Interplay between host humoral pattern recognition molecules controls undue immune responses against Aspergillus fumigatus

Abstract

Pentraxin 3 (PTX3), a long pentraxin and a humoral pattern recognition molecule (PRM), has been demonstrated to be protective against Aspergillus fumigatus, an airborne human fungal pathogen. We explored its mode of interaction with A. fumigatus, and the resulting implications in the host immune response. Here, we demonstrate that PTX3 interacts with A. fumigatus in a morphotype-dependent manner: (a) it recognizes germinating conidia through galactosaminogalactan, a surface exposed cell wall polysaccharide of A. fumigatus, (b) in dormant conidia, surface proteins serve as weak PTX3 ligands, and (c) surfactant protein D (SP-D) and the complement proteins C1q and C3b, the other humoral PRMs, enhance the interaction of PTX3 with dormant conidia. SP-D, C3b or C1q opsonized conidia stimulated human primary immune cells to release pro-inflammatory cytokines and chemokines. However, subsequent binding of PTX3 to SP-D, C1q or C3b opsonized conidia significantly decreased the production of pro-inflammatory cytokines/chemokines. PTX3 opsonized germinating conidia also significantly lowered the production of pro-inflammatory cytokines/chemokines while increasing IL-10 (an anti-inflammatory cytokine) released by immune cells when compared to the unopsonized counterpart. Overall, our study demonstrates that PTX3 recognizes A. fumigatus either directly or by interplaying with other humoral PRMs, thereby restraining detrimental inflammation. Moreover, PTX3 levels were significantly higher in the serum of patients with invasive pulmonary aspergillosis (IPA) and COVID-19-associated pulmonary aspergillosis (CAPA), supporting previous observations in IPA patients, and suggesting that it could be a potential panel-biomarker for these pathological conditions caused by A. fumigatus.

Fig. 1. Upon immunolabelling and confocal microscopy, swollen and germinating conidia of A. fumigatus, but not dormant conidia, show positive immunolabelling for PTX3.

Dormant, swollen and germinating conidia were blocked for 1 h with PBS containing 1% BSA, incubated with PTX3 [1 μg/mL in HEPES buffer (pH 7.4) supplemented with CaCl₂ and MgCl₂] followed by sequential incubations with primary anti-human PTX3 polyclonal antibodies (raised in rabbit, 1:500 dilution) and secondary Alexa Fluor-647 labeled anti-rabbit IgG (with PBS washing between these two incubations) followed by confocal microscopy. Only swollen and germinating conidia showed PTX3 binding, but not dormant conidia of A. fumigatus (controls: treated like other samples, but without incubation with PTX3). Immunolabelling was performed with three different cultures of A. fumigatus (biological replicates), and every time at least five images were taken for each assay condition.

Fig. 2. Galactosaminogalactan (GAG), a cell-wall polysaccharide synthesized by A. fumigatus during germination, is the PTX3 ligand.

ELISA was performed by coating A. fumigatus cell wall components into ELISA plate wells, adding biotinylated PTX3 (1 μg/mL) followed by horseradish peroxidase (HRP) conjugated to streptavidin, and revealing by O-phenylenediamine method. A PTX3 interacts with alkali-soluble (AS) fractions from swollen (SC) and germinating (GC) conidia, but not dormant conidia (DC). B PTX3 binds to GC-AS fraction and GAG, but not other cell wall components (10 μg/mL) or DC specific RodAp and melanin pigments. C PTX3 binds to GC-AS fraction in a concentration dependent manner. D Irrespective of the batches of preparations, PTX3 shows better affinity towards insoluble galactosaminogalactan (GAG) (10 μg/mL). E PTX3 binds to native and completely deacetylated GAGs, but not with acetylated GAG. F Germinating conidia were incubated with PTX3, followed by immunolabelling for GAG with mouse monoclonal anti-GAG antibodies and anti-mouse IgG-TRITC and for PTX3 with monoclonal anti-PTX3 antibodies raised in rat and Alexa Fluor 405 conjugated rat IgG; merged image (in pink) suggests the colocalization of GAG and PTX3. AS and AI fractions extracted from two independent batches of dormant, swollen and germinating conidia were used in the assays, with technical replicates (2A, B, C). A recombinant RodA-protein, melanin (the DC components), β−1,3-glucan, chitin, α−1,3-glucan, galactomannan (GM) and GAG extracted from two independent mycelial cultures of A. fumigatus, two batches of native GAG, as well as acetylated and deacetylated GAGs derivatized from native GAGs were used for the binding assays (2B, D, E). Data are presented as mean values ± SD. PTX3-GAG double immunolabelling was performed with two independent cultures of A. fumigatus (biological replicates); each time at least five images were captured for a condition presented (2F). Source data (2A–E) are provided as a Source data file.

Fig. 3. PTX3 binds to dormant A. fumigatus conidia in presence of serum factors.

A Upon opsonization with pooled normal human serum (NHS) or heat-inactivated (HI) NHS followed by incubation with primary human anti-PTX3 polyclonal antibodies (rabbit, at 1:500 dilution) and FITC-conjugated anti-rabbit IgG, dormant conidia showed positive labeling, suggesting that serum factor(s) mediate PTX3-conidia interaction. Positive labeling with inactivated NHS suggested that the mediating serum factor(s) are heat stable. B Humoral pattern-recognition molecules (PRMs) reported to interact with A. fumigatus conidia were coated on 96-well ELISA plate, followed by sequential incubations with biotinylated PTX3 (1 μg/mL), streptavidin-horseradish peroxidase (HRP), and 3,3’,5,5’-tetramethylbenzidine (TMB; HRP substrate) followed by reading the optical densities at 450 nm (with a reference at 530 nm). Among these humoral PRMs, surfactant protein D (SP-D), and complement proteins C1q, C3b showed PTX3 interaction. Control has been subtracted to plot the graph. SP-A: Surfactant protein-A, C4b: complement protein C4b, MBL: mannose binding lectin, FCN: Ficolin. Immunolabelling was performed two times with independent cultures of A. fumigatus (biological replicates); each time, at least five images were captures for an assay condition (3 A). PTX3 and other PRM interaction study (3B) was performed in triplicate (technical replicate); data are presented as mean values ± SD. Source data are provided as a Source data file.

Fig. 4. Complexing with SP-D, C1q or C3b enhance the interaction of PTX3 with dormant conidia of A. fumigatus.

A Bar-graph representing the percentage of conidia positive for SP-D, C1q or C3b binding, as determined by flow-cytometry. B Bar-graph showing conidia positive for PTX3 binding alone or upon its complexing with SP-D, C1q or C3b (flow-cytometry). PTX3 binding was detected by anti-human PTX3-FITC. Median values are presented. Statistical analysis was performed with two-sided paired t-test (*p < 0.05). C Histograms (flow-cytometry data) showing the conidial binding pattern of PTX3, directly or after complexing with SP-D, C1q or C3b. Data acquired from one experiment is presented; therefore, the control and PTX3 counts were the same in the SP-D + PTX3, C1q + PTX3 and C3b + PTX3 panels, while the binding assay was repeated with two independent batches of A. fumigatus (biological replicates), each time in duplicates (technical replicates). Source data are provided as a Source data file.

Fig. 5. Cytokines and chemokine released by human monocyte-derived macrophages (hMDMs) and neutrophils when stimulated with (i) unopsonized dormant conidia, (ii) dormant conidia opsonized with SP-D, C1q, C3b, or PTX3, (iii) dormant conidia opsonized with SP-D, C1q or C3b, and then incubated with PTX3, and (iii) unopsonized or PTX3 opsonized germinating conidia.

PFA-fixed dormant/germinating conidia were used; hMDMs and neutrophils were interacted with these fungal samples for 24 h and 20 h, respectively, in a CO₂ incubator at 37^oC. hMDMs or neutrophils cultured only with medium served as the control, stimulated with lipopolysaccharide (LPS) served as a positive control. Individual humoral PRMs alone were also added to hMDMs/neutrophils that served as another set of controls. Supernatants of these cultures were collected after indicated times of interaction and analyzed for cytokines/chemokine; (A) hMDMs (number of donors, n = 8) and (B) neutrophils (n = 10). Median values are presented for each assay condition. Statistical analysis was performed by one-way ANOVA with Tukey’s multiple comparison test (ns-nonsignificant). At a time, Neutrophils or hMDMs isolated/obtained from two donors were subjected to conidial interaction assays; accordingly, 4–5 independent cultures of A. fumigatus conidia (biological replicates) were used for this entire study. Source data are provided as a Source data file.

Fig. 6. PTX3, TNF-α and IL-10 released/produced by neutrophils isolated from human whole blood samples and human monocyte-derived macrophages (hMDMs) stimulated with A. fumigatus conidia.

Neutrophils and hMDMs were incubated with metabolically active (live) A. fumigatus conidia in a medium without or supplemented with normal human serum (NHS) at 37^oC in a CO₂ incubator. The culture supernatants were collected at intervals of 0, 4, 6, 8, and 24 h post-stimulation. In the collected culture supernatants, PTX3 (both neutrophils and hMDMs), TNF-α (for hMDMs) and IL-10 (for hMDMs) levels were quantified by ELISA, using respective detection kits. A, B PTX3 released by neutrophils stimulated with conidia in medium without or with NHS; C, D PTX3 produced by hMDMs stimulated with conidia in medium without or with NHS; E, F TNF-α and IL-10 secreted by hMDMs stimulated with conidia in medium supplemented with NHS; (number of donors = 4). Data are presented as mean values ± SEM. Source data are provided as a Source data file.

Fig. 7. Exploring PTX3 as a diganostic marker of A. fumigatus infection.

A PTX3 and B IL-10 levels were higher in the serum from patients with invasive pulmonary aspergillosis (IPA) and COVID-19-associated pulmonary aspergillosis (CAPA; only PTX3 level). Serum samples were retrospectively collected from the patients with IPA (n = 19), chronic pulmonary aspergillosis (CPA; n = 12), CAPA (n = 6), candidemia (n = 5), mucormycosis (n = 6) and healthy controls (n = 11). Median values are presented. Statistical analysis was performed by two-tailed Mann Whitney test for comparison of control with infected patient groups and by unpaired t-test between two infected patient groups. Source data are provided as a Source data file.

Fig. 8. Schematic model for the release of and the immunomodulatory function exerted by PTX3.

Inhaled conidia opsonized with SP-D, C1q or C3b will be phagocytosed by alveolar macrophages, which stimulate macrophages to release pro-inflammatory cytokines TNF-α, IL-1β, chemokine IL-8 as well as other cytokines. IL-8 recruits neutrophils, TNF-α, IL-1β stimulate macrophages to synthesize-secrete PTX3 and neutrophils to release PTX3. As conidial inhalation is a constant process, additional conidia entering lung alveoli encounter immune environment with existing pro-inflammatory cytokines and chemokine. While these conidia will be phagocytosed through PTX3 that complexes with SP-D, C1q or C3b but without stimulating additional cytokine/chemokine secretions. Conidia that occasionally escape from phagocytes and germinating in the alveoli will be opsonized by PTX3 and killed by phagocytes. PTX3 mediated phagocytosis prevents additional/undue immune activation, maintaining immune homeostasis [Image created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs (CC-BY-NC-ND) 4.0 International license].