Susceptibility of Germfree Phagocyte Oxidase- and Nitric Oxide Synthase 2-Deficient Mice, Defective in the Production of Reactive Metabolite
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感染与免疫杂志 2005年第3期
Department of Microbiology and Immunology
Department of Stomatology, Medical University of South Carolina, Charleston, South Carolina
Department of Surgical Pathology, University of Wisconsin Medical School, Madison, Wisconsin
ABSTRACT
Mice deficient for phagocyte oxidase (Phox) and nitric oxide synthase 2 (NOS2) (gp91phox–/–/NOS2–/–), defective in the production of both reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), were used to investigate the role of phagocytic cells during mucosal and systemic candidiasis of endogenous origin. The alimentary tracts of germfree mice were colonized with Candida albicans wild type or each of two hyphal signaling-defective mutants (efg1/efg1 and efg1/efg1 cph1/cph1). All Candida-colonized gp91phox–/–/NOS2–/– mice were moribund within 12 to 15 days after oral inoculation. C. albicans wild-type and mutant strains colonized the alimentary tracts equally well and were able to translocate, most likely via Peyer's patches and mesenteric lymph nodes, to the internal organs and trigger the formation of abscesses; however, the wild-type and mutant strains did not survive in the abscessed murine tissues. Surprisingly, there was no significant difference in the ability of peritoneal exudate cells from gp91phox–/–/NOS2–/–, NOS2–/–, gp91phox–/–, or immunocompetent C57BL/6 mice to kill C. albicans in vitro. This suggests that anti-Candida factors other than ROI and RNI can control the growth of C. albicans and that gp91phox–/–/NOS2–/– mice die due to the inability of the host to control its inflammatory response to Candida. Correspondingly, reverse transcription-PCR analysis showed increased expression of the cytokines gamma interferon, tumor necrosis factor alpha, and the chemokines MIP-2 and KC at the site of infection, while interleukin-15 expression remained relatively unchanged between germfree and infected tissues. These studies indicate that defects in ROI and RNI enabled C. albicans to translocate and disseminate to the internal organs, resulting in an uncontrolled immune response, severe pathology, and death; however, ROI and RNI were not required for the killing of phagocytized C. albicans, indicating that other anti-Candida factors either compensate or are sufficient for the killing of phagocytized Candida.
INTRODUCTION
Superficial and invasive candidiasis continues to be a problem for an ever-increasing population of immunocompromised patients (10, 24). Clinical studies have demonstrated that Candida species are the fourth most common cause of bloodstream infections. Alarmingly, these opportunistic yeasts are becoming more resistant to antifungal agents, and mortality rates associated with hospital-acquired infections are increasing (13, 27).
Phagocytic cells play a key role in both innate and acquired resistance to mucosal and systemic candidiasis (4, 15, 34). Professional and nonprofessional phagocytes produce a number of metabolites and peptides that can kill or interfere with the growth of Candida species (16, 34); however, the generation of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) by professional phagocytes is thought to play a critical role in resistance to mucosal and systemic candidiasis (34). Among the latter metabolites, superoxide, hypochlorous acid, nitric oxide, peroxynitrite, and perhaps ozone (4, 34, 38) appear to be important products that can kill invading microbes.
The importance of phagocyte oxidase in resistance to invasive candidiasis is suggested by the increased susceptibility of chronic granulomatous disease patients to systemic candidiasis (12, 18, 19). Also, mice genetically engineered to be deficient in phagocyte oxidase (Phox) activity are more susceptible to acute systemic candidiasis than their Phox-sufficient counterparts (1-3). Nitric oxide synthase 2 (NOS2) knockout mice have not yet been assessed for their susceptibility to mucosal or systemic candidiasis of endogenous origin; however, mice with chemically induced deficiencies in RNI demonstrated an increased susceptibility to invasive and mucosal candidiasis (37), and RNI has been shown to be important for the candidacidal activity of macrophages (34-36). Intriguingly, recent studies indicate Candida albicans can suppress the production of RNI metabolites produced by murine phagocytic cells in vitro (9, 30). Thus, defects in both ROI and RNI could exist in Candida-infected ROI-defective hosts; however, murine susceptibility to mucosal and systemic candidiasis in animals defective in both ROI and RNI has not been assessed. In order to learn more about the relative importance of combined defects in both ROI and RNI on resistance to mucosal and systemic candidiasis of endogenous origin, we colonized (alimentary tract) germfree Phox- and NOS2-deficient (gp91phox–/–/NOS2–/–) mice (31) with C. albicans wild type and two hyphal signaling-defective mutants (efg1/efg1 and efg1/efg1 cph1/cph1) (20).
MATERIALS AND METHODS
Mice. The germfree mice used in this study were derived (cesarean section) into the germfree state and maintained at the University of Wisconsin Gnotobiotic Laboratory (Madison). The germfree colony of gp91phox–/–/NOS2–/– animals was derived from breeders kindly supplied by C. Nathan (Cornell Medical Center, New York, N.Y.).
Microorganisms. C. albicans SC5314 was maintained by monthly transfer on Sabouraud glucose agar (SGA; Difco), whereas the two mutants (efg1/efg1 and efg1/efg1 cph1/cph1) were maintained by monthly transfer on minimal synthetic dropout plates (BD Biosciences). C. albicans SC5314 was obtained from J. Dolan (Nashville State Community College, Nashville, Tenn.), and the efg1/efg1 and efg1/efg1 cph1/cph1 strains were provided by G. Fink (Massachusetts Institute of Technology, Boston).
Oral colonization with C. albicans. Germfree adult (6 to 8 weeks of age) gp91phox–/–/NOS2–/– mice were transferred into sterile isolators, where they were colonized (alimentary tract) with a pure culture of C. albicans wild-type or mutant strains. A separate isolator was used for each wild-type and mutant strain. A viable C. albicans inoculum (106 cells/ml), harvested from a 24-h Sabouraud glucose broth culture incubated at 37°C, was swabbed into the oral cavity and onto the ano-rectal area of each mouse. After one inoculation, the germfree mice were quickly colonized with the C. albicans strains, since 24-h fecal samples contained viable C. albicans (7 to 8 log10 CFU/g). Alimentary tract colonization with the wild-type and mutant strains of C. albicans was quantified when moribund Candida-monoassociated mice were euthanized. Dilutions of intestinal contents and homogenized tissues were inoculated onto SGA. The number of viable C. albicans was determined following 24 h of incubation at 37°C. Data are presented as the log10 number of viable C. albicans CFU per gram (dry weight) of tissue.
Systemic candidiasis of endogenous origin. Internal organs from moribund mice were mixed with 10 ml of sterile distilled water and homogenized using the Stomacher II (Fisher Scientific). The homogenized tissues were serially diluted and inoculated (100 μl) onto SGA. The number of viable C. albicans organisms in tissue homogenates was determined following 24 h of incubation at 37°C.
Histopathology. Tissue samples were collected aseptically, and tongue, palate, esophagus, stomach, large and small intestines, spleen, Peyer's patches, mesenteric lymph nodes, liver, and lungs were fixed in 10% formaldehyde in phosphate-buffered saline (PBS). The tissues were processed in graded (100, 95, 80, and 70%) alcohol and xylene solutions and embedded in paraffin. Tissue sections (4 μm) were stained with hematoxylin and eosin or Gomori stain for fungi.
RNA extraction and RT-PCR. Tissues from germfree control and C. albicans-infected mice were homogenized in RNAwiz (Ambion) using a Tekmar tissumizer, and the RNA was extracted according to the manufacturer's instructions. The RNA preparation was treated with DNase I, and the quantity and quality of RNA was measured spectrophotometrically at absorbances of 260 and 280 nm. Equal amounts of total RNA (2 μg) from infected and germfree control tissues were reverse transcribed into cDNA using the Retroscript kit (Ambion) according to the manufacturer's instructions. PCRs were performed using the following forward and reverse primers, respectively: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CCAAAAGGGTCATCATCTCC and 5'-ACAGTCTTCTGGGTGGCAG; MIP-2, 5'-AAGTCATAGCCACTCTCAGG and 5'-AGCGAGGCACATCAGGTAC; KC, 5'-GCTCGCTTCTCTGTGCAG and 5'-GGAGCTTCAGGGTCAAGG; interleukin-10 (IL-10), 5'-GCTTCTATTCTAAGGCTGGC and 5'-GAGCTGCTGCAGGAATGATC; IL-15, 5'-GGGCTGTGTCAGTGTAGGT and 5'-ATTTGGACAATGCGTATAAAG. Primers for gamma interferon (IFN-), tumor necrosis factor alpha (TNF-), and IL-12 (p40) were purchased from BioSource (Camarillo, Calif.). After an initial denaturation at 95°C for 2 min, the samples were subjected to 30 (GAPDH) or 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C (GAPDH, TNF-, IFN-, MIP-2, IL-10, and IL-15), 60°C (IL-12), or 62°C (KC) for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 10 min. The predicted sizes (in base pairs) of reverse transcription-PCR (RT-PCR) products were as follows: GAPDH, 217; TNF-, 298; MIP-2, 214; KC, 226; IFN-, 937; IL-10, 172; IL-12, 331; IL-15, 362. The identities of the RT-PCR products were confirmed by DNA sequencing. Reaction mixtures lacking the addition of reverse transcriptase were also subjected to PCR amplification to check for the presence of genomic DNA. In addition, primers for KC, IFN-, and TNF- amplification were designed to flank an intron. Consequently, this ensured that KC, IFN-, and TNF- PCR products were derived from cDNA as opposed to genomic DNA. The preparation of C. albicans RNA from infected tissues and the primers and PCR conditions for EFB1, PLB1, and PLB2 have been described previously (29).
PECs: phagocytosis and killing assay. The candidacidal activity of peritoneal exudate cells (PECs) was assessed as described previously (35). Briefly, thioglycollate-elicited (3%; 24 h) PECs were cultured (RPMI medium, supplemented with 10% heat-inactivated fetal calf serum and 0.1% minimal essential medium) with C. albicans at a PECs-to-Candida ratio of 10:1. After 2 h of incubation at 37°C with 5% CO2, the number of viable C. albicans organisms able to form a colony (CFU) was scored after overnight incubation on SGA plates at 37°C. The candidacidal activity was calculated as follows: {[CFU from control (no PECs) – CFU from test (PECs + C. albicans)]/(CFU from control) } x 100. Results represent the average of three or more independent experiments with PECs collected from six to eight mice per experiment.
Flow cytometry. Cell phenotyping of the PECs was performed using the manufacturer's recommended methodology (BD Pharmingen). Briefly, thioglycollate-elicited PECs were adjusted to a concentration of 5 x 106/ml in PBS (pH 7.4) supplemented with 0.5% glucose. The PEC suspension (200 μl) was labeled with phycoerythrin- or fluorescein-5-isothiocyanate-conjugated monoclonal antibody (Mac3 for macrophages or GR-1 for granulocytes). The percentage of positive cells within the PEC population was measured using an Ortho Cytoron Absolute flow cytometer (Raritan) to record 3,000 to 5,000 events for each assay.
For the phagocytosis assays, C. albicans was harvested from Sabouraud glucose broth after overnight incubation at 37°C. After washing the cells twice with PBS, C. albicans cells were resuspended in PBS supplemented with 0.5% glucose at a concentration of 5 x 106/ml and labeled with calcein-AM (10 μM; Molecular Probes) for 1 h at 37°C. PECs and calcein-AM-labeled C. albicans (1:1 ratio) were incubated at 37°C for 2 h with 5% CO2. The mixture of PECs and calcein-AM-labeled C. albicans was then reacted with PE-conjugated monoclonal antibody to GR-1. The percentage of double-positive PECs, labeled with GR-1 (orange fluorescence) and calcein-AM (green fluorescence), was recorded as the percentage of GR-1-positive PECs phagocytizing C. albicans (26).
For the C. albicans killing assays, ethidium homodimer 1 (Molecular Probes) was used to assess the percentage of dead C. albicans cells (red fluorescence). Briefly, PECs and C. albicans cells (1:1 ratio) were incubated at 37°C with 5% CO2 as described previously (26). After a 2-h incubation, 2 μM ethidium homodimer 1 (400 μl) was added to the PECs-C. albicans mixture (200 μl) and incubated at room temperature for 10 min. PBS supplemented with 0.5% glucose (700 μl) was then added, and the tubes were placed on ice and read immediately. The percentage of positive red fluorescent cells was obtained by flow cytometry (5,000 events).
RESULTS
Alimentary tract colonization and survival. The alimentary tracts of germfree gp91phox–/–/NOS2–/– mice were quickly (by 24 h) colonized by a C. albicans wild-type strain (SC5314) and both mutant strains (efg1/efg1 and efg1/efg1 cph1/cph1) (Table 1). At 24 h after oral inoculation, their alimentary tracts contained 6 to 8 log10 CFU of viable C. albicans/g in the intestinal contents and the mice remained chronically colonized with a pure culture of the C. albicans strains for the duration of the study (Table 1). Table 1 also shows that no viable C. albicans cells were recovered from the internal organs (kidney, liver, and spleen) of moribund gp91phox–/–/NOS2–/– mice that were colonized with either the wild-type or each of the two mutant strains. The C. albicans wild-type and mutant strains were both lethal for gp91phox–/–/NOS2–/– mice. The Candida-colonized mice did not survive beyond 16 days after oral colonization (Table 1).
Pathology of gp91phox–/–/NOS2–/– mice colonized with C. albicans wild-type (SC5314) or mutant strains (efg1/efg1 and efg1/efg1 cph1/cph1). Gross pathology was very evident in moribund gp91phox–/–/NOS2–/– mice colonized with C. albicans wild-type or either of the two mutant strains. Moribund mice had enlarged, pale yellow mesenteric lymph nodes (four to five times normal size) and, in several mice, cervical lymph nodes. In mice colonized with the SC5314 strain, numerous lung nodules were visible (Fig. 1). The spleen was often enlarged and abscessed (Fig. 1). Peyer's patches were also prominent, and some appeared yellow. The livers and kidneys appeared normal in size and were without grossly visible lesions (data not shown).
Microscopic examination of the hematoxylin and eosin-stained tissue sections revealed multiple collections of polymorphonuclear leukocytes (PMNs) in most internal organs. Abscesses were detected in the Peyer's patches, lymph nodes, liver, lungs, spleen, kidneys and, to a lesser extent, in the submucosa of the stomach, esophagus, tongue, and palate (Fig. 2A; Table 2). Except for the stomachs of mice colonized with SC5314, superficial candidiasis of keratinized mucosae was often absent, albeit in the presence of submucosal lesions in these same sites and in major organs of the same animal (Table 3). Hyphae, pseudohyphae, and blastoconidia, when detected in the abscesses, were usually present in small numbers (Fig. 2B). Although originally described as being "locked" in the yeast form (20), the finding that the efg1/efg1 cph1/cph1 double mutant was able to form hyphae in vivo is in agreement with previous observations that this strain can indeed form hyphae during infection and under specific conditions in vitro (8, 25). C. albicans was also rare in lesions of Peyer's patches from all three groups of colonized mice; however, viable Candida could not be cultured from abscessed internal organs, suggesting that the Candida cells present in the tissues were dead. Interestingly, the efg1/efg1 cph1/cph1 double mutant strain, while less invasive in gastric tissues than the wild-type SC5314 strain, appeared to be more invasive for oroesophageal (stomach and esophagus) tissues than the efg1/efg1 mutant (Table 3). Since C. albicans phospholipase B (PLB) has been shown to be required for the invasion of gastric mucosa and the translocation from the gastrointestinal tract (22), we examined PLB1 and PLB2 expression in gastric tissues from mice colonized with either SC5314 or each of the two mutant strains. PLB1 and PLB2 transcripts were detected for all three strains in vivo; however, there was no discernible difference in the ability of the different strains to express PLB1 and PLB2 (Fig. 3).
The vast majority of leukocytes in lesions were PMNs in various stages of degeneration. A small macrophage component was evident in the periphery of most soft tissue abscesses; however, in the lung, macrophages comprised approximately 5% of the infiltrating cells, and they were dispersed throughout collections of PMNs. Centrally, in some of the larger abscesses, areas of PMN necrosis, or liquefaction of PMNs, were evident. PMNs were represented by both mature polylobated forms and immature band forms. Granulomas with giant cells, epithelial cells, and lymphocytes were not present in involved tissues. A consistent observation from the three experimental groups 2 weeks after colonization was the pathology present in the Peyer's patches and mesenteric lymph nodes; usually, the involved lymph node was completely destroyed by infiltrating phagocytic cells.
Cytokine and chemokine expression in response to colonization and infection. ROI and RNI play important roles in mediating innate and acquired immune responses (6, 11, 17, 23). Impairment of ROI- and RNI-dependent signaling and immune responses could have contributed to the observed lethality. We investigated the capacity of these ROI- and RNI-deficient mice to express several cytokine (TNF-, IFN-, IL-10, IL-12, and IL-15) and chemokine (MIP-2 and KC) genes in response to C. albicans colonization and infection by using relative RT-PCR (Fig. 4). The relative expression levels of TNF- and IFN- were increased in gastric tissues from mice infected with either C. albicans SC5314 or efg1/efg1 cph1/cph1 strains compared to germfree controls. Similarly, expression levels of the chemoattractants MIP-2 and KC were increased in gastric tissues from the ROI- and RNI-deficient mice. In contrast, expression of IL-15, and to a lesser extent IL-10 and IL-12, were relatively similar in both infected and germfree control gastric tissues (Fig. 4). Since Toll-like receptors (TLR) play an important role in activating innate defenses and, in particular, inflammatory responses (14), we examined TLR1 to -10 mRNA expression in germfree and Candida-infected gastric tissues. Surprisingly, TLR1 to -10 were all readily detected in germfree control tissues (in the absence of a viable microbial flora), and an increase in TLR1 to -10 expression was not detected in infected tissues (data not shown).
Phagocytosis and killing of C. albicans. Shiloh et al. (31) reported that macrophages from gp91phox–/–/NOS2–/– mice were unable to kill a virulent strain of Listeria monocytogenes in vitro but were able to kill Salmonella enterica serovar Typhimurium, Escherichia coli, and an avirulent strain of L. monocytogenes. PECs (thioglycollate elicited) from gp91phox–/–/NOS2–/– mice and from single knockout mice, gp91phox–/– or NOS2–/–, were able to phagocytize and kill C. albicans as well as PECs from immunocompetent C57BL/6 mice in an in vitro assay (Table 4). The PECs from the immunocompetent and immunodeficient mice killed approximately 50% of the C. albicans cells within 2 h at 37°C. In addition, there was no significant difference in the percentage of macrophages or granulocytes in the PEC population retrieved from the immunocompetent C57BL/6 mice compared to that from the deficient NOS2–/–, gp91phox–/–, or gp91phox–/–/NOS2–/– mice.
DISCUSSION
This study demonstrated that mice with defects in their capacity to produce metabolites of both oxygen and nitrogen are susceptible to lethal candidiasis of endogenous (alimentary tract) origin. gp91phox–/–/NOS2–/– mice were found to be susceptible (lethal) to C. albicans wild type and to each of the two mutant strains that have previously been reported to exhibit significantly reduced virulence by acute systemic challenge (20). Our study demonstrated again, as is well known from many clinical candidiasis cases, that immunocompetence is an important component of a host's susceptibility to Candida species.
It became obvious in this study that the moribund ROI- and RNI-deficient mice were not dying from an overwhelming C. albicans sepsis. We observed (grossly and histologically) exaggerated and tissue-destructive abscess formation in most internal organs of the C. albicans-colonized ROI- and RNI-deficient mice. The abscesses were most prominent in mice colonized with the C. albicans wild-type strain. Although C. albicans hyphae, and to a lesser extent yeast cells, could be seen within the abscesses that formed in the C. albicans-colonized and -infected mice, we were unable to recover viable C. albicans from homogenized abscessed organs. In contrast, viable Candida organisms were readily isolated from the alimentary tract. Apparently, host defense mechanisms (antibody, complement, antibacterial proteins, etc.) other than ROI and RNI were still functional in these immunodeficient mice. The anti-Candida factors likely came from infiltrating phagocytic cells and killed the C. albicans cells that translocated to internal organs from the alimentary tract. Histopathology indicated that the most likely route into the internal organs was via Peyer's patches and mesenteric lymph nodes, which were almost completely destroyed by infiltrating phagocytic cells. Thus, even with deficiencies in the production of reactive metabolites of both oxygen and nitrogen, the gp91phox–/–/NOS2–/– mice still possessed anti-Candida mechanisms that killed C. albicans in abscessed internal organs; however, the mice were obviously unable to regulate an exaggerated and tissue-destructive inflammatory response to the Candida that translocated from the intestinal tract. Most important, the anti-Candida factors present in the ROI- and RNI-deficient mice were not able to prevent the animals' demise.
Shiloh et al. (31) reported that gp91phox–/–/NOS2–/– mice developed abscesses that contained viable commensal, mostly enteric, bacteria. Mice with a deficiency in either ROI or RNI were reported to be resistant to the commensal infections that afflicted the mice with combined ROI and RNI deficiencies (31). The latter report and our data with C. albicans demonstrated that combined deficiencies in ROI and RNI predispose mice to systemic infections by commensal prokaryotic and eukaryotic intestinal microorganisms. The ROI- and RNI-deficient mice, which apparently lack mechanisms to kill the prokaryotic commensals, retained their candidacidal activity. The germfree mice used in our study were not fed antibiotics, and germfree controls, which were not colonized with Candida, had no abscessed tissues. Shiloh et al. (31), in an attempt to control the commensal bacterial infections, administered oral antibiotics to the ROI- and RNI-deficient mice. The addition of antibiotics to the drinking water and housing these mice in microisolator cages with sterile food, water, and bedding (specific-pathogen-free conditions) did not prevent endogenous infections by commensal bacteria (31). It is noteworthy that Shiloh et al. (31) isolated Candida guillermondii from a few abscesses in their antibiotic-treated ROI- and RNI-deficient mice. The latter data suggest that the candidacidal mechanisms that persist in mice with combined ROI and RNI deficiencies may not be able to kill all Candida species. The latter could be associated with an immunodeficient host's susceptibility to opportunistic infections by different Candida species.
We have previously reported that the lethality of C. albicans for two other strains of immunodeficient mice, Tg26 (5, 33) and beige-nude (7) mice, was very likely due to severe, occlusive, esophageal candidiasis. We observed no overwhelming Candida sepsis or abscess formation in the Peyer's patches, lymph nodes, or internal organs of the former mice (5, 33). In contrast, occlusive oroesophageal candidiasis did not appear to be associated with lethality in the ROI- and RNI-deficient mice. In fact, mucosal tissues often had abscesses in the absence of obvious mucosal candidiasis. The mice with combined ROI and RNI defects had no problems in signaling phagocytic cells to the Candida-infected tissues, since MIP-2 and KC mRNAs were present in the infected gastric tissues. The exaggerated immune response apparently caused the destructive tissue pathology in the Peyer's patches and mesenteric lymph nodes that contributed to their rapid demise.
It is well known that ROI and RNI play important roles in signaling and regulating inflammation and immunity to infectious diseases (11, 17, 21, 23). For example, it has been reported that NO stimulates MIP-2 expression (32). In our study with mice that were deficient in NO, we detected basal levels of MIP-2 mRNA in germfree control tissues and also increased levels of MIP-2 mRNA in Candida-infected gastric tissues. The ROI- and RNI-deficient mice were also able to produce the mRNAs for the important inflammatory cytokines IFN- and TNF-, which may have contributed to the pathology in the ROI- and RNI-deficient mice; however, our previous studies indicated that ROI- and RNI-deficient mice were defective in their capacity to produce the antimicrobial peptide -defensin 4 (mBD-4) (28). ROI- and RNI-deficient mice, compared to immunocompetent controls, expressed lower levels of mBD-4 and were unable to increase mBD-4 expression in response to a Candida infection. This suggests that ROI and/or RNI may be required for maximal production of mBD-4.
In summary, the gp91phox–/–/NOS2–/– mice offer an exciting new model to study many facets of resistance and susceptibility to candidiasis.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health (grant DE-13968).
We thank Kimberly Bauer, Andrea Boan, and April Harkins for critical reading of the paper.
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Department of Stomatology, Medical University of South Carolina, Charleston, South Carolina
Department of Surgical Pathology, University of Wisconsin Medical School, Madison, Wisconsin
ABSTRACT
Mice deficient for phagocyte oxidase (Phox) and nitric oxide synthase 2 (NOS2) (gp91phox–/–/NOS2–/–), defective in the production of both reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI), were used to investigate the role of phagocytic cells during mucosal and systemic candidiasis of endogenous origin. The alimentary tracts of germfree mice were colonized with Candida albicans wild type or each of two hyphal signaling-defective mutants (efg1/efg1 and efg1/efg1 cph1/cph1). All Candida-colonized gp91phox–/–/NOS2–/– mice were moribund within 12 to 15 days after oral inoculation. C. albicans wild-type and mutant strains colonized the alimentary tracts equally well and were able to translocate, most likely via Peyer's patches and mesenteric lymph nodes, to the internal organs and trigger the formation of abscesses; however, the wild-type and mutant strains did not survive in the abscessed murine tissues. Surprisingly, there was no significant difference in the ability of peritoneal exudate cells from gp91phox–/–/NOS2–/–, NOS2–/–, gp91phox–/–, or immunocompetent C57BL/6 mice to kill C. albicans in vitro. This suggests that anti-Candida factors other than ROI and RNI can control the growth of C. albicans and that gp91phox–/–/NOS2–/– mice die due to the inability of the host to control its inflammatory response to Candida. Correspondingly, reverse transcription-PCR analysis showed increased expression of the cytokines gamma interferon, tumor necrosis factor alpha, and the chemokines MIP-2 and KC at the site of infection, while interleukin-15 expression remained relatively unchanged between germfree and infected tissues. These studies indicate that defects in ROI and RNI enabled C. albicans to translocate and disseminate to the internal organs, resulting in an uncontrolled immune response, severe pathology, and death; however, ROI and RNI were not required for the killing of phagocytized C. albicans, indicating that other anti-Candida factors either compensate or are sufficient for the killing of phagocytized Candida.
INTRODUCTION
Superficial and invasive candidiasis continues to be a problem for an ever-increasing population of immunocompromised patients (10, 24). Clinical studies have demonstrated that Candida species are the fourth most common cause of bloodstream infections. Alarmingly, these opportunistic yeasts are becoming more resistant to antifungal agents, and mortality rates associated with hospital-acquired infections are increasing (13, 27).
Phagocytic cells play a key role in both innate and acquired resistance to mucosal and systemic candidiasis (4, 15, 34). Professional and nonprofessional phagocytes produce a number of metabolites and peptides that can kill or interfere with the growth of Candida species (16, 34); however, the generation of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) by professional phagocytes is thought to play a critical role in resistance to mucosal and systemic candidiasis (34). Among the latter metabolites, superoxide, hypochlorous acid, nitric oxide, peroxynitrite, and perhaps ozone (4, 34, 38) appear to be important products that can kill invading microbes.
The importance of phagocyte oxidase in resistance to invasive candidiasis is suggested by the increased susceptibility of chronic granulomatous disease patients to systemic candidiasis (12, 18, 19). Also, mice genetically engineered to be deficient in phagocyte oxidase (Phox) activity are more susceptible to acute systemic candidiasis than their Phox-sufficient counterparts (1-3). Nitric oxide synthase 2 (NOS2) knockout mice have not yet been assessed for their susceptibility to mucosal or systemic candidiasis of endogenous origin; however, mice with chemically induced deficiencies in RNI demonstrated an increased susceptibility to invasive and mucosal candidiasis (37), and RNI has been shown to be important for the candidacidal activity of macrophages (34-36). Intriguingly, recent studies indicate Candida albicans can suppress the production of RNI metabolites produced by murine phagocytic cells in vitro (9, 30). Thus, defects in both ROI and RNI could exist in Candida-infected ROI-defective hosts; however, murine susceptibility to mucosal and systemic candidiasis in animals defective in both ROI and RNI has not been assessed. In order to learn more about the relative importance of combined defects in both ROI and RNI on resistance to mucosal and systemic candidiasis of endogenous origin, we colonized (alimentary tract) germfree Phox- and NOS2-deficient (gp91phox–/–/NOS2–/–) mice (31) with C. albicans wild type and two hyphal signaling-defective mutants (efg1/efg1 and efg1/efg1 cph1/cph1) (20).
MATERIALS AND METHODS
Mice. The germfree mice used in this study were derived (cesarean section) into the germfree state and maintained at the University of Wisconsin Gnotobiotic Laboratory (Madison). The germfree colony of gp91phox–/–/NOS2–/– animals was derived from breeders kindly supplied by C. Nathan (Cornell Medical Center, New York, N.Y.).
Microorganisms. C. albicans SC5314 was maintained by monthly transfer on Sabouraud glucose agar (SGA; Difco), whereas the two mutants (efg1/efg1 and efg1/efg1 cph1/cph1) were maintained by monthly transfer on minimal synthetic dropout plates (BD Biosciences). C. albicans SC5314 was obtained from J. Dolan (Nashville State Community College, Nashville, Tenn.), and the efg1/efg1 and efg1/efg1 cph1/cph1 strains were provided by G. Fink (Massachusetts Institute of Technology, Boston).
Oral colonization with C. albicans. Germfree adult (6 to 8 weeks of age) gp91phox–/–/NOS2–/– mice were transferred into sterile isolators, where they were colonized (alimentary tract) with a pure culture of C. albicans wild-type or mutant strains. A separate isolator was used for each wild-type and mutant strain. A viable C. albicans inoculum (106 cells/ml), harvested from a 24-h Sabouraud glucose broth culture incubated at 37°C, was swabbed into the oral cavity and onto the ano-rectal area of each mouse. After one inoculation, the germfree mice were quickly colonized with the C. albicans strains, since 24-h fecal samples contained viable C. albicans (7 to 8 log10 CFU/g). Alimentary tract colonization with the wild-type and mutant strains of C. albicans was quantified when moribund Candida-monoassociated mice were euthanized. Dilutions of intestinal contents and homogenized tissues were inoculated onto SGA. The number of viable C. albicans was determined following 24 h of incubation at 37°C. Data are presented as the log10 number of viable C. albicans CFU per gram (dry weight) of tissue.
Systemic candidiasis of endogenous origin. Internal organs from moribund mice were mixed with 10 ml of sterile distilled water and homogenized using the Stomacher II (Fisher Scientific). The homogenized tissues were serially diluted and inoculated (100 μl) onto SGA. The number of viable C. albicans organisms in tissue homogenates was determined following 24 h of incubation at 37°C.
Histopathology. Tissue samples were collected aseptically, and tongue, palate, esophagus, stomach, large and small intestines, spleen, Peyer's patches, mesenteric lymph nodes, liver, and lungs were fixed in 10% formaldehyde in phosphate-buffered saline (PBS). The tissues were processed in graded (100, 95, 80, and 70%) alcohol and xylene solutions and embedded in paraffin. Tissue sections (4 μm) were stained with hematoxylin and eosin or Gomori stain for fungi.
RNA extraction and RT-PCR. Tissues from germfree control and C. albicans-infected mice were homogenized in RNAwiz (Ambion) using a Tekmar tissumizer, and the RNA was extracted according to the manufacturer's instructions. The RNA preparation was treated with DNase I, and the quantity and quality of RNA was measured spectrophotometrically at absorbances of 260 and 280 nm. Equal amounts of total RNA (2 μg) from infected and germfree control tissues were reverse transcribed into cDNA using the Retroscript kit (Ambion) according to the manufacturer's instructions. PCRs were performed using the following forward and reverse primers, respectively: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-CCAAAAGGGTCATCATCTCC and 5'-ACAGTCTTCTGGGTGGCAG; MIP-2, 5'-AAGTCATAGCCACTCTCAGG and 5'-AGCGAGGCACATCAGGTAC; KC, 5'-GCTCGCTTCTCTGTGCAG and 5'-GGAGCTTCAGGGTCAAGG; interleukin-10 (IL-10), 5'-GCTTCTATTCTAAGGCTGGC and 5'-GAGCTGCTGCAGGAATGATC; IL-15, 5'-GGGCTGTGTCAGTGTAGGT and 5'-ATTTGGACAATGCGTATAAAG. Primers for gamma interferon (IFN-), tumor necrosis factor alpha (TNF-), and IL-12 (p40) were purchased from BioSource (Camarillo, Calif.). After an initial denaturation at 95°C for 2 min, the samples were subjected to 30 (GAPDH) or 35 cycles of denaturation at 95°C for 30 s, annealing at 58°C (GAPDH, TNF-, IFN-, MIP-2, IL-10, and IL-15), 60°C (IL-12), or 62°C (KC) for 30 s, and extension at 72°C for 30 s, with a final extension at 72°C for 10 min. The predicted sizes (in base pairs) of reverse transcription-PCR (RT-PCR) products were as follows: GAPDH, 217; TNF-, 298; MIP-2, 214; KC, 226; IFN-, 937; IL-10, 172; IL-12, 331; IL-15, 362. The identities of the RT-PCR products were confirmed by DNA sequencing. Reaction mixtures lacking the addition of reverse transcriptase were also subjected to PCR amplification to check for the presence of genomic DNA. In addition, primers for KC, IFN-, and TNF- amplification were designed to flank an intron. Consequently, this ensured that KC, IFN-, and TNF- PCR products were derived from cDNA as opposed to genomic DNA. The preparation of C. albicans RNA from infected tissues and the primers and PCR conditions for EFB1, PLB1, and PLB2 have been described previously (29).
PECs: phagocytosis and killing assay. The candidacidal activity of peritoneal exudate cells (PECs) was assessed as described previously (35). Briefly, thioglycollate-elicited (3%; 24 h) PECs were cultured (RPMI medium, supplemented with 10% heat-inactivated fetal calf serum and 0.1% minimal essential medium) with C. albicans at a PECs-to-Candida ratio of 10:1. After 2 h of incubation at 37°C with 5% CO2, the number of viable C. albicans organisms able to form a colony (CFU) was scored after overnight incubation on SGA plates at 37°C. The candidacidal activity was calculated as follows: {[CFU from control (no PECs) – CFU from test (PECs + C. albicans)]/(CFU from control) } x 100. Results represent the average of three or more independent experiments with PECs collected from six to eight mice per experiment.
Flow cytometry. Cell phenotyping of the PECs was performed using the manufacturer's recommended methodology (BD Pharmingen). Briefly, thioglycollate-elicited PECs were adjusted to a concentration of 5 x 106/ml in PBS (pH 7.4) supplemented with 0.5% glucose. The PEC suspension (200 μl) was labeled with phycoerythrin- or fluorescein-5-isothiocyanate-conjugated monoclonal antibody (Mac3 for macrophages or GR-1 for granulocytes). The percentage of positive cells within the PEC population was measured using an Ortho Cytoron Absolute flow cytometer (Raritan) to record 3,000 to 5,000 events for each assay.
For the phagocytosis assays, C. albicans was harvested from Sabouraud glucose broth after overnight incubation at 37°C. After washing the cells twice with PBS, C. albicans cells were resuspended in PBS supplemented with 0.5% glucose at a concentration of 5 x 106/ml and labeled with calcein-AM (10 μM; Molecular Probes) for 1 h at 37°C. PECs and calcein-AM-labeled C. albicans (1:1 ratio) were incubated at 37°C for 2 h with 5% CO2. The mixture of PECs and calcein-AM-labeled C. albicans was then reacted with PE-conjugated monoclonal antibody to GR-1. The percentage of double-positive PECs, labeled with GR-1 (orange fluorescence) and calcein-AM (green fluorescence), was recorded as the percentage of GR-1-positive PECs phagocytizing C. albicans (26).
For the C. albicans killing assays, ethidium homodimer 1 (Molecular Probes) was used to assess the percentage of dead C. albicans cells (red fluorescence). Briefly, PECs and C. albicans cells (1:1 ratio) were incubated at 37°C with 5% CO2 as described previously (26). After a 2-h incubation, 2 μM ethidium homodimer 1 (400 μl) was added to the PECs-C. albicans mixture (200 μl) and incubated at room temperature for 10 min. PBS supplemented with 0.5% glucose (700 μl) was then added, and the tubes were placed on ice and read immediately. The percentage of positive red fluorescent cells was obtained by flow cytometry (5,000 events).
RESULTS
Alimentary tract colonization and survival. The alimentary tracts of germfree gp91phox–/–/NOS2–/– mice were quickly (by 24 h) colonized by a C. albicans wild-type strain (SC5314) and both mutant strains (efg1/efg1 and efg1/efg1 cph1/cph1) (Table 1). At 24 h after oral inoculation, their alimentary tracts contained 6 to 8 log10 CFU of viable C. albicans/g in the intestinal contents and the mice remained chronically colonized with a pure culture of the C. albicans strains for the duration of the study (Table 1). Table 1 also shows that no viable C. albicans cells were recovered from the internal organs (kidney, liver, and spleen) of moribund gp91phox–/–/NOS2–/– mice that were colonized with either the wild-type or each of the two mutant strains. The C. albicans wild-type and mutant strains were both lethal for gp91phox–/–/NOS2–/– mice. The Candida-colonized mice did not survive beyond 16 days after oral colonization (Table 1).
Pathology of gp91phox–/–/NOS2–/– mice colonized with C. albicans wild-type (SC5314) or mutant strains (efg1/efg1 and efg1/efg1 cph1/cph1). Gross pathology was very evident in moribund gp91phox–/–/NOS2–/– mice colonized with C. albicans wild-type or either of the two mutant strains. Moribund mice had enlarged, pale yellow mesenteric lymph nodes (four to five times normal size) and, in several mice, cervical lymph nodes. In mice colonized with the SC5314 strain, numerous lung nodules were visible (Fig. 1). The spleen was often enlarged and abscessed (Fig. 1). Peyer's patches were also prominent, and some appeared yellow. The livers and kidneys appeared normal in size and were without grossly visible lesions (data not shown).
Microscopic examination of the hematoxylin and eosin-stained tissue sections revealed multiple collections of polymorphonuclear leukocytes (PMNs) in most internal organs. Abscesses were detected in the Peyer's patches, lymph nodes, liver, lungs, spleen, kidneys and, to a lesser extent, in the submucosa of the stomach, esophagus, tongue, and palate (Fig. 2A; Table 2). Except for the stomachs of mice colonized with SC5314, superficial candidiasis of keratinized mucosae was often absent, albeit in the presence of submucosal lesions in these same sites and in major organs of the same animal (Table 3). Hyphae, pseudohyphae, and blastoconidia, when detected in the abscesses, were usually present in small numbers (Fig. 2B). Although originally described as being "locked" in the yeast form (20), the finding that the efg1/efg1 cph1/cph1 double mutant was able to form hyphae in vivo is in agreement with previous observations that this strain can indeed form hyphae during infection and under specific conditions in vitro (8, 25). C. albicans was also rare in lesions of Peyer's patches from all three groups of colonized mice; however, viable Candida could not be cultured from abscessed internal organs, suggesting that the Candida cells present in the tissues were dead. Interestingly, the efg1/efg1 cph1/cph1 double mutant strain, while less invasive in gastric tissues than the wild-type SC5314 strain, appeared to be more invasive for oroesophageal (stomach and esophagus) tissues than the efg1/efg1 mutant (Table 3). Since C. albicans phospholipase B (PLB) has been shown to be required for the invasion of gastric mucosa and the translocation from the gastrointestinal tract (22), we examined PLB1 and PLB2 expression in gastric tissues from mice colonized with either SC5314 or each of the two mutant strains. PLB1 and PLB2 transcripts were detected for all three strains in vivo; however, there was no discernible difference in the ability of the different strains to express PLB1 and PLB2 (Fig. 3).
The vast majority of leukocytes in lesions were PMNs in various stages of degeneration. A small macrophage component was evident in the periphery of most soft tissue abscesses; however, in the lung, macrophages comprised approximately 5% of the infiltrating cells, and they were dispersed throughout collections of PMNs. Centrally, in some of the larger abscesses, areas of PMN necrosis, or liquefaction of PMNs, were evident. PMNs were represented by both mature polylobated forms and immature band forms. Granulomas with giant cells, epithelial cells, and lymphocytes were not present in involved tissues. A consistent observation from the three experimental groups 2 weeks after colonization was the pathology present in the Peyer's patches and mesenteric lymph nodes; usually, the involved lymph node was completely destroyed by infiltrating phagocytic cells.
Cytokine and chemokine expression in response to colonization and infection. ROI and RNI play important roles in mediating innate and acquired immune responses (6, 11, 17, 23). Impairment of ROI- and RNI-dependent signaling and immune responses could have contributed to the observed lethality. We investigated the capacity of these ROI- and RNI-deficient mice to express several cytokine (TNF-, IFN-, IL-10, IL-12, and IL-15) and chemokine (MIP-2 and KC) genes in response to C. albicans colonization and infection by using relative RT-PCR (Fig. 4). The relative expression levels of TNF- and IFN- were increased in gastric tissues from mice infected with either C. albicans SC5314 or efg1/efg1 cph1/cph1 strains compared to germfree controls. Similarly, expression levels of the chemoattractants MIP-2 and KC were increased in gastric tissues from the ROI- and RNI-deficient mice. In contrast, expression of IL-15, and to a lesser extent IL-10 and IL-12, were relatively similar in both infected and germfree control gastric tissues (Fig. 4). Since Toll-like receptors (TLR) play an important role in activating innate defenses and, in particular, inflammatory responses (14), we examined TLR1 to -10 mRNA expression in germfree and Candida-infected gastric tissues. Surprisingly, TLR1 to -10 were all readily detected in germfree control tissues (in the absence of a viable microbial flora), and an increase in TLR1 to -10 expression was not detected in infected tissues (data not shown).
Phagocytosis and killing of C. albicans. Shiloh et al. (31) reported that macrophages from gp91phox–/–/NOS2–/– mice were unable to kill a virulent strain of Listeria monocytogenes in vitro but were able to kill Salmonella enterica serovar Typhimurium, Escherichia coli, and an avirulent strain of L. monocytogenes. PECs (thioglycollate elicited) from gp91phox–/–/NOS2–/– mice and from single knockout mice, gp91phox–/– or NOS2–/–, were able to phagocytize and kill C. albicans as well as PECs from immunocompetent C57BL/6 mice in an in vitro assay (Table 4). The PECs from the immunocompetent and immunodeficient mice killed approximately 50% of the C. albicans cells within 2 h at 37°C. In addition, there was no significant difference in the percentage of macrophages or granulocytes in the PEC population retrieved from the immunocompetent C57BL/6 mice compared to that from the deficient NOS2–/–, gp91phox–/–, or gp91phox–/–/NOS2–/– mice.
DISCUSSION
This study demonstrated that mice with defects in their capacity to produce metabolites of both oxygen and nitrogen are susceptible to lethal candidiasis of endogenous (alimentary tract) origin. gp91phox–/–/NOS2–/– mice were found to be susceptible (lethal) to C. albicans wild type and to each of the two mutant strains that have previously been reported to exhibit significantly reduced virulence by acute systemic challenge (20). Our study demonstrated again, as is well known from many clinical candidiasis cases, that immunocompetence is an important component of a host's susceptibility to Candida species.
It became obvious in this study that the moribund ROI- and RNI-deficient mice were not dying from an overwhelming C. albicans sepsis. We observed (grossly and histologically) exaggerated and tissue-destructive abscess formation in most internal organs of the C. albicans-colonized ROI- and RNI-deficient mice. The abscesses were most prominent in mice colonized with the C. albicans wild-type strain. Although C. albicans hyphae, and to a lesser extent yeast cells, could be seen within the abscesses that formed in the C. albicans-colonized and -infected mice, we were unable to recover viable C. albicans from homogenized abscessed organs. In contrast, viable Candida organisms were readily isolated from the alimentary tract. Apparently, host defense mechanisms (antibody, complement, antibacterial proteins, etc.) other than ROI and RNI were still functional in these immunodeficient mice. The anti-Candida factors likely came from infiltrating phagocytic cells and killed the C. albicans cells that translocated to internal organs from the alimentary tract. Histopathology indicated that the most likely route into the internal organs was via Peyer's patches and mesenteric lymph nodes, which were almost completely destroyed by infiltrating phagocytic cells. Thus, even with deficiencies in the production of reactive metabolites of both oxygen and nitrogen, the gp91phox–/–/NOS2–/– mice still possessed anti-Candida mechanisms that killed C. albicans in abscessed internal organs; however, the mice were obviously unable to regulate an exaggerated and tissue-destructive inflammatory response to the Candida that translocated from the intestinal tract. Most important, the anti-Candida factors present in the ROI- and RNI-deficient mice were not able to prevent the animals' demise.
Shiloh et al. (31) reported that gp91phox–/–/NOS2–/– mice developed abscesses that contained viable commensal, mostly enteric, bacteria. Mice with a deficiency in either ROI or RNI were reported to be resistant to the commensal infections that afflicted the mice with combined ROI and RNI deficiencies (31). The latter report and our data with C. albicans demonstrated that combined deficiencies in ROI and RNI predispose mice to systemic infections by commensal prokaryotic and eukaryotic intestinal microorganisms. The ROI- and RNI-deficient mice, which apparently lack mechanisms to kill the prokaryotic commensals, retained their candidacidal activity. The germfree mice used in our study were not fed antibiotics, and germfree controls, which were not colonized with Candida, had no abscessed tissues. Shiloh et al. (31), in an attempt to control the commensal bacterial infections, administered oral antibiotics to the ROI- and RNI-deficient mice. The addition of antibiotics to the drinking water and housing these mice in microisolator cages with sterile food, water, and bedding (specific-pathogen-free conditions) did not prevent endogenous infections by commensal bacteria (31). It is noteworthy that Shiloh et al. (31) isolated Candida guillermondii from a few abscesses in their antibiotic-treated ROI- and RNI-deficient mice. The latter data suggest that the candidacidal mechanisms that persist in mice with combined ROI and RNI deficiencies may not be able to kill all Candida species. The latter could be associated with an immunodeficient host's susceptibility to opportunistic infections by different Candida species.
We have previously reported that the lethality of C. albicans for two other strains of immunodeficient mice, Tg26 (5, 33) and beige-nude (7) mice, was very likely due to severe, occlusive, esophageal candidiasis. We observed no overwhelming Candida sepsis or abscess formation in the Peyer's patches, lymph nodes, or internal organs of the former mice (5, 33). In contrast, occlusive oroesophageal candidiasis did not appear to be associated with lethality in the ROI- and RNI-deficient mice. In fact, mucosal tissues often had abscesses in the absence of obvious mucosal candidiasis. The mice with combined ROI and RNI defects had no problems in signaling phagocytic cells to the Candida-infected tissues, since MIP-2 and KC mRNAs were present in the infected gastric tissues. The exaggerated immune response apparently caused the destructive tissue pathology in the Peyer's patches and mesenteric lymph nodes that contributed to their rapid demise.
It is well known that ROI and RNI play important roles in signaling and regulating inflammation and immunity to infectious diseases (11, 17, 21, 23). For example, it has been reported that NO stimulates MIP-2 expression (32). In our study with mice that were deficient in NO, we detected basal levels of MIP-2 mRNA in germfree control tissues and also increased levels of MIP-2 mRNA in Candida-infected gastric tissues. The ROI- and RNI-deficient mice were also able to produce the mRNAs for the important inflammatory cytokines IFN- and TNF-, which may have contributed to the pathology in the ROI- and RNI-deficient mice; however, our previous studies indicated that ROI- and RNI-deficient mice were defective in their capacity to produce the antimicrobial peptide -defensin 4 (mBD-4) (28). ROI- and RNI-deficient mice, compared to immunocompetent controls, expressed lower levels of mBD-4 and were unable to increase mBD-4 expression in response to a Candida infection. This suggests that ROI and/or RNI may be required for maximal production of mBD-4.
In summary, the gp91phox–/–/NOS2–/– mice offer an exciting new model to study many facets of resistance and susceptibility to candidiasis.
ACKNOWLEDGMENTS
This work was supported by the National Institutes of Health (grant DE-13968).
We thank Kimberly Bauer, Andrea Boan, and April Harkins for critical reading of the paper.
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