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

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.

[Research advances on the mechanism of immune regulation of interferon in Aspergillus infection].

Pulmonary aspergillosis is a serious pulmonary fungal infectious disease. It is difficult to manage and has limited treatment options. Existing anti-aspergillus medications have high rates of treatment failure and increased drug resistance, making it difficult to meet the clinical requirements. Therefore, the development of new, effective treatment programs is critical. According to research, interferons play an important role in the body's immune response to bacterial and viral infectious diseases. Inadequate interferon expression or dysfunction can put the body at risk for certain infectious diseases. Interferon has been used in clinical trials to prevent or treat infectious diseases. In recent years, researchers have focused on the immunological role of interferon in Aspergillus infections and its potential for clinical application. This review summarized the most recent advances in the immunoregulatory mechanisms of interferon and its clinical application in Aspergillus infections.

Identification and validation of diagnostic cut-offs of the ELISpot assay for the diagnosis of invasive aspergillosis in high-risk patients.

OBJECTIVE: We investigated the performance of enzyme linked immunospot (ELISpot) assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematologic malignancies. METHODS: We prospectively enrolled two cohorts of patients undergoing intensive myelosuppressive or immunosuppressive treatments at high risk for IA. ELISpot was performed to detect Aspergillus-specific T cells producing Interleukin-10. RESULTS: In the discovery cohort, a derived cut-off of 40 spot forming cells (SFCs)/106 PBMCs has shown to correctly classify IA cases with a sensitivity and specificity of 89.5% and 88.6%, respectively. This cut-off is lowered to 25 SFC when considering the subset of possible IA patients, with sensitivity and specificity of 76% and 93%, respectively. The application of the 40 SFCs cut-off to the validation cohort resulted in a positivity rate of 83.3% in proven/probable cases and a negativity rate of 92.5% in possible/non-IA cases. Adopting the 25 SCFs cut-off, the assay resulted positive in 83.3% of proven/probable cases while it resulted negative in 66.7% of possible/non-IA cases. CONCLUSIONS: ELISpot shows promises in the diagnosis of IA and the possibility to use two distinct cut-offs with similar diagnostic performances according to patients' different pre-test probability of infection can widen its use in patients at risk.

The Role of LC3-Associated Phagocytosis Inhibits the Inflammatory Response in Aspergillus fumigatus Keratitis.

Purpose: The purpose of this study was to investigate the role and mechanism of microtubule-associated protein light chain-3 (LC3)-associated phagocytosis (LAP) in the immune response to Aspergillus fumigatus (A. fumigatus) keratitis. Methods: The formation of single-membrane phagosomes was visualized in the corneas of healthy or A. fumigatus-infected humans and C57BL/6 mice using transmission electron microscopy (TEM). Rubicon siRNA (si-Rubicon) was used to block Rubicon expression. RAW 264.7 cells or mice corneas were infected with A. fumigatus with or without pretreatment of si-Rubicon and scrambled siRNA. RAW 264.7 cells were pretreated with Dectin-1 antibody or Dectin-1 overexpressed plasmid and then stimulated with A. fumigatus. Flow cytometry was used to label macrophages in normal and infected corneas of mice. In mice with A. fumigatus keratitis, the severity of the disease was assessed using clinical scores. We used lentiviral technology to transfer GV348-Ubi-GFP-LC3-II-SV40-Puro Lentivirus into the mouse cornea. The GFP-LC3 fusion protein was visualized in corneal slices using a fluorescence microscope. We detected the mRNA and protein expressions of the inflammatory factors IL-6, IL-1ß, and IL-10 using real-time PCR (RT-PCR) and ELISA. We detected the expression of LAP-related proteins Rubicon, ATG-7, Beclin-1, and LC3-II using Western blot or immunofluorescence. Results: Accumulation of single-membrane phagosomes within macrophages was observed in the corneas of patients and mice with A. fumigatus keratitis using TEM. Flow cytometry (FCM) analysis results show that the number of macrophages in the cornea of mice significantly increases after infection with A. fumigatus. LAP-related proteins were significantly elevated in the corneas of mice and RAW 264.7 cells after infection with A. fumigatus. The si-Rubicon treatment elevated the clinical score of mice. In A. fumigatus keratitis mice, the si-Rubicon treated group showed significantly higher expression of IL-6 and IL-1ß and lower expression of IL-10 and LC3-II compared to the control group. In RAW 264.7 cells, treatment with the Dectin-1 overexpressed plasmid upregulated the expression of LAP-related proteins, a process that was significantly inhibited by the Dectin-1 antibody. Conclusions: LAP participates in the anti-inflammatory immune process of fungal keratitis (FK) and exerts an anti-inflammatory effect. LAP is regulated through the Dectin-1 signaling pathway in A. fumigatus keratitis.

Anti-Inflammatory Efficacy of Nanobody Specific to ß-Glucan on a Fungal Cell Wall in a Murine Model of Fungal Keratitis.

Purpose: to explore the anti-inflammatory effects of a nanobody (Nb) specific to ß-glucan on fungal keratitis (FK). Methods: in order to verify the therapeutic and anti-inflammatory efficacy of Nb in FK, the severity of inflammation was assessed with inflammatory scores, hematoxylin-eosin (HE) staining, and myeloperoxidase (MPO) assays. In corneas of mice of FK model and human corneal epithelial cells stimulated by fungal hyphae, real-time reverse transcriptase polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines and pattern recognition receptors (PRRs). In vivo, macrophages and neutrophils infiltration in the cornea stroma was detected by immunofluorescence (IFS) staining. Results: In murine models infected with Aspergillus fumigatus (A. fumigatus), Nb treatment could reduce the inflammatory scores. HE staining and MPO results showed Nb significantly alleviated corneal edema and reduced inflammatory cell infiltration 3 days post-infection. In addition, the expression levels of LOX-1 and Dectin-1 were significantly decreased in the Nb group in vivo. The expression of chemokines CCL2 and CXCL2 also decreased in the Nb group. Compared with the PBS group, the number of macrophages and neutrophils in the Nb group was significantly decreased, which was shown in IFS results. Moreover, Nb attenuated the expression of Dectin-1, LOX-1, and inflammatory mediators, including IL-6 and IL-8 in vitro. Conclusion: our study showed that Nb could alleviate FK by downregulating the expression of PRRs and inflammatory factors as well as reducing the infiltration of macrophages and neutrophils.

New developments in Aspergillus fumigatus and host reactive oxygen species responses.

Aspergillus fumigatus is a filamentous fungus abundant in the environment and the most common causative agent of a spectrum of human diseases collectively termed aspergillosis. Invasive pulmonary aspergillosis is caused by deficiencies in innate immune function that result in the inability of the host to clear inhaled Aspergillus conidia that then germinate and form invasive hyphae. Myeloid cells, and their ability to generate reactive oxygen species (ROS), are essential for conidia clearance from the host. To combat ROS, A. fumigatus employs an expansive antioxidant system, though how these canonical antioxidant mechanisms contribute to infection initiation and disease progression remain to be fully defined. Recent research has identified noncanonical pathways in the A. fumigatus ROS response and new host populations with ROS deficiencies that are at-risk for invasive aspergillosis. Here, we highlight recent developments in the understanding of ROS at the interface of the dynamic A. fumigatus-host interaction.

CD56-mediated activation of human natural killer cells is triggered by Aspergillus fumigatus galactosaminogalactan.

Invasive aspergillosis causes significant morbidity and mortality in immunocompromised patients. Natural killer (NK) cells are pivotal for antifungal defense. Thus far, CD56 is the only known pathogen recognition receptor on NK cells triggering potent antifungal activity against Aspergillus fumigatus. However, the underlying cellular mechanisms and the fungal ligand of CD56 have remained unknown. Using purified cell wall components, biochemical treatments, and ger mutants with altered cell wall composition, we herein found that CD56 interacts with the A. fumigatus cell wall carbohydrate galactosaminogalactan (GAG). This interaction induced NK-cell activation, degranulation, and secretion of immune-enhancing chemokines and cytotoxic effectors. Supernatants from GAG-stimulated NK cells elicited antifungal activity and enhanced antifungal effector responses of polymorphonuclear cells. In conclusion, we identified A. fumigatus GAG as a ligand of CD56 on human primary NK cells, stimulating potent antifungal effector responses and activating other immune cells.

Rethinking Aspergillosis in the Era of Microbiota and Mycobiota.

Aspergillosis encompasses a wide range of clinical conditions based on the interaction between Aspergillus and the host. It ranges from colonization to invasive aspergillosis. The human lung provides an entry door for Aspergillus. Aspergillus has virulence characteristics such as conidia, rapid growth at body temperature, and the production of specific proteins, carbohydrates, and secondary metabolites that allow A. fumigatus to infiltrate the lung's alveoli and cause invasive aspergillosis. Alveolar epithelial cells play an important role in both fungus clearance and immune cell recruitment via cytokine release. Although the innate immune system quickly clears conidia in immunocompetent hosts, A. fumigatus has evolved multiple virulence factors in order to escape immune response such as ROS detoxifying enzymes, the rodlet layer, DHN-melanin and toxins. Bacterial co-infections or interactions can alter the immune response, impact Aspergillus growth and virulence, enhance biofilm formation, confound diagnosis, and reduce treatment efficacy. The gut microbiome's makeup influences pulmonary immune responses generated by A. fumigatus infection and vice versa. The real-time PCR for Aspergillus DNA detection might be a particularly useful tool to diagnose pulmonary aspergillosis. Metagenomics analyses allow quick and easy detection and identification of a great variety of fungi in different clinical samples, although optimization is still required particularly for the use of NGS techniques. This review will analyze the current state of aspergillosis in light of recent discoveries in the microbiota and mycobiota.

Orbital aspergillosis in an immunocompromised man with no history of trauma: a case report.

Fungal orbital cellulitis is usually seen in immunocompromised individuals, and opportunistic pathogens are the main etiology. We herein report a case of fungal orbital cellulitis due to Aspergillus in a patient with no history of trauma. A 48-year-old man presented to the emergency room of our hospital with a 2-week history of periorbital swelling, conjunctival hyperemia, and chemosis of his right eye. The visual acuity of his right eye was 6/20, and the intraocular pressure was 44 mmHg. The main clinical findings were proptosis of the right ocular globe with conjunctival hyperemia and a palpable infratemporal orbital mass. Laboratory testing failed to detect the presence of a pathogenic infection, and the lesions on computed tomography images resembled those of a malignant tumor of the orbit. The diagnosis was finally confirmed by postoperative pathological examination, and the patient responded favorably to debridement combined with antifungal therapy. Histopathological examination may help to reveal the nature of this disease. Surgical removal of inflammatory lesions can serve as an important diagnostic and treatment method for fungal orbital cellulitis.

The Inflammatory Response Induced by Aspergillus fumigatus Conidia Is Dependent on Complement Activation: Insight from a Whole Blood Model.

INTRODUCTION: We aimed to elucidate the inflammatory response of Aspergillus fumigatus conidia in a whole-blood model of innate immune activation and to compare it with the well-characterized inflammatory reaction to Escherichia coli. METHODS: Employing a human lepirudin whole-blood model, we analyzed complement and leukocyte activation by measuring the sC5b-9 complex and assessing CD11b expression. A 27-multiplex system was used for quantification of cytokines. Selective cell removal from whole blood and inhibition of C3, C5, and CD14 were also applied. RESULTS: Our findings demonstrated a marked elevation in sC5b-9 and CD11b post-A. fumigatus incubation. Thirteen cytokines (TNF, IL-1ß, IL-1ra, IL-4, IL-6, IL-8, IL-17, IFNγ, MCP-1, MIP-1α, MIP-1ß, FGF-basic, and G-CSF) showed increased levels. A generally lower level of cytokine release and CD11b expression was observed with A. fumigatus conidia than with E. coli. Notably, monocytes were instrumental in releasing all cytokines except MCP-1. IL-1ra was found to be both monocyte and granulocyte-dependent. Pre-inhibiting with C3 and CD14 inhibitors resulted in decreased release patterns for six cytokines (TNF, IL-1ß, IL-6, IL-8, MIP-1α, and MIP-1ß), with minimal effects by C5-inhibition. CONCLUSION: A. fumigatus conidia induced complement activation comparable to E. coli, whereas CD11b expression and cytokine release were lower, underscoring distinct inflammatory responses between these pathogens. Complement C3 inhibition attenuated cytokine release indicating a C3-level role of complement in A. fumigatus immunity.

BTK inhibitor-induced defects in human neutrophil effector activity against Aspergillus fumigatus are restored by TNF-α.

Inhibition of Bruton's tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the effect of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNF-α fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNF-α did not affect ROS production in healthy neutrophils but prevented exogenous TNF-α from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNF-α was independent of transcription. Moreover, the addition of TNF-α immediately rescued ROS production in IBT-treated neutrophils, indicating that TNF-α worked through a BTK-independent signaling pathway. Finally, TNF-α restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNF-α rescued the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.

Evolution of the pathogenic mold Aspergillus fumigatus on high copper levels identifies novel resistance genes.

Aspergillus fumigatus is the leading cause of severe mold infections in immunocompromised patients. This common fungus possesses innate attributes that allow it to evade the immune system, including its ability to survive the high copper (Cu) levels in phagosomes. Our previous work has revealed that under high Cu levels, the A. fumigatus transcription factor AceA is activated, inducing the expression of the copper exporter CrpA to expel excess Cu. To identify additional elements in Cu resistance, we evolved A. fumigatus wild-type and mutant ΔaceA or ΔcrpA strains under increasing Cu concentrations. Sequencing of the resultant resistant strains identified both shared and unique evolutionary pathways to resistance. Reintroduction of three of the most common mutations in genes encoding Pma1 (plasma membrane H+-ATPase), Gcs1 (glutamate cysteine-ligase), and Cpa1 (carbamoyl-phosphate synthetase), alone and in combination, into wild-type A. fumigatus confirmed their additive role in conferring Cu resistance. Detailed analysis indicated that the pma1 mutation L424I preserves Pma1 H+-ATPase activity under high Cu concentrations and that the cpa1 mutation A37V confers a survival advantage to conidia in the presence of Cu. Interestingly, simultaneous mutations of all three genes did not alter virulence in infected mice. Our work has identified novel Cu-resistance pathways and provides an evolutionary approach for dissecting the molecular basis of A. fumigatus adaptation to diverse environmental challenges.IMPORTANCEAspergillus fumigatus is the most common mold infecting patients with weakened immunity. Infection is caused by the inhalation of mold spores into the lungs and is often fatal. In healthy individuals, spores are engulfed by lung immune cells and destroyed by a combination of enzymes, oxidants, and high levels of copper. However, the mold can protect itself by pumping out excess copper with specific transporters. Here, we evolved A. fumigatus under high copper levels and identified new genetic mutations that help it resist the toxic effects of copper. We studied how these mutations affect the mold's ability to resist copper and how they impact its ability to cause disease. This is the first such study in a pathogenic mold, and it gives us a better understanding of how it manages to bypass our body's defenses during an infection.

Serum Aspergillus fumigatus-Specific IgG as a Complementary Biomarker in Differentiating Endotypes of Chronic Rhinosinusitis: A Study on Its Role and Diagnostic Efficacy in Type 2 Inflammation.

BACKGROUND: Aspergillus is one of the most common pathogens causing fungal allergy in the respiratory tract. Serum Aspergillus fumigatus-specific immunoglobulin G (Af-sIgG) levels have been used as a biomarker for the diagnosis and treatment response monitoring in airway allergic diseases such as allergic bronchopulmonary aspergillosis and allergic fungal rhinosinusitis. However, its role in common primary chronic rhinosinusitis (CRS) was unclear. OBJECTIVE: This study aims to evaluate whether serum Af-sIgG level could serve as a biomarker for the disease presentation of primary CRS. METHODS: We obtained serum Af-sIgG levels from patients diagnosed as bilateral primary CRS refractory to medical treatment and evaluated the correlations between serum Af-sIgG levels and disease severity in patients with type 2 (T2) and non-T2 CRS. RESULTS: Patients with T2 CRS exhibited significantly higher serum Af-sIgG levels than non-T2 CRS patients. The cut-off value of serum Af-sIgG in T2 CRS was 20.9 mg/L, with an odds ratio of 3.8 (95% CI 1.17-12.20, P = .026). Furthermore, serum Af-sIgG levels were positively correlated with symptom scores evaluated by the Sino-Nasal Outcome Test-22 (SNOT-22) scores in T2 patients (P = .009). While stratified by SNOT-22 total scores, patients with severe disease had higher serum Af-sIgG levels only in T2 CRS (P = .034). In individual domains of SNOT-22 analysis, serum Af-sIgG levels showed a significant correlation with "ear/facial" symptom scores in the T2 group (P < .001). CONCLUSIONS: Serum Af-sIgG levels may serve as a supplementary objective biomarker that correlates with identification and subjective measurements of T2 CRS, and may be associated with symptoms arising from Eustachian tube dysfunction.

PET imaging of Aspergillus infection using Zirconium-89 labeled anti-ß-glucan antibody fragments.

PURPOSE: Invasive fungal diseases, such as pulmonary aspergillosis, are common life-threatening infections in immunocompromised patients and effective treatment is often hampered by delays in timely and specific diagnosis. Fungal-specific molecular imaging ligands can provide non-invasive readouts of deep-seated fungal pathologies. In this study, the utility of antibodies and antibody fragments (Fab) targeting ß-glucans in the fungal cell wall to detect Aspergillus infections was evaluated both in vitro and in preclinical mouse models. METHODS: The binding characteristics of two commercially available ß-glucan antibody clones and their respective antigen-binding Fabs were tested using biolayer interferometry (BLI) assays and immunofluorescence staining. In vivo binding of the Zirconium-89 labeled antibodies/Fabs to fungal pathogens was then evaluated using PET/CT imaging in mouse models of fungal infection, bacterial infection and sterile inflammation. RESULTS: One of the evaluated antibodies (HA-ßG-Ab) and its Fab (HA-ßG-Fab) bound to ß-glucans with high affinity (KD = 0.056 & 21.5 nM respectively). Binding to the fungal cell wall was validated by immunofluorescence staining and in vitro binding assays. ImmunoPET imaging with intact antibodies however showed slow clearance and high background signal as well as nonspecific accumulation in sites of infection/inflammation. Conversely, specific binding of [89Zr]Zr-DFO-HA-ßG-Fab to sites of fungal infection was observed when compared to the isotype control Fab and was significantly higher in fungal infection than in bacterial infection or sterile inflammation. CONCLUSIONS: [89Zr]Zr-DFO-HA-ßG-Fab can be used to detect fungal infections in vivo. Targeting distinct components of the fungal cell wall is a viable approach to developing fungal-specific PET tracers.

Global Transcriptomic Profiling of Innate and Adaptive Immunity During Aspergillus flavus Endophthalmitis in a Murine Model.

Purpose: Fungal endophthalmitis is characterized by chronic inflammation leading to the partial or complete vision loss. Herein, we analyzed the transcriptomic landscape of Aspergillus flavus (A. flavus) endophthalmitis in C57BL/6 mice to understand the host-pathogen interactions. Methods: Endophthalmitis was induced by intravitreal injection of A. flavus spores in C57BL/6 mice and monitored for disease progression up to 72 hours. The enucleated eyeballs were subjected to histopathological analysis and mRNA sequencing using the Illumina Nextseq 2000. Pathway enrichment analysis was performed to further annotate the functions of differentially expressed genes (DEGs) and validation of cytokines was performed in vitreous of patients with fungal endophthalmitis using multiplex ELISA. Results: Transcriptomic landscape of A. flavus endophthalmitis revealed upregulated T-cell receptor signaling, PI3K-AKT, MAPK, NF-κB, JAK-STAT, and NOD like receptor signaling pathways. We observed significant increase in the T-cells during infection especially at 72 hours infection along with elevated expression levels of IL-6, IL-10, IL-12, IL-18, IL-19, IL-23, CCR3, and CCR7. Furthermore, host-immune response associated genes, such as T-cell interacting activating receptor, TNF receptor-associated factor 1, TLR1, TLR9, and bradykinin receptor beta 1, were enriched. Histopathological assessment validated the significant increase in inflammatory cells, especially T-cells at 72 hours post-infection along with increased disruption in the retinal architecture. Additionally, IL-6, IL-8, IL-17, TNF-α, and IL-1ß were also significantly elevated, whereas IL-10 was downregulated in vitreous of patients with Aspergillus endophthalmitis. Conclusions: Regulating T-cell influx could be a potential strategy to modulate the excessive inflammation in the retina and potentially aid in better vision recovery in fungal endophthalmitis.

Formononetin protects against Aspergillus fumigatus Keratitis: Targeting inflammation and fungal load.

PURPOSE: To investigate the potential treatment of formononetin (FMN) on Aspergillus fumigatus (A. fumigatus) keratitis with anti-inflammatory and antifungal activity. METHODS: The effects of FMN on mice with A. fumigatus keratitis were evaluated through keratitis clinical scores, hematoxylin-eosin (HE) staining, and plate counts. The expression of pro-inflammatory factors was measured using RT-PCR, ELISA, or Western blot. The distribution of macrophages and neutrophils was explored by immunofluorescence staining. The antifungal properties of FMN were assessed through minimum inhibitory concentration (MIC), propidium iodide (PI) staining, fungal spore adhesion, and biofilm formation assay. RESULTS: In A. fumigatus keratitis mice, FMN decreased the keratitis clinical scores, macrophages and neutrophils migration, and the expression of TNF-α, IL-6, and IL-1ß. In A. fumigatus-stimulated human corneal epithelial cells (HCECs), FMN reduced the expression of IL-6, TNF-α, IL-1ß, and NLRP3. FMN also decreased the expression of thymic stromal lymphopoietin (TSLP) and thymic stromal lymphopoietin receptor (TSLPR). Moreover, FMN reduced the levels of reactive oxygen species (ROS) induced by A. fumigatus in HCECs. Furthermore, FMN inhibited A. fumigatus growth, prevented spore adhesion and disrupted fungal biofilm formation in vitro. In vivo, FMN treatment reduced the fungal load in mice cornea at 3 days post infection (p.i.). CONCLUSION: FMN demonstrated anti-inflammatory and antifungal properties, and exhibited a protective effect on mouse A. fumigatus keratitis.

Serological Cross-Reactivity of Various Aspergillus spp. with Aspergillus fumigatus: A Diagnostic Blind Spot.

INTRODUCTION: Aspergillus fumigatus is the most common airborne allergen of the Aspergillus family. However, allergies to Aspergillus spp. are increasing, and subsequently, allergies to Aspergillus species other than fumigatus are also on the rise. Commercial diagnostic tools are still limited to Aspergillus fumigatus. Hence, there is a need for improved tests. We decided to investigate the correlation between serological sensitization to A. fumigatus and other Aspergillus species. METHODS: Hundred and seven patients with positive skin prick tests to A. fumigatus were included in this study. Immunoglobulin E (IgE) concentrations against A. fumigatus, A. terreus, A. niger, A. flavus, and A. versicolor were measured from specimens by fluorescent enzyme-linked immunoassays. RESULTS: Patients showed considerably higher IgE concentrations against A. fumigatus (6.00 ± 15.05 kUA/L) than A. versicolor (0.30 ± 1.01 kUA/L), A. niger (0.62 ± 1.59 kUA/L), A. terreus (0.45 ± 1.12 kUA/L), or A. flavus (0.41 ± 0.97 kUA/L). Regression analysis yielded weak positive correlations for all Aspergillus spp., but low r2 values and heteroscedastic distribution indicate an overall poor fit of the calculated models. CONCLUSION: Serological sensitization against A. fumigatus does not correlate with sensitization against other Aspergillus spp. To detect sensitization against these, other diagnostic tools like a skin prick test solution of different Aspergillus spp. are needed.

Siglec-F Promotes IL-33-Induced Cytokine Release from Bone Marrow-Derived Eosinophils Independently of the ITIM and ITIM-like Motif Phosphorylation.

Eosinophils are potent innate effector cells associated mainly with type 2 immune responses elicited by helminths and allergens. Their activity needs to be tightly controlled to prevent severe inflammation and tissue damage. Eosinophil degranulation and secretion of inflammatory effector molecules, including cytokines, chemokines, and lipid mediators, can be regulated by activating and inhibitory receptors on the cell surface. In this study, we investigated the modulation of proliferation, apoptosis, gene expression, and cytokine/chemokine secretion from IL-33-activated Mus musculus eosinophils on cross-linking of the transmembrane receptor Sialic acid-binding Ig-like lectin F (Siglec-F). Siglec-F contains an ITIM plus an ITIM-like motif in its intracellular tail and is mainly regarded as an inhibitory and apoptosis-inducing receptor. In vitro costimulation of bone marrow-derived eosinophils with anti-Siglec-F and IL-33 compared with treatment with either alone led to enhanced STAT6 phosphorylation, stronger induction of hypoxia/glycolysis-related proinflammatory genes, and elevated secretion of type 2 cytokines (IL-4, IL-13) and chemokines (CCL3, CCL4) with only minor effects on proliferation and apoptosis. Using a competitive mixed bone marrow chimera approach with wild-type and Siglec-F-deficient eosinophils, we observed no evidence for Siglec-F-regulated inhibition of Aspergillus fumigatus-elicited lung eosinophilia. Truncation of the Siglec-F cytoplasmic tail, but not mutation of the ITIM and ITIM-like motifs, ablated the effect of enhanced cytokine/chemokine secretion. This provides evidence for an ITIM phosphorylation-independent signaling pathway from the cytoplasmic tail of the Siglec-F receptor that enhances effector molecule release from activated eosinophils.

IL-27 Signaling Promotes Th1 Responses and Is Required to Inhibit Fungal Growth in the Lung during Repeated Exposure to Aspergillus fumigatus.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes a wide spectrum of diseases in humans, including life-threatening invasive infections as well as several hypersensitivity respiratory disorders. Disease prevention is predicated on the host's ability to clear A. fumigatus from the lung while also limiting inflammation and preventing allergic responses. IL-27 is an important immunoregulatory cytokine, but its role during A. fumigatus infection remains poorly understood. In contrast to most infection settings demonstrating that IL-27 is anti-inflammatory, in this study we report that this cytokine plays a proinflammatory role in mice repeatedly infected with A. fumigatus We found that mice exposed to A. fumigatus had significantly enhanced secretion of IL-27 in their lungs. Genetic ablation of IL-27Rα in mice resulted in significantly higher fungal burdens in the lung during infection. The increased fungal growth in IL-27Rα-/- mice was associated with reduced secretion of IL-12, TNF-α, and IFN-γ, diminished T-bet expression, as well as a reduction in CD4+ T cells and their activation in the lung, demonstrating that IL-27 signaling promotes Th1 immune responses during repeated exposure to A. fumigatus In addition, infected IL-27Rα-/- mice displayed reduced accumulation of dendritic cells and exudate macrophages in their lungs, and these cells had a lower expression of MHC class II. Collectively, this study suggests that IL-27 drives type 1 immunity and is indispensable for inhibiting fungal growth in the lungs of mice repeatedly exposed to A. fumigatus, highlighting a protective role for this cytokine during fungal infection.