INFECTION AND IMMUNITY, May 2010, p. 2342–2352 Vol. 78, No. 50019-9567/10/$12.00 doi:10.1128/IAI.01140-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Role of Host Sphingosine Kinase 1 in the LungResponse against Cryptococcosis�

Travis McQuiston,1 Chiara Luberto,1 and Maurizio Del Poeta1,2,3,4*Department of Biochemistry and Molecular Biology,1 Department of Microbiology and Immunology,2 Department of Craniofacial Biology,3

and Division of Infectious Diseases,4 Medical University of South Carolina, Charleston, South Carolina

Received 9 October 2009/Returned for modification 24 November 2009/Accepted 19 February 2010

Cryptococcus neoformans is a fungal pathogen causing pulmonary infection and a life-threatening meningo-encephalitis in human hosts. The fungus infects the host through inhalation, and thus, the host response in thelung environment is crucial for containment or dissemination of C. neoformans to other organs. In the lung,alveolar macrophages (AMs) are key players in the host lung immune response, and upon phagocytosis, theycan kill C. neoformans by evoking an effective immune response through a variety of signaling molecules. Onthe other hand, under conditions not yet fully defined, the fungus is able to survive and proliferate withinmacrophages. Since the host sphingosine kinase 1 (SK1) regulates many signaling functions of immune cells,particularly in macrophages, in this study we determined the role of SK1 in the host response to C. neoformansinfection. Using wild-type (SK1/2�/�) and SK1-deficient (SK1�/�) mice, we found that SK1 is dispensableduring infection with a facultative intracellular wild-type C. neoformans strain. However, SK1 is required toform a host lung granuloma and to prevent brain infection by a C. neoformans mutant strain lacking the cellwall-associated glycosphingolipid glucosylceramide (�gcs1), previously characterized as a mutant able toreplicate only intracellularly. Specifically, in contrast to those from SK1/2�/� mice, lungs from SK1�/� micehave no collagen deposition upon infection with C. neoformans �gcs1, and AMs from these mice containsignificantly more C. neoformans cells than AMs from SK1/2�/� mice, suggesting that under conditions inwhich C. neoformans is more internalized by AMs, SK1 may become important to control C. neoformansinfection. Indeed, when we induced immunosuppression, a host condition in which wild-type C. neoformans cellsare increasingly found intracellularly, SK1�/� survived significantly less than SK1/2�/� mice infected with afacultative intracellular wild-type strain, suggesting that SK1 has an important role in controlling C. neofor-mans infection under conditions in which the fungus is predominantly found intracellularly.

Cryptococcus neoformans is the etiological agent of the mostcommon form of fungal meningoencephalitis worldwide in im-munocompromised individuals. Upon environmental expo-sure, desiccated yeasts or basidiospores are inhaled into thealveolar spaces of the host lung. In the lung, C. neoformans cansurvive and replicate in the extracellular environment of alve-olar spaces and/or, following phagocytosis, intracellularlywithin the phagolysosome of the alveolar macrophages (AMs)(13). Following internalization of C. neoformans, AMs initiatean immune response resulting in granuloma formation, whichcontains the infection in the lung, thereby preventing fungaldissemination to other organs or tissues. Hence, the high inci-dence of cryptococcal meningitis in patients with impairedcell-mediated immunity clearly links dissemination from thelungs to the lack of or impaired formation of a granuloma. Thehost cellular pathway(s) controlling the formation of a granu-loma response has not been elucidated, mainly because a gran-ulomatous response is very weakly evoked in mouse models ofcryptococcosis. Recent studies, however, using a mutant strainof C. neoformans lacking the glucosylceramide synthase 1(GCS1) gene have shown that immunocompetent but not im-munodeficient mice are able to produce a granuloma that

successfully contains this mutant strain within the lung tissue,thus preventing its dissemination to the brain and the devel-opment of meningoencephalitis (24, 36).

Sphingosine kinases 1 and 2 (SK1 and SK2) are two enzymesof the sphingolipid pathway that phosphorylate sphingosine toproduce sphingosine-1-phosphate (S1P), a bioactive signalingmolecule. In mammalian cells, S1P modulates numerous cel-lular processes, including immune cell development, differen-tiation, activation, and migration (14, 25). The predominantisoform expressed in the lung is SK1, and the regulation of SK1activity is thought to have a central role in the lung immuneresponse (1, 4, 31). For instance, S1P levels increase in thebronchoalveolar lavage (BAL) fluid upon antigen challenge(2); S1P modulates pulmonary epithelial and endothelial cellfunction, including the expression of adhesion molecules re-quired for immune cell migration (35); and binding of S1P totheir respective cell surface receptors (S1PRs) stimulates SK1activity and the proinflammatory response in macrophages (20,30, 32, 50). Importantly, SK1 also has a role in the antimicro-bial activities of AMs against internalized pathogens, such asthe Mycobacterium species, where its activity is required forphagosome maturation of the pathogen-containing vesicle intothe microbicidal phagolysosome (17, 29, 46). Exogenous S1Phas also been found to induce a Th1-associated phenotype,intracellular killing, and antigen presentation by human mono-cytes and macrophages containing intracellular Mycobacteriumin vitro (16, 17, 40) and ex vivo (16). Furthermore, intravenousadministration of S1P decreases bacterial burden and improves

* Corresponding author. Mailing address: Department of Biochem-istry and Molecular Biology, Medical University of South Carolina, 173Ashley Avenue, BSB 512A, Charleston, SC 29425. Phone: (843) 792-8381. Fax: (843) 792-8565. E-mail: delpoeta@musc.edu.

� Published ahead of print on 1 March 2010.

2342

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

histopathology in the lungs of Mycobacterium-challenged miceduring acute infection (16, 17, 39). Whereas these data clearlyshow that SK1 regulates facets of host inflammation in vitroand that its product, S1P, evokes antimycobacterial actions invitro and in vivo, the role of SK1 during pulmonary infection invivo and its effect on the antimicrobial actions of AMs againstother facultative intracellular pathogens, such as C. neofor-mans, are unknown.

In this research, the effect of SK1 on the host immuneresponse during pulmonary cryptococcosis and on the viru-lence of C. neoformans was examined. Using mice deficient inSK1, we obtained data demonstrating that SK1 modulates thehost immune response involved in the formation of granulo-mas. We also show that SK1 is vital for the containment of C.neoformans during pulmonary infection but only when the fun-gus predominantly replicates intracellularly.

MATERIALS AND METHODS

Mouse strains. Five- to 7-week-old wild-type C57BL/6J mice (The JacksonLaboratory, Bar Harbor, ME), SK1-deficient mice (SK1�/�), and SK2-deficientmice (SK2�/�) were used for this research. SK1�/� and SK2�/� mice werepreviously generated and colonies were maintained as previously described (1).SK1�/� and SK2�/� mice were available to us through the MUSC COBREAnimal Core Facility, directed by T. Kawamori, who provided breeding pairs.Travis J. McQuiston performed all breeding, weaning, and genotyping (data notshown). For all experiments, SK1�/� and SK2�/� mice were age and sexmatched with SK1/2�/� wild-type mice (C57BL/6).

Isolation and cell culturing of AMs. AMs were isolated from the lungs of miceusing 1� sterile phosphate-buffered saline (PBS) at pH 7.0 by bronchoalveolarlavage (BAL). BAL fluid was subjected to centrifugation at 500 � g for 5 min.Cell pellets were resuspended in serum-free RPMI medium supplemented with0.1% penicillin-streptomycin, and cell number was determined using a hemato-cytometer. For all coincubation assays, 1 � 105 cells were plated on the glassportion of a poly-D-lysine-coated glass-bottom confocal cell dish (MatTek Cor-poration, Ashland, MA). AMs were allowed to adhere for 30 min before the celldishes were washed three times and fresh medium was added.

C. neoformans strains and growing media. C. neoformans var. grubii serotype Astrain H99 (wild type [WT]) and a mutant C. neoformans strain lacking GCS1(�gcs1) (36, 42) were used in this study. Both strains were grown in yeastextract-peptone-dextrose (YPD) medium for 16 to 18 h at 30°C in a shaking cellculture incubator.

Real-time reverse transcriptase (RT)-PCR. mRNA was isolated from AMsusing the RNeasy minikit from Qiagen. cDNA was generated from 0.5 �g RNAby using random hexamer primers and the SuperScript III First Strand cDNAsynthesis system from Invitrogen (Carlsbad, CA). Real-time RT-PCR was con-ducted using a Bio-Rad iCycler to quantify mRNA levels of SK1 and SK2 or ofthe S1PRs, respectively. The standard real-time RT-PCR volume was 25 �l,which comprised 12.5 �l SYBR green PCR reagents (Bio-Rad, Hercules, CA), 5�l cDNA template, 1 �l forward primer (4 �M), 1 �l reverse primer (4 �M), and5.5 �l water. The sequences of primer pairs for SK isoforms, along with theRT-PCR steps for amplification, were described previously (3, 44). All reactionswere performed in triplicate. Q-Gene software was used to analyze data, whichwere then expressed as the change (fold) between the mean normalized expres-sion and the control value. The mean normalized expression is directly propor-tional to the amount of mRNA of the target gene relative to the amount ofmRNA of the reference gene, the �-actin gene. Melting curves were also exam-ined to ensure that the data corresponded to production of the single desiredRT-PCR fragment for each target gene. Data are the averages from threeseparate experiments.

In vitro phagocytosis assay. AMs were plated as described above. C. neofor-mans cultures were subjected to centrifugation at 500 � g for 10 min. YPDmedium was removed, and the cell pellet was washed three times with sterilewater. After washing, C. neoformans cells were resuspended in the desired cellmedium, and cell number was calculated using a hematocytometer. Next, 1 � 106

C. neoformans cells were opsonized in 1 ml (final volume) RPMI mediumcontaining either 10% fresh mouse serum, 10 �g/ml of the anti-glucuronoxylo-mannan (GXM) monoclonal IgG1 antibody 18B7 (kindly provided by ArturoCasadevall, Albert Einstein College of Medicine, Bronx, NY), or both. Serum

was obtained from C57BL/6J mice immediately before the assay. These opso-nization solutions were then vortexed vigorously and incubated for 20 min at37°C. After opsonization of C. neoformans, the medium from the confocal dishescontaining the AMs was removed and replaced with 100 �l opsonized C. neo-formans solution containing 1 � 105 C. neoformans cells, thereby making themultiplicity of infection (MOI) 1:1. After 2 h of coincubation, the medium wasremoved, the plates were washed three times with PBS, fixed in ice-cold meth-anol, and stained with Giemsa for analysis by light microscopy using a 100�objective under oil immersion. For each confocal dish, a minimum of 500 mac-rophages were examined for C. neoformans internalization. As previously de-scribed, the phagocytic index is the percentage of macrophages with internalizedfungal cells multiplied by the average number of internalized fungal cells (45).

In vitro C. neoformans intracellular growth assay. The ability of internalized C.neoformans cells to replicate in a coculture within AMs was examined after 4 hof coincubation. C. neoformans opsonized cells with 10% fresh mouse serum andthe anti-GXM IgG1 antibody 18B7 were coincubated with AMs, as describedabove for the in vitro phagocytosis assay. To determine the intracellular growthof C. neoformans, the medium from coincubations was removed after 2 h andplates were washed three times to remove any extracellular C. neoformans. Freshserum-free RPMI medium was added for an additional 2 h. After a total of 4 h,plates were processed for light microscopy to allow visualization of daughtercells, also known as buds. A minimum of 100 internalized C. neoformans cells perplate were inspected for budding, and intracellular growth was calculated as thepercentage of total internalized C. neoformans cells that exhibited buds.

Immunosuppression. Mice received intraperitoneal injections of 125 mg/kg ofcortisone acetate (CA; Sigma Chemical Co., St. Louis, MO) 24 h prior to,immediately prior to, and at days 1, 4, and 7 after intranasal challenge with 5 �105 C. neoformans cells. The CA regimen was carried out as described in previousstudies examining pulmonary fungal infections and virulence (5, 8). Controlanimals received CA injections but were not challenged with C. neoformans.

WBC analysis. Blood samples were collected from CA-treated mice on thesame days immediately prior to CA treatments to assess the effect of the corti-costeroid on the white blood cell (WBC) population using C. neoformans infec-tion. Blood was obtained by lancing the saphenous vein with a 23 1/2-gaugeneedle, and approximately 100 �l of blood was collected from each mouse. Theblood from three mice was pooled, totaling 300 �l in an EDTA-coated Micro-tainer collection tube (catalog no. 365973; Becton Dickinson, Franklin Lakes,NJ). A complete blood count (CBC) was conducted on each pooled sample todetermine the total WBC count.

Survival studies in mouse models of pulmonary cryptococcosis. Mice wereanesthetized with an intraperitoneal injection of 60 �l of a xylazine-ketaminemixture containing 5 mg xylazine and 95 mg ketamine per kg body weight. Allstrains of C. neoformans were grown in YPD medium for 16 to 18 h at 30°C. C.neoformans cells were washed and resuspended in PBS. Mice were challengedintranasally with 20 �l of the inoculum solution containing 5 � 105 C. neoformanscells. Mice were fed ad libitum and monitored twice a day for signs of appearingmoribund or in pain and for any clinical signs suggesting meningoencephalitis.Mice showing any of these signs were immediately sacrificed using CO2 inhala-tion followed by cervical dislocation.

Histological analyses. Organs were harvested and fixed overnight in 37%formaldehyde, paraffin embedded, sectioned, and stained with hematoxylin andeosin (H&E), Movat, mucicarmine, Verhoeff-Van Gieson (VVG), or Giemsa foridentification and visualization of host components and C. neoformans cells usinglight microscopy. The Histology Core Facility at the Medical University of SouthCarolina performed all staining and tissue processing. Russell Harley and MashaBilic performed histological analyses on separate occasions.

Statistics. Data from each experimental group were subjected to an analysis ofnormality and variance. The statistical significance of the difference between themeans of two experimental data sets composed of normally distributed valueswas analyzed using Student’s two-tailed t test. Nonparametric evaluation ofindependent data sets was performed with the Mann-Whitney rank sum test (alsoknown as the Wilcoxon rank sum test). For both types of analyses, P values lessthan 0.05 were considered significant.

RESULTS

Effect of host SK on the virulence of C. neoformans. Previousstudies have suggested that SK1 activity is essential to thekilling of internalized Mycobacterium cells by human mono-cytes and macrophages in vitro (29, 46). Thus, we wondered

VOL. 78, 2010 ROLE OF SK1 AGAINST C. NEOFORMANS 2343

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

whether SK1 has a role during C. neoformans infection, as C.neoformans is a facultative intracellular pathogen.

To determine if SK1 affects host susceptibility to cryptococ-cosis, SK1/2�/� and SK1�/� mice were infected intranasallywith 5 � 105 C. neoformans cells and monitored for morbidity.This well-established mouse model of cryptococcal meningitiswas chosen because inhalation is the most common route ofinfection following environmental exposure and, therefore,provides the best infection model to examine the pathogenicityof C. neoformans as it relates to the clinical setting. Figure 1shows that there was no significant difference in the survival ofSK1/2�/� and SK1�/� mice infected with WT C. neoformansstrain H99 cells (19.6 � 1.5 days versus 20.6 � 1.2 days). Sincemice are innately susceptible to cryptococcosis, it is possiblethat the virulence of this WT strain of C. neoformans may maskany effect that SK1 has on the development of cryptococcosis.Thus, to determine if SK1 affects the dissemination of WT C.neoformans and disease progression, the fungal burdens in theorgans from H99-infected SK1/2�/� and SK1�/� mice weredetermined at days 5, 10, and 15 after C. neoformans challenge.Tissues from three mice were isolated and processed to deter-mine CFU at each time point. There were no significant dif-ferences in the fungal burdens in the lungs (Fig. 2A) and brains(Fig. 2B) of H99-infected SK1/2�/� and SK1�/� mice at anytime point inspected. No differences were found in the fungalburdens of the liver, spleen, and kidneys (data not shown).These results are not surprising, as there was no difference inthe symptoms between SK1/2�/� and SK1�/� mice challengedwith WT C. neoformans. Since no differences in organ fungalburden or symptoms were found, histopathology was not per-formed on the organs from H99-infected SK1/2�/� andSK1�/� mice. Together, Fig. 1 and 2 suggest that SK1 does notplay a role in the susceptibility of an immunocompetent mouseto a WT (e.g., facultatively intracellular) C. neoformans strain.

SK1 is required for the antimicrobial activity of macro-phages against internalized Mycobacterium (16, 17, 29, 39, 46).To investigate the effect of SK1 on the pathogenicity of intra-cellular C. neoformans, we used a C. neoformans strain in whichthe gene encoding glucosylceramide synthase 1 (GCS1) is de-leted (�gcs1) (24, 36). C. neoformans �gcs1 cells do not repli-cate in the neutral or alkaline pH and physiological concen-trations of CO2 characteristic of extracellular environments,

such as in alveolar spaces and the bloodstream. However, thismutant strain lacking the GCS1 gene has no growth defectunder acidic conditions, such as within the phagolysosome(36). It is important to note that the �gcs1 strain does notdisseminate and is avirulent in immunocompetent mice (24,36). Therefore, we refer to C. neoformans �gcs1 as obligatelyintracellular for replication in vivo. Thus, SK1/2�/� andSK1�/� mice were infected intranasally with C. neoformans�gcs1, and mouse survival and tissue burden cultures wereexamined. All SK1/2�/� and SK1�/� mice infected with �gcs1survived 100 days postchallenge (Fig. 3A). At this time point,the lungs and brains of these mice were extracted and pro-cessed either to determine fungal burden using CFU or forhistopathological analysis using light microscopy. SK1�/� micehad a significantly greater number of �gcs1 cells in the lungsthan SK1/2�/� mice (P � 0.05) (Fig. 3B). Tissue burden cul-ture of brains showed that none of the seven brains fromSK1/2�/� mice contained �gcs1 cells, whereas all seven brainsfrom SK1�/� mice contained C. neoformans cells, representinga statistically significant difference (P � 0.005) (Fig. 3B). Or-gan fungal burdens in the livers, spleens, and kidneys were alsoexamined using CFU. C. neoformans cells were not foundwithin the livers, spleens, and kidneys of �gcs1 mutant-infectedSK1/2�/� mice, while the �gcs1 strain was present in thespleens of two SK1�/� mice but absent in all other organsexamined (data not shown). Together, these data suggest thatSK1 protects the host from pulmonary C. neoformans infectionand prevents dissemination to the central nervous system onlywhen dealing with a C. neoformans strain that can replicateonly intracellularly.

Role of SK1 in host immune response during pulmonarycryptococcosis. Histopathology of the lungs of �gcs1-infectedSK1/2�/� mice compared to �gcs1-infected SK1�/� mice cor-roborated the hypothesis that SK1 was instrumental in thehost’s ability to prevent dissemination of C. neoformans �gcs1from the lung to the brain. The lungs of three SK1/2�/� andSK1�/� mice were excised, sectioned, mounted onto micro-scope slides, and subjected to various stainings for inspectionof the host immune response at 100 days after intranasal chal-lenge with C. neoformans �gcs1. We found that SK1/2�/� mice

FIG. 1. SK1 is dispensable for virulence of a facultative intracellu-lar C. neoformans wild-type strain. C57BL/6J wild-type mice (SK1/2�/�) and sphingosine kinase 1 knockout mice (SK1�/�) were chal-lenged intranasally with 5 � 105 C. neoformans wild-type strain H99cells and monitored daily. Data are representative of three individualexperiments. There was no significant difference in survival betweenSK1/2�/� and SK1�/� mice (19.6 � 1.4 days versus 20.9 � 1.0 days).

FIG. 2. SK1 deficiency does not affect fungal organ burden in micechallenged with a facultative intracellular C. neoformans wild-typestrain. C57BL/6J wild-type mice (SK1/2�/�, n 9) and sphingosinekinase 1 knockout mice (SK1�/�, n 9) were challenged intranasallywith 5 � 105 C. neoformans wild-type strain H99 cells and monitoreddaily. Three mice from each group were sacrificed at days 5, 10, and 15after C. neoformans challenge. Lungs (A) and brains (B) from thesemice were processed and analyzed for CFU. There were no significantdifferences in fungal burden in the lungs or brains of SK1/2�/� andSK1�/� mice at any of the time points examined.

2344 MCQUISTON ET AL. INFECT. IMMUN.

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

challenged intranasally with �gcs1 cells showed a granuloma inthe lungs that, in light of the data presented in Fig. 3B, suc-cessfully contained and prevented dissemination. These re-sults, obtained with C57BL/6J immunocompetent mice, aresimilar to previous findings with CBA/J immunocompetentmice (36). Figure 4A shows that C. neoformans cells, stainedwith Alcian blue by Movat staining, are contained within thecenter region of a well-organized granuloma, where the hostcellular components are stained shades of red. Activated fibro-blasts deposited extracellular matrix, including significantamounts of collagen, which is stained pink by VVG staining, toform a thick fibrotic tissue encapsulating the �gcs1 strain-containing granuloma, thereby acting as a physical barrier pre-venting dissemination from the lungs (Fig. 4B). The encapsu-lation of the fibrotic tissue is further demonstrated with Movatstaining, which showed that fibrotic tissues (stained light red)and the leukocyte infiltrate (stained deep red) prevent theegress of �gcs1 cells from the lungs (Fig. 4C). Mixed popula-tions of leukocytes were observed to reside between the fi-brotic tissue and the region containing C. neoformans cells(Fig. 4B, D, and E). When H&E and Giemsa stains were usedto examine the cell types comprising the leukocyte infiltrationduring pulmonary cryptococcosis (9, 37), neutrophils wereidentified as the dominant granulocyte cell type, whereasplasma cells were also present (Fig. 4D and E). Althougheosinophils have been shown to be a common component ofthe pulmonary inflammatory response of C57BL/J6 mice towild-type C. neoformans challenge through 28 days (9, 11, 21),eosinophils were rarely observed in the granulomas within thelungs of �gcs1 strain-infected SK1/2�/� mice and do not rep-resent a significant cell type in the inflammatory response at

100 days after C. neoformans �gcs1 challenge. However, crys-tal-like structures were observed both extracellularly and in-tracellularly in host phagocytes with and without internalizedC. neoformans throughout the granulomas in the lungs from�gcs1 strain-challenged SK1/2�/� mice (Fig. 4D and F). Theseunique structures are mostly likely derived from the eosinophilchemotactic factors Ym1 and/or Ym2, whose formation com-monly occurs in C. neoformans-challenged C57BL/J6 mice (11,21). The presence of these crystals indirectly suggests eosino-phil involvement at some junction during the host inflamma-tory response.

C. neoformans cells were localized to the center of the gran-ulomas in the lungs from �gcs1 strain-challenged SK1/2�/�

mice (Fig. 4A and F). In developing granulomas, C. neofor-mans cells were found to reside mostly intracellularly withinhost phagocytes. The center regions of the more developedgranulomas were necrotic and thus contained cellular debris(Fig. 4F). Numerous extracellular C. neoformans cells werealso found within the necrotic cores of these advanced granu-lomas (Fig. 4F). Since the vast majority of C. neoformans �gcs1cells are observed intracellularly in developing granulomas,while the more-developed granulomas contain cellular debrisand extracellular �gcs1 cells, it is likely that the extracellular C.neoformans cells in the necrotic regions are �gcs1 cells that hadbeen previously internalized by macrophages and then re-leased following lysis of the host phagocytes.

In contrast, the inflammatory response to the �gcs1 strain inlungs of SK1�/� mice did not result in the formation of well-developed granulomas, as observed in �gcs1 strain-challengedSK1/2�/� mice (Fig. 4G). The nodule-like structures in thelungs of �gcs1 strain-challenged SK1�/� mice lacked necrotic

FIG. 3. SK1 prevents dissemination of an obligately intracellular C. neoformans strain for replication. C57BL/6J wild-type mice (SK1/2�/�) andsphingosine kinase 1 knockout mice (SK1�/�) were infected intranasally with 5 � 105 C. neoformans strain �gcs1 cells and monitored daily. (A) Allmice survived until 100 days postchallenge, at which time the fungal burden in the lungs, livers, spleens, kidneys, and brain of 7 mice from eachgroup was determined. (B) Lungs and brains of SK1�/� mice contained statistically significantly greater numbers of C. neoformans cells than lungsand brains from SK1/2�/� mice.

VOL. 78, 2010 ROLE OF SK1 AGAINST C. NEOFORMANS 2345

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

centers, defined rings of recruited immune cells, and encapsu-lating fibrotic tissue (Fig. 4G, H, and I). Although collagendeposition was visible in some areas, it was present in relativelylow abundance and did not surround the outer perimeter ofthe nodule-like structure (Fig. 4H). In comparison to the leu-kocyte infiltration of the lungs from SK1/2�/� mice, signifi-cantly more foamy histiocytes, giant macrophages, and lym-phocytes were observed in lungs from SK1�/� mice (Fig. 4Iand J). In addition, �gcs1 cells were almost exclusively local-

ized intracellularly within macrophages (Fig. 4L). Thesephagocytic cells also contained numerous C. neoformans cellsand/or a copious amount of capsule fragments (Fig. 4L). In-terestingly, these �gcs1 mutant-containing host cells wereabundantly distributed throughout the tissue and were fre-quently found near blood vessels (Fig. 4I and L), an observa-tion possibly foreshadowing the escape from the host defenseperimeter, leading to dissemination. Neutrophils were lessprevalent in the inflammatory response of SK1�/� mice to the

FIG. 4. SK1 modulates the granulomatous inflammatory response to an obligately intracellular C. neoformans strain for replication. Histologyof lungs of C57BL/6J wild-type (SK1/2�/�) mice (A to F) and sphingosine kinase 1 knockout (SK1�/�) mice (G to L) challenged intranasally with5 � 105 C. neoformans �gcs1 cells was analyzed after 100 days postchallenge using Movat staining (A, C, G, I, and L), Verhoeff-van Gieson (VVG)staining (B and H), hematoxylin and eosin (H&E) staining (D, F, and J), and Giemsa staining (E and K). Boxed areas in A and G are magnifiedin the other representative images. (A) White arrows indicate �gcs1 cells stained with Alcian blue in necrotic regions; black arrows indicate thefibrotic tissue encapsulating the well-developed granulomas (magnification, �2). (B) Black arrows indicate the collagen (stained pink/purple)deposited in the fibrotic tissues. White arrows indicate recruited leukocytes inside the fibrotic tissue and within the necrotic core of the granuloma(magnification, �20). (C) White arrows indicate leukocytes (stained deep red) which reside between the fibrotic tissues and within the necroticregion; black arrows indicate intracellular C. neoformans cells (Alcian blue-stained), whereas black arrowheads indicate extracellular C. neoformanscells (magnification, �20). (D) H&E staining reveals a mixed population of leukocytes (stained dark purple) representing the host inflammatoryresponse. Neutrophils (black arrowheads) are the most represented granulocyte cell type at 100 days after C. neoformans challenge and are oftenproximal to AMs containing the intracellular �gcs1 strain (black arrowheads). Note the absence of eosinophils, which are easily identified usingH&E by their bilobed nuclei and reddish-stained cytosol. Crystal-like structures (white arrows; stained pink), which are mostly Ym1 and/or Ym2derived, suggest eosinophil involvement during some junction of the host immune response (magnification, �40). (E) Giemsa staining showsleukocytes, which are stained dark blue-purple, located on the periphery of the granuloma (black arrows). Neutrophils (black arrowheads) are thepredominant leukocytes present in the leukocyte infiltration. (F) The necrotic core of well-developed granulomas contained giant macrophages(black arrows) often containing the crystal-like structures, which are stained pink. Mixed with cellular debris are numerous extracellular �gcs1 cells(white arrowheads) and some intact neutrophils (black arrowheads) (magnification, �100). (G to L) Histology of the lungs of �gcs1 strain-challenged SK1�/� mice. (G) �gcs1 cells are found throughout the lung section and are stained lightly with Alcian blue (magnification, �2). Notethat in both panels G and H, there is no fibrotic tissue with collagen deposition, as observed in panels A and B. The bright red structure in thelower right corner of H is a blood vessel. Note the lack of fibrotic tissue and the giant macrophages containing intracellular �gcs1 cells (blackarrowheads) (magnification, �20). (I) Intracellular and abundant amounts of capsule are stained lightly with Alcian blue (black arrows). The deepred sections correspond to large numbers of leukocytes (white arrows), which are dispersed throughout the section. (J) Foamy histiocytes and giantmacrophages (upper) containing intracellular �gcs1 cells (black arrows) comprise much of the infected lung tissue from �gcs1 strain-challengedSK1�/� mice. Large numbers of leukocytes (lower), specifically neutrophils (white arrows) and lymphocytes (white arrowheads), are located nearphagocytes containing intracellular fungal cells (magnification, �40). (K) Giemsa staining, which stains the nuclei of leukocytes dark blue-purple,shows the presence of increased leukocyte infiltration (compare panel E). This influx of leukocytes comprised predominately neutrophils andlymphocytes. More lymphocytes (black arrows), which have purple-stained cytosols (K), were observed in the host inflammatory response of �gcs1strain-challenged SK1�/� mice than in �gcs1 strain-challenged SK1/2�/� mice (E). (L) Macrophages are filled with Alcian blue-stained intracel-lular �gcs1 cells (white arrows) with large numbers of leukocytes bordering them (black arrowheads).

2346 MCQUISTON ET AL. INFECT. IMMUN.

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

�gcs1 mutant, whereas the lymphocyte infiltration occurred toa much greater extent than in SK1/2�/� mice (Fig. 4J and H).As with �gcs1 strain-challenged SK1/2�/� mice, eosinophilswere rarely observed in the inflammatory response of �gcs1strain-challenged SK1�/� mice. However, the crystal forma-tion commonly found in the lung granulomas of SK1/2�/�micewas not present in the lung granulomas of SK1�/�mice. Theseobservations suggest that SK1 affects host factors influencinggranulocyte recruitment and lymphocyte infiltration. As a re-sult, the inflammatory response in SK1�/� mice to the �gcs1strain does not promote the formation of a proper granu-loma, leading to increased lung fungal burden and ulti-mately resulting in C. neoformans �gcs1 dissemination to thebrain (Fig. 2B).

Role of SK1 in the antimicrobial activities of AMs against C.neoformans. Phagocytosis is an essential component of the ef-fector activities of AMs against C. neoformans. Histopathologyof the lungs from SK1/2�/� and SK1�/� mice challenged withthe �gcs1 mutant revealed a much greater number of intracel-lular C. neoformans cells residing within AMs from SK1�/�

mice than within AMs from SK1/2�/� mice, suggesting thatSK1 may affect the internalization and/or intracellular growthof C. neoformans. Analysis of the mRNA expression of AMsfrom SK1/2�/� mice using real-time RT-PCR revealed thatthese phagocytes express both SK1 and SK2 genes, althoughSK1 was the predominant isoform, as its expression was ap-proximately 2.7-fold times higher than that of SK2 (data notshown). To determine the specific role of SK1 in phagocytosis

of C. neoformans, AMs from SK1/2�/�, SK1�/�, and SK2�/�

mice were isolated, coincubated with C. neoformans at a MOIof 1:1 for 2 h, and processed for light microscopy to determinethe number of internalized C. neoformans cells. Prior to coin-cubation with AMs, C. neoformans cells were opsonized witheither complement from fresh mouse serum, the anti-GXMmonoclonal IgG1 antibody 18B7, or both. Figure 5A shows thephagocytic index of complement-opsonized C. neoformans�gcs1 cells by AMs isolated from SK1/2�/�, SK1�/�, andSK2�/� mice. All three groups of AMs had low phagocyticindices, and no differences were observed. However, AMsfrom SK1�/� mice had significantly greater internalization ofantibody-opsonized �gcs1 cells than AMs from SK1/2�/� mice(65.5 � 9.9 versus 27.5 � 8.5, P � 0.05) (Fig. 5B). The phago-cytic index of the �gcs1 strain by SK2�/� AMs was approxi-mately the same as the phagocytic index of AMs possessingboth SK isoforms (34.5 � 14.5 versus 27.5 � 8.5) (Fig. 5B).Similar results were found when �gcs1 cells were opsonizedwith both complement and antibody (Fig. 5C). These datareveal that the deficiency in SK1 increases phagocytosis of C.neoformans �gcs1 when the fungal cells have been opsonizedwith an IgG1 antibody. The phagocytic indices of AMs isolatedfrom SK1/2�/�, SK1�/�, and SK2�/� mice were also analyzedfor C. neoformans wild-type strain H99. As was observed withthe phagocytosis of the �gcs1 mutant, complement-opsonizedH99 cells were poorly internalized, with no differences betweengroups of AMs. We found that the phagocytic indices of anti-body-opsonized wild-type C. neoformans cells significantly in-

FIG. 5. SK1 deficiency in AMs increases the phagocytosis of C. neoformans. AMs from C57BL/6J wild-type (SK1/2�/�) and sphingosine kinase1 (SK1�/�) and 2 (SK2�/�) knockout mice were incubated with either the C. neoformans �gcs1 strain (A, B, and C) or the wild-type (WT) strainH99 (D, E, and F) at an MOI of 1:1. Cocultures were stopped after 2 h by removal of the medium and fixation with MeOH. Cells were stainedwith Giemsa, and internalization of C. neoformans was determined using light microscopy. A minimum of 500 AMs was examined per cocultureto determine and calculate the phagocytic index. Data are averages � standard deviations from five separate experiments. Student’s t test wasconducted to determine significance (�, P � 0.05).

VOL. 78, 2010 ROLE OF SK1 AGAINST C. NEOFORMANS 2347

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

creased, similarly to those of C. neoformans �gcs1, upon dele-tion of SK1 (Fig. 5E and F), suggesting that this increase isdue solely to the lack of SK1 and not to the lack of GlcCer.Additionally, AMs from SK2�/� mice had phagocytic indi-ces that were comparable to those of AMs from SK1/2�/�

mice, demonstrating that the increased internalization of C.neoformans cells by AMs from SK1�/� mice is specific to thedeficiency of SK1.

To determine if SK1 modulates the ability of AMs to controlthe initial growth of internalized C. neoformans wild-type and�gcs1 mutant cells, the presence of daughter cells, also re-ferred to as budding, was analyzed in C. neoformans cells

internalized within SK1/2�/�, SK1�/�, and SK2�/� AMs. Toenable phagocytosis, C. neoformans cells were opsonized withboth complement from fresh mouse serum and the anti-GXMIgG1 antibody 18B7, which was used in the in vivo phagocyticassays. After 4 h of coincubation, there was no difference inintracellular growth of the C. neoformans �gcs1 or wild-typestrain among different types of AMs (Fig. 6A and B), suggest-ing that SK1 and SK2 do not have roles in controlling C.neoformans intracellular growth.

Effect of SK1 on the virulence of a facultative intracellularC. neoformans strain in an immunosuppressed host. Severallines of evidence suggest that AMs can promote and exacer-bate dissemination, particularly when they are not activated byTh1-specific cytokines (6, 12, 24, 28, 36). Very interestingly,downregulation of SK1, but not SK2, by small interfering RNA(siRNA) in mice leads to a suppression of collagen-specificproinflammatory/Th1 cytokines (e.g., IL-6, TNF-, and IFN-�)(26), suggesting that SK1 plays a role in regulating the immuneresponse to the granuloma formation. Since we found thatlack of SK1 does not affect the infection of C. neoformansWT H99 under conditions of immunocompetence, we won-dered whether we could exacerbate C. neoformans infection inSK1�/� compared to SK1/2�/� mice by inducing immunode-ficiency. Thus, SK1/2�/� and SK1�/� mice were treated with aregimen of the corticosteroid cortisone acetate (CA) and chal-lenged intranasally with a C. neoformans WT strain. Cortico-steroids suppress inflammation by inhibiting the generation ofTh1 response-associated cytokines, which results in reducedT-cell proliferation and numbers. To make sure of the effec-tiveness of the CA regimen (decrease in the number and pro-liferation of circulating lymphocytes), we performed pilot ex-periments to measure the effect of the immunosuppression onwhite blood cell (WBC) count in our mouse model of pulmo-nary cryptococcosis. We found that treatment with CA in miceprofoundly reduced the WBC count (Fig. 7A), specifically that

FIG. 6. SK1 deficiency in AMs does not affect intracellular growthof C. neoformans. AMs from C57BL/6J wild-type (SK1/2�/�) andsphingosine kinase 1 (SK1�/�) and 2 (SK2�/�) knockout mice wereincubated with either the C. neoformans �gcs1 strain (A) or the wild-type (WT) strain H99 (B) at an MOI of 1:1. Medium from cocultureswas removed after 2 h, and cocultures were washed to remove extra-cellular C. neoformans. Fresh medium was then added for an addi-tional 2 h. After a total of 4 h, the medium was removed and MeOHwas added for fixation. Cells were stained with Giemsa, and internal-ization of C. neoformans was determined using light microscopy. Aminimum of 100 internalized C. neoformans cells for each coincubationwere examined per coculture to determine the number of buds. Dataare averages � standard deviations from five separate experiments.Student’s t test was conducted to determine significance (�, P � 0.05).

FIG. 7. Corticosteroid significantly decreases total white blood cells and lymphocytes in WT and SK1�/� mice. C57BL/6J wild-type mice(SK1/2�/�, n 9) and sphingosine kinase knockout mice (SK1�/�, n 9) received intraperitoneal injections of 125 mg/kg of cortisone acetate(CA) 24 h prior to, immediately prior to, and at days 1, 4, and 7 after intranasal challenge with 5 � 105 C. neoformans cells. Blood was collectedfrom the saphenous vein immediately prior to injection with CA. Data are averages � standard deviations from three different pools of blood fromthree mice. Student’s t test was conducted to determine significance (�, P � 0.05; §, P � 0.01; €, P � 0.001; $, P � 0.0001; ¢, P � 0.00001).

2348 MCQUISTON ET AL. INFECT. IMMUN.

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

of lymphocytes (Fig. 7B). Circulating lymphocytes account for60 to 90% (depending on mouse strains) of all WBCs (34, 41),and thus, the observed decrease in the WBC counts upon CAregimen results in a specific depletion of lymphocytes as thenumber of the other cell types examined (e.g., neutrophils,monocytes, eosinophils, and basophils) was not significantlyaffected by CA treatment (data not shown). Interestingly, un-treated SK1/2�/� mice had a significantly greater number ofWBCs and lymphocytes than untreated SK1�/� mice (P �0.05) (Fig. 7), which appears to be in contrast with previousstudies in which no abnormalities in the number of lympho-cytes between SK1/2�/� and SK1�/� mice were reported (1).However, it is possible that this discrepancy may be due to thedifferent sites of blood collection (e.g., venous versus heart), asWBC count varies according to which blood vessel is utilizedfor collection (34, 41). Regardless of this, CA treatment clearlyresults in a decrease in WBCs, and more specifically in lym-phocytes, in both SK1/2�/� and SK1�/� mice.

As expected, we found that CA-treated mice were signifi-cantly more susceptible than untreated mice to the infection byC. neoformans WT strain (Fig. 8 versus Fig. 1). Interestingly,Fig. 8 reveals that CA-treated SK1�/� mice showed a signifi-cantly greater susceptibility than CA-treated SK1/2�/� mice(14.2 � 3.4 days versus 17.5 � 2.3 days, P � 0.05). Theseresults suggest that, under conditions of immunosuppression,SK1 may have a role in controlling infection by a wild-type C.neoformans strain.

DISCUSSION

In this study, we show that SK1 is involved in the regulationof the host immune response to a fungal human pathogen.More specifically, we found that SK1 plays an essential role inthe granuloma formation required to prevent dissemination ofthe C. neoformans �gcs1 strain. In fact, in lungs of SK1�/�

mice infected with the �gcs1 mutant, we found a significantlyincreased fungal burden and lack of granuloma. Thus, the C.neoformans �gcs1 strain was able to disseminate to the brains

of SK1�/� mice. In addition, in vitro experiments revealed thatthe deficiency in SK1 increases phagocytosis of C. neoformansWT and �gcs1 by AMs but does not promote intracellularfungal growth. Interestingly, this increased internalization ofC. neoformans was dependent on antibody- and not comple-ment-mediated opsonization. Finally, we found that the effectof SK1 on host susceptibility to cryptococcosis is more pro-nounced when the host is immunocompromised. To ourknowledge, this is the first report implicating SK1 in the reg-ulation of phagocytosis and virulence of a fungal pathogen.

Evidence supporting a role of SK1 during infection is pro-vided by previous studies showing that the SK1/S1P pathwayregulates immune cell function in vitro and by the fact that thispathway is of particular importance in the lung, as S1P level ismodulated in lung diseases (16, 22, 38). Additional support isalso provided by investigations examining the effects of intra-venous administration of S1P on the histopathology of Myco-bacterium (16, 17, 39). However, to date, the role of SK1, whichis the predominant SK isoform expressed in lung tissue, in themodulation of the host immune response during pulmonaryinfection has not been directly examined.

Using SK1/2�/� and SK1�/� mice as models of pulmonarycryptococcosis, we sought to determine if SK1 was involved inregulating the host immune response to the human pathogenicfungus C. neoformans. We found that SK1 did not affect thevirulence of the WT C. neoformans strain H99, a facultativeintracellular pathogen. In previous studies, histopathology ofpulmonary cryptococcosis in mice revealed that the changes inthe morphology of C. neoformans cells (capsule structure andsize, cell wall thickness, and fungal cell size) occur throughoutthe course of infection and correspond to changes in the lo-calization of the fungal cells (extracellular versus intracellular)within the host (10, 12, 15). Hence, the ability of WT C.neoformans cells to reside both extracellularly and intracellu-larly could be a result of an adaptive advantage toward anormal host immune response and, as a consequence, it mayneglect the role of SK1 in this particular host condition.

Since SK1 and its specific production of S1P regulate anti-microbial actions of macrophages against internalized Myco-bacterium, we hypothesized that SK1 may affect the pathogen-esis and virulence of C. neoformans under conditions that allowthe fungus to replicate only intracellularly within host phago-cytes. To investigate this hypothesis, we examined the effect ofSK1 during pulmonary infection with the C. neoformans �gcs1strain, which cannot replicate at the pH (7.0) and carbon di-oxide concentration (5%) that characterize the extracellularspaces of the lungs. Therefore, this mutant strain of C. neofor-mans must reside intracellularly to replicate in vivo (24, 36).Under normal host conditions (e.g., wild-type mice), internal-ization of �gcs1 cells stimulates an anticryptococcal responsethat leads to containment and killing of the fungus by AMs. Inthis study, comparison of histopathology specimens of lungsfrom SK1/2�/� and SK1�/� mice reveals that there are vastdifferences in the host immune response to the �gcs1 strainafter 100 days of infection. In SK1/2�/� mice, but not inSK1�/� mice, the host inflammatory response contains �gcs1cells within granulomas, thereby limiting disease progression(e.g., organ fungal burden and dissemination).

An abundance of crystal-like structures was observedthroughout the granulomas in the lungs of �gcs1 strain-in-

FIG. 8. Role of SK1 during immunosuppression in host suscepti-bility to a facultative intracellular C. neoformans wild-type strain.C57BL/6J wild-type mice (SK1/2�/�) and sphingosine kinase 1 knock-out mice (SK1�/�) were administered a regimen of cortisone acetate(CA) to induce immunosuppression and challenged intranasally with5 � 105 C. neoformans strain H99 cells, as described in Materials andMethods. The Wilcoxon rank sum test was used to determine signifi-cance (�, P � 0.05).

VOL. 78, 2010 ROLE OF SK1 AGAINST C. NEOFORMANS 2349

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

fected SK1/2�/� mice, particularly in the extracellular space ofthe necrotic core of well-developed granulomas. These crystal-like structures derived from Ym1 and/or Ym2, eosinophil che-motactic factors possessing chitinase activity (19, 33). Thesecrystal structures have been previously described to occur inthe different murine lung diseases, including pulmonary cryp-tococcosis (11, 21). Here, we observed crystal formation inSK1/2�/� mice following challenge with the �gcs1 mutantupon a well-developed granulomatous response. Interestingly,these crystal-like structures were not observed in the lungs ofSK1-deficient mice, which do not show granulomas. Addition-ally, we did not observe granuloma formation or the presenceof these crystal structures in the lungs of SK1/2�/� or SK1�/�

mice infected with the wild-type strain C. neoformans H99(data not shown). Together, these results suggest that lack ofthe GCS1 gene (microbial side) and presence of SK1 (hostside) may have a role in contributing to the production of thesecrystals.

Since SK1 mediates proinflammatory stimuli and its activityis required for cytokine generation and secretion (20, 30, 32,50), it is hypothesized that SK1 promotes the ability of AMs torecruit other effector immune cells and is therefore essential tothe host immune response during pulmonary cryptococcosis.The decreased presence of neutrophils at the site of the infec-tion observed in the lungs from SK1�/� mice compared toSK1/2�/� mice supports this hypothesis. Intriguingly, neutro-phils and not eosinophils were the most represented granulo-cyte population in the host inflammatory response to C. neo-formans �gcs1 in both SK1/2�/� and SK1�/� mice (Fig. 4D, E,J, and K). This is seemingly contradictory to other studies usingC57BL/6J mice in models of pulmonary cryptococcosis, whereeosinophils are the major granulocyte cell type representedduring the host inflammatory response (9, 21). However, thereare two major differences between previous reports and thedata presented here. First, in previous studies, WT C. neofor-mans strains H99 (serotype A) and ATCC 24067 (serotype D)were used. Both of these strains are well established as virulentC. neoformans strains in mouse models of pulmonary crypto-coccosis. The strain used in our mouse model of pulmonarycryptococcosis for this experiment, the C. neoformans �gcs1strain, is an avirulent mutant strain that evokes a granuloma-tous inflammatory response to a degree that has not beendescribed for infections with WT C. neoformans strains. Im-portantly, WT C. neoformans strains are facultative intracellu-lar pathogens, whereas the �gcs1 strain is obligately intracel-lular for replication in vivo. Therefore, the �gcs1 strain elicitsan immune response that is different than that of the WT C.neoformans strain used in previous studies. Second, previousstudies analyzed the host inflammatory response at earlier timepoints (mostly less than 30 days after C. neoformans challenge).Here, we examined the host inflammatory response at 100 daysafter C. neoformans challenge. Thus, although eosinophils mayhave been involved at an earlier junction of the host immuneresponse, eosinophils do not represent a major component ofthe lung host immune response at the time point we examined.Furthermore, new data are emerging that suggest a concertedaction of neutrophils and macrophages in the induction ofadaptive immunity against intracellular pathogens, includingMycobacterium tuberculosis, Toxoplasma gondii, Listeria mono-cytogenes, and Salmonella species (43). It is interesting to spec-

ulate that, under conditions where C. neoformans cells arepressured to reside intracellularly, neutrophils may replaceeosinophils as the main granulocyte recruited to the site ofinfection.

Conditions facilitating intracellular growth of C. neoformanswithin AMs transform the host phagocytes into a niche thatmay actually promote fungal dissemination. Furthermore, thelack of fibrotic tissue surrounding the granuloma in lungs ofSK1�/� mice may enable the dissemination of �gcs1 cells fromthe lungs. Interestingly, SK1 activity and S1P induce humanlung fibroblasts to differentiate into myofibroblasts producingextracellular matrix (ECM) (47), and in several tissue-specificfibroblasts, they increase collagen synthesis, which is requiredfor proper granuloma formation (18, 49). In addition, studieshave recently suggested that SK1 is involved in promoting thestimulation of collagen-specific proinflammatory Th1 cyto-kines, such as IL-6, TNF-, and IFN-� (26). Also, treatmentwith SK1 inhibitors suppresses the production of IFN-� by Tcells and IL-12 by dendritic cells (23), suggesting that SK1 is aregulator of the Th1 immune response. These studies couldexplain why the formation of fibrotic tissues and collagendeposition was almost absent in nodule-like structures found inlungs of SK1�/� mice, further exemplifying the importance ofSK1 in granuloma formation. It is important to emphasize thatthis phenomenon (granuloma formation) cannot be studiedusing C. neoformans wild-type strain H99 because the strain istoo virulent in mice and the animals succumb before a lunggranuloma can be formed.

Unlike many intracellular pathogenic microorganisms, C.neoformans does not possess a mechanism to actively enterhost phagocytes. Internalization of C. neoformans by hostphagocytes, including AMs, occurs through receptor-mediatedpathways, which mostly involve complement and antibody op-sonization (27). We found that AMs from SK1�/� miceshowed significantly increased phagocytosis of both the C. neo-formans WT and �gcs1 strains in comparison to AMs fromSK1/2�/� mice in vitro. In our assays, SK1 deficiency increasedthe internalization of C. neoformans cells only when opsoniza-tion included antibody. Importantly, we found no differences inphagocytosis between C. neoformans wild-type and �gcs1strains in SK1/2�/� and SK1�/� AMs when fungal cells wereopsonized only with complement (Fig. 5A and D). Thus, SK1may have a role in the phagocytosis of C. neoformans not in thetime period shortly following inhalation but rather only afterthe production of C. neoformans-specific antibodies. From ourin vitro phagocytosis assay data, we hypothesize that SK1 mayregulate the expression of cell surface receptors on AMs thatmediate phagocytosis, specifically Fc�Rs that recognize IgGmolecules. This hypothesis is supported by studies showingthat inhibition of SK1, but not SK2, regulates the expression ofsurface molecules such as CD40, CD80, CD86, and MHC classII in certain immune cells (23), suggesting that SK1 may alsobe important for the expression of receptors in AMs involvedin the phagocytosis of C. neoformans. Additionally, treatmentwith exogenous S1P has been shown to increase Fc�RII ex-pression in macrophages (7). On the other hand, a possibilityexists that SK1 regulates phagocytosis of C. neoformans afterhost recognition. In macrophages, the interaction of IgG withFc�RI triggers SK1 activity, which then evokes a signalingcascade involving PI-PLC, cPKC, ERK1/2, and PI 3-kinase,

2350 MCQUISTON ET AL. INFECT. IMMUN.

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

which has been suggested to modulate phagocytosis of Myco-bacterium species (48). Our results, along with these previousreports, suggest that SK1 and its product, S1P, may have a rolein controlling the antibody-mediated phagocytosis of C. neo-formans.

As an opportunistic pathogen, C. neoformans most com-monly infects individuals with impaired cellular immunitycaused by human immunodeficiency virus (HIV) infection,solid organ transplantation, or administration of potent immu-nosuppressive regimens. We found that loss of SK1 increasedsusceptibility to WT C. neoformans when a corticosteroid reg-imen drastically reduced the number of circulating lympho-cytes, suggesting that SK1 may affect host susceptibility to WTC. neoformans (i.e., a facultative intracellular pathogen) duringan immunocompromised state. When immunosuppression oc-curs, AMs are not activated and, therefore, are unable to killinternalized C. neoformans, which can then replicate intracel-lularly to exacerbate disease progression and eventually lead todecreased mouse survival. Thus, the difference in susceptibilityto WT C. neoformans between CA-treated SK1/2�/� mice andCA-treated SK1�/� mice (Fig. 8) may result from the highernumber of C. neoformans cells found intracellularly in thelungs of SK1-deficient mice (Fig. 4).

In conclusion, in this research, we show that SK1 is dispens-able in mice infected with a facultative intracellular C. neofor-mans wild-type strain, but it is required for total containmentof an obligate C. neoformans intracellular pathogen for repli-cation. We also show that SK1 may be important during theinfection caused by a C. neoformans wild-type strain underconditions of immunodeficiency.

ACKNOWLEDGMENTS

We thank all members of M. Del Poeta’s and C. Luberto’s labora-tories for helpful and constructive discussion. We are particularlygrateful to Russell Harley and Masha Bilic for helping with histologyanalysis.

This work was supported by grants AI56168 and AI72142 (toM.D.P.) and was conducted in a facility constructed with support fromthe National Institutes of Health, grant number C06 RR015455 fromthe Extramural Research Facilities Program of the National Center forResearch Resources. T. McQuiston was supported by the GraduateAssistance in Areas of National Need (GAANN) training grant inLipidology and New Technologies (to M.D.P.) from the United StatesDepartment of Education. Maurizio Del Poeta is a Burroughs Well-come New Investigator in Pathogenesis of Infectious Diseases.

REFERENCES

1. Allende, M. L., T. Sasaki, H. Kawai, A. Olivera, Y. Mi, G. van Echten-Deckert, R. Hajdu, M. Rosenbach, C. A. Keohane, S. Mandala, S. Spiegel,and R. L. Proia. 2004. Mice deficient in sphingosine kinase 1 are renderedlymphopenic by FTY720. J. Biol. Chem. 279:52487–52492.

2. Ammit, A. J., A. T. Hastie, L. C. Edsall, R. K. Hoffman, Y. Amrani, V. P.Krymskaya, S. A. Kane, S. P. Peters, R. B. Penn, S. Spiegel, and R. A.Panettieri, Jr. 2001. Sphingosine 1-phosphate modulates human airwaysmooth muscle cell functions that promote inflammation and airway remod-eling in asthma. FASEB J. 15:1212–1214.

3. Argraves, K. M., P. J. Gazzolo, E. M. Groh, B. A. Wilkerson, B. S. Matsuura,W. O. Twal, S. M. Hammad, and W. S. Argraves. 2008. High density lipopro-tein-associated sphingosine 1-phosphate promotes endothelial barrier func-tion. J. Biol. Chem. 283:25074–25081.

4. Billich, A., F. Bornancin, P. Devay, D. Mechtcheriakova, N. Urtz, and T.Baumruker. 2003. Phosphorylation of the immunomodulatory drug FTY720by sphingosine kinases. J. Biol. Chem. 278:47408–47415.

5. Clancy, C. J., M. H. Nguyen, R. Alandoerffer, S. Cheng, K. Iczkowski, M.Richardson, and J. R. Graybill. 2006. Cryptococcus neoformans var. grubiiisolates recovered from persons with AIDS demonstrate a wide range ofvirulence during murine meningoencephalitis that correlates with the expres-sion of certain virulence factors. Microbiology 152:2247–2255.

6. Diamond, R. D., and J. E. Bennett. 1973. Disseminated cryptococcosis inman: decreased lymphocyte transformation in response to Cryptococcusneoformans. J. Infect. Dis. 127:694–697.

7. Duong, C. Q., S. M. Bared, A. Abu-Khader, C. Buechler, A. Schmitz, and G.Schmitz. 2004. Expression of the lysophospholipid receptor family and in-vestigation of lysophospholipid-mediated responses in human macrophages.Biochim. Biophys. Acta 1682:112–119.

8. Duong, M., N. Ouellet, M. Simard, Y. Bergeron, M. Olivier, and M. G.Bergeron. 1998. Kinetic study of host defense and inflammatory response toAspergillus fumigatus in steroid-induced immunosuppressed mice. J. Infect.Dis. 178:1472–1482.

9. Feldmesser, M., A. Casadevall, Y. Kress, G. Spira, and A. Orlofsky. 1997.Eosinophil-Cryptococcus neoformans interactions in vivo and in vitro. Infect.Immun. 65:1899–1907.

10. Feldmesser, M., Y. Kress, and A. Casadevall. 2001. Dynamic changes in themorphology of Cryptococcus neoformans during murine pulmonary infec-tion. Microbiology 147:2355–2365.

11. Feldmesser, M., Y. Kress, and A. Casadevall. 2001. Intracellular crystalformation as a mechanism of cytotoxicity in murine pulmonary Cryptococcusneoformans infection. Infect. Immun. 69:2723–2727.

12. Feldmesser, M., Y. Kress, P. Novikoff, and A. Casadevall. 2000. Cryptococcusneoformans is a facultative intracellular pathogen in murine pulmonary in-fection. Infect. Immun. 68:4225–4237.

13. Feldmesser, M., S. Tucker, and A. Casadevall. 2001. Intracellular parasitismof macrophages by Cryptococcus neoformans. Trends Microbiol. 9:273–278.

14. Futerman, A. H., and Y. A. Hannun. 2004. The complex life of simplesphingolipids. EMBO Rep. 5:777–782.

15. Garcia-Hermoso, D., F. Dromer, and G. Janbon. 2004. Cryptococcus neofor-mans capsule structure evolution in vitro and during murine infection. Infect.Immun. 72:3359–3365.

16. Garg, S. K., M. B. Santucci, M. Panitti, L. Pucillo, M. Bocchino, F. Okajima,P. S. Bisen, C. Saltini, and M. Fraziano. 2006. Does sphingosine 1-phosphateplay a protective role in the course of pulmonary tuberculosis? Clin. Immu-nol. 121:260–264.

17. Garg, S. K., E. Volpe, G. Palmieri, M. Mattei, D. Galati, A. Martino, M. S.Piccioni, E. Valente, E. Bonanno, P. De Vito, P. M. Baldini, L. G. Spagnoli,V. Colizzi, and M. Fraziano. 2004. Sphingosine 1-phosphate induces antimi-crobial activity both in vitro and in vivo. J. Infect. Dis. 189:2129–2138.

18. Gellings Lowe, N., J. S. Swaney, K. M. Moreno, and R. A. Sabbadini. 2009.Sphingosine-1-phosphate and sphingosine kinase are critical for transform-ing growth factor-beta-stimulated collagen production by cardiac fibroblasts.Cardiovasc. Res. 82:303–312.

19. Guo, L., R. S. Johnson, and J. C. Schuh. 2000. Biochemical characterizationof endogenously formed eosinophilic crystals in the lungs of mice. J. Biol.Chem. 275:8032–8037.

20. Hammad, S. M., H. G. Crellin, B. X. Wu, J. Melton, V. Anelli, and L. M.Obeid. 2008. Dual and distinct roles for sphingosine kinase 1 and sphingosine1 phosphate in the response to inflammatory stimuli in RAW macrophages.Prostaglandins Other Lipid Mediat. 85:107–114.

21. Huffnagle, G. B., M. B. Boyd, N. E. Street, and M. F. Lipscomb. 1998. IL-5is required for eosinophil recruitment, crystal deposition, and mononuclearcell recruitment during a pulmonary Cryptococcus neoformans infection ingenetically susceptible mice (C57BL/6). J. Immunol. 160:2393–2400.

22. Jolly, P. S., H. M. Rosenfeldt, S. Milstien, and S. Spiegel. 2002. The roles ofsphingosine-1-phosphate in asthma. Mol. Immunol. 38:1239–1245.

23. Jung, I. D., J. S. Lee, Y. J. Kim, Y. I. Jeong, C. M. Lee, T. Baumruker, A.Billlich, Y. Banno, M. G. Lee, S. C. Ahn, W. S. Park, J. Han, and Y. M. Park.2007. Sphingosine kinase inhibitor suppresses a Th1 polarization via theinhibition of immunostimulatory activity in murine bone marrow-deriveddendritic cells. Int. Immunol. 19:411–426.

24. Kechichian, T. B., J. Shea, and M. Del Poeta. 2007. Depletion of alveolarmacrophages decreases the dissemination of a glucosylceramide-deficientmutant of Cryptococcus neoformans in immunodeficient mice. Infect. Im-mun. 75:4792–4798.

25. Kim, R. H., K. Takabe, S. Milstien, and S. Spiegel. 2009. Export and func-tions of sphingosine-1-phosphate. Biochim. Biophys. Acta 1791:692–696.

26. Lai, W. Q., A. W. Irwan, H. H. Goh, A. J. Melendez, I. B. McInnes, and B. P.Leung. 2009. Distinct roles of sphingosine kinase 1 and 2 in murine collagen-induced arthritis. J. Immunol. 183:2097–2103.

27. Levitz, S. M. 2002. Receptor-mediated recognition of Cryptococcus neofor-mans. Nippon Ishinkin Gakkai Zasshi 43:133–136.

28. Levitz, S. M., S. H. Nong, K. F. Seetoo, T. S. Harrison, R. A. Speizer, andE. R. Simons. 1999. Cryptococcus neoformans resides in an acidic phagoly-sosome of human macrophages. Infect. Immun. 67:885–890.

29. Malik, Z. A., C. R. Thompson, S. Hashimi, B. Porter, S. S. Iyer, and D. J.Kusner. 2003. Cutting edge: Mycobacterium tuberculosis blocks Ca2� sig-naling and phagosome maturation in human macrophages via specific inhi-bition of sphingosine kinase. J. Immunol. 170:2811–2815.

30. Melendez, A., R. A. Floto, D. J. Gillooly, M. M. Harnett, and J. M. Allen.1998. Fc�RI coupling to phospholipase D initiates sphingosine kinase-me-diated calcium mobilization and vesicular trafficking. J. Biol. Chem. 273:9393–9402.

VOL. 78, 2010 ROLE OF SK1 AGAINST C. NEOFORMANS 2351

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

31. Melendez, A. J., E. Carlos-Dias, M. Gosink, J. M. Allen, and L. Takacs. 2000.Human sphingosine kinase: molecular cloning, functional characterizationand tissue distribution. Gene 251:19–26.

32. Melendez, A. J., and F. B. Ibrahim. 2004. Antisense knockdown of sphin-gosine kinase 1 in human macrophages inhibits C5a receptor-dependentsignal transduction, Ca2� signals, enzyme release, cytokine production, andchemotaxis. J. Immunol. 173:1596–1603.

33. Milam, J. E., A. C. Herring-Palmer, R. Pandrangi, R. A. McDonald, G. B.Huffnagle, and G. B. Toews. 2007. Modulation of the pulmonary type 2 T-cellresponse to Cryptococcus neoformans by intratracheal delivery of a tumornecrosis factor alpha-expressing adenoviral vector. Infect. Immun. 75:4951–4958.

34. Nemzek, J. A., G. L. Bolgos, B. A. Williams, and D. G. Remick. 2001.Differences in normal values for murine white blood cell counts and otherhematological parameters based on sampling site. Inflamm. Res. 50:523–527.

35. Oskeritzian, C. A., S. Milstien, and S. Spiegel. 2007. Sphingosine-1-phos-phate in allergic responses, asthma and anaphylaxis. Pharmacol. Ther. 115:390–399.

36. Rittershaus, P. C., T. B. Kechichian, J. C. Allegood, A. H. Merrill, Jr., M.Hennig, C. Luberto, and M. Del Poeta. 2006. Glucosylceramide synthase isan essential regulator of pathogenicity of Cryptococcus neoformans. J. Clin.Invest. 116:1651–1659.

37. Rivera, J., and A. Casadevall. 2005. Mouse genetic background is a majordeterminant of isotype-related differences for antibody-mediated protectiveefficacy against Cryptococcus neoformans. J. Immunol. 174:8017–8026.

38. Rosenfeldt, H. M., Y. Amrani, K. R. Watterson, K. S. Murthy, R. A. Panet-tieri, Jr., and S. Spiegel. 2003. Sphingosine-1-phosphate stimulates contrac-tion of human airway smooth muscle cells. FASEB J. 17:1789–1799.

39. Sali, M., G. Delogu, E. Greco, S. Rocca, V. Colizzi, G. Fadda, and M.Fraziano. 2009. Exploiting immunotherapy in Mycobacterium tuberculosis-infected mice: sphingosine 1-phosphate treatment results in a protective ordetrimental effect depending on the stage of infection. Int. J. Immunopathol.Pharmacol. 22:175–181.

40. Santucci, M. B., E. Greco, M. De Spirito, G. Arcovito, G. De Angelis, R.Cauda, and M. Fraziano. 2007. Sphingosine 1-phosphate promotes antigen

processing and presentation to CD4� T cells in Mycobacterium tuberculo-sis-infected monocytes. Biochem. Biophys. Res. Commun. 361:687–693.

41. Schnell, M. A., C. Hardy, M. Hawley, K. J. Propert, and J. M. Wilson. 2002.Effect of blood collection technique in mice on clinical pathology parame-ters. Hum. Gene Ther. 13:155–161.

42. Shea, J. M., and M. Del Poeta. 2006. Lipid signaling in pathogenic fungi.Curr. Opin. Microbiol. 9:352–358.

43. Silva, M. T. 28 January 2010, posting date. Neutrophils and macrophageswork in concert as inducers and effectors of adaptive immunity againstextracellular and intracellular microbial pathogens. J. Leukoc. Biol. [Epubahead of print.]

44. Snider, A. J., T. Kawamori, S. G. Bradshaw, K. A. Orr, G. S. Gilkeson, Y. A.Hannun, and L. M. Obeid. 2009. A role for sphingosine kinase 1 in dextransulfate sodium-induced colitis. FASEB J. 23:143–152.

45. Taborda, C. P., and A. Casadevall. 2002. CR3 (CD11b/CD18) and CR4(CD11c/CD18) are involved in complement-independent antibody-mediatedphagocytosis of Cryptococcus neoformans. Immunity 16:791–802.

46. Thompson, C. R., S. S. Iyer, N. Melrose, R. VanOosten, K. Johnson, S. M.Pitson, L. M. Obeid, and D. J. Kusner. 2005. Sphingosine kinase 1 (SK1) isrecruited to nascent phagosomes in human macrophages: inhibition of SK1translocation by Mycobacterium tuberculosis. J. Immunol. 174:3551–3561.

47. Urata, Y., Y. Nishimura, T. Hirase, and M. Yokoyama. 2005. Sphingosine1-phosphate induces alpha-smooth muscle actin expression in lung fibro-blasts via Rho-kinase. Kobe J. Med. Sci. 51:17–27.

48. Yadav, M., L. Clark, and J. S. Schorey. 2006. Macrophage’s proinflammatoryresponse to a mycobacterial infection is dependent on sphingosine kinase-mediated activation of phosphatidylinositol phospholipase C, protein kinaseC, ERK1/2, and phosphatidylinositol 3-kinase. J. Immunol. 176:5494–5503.

49. Youm, J. K., H. Jo, J. H. Hong, D. M. Shin, M. J. Kwon, S. K. Jeong, B. D.Park, E. H. Choi, and S. H. Lee. 2008. K6PC-5, a sphingosine kinase acti-vator, induces anti-aging effects in intrinsically aged skin through intracellu-lar Ca2� signaling. J. Dermatol. Sci. 51:89–102.

50. Zhi, L., B. P. Leung, and A. J. Melendez. 2006. Sphingosine kinase 1 regu-lates proinflammatory responses triggered by TNF in primary humanmonocytes. J. Cell. Physiol. 208:109–115.

Editor: G. S. Deepe, Jr.

2352 MCQUISTON ET AL. INFECT. IMMUN.

on October 21, 2020 by guest

http://iai.asm.org/

Dow

nloaded from