High-Dose Ascorbate with Low-Dose Amphotericin B Attenuates Severity of Disease in a Model of the Reappearance of Candidemia During Sepsis in the Mouse

Am J Physiol Regul Integr Comp Physiol 309: R223–R234, 2015. First published May 20, 2015; doi:10.1152/ajpregu.00238.2014.

High-dose ascorbate with low-dose amphotericin B attenuates severity of disease in a model of the reappearance of candidemia during sepsis in the mouse

Asada Leelahavanichkul,1,2 Poorichaya Somparn,3 Tanabodee Bootprapan,1 Hongbin Tu,4 Pattarin Tangtanatakul,1 Ratchanok Nuengjumnong,2 Navaporn Worasilchai,1 Khajohn Tiranathanagul,5 Somchai Eiam-ong, Mark Levine,4 Ariya Chinampon,1 and Nattachai Srisawat5 1Department of Microbiology, Chulalongkorn University, Bangkok, Thailand; 2Center of Excellence in Immunology and Immune-Mediated Diseases, Department of Microbiology, Faculty of Medicine and STAR on Craniofacial and Skeletal Disorders, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand; 3Research Affairs, Department of Medicine, Chulalongkorn University, Bangkok, Thailand; 4National Institutes of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; and 5Nephrology Unit, Department of Medicine, Chulalongkorn University, Bangkok, Thailand Submitted 16 June 2014; accepted in ﬁnal form 5 May 2015

Leelahavanichkul A, Somparn P, Bootprapan T, Tu H, Tang- immune response in sepsis may present as a hyperimmune tanatakul P, Nuengjumnong R, Worasilchai N, Tiranathanagul characteristic with a rapid natural course of disease and/or an K, Eiam-Ong S, Levine M, Chinampon A, Srisawat N. immunosuppression phase responsible for nosocomial infec- High-dose ascorbate with low-dose amphotericin B attenuates tion (55, 62). Candidiasis, especially resulting from Candida severity of disease in a model of the reappearance of candidemia albicans, is one of the leading worldwide nosocomial infec- during sepsis in the mouse. Am J Physiol Regul Integr Comp Physiol 309: R223–R234, 2015. First published May 20, 2015; tions occurring after the onset of sepsis (30, 49). The mortality doi:10.1152/ajpregu.00238.2014.—Amphotericin B (Ampho B) is rate of patients with postsepsis systemic candidiasis is more a fungicidal drug that causes cell wall injury. Pharmacological than 50%, with more than 25% of cases of candidiasis occur- ascorbate induces the extracellular prooxidants, which might enter ring in patients who had developed sepsis (17, 22). Although the Ampho B-induced cell wall porosity and act synergistically. the incidences of infection from other fungal species are We tested low-dose Ampho B with a short course of pharmaco- increasing in patients in the intensive care unit, C. albicans still logical ascorbate using a mouse model of sepsis preconditioned remains the leading cause of nosocomial fungal infection (22, with an injection of Candida albicans 6 h prior to cecal ligation 48, 51). The increasing prevalence of nosocomial fungal in- and puncture (CLP). In this model, candidemia reappeared as early fection in cases of sepsis may be due to the growing use of as 6 h after CLP with a predictably high mortality rate. This broad-spectrum antibiotics coupled with the advancement of characteristic mimics sepsis in the phase of immunosuppression in patients. Using the model, at 12- and 18-h post-CLP, we admin- intensive care supports (8, 29, 37, 43, 59). istered isotonic (pH neutralized) pharmacological ascorbate intra- Amphotericin B (Ampho B) is a traditional broad-spectrum, venously with low-dose Ampho B or sodium deoxycholate, vehi- dose-dependent fungicidal drug, which is a pore-forming agent cle-controlled, administered IP. The survival rate of low-dose that acts though ergosterol inhibition. Ampho B, unfortunately, Ampho B plus ascorbate was 53%, compared with Ͻ11% for has substantial dose-dependent nephrotoxicity, and liposomal low-dose Ampho B or high-dose Ampho B alone. In addition, a amphotericin B is expensive (11). If Ampho B can be used at beneficial effect was demonstrated in terms of kidney damage, a lower dose in combination with another agent, perhaps liver injury, spleen histopathology, and serum markers at 24 h after nephrotoxicity and cost will be reduced. When given intrave- CLP. Kidney injury was less severe in low-dose Ampho B plus nously as a pharmacological agent, ascorbate (ascorbic acid or ascorbate combination therapy due to less severe sepsis. Moreover, vitamin C) demonstrates antineoplastic actions via the gener- ascorbate enhanced the effectiveness of phagocytosis against C. albicans in human phagocytic cells. Taken together, the data ation of extracellular hydrogen peroxide and downstream re- indicate that the new mouse model simulates sepsis-induced im- active oxygen species (ROS) (5–7, 37, 38). ROS formed in the munosuppression and that the combination of pharmacological presence of pharmacological ascorbate are less toxic to the host ascorbate with an antifungal drug is a potentially effective treat- tissue, but are very toxic to cancer cells in vitro and in vivo. We ment that may reduce nephrotoxicity, and perhaps also increase hypothesized that extracellular hydrogen peroxide generated fungicidal activity in patients with systemic candidiasis caused by by pharmacological ascorbate could enter the fungal cell wall, Candida albicans. which is injured or made vulnerable by the fungicidal pore- Candida albicans; ascorbate; sepsis; amphotericin B forming agent and enhanced antifungal therapeutic effectiveness (4–7). It should be noted that lower doses of ascorbate, given as a drug, may attenuate bacterial sepsis via additional SEPSIS IS A SERIOUS, GENERALIZED immune response to systemic mechanisms (1, 58, 63–65, 67). If ascorbate demonstrates infection, independent of the infecting organisms (42). The actions that support antibacterial and antifungal activities, then ascorbate in combination with Ampho B may be an effective combined therapy. Address for reprint requests and other correspondence: A. Leelahavanich- kul, Immunology Unit, Dept. of Microbiology, Chulalongkorn Univ., Bangkok To study ascorbate in combination with antifungal agents, an 10330, Thailand. animal model system is necessary. The available model for http://www.ajpregu.org R223 Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (079.016.061.055) on May 28, 2018. Copyright © 2015 American Physiological Society. All rights reserved. R224 HIGH-DOSE ASCORBATE Table 1. The minimal inhibitory concentration that BioTek, Shoreline, WA) by the optical density at 630 nm at 0.45 (OD demonstrated antifungal resistance to the Candida albicans 630 nm at 0.45). The procedure for LD50 was described in detail in the used in this study animal section below. For the germ tube test, 300 blastospores of C. albicans, as determined by hemocytometer, were placed in 50 ␮lof Drug MIC, ng/ml serum from a septic mouse (24 h after CLP) or serum from a sham mouse, incubated at 37°C for 3 h, then counted by hemocytometer. Amphotericin B 125 Fluconazole 125 The percentage of the ratio of blastospores with germ tube formation Itraconazole 31.3 to total blastospores was calculated. Ketonazole 31.3 Posaconazole 31.3 Animal and Animal Models Voriconazole 31.3 Anidulafungin 15 The U.S. National Institutes of Health (NIH) criteria and protocols Caspofungin 15 for the use and treatment of laboratory animals were followed (NIH MIC, minimal inhibitory concentration. publication protocols no. 85-23, revised 1985). Male, 8–10-wk old BalB/C mice (National Laboratory Animal Center, Nakornpathom, Thailand) were used. The animal protocols were approved by the Candida infection, characterized by Candida injection after Institutional Animal Care and Use Committee of the Faculty of sepsis (9), may not reflect Candida infections in humans, Medicine, Chulalongkorn University, Bangkok, Thailand. All proce- which mostly resulted from endogenous sources (50). As a dures were performed under isoflurane anesthesia. result, we developed a new sepsis-induced candidemia reap- Candida albicans injection model. Resuspended C. albicans in normal saline solution (NSS) adjusted into different doses of optical pearance model that is characterized by the reactivation of an density at 630 nm in the same volume of each preparation (100 ␮l) exogenous Candida administration prior to the onset of sepsis. was injected through the tail vein. The LD50 was extrapolated from We used this new system to test Ampho B administered alone the correlation equation of the graph of the 7-day mortality of mice, and in combination with pharmacological ascorbate. which was injected with C. albicans in different OD 630-nm doses (0.8, 0.6, 0.4, 0.2, and 0.1). The Candida dose with 50% mortality rate METHODS was calculated and identified as LD50. C. albicans Preparation, Characterization, and Germ Tube Test Cecal ligation and puncture sepsis model. The cecal ligation and puncture (CLP) procedure was described in detail previously (34), C. albicans was isolated from blood samples (Mycology Unit, King with some modifications. Briefly, the cecum was ligated at 12 mm Chulalongkorn Memorial Hospital), identified by morphology to- from the cecal tip and punctured twice with a 23-gauge needle, gether with the API 20C AUX yeast identification kit (Biomuriex, leaving a small amount of fecal material exposed. The abdominal Lyon, France), and stored in Sabouraud dextrose agar (SDA) (Thermo incision was closed in two layers with 6-0 nylon sutures. Antibiotic Scientific, Hampshire, UK) at Ϫ80°C. Before conducting all of the (imipenem/cilastatin 7 mg/kg in 0.3 ml NSS) was administered experiments, C. albicans was subcultured in SDA at 35°C for 24 h. subcutaneously at 3 h and 8 h after CLP for 24-h end-point experi- The characteristics of the clinically isolated C. albicans were demon- ments. Additional antibiotic doses were administered at days 2, 4, and strated by the minimal inhibitory concentration (MIC) (Table 1) and 6 after operation for 7-day survival experiments (Fig. 1). 50% lethal dose (LD50). MIC analysis followed a standard protocol A model of reappearance of candidemia during sepsis (43), with all reagents purchased from Sigma-Aldrich (St. Louis, (candidaϩCLP). One hundred microliters of C. albicans with the dose MO). The Clinical Laboratory Standard Institute recommended strains of OD 630 nm at 0.3 (ϳ2 ϫ 105 blastospores) or reciprocal NSS was Candida krusei American Type Culture Collection (ATCC) 6258 and injected through the tail vein at the Ϫ6 h time point (Fig. 1). Then at Candida parapsilosis ATCC 22019 were used for quality assurance. 0 h, the CLP operation or sham laparotomy was performed. The 5 The LD50 of selected C. albicans was 4 ϫ 10 blastospores as counted schedule of antibiotic administration was demonstrated in Fig. 1. In by hemocytometer (Bright-Line, Denver, CO), which was equal to the parallel, a reciprocal volume of NSS was injected in the sham group. value measured by a spectrophotometer (ELϫ808 absorbance reader; Blood was collected through the right retroorbital plexus under

Fig. 1. Timeline of a model of reappearance of candidemia in sepsis [Candidaϩcecal ligation and puncture (CLP)]. In 24-h experiments, Candida albicans was intravenously injected at 6 h before cecal ligation and puncture (CLP) (Ϫ6 h), then subcutaneously administered antibiotic (ATB sc) was administered at 3 h and 8 h. Subsequently, the intravenous (tail vein) ascorbate (Asc) or normal saline (NSS) plus intraperitoneal amphotericin B 0.1 mg·kgϪ1·doseϪ1 (0.1 MK) or 1 mg·kgϪ1·doseϪ1 (1 MK) or intraperitoneal deoxyglycolate (De-cholate) were administered at 12 h and 18 h after CLP. The mice were euthanized at 24 h after CLP. In the 7-day survival experiment, additional ATBs were injected subcutaneously at 1, 2, 4, and 6 days after CLP, and intraperitoneal amphotericinB1 mg·kgϪ1·doseϪ1 (Ampho B 1 MK) or De-cholate was given at 2, 4, and 6 days after CLP.

Polymorphonuclear Cell Count, Blood Chemistry, and Cytokine Phagocytosis and Killing Assays Concentrations Human phagocytes from healthy donors were separated by 6% ␮ ␮ A volume of 15 l of blood was mixed with 250 l of 3% acetic dextran solution, as previously described (41). Coincubation of 3 ϫ acid, a hemolytic solution, and the total number of leukocytes was 103 phagocytes/ml with 3 ϫ 103 blastospores/ml of C. albicans in ␮ counted with a hemocytometer. Simultaneously, 10 l of blood was PBS with the serial concentrations of ascorbate (10–40 mM) adjusted ϫ smeared on a glass slide for Wright stain and counted with 100 to pH 7 at 37°C for 1 h was performed. Then, 100 ␮l of well-mixed magnification in 100 fields to determine the percentage of polymor- solution was isolated and stained with Wright’s stain according to the phonuclear cells (PMN). The total number of PMN was calculated by standard protocol. Phagocytic activity was determined by the percent- total leukocyte count from hemocytometer multiplied by the percentage of phagocytes with Candida blastospores inside to the total age of PMN from the Wright stain glass slide. Serum ascorbate (Asc) phagocyte count at ϫ400 magnification from 100 fields per slide. and alanine transaminase (ALT) were measured with colorimetric Concurrently, to determine the phagocyte-killing function, 150 ␮lof detection by ascorbic acid assay (BioAssay U.S. Biological, Salem, the solution was centrifuged to separate phagocytes, then washed MS) and ALT assay (DiaSys Diagnostic Systems, Holzheim, Ger- twice with PBS, recentrifuged, and filled with 100 ␮l of a nutritionally many), respectively. Serum creatinine (Scr) was measured by HPLC rich medium (Lysogeny broth; Sigma-Aldrich) to initiate complete (32–34). Serum TNF-␣, IL-6, IL-10, and IL-17 were determined by cell disruption and the release of intracellular blastospores. The the Luminex xMap-based multiplex technology MILLIPLEX MAP (multianalyte panels) 4-plex cytokine kit (Millipore, Billerica, MA), solution was then put in SDA at 35°C for 24 h and counted for fungal according to the manufacturer’s recommended procedure. colonies (16). All experiments were performed in triplicate.

Microorganism Burdens in Blood, Peritoneal Fluid, and Internal Statistical and Survival Analysis Organs SPSS version 17.0 (SPSS, Chicago, IL) was used for all statistical Blood and peritoneal fluid in serial volume was immediately analyses. Data are presented as mean values and the standard error of Ϯ diluted in PBS, plated in SDA for 24 h at 35°C for fungal colony the means (means SE). Differences among groups of results were counting, and reciprocally placed in blood agar (Oxoid, Hampshire, examined using one-way ANOVA, followed by Bonferroni analysis. UK) for 24 h at 37°C for bacterial colony counting. Internal organs The two-tailed Student’s t-test was used to examine the difference of were washed twice in PBS, weighed, homogenized, diluted in PBS, the optical density at 630 nm between Asc and Ampho BϩAsc and and incubated in SDA. The colony count in SDA after 24 h at 35°C also used to demonstrate the difference in serum creatinine between was defined as the C. albicans burden. Moreover, to test whether there Candida plus CLP and sham. A P value Ͻ0.05 was considered was C. albicans in feces and gastrointestinal tissues of mice without statistically significant. Survival analyses were evaluated using the a preconditioning Candida injection, the different amounts of fecal log-rank test by observation and recorded every 6–12 h for the first 72 contents were collected. Serial dilutions of gastrointestinal contents h after Candida injection or CLP surgery, and then once daily to 7 and and tissue from cecum, colon, and recto-sigmoid area were collected 14 days for CLP survival and Candida injection survival, respectively. from normal mice and septic mice at 24 h after CLP. Feces, well- Mice exhibiting extreme morbidity were euthanized. Some of the mixed in PBS, and tissues were plated directly in SDA with 0.1% mice with the manifestations of the extreme morbidity, such as chloramphenicol (Bio-Rad, Hercules, CA), Sabouraud dextrose broth abnormal breathing patterns or no response to stimuli, were eutha- (SDB), and SDB with 0.05 g/l chloramphenicol (Sigma-Aldrich). nized, and their internal organs were examined for organ histology.

Characteristics of a Model of Reappearance of Candidemia During Sepsis (CandidaϩCLP) The goal of a superimposed Candida infection in a sepsis model is to have reappearance of Candida, so as to mimic Candida infection as it occurs after sepsis in humans. There- fore, the reappearance of candidemia was demonstrated. Can- didemia spontaneously reappeared from the 4th day after Candida injection alone (Fig. 2B), but reappeared as early as 6 h after CandidaϩCLP (Fig. 3A), ϳ93 h earlier. Moreover, candidemia in CandidaϩCLP increased very rapidly from 34 Ϯ 16 CFU/ml at3hto2,143 Ϯ 434 CFU/ml at 24 h after CLP (Fig. 3A). In contrast, Candida injection alone without CLP showed a delayed and more modest increase in candidemia from 15 Ϯ 10 CFU/ml at day 4 to 1,650 Ϯ 897 CFU/ml at day 7 after injection (Fig. 2B). Of note, CLP alone did not produce candidemia at 24 h after CLP (Fig. 3A) and at 7 days (data not shown). Moreover, C. albicans could not be detected by culture (see METHODS) of cecal contents and gastrointestinal tissues of normal and septic mice. Sepsis from CLP without Candida, coupled with postsurgery treatment, showed a survival rate of 73% (11 of 15 mice) at day 7 (NSSϩCLP), indicating a modest severity of sepsis in our CLP model (Fig. 3B). The addition of Candida infection before sepsis (CandidaϩCLP) produced 100% mortality at 5 days (Fig. 3B). Fig. 2. Characteristics of Candida injection model. Blood candidemia after Moreover, mice with CandidaϩCLP died rapidly, with Ͼ50% administration of various doses of Candida at 24 h is demonstrated. A, inset: mortality within 1 day and Ͼ67% mortality within 2 days (Fig. graph magnification is shown that indicates zero Candida blood level. B: ϩ candidemia at 7 days observation (B)(n ϭ 5/time point). Candida burdens in 3B). Rapid mortality in the Candida CLP model is likely due different organs at 6 h and 24 h after Candida injection at the dose of optical to the severity of sepsis. Compared with controls, within the density (OD) 630 nm 0.3 are shown (C)(n ϭ 4–6/group). Measured param- first 24-h CandidaϩCLP mice had higher cytokines and more eters (means Ϯ SE) were significantly different among the absorbance for fixed pronounced neutropenia than CLP alone (Fig. 3, C–G), as well time point using one-way ANOVA followed by Bonferroni analysis with alpha set at 0.05 and four comparisons for A and B and eight comparisons for C. as higher fungal burden (Fig. 3A). Limited leukocytosis and/or (a,b,cP Ͻ 0.05; a is OD 1.2 vs. OD 0.8, b is OD 1.2 vs. OD 0.6, and c is OD rapid neutropenia could be factors in the severity of 1.2 vs. OD 0.3). CandidaϩCLP. In contrast, germ tube production in septic serum was lower than in normal serum (Fig. 3H). High blood Candida levels in CLP mice (Fig. 3A), despite less Candida RESULTS growth in vitro with septic serum, is consistent with an immu- nologic cell role in the control of candidemia. CandidaϩCLP Characteristics of Candida Injection Model mice at 3 days after injection showed multiple Candida After the injection of different doses of Candida, candi- pseudohyphae lesions on the heart, renal glomeruli, and demia presented after all of the selected doses of the injection. renal interstitium (Fig. 4). These findings are consistent with Candidemia became undetectable after 3–6 h for low-dose disseminated candidiasis. The reappearance of Candida injections (OD 630 nm 0.3 and 0.6) (Fig. 2A, inset) and then from internal sources in the CandidaϩCLP mice resembles reappeared (Fig. 2B). Despite the disappearance of Candida human sepsis in terms of the reactivation of latent Candida, from the blood after 6 h and 24 h after injection with a dose of although from different sources of Candida (21, 50). Sub- OD 630 nm 0.3, Candida was present in all measured organs sequently, we used the CandidaϩCLP model for further

Fig. 3. Characteristics of a model of reappearance of candidemia in sepsis (CandidaϩCLP). Serial blood candidemia within 24 h after Can- dida injection at 6 h before CLP (CandidaϩCLP) or NSS injection then CLP (NSSϩCLP) or Candida injection with sham operation (CandidaϩSham) (A)(n ϭ 7/group) was demonstrated. The survival of different groups (B)(n ϭ 15/group), cytokine responses including, proinflammatory cytokines (TNF-␣, IL-6) (C and D), anti-inflammatory cytokines (IL-10) (E), fungal response cytokines (IL-17) (F)(n ϭ 7/group), neutrophil count (G)(n ϭ 5/group) are shown. Additionally, the comparison of germ tube growth in normal mouse serum and septic mouse serum is shown (H)(n ϭ 3–5/group). Measured parameters (mean Ϯ SE) were significantly different among the CandidaϩCLP, NSSϩCLP, and CandidaϩSham groups for fixed time point using one-way ANOVA followed by Bonfer- roni analysis with alpha set at 0.05 and three comparisons. (a,b,cP Ͻ 0.05, *,#,$P Ͻ 0.005, **,##,$$P Ͻ 0.0005; a, *, and ** denote CandidaϩCLP vs. NSSϩCLP; b, #, and ## denote CandidaϩCLP vs. CandidaϩSham; and c, $, and $$ denote NSSϩCLP vs. CandidaϩSham).

studies due to the Candida reactivation property of the inhibition of ϳ85% (Fig. 5). Alternatively, there was no model. synergistic effect of ascorbate at these concentrations with fluconazole at a dose 10 times lower than the MIC (125 ng/ml) The In Vitro Synergistic Effect of Amphotericin B with (data not shown). It appears that the synergistic effects of Ascorbate and In Vivo Ascorbate Concentrations high-dose ascorbate may be limited to only specific antifungal We used the CandidaϩCLP model to test the synergistic drugs. properties of Ampho B and ascorbate, beginning with in vitro Subsequently, ascorbate concentration at or above 10 mM experiments. The Ampho B concentration was selected at 10 was chosen as the target level of serum ascorbate for further times lower than the minimal inhibitory concentration (MIC), experiments. We take advantage of renal dysfunction in the and incubations were performed with different pharmacologi- CandidaϩCLP model to maintain serum ascorbate (Fig. 6A). cal ascorbate concentrations. The Ampho B concentration was While ascorbate is rapidly cleared in mice without renal insuf- chosen such that no fungicidal effect was predicted with its use ficiency, ascorbate concentrations are maintained at or above alone. Also, a very low dose of Ampho B could reduce 10 mM for many hours in CandidaϩCLP mice with ascorbate nephrotoxicity in vivo. Ascorbate concentrations were chosen injection at 12 h and 18 h after surgery (Fig. 6B). Ascorbate to reflect those measured in humans after intravenous admin- injections administered prior to 12 h post-CLP were cleared istration (66, 67). As predicted, neither low-dose Ampho B more rapidly (data not shown). In addition, the treatment of (125 ng/ml) alone nor ascorbate alone had any effect on patients is usually performed after the onset of the diseases, Candida growth. Ampho B combined with ascorbate concen- which is similar to the delayed treatment in the mouse model. trations ranging from 10 to 40 mM demonstrated growth For these reasons, we conducted experiments with treatment

Fig. 4. Representative histology from a model of reappearance of candidemia in sepsis (CandidaϩCLP). Pseudohyphae and mononu- clear inﬁltration at ϫ200 and ϫ400 magniﬁ- cation by periodic acid-Schiff (PAS) stain in cardiac tissue in (A and B), renal interstitium (C and D), and renal glomeruli (E and F) from mice that died after 3 days of a model of reappearance of candidemia in sepsis (CandidaϩCLP).

beginning 12 h after sepsis to test the synergy of pharmaco- was not statistically significant (AscϩDe-cholate vs. logic ascorbate with low-dose amphotericin B. NSSϩDecholate) (Fig. 7C). Although Ampho B administration in CLP mice did not noticeably worsen the severity of Low-Dose Amphotericin B Plus Pharmacologic Ascorbate In sepsis (NSSϩDe-cholate vs. NSSϩAmpho B 1 MK), it Vivo: Effects in a Model of Reappearance of Candidemia ϩ seemed to neutralize the potential benefit of ascorbate admin- During Sepsis (Candida CLP Model) istration (AscϩAmpho B) (Fig. 7C). To determine the micro- The objective of these experiments was to test the therapeu- organism loads, blood and peritoneal fluid were collected at 24 tic effects of low-dose Ampho B (0.1 mg·kgϪ1·doseϪ1, which h for Candida and bacterial cultures. As expected, candidemia is 10 times lower than the MIC) combined with pharmaco- and peritoneal Candida was lower with low-dose Ampho B logical ascorbate. As controls, deoxycholate alone plus ascorbate, but not with low-dose Ampho B alone (Fig. 8, (NSSϩDe-cholate), pharmacologic ascorbate with deoxy- A and B), consistent with the in vitro data (Fig. 5). However, cholate (AscϩDe-cholate), low-dose Ampho B alone bacterial burden was not different between groups (data not (NSSϩAmpho B 0.1 MK), and full-dose Ampho B alone shown). In addition, we evaluated sepsis severity in the (NSSϩAmpho B 1 MK) showed no survival benefit at 7 CandidaϩCLP model at 24 h, based on the following serum days (Fig. 7, A and B). Only ascorbate combined with markers: Scr for renal function, ALT for liver function, and low-dose Ampho B (AscϩAmpho B 0.1 MK) improved the cytokines for immune responsiveness. We also obtained histo- 7-day survival, from 9% (2/22) to 54% (12/22) (Fig. 7B). It pathology data of the kidney, spleen, and heart (Fig. 9). is both notable and interesting that full-dose Ampho B alone Low-dose Ampho B combined with pharmacological ascorbate improved the survival in days 1 and 2, but was reversed on attenuated organ injury: kidney injury as determined by Scr day 3, leading to high mortality and a low 7-day survival rate (Fig. 8C) and renal histopathology (Fig. 9H), liver injury as (Fig. 7B). In bacterial sepsis alone without Candida injection measured by ALT (Fig. 8D), and spleen injury as evaluated by (CLP), there was a tendency toward improved sepsis survival vacuole-like lesions in the white pulp of the spleen (Fig. 9I). in the ascorbate administration group, but the improvement Moreover, there was cardiac vacuolization in some mice re-

Fig. 5. In vitro synergy of low-dose amphotericin B and high-dose ascorbate. Synergistic effect of nontherapeutic dose Amphotericin B with different ascorbate concentration, in vitro, is shown. Data (means Ϯ SE) were obtained from ﬁve to seven independent experiments. P values were compared between doses using the independent Student’s t-test. ceiving low-dose Ampho B treatment alone, but not in mice with the combined treatment (Fig. 9G). Ampho B combined with pharmacological ascorbate reduced IL-6, TNF-␣, and IL-10, but not IL-17 (Fig. 8, E–H). Higher Ascorbate Concentration Improved Phagocytosis and Intracellular Killing of Candida by Human Phagocytic Cells Effective phagocytosis and phagocyte killing are mechanisms for the control of candidiasis, especially in the setting of neutropenia (32). The phagocyte killing depends on oxidative stress production, which might be associated with ascorbate

Fig. 7. Low-dose amphotericin B with high-dose ascorbate improved survival of a model of reappearance of candidemia in sepsis (CandidaϩCLP). Survival analysis of mice treated with high-dose ascorbate alone (AscϩDe-cholate), saline alone [normal saline solution (NSS)ϩDe-cholate], and a therapeutic dose of amphotericin B (NSSϩAmpho B 1 MK) (A) and the treatment with low-dose amphotericin B with and without ascorbate (AscϩAmpho B 0.1 MK and NSSϩAmpho B 0.1 MK) or full-dose Ampho B without ascorbate (NSSϩAmpho B 1 MK) in CandidaϩCLP model (B) is demonstrated. In addition, survival analysis is shown of reciprocal treatment in CLP mice in the absence of prior C. albicans inoculation (C) using the log-rank (Mantel-Cox) test (n ϭ 20–22/group) *P Ͻ 0.01, #P Ͻ 0.05 between indicated time point.

(61). We tested the ability of human phagocytic cells to phagocytize and kill Candida, in the presence of different ascorbate concentrations. Phagocytic activity was enhanced with pharmacological ascorbate concentrations of 30 and 40 mM (Fig. 10A), and Candida killing activity improved at concentrations of 20 and 40 mM (Fig. 10B).

DISCUSSION

Fig. 6. Time course of serum creatinine (serum Cr) and in vivo ascorbate level Prior mouse models of Candida used Candida injection after after intravenous injection. With CandidaϩCLP and CandidaϩSham model, sepsis (9), but this may not be the ideal method to mimic progression of kidney injury as measured by serum Cr (A), and serum reactivation of candidemia. In this study, we injected Candida ascorbate (serum Asc) level at baseline and time course after injection (B)is shown (n ϭ 5 or 6/time point). Of note, serum ascorbate level at 24 h was before sepsis, which was induced by CLP surgery 6 h after 12.8 Ϯ 1.2 mM. P values of the data (means Ϯ SE) were compared between Candida injection. We demonstrated that sepsis reactivated doses by the independent Student’s t-test. candidemia, resembling the alteration of intrinsic Candida

Fig. 8. Low-dose Ampho B with high-dose Asc attenuated severity of a model of reappearance of candidemia in sepsis (CandidaϩCLP). Sepsis severity parameters at 24 h of sham surgery models (CandidaϩSham, NSS administered at 12 h and 18 h), Asc, or NSS administered with CLP surgery models (CandidaϩCLP with NSS or Asc administered at 12 h and 18 h), as measured by candidemia (A), peritoneal Candida (B), serum creatinine (Scr) (C), alanine transminase (ALT) (D), cytokines [TNF-␣ (E), IL-6 (F) IL-10 (G), and IL-17 (H)] are demonstrated (n ϭ 5–7/ group). Blood ascorbate level at 24 h of Acs injection was 15.2 Ϯ 3.1 mM. Measured parameters (means Ϯ SE) were signiﬁcantly different among the CandidaϩSham with NSS, CandidaϩCLP with Asc, and CandidaϩCLP with NSS groups for a ﬁxed time point using one-way ANOVA, followed by Bonferroni analysis with alpha set at 0.05 and making three comparisons.

microbiota in patients with candidiasis (50). We found more 66). For these reasons, low-dose injections seemed to suitably severe injuries and a much higher mortality rate in mimic the immunosuppression phase, despite the more rapid CandidaϩCLP, compared with CLP alone. Using this model, natural course in mice, compared with human patients with we were able to investigate a new treatment modality and the sepsis. It is interesting to note that Candida injection alone combination of pharmacological ascorbate with low-dose am- without CLP did not cause renal injury at 24 h after injection, photericin B (Ampho B). This combination improved survival as measured by serum creatinine (Scr) (Figs. 8C and 9H), and attenuated disease severity, compared with the use of despite Candida being detected in the kidney at 24 h (Fig. 2C). either modality alone. Decreasing the time interval needed for Candida reappear- The severity of intravenous Candida albicans infection ance would improve the experimental model of sepsis immu- depends on the injected doses and the properties of C. albicans nosuppression. Candida reappeared as early as 6 h after sepsis used in different studies (36, 56). With high-dose Candida initiation in a model of reappearance of candidemia during injection, candidemia persisted at all time points. As for low- sepsis (Fig. 3A). In comparison, more than 4 days were needed dose Candida injection, mice cleared up candidemia very in nonseptic mice for Candida reappearance (Fig. 2B). Several rapidly within the ﬁrst 24 h after injection, with no spontaneous sources of Candida reactivation were demonstrated in several reappearance until after 4 days. Even with rapid clearance of organs (Fig. 2C) for mice in the CandidaϩCLP model, which candidemia in the low-dose model, tissue Candida persisted in was different from the translocation across the gut mucosa in the lung, kidney, and liver, consistent with other reports (36, patients with superimposed candidiasis (14). However, the

Fig. 9. Low-dose Ampho B with high-dose Asc attenuated organ histology of a model of reappearance of candidemia in sepsis (CandidaϩCLP). The representative figures of renal histology at 24 h of the model with PAS staining in sham surgery models (CandidaϩSham, NSS administered at 12 h and 18 h) (A), Asc or NSS administered with CLP surgery models (CandidaϩCLP with Asc or NSS administered at 12 h and 18 h; B and C). In parallel, spleen histology in individual group (D–F) and cardiac histology in non-Asc-treated CandidaϩCLP (G) is demonstrated. The original magnification in all of histology figures was ϫ400. The histological scoring from renal (H) and spleen histology (I) is shown (n ϭ 5 or 6/group). Measured parameters (means Ϯ SE) were significantly different among the CandidaϩSham with NSS, CandidaϩCLP with Asc, and CandidaϩCLP with NSS groups for fixed time point using one-way ANOVA, followed by Bonferroni analysis with alpha set at 0.05 and three comparisons. reactivation of the existing internal sources of Candida may phase. The parallel theory states that immunosuppression is more closely resemble the human condition, compared with concomitant with hyperinflammation (3, 52). Generally, im- other current models. Sepsis immunosuppression is initially mune compromise after sepsis can be manifested by the reac- defined as a condition in which patients or experimental ani- tivation of endogenous infections, such as cytomegalovirus, mals are susceptible to opportunistic infection in sepsis patients herpes virus, mycobacteria, and candidosis. We used candi- (40, 55). There are at least two hypotheses related to the demia reactivation as a marker of sepsis immunosuppression. It sepsis-immunosuppressive phase. The serial theory states that was necessary to inject Candida to be able to study its reacti- immunosuppression takes place after the hyperinflammatory vation as part of sepsis immunosuppression. Without prior

Fig. 10. Ascorbate-enhanced effective phagocytosis of human phagocytes. The positive phagocytic cells after incubation with Can- dida blastospore (A) and the Candida killing activity (B) with different ascorbate concentrations are shown. Data (means Ϯ SE) were obtained from five or six independent experiments. Measured parameters were significantly different among the ascorbate concentration groups for using one-way ANOVA, followed by Bonferroni analysis with alpha set at 0.05 and five comparisons.

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00238.2014 • www.ajpregu.org Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (079.016.061.055) on May 28, 2018. Copyright © 2015 American Physiological Society. All rights reserved. R232 HIGH-DOSE ASCORBATE Candida injection, we could not identify C. albicans by culture In animals and humans, concentrations of ascorbate (vitamin methodology in gastrointestinal tissue and contents, the natural C) are normally tightly controlled when it is ingested orally, reservoir of Candida (14), from either normal or septic mice. via transporter saturation (45–47). In humans, intestinal trans- Several explanations for this failure to identify C. albicans are porters saturate at doses above 200 mg, with decreased bio- possible, including 1) conventional culturing methods may be availability. Tissue transporters saturate when ingested doses insufficient to detect Candida as part of normal mouse flora; 2) are above 100 mg. Likewise, the renal reabsorptive ascorbate Candida may not be part of normal mouse flora; or 3) the transporter saturates at doses above 100 mg, with urinary presence of Candida may be dependent on mouse environment excretion of unabsorbed ascorbate. Together, these mecha- or strain. More sensitive detection methods and/or longer nisms produce orchestrated ascorbate concentrations. When observation times after sepsis (60) might be necessary to ascorbate is administered either intraperitoneally in animals or demonstrate spontaneous candidemia after sepsis alone. Nev- intravenously in animals or humans, tight control mechanisms ertheless, the reactivation of candidemia in CandidaϩCLP was are transiently saturated, until renal excretion restores homeo- very rapid, within hours after sepsis, compared with days or stasis. With parenteral administration of pharmacological weeks in patients in the intensive care unit. This difference doses of ascorbate, plasma concentrations can reach 500 times might, at least in part, be due to more focused and appropriate those possible with oral administration (47). Only these phar- treatments and life support systems for patients, a notable macological concentrations produce hydrogen peroxide in the absence in experimental studies using mice. extracellular fluid, which serves as a pro-drug for the formation The Candida injection changed a modest severity of CLP of reactive oxygen species that are toxic to cancer cells, but model with a 30% mortality rate into more severe injury with harmless to normal tissues (4, 7, 23). It has been postulated that a 100% mortality rate (Fig. 3B). A number of factors could be ROS formed from pharmacological ascorbate could also be responsible. The germ tube findings that we report here show harmful to bacteria (6). Lower doses of pharmacological ascor- that the serum of sepsis mice was less effective than normal bate have effects that attenuate the severity of sepsis (1, 2, 58, mouse serum as for fungal growth, despite an exponential 63–65, 67) that are not bactericidal. increase in Candida in sepsis. This finding suggests that Our findings showed that while pharmacological ascorbate changes in immune response elements could be responsible for alone was ineffective for candidemia, pharmacological ascor- accelerated candidemia in CandidaϩCLP septic mice. One bate combined with Ampho B, even when the latter was contributing factor may be a decreased neutrophil response in reduced to 10 times lower than the MIC, was more effective than Ampho B alone. Furthermore, full-dose Ampho B showed CandidaϩCLP compared with CLP alone, consistent with the minimal survival at 7 days in the Candida CLP model, com- neutrophil depletion occurring more rapidly when sepsis is pared with more than 50% survival at 7 days using pharmaco- combined with candidemia (31, 32). Rapid neutrophil deple- logical ascorbate with low-dose Ampho B. In vitro, for maxi- tion in CandidaϩCLP might be due to the higher sepsis mum synergy with low-dose Ampho B, 10 mM ascorbate was severity, which leads to the higher rate of neutrophil apoptosis, sufficient (Fig. 5). This concentration was not only easily as previously mentioned (18). Another factor may be associ- ϩ achieved in mice but also achieved easily and safely in humans ated with IL-17 responsiveness in Candida CLP mice, as (23). Synergy between pharmacological ascorbate and Ampho IL-17 is a cytokine responsible for the fungal infection (25, ϩ B may be a consequence of each having separate actions on 26). While Candida CLP mice had an increase in IL-17 Candida membranes. Ampho B is a pore-forming agent that compared with CLP mice, the increase may have been insuf- destabilizes the Candida membrane. Reactive oxygen species ficient for host protection. IL-17 was the only cytokine, among generated by pharmacological ascorbate from hydrogen perox- ϩ others we tested, with a similar level at 24 h in Candida CLP, ide, could also act independently to destabilize Candida mem- compared with CLP alone. Moreover, IL-17 was the earliest branes. In addition, hydrogen peroxide may also diffuse into ϩ cytokine to increase as early as 3 h after Candida CLP, but Candida, with the subsequent formation of internal reactive IL-17 did not increase in CLP alone. This characteristic sug- oxygen species leading to internal membrane or protein dam- gested the importance of IL-17 in controlling the severity of age. These potential actions of pharmacological ascorbate are ϩ sepsis in the Candida CLP model. We propose that IL-17 independent of Ampho B, and synergy is reasonably predict- synthesis in CandidaϩCLP was more strongly stimulated by able. Additional independent effects of ascorbate on neutro- both bacterial and fungal infection (25, 26). The dual impor- phils, phagocytosis, and killing could also contribute to the tance of IL-17 in both bacterial and fungal infection was benefits of pharmacological ascorbate (15, 27, 53, 61). previously published (19, 20, 25, 28). Still another contributing Because Ampho B is nephrotoxic, lower-dose regimens are factor of high mortality in CandidaϩCLP mice could be injury preferable. Pharmacological ascorbate was permissive for to the spleen. In this study, we found groups of apoptotic/ lower dosing of Ampho B in the CandidaϩCLP model. The necrotic cells in the white pulp of the spleen, previously CLP model induces renal insufficiency, such that only two described as a “vacuole-like area”, were more prominent in doses of intravenous ascorbate as the delayed treatment were CandidaϩCLP spleens, consistent with the description in se- sufficient. In contrast, most of the successful sepsis treatments vere sepsis (44). Apoptosis of immune cells, especially apo- in mouse models need to be administered at the onset or the ptosis in the spleen as reported in patients with sepsis (24) and early phase of sepsis (10, 12, 13, 33–35). mouse models of sepsis (34, 35, 44), was hypothesized to be A concern relating to this model is whether or not additional one of the responsible factors for the immunosuppression and nephrotoxicity is induced by pharmacologic ascorbate. In the more severe injury in sepsis. The severe destruction of the setting of renal insufficiency, intrarenal oxalate formation and spleen might be an additional contributing factor to the immu- crystallization are concerns with pharmacological ascorbate. nosuppression observed in the CandidaϩCLP model. Oxalic acid, an end product of ascorbate metabolism, can

AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00238.2014 • www.ajpregu.org Downloaded from www.physiology.org/journal/ajpregu by ${individualUser.givenNames} ${individualUser.surname} (079.016.061.055) on May 28, 2018. Copyright © 2015 American Physiological Society. All rights reserved. HIGH-DOSE ASCORBATE R233 crystallize as calcium oxalate in urine and renal interstitial 4. Chen P, Yu J, Chalmers B, Drisko J, Yang J, Li B, Chen Q. tissues. In CandidaϩCLP mice treated with ascorbate, we did Pharmacological ascorbate induces cytotoxicity in prostate cancer cells not find any crystal deposits in kidney histology, after 1 day through ATP depletion and induction of autophagy. Anticancer Drugs 23: 437–444, 2012. and from autopsy (data not shown). We also found that serum 5. Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner creatinine was lower in mice treated with pharmacological GR, Shacter E, Levine M. Pharmacologic ascorbic acid concentrations ascorbate combined with low-dose Ampho B, compared with selectively kill cancer cells: action as a pro-drug to deliver hydrogen Ampho B alone. Moreover, we demonstrated that ascorbate peroxide to tissues. Proc Natl Acad Sci USA 102: 13,604–13,609, 2005. injection in CandidaϩCLP did not alter the kidney function. 6. Chen Q, Espey MG, Sun AY, Lee JH, Krishna MC, Shacter E, Choyke PL, Pooput C, Kirk KL, Buettner GR, Levine M. Ascorbate in On the other hand, full-dose Ampho B improved the mortality pharmacologic concentrations selectively generates ascorbate radical and rate at day 2 of survival but not at day 7 of survival, a finding hydrogen peroxide in extracellular fluid in vivo. Proc Natl Acad Sci USA that suggests its toxicity in full-dose treatment (Fig. 6B). The 104: 8749–8754, 2007. findings from the combined treatment group demonstrate that 7. Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, the treatment effects of adding pharmacological ascorbate were Khosh DB, Drisko J, Levine M. Pharmacologic doses of ascorbate act as beneficial for renal function. Another concern involved the a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci USA 105: 11,105–11,109, 2008. limitations of mouse models and whether or not they are able 8. Collin B, Clancy CJ, Nguyen MH. Antifungal resistance in non-albicans to sufficiently represent human patient conditions and re- Candida species. Drug Resist Updat 2: 9–14, 1999. sponses. The debate on this topic is still ongoing (54, 57). 9. Davis CG, Chang K, Osborne D, Walton AH, Dunne WM, Muenzer Nevertheless, we postulated that our two-hit models are more JT. Increased susceptibility to Candida infection following cecal ligation complex and might more accurately mimic the patient’s con- and puncture. Biochem Biophys Res Commun 414: 37–43, 2011. 10. Dear JW, Leelahavanichkul A, Aponte A, Hu X, Constant SL, Hewitt ditions. However, validation in patients with reciprocal sepsis SM, Yuen PS, Star RA. Liver proteomics for therapeutic drug discovery: condition is required. inhibition of the cyclophilin receptor CD147 attenuates sepsis-induced acute renal failure. Crit Care Med 35: 2319–2328, 2007. Perspectives and Significance 11. Deray G. Amphotericin B nephrotoxicity. J Antimicrob Chemother 49 Suppl 1: 37–41, 2002. In our new mouse model demonstrating an immunosuppres- 12. Doi K, Hu X, Yuen PS, Leelahavanichkul A, Yasuda H, Kim SM, sive phase with Candida reappearance, a synergistic effect of Schnermann J, Jonassen TE, Frokiaer J, Nielsen S, Star RA. AP214, an analogue of alpha-melanocyte-stimulating hormone, ameliorates sep- pharmacological ascorbate with low-dose Ampho B was dem- sis-induced acute kidney injury and mortality. Kidney Int 73: 1266–1274, onstrated in vitro and in vivo, without apparent toxicity. Taken 2008. together, the data provide a proof of concept for pharmacolog- 13. Doi K, Leelahavanichkul A, Hu X, Sidransky KL, Zhou H, Qin Y, ical ascorbate as an adjuvant for antifungal treatment. Pharma- Eisner C, Schnermann J, Yuen PS, Star RA. Pre-existing renal disease cological ascorbate is inexpensive and can be easily available. promotes sepsis-induced acute kidney injury and worsens outcome. Kid- Pharmacological ascorbate in combination with existing anti- ney Int 74: 1017–1025, 2008. 14. Donskey CJ. The role of the intestinal tract as a reservoir and source for fungals may indicate the use of lower doses with reduced transmission of nosocomial pathogens. Clin Infect Dis 39: 219–226, 2004. toxicity. 15. Dwenger A, Pape HC, Bantel C, Schweitzer G, Krumm K, Grotz M, Lueken B, Funck M, Regel G. Ascorbic acid reduces the endotoxin- ACKNOWLEDGMENTS induced lung injury in awake sheep. Eur J Clin Invest 24: 229–235, 1994. The authors would like to gratefully acknowledge the Center of Excellence 16. Du C, Calderone RA. Phagocytosis and killing assays for Candida in Immunology and Immune-Mediated Diseases, Department of Microbiology, species. Methods Mol Biol 499: 17–26, 2009. Faculty of Medicine, Chulalongkorn University for its support of this research 17. Falagas ME, Apostolou KE, Pappas VD. Attributable mortality of study. This study was funded by the following grants: the Ratchadapiseksom- candidemia: a systematic review of matched cohort and case-control potch Fund, Faculty of Medicine, Chulalongkorn University; Grant for Devel- studies. Eur J Clin Microbiol Infect Dis 25: 419–425, 2006. opment of New Faculty Staff, Chulalongkorn University, as well as a grant for 18. Fialkow L, Fochesatto Filho L, Bozzetti MC, Milani AR, Rodrigues Chula Research Scholar, Ratchadaphiseksomphotch Endowment Fund. We Filho EM, Ladniuk RM, Pierozan P, de Moura RM, Prolla JC, also thank Kevin P. 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