The Therapeutic Role and Mechanism of Glabridin Under Aspergillus fumigatus Infection.

Purpose: To characterize the efficiency of glabridin alone and in combination with clinical antifungals in Aspergillus fumigatus keratitis. Methods: The broth microdilution method was performed to investigate whether glabridin exerted an antifungal role on planktonic cells and immature and mature biofilm. Antifungal mechanism was evaluated by Sorbitol and Ergosterol Assays. The synergistic effect of glabridin and antifungals was assessed through the checkerboard microdilution method and time-killing test. Regarding anti-inflammatory role, inflammatory substances induced by A. fumigatus were assessed by real-time quantitative polymerase chain reaction, western blot, and enzyme-linked immunosorbent assay. Drug toxicity was assessed by Draize test in vivo. Macrophage phenotypes were examined by flow cytometry. Results: Regarding antifungal activity, glabridin destroyed fungal cell wall and membrane on planktonic cells and suppressed immature and mature biofilm formation. After combining with natamycin or amphotericin B, glabridin possessed a potent synergistic effect against A. fumigatus. Regarding anti-inflammatory aspects, Dectin-1, toll­like receptor (TLR)-2 and TLR-4 expression of human corneal epithelial cells were significantly elevated after A. fumigatus challenge and reduced by glabridin. The elevated expression of interleukin-1ß and tumor necrosis factor-alpha induced by A. fumigatus or corresponding agonists were reversed by glabridin, equivalent to the effect of corresponding inhibitors. Glabridin could also contribute to anti-inflammation by downregulating inflammatory mediator expression to suppress macrophage infiltration. Conclusions: Glabridin contributed to fungal clearance by destroying fungal cell wall and membrane, and disrupting biofilm. Combining glabridin with clinical antifungals was superior in reducing A. fumigatus growth. Glabridin exerted an anti-inflammatory effect by downregulating proinflammatory substance expression and inhibiting macrophage infiltration, which provide a potential agent and treatment strategies for fungal keratitis.

Macrophages inhibit extracellular hyphal growth of A. fumigatus through Rac2 GTPase signaling.

Macrophages act as a first line of defense against pathogens. Against Aspergillus fumigatus, a fungus with pathogenic potential in immunocompromised patients, macrophages can phagocytose fungal spores and inhibit spore germination to prevent the development of tissue-invasive hyphae. However, the cellular pathways that macrophages use to accomplish these tasks and any roles macrophages have later in infection against invasive forms of fungi are still not fully known. Rac-family Rho GTPases are signaling hubs for multiple cellular functions in leukocytes, including cell migration, phagocytosis, reactive oxygen species (ROS) generation, and transcriptional activation. We therefore aimed to further characterize the function of macrophages against A. fumigatus in an in vivo vertebrate infection model by live imaging of the macrophage behavior in A. fumigatus-infected rac2 mutant zebrafish larvae. While Rac2-deficient zebrafish larvae are susceptible to A. fumigatus infection, Rac2 deficiency does not impair macrophage migration to the infection site, interaction with and phagocytosis of spores, spore trafficking to acidified compartments, or spore killing. However, we reveal a role for Rac2 in macrophage-mediated inhibition of spore germination and control of invasive hyphae. Re-expression of Rac2 under a macrophage-specific promoter rescues the survival of A. fumigatus-infected rac2 mutant larvae through increased control of germination and hyphal growth. Altogether, we describe a new role for macrophages against extracellular hyphal growth of A. fumigatus and report that the function of the Rac2 Rho GTPase in macrophages is required for this function.

0.01% Hypochlorous Acid Treats Aspergillus fumigatus Keratitis in Rats by Reducing Fungal Load and Inhibiting the Inflammatory Response.

Purpose: To investigate the antifungal and anti-inflammatory effects of 0.01% hypochlorous acid (HCLO) on rats with Aspergillus fumigatus keratitis. Methods: The time-kill assay and broth microdilution procedures were used in vitro to demonstrate that 0.01% HCLO was fungicidal and fungistatic. The severity of the disease was evaluated in vivo using a clinical score and slit-lamp photographs. Fungal load, polymorphonuclear neutrophil infiltration, and the production of related proteins were determined using colony plate counting, in vivo confocal microscopy, periodic acid-Schiff staining, fungal fluorescence staining, immunofluorescence staining, myeloperoxidase assay, and Western blotting. Result: In vitro, 0.01% HCLO can destroy A. fumigatus spores in 1 minute. The optical density of the 0.01% HCLO group was significantly lower than that of the phosphate-buffered saline control group (P < 0.01), and no visible mycelium was observed using a fluorescence microscope. 0.01% HCLO reduced the severity of A. fumigatus keratitis in rats by decreasing the clinical score, fungal loading (periodic acid-Schiff, plate count, and fungal fluorescence staining), and inhibiting neutrophil infiltration and activity (immunofluorescence staining and myeloperoxidase). Furthermore, the Western blot analysis revealed that 0.01% HCO decreased protein expression levels of tumor necrosis factor-α and IL-1ß. Conclusions: According to our findings, 0.01% HCLO can kill A. fumigatus spores in vitro. It has antifungal and anti-inflammatory effects on A. fumigatus keratitis in rats. It also inhibited A. fumigatus growth; decreased neutrophil infiltration, tumor necrosis factor-α, and IL-1ß expression; and provided a potential treatment for fungal keratitis. Translational Relevance: This study provides a potential treatment for fungal keratitis in the clinic.

Resolvin D1 protects against Aspergillus fumigatus keratitis in diabetes by blocking the MAPK-NF-κB pathway.

Fungal keratitis (FK) is one of the main causes of blindness in China. People with diabetes are susceptible to corneal epithelial disease, even fungal keratitis. At present, there are few studies on this disease. Resolvins (Rv) has been reported as a mediators that exert crucial anti-inflammatory and immune regulation roles in serval diseases. In order to investigate the roles and underlying mechanism of Resolvins D1 (RvD1) on the Aspergillus fumigatus (A. fumigatus) keratitis in diabetes, we established in vivo and in vitro models of A. fumigatus keratitis, which were then exposed to high glucose. The expression levels of RvD1, 5-lipoxygenase (5-LOX), and 15-lipoxygenase (15-LOX) in A. fumigatus keratitis patients with diabetes were determined through Enzyme Linked Immunosorbent Assay (ELISA), Western blot and immunohistochemistry. Reactive Oxygen Species (ROS) production, ELISA, flow cytometry, Hematoxylin-Eosin (HE) staining and fungal loading determination were conducted to evaluate the severity of A. fumigatus infection. Lymphangiogenesis and angiogenesis were examined by immunofluorescence assay. Western blot was applied to detect the proteins of the MAPK-NF-κB pathway. The results showed that RvD1 diminished the high glucose-induced oxidative stress and inflammatory response, as evidenced by the reduction of ROS production, Interleukin-6 (IL-6), Interleukin-8 (IL-8), Heme Oxygenase-1 (HMOX-1), and the elevation of Cyclooxygenase-2 (COX2), Superoxide Dismutase (SOD-1), and Glutathione Peroxidase-2 (GPX2) levels in A. fumigatus-infected Human Corneal Endothelial Cells (HCECs). Additionally, lymphangiogenesis and angiogenesis prominently decreased after intervention with RvD1. Furthermore, RvD1 significantly reduced the levels of p-MEK1/2 and p-ERK1/2, and restrained the NF-κB and GPR32 activation. The above results showed that RvD1 protects against A. fumigatus keratitis in diabetes by suppressing oxidative stress, inflammatory response, fungal growth, and immunoreaction via modulating MAPK-NF-κB pathway. RvD1 provides clues for the therapeutic targets of Fungal keratitis complicated with diabetes.

Kaempferol ameliorate the prognosis of Aspergillus fumigatus keratitis by reducing fungal load and inhibiting the Dectin-1 and p38 MAPK pathway.

Fungal keratitis is one of leading reasons for blindness in the world, which causes corneal blindness mainly due to excessive inflammatory responses. Kaempferol (KAE) is a natural flavonoid which has potent anti-inflammatory effects. However, whether KAE plays protective roles in fungal keratitis and the potentially protective mechanisms are unrevealed. Here we first investigated the anti-inflammatory and antifungal effects of KAE on Aspergillus fumigatus (A. fumigatus) keratitis in C57BL/6 mice. We found that treatment of KAE ameliorated the severity of keratitis, inhibited macrophages and neutrophils recruitment, depressed corneal fungal load, and declined the expression of TLR4 and Dectin-1 in A. fumigatus infected mice corneas. And in activated hyphae or Curdlan stimulated macrophages, pretreatment of KAE also significantly decreased the mRNA and protein expression of IL-1ß, TNF-α, MIP-2 and the phosphorylated-p38 (p-p38)/p38 MAPK ratio. In summary, KAE ameliorated the prognosis of fungal keratitis in C57BL/6 mice by reducing corneal fungal load, depressing the inflammatory cells recruitment, and downregulating the expression of inflammatory factors, and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway.

α- MSH Plays Anti-inflammatory and Anti-fungal Role in Aspergillus Fumigatus Keratitis.

PURPOSE: To investigate the anti-inflammatory and antifungal role of α-melanocyte stimulating hormone (α-MSH) in Aspergillus Fumigatus (A. fumigatus) keratitis. METHOD: Corneas of C57BL/6 mice were infected with A. Fumigatus. α-MSH (5 ul, 1×10-4 mmol/ml) was given by subconjunctival injection from day 1 to day 3 post infection (p.i.). After 3 days p.i., clinical score was recorded and HE staining was tested. Fungal load in mice corneas was observed by plate counting. Proinflammatory mediators and pattern recognition receptors (PRRs) were detected. The number of neutrophils and macrophages was tested by immunofluorescence staining. The role of α-MSH in RAW264.7 cells after A. fumigatus stimulation were evaluated by PCR and Western blot, and MPKA protein levels including total-JNK (T-JNK), phosphorylated-JNK (P-JNK), total-ERK (T-ERK), and phosphorylated-ERK (P-ERK) were tested via Western blot with or without α-MSH treatment. RESULTS: Compared with PBS control group, α-MSH treatment alleviated disease response and decreased clinical score at 3 days p.i. HE staining showed less infiltration in corneal tissue after α-MSH treatment. Plate counting experiment showed that number of viable fungus in corneas of α-MSH treated group was less than control group. mRNA levels of IL-1ß, TNF-α, IL-6, MIP-2, LOX-1, Dectin-1, and iNOS were decreased. Protein levels of IL-1ß, TNF-α, IL-6, and Dectin-1 were decreased. α-MSH treatment also decreased the infiltrating neutrophils and macrophages. The levels of proinflammatory cytokines, Dectin-1 and LOX-1 stimulated by A. fumigatus, were also suppressed by pretreatment of α-MSH in RAW264.7 cells. The ratio of P-JNK/T-JNK and P-ERK/T-ERK was downregulated in α-MSH group compared with PBS control group. CONCLUSION: α-MSH alleviates the severity and decreases fungal load of A. fumigatus keratitis in mice. Migration of neutrophils and macrophages are restrained. α-MSH downregulates the expression of dectin-1 and the ratio of P-JNK/T-JNK and P-ERK/T-ERK in A. fumigatus infection.

Wnt-ß-Catenin Signaling in Human Dendritic Cells Mediates Regulatory T-Cell Responses to Fungi via the PD-L1 Pathway.

The signaling pathways activated following interaction between dendritic cells (DCs) and a pathogen determine the polarization of effector T-cell and regulatory T-cell (Treg) responses to the infection. Several recent studies, mostly in the context of bacterial infections, have shown that the Wnt/ß-catenin pathway plays a major role in imparting tolerogenic features in DCs and in promotion of Treg responses. However, the significance of the Wnt/ß-catenin pathway's involvement in regulating the immune response to the fungal species is not known. Using Aspergillus fumigatus, a ubiquitous airborne opportunistic fungal species, we show here that fungi activate the Wnt/ß-catenin pathway in human DCs and are critical for mediating the immunosuppressive Treg responses. Pharmacological inhibition of this pathway in DCs led to inhibition of maturation-associated molecules and interleukin 10 (IL-10) secretion without affecting the majority of the inflammatory cytokines. Furthermore, blockade of Wnt signaling in DCs suppressed DC-mediated Treg responses in CD4+ T cells and downregulated both tumor necrosis factor alpha (TNF-α) and IL-10 responses in CD8+ T cells. Mechanistically, induction of ß-catenin pathway by A. fumigatus required C-type lectin receptors and promoted Treg polarization via the induction of programmed death-ligand 1 on DCs. Further investigation on the identity of fungal molecular patterns has revealed that the cell wall polysaccharides ß-(1, 3)-glucan and α-(1, 3)-glucan, but not chitin, possess the capacity to activate the ß-catenin pathway. Our data suggest that the Wnt/ß-catenin pathway is a potential therapeutic target to selectively suppress the Treg response and to sustain the protective Th1 response in the context of invasive aspergillosis caused by A. fumigatus. IMPORTANCE The balance between effector CD4+ T-cell and immunosuppressive regulatory T-cell (Treg) responses determines the outcome of an infectious disease. The signaling pathways that regulate human CD4+ T-effector versus Treg responses to the fungi are not completely understood. By using Aspergillus fumigatus, a ubiquitous opportunistic fungal species, we show that fungi activate the Wnt/ß-catenin pathway in human dendritic cells (DCs) that promotes Treg responses via induction of immune checkpoint molecule programmed death ligand 1 on DCs. Blockade of the Wnt/ß-catenin pathway in DCs led to the selective inhibition of Treg without affecting the Th1 response. Dissection of the identity of A. fumigatus pathogen-associated molecular patterns (PAMPs) revealed that cell wall polysaccharides exhibit selectivity in their capacity to activate the ß-catenin pathway in DCs. Our data thus provide a pointer that Wnt/ß-catenin pathway represents potential therapeutic target to selectively suppress Treg responses and to sustain protective a Th1 response against invasive fungal diseases.

A Shifted Composition of the Lung Microbiota Conditions the Antifungal Response of Immunodeficient Mice.

The microbiome, i.e., the communities of microbes that inhabit the surfaces exposed to the external environment, participates in the regulation of host physiology, including the immune response against pathogens. At the same time, the immune response shapes the microbiome to regulate its composition and function. How the crosstalk between the immune system and the microbiome regulates the response to fungal infection has remained relatively unexplored. We have previously shown that strict anaerobes protect from infection with the opportunistic fungus Aspergillus fumigatus by counteracting the expansion of pathogenic Proteobacteria. By resorting to immunodeficient mouse strains, we found that the lung microbiota could compensate for the lack of B and T lymphocytes in Rag1-/- mice by skewing the composition towards an increased abundance of protective anaerobes such as Clostridia and Bacteroidota. Conversely, NSG mice, with major defects in both the innate and adaptive immune response, showed an increased susceptibility to infection associated with a low abundance of strict anaerobes and the expansion of Proteobacteria. Further exploration in a murine model of chronic granulomatous disease, a primary form of immunodeficiency characterized by defective phagocyte NADPH oxidase, confirms the association of lung unbalance between anaerobes and Proteobacteria and the susceptibility to aspergillosis. Consistent changes in the lung levels of short-chain fatty acids between the different strains support the conclusion that the immune system and the microbiota are functionally intertwined during Aspergillus infection and determine the outcome of the infection.

NADPH Oxidase Limits Collaborative Pattern-Recognition Receptor Signaling to Regulate Neutrophil Cytokine Production in Response to Fungal Pathogen-Associated Molecular Patterns.

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by genetic defects in leukocyte NADPH oxidase, which has both microbicidal and immunomodulatory roles. Hence, CGD is characterized by recurrent bacterial and fungal infections as well as aberrant inflammation. Fungal cell walls induce neutrophilic inflammation in CGD; yet, underlying mechanisms are incompletely understood. This study investigated the receptors and signaling pathways driving aberrant proinflammatory cytokine production in CGD neutrophils activated by fungal cell walls. Although cytokine responses to ß-glucan particles were similar in NADPH oxidase-competent and NADPH oxidase-deficient mouse and human neutrophils, stimulation with zymosan, a more complex fungal particle, induced elevated cytokine production in NADPH oxidase-deficient neutrophils. The dectin-1 C-type lectin receptor, which recognizes ß-glucans (1-3), and TLRs mediated cytokine responses by wild-type murine neutrophils. In the absence of NADPH oxidase, fungal pathogen-associated molecular patterns engaged additional collaborative signaling with Mac-1 and TLRs to markedly increase cytokine production. Mechanistically, this cytokine overproduction is mediated by enhanced proximal activation of tyrosine phosphatase SHP2-Syk and downstream Card9-dependent NF-κB and Card9-independent JNK-c-Jun. This activation and amplified cytokine production were significantly decreased by exogenous H2O2 treatment, enzymatic generation of exogenous H2O2, or Mac-1 blockade. Similar to zymosan, Aspergillus fumigatus conidia induced increased signaling in CGD mouse neutrophils for activation of proinflammatory cytokine production, which also used Mac-1 and was Card9 dependent. This study, to our knowledge, provides new insights into how NADPH oxidase deficiency deregulates neutrophil cytokine production in response to fungal cell walls.

Virus Infection of Aspergillus fumigatus Compromises the Fungus in Intermicrobial Competition.

Aspergillus and Pseudomonas compete in nature, and are the commonest bacterial and fungal pathogens in some clinical settings, such as the cystic fibrosis lung. Virus infections of fungi occur naturally. Effects on fungal physiology need delineation. A common reference Aspergillus fumigatus strain, long studied in two (of many) laboratories, was found infected with the AfuPmV-1 virus. One isolate was cured of virus, producing a virus-free strain. Virus from the infected strain was purified and used to re-infect three subcultures of the virus-free fungus, producing six fungal strains, otherwise isogenic. They were studied in intermicrobial competition with Pseudomonasaeruginosa. Pseudomonas culture filtrates inhibited forming or preformed Aspergillus biofilm from infected strains to a greater extent, also seen when Pseudomonas volatiles were assayed on Aspergillus. Purified iron-chelating Pseudomonas molecules, known inhibitors of Aspergillus biofilm, reproduced these differences. Iron, a stimulus of Aspergillus, enhanced the virus-free fungus, compared to infected. All infected fungal strains behaved similarly in assays. We show an important consequence of virus infection, a weakening in intermicrobial competition. Viral infection may affect the outcome of bacterial-fungal competition in nature and patients. We suggest that this occurs via alteration in fungal stress responses, the mechanism best delineated here is a result of virus-induced altered Aspergillus iron metabolism.

Quercetin amelioratesAspergillus fumigatuskeratitis by inhibiting fungal growth, toll-like receptors and inflammatory cytokines.

PURPOSE: To investigate the antifungal and anti-inflammatory effects of quercetin on Aspergillus fumigatus (A. fumigatus) keratitis. METHODS: Human corneal epithelial cells (HCECs) and C57BL/6 mice were stimulated by A. fumigatus and treated with quercetin or dimethyl sulfoxide (DMSO) after infection. In HCECs, minimum inhibitory concentration (MIC) and cytotoxicity tests (CCK-8) were used to detect the antifungal effect and cytotoxicity of quercetin. In mice with A. fumigatuskeratitis, clinical score, plate counting and hematoxylin-eosin (HE) staining were performed to evaluate the effects of quercetin in vivo. Myeloperoxidase (MPO) assay and immunofluorescence staining were applied to assess neutrophil recruitment and infiltration. Real time PCR (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and western blot were used to detect the mRNA and protein expressions of inflammatory mediators. RESULTS: Compared with DMSO control, quercetin (16-64 µM) significantly inhibited the growth of A. fumigatus in a concentration-dependent manner without affecting cell viability in HCECs. In corneas of mice with A. fumigatuskeratitis, quercetin decreased clinical score and fungal load, and reduced neutrophil recruitment and infiltration to the corneal stroma. Moreover, quercetin attenuated the expression of inflammatory mediators including toll-like receptor-4 (TLR-4), TLR-2, interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α) and high mobility group box 1 (HMGB1) in vitro and in vivo. CONCLUSIONS: Our study demonstrated that quercetin treatment can ameliorate A. fumigatus keratitis by inhibiting the growth of A. fumigatus, decreasing neutrophil recruitment and infiltration, and downregulating the productions of TLR-4, TLR-2, TNF-α, IL-1ß and HMGB1, indicating quercetin is likely to become a potential therapeutic agent in FK treatment.

The infectious propagules of Aspergillus fumigatus are coated with antimicrobial peptides.

Fungal spores are unique cells that mediate dispersal and survival in the environment. For pathogenic fungi encountering a susceptible host, these specialised structures may serve as infectious particles. The main causative agent of the opportunistic disease aspergillosis, Aspergillus fumigatus, produces asexual spores, the conidia, that become dissipated by air flows or water currents but also serve as propagules to infect a susceptible host. We demonstrate that the defX gene of this mould encodes putative antimicrobial peptides resembling cysteine-stabilised (CS)αß defensins that are expressed in a specific spatial and temporal manner in the course of asexual spore formation. Localisation studies on strains expressing a fluorescent proxy or tagged defX alleles expose that these antimicrobial peptides are secreted to coat the conidial surface. Deletion mutants reveal that the spore-associated defX gene products delay the growth of Gram-positive Staphylococcus aureus and demonstrate that the defX gene and presumably its encoded spore-associated defensins confer a growth advantage to the fungal opponent over bacterial competitors. These findings have implications with respect to the ecological niche of A. fumigatus that serves as a 'virulence school' for this human pathogenic mould; further relevance is given for the infectious process resulting in aspergillosis, considering competition with the host microbiome or co-infecting microorganisms to break colonisation resistance at host surfaces.

Invasive pulmonary aspergillosis in the COVID-19 era: An expected new entity.

OBJECTIVES: Information on the recently COVID-19-associated pulmonary aspergillosis (CAPA) entity is scarce. We describe eight CAPA patients, compare them to colonised ICU patients with coronavirus disease 2019 (COVID-19), and review the published literature from Western countries. METHODS: Prospective study (March to May, 2020) that included all COVID-19 patients admitted to a tertiary hospital. Modified AspICU and European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria were used. RESULTS: COVID-19-associated pulmonary aspergillosis was diagnosed in eight patients (3.3% of 239 ICU patients), mostly affected non-immunocompromised patients (75%) with severe acute respiratory distress syndrome (ARDS) receiving corticosteroids. Diagnosis was established after a median of 15 days under mechanical ventilation. Bronchoalveolar lavage was performed in two patients with positive Aspergillus fumigatus cultures and galactomannan (GM) index. Serum GM was positive in 4/8 (50%). Thoracic CT scan findings fulfilled EORTC/MSG criteria in one case. Isavuconazole was used in 4/8 cases. CAPA-related mortality was 100% (8/8). Compared with colonised patients, CAPA subjects were administered tocilizumab more often (100% vs. 40%, p = .04), underwent longer courses of antibacterial therapy (13 vs. 5 days, p = .008), and had a higher all-cause mortality (100% vs. 40%, p = .04). We reviewed 96 similar cases from recent publications: 59 probable CAPA (also putative according modified AspICU), 56 putative cases and 13 colonisations according AspICU algorithm; according EORTC/MSG six proven and two probable. Overall, mortality in the reviewed series was 56.3%. CONCLUSIONS: COVID-19-associated pulmonary aspergillosis must be considered a serious and potentially life-threatening complication in patients with severe COVID-19 receiving immunosuppressive treatment.

Reconstituting NK Cells After Allogeneic Stem Cell Transplantation Show Impaired Response to the Fungal Pathogen Aspergillus fumigatus.

Delayed natural killer (NK) cell reconstitution after allogeneic stem cell transplantation (alloSCT) is associated with a higher risk of developing invasive aspergillosis. The interaction of NK cells with the human pathogen Aspergillus (A.) fumigatus is mediated by the fungal recognition receptor CD56, which is relocated to the fungal interface after contact. Blocking of CD56 signaling inhibits the fungal mediated chemokine secretion of MIP-1α, MIP-1ß, and RANTES and reduces cell activation, indicating a functional role of CD56 in fungal recognition. We collected peripheral blood from recipients of an allograft at defined time points after alloSCT (day 60, 90, 120, 180). NK cells were isolated, directly challenged with live A. fumigatus germ tubes, and cell function was analyzed and compared to healthy age and gender-matched individuals. After alloSCT, NK cells displayed a higher percentage of CD56brightCD16dim cells throughout the time of blood collection. However, CD56 binding and relocalization to the fungal contact side were decreased. We were able to correlate this deficiency to the administration of corticosteroid therapy that further negatively influenced the secretion of MIP-1α, MIP-1ß, and RANTES. As a consequence, the treatment of healthy NK cells ex vivo with corticosteroids abrogated chemokine secretion measured by multiplex immunoassay. Furthermore, we analyzed NK cells regarding their actin cytoskeleton by Structured Illumination Microscopy (SIM) and flow cytometry and demonstrate an actin dysfunction of NK cells shown by reduced F-actin content after fungal co-cultivation early after alloSCT. This dysfunction remains until 180 days post-alloSCT, concluding that further actin-dependent cellular processes may be negatively influenced after alloSCT. To investigate the molecular pathomechansism, we compared CD56 receptor mobility on the plasma membrane of healthy and alloSCT primary NK cells by single-molecule tracking. The results were very robust and reproducible between tested conditions which point to a different molecular mechanism and emphasize the importance of proper CD56 mobility.

BTK Inhibition Impairs the Innate Response Against Fungal Infection in Patients With Chronic Lymphocytic Leukemia.

Infections represent a cause of morbidity and mortality in patients affected by chronic lymphocytic leukemia (CLL). Introduction of new drugs in CLL clinical practice has showed impressive efficacy, in particular those targeting BTK. Among the consistent clinical data, an increasing number of reports describing the occurrence of unexpected opportunistic fungal infections has been reported during treatment with ibrutinib in the first 6 months of treatment. The reason underlying manifestations of invasive fungal infections in patients treated with ibrutinib is still under investigation. Our study aimed to understand the impact of BTK inhibition on immune response to fungal infection mediated by macrophages and CD14+ monocytic population obtained from CLL patients. Exposure to ibrutinib and acalabrutinib reduced signaling pathways activated by Aspergillus fumigatus determining an exacerbation of an immunosuppressive signature, a reduction of phagocytosis and a significant deficit in the secretion of inflammatory cytokines either in macrophages and monocytes isolated from CLL patients and healthy donors. These effects lead to a failure in completely counteracting conidia germination. In addition we investigated the biological effects of ibrutinib on monocyte counterpart in patients who were undergoing therapy. A significant impairment in cytokine secretion and a deficit of phagocytosis in circulating monocytes were detected after 3 months of treatment. Thus, our results uncover modifications in the innate response in CLL patients induced by ibrutinib that may impair the immunological response to fungal infection. KEYPOINTS: •BTK inhibition affects a productive immune response of CLL-associated macrophages (NLC) during Aspergillus fumigatus infection.•Reduction of TNF-α secretion and phagocytosis are detected in monocytes isolated from CLL patients during ibrutinib therapy.

The Search for Antifungal Prophylaxis After Artificial Corneal Surgery-An In Vitro Study.

PURPOSE: To evaluate the antifungal properties of topical antibiotics (already being used successfully to prevent bacterial endophthalmitis) and some promising antiseptics for antifungal prophylaxis in the setting of artificial corneal implantation. METHODS: Several commonly used antibiotics for antimicrobial prophylaxis after artificial corneal implantation, in addition to antiseptics [benzalkonium chloride (BAK), povidone-iodine (PI), and some ionic liquids (ILs)], were tested in vitro against Candida albicans, Fusarium solani, and Aspergillus fumigatus. The time-kill activity was determined. Toxicity was assayed in vitro on human corneal epithelial cultures using trypan blue. Adhesion and tissue invasion experiments were also carried out on porcine corneas and commonly used contact lenses, with or without gamma irradiation, and by analysis with fluorescence microscopy. RESULTS: Polymyxin B (PMB)/trimethoprim/BAK (Polytrim), PMB alone, gatifloxacin with BAK (Zymaxid), and same-concentration BAK alone exhibited antifungal activity in vitro. Moxifloxacin (MOX) or gatifloxacin without BAK-as well as trimethoprim, vancomycin, and chloramphenicol-had no effect. 1% PI and ILs had the highest efficacy/toxicity ratios (>1), and Polytrim was species dependent. Subfungicidal concentrations of Polytrim reduced adhesion of C. albicans to Kontur contact lenses. Gamma-irradiated corneas showed enhanced resistance to fungal invasion. CONCLUSIONS: Of antibiotic preparations already in use for bacterial prophylaxis after KPro surgery, Polytrim is a commonly used antibiotic with antifungal effects mediated by both PMB and BAK and may be sufficient for prophylaxis. PI as a 1% solution seems to be promising as a long-term antifungal agent. Choline-undecanoate IL is effective and virtually nontoxic and warrants further development.

Allicin reduces inflammation by regulating ROS/NLRP3 and autophagy in the context of A. fumigatus infection in mice.

OBJECTIVES: Inhibitory effect of allicin with broad-spectrum antimicrobial activity on A. fumigatus and the regulation mechanism of inflammation and autophagy in vitro and in vivo. METHODS: The corresponding concentration of allicin was prepared according to the needs of the experiment. In vitro, 2 ml 5 × 104 of fungal spores suspension was added to the 6-well plate per hole, and different final concentrations of allicin (1 µl/ml, 2.5 µl/ml, 5 µl/ml, 10 µl/ml, 20 µl/ml, 30 µl/ml) were added. The fungal spores were stained by fluorescent dye SYTO 9 (green) every day, and the spore germination inhibition was detected by flow cytometry in different PH. RAW264.7 cells were cultured and stimulated by A. fumigatus spores for 3 h, then allicin solution was added. Then some cells were stained with ROS probe (green) and hochest33342 (blue). The effect of allicin on ROS was observed by fluorescence microscope. The other part of cells extracted protein from cell lysate and detected the effect of allicin on inflammatory factors and autophagy by Western-blotting. The green and red spots of RAW264.7 cells stably transfected with GFP-RFP-LC3 were observed by fluorescence microscopy. In vivo, A. fumigatus spore was injected intratracheally into mice, then allicin was injected intravenously at a concentration of 5 mg/kg/day for 7 consecutive days. The survival status, pulmonary fungal load and weight of mice was recorded continuously for 30 days and detected the changes of lung by pathological examination and immunohistochemistry. RESULTS: In vitro, allicin significantly inhibited the spore germination of A. fumigatus within 24 h in a dose-dependent manner and it had a stable inhibition on the spore germination of A. fumigatus in acidic environment. Cell experiments showed that allicin inhibited intracellular spore germination by inhibiting ROS production, inflammation and autophagy. In the animal experiment, the survival rate and body weight of allicin injection group were higher than that of non injection group, while the spore load of lung was lower than that of non injection group (P < 0.05). CONCLUSIONS: These results support that allicin reduces inflammation and autophagy resistance to A. fumigatus infection, It also provides a possible treatment for Aspergillus infectious diseases, i.e. early anti-inflammation, antibiotics or drugs that inhibit excessive autophagy.

Duration of posaconazole therapy for Aspergillus fumigatus osteomyelitis dictated by serial monitoring of 1,3-beta-D glucan.

Here, we present a case of an immunocompetent 37-year old male who developed Aspergillus fumigatus osteomyelitis 16 years after extensive chest wall reconstructive surgery for Ewing sarcoma. His treatment course was complicated by a severe adverse drug reaction to voriconazole, requiring the use of oral posaconazole therapy. Serum 1,3-beta-D glucan assay was utilized to dictate the duration of posaconazole therapy. The patient successfully completed 9 months of oral posaconazole therapy and has not had clinical recurrence for 9 months off antifungal therapy.

Influenza Suppresses Neutrophil Recruitment to the Lung and Exacerbates Secondary Invasive Pulmonary Aspergillosis.

Aspergillus fumigatus is an environmental fungus that can cause invasive pulmonary aspergillosis when spores are inhaled into the respiratory tract and invade airway or lung tissue. Influenza is a common respiratory virus that can cause severe respiratory disease, and postinfluenza invasive pulmonary aspergillosis, which is becoming a well-recognized clinical problem, typically occurs in critically ill patients. Mice challenged with influenza A PR/8/34 H1N1 and subsequently challenged with A. fumigatus had increased fungal burden, viral burden, inflammation, and mortality compared with single infected mice. Neutrophil recruitment in the lung of superinfected mice was decreased; however, mice were not neutropenic, and there was no difference in absolute blood neutrophils between groups. Additionally, CXCL1 and CXCL2 were decreased in lungs of superinfected mice compared with controls. IFN levels were increased in mice that received influenza, and deletion of STAT1 resulted in decreased fungal burden, increased airway and lung neutrophils, and increased CXCL1 compared with wild-type mice, whereas deletion of STAT2 did not change fungal burden or airway neutrophilia compared with wild-type mice. These data demonstrate a mechanism by which influenza A-induced STAT1 signaling inhibits neutrophil recruitment and increases susceptibility to postinfluenza invasive pulmonary aspergillosis.

Vitamin D reduces autophagy by regulating NF-κB resistance to Aspergillus fumigatus infection.

Vitamin D is one of the indispensable nutrients of human body. When vitamin D is deficient, it can cause a series of related diseases, such as respiratory tract infection. The regulatory role of vitamin D in inflammatory immune response and defense has attracted more and more attention. However, few studies have shown that vitamin D regulates inflammation and autophagy in Aspergillus fumigatus infected lungs. In this study, we will explain the mechanism of vitamin D regulating inflammation and autophagy in Aspergillus fumigatus infected lungs. METHODS: Different concentrations of Aspergillus fumigatus spores were injected into mice with deficien diets (VitD-) or sufficient vitamin D (VitD + ) , and the survival rates were recorded. Then, the weight changes of rats were measured every other time. At the same time, a gauze was used to filter the lapped lung tissue to get the pulmonary spores and measured the amount of the spores. The mice with the same concentration of Aspergillus fumigatus infected were cut off and the lung tissue for pathological examination in the deficien diets (VitD-) group or sufficient vitamin D (VitD + ) group. Moreover, the expression of inflammation related factors TNF-α, IL-1ß, IL-6 in lung was measured by immunohistochemical method. The expression of TNF-α, IL-1ß, IL-6 in the serum of vitamin D deficiency and sufficient mice were measured by ELISA. In vitro, we obtained macrophages from healthy mice and mixed cultures of Aspergillus fumigatus spores and lung macrophages in medium with or without vitamin D. After the cells were infected with Aspergillus fumigatus spores, the expressions of NF-κB and IL-8 were detected by RT-PCR and Western blot. The RAW264.7 cells transfected with GFP-LC3BII were mixed with Aspergillus fumigatus spores, and the expression of cell fluorescence was observed by the fluorescence microscope with or without chloroquine and rapamycin , and the autophagy flow of the cells was measured by Western blot. In the RAW264.7 cells, Lentivirus transfection and SiRNA technologies were used to enhance or reduce the expression of the NF-κB gene (siNF-κB) for investgating the influence of high or low expression of NF-κB in the autophagic flow of vitamin D + or vitamin D-treated RAW264.7 cells. RESULTS: The survival rate of vitamin D deficient mice infected Aspergillus fumigatus was significantly lower than that of vitamin D sufficient mice, while the number of spores, spore activity, pathological changes of lungs and inflammation in the lungs of vitamin D deficient mice were more severe than that of vitamin D sufficient mice. In vitro cell experiments, when cell was stimulated with vitamin D, the expressions of NF-κB and IL-8 in cells were lower. The autophagic flux and TNF-α, IL-1ß, IL-6 and LC3BII in vitamin D group were significantly lower than those in vitamin D deficiency group. CONCLUSIONS: Vitamin D deficiency can aggravate the inflammatory damage in the lungs of Aspergillus fumigatus. When the body is sufficient in vitamin D, if the lungs infect Aspergillus fumigatus spores, the body may resist the infection of Aspergillus fumigatus by reducing the expression of NF-κB, inflammatory factors and autophagy.