Immune Response of Human Polymorphnuclear Neutrophils to Aspergillus Fumigatus

Abstract Invasive fungal infections with Aspergillus fumigatus show an increasing incidence due to the growing number of severely immunocompromised patients. Polymorphonuclear neutrophils (PMNs), as part of the innate immune system, are key players in antifungal immune responses, recognizing fungal pathogens at an early step of infection. Besides phagocytic mechanisms, PMNs kill pathogens by the release of reactive oxygen species (ROS). Human PMNs were isolated from blood of healthy donors using Biocoll separation. Cells were co-cultivated with conidia, germlings and hyphae of the clinical strain ATCC 9197. The oxidative burst was determined in a kinetic measurement quantifying dichlorfluorescein production. Chemotaxis was analyzed by transwell assays. Furthermore, total RNA was extracted and gene expression profiling was performed using Affymetrix U133Plus2.0 arrays. We could demonstrate that A. fumigatus represents a strong stimulus releasing ROS, depending on the morphotype; germlings revealed high ROS release, whereas resting conidia showed low stimulation capacity. PMNs actively tracked germlings and directly attached to fungi as demonstrated by real-time microscopy. Transwell assays revealed that chemotaxis of PMN is strongly dependent on the fungal morphotype; PMN showed only weak chemotaxis in the presence of conidia, whereas in the presence of germlings, high chemotactic activity was achieved. Furthermore, after 6h co-cultivation of PMNs and A. fumigatus germ tubes, 195 (1.273) genes showed an at least 4fold (2fold) altered gene expression. We observed upregulation of genes (hemoxygenase, heat shock 70kDa protein, HSPA8, HSPA1B, HSP90AB1, Ferritin) involved in self-protection against radicals. Additionally, genes involved in inflammatory responses (IL-8, CCL3, CXCL2, IL1RN) were significantly upregulated. Luminex analysis was performed for TNF-a, IL-12, GM-CSF, IFN-γ, IL-6, IL-8, IL-10 and IL-1b to identify secreted cytokines, thereby confirming array data. In conclusion, A. fumigatus had substantial effects on the activation of human PMNs. Various defence strategies were initiated, including phagocytosis, ROS release and mobilization of other immune effector cells by secretion of chemoattractants.

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Analyses of Immune Defense Mechanisms of Human Polymorphonuclear Neutrophils Co-Cultivated with Aspergillus fumigatus Germ Tubes.

Blood ◽

10.1182/blood.v110.11.3848.3848 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3848-3848

Author(s):

Juergen Loeffler ◽

Markus Mezger ◽

Hermann Einsele

Keyword(s):

Aspergillus Fumigatus ◽

Defense Mechanisms ◽

Fungal Pathogens ◽

Fungal Infections ◽

Early Stage ◽

Immune Defense ◽

Polymorphonuclear Neutrophils ◽

Number Of Patients ◽

Human Pmns ◽

Germ Tubes

Abstract Invasive fungal infections with the opportunistic pathogen Aspergillus fumigatus show an increasing incidence due to a higher number of patients with hematological malignancies. Polymorphonuclear neutrophils (PMNs), as part of the innate immunity, recognize fungal pathogens at an early stage after infiltration. Besides phagocytotic mechanisms, PMNs kill pathogens by the release of reactive oxygen species (ROS). Human PMNs were isolated from blood of healthy donors and co-cultivated with A. fumigatus germ tubes for defined time points. Oxidative burst was determined in a kinetic measurement by the use of dichlorfluorescein. In parallel, PMNs were co-cultivated with A. fumigatus germ tubes, followed by whole genome expression analyses (Affymetrix U133 Plus 2.0 Array). We could demonstrate that A. fumigatus germlings of the clinical relevant strain ATCC 9197 represented a strong stimulus for the release of ROS. PMNs actively tracked germlings and directly attached to fungi as demonstrated by real-time microscopy. In addition, co-cultivation of PMNs with A. fumigatus germ tubes resulted in a strong upregulation of genes involved in self-protection against radicals (hämoxygenase, heat shock 70kDa protein HSPA8, thioredoxin, HSPA1B, HSP90AB1, Ferritin). After 6h of co-cultivation, 195 genes showed an at least 4fold altered gene expression. Therein, 4 genes encoding for cytokines and chemokines (IL-8, CCL3, CXCL2, IL1RN) were significantly upregulated. Luminex ELISA analyses confirmed array data and revealing IL-8 to be strongly released (5fold) by PMNs after fungal co-culturing. In conclusion, A. fumigatus had a substantial effect on the activity of human PMNs. In consequence, various defence strategies were activated, including phagocytosis, ROS release and mobilization of other immune effector cells by secretion of chemoattractant cytokines. A better understanding of innate immune defense mechanisms may provide new directions for antifungal therapies.

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Physiology of Midkine and Its Potential Pathophysiological Role in COVID-19

Frontiers in Physiology ◽

10.3389/fphys.2020.616552 ◽

2020 ◽

Vol 11 ◽

Author(s):

Giulia Sanino ◽

Martino Bosco ◽

Giuseppe Terrazzano

Keyword(s):

Inflammatory Responses ◽

Pathological Condition ◽

Severe Pneumonia ◽

Immune Effector ◽

Effector Cells ◽

Coronary Syndrome ◽

Clinical Complications ◽

Mediators Of Inflammation ◽

Autoimmune Conditions ◽

Pathogen Clearance

SARS-CoV2 infection not only causes abnormal severe pneumonia but also induces other relevant pathophysiological effects on several tissues and organs. In this regard, the clinical complications observed in COVID-19 include acute coronary syndrome, pulmonary thromboembolism, myocarditis and, in the severe cases, the occurrence of disseminated intravascular coagulation. Literature on COVID-19 highlighted the central role of the Renin Angiotensin Aldosterone System in the determinism of SARS-CoV2 cellular internalization in the target tissues. Lung degeneration and respiratory distress appear to be dependent on the perturbance of physiological mechanisms, such as the uncontrolled release of pro-inflammatory cytokines, a dysregulation of the fibrinolytic coagulative cascade and the hyperactivation of immune effector cells. In this mini review, we address the physiology of Midkine, a growth factor able to bind heparin, and its pathophysiological potential role in COVID-19 determinism. Midkine increases in many inflammatory and autoimmune conditions and correlates with several dysfunctional immune-inflammatory responses that appear to show similarities with the pathophysiological elicited by SARS-CoV2. Midkine, together with its receptor, could facilitate the virus entry, fostering its accumulation and increasing its affinity with Ace2 receptor. We also focus on Netosis, a particular mechanism of pathogen clearance exerted by neutrophils, which under certain pathological condition becomes dysfunctional and can cause tissue damage. Moreover, we highlight the mechanism of autophagy that the new coronavirus could try to escape in order to replicate itself, as well as on pulmonary fibrosis induced by hypoxia and on the release of cytokines and mediators of inflammation, correlating the interplay between Midkine and SARS-CoV2.

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A Regulatory Role for Interleukin 4 in Differential Inflammatory Responses in the Lung following Infection of Mice Primed with Th1- or Th2-Inducing Pertussis Vaccines

Infection and Immunity ◽

10.1128/iai.68.3.1383-1390.2000 ◽

2000 ◽

Vol 68 (3) ◽

pp. 1383-1390 ◽

Cited By ~ 24

Author(s):

Peter McGuirk ◽

Kingston H. G. Mills

Keyword(s):

Inflammatory Response ◽

Inflammatory Responses ◽

Th2 Cells ◽

Infection Model ◽

Local Production ◽

Local Inflammatory Response ◽

Immune Effector ◽

Effector Cells ◽

Immune Effector Cells ◽

Th1 And Th2

ABSTRACT Protection against infectious pathogens at mucosal surfaces is dependent on local antibody responses, production of inflammatory mediators, and recruitment of immune effector cells to the site of infection. Since Th1 and Th2 cells produce cytokines with pro- and anti-inflammatory activities, immunization with vaccines that induce these T-cell subtypes may regulate the subsequent inflammatory response to infection. We have demonstrated that immunization of mice with pertussis whole-cell or acellular vaccines (Pw or Pa) selectively induces Th1 and Th2 cells, respectively. In this study we have used a murine respiratory-infection model to demonstrate that priming with a Th1- or Th2-inducing pertussis vaccine can influence the local inflammatory response and immune effector cells in the lung following aerosol challenge with Bordetella pertussis. Analysis of bronchoalveolar lavage (BAL) fluid taken during the course of B. pertussis infection of naı̈ve mice or mice immunized with Pw revealed an early influx of neutrophils and local production of interleukin 1β (IL-1β) in the lungs. In contrast, neutrophil infiltration and IL-1β production were not observed following challenge of mice immunized with the Th2-inducing Pa. Conversely, during infection local production of IL-6 and IL-1ra was significantly greater in mice immunized with Pa than in those immunized with Pw. Studies of knockout mice revealed neutrophil and lymphocyte infiltration in the lungs following B. pertussis infection of IL-4-defective (IL-4−/−) mice but not in wild-type mice immunized with Pa. Furthermore, the levels of IL-1β, IL-6, and IL-1ra in Pa-immunized IL-4−/− mice were comparable to those in mice immunized with Pw. These results demonstrate distinct influences of Th1- and Th2-inducing vaccines on the protective inflammatory responses in the lungs following challenge with B. pertussis and implicate IL-4 as an important regulator of inflammatory-cell recruitment.

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A novel glyco-conjugate vaccine against fungal pathogens

Journal of Experimental Medicine ◽

10.1084/jem.20050749 ◽

2005 ◽

Vol 202 (5) ◽

pp. 597-606 ◽

Cited By ~ 311

Author(s):

Antonella Torosantucci ◽

Carla Bromuro ◽

Paola Chiani ◽

Flavia De Bernardis ◽

Francesco Berti ◽

...

Keyword(s):

Conjugate Vaccine ◽

Fungal Pathogens ◽

Pathogenic Fungi ◽

Immune Serum ◽

Vaginal Fluid ◽

Vaginal Infections ◽

Lethal Challenge ◽

Immune Effector ◽

Effector Cells

To generate a vaccine to protect against a variety of human pathogenic fungi, we conjugated laminarin (Lam), a well-characterized but poorly immunogenic β-glucan preparation from the brown alga Laminaria digitata, with the diphtheria toxoid CRM197, a carrier protein used in some glyco-conjugate bacterial vaccines. This Lam-CRM conjugate proved to be immunogenic and protective as immunoprophylactic vaccine against both systemic and mucosal (vaginal) infections by Candida albicans. Protection probably was mediated by anti-β-glucan antibodies as demonstrated by passive transfer of protection to naive mice by the whole immune serum, the immune vaginal fluid, and the affinity-purified anti-β-glucan IgG fractions, as well as by administration of a β-glucan–directed IgG2b mAb. Passive protection was prevented by adsorption of antibodies on Candida cells or β-glucan particles before transfer. Anti-β-glucan antibodies bound to C. albicans hyphae and inhibited their growth in vitro in the absence of immune-effector cells. Remarkably, Lam-CRM–vaccinated mice also were protected from a lethal challenge with conidia of Aspergillus fumigatus, and their serum also bound to and markedly inhibited the growth of A. fumigatus hyphae. Thus, this novel conjugate vaccine can efficiently immunize and protect against two major fungal pathogens by mechanisms that may include direct antifungal properties of anti-β-glucan antibodies.

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The Host Cytokine Responses and Protective Immunity in Oropharyngeal Candidiasis

Journal of Dental Research ◽

10.1177/154405910508401101 ◽

2005 ◽

Vol 84 (11) ◽

pp. 966-977 ◽

Cited By ~ 90

Author(s):

A. Dongari-Bagtzoglou ◽

P.L. Fidel

Keyword(s):

T Cells ◽

Epithelial Cells ◽

Fungal Infections ◽

Immunocompromised Host ◽

Oral Infection ◽

Immune Protection ◽

Oropharyngeal Candidiasis ◽

Immune Effector ◽

Effector Cells ◽

Therapeutic Implications

Over the last three decades, the prevalence of oropharyngeal fungal infections has increased enormously, mainly due to an increasing population of immunocompromised patients, including individuals with HIV infection, transplant recipients, and patients receiving cancer therapy. The vast majority of these infections are caused by Candida species. The presence of cytokines in infected tissues ultimately dictates the host defense processes that are specific to each pathogenic organism. During oral infection with Candida, a large number of pro-inflammatory and immunoregulatory cytokines are generated in the oral mucosa. The main sources of these cytokines are oral epithelial cells, which maintain a central role in the protection against fungal organisms. These cytokines may drive the chemotaxis and effector functions of innate and/or adaptive effector cells, such as infiltrating neutrophils and T-cells in immunocompetent hosts, and CD8+ T-cells in HIV+ hosts. Epithelial cells also have direct anti- Candida activity. Several studies have provided a potential link between lower levels of certain pro-inflammatory cytokines and susceptibility to oral C. albicans infection, suggesting that such cytokines may be involved in immune protection. The exact role of these cytokines in immune protection against oropharyngeal candidiasis is still incompletely understood and requires further investigation. Identification of such cytokines with the ability to enhance anti-fungal activities of immune effector cells may have therapeutic implications in the treatment of this oral infection in the severely immunocompromised host.

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Neutrophil and Eosinophil DNA Extracellular Trap Formation: Lessons From Pathogenic Fungi

Frontiers in Microbiology ◽

10.3389/fmicb.2021.634043 ◽

2021 ◽

Vol 12 ◽

Author(s):

Juliana da Costa Silva ◽

Glaucia de Azevedo Thompson-Souza ◽

Marina Valente Barroso ◽

Josiane Sabbadini Neves ◽

Rodrigo Tinoco Figueiredo

Keyword(s):

Fungal Pathogens ◽

Fungal Infections ◽

Pathogenic Fungi ◽

Allergic Diseases ◽

Extracellular Vesicle ◽

Fungal Colonization ◽

Polymorphonuclear Cells ◽

Effector Cells ◽

Inflammatory Stimuli ◽

Extracellular Trap

Fungal infections represent a worldwide health problem. Fungal pathogens are responsible for a variety of conditions, including superficial diseases, allergic pathologies and potentially lethal invasive infections. Neutrophils and eosinophils have been implicated as effector cells in several pathologies. Neutrophils are major effector cells involved in the control of fungal infections and exhibit a plethora of antifungal mechanisms, such as phagocytosis, reactive oxygen species production, degranulation, extracellular vesicle formation, and DNA extracellular trap (ET) release. Eosinophils are polymorphonuclear cells classically implicated as effector cells in the pathogenesis of allergic diseases and helminthic infections, although their roles as immunomodulatory players in both innate and adaptive immunity are currently recognized. Eosinophils are also endowed with antifungal activities and are abundantly found in allergic conditions associated with fungal colonization and sensitization. Neutrophils and eosinophils have been demonstrated to release their nuclear and mitochondrial DNA in response to many pathogens and pro-inflammatory stimuli. ETs have been implicated in the killing and control of many pathogens, as well as in promoting inflammation and tissue damage. The formation of ETs by neutrophils and eosinophils has been described in response to pathogenic fungi. Here, we provide an overview of the mechanisms involved in the release of neutrophil and eosinophil ETs in response to fungal pathogens. General implications for understanding the formation of ETs and the roles of ETs in fungal infections are discussed.

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The Siderophore Transporters Sit1 and Sit2 Are Essential for Utilization of Ferrichrome-, Ferrioxamine- and Coprogen-Type Siderophores in Aspergillus fumigatus

Journal of Fungi ◽

10.3390/jof7090768 ◽

2021 ◽

Vol 7 (9) ◽

pp. 768

Author(s):

Mario Aguiar ◽

Thomas Orasch ◽

Matthias Misslinger ◽

Anna-Maria Dietl ◽

Fabio Gsaller ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Filamentous Fungi ◽

Fungal Pathogens ◽

Fungal Infections ◽

The Novel ◽

Human Pathogen ◽

Substrate Specificities ◽

Triacetylfusarinine C ◽

Opportunistic Human Pathogen ◽

Siderophore Transporter

Siderophore-mediated acquisition of iron has been shown to be indispensable for the virulence of several fungal pathogens, the siderophore transporter Sit1 was found to mediate uptake of the novel antifungal drug VL-2397, and siderophores were shown to be useful as biomarkers as well as for imaging of fungal infections. However, siderophore uptake in filamentous fungi is poorly characterized. The opportunistic human pathogen Aspergillus fumigatus possesses five putative siderophore transporters. Here, we demonstrate that the siderophore transporters Sit1 and Sit2 have overlapping, as well as unique, substrate specificities. With respect to ferrichrome-type siderophores, the utilization of ferrirhodin and ferrirubin depended exclusively on Sit2, use of ferrichrome A depended mainly on Sit1, and utilization of ferrichrome, ferricrocin, and ferrichrysin was mediated by both transporters. Moreover, both Sit1 and Sit2 mediated use of the coprogen-type siderophores coprogen and coprogen B, while only Sit1 transported the bacterial ferrioxamine-type xenosiderophores ferrioxamines B, G, and E. Neither Sit1 nor Sit2 were important for the utilization of the endogenous siderophores fusarinine C and triacetylfusarinine C. Furthermore, A. fumigatus was found to lack utilization of the xenosiderophores schizokinen, basidiochrome, rhizoferrin, ornibactin, rhodotorulic acid, and enterobactin. Taken together, this study characterized siderophore use by A. fumigatus and substrate characteristics of Sit1 and Sit2.

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Immunological Aspects ofCandidaandAspergillusSystemic Fungal Infections

Interdisciplinary Perspectives on Infectious Diseases ◽

10.1155/2013/102934 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 6

Author(s):

Christoph Mueller-Loebnitz ◽

Helmut Ostermann ◽

Anke Franzke ◽

Juergen Loeffler ◽

Lutz Uharek ◽

...

Keyword(s):

High Risk ◽

Defense Mechanisms ◽

Fungal Infections ◽

Current Knowledge ◽

Immunosuppressive Drugs ◽

Immune Defense ◽

Immunological Mechanism ◽

Immune Effector ◽

Effector Cells ◽

Risk Patients

Patients with allogeneic stem cell transplantation (SCT) have a high risk of invasive fungal infections (IFIs) even after neutrophil regeneration. Immunological aspects might play a very important role in the IFI development in these patients. Some data are available supporting the identification of high-risk patients with IFI for example patients receiving stem cells from TLR4 haplotype S4 positive donors. Key defense mechanisms against IFI include the activation of neutrophils, the phagocytosis of germinating conidia by dendritic cells, and the fight of the cells of the innate immunity such as monocytes and natural killer cells against germlings and hyphae. Furthermore, immunosuppressive drugs interact with immune effector cells influencing the specific fungal immune defense and antimycotic drugs might interact with immune response. Based on the current knowledge on immunological mechanism inAspergillus fumigatus, the first approaches of an immunotherapy using human T cells are in development. This might be an option for the future of aspergillosis patients having a poor prognosis with conventional treatment.

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