Medical Mycology, 2015, 00, 1–10doi: 10.1093/mmy/myv021

Advance Access Publication Date: 0 2015Original Article

Original Article

Candida parapsilosis complex induces local

inflammatory cytokines in immunocompetent

mice

Rogelio de J. Trevino-Rangel1, Gloria M. Gonzalez1, Azalia M.

Martınez-Castilla2, Jaime Garcıa-Juarez3, Efren R. Robledo-Leal4,

Jose G. Gonzalez5 and Adrian G. Rosas-Taraco2,∗

1Departamento de Microbiologıa, Facultad de Medicina, Universidad Autonoma de Nuevo Leon. NuevoLeon, Mexico, 2Departamento de Inmunologıa, Facultad de Medicina, Universidad Autonoma de NuevoLeon. Nuevo Leon, Mexico, 3Departamento de Histologıa, Facultad de Medicina, Universidad Autonomade Nuevo Leon. Nuevo Leon, Mexico, 4Facultad de Ciencias Biologicas, Universidad Autonoma de NuevoLeon. Nuevo Leon, Mexico and 5Hospital Universitario, Universidad Autonoma de Nuevo Leon. NuevoLeon, Mexico

*To whom correspondence should be addressed. Adrian G. Rosas-Taraco, Departamento de Inmunologıa, Facultad deMedicina, Universidad Autonoma de Nuevo Leon, Ave. Gonzalitos 235 Nte., Mitras Centro, 64460, Monterrey, Nuevo Leon,Mexico. Tel: +52 (81) 83294211, Fax: +52 (81) 83331058; E-mail: adrian.rosastr@uanl.edu.mx

Received 6 November 2014; Revised 12 January 2015; Accepted 22 February 2015

Abstract

Despite the increasing incidence of the Candida parapsilosis complex in the clinical set-ting and high mortality rates associated with disseminated infection, the host-fungusinteractions regarding Candida parapsilosis sensu stricto and the closely related speciesC. orthopsilosis and C. metapsilosis remains blurred. In this study, we analyzed inflam-matory cytokines levels and histopathology as well as fungal burden in spleen, kidneyand lung of mice infected with six strains of the “psilosis” group with different enzymaticprofiles. Strong interleukin 22 (IL-22) and tumor necrosis factor α (TNF-α) responses wereobserved in analyzed organs from infected mice (P < .0001) regardless of the species andenzymatic profile. TNF-α and IL-22 levels were related with spleen inflammation and fun-gal load. Fungal cells were detected only in spleen and kidney of infected mice, especiallyby day 2 post-challenge. The kidney showed glomerular retraction and partial destructionof renal tubules. Our data suggest that a strong inflammatory response, mainly of IL-22and TNF-α, could be involved in Candida parapsilosis complex infection control.

Key words: Candida parapsilosis complex, cytokines response, IL-22, TNF-α, IFN-γ , IL-17A.

Introduction

Candida parapsilosis sensu stricto has been recognized asone of the most common Candida species that originatecandidemia [1,2]. Along with Candida orthopsilosis and

Candida metapsilosis, these three phenotypically indistin-guishable cryptic yeasts belong to the so-called psilosisgroup [3]. The administration of parenteral hyperalimenta-tion solutions, as well as the use of intravascular devices and

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Medical Mycology Advance Access published April 23, 2015

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Table 1. Enzymatic activities of the strains used to infect BALB/c mice though tail injection of 1.5 × 107 CFU/mouse inocula.

Enzymatic activitya

Strain Clinical origin Aspartyl proteinase Phospholipase Esterase Hemolysin

Candida parapsilosisc/c 105 Peritoneal fluid − +++ ++++ +++H-124 Blood − − − −Candida orthopsilosisHP-179 Blood − ++++ ++++ +++H-152 Blood +++ − − −Candida metapsilosisM-18 Skin ++++ +++ − +++ATCC 96144 Skin − ++ − −aSemiquantitative estimation according to the Pz index (colony diameter/total diameter of the colony plus precipitation or halo zone). Very strong (++++), strong(+++), mild (++), weak (+), and negative (−) enzymatic activity.

prosthetic materials represent important risk factors thatcould lead to infection by these opportunistic pathogens insusceptible hosts [2,4].

The incidence of C. parapsilosis species complex hasunacceptably increased in recent decades, a fact that hasmotivated many studies regarding their epidemiology [4,5],antifungal susceptibility [6], virulence [7], and pathogen-esis [8], even in nonmurine novel infection models [9].Otherwise, the model organism Candida albicans has ledsome progress in the field of host-fungus interactions. Inthis sense, the immune response elicited by C. albicans in-fection is driven by inflammatory mediators, particularlyinflammasome-derived interleukin 1β (IL-1β), and is char-acterized by the production of interferon γ (IFN-γ ) fromTh1 cells and interleukin 17A (IL-17A) from Th17 cells[10,11]. In disseminated C. albicans infection, Th1 cells areassociated with protection from disease, while a predomi-nance of Th2 cells promotes susceptibility [11]. However, acomprehensive tissue-specific analysis of the cytokine levelsparticipating in the anti-Candida host defense to dissemi-nated disease is lacking and there is a large gap in the currentknowledge of the immunity against infection by C. parap-silosis sensu stricto, C. orthopsilosis and C. metapsilosis.

Recent evidence suggests that the three species of the“psilosis” group possess a similar pathogenic potential indisseminated candidiasis regardless of their particular invitro enzymatic profiles [8]. The aim of this study was toanalyze the levels of inflammatory cytokines in three or-gans (spleen, kidney, and lung) in immunocompetent miceinfected with six strains of the psilosis group with differentenzymatic profiles.

Materials and methods

Ethics statement

Murine experiments were performed with the approvalof the Ethics and Research Committee of the School of

Medicine of the Universidad Autonoma de Nuevo Leon(registration code: MB12-002). The animal sacrifices werecarried out by cervical dislocation, and all efforts were madeto minimize suffering. The experimental protocol was de-signed in conformity with the International Review Boardregulations, following the recommendations of the Guide-lines for the Care and Use of Laboratory Animals, and inagreement with Good Laboratory Practices. Care, main-tenance, and handling of the animals were in accordancewith the Mexican regulations for animal experimentation(NOM-062-ZOO-1999).

Mice

Male BALB/c mice, 5 weeks old (22–24 g weight) werepurchased from Harlan (Harlan Mexico, S.A. de C.V.,Mexico). A total of 132 animals were used; these werehoused in ventilated cages of five mice each under spe-cific pathogen-free conditions at the Animal Facility of theDepartment of Microbiology. All mice were given sterilewater and Purina rodent food ad libitum and were moni-tored daily for 15 days. The day/night cycle was 12 h/12h. Before use, the animals were allowed to acclimatize for5 days.

Fungal strains

The strains used in this study were previously utilized inour laboratory [8]: C. parapsilosis sensu stricto (c/c 105and H-124), C. orthopsilosis (HP-179 and H-152), andC. metapsilosis (MEX-18 and ATCC-96144). Details re-garding the enzymatic activities of the strains are shown inTable 1. The strains were stored as suspensions in steriledistilled water at room temperature and cultured for 48 hon Sabouraud-dextrose agar (SDA) slants (Difco, Detroit,MI, USA) at 37◦C before use.

Trevino-Rangel et al. 3

Experimental infection

For inocula preparation, the six strains were passaged atleast twice on SDA plates to check purity and viability of thecultures. After 48 h of incubation at 37◦C yeast cells wereharvested, washed twice in sterile saline, quantified with ahemocytometer, and adjusted to the desired concentration.To corroborate the yeast cell counts, serial fold dilutionswere cultured on SDA at 37◦C for 48 h. Systemic candidiasiswas induced in mice by injecting an inoculum of 1.5 × 107

CFU/mouse through the lateral tail vein in 200 μl of ayeast suspension in groups of 20 animals per strain. Threeuninfected mice to which sterile saline were intravenouslyadministered were used as controls per experimental day.No immunosuppressive scheme was used.

Kidneys are the target organ of murine disseminated can-didiasis and therefore one of the most studied tissues invarious works [12–14], so we considered analyzing bothfungal tissue burden and inflammatory cytokine levels lo-cally expressed in this organ and in the spleen and lungs.The fungal tissue burden determinations were previouslypublished [8]. Briefly, five mice per strain were humanelykilled at days 2, 5, 10, and 15 post-challenge. After sac-rifice, the kidneys, spleen, and lungs of each mouse wereswiftly aseptically removed, weighed, and placed in 2.5 mlof sterile 10 mM phosphate-buffered saline (PBS), pH 7.2to 7.4. The organs were homogenized in a tissue grinder(Polytron-Aggregate, Kinematica) and serially diluted 1:10in sterile saline. Aliquots of 0.1 ml of the undiluted and di-luted homogenates were then plated twice onto SDA plates,and colony counts were performed after 48 h of incubationat 37◦C. At par, splenic inflammation was monitored ateach time point indicated during the course of the studyby measuring the length, width, and height of the spleenswith a sterile Vernier scale and applying the formula forthe volume of an ellipse. The entire in vivo experiment wasperformed twice.

Cytokine assays

Four mouse cytokines were assayed in supernatants fromtissue homogenates. The cytokines measured were: TNF-α,IFN-γ , IL-17A, and IL-22. Determinations were performedby sandwich enzyme-linked immunosorbent assay (ELISA)according to the manufacturer’s protocol for TNF-α,IFN-γ (PeproTech, Mexico City, Mexico), IL-17A, and IL-22 (eBioscience, San Diego, CA, USA), using the iMarkmicroplate reader (Bio-Rad Inc., Hercules, CA, USA). Thequantitation ranges were 32–2000 pg/ml, 16–2000 pg/ml,4–500 pg/ml, and 8–1000 pg/ml for TNF-α, IFN-γ , IL-17A, and IL-22, respectively. Each sample was analyzedtwice.

Histology

For histopathological analysis, half of each organ was fixedwith 10% buffered formalin. Samples were dehydrated,paraffin embedded, and sliced into 5-μm sections, whichwere then stained with periodic acid-Schiff (PAS) and ex-amined in a blinded fashion by light microscopy.

Statistical analysis

The Kruskal-Wallis and Dunn’s multiple comparison testswere applied to verify statistical significance among exper-imental groups. Pearson’s correlations between splenic in-flammation and fungal load with local cytokine levels wereadditionally made. Calculations and graphics were per-formed using GraphPad Prism version 5.03 for Windows(GraphPad Software, Inc., La Jolla, CA, USA). P values≤ .05 were considered significant.

Results

Inflammatory cytokines were detected since earlystage of infection

We studied whether C. parapsilosis complex strains withdifferent enzymatic profiles induce a specific cytokine pro-file in kidney, spleen, and lung. All C. parapsilosis com-plex strains induced TNF-α and IL-22 production in in-fected mice (P < .0001). The concentration of TNF-α washigher compared with other analyzed cytokines in mice in-fected with C. parapsilosis complex. TNF-α levels in kid-ney were > 5–10 times fold compared with lung or spleen(Fig. 1). In kidney, C. metapsilosis ATCC 96144 strainwas a high inducer of TNF-α, while C. metapsilosis M-18 induced low TNF-α levels at 2–10 days post-infection(Fig. 1A). In lung, C. parapsilosis sensu stricto c/c 105,C. orthopsilosis HP-179, and C. metapsilosis M-18 werethe most potent inducers of TNF-α at 10 and 15 dayspost-infection (Fig. 1B). A nonspecific pattern was foundin spleen between cytokine levels and C. parapsilosis com-plex strains (Fig. 1C). Similar to TNF-α, IL-22 levels werehigher in kidney (>10 fold) compared with lung and spleen(Fig. 2). In kidney, C. parapsilosis sensu stricto H-124, C.orthopsilosis H-152, and C. metapsilosis ATCC-96144 in-duced high levels of IL-22 in early infection (Fig. 2A). Inlung, C. parapsilosis sensu stricto c/c 105 and C. metap-silosis M-18 induced high levels of IL-22 at 2 and 5 dayspost-infection. However, in the next post-infection periods,this cytokine was elevated in all infected mice regardless ofthe species and enzymatic profile (Fig. 2B). In spleen, C.metapsilosis strains were the most potent inducers of IL-22at 5 and 10 days post-challenge (Fig. 2C). C. parapsilo-sis complex strains downregulated IFN-γ levels in kidney

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Figure 1. Inflammatory cytokines were detected since early stage of infection. Graphs show TNF-α locally expressed in kidney (A), lung (B), andspleen (C). The data represent the means of five mice per group ± SD. Asterisks represent significant differences compared to controls. TNF-α levelsin kidney were found > 5–10-fold compared with lung or spleen.

at 2, 5 and 10 days post-infection. In lung, C. parapsilosissensu stricto c/c 105 and H124 strains were the most potentinducers of IFN-γ at 2 and 5 days post-infection, respec-tively; however, in the next days it was reduced (Fig. 3B).IL-17A production was downregulated in the kidneys andlungs of mice infected with C. parapsilosis complex strainsat 2, 5, and 10 days, respectively (Fig. 4A–B). In spleen, C.parapsilosis sensu stricto H124 strain induced high levelsof IL-17A at 5 and 10 days post-infection (Fig. 4C).

Spleen inflammation is paralleled with localTNF-α and IL-22 levels

The splenomegaly was measured throughout the infectionwith a vernier, and the inflammation score was calcu-lated using the ellipsoid equation. Mice infected with C.parapsilosis complex strains exhibited spleen inflamma-tion, and this was in accord with TNF-α and IL-22 levels,

especially with C. parapsilosis sensu stricto and C. metap-silosis strains.

TNF-α and IL-22 could control fungal load

TNF-α and IL-22 are essential cytokines in the control offungal infections. TNF-α induces oxidative and nitrosativedistress in the phagolysosome microenvironment and con-tribute to the inflammatory process [15]. On the other hand,IL-22 limits fungal growth due to induction of antimicrobialpeptides [16]. Thus, we questioned if the levels of TNF-αand IL-22 are related with the decrease in fungal load. Theseanalyses demonstrated that both cytokines increase in kid-ney from mice infected with most C. parapsilosis complexstrains, showing > 1 log reduction in fungal load. C. orthop-silosis H-152 (a biofilm former strain) was the lowest af-fected of the studied strains in the kidney, showing < 0.5 logreduction. In spleen and lung, fungal load was reduced > 2log in most C. parapsilosis complex strains, where high

Trevino-Rangel et al. 5

Figure 2. Inflammatory cytokines were detected since early stage of infection. Graphs show IL-22 locally expressed in kidney (A), lung (B), and spleen(C). The data represent the means of five mice per group ± SD. Asterisks represent significant differences compared to controls. IL-22 levels werehigher in kidney (>10 fold) compared with lung and spleen.

levels of TNF-α and IL-22 were presented (data not shown);however, when a correlation was analyzed it was not sig-nificant.

The kidney is the most affected organ duringC. parapsilosis complex infection

During the course of the infections, histological alterationsin spleen and lung were not found compared with controlmice. However, in the spleen, the presence of isolated yeastsnear the cells of the Billroth cords and in the red pulp sinu-soids were evident, especially at day 2 post-challenge. Therewas no evidence of fungal structures in lungs of infectedmice. On the other hand, abundant yeast cells at day 2 weredetected in the kidney after systemic infection, mainly in therenal intersitium, and these tended to gradually decrease byday 15. In addition, the presence of acute inflammatory in-filtrate foci surrounding yeasts in kidney was evident at day

5 and 10 post-challenge (Table 2). Unlike the spleen andlung, the kidney of infected mice showed some histologicalalterations, glomerular retraction, and destruction of renaltubules, which were the principal findings compared withuninfected controls, and these were detected since day 2post-challenge (Fig. 5 and Table 2). Importantly, a particu-lar histopathological pattern associated to a specific speciesor enzymatic profile among the analyzed strains was notfound.

Discussion

Diverse animal models of infection have been developedto investigate the different clinical forms of candidiasis,being murine models the gold standard to study patho-genesis as well as efficacy of antifungal agents alone orin multiple combinations [9]. The murine intravenous(IV) challenge model mimics human bloodstream-derived

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Figure 3. Inflammatory cytokines were detected since early stage of infection. Graphs show IFN-γ locally expressed in kidney (A), lung (B), andspleen (C). The data represent the means of five mice per group ± SD. Asterisks represent significant differences compared to controls. IFN-γ wasdown-regulated in lung and spleen with respect to kidney.

candidiasis, and due to its highly reproducibility it re-mains as the most common long-standing model used toinvestigate C. albicans virulence and to determine partic-ular aspects of host-fungus interactions [17–19]. Duringthe systemic infection, the bloodstream and the majority oforgans (spleen, lungs, and liver) are gradually cleared of thepathogen, but this scenario is quite different in the kidney,the principal target organ for IV challenge in the mouse, inwhich the increase of fungal burden is accompanied by in-creasing levels of renal cytokines and chemokines [13,14],with an existing correlation between the increased renal cy-tokine levels and the lesion severity with the consequentinfection outcome [14,19]. Yet, the cellular and molecularfactors that determine differential organ-specific control ofCandida remain largely unknown.

During pathogenic C. albicans infection, the inflamma-tory mediators of the immune response are driven by Th1and the recently implicated involvement of Th17 cells. Thestimulated Th1 lymphocytes produce IFN-γ , which con-tributes to anti-Candida host defense by inducing nitricoxide (NO) production by macrophages [20]. Moreover,Th17 cells are characterized by the production of IL-17A,IL-17F, IL-21, and IL-22 [21]. IL-17A interconnect lym-phoid and myeloid host defense [22] through the infiltra-tion induction of neutrophilic granulocytes at the site ofinfection and activation of macrophages [21]. Meanwhile,

IL-22 is a member of the IL-10 family of cytokines, whichhas been confounded by data suggesting both pro- and anti-inflammatory functions. However, recent evidence suggeststhat this cytokine accounts for innate resistance to candidi-asis at the early stages of infection in the kidney and gutby critically controlling initial fungal growth and epithe-lial homeostasis in the relative absence of Th1 immunity[23]. Interestingly, current findings suggests that IL-22 andTNF-α represent a potent synergistic cytokine combinationfor skin immunity, efficiently conserving epidermal barrierintegrity in a skin infection model compared with IFN-γ ,IL-17, IL-22, or TNF-α alone [24]. In disseminated C. al-bicans infection, Th1 cells are associated with protectionfrom disease, while a predominance of Th2 cells promotessusceptibility [11]. Finally, the balance in innate responsesbetween fungicidal (beneficial) immunity and immunoregu-latory (detrimental) compensative mechanisms determinesthe scope of tissue damage in fungal infections [16].

The tissue specificity of mouse cytokine host responsesin hematogenously C. albicans infection was previouslydemonstrated by Spellberg et al. [25] They examined therelationship between host survival and local immune re-sponses in kidney and spleen during candidiasis with dif-ferent inocula, concluding that this pathogen induced type2 splenocyte responses with both fatal and nonfatal inoc-ula. Conversely, the nature of immune polarization in the

Trevino-Rangel et al. 7

Figure 4. Inflammatory cytokines were detected since early stage of infection. Graphs show IL-17A locally expressed in kidney (A), lung (B), andspleen (C). The data represent the means of five mice per group ± SD. Asterisks represent significant differences compared to controls. IL-17A wasdown-regulated in kidneys and lungs at days 2, 5, and 10 post-challenge.

kidney correlated with host survival, with IFN-γ -dominanttype 1 responses causing no or low mortality and type 2or IL-10-dominant responses during 100% fatal infection.Subsequently, MacCallum et al. [14] analyzed cytokineand chemokine levels in infected organs elucidating organ-specific responses, with high cytokine and chemokine levelsin infected kidneys but reduced responses in the spleen.They concluded that keratinocyte-derived chemokine is animportant early mediator of overall outcome and correlatealong with IL-6 and MIP-1β with lesion severity, whereasGM-CSF and IL-10 showed inverse correlations with his-tological damage. These differences were also reflected atthe transcriptional level, with differential expression of cy-tokine genes in both organs [26]. Since our interest wasfocused in local responses to infection, the experimental de-sign we adopted did not include measurement of serum cy-tokine levels. Of the assayed organs in this study, the kidneyshowed the highest fungal load, as well as pro-inflammatory

cytokine levels, and it was the only tissue that showed his-tological alterations due to the systemic infection (Fig. 7).Recently, Lionakis et al. [27] characterized the immune cellpopulations in infected organs during the progression ofdisseminated candidiasis in a mouse model. They foundneutrophils accumulated in all infected organs, but a de-lay in their appearance in the kidneys, leaving these organsunprotected during the first 24 h post-challenge. Furtherincreases in neutrophils occurred in these organs as diseaseprogressed. The kidney inflammation could be explainedin part by the CCR1 neutrophils, which play a pathogenicrole in invasive candidiasis mediating renal immunopathol-ogy via excessive neutrophil recruitment from the bloodinto the kidney [12]. The role of IL-22 in neutrophil re-cruitment in peripheral tissue has been already reported inmurine cytomegalovirus [28]. We found downregulation ofIFN-γ production due to C. parapsilosis complex infection,agreeing with Carvalho et al., who also reported this finding

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Figure 5. Representative histopathological periodic acid-Schiff (PAS)-stained tissue sections. A, C, and E represent uninfected controls of kidney,spleen and lung, respectively. On the other hand, B, D, and F correspond to randomly selected sections of infected kidney, spleen, and lung,respectively by day 5 post-challenge. In B it was depicted PAS positive yeast cells (arrows) surrounded by inflammatory infiltrate focus, as well aspresence of yeasts in the convoluted tubule (arrowhead). In D it was observed yeasts between the cells of splenic cords (arrow). Original magnification×400, metric bar 20 μm.

in some patients with recurrent vaginal candidiasis (RVC)[29]; this could probably be coupled to the absence of IL-6in response to the fungus on epithelial surfaces [30]. Onthe other hand, high levels of IL-22 found in kidney couldbe involved in neutrophil recruitment in this tissue. In lungand spleen were not observed neutrophil indeed IL-22 in-crement during C. parapsilosis complex infection, it may beto absent or low number of yeasts in these tissues. Finally,a significant correlation between splenic inflammation andfungal load with local cytokine levels was not found.

Overall, this study demonstrates that male BALB/c miceexperimentally infected with strains of the “psilosis” groupexhibiting different in vitro enzymatic profiles irrespec-tively induce a pronounced inflammatory response thatis essential for protective antifungal immunity and drivepathology during disseminated infection, principally in thekidney. Although a correlation between cytokine levels

and enzymatic activity of the strains was not found, therole of these enzymes in systemic disease caused by theC. parapsilosis sensu lato group should not be underes-timated. In this regard, further studies with more char-acterized strains, as well as homozygous mutants for the

Table 2. Histopathological findings in kidney of mice infected

with six strains of Candida parasilopsis complex.

Day post-infection (dpi)

Finding Control 2 5 10 15

Glomerular retraction − −+ + ++ ++Neutrophil infiltration − −+ ++ +++ +Destruction of renal tubules − −+ + ++ +

Note: Values: − Absent, −+ Variable, + Present, ++ Moderate, +++ Abun-dant.

Trevino-Rangel et al. 9

genes involved in each enzyme, become essential in order toelucidate their particular contribution to the pathogenesisby these opportunistic pathogens. In general, this knowl-edge have implications in the development of novel im-munotherapy strategies, which aims improve host defenseagainst C. parapsilosis complex, providing a solution toprevent and reduce the high mortality rates of the invasivecandidiasis.

Acknowledgments

We thank Sergio Lozano-Rodriguez, M.D. of the “Dr. Jose EleuterioGonzalez” University Hospital (Monterrey, Mexico), for his reviewof the manuscript prior to submission.

Declaration of interest

The authors report no conflicts of interest. The authors alone areresponsible for the content and writing of the paper.

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