Medical Mycology, 2014, 52, 847–852 doi: 10.1093/mmy/myu048 Advance Access Publication Date: 17 September 2014 Original Article

Original Article

Vitamin D and experimental invasive aspergillosis Downloaded from http://mmy.oxfordjournals.org/ at University of California, Santa Barbara on November 24, 2014

Christopher Sirivoranankul1,2 , Marife Martinez1 , Vicky Chen1 , Karl V. Clemons1,3,∗ and David A. Stevens1,3 1

Infectious Diseases Research Laboratory, California Institute for Medical Research, San Jose, CA, USA, College of Letters and Sciences, University of California at Los Angeles, Los Angeles, CA, USA and 3 Division of Infectious Diseases and Geographic Medicine, Stanford University, Stanford, CA 2

*To whom correspondence should be addressed. Karl V. Clemons, PhD, California Institute for Medical Research, 2260 Clove Dr., San Jose, California 95128. Tel: +408-998-4554; Fax: +408-998-2723; E-mail: [email protected] Received 30 June 2014; Revised 1 April 2014; Accepted 21 July 2014

Abstract Immune cells express the vitamin D receptor and vitamin D metabolizing enzymes. Favorable vitamin D effects have been indicated in tuberculosis. Vitamin D deficiency increases T helper (Th) 2 responses to Aspergillus, and it suppresses Th2 responses in cystic fibrosis–allergic bronchopulmonary aspergillosis. Can vitamin D modulate the proinflammatory effects of amphotericin B (AmB) therapy in aspergillosis? Groups of mice were infected intravenously (IV) with 3–8 × 106 Aspergillus fumigatus conidia. In six experiments, doses of 0.08, 2, or 4 µg/kg calcitriol (active form of vitamin D) were given intraperitoneally +/− AmB–deoxycholate (AmBd) at 0.4, 0.8, 1.2, 1.8, 3.3, or 4.5 mg/kg or 0.8 or 1.2 mg/kg IV. Calcitriol doses were selected to range from doses used in humans to those just below doses shown to decalcify murine bones. In most experiments, doses of calcitriol and AmBd (or control diluents) were given five times, on alternate days, to minimize drug–drug interactions. Calcitriol treatment began on the day of challenge, and survival assessed for 10 days. In no experiments did calcitriol alone significantly worsen or enhance survival or affect residual infection in survivors. Calcitriol also did not affect the efficacy of AmBd. In a representative experiment, AmBd at 0.8 or 1.2 mg/kg IV alone +/− calcitriol at 2 µg/kg enhanced survival (P ≤ 0.01). However, the AmBd regimens with calcitriol were not different than those without, and calcitriol alone was identical to controls. In disseminated invasive aspergillosis, calcitriol did not affect outcome nor influence antifungal efficacy. Key words: vitamin D, Aspergillus fumigatus, murine model, amphotericin B, systemic aspergillosis.

Introduction Vitamin D is a fat-soluble vitamin that is crucial for the development and maintenance of bones [1]. Through vitamin D receptor (VDR) signaling, vitamin D increases calcium absorption to promote bone mineralization. As

a “nonessential” vitamin, the vitamin D precursor is created by the human skin through exposure to sunlight; more specifically, ultraviolet B rays convert stored 7-dehydrocholesterol into cholecalciferol (vitamin D3 ). Vitamin D3 is transferred via the bloodstream to the liver

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ciated with different manifestations of coccidioidomycosis in patients [23]. This study focused on the idea that vitamin D appears to favorably modulate bacterial infections such as tuberculosis [24], that may be similar to fungal infections, possibly through induction of cathelicidin by monocytic cells, which improves killing by the phagocytes [10]. Vitamin D deficiency increases Th2 responses to Aspergillus fumigatus, and vitamin D has been shown to downregulate Aspergillusinduced Th2 cytokines, such as IL-5 and IL-13, from cells of allergic bronchopulmonary aspergillosis patients. However, gliotoxin, which is produced by the fungus, has been shown to overcome the vitamin D/VDR downregulation of IL-5 and IL-13 [25]. In addition, because amphotericin B toxicity correlates with induction of proinflammatory cytokines, vitamin D may act to moderate amphotericin B toxicity [26]. In this study, a murine model was used to test the combination of calcitriol with amphotericin B as a therapy against systemic aspergillosis.

Methods Organism Aspergillus fumigatus 10AF was thawed from −80o C storage and grown for 3 days at 37o C on potato dextrose agar plates [27]. The conidia were harvested, washed by centrifugation, and suspended in 0.05% Tween 80 saline solution. Conidia were enumerated by plating dilutions and further confirmed by counting in a hemacytometer. The conidia were then diluted in 0.05% Tween 80 saline solution.

Infection model and therapy Groups of ten 38-day-old female CD-1 mice (Charles River Laboratories, Hollister, CA, USA) were infected intravenously (IV) with (3–8) × 106 A. fumigatus conidia as described previously [28]. All animal experiments were done under a protocol approved by the Institutional Animal Care and Use Committee of the California Institute for Medical Research. In six experiments, doses of calcitriol at 0.08, 2, or 4 µg/kg (CAS 32222–06–3; Sigma Aldrich, St. Louis, MO, USA) were given intraperitoneally (IP). Amphotericin B–deoxycholate (AmBd; X-Gen, Big Flats, NY, USA) was given at doses of 0.4, 0.8, 1.2, 1.8, 3.3, or 4.5 mg/kg IP or 0.8 or 1.2 mg/kg IV; all AmBd doses were given in sterile 5% dextrose water (D5W). In one experiment, 5 mg/kg posaconazole (Merck, Whitehouse Station, NJ, USA) given via oral gavage daily was also similarly studied with or without calcitriol.

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where it is converted to calcidiol; this metabolite is used as a measure of the serum concentration of vitamin D of patients. Calcidiol is enzymatically converted in the kidneys to its active form, calcitriol (1,25-dihydroxyvitamin D3 ). Without sufficient active vitamin D, serum calcium levels would drop, causing bones to demineralize. In addition to its endocrinological benefits, vitamin D has been shown to enhance innate immunity [2]. Our interest in the effect of vitamin D on aspergillosis stems from the demonstrated vitamin D effects, in humans and mice, on T and B cells, natural killer cells, cytokine production, macrophages, antigen-presenting cells, antimicrobial peptides, reactive nitrogen, and oxygen species; Tolllike, dectin-1 and mannose receptors; and microbial killing, all relevant to the importance of T helper (Th)1/Th2 and effector phagocytes in aspergillosis [3]. The initial thought that vitamin D may boost immunity came with seasonal waves of influenza and low levels of vitamin D [4], indicating a correlation [5]. Recently, calcitriol was found to downregulate the proinflammatory responses of Th1 and Th17 cells [6], while upregulating Th2 cytokines [7], such as interleukin (IL)-4, and responses [8,9]. Along with these antiinflammatory actions, vitamin D also induces the production of cathelicidin, an antimicrobial peptide, through a VDR complex [10]. In addition, vitamin D precursors inhibit dendritic cell maturation and subsequent Th cell proliferation and also increase the numbers of transforming growth factor–β producing Treg cells, which modulate the general immune response [10,11]. Such potential defense against autoimmune and microbial diseases has prompted various studies on vitamin D and immunity [12,13]. Calcitriol and VDR decrease the likelihood of autoimmune disorders such as multiple sclerosis and inflammatory bowel disease by decreasing the function of Th1 cells [14], while increasing that of Th2 responses [15]. More relevant to infections, cathelicidin, induced by calcitriol through the actions of IL-37 [16,17], has been shown to inhibit Mycobacterium tuberculosis growth in vitro, which may explain why patients with tuberculosis have lower serum vitamin D levels [18,19]. However, few studies have been done with vitamin D in fungal infection. Although it was shown that cathelicidin has the ability to penetrate and inhibit growth of Candida albicans, no benefit of exogenous vitamin D in an in vivo model was found [20]. Similarly, vitamin D was shown to prolong graft survival and increase bone density of treated mice, but it did not alter susceptibility to infection with C. albicans [21]. Others have shown that vitamin D modulated host responses by downregulating the proinflammatory response to C. albicans both in vivo and ex vivo [22]. Lastly, differences in serum concentrations of vitamin D were not asso-

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Table 1. Table of calcitriol and amphotericin B–deoxycholate regimens studied. Calcitriol alone, µg/kg 0.08 QOD × 5 2 QOD × 4 2 QOD × 5∗ 4 QD × 9 Calcitriol with amphotericin B Amphotericin B–deoxycholate, mg/kg

0.08 QOD × 5 2 QOD × 4 2 QOD × 4 2 QOD × 5 2 QOD × 5 2 QOD × 5 2 QOD × 5 2 QOD × 5 2 QOD × 5 2 QOD × 5

0.4 IP QOD × 5 1.2 IP QOD × 3 1.8 IP QOD × 3 0.4 IP QOD × 5† 0.8 IP QOD × 5 3.3 IP QOD × 5† 3.3 IP QD × 10 4.5 IP QOD × 5 0.8 IV QOD × 5† 1.2 IV QOD × 5

The number in the table preceding frequency (QD or QOD) is the calcitriol dose, in μg/kg, or amphotericin B dose, in mg/kg; appearing after QD or QOD is the number of total doses given. For example, “calcitriol 2 QOD × 5” is calcitriol 2 μg/kg given every other day to a total of five doses. Calcitriol and amphotericin B were given on alternate days, with calcitriol given on the day of infection. All calcitriol treatments were given IP. IP, intraperitoneally; IV, intravenously; QD, daily; QOD, every other day. ∗ This regimen was studied five times. † These combined regimens were studied twice each.

Calcitriol doses were selected to range from doses used in humans up to those just below doses shown to decalcify murine bones. Doses of calcitriol and AmBd (or control diluents) were given five times each, every other day (QOD), to minimize drug–drug interactions in groups given both calcitriol and AmBd. In single experiments, calcitriol was also given daily for 10 days or QOD for four doses, or AmBd was given daily for 10 days or QOD for three doses. The various calcitriol–AmBd regimens studied are shown in Table 1. Calcitriol treatment began on the day of challenge, 2 hours after infection. The calcitriol or AmBd dose was adjusted according to the average weight of the treatment group. Saline with 0.1% ethanol (EtOH) was used for vitamin D dilutions (originally stored at −20o C in 100% EtOH), and D5W was the diluent for AmBd. Calcitriol and 0.1% EtOH treatments were given IP in 0.1-ml volumes and AmBd doses were given in 0.25-ml volumes IP or IV. Survival was followed through 10 days of infection. All surviving mice were euthanized, and kidneys of each mouse were harvested to determine the residual fungal burden, given as log10 colony-forming units (CFUs) per kidney pair on day 10 post-infection. Mice that succumbed to A.

Results The study spanned six trials, using a variety of treatment combinations. The AmBd–calcitriol trials are tabulated in Table 1. Despite these broad variations, in no experiments did any dose of calcitriol alone significantly worsen or enhance survival or affect residual CFU in survivors. Additionally, calcitriol did not affect antifungal efficacy when coupled with different doses of AmBd. These were also the conclusions of one experiment with posaconazole (data not shown). A representative experiment is presented in detail to illustrate the findings of this study. In the specific experiment shown, 60 female CD-1 mice (38-days-old) were infected IV with 3.51 × 106 conidia of A. fumigatus 10AF. The various treatment groups, days of dosing, doses, and routes of dosage are shown in Table 2. The survival of mice infected with A. fumigatus and treated with calcitriol alone or in combination with AmBd is shown in Figure 1. Two groups, control and calcitriol at 2 µg/kg alone, had 100% mortality by day 7 post-infection. Mice treated with AmBd at 1.2 mg/kg in combination with calcitriol at 2 µg/kg had 50% survival on day 10 postinfection. The group given AmBd at 0.8 mg/kg plus calcitriol at 2 µg/kg had 60% survival. The groups with the longest survival were given AmBd alone, with 70% survival for mice given either 0.8 mg/kg or 1.2 mg/kg. All treatments with AmBd were statistically significant compared with both the diluent-treated control and calcitriol-alone group (P = 0.013 to 0.05). Also, there were no significant differences between the control and calcitriolalone groups (P > 0.05). The 25 surviving mice were euthanized on day 10 postinfection, and kidneys of each mouse were harvested to determine the residual fungal burden. The median, represented by the horizontal lines in Figure 2, of groups with survivors were as follows: AmBd 0.8 mg/kg = 2.6, AmBd 1.2 mg/kg = 2.7, AmBd 0.8 mg/kg + calcitriol = 4.3, and

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Calcitriol, µg/kg

fumigatus infection were assigned a log10 CFU value of 5 for the fungal burden in the kidneys, thus according death a worse outcome than survival with any burden, as recommended for measurement of censored data [29,30]. Using the nonparametric Mann–Whitney test, statistical significance for log10 CFU was calculated. For survival curves, log-rank tests were used to determine the statistical significance between groups. Statistical significance was set as P < 0.05.

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Table 2. Groups and regimens of treatment of representative experiment. Group

Treatment 0

1 Control 2 Vitamin D 3 AmBd 0.8 4 AmBd 1.2 5 Vitamin D + AmBd 0.8 6 Vitamin D + AmBd 1.2

0.1% ethanol (IP) 5% dextrose water (IV) 2 µg/kg vitamin D (IP) 0.8 mg/kg AmBd (IV) 1.2 mg/kg AmBd (IV) 2 µg/kg vitamin D (IP) 0.8 mg/kg AmBd (IV) 2 µg/kg vitamin D (IP) 1.2 mg/kg AmBd (IV)

1

2

X

3

X X

X

X X

X X X

X

X X

X

X X

X X X

X X

X

X X

X X

X

9

X

X

X

8

X

X X

X

7

X

X

X

6

X

X X

X X

Treatment days 4 5

X X

X

X

Figure 1. Survival curves of CD-1 female mice infected with Aspergillus fumigatus 10AF and treated with varying regimens of amphotericin B– deoxycholate and calcitriol (vitamin D). Vitamin D treatment dose was 2 µg/kg. Vitamin D and ethanol (EtOH) were given intraperitoneally. Treatments were given every other day, with vitamin D starting on day 0 and amphotericin B (AmBd) starting on day 1 post-infection. All AmBd regimens were statistically significant compared with both 0.1% EtOH + sterile 5% dextrose water (D5W; control) and vitamin D (2 µg/kg; P = 0.013 to 0.05). IV, intravenously.

AmBd 1.2 mg/kg + calcitriol = 2.1. All AmBd regimens with and without calcitriol had significantly reduced fungal burdens compared with those given 0.1% EtOH + D5W or only calcitriol at 2µg/kg (P = 0.036–0.0039, dependent on comparison). As seen in the survival data, there was similar lack of significant differences in the CFU data in the AmBd alone vs. AmBd–calcitriol groups or calcitriol vs. controls.

Discussion Vitamin D has been widely studied in recent years, owing to potential benefits relating to autoimmune diseases as well as bacterial infections such as those caused by M. tuberculosis. Our goal in this study was to test the effect of vitamin D, as sole or adjunctive therapy, in modulating the innate immunity of the host, as well as possibly affecting the inflammatory responses that systemic aspergillosis and

AmBd induce. Mice treated with AmBd with or without vitamin D had longer survival. However, there was no statistical difference between groups treated with only AmBd and those treated with AmBd and vitamin D. Vitamin D alone showed no benefits or adverse effects on the survival of mice with systemic aspergillosis. The lack of an effect of vitamin D may be caused by gliotoxin, which is produced by the A. fumigatus used to initiate the infection. Gliotoxin downregulates VDRs, thus possibly lowering the modulating effect of supplementary vitamin [31]. Before concluding a lack of effect of vitamin D on aspergillosis, studies of other aspergillosis models, such as pulmonary aspergillosis, would be of interest. Our model mimics the clinical situation of disseminated aspergillosis, which is all too common in invasive human disease. Challenge by the respiratory route would focus on the pulmonary immune system, and modulation of that system might

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AmBd, amphotericin B–deoxycholate; IP, intraperitoneally; IV, intravenously.

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3. 4. 5. 6.

7.

Figure 2. Colony-forming units of CD-1 female mice 10 days postinfection. Horizontal lines represent the median of each group. Treatment regimens and abbreviations are as in Figure 1. Using the nonparametric Mann–Whitney test, all amphotericin B regimens with and without vitamin D were statistically significant compared with both 0.1% ethanol + sterile 5% dextrose water (D5W) and vitamin D–only groups (P = 0.036–0.0039, dependent on comparison). AmBd, amphotericin B–deoxycholate; CFU, colony-forming unit; EtOH, ethanol.

9. 10. 11. 12. 13.

produce results that cannot necessarily be predicted from our model. Another potentially useful study would be that of using vitamin D–deficient mice infected with A. fumigatus. Vitamin D–deficient mice might be more susceptible to infection with A. fumigatus and be unable to counteract the inflammatory effects of amphotericin B. However, pursuing this avenue is problematic in that there are inadequate data on what constitutes a vitamin D–sufficient mouse diet [32]. It would also be interesting to test the efficacy of vitamin D against a strain of A. fumigatus known to not produce gliotoxin, whether it be a mutant or wild-type strain. Such a study could provide insight as to whether calcitriol is ineffective or whether it simply cannot overcome the downregulation of VDRs by gliotoxin. Overall, our study has shown that adjunctive vitamin D therapy has no beneficial or deleterious effects on systemic aspergillosis and also does not alter the efficacy of AmBd in the treatment of this disease.

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

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Medical Mycology, 2014, Vol. 52, No. 8

Vitamin D and experimental invasive aspergillosis.

Immune cells express the vitamin D receptor and vitamin D metabolizing enzymes. Favorable vitamin D effects have been indicated in tuberculosis. Vitam...
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