatus. We also found that the other genes of the cluster that is required for GAG synthesis10 were not downregulated in the absence of GT4C compared with their expression in wild-type A. fumigatus (Extended Data Fig. 1g).
To define the role of GAG in innate immunity and activation of the inflammasome, we first confirmed that the GAG biosynthetic gene cluster was expressed and that GAG was synthesized by wild-type A. fumigatus and present in the cytosol during infection of bone-marrow-derived macrophages (BMDMs) (Fig. 2a, b). Cleavage of caspase 1 and the release of IL-1β were impaired in unprimed BMDMs infected with the ∆gt4c strain compared with those infected with wild-type A. fumigatus, whereas the secretion of inflammasome-independent cytokines was not affected (Fig. 2c, d, Extended Data Fig. 2). We further confirmed that this impaired activation of the inflammasome was due to the absence of GAG by using another A. fumigatus strain (∆uge3), which cannot produce GAG owing to the deletion of UDP-glucose-4-epimerase (UGE3)7 (Extended Data Fig. 3a, b).
We next determined the ability of the ∆gt4c and ∆uge3 strains of A. fumigatus to activate the inflammatory signalling required for priming of the inflammasome. Phosphorylation of extracellular signal-regulated kinases 1 and 2 (hereafter, ERK1/2) and IκBα was not reduced during infection with ∆gt4c and ∆uge3 A. fumigatus compared with the wild type (Fig. 2e, f, Extended Data Fig. 3c). Consistently, wild-type, ∆gt4c and ∆uge3 A. fumigatus induced comparable NLRP3 and pro-IL-1β protein expression after infection (Fig. 2g, Extended Data Fig. 3d). Therefore, A.-fumigatus-induced inflammasome priming was independent of GAG, possibly owing to other PAMPs on the surface of A. fumigatus and the contribution of other polysaccharides such as β-glucan12. There were no substantial differences in the expression of Nlrp3, Il1b and Tnf in BMDMs infected with wild-type A. fumigatus or the ∆gt4c or ∆uge3 strains (Fig. 2h–j, Extended Data Fig. 3e–g). Furthermore, priming with lipopolysaccharide did not rescue the defect in caspase 1 activation induced by the ∆gt4c strain (Fig. 2k), which further confirms that the defective inflammasome response was independent of priming but dependent on inflammasome activation itself.
We also found that BMDMs infected with the ∆ugm1 strain of A. fumigatus, which produces more GAG than does the parental A. fumigatus7 (Extended Data Fig. 4a), showed enhanced cleavage of caspase 1 and IL-1β release compared with wild-type A. fumigatus (Extended Data Fig. 4b, c). This confirmed the crucial role of GAG in inflammasome activation mediated by A. fumigatus.
It has recently been shown that cytosolic delivery of β-glucan activates the inflammasome2. Upon infection, Aspergillus conidia inflate and germinate inside the host cell, which releases GAG into the cytosol (Fig. 2b, Extended Data Fig. 4a). Cytosolic delivery of GAG or flagellin induced caspase 1 cleavage and cell death in BMDMs, which confirms the ability of cytosolic GAG to induce activation of the inflammasome (Fig. 3a–c, Extended Data Fig. 5a, b). A. fumigatus induces activation of both the NLRP3 and AIM2 inflammasomes3. However, with GAG transfection, NLRP3 was required for caspase 1 cleavage and cell death—but AIM2 was not (Fig. 3d, e). This shows that GAG specifically induces activation of the NLRP3 inflammasome. In addition, GSDMD was dispensable for activation of the NLRP3 inflammasome during GAG transfection (Extended Data Fig. 5c).
The bioactivity of bacterial or fungal exopolysaccharides such as GAG is highly dependent on their degree of acetylation11. We observed that acetylated GAG (Ac-GAG) did not induce notable caspase 1 cleavage and cell death whereas deacetylated GAG (d-GAG) did (Fig. 3f, g), which suggested that the galactosamine subunit of GAG is required for inflammasome activation and cell death. To further confirm the role of d-GAG, we infected BMDMs with the ∆agd3 strain of A. fumigatus, which produces only acetylated GAG owing to loss of N-acetyl glucosamine deacetylase11. Similar to the effect of treatment with Ac-GAG (Fig. 3f), the ∆agd3 strain did not induce caspase 1 cleavage (Fig. 3h)—further confirming that the presence of galactosamine in GAG is crucial for activation of the inflammasome.
We then identified ribosomal proteins as interacting partners of GAG (Fig. 3i, Supplementary Table 1) but not of β-glucan (Extended Data Fig. 5d). We validated that RPL14 interacted specifically with GAG and not with β-glucan (Fig. 3j). Furthermore, d-GAG—but not Ac-GAG or β-glucan—interacted with the ribosomal proteins that we found in our mass spectrometry analysis (RPL6, RPL7a and RPL14) (Fig. 3k). The addition of NaCl inhibited this interaction, which suggests that charge–charge interactions are responsible for the binding (Extended Data Fig. 5e). To confirm this, we complemented the reaction medium with NaCl during GAG transfection; this inhibited the GAG–ribosome interaction and protected against inflammasome activation and cell death (Extended Data Fig. 5f–h). We also observed that purified ribosomes incubated with GAG became clumped (Extended Data Fig. 5i). Together, these data suggest that GAG traps ribosomes and inhibits translation elongation and/or termination. These findings also confirmed that the galactosamine moiety is responsible for GAG trapping ribosomal proteins through charge–charge interactions, resulting in activation of the inflammasome.
Because aberrations in ribosomes and translation inhibition can drive cell death13, we quantified the total translation activity in BMDMs transfected with GAG. Both GAG and d-GAG rapidly and strongly inhibited translation (Fig. 3l, Extended Data Fig. 6a), whereas Ac-GAG was unable to inhibit translation—similar to flagellin and double-stranded DNA, two other known triggers of the inflammasome (Fig. 3l, m, Extended Data Fig. 6b). Infection with A. fumigatus also inhibited translation (Extended Data Fig. 6c).
Using polysome profiling to investigate how GAG inhibited translation, we observed that the size of the 80S peak remained small in the GAG-transfected samples compared with samples transfected with N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP) alone, suggesting stabilization of the polysomes in the former (Extended Data Fig. 6d). A fraction of the ribosomal proteins precipitated out with GAG during the analysis (Extended Data Fig. 6e). This may explain the similar polysome peak sizes we observed in the GAG-transfected and DOTAP-transfected samples (Extended Data Fig. 6d), as larger polysomes are more likely to form GAG–ribosome precipitates. The polysome-to-monosome ratio also increased during GAG transfection (Extended Data Fig. 6f). These data suggest that GAG immobilizes the ribosome to inhibit translation (Fig. 3l, Extended Data Fig. 6d–f).
To confirm that inhibition of translation triggers activation of the inflammasome, we stimulated BMDMs with the translation inhibitors anisomycin, puromycin or cycloheximide14. BMDMs treated with any of the three classes of inhibitors (initiation, elongation or termination inhibitors) showed caspase 1 cleavage and pyroptosis in an NLRP3-dependent manner, as we also observed with GAG (Fig. 3n–p, Extended Data Fig. 6g–i). Collectively, these data show that the galactosamine unit of GAG interacts with ribosomal proteins to shut down translation, and thus activate the NLRP3 inflammasome.
Both ribosomes and the endoplasmic reticulum are central to protein synthesis and quality control. Defects in these processes can lead to endoplasmic reticulum stress, which mediates activation of the NLRP3 inflammasome15. We observed that GAG transfection induced PERK phosphorylation and the expression of IRE1α, two markers of endoplasmic reticulum stress (Extended Data Fig. 6j); similar induction of PERK activation was observed upon treatment with translation inhibitors (Extended Data Fig. 6k). Additionally, GAG induced protein polyubiquitination (Extended Data Fig. 6l), which suggests an accumulation of misfolded proteins that should be degraded via the unfolded protein response and proteasome16. Inhibition of proteasome activity during GAG transfection abolished caspase 1 activation (Extended Data Fig. 6m), which suggests a mechanism that is dependent on the unfolded protein response for inflammasome activation in response to GAG. Together, these data suggest that GAG inhibits translation and induces endoplasmic reticulum stress, which activates the unfolded protein response to mediate activation of the NLRP3 inflammasome.
It has recently been shown that—in response to stressors—cell fate is decided by the localization of DDX3X molecules to stress granules (prosurvival) or the NLRP3 inflammasome (propyroptosis)17. GAG did not induce stress granules containing G3BP1 and DDX3X, nor did A. fumigatus infection (Extended Data Fig. 7a–c). DDX3X was required for inflammasome activation in response to GAG transfection and A. fumigatus infection (Extended Data Fig. 7d–f).
To investigate whether the protection provided by the inflammasome during aspergillosis2,3,18 is mediated by GAG, we infected immunocompromised wild-type mice with wild-type or ∆ugm1 (GAG-overproducing strain) A. fumigatus strains. As expected, mice infected with the ∆ugm1 strain showed a survival rate higher than that of mice infected with wild-type A. fumigatus (Fig. 4a). Casp1−/−Casp11−/− (Casp11 is also known as Casp4) mice, which lack components of the inflammasome, showed a higher mortality rate than wild-type mice when infected with either wild-type A. fumigatus or the ∆ugm1 A. fumigatus strain (Fig. 4b). Although the ∆ugm1 strain was less virulent than wild-type A. fumigatus in wild-type mice, the two strains were equally virulent in Casp1−/−Casp11−/− mice (Fig. 4b). The ∆ugm1 strain also induced more IL-1β release in wild-type mice than did the wild-type strain (Extended Data Fig. 8a, b). Together, these data suggest that activation of the inflammasome in response to GAG limits the virulence of the ∆ugm1 strain in wild-type mice.
The survival rates of immunocompromised wild-type mice infected with wild-type and ∆gt4c A. fumigatus strains were similar (Fig. 4c). However, mice infected with the ∆gt4c strain looked more hunched than those infected with the wild-type strain (data not shown). Moreover, immunocompetent wild-type mice lost more body weight when infected with the ∆gt4c strain as compared to wild-type A. fumigatus (Fig. 4d). In a systemic model, direct injection of ∆gt4c-mutant conidia into the blood stream showed significantly higher virulence compared to wild-type A. fumigatus conidia, whereas the ∆ugm1 mutant was less virulent (Fig. 4e, Extended Data Fig. 8c). In addition, IL-1β and IL-18 release were reduced in the liver of mice infected with the ∆gt4c strain as compared with wild-type A. fumigatus (Extended Data Fig. 8d, e). These results collectively demonstrate that GAG-mediated activation of the inflammasome is required for host defence against aspergillosis in mice.
We further extended our study of the role and physiological relevance of GAG-mediated activation of the inflammasome using a model of colitis induced by dextran sulfate sodium, in which inflammasome activation is largely protective19,20. We observed that wild-type mice treated with GAG did not lose body weight and were protected from inflammation (Fig. 4f–k). Although levels of pro-inflammatory cytokines secreted in the colon were reduced in GAG-treated mice compared with levels in vehicle-treated mice (Extended Data Fig. 8f–k), levels of the inflammasome-dependent cytokine IL-18 were increased in both serum and colon lysates upon GAG treatment (Fig. 4l, m). In addition, GAG treatment did not provide protection to Il18−/− mice during DSS-induced colitis (Fig. 4n–p), which suggests that GAG-induced IL-18 production is crucial for protection against colitis.
In summary, we have identified the d-GAG of A. fumigatus as a PAMP that activates the NLRP3 inflammasome. Mechanistically, the galactosamine subunit interacts with ribosomal proteins through charge–charge interactions to inhibit translation and induce endoplasmic reticulum stress, and thus causes activation of the inflammasome. Ribosomes are the most abundant organelle in the cell and are required for translation and cellular homeostasis. Aberrant protein synthesis is therefore a danger signal. Ribosomes may act as cytosolic sensors of intracellular GAG that initiate activation of a protective NLRP3-inflammasome response. Ribosomes have previously been proposed as a metabolic sensor in bacteria that allows them to adapt in response to the environment21,22. Although aberrant cellular ionic exchange, lysosomal rupture, mitochondria destabilization, endoplasmic reticulum stress23 and trans-Golgi network disassembly24 have each been proposed as mechanisms that contribute to activation of the NLRP3 inflammasome, the exact mechanism is debated. A loss of cellular potassium—an event that often occurs during the activation of the NLRP3 inflammasome—induces translation inhibition via ribotoxic stress25. In addition, reactive oxygen species and cellular oxidative stress inhibit translation26,27. Reactive oxygen species and mitochondria are associated with activation of the NLRP3 inflammasome. These observations suggest that ribosomes and translation inhibition could be an important mechanism for the activation of the NLRP3 inflammasome. However, the role of ribosomes and translation during activation of the NLRP3 inflammasome by other triggers and pathogens needs further investigation. Our data provide a paradigm in which ribosomes have a crucial role in driving inflammasome activation and promoting an effective innate immune response against fungal pathogens.
No statistical methods were used to predetermine sample size. Mice from the same cage were randomly selected for different treatments. For in vitro experiments, cells from the same pool of BMDMs were randomly split into separate wells and subjected to the treatments. The pathologist was blinded for examination of histological analyses. For survival and body weight analyses, blinding was not performed.
Aspergillus strain and culture
Aspergillus fumigatus A1160, ∆gt4c, ∆ugm128, AF293 (NCPF 7367), ∆agd311 and ∆uge37 strains were grown on 2% (w/v) malt/2% (w/v) agar slants for 1 week. Conidia were collected in PBS containing 0.1% (v/v) Tween 2029.
Casp1–/–Casp11–/– (ref. 30), Nlrp3–/– (ref. 31), Aim2–/– (ref. 32), Il18–/– (ref. 3), Nlrp3–/–Aim2–/– (ref. 3), Gsdmd–/– (ref. 33) and Ddx3xfl/flLysMcre (ref. 17) mice have previously been described. All mice were backcrossed to the C57BL/6 background. Mice were bred at St Jude Children’s Research Hospital (St Jude). Animal studies were conducted under protocols approved by the St Jude Animal Care and Use Committee. Mice were kept with a 12:12 light:dark cycle. Humidity was maintained between 30 and 70% and the temperature ranged from 20 to 23.3 °C. Prior sample size determination was not done. Animals from the same cage were randomly selected for different treatments. The pathologist was blinded for examination of histological analyses.
Generation of A. fumigatus Δgt4c deletion strains
The deletion mutants were constructed in the A1160 background29 using the β-rec/six site-specific recombination system34. The self-excising β-rec/six blaster cassette containing the hygromycin resistance marker was released from the plasmid pSK529 via FspI restriction enzyme digestion. Using GeneArt Seamless Cloning and Assembly (Life Technologies), the GT4 replacement cassette containing the marker module flanked by 5′ and 3′ homologous regions of the target gene generated by PCR was cloned into the pUC19 vector. The corresponding replacement cassettes were released from the resulting vector via EcoRV or DraI digestion, respectively. The A1160 parental strain was transformed with the GT4C replacement cassettes by electroporation to generate the single-deletion mutants. Transformants obtained were analysed by Southern blot using the DIG probe protocol (Roche Diagnostics) (Extended Data Fig. 1e, f, Extended Data Table 1).
Cell-inert surface assay
The cell-inert surface adhesion assay was performed on an abiotic surface (tissue culture test plate 24, TPP). Liquid medium (1 ml) containing 104 A. fumigatus conidia was incubated at 37 °C for 20 h. After washing with water, adherent mycelium was estimated, as previously described, with Crystal violet staining35.
Carbohydrate analysis of the cell wall and culture supernatant fractions
After 48 h of growth in Brian liquid medium shaking at 37 °C and 150 rpm, mycelia and culture supernatant were separated by filtration. Macromolecules from the supernatant were precipitated by 3 volumes of ethanol at 4 °C overnight and collected by centrifugation (5 min, 4,000g). Cell wall fractions were obtained after mycelium disruption and centrifugation as previously described36. Polysaccharides from the cell wall were separated on the basis of their alkali solubility36. Neutral hexoses were estimated by the phenolsulfuric method using glucose as a standard37. Osamines were quantified by high-performance liquid chromatography after acid hydrolysis by 6 N HCl at 100 °C for 6 h38. The amount of de-N-acetylated galactosamine in the GAG fraction was estimated by assaying for anhydrotalose formed after nitrous deamination39. In brief, 500 μl of sample containing up to 40 μg of osamine was treated with 500 μl of 5% KHSO4 and 500 μl of 5% NaNO2 for 2 h at 50 °C. After cooling, the excess nitrous acid was destroyed by 500 μl of 12.5% ammonium sulfamate for 5 min at room temperature. Then, 500 μl of 0.5% 3-methyl-2-benzothiazolinone hydrazone monohydrate (Sigma) was added and the mixture was allowed to stand for 30 min at 37 °C before the addition of 500 μl of 0.5% FeCl3. The blue colour was allowed to develop for 30 min at room temperature and the absorbance was measured at 650 nm. Proteins were quantified by the BCA assay (Thermo Fisher Scientific) using BSA as a standard. Monosaccharides were identified and quantified by gas–liquid chromatography after acid hydrolysis with 4 N trifluoroacetic acid at 100 °C for 4 h36. Glycosidic linkage analysis was performed by methylation using the lithium methyl sulfinyl carbanion procedure and identified as partially methylated acetate alditol by gas chromatography–mass spectrometry, as previously described40.
GAG purification and biochemical modifications (de-N-acetylation and acetylation)
GAG production and chemical modifications were carried out as previously described6,41. For de-N-acetylation, GAG was suspended in 3 ml of 10 mM HCl at a final concentration of 3.33 mg/ml by sonication in plastic tubes. The de-N-acetylation was started by addition of 3.4 ml 18.8 M NaOH, and the mixture was incubated at 100 °C for up to 4−5 h. The tubes were vortexed every hour. The reaction was stopped on ice and neutralized with 12 N HCl. De-N-acetylated GAG was dialysed against milli-Q water and finally lyophilized. For acetylation, GAG was dissolved in 25 μl of 400 mM acetic acid and 100 μl CH3OH. The mixture was preincubated for 1 h at ambient temperature with agitation at 300 rpm. Acetylation was initiated by addition of 3 μl acetic anhydride for 1 h. After dialysis with milli-Q water, the sample was lyophilized.
In vitro stimulation of BMDMs
Primary BMDMs were grown for 6 days in IMDM (12440-053, Gibco) supplemented with 10% FBS (S1620, lot number 221C16, Biowest), 30% L929-conditioned medium and 1% penicillin and streptomycin. BMDMs (1 × 106) were seeded in 12-well cell culture plates (3513, Costar) in DMEM (11995-065, Gibco) supplemented with 10% FBS and 1% penicillin–streptomycin before stimulation with ligands. For infection, granulocyte macrophage colony-stimulating factor-derived bone marrow cells (denoted as BMDMs) were prepared as previously described2 and infected with A. fumigatus conidia at a MOI of 10 for 18 h. For priming, BMDMs were incubated with 100 ng/ml of LPS (tlrl-smlps, Invivogen) for 3 h and washed before infecting with A. fumigatus conidia2.
For stimulation with translation inhibitors, BMDMs were incubated with 1 μg/ml of Pam3CSK4 (tlrl-pms, Invivogen) for 4 h, washed and incubated with 25 μg/ml of anisomycin (11308, Cayman Chemical), 50 μg/ml of cycloheximide (01810, Sigma-Aldrich) or 50 μg/ml of puromycin (ant-pr-1, Invivogen).
Ligand transfection into BMDMs
For polysaccharide (GAG or β-glucan (also known as curdlan); C7821, Sigma) or flagellin (tlrl-epstfla, Invivogen) transfection, BMDMs were incubated with 1 μg/ml of Pam3CSK4 (tlrl-pms, Invivogen) for 4 h, washed and incubated for 1 h before transfection in HBSS/Modified (HyClone, SH30031.02). Then, 20 μg/ml GAG, Ac-GAG, d-GAG or β-glucan were resuspended in HCl 0.01 N or 2 μg/ml flagellin was resuspended in LAL water (Invivogen) and mixed with DOTAP (Roche, 11202375001) as per the manufacturer’s protocol.
For poly(dA:dT) (tlrl-patn, Invivogen) transfection, the BMDMs were incubated with 1 μg/ml of Pam3CSK4 for 4 h, washed and then incubated 1 h before transfection in Opti-MEM (31985-070, Thermo Fisher Scientific). One μg/ml of poly(dA:dT) was mixed with Xfect (631318, Takara) as per the manufacturer’s protocol. The BMDMs were stimulated with mixed solutions for 3 h before cell lysate collection.
For inhibition of the proteasome or salt treatment during GAG transfection, the medium was complemented 10 min before transfection with 30 μM of MG132 (M8699, Sigma Aldrich) or 200 mM of NaCl (BP358, Fisher).
To measure polyubiquitination, the cells were washed with PBS and collected with RIPA buffer complemented with 10 μM of N-ethylmaleimide (NEM).
Real-time cell-death analysis
Real-time cell-death assays were performed using a two-colour IncuCyte S3 incubator imaging system (Essen Biosciences). BMDMs (80,000 per well) were seeded in 96-well tissue culture plates (3596, Costar) in the presence of 20 nM Sytox Green (Thermo Fisher Scientific, S7020) in the corresponding medium or buffer used for the ligand transfection method (as described in ‘Ligand transfection into BMDMs’). The images were acquired every 30 min for 3 h at 37 °C and 5% CO2. The resulting images were analysed using the software package supplied with the IncuCyte imager (IncuCyte S3, v.2018C), which counts the number of Sytox-Green-positive BMDM nuclei (Sytox+ BMDM nuclei) present in each image.
Translation rate of BMDMs
For translation rate quantification, BMDMs were transfected as described in ‘Ligand transfection into BMDMs’ with GAGs, flagellin or poly(dA:dT) or infected with A. fumigatus. For translation inhibition controls, the cells were stimulated with 50 μg/ml anisomycin or incubated in PBS. Ten minutes before the collection of cell lysates, 10 μg/ml of puromycin (ant-pr, Invivogen) was added. BMDMs were lysed in 1× RIPA buffer and sample loading buffer for immunoblotting analysis.
Polysaccharide pull-down analysis
For identification of proteins interacting with GAG, the BMDMs were lysed in NP40 buffer (50 mM HEPES, 150 mM NaCl, 1% NP40, 50 mM EDTA, pH 7.4 ± 0.2) and centrifuged for 10 min at 10,000g to remove cell debris. Cell lysate and GAGs (GAG, Ac-GAG and d-GAG) or β-glucan were incubated for 2 h with rotation at room temperature with or without 1.2 M NaCl (BP358, Fisher). The samples were washed 6 times with NP40 buffer after 10 min of 10,000g centrifugation at 4 °C. After washes, the polysaccharides pulled down were eluted in sample buffer and used for immunoblotting analysis or mass spectrometry analysis on the Q-Exactive mass spectrometer as previously described17.
BMDMs (12 × 106) were transfected with DOTAP + GAG or DOTAP alone and incubated for 1 h. Polysome profiling was performed as previously described42, with minor modifications. In brief, clarified cell lysates were loaded on top of a 5–50% sucrose gradient and spun at 133,000g in an SW-60 rotor for 2 h at 4 °C. Absorbance at UV 260 nm was used to track separation of the polysomes in the sucrose gradient. Area under the curve was calculated in GraphPad Prism 8.0.
The ribosomes of BMDMs were purified as previously described42, with minor modifications. In brief, 20 × 106 wild-type BMDMs were collected and washed 3 times with PBS. Then, the cells were lysed in standard buffer (10 mM Tris pH 7.4, 5 mM BME, 50 mM ammonium chloride and 5 mM magnesium acetate), and the lysate was clarified by centrifugation at 20,000g for 30 min at 4 °C. The clarified lysate was loaded on a discontinuous sucrose gradient (5% and 20% sucrose made in wash buffer (10 mM Tris pH 7.4, 5 mM BME, 500 mM ammonium chloride and 100 mM magnesium acetate)). The cells were spun at 60,000g in an SW-55Ti rotor for 16 h at 4 °C. The ribosome pellet was resuspended in standard buffer and spun at 10,000g for 10 min at 4 °C to remove insoluble debris.
In vivo A. fumigatus infection
Mice were infected with A. fumigatus as previously described18. In brief, cyclophosphamide monohydrate (C0768, Sigma) was dissolved in sterile PBS and administered intraperitoneally (150 mg per kg of body weight). Cortisone 21-acetate (C3130, Sigma) was suspended in 0.1% Tween 20 in PBS and subcutaneously injected (112 mg per kg of body weight). Male and female mice aged 7 to 8 weeks were used for this study. Mice were given a combination of cyclophosphamide and cortisone acetate 2 days before infection and on the day of infection for the immunocompromised model of pulmonary aspergillosis or directly infected without cyclophosphamide and cortisone acetate treatment for the immunocompetent model. Mice were anaesthetized by isoflurane inhalation and inoculated intranasally with 1 × 105 to 1 × 106 conidia of A. fumigatus in 30 μl of 0.1% Tween 20 in PBS for the immunocompromised model or 108 conidia of A. fumigatus in 30 μl 0.1% Tween 20 in PBS for the immunocompetent pulmonary model. For the immunocompetent systemic model, mice were injected in the retro-orbital vein with 106 conidia in 150 μl 0.1% Tween 20 in PBS. Fungal burden was quantified by RT–PCR as previously described43. For the measurement of in vivo cytokines released during pulmonary aspergillosis (wild-type and ∆ugm1 A. fumigatus strains), bronchioalveolar lavage was processed with 1 ml of PBS 2 days after infection, and the cytokines were analysed. For the systemic model (wild-type and ∆gt4c A. fumigatus strains), the livers of infected mice were extracted, and the cytokines in the liver lysate were measured 16 h and 24 h after infection for IL-1β and IL-18, respectively.
In vivo DSS-induced colitis
The DSS-induced colitis model was carried out as previously described44. In brief, male and female mice aged 7 to 8 weeks were supplemented with 3% DSS (9011-18-1, Thermo Fisher Scientific) in the drinking water for 7 days and injected daily intraperitoneally with 1 mg/kg mouse weight GAG or vehicle control for 7 days (concurrent with DSS treatment) followed by regular drinking water for 3 days. For the measurement of in vivo cytokines released in the DSS-induced colitis model, serum and colon lysates were collected at day 10.
To check for caspase 1 cleavage by western blot analysis, cells and supernatants were lysed in caspase lysis buffer and sample loading buffer containing SDS and 100 mM dithiothreitol. To check for signalling molecules, BMDMs were lysed in 1× RIPA buffer and sample loading buffer as previously described3. Proteins (15 μg) were separated on 8–10% or 12% polyacrylamide gels. After electrophoretic transfer of protein onto PVDF membranes (EMD Millipore), membranes were blocked in 5% skim milk and incubated with primary antibodies against caspase 1 (AG-20B-0042, Adipogen, 1:1,000), p-IκBα (2859, Cell Signaling, 1:1,000), total IκBα (9242, Cell Signaling, 1:1,000), p-ERK (9101, Cell Signaling, 1:1,000), total ERK (9102, Cell Signaling, 1:1,000), NLRP3 (AG-20B-0006, Adipogen, 1:1,000), pro-IL-1β (12507, Cell Signaling, 1:1,000), β-actin (8H10D10, Cell Signaling, 1:1,000), RPL6 (A15094, ABclonal, 1:1,000), RPL7a (A14060, ABclonal, 1:1,000), RPL14 (A6724, ABclonal, 1:1,000), p-PERK (3179S, Cell Signaling, 1:750–1:1,000), total PERK (3192, Cell Signaling, 1:1,000), IRE1α (3294S, Cell Signaling, 1:1,000), ubiquitin (3933S, Cell Signaling, 1:1,000) and puromycin (MABE343, EMD Millipore, 1:25,000) followed by secondary anti-rabbit, anti-mouse or anti-goat HRP antibodies (111-035-047, 315-035-047 and 705-035-003, respectively, from Jackson ImmunoResearch Laboratories). Membranes were developed with an Amersham imager and analysed with Fiji for MacOS X (version 2.0.0-rc-67/1.52c)45.
Quantitative RT–PCR analysis
BMDMs infected with A. fumigatus as described in ‘In vitro stimulation of BMDMs’ for the indicated time were collected in TRIzol (15596026, Thermo Fisher Scientific) for RNA extraction. Aspergillus fumigatus mycelium grown for 8 h in LB medium at 37 °C and 250 rpm were washed with PBS and collected in TRIzol. RNA was converted into cDNA by using the High-Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems). Real-time qPCR was performed on an ABI 7500 real-time PCR instrument with 2× SYBR Green (4368706, Applied Biosystems). Macrophage gene quantifications and A. fumigatus gene quantifications are presented relative to Actb and TEF1, respectively.
Cytokine levels were determined by multiplex enzyme-linked immunosorbent assay (ELISA) (MCYTOMAG-70K, Millipore) according to the manufacturer’s instructions. For the in vivo measurement of IL-1β and IL-18, the mouse IL-1β uncoated ELISA kit (88-703-88, Invitrogen) and mouse IL-18 ELISA Kit (BMS618-3TEN, Invitrogen) were used according to the manufacturer’s instructions.
Mycelia of Δgt4c and parental strains were paraformaldehyde (PFA)-fixed (2.5% (v/v) PFA in PBS) overnight at 4 °C, washed 3 times with 0.1 N NH4Cl in PBS, once with PBS and then incubated with a specific GAG monoclonal antibody, as previously described previously6. For GAG immunofluorescence staining in BMDMs, 1 × 106 BMDMs were seeded on 4-well, 15-μm slides (80426, Ibidi) in DMEM supplemented with 10% FBS and 1% penicillin–streptomycin and infected with A. fumigatus conidia at 10 MOI for the indicated time. Cells were washed 3 times and fixed in 4% paraformaldehyde for 15 min at room temperature, followed by blocking in PBS containing 10% goat serum (PCN5000, Life Technologies) supplemented with 0.2% saponin (47036, Sigma Aldrich) for 1 h at room temperature. Then cells were incubated with a specific GAG monoclonal antibody as previously described6. Next, cells were washed 4 times with PBS and stained with an anti-rabbit secondary Alexa Fluor 488 antibody (1:250 dilution, A32723, Invitrogen) and 5 μg/ml DAPI (Biotium) in PBS containing 10% goat serum supplemented with 0.2% saponin for 1 h at room temperature. Cells were visualized and imaged using a Nikon C2 confocal microscope. For stress granule staining, the BMDMs were transfected with GAG or incubated with 50 μM sodium (meta)arsenite (S7400, Sigma Aldrich) for 1 h or infected with A. fumigatus for 15 h before being fixed with 4% PFA for 15 min. Then, the cells were processed as previously described for DDX3X–G3BP1 staining17. Antibodies used were anti-DDX3X (A300-474A, Bethyl Laboratories, 1:200) and anti-G3BP1 (66486-1-Ig, Proteintech, 1:250).
Scanning electron microscopy
Cells were grown in Brian medium at 30 °C for 20 h on 1.2-mm coverslips coated with a carbon tap. Samples were fixed in 2.5% glutaraldehyde in 0.1 M Hepes buffer (pH 7.4) overnight at 4 °C, then washed for 5 min 3 times in 0.1 M Hepes buffer (pH 7.2), post-fixed for 1 h in 1% osmium and rinsed with distilled water. Cells were dehydrated through a graded ethanol series followed by critical point drying with CO2. Dried specimens were gold and palladium sputter-coated with a gun ionic evaporator PEC 682. The samples were imaged in a JEOL JSM 6700F field emission scanning-electron microscope operating at 5 kV.
GraphPad Prism 8.0 was used for data analysis. Data are represented as mean ± s.e.m. P < 0.05 was considered statistically significant.
Further information on research design is available in the Nature Research Reporting Summary linked to this paper.
The datasets generated and analysed in this study are contained within the Article, and its Supplementary Information; any other relevant data are available from the corresponding author upon reasonable request. Source data are provided with this paper.
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We thank members of the Kanneganti laboratory for their comments, suggestions and technical assistance; R. Tweedell for scientific editing of the manuscript; the St Jude Children’s Research Hospital Veterinary Pathology Core, SJCRH Center for Proteomics and Metabolomics and SJCRH Cell and Tissue Imaging Center (supported by the NCI P30 CA021765); D. Sheppard for sharing the A. fumigatus deletion mutant ∆agd; and V. M. Dixit and N. Kayagaki for the Casp1−/−Casp11−/− mutant mouse strain. T.-D.K. is supported by NIH grants AI101935, AI124346, AR056296 and CA253095 and by the American Lebanese Syrian Associated Charities. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. J.-P.L. is supported by the Aviesan project Aspergillus, the French Government’s Investissement d’Avenir program, Laboratoire d’Excellence ‘Integrative Biology of Emerging Infectious Diseases’ (grant number ANR-10-LABX-62-IBEID) and la Fondation pour la Recherche Médicale (DEQ20150331722 LATGE Equipe FRM 2015).
The authors declare no competing interests.
Peer review information Nature thanks Gordon D. Brown, Osamu Takeuchi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Schematic of GAG synthase cluster. b, RNA sequencing analysis during A. fumigatus growth (0, 4 and 8 h) from ref. 46; gene expression is represented by mean of normalized read count per gene for the GT4C cluster. n = 3 biologically independent samples. Data are mean ± s.e.m. c, Heat map showing differential gene expression of A. fumigatus at 4 h (swollen conidia) and 8 h (germinated conidia) compared to 0 h (resting conidia). d, Schematic of the GT4C protein with transmembrane regions (black), α-glycosyltransferase domains (green) and major facilitator superfamily domain (MFS) predicted from amino acid sequence with InterProscan 5. e, Schematic of wild-type (WT) and Δgt4c locus with NcoI restriction sites and Southern blot probe used to control the GT4C gene deletion. f, Southern blot using GT4C probe with WT and Δgt4c purified DNA from one experiment. g, RT–PCR analysis of GT4C, AGD3, EGA3, SPH3 and UGE3 A. fumigatus genes in WT and Δgt4c strains (8 h in LB medium, 37 °C and 250 rpm) presented relative to that of TEF1. n.d., not detected. n = 3 biologically independent samples. Data are mean ± s.e.m.
Extended Data Fig. 2 Absence of GAG does not affect release of non-inflammasome dependent cytokines.
a, b, Release of IL-6 (a) and TNF (b) from unprimed BMDMs left uninfected (med.) or assessed 20 h after infection with A. fumigatus wild type (WT) or Δgt4c strain (MOI of 10). n = 3 independent biological samples. Data are mean ± s.e.m.
a, Assessment of biofilm formation on an abiotic surface with A. fumigatus (A. f) wild type (WT) and deletion mutant strain Δuge3. b, Immunoblot analysis of pro-caspase 1 (pro-Casp1; p45) and the active caspase 1 subunit (p20) from unprimed BMDMs left untreated (medium alone (med.)) or measured 20 h after infection with the indicated A. fumigatus live resting conidia (MOI of 10). Representative images. n ≥ 3 independent experiments. c, Immunoblot analysis of phospho-IκBα and total IκBα (t-IκBα) or phospho-ERK1/2 (p-ERK) and total ERK1/2 (t-ERK) from unprimed WT BMDMs 0–8 h after infection with WT or Δuge3 mutant A. fumigatus live resting conidia (MOI of 10). Representative images. n ≥ 3 independent experiments. d, Immunoblot analysis of pro-IL-1β from unprimed BMDMs 0–8 h after infection with WT or Δuge3 mutant A. fumigatus live resting conidia (MOI of 10). Representative images. n ≥ 3 independent experiments. e–g, RT–PCR analysis of Nlrp3, Il1b and Tnf genes from WT BMDMs 0–8 h after infection with WT or Δuge3 mutant A. fumigatus live resting conidia presented relative to that of the gene encoding β-actin. n = 4 biologically independent samples. Data are mean ± s.e.m.
a, Immunofluorescence staining of A. fumigatus GAG (green) and BMDM nuclei (blue) in unprimed BMDMs 4 h after infection with A. fumigatus wild-type (WT) or Δugm1 resting conidia (MOI of 10). Scale bars, 10 μm. Representative images. n ≥ 3 independent experiments. b, Immunoblot analysis of pro-caspase 1 (pro-Casp1; p45) and the active caspase 1 subunit (p20) of unprimed BMDMs left untreated (medium alone (med.)) or assessed 20 h after infection with the indicated live A. fumigatus resting conidia genotype (WT or A. fumigatus deletion mutant Δugm1) (MOI of 10). Representative image. n ≥ 3 independent experiments. c, Release of IL-1β from unprimed BMDMs left uninfected (med.) or assessed 20 h after infection with A. fumigatus (MOI of 10). **P = 0.0046. Unpaired two-tailed t-test. n = 4 biologically independent samples. Data are mean ± s.e.m.
Extended Data Fig. 5 GAG induces caspase 1 cleavage in a dose- and charge–charge interaction-dependent manner and interacts with ribosomes.
a, Representative images of BMDMs in medium (med.) or during treatment with DOTAP alone (green fluorescence corresponds to Sytox green nuclei, and Sytox green-positive nuclei are marked with a red circle). Scale bars, 10 μm. Representative images. n ≥ 3 independent experiments. b, Immunoblot analysis of pro-caspase 1 (pro-Casp1; p45) and the active caspase 1 subunit (p20) of BMDMs left untreated (medium alone (med.)) or assessed 3 h after transfection with increasing concentrations of GAG or vehicle alone (DOTAP). Representative image. n ≥ 3 independent experiments. c, Immunoblot analysis of caspase 1 during transfection with GAG in wild-type (WT) and Gsdmd−/− BMDMs. Representative image. n ≥ 3 independent experiments. d, Volcano plot of the polysaccharide pull-down mass spectrometry analysis of the β-glucan interactome with BMDM cytosolic proteins versus control. Proteins with P < 0.005 are highlighted in orange and proteins with P < 0.005 and log2-transformed fold change > 7 compared to control are highlighted in red (none identified); P value was determined by the G test; exact P values are presented in Supplementary Table 1. e, Immunoblot analysis of ribosomal proteins interacting with GAG, Ac-GAG, d-GAG or β-glucan or vehicle with or without NaCl. Representative images. n ≥ 3 independent experiments. f, Immunoblot analysis of caspase 1 from BMDMs assessed after 3 h incubation with GAG, Ac-GAG or d-GAG, with or without NaCl. Representative image. n ≥ 3 independent experiments. g, h, Measurement of cell death by Sytox green staining during GAG and d-GAG treatment, with or without NaCl. n = 3 biologically independent samples. Data are mean ± s.e.m. i, Electron microscopy pictures of ribosomes, GAG + ribosomes and chitin + ribosomes with negative staining; data from one experiment. Scale bars, 100 nm.
a–c Immunoblot analysis of translation rate in BMDMs by puromycin integration into proteins during vehicle (DOTAP) or PBS incubation (a), poly(dA:dT) transfection (b) or A. fumigatus infection and pro-caspase 1 (pro-Casp1; p45) and the active caspase 1 subunit (p20) during A. fumigatus infection (c). Representative images. n ≥ 2 independent experiments. d, Polysome profiling during DOTAP or DOTAP + GAG treatments. e, Immunoblot analysis of the cell pellet after polysome profiling. Representative images. n ≥ 2 independent experiments. f, Polysome:monosome ratio during DOTAP or DOTAP + GAG treatments. Data are mean ± s.e.m. *P = 0.0366. Paired two-tailed t-test. n = 3 biologically independent samples. g−i, Immunoblot analysis of pro-caspase 1 and the active caspase 1 subunit of wild-type (WT) or Nlrp3−/− BMDMs assessed after 16 h incubation with 25 μg ml−1 anisomycin (aniso) (g), 50 μg ml−1 puromycin (puro) (h) or 50 μg ml−1 cycloheximide (CHX) (i). Representative images. n ≥ 2 independent experiments. j, k, Immunoblot analysis of PERK activation (p-PERK) and IRE1α induction during GAG transfection (j) or PERK activation during treatment with translation inhibitors (k). Representative images. n ≥ 2 independent experiments. l, Immunoblot analysis of proteins ubiquitinated during GAG transfection. Representative images. n ≥ 2 independent experiments. m, Immunoblot analysis of caspase 1 of BMDMs left untreated (medium alone (med.)) or assessed 3 h after transfection with DOTAP alone, GAG or GAG with MG132 treatment. Representative image. n ≥ 2 independent experiments.
a, Immunofluorescence staining of G3BP1 (green), DDX3X (red) and BMDM nuclei (blue) in unprimed BMDMs 40 min after transfection with GAG or incubation with arsenite (Ars). Representative images. n ≥ 2 independent experiments. b, Quantification of the percentage of stress-granule-positive cells after transfection with GAG, vehicle (DOTAP) alone or Ars. n > 10 biologically independent fields of cells. Data are mean ± s.e.m. c, Immunofluorescence staining of G3BP1 (green), DDX3X (red) and BMDM nuclei (blue) in unprimed BMDMs 15 h after infection with A. fumigatus. Representative images. n ≥ 2 independent experiments. d, e, Immunoblot analysis of pro-caspase 1 (pro-Casp1; p45) and the active caspase 1 subunit (p20) of BMDMs assessed 3 h after transfection with vehicle (DOTAP), GAG (d) or d-GAG (e). Representative images. n ≥ 2 independent experiments. f, Immunoblot analysis of caspase 1 from BMDMs left untreated (medium alone (med.)) or infected with A. fumigatus wild-type (WT) or deletion mutant ∆gt4c (MOI of 10). Representative image. n ≥ 2 independent experiments. Scale bars, 10 μm (a, c).
Extended Data Fig. 8 GAG-induced pro-inflammatory cytokine secretion during aspergillosis and DSS-induced colitis.
a, b, Level of IL-1β (a) and IL-18 (b) in bronchioalveolar lavage 2 days after infection with wild-type (WT) or Δugm1 strains of A. fumigatus. a, *P = 0.036. Unpaired two-tailed t-test. n = 6 independent samples. Data are mean ± s.e.m. c, Survival of 7–8-week-old immunocompetent WT mice infected intravenously with 1 × 106 A. fumigatus resting conidia (WT or Δugm1). **P = 0.0014. Log-rank (Mantel–Cox) test. d, e, Levels of IL-1β (d) and IL-18 (e) in liver homogenates after infection with WT or Δgt4c strains. d, *P = 0.0209; e, **P = 0.0041. Unpaired two-tailed t-test. n = 5 independent samples. Data are mean ± s.e.m. f–k, Concentration of cytokines in colon homogenates after DSS water supplementation and treatment with GAG or vehicle (vehicle and GAG, n = 10 mice each). f, *P = 0.0336; g, *P = 0.0181; h, *P = 0.0188; i, *P = 0.0115; j, **P = 0.0066; k, *P = 0.0154. Unpaired two-tailed t-test. Data are mean ± s.e.m.
Supplementary Table 1: Spectral count comparison of proteins identified from the Control and GAG-treated samples. P values were determined by the G-test.
Supplementary Figure 1: Uncropped blots with molecular weight and size markers and an indication of how the images were cropped.
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Briard, B., Fontaine, T., Samir, P. et al. Galactosaminogalactan activates the inflammasome to provide host protection. Nature 588, 688–692 (2020). https://doi.org/10.1038/s41586-020-2996-z
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