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Opportunistic pathogen Candida albicans elicits a temporal response in primary human mast cells
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Stockholm, Sweden.
Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Clinical Bacteriology.
2015 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 12287Article in journal (Refereed) Published
Abstract [en]

Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal, colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. However, mast cell-fungus interaction remains a neglected area of study. Here we show that human mast cells mounted specific responses towards C. albicans. Collectively, mast cell responses included the launch of initial, intermediate and late phase components determined by the secretion of granular proteins and cytokines. Initially mast cells reduced fungal viability and occasionally internalized yeasts. C. albicans could evade ingestion by intracellular growth leading to cellular death. Furthermore, secreted factors in the supernatants of infected cells recruited neutrophils, but not monocytes. Late stages were marked by the release of cytokines that are known to be anti-inflammatory suggesting a modulation of initial responses. C. albicans-infected mast cells formed extracellular DNA traps, which ensnared but did not kill the fungus. Our results suggest that mast cells serve as tissue sentinels modulating antifungal immune responses during C. albicans infection. Consequently, these findings open new doors for understanding fungal pathogenicity.

Place, publisher, year, edition, pages
Nature Publishing Group, 2015. Vol. 5, article id 12287
National Category
Microbiology in the medical area
Identifiers
URN: urn:nbn:se:umu:diva-106777DOI: 10.1038/srep12287ISI: 000358152500001PubMedID: 26192381OAI: oai:DiVA.org:umu-106777DiVA, id: diva2:847512
Available from: 2015-08-20 Created: 2015-08-07 Last updated: 2018-09-07Bibliographically approved
In thesis
1. Pharmaceutical And Immunollogical Challenge Of Fungal Pathogens
Open this publication in new window or tab >>Pharmaceutical And Immunollogical Challenge Of Fungal Pathogens
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Incidences of fungal infections are on the rise in immunosuppressed people. Predominant causative agents for these mycoses are species of the genus Candida, including Candida albicans, Candida glabrata and Candida dublieniensis. Despite a wide range of emerging pathogens, C. albicans remains the leading cause. According to recent epidemiological studies, blood stream infections with C. albicans cause annually ~55% mortality in approximately 300,000 patients from intensive care units worldwide. Furthermore, the percentage of morbidity linked to oral, esophageal and vulvovaginal mycoses cause by C. albicans reach up to 90%. Reasons for these medical concerns are the lack of efficient diagnostics and antifungal therapy.

Here, we therefore sought to find novel antifungal strategies inspired by innate immune cells, such as neutrophils. These phagocytes are able to block the fungal pathogenicity. Neutrophils are bloodstream leukocytes serving as the first line of defense against pathogenic microbes. It has been shown that neutrophils have a strong antifungal activity by impairing the conversion of the dimorphic C. albicans from yeast to hyphal form (Y-H). Consequently, we raised the question whether other immune cells, such as mast cells, with less phagocytic cabapilities may have similar activity to neutrophils.

Mast cells are tissue-dwelling cells. Mucosal tissue is rich in mast cells and usually constitutes the entry ports for fungal pathogens into the human body. A contribution of mast cells in antifungal defense is, thus, very likely. We human explored mast cell functions upon encounter with fungal pathogens. Interestingly, human mast cells show a transient potential to impair fungal viability. To understand the mechanism behind this impairment we analyzed the human mast cell functions in more detail. We found that human mast cells challenged with C. albicans, immediately degranulate and secrete distinct cytokines and chemokines in an orchestrated manner. The chemokines secreted attract neutrophils. Mast cells moreover are able to internalize fungal cells and to ‘commit suicide’ by releasing extracellular DNA traps that ensnare the pathogen.

 

The effectiveness of future antifungals is depended on targeting the pathogen virulence with more efficiency.

The dimorphism of C. albicans is proven to be essential its virulence. Blockage of this switching ability could render the pathogen avirulent. Consequently, we screened for compounds that mimic the neutrophils anti-dimorphic activity by screening small chemical molecule libraries that block Y-H transition. The screening of big chemical libraries requires a reliable, reproducible and rapid high-throughput screening assay (HTS). We developed an HTS assay based on automated microscopy and image analysis, thereby allowing to distinguish between yeast and filamentous forms. In order to find the ideal Y-H blocker, we also evaluated the cell viability via the count of ATP levels when challenged with the respective small chemical molecules.

 

Drug development is an elaborate and expensive process. We therefore applied our screening setup to identify antidimorphic/antifungal activity in compounds from two different chemical libraries including FDA-approved drugs. The study disclosed 7 off-patent antifungal drugs that have potent antimycotic activity, including 4 neoplastic agents, 2 antipsychotic drugs and 1 antianemic medication.

In a nutshell, we aimed to mimic the anti-dimorphic/antifungal activity of neutrophils with small chemical molecules. Furthermore, we elucidated how immune cells contribute to antifungal defense to exploit these mechanisms for the development of novel antifungal therapies. Thus, this thesis provides novel tools for the discovery of more efficient compounds, identifies previously unknown antifungal aspect of off-patent FDA-approved drugs and highlights the interplay of mast cells with pathogenic fungi with the aim to define new screening strategies.

Place, publisher, year, edition, pages
Umeå Universitet, 2015. p. 52
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1735
Keywords
mast cells, Candida albicans, yeast to hyphal form, antifungal drugs, repurposed drugs, HTS
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:umu:diva-107713 (URN)978-91-7601-308-3 (ISBN)
Public defence
2015-09-25, sal Eo4, byggnad 6E, NUS, Norrlands universitetssjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2015-09-04 Created: 2015-08-27 Last updated: 2018-06-07Bibliographically approved
2. Candida albicans adaption to host microenvironments drives immune evasion
Open this publication in new window or tab >>Candida albicans adaption to host microenvironments drives immune evasion
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Immunosuppressed patients are frequently afflicted with severe mycoses caused by opportunistic fungal pathogens. Besides being a commensal colonizing predominantly skin and mucosal surfaces, Candida albicans is the most common human fungal pathogen. Mast cells are present in tissues prone to fungal colonization being expectedly among the first immune cells to get into contact with C. albicans. Here we describe how mast cells acted as tissue sentinels and modulated initial antifungal immune responses. Mast cells response was able to reduce fungal viability and signaled for neutrophil infiltration to the tissue. Upon chemokine sensing circulating neutrophils are rapid infiltrating to the mucosal to help fight infection. A high number of infiltrating cells coupled with the formation of multicellular structures such as biofilm comes with induction of hypoxic and anoxic micro niches. We found that a persistence anoxia hampered neutrophil responses by affecting fungi sensing and consequent antifungal due to cell wall masking. Adaption to low oxygen seems is important for a successful host infection. Hypoxic and anoxic environments do not allow neutrophils to efficiently produce ROS. Neutrophil oxidative burst is essential for antifungal activity and many fungal pathogens evolved antioxidative factors to mediate survival during infection. We reasoned that targeting of fungal redox balances could be a new therapy approach. We have tested tempol, a redox-cycling nitroxide Tempol as a new antifungal drug. Tempol proved an efficient compound in our testing. We found that Tempol affected fundamental pathways for fungal homeostases such as glycolysis and steroid biosynthesis. Additionally, Tempol helped curve fungal infectivity in a mouse model and leads for an enhanced immune system cytokine profile in human blood. The results obtained proposed tempol as a valid new antifungal compound and open new opportunities for the future development of therapies. Efficient antifungal therapies are still urgent since only 6 classes of antimycotics exist and all with few restricted fungal targets. Since primarily fungal infections affect patients with other immunosuppressive conditions, which are undergoing treatment, we reasoned that repurposing drugs could offer clinical benefits. We performed a screening of two US Food and Drug Administration (FDA)–approved compound libraries for compounds with anti-Candida activity. From 844 drugs, 26 agents showed activity against C. albicans. We identified 7 new off-target drugs all with potent anti- C. albicans activity. The use of these new drugs could be prophylactic or to treat both conditions simultaneously offering, therefore the intended benefit.

Overall, in this thesis work, we have focused on the sensing clearing and management of fungal pathogens. These findings open new doors for understanding better fungal pathogenicity and purpose valid new antifungal compounds that pave the way for future development of therapies.

Abstract [sv]

Den mänskliga kroppen är ständigt utsatt för angrepp från mikrober i vår miljö. Varje dag träffar vår kropp på organismer som potentiellt kan infektera oss, så kallade patogener så som bakterier, virus, parasiter och svampar. Det är immunsystemets uppgift att befria oss från oönskade mikroorganismer. Vårt immunförsvar är mycket effektivt vilket framgår av att trots den ständiga närvaron av mikroorganismer så blir vi sällan allvarligt sjuka. Men, människor med nedsatt immunförsvar kan utveckla allvarliga infektioner från opportunister patogener så som svampen Candida albicans. C. albicans är en viktig orsak till sjukdom och död över hela världen. Syftet med denna avhandling är att bättre förstå samspelet mellan dessa svampar och vårt immunförsvar. Vi fokuserar på det medfödda immunförsvaret och försöker identifiera de delar som är nödvändiga för kontroll under en infektion. Vi studerar också hur svamparna utvecklar mekanismer för att möta värdorganismens försvar och vad som reglerar dessa mekanismer. Det medfödda immunförsvaret finns i hela kroppen och består bland annat av vävnader - huden och slemhinnorna - och en mängd olika celltyper. Mastcellerna är en del av det medfödda immunförsvaret och finns utspridda på kroppens ytor mot omvärlden. Främst finns de i huden, tarmens slemhinnor och luftvägarna och det är också här som patogena svampar förekommer i kroppen. Mastcellerna är därför strategiskt placerade för att snabbt kunna möta infekterande mikroorganismer. Här har vi visat att mastceller är kroppens vaktposter och mycket viktiga vid svampinfektioner, men de kan bara tillfälligt kontrollera C. albicans. Mastcellerna aktiveras när de möter C. albicans och släpper omedelbart ut inflammatoriska mediatorer som är lagrade i cellernas granula. Denna degranulering kan också rekrytera fler immunceller så som neutrofiler. De kan även släppa ut strukturer som kallas extracellulära fällor (MCETs), som fångar in inkräktarna för nedbrytning. Med dessa metoder kan Mastcellerna ta upp och oskadliggöra invaderande mikroorganismer, men här har även C. albicans utvecklat ett eget försvar mot Mastcellerna. När C. albicans tas upp av mastcellerna kan de undkomma avdödning och utnyttjar då istället den skyddade intracellulära miljön för döda mastcellerna inifrån.

Neutrofiler är också en del av det medfödda immunförsvaret och de är den vanligaste typen av vita blodkroppar. Dessa celler cirkulerar i blodet men reagerar snabbt på hot och migrerar då från blodet in i vävnaden där bekämpandet av svamparna sker. De är oftast först på plats vid en infektion.

I det andra projektet har vi tittat närmare på neutrofilers försvar mot C. albicans infektioner och hur detta skiljer sig mellan syrerika och syrefattiga miljöer. Vi fann att neutrofilernas försvarsförmåga är kraftigt minskad under syrefattiga förhållanden. Både formationen av neutrofila extracellulära fällor (NETs) och även fagocytering, eller förtäring är kraftigt nedsatta under dessa förhållanden och detta kan ha en stor betydelse för immunförsvarets förmåga att bekämpa C. albicans infektioner. Bekämpandet av mikroorganismer är en aktiv process, driven av neutrofilernas snabba migration från blodet ut till vävnaden där mycket av syret förbrukas. I syrefattiga miljöer kan C. albicans också ändra cellväggens komposition och det ändrar i sin tur hur immunförsvaret reagerar mot dem.

Produktion av reaktiva syreradikaler är en av de mest påverkade försvarsmekanismerna i syrefattig miljö. Neutrofilerna använder reaktiva syreradikaler som en robust och effektiv kontroll-mekanism mot svamppatogener. I det tredje projektet undersöker vi om vi kan förhindra spridning av svampceller genom att påverka redoxpotentialen. Vi använder Tempol som är ett ämne med bevisad påverkan på metabolism och cellväggens komposition och har även inflammatorisk effekt. Behandling av svampcellerna med Tempol gav en framgångsrik anti-Candida effekt som karakteriserades med hjälp av RNA-sekvensering och metaboliska tester.

Idag finns det ett mycket stort behov av nya och bättre läkemedel mot systemisk svampinfektion. Opportunistiska svampinfektioner är ett stort hot mot patienter med nedsatt immunförsvar e.g patienter med leukemi eller som fått organtransplantationer. I projekt nummer fyra tänker vi oss att dessa patienter kanske skulle kunna får endast ett läkemedel mot både sjukdomarna. Därför har vi undersökt 844 godkänt-FDA läkemedlen för effekt mot C. albicans. Vi identifierat 7 nya molekyler med god effekt mot C. albicans. Vi har karakteriserad dessa läkemedlen och visat att de är lika effektiva som kommersiellt tillgängliga läkemedel.

Sammanfattningsvis visar våra resultat nödvändigheten till en fördjupad förståelse om immunceller och svampars samspel i mikromiljöer, så som de syrefattiga. En mer detaljerad förståelse kan därmed bidra till att utveckla bättre diagnostiska och terapeutiska verktyg för svampinfektioner.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2018. p. 64+4
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1977
Keywords
fungi, candida albicans, neutrophils, mast cells, immune evasion, mycoses, infectious diseases
National Category
Basic Medicine
Research subject
Immunology; Infectious Diseases; Molecular Immunology
Identifiers
urn:nbn:se:umu:diva-151598 (URN)978-91-7601-919-1 (ISBN)
Public defence
2018-10-05, Major Groove, Universitetssjukhuset 6L, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2018-09-14 Created: 2018-09-07 Last updated: 2018-09-19Bibliographically approved

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