Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model

Niloofar Najarzadegan; Mahboobeh Madani; Masoud Etemadifar; Nahad Sedaghat

doi:10.1371/journal.pone.0278488

. 2022 Dec 7;17(12):e0278488. doi: 10.1371/journal.pone.0278488

Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model

Niloofar Najarzadegan ¹, Mahboobeh Madani ^1,^*, Masoud Etemadifar ², Nahad Sedaghat ^3,⁴

Editor: Aijaz Ahmad⁵

PMCID: PMC9728862 PMID: 36477491

Abstract

Fingolimod (FTY720) is a drug derived from the fungicidal compound myriocin. As it was unclear whether FTY720 has antifungal effects as well, we aimed to characterize its effect on Candida albicans in vitro and in a mouse candidiasis model. First, antifungal susceptibility testing was performed in vitro. Then, a randomized, six-arm, parallel, open-label trial was conducted on 48 mice receiving oral FTY720 (0.3 mg/kg/day), intraperitoneal C. albicans inoculation, or placebo with different combinations and chorological patterns. The outcome measures of the trial included serum concentrations of interleukin-10 and interferon-gamma, absolute lymphocyte counts, and fungal burden values in the mice’s livers, kidneys, and vaginas. Broth microdilution assay revealed FTY720’s minimum inhibitory concentration (MIC₉₉) to be 0.25 mg/mL for C. albicans. The infected mice treated with FTY720 showed lower fungal burden values than the ones not treated with FTY720 (p<0.05). As expected, the mice treated with FTY720 showed a less-inflammatory immune profile compared to the ones not treated with FTY720. We hypothesize that FTY720 synergizes the host’s innate immune functions by inducing the production of reactive oxygen species. Further studies are warranted to unveil the mechanistic explanations of our observations and clarify further aspects of repurposing FTY720 for clinical antifungal usage.

1. Introduction

Fingolimod (FTY720) is an immunomodulatory drug currently indicated and approved by the United States Food and Drug Administration (FDA) for treatment of the relapsing forms of multiple sclerosis (MS)–an autoimmune entity involving the central nervous system. It is used off-label for progressive MS and other autoimmune neuropathies, while being evaluated for extra-nervous pathologies as well [1–3]. FTY720 is considered a prodrug of its phosphorylated form fingolimod-phosphate (FTY720-P). Being a modulator of the sphingosine-1-phosphate receptors (S1PR) by structural analogy, FTY720-P induces internalization of S1PR from the surface of lymphocytes, hindering their trafficking outside the primary lymphatics and, therefore, keeping them away from inflammatory sites in end organs [4].

While being associated with beneficial outcomes and reasonable tolerability among people with immune-mediated conditions such as MS [5, 6], safety and toxicity studies have shown both dose-dependent and time-dependent adverse effects associated with FTY720 [7]. The time-dependent adverse effects have been opportunistic infections, reactivation of latent viruses, malignancies, etc.; these are mostly deemed to be due to chronic immunosuppression [7]. Among the dose-dependent adverse effects, the most common has been cardiotoxicity [5, 6]. S1PRs are highly expressed on cardiomyocytes; the initial agonistic effect of FTY720-P on S1PRs before inducing their internalization seems to be responsible for its dose-dependent cardiotoxic effects [8]. Hepatotoxic dose-dependent effects have also been reported [5–7], deemed to be associated with FTY720’s metabolic burden on the liver [9]. Other dose-dependent unwanted and toxic effects of FTY720 due to S1PR agonism, e.g., macular edema [10], unwanted loss of weight and appetite [11], etc. have been reported.

FTY720 was derived from myriocin (ISP-1), which is a secondary metabolite of the entomopathogenic fungus Isaria sinclairii and a potent inhibitor of the serine palmitoyltransferase (SPT) enzyme in the de novo sphingolipid biosynthesis pathway [12]. Myriocin alters a metabolic pathway in eukaryotic cells vital for their stability and proliferation. Consistently, it has shown to have antifungal effects e.g., against Candida and Aspergillus spp. [13, 14].

While FTY720 has no activity against the SPT, emerging studies [3, 15] are pointing towards S1PR-independent anti-proliferative effects of FTY720 in its non-phosphorylated form. Meanwhile, several cases of opportunistic fungal infections are reported among people receiving FTY720 chronically [16, 17]. Although these reports describe case studies and population-based evidence is lacking in this regard, it seems unclear whether FTY720 facilitates fungal expansion in vivo by weakening the immune system, or has retained the antifungal effect of its molecular ancestor.

The subject of identifying and confirming the possible antifungal properties of substances like FTY720 –which could be administered safely in humans–gains more relevance when considering the rapid emergence of serious mycosis cases resistant to our usual antifungal armamentarium. Among the candidiasis cases, resistance to drugs is classically attributed to the less prevalent infections with non-albicans species, however, recent studies are documenting an alarming increase in the antifungal resistance of Candida albicans–the dominant pathogen in candidiasis cases [18, 19]. Considering that the incidence of opportunistic candidiasis is rising, and that commensal C. albicans has a high prevalence [20], its resistance to drugs could be a major complicating factor for the future candidiasis cases.

Hence, in order to address our gaps of knowledge, we first aimed to determine whether FTY720 has antifungal effects in vitro using antifungal susceptibility testing. Then, we aimed to characterize FTY720’s effects on the immunological profiles, and on the fungal burden values in mice infected with C. albicans. We hereby report our studies in accordance with the Animal Research: Reporting of In Vivo Experiments 2.0 (ARRIVE 2.0) guidelines (available at: https://arriveguidelines.org/).

2. Methods

2.1 Design

The present studies were conducted from October 2019 until July 2020 in the Islamic Azad University of Falavarjan, Isfahan, Iran. As mentioned, a study was done in vitro to measure FTY720’s minimum inhibitory concentration (MIC) for C. albicans, and a study was done in vivo to characterize FTY720’s effect on the immunological profiles and fungal burden values in murine candidiasis models. The later study (from now on referred to as the in vivo study) was designed with the following formulation of research question:

Population: Female C57BL/6 mice.
Interventions:
- ○ Oral FTY720
- ○ Intraperitoneal (IP) C. albicans inoculation
Comparison:
- ○ Oral normal saline (oral placebo)
- ○ IP normal saline (injectable placebo)
Outcomes and rationale of each:
- ○Complete blood count with differential (CBC diff) and serum concentrations of interferon-gamma (IFNγ) and interleukin-10 (IL10) after 21 days of receiving oral intervention: to characterize the immunological profiles of the mice.
- ○Colony count of C. albicans in the vaginas, kidneys, and livers of the mice after 21 days of receiving oral intervention: to measure the fungal burden [21].

Based on the formulation of the research question, the in vivo study was conducted with six arms (Fig 1):

Control: Six mice receiving oral and injectable placebos.
Only FTY720: Six mice, each treated with oral FTY720 and injectable placebo.
Only C. albicans: Six mice, each treated with oral placebo and an IP C. albicans inoculation.
FTY720 after C. albicans: Ten mice, each treated with oral FTY720 starting from two days after receiving an IP C. albicans inoculation.
FTY720 with C. albicans: Ten mice, each treated with oral FTY720 starting from the same day as receiving an IP C. albicans inoculation.
FTY720 before C. albicans: Ten mice, each treated with oral FTY720 starting from two days before receiving an IP C. albicans inoculations.

The only exclusion criterion for the in vivo study was death due to an unrelated reason. No other eligibility criteria were considered.

2.2 In vitro antifungal susceptibility testing

2.2.1 Well diffusion assay

We first used a qualitative well diffusion assay in order to i) estimate the probable in vitro MIC of FTY720 for C. albicans and ii) to have an estimation of the serial drug concentration measures required for the subsequent broth microdilution assay. For the well diffusion assay, 0.5 McFarland standard (1–5 x 10⁶ colony forming units (CFU)/mL) preparations of C. albicans (CBS2747, Tehran University, Iran) were spread on three plates of Sabouraud dextrose agar (SDA) (Scharlau, Turkey). A total of eleven sample wells (four wells in two plates, and three wells in one plate), each with a diameter of six millimeters, were created in the plates using a sterile short (2 mL) Pasteur pipette. Then, using a standard sampler, one well was filled with normal saline as control and the others were filled with 200 μL suspensions of FTY720 with serial concentrations from 0.005 to 1.5 mg/mL. The presence or absence of a zone of inhibition around each well was determined without visual aid, after a 30-hour incubation of the plates at 37°C. The lowest concentration for which a zone of inhibition surrounded the corresponding well was considered as the probable MIC.

2.2.2 Broth microdilution assay

After obtaining the probable MIC using the well diffusion assay, a broth microdilution assay was performed following the methods described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) [22]. Briefly, microtubes in a sterile microtube plate were filled with 10 μL inocula of C. albicans (0.5 McFarland standard; 1–5 x 10⁶ CFU/mL), 90 μL of Sabouraud dextrose broth (SDB) (Scharlau, Turkey), and 100 μL of serially-diluted suspensions of FTY720, from 0.0005 to 0.5 mg/mL (i.e., the microtubes finally contained 0.5–2.5 x 10⁵ CFU/mL of C. albicans in SDB with serial dilutions of FTY720 from 0.00025 to 0.25 mg/mL). One microtube was added by normal saline instead of FTY720 (positive control), and one was filled with sterile SDB without the C. albicans inoculum (negative control). The optical density (OD) of each microtube was read and documented at 640 nm immediately after preparation, and after a 24-hour incubation at 37°C. The minimum drug concentrations for which the post-incubation change in OD was <1% and <50% were considered as the MIC₉₉ and the MIC₅₀, respectively.

2.3 Preparation, allocation, and follow-up of the mice

For the in vivo study, 48 female C57BL/6 mice (Royan Institute, Iran), each weighing 22 (±3) grams, were prepared in accordance with the National Research Council guidelines for laboratory animal care [23]. From one week before randomization, all mice were kept in standard cages in a humidity-controlled room at a 25°C (±2) temperature and a 12 hours daylight, 12 hours darkness cycle. Thereafter, each mouse was identification-marked by an ear tag, and allocated to each one of the study arms based on a random sequence generated by the NumPy software package (for Python version 2.7 on MacOS). They received their interventions in an open-label manner and were followed up daily until the endpoint of the study. All mice were treated with 100 μL liquid preparations of FTY720 (0.3 mg/kg/day) or normal saline, administered daily by gastric gavage for 21 days. This particular regimen of FTY720 was selected as it has been frequently used in previous studies, proven to bear immunomodulatory effects, and associated with minimal adverse reactions in C57BL/6 mice [24–30]. The mice assigned to be infected with C. albicans were inoculated with C. albicans through IP injections, with each inoculum containing 1.5 x 10⁵ CFU of C. albicans; the other mice were injected with the same amounts of normal saline. At the end of the follow-up, the mice were sedated with ketamine-xylazine (100–10 mg/kg) and 1 mL of blood was taken from each through cardiac catheterization. The blood samples were taken in anticoagulated tubes, and were promptly sent to be prepared for CBC diff and cytokine assays. Then, through sterile procedures, the mice’s livers, kidneys, and vaginal samples were extracted and sent to be prepared for fungal culture. The mice were finally euthanized with anesthetic overdose per American Veterinary Medical Association (AVMA) guidelines [31].

2.4 Analyses of samples

In order to characterize the mice’s immune responses to the fungal infection, absolute WBC and lymphocyte counts, serum IL10 (an anti-inflammatory cytokine), and IFNγ (a proinflammatory cytokine) concentrations were measured. CBC diff was performed on whole blood samples manually (i.e., using a Neubauer chamber) [32]. IFNγ and IL10 concentrations were quantified using an enzyme-linked immunosorbent assay (ELISA) (BioAssay™ kit for mouse, USBiological, USA) in accordance with the manufacturer’s instructions. For fungal culture, the vaginal samples were directly spread on SDA plates. The livers and kidneys were first homogenized and filtered (using sterile techniques and equipment), were diluted in ten serial concentrations, and then spread on SDA plates. After a 30-hour incubation at 37°C, the numbers of C. albicans colonies were counted and documented.

2.5 Statistical methods

2.5.1 Data preparation and determination of appropriate tests

The distributions of immune response measures (IFNγ, IL10. WBC, and ALC) were assumed to be lognormal; these measures were log₁₀-transformed before the analyses. The distributions of fungal burden values were assumed to be normal. The normality/lognormality assumptions were tested using the Kolmogorov-Smirnov method [33, 34]; if the assumptions were confirmed by alpha>0.05, parametric tests (e.g., one-way analysis of variance [ANOVA]), and if not, non-parametric tests (e.g., Kruskal-Wallis) were used for those measures. Post hoc analysis and correction for multiple comparisons were performed by controlling the false discovery rate, using the two-stage step-up method of Benjamini, Krieger, and Yakutielli [35].

In order to determine whether a single pooled variance could be used in the parametric comparisons, the group variances were compared using the Brown-Forsyth test [36]. In cases of insignificant (p>0.05) difference among variances of the arms, a single pooled variance was calculated and used; otherwise, the Welch and Brown-Forsyth method [37] was used (variances were calculated for individual arms separately).

2.5.2 Comparisons and their rationale

To confirm the sufficiency of the follow-up period for FTY720 to implement its immunological effects, the IFNγ, IL10, WBC, and ALC measures were compared between the control mice (arm 1) and the mice receiving FTY720 with placebo injection (arm 2).

To confirm the sufficiency of the follow-up period for the mice to develop an immune response against the fungal infection, the IFNγ, IL10, WBC, and ALC measures in the control mice (arm 1) were compared with the mice receiving C. albicans inoculation with oral placebo (arm 3).

To confirm the sufficiency of the follow-up period for in vivo dissemination of C. albicans, the fungal burden values in the control mice (arm 1) were compared with the arm receiving C. albicans inoculation with oral placebo (arm 3).

To characterize the effect of FTY720 on mice’s immunological response to C. albicans, the IFNγ, IL10, WBC, and ALC measures in the arms 4,5,6 (i.e., the mice receiving both FTY720 and C. albicans inoculation) were once compared with the mice receiving FTY720 with placebo injection (arm 2), and once with the mice receiving C. albicans inoculation with oral placebo (arm 3).

In order to characterize the effect of FTY720 on the in vivo fungal burden, the liver, kidney, and vaginal fungal burden values in arms 4,5,6 (i.e., the mice receiving both FTY720 and C. albicans inoculation) were compared with the mice receiving C. albicans inoculation with oral placebo (arm 3).

2.5.3 Software

The Prism software (version 9.0.2 for MacOS; GraphPad Software LLC.) was used for statistical analysis and graphing.

2.6 Reproducibility, safety and ethical considerations

In order to ensure the reproducibility of the results, the Enhancing the QUAlity and Transparency Of health Research (EQUATOR) guidelines [38] were followed to report the study, enabling all researchers across the globe to repeat the experiments, obtain, compare, and validate the results.

The laboratory researchers followed strict regulatory standards in terms of safety. Microbiological Petri dishes were not handled without gloves during any of the experiments. The laboratory researchers wore sterile gowns, protective face-shields and masks, and followed standard hand-washing protocols with povidone iodine before entering and after exiting the laboratory. All of the safety procedures were approved by the regulatory scientific review board who were monitoring the experiments through their surveillance systems.

The animal subjects of this study were kept in standard cages and were not restricted in terms of space, water, and food. All procedures on animal subjects were executed in accordance with national ethical guidelines. This study was approved by the ethics committee of the Islamic Azad University Falavarjan branch (Approval ID: IR.IAU.FALA.REC.1401.010).

3. Results

3.1 In vitro antifungal susceptibility tests

In the well diffusion assay, the lowest concentration of FTY720 for which a visible zone of inhibition surrounded its corresponding well was 0.5 mg/mL (well number 4 in Fig 2B), hence, the probable MIC of FTY720 was estimated to be 0.5 mg/mL or less. As mentioned, we then used the resulted qualitative measures for precise and standard MIC measurement with a broth microdilution assay, in which the MIC₉₉ was measured to be 0.25 mg/mL, and the MIC₅₀ was measured to be 0.12 mg/mL (Fig 3). These results confirmed that FTY720 comprises antifungal effects in vitro.

Fig 2 — First, standard C. *albicans* preparations (0.5 McFarland, 1–5 x 10⁶ CFU/mL) were spread on SDA plates and eleven sample wells were created. The wells numbered from 1 to 10 in the photographs **(a)**, **(b)**, and **(c)** were then filled with serial concentrations of FTY720 from 1.5 to 0.005 mg/mL respectively. The well labelled as “CONT” in photograph **(c)** was filled with normal saline. Then, the plates were incubated for 30 hours in 37°C, after which, the results were documented and the depicted photographs were taken. Zones of inhibition (white arrowheads) are visible surrounding the wells numbered from 1 to 4 in the photographs **(a)** and **(b)**, corresponding to serial concentrations of FTY720 from 1.5 to 0.5 mg/mL. Abbreviations: SDA, Sabouraud dextrose agar; CONT, control; CFU, colony forming unit.

Fig 3 — First, microtubes were filled with standard C. *albicans* preparations in SDB and serial concentrations of FTY720. Then, the background OD’s were measured at 640 nm and the microtubes were incubated for 24 hours at 37°C. After that, the OD’s were measured again at 640 nm and the results were documented. The measures plotted on the Y-axis are resulted from subtracting the pre-incubation (background) OD’s from the post-incubation OD’s. The hyphenated line corresponds to the best-fit regression model. Abbreviations: MIC, minimum inhibitory concentration; SDB, Sabouraud dextrose broth; OD, optical density.

3.2 Overview and validation of the in vivo study

The study was conducted per protocol; no unintended events happened during the study. The mice were randomized and allocated to the study arms two days before receiving their oral interventions (T -2). All of the randomized mice were followed up for 23 days (until T +21), except for one mouse in the control arm (arm 1) which died due to an unknown reason in the second day of oral normal saline consumption (T +2, four days after randomization). An investigative dissection of the mouse was conducted to identify the reason of death, but no gross abnormalities were detected. Samples from the normal saline administered to the mouse were cultured, resulting in growth of no microorganisms after one week. Apart from the signs of sepsis in the ones receiving the IP C. albicans inoculation, the mice experienced no serious adverse events during their follow-up.

At the end of the study, the immunological profile of the mice treated with FTY720 and injectable placebo (arm 2) significantly differed from the controls (arm 1) (Fig 4A), indicating a sufficient follow-up period for FTY720 to execute its immunomodulatory effects. FTY720 significantly decreased IFNγ, increased IL10, and decreased white blood cell (WBC) and absolute lymphocyte (AL) counts (Fig 4A).

Fig 4 — **(a)** comparison of immune response measures between the control mice (arm 1), the mice receiving FTY720/placebo injection (arm 2), and the ones receiving C. *albicans* inoculation/oral placebo (arm 3). These measures confirm that the follow-up duration of the *in vivo* study was sufficient. **(b)** comparison of immune response measures between the mice receiving either FTY720/placebo injection (arm 2) or C. *albicans* inoculation/oral placebo (arm 3), and the mice receiving both FTY720 and C. *albicans* inoculation (arms 4,5,6); only comparisons with p<0.05 are shown. ns: p>0.05; *0.01<p<0.05; **0.001<p<0.01; ***0.0001<p<0.001; ****p<0.0001. Abbreviations: IFNγ, interferon-gamma; IL10, interleukin-10; WBC, white blood cell; ALC, absolute lymphocyte count; IP, intraperitoneal; C. alb, *Candida albicans*.

The C. albicans-infected mice treated with oral normal saline (arm 3) showed significantly-different immunological profiles and fungal burden measures compared to the controls (arm 1) (Fig 4A); this indicated that the follow-up period was sufficient for in vivo dissemination of C. albicans, and for an antifungal immunological response to develop. Disseminated candidiasis significantly increased IFNγ, WBC, and AL counts but affected IL10 insignificantly (Fig 4A). Cultures of livers, kidneys, and vaginal samples of the mice not receiving IP C. albicans inoculation showed no growth of fungi, whereas the cultures from all of the C. albicans-injected ones showed growth of C. albicans–confirmed with gross visual examination and direct smear microscopy (Figs 5 and 6). In two cultures (one kidney culture from a mouse in arm 3 and one vaginal sample culture from a mouse in arm 6) multiple microorganisms were colonized. As this was highly indicative of procedural contamination, the mentioned cultures were excluded from the analyses.

Fig 5 — First, the mice’s organs were extracted and prepared through sterile procedures and spread on SDA plates. Then, the plates were incubated for 30 hours in 37°C, after which, the colonies were counted, the results were documented and the depicted photographs were taken. It should be noted that the photographs were taken *after* completion of the experiments and documentation of their results, and that *during* the experiments, none of the microbiological Petri dishes were handled without gloves as seen in these photographs. **(a)** Vaginal sample from a mouse in arm 5; **(b)** Liver sample from a mouse in arm 6; **(c)** Vaginal sample from a mouse in arm 2 (above) and a mouse in arm 3 (below); **(d)** Kidney sample from a mouse in arm 3. Abbreviations: SDA, Sabouraud dextrose agar.

Fig 6 — The depicted micrographs were taken from gram-stained slides prepared with the colonies from mice’s vaginal sample cultures on SDA plates. C. *albicans* yeasts are seen undergoing filamentation in the depicted micrographs. Abbreviations: SDA, Sabouraud dextrose agar.

3.3 Effect of FTY720 on mice’s immune response to C. albicans

In order to characterize the effect of FTY720 on the mice’s immune response to C. albicans, we compared the mice receiving the IP C. albicans inoculation before, after, or simultaneously with initiation of FTY720 (arms 4,5,6), with the ones receiving FTY720/injectable placebo or C. albicans/oral placebo (arms 2,3) (Fig 1).

Similar to the mice receiving C. albicans inoculation/oral placebo (arm 3), the mice receiving C. albicans inoculation before or simultaneously with initiation of FTY720 (arms 4,5) showed significantly increased IFNγ levels compared to the mice receiving FTY720/placebo injection (arm 2) (Fig 4B) (eTable 1 in S1 File). However, the mice receiving C. albicans inoculation after two days of FTY720 consumption (in arm 6) showed only insignificantly higher levels of IFNγ compared to the mice receiving FTY720/placebo injection (arm 2); IFNγ levels were significantly lower in these mice compared to the mice receiving C. albicans inoculation/oral placebo (arm 3) (Fig 4B) (eTable 1 in S1 File). In simple words, at the time of fungal inoculation, being on FTY720 for at least two days hindered the IFNγ response of the mice to the disseminating fungal infection, while initiating FTY720 in later timepoints did not.

The mice infected before initiation of FTY720 (arm 4) had significantly lower levels of IL10 compared to the ones receiving FTY720/placebo injection (arm 2), while being comparable to the ones receiving C. albicans inoculation/oral placebo (arm3) (Fig 4B) (eTable 1 in S1 File). It could be interpreted that the prior fungal infection hindered the IL10-increasing effect of FTY720. Induction of candidiasis simultaneously with, or later than FTY720 initiation (in arms 5,6) did not affect the IL10-increasing effect of FTY720 as shown in Fig 4B.

As interpreted before, FTY720 significantly decreased, and candidiasis significantly increased WBC and AL counts in mice (Fig 4A). The mice which started taking FTY720 before or after C. albicans inoculation (arms 4,6) showed significantly lower WBC and AL counts than the ones receiving C. albicans inoculation/oral placebo (arm 2) (Fig 4B). Surprisingly, the mice which initiated FTY720 and were inoculated with C. albicans on the same day still showed increased WBC and AL counts–significantly higher than the mice receiving FTY720/injectable placebo (arm 2), and comparable to the ones receiving C. albicans/oral placebo (arm 3) (Fig 4B) (eTable 1 in S1 File).

3.4 Effect of FTY720 on fungal burden in vivo

To demonstrate the antifungal effect of FTY720 in vivo, after 21 days of receiving their oral interventions, we cultured the mice’s livers, kidneys, and vaginal samples, and compared the resulted fungal burden values between the mice receiving C. albicans inoculation/oral placebo (arm 3), and the ones receiving both FTY720 and the C. albicans inoculation (arms 4,5,6). The mice initiating FTY720 before, after, or on the same day as C. albicans inoculation showed significantly lower fungal burden measures in their livers and kidneys (eTable 1 in S1 File) (Fig 7); interestingly, the ones initiating FTY720 and receiving the C. albicans inoculation on the same day (arm 5)–which also showed the high AL counts before–had the lowest liver/kidney fungal burdens–significantly lower than all of the other mice in other arms. Regarding the vaginal burdens, only the mice initiating FTY720 before receiving the C. albicans inoculation (arm 6) showed lower values than the ones receiving C. albicans inoculation/oral placebo (arm 3) (eTable 1 in S1 File) (Fig 7).

Fig 7 — ns: p>0.05; *0.01 <p<0.05; **0.001<p<0.01; ***0.0001<p<0.001; ****p<0.0001. Abbreviations: C. alb, *Candida albicans*.

4. Discussion

Our results demonstrated that FTY720 has antifungal effects against C. albicans–both in vitro and in vivo. In the mice models of disseminated candidiasis, this effect varied based on the chronological relation of FTY720 initiation and C. albicans inoculation. Our report is the first describing the in vivo antifungal effect of FTY720 as far as we know.

In line with our study, a recent ex vivo study by Wei and colleagues pointed to the fungicidal effect of FTY720, which synergized the effect of amphotericin B [39]. Wei et al. showed that this fungicidal effect is present against non-albicans Candida spp., Saccharomyces cerevisiae, and Cryptococcus neoformans as well as C. albicans [39]. They also showed that 0.011 mg/mL of FTY720 affects C. albicans’ growth rate similar to 0.08 mg/L of amphotericin B [39]. In an attempt to characterize the mechanism of this effect, they demonstrated that FTY720 induces production and hyperaccumulation of reactive oxygen species (ROS) in a dose-dependent manner. Scavenging the ROS from the samples using N-acetyl cysteine compromised the fungicidal effect of FTY720 significantly, therefore, Wei et al. suggested hyperaccumulation of ROS as the mechanism of FTY720’s fungicidal effect [39]. ROS accumulation in fungi induced by unphosphorylated FTY720 has been observed in previous studies as well [15, 40], the upstream processes of which are areas of active investigation.

Administration of FTY720 at 1.25 mg/day in healthy adults–above which has proved unbeneficial for people with MS [7]–results in a maximum blood concentration (C_max) of 10.2 (±2.7) ng/mL at steady-state [9]; this concentration is 25000 times lower than its MIC₉₉ for C. albicans. Maximum concentrations of FTY720 in kidney and liver tissues reach approximately 40 times the C_max [41]–still much less than its in vitro MIC₉₉. Therefore, the current FTY720 regimens in people with MS are doubted to directly restrict C. albicans infection. In our study in vivo, the mice received 0.3 mg/kg/day of FTY720. A single 0.3 mg/kg dose of FTY720 is associated with a maximum liver and kidney concentration of around 1.5 μg/mL [41]. Considering a 10-fold steady-state concentration reached by daily administration, the maximum concentration of FTY720 in liver and kidney reaches 15 μg/mL– 17 times less than its in vitro MIC₉₉ and 8 times less than its MIC₅₀. Despite this fact, we did observe the antifungal effect of FTY720 in vivo. Hence, we speculate that FTY720 had a synergistic effect with host’s innate immunity against C. albicans. The yeast could antagonize oxidative stress up to a specific threshold by inducing antioxidant metabolic pathways [42]. The innate immunity is absent in vitro, therefore, higher concentrations of FTY720 are needed to raise the ROS levels up to the threshold intolerable by the yeast. The ROS generation induced by FTY720 in vivo, although may not be adequate by itself, is added by the ROS produced by the innate immune cells, attracts more cells to the frontline [43], facilitates phagocytizing, and eases the killing of fungi by the phagocytes [39]. Nevertheless, countless processes are involved in fungal proliferation and expansion in vivo; determination of our observations’ mechanistic background could be an interesting subject for future studies.

As mentioned in the introduction, some case studies focusing on atypical cryptococcal infections [16, 17] hypothesize that people with MS receiving FTY720 may be generally more susceptible to fungal infections. However, Wei et al. demonstrated that the ex vivo fungicidal effect of FTY720 against Cryptococcus neoformans is similar to its effect on C. albicans, and we demonstrated that the in vitro antifungal effect of FTY720 against C. albicans is also present in vivo. The dose-dependency of FTY720’s antifungal effect may be the explanation behind this discrepancy. It could be hypothesized that FTY720’s immunosuppressive effect when administered chronically with low doses–as in people with MS–facilitates fungal infections, while its antifungal effects are absent in concentrations reached with those regimens (discussed above).

Another notable observation in our study was the mice with simultaneous C. albicans inoculation and initiation of FTY720 (arm 5) had a prominent increase in AL counts, similar to the mice receiving C. albicans inoculation/oral placebo (arm 3); however, their kidney and liver samples revealed significantly lower fungal burdens. The decreased fungal burden values in these mice could not merely be explained by the increased AL counts, otherwise, the mice receiving C. albicans inoculation/oral placebo (arm 3) who had similar AL counts would have similarly had low fungal burdens in their organs. Further fundamental work is warranted to explain this interesting observation.

5. Conclusion

We showed that the immunomodulatory drug FTY720 has an antifungal effect in vitro, which is present at even lower concentrations in vivo. The facilitation of ROS hyperaccumulation by FTY720 –which assists the innate immune system in creating an antifungal environment–may explain our observations. Future studies are warranted to evaluate further aspects of repositioning FTY720 for antifungal use in clinical settings.

6. Limitations

In order to ensure a sterile and prompt procedure of homogenization and fungal culturing, we regret that we did not perform any sectioning, staining, and direct microscopy of the livers, kidneys, and vagina samples to provide visual confirmation of C. albicans colonization within the tissues of the mice in treated and control groups. Also, mainly due to limited resources, the in vitro effect of FTY720 on important virulence traits of C. albicans, including filamentation, adhesion, and biofilm formation was not investigated in any standardized manner. Furthermore, the photographic documentations of the experiments which are provided in this report may not be considered in accordance with research standards; therefore, future studies are advised to replicate our experiments with research-grade photographic documentations to ensure the reproducibility of the results. Other limitations included outcome measurement at a single timepoint rather than several timepoints during the study, usage of limited number of assays, relatively short follow-up period, open-label fashion, not measuring drug concentrations in blood and tissues after administration, not measuring the ROS concentrations in vitro or in vivo, and other possible unnoticed biases. Further replicative studies are encouraged to add to our knowledge and account for the limitations of our study.

Supporting information

S1 File. Contains eTable 1.

(DOCX)

Click here for additional data file.^{(24.6KB, docx)}

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

References

1.Argollo M, Furfaro F, Gilardi D, Roda G, Allocca M, Peyrin-Biroulet L, et al. Modulation of sphingosine-1-phosphate in ulcerative colitis. Expert Opin Biol Ther. 2020;20(4):413–20. doi: 10.1080/14712598.2020.1732919 [DOI] [PubMed] [Google Scholar]
2.Baker KF, Isaacs JD. Novel therapies for immune-mediated inflammatory diseases: What can we learn from their use in rheumatoid arthritis, spondyloarthritis, systemic lupus erythematosus, psoriasis, Crohn’s disease and ulcerative colitis? Ann Rheum Dis. 2018;77(2):175–87. doi: 10.1136/annrheumdis-2017-211555 [DOI] [PubMed] [Google Scholar]
3.Zhang L, Wang H-D, Ji X-J, Cong Z-X, Zhu J-H, Zhou Y. FTY720 for cancer therapy. Oncol Rep. 2013;30(6):2571–8. [DOI] [PubMed] [Google Scholar]
4.Roy R, Alotaibi AA, Freedman MS. Sphingosine 1-Phosphate Receptor Modulators for Multiple Sclerosis. CNS Drugs. 2021;35(4):385–402. doi: 10.1007/s40263-021-00798-w [DOI] [PubMed] [Google Scholar]
5.Kappos L, Radue E-W, O’Connor P, Polman C, Hohlfeld R, Calabresi P, et al. A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med. 2010;362(5):387–401. doi: 10.1056/NEJMoa0909494 [DOI] [PubMed] [Google Scholar]
6.La Mantia L, Tramacere I, Firwana B, Pacchetti I, Palumbo R, Filippini G. Fingolimod for relapsing-remitting multiple sclerosis. Cochrane Database Syst Rev. 2016;4:Cd009371. doi: 10.1002/14651858.CD009371.pub2 [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Wu X, Xue T, Wang Z, Chen Z, Zhang X, Zhang W, et al. Different doses of fingolimod in relapsing-remitting multiple sclerosis: a systematic review and meta-analysis of randomized controlled trials. Front Pharmacol. 2021;12:1132. doi: 10.3389/fphar.2021.621856 [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Vargas WS, Perumal JS. Fingolimod and cardiac risk: latest findings and clinical implications. Ther Adv Drug Saf. 2013;4(3):119–24. doi: 10.1177/2042098613481023 [DOI] [PMC free article] [PubMed] [Google Scholar]
9.David OJ, Kovarik JM, Schmouder RL. Clinical pharmacokinetics of fingolimod. Clin Pharmacokinet. 2012;51(1):15–28. doi: 10.2165/11596550-000000000-00000 [DOI] [PubMed] [Google Scholar]
10.Cugati S, Chen CS, Lake S, Lee AW. Fingolimod and macular edema: Pathophysiology, diagnosis, and management. Neurol Clin Pract. 2014;4(5):402–9. doi: 10.1212/CPJ.0000000000000027 [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Etemadifar M, Khorvash R, Zolfagharifard H, Sedaghat N, Nouri H, Salari M. Weight Loss and Loss of Appetite as a Side Effect of Fingolimod (Preprint). Res Sq. 2022. doi: 10.21203/rs.3.rs-1870526/v1 [DOI] [Google Scholar]
12.Chiba K, Adachi K. Discovery of fingolimod, the sphingosine 1-phosphate receptor modulator and its application for the therapy of multiple sclerosis. Future Med Chem. 2012;4(6):771–81. doi: 10.4155/fmc.12.25 [DOI] [PubMed] [Google Scholar]
13.Perdoni F, Signorelli P, Cirasola D, Caretti A, Galimberti V, Biggiogera M, et al. Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm. BMC Microbiol. 2015;15(1):1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Yang X, Pei Z, Hu R, Zhang Z, Lou Z, Sun X. Study on the Inhibitory Activity and Possible Mechanism of Myriocin on Clinically Relevant Drug-Resistant Candida albicans and Its Biofilms. Biol Pharm Bull. 2021;44(3):305–15. doi: 10.1248/bpb.b20-00246 [DOI] [PubMed] [Google Scholar]
15.Takasaki T, Hagihara K, Satoh R, Sugiura R. More than just an immunosuppressant: the emerging role of FTY720 as a novel inducer of ROS and apoptosis. Oxid Med Cell Longev. 2018;2018. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Carpenter AF, Goodwin SJ, Bornstein PF, Larson AJ, Markus CK. Cutaneous cryptococcosis in a patient taking fingolimod for multiple sclerosis: here come the opportunistic infections? Mult Scler. 2017;23(2):297–9. doi: 10.1177/1352458516670732 [DOI] [PubMed] [Google Scholar]
17.Del Poeta M, Ward BJ, Greenberg B, Hemmer B, Cree BA, Komatireddy S, et al. Cryptococcal Meningitis Reported With Fingolimod Treatment: Case Series. Neurol Neuroimmunol Neuroinflamm. 2022;9(3). doi: 10.1212/NXI.0000000000001156 [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD. Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front Microbiol. 2017;7:2173. doi: 10.3389/fmicb.2016.02173 [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Coste AT, Kritikos A, Li J, Khanna N, Goldenberger D, Garzoni C, et al. Emerging echinocandin-resistant Candida albicans and glabrata in Switzerland. Infection. 2020;48(5):761–6. doi: 10.1007/s15010-020-01475-8 [DOI] [PMC free article] [PubMed] [Google Scholar]
20.Romo JA, Kumamoto CA. On Commensalism of Candida. J Fungi (Basel). 2020;6(1). doi: 10.3390/jof6010016 [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Conti HR, Huppler AR, Whibley N, Gaffen SL. Animal models for candidiasis. Curr Protoc Immunol. 2014;105:19.6.1-.6.7. doi: 10.1002/0471142735.im1906s105 [DOI] [PMC free article] [PubMed] [Google Scholar]
22.EUCAST DEFINITIVE DOCUMENT E.DEF 7.3.1 Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf (2017). Accessed November 2022.
23.National Research Council. Guide for the care and use of laboratory animals. 8th ed. National Academies Press (US); 2011. [PubMed] [Google Scholar]
24.Abdullah CS, Li Z, Wang X, Jin ZQ. Depletion of T lymphocytes ameliorates cardiac fibrosis in streptozotocin-induced diabetic cardiomyopathy. Int Immunopharmacol. 2016;39:251–64. doi: 10.1016/j.intimp.2016.07.027 [DOI] [PubMed] [Google Scholar]
25.Bonfiglio T, Olivero G, Merega E, Di Prisco S, Padolecchia C, Grilli M, et al. Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis. PLoS One. 2017;12(1):e0170825. doi: 10.1371/journal.pone.0170825 [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Cheng Q, Li D, Liang H, Yang H, Lei D, Gao D, et al. Effects of long-term administration of low-dose FTY720 on survival of murine cardiac allograft. J Huazhong Univ Sci Technolog Med Sci. 2012;32(2):199–204. doi: 10.1007/s11596-012-0035-5 [DOI] [PubMed] [Google Scholar]
27.Efstathopoulos P, Kourgiantaki A, Karali K, Sidiropoulou K, Margioris AN, Gravanis A, et al. Fingolimod induces neurogenesis in adult mouse hippocampus and improves contextual fear memory. Transl Psychiatry. 2015;5(11):e685. doi: 10.1038/tp.2015.179 [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Hwang MW, Matsumori A, Furukawa Y, Ono K, Okada M, Iwasaki A, et al. FTY720, a new immunosuppressant, promotes long-term graft survival and inhibits the progression of graft coronary artery disease in a murine model of cardiac transplantation. Circulation. 1999;100(12):1322–9. doi: 10.1161/01.cir.100.12.1322 [DOI] [PubMed] [Google Scholar]
29.Pinschewer DD, Ochsenbein AF, Odermatt B, Brinkmann V, Hengartner H, Zinkernagel RM. FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory. J Immunol. 2000;164(11):5761–70. doi: 10.4049/jimmunol.164.11.5761 [DOI] [PubMed] [Google Scholar]
30.Wang G, Kim RY, Imhof I, Honbo N, Luk FS, Li K, et al. The immunosuppressant FTY720 prolongs survival in a mouse model of diet-induced coronary atherosclerosis and myocardial infarction. J Cardiovasc Pharmacol. 2014;63(2):132–43. doi: 10.1097/FJC.0000000000000031 [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Underwood W, Anthony R. AVMA guidelines for the euthanasia of animals. 2020 ed. American Veterinary Medical Association; 2020. [Google Scholar]
32.JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Using a Hemacytometer to Count Cells.: https://www.jove.com/v/5048/using-a-hemacytometer-to-count-cells (2022). Accessed November 2022.
33.Smirnov N. Table for Estimating the Goodness of Fit of Empirical Distributions. Ann Math Statist. 1948;19(2):279–81. [Google Scholar]
34.Kolmogorov A. Sulla determinazione empirica di una lgge di distribuzione. Giornale dell’Istituto Italiano degli Attuari. 1933;4:83–91. [Google Scholar]
35.Benjamini Y, Krieger AM, Yekutieli D. Adaptive linear step-up procedures that control the false discovery rate. Biometrika. 2006;93(3):491–507. [Google Scholar]
36.Brown MB, Forsythe AB. Robust tests for the equality of variances. J Am Stat Assoc. 1974;69(346):364–7. [Google Scholar]
37.Welch BL. The Generalization of ‘Student’s’ Problem When Several Different Population Variances are Involved. Biometrika. 1947;34(1–2):28–35. doi: [DOI] [PubMed] [Google Scholar]
38.Groves T. Enhancing the quality and transparency of health research. BMJ. 2008;337. doi: 10.1136/bmj.a718 [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Wei L-Q, Tan J-C, Wang Y, Mei Y-K, Xue J-Y, Tian L, et al. Fingolimod potentiates the antifungal activity of amphotericin B. Front Cell Infect Microbiol. 2021;11:346. doi: 10.3389/fcimb.2021.627917 [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Hagihara K, Mizukura A, Kitai Y, Yao M, Ishida K, Kita A, et al. FTY 720 stimulated ROS generation and the S ty1/A tf1 signaling pathway in the fission yeast S chizosaccharomyces pombe. Genes Cells. 2014;19(4):325–37. doi: 10.1111/gtc.12134 [DOI] [PubMed] [Google Scholar]
41.Meno-Tetang GM, Li H, Mis S, Pyszczynski N, Heining P, Lowe P, et al. Physiologically based pharmacokinetic modeling of FTY720 (2-amino-2 [2-(-4-octylphenyl) ethyl] propane-1, 3-diol hydrochloride) in rats after oral and intravenous doses. Drug Metab Dispos. 2006;34(9):1480–7. doi: 10.1124/dmd.105.009001 [DOI] [PubMed] [Google Scholar]
42.Garcerá A, Casas C, Herrero E. Expression of Candida albicans glutathione transferases is induced inside phagocytes and upon diverse environmental stresses. FEMS Yeast Res. 2010;10(4):422–31. doi: 10.1111/j.1567-1364.2010.00613.x [DOI] [PubMed] [Google Scholar]
43.Brothers KM, Gratacap RL, Barker SE, Newman ZR, Norum A, Wheeler RT. NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality. PLoS Pathog. 2013;9(10):e1003634. doi: 10.1371/journal.ppat.1003634 [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0278488.r001

Decision Letter 0

Aijaz Ahmad

7 Jul 2022

PONE-D-22-15968Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis modelPLOS ONE

Dear Dr Nahad Sedaghat

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please submit your revised manuscript by 07/08/2022. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Aijaz Ahmad, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements;

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. In your Methods section, please provide additional information on the animal research and ensure you have included details on : (1) methods of sacrifice (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

3. Your ethics statement should only appear in the Methods section of your manuscript. If your ethics statement is written in any section besides the Methods, please move it to the Methods section and delete it from any other section. Please ensure that your ethics statement is included in your manuscript, as the ethics statement entered into the online submission form will not be published alongside your manuscript.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Comments after review:

1. The authors are advised to go through the entire manuscript and correct it for grammatical errors.

2. Authors should mention protocol number approved by Institutional Ethics Committee (IEC) in the manuscript.

3. In in-vitro well diffusion assay, how did the authors manage to load 200ml of sample in the wells. Also, please mention the zone of inhibition values obtained.

4. Please mention the histopathological images of livers, kidneys and vagina obtained after 21 days of treatment to provide visual analysis of Candida colonization between the treated and control groups.

5. Authors have mentioned that Fingolimod restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model. Have the authors investigated the in vitro effect of this compound on important virulence traits mainly- hydrolytic enzymes, yeast to hyphae transition, adhesion and biofilm formation. If yes, please mention this in manuscript.

Reviewer #2: Overall the necessary experiments were performed in this study. However some structural changes are needed.

1. the in vitro results should precede the in vivo. The figures should follow the same logic.

2. L187-190. Please provide data/figure against this observation.

3. L 106, was it 200 ul of suspension? Figures against the well diffusion assay need to be added.

4. What is the reasoning behind performing the well diffusion assay when the authors performed a more sensitive broth dilution assay?

5. Please stick to the convention of using IFNγ.

6. What is the significance of measuring IFNg, IL10, and WBC, and ALC, What role do they play against infection?

7. If FTY720 has antifungal activity both in vitro and in vivo then what causes fungal infections are reported among people receiving FTY720, mentioned in the introduction?

Reviewer #3: The manuscript entitled “Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model” is an interesting work done by the authors. However, the manuscript has several flaws starting from the way of writing to the technicality in the method section

1. The introduction lacks information about the severity of Candida albicans in present world and why authors are only targeting Candida albicans and not non-albicans Candida.

2. The method section is very confusing and should be re-written for better understanding of readers.

3. All the methods lack relevant references

4. Line number 106, should be rechecked for the volume, 200 ml is not correct.

5. Line number 108, justification for 30-hour incubation which is not as per standard guidelines.

6. Section 2.3, which labelling technique was used, which program was used and what was the basis for dose determination.

7. Section 2.3, is it possible to withdraw 20 ml of blood from the mice (line 133).

8. Use abbreviations for Sabouraud dextrose agar and other reagents after mentioning at place.

9. Statistical analysis should be re-written for proper understanding

10. Section 3.2, line 194, broth dilution assay should be replaced by microbroth dilution assay.

11. The results need to be discussion with reference to the relevant literature.

12. The manuscript lacks conclusion.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: SAIEMA AHMEDI Department of Biosciences, Jamia Millia Islamia, New Delhi-110025

Reviewer #2: No

Reviewer #3: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

Attachment

Submitted filename: Comments.pdf

Click here for additional data file.^{(10KB, pdf)}

PLoS One. 2022 Dec 7;17(12):e0278488. doi: 10.1371/journal.pone.0278488.r002

Author response to Decision Letter 0

8 Aug 2022

Response to the reviewer comments

Dear Editor

Thank you for having our manuscript considered and reviewed. We carefully considered each of the reviewer comments, revised and improved our manuscript accordingly. The changes were tracked in a marked version of the revised manuscript, which is uploaded along the clean version. Below, you can find the reviewer comments, along with our responses below each. Please let us know if any further action is required from us. Thank you in advance for your consideration; we hope to hear from you soon.

Best regards

Nahad Sedaghat on behalf of the authors

Reviewer #1

1. “The authors are advised to go through the entire manuscript and correct it for grammatical errors.”

Response: Thank you for reviewing our manuscript and for your constructive suggestions. Per your request, the manuscript was reviewed and edited by a native English scientific writer to ensure proper understanding of the readers.

2. “Authors should mention protocol number approved by Institutional Ethics Committee (IEC) in the manuscript.”

Response: Thank you for noticing. The approval number issued by the ethics committee was added under the “Ethical considerations” section.

3. “In in-vitro well diffusion assay, how did the authors manage to load 200ml of sample in the wells. Also, please mention the zone of inhibition values obtained.”

Response: We loaded 200 “μL” of sample to the wells. Thank you for noticing this error, which is now corrected. Furthermore, it should be mentioned that the well diffusion assay was performed qualitatively only to give us an estimate of the MIC and the required concentrations for the more sensitive and objective broth microdilution assay. In other words, only the qualitative presence of the zones was important to us, as we did not aim to report the MICs based on zone of inhibition cut-offs in well diffusion assays. For the same reason, you could interpret that we did not follow the standard 24-hour incubation protocol to enable MIC estimation based on zone cut-offs. We mistakenly mentioned that the diameters of the zones were measured for MIC determination; this point was corrected. The section on fungal susceptibility assays was reworded while explicitly clarifying and emphasizing the mentioned points to avoid any confusion for the readers.

4. “Please mention the histopathological images of livers, kidneys and vagina obtained after 21 days of treatment to provide visual analysis of Candida colonization between the treated and control groups.”

Response: In order to ensure a sterile and prompt procedure of homogenization and fungal culturing, we regret that we did not perform any histopathological sectioning of the tissues to provide the requested visual analysis of Candida colonization between the treated and control groups. This point was mentioned in the limitations section. To validate the colonization of C. albicans, we did perform staining and direct microscopy of the colonies after culturing; the available gross and microscopic photographs were added to the supplemental contents.

5. “Authors have mentioned that Fingolimod restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model. Have the authors investigated the in vitro effect of this compound on important virulence traits mainly- hydrolytic enzymes, yeast to hyphae transition, adhesion and biofilm formation. If yes, please mention this in manuscript.”

Response: Unfortunately, mainly due to limited resources, the in-vitro effect of FTY720 on important virulence traits of Candida albicans was not investigated in any standardized manner. This limitation is now explicitly stated in the limitations section. Thank you once more for dedicating your time to review and improve our manuscript.

Reviewer #2

0. “Overall the necessary experiments were performed in this study. However some structural changes are needed.”

Response: Thank you for reviewing our manuscript and providing us with your valuable comments. We hope that we addressed each of your comments sufficiently.

1. “the in vitro results should precede the in vivo. The figures should follow the same logic.”

Response: Thank you for your suggestion. The figures followed the order they were mentioned in the text in line with the journal requirements. Per your suggestion, we changed the order of the sections 3.1 and 3.2. In the revised manuscript, you could find the in-vitro results in section 3.1, prior to the first description of the in-vivo results in section 3.2. Their corresponding figures also follow the same order in the revised manuscript.

2. “L187-190. Please provide data/figure against this observation.”

Response: We mentioned that “Cultures from liver, kidney, and vaginal samples from all of the control mice showed no growth of fungi, whereas the samples from all of the C. albicans-injected ones showed growth of C. albicans. Results of two cultures (one kidney sample from an untreated infected mouse and one vaginal sample from a mouse in arm 6) indicated contamination; they were excluded from analyses.” The data showing the fungal colony counts in the cultures of infected mice are interpretable from the diagram presented in Figure 4; raw data is also available upon request (now mentioned in the new “data availability” section). We unfortunately did not follow a standardized manner in photographic documentation of our experiments, nevertheless, some photographs of the culture plates were taken for the record; they are now added to the supplemental contents. Our limitation in providing standardized photographic documentation of the experiments was explicitly stated in the limitations section. The contaminations we mentioned were concluded due to colonization of multiple organisms in the culture plates. This point was added to the manuscript.

3. “L 106, was it 200 ul of suspension? Figures against the well diffusion assay need to be added.”

Response: Yes, thank you for noticing our error, which is now corrected. In the supplemental contents, we uploaded a series of pictures taken for the record from the well diffusion plates; the zone of inhibition around the wells is visible in those images, but the pictures may not be considered per research standards. As mentioned, we disclosed our limitation in providing standard photographic documentation of the experiments, and also encouraged further replication following the detailed description of methods we provided, while also considering visual validation with guideline-recommended photographic documentations.

4. “What is the reasoning behind performing the well diffusion assay when the authors performed a more sensitive broth dilution assay?”

Response: We performed a qualitative well diffusion assay to have an estimate of the probable MIC measure, in order to determine the concentrations required for the more precise broth microdilution assay. Precise and objective MIC measurement was not intended to be done using zone of inhibition cut-offs in well diffusion assays, which was also the reason we did not follow the standard 24-hour incubation procedure per standard guidelines, and did not measure the MIC based on the zone diameters (as mistakenly stated in the initial draft), but visually assessed the presence of zones. Precise and standard MIC measurement was performed with guideline-recommended procedures using broth microdilution. These points were disclosed explicitly in the revised manuscript.

5. “Please stick to the convention of using IFNγ.”

Response: Noted. IFNg was changed to IFNγ throughout the manuscript.

6. “What is the significance of measuring IFNg, IL10, and WBC, and ALC, What role do they play against infection?”

Response: The mentioned measures were obtained as markers of extent and profile of the systemic immune response to the fungal infection. This point was clarified in the revised manuscript.

7. “If FTY720 has antifungal activity both in vitro and in vivo then what causes fungal infections are reported among people receiving FTY720, mentioned in the introduction?”

Response: We discussed and elaborated the mentioned point in the revised manuscript. Thank you once more for your time dedicated to reviewing and contributing to the coherence of our manuscript.

Reviewer #3

0. “The manuscript entitled “Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model” is an interesting work done by the authors. However, the manuscript has several flaws starting from the way of writing to the technicality in the method section”

Response: Thank you for dedicating your time to review our manuscript and highlight our errors. We hope we were able to address your comments in a sufficient manner.

1. “The introduction lacks information about the severity of Candida albicans in present world and why authors are only targeting Candida albicans and not non-albicans Candida.”

Response: Thank you for your suggestion. We elaborated more on the severity of Candida albicans infection in the present world, and the relevance of determining the effect of FTY720 on Candida albicans in our experiment.

2. “The method section is very confusing and should be re-written for better understanding of readers.”

Response: Thank you for mentioning this important point. To ensure correct understanding of the readers and reproducibility of our work, we have reworded the methods section, this time aiming to choose our words in the least confusing manner, while providing as much detail as possible.

3. “All the methods lack relevant references”

Response: Thank you for noticing. Several relevant references were added to the methods section per your request. Please do not hesitate to let us know more specifically if we are still missing any essential referencing.

4. “Line number 106, should be rechecked for the volume, 200 ml is not correct.”

Response: Indeed. Thank you for noticing. The mentioned error was corrected.

5. “Line number 108, justification for 30-hour incubation which is not as per standard guidelines.”

Response: Thank you for asking. We did not aim to measure the MIC based on the guideline-recommended zone diameter cut-offs after a 24-hour incubation of the well diffusion plates. We performed a qualitative well diffusion assay in order to estimate the probable MIC and obtain the concentration measures required for the more precise broth microdilution assay. Therefore, as only qualitative visibility of inhibition zones was of value, we incubated the plates for an additional 6 hours. As interpreted, the samples were incubated for 24 hours before precise measurement of MIC, as per EUCAST guideline for the broth microdilution assays. These points were disclosed in the revised manuscript to prevent any confusion.

6. “Section 2.3, which labelling technique was used, which program was used and what was the basis for dose determination.”

Response: The mice were labeled (ie ID-marked) using ear tags. The NumPy package for Python was used for generating a random sequence. For dose determination we decided to obtain a value frequently used in previous studies and proven to have immunomodulatory effects while being safe for the C57BL/6 mice. All of these points were disclosed in the revised manuscript.

7. “Section 2.3, is it possible to withdraw 20 ml of blood from the mice (line 133).”

Response: Certainly not. We sincerely apologize for and corrected the mentioned error.

8. “Use abbreviations for Sabouraud dextrose agar and other reagents after mentioning at place.”

Response: Thank you for your suggestion. We used abbreviations for the reagents after their first mention.

9. “Statistical analysis should be re-written for proper understanding”

Response: Along the other parts of the methods section, the statistical analysis section was reworded to ensure proper understanding of the readers.

10. “Section 3.2, line 194, broth dilution assay should be replaced by microbroth dilution assay.”

Response: Thank you for mentioning. We replaced the term “broth dilution” with “microbroth dilution”.

11. “The results need to be discussion with reference to the relevant literature.”

Response: Per your suggestion, the relevant literature was reviewed and discussed more extensively.

12. “The manuscript lacks conclusion.”

Response: A “conclusion” section was added at the end of the manuscript. Thank you sincerely for reviewing our manuscript and providing your critical appraisal of our work; your contribution is very much appreciated.

Attachment

Submitted filename: Res2Rev.docx

Click here for additional data file.^{(31.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0278488.r003

Decision Letter 1

Aijaz Ahmad

29 Aug 2022

PONE-D-22-15968R1Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis modelPLOS ONE

Dear Dr. Sedaghat

Please submit your revised manuscript by Oct 13 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Aijaz Ahmad, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: 1. Scientific notation of writing Candida albicans is not correct in abstract. Please check. It should be uniform throughout the manuscript.

2. Please incorporate the images of well diffusion assay in the manuscript.

3. Authors should keep space between values and their units throughout the manuscript and

used SI units.

4. Authors are advised to incorporate images to get better picture of their results.

5. Please incorporate toxicity related studies in the manuscript. If already published by others cite that reference in the manuscript.

Reviewer #2:

The authors have answered have all the questions raised by me. However, as the authors mentioned in the article that there are some significant limitations to the reproducibility of the experiments. The supplementary figures are also not as per scientific standards, especially handling microbiological Petri dishes without gloves is a serious concern. There are still a few typos in the manuscript (eg. Line 63 homo-, IFNg instead of γ in the etable etc.). Therefore, I recommend to resubmit the article with proper figures.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

Attachment

Submitted filename: comments 26 August 2022.docx

Click here for additional data file.^{(12.7KB, docx)}

PLoS One. 2022 Dec 7;17(12):e0278488. doi: 10.1371/journal.pone.0278488.r004

Author response to Decision Letter 1

28 Sep 2022

Reviewer #1:

1. Scientific notation of writing Candida albicans is not correct in abstract. Please check. It should be uniform throughout the manuscript.

Response: Thank you for noticing. We uniformly changed all the instances in the abstract to “Candida albicans”.

2. Please incorporate the images of well diffusion assay in the manuscript.

Response: Thank you for your suggestion. All eFigures were moved to the main manuscript.

3. Authors should keep space between values and their units throughout the manuscript and used SI units.

Response: Thank you for mentioning. We added the required space between the values and units. About using SI units, as far as we checked, we did not use non-SI units anywhere in the manuscript. In case we overlooked an instance of using any non-SI unit, we would appreciate if you could point to the line, so we could correct that instance.

4. Authors are advised to incorporate images to get better picture of their results.

Response: Thank you for your suggestion. As mentioned, we moved all the eFigures to the main manuscript. We unfortunately have a limitation in presenting further images apart from what is already presented, as the experiments are no longer in progress. We mentioned this point in the limitations section and advised future studies to account for this limitation.

5. Please incorporate toxicity related studies in the manuscript. If already published by others cite that reference in the manuscript.

Response: Thank you for your attention. We added and discussed toxicity related studies as well in the revised manuscript. We hope the changes and improvements satisfy your concerns.

Reviewer #2:

0. The authors have answered have all the questions raised by me.

Response: We are pleased to hear that. Thank you for dedicating your time to our manuscript.

1. However, as the authors mentioned in the article that there are some significant limitations to the reproducibility of the experiments.

Response: Thank you for mentioning your concern about the replicability of the study and reproducibility of the results. In case your concern is due to the lack of standard photographic documentation of the experiments and results, we made the following efforts to account for this limitation and make our report of merit for publication. First of all, we described and disclosed all of the experiments in full detail and in complete accordance with the EQUATOR guidelines, in order to allow their complete repetition by any scientist across the globe. Therefore, replication of the experiments and reproduction of the results would be possible for any team concerned about the lack of proper photographic documentations in the present report. Additionally, all of the experiments were performed in compliance with strict regulations and were being monitored through surveillance systems by independent scientific, safety, and ethical review boards, consisting of anonymous nationally-certified scientists. Finally, we shared and deposited our data for all researchers across the globe to review and validate. We would also welcome any further suggestions to improve our work.

2. The supplementary figures are also not as per scientific standards.

Response: Thank you for mentioning your concern. We completely agree that the presented figures are not in compliance with scientific standards, as we unfortunately, did not intend to keep scientific photographic documentations at the beginning. The reason was that in presence of strict regulations and standard guidelines, we erroneously considered photographic documentations to be optional. Now, as the experiments are no longer in process, we are unfortunately limited in terms of presenting standard figures. However, describing the experiments with full detail allows their repetition by any researcher concerned about the reproducibility of the results. In the limitations section, we explicitly stated this point, and strongly encouraged further replication of the experiments using the detailed description of the methods we provided. We further stated that due to our limitation in providing standard figures, our conclusions are subject to reproduction of the results by future studies with standard photographic documentations.

3. especially handling microbiological Petri dishes without gloves is a serious concern.

Response: Thank you for your punctiliousness. We completely acknowledge your concern. We should mention that the photograph you are referring to was taken after completion of the experiments and documentation of their results. During the experiments, the laboratory researchers followed strict regulatory standards in terms of their safety; microbiological Petri dishes were not handled without gloves during any of the experiments, and the laboratory researchers wore sterile gowns, protective face-shields and masks, as well as following standard hand-washing protocols with povidone iodine solution before entering the laboratory. This has been evidenced by absence of biological and non-biological contamination in all of the Petri dishes except for one, and by the approval of the safety procedures by the regulatory scientific review board. These points were clarified in the revised manuscript and in the legend of the mentioned figure to appreciate any concern.

4. There are still a few typos in the manuscript (eg. Line 63 homo-, IFNg instead of γ in the etable etc.).

Response: We apologize for overseeing the mentioned typos in our reviews. The Latin phrase “in homo” means “in man” in English; we exchanged “in homo” with the standard English term “in humans” to avoid any confusion. IFNg was changed to IFNγ in the supplementary table as well. Furthermore, we reviewed the revised manuscript several more times to ensure proper usage of language for the readers. We would certainly appreciate any further suggestions for improving our report.

5. Therefore, I recommend to resubmit the article with proper figures.

Response: Thank you for your recommendation. As mentioned previously, the experiments are no longer in progress, therefore, we are unfortunately limited in presenting standard photographic documentations. We truly hope our explicit and detailed disclosure of the experimental procedures according to the EQUATOR guidelines and encouragement of future replicative studies could at least, partially resolve your concerns about the reproducibility of the results. We are also, hoping that the publication of our study, if considered of merit based on novelty and relevance, leads to conduction of further studies on the subject accounting for our limitations (including provision of standard figures) and validating the results. Once again, we would like to appreciate your punctiliousness and explicitly in critically appraising our study, and thank you for your time and consideration.

Attachment

Submitted filename: Res2Rev2.docx

Click here for additional data file.^{(27.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0278488.r005

Decision Letter 2

Aijaz Ahmad

26 Oct 2022

PONE-D-22-15968R2Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis modelPLOS ONE

Dear Dr. Nahad Sedaghat

Please submit your revised manuscript by Dec 10 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Aijaz Ahmad, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments:

Reviewer 1:

1. The annotation of figures is not explicit enough, for example in the legend of figure 2 and 5, no information is provided for the images such as what type of images these are, how processed and the conditions. Please mention.

2. Quality of all disc/plates images is not good. Zone of inhibition is not visible in Figure 2 b and c. Better to crop the image, put it on black or dark background then take the image.

3. Authors are advised to thoroughly check the manuscript for grammatical errors and formatting.

Reviewer 2:

Reviewer 3:

The manuscript need minor changes before it gets accepted for publication.

I would urge the authors to send their paper for English editing because it has significant grammatical errors that make it difficult to read the work as a whole. There are also several errors that might have been corrected with ease. The examples I provided, which can be found in the mark-up document, are by no means exhaustive.

Green colour- revise whole sentence

Yellow colour - check for the consistency throughout the manuscript

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

**********

6. Review Comments to the Author

Reviewer #1: 1. The annotation of figures is not explicit enough, for example in the legend of figure 2 and 5, no information is provided for the images such as what type of images these are, how processed and the conditions. Please mention.

2. Quality of all disc/plates images is not good. Zone of inhibition is not visible in Figure 2 b and c. Better to crop the image, put it on black or dark background then take the image.

3. Authors are advised to thoroughly check the manuscript for grammatical errors and formatting.

Reviewer #2: The authors have responded to all my concerns. However, it is always a good laboratory practice to properly document all the data and store it properly.

Reviewer #3: The manuscript need minor changes before it gets accepted for publication.

Green colour- revise whole sentence

Yellow colour - check for the consistency throughout the manuscript

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

Attachment

Submitted filename: oct-PONE-D-22-15968_R2_reviewer.pdf

Click here for additional data file.^{(3.9MB, pdf)}

Attachment

Submitted filename: Plos comments 23 Oct 2022.docx

Click here for additional data file.^{(13.2KB, docx)}

PLoS One. 2022 Dec 7;17(12):e0278488. doi: 10.1371/journal.pone.0278488.r006

Author response to Decision Letter 2

9 Nov 2022

Reviewer #1:

1. “The annotation of figures is not explicit enough, for example in the legend of figure 2 and 5, no information is provided for the images such as what type of images these are, how processed and the conditions. Please mention.”

Response: Thank you for your notice. Further information including but not limited to the points you mentioned were added to the figure legends to ensure adequate understanding of the readers.

2. “Quality of all disc/plates images is not good. Zone of inhibition is not visible in Figure 2 b and c. Better to crop the image, put it on black or dark background then take the image.”

Response: Thank you sincerely for your concern and for your suggestion. Following your suggestion and for the zones of inhibition to be visible for the readers, backgrounds were darkened in Figure 2 photographs (showing the results of the well diffusion antifungal susceptibility assay) and the zones of inhibition were marked with white arrowheads. Please note that, no zone of inhibition was present around wells number 5 to 10 which are depicted in Figure 2 b and c, and that no “discs” were used on the plates, but wells were created in the medium and filled with drug suspensions.

3. “Authors are advised to thoroughly check the manuscript for grammatical errors and formatting. “

Response: Thank you for once again for your suggestion. The manuscript was checked by a native English scientific copyeditor to ensure proper usage of language and formatting.

Reviewer #3:

0. “The manuscript need minor changes before it gets accepted for publication. I would urge the authors to send their paper for English editing because it has significant grammatical errors that make it difficult to read the work as a whole. There are also several errors that might have been corrected with ease. The examples I provided, which can be found in the mark-up document, are by no means exhaustive. Green colour- revise whole sentence Yellow colour - check for the consistency throughout the manuscript”

Response: We would like to sincerely thank you for providing us with the mark-up document, which indeed, alleviated the process of further improving our language. All of the points in the mark-up document were addressed in our revision. Also, the manuscript was reviewed by a native English scientific copyeditor to ensure proper usage of syntax and formatting.

PLoS One. doi: 10.1371/journal.pone.0278488.r007

Decision Letter 3

Aijaz Ahmad

17 Nov 2022

Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model

PONE-D-22-15968R3

Dear Dr. Nahad Sedaghat

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Aijaz Ahmad, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0278488.r008

Acceptance letter

Aijaz Ahmad

28 Nov 2022

PONE-D-22-15968R3

Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast <i>Candida albicans in vitro<i> and in a mouse candidiasis model

Dear Dr. Madani:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Aijaz Ahmad

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 File. Contains eTable 1.

(DOCX)

Click here for additional data file.^{(24.6KB, docx)}

Attachment

Submitted filename: Comments.pdf

Click here for additional data file.^{(10KB, pdf)}

Attachment

Submitted filename: Res2Rev.docx

Click here for additional data file.^{(31.8KB, docx)}

Attachment

Submitted filename: comments 26 August 2022.docx

Click here for additional data file.^{(12.7KB, docx)}

Attachment

Submitted filename: Res2Rev2.docx

Click here for additional data file.^{(27.9KB, docx)}

Attachment

Submitted filename: oct-PONE-D-22-15968_R2_reviewer.pdf

Click here for additional data file.^{(3.9MB, pdf)}

Attachment

Submitted filename: Plos comments 23 Oct 2022.docx

Click here for additional data file.^{(13.2KB, docx)}

Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

[pone.0278488.ref001] 1.Argollo M, Furfaro F, Gilardi D, Roda G, Allocca M, Peyrin-Biroulet L, et al. Modulation of sphingosine-1-phosphate in ulcerative colitis. Expert Opin Biol Ther. 2020;20(4):413–20. doi: 10.1080/14712598.2020.1732919 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref002] 2.Baker KF, Isaacs JD. Novel therapies for immune-mediated inflammatory diseases: What can we learn from their use in rheumatoid arthritis, spondyloarthritis, systemic lupus erythematosus, psoriasis, Crohn’s disease and ulcerative colitis? Ann Rheum Dis. 2018;77(2):175–87. doi: 10.1136/annrheumdis-2017-211555 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref003] 3.Zhang L, Wang H-D, Ji X-J, Cong Z-X, Zhu J-H, Zhou Y. FTY720 for cancer therapy. Oncol Rep. 2013;30(6):2571–8. [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref004] 4.Roy R, Alotaibi AA, Freedman MS. Sphingosine 1-Phosphate Receptor Modulators for Multiple Sclerosis. CNS Drugs. 2021;35(4):385–402. doi: 10.1007/s40263-021-00798-w [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref005] 5.Kappos L, Radue E-W, O’Connor P, Polman C, Hohlfeld R, Calabresi P, et al. A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis. N Engl J Med. 2010;362(5):387–401. doi: 10.1056/NEJMoa0909494 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref006] 6.La Mantia L, Tramacere I, Firwana B, Pacchetti I, Palumbo R, Filippini G. Fingolimod for relapsing-remitting multiple sclerosis. Cochrane Database Syst Rev. 2016;4:Cd009371. doi: 10.1002/14651858.CD009371.pub2 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref007] 7.Wu X, Xue T, Wang Z, Chen Z, Zhang X, Zhang W, et al. Different doses of fingolimod in relapsing-remitting multiple sclerosis: a systematic review and meta-analysis of randomized controlled trials. Front Pharmacol. 2021;12:1132. doi: 10.3389/fphar.2021.621856 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref008] 8.Vargas WS, Perumal JS. Fingolimod and cardiac risk: latest findings and clinical implications. Ther Adv Drug Saf. 2013;4(3):119–24. doi: 10.1177/2042098613481023 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref009] 9.David OJ, Kovarik JM, Schmouder RL. Clinical pharmacokinetics of fingolimod. Clin Pharmacokinet. 2012;51(1):15–28. doi: 10.2165/11596550-000000000-00000 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref010] 10.Cugati S, Chen CS, Lake S, Lee AW. Fingolimod and macular edema: Pathophysiology, diagnosis, and management. Neurol Clin Pract. 2014;4(5):402–9. doi: 10.1212/CPJ.0000000000000027 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref011] 11.Etemadifar M, Khorvash R, Zolfagharifard H, Sedaghat N, Nouri H, Salari M. Weight Loss and Loss of Appetite as a Side Effect of Fingolimod (Preprint). Res Sq. 2022. doi: 10.21203/rs.3.rs-1870526/v1 [DOI] [Google Scholar]

[pone.0278488.ref012] 12.Chiba K, Adachi K. Discovery of fingolimod, the sphingosine 1-phosphate receptor modulator and its application for the therapy of multiple sclerosis. Future Med Chem. 2012;4(6):771–81. doi: 10.4155/fmc.12.25 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref013] 13.Perdoni F, Signorelli P, Cirasola D, Caretti A, Galimberti V, Biggiogera M, et al. Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm. BMC Microbiol. 2015;15(1):1–8. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref014] 14.Yang X, Pei Z, Hu R, Zhang Z, Lou Z, Sun X. Study on the Inhibitory Activity and Possible Mechanism of Myriocin on Clinically Relevant Drug-Resistant Candida albicans and Its Biofilms. Biol Pharm Bull. 2021;44(3):305–15. doi: 10.1248/bpb.b20-00246 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref015] 15.Takasaki T, Hagihara K, Satoh R, Sugiura R. More than just an immunosuppressant: the emerging role of FTY720 as a novel inducer of ROS and apoptosis. Oxid Med Cell Longev. 2018;2018. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref016] 16.Carpenter AF, Goodwin SJ, Bornstein PF, Larson AJ, Markus CK. Cutaneous cryptococcosis in a patient taking fingolimod for multiple sclerosis: here come the opportunistic infections? Mult Scler. 2017;23(2):297–9. doi: 10.1177/1352458516670732 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref017] 17.Del Poeta M, Ward BJ, Greenberg B, Hemmer B, Cree BA, Komatireddy S, et al. Cryptococcal Meningitis Reported With Fingolimod Treatment: Case Series. Neurol Neuroimmunol Neuroinflamm. 2022;9(3). doi: 10.1212/NXI.0000000000001156 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref018] 18.Whaley SG, Berkow EL, Rybak JM, Nishimoto AT, Barker KS, Rogers PD. Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species. Front Microbiol. 2017;7:2173. doi: 10.3389/fmicb.2016.02173 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref019] 19.Coste AT, Kritikos A, Li J, Khanna N, Goldenberger D, Garzoni C, et al. Emerging echinocandin-resistant Candida albicans and glabrata in Switzerland. Infection. 2020;48(5):761–6. doi: 10.1007/s15010-020-01475-8 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref020] 20.Romo JA, Kumamoto CA. On Commensalism of Candida. J Fungi (Basel). 2020;6(1). doi: 10.3390/jof6010016 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref021] 21.Conti HR, Huppler AR, Whibley N, Gaffen SL. Animal models for candidiasis. Curr Protoc Immunol. 2014;105:19.6.1-.6.7. doi: 10.1002/0471142735.im1906s105 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref022] 22.EUCAST DEFINITIVE DOCUMENT E.DEF 7.3.1 Method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts. https://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf (2017). Accessed November 2022.

[pone.0278488.ref023] 23.National Research Council. Guide for the care and use of laboratory animals. 8th ed. National Academies Press (US); 2011. [PubMed] [Google Scholar]

[pone.0278488.ref024] 24.Abdullah CS, Li Z, Wang X, Jin ZQ. Depletion of T lymphocytes ameliorates cardiac fibrosis in streptozotocin-induced diabetic cardiomyopathy. Int Immunopharmacol. 2016;39:251–64. doi: 10.1016/j.intimp.2016.07.027 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref025] 25.Bonfiglio T, Olivero G, Merega E, Di Prisco S, Padolecchia C, Grilli M, et al. Prophylactic versus Therapeutic Fingolimod: Restoration of Presynaptic Defects in Mice Suffering from Experimental Autoimmune Encephalomyelitis. PLoS One. 2017;12(1):e0170825. doi: 10.1371/journal.pone.0170825 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref026] 26.Cheng Q, Li D, Liang H, Yang H, Lei D, Gao D, et al. Effects of long-term administration of low-dose FTY720 on survival of murine cardiac allograft. J Huazhong Univ Sci Technolog Med Sci. 2012;32(2):199–204. doi: 10.1007/s11596-012-0035-5 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref027] 27.Efstathopoulos P, Kourgiantaki A, Karali K, Sidiropoulou K, Margioris AN, Gravanis A, et al. Fingolimod induces neurogenesis in adult mouse hippocampus and improves contextual fear memory. Transl Psychiatry. 2015;5(11):e685. doi: 10.1038/tp.2015.179 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref028] 28.Hwang MW, Matsumori A, Furukawa Y, Ono K, Okada M, Iwasaki A, et al. FTY720, a new immunosuppressant, promotes long-term graft survival and inhibits the progression of graft coronary artery disease in a murine model of cardiac transplantation. Circulation. 1999;100(12):1322–9. doi: 10.1161/01.cir.100.12.1322 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref029] 29.Pinschewer DD, Ochsenbein AF, Odermatt B, Brinkmann V, Hengartner H, Zinkernagel RM. FTY720 immunosuppression impairs effector T cell peripheral homing without affecting induction, expansion, and memory. J Immunol. 2000;164(11):5761–70. doi: 10.4049/jimmunol.164.11.5761 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref030] 30.Wang G, Kim RY, Imhof I, Honbo N, Luk FS, Li K, et al. The immunosuppressant FTY720 prolongs survival in a mouse model of diet-induced coronary atherosclerosis and myocardial infarction. J Cardiovasc Pharmacol. 2014;63(2):132–43. doi: 10.1097/FJC.0000000000000031 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref031] 31.Underwood W, Anthony R. AVMA guidelines for the euthanasia of animals. 2020 ed. American Veterinary Medical Association; 2020. [Google Scholar]

[pone.0278488.ref032] 32.JoVE Science Education Database. Basic Methods in Cellular and Molecular Biology. Using a Hemacytometer to Count Cells.: https://www.jove.com/v/5048/using-a-hemacytometer-to-count-cells (2022). Accessed November 2022.

[pone.0278488.ref033] 33.Smirnov N. Table for Estimating the Goodness of Fit of Empirical Distributions. Ann Math Statist. 1948;19(2):279–81. [Google Scholar]

[pone.0278488.ref034] 34.Kolmogorov A. Sulla determinazione empirica di una lgge di distribuzione. Giornale dell’Istituto Italiano degli Attuari. 1933;4:83–91. [Google Scholar]

[pone.0278488.ref035] 35.Benjamini Y, Krieger AM, Yekutieli D. Adaptive linear step-up procedures that control the false discovery rate. Biometrika. 2006;93(3):491–507. [Google Scholar]

[pone.0278488.ref036] 36.Brown MB, Forsythe AB. Robust tests for the equality of variances. J Am Stat Assoc. 1974;69(346):364–7. [Google Scholar]

[pone.0278488.ref037] 37.Welch BL. The Generalization of ‘Student’s’ Problem When Several Different Population Variances are Involved. Biometrika. 1947;34(1–2):28–35. doi: [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref038] 38.Groves T. Enhancing the quality and transparency of health research. BMJ. 2008;337. doi: 10.1136/bmj.a718 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref039] 39.Wei L-Q, Tan J-C, Wang Y, Mei Y-K, Xue J-Y, Tian L, et al. Fingolimod potentiates the antifungal activity of amphotericin B. Front Cell Infect Microbiol. 2021;11:346. doi: 10.3389/fcimb.2021.627917 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0278488.ref040] 40.Hagihara K, Mizukura A, Kitai Y, Yao M, Ishida K, Kita A, et al. FTY 720 stimulated ROS generation and the S ty1/A tf1 signaling pathway in the fission yeast S chizosaccharomyces pombe. Genes Cells. 2014;19(4):325–37. doi: 10.1111/gtc.12134 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref041] 41.Meno-Tetang GM, Li H, Mis S, Pyszczynski N, Heining P, Lowe P, et al. Physiologically based pharmacokinetic modeling of FTY720 (2-amino-2 [2-(-4-octylphenyl) ethyl] propane-1, 3-diol hydrochloride) in rats after oral and intravenous doses. Drug Metab Dispos. 2006;34(9):1480–7. doi: 10.1124/dmd.105.009001 [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref042] 42.Garcerá A, Casas C, Herrero E. Expression of Candida albicans glutathione transferases is induced inside phagocytes and upon diverse environmental stresses. FEMS Yeast Res. 2010;10(4):422–31. doi: 10.1111/j.1567-1364.2010.00613.x [DOI] [PubMed] [Google Scholar]

[pone.0278488.ref043] 43.Brothers KM, Gratacap RL, Barker SE, Newman ZR, Norum A, Wheeler RT. NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality. PLoS Pathog. 2013;9(10):e1003634. doi: 10.1371/journal.ppat.1003634 [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Immunomodulatory drug fingolimod (FTY720) restricts the growth of opportunistic yeast Candida albicans in vitro and in a mouse candidiasis model

Niloofar Najarzadegan

Mahboobeh Madani

Masoud Etemadifar

Nahad Sedaghat

Roles

Abstract

1. Introduction

2. Methods

2.1 Design

Fig 1. Schematic overview of the parallel intervention arms of the in vivo study.

2.2 In vitro antifungal susceptibility testing

2.2.1 Well diffusion assay

2.2.2 Broth microdilution assay

2.3 Preparation, allocation, and follow-up of the mice

2.4 Analyses of samples

2.5 Statistical methods

2.5.1 Data preparation and determination of appropriate tests

2.5.2 Comparisons and their rationale

2.5.3 Software

2.6 Reproducibility, safety and ethical considerations

3. Results

3.1 In vitro antifungal susceptibility tests

Fig 2. Final results of the well diffusion assay.

Fig 3. Results of the broth microdilution assay for determining FTY720’s MIC for C. albicans.

3.2 Overview and validation of the in vivo study

Fig 4. Immune profile of the mice at the end of the in vivo study stratified by study arms.

Fig 5. Final results of four selected mice’s organ cultures.

Fig 6. Micrographs of gram-stained smears of colonies in vaginal sample cultures of two mice in arm 5.

3.3 Effect of FTY720 on mice’s immune response to C. albicans

3.4 Effect of FTY720 on fungal burden in vivo

Fig 7. Colony counts of C. albicans in kidneys, livers, and vaginas of the mice inoculated with C. albicans at the end of the in vivo study, i.e., after 21 days of oral intervention.

4. Discussion

5. Conclusion

6. Limitations

Supporting information

Data Availability

Funding Statement

References

Decision Letter 0

Aijaz Ahmad

Roles

Author response to Decision Letter 0

Decision Letter 1

Aijaz Ahmad

Roles

Author response to Decision Letter 1

Decision Letter 2

Aijaz Ahmad

Roles

Author response to Decision Letter 2

Decision Letter 3

Aijaz Ahmad

Roles

Acceptance letter

Aijaz Ahmad

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

Similar articles

Cited by other articles

Links to NCBI Databases