STAT1 Signaling Is Essential for Protection against neoformans Infection in Mice

This information is current as Chrissy M. Leopold Wager, Camaron R. Hole, Karen L. of September 28, 2021. Wozniak, Michal A. Olszewski and Floyd L. Wormley, Jr. J Immunol 2014; 193:4060-4071; Prepublished online 8 September 2014; doi: 10.4049/jimmunol.1400318

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2014 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

STAT1 Signaling Is Essential for Protection against Cryptococcus neoformans Infection in Mice

Chrissy M. Leopold Wager,*,† Camaron R. Hole,*,† Karen L. Wozniak,*,† Michal A. Olszewski,‡,x and Floyd L. Wormley, Jr.*,†

Nonprotective immune responses to highly virulent Cryptococcus neoformans strains, such as H99, are associated with Th2-type cytokine production, alternatively activated macrophages, and inability of the host to clear the . In contrast, experimental studies show that protective immune responses against are associated with Th1-type cytokine production and classical macrophage activation. The protective response induced during C. neoformans strain H99g (C. neoformans strain H99 engineered to produce murine IFN-g) infection correlates with enhanced phosphorylation of the transcription factor STAT1 in macrophages; however, the role of STAT1 in protective immunity to C. neoformans is unknown. The current studies examined the effect of STAT1 deletion in murine models of protective immunity to C. neoformans. Survival and fungal burden were evaluated in Downloaded from wild-type and STAT1 knockout (KO) mice infected with either strain H99g or C. neoformans strain 52D (unmodified clinical isolate). Both strains H99g and 52D were rapidly cleared from the lungs, did not disseminate to the CNS, or cause mortality in the wild-type mice. Conversely, STAT1 KO mice infected with H99g or 52D had significantly increased pulmonary fungal burden, CNS dissemination, and 90–100% mortality. STAT1 deletion resulted in a shift from Th1 to Th2 cytokine bias, pronounced lung inflammation, and defective classical macrophage activation. Pulmonary macrophages from STAT1 KO mice exhibited defects in

NO production correlating with inefficient inhibition of fungal proliferation. These studies demonstrate that STAT1 signaling is http://www.jimmunol.org/ essential not only for regulation of immune polarization but also for the classical activation of macrophages that occurs during protective anticryptococcal immune responses. The Journal of Immunology, 2014, 193: 4060–4071.

ryptococcus neoformans, the predominant etiological therapies are oftentimes rendered ineffective because of the devel- agent of cryptococcosis, is an opportunistic fungal path- opment of drug resistance or an inability of the host’s immune C ogen and frequent cause of life-threatening infection in system to participate in eradicating the (17–19), necessi- individuals with impaired T cell function (i.e., persons with AIDS, tating new therapeutic strategies that consider the immune status of lymphoid malignancies, and recipients of immunosuppressive the host population commonly impacted by cryptococcal disease. by guest on September 28, 2021 therapies) (1–13). Cryptococcosis is considered an AIDS-defining C. neoformans is ubiquitous in the environment and exposure illness and the most common mycological cause of morbidity and via inhalation of desiccated or into the lung mortality among AIDS patients (14). Global estimates show that alveoli often occurs in early childhood (20–22). The over- 1 million cases of cryptococcal occur in AIDS patients whelming majority of exposures result in asymptomatic disease each year, resulting in ∼625,000 deaths (14). C. neoformans also and possible latency in immunocompetent individuals. However, remains the third most common invasive fungal infection among in immunocompromised individuals, C. neoformans can spread organ transplant recipients (13, 15, 16). Current antifungal drug from the lungs to the CNS, resulting in life-threatening menin- goencephalitis (23). Because inhalation is the principal route of *Department of Biology, University of Texas, San Antonio, TX 78249; †South Texas entry, the host is dependent on resident pulmonary , Center for Emerging Infectious Diseases, University of Texas, San Antonio, TX 78249; such as macrophages, to contain the pathogen and prevent dis- ‡Veterans Affairs Ann Arbor Health System, University of Michigan Health System, Ann Arbor, MI 48109; and xDivision of Pulmonary and Critical Care Medicine, De- semination from the lungs. Studies using various experimental partment of Internal Medicine, University of Michigan Health System, Ann Arbor, models of pulmonary cryptococcosis indicate that protection MI 48109 against C. neoformans infection is associated with the induction of Received for publication February 4, 2014. Accepted for publication August 6, 2014. Th1-type cytokine responses (IL-2, IL-12, IFN-g, and TNF-a), This work was supported by Research Grant 2RO1 AI071752 from the National increased lymphocyte infiltration, and classical macrophage (M1) Institute of Allergy and Infectious Diseases of the National Institutes of Health (to F.L.W.), Grant W911NF-11-1-0136 from the Army Research Office of the Depart- activation (24–30). In contrast, Th2-type cytokine responses (IL-4, ment of Defense (to F.L.W.), and Department of Veterans Affairs Merit Review IL-5, and IL-13) are detrimental to the host and are associated Grant 1I01BX000656 (to M.A.O.). with alternative macrophage (M2) activation (31–33). M1 mac- The content of this article is solely the responsibility of the authors and does not rophages are known for their capacity to efficiently kill phago- necessarily represent the official views of the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, or the University of Texas at cytized organisms through the production of reactive oxygen and San Antonio. nitrogen species (25, 31, 34–36). M2 macrophages, however, are Address correspondence and reprint requests to Dr. Floyd L. Wormley, Jr., Depart- associated with wound healing and are not efficiently microbicidal ment of Biology, University of Texas at San Antonio, One UTSA Circle, San Antonio, TX 78249-0062. E-mail address: fl[email protected] against C. neoformans (25, 31, 34–36). M1 macrophages produce L Abbreviations used in this article: Arg1, arginase 1; FIZZ1, found in inflammatory NO and -citrulline via inducible NO synthase (iNOS) by acting zone 1; H99g,IFN-g–producing Cryptococcus neoformans strain; iNOS, inducible on the substrate L-arginine. Arginase-1 (Arg1) in M2 macrophages NO synthase; KO, knockout; ROS, reactive oxygen species; WT, wild-type; YM1, competes with iNOS for L-arginine to produce L-ornithine and chitinase 3-like 3; YPD, yeast extract/peptone/dextrose. urea (37), which is not fungicidal against C. neoformans (38, 39). Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 Therefore, macrophage polarization toward an M2 phenotype, www.jimmunol.org/cgi/doi/10.4049/jimmunol.1400318 The Journal of Immunology 4061 which occurs during infection with wild-type (WT) C. neoformans inoculation with 1 3 104 CFU C. neoformans strains H99g or 52D in 50 ml strain H99, results in uncontrolled fungal growth, dissemination, sterile PBS. The inocula used for nasal inhalation were verified by quan- and exacerbation of disease (31, 35, 40, 41). titative culture on YPD agar. Mice were euthanized on predetermined days by CO2 inhalation, followed by cervical dislocation, and lung tissues were Experimental pulmonary infection in mice with a C. neoformans excised using aseptic technique. For survival analyses, mice were inoculated strain engineered to express IFN-g, designated H99g, results in as stated above and monitored twice daily for up to 60 d postinoculation. Th1-type and IL-17A cytokine responses, M1 macrophage acti- Fungal burden vation, and resolution of the acute infection (26, 42). In addition, mice protectively immunized with C. neoformans strain H99g For fungal burden analysis, the left lobe of the lung was removed and developed an M1 macrophage activation phenotype during sec- homogenized in 1 ml sterile PBS. A 50-ml aliquot was removed, serially diluted in sterile PBS, and plated on YPD agar supplemented with chlor- ondary challenge with a non–IFN-g–producing WT C. neofor- amphenicol. Plates were incubated for 48 h at 30˚C, and fungal colonies mans strain (H99) and displayed enhanced fungistasis and NO were recorded. production compared with macrophages from nonprotectively Cytokine analysis immunized mice following secondary challenge (24). Specifically, resolution of pulmonary C. neoformans infection in protectively Cytokine production in lung tissues was analyzed using the Bio-Plex protein immunized mice was associated with M1 macrophage activation array system (Luminex-based technology; Bio-Rad Laboratories, Hercules, CA). Briefly, the left lobe of the lung was excised and homogenized in ice- that coincided with robust STAT1 phosphorylation (24). STAT1 cold sterile PBS (1 ml). An aliquot (50 ml) was taken to quantify the pulmonary signaling via the IFN-g-JAK1/2-STAT1 pathway leads to the fungal burden and an antiprotease buffer solution containing PBS, protease formation of phospho-STAT1 homodimers that translocate to the inhibitors (inhibiting cysteine, serine, and other metalloproteinases), and nucleus, bind to g-activated sequences, and promote the tran- 0.05% Triton X-100 was added to the homogenate and then clarified by Downloaded from centrifugation (800 3 g) for 10 min. Supernatants from pulmonary homoge- scription of STAT1 target genes (reviewed in Ref. 43). STAT1 sig- nates were assayed for the presence of IL-1a,IL-1b, IL-2, IL-3, IL-4, IL-5, naling and its downstream products have been tied to the generation IL-6, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13, IL-17A, CCL5/RANTES, of Th1-type immune responses (44–46) that are protective against Eotaxin, CXCL1/KC, CCL3/MIP-1a,CCL4/MIP-1b, CCL2/MCP-1, G-CSF, cryptococcosis and associated with M1 macrophage activation GM-CSF, TNF-a,andIFN-g. (24–30, 32). Little is known about the role of STAT1 in antifungal Pulmonary leukocyte isolation immunity. Specific mutations that alter STAT1 function (loss-of- http://www.jimmunol.org/ function or gain-of-function mutations) have been shown to increase Lungs were excised on days 7 and 10 postinoculation and digested en- zymatically at 37˚C for 30 min in 10 ml digestion buffer (RPMI 1640 susceptibility/interfere with host defenses to fungal infections such medium and 1 mg/ml collagenase type IV [Sigma-Aldrich, St. Louis, as chronic mucocutaneous (47, 48), , MO]) with intermittent (every 10 min) stomacher homogenizations. The and (49). The role of STAT1 signaling during digested tissues were then successively filtered through sterile 70- and Cryptococcus infections has not been studied previously. 40-mm nylon filters (BD Biosciences, San Diego, CA) to enrich for leu- kocytes, and then, cells were washed with sterile HBSS. Erythrocytes were The objective of the studies presented in this paper was to de- lysed by incubation in NH4Cl buffer (0.859% NH4Cl, 0.1% KHCO3, and termine the role of STAT1 in the generation of protective immunity 0.0372% Na2EDTA [pH 7.4]; Sigma-Aldrich) for 3 min on ice, followed against C. neoformans, including its role in mediating M1 mac- by a 2-fold excess of PBS. The resulting leukocyte population was then rophage activation and antifungal activity in mice. Using STAT1 enriched for macrophages by positive selection using magnetic beads la- by guest on September 28, 2021 knockout (KO) mice, we demonstrated that STAT1 signaling pro- beled with F4/80 Ab (Miltenyi Biotec, Auburn, CA), according to the manufacturer’s recommendations. motes Th1-type immune responses but, most importantly, is required for M1 macrophage activation and subsequent protection against Real-time PCR analysis experimental pulmonary infection with C. neoformans strain H99g. Total RNA was isolated from purified F4/80+ cells using TRIzol reagent To our knowledge, these data provide the first evidence of an effector (Invitrogen, Carlsbad, CA) and then DNase (Qiagen, Germantown, MD) cell population and the intracellular signaling mechanism by which treated to remove possible traces of contaminating DNA, according to the the host mounts a protective immune response to C. neoformans. manufacturer’s instructions. Total RNA was subsequently recovered using the Qiagen RNeasy kit. cDNA was synthesized from 1 mg total RNA using the oligo(dT) primer and reagents supplied in the SuperScript III RT kit Materials and Methods (Invitrogen), according to the manufacturer’s instructions. The cDNA was Mice used as a template for real-time PCR analysis using the TaqMan gene ex- 1tm1Rds pression assay (Applied Biosystems, Foster City, CA), according to the Female STAT1 KO (129S6/SvEv-Stat )andWTcontrolmice manufacturer’s instructions. All real-time PCRs were performed using the (129S6/SvEvTac) from Taconic Farms (Germantown, NY) and female 7300 real-time PCR system (Applied Biosystems). For each real-time PCR, IFN-g KO (C.129S7(B6)-Ifngtm1Ts/J) and WT control mice (BALB/cJ) a master mix was prepared on ice with TaqMan gene expression assays spe- from The Jackson Laboratory (Bar Harbor, ME) were used throughout cific for iNOS, IFN-g,TNF-a, CXCL9, CXCL10, IRF-1, suppressor of cy- these studies. Mice were housed at the University of Texas at San Antonio tokine signaling-1, IL-17A, chitinase 3-like 3 (YM1), found in inflammatory Small Laboratory Vivarium and handled according to guidelines zone 1 (FIZZ1), Arg1, IL-4, IL-13, and CD206 (Applied Biosystems). Taq- approved by the Institutional Animal Care and Use Committee. Man rodent GAPDH (Applied Biosystems) was used as an internal control. Strains and media The thermal cycling parameters contained an initial denaturing cycle of 95˚C for 10 min, followed by 40 cycles of 95˚C for 15s and 60˚C for 60s. Results of C. neoformans strain H99g (derived from H99 serotype A, mating type a) the real-time PCR data were derived using the comparative Ct method to (50) and C. neoformans strain 52D (serotype D) (a gift from Dr. Brian detect relative gene expression as described previously (26). Wickes, University of Texas Health Science Center at San Antonio, San Antonio, TX) were recovered from a 15% glycerol stock stored at 280˚C Immunohistochemistry and histology prior to use in the experiments described in this study. The strain was Mice were sacrificed according to approved protocols, and the lungs were maintained on yeast extract/peptone/dextrose (YPD) medium agar plates immediately perfused with sterile PBS by transcardial perfusion through the (BD Biosciences, Sparks, MD). Yeast cells were grown for 16–18 h at 30˚C right ventricle. The pericardium and trachea were exposed by dissection, with shaking in liquid YPD broth, collected by centrifugation, washed three and an incision was made in the trachea for the insertion of a sterile flexible times with sterile PBS, and viable were quantified using trypan blue cannula attached to a 3-ml syringe. The lungs were slowly inflated with 0.5– dye exclusion on a hemacytometer. 0.7 ml 10% ultrapure formaldehyde (Polysciences, Warrington, PA), ex- Pulmonary cryptococcal infections cised, and immediately fixed in 10% ultrapure formaldehyde for 24–48 h. The lungs were then transferred to 70% ethanol and subsequently mounted Mice were anesthetized with 2% isoflurane using a rodent anesthesia device into cassettes and paraffin embedded by personnel at the University of (Eagle Eye Anesthesia, Jacksonville, FL) and then given an intranasal Texas Health Science Center at San Antonio Histology and Immunohis- 4062 PROTECTIVE MACROPHAGE RESPONSES AGAINST C. NEOFORMANS tochemistry Laboratory. After paraffin embedding, 5-mm sections were cut and A/Jcr mice (24, 26, 50–52) and C129 mice (data not shown) and stained using H&E with mucicarmine at McClinchey Histology Labs results in clearance of the acute infection and the induction of (Stockbridge, MI). Sections were analyzed with light microscopy using protective immunity against a subsequent challenge with a WT a Nikon microscope and microphotographs taken using Digital Micro- photography system DFX1200 with ACT-1 software (Nikon, Tokyo, C. neoformans strain that does not produce IFN-g. Protection in Japan) at the Ann Arbor VA Health System. H99g-immunized mice against WT C. neoformans challenge oc- Macrophage anticryptococcal assay, reactive oxygen species curred concurrent to the induction of STAT1 signaling within detection assay, and NO production assay macrophages and M1 macrophage activation (24). To determine the requirement for STAT1 signaling in mediating protection + Cell viability and quantity of F4/80 -enriched macrophage populations against pulmonary cryptococcosis, STAT1 KO (129S6/SvEv- were assessed using trypan blue exclusion in a hemacytometer. Macro- tm1Rds phages were cultured at a density of 5 3 105 cells per well in a 96-well Stat1 ) and WT controls (129S6/SvEvTac) were intranasally tissue culture plate in RPMI 1640 medium (without phenol red) (Life inoculated with H99g. Mice survival (morbidity) was monitored Technologies, Grand Island, NY) supplemented with 10% heat-inactivated for 60 d postinoculation in one subgroup, whereas pulmonary FBS, 2mM L-glutamine, and 100 mg/ml penicillin-streptomycin (complete fungal burden was quantified in another subgroup of infected medium). Macrophages were incubated in complete medium at 37˚C with at selected time points. WT mice had a 90% survival rate 5% CO2. Initial fungal burden was determined by lysis of the macrophages using sterile deionized water, followed by serial dilution and plating on at day 60 postinoculation with H99g (Fig. 1A). In comparison, YPD agar supplemented with chloramphenicol (Mediatech, Manassas, VA) STAT1 KO mice began to succumb to H99g infection on day 12 for 48 h at 30˚C. After 24 h of incubation, cell supernatants were collected, postinoculation and had only a 10% survival rate by day 60 and an aliquot (50 ml) was used to determine NO production with Griess postinoculation (p , 0.001; Fig. 1A) with a median survival time reagent (Sigma-Aldrich), according to the manufacturer’s instructions. Light absorbance values were measured at 540 nm using a BioTek Elx808 of 18 d. The fungal burden was evaluated on days 7 and 10 Downloaded from absorbance microplate reader with Gen5 version 1.04.5 software. Alter- postinoculation with H99g (i.e., at the time points prior to the natively, F4/80+ macrophages were isolated and adjusted to 5 3 105 cells/ onset of mortality in STAT1 KO mice), which ensured the most well as described above and reactive oxygen species (ROS) measured accurate statistical analysis of the fungal burden. STAT1 KO mice using CellROX Deep Red Reagent (Life Technologies), according to showed significantly higher pulmonary fungal burden on day 10 manufacturer’s instructions and measured using BD FACSArray software , on a BD FACSArray flow cytometer (BD Biosciences). postinoculation compared with infected WT mice (p 0.001;

Fig. 1B). Fungal burden in the WT mice did not increase from http://www.jimmunol.org/ Flow cytometry days 7 to 10, indicating that the host was able to control the Standard methodology was used for the direct immunofluorescence of growth of the yeast. However, fungal burden in STAT1 KO mice pulmonary leukocytes. Briefly, in 96-well U-bottom plates, 100-ml con- 3 6 was significantly increased on day 10 compared with day 7 in taining 1 10 cells in PBS plus 2% FBS (FACS buffer) were incubated , with 50 ml Fc block (BD Biosciences) diluted in FACS buffer for 5 min to these mice (p 0.001), revealing that they were unable to control block nonspecific binding of Abs to cellular FcRs. Subsequently, an op- proliferation of C. neoformans in the lungs. Therefore, STAT1 timal concentration of fluorochrome-conjugated Abs (between 0.06 and signaling is required for control of pulmonary infection with 6 0.5 mg/1 3 10 cells in 50 ml FACS buffer) was added in various com- C. neoformans strain H99g in mice. binations to allow for dual or triple staining experiments, and cells were To determine the requirement of STAT1 signaling for protection incubated for 30 min at 4˚C. Following incubation, the cells were washed by guest on September 28, 2021 three times with FACS buffer and were fixed in 200 ml 2% ultrapure against infection with a WT strain of C. neoformans, WT and formaldehyde (Polysciences) diluted in FACS buffer (fixation buffer). STAT1 KO mice were intranasally inoculated with an unmodified Cells incubated with either FACS buffer alone or single fluorochrome- clinical isolate, C. neoformans strain 52D, and survival monitored conjugated Abs were used to determine positive staining and spillover/ for 45 d postinfection. Inoculation with this strain is generally compensation calculations, and the flow cytometer determined back- ground fluorescence. The samples were analyzed using BD FACSArray controlled in BALB/c mice; however, it is capable of inducing software on a BD FACSArray flow cytometer (BD Biosciences). Dead a chronic infection in C57BL/6 mice (53, 54). All WT mice cells were excluded on the basis of forward angle and 90˚ light scatter. For infected with C. neoformans strain 52D were alive and appeared data analyses, 30,000 events (cells) were evaluated from a predominantly + healthy upon conclusion of the experiment (Fig. 2A). In contrast, leukocyte population identified by backgating from CD45 -stained cells. all STAT1 KO mice succumbed to the infection or were moribund The absolute number of total leukocytes was quantified by multiplying the total number of cells observed by hemacytometer counting by the by day 28 with a median survival time of 26.5 d. Postmortem percentage of CD45+ cells determined by flow cytometry. The absolute analysis showed that moribund STAT1 KO mice had significantly number of leukocytes (CD45+ cells), CD4+/CD3+ T cells, CD8+/CD3+ higher pulmonary fungal burden compared with WT mice (p , + + + + + + T cells, CD19 /B220 B cells, PMNs (1A8 /CD45 ), MFs(F4/80/CD45 ), 0.01; Fig. 2B). In addition, STAT1 KO mice showed evidence of DCs (CD11bint/CD11c+/CD45+), NK cells (Nkp46+/DX5+), NKT cells (Nkp46+/DX5+/CD4+), and eosinophils (SiglecF+/CD11bint) was deter- massive CNS dissemination (Fig. 2B) unlike the WT mice, which mined by multiplying the percentage of each gated population by the total were negative for CNS fungal cultures with the exception of 1 of number of CD45+ cells. 10 WT mice that had detectable (one colony) C. neoformans in the Statistical analysis brain. These results indicate that STAT1 signaling is required for the induction of immune protection against C. neoformans and The unpaired Student t test was used to analyze fungal burden, cytokine/ prevention of fungal dissemination into the CNS. chemokine levels, pulmonary cell populations, ROS, and NO assays (two- tailed) using GraphPad Prism version 5.00 for Windows (GraphPad Soft- STAT1 deletion results in the development of severe pathology ware, San Diego, CA) to detect statistically significant differences. The One-Way ANOVA with Tukey’s posttest was used for real-time PCR in C. neoformans strain H99g-infected lungs analysis (GraphPad Software) to detect significant differences. Survival To further characterize the defect in pulmonary control of cryp- data were analyzed using the log-rank test (GraphPad Software). Signifi- cant differences were defined as *p , 0.05, **p , 0.01, or ***p , 0.001. tococcal growth in STAT1 KO compared with WT mice, we ex- amined histological sections of the infected and uninfected mouse lungs at day 10 postinoculation. Although the uninfected lungs of Results WT and STAT1 KO mice showed normal morphology (Fig. 3A, STAT1 is critical for the induction of protection against 3B), histological examination of infected lungs at day 10 postin- C. neoformans infection in mice oculation revealed the development of severe lung pathology in Previous studies in our laboratory demonstrated that experimental the absence of STAT1 expression (Fig. 3D, 3F). The inflamed pulmonary infection with C. neoformans strain H99g in BALB/c areas in the lungs of the WT mice at day 10 show that both in- The Journal of Immunology 4063

FIGURE 1. STAT1 signaling is required for control of pulmonary fungal burden and survival following inoculation with C. neoformans strain H99g. 129S6/SvEv (WT) and STAT1 KO mice were given an intranasal inoculation with 1 3 104 CFU C. neoformans strain H99g. Mice were observed for up to 60 d for survival analysis (A) or pulmonary fungal burden was analyzed at days 7 and 10 postinoculation (B). Experiments are cumulative of two experiments using five mice per group per time point (A) or two experiments using four mice per group per time point (B). **p , 0.01, ***p , 0.001. fection and the inflammatory response are well contained within flammatory response were caused predominantly by differential granulomatous foci, whereas the majority of the lung tissue distribution/density of the inflammatory infiltrates, rather than the remains clear of the infection/inflammation (Fig. 3C). In contrast, major changes in total leukocyte number. In addition, there were virtually all lung tissue from STAT1 KO mice is severely laden no significant differences in the numbers of infiltrating leukocyte with cryptococcal organisms and infiltrated with inflammatory subsets including macrophages, dendritic cells, CD4+ and CD8+ Downloaded from cells (Fig. 3D), consistent with uncontrolled growth of the T cells, B cells, and neutrophils (Fig. 4B–G) at days 7 and 10 microbe and a nonprotective immune/inflammatory response. postinoculation with H99g in STAT1 KO mice compared with WT Analysis of histological sections under high-power magnification mice. However, the absolute number of NK cells in STAT1 KO demonstrates that, in addition to major differences in fungal mice was significantly lower (p , 0.05; Fig. 4I), and the absolute burden, cellular composition of inflammatory infiltrates varies number of CD4+ T cells in STAT1 KO mice trended lower (p , between the WT and STAT1 KO mice. Minimal presence of 0.08; Fig. 4D) at day 10 postinoculation compared with WT http://www.jimmunol.org/ cryptococcal organisms and predominance of lymphoid cells and mice. In contrast, eosinophils, which are associated with a Th2- macrophage-type cells within the infiltrates of WT mice is con- type immune response and a hallmark of disease progression, sistent with the development of a protective immune response were significantly increased in STAT1 KO mice at days 7 and (Fig. 3E). In contrast, the abundance of cryptococcal organisms 10 postinoculation compared with WT mice (p , 0.05; Fig. 4H). accompanied by mixed inflammatory infiltrates with enhanced Neutrophils also showed an increasing trend (p , 0.146; Fig. 4G) presence of granulocytes (eosinophils and PMNs) and lower fre- in STAT1 KO mice at day 10 postinoculation. Collectively, these quency of lymphoid cells characterize the lesions within the data suggest that STAT1 is not required for induction of the in- infected lungs of STAT1 KO mice (Fig. 3F). Such severe pneu- flammatory response/recruitment of leukocytes into the lungs; by guest on September 28, 2021 monia and lung consolidation could explain the mortality ob- however, STAT1 ablation does impact the phenotype of the cel- served in STAT1 KO mice and indicate that the immune response lular response to C. neoformans strain H99g. in the lungs of STAT1 KO mice, albeit robust, is nonprotective. STAT1 signaling does not affect the induction of a STAT1 deletion results in altered inflammatory cell cell-mediated immune response but does contribute to an accumulation in C. neoformans strain H99g-infected lungs increased proinflammatory cytokine response and a shift in Our histology data suggest that STAT1 deletion results in a sig- Th1-type/Th2-type cytokine bias nificantly altered immune/inflammatory response in H99g-infected The changes in fungal control and cellular composition of the lungs. To further characterize and quantify these differences, flow inflammatory infiltrates suggested that STAT1 expression affected cytometry analysis of isolated pulmonary leukocytes from enzy- the type of immune response/cytokine response during pulmonary matically dispersed lungs of WT and STAT1 KO mice was per- inoculation with C. neoformans strain H99g. We next determined formed at days 7 and 10 postinoculation. A trend toward increased how STAT1 deletion impacted cytokine expression in the infected leukocyte infiltration to the lungs of the STAT1 KO mice has been lungs by analyzing lung homogenates from STAT1 KO and WT noted at day 10. However, we observed no statistically significant mice on days 7 and 10 postinoculation with C. neoformans strain differences in total CD45+ leukocyte numbers (Fig. 4A), sug- H99g for cytokine protein content. The levels of Th1-type cyto- gesting that the differences in histological appearance of the in- kines IL-2, IL-12p40, and IL-12p70 in STAT1 KO mice were

FIGURE 2. Control of pulmonary fungal burden, CNS dissemination, and survival during WT C. neoformans strain 52D inoculation are dependent on STAT1 signaling. 129S6/SvEv (WT) and STAT1 KO mice were given an intranasal inoculation with 1 3 104 CFU C. neoformans strain 52D. Mice were observed for up to 45 d for survival analysis (A), and pulmonary and brain fungal burden were analyzed postmortem (B). Experiments are cumulative of two experiments using five mice per group per time point. **p , 0.01, ***p , 0.001. 4064 PROTECTIVE MACROPHAGE RESPONSES AGAINST C. NEOFORMANS

FIGURE 3. Uncontrolled C. neoformans expansion and severe lung pathology develop in H99g-infected STAT1 KO mice by day 10 postinfection. 129S6/SvEv (WT) and STAT1 KO mice were given an intranasal inoculation with 1 3 104 CFU C. neoformans strain H99g or left uninfected. Lungs of Downloaded from uninfected (A and B) and H99g-infected (C–F), WT (upper row), and STAT1 KO mice (lower row) were collected on day 10 postinoculation, processed, and analyzed using light microscope. Deletion of STAT1 that did not affect morphology of uninfected lungs has resulted in the development of severe lung pathology and massive expansion of fungus by day 10 postinoculation. Note that the infection and inflammatory response are contained within portions of the lungs in the WT mice (C), whereas virtually all lungs are infected and consolidated in STAT1 KO mice (D). High-power images show minimal presence of cryptococcal organisms and predominantly mononuclear cell infiltrates with lymphoid and macrophage-type morphologies within infected lungs areas in the WT mice (E), contrasting with granulocyte-enriched mixed cellular infiltrate surrounding clusters of proliferating cryptococci (green arrows) and in the http://www.jimmunol.org/ lungs of STAT1 KO mice (F). Histological slides were stained with H&E and mucicarmine, and images were taken at original magnification 310 (A–D) and original magnification 340 (E and F), objective power. Images are representative of images derived from two experiments using three mice per group. similar relative to WT mice on day 7 postinoculation (Table I). STAT1 KO and WT mice were not significantly different during IL-12p70 was significantly increased in the STAT1 KO mice the time course evaluated. Overall, the cytokine milieu indicates compared with WT mice at day 10 postinoculation (p , 0.05); that a drift in Th1/Th2 cytokine balance away from Th1 and to- however, IL-12p40 levels were significantly decreased at day 10 ward Th2 occurred early in C. neoformans strain H99g-inoculated postinoculation in STAT1 KO mice compared with WT mice (p , STAT1-deficient mice. However, the subsequent increases in 0.01). IFN-g levels were significantly decreased at days 7 (p , proinflammatory cytokine and chemokine production in the lungs by guest on September 28, 2021 0.05) and 10 (p , 0.01) postinoculation in STAT1 KO mice of STAT1 KO mice compared with WT type mice during H99g compared with WT mice, despite the increase in pulmonary fungal infection were much more dramatic, an indication of aberrant burden of the IFN-g–producing C. neoformans strain in the lungs immune responses. of STAT1 KO mice (Fig. 1B). The available methodology does not We observed significant increases in IL-17A at days 7 and 10 allow us to directly distinguish what portion of the IFN-g mea- postinoculation (p , 0.001) and of IL-1b, IL-6, G-CSF, and TNF-a sured in vivo is derived from the IFN-g–producing C. neoformans at day 10 postinoculation (p , 0.05) within lung homogenates of strain versus the host leukocytes. To evaluate whether differences STAT1 KO mice compared with WT mice during C. neoformans in IFN-g levels are due to changes in production by the host or the strain H99g infection (Table I). IL-1a levels were also increased, IFN-g–producing C. neoformans strain, IFN-g KO (C.129S7(B6)- although not significantly, at day 10 postinoculation in the STAT1 Ifngtm1Ts/J), and WT controls (BALB/cJ) were inoculated with KO mice compared with WT mice. In addition, we observed C. neoformans strain H99g as described above, and IFN-g protein significantly decreased CCL5 production (p , 0.001) at days 7 levels were quantified from lung homogenates at day 7 postin- and 10 postinoculation in STAT1 KO mice compared with WT oculation (four to six mice per group). There was no significant mice but significantly increased CXCL1 and CCL2 production difference in pulmonary IFN-g production in WT mice (17.6 6 (p , 0.01) at day 10 postinoculation in STAT1 KO mice com- 1.947 pg/ml) compared with IFN-g KO mice (11.00 6 2.203 pared with WT mice. These data show that more robust proin- pg/ml), and overall amounts of IFN-g were low. This comparison flammatory and altered chemokine responses occur in STAT1 suggests that the majority of IFN-g detected in the STAT1 KO KO mice compared with the WT mice during infection with mice was produced by the microbe. In contrast, H99g-infected C. neoformans strain H99g. This increase in inflammatory cytokines WT mice have greater levels of IFN-g in the lungs, indicating that was highly pronounced at day 10, consistent with an increas- the host’s cells produce a detectable amount of IFN-g (p , 0.05) ingly dysregulated inflammatory response promoting inflam- above what is produced by the microbe alone and that this amount matory pathology in response to the expansion of C. neoformans contributes to the global level of IFN-g in the infected lungs of the in the lungs. WT mice. Thus, the data acquired from IFN-g KO mice suggest that the pulmonary leukocytes in STAT1 KO mice have dimin- STAT1 signaling is required for the induction of classically ished IFN-g production when infected with the IFN-g–producing activated macrophages C. neoformans strain. Although STAT1 signaling was required to mediate protection The STAT1 KO mice demonstrated significantly higher levels against pulmonary cryptococcosis, the changes in inflammatory of Th2-type cytokine IL-4 in the lungs at day 7 postinoculation responses and cytokine profiles could only partially explain the compared with WT mice (p , 0.05). However, IL-4 levels on day severe defect in fungal clearance of STAT1 KO mice. We sought to 10 and levels of IL-5 and immune-regulatory cytokine IL-10 in establish a causal link between STAT1 signaling and M1 macro- The Journal of Immunology 4065 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021 FIGURE 4. STAT1 is not required for pulmonary infiltration by immune cells in response to C. neoformans strain H99g infection. 129S6/SvEv (WT) and STAT1 KO mice were given a pulmonary inoculation with 1 3 104 C. neoformans strain H99g yeast cells. At days 7 and 10 postinoculation, lungs were excised, tissue was digested, and pulmonary infiltrates were analyzed by flow cytometry. Leukocytes were labeled with anti-CD45 Abs for total leukocytes (A) or dual labeled with anti-CD45 and Abs for specific cell types (B–I) and analyzed by flow cytometry. Data shown are the mean 6 SEM of absolute cell numbers from three independent experiments performed using four mice per group per experiment. *p , 0.05. phage activation, which we have previously shown correlates with WT mice, although not significantly, compared with macrophages disease resolution (24). Therefore, to examine macrophage po- from STAT1 KO mice at days 7 and 10; however no difference larization, we quantified the expression of genes associated with was observed in the expression of TNF-a or IL-17A. Gene ex- M1 and M2 macrophage activation in macrophages isolated from pression for cytokines IL-4 and IL-13, which are associated with H99g infected lungs of STAT1 KO compared with WT mice. a nonprotective Th2-type response and M2 macrophages, was not Pulmonary F4/80+ macrophages were isolated at days 7 and 10 different in macrophages from STAT1 KO mice compared with postinoculation with H99g, and total RNA was evaluated for the WT mice. Also, gene expression of CXCL9 and CXCL10, down- expression of genes commonly associated with macrophage acti- stream chemokines in the STAT1 signaling pathway, was signifi- vation using real-time PCR (Fig. 5). Gene expression of the M1 cantly decreased in macrophages from STAT1 KO mice compared activation marker iNOS was significantly decreased in STAT1 KO with WT mice (p , 0.001; Fig. 5A). Gene expression of CXCL9 mice compared with WT mice at day 7 by 200-fold (p , 0.001; was increased by ∼13,000-fold, and CXCL10 was increased by Fig. 5A) and at day 10 by 10-fold (p , 0.01). Conversely, mRNA 300-fold at day 7 postinoculation, suggesting an increase in che- expression for M2 macrophage activation markers Arg1 and mokine-induced infiltration of leukocytes to the site of infection. CD206 were significantly increased at days 7 (p , 0.05 and p , Furthermore, gene expression for transcription factor IRF-1, which 0.01, respectively; Fig. 5B) and 10 (p , 0.05) postinoculation in is induced by STAT1, was significantly decreased in macrophages STAT1 KO mice compared with WT mice. M2 activation marker from STAT1 KO mice compared with macrophages WT from mice found in inflammatory zone 1 (FIZZ1) expression also was in- (p , 0.001; Fig. 5A). Altogether, the gene expression profile creased in macrophages from STAT1 KO mice, although not demonstrates that STAT1 signaling is required for M1 macrophage significantly, and chitinase 3-like 3 (Ym1) expression was sig- polarization during pulmonary C. neoformans H99g infection. nificantly increased in macrophages from STAT1 KO mice at day 7 postinoculation (p , 0.01; Fig. 5B) and increased, although not STAT1 signaling is important for optimal macrophage NO significantly, at day 10 postinoculation compared with expression production and antifungal activity against C. neoformans in macrophages from WT mice. Gene expression of the proin- NO and ROS are byproducts of M1 activated macrophages and flammatory cytokine IFN-g was increased in macrophages from these molecules together account for the oxidative microbicidal 4066 PROTECTIVE MACROPHAGE RESPONSES AGAINST C. NEOFORMANS

Table I. Cytokine analysis of lung homogenates in WT mice compared with STAT1 KO mice

Level (pg/ml) in Pulmonary Homogenate Tissue at Day

710

Cytokine or Chemokine WT STAT 1 KO WT STAT 1 KO Th1-type cytokine IFN-g 21.1 6 3.89 12.48 6 0.74* 20.48 6 1.96 13.74 6 0.79** IL-2 32.57 6 3.19 46.33 6 3.47 23.34 6 1.47 20.12 6 1.39 IL-12 p40 81.27 6 9.62 53.26 6 12.15 227.46 6 18.73 147.26 6 18.48** IL-12 p70 78.98 6 12.20 88.52 6 13.28 106.59 6 15.52 153.50 6 10.94* Th2-type cytokine IL-4 18.37 6 5.98 41.31 6 5.00* 10.01 6 2.08 12.67 6 1.58 IL-5 7.35 6 1.02 18.02 6 3.75 7.06 6 0.85 8.14 6 0.68 IL-10 31.75 6 2.94 31.95 6 1.74 48.52 6 4.23 47.80 6 2.81 IL-13 101.54 6 14.09 106.21 6 5.39 96.84 6 8.14 109.80 6 4.60 Proinflammatory cytokine IL-1a 461.78 6 114.37 310.38 6 95.16 842.92 6 185.14 1340.21 6 153.47 IL-1b 2114.39 6 549.41 2759.83 6 845.82 3403.94 6 654.74 5891.87 6 611.89* IL-6 112.10 6 23.18 155.52 6 59.42 72.76 6 15.66 453.48 6 86.54*** 6 6 6 6

IL-17A 26.32 3.63 240.85 59.96*** 20.15 1.96 209.17 33.27*** Downloaded from TNF-a 130.3 6 21.03 157.33 6 17.90 239.29 6 59.03 458.83 6 37.63* G-CSF 348.32 6 43.58 909.00 6 281.48 294.15 6 54.97 2709.22 6 214.13** GM-CSF 102.30 6 8.10 103.72 6 10.42 100.22 6 3.59 129.84 6 2.87 Chemokine Eotaxin 719.54 6 171.16 606.13 6 87.17 647.81 6 35.75 705.59 6 32.28 CXCL1/KC 765.54 6 109.51 955.89 6 213.18 729.03 6 141.35 1838.48 6 178.00*** CCL5/RANTES 715.31 6 160.53 212.17 6 42.91*** 1058.68 6 110.91 132.78 6 24.12***

CCL3/MIP-1a 770.42 6 160.53 1114.23 6 301.66 1215.11 6 314.48 2080.93 6 254.31 http://www.jimmunol.org/ CCL4/MIP-1b 175.68 6 41.64 157.79 6 38.92 255.24 6 43.82 305.41 6 39.42 CCL2/MCP-1 924.17 6 98.47 497.23 6 98.31** 883.19 6 146.02 1874.04 6 192.94** Data shown are expressed as mean 6 SEM and are cumulative of two experiments using four mice per group per time point. *p , 0.05, **p , 0.01, ***p , 0.001 compared with the infected counterpart on the same day postinoculation. arsenal of phagocytes. Both NO and ROS are thought to be im- were examined for ROS production or cultured for 24 h in vitro, portant for C. neoformans killing by host cells (24, 55–60). To and nitrite production was measured in the culture supernatants. assess the antifungal activity of STAT1-deficient macrophages in Additional C. neoformans cells were not added to the cell culture by guest on September 28, 2021 response to C. neoformans strain H99g, we isolated pulmonary medium because the macrophages were already infected in vivo. macrophages from STAT1 KO and WT mice at days 7 and 10 We observed no significant difference in the number of cryp- postinoculation. The macrophages were lysed to release any in- tococci found within macrophages obtained from STAT1 KO and tracellular cryptococci and the fungal burden was determined to WT mice at day 7 postinoculation; however, significantly more quantify antifungal efficiency of macrophages. Macrophages also yeast were found within macrophages isolated from STAT1-

FIGURE 5. STAT1 signaling is required for polari- zation of M1 macrophages in response to Cryptococcus strain H99g infection. 129S6/SvEv (WT) and STAT1 KO mice were inoculated with 1 3 104 CFU C. neo- formans strain H99g. Pulmonary F4/80+ macrophages were isolated from the lungs of WT and STAT1 KO mice at days 7 and 10 postinoculation with C. neofor- mans strain H99g. Total RNA was extracted and tran- scripts were analyzed for markers and cytokines associated with M1 (A) and M2 (B) macrophage activa- tion. The indicated mRNA levels were normalized to GAPDH. Results are expressed as mean 6 SEM and are cumulative of three experiments using five mice per group per time point. *p , 0.05, **p , 0.01, ***p , 0.001. The Journal of Immunology 4067 deficient mice compared with macrophages from WT mice at day NO production and hallmark marker of M1 macrophage activa- 10 postinoculation (p , 0.01; Fig. 6A). Furthermore, the mor- tion, in macrophages from STAT1 KO mice compared with WT phology of intracellular organisms was different between WT and mice (Fig. 5A). Thus, loss of STAT1 signaling results in profound STAT1 KO mice. By day 10, macrophages from WT mice con- defect in NO production by macrophages in response to C. neo- tained predominantly degraded organisms as documented by the formans H99g infection, which correlates with decreased anti- presence of vacuoles that contained contracted and/or fragmented fungal efficiency and increased intramacrophage residence of yeasts or the capsular material alone (Fig. 6B). In contrast, mac- cryptococci. rophages from STAT1 KO mice contained large and heavily capsulated yeast cells that grouped in multicellular clusters and Discussion robustly stained with mucicarmine (Fig. 6B), consistent with vi- The identification of specific mechanisms that mediate the in- able and intracellular proliferating yeasts and illustrating the di- duction of protective immune responses following C. neoformans minished capacity of the macrophages for control of intracellular infection are likely to aid in the development of novel immuno- fungal proliferation. therapies for treatment of cryptococcosis. We have developed To further demonstrate a molecular basis for the diminished a murine infection model with C. neoformans strain H99g, a strain control of intracellular cryptococci by STAT1 KO mice, we that secretes murine IFN-g, which rapidly induces 1) a protective assessed ROS and NO production by macrophages isolated from Th1-type immune response in mice, 2) classical, M1, macrophage WT and STAT1 KO mice at days 7 and 10 postinoculation. At day activation, and 3) resolution of infection with a protective memory 7 postinoculation, a small but significant reduction of ROS was (24, 26, 42, 50–52). This immune response to C. neoformans detected in macrophages from STAT1 KO mice compared with WT strain H99g results in upregulation of the STAT1 pathway in Downloaded from mice (p , 0.001; Fig. 6C). However, at day 10 postinoculation, macrophages. STAT1 has been shown to play a major role in there was no difference in the ROS production by macrophages mediating immune and proinflammatory actions of IFN-g (61), between the two groups. This suggests that STAT1 plays only a cytokine important for regulation of the immune response a minor role in induction of ROS production by macrophages and (reviewed in Ref. 62). Signaling via the IFN-g-JAK1/2-STAT1 that ROS generation is insufficient for control of C. neoformans pathway is imperative for resistance to various intracellular

proliferation within macrophages (Fig. 6A). NO production was including Listeria monocytogenes and Toxoplasma http://www.jimmunol.org/ assessed by measuring nitrite accumulation in the supernatants gondii (61, 63–65). Previous studies have shown that loss of of the 24-h macrophage cell cultures. Macrophages isolated from STAT1 signaling in mice results in a complete lack of respon- STAT1 KO mice inoculated with C. neoformans strain H99g at siveness to IFN-g and IFN-a, correlating with high sensitivity to days 7 and 10 postinoculation had a profound decrease in nitrite infection by some microbial pathogens (61, 65, 66). However, the accumulation following 24 h of culture compared with macro- role of STAT1 in the development of protective immunity to phages from H99g-infected WT mice (p , 0.01, p , 0.001, re- C. neoformans has not been investigated. In this paper, to our spectively; Fig. 6D). These data correlate with the significant knowledge, we present for the first time that STAT1 is required for decrease in gene expression for iNOS, the enzyme that catalyzes the generation of immune protection against C. neoformans. Our by guest on September 28, 2021

FIGURE 6. Anticryptococcal activity of pulmonary macrophages requires STAT1-dependent NO production. 129S6/SvEv (WT) and STAT1 KO mice were intranasally inoculated with 1 3 104 CFU of C. neoformans strain H99g. Macrophages were isolated from the lungs of WT and STAT1 KO mice at days 7 and 10 postinoculation and intracellular cryptococci enumerated (A). Lungs of H99g-infected WT and STAT1 KO mice at day 10 postinoculation were processed and analyzed using a light microscope (B). Note that pulmonary macrophages in the WT mice contain small vacuoles/inclusions consistent with degraded cryptococci and no evidence of intracellular growth. Macrophages in STAT1 KO mice harbor large cryptococci with well-developed capsules grouped in clusters of newly divided cells. Isolated pulmonary macrophages were examined for ROS production (C) or cultured ex vivo for 24 h, and supernatants were analyzed for the presence of NO (D). Data are expressed as the mean 6 SEM and are cumulative of three experiments using three to fice mice per group per time point (A, C,andD) or are representative images derived from two experiments using three mice per group stained with H&E and mucicarmine and photographed at original magnification 3100, objective power (B). **p , 0.01, ***p , 0.001. 4068 PROTECTIVE MACROPHAGE RESPONSES AGAINST C. NEOFORMANS data further highlight that STAT1 is not absolutely required for the and IFN-g induced chemokines that play a role in attracting other development of T cell–mediated immune responses; however, it immune cells including Ag-specific Th1 cells and NK cells (46, contributes to Th1 development and is critical for M1 polarization 75). This may partially explain the decreasing trend in CD4+ of macrophages and their fungicidal function. T cells and the significant decrease in NK cells trafficking into the The present studies were designed to evaluate a putative role of lungs of the STAT1-deficient mice at day 10 postinoculation. The STAT1 signaling in mediating protection against pulmonary in- necessity of NK cell activity during cryptococcal infection is fection with C. neoformans. We previously examined the JAK/ unclear. Although it has been demonstrated that the absence of NK STAT pathway and TLR pathways in which we observed in- cells results in a reduction of fungal clearance from the lungs and creased gene expression of members of the IFN-gR pathway, in- spleens (76, 77), studies conducted in our laboratory show that cluding STAT1, IRF-1, CXCL9, and CXCL10 (24). However, we mice depleted of NK cells maintain a protective immune response detected no changes in expression of other STAT genes, MAPK to C. neoformans strain H99g (data not shown). In addition, genes, or genes within the TLR pathways (24). Our results dem- CXCL9 has been reported to inhibit eosinophil infiltration (78, onstrate that STAT1 deficiency in mice results in increased fungal 79); therefore, the decrease in CXCL9 gene expression by mac- burden and decreased survival when inoculated with C. neofor- rophages may be, to some degree, responsible for the increased mans strain 52D and C. neoformans strain H99g, whereas WT eosinophilia observed in the STAT1 KO mice. mice were able to clear infection with both of these strains. Fur- It is very important for the host to be able to respond to an in- thermore, inoculation with C. neoformans strain 52D resulted in vading pathogen with a balance between pro- and anti-inflammatory increased dissemination to the brain in STAT1 KO mice compared factors because excessive inflammation can result in severe lung with WT mice. A similar trend occurred during inoculation with pathology and ultimately death (80, 81). Cytokine analysis of Downloaded from C. neoformans strain H99g where lack of STAT1 signaling also pulmonary tissue from STAT1 KO mice showed increased levels resulted in increased trafficking of the yeast to the brain (data not of proinflammatory cytokines and a substantial increase in pul- shown). The increased trafficking to the CNS in the STAT1- monary leukocyte infiltrates compared with WT mice; however, deficient mice may be due to the loss of control of the pulmo- the STAT1-deficient mice were unable to eradicate the yeast and nary infection, which then leads to dissemination. Altogether, permitted rapid fungal proliferation. This increase in pulmonary

these data indicate that STAT1 signaling has a role in prevention fungal burden could contribute to the increased production of http://www.jimmunol.org/ of dissemination to the CNS. Collectively, these results indicate proinflammatory factors leading to increased inflammation. Taken that STAT1 signaling is essential for the generation of the pro- together, these studies demonstrate that although STAT1 is not tective immune response to C. neoformans infection in mice. required for leukocyte recruitment in response to C. neoformans The data further show that loss of STAT1 signaling did not result strain H99g infection, they strongly suggest that STAT1 signaling in deficient leukocyte accumulation in the lungs. A trend toward may be directly (through the regulatory negative feedback loops) increased leukocyte recruitment and an increase in the production and/or indirectly (increased fungal burden) responsible for limit- of some but not all proinflammatory cytokines and chemokines ing damage-inducing inflammation. occurred within the lungs of STAT1 KO mice. These data imply It is accepted that macrophage activation phenotype is important that the deficient cryptococcal clearance from the lungs of inoc- for host defense against Cryptococcus infection (24–26, 35, 36, 42, by guest on September 28, 2021 ulated STAT1 KO mice is a result of a dysregulated immune re- 82). Our data show that when STAT1 signaling is ablated in the sponse. Consistent with this view, the overall magnitude of the host, there is an increase in Arg1 gene expression and a decrease T cell response did not appear to be defective in the STAT1 KO in iNOS gene expression compared with WT mice. Overall, this mice. IFN-g and STAT1 induce the transcription factor T-bet, indicates a shift in the Arg1:iNOS ratio favoring Arg1 and M2 which in turn promotes Th-1 type T cell development (67, 68). macrophage activation, suggesting that the loss of STAT1 sig- In addition, T-bet suppresses GATA3, a transcription factor that naling leads to alternative activation of macrophages that are as- aids in regulation of Th2-type immune responses (69–72). Inter- sociated with nonprotective responses to C. neoformans. Although estingly, although some decrease in Th1 cytokines was observed, loss of STAT1 signaling does not significantly alter the total the levels of Th2-associated cytokines IL-4, IL-5, and IL-13 were numbers of macrophages trafficking into the lungs during inocu- not dramatically different between WT and STAT1-deficient mice, lation with C. neoformans strain H99g, it appears that it is the except for IL-4 at day 7 postinoculation. The lack of major quality and not quantity of macrophages that aids in eradication of changes in expression of these cytokines that are transcriptionally the yeast. Assays measuring macrophage production of ROS and enhanced by GATA3 suggests that STAT1 deletion did not sig- NO, molecules associated with anticryptococcal activity, reveal nificantly affect GATA3 levels. These findings suggest that STAT1 that STAT1 has minimal effect in promoting ROS generation but is more important for execution of downstream effector mecha- has a profound effect on NO production. Although sufficient in nisms, such as M1 macrophage activation, rather than the up- ROS generation, STAT1-deficient macrophages produced signifi- stream regulation of T cell polarizing responses during cryptococcal cantly less NO compared with WT macrophages, which correlated infection. However, further studies including T-bet and GATA3 with a defect in cryptococcal killing. These effects are attributed expression levels by T cells during cryptococcal infection in STAT1 to dramatically decreased iNOS expression but also significantly KO mice are needed to definitively address this point. increased Arg1. Arg1 competes with iNOS for the substrate Apart from the increased cryptococcal Ag load, the uncontrolled L-arginine (37); therefore, the observed decrease in NO production inflammation may be a result of the loss of regulatory proteins that in STAT1-deficient macrophages could be linked to the changes in are induced downstream of STAT1 signaling. We observed that both of these enzymes. In support of this theory, previous studies transcripts for IRF-1, a regulatory protein induced by STAT1 (62, have shown that the ratio Arg1/iNOS is predictive of the fungi- 73, 74), were decreased in macrophages from STAT1 KO mice cidal activity of macrophages during C. neoformans infection (31, compared with WT mice. Although IRF-1 participates in regula- 54, 83). Our results showing that STAT1 deficiency result in tion of the IFN-g/STAT1 pathway, this protein also plays a role in a .100-fold change in Arg1/iNOS ratio at days 7 and 10 post- other pathways to restrain the proinflammatory immune response, infection is consistent with the diminished fungicidal capacity thus limiting damage to the host. Macrophages from STAT1 KO observed by macrophages from STAT1 KO mice infected with mice also had a decrease in transcripts for CXCL9 and CXCL10, C. neoformans strain H99g. Collectively, our data indicate that the The Journal of Immunology 4069 most important function of STAT1 in anticryptococcal immunity 9. Collins, V. P., A. Gellhorn, and J. R. Trimble. 1951. The coincidence of cryp- is to mediate M1 polarization of macrophages, which is then tococcosis and disease of the reticulo-endothelial and lymphatic systems. Cancer 4: 883–889. crucial for protection against pulmonary cryptococcosis by the 10. Gendel, B. R., M. Ende, and S. L. Norman. 1950. Cryptococcosis; a review with production of NO with possible contribution of other factors such special reference to apparent association with Hodgkin’s disease. Am. J. Med. 9: 343–355. as activation of lysosome enzymes, which was not investigated at 11. Keye, J. D., Jr., and W. E. Magee. 1956. Fungal diseases in a general hospital; this time. Our new data combined with our previous observation a study of 88 patients. Am. J. Clin. Pathol. 26: 1235–1253. of increased STAT1 phosphorylation in macrophages from pro- 12. Lewis, J. L., and S. Rabinovich. 1972. The wide spectrum of cryptococcal infections. Am. J. Med. 53: 315–322. tectively immunized mice strongly suggest that this is an intrinsic 13. Husain, S., M. M. Wagener, and N. Singh. 2001. Cryptococcus neoformans in- effect of STAT1 determining the fate of macrophage polarization fection in organ transplant recipients: variables influencing clinical character- downstream of IFN-g signaling. One caveat to this conclusion is istics and outcome. Emerg. Infect. Dis. 7: 375–381. 14. Park, B. J., K. A. Wannemuehler, B. J. Marston, N. Govender, P. G. Pappas, and that the general STAT1 deletion also resulted in diminished IFN-g T. M. Chiller. 2009. Estimation of the current global burden of cryptococcal production in the infected lungs. Because IFN-g is the major factor meningitis among persons living with HIV/AIDS. AIDS 23: 525–530. driving M1 polarization, some of the in vivo effects of the STAT1 15. Singh, N., O. Lortholary, B. D. Alexander, K. L. Gupta, G. T. John, K. Pursell, P. Munoz, G. B. Klintmalm, V. Stosor, R. del Busto, et al. 2005. An immune deletion could be associated with an indirect effect of STAT1 on reconstitution syndrome-like illness associated with Cryptococcus neoformans IFN-g production. Future studies using transgenic mice with cell- infection in organ transplant recipients. Clin. Infect. Dis. 40: 1756–1761. restricted STAT1 abrogation will be useful tools to specifically 16. Pappas, P. G., B. D. Alexander, D. R. Andes, S. Hadley, C. A. Kauffman, A. Freifeld, E. J. Anaissie, L. M. Brumble, L. Herwaldt, J. Ito, et al. 2010. In- address this point. vasive fungal infections among organ transplant recipients: results of the The applications of these findings have the potential to spread Transplant-Associated Infection Surveillance Network (TRANSNET). Clin. In- beyond the scope of cryptococcosis. IFN-g activated macrophages fect. Dis. 50: 1101–1111. Downloaded from 17. Coker, R. J., M. Viviani, B. G. Gazzard, B. Du Pont, H. D. Pohle, S. M. Murphy, have been shown to have fungicidal activity against Histoplasma J. Atouguia, J. L. Champalimaud, and J. R. Harris. 1993. Treatment of crypto- capsulatum (84–86), whereas presence of IL-4 and IL-10 results in coccosis with liposomal (AmBisome) in 23 patients with AIDS. inhibition of apoptosis and increase in disease severity (87). 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