Candida parapsilosis complex induces local inflammatory cytokines in immunocompetent mice

Medical Mycology Advance Access published April 23, 2015 Medical Mycology, 2015, 00, 1–10 doi: 10.1093/mmy/myv021 Advance Access Publication Date: 0 2015 Original Article

Original Article

Candida parapsilosis complex induces local inflammatory cytokines in immunocompetent mice 1 ˜ ´ 1 , Azalia M. Rogelio de J. Trevino-Rangel , Gloria M. Gonzalez 3 ´ ´ R. Robledo-Leal4 , , Efren Mart´ınez-Castilla2 , Jaime Garc´ıa-Juarez 5 ´ and Adrian G. Rosas-Taraco2,∗ Jose´ G. Gonzalez 1

´ ´ Nuevo Departamento de Microbiolog´ıa, Facultad de Medicina, Universidad Autonoma de Nuevo Leon. ´ ´ Mexico, 2 Departamento de Inmunolog´ıa, Facultad de Medicina, Universidad Autonoma de Nuevo Leon, ´ ´ Nuevo Leon, ´ Mexico, 3 Departamento de Histolog´ıa, Facultad de Medicina, Universidad Autonoma Leon. ´ ´ ´ Nuevo Leon, ´ Mexico, 4 Facultad de Ciencias Biologicas, Universidad Autonoma de Nuevo de Nuevo Leon. ´ ´ Nuevo ´ Nuevo Leon, ´ Mexico ´ de Nuevo Leon. Leon. and 5 Hospital Universitario, Universidad Autonoma ´ Mexico Leon,

*To whom correspondence should be addressed. Adrian G. Rosas-Taraco, Departamento de Inmunolog´ıa, Facultad de ´ ´ Ave. Gonzalitos 235 Nte., Mitras Centro, 64460, Monterrey, Nuevo Leon, ´ Medicina, Universidad Autonoma de Nuevo Leon, Mexico. Tel: +52 (81) 83294211, Fax: +52 (81) 83331058; E-mail: [email protected] 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 setting and high mortality rates associated with disseminated infection, the host-fungus interactions regarding Candida parapsilosis sensu stricto and the closely related species C. orthopsilosis and C. metapsilosis remains blurred. In this study, we analyzed inflammatory cytokines levels and histopathology as well as fungal burden in spleen, kidney and lung of mice infected with six strains of the “psilosis” group with different enzymatic profiles. Strong interleukin 22 (IL-22) and tumor necrosis factor α (TNF-α) responses were observed in analyzed organs from infected mice (P < .0001) regardless of the species and enzymatic profile. TNF-α and IL-22 levels were related with spleen inflammation and fungal load. Fungal cells were detected only in spleen and kidney of infected mice, especially by day 2 post-challenge. The kidney showed glomerular retraction and partial destruction of renal tubules. Our data suggest that a strong inflammatory response, mainly of IL-22 and 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 as one of the most common Candida species that originate candidemia [1,2]. Along with Candida orthopsilosis and

Candida metapsilosis, these three phenotypically indistinguishable cryptic yeasts belong to the so-called psilosis group [3]. The administration of parenteral hyperalimentation solutions, as well as the use of intravascular devices and

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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 Candida parapsilosis c/c 105 H-124 Candida orthopsilosis HP-179 H-152 Candida metapsilosis M-18 ATCC 96144

Clinical origin

Aspartyl proteinase

Phospholipase

Esterase

Hemolysin

Peritoneal fluid Blood

− −

+++ −

++++ −

+++ −

Blood Blood

− +++

++++ −

++++ −

+++ −

Skin Skin

++++ −

+++ ++

− −

+++ −

a Semiquantitative 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 that could lead to infection by these opportunistic pathogens in susceptible hosts [2,4]. The incidence of C. parapsilosis species complex has unacceptably increased in recent decades, a fact that has motivated many studies regarding their epidemiology [4,5], antifungal susceptibility [6], virulence [7], and pathogenesis [8], even in nonmurine novel infection models [9]. Otherwise, the model organism Candida albicans has led some progress in the field of host-fungus interactions. In this sense, the immune response elicited by C. albicans infection is driven by inflammatory mediators, particularly inflammasome-derived interleukin 1β (IL-1β), and is characterized by the production of interferon γ (IFN-γ ) from Th1 cells and interleukin 17A (IL-17A) from Th17 cells [10,11]. In disseminated C. albicans infection, Th1 cells are associated with protection from disease, while a predominance of Th2 cells promotes susceptibility [11]. However, a comprehensive tissue-specific analysis of the cytokine levels participating in the anti-Candida host defense to disseminated disease is lacking and there is a large gap in the current knowledge of the immunity against infection by C. parapsilosis sensu stricto, C. orthopsilosis and C. metapsilosis. Recent evidence suggests that the three species of the “psilosis” group possess a similar pathogenic potential in disseminated candidiasis regardless of their particular in vitro enzymatic profiles [8]. The aim of this study was to analyze the levels of inflammatory cytokines in three organs (spleen, kidney, and lung) in immunocompetent mice infected with six strains of the psilosis group with different enzymatic profiles.

Materials and methods Ethics statement Murine experiments were performed with the approval of the Ethics and Research Committee of the School of

Medicine of the Universidad Autonoma de Nuevo Leon ´ ´ (registration code: MB12-002). The animal sacrifices were carried out by cervical dislocation, and all efforts were made to minimize suffering. The experimental protocol was designed in conformity with the International Review Board regulations, following the recommendations of the Guidelines for the Care and Use of Laboratory Animals, and in agreement with Good Laboratory Practices. Care, maintenance, and handling of the animals were in accordance with the Mexican regulations for animal experimentation (NOM-062-ZOO-1999).

Mice Male BALB/c mice, 5 weeks old (22–24 g weight) were purchased from Harlan (Harlan M´exico, S.A. de C.V., Mexico). A total of 132 animals were used; these were housed in ventilated cages of five mice each under specific pathogen-free conditions at the Animal Facility of the Department of Microbiology. All mice were given sterile water and Purina rodent food ad libitum and were monitored daily for 15 days. The day/night cycle was 12 h/12 h. Before use, the animals were allowed to acclimatize for 5 days.

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

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Experimental infection

Histology

For inocula preparation, the six strains were passaged at least twice on SDA plates to check purity and viability of the cultures. After 48 h of incubation at 37◦ C yeast cells were harvested, washed twice in sterile saline, quantified with a hemocytometer, and adjusted to the desired concentration. To corroborate the yeast cell counts, serial fold dilutions were cultured on SDA at 37◦ C for 48 h. Systemic candidiasis was induced in mice by injecting an inoculum of 1.5 × 107 CFU/mouse through the lateral tail vein in 200 μl of a yeast suspension in groups of 20 animals per strain. Three uninfected mice to which sterile saline were intravenously administered were used as controls per experimental day. No immunosuppressive scheme was used. Kidneys are the target organ of murine disseminated candidiasis and therefore one of the most studied tissues in various works [12–14], so we considered analyzing both fungal tissue burden and inflammatory cytokine levels locally expressed in this organ and in the spleen and lungs. The fungal tissue burden determinations were previously published [8]. Briefly, five mice per strain were humanely killed at days 2, 5, 10, and 15 post-challenge. After sacrifice, the kidneys, spleen, and lungs of each mouse were swiftly aseptically removed, weighed, and placed in 2.5 ml of sterile 10 mM phosphate-buffered saline (PBS), pH 7.2 to 7.4. The organs were homogenized in a tissue grinder (Polytron-Aggregate, Kinematica) and serially diluted 1:10 in sterile saline. Aliquots of 0.1 ml of the undiluted and diluted homogenates were then plated twice onto SDA plates, and colony counts were performed after 48 h of incubation at 37◦ C. At par, splenic inflammation was monitored at each time point indicated during the course of the study by measuring the length, width, and height of the spleens with a sterile Vernier scale and applying the formula for the volume of an ellipse. The entire in vivo experiment was performed twice.

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

Cytokine assays Four mouse cytokines were assayed in supernatants from tissue homogenates. The cytokines measured were: TNF-α, IFN-γ , IL-17A, and IL-22. Determinations were performed by sandwich enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol for TNF-α, IFN-γ (PeproTech, Mexico City, Mexico), IL-17A, and IL22 (eBioscience, San Diego, CA, USA), using the iMark microplate reader (Bio-Rad Inc., Hercules, CA, USA). The quantitation ranges were 32–2000 pg/ml, 16–2000 pg/ml, 4–500 pg/ml, and 8–1000 pg/ml for TNF-α, IFN-γ , IL17A, and IL-22, respectively. Each sample was analyzed twice.

Statistical analysis The Kruskal-Wallis and Dunn’s multiple comparison tests were applied to verify statistical significance among experimental groups. Pearson’s correlations between splenic inflammation and fungal load with local cytokine levels were additionally made. Calculations and graphics were performed 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 early stage of infection We studied whether C. parapsilosis complex strains with different enzymatic profiles induce a specific cytokine profile in kidney, spleen, and lung. All C. parapsilosis complex strains induced TNF-α and IL-22 production in infected mice (P < .0001). The concentration of TNF-α was higher compared with other analyzed cytokines in mice infected with C. parapsilosis complex. TNF-α levels in kidney were > 5–10 times fold compared with lung or spleen (Fig. 1). In kidney, C. metapsilosis ATCC 96144 strain was a high inducer of TNF-α, while C. metapsilosis M18 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 were the most potent inducers of TNF-α at 10 and 15 days post-infection (Fig. 1B). A nonspecific pattern was found in spleen between cytokine levels and C. parapsilosis complex strains (Fig. 1C). Similar to TNF-α, IL-22 levels were higher 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 induced high levels of IL-22 in early infection (Fig. 2A). In lung, C. parapsilosis sensu stricto c/c 105 and C. metapsilosis M-18 induced high levels of IL-22 at 2 and 5 days post-infection. However, in the next post-infection periods, this cytokine was elevated in all infected mice regardless of the species and enzymatic profile (Fig. 2B). In spleen, C. metapsilosis strains were the most potent inducers of IL-22 at 5 and 10 days post-challenge (Fig. 2C). C. parapsilosis 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), and spleen (C). The data represent the means of five mice per group ± SD. Asterisks represent significant differences compared to controls. TNF-α levels in kidney were found > 5–10-fold compared with lung or spleen.

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

Spleen inflammation is paralleled with local TNF-α and IL-22 levels The splenomegaly was measured throughout the infection with a vernier, and the inflammation score was calculated using the ellipsoid equation. Mice infected with C. parapsilosis complex strains exhibited spleen inflammation, and this was in accord with TNF-α and IL-22 levels,

especially with C. parapsilosis sensu stricto and C. metapsilosis strains.

TNF-α and IL-22 could control fungal load TNF-α and IL-22 are essential cytokines in the control of fungal infections. TNF-α induces oxidative and nitrosative distress in the phagolysosome microenvironment and contribute to the inflammatory process [15]. On the other hand, IL-22 limits fungal growth due to induction of antimicrobial peptides [16]. Thus, we questioned if the levels of TNF-α and IL-22 are related with the decrease in fungal load. These analyses demonstrated that both cytokines increase in kidney from mice infected with most C. parapsilosis complex strains, showing > 1 log reduction in fungal load. C. orthopsilosis H-152 (a biofilm former strain) was the lowest affected of the studied strains in the kidney, showing < 0.5 log reduction. In spleen and lung, fungal load was reduced > 2 log in most C. parapsilosis complex strains, where high

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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 were higher 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 significant.

The kidney is the most affected organ during C. parapsilosis complex infection During the course of the infections, histological alterations in spleen and lung were not found compared with control mice. However, in the spleen, the presence of isolated yeasts near the cells of the Billroth cords and in the red pulp sinusoids were evident, especially at day 2 post-challenge. There was no evidence of fungal structures in lungs of infected mice. On the other hand, abundant yeast cells at day 2 were detected in the kidney after systemic infection, mainly in the renal intersitium, and these tended to gradually decrease by day 15. In addition, the presence of acute inflammatory infiltrate foci surrounding yeasts in kidney was evident at day

5 and 10 post-challenge (Table 2). Unlike the spleen and lung, the kidney of infected mice showed some histological alterations, glomerular retraction, and destruction of renal tubules, which were the principal findings compared with uninfected controls, and these were detected since day 2 post-challenge (Fig. 5 and Table 2). Importantly, a particular histopathological pattern associated to a specific species or enzymatic profile among the analyzed strains was not found.

Discussion Diverse animal models of infection have been developed to investigate the different clinical forms of candidiasis, being murine models the gold standard to study pathogenesis as well as efficacy of antifungal agents alone or in 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), and spleen (C). The data represent the means of five mice per group ± SD. Asterisks represent significant differences compared to controls. IFN-γ was down-regulated in lung and spleen with respect to kidney.

candidiasis, and due to its highly reproducibility it remains as the most common long-standing model used to investigate C. albicans virulence and to determine particular aspects of host-fungus interactions [17–19]. During the systemic infection, the bloodstream and the majority of organs (spleen, lungs, and liver) are gradually cleared of the pathogen, but this scenario is quite different in the kidney, the principal target organ for IV challenge in the mouse, in which the increase of fungal burden is accompanied by increasing levels of renal cytokines and chemokines [13,14], with an existing correlation between the increased renal cytokine levels and the lesion severity with the consequent infection outcome [14,19]. Yet, the cellular and molecular factors that determine differential organ-specific control of Candida remain largely unknown. During pathogenic C. albicans infection, the inflammatory mediators of the immune response are driven by Th1 and the recently implicated involvement of Th17 cells. The stimulated Th1 lymphocytes produce IFN-γ , which contributes to anti-Candida host defense by inducing nitric oxide (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 lymphoid and myeloid host defense [22] through the infiltration induction of neutrophilic granulocytes at the site of infection and activation of macrophages [21]. Meanwhile,

IL-22 is a member of the IL-10 family of cytokines, which has been confounded by data suggesting both pro- and antiinflammatory functions. However, recent evidence suggests that this cytokine accounts for innate resistance to candidiasis at the early stages of infection in the kidney and gut by critically controlling initial fungal growth and epithelial homeostasis in the relative absence of Th1 immunity [23]. Interestingly, current findings suggests that IL-22 and TNF-α represent a potent synergistic cytokine combination for skin immunity, efficiently conserving epidermal barrier integrity in a skin infection model compared with IFN-γ , IL-17, IL-22, or TNF-α alone [24]. In disseminated C. albicans infection, Th1 cells are associated with protection from disease, while a predominance of Th2 cells promotes susceptibility [11]. Finally, the balance in innate responses between fungicidal (beneficial) immunity and immunoregulatory (detrimental) compensative mechanisms determines the scope of tissue damage in fungal infections [16]. The tissue specificity of mouse cytokine host responses in hematogenously C. albicans infection was previously demonstrated by Spellberg et al. [25] They examined the relationship between host survival and local immune responses in kidney and spleen during candidiasis with different inocula, concluding that this pathogen induced type 2 splenocyte responses with both fatal and nonfatal inocula. Conversely, the nature of immune polarization in the

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Figure 4. Inflammatory cytokines were detected since early stage of infection. Graphs show IL-17A 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-17A was down-regulated in kidneys and lungs at days 2, 5, and 10 post-challenge.

kidney correlated with host survival, with IFN-γ -dominant type 1 responses causing no or low mortality and type 2 or IL-10-dominant responses during 100% fatal infection. Subsequently, MacCallum et al. [14] analyzed cytokine and chemokine levels in infected organs elucidating organspecific responses, with high cytokine and chemokine levels in infected kidneys but reduced responses in the spleen. They concluded that keratinocyte-derived chemokine is an important early mediator of overall outcome and correlate along with IL-6 and MIP-1β with lesion severity, whereas GM-CSF and IL-10 showed inverse correlations with histological damage. These differences were also reflected at the transcriptional level, with differential expression of cytokine genes in both organs [26]. Since our interest was focused in local responses to infection, the experimental design we adopted did not include measurement of serum cytokine levels. Of the assayed organs in this study, the kidney showed the highest fungal load, as well as pro-inflammatory

cytokine levels, and it was the only tissue that showed histological alterations due to the systemic infection (Fig. 7). Recently, Lionakis et al. [27] characterized the immune cell populations in infected organs during the progression of disseminated candidiasis in a mouse model. They found neutrophils accumulated in all infected organs, but a delay in their appearance in the kidneys, leaving these organs unprotected during the first 24 h post-challenge. Further increases in neutrophils occurred in these organs as disease progressed. The kidney inflammation could be explained in part by the CCR1 neutrophils, which play a pathogenic role in invasive candidiasis mediating renal immunopathology via excessive neutrophil recruitment from the blood into the kidney [12]. The role of IL-22 in neutrophil recruitment in peripheral tissue has been already reported in murine cytomegalovirus [28]. We found downregulation of IFN-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 as presence 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-6 in response to the fungus on epithelial surfaces [30]. On the other hand, high levels of IL-22 found in kidney could be involved in neutrophil recruitment in this tissue. In lung and spleen were not observed neutrophil indeed IL-22 increment during C. parapsilosis complex infection, it may be to absent or low number of yeasts in these tissues. Finally, a significant correlation between splenic inflammation and fungal load with local cytokine levels was not found. Overall, this study demonstrates that male BALB/c mice experimentally infected with strains of the “psilosis” group exhibiting different in vitro enzymatic profiles irrespectively induce a pronounced inflammatory response that is essential for protective antifungal immunity and drive pathology during disseminated infection, principally in the kidney. Although a correlation between cytokine levels

and enzymatic activity of the strains was not found, the role of these enzymes in systemic disease caused by the C. parapsilosis sensu lato group should not be underestimated. In this regard, further studies with more characterized 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, +++ Abundant.

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genes involved in each enzyme, become essential in order to elucidate their particular contribution to the pathogenesis by these opportunistic pathogens. In general, this knowledge have implications in the development of novel immunotherapy strategies, which aims improve host defense against C. parapsilosis complex, providing a solution to prevent and reduce the high mortality rates of the invasive candidiasis.

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

Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

References 1. Pfaller MA, Moet GJ, Messer SA et al. Candida bloodstream infections: comparison of species distributions and antifungal resistance patterns in community-onset and nosocomial isolates in the sentry antimicrobial surveillance program, 2008–2009. Antimicrob Agents Chemother 2011; 55: 561–566. 2. Trofa D, Gacser A, Nosanchuk JD. Candida parapsilosis, an ´ emerging fungal pathogen. Clin Microbiol Rev 2008; 21: 606– 625. 3. Tavanti A, Davidson AD, Gow NA et al. Candida orthopsilosis and Candida metapsilosis spp. nov. to replace Candida parapsilosis groups II and III. J Clin Microbiol 2005; 43: 284– 292. 4. van Asbeck EC, Clemons KV, Stevens DA. Candida parapsilosis: a review of its epidemiology, pathogenesis, clinical aspects, typing and antimicrobial susceptibility. Crit Rev Microbiol 2009; 35: 283–309. 5. Lockhart SR, Messer SA, Pfaller MA et al. Geographic distribution and antifungal susceptibility of the newly described species Candida orthopsilosis and Candida metapsilosis in comparison to the closely related species Candida parapsilosis. J Clin Microbiol 2008; 46: 2659–2664. 6. Trevino-Rangel Rde J, Garza-Gonzalez E, Gonzalez JG et al. ˜ ´ ´ Molecular characterization and antifungal susceptibility of the Candida parapsilosis species complex of clinical isolates from Monterrey, Mexico. Med Mycol 2012; 50: 781–784. 7. Trevino-Rangel Rde J, Gonzalez JG, Gonzalez GM. Aspartyl ˜ ´ ´ proteinase, phospholipase, esterase and hemolysin activities of clinical isolates of the Candida parapsilosis species complex. Med Mycol 2013; 51: 331–335. 8. Trevino-Rangel Rde J, Rodr´ıguez-Sanchez IP, Elizondo˜ ´ Zertuche M et al. Evaluation of in vivo pathogenicity of Candida parapsilosis, Candida orthopsilosis, and Candida metapsilosis with different enzymatic profiles in a murine model of disseminated candidiasis. Med Mycol 2014; 52: 240– 245.

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9. Jacobsen ID. Galleria mellonella as a model host to study virulence of Candida. Virulence 2014; 5: 237–239. 10. Cheng SC, van de Veerdonk FL, Lenardon M et al. The dectin1/inflammasome pathway is responsible for the induction of protective t-helper 17 responses that discriminate between yeasts and hyphae of Candida albicans. J Leukoc Biol 2011; 90: 357– 366. 11. Romani L, Mencacci A, Grohmann U et al. Neutralizing antibody to interleukin 4 induces systemic protection and T helper type 1-associated immunity in murine candidiasis. J Exp Med 1992; 176: 19–25. 12. Lionakis MS, Fischer BG, Lim JK et al. Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis. PLoS Pathog 2012; 8: e1002865. 13. MacCallum DM. Massive induction of innate immune response to Candida albicans in the kidney in a murine intravenous challenge model. FEMS Yeast Res 2009; 9: 1111–1122. 14. MacCallum DM, Castillo L, Brown AJ et al. Early-expressed chemokines predict kidney immunopathology in experimental disseminated Candida albicans infections. PLoS One 2009; 4: e6420. 15. Brown GD. Innate antifungal immunity: the key role of phagocytes. Annu Rev Immunol 2011; 29: 1–21. 16. Romani L. Immunity to fungal infections. Nat Rev Immunol 2011; 11: 275–288. 17. Szabo EK, MacCallum DM. The contribution of mouse models to our understanding of systemic candidiasis. FEMS Microbiol Lett 2011; 320: 1–8. 18. MacCallum DM, Odds FC. Temporal events in the intravenous challenge model for experimental Candida albicans infections in female mice. Mycoses 2005; 48: 151–161. 19. Spellberg B, Ibrahim AS, Edwards JE, Jr. et al. Mice with disseminated candidiasis die of progressive sepsis. J Infect Dis 2005; 192: 336–343. 20. Kaposzta R, Tree P, Marodi L et al. Characteristics of invasive candidiasis in gamma interferon- and interleukin-4-deficient mice: role of macrophages in host defense against Candida albicans. Infect Immun 1998; 66: 1708–1717. 21. Ouyang W, Kolls JK, Zheng Y. The biological functions of T helper 17 cell effector cytokines in inflammation. Immunity 2008; 28: 454–467. 22. Ye P, Rodriguez FH, Kanaly S et al. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. J Exp Med 2001; 194: 519–527. 23. De Luca A, Zelante T, D’Angelo C et al. Il-22 defines a novel immune pathway of antifungal resistance. Mucosal Immunol 2010; 3: 361–373. 24. Eyerich S, Wagener J, Wenzel V et al. IL-22 and TNF-α represent a key cytokine combination for epidermal integrity during infection with Candida albicans. Eur J Immunol 2011; 41: 1894–1901. 25. Spellberg B, Johnston D, Phan QT et al. Parenchymal organ, and not splenic, immunity correlates with host survival during disseminated candidiasis. Infect Immun 2003; 71: 5756–5764. 26. Brieland J, Essig D, Jackson C et al. Comparison of pathogenesis and host immune responses to Candida glabrata and Candida albicans in systemically infected immunocompetent mice. Infect Immun 2001; 69: 5046–5055.

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27. Lionakis MS, Lim JK, Lee CC et al. Organ-specific innate immune responses in a mouse model of invasive candidiasis. J Innate Immun 2011; 3: 180–199. 28. Stacey MA, Marsden M, Pham NT et al. Neutrophils recruited by IL-22 in peripheral tissues function as TRAIL-dependent antiviral effectors against MCMV. Cell Host Microbe 2014; 15: 471–483.

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29. Carvalho LP, Bacellar O, Neves N et al. Downregulation of IFN-gamma production in patients with recurrent vaginal candidiasis. J Allergy Clin Immunol 2002; 109: 102– 105. 30. Fidel PL, Jr. The protective immune response against vaginal candidiasis: lessons learned from clinical studies and animal models. Int Rev Immunol 2002; 21: 515–548.