Retrospective Comparison of Two Aspergillus IgG Enzyme Immunoassays for Diagnosing Pulmonary Aspergillosis.

Aspergillus-specific antibodies are diagnostic indicators of allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis (CPA). Tests for detecting Aspergillus-specific antibodies were not used clinically in Japan, and the production of the Aspergillus precipitin test was discontinued. Thus, alternative tests for diagnosing aspergillosis are urgently needed. We retrospectively evaluated 64 patients with suspected ABPA and CPA who underwent precipitin antibody testing. Serum Aspergillus IgG levels were measured and compared using the Bordier Aspergillus fumigatus ELISA and the Platelia Aspergillus IgG (Bio-Rad) kits. Of the participants, 18 were diagnosed with CPA, and 8 were diagnosed with ABPA. Both the Bordier and Bio-Rad kits showed high sensitivity and specificity for CPA and ABPA. The area under the receiver operating characteristic curves for the Bordier and Bio-Rad kits were 0.97 and 0.95, respectively, for CPA, and 0.89 and 0.91, respectively, for ABPA. In contrast to the Bordier kit, the Bio-Rad kit showed relatively low anti-Aspergillus IgG levels and lower sensitivity to non-fumigatus Aspergillus infections. The Aspergillus-specific IgG ELISA tests showed sufficient diagnostic accuracy. Therefore, these assays are recommended as alternatives to the precipitin kit for diagnosing aspergillosis in clinical settings in Japan.

Novel acute hypersensitivity pneumonitis model induced by airway mycosis and high dose lipopolysaccharide.

BACKGROUND: Inhalation of fungal spores is a strong risk factor for severe asthma and experimentally leads to development of airway mycosis and asthma-like disease in mice. However, in addition to fungal spores, humans are simultaneously exposed to other inflammatory agents such as lipopolysaccharide (LPS), with uncertain relevance to disease expression. To determine how high dose inhalation of LPS influences the expression of allergic airway disease induced by the allergenic mold Aspergillus niger (A. niger). METHODS: C57BL/6J mice were intranasally challenged with the viable spores of A. niger with and without 1 µg of LPS over two weeks. Changes in airway hyperreactivity, airway and lung inflammatory cell recruitment, antigen-specific immunoglobulins, and histopathology were determined. RESULTS: In comparison to mice challenged only with A. niger, addition of LPS (1 µg) to A. niger abrogated airway hyperresponsiveness and strongly attenuated airway eosinophilia, PAS+ goblet cells and TH2 responses while enhancing TH1 and TH17 cell recruitment to lung. Addition of LPS resulted in more severe, diffuse lung inflammation with scattered, loosely-formed parenchymal granulomas, but failed to alter fungus-induced IgE and IgG antibodies. CONCLUSIONS: In contrast to the strongly allergic lung phenotype induced by fungal spores alone, addition of a relatively high dose of LPS abrogates asthma-like features, replacing them with a phenotype more consistent with acute hypersensitivity pneumonitis (HP). These findings extend the already established link between airway mycosis and asthma to HP and describe a robust model for further dissecting the pathophysiology of HP.

The IL-1 Receptor Is Required to Maintain Neutrophil Viability and Function During Aspergillus fumigatus Airway Infection.

Aspergillus fumigatus airway infections are associated with increased rates of hospitalizations and declining lung function in patients with chronic lung disease. While the pathogenesis of invasive A. fumigatus infections is well studied, little is known about the development and progression of airway infections. Previous studies have demonstrated a critical role for the IL-1 cytokines, IL-1α and IL-1ß in enhancing pulmonary neutrophil recruitment during invasive aspergillosis. Here we use a mouse model of A. fumigatus airway infection to study the role of these IL-1 cytokines in immunocompetent mice. In the absence of IL-1 receptor signaling, mice exhibited reduced numbers of viable pulmonary neutrophils and increased levels of neutrophil apoptosis during fungal airway infection. Impaired neutrophil viability in these mice was associated with reduced pulmonary and systemic levels of G-CSF, and treatment with G-CSF restored both neutrophil viability and resistance to A. fumigatus airway infection. Taken together, these data demonstrate that IL-1 dependent G-CSF production plays a key role for host resistance to A. fumigatus airway infection through suppressing neutrophil apoptosis at the site of infection.

Diagnostic accuracy of Aspergillus-specific antibodies for chronic pulmonary aspergillosis: A systematic review and meta-analysis.

We performed this study to provide the latest evidence of the diagnostic accuracy of all Aspergillus antibodies for chronic pulmonary aspergillosis (CPA). In this meta-analysis, we searched the Cochrane Central Register of Controlled Trials, MEDLINE, Embase, and other databases, until 19 March 2020, for studies that examined the diagnostic accuracy of each Aspergillus-specific antibody for CPA and assessed the risk of bias using the revised Quality Assessment of Diagnostic Accuracy Studies-2 tool. We integrated the results using a hierarchical summary receiver operating characteristic (HSROC) model and calculated the point estimates of specificity with sensitivity fixed at 0.90 using the HSROC curve. We identified 32 published and one unpublished studies, including 75 studies on five antibody test types: 18 of precipitin test (2810 participants), 46 of IgG (8197), three of IgA (283), six of IgM (733) and two of combined IgG and IgM (IgG + IgM) (920). The results of specificity with sensitivity fixed at 0.90 were as follows: precipitin test, 0.93 (95% credible intervals: 0.86, 1.00); IgG, 0.90 (0.86, 0.95); IgA, 0.74 (0.00, 1.00); IgM, 0.50 (0.37, 0.53); IgG + IgM, 0.47 (0.00, 1.00). However, the precipitin test showed imprecision and instability in the sensitivity analysis. Most studies had a high risk of bias due to the case-control design. Although there is lack of applicability for malignancy or immunosuppressive patients, our study suggests a preference for IgG over other antibody tests in CPA screening. Particularly, IgG should be used as an adjunct when ruling out CPA.

A Murine Model for Chronic A. fumigatus Airway Infections.

In addition to causing acute invasive infections in immunocompromised patients, the mold Aspergillus fumigatus causes chronic infections in patients with chronic pulmonary conditions such as cystic fibrosis. Here we describe a non-lethal model of chronic pulmonary aspergillosis in which immunocompetent mice are endotracheally infected with A. fumigatus conidia embedded in agar beads. This approach results in the establishment of hyphal infection within the airways of mice for up to a 28-day period and is amenable to the study of innate and adaptive antifungal responses, fungal mutant strains, and antifungal agents.

NLRX1 is a key regulator of immune signaling during invasive pulmonary aspergillosis.

Aspergillus fumigatus is an opportunistic fungal pathogen of immunocompromised patient populations. Mortality is thought to be context-specific and occurs via both enhanced fungal growth and immunopathogenesis. NLRX1 is a negative regulator of immune signaling and metabolic pathways implicated in host responses to microbes, cancers, and autoimmune diseases. Our study indicates loss of Nlrx1 results in enhanced fungal burden, pulmonary inflammation, immune cell recruitment, and mortality across immuno-suppressed and immuno-competent models of IPA using two clinically derived isolates (AF293, CEA10). We observed that the heightened mortality is due to enhanced recruitment of CD103+ dendritic cells (DCs) that produce elevated amounts of IL-4 resulting in a detrimental Th2-mediated immune response. Adoptive transfer of Nlrx1-/- CD103+ DCs in neutropenic NRG mice results in enhanced mortality that can be ablated using IL-4 neutralizing antibodies. In vitro analysis of CD103+ DCs indicates loss of Nlrx1 results in enhanced IL-4 production via elevated activation of the JNK/JunB pathways. Interestingly, loss of Nlrx1 also results in enhanced recruitment of monocytes and neutrophils. Chimeras of irradiated Nlrx1-/- mice reconstituted with wild type bone marrow have enhanced neutrophil recruitment and survival during models of IPA. This enhanced immune cell recruitment in the absence of Nlrx1 is mediated by excessive production of CXCL8/IL-8 family of chemokines and IL-6 via early and enhanced activation of P38 in response to A. fumigatus conidia as shown in BEAS-2B airway epithelial cells. In summary, our results point strongly towards the cell-specific and contextual function of Nlrx1 during invasive pulmonary aspergillosis and may lead to novel therapeutics to reduce Th2 responses by CD103+ DCs or heightened recruitment of neutrophils.

A complex COVID-19 case with rheumatoid arthritis treated with tocilizumab.

Recurrences of COVID-19 were observed in a patient with long-term usage of hydroxychloroquine, leflunomide, and glucocorticoids due to her 30-year history of rheumatoid arthritis (RA). Tocilizumab was applied and intended to target both COVID-19 and RA. However, disease of this patient aggravated after usage of tocilizumab. After the discussion of a multiple disciplinary team (MDT) including rheumatologists, antimicrobial treatments were applied to target the potential opportunistic infections (Pneumocystis jirovecii and Aspergillus fumigatus), which were authenticated several days later via high throughput sequencing. As an important cytokine in immune responses, IL-6 can be a double-edged sword: interference in the IL-6-IL-6 receptor signaling may save patients from cytokine release storm (CRS), but can also weaken the anti-infectious immunity, particularly in rheumatic patients, who may have received a long-term treatment with immunosuppressive/modulatory agents. Thus, we suggest careful considerations before and close monitoring in the administration of tocilizumab in rheumatic patients with COVID-19. Besides tocilizumab, several disease-modifying antirheumatic drugs (DMARDs) can also be applied in the treatment of COVID-19. Therefore, we also reviewed and discussed the application of these DMARDs in COVID-19 condition.

Platelets are critical for survival and tissue integrity during murine pulmonary Aspergillus fumigatus infection.

Beyond their canonical roles in hemostasis and thrombosis, platelets function in the innate immune response by interacting directly with pathogens and by regulating the recruitment and activation of immune effector cells. Thrombocytopenia often coincides with neutropenia in patients with hematologic malignancies and in allogeneic hematopoietic cell transplant recipients, patient groups at high risk for invasive fungal infections. While neutropenia is well established as a major clinical risk factor for invasive fungal infections, the role of platelets in host defense against human fungal pathogens remains understudied. Here, we examined the role of platelets in murine Aspergillus fumigatus infection using two complementary approaches to induce thrombocytopenia without concurrent neutropenia. Thrombocytopenic mice were highly susceptible to A. fumigatus challenge and rapidly succumbed to infection. Although platelets regulated early conidial phagocytosis by neutrophils in a spleen tyrosine kinase (Syk)-dependent manner, platelet-regulated conidial phagocytosis was dispensable for host survival. Instead, our data indicated that platelets primarily function to maintain hemostasis and lung integrity in response to exposed fungal antigens, since thrombocytopenic mice exhibited severe hemorrhage into the airways in response to fungal challenge in the absence of overt angioinvasion. Challenge with swollen, heat-killed, conidia was lethal in thrombocytopenic hosts and could be reversed by platelet transfusion, consistent with the model that fungus-induced inflammation in platelet-depleted mice was sufficient to induce lethal hemorrhage. These data provide new insights into the role of platelets in the anti-Aspergillus host response and expand their role to host defense against filamentous molds.

Clinical relevance of Aspergillus fumigatus sensitization in cystic fibrosis.

RATIONALE: The clinical relevance of sensitization to Aspergillus (A) fumigatus in cystic fibrosis (CF) is unclear. Some researchers propose that specific A fumigatus IgE is an innocent bystander, whereas others describe it as the major cause of TH-2-driven asthma-like disease. OBJECTIVES: Lung function parameters in mild CF patients may be different in patients with and without A fumigatus sensitization. We aimed to ascertain whether allergen exposure to A fumigatus by bronchial allergen provocation (BAP) induces TH-2 inflammation comparable to an asthma-like disease. METHODS: A total of 35 patients, aged 14.8 ± 8.5 years, and 20 healthy controls were investigated prospectively. The patients were divided into two groups: group 1 (n = 18): specific (s)IgE negative, and group 2 (n = 17): sIgE positive (≥0.7 KU/L) for A fumigatus. Lung function, exhaled NO, and induced sputum were analysed. All sensitized patients with an FEV1 > 75% (n = 13) underwent BAP with A fumigatus, and cell counts, and the expression of IL-5, IL-13, INF-γ, and IL-8 as well as transcription factors T-bet, GATA-3, and FoxP3, were measured. RESULTS: Lung function parameters decreased significantly compared to controls, but not within the CF patient group. After BAP, 8 of 13 patients (61%) had a significant asthmatic response and increased eNO 24 hours later. In addition, marked TH-2-mediated inflammation involving eosinophils, IL-5, IL-13, and FoxP3 became apparent in induced sputum cells. CONCLUSION: Our study demonstrated the clinical relevance of A fumigatus for the majority of sensitized CF patients. A distinct IgE/TH-2-dominated inflammation was found in induced sputum after A fumigatus exposure.

Analysis of the Clinical Features of Tracheobronchial Fungal Infections with Tumor-Like Lesions.

BACKGROUND: Tracheobronchial fungal infections (TBFI) cause life-threatening complications in immunocompromised hosts but are rarely reported. Misdiagnosis and delayed antifungal treatment are associated with the high mortality rate of patients with TBFI. OBJECTIVES: This study analyzed the bronchoscopic features of TBFI and their roles in the early diagnosis of TBFI. METHODS: The demographic, clinical, radiologic, and bronchoscopic data of 53 patients diagnosed with TBFI in our department during a 15-year period were retrospectively analyzed. RESULTS: Most of the TBFI patients were male, and mass was the most common radiologic abnormality. Obvious predilection in primary bronchus distributions was observed. 41.9% of the 43 Aspergillus tracheobronchitis (AT) patients, 70% of the 10 tracheobronchial mucormycosis (TM) patients, and 100% of the 3 endobronchial cryptococcosis patients had been misdiagnosed as having cancer on bronchoscopy because of the presence of tumor-like lesions. The most common features of AT were bronchial occlusion with a mass or mucosal necrosis, bronchial stenosis with mucosal hyperplasia, or uneven mucosa. The main descriptions of TM were bronchial stenosis or obstruction due to mucosal necrosis, uneven mucosa, or a mass. The endoscopic characteristics of endobronchial cryptococcosis included occlusion due to uneven mucosa or mass, or external compressive stricture. CONCLUSION: Immunocompromised patients and immunocompetent patients with underlying disease displaying tumor-like lesions on bronchoscopy should be differentially diagnosed with cancer. Bronchial biopsy is indispensable for the early diagnosis of TBFI.

Aspergillus colonization and antifungal immunity in cystic fibrosis patients.

Cystic fibrosis (CF), caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, is the most common inherited life-limiting disease in North European people affecting 90,000 people worldwide. Progressive lung damage caused by recurrent infection and chronic airway inflammation is the major determinant of survival with a median age at death of 29 years. Approximately 60% of CF patients are infected with Aspergillus fumigatus, a ubiquitous environmental fungus, and its presence has been associated with accelerated lung function decline. Half of the patients infected with Aspergillus are <18 years of age. Yet time of acquisition of this fungus and determinants of CF-related Aspergillus disease severity and progression are not known. CFTR expression has been demonstrated in cells of the innate and adaptive immune system and has shown to be critical for normal function. Research delineating the role of CFTR-deficient phagocytes in Aspergillus persistence and infection in the CF lung, has only recently received attention. In this concise review we aim to present the current understanding with respect to when people with CF acquire infection with A. fumigatus and antifungal immune responses by CF immune cells.

The Serum Level of IL-1B Correlates with the Activity of Chronic Pulmonary Aspergillosis.

Background: Until now, there have been no objective criteria to determine the activity of chronic pulmonary aspergillosis (CPA). This study aims to analyze the correlation between serum level of IL-1B and the activity of CPA and to determine whether serum IL-1B could be used to assess the activity of CPA. Methods: A total of 469 newly diagnosed CPA patients were enrolled. Correlation analysis in the whole subjects showed that only IL-1B level was associated with the activity of CPA. Then, 381 cases with factors significantly affecting IL-1B expression was excluded through multiple linear regression; the remaining 88 patients were divided into high IL-1B group and low IL-1B group, according to the median value of serum IL-1B, for subgroup analysis. A retrospective comparative analysis was subsequently performed between the two groups, including the clinical manifestation, microbiology and laboratory tests results, and imaging findings. We further investigated the relationship between IL-1B levels and CT characteristic which acted as the indicator of CPA activity, as well as changes in IL-1B level before and after surgery. Results: For all patients, correlation analysis revealed that IL-1B level correlated with both cavitary diameter (P=0.035) and aspergilloma size (P<0.047) but not with the thickness of the cavity (P=0.479). In subgroup comparative analysis, CT characteristics suggested that high activity of CPA, such as cavitary (27/44 vs 13/44, P=0.003) and aspergilloma lesions (25/44 vs. 11/44, P<0.002), were more frequently found in high IL-1B group. The cavity diameter (P<0.001), aspergilloma size (P=0.006), and cavity wall thickness (P=0.023) were significantly different between the two groups. When Spearman correlation analysis was performed once again in subgroup, an even stronger relationship of serum IL-1B with the cavity diameter (Rs=0.501, P=0.002) and aspergilloma size (Rs=0.615, P=0.001) was observed. Interestingly, a significant reduction of IL-1B level was observed after successful resection of CPA lesions. Conclusion: Higher level of serum IL-1B is associated with more severe cavitary and aspergilloma lesions, which are indicative of more active CPA. In addition, IL-1B level reduced accordingly after lesion resection. Measuring IL-1B level therefore could be served as a convenient method to monitor the activity of CPA and be a potential predictive/prognostic marker for treatment response.

Immunological corollary of the pulmonary mycobiome in bronchiectasis: the CAMEB study.

Understanding the composition and clinical importance of the fungal mycobiome was recently identified as a key topic in a "research priorities" consensus statement for bronchiectasis.Patients were recruited as part of the CAMEB study: an international multicentre cross-sectional Cohort of Asian and Matched European Bronchiectasis patients. The mycobiome was determined in 238 patients by targeted amplicon shotgun sequencing of the 18S-28S rRNA internally transcribed spacer regions ITS1 and ITS2. Specific quantitative PCR for detection of and conidial quantification for a range of airway Aspergillus species was performed. Sputum galactomannan, Aspergillus specific IgE, IgG and TARC (thymus and activation regulated chemokine) levels were measured systemically and associated to clinical outcomes.The bronchiectasis mycobiome is distinct and characterised by specific fungal genera, including Aspergillus, Cryptococcus and ClavisporaAspergillus fumigatus (in Singapore/Kuala Lumpur) and Aspergillus terreus (in Dundee) dominated profiles, the latter associating with exacerbations. High frequencies of Aspergillus-associated disease including sensitisation and allergic bronchopulmonary aspergillosis were detected. Each revealed distinct mycobiome profiles, and associated with more severe disease, poorer pulmonary function and increased exacerbations.The pulmonary mycobiome is of clinical relevance in bronchiectasis. Screening for Aspergillus-associated disease should be considered even in apparently stable patients.

Clinical efficacy of sequential therapy with voriconazole on COPD patients in acute phase with pulmonary aspergillosis and effects on cytokines and pulmonary functions.

OBJECTIVE: To explore the clinical efficacy of sequential therapy with voriconazole on chronic obstructive pulmonary disease (COPD) patients in acute phase with pulmonary aspergillosis and its effects on cytokines and pulmonary functions. PATIENTS AND METHODS: A total of 110 COPD patients in acute phase with pulmonary aspergillosis who were admitted to the hospital between February 2015 and November 2016 were enrolled. We divided them randomly into two groups, i.e., the control group (n = 55) and the treatment group (n = 55). Patients in the control group took itraconazole capsules orally (200 mg/time, twice per day for three days followed by once per day). Patients in treatment group underwent sequential treatment with voriconazole through intravenous infusion at a dose of 5 mg/kg/time twice a day for 3 days followed by a dose of 4 mg/kg/time, twice a day for 8 days. Then, patients took voriconazole orally at a dose of 150 mL/time, twice a day for 6 days. Patients in two groups received the treatment for a total of 14 days. After treatment, we evaluated the levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-8. The total lung capacity (TLC), diffusing capacity of the lung for carbon monoxide (DLco), and arterial oxygen saturation (SaO2), were measured as well. RESULTS: The total effectiveness rates of the treatment group and the control group were 83.63% and 61.82%. The differences had statistical significance (p < 0.01). After treatment, the incidence of chest pain, cough, sputum-coughing, hemoptysis, cyanosis, and dyspnea in the treatment group was significantly fewer than that in the control group (p < 0.05). TCL, DLco, and SaO2 in the two groups were significantly ameliorated by treatment (p < 0.05). The amelioration in the treatment group was more prominent than that in the control group (p < 0.05). The levels of TNF-α, IL-8, and IL-6 in the two groups were decreased dramatically by the treatments. The decrease in the treatment group was significantly lower than those in the control group (p < 0.05). Occurrence of adverse reactions in treatment group and control group were 8.33% and 6.25%, respectively; (p > 0.05). CONCLUSIONS: Sequential therapy with voriconazole exhibits promising clinical efficacy in COPD patients in acute phase with pulmonary aspergillosis. The treatment ameliorated the clinical symptoms and vital signs of patients significantly. It also improved the pulmonary functions and inhibited the inflammatory responses of patients with evident clinical efficacy.

Sensitization to Aspergillus fumigatus as a risk factor for bronchiectasis in COPD.

BACKGROUND: Bronchiectasis-chronic obstructive pulmonary disease (COPD) overlap presents a possible clinical phenotype of COPD, but it is unclear why it develops in a subset of patients. We hypothesized that sensitization to Aspergillus fumigatus (A fum) is associated with bronchiectasis in COPD and occurs more frequently in vitamin D-deficient patients. METHODS: This observational study investigated sensitization to A fum in an outpatient clinical cohort of 300 COPD patients and 50 (ex-) smoking controls. Total IgE, A fum-specific IgE against the crude extract and against the recombinant antigens and A fum IgG were measured using ImmunoCAP fluoroenzyme immunoassay. Vitamin D was measured by radioimmunoassay, and computed tomography images of the lungs were scored using the modified Reiff score. RESULTS: Sensitization to A fum occurred in 18% of COPD patients compared to 4% of controls (P=0.0110). In all, 31 COPD patients (10%) were sensitized to the crude extract and 24 patients (8%) had only IgE against recombinant antigens. A fum IgG levels were significantly higher in the COPD group (P=0.0473). Within COPD, A fum-sensitized patients were more often male (P=0.0293) and more often had bronchiectasis (P=0.0297). Pseudomonas aeruginosa and Serratia marcescens were more prevalent in historical sputum samples of A fum-sensitized COPD patients compared to A fum-non-sensitized COPD patients (P=0.0436). Vitamin D levels were comparable (P=0.2057). Multivariate analysis demonstrated that sensitization to recombinant f1 or f3 had a 2.8-fold increased risk for bronchiectasis (P=0.0030). CONCLUSION: These results highlight a potential role for sensitization to A fum in COPD-related bronchiectasis.

Krüppel-like Factor 4 modulates interleukin-6 release in human dendritic cells after in vitro stimulation with Aspergillus fumigatus and Candida albicans.

Invasive fungal infections are associated with high mortality rates and are mostly caused by the opportunistic fungi Aspergillus fumigatus and Candida albicans. Immune responses against these fungi are still not fully understood. Dendritic cells (DCs) are crucial players in initiating innate and adaptive immune responses against fungal infections. The immunomodulatory effects of fungi were compared to the bacterial stimulus LPS to determine key players in the immune response to fungal infections. A genome wide study of the gene regulation of human monocyte-derived dendritic cells (DCs) confronted with A. fumigatus, C. albicans or LPS was performed and Krüppel-like factor 4 (KLF4) was identified as the only transcription factor that was down-regulated in DCs by both fungi but induced by stimulation with LPS. Downstream analysis demonstrated the influence of KLF4 on the interleukine-6 expression in human DCs. Furthermore, KLF4 regulation was shown to be dependent on pattern recognition receptor ligation. Therefore KLF4 was identified as a controlling element in the IL-6 immune response with a unique expression pattern comparing fungal and LPS stimulation.

Aspergillus infections in cystic fibrosis.

Patients with cystic fibrosis (CF) suffer from chronic lung infection and airway inflammation. Respiratory failure secondary to chronic or recurrent infection remains the commonest cause of death and accounts for over 90% of mortality. Bacteria as Staphylococcus aureus, Pseudomonas aeruginosa and Burkholderia cepacia complex have been regarded the main CF pathogens and their role in progressive lung decline has been studied extensively. Little attention has been paid to the role of Aspergillus spp. and other filamentous fungi in the pathogenesis of non-ABPA (allergic bronchopulmonary aspergillosis) respiratory disease in CF, despite their frequent recovery in respiratory samples. It has become more apparent however, that Aspergillus spp. may play an important role in chronic lung disease in CF. Research delineating the underlying mechanisms of Aspergillus persistence and infection in the CF lung and its link to lung deterioration is lacking. This review summarizes the Aspergillus disease phenotypes observed in CF, discusses the role of CFTR (cystic fibrosis transmembrane conductance regulator)-protein in innate immune responses and new treatment modalities.

Calcineurin Orchestrates Lateral Transfer of Aspergillus fumigatus during Macrophage Cell Death.

RATIONALE: Pulmonary aspergillosis is a lethal mold infection in the immunocompromised host. Understanding initial control of infection and how this is altered in the immunocompromised host are key goals for comprehension of the pathogenesis of pulmonary aspergillosis. OBJECTIVES: To characterize the outcome of human macrophage infection with Aspergillus fumigatus and how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants. METHODS: We defined the outcome of human macrophage infection with A. fumigatus, as well as the impact of calcineurin inhibitors, through a combination of single-cell fluorescence imaging, transcriptomics, proteomics, and in vivo studies. MEASUREMENTS AND MAIN RESULTS: Macrophage phagocytosis of A. fumigatus enabled control of 90% of fungal germination. However, fungal germination in the late phagosome led to macrophage necrosis. During programmed necroptosis, we observed frequent cell-cell transfer of A. fumigatus between macrophages, which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein envelope. Its relevance to the control of fungal germination was also shown by direct visualization in our zebrafish aspergillosis model in vivo. The calcineurin inhibitor FK506 (tacrolimus) reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and also resulted in hyphal escape. CONCLUSIONS: These observations identify programmed, necrosis-dependent lateral transfer of A. fumigatus between macrophages as an important host strategy for controlling fungal germination. This process is critically dependent on calcineurin. Our studies provide fundamental insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.

FleA Expression in Aspergillus fumigatus Is Recognized by Fucosylated Structures on Mucins and Macrophages to Prevent Lung Infection.

The immune mechanisms that recognize inhaled Aspergillus fumigatus conidia to promote their elimination from the lungs are incompletely understood. FleA is a lectin expressed by Aspergillus fumigatus that has twelve binding sites for fucosylated structures that are abundant in the glycan coats of multiple plant and animal proteins. The role of FleA is unknown: it could bind fucose in decomposed plant matter to allow Aspergillus fumigatus to thrive in soil, or it may be a virulence factor that binds fucose in lung glycoproteins to cause Aspergillus fumigatus pneumonia. Our studies show that FleA protein and Aspergillus fumigatus conidia bind avidly to purified lung mucin glycoproteins in a fucose-dependent manner. In addition, FleA binds strongly to macrophage cell surface proteins, and macrophages bind and phagocytose fleA-deficient (∆fleA) conidia much less efficiently than wild type (WT) conidia. Furthermore, a potent fucopyranoside glycomimetic inhibitor of FleA inhibits binding and phagocytosis of WT conidia by macrophages, confirming the specific role of fucose binding in macrophage recognition of WT conidia. Finally, mice infected with ΔfleA conidia had more severe pneumonia and invasive aspergillosis than mice infected with WT conidia. These findings demonstrate that FleA is not a virulence factor for Aspergillus fumigatus. Instead, host recognition of FleA is a critical step in mechanisms of mucin binding, mucociliary clearance, and macrophage killing that prevent Aspergillus fumigatus pneumonia.