The anthelmintic drug Praziquantel promotes human Tr1

differentiation

Running title: Immunoregulatory role of praziquantel

Enwono Eyoh1, Patrick McCallum1, Justin Killick2, Seth Amanfo1, Francisca Mutapi1,3,†,*,

Anne L. Astier2,4,†,*

1Institute of Immunology & Infection Research and Centre for Immunity, Infection and

Evolution, School of Biological Sciences, University of Edinburgh, Ashworth

Laboratories, King's Buildings, Edinburgh, United Kingdom

2The MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

3NIHR Global Health Research Unit Tackling Infections to Benefit Africa (TIBA),

University of Edinburgh, Ashworth Laboratories, King's Buildings, Edinburgh, United

Kingdom

4Centre de Physiopathologie Toulouse-Purpan (CPTP) INSERM U1043, CNRS U5282,

Université de Toulouse, Toulouse, France

† co-senior authorship

*Corresponding authors: F.Mutapi@ed.ac.uk and anne.astier@inserm.fr

Keywords: Praziquantel; regulatory T cells; CD46; human CD4+ T cells; immune

regulation.

1

ABSTRACT

Praziquantel (PZQ) is an anthelminthic human and veterinary drug used to treat trematode

and cestode worms. Changes in immune responses have been demonstrated in humans

following curative PZQ treatment of schistosome infections. These changes have been

attributed to the removal of immunosupressive worms and immune responses to parasite

antigens exposed from dying worms. To date, there has been no study investigating the

potential direct effect of PZQ on the host immune cells. Herein, we analyzed the effect of

PZQ on human CD4+ T cells classically costimulated by CD3/CD28 or costimulated by the

complement regulator CD46 to induce Type 1 regulatory T cells (Tr1). Our results show

that PZQ enhanced T cell proliferation, increased secretion of IL-17 and IL-10 but had no

effect on secretion of GM-CSF or IFN. Moreover, PZQ increased the co-expression of

CD49b and LAG-3, a hallmark of Tr1 cells, suggesting increased Tr1 differentiation.

Indeed, supernatants from PZQ-treated cells were able to decrease bystander T cell

activation, and this was partly reduced when blocking IL-10. Hence, our study

demonstrates that PZQ directly modulates human T cell activation and promotes Tr1

differentiation, suggesting that PZQ may have immunomodulatory functions in parasite-

unrelated human inflammatory diseases.

2

INTRODUCTION

Praziquantel (PZQ) is a pyrazinoisoquinoline derivative with low toxicity and side effects

used to treat humans and animals for trematodes and cestodes infections 1. Experimental

studies show that PZQ directly acts on the parasites, by increasing the permeability of the

parasite membrane, leading to enhanced calcium ion influx. In schistosomes, PZQ also has

a disruptive effect on the tegument of the worms. This protective tegument layer shields

antigens from interaction with the host immune system, thereby allowing the helminth to

evade detection and immune destruction. Upon PZQ treatment, morphological disruption

of the tegument occurs, with blebbing and vacuolization resulting in helminth antigens

becoming exposed to the host immune system 2 and thus inducing parasite-specific

immune responses. In humans, significant changes in parasite-specific as well as

unspecific cellular and humoral immune responses are observed following curative PZQ

treatment of worm infections, and these changes were due to the removal of worms 3-5.

Studies performed in fish showed that PZQ upregulated transcriptional expression of T cell

receptors and IL-8 with an increase in antiviral signaling and IL-1β production, suggesting

that PZQ is able to act as an immune-stimulant in these fish species 6. In mice, PZQ was

shown to increase the efficacy of influenza A and hepatitis B vaccines by enhancing the

induction of pro-inflammatory CD8+ T cells and effector cytokines 7, 8.

Importantly, a recent study identified a serotonin receptor as a cellular receptor for PZQ 9.

PZQ bound specifically to the 5-HT2BR and elicited contraction of mouse mesenteric

vasculature. Moreover, PZQ could also act as an agonist of the human receptor 9, which

demonstrates the potential ability of PZQ to modulate human cells directly. However, little

is known of the direct effects of PZQ on human immune cells.

CD4+ T cells play a critical role in maintaining immune homeostasis. They differentiate

into different effector Th subsets depending on the stimuli they receive. Activation of the

3

effector cells is counterbalanced by regulatory T cells, which are themselves subdivided

into different subsets depending on their mode of suppression. Among the latter, Type 1

regulatory cells (Tr1) are generated in the periphery and are involved in the maintenance

of peripheral tolerance 10, 11. Tr1 cells suppress bystander effector cell activation by

secretion of IL-10, and they can be induced by the ligation of the complement regulator

CD46 12, 13. While initial activation of CD46 induces a Th1 phenotype characterized by

secretion of IFN, as IL-2 accumulates, CD46 promotes a switch of these cells to a Tr1

phenotype secreting IL-10. This Th1-Tr1 switch is dysregulated in a number of patients

with chronic inflammatory diseases highlighting the importance of this pathway in

maintaining immune homeostasis 13-15. In this study, we aimed to characterize the potential

immunomodulatory effects of PZQ on human CD4+ T cells in the absence of a helminth

infection and focused on both effector Th cells and regulatory Tr1 cells.

4

RESULTS

PZQ promotes T cell proliferation

We first assessed whether the addition of PZQ to CD4+ T cells modulated their

proliferation. CD4+ T cells were isolated from healthy volunteers and activated by

immobilized anti-CD3/CD28 or anti-CD3/CD46 antibodies in the presence of increasing

concentrations of PZQ that was biologically relevant 9, 16 (Supplementary figure 1). The

addition of PZQ significantly increased both the percentage of proliferation (figure 1a,b)

and the proliferation index (Figure 1c) of anti-CD3/CD28-costimulated T cells. We did not

observe a significant effect in anti-CD3/CD46-costimulated T cells for most donors as

their proliferation was maximal compared to cells costimulated by CD28. However, we

could detect a mild increase in proliferation after addition of PZQ for the donors who

exhibited a lower proliferation with CD46 (Figure 1b). Hence, PZQ promotes human CD4+

T cell proliferation.

PZQ modulates cytokine production

We next assessed whether PZQ modulated cell activation by measuring the expression of

the activation markers CD25, CD69 and PD-1, and of CD46 after either 1 or 5 days of

activation (supplementary Figure 2, Figure 2). As expected, CD46 costimulation led to a

strong decrease in CD46 surface expression due to the shedding of its ectodomain 17, 18.

Addition of PZQ had no significant effect on the expression of CD46 and did not

significantly modulate the induction of CD69, CD25 and PD-1. PZQ has been shown to

act as an adjuvant for vaccination, correlating with an increase in IL-17 and IFN

production by murine CD8+ T cells 7, 8. Hence, the direct effect of PZQ on cytokine

production by human CD4+ T cells was next assessed. The amounts of the effector

5

cytokines IL-17, GM-CSF and IFN and of the anti-inflammatory cytokine IL-10 were

measured by ELISA. Addition of PZQ led to a moderate increase in IL-17 secretion and

enhanced the production of IL-10 by CD46-costimulated T cells after 5 days (Figure 3). In

contrast, PZQ had no effect on the production of GM-CSF or IFN. As we observed that

PZQ enhanced T cell proliferation, the increased secretion of cytokines may simply reflect

the increase in cell numbers. However, the lack of effect on the production of IFNγ and

GM-CSF suggests that PZQ skews the profile of cytokines secreted. Of note, we also

assessed the effects of PZQ on secretion of IL-10 and IFN earlier, after 3 days of

activation (supplementary figure 3). While no effect was observed on IFN, an increased

production of IL-10 was also observed for the 5 donors tested.

PZQ promotes Tr1 differentiation

Our data show that PZQ promoted CD4+ T cell proliferation and cytokine production,

especially of IL-10, while it did not affect secretion of IFN. As increased production of

IL-10 and low IFN are characteristics of Tr1 differentiation and typically of the CD46-

mediated Th1-Tr1 differentiation switch 13, we analyzed the induction of LAG3 and

CD49b, two markers whose co-expression are a hallmark of Tr1 cells 19. PZQ significantly

increased expression of LAG3 and the percentage of cells co-expressing both markers,

suggesting that PZQ enhances Tr1 differentiation (Figure 4). To confirm that PZQ

promoted Tr1 differentiation, we next examined whether the supernatants of PZQ-treated

cells were suppressive. We activated EF670-pre-labelled CD4+ T cells by CD3/CD28 in

the presence of supernatants of T cells activated by CD3/CD46 in the presence or absence

of PZQ (Figure 5). Supernatants of cells activated in the presence of PZQ were indeed

highly suppressive, indicating that PZQ enhanced Tr1 differentiation with suppressive

function (Figure 5b). As Tr1 cells mainly exert their suppressive function by secretion of

6

IL-10, we next added a blocking anti-IL-10 antibody or IgG1 control (Figure 5c). Blocking

IL-10 partly restored proliferation, indicating that IL-10 was at least partly involved in the

suppression of proliferation.

7

DISCUSSION

This study is the first to investigate PZQ’s direct modulatory effects in human CD4+ cells

in vitro. We found that PZQ promoted CD4+ T cell proliferation and cytokine production,

especially of IL-10, while it did not affect secretion of IFN. As increased production of

IL-10 but reduction of IFN are hallmarks of Tr1 differentiation, and typically of CD46-

mediated Th1 to Tr1 differentiation, we analyzed the induction of LAG3 and CD49b, two

markers whose co-expression are found on Tr1 cells 19. Indeed, PZQ increased the

percentage of cells expressing both markers, suggesting that it promoted Tr1

differentiation. We further demonstrated this as supernatants of PZQ-treated cells strongly

suppressed proliferation of bystander T cells, and this was partly restored by blocking IL-

10. Interestingly, a recent study showed that PZQ acted as an agonist for the human

serotonin receptor 5-HT2BR, demonstrating its ability to modulate human cells 9. T cells

express serotonin receptors upon their activation 20 and it is theoretically possible that PZQ

may utilize 5-HT2BR to exert its action on human T cells.

Schistosome infection is associated with elevated levels of circulating regulatory T cells,

with PZQ treatment resulting in a decrease in Treg levels and concurrent increase of

immune response against the pathogen 21. Although induction of Tr1 cells by PZQ may

initially seem to be contradictory to the known effects of PZQ, PZQ may directly be

involved in reversing the inflammatory symptoms of the disease upon treatment by

activating the CD46 pathway that first induces Th1 cells and then their switch to Tr1

differentiation 13, 22. After PZQ administration, helminth eggs are no longer being shed and

so further inflammatory responses do not occur. This decreased inflammation may also be

8

Hence, looking at the body of work from the previous schistosome immunoepidemiology

studies, PZQ may have both a direct and indirect effect on the immune shifts observed

following schistosomiasis treatment, facilitating the reversal of inflammatory disease

symptoms, Thus this study reports for the first time, evidence for an immunoregulatory

role of PZQ which may lead to the possibility of new applications for the established drug.

This possibility warrants further detailed investigations particularly those on the drug’s

effects in other human immune cell subsets.

This study reports for the first time evidence for an immunoregulatory role of PZQ. This

may lead to the possibility of new applications for the established drug, especially as

manipulation of Tr1 as potential therapeutic agents to restore tolerance is actively

investigated 23, 24. This warrants further detailed investigations particularly those on the

drug’s effects in other human immune cell subsets.

9

METHODS

CD4+ T cells isolation. Human peripheral blood was obtained from healthy donors after

informed consent. Ethical approval was obtained from the Lothian Health Board Ethics

Committee (ethical approval 15-HV-013). Peripheral blood mononuclear cell (PBMCs)

were isolated using Ficoll-Hypaque gradients (Merck, Southampton, UK). CD4+ T cells

were purified by negative selection using EasySepTM Human CD4+ T cell isolation kits

(STEMCELL Technologies, Cambridge, UK), and cells were resuspended in complete

RPMI 10%FCS (Merck)(representative purification in Supplementary figure 3).

T cell activation. CD4+ T cells were left unstimulated or were activated by plating 5 x 105

T-cells on wells pre-coated for 2 hours with anti-CD3 (OKT3, 5 μg mL-1, Biolegend,

London, UK), anti-CD3/anti-CD28 (CD28.2; 5 μg mL-1, Biolegend) or anti-CD3/anti-

CD46 (MCI20.6, 10 μg mL-1) to induce Tr1 regulatory cells 13, 14, 22. 10 U/ml of IL-2 was

added to all activating conditions. PZQ (1 μg.mL-1, a biologically relevant concentration 16,

or between 1-0.04 μg.mL-1 for Supplementary figure 1, Sigma-Aldrich, Merck, Dorset,

UK) was added into the PZQ wells and ethanol was used as a vehicle control.

Flow cytometry. After 5 days, CD4+ T cells were harvested and stained with anti-CD46

(FITC), anti-PD1 (PE), anti-CD25 (APC), anti-CD49b (FITC), anti-LAG-3 (PE) and anti-

CD69 (APC) mAbs (Biolegend)(representative flow histograms in Supplementary figure

2), and analyzed with a FACSCalibur. FlowJo® 10 was used to analyze the data. Day 3 or

5 supernatants were stored at -20°C until needed for ELISA.

Proliferation assay. CD4+ T cells were isolated as previously stated and washed with PBS.

A 5 µM solution of Cell Proliferation Dye eFluor™ 670 (eBioscience, ThermoFisher

Scientific, Loughborough, UK) was prepared in PBS, according to the manufacturer’s

instructions. Briefly, this solution was mixed with 2 x 106 CD4+ T cells and incubated in

the dark at 37°C for 10 min. RPMI media was added to the cells and incubated on ice for 5

10

min to stop the reaction. The cells were washed with RPMI and 10 x 104 eFluor™ 670

stained cells were activated in pre-coated wells and 1 μg mL-1 of PZQ or ethanol as a

vehicle control was added. Proliferation was assessed after 3 or 5 days of proliferation by

flow cytometry.

ELISA. For detection of IFNγ, IL-17, GM-CSF and IL-10 capture ELISAs were

performed on day 5 supernatants, in duplicates. IL-10 and IFNγ secretion was determined

by ELISA specific for human IL-10 (BD Pharmingen, Oxford, UK) and IFNγ (Thermo-

Fisher) as previously published 17, 25. ELISAs for IL-17 and GMCSF were purchased from

BioLegend (ELISA MAX Deluxe) and were performed according to the manufacturer’s

instructions.

Suppression assays. CD4+ T cells were isolated and stained with the Cell Proliferation Dye

eFluor™ 670 as previously stated. Pre-labeled cells (10 x 104) were then plated in wells

which had been pre-coated with anti-CD3/anti-CD28 antibodies, in the presence of 100 l

of day 5 supernatants harvested from cells that were activated by CD3/CD46 in the

presence of PZQ or vehicle control (Ethanol). Proliferation at day 3 to 5 was assessed by

flow cytometry and analyzed by FlowJo® 10. In some experiments a blocking anti-hIL-10

or control IgG1 (10 g.mL-1, Biolegend) were added to the culture.

Statistical Analysis. The statistical significance has been calculated using a non-parametric

Friedman test with Dunn's correction for multiple comparisons using the Prism 7

Graphpad software. The non-parametric paired Wilcoxon t-test was performed when

comparing two paired samples. All analyses were performed at a 95% Ci and P-values <

0.05 were considered significant. *P < 0.05; ** P < 0.01; *** P < 0.001.

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ACKNOWLEDGMENTS

The authors are grateful to all the volunteers who donated their blood and the members of

Parasite Immunology group (University of Edinburgh) as well as Roland Liblau (CPTP,

Toulouse) for their critical reading of the manuscript. We thank Shonna Johnston and Will

Ramsay (MRC Centre for Inflammation Research, University of Edinburgh) and Dr.

Martin Waterfall (IIIR, University of Edinburgh) for their help with the flow cytometry.

This study was partly supported by a research grant to ALA from the Multiple Sclerosis

Society (MS 41).

CONFLICT OF INTEREST

The authors do not have any conflicts of interest.

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Figure legends

Figure 1. PZQ promotes proliferation.

(a) Purified human CD4+ T cells were pre-labeled with EF670 and left unstimulated (US)

or were activated with immobilized anti-CD3/CD28 or anti-CD3/CD46 antibodies as

indicated, in the presence of PZQ (1ug.mL-1) or ethanol as a vehicle control. The

proliferation was determined by flow cytometry after 5 days of activation. (b) Percentage

of cells proliferation from all donors (mean ± SEM, n=12, non-parametric Friedman test

with Dunn's correction for multiple comparisons). (c) Proliferation index of CD3/CD28

costimulated T cells.

Figure 2. PZQ does not modulate the phenotype of activated T cells.

Human CD4+ T cells were left unstimulated (US) or were stimulated with immobilized

anti-CD3/CD28 or anti-CD3/CD46 antibodies in the presence of PZQ (1ug.mL-1) or

ethanol as a vehicle control. The expression of CD46, CD25, CD69 and PD-1 was assessed

by flow cytometry (mean ± SEM, n=16, non-parametric Friedman test with Dunn's

correction for multiple comparisons) after (a) 1 and (b) 5 days of culture.

Figure 3. PZQ modulates cytokine production by activated T cells.

Human CD4+ T cells were left unstimulated (US) or were stimulated with immobilized

anti-CD3/CD28 or anti-CD3/CD46 antibodies in the presence of PZQ (1ug.mL-1) or

ethanol as a vehicle control. The secretion of IFN, IL-10, IL-17 and GMCSF in the

supernatants were assessed by ELISA (mean ± SEM, n=13 for IL-10 and IFN and n=7

for IL-17 and GMCSF; non-parametric Friedman test with Dunn's correction for multiple

comparisons).

15

Figure 4. PZQ promotes the co-expression of the Tr1 markers, LAG3 and CD49d.

Human CD4+ T cells were left unstimulated (US) or were stimulated with immobilized

anti-CD3/CD28 or anti-CD3/CD46 antibodies for 5 days in the presence of PZQ (1ug.mL-

1) or ethanol as a vehicle control. The expression of LAG3 (a) and CD49b (b) was then

assessed by flow cytometry. (c) represents the data for all donors assessed (mean ± SEM,

n=16, non-parametric Friedman test with Dunn's correction for multiple comparisons).

Figure 5. PZQ promotes Tr1 differentiation.

Human CD4+ T cells were pre-labeled with EF670 and stimulated with immobilized anti-

CD3/CD28 in the presence of supernatants of CD3/CD46 costimulated T cells cultured in

the presence of PZQ (1ug.mL-1) (PZQ) or or ethanol as a vehicle control (Ctrl). (a) shows

the data obtained for one representative donor and (b) the data obtained for all donors are

represented (mean ± SEM, n=10, Wilcoxon matched paired ranked test). (c) a blocking

anti-hIL-10 or IgG1 control (10 g.mL-1) was added to the culture. Representative of 2

experiments.

16