Endothermy and innate antifungal immunity

13 Pages·2015·0.2 MB·English

Polish Journal of Veterinary Sciences Vol. 17, No. 3 (2014),555–567
DOI 10.2478/pjvs-2014-0084
Review
Two fundamentals of mammalian defense
in fungal infections: Endothermy and innate
antifungal immunity
M.J. Biegańska
DepartmentofPreclinicalSciences,FacultyofVeterinaryMedicine,WarsawUniversityofLifeSciences–SGGW,
Ciszewskiego8, 02-786 Warsaw,Poland
Abstract
Theenvironmentofanimalsisinhabitedbyenormousfungalspecies,butonlyafewhundredsare
pathogenic for mammals. Most of potentially pathogenic fungal species, excluding dermatophytes,
seldomcausethediseaseinimmunocompetenthosts.Datafromliteratureindicate,thatanimmune
system and endothermy are foundations for this mammalian relative resistance to fungal systemic
infections. Stable and high temperature of the body restricts invasion and growth of potentially
pathogenic fungi. Together with elevated metabolism it supports the effectiveness of mammalian
immunity. The innate immunity is assigned to prevent the invasion of various microbes (including
fungi) to the hosts’ organism. It consists of cellular receptors and several humoral factors as the
AntimicrobialPeptides.Ifthephysicalbarriersfailinstoppingtheinvader,itisrecognizedas“alien”
by multiple Pattern Recognition Receptors (PRRs) like Toll Like Receptors (TLRs) expressed by
cellsofinnateimmunityand/orC-typelectins.Atthesametimeinnateinflammationbeginsandthe
complement cascade is activated. These mechanisms are able to stop and clear some fungal infec-
tions.Duringexistinginfectiontheadaptiveimmunityisinduced.Thisreviewaimstoshowtheroleof
mammalianendothermyandtopointthemostimportantelementsofinnateantifungalimmunity.
Key words: fungalinfections,mammals,PAMPs,PRRs,innateimmunity,endothermy
Introduction only a few hundreds are considered to be pathogenic
for humans and other mammals (Taylor et al. 2001,
Fungi are highly diversified organisms, classified Casadevall 2005, Robert and Casadevall 2009). The
as a separate taxonomic kingdom. They colonize list of human and mammalian fungal pathogens en-
a number of various habitats. The quantity of fungal closes:Candidaspp.,Cryptococcusneoformans,Asper-
specieswasestimatedat1.5million,but somepredic- gillus spp., Histoplasma capsulatum, Coccdioides im-
tions, based on the effects of modern sequencing mitis, C. posadsii, Blastomyces dermatitidis, Paracoc-
methods,anticipate the numberof 5.1 millionspecies cidioides brasiliensis, dermatophytes, some represen-
existing on the Earth (Blackwell 2011). Among them tatives of Hyphomycetes (e.g. Penicilium, Paeciliomy-
Correspondenceto:M.J.Biegańska,e-mail:malgorzata–[email protected]
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556 M.J. Biegańska
ces, Scopulariopsis, Fusarium or Exophiala) and Krutkiewicz 2010)ormay contribute with fungalcell
some Zygomycetes (e.g. Mucor or Rhizopus) (Burg- integrity, also inside the phagocyte (e.g. C. neofor-
eois and Kuchler 2012). The majority of fungal spe- mans’ laccase) (Steenbergen and Casadevall 2003).
cies, excluding dermatophytes, rarely cause the dis- Furthermore,adherenceabilities,molecularmimicry
ease in hosts with efficient mechanisms of defense. and thermaltoleranceareimportant forvariousfun-
The main factors predisposing humans and other gi. The crucial virulence factor for fungal opportun-
mammals to severe fungal diseases are primary and ists like C. albicans and Penicilium marneffei is their
secondary immunodeficiences, as indicate multiple abilitytoswitchthemorphologyduringthetransition
cases of life-threatening mycoses, observed among from a commensal or environmental organism into
immunocompromised hosts (Batura-Gabryel 2003, pathogen (Cooper and Haycocks 2000, An-
Soboletal.2009).Thuscomplexinnateandadaptive drianopoulos 2002, Cooney and Klein 2008, Krut-
mechanisms of the mammalian immune system are kiewicz2010).Dimorphismisalsoanimportantviru-
fundamentals in resistance to fungal infections. lence factor of B. dermatitidis, H. capsulatum and C.
The vast majority of publications about fungal immitis. Besides changes in the enzymatic activity,
diseases in animals concerns cold-blooded verte- thistransformationmodifiesalsofungalcellwalland
brates, like Amphibia and Pisces. Papers referring to aggravate the recognition of fungus (Cooney and
the cases of severe amphibian mycoses caused by Klein 2008, LeibundGut-Landmann et al. 2012).
Batrachochytrium dendrobatidis may serve as the Interactions between the host and potential
example. These infections have resulted in large de- pathogen are modified by the hosts’ immunity. Ani-
cline or extinction of about 200of frogs’ populations mal organisms had evolved complex mechanisms of
and species(Skerratt et al.2007,Rollins-Smithet al. defense,whichareabletopreventtheinfectionorto
2011). Moreover, phaeohyphomycoses and saproleg- terminate the infection before the disease occurs.
nioses of fish and their eggs are noted quite fre- Someoftheme.g.theantimicrobialinnateimmunity
quently (Hussein et al. 2001, Faisal et al. 2007, Cao were established very early during evolution and are
et al. 2012). Numerous data may lead to the con- present also in the invertebrates. The mammalian
clusion, that poikilothermic animals are more sus- immune system is more sophisticated and more ef-
ceptible to fungal infections. Additionally, the list of fective. The entire protection against most of fungal
mammalianandhumanfungalpathogensmentioned pathogens, requires the cooperation between the in-
above, seems to be quite short, when compared to nate response provided by phagocytes (macrophages
the thousands of species pathogenic to lower verte- and neutrophils) and dendritic cells, and adaptive
brates.Themain differencebetween thesegroups of mechanisms directed by immunocompetent cells
animals is their immunity and mammalian en- (Brown2011).Theadvantageofalmostinstantlyact-
dothermy. Some experiments points that high body inginnatedefensemechanismsisdetectionandelim-
temperatureis the second foundation ofmammalian ination of several various pathogens. These mechan-
resistance to fungal infections (Robert and isms are also involved in activation of the specific,
Casadevall 2009, Bergman and Casadevall 2010). adaptive (acquired) response, involving cell me-
diated immunity (CMI) and humoral immunity (HI)
(Weinberg et al. 1998, Zelante et al. 2007, Blanco
Interaction between the host and Garcia 2008). That immunity may be also pro-
and pathogenic fungus tective during reinfection. If innate mechanisms are
overcome by pathogenic fungus, CMI enclosing the
To use a mammalian organism as the source of Th1 type of the immune response is the main mech-
nutrients, potentially pathogenic microbes have to anism of defense. Moreover, the production of cer-
colonizethehost,invadethehosts’tissuesandestab- tain types of antibodies is important in antifungal
lishthemselvesinsideitsorganism.Thussomefungal protection and in pathogen clearing (Blanco and
specieshaveevolvedandformedmanyvirulencefac- Garcia 2008). However, Th2 type of the immune re-
tors e.g. polysaccharide capsule of Cryptococcus sponsemaycausethehypersensitivity,frequentlyob-
neoformans inhibits phagocytosis and enhances the served during fungal diseases. Also, certain im-
dissemination of the fungus. Also fungal enzymes munoglobulins in some fungal infections may con-
such as phospholipases and keratinases are used to tribute to the pathogenesis (e.g. in C. neoformans or
overcome the hosts’ immunity and togain theaccess C. albicans infections) (Casadevall 1995, Blanco and
to the tissues and cells (Steenbergen and Casadevall Garcia 2008). In both CMI and HI, the role of par-
2003, Dworecka-Kaszak 2008, Krutkiewicz 2010). ticular elements depends on the host and on the in-
Some of them like SAP1, SAP2, SAP3 proteases of vadingfungus.Althoughmanyofthepathwaysofthe
C. albicans serve as adhesins (Monod et al. 2002, adaptive immunity has already been explained, the
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complexity of all adaptive mechanisms and differen- IgA and carbohydrate-digesting lysozyme. Lysozyme
ces observed among various hosts does not allow for has been found in secretions such as tears and saliva
generalization. and in some body fluids. It is also present in
granulocytes, macrophages and monocytes. This en-
zymehaswellknownantibacterialactivity,butitisalso
The basis of the relative mammalian able to kill or to impede the growth of various fungi
resistance to fungal infections e.g. Candida, Cryptococcus, Histoplasma, Aspergillus
and Paracoccidioides (Brown 2011). Mucosal mem-
During their life all animals have permanent con- branesarealso“self-cleaned”bytheirnaturalfunction-
tact with various hostile microorganisms. The effec- alitye.g.bytheGIperistalsis,urinefloworbycongeni-
tiveresistancetotheirconstantattacksisessentialfor talreactions like coughing and sneezing.
organisms’survival.Itbeginswithphysical“obstacles” As it is known, some fungi (e.g. Candida yeasts)
which eliminate some of microbes. The next step is participate in the oral, gastrointestinal and vaginal bi-
properrecognitionofattackingfungifollowedbyacti- ota of healthy individuals (Jouault et al. 2006). This
vation of defense mechanisms capable of destroying well adapted biota is following defensive barrier, play-
the invader. All of this is provided by the innate im- ing the supportive role in innate immunity, because it
munityenclosingtheabilitiesofphagocyticcells(neu- impedes the settlement of pathogens and counteracts
trophilsandmacrophages),theactivityofthecomple- theinfection(BlancoandGarcia2008).Thefungaldis-
ment system and inflammatory response. Additional ease develops, if some of mentioned mechanisms are
advantage of mammals is their endothermy. This disturbed or debilitated. For example, vulvovaginal
achievementenablesproperactionoftheimmunesys- candydosis frequently occurs when the natural biota is
temandeliminatesmanyofthefungalpathogens,un- disrupted after antibiotic therapy. The efficiency of
able to grow in elevated, stable temperature of the physico-chemical barriers, supported with immunity
mammalian body. If the complete elimination of seems to be quite high, considering how many fungal
pathogens is not possible, the disease occurs. species encounter an animal during its’ life and how
many fungalelements it inhales or ingests.
Simultaneously with stopping many of encounter-
Innate defense mechanisms ing microbes the innate response is capable of more
sophisticated reactions. The important assignment of
Innatedefensemechanismsweretraditionallyclas- innate immunity is the protection against the
sified as constitutive and inducible mechanisms. While microbes breaking through the physico-chemical bar-
the constitutive mechanisms are inherent barrier pres- riers. This kind of response is almost immediate be-
ent in the “natural doorways” of the body, constantly cause the first reactions are observed within minutes.
confronted with the fungi existing in an environment, Innate defense mechanisms detect and recognize un-
induciblepathwaysareactivatedduringtherecognition familiar,chemicalstructurescommontomicrobesand
of fungi (Zelante et al. 2007). The physico-chemical absent in animals (Zelante et al. 2007). Generally,
barriers of the skin and mucosal membranes of the they identify “invaders” on molecular level and react
gastrointestinal, genitourinary and respiratory tracts with specialized sentinel and defensive cells. During
are the first line of defense against fungi, able to stop inflammation the innate response is mediated by the
many of them (Tizard 2004, Blanco and Garcia 2008). phagocytes mainly macrophages, neutrophils and
The intact skin epidermis covered with sebum rich in monocytes.Itiseffectiveincontrollingofsomefungal
short-chainfatty acids and high concentration ofNaCl infections, when those specialized cells, are able to
is the physical barrier difficult to cross. Excluding the phagocytose and destroy fungal elements. The multi-
dermatophytes, able to penetrate it with keratinolytic factorial inflammatory response and the activated
enzymes, other fungi (e.g. Zygomycetes) to cause the complement cascade are additional, accompanying
clinical mycosis have to be implemented into deeper processes involved (Tizard 2004, Zelante et al. 2007,
skin layers or to the tissues during the trauma. Some- Brown 2011, Santamaria et al. 2011).
times the fungal infection develops in the skin areas
debilitatedduetoextensiveburnsorindiabeticlesions.
Frequently,mycosesaretheconsequencesofotherdis- The firstelement of defense – the recognition
ordersaffectingtheskine.g.atopicdermatitisorlethal of pathogen: PAMPsand PRRs
acrodermatitis in dogs (McEwan 2001).
Besides the skin, other entry to the mammalian Activation of innate antifungal defense mechan-
body are the mucous membranes. Their continuity is isms depends on the recognition of the PAMP struc-
supported by the presence of antimicrobial peptides, tures (Pathogen-Associated Molecular Patterns),
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558 M.J. Biegańska
present on the microbes invading the animal. These Galectin-3, membrane scavenger receptors (CD5,
structuresareevolutionaryconservedandinvariantin CD36) and the family of C-type lectin receptors
many groups of pathogens, including fungi. The ma- (CLRs),suchasdectin-1,dectin-2andMincle(Brown
jorityofdataaboutfungalPAMPsreferstoopportun- 2011, Burgeoisand Kuchler 2012, LeibundGut-Land-
istic or pathogenic species, frequently isolated from mann et al. 2012). Other PRRs are the receptors for
severehumanmycoses.Thesestructuresinotherfun- mannosyl/fucosyl glycoconjugate ligands (MRs)
giareless known.In general,therecognitionoffungi (Zelante et al. 2007, Brown 2011). Because of
is based on the detection of cell wall components, multiple PAMPs associated with various pathogenic
mostlycarbohydratepolymersandmannosylatedpro- fungi, to start the specific immune response against
teins, by specialized corresponding receptors. In C. differentfunginumerousPRRsareactivatedsimulta-
albicans glucuronoxylomannan (GXM), mannan, neously or sequentially.
β1,3- and β1,6-glucan and phospholipomannan The main group of PRRs involved in recognition
(PLM) of cell wall were identified as PAMPs. Also, of fungi are Toll-likereceptors, but distinct hosts can
GXM of C. neoformans, C. gattii and A. fumigatus usedifferent TLRto identify thesamefungus. More-
were recognized as PAMPs (Fonseca et al. 2010, over,theengagementofsingleTLRvariesdepending
Brown 2011, Burgeois and Kuchler 2012). Addition- on fungal morphotype and the route of infection
ally, some fungal proteins can be sensed e.g. one of (Bourgeoisetal.2010,Santamariaetal.2011).Some-
adhesins – BAD-1 in B. dermatitidis or one of heat timessingleTLRismore“universal”andbindsdiffer-
shockproteins–HSP60,presentinthe cellwallofH. entPAMPsofvariousfungi(Neteaetal.2006a,Burg-
capsulatum (Long et al. 2003, Brown 2011). eoisandKuchler2012).Forexample,TLR2identifies
Pathogenic C. albicans is recognized by proteins phospholipomannan(PLM)ofC.albicans(Jouaultet
glycosylated with O- or N- linked mannosyl chains, al.2006)andunknownligandsofhyphaeorconidiaof
present in outerlayer ofcell wall(Neteaet al. 2006b). A. fumigatus (Netea et al. 2003). TLR2 also recog-
SomepublicationsindicatethatfungalPAMPsarealso nizesβ-glucanofCoccidioidesposadsii(Viriyakosolet
nucleicacidse.g.DNAandRNA of Candidaspp.and al.2005),C.albicans(Neteaetal.2006a)andH.cap-
A. fumigatus alike DNA of C. neoformans and Malas- sulatum (Sorgi et al. 2009). Next, TLR4 is stimulated
seziafurfur(Fonsecaetal.2010,Brown2011,Burgeois by mannanosylated proteins (O-linked mannosylated
andKuchler2012).However,nucleicacidsareexposed proteins)ofC.albicans(Neteaetal.2006a),byGXM
for recognition only after the fungus is destroyed or ofC.neoformans (Shohametal.2001)andby ligands
phagocytosed.Theytriggeralternativepathwaysofthe presentonconidiaofA.fumigatus(Neteaetal.2003).
immuneresponsethroughmechanismsactivatedbythe The ligand for TLR3 is double-stranded RNA, re-
PAMPs of fungal cell wall (Brown 2011, Leibun- leased within the phagosome from conidia of A.
dGut-Landmann et al. 2012). It is also presumed, that fumigatus, while TLR7 is activated by single-stranded
chitinsimilarlytoothersurfacepolysaccharidesmaybe RNA from C. albicans. The recognition of genomic
fungal PAMP, but neither corresponding receptor nor DNA by endosomal or cytoplasmic TLR9 is common
its location is established(Brown 2011). forseveralfungalspecies,buttheidentifiedmotifsare
To identify PAMPs, the pattern recognition re- different(Burgeoisand Kuchler 2012).In some cases
ceptors (PRRs) are used by multiple hosts’ cells, TLRsmayformheterodimerstoenhancethepossibil-
mainly macrophages, monocytes, dendritic cells and ity of fungal recognition in different hosts. Hetero-
endothelialcells(Zelanteetal.2007,BlancoandGar- dimersofTLR2/TLR6andTLR2/TLR1arereceptors
cia2008).PRRs activatedby equivalentPAMPsiniti- for cryptococcal GXM and some ligands of A.
ate multiple cellularprocesses,leading to the specific fumigatus, but the first one recognizes A. fumigatus
immune response against attacking fungus. These re- only in mice, whereas the second can identify that
ceptors may be bounded with the phagocytes mem- fungus both in mice and in humans (Burgeois and
brane and directly recognize the fungi (e.g. Toll-like Kuchler 2012, Rubino et al. 2012).
receptors – TLRs, dectin-1). Some of them are pres- ThesecondimportantgroupofPRRsisthefamily
ent in cytoplasm or endosomal vesicle (TLR9), other of C-type lectins (CRLs) enclosingdectin-1, dectin-2,
like collectins are secreted by both phagocytic and Mincle, pentraxin-3, DC-SIGN (CD209) and man-
non-phagocytic cells as soluble PRRs. They attach to nose receptors (MRs, CD206, CD280) (Bourgeois et
the pathogen, opsonize it and allow to identify the al. 2010, LeibundGut-Landmann et al. 2012). CRL
fungus by membrane-bounded opsonic receptors e.g. are present in soluble form in plasma (collectins) or
complement receptors (CRs) (Brown 2011, Leibun- onthesurfaceofendothelialcellsandleukocytes(se-
dGut-Landmann et al. 2012). Among PRRs respon- lectins). Generally, mammalian lectins are proteins
sible for the identification of fungal PAMPs are the able to identify bacterial and fungal carbohydrates
receptorsforcomplementcomponents(CRs),soluble like mannans, glucans, lipophosphoglycans or
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Table1.TheoccurrenceofPathogen-AssociatedMolecularPatterns(PAMPs)inchosenfungiandtheirrecognitionbyPattern
RecognitionReceptors(PRRs).
PRRsco-receptorswith
RecognitionoffungalPAMPs
Fungalspecies FungalPAMPs confirmedphysical
byPRRsandco-receptors
interactions
GXM–glucuronoxyloman TLR4 Galectin-3;
N-linkedmannan TLR4 Dectin-1;
O-linkedmannan TLR4 SIGNR1
Candidaalbicans PLM–phospholipomannan TLR2;TLR4
β-glucan TLR2/Dectin-1
genomicDNA TLR9
ssRNA TLR7
GXM–glucuronoxyloman TLR4;TLR1/TLR2;
TLR2/TLR6(onlyinmice)
PLM–phospholipomannan TLR2/TLR1;TLR2/TLR6
Aspergillusfumigatus Undefinedligandsonhyphae TLR2
andconidia
dsRNA TLR3
Histoplasmacapsulatum β-glucan TLR2/Dectin-1 Dectin-1
Coccidioidesposadsii β-glucan TLR2/Dectin-1 Dectin-1
Cryptococcusgattii GXM-glucuronoxyloman TLR2/TLR1;TLR2/TLR6
GXM–glucuronoxyloman TLR2/TLR1;TLR2/TLR6;TLR4
Cryptococcus neoformans genomicDNA TLR9
PLM–phospholipomannan TLR2/TLR1;TLR2/TLR6
glycoinositol-phospholipids with mannose, glucose receptor starts the response against various fungi in-
andN-acetyloglucosaminedomains(Ramkumaretal. cluding Aspergillus, Candida, Pneymocystis and Coc-
2003,Tizard2004).Oneofimportantlectinsissoluble cidioides(Burgeoiset al.2010,LeibundGut-Landman
mannose binding lectin (MBL) (Brown 2011). It rec- 2012). Sometimes the recognition of fungal β-glucans
ognizes mannose and N-acetylglucosamine on many viadectin-1islimited,becausethemorphologyoffun-
microorganisms, what enables its binding to multiple gus has been changed during phase transformation.
bacteria and yeasts, including C. albicans and C. AgoodexampleisC.albicans.Duringits’yeastphase,
neoformans.MBLisanimportantfactorofinnateim- β-glucan is exposed only in budding scars, but on the
munity because it may serve as opsonin and activate surface of hyphal form of C. albicans this structure is
the complement alternate pathway (Tizard 2004). covered by the mannan layer (Brown 2011, Leibun-
Mannose receptor is also present in the cell mem- dGut-Landman 2012). If β-glucan is masked by other
brane of mature macrophages and immature myeloid components like α-glucan layer of H. capsulatum or
dendritic cells. Besides tissue macrophages the Mac- by external hydrophobin of A. fumigatus’ resting
rophage Mannose Receptor (MMR), also known as conidia, to recognize the fungus dectin-1 cooperates
CD206 and MRC1 (mannose receptor C, type 1) is with TLRs or with other CLRs e.g. DC-SIGN (Burg-
expressed on lymphatic endothelial and on liver cells eois et al. 2010, Brown 2011).
(Ramkumar et al. 2003, Santamaria et al. 2011). Be- Dectin-2 identifies the mannose rich motifs of
sides N-mannan of C. albicans it identifies also various pathogenic and opportunistic fungi including
N-acetyl-D-glucosamine and glycoprotein A of Pneu- H. capsulatum, P. brasiliensis, A. fumigatus, C. albi-
mocystis carinii (Brown 2011). cans and Trichophyton rubrum. Together with de-
Other receptors from C-type lectin family are de- ctin-1 it influences the activation of dendritic cells by
ctin-1 and dectin-2 (dendritic cells-associated C-type fungal pathogens (Burgeois et al. 2010).
lectins). Dectin-1 – the main receptor for β-glucan is ThethirdCtypelectin–Mincleistypicalformac-
expressed in myeloid dendritic cells. It initiates their rophages. It identifies α-mannose. Although, this re-
endocytosis. Recognition of β-1-3 glucan by this ceptor recognizes C. albicans and initiates the potent
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560 M.J. Biegańska
inflammatory response, it is not necessary for the neutrophils. The accumulation of complement com-
phagocytosis of this yeast. Mincle is also involved in ponents in neutrophils, macrophages and in body
Malasseziaidentification.TogetherwiththeFcγR(the fluidsmakesthemeasilyaccessibleforthequickreac-
receptor for Fc fragment of Igs) it also activates the tion to invaders (Tizard 2004).
response to Fonsecaea pedrosi, although this reaction Regardless of its innate nature the complement
results in chronic infection (Burgeois et al. 2010, system is activated by both innate and acquired im-
Brown 2011, LeibundGut-Landmann et al. 2012). munemechanisms.Theclassicalactivationpathwayis
Cooperation between several PRRs (also those initiated during fungal infection by immune com-
fromdifferentfamilies)enablestheadjustmentofthe plexes made by antibodies bound to fungal antigens.
host response. It also improves the recognition of The second way of complement activation is alterna-
PAMPs and increases the specific pathogen identifi- tive pathway, induced by direct contact of comple-
cation (Santamaria et al. 2011, Burgeois and Kuchler ment component C3 with the carbohydrates and
2012). For example, pathogenic C. albicans is detec- lipopolysaccharides in microbial cell wall. The third
ted by at least three PRRs identifying three sur- way of complement activation is the lectin pathway.
face-associated PAMPs. At first mannose receptor This innate mechanism is mediated by soluble man-
binds to N-linked, branched, long mannose chains of nose binding lectin (MBL) – the PRR mentioned
cell wall proteins, but TLR4 recognizes short, O-lin- above. All of three complement activation pathways
ked mannose residues of mentioned proteins. Addi- lead to formation of membrane attack complex
tionally, complex of Dectin-1/TLR2 identifies (MAC) and initiate the pathogen killing (Tizard
β-glucan. First two PAMPs are present in the outer 2004). In fungal infections the complement may be
part of the cell wall, but β1,3-glucan is situated in its’ activated by various PAMPs like exposed β-glucan.
innerlayer.Itisaccessibleonlywhensurfacemannop- Moreover, the recognition of fungal PAMPs may re-
roteins are damaged or in the budding scars of C. sultinopsonizationbysolublePRRse.g.complement
albicans(Neteaetal.2006b).Also,TLR2cancooper- componentslikeC3 orMBL. Subsequent recognition
ate with galectin-3 to differentiate C. albicans from and phagocytosis is mediated by appropriate phago-
non-pathogenicSaccharomycescerevisiae(Burgeoiset cyte receptors including complement receptor
al. 2010). 3(CR3). Oneof solublePRRsisrespiratorycollectin
All of mentioned pathogen recognition receptors –the pentraxin-3 present inthe respiratory portalsof
activate innate effector cells, although the response entry.ItopsonizetheinhaledconidiaofA.fumigatus,
induced by them may involve many different path- activatingthecomplementdeposition.Thefinalresult
ways. The result of PRRs stimulation by fungal of this process is the neutrophilic phagocytosis of
PAMPs also leads to the production of various conidia (LeibundGut-Landmann et al. 2012). Among
cytokines, including those triggering the adaptive im- the end-effect of complement activation is also the
munity and T cells differentiation or influencing the productionofchemotacticpeptidesincludingC5aand
antibody response (Burgeois et al. 2010, Brown 2011, the complex C5b67. The first one attracts the neu-
LeibundGut-Landmann et al. 2012). The occurrence trophils and eosinophils, while the second factor is
of various fungal PAMPs in chosen species of chemotactic for mentioned cells and also for macro-
pathogenicand opportunisticfungi andtheirrecogni- phagesand basophils(Tizard2004).Bothneutrophils
tion by different PRRs is shown in Table 1. and macrophages are the main phagocytic cells in-
cluded in the innate immune response for fungal in-
fections (Brown 2011).
The second step – neutralizationof pathogen: The complement activation is also strongly con-
complement activation,the inflammatory nected with acute inflammatory response, because
responseand the phagocyticcells some of its components degranulate mast cells and
influence the release of vasoactive molecules from
In order to protect the host, fungal detection and platelets(Tizard2004).Asitisknownoneoftheclini-
recognitionmust be followed by neutralization of the cal signs of dermatophytosis is strong inflammatory
invader. One of the most potent host-protecting sys- response observed as “the ring worm”. In all fungal
tems is the complement system able to neutralize infectionsinflammatoryresponseiscontrolledbyvari-
some pathogens. In uninfected hosts this system re- ous PRRs, which may either stimulate the inflamma-
mains inactive, but during the infection complement tion processes or initiate the anti-inflammatory re-
cascade is instantly activated to destroy the invading sponse. One result of activation of TLR2/TLR1 and
microorganisms. Complement components – enzy- TLR2/TLR6 heterodimers is the cytokine production
matic and receptor proteins are produced by hepa- e.g. pro-inflammatory tumor necrosis factor α
tocytesandmacrophages.Someofthemarestoredin (TNF-α) and Interleukin-12 (IL-12), that stimulates
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adaptiveresponseinvolvingTh-1cell.Thisreactionis formsoffungusandintheiruptakebydendriticcells.
required for clearance of some fungal infection DCs can internalize conidia of Aspergillus and Can-
(Blanco and Garcia 2008, Burgeois and and Kuchler dida yeasts by coiling mechanism, while hyphal forms
2012). Additionally, the same heterodimers may acti- of both fungi are phagocytosed by zipper-type mech-
vate slightly different signaling pathway, using other anism. Additionally, phagocytes and dendritic cells
intracellular adaptors or transcription factors and re- canusespecialstructuresbasedonactintointernalize
sultinginproductionofIL-10andTGF-β(Transform- thefungus. Macrophagesingest conidia of Aspergillus
ing growth factor-β) (Burgeois and and Kuchler by “ruffle-like” structure, while C. parapsilosis can be
2012). Both of mentioned cytokines are immunosup- internalized by DCs and macrophages with special
pressive. IL-10 suppresses the production of IL-2, structurescalled“fungipods”.Aftertheuptakeoffun-
IFN-γandTNF-βandinhibitsthesecretionofTNF-α, gal elements dendritic cells induce the adaptive im-
IL-1andIL-6.Italsoregulatesthefunctionofmacro- munity, because similarly to other APCs they process
phages and Th-1 cells and activity of B and NK cells. and present fungal antigens (Brown 2011).
TGF-βinfluencesthe differentiationofBandTcells, Killingof theinvading fungus may proceedeither
macrophagesand dendriticcells(Tizard2004).Other outsidethephagocyte,whenthefungalelementscan-
PRRs activate the synthesis of different cytokines, not be phagocytosed or within the phagolysosomein-
chemokinesand other factors promotingtheantigens sidethe phagocytic cell. The whole process of phago-
processingbyantigenpresentingcells(APCs),includ- some maturation and phagolysosome formation is
ing dendritic cells (DCs) (Bourgeois et al. 2010, controlled by some TLRs, opsonic and non-opsonic
LeibundGut-Landmann et al. 2012). Although TLRs receptors (Brown 2011). The major mechanism of
are essential for both innate and adaptive immunity, fungal destruction by phagocytes is the respiratory
activation of some of these receptors may be also (oxidative) burst, mediated by reactive oxygen inter-
harmfulforthehost,becausetheyenhancethehyper- mediates (ROI). The protein complex of phagocyte
production of pro-inflammatory cytokines in autoim- component NOX2 (gp91phox) forming NADPH
muneorchronicinflammatorydiseases(Zelanteetal. oxidaseisessentialinthisprocess. Itassemblesat the
2007). membraneof phagosome.Ifthe fungalparticleswere
Besides the detection and recognition of fungal not ingested, it is formed at the plasma membraneof
pathogenstheinnateimmunityisalsoinvolvedinneu- the phagocyte. NADPH oxidase produces the
tralizationofattackingfungi.Thefungusenteringthe superoxide, subsequently converted into toxic, reac-
hosts’ tissues activates the synthesis of chemotactic tive oxygen intermediates e.g. hydrogen peroxide or
factors able to attract the leukocytes and all other hydroxylradicals.Otherintermediatesarehypoiodous
effectorimmunecells.Amongchemotacticfactorsare and hypochlorous acids, produced by
complement activation products mentioned above, myeloperoxidase (MPO) of monocytes and neu-
then APs – antimicrobial peptides (described below) trophils.MacrophagesaredeficientinMPO,butthey
and leukotriens. All these factors attract the cells in- may acquire this enzyme through mannose receptors
volved in host defense mechanisms e.g. neutrophils, and transport it into phagolysosome (Brown 2011).
monocytes, macrophages, dendritic cells, natural Genetic deficiency of phagocytic NADPH oxidase
killercellsandT-γδcells.Aftertherecognitionoffun- (e.g. humans with Chronic Granulomatous Disease
gus by various PRRs phagocytic cells can ingest and –CGD)orthelossofMPOsignificantlyincreasesthe
destroy fungal elements. Main cells involved in the risk of severe mycoses including invasive aspergilosis
phagocytosis and killing are macrophages and neu- or candydosis (Brown 2011). Production of reactive
trophils, whereas dendritic cells internalize, process oxygen intermediates is activated by FcγR and by
and present fungal antigens (Brown 2011). In differ- multiple PRRs including TLRs, soluble galectin-3 or
ent infections various primary effector cells are ac- membranedectin-1 (Brown 2011).
tivated: neutrophils are involved in killing of C. albi- LikewiseROI, theantifungal effect havealsoRe-
cansandA.fumigatus,whilemacrophagesarethepri- activeNitrogenIntermediates(RNI),producedbyin-
mary phagocytic cells during Cryptococcus or Pneu- ducible nitric oxide synthase (iNOS, NOS2). This en-
mocystis infections (Blanco and Garcia 2008). zyme removes the amino group from L-arginine and
During the phagocytosis, the cellular membrane produces nitric oxide (NO), having a small antimic-
encloses the fungus in the intracellular phagosome. robialactivityofitsown.Furtherreactionofthismol-
Thisprocessdependsonactin,butisalsoconditioned ecule with superoxide leads to peroxynitrite, which is
by phagocyte type, the chemistry of fungal cell wall thepotentfungicide.TheroleofRNIisstilluncertain
and the presence of serum opsonins,which facilitates and many aspects of this process remain to be ex-
phagocytosis. Moreover, different mechanisms are plained. However, the data obtained from experi-
employed in phagocytosis of different morphological ments on iNOS-deficient mice suggest, that RNI are
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562 M.J. Biegańska
importantintheoutcomeofC.neoformansinfections. β- and θ-defensins. Alfa-defensins were discovered in
Atthesametime,micedeficientinbothiNOSandin some mammalian species like humans, mice, rhesus
NOX2 (gp91phox) show extreme susceptibility to macaquesandhorses,buttheywerenotfoundindogs
candydosis,but in vitro their phagocytes maintain the and cattle. They are produced in neutrophils and in
ability to kill Candida. It suggests that another mech- secretory,epithelialPanethcellsofthesmallintestine.
anism is involved in killing of pathogenic fungi in The protective role of four different α-defensins
phagocytes (Brown 2011). RED1, 2, 3 and RED4, secreted in the gastrointes-
Phagocytic cells have also other, non-oxidative tinal tract of the rhesus macaque has been observed.
antifungalmechanismstokillextracellularfungalpar- In horses thirty eight transcripts of intestinal h-de-
ticlesaswell as phagocytosed fungi. Amongthem are fensins have been found, and at least twenty of them
gelatinase-associatedlipocalinfoundinneutrophilsor may code functional peptides (Bruhn et al. 2009). In
specialhydrolases–serprocidins.Thisgroupofserine humans six α-defensins have been described as the
partofthesystemicinnatedefense:fourHumanNeu-
proteasesenclosescathepsinG,proteinase-3andelas-
trophil Peptides (HNP) and two Human Defensins
tase. All of them are considered the important anti-
–HD5 and HD6.They arecontributing to the innate
microbial factors. These enzymes have antifungal ac-
immunity of gastrointestinal mucosal surfaces (Cun-
tivity e.g. against Histoplasma, Candida and Aspergil-
liffe 2003). Human neutrophilic α-defensins
lus (Brown 2011). Other elements of phagocyte-me-
HNP1-HNP4 have antifungal effect against A.
diated antifungal immunity are Antimicrobial Pept-
fumigatus, C. albicans, C. neoformans and H. cap-
ides (AMPs) produced by phagocytes and epithelial
sulatum, but the mechanism of this fungicidal activity
cells,described below.
is not explained (Brown 2011). Well-studied mice
h-defensins are the cryptdins and cryptdins-related
entericpeptides.Twentythreedifferentpeptideshave
Humoralfactorsof the innate antimicrobial
been described in the small intestine, cecum, colon
and antifungalimmunity
and rectum of these animals. It is presumed, that
these defensins regulate local bacterial colonization
Asitwasmentionedthefirstbarriersforfungiare
and play an important role in the protection against
skinand mucous membranes.Theirroleasthe physi-
theinfectionof givenGIsections(Bruhnetal.2009).
cal obstacle for pathogens was described above, but
In guinea pigs defensins called guinea pig neutrophil
there are some additional factors enhancing the first
peptides (GPNPs) have revealed fungicidal effect
line of immunity: the antimicrobial peptides (APs;
against yeasts – C. parapsilosis and C. neoformans
AMPs). APs are gathered in these sites, where the
(Selsted and Harwig 1987). Defensins obtained from
microbial invaders encounter preferably and in the
hamsters’ neutrophils HaNP-1, 2, 3 and HaNP-4
natural “doorways” like the gastrointestinal tract or
(Hamster Neutrophil Peptides) have various antimic-
the airways. Various mammalian cells e.g. epithelial
robial activity depending on the peptide and its
cellsofmucosaltissues,leukocytes,circulatingphago-
amount (Mak et al. 1996).
cytesandothercellscanproducesomeAMPs,varying Beta-defensins being a part of humoral innate
with chemicalstructures,antimicrobialpotentialsand mechanismshavebeenfound inmucus, in severaltis-
effects on the host cells (Marshal and Arenas 2003, suesofdifferent mammalsandalsointhe udder.It is
Brandenburg et al. 2012). Antimicrobial peptides are believed, that they are involved in maintaining the
present in cytoplasmic granules inside some cells like natural biota associated with multiple, different
neutrophils and lymphocytes, including NK-cells and niches such as skin, the airways, the GI tract starting
cytotoxic T-cells (Tizard 2004). from the oral cavity and genitourinary tract (Wein-
In mammals AMPs were found in humans, pigs, bergetal.1998,Bruhnetal.2009,Brandenburgetal.
cattle, horses (Brown 2011, Brandenburg et al. 2012) 2012).Invivo β-defensinsshowanti-microbialactivity
and in rhesus macaques, mice, rats, hamsters, guinea againstfungi e.g. C. albicans, some envelopedviruses
pigs,rabbits.GenesencodingAMPs were alsoidenti- andG+andG-bacteria(Bruhnetal.2009,Branden-
fied in elephant, opossum and hedgehogs (Bruhn et burg et al. 2012).
al. 2009). Basing on their structural differences APs Theta-defensins were isolated from leukocytes
were divided into three families: defensins, and from the bone marrow of rhesus macaques. In
cathelicidinsandhistatins.Defensinsarecationic,cys- non-human Primatesthey display antifungal, antibac-
teine-rich antimicrobial peptides observed in plants, terialandantiviralactivity.InhumansmRNAsencod-
invertebrates and vertebrates. Six cysteine residues, ingtwoθ-defensinsareexpressedinthebonemarrow,
formingdisulfidebondsarecharacteristicelementsof but functional θ-defensins are missing (Selsted 2004).
their chemical structure (Bruhn et al. 2009). Mam- It is presumed that it may influence the humans’ sus-
maliandefensinswereclassifiedinthreefamilies:aα-, ceptibility to HIV infections (Nguyen et al. 2003).
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Two fundamentals of mammalian defense in fungal infections:... 563
Table2.Theoccurrenceofvariousantimicrobialpeptides(APs)andtheiractivitiesindifferentmammals.
Characteristic
APfamily/sub-family Mamalianspecies Antimicrobialactivity/otheractivity
elementofstructure
cryptdinsand regulationoflocalbacterial
cryptdins-related mice colonization;protectionagainstthe
entericpeptides infectionofselectedgastrointestinal
(23peptides) tract(GI)sections
HumanNeutrophil
Peptides–HNP humans inductionofchemokinesynthesis;
(4peptides)
HumanDefensins
α-defensins –HD5andHD6 6cysteineresidues, humans inductionofchemokinesynthesis
4α-defensins: formingdisulfide
bonds,position
RED1,RED2, rhesusmacaques protectionofgastrointestinaltract(GI);
ofthecysteinsvary
Defensins RED3andRED4
insub-families
DEFAIand horses probableprotectionofintestines;
~20otherpeptides antimicrobialactivity
rats, 1GPNPs–microbicidalforselected
rabbits, bacteria,antifungalagainstC.albicans
guineapigs1, andC.neoformans
hamsters2 2HaNP-1,2,3,4–antibacterialactivity
dependingonpeptideandits
concentration
anti-microbialactivityagainstfungie.g.
C.albicans,G+andG-bacteriaand
β-defensins variousspecies someenvelopedviruses;maintainingthe
naturalbiotaofdifferentniches(e.g.
skin,oralcavityandairways,the
intestines,genito-urinarytract)
θ-defensins rhesusmacaques, antifungal,antibacterialandantiviral
primates
Cathelicidins N-terminalcathelin humans,rodents, activityagainstparasites,bacteria
domain horses,pigsand orfungi;
cattle. cytotoxiceffectoneukaryoticcells;
inductionofchemokinesynthesis
Histatins cationicpeptides, humans,primates maintainingoforalhealth;
richinhistidine antibacterialandantifungalactivity
(e.g.Histatin5activityagainst
C.albicans,C.neoformans
andA.fumigatus)
The second group of AMPs are cathelicidins se- Cryptococcus and Candida, but it has no effect on
cretedbyleukocytes.Theyhavebeenidentifiedinhu- filamentousfungi.Bothcathelicidinsanddefensinsin-
mans, rodents, horses, pigs and cattle. Their express- ducechemokinesynthesis(Brown2011, Brandenburg
ion/presenceisnotrestrictedonlytolymphoidorgans, et al. 2012).
becausetheyhavebeenalsofoundintheskin,intesti- Histatins, the third group of mammalian APs, re-
nal epithelium and in the brain. Cytotoxic effect on vealantibacterialandantifungalactivityandmaintain
eukaryoticcellsandactivityagainstparasites,bacteria oral health. These peptides are secreted by the
or fungi has been demonstrated for cathelicidins. In parotid,sublingualandsubmandibularsalivaryglands.
humansthereis the only onecathelicidin–hCAP-18. Thestrongactivityofhistatin5againstC.albicans,C.
Ithasbeenfoundinmonocytes,lymphocytesandNK neoformans and A. fumigatus has been already
cells, in neutrophil granules and in epithelial cells. proven. Antifungal activity of histatins encloses dis-
ThehCAP-18hasbeenalsodetectedinplasmaandin ruption of outer cellular membrane followed by cell
the secretions such as seminal fluid or sweat. This lysis, dysregulation of the cell wall synthesis and de-
cathelicidin is able to disrupt cellular membranes of polymerysation of actin cytoskeleton (Brandenburg
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564 M.J. Biegańska
et al. 2012). Table 2 shows the occurrence of mam- parison of mammalian temperature with thermal
malianAPsandtheirknownactivityinchosenanimal growth tolerances of chosen ascomycetous and
species. basidiomycetousspecies,examinedforgrowthintem-
Epithelial antimicrobial peptides, called small peratures ranging from +4oC to +45oC. Statistical
Cationic Antimicrobial Peptides (CAPs) have been analysis revealed that most of the investigated fungi
found in various mammals, birds and other animals can grow at temperature range 12-30oC, but their
– insects and amphibians. They have strong cationic thermal tolerance decreases rapidly if the tempera-
charge, because of multiple arginine and lysine resi- tures exceeds 35oC. Moreover, the authors noticed
dues. Among them are CAPs found in pigs – cec- thatevery1oCincreaseofthecultivationtemperature,
ropins and protegrins (Weinberget al. 1998). excluded the growth of further ~6% of strains. Addi-
Besidesantimicrobialactivityimportantfeatureof tionally,theproportionofstrainsgrowingat25oCwas
AMPsistheirinfluenceontheimmunesystem.These higher among the isolates from living hosts, than
amongthoserecoveredfromsoil.At37oCthepropor-
peptides are chemotactic factors for immune and
tionofgrowingstrainswassignificantlyhigherforiso-
non-immunecells(e.g.macrophages,neutrophils,de-
lates from endothermic animals (Robert and
ndritic cells, T-cells). They act like adjuvants in the
Casadevall 2009). The authors have suggested, that
inflammatory response. Also it has been proven, that
temperaturesinthe30-40oCrangegenerate“thether-
AMPscanmodifytheexpressionofpro-inflammatory
cytokinessuchasTNF-αorIL-1βandanti-inflamma- mal exclusionary zone”, which can protect mammals
against many environmental pathogens. According to
tory cytokines like IL-10, at the same time. It indi-
presentedopinion,theinabilityofmostfungalspecies
cates, that APs maintain the balance between the in-
togrowattherangeofmammalianbodytemperature,
duction of inflammation and the protection of host
reflects the relative paucity of fungal diseases in the
from harmful effects of the excessive inflammatory
immunocompetent mammals. Moreover, fever occur-
response (Brandenburg et al. 2012). During infection
ring as the systemic reaction during infection, adds
their activity intensify innate and adaptive responses.
further 1-3oC to the normal body temperature, what
Theirimpact on the host defense involvesthe enhan-
canalsoreducetheproportionoffungiinhabitingand
cementofphagocytosis.Also,AMPsplayaroleinthe
surviving in the host (Robert and Casadevall 2009,
recruitment and maturation of antigen presenting
Bergmanand Casadevall 2010).
cellse.g.dendriticcells(Brandenburgetal.2012).All
More data confirming mentioned hypothesis can
collected data demonstrate, that antimicrobial pept-
be found in the literature. Normal body temperature
idesaresignificantelementsofhost-pathogeninterac-
of rabbits is 39-40oC. In these animals the systemic
tions and may determine their outcome.
infectionwithC.neoformansisdifficulttoobtaineven
bydirectinjectionoffungus.Probablytheirhighbody
temperaturemakesthemrelativelyresistanttothein-
Endothermy
fection.However,the diseasemay be induced experi-
mentally under two conditions: firstly, the fungus has
Besides the complexity of their immunity, the tobe inoculateddirectlytothetestes,wherethe tem-
mammalian resistance to pathogenic fungi is strongly peratureislowerandsecondly,theanimals’immunity
connected with the endothermy of these animals should be suppressed e.g. with administration of cor-
(Robert and Casadeval 2009, Bergman and ticosteroids (Casadevall2005). The influence of body
Casadevall2010).Thisisanevolutionaryachievement temperaturemayalsobeobservedinWhiteNoseSyn-
of warm-blooded mammals and birds. Despite all drome (WNS) of bats, caused by Geomyces destruc-
metabolic costs elevated rates of resting metabolism tans, a psychrophilic fungus, colonizing the skin. The
determine high and stable body temperature and en- disease occurs in different species, but exclusively in
sure high, prolonged activity of these animals. Also, hibernating animals, when their body temperature is
endothermic animals are independent of ambient thelowest(Casadevall2005,Blehertetal.2009).The
temperature and capable of inhabiting various envi- recoveryfromWNSispossiblewhenbatsareawoken
ronments, including habitats with low or highly vari- from hibernation, properly treated and kept in the
abletemperatures(Hilleniusand Ruben2004).Addi- temperature range 18-23oC (Meteyer et al. 2011,
tionally, it is suggested that these physiological adap- Casadevall2012).Also,itwasobserved,thatraisingof
tations were selected during evolution, because they the temperature in terrarium to the 37oC, along with
had provided better protection against many patho- the body temperature of frogs infected by
gens (Casadevall 2005). Data confirming this were Batarachochytrium dendrobatidis–enables the cure
presented in some experiments, done by Casadevall’s (Casadevall 2005). In general, it can be stated, that
groups (Robert and Casadevall 2009, Bergman and the increase of temperature may influence the posi-
Casadevall 2010). The tests were based on the com- tive outcome of the fungal disease.
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