[In silico analysis of molecular mimicry between human aquaporin 3, Aspergillus fumigatus aquaporin and aquaporins from allergic sources]. / Análisis en silico del mimetismo molecular entre acuaporina 3 humana y Aspergillus fumigatus: implicaciones para el potencial de reactividad cruzada.

OBJECTIVE: Conduct an in-silico assessment of potential molecular mimicry between human aquaporins, A. fumigatus, and diverse allergenic sources. METHODS: Amino acid sequences of human AQP3 and A. fumigatus aquaporin were compared through multiple alignments with 25 aquaporins from diverse allergenic sources. Phylogenetic analysis and homology-based modeling were executed, and the ElliPro server predicted conserved antigenic regions on 3D structures. RESULTS: Global identity among studied aquaporins was 32.6%, with a specific conserved local region at 71.4%. Five monophyletic clades (A-E) were formed, and Group B displayed the highest identity (95%), including 6 mammalian aquaporins, notably AQP3. A. fumigatus aquaporin exhibited the highest identity with Malassezia sympodialis (35%). Three linear and three discontinuous epitopes were identified in both human and A. fumigatus aquaporins. The Root Mean Square Deviation (RMSD) from overlapping aquaporin structures was 1.006. CONCLUSION: Identification of potential linear and conformational epitopes on human AQP3 suggests likely molecular mimicry with A. fumigatus aquaporins. High identity in a specific antigenic region indicates potential autoreactivity and a probable antigenic site involved in cross-reactivity. Validation through in vitro and in vivo studies is essential for further understanding and confirmation.

OBJETIVO: Realizar una evaluación in silico del posible mimetismo molecular entre las acuaporinas humanas, A. fumigatus y diversas fuentes alergénicas. MÉTODOS: Se compararon secuencias de aminoácidos de AQP3 humana y acuaporina de A. fumigatus mediante alineamientos múltiples con 25 acuaporinas de diversas fuentes alergénicas. Se ejecutaron análisis filogenéticos y modelos basados en homología, y el servidor ElliPro predijo regiones antigénicas preservadas en estructuras 3D. RESULTADOS: La identidad global entre las acuaporinas estudiadas fue del 32.6%, con una región local específica preservada en el 71.4%. Se formaron cinco clados monofiléticos (A-E), y el grupo B mostró la identidad más alta (95%), incluidas 6 acuaporinas de mamíferos, en particular AQP3. A. fumigatus aquaporin exhibió la mayor identidad con Malassezia sympodialis (35%). Se identificaron tres epítopos lineales y tres discontinuos en acuaporinas tanto humanas como de A. fumigatus. La desviación cuadrática media (RMSD) de las estructuras de acuaporinas superpuestas fue de 1,006. CONCLUSIÓN: La identificación de posibles epítopos lineales y conformacionales en AQP3 humano sugiere un probable mimetismo molecular con acuaporinas de A. fumigatus. La identidad alta en una región antigénica específica indica autorreactividad potencial y un sitio antigénico probable implicado en la reactividad cruzada. La validación mediante estudios in vitro e in vivo es desicivo para una mayor comprensión y confirmación.

Identification of allergens from Aspergillus fumigatus-Potential association with lung damage in asthma.

BACKGROUND: Component-resolved diagnosis allows detection of IgE sensitization having the advantage of reproducibility and standardization compared to crude extracts. The main disadvantage of the traditional allergen identification methods, 1- or 2-dimensional western blotting and screening of expression cDNA libraries with patients' IgEs, is that the native structure of the protein is not necessarily maintained. METHODS: We used a novel immunoprecipitation technique in combination with mass spectrometry to identify new allergens of Aspergillus fumigatus. Magnetic Dynabeads coupled with anti-human IgE antibodies were used to purify human serum IgE and subsequently allergens from A. fumigatus protein extract. RESULTS: Of the 184 proteins detected by subsequent mass peptide fingerprinting, a subset of 13 were recombinantly expressed and purified. In a panel of 52 A. fumigatus-sensitized people with asthma, 23 non-fungal-sensitized asthmatics and 18 healthy individuals, only the former showed an IgE reaction by immunoblotting and/or ELISA. We discovered 11 proteins not yet described as A. fumigatus allergens, with fructose-bisphosphate aldolase class II (FBA2) (33%), NAD-dependent malate dehydrogenase (31%) and Cu/Zn superoxide dismutase (27%) being the most prevalent. With respect to these three allergens, native versus denatured protein assays indicated a better recognition of the native proteins. Seven of 11 allergens fulfilled the WHO/IUIS criteria and were accepted as new A. fumigatus allergens. CONCLUSION: In conclusion, we introduce a straightforward method of allergen identification from complex allergenic sources such as A. fumigatus by immunoprecipitation combined with mass spectrometry, which has the advantage over traditional methods of identifying allergens by maintaining the structure of the proteins.

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.

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.

High Frequency of Allergic Bronchopulmonary Aspergillosis in Bronchiectasis-COPD Overlap.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) is associated with frequent exacerbations and poor outcomes in chronic respiratory disease, but remains underdiagnosed. The role of fungal sensitization in bronchiectasis-COPD overlap (BCO) is unknown. RESEARCH QUESTION: What is the occurrence and clinical relevance of Aspergillus sensitization and ABPA in BCO when compared with individuals with COPD or bronchiectasis without overlap? STUDY DESIGN: Prospective, observational, cross-sectional study. METHODS: We prospectively recruited 280 patients during periods of clinical stability with bronchiectasis (n = 183), COPD (n = 50), and BCO (n = 47) from six hospitals across three countries (Singapore, Malaysia, and Scotland). We assessed sensitization responses (as specific IgE) to a panel of recombinant Aspergillus fumigatus allergens and the occurrence of ABPA in relationship to clinical outcomes. RESULTS: Individuals with BCO show an increased frequency and clinical severity of ABPA compared with those with COPD and bronchiectasis without overlap. BCO-associated ABPA is associated with more severe disease, higher exacerbation rates, and lower lung function when compared with ABPA occurring in the absence of overlap. BCO with a severe bronchiectasis severity index (BSI; > 9) is associated significantly with the occurrence of ABPA that is unrelated to underlying COPD severity. CONCLUSIONS: BCO demonstrates a high frequency of ABPA that is associated with a severe BSI (> 9) and poor clinical outcomes. Clinicians should maintain a high index of suspicion for the potential development of ABPA in patients with BCO with high BSI.

Disulfiram inhibits IL-1ß secretion and inflammatory cells recruitment in Aspergillus fumigatus keratitis.

PURPOSE: Disulfiram, an inhibitor of gasdermin D-induced pore formation, is known to suppress interleukin (IL)-1ß secretion and pyroptosis. However, its effects on fungal keratitis remain unknown. Therefore, we investigated the role of disulfiram in Aspergillus fumigatus keratitis. METHODS: In vitro, Cell Count Kit-8 (CCK8) assay and cell scratch test were performed to determine optimal concentration. In vivo and in vitro experiments were conducted in a mouse model, human neutrophils, and mouse peritoneal macrophages. We pre-treated the mice or cells with disulfiram and infected them with A. fumigatus at specific times. We subsequently evaluated the development of fungal keratitis lesions, the recruitment of inflammatory cells, and the production of inflammatory cytokines using slit lamp microscopy, clinical evaluation, quantitative reverse transcription polymerase chain reaction, immunofluorescence staining, enzyme-linked immunosorbent assay, and western blotting. We also used slit lamp microscopy and clinical evaluation to assess the effect of natamycin with or without disulfiram. RESULTS: Disulfiram at 20 µM has no significant cytotoxic effect and does not affect cell migration. In the mouse model, disulfiram significantly suppressed inflammatory responses, reduced neutrophil and macrophage recruitment, and down-regulated myeloperoxidase and nitric oxide synthase levels at earlier stages of infection. Disulfiram had no effect on IL-1ß production and maturation, but it inhibited IL-1ß secretion in macrophages. Disulfiram combined with natamycin significantly increased corneal transparency in the mice model. CONCLUSION: Overall, disulfiram reduced the host immune response in fungal keratitis by attenuating neutrophil and macrophage recruitment and inhibiting IL-1ß secretion in macrophages. Disulfiram in combination with antifungal agents may serve as a novel therapeutic method for reducing corneal opacity in fungal keratitis.

Complementary Roles of Short and Long Pentraxins in the Complement-Mediated Immune Response to Aspergillus fumigatus Infections.

The ubiquitous mold Aspergillus fumigatus is the major etiologic agent of invasive aspergillosis, a life-threatening infection amongst immune compromised individuals. An increasing body of evidence indicates that effective disposal of A. fumigatus requires the coordinate action of both cellular and humoral components of the innate immune system. Early recognition of the fungal pathogen, in particular, is mediated by a set of diverse soluble pattern recognition molecules (PRMs) that act as "ancestral antibodies" inasmuch as they are endowed with opsonic, pro-phagocytic and killing properties. Pivotal is, in this respect, the contribution of the complement system, which functionally cooperates with cell-borne pattern recognition receptors (PRRs) and other soluble PRMs, including pentraxins. Indeed, complement and pentraxins form an integrated system with crosstalk, synergism, and regulation, which stands as a paradigm of the interplay between PRMs in the mounting and orchestration of antifungal immunity. Following upon our past experience with the long pentraxin PTX3, a well-established immune effector in the host response to A. fumigatus, we recently reported that this fungal pathogen is targeted in vitro and in vivo by the short pentraxin Serum Amyloid P component (SAP) too. Similar to PTX3, SAP promotes phagocytosis and disposal of the fungal pathogen via complement-dependent pathways. However, the two proteins exploit different mechanisms of complement activation and receptor-mediated phagocytosis, which further extends complexity and integration of the complement-pentraxin crosstalk in the immune response to A. fumigatus. Here we revisit this crosstalk in light of the emerging roles of SAP as a novel PRM with antifungal activity.

Innate Type 2 Response to Aspergillus fumigatus in a Murine Model of Atopic Dermatitis-like Skin Inflammation.

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease mediated by T helper type 2 (Th2) cells in acute phase. Group 2 innate lymphoid cells (ILCs) play a role in the initiation of the Th2 response. Although mold exposure is associated with the development of AD, studies on the underlying mechanisms are lacking. This study investigated whether group 2 ILCs are involved in inflammation in AD-like skin induced by Aspergillus fumigatus (Af). METHODS: We investigated changes of group 2 ILCs population in Af-induced AD-like skin lesions. To induce AD-like skin lesions, Af extracts were applied to the dorsal skin of BALB/c and Rag1-/- mice five times per week, with repeat exposures at 2-week intervals. RESULTS: The clinical parameters were higher in the Af-treated group than in the control group. Histologic findings revealed epiderrmal and dermal thickening as well as eosinophil and mast cell infiltration into the skin of Af-treated mice. Populations of group 2 ILCs in the skin were also significantly higher in the Af-treated group. In addition, interleukin-33 mRNA expression was significantly higher in the skin lesions of the Af-treated mice. In the Rag1-/- mice lacking mature lymphocytes, AD-like skin lesions were still induced by Af and ILCs depletion using an anti-CD90.2 mAb lowered the Af-induced inflammatory response. CONCLUSIONS: Group 2 ILCs may play a role in a murine model of Af-induced AD-like skin lesions.

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.

Serum levels of IgG antibodies against Aspergillus fumigatus and the risk of hypersensitivity pneumonitis and other interstitial lung diseases.

Hypersensitivity pneumonitis (HP) is an interstitial lung disease (ILD) caused by the inhalation of antigens. Antigen-specific IgG antibodies (sIgG) are used as biomarkers of exposure when diagnosing HP, but little is known about the longitudinal relation between antibody levels and risk of HP or other ILD. In a follow-up design, we explored the relationship between sIgG antibodies against Aspergillus fumigatus and the diagnosis of HP in 647 subjects suspected of HP. We showed that IgG levels above the reference value resulted in a hazard ratio of 9.5 for subsequent HP. Our findings support a relationship between high levels of sIgG against A. fumigatus and risk of HP.

Aspergillus fumigatus Influences Gasdermin-D-Dependent Pyroptosis of the Lung via Regulating Toll-Like Receptor 2-Mediated Regulatory T Cell Differentiation.

PURPOSE: Aspergillus fumigatus, as an opportunistic fungus, has developed a series of escape mechanisms under the host's immune response to obtain nutrients and promote fungal growth in the hostile environment. The immune escape of pathogens may be through suppressing the inflammatory response mediated by regulatory T cells (Tregs). The aim of this study was to explore whether A. fumigatus influences Gasdermin-D-dependent pyroptosis of the lung by regulating Toll-like receptor 2-mediated regulatory T cell differentiation. METHODS: Collect peripheral blood from patients with A. fumigatus. ELISA kits we used to detect the expression levels of IL-1ß, IL-6, IL-2R, and IL-10 in the serum and flow cytometry to detect the percentage of CD4+CD25+Foxp3+ Tregs in the patients' peripheral blood mononuclear cells (PBMCs). The mouse model of A. fumigatus infection was constructed by tracheal instillation. The pathological changes in the lungs of the mice were observed under a microscope. The fungal load in the lung tissue was determined by the plate colony count. ELISA kit was used to detect the lung tissue homogenate proinflammatory cytokines TNF-α, IL-6, CCL2, and VEGF. Q-PCR was used for the detection of the expression of Foxp3 and TLR2 genes in the lung. Western blot was used for the detection of the expression of TLR2, Gasdermin-D (GSDMD), IL-1α, and IL-1ß in the lung. Flow cytometry was used to detect splenic CD4+CD25+FOXP3+ Tregs. Using magnetic beads to extract CD4+ T cells from mice spleen, the effects of A. fumigatus conidia or TLR2 inhibitor (C29) to differentiate CD4+ T cells in vitro were tested. RESULTS: The expression of Foxp3 and TLR2 in the lung tissue of mice infected with A. fumigatus increased, and we observed that the proportion of Tregs in both A. fumigatus infection patients and mice was upregulated. After using the CD25 neutralizing antibody, the number of Tregs in the mice spleen was significantly reduced. However, lung damage was reduced and the ability to clear lung fungi was enhanced. We found that the Tregs in TLR2-/- mice were significantly reduced and the nonlethal dose of A. fumigatus conidia did not cause severe lung damage in TLR2-/- mice. Compared with that of wild-type mice, the fungal burden in the lung of TLR2-deficient mice was reduced and the knockout of TLR2 changed the expression of GSDMD, IL-1α, and IL-1ß in A. fumigatus. In in vitro experiments, we found that the inhibition of TLR2 can reduce Treg differentiation. CONCLUSIONS: A. fumigatus triggers CD4+CD25+FOXP3+ Treg proliferation and differentiation by activating the TLR2 pathway, which may be a potential mechanism for evading host defenses in A. fumigatus. This effect can modulate GSDMD-dependent pyroptosis and may partly involve TRL2 signaling.

Vaccine-Induced Protection in Two Murine Models of Invasive Pulmonary Aspergillosis.

Life-threatening, invasive fungal infections (IFIs) cause over 1.5 million deaths worldwide and are a major public health concern with high mortality rates even with medical treatment. Infections with the opportunistic fungal pathogen, Aspergillus fumigatus are among the most common. Despite the growing clinical need, there are no licensed vaccines for IFIs. Here we evaluated the immunogenicity and protective efficacy of an A. fumigatus recombinant protein vaccine candidate, AF.KEX1, in experimental murine models of drug-induced immunosuppression. Immunization of healthy mice with AF.KEX1 and adjuvant induced a robust immune response. Following AF.KEX1 or sham immunization, mice were immunosuppressed by treatment with either cortisone acetate or hydrocortisone and the calcineurin inhibitor, tacrolimus. To test vaccine efficacy, immunosuppressed mice were intranasally challenged with A. fumigatus conidia (Af293) and weight and body temperature were monitored for 10 days. At study termination, organism burden in the lungs was evaluated by quantitative PCR and Gomori's methanamine silver staining. In both models of immunosuppression, AF.KEX1 vaccinated mice experienced decreased rates of mortality and significantly lower lung organism burden compared to non-vaccinated controls. The lung fungal burden was inversely correlated with the peak anti-AF.KEX1 IgG titer achieved following vaccination. These studies provide the basis for further evaluation of a novel vaccine strategy to protect individuals at risk of invasive aspergillosis due to immunosuppressive treatments.

Serum amyloid P component is an essential element of resistance against Aspergillus fumigatus.

Serum amyloid P component (SAP, also known as Pentraxin 2; APCS gene) is a component of the humoral arm of innate immunity involved in resistance to bacterial infection and regulation of tissue remodeling. Here we investigate the role of SAP in antifungal resistance. Apcs-/- mice show enhanced susceptibility to A. fumigatus infection. Murine and human SAP bound conidia, activate the complement cascade and enhance phagocytosis by neutrophils. Apcs-/- mice are defective in vivo in terms of recruitment of neutrophils and phagocytosis in the lungs. Opsonic activity of SAP is dependent on the classical pathway of complement activation. In immunosuppressed mice, SAP administration protects hosts against A. fumigatus infection and death. In the context of a study of hematopoietic stem-cell transplantation, genetic variation in the human APCS gene is associated with susceptibility to invasive pulmonary aspergillosis. Thus, SAP is a fluid phase pattern recognition molecule essential for resistance against A. fumigatus.

CcpA- and Shm2-Pulsed Myeloid Dendritic Cells Induce T-Cell Activation and Enhance the Neutrophilic Oxidative Burst Response to Aspergillus fumigatus.

Aspergillus fumigatus causes life-threatening opportunistic infections in immunocompromised patients. As therapeutic outcomes of invasive aspergillosis (IA) are often unsatisfactory, the development of targeted immunotherapy remains an important goal. Linking the innate and adaptive immune system, dendritic cells are pivotal in anti-Aspergillus defense and have generated interest as a potential immunotherapeutic approach in IA. While monocyte-derived dendritic cells (moDCs) require ex vivo differentiation, antigen-pulsed primary myeloid dendritic cells (mDCs) may present a more immediate platform for immunotherapy. To that end, we compared the response patterns and cellular interactions of human primary mDCs and moDCs pulsed with an A. fumigatus lysate and two A. fumigatus proteins (CcpA and Shm2) in a serum-free, GMP-compliant medium. CcpA and Shm2 triggered significant upregulation of maturation markers in mDCs and, to a lesser extent, moDCs. Furthermore, both A. fumigatus proteins elicited the release of an array of key pro-inflammatory cytokines including TNF-α, IL-1ß, IL-6, IL-8, and CCL3 from both DC populations. Compared to moDCs, CcpA- and Shm2-pulsed mDCs exhibited greater expression of MHC class II antigens and stimulated stronger proliferation and IFN-γ secretion from autologous CD4+ and CD8+ T-cells. Moreover, supernatants of CcpA- and Shm2-pulsed mDCs significantly enhanced the oxidative burst in allogeneic neutrophils co-cultured with A. fumigatus germ tubes. Taken together, our in vitro data suggest that ex vivo CcpA- and Shm2-pulsed primary mDCs have the potential to be developed into an immunotherapeutic approach to tackle IA.

Thymic stromal lymphopoietin participates in the TLR2-and TLR4-dependent immune response triggered by Aspergillus fumigatus in human corneal cells.

Fungal keratitis constitutes a serious vision-threatening disease. Toll-like receptors (TLRs) comprise key mediators of innate immunity triggered by Aspergillus fumigatus (AF) in the cornea, but the messenger between innate and adaptive immunity remained unknown. Thymic stromal lymphopoietin (TSLP) represents a critical factor of adaptive immunity. Here we investigated the expression of TSLP in corneal epithelial and stromal cells challenged by AF and its relationship with TLRs. We stimulated corneal cells with TLR ligands zymosan or lipopolysaccharide (LPS), human recombinant TSLP, or AF hyphae for various periods, with or without prior TLR2, TLR4, or TSLP inhibition. TLR2, TLR4, TSLP, IL-8, and TNF-α release and expression were measured via enzyme-linked immunosorbent analysis, quantitative polymerase chain reaction, or western blot. Corneal cell stimulation with zymosan or LPS induced up-regulated TSLP expression. Enhanced TSLP expression was associated with AF treatment in human corneal cells; TLR2 or TLR4 inhibition impaired the AF-induced TSLP levels. Human recombinant TSLP augmented TLR2 and TLR4 expression; RNA interference of TSLP attenuated TLR, IL-8, and TNF-α expression stimulated by AF hyphae. These findings indicated that TSLP participates in the immune response of corneal cells triggered by AF, which is closely related to TLR function, and the innate immunity mediated by TLRs could be enhanced by TSLP. Innate immunity may therefore transmit inflammatory signals to adaptive immunity through activation of TSLP; in turn, adaptive immunity likely exerts certain regulatory effects on innate immunity via TSLP. That is, TSLP could interact with innate immunity mediated by TLR2 and TLR4 in human corneal cells challenged by AF and thus may serve as a messenger between the innate and adaptive immune responses in AF keratitis.

Opaque hemithorax - An interesting case.

OBJECTIVE: To present an interesting case of left opaque hemithorax in an adult female and discuss its assessment and management. METHODS: Design: Case Report. SETTING: Tertiary care hospital. PATIENT: One. RESULTS: 44yrs retropositive female admitted with complaints of acute onset dry cough since 15-20 days, sudden breathlesness since 5 days which was progressive in nature, left sided heaviness in chest since 5 days. CECT Thorax showed complete collapse of left lung with cut off of left main bronchus while video bronchoscopy showed left main bronchus completely blocked with very thick necrotic mass and was difficult to dislodge. Debulking with cryo probe was done and left main bronchus was completely cleared off. Allergen panel showed very high serum IgE, high S.IgE against aspergillus and high specific S.IgG against aspergillus. Patient and her Chest X-ray showed significant improvement post cryo debulking and was discharged satisfactorily on oral voriconazole therapy. CONCLUSION: Endobronchial aspergillosis is characterized by massive intrabronchial overgrowth of the aspergillus species, mainly aspergillus fumigatus. Most patients with chronic pulmonary aspergillosis, including those with simple aspergillomas and Aspergillus nodules, have positive Aspergillus IgG antibodies in the blood. We hereby present a case of 44 yrs female presenting with complaints of dry cough and dyspnea and was diagnosed with endobronchial aspergillosis with complete obliteration of left main bronchus by fungal debris in which cryo debulking was done which relieved the symptoms significantly and was discharged in satisfactory condition on oral voriconazole therapy.

Mitochondrial Reactive Oxygen Species Enhance Alveolar Macrophage Activity against Aspergillus fumigatus but Are Dispensable for Host Protection.

Aspergillus fumigatus is the most common cause of mold pneumonia worldwide, and a significant cause of infectious morbidity and mortality in immunocompromised individuals. The oxidative burst, which generates reactive oxidative species (ROS), plays a pivotal role in host defense against aspergillosis and induces regulated cell death in Aspergillus conidia, the infectious propagules. Beyond the well-established role of NADP (NADPH) oxidase in ROS generation by neutrophils and other innate effector cells, mitochondria represent a major ROS production site in many cell types, though it is unclear whether mitochondrial ROS (mtROS) contribute to antifungal activity in the lung. Following A. fumigatus infection, we observed that innate effector cells, including alveolar macrophages (AMs), monocyte-derived dendritic cells (Mo-DCS), and neutrophils, generated mtROS, primarily in fungus-infected cells. To examine the functional role of mtROS, specifically the H2O2 component, in pulmonary host defense against A. fumigatus, we infected transgenic mice that expressed a mitochondrion-targeted catalase. Using a reporter of fungal viability during interactions with leukocytes, mitochondrial H2O2 (mtH2O2) was essential for optimal AM, but not for neutrophil phagocytic and conidiacidal activity in the lung. Catalase-mediated mtH2O2 neutralization did not lead to invasive aspergillosis in otherwise immunocompetent mice and did not shorten survival in mice that lack NADPH oxidase function. Collectively, these studies indicate that mtROS-associated defects in AM antifungal activity can be functionally compensated by the action of NADPH oxidase and by nonoxidative effector mechanisms during murine A. fumigatus lung infection. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes invasive disease in humans with defects in immune function. Airborne conidia, the infectious propagules, are ubiquitous and inhaled on a daily basis. In the respiratory tree, conidia are killed by the coordinated actions of phagocytes, including alveolar macrophages, neutrophils, and monocyte-derived dendritic cells. The oxidative burst represents a central killing mechanism and relies on the assembly of the NADPH oxidase complex on the phagosomal membrane. However, NADPH oxidase-deficient leukocytes have significant residual fungicidal activity in vivo, indicating the presence of alternative effector mechanisms. Here, we report that murine innate immune cells produce mitochondrial reactive oxygen species (mtROS) in response to fungal interactions. Neutralizing the mtROS constituent hydrogen peroxide (H2O2) via a catalase expressed in mitochondria of innate immune cells substantially diminished fungicidal properties of alveolar macrophages, but not of other innate immune cells. These data indicate that mtH2O2 represent a novel AM killing mechanism against Aspergillus conidia. mtH2O2 neutralization is compensated by other killing mechanisms in the lung, demonstrating functional redundancy at the level of host defense in the respiratory tree. These findings have important implications for the development of host-directed therapies against invasive aspergillosis in susceptible patient populations.

Evaluation of an Aspergillus IgG/IgM lateral flow assay for serodiagnosis of fungal asthma in Uganda.

BACKGROUND: Diagnosis of fungal allergies in asthma remains problematic in low-and middle-income countries due to non-availability of point-of-care testing. In this study, we aimed to evaluate the performance of an Aspergillus immunochromatographic technology (ICT) IgG/M lateral flow device (LFD) for the serological diagnosis of allergic bronchopulmonary aspergillosis (ABPA) and severe asthma with fungal sensitisation (SAFS) among Ugandan adult asthmatics. METHODS: 374 adult (aged ≥18years) asthmatics in the African Severe Asthma Program study, Ugandan site constituted the study population. ABPA and SAFS were diagnosed according to standard criteria. Asthmatics who did not meet the above criteria constituted a control group. The LFD tests were performed and read according to manufacturer's instructions. RESULTS: ABPA was found in 12/374 (3.2%) and SAFS in 60/374 (16%) participants. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for the Aspergillus ICT for the diagnosis of ABPA were 0.0%, 96.4%, 0.0% and 96.7% respectively, and for SAFS 6.7%, 97.1%, 30.8% and 84.5% respectively. False positive and negative rates were 3.5% and 3.2% for ABPA and 2.4% and 14.9% for SAFS, respectively. Patients with a positive LFD significantly had higher median Aspergillus fumigatus-specific IgE levels compared to those with negative LFD (median: 0.06 kUA/l VS 0.03 kUA/L, P = 0.011). CONCLUSION: The Aspergillus ICT IgG/M LFD had a poor diagnostic performance for the diagnosis of both ABPA and SAFS. Its greatest value may be in distinguishing chronic and allergic aspergillosis in Africa.

Microphysiological Systems for Studying Cellular Crosstalk During the Neutrophil Response to Infection.

Neutrophils are the primary responders to infection, rapidly migrating to sites of inflammation and clearing pathogens through a variety of antimicrobial functions. This response is controlled by a complex network of signals produced by vascular cells, tissue resident cells, other immune cells, and the pathogen itself. Despite significant efforts to understand how these signals are integrated into the neutrophil response, we still do not have a complete picture of the mechanisms regulating this process. This is in part due to the inherent disadvantages of the most-used experimental systems: in vitro systems lack the complexity of the tissue microenvironment and animal models do not accurately capture the human immune response. Advanced microfluidic devices incorporating relevant tissue architectures, cell-cell interactions, and live pathogen sources have been developed to overcome these challenges. In this review, we will discuss the in vitro models currently being used to study the neutrophil response to infection, specifically in the context of cell-cell interactions, and provide an overview of their findings. We will also provide recommendations for the future direction of the field and what important aspects of the infectious microenvironment are missing from the current models.