A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs

The development of new drugs to disrupt malaria transmission requires the establishment of an in vivo model to address the biology of Plasmodium falciparum sexual stages (gametocytes). Herein we show that chemically immune-modulated NSG mice grafted with human erythrocytes support complete sexual development of P. falciparum parasites and generate high gametocytemia. Immunohistochemistry and RT-qPCR analyses indicate an enrichment of immature gametocytes in the bone marrow and the spleen, suggesting a sequestration mechanism reminiscent to that observed in humans. Upon primaquine treatment, elimination of gametocytes from peripheral blood and from sequestration sites was observed, providing a proof of concept that these mice can be used for testing drugs. Therefore, this model allows the investigation of P. falciparum sexual commitment, gametocyte interactions with the bone marrow and spleen and provides the missing link between current in vitro assays and Phase I trials in humans for testing new malaria gametocytidal drugs

cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission.

Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites

Plasmodium falciparum STEVOR phosphorylation regulates host erythrocyte deformability enabling malaria parasite transmission

Key Points P falciparum STEVORs interact with the erythrocyte cytoskeletal ankyrin complex. Infected erythrocyte deformability is regulated by PKA-mediated phosphorylation of STEVOR cytoplasmic domain.</jats:p

A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs

The development of new drugs to disrupt malaria transmission requires the establishment of an in vivo model to address the biology of Plasmodium falciparum sexual stages (gametocytes). Herein we show that chemically immune-modulated NSG mice grafted with human erythrocytes support complete sexual development of P. falciparum parasites and generate high gametocytemia. Immunohistochemistry and RT-qPCR analyses indicate an enrichment of immature gametocytes in the bone marrow and the spleen, suggesting a sequestration mechanism reminiscent to that observed in humans. Upon primaquine treatment, elimination of gametocytes from peripheral blood and from sequestration sites was observed, providing a proof of concept that these mice can be used for testing drugs. Therefore, this model allows the investigation of P. falciparum sexual commitment, gametocyte interactions with the bone marrow and spleen and provides the missing link between current in vitro assays and Phase I trials in humans for testing new malaria gametocytidal drugs

Sildenafil impairs mature GIE filterability.

<p><b>A</b>. Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 100 μM sildenafil, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out five times. Error bars denote the standard error of the mean. **Highly significant differences in retention rates compared to control (DMSO) (<i>P <</i> 0.01). <b>B.</b> Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 100μM sildenafil, or 0.1% DMSO (Control). <b>C</b>. Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. <b>D</b>. Retention rates in microsphilters of stage V GIE (light grey) and uninfected red blood cells (uRBC, dark grey) pre-incubated 30 min at 37°C with different concentrations of sildenafil. Error bars denote the standard error of the mean. Outliers are shown as open circles. *** and **Highly significant differences in retention rates compared to control (**<i>P <</i> 0.01; ***<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments.</p

The effect of PDE inhibitors on particulate fractions isolated from <i>P</i>. <i>falciparum</i> parasites.

<p>IC₅₀ values (the concentration of compound that gave 50% inhibition of native PDE activity) are mean ± SEM in μM and were calculated using the Prism software package (GraphPad Software, Inc.). Percentage inhibition data were fitted to a sigmoidal dose-response curve using non-linear regression. Bovine control PDE column displays cAMP dependent activity. All TbPDE1 and most Human values obtained from Kunz, <i>et al</i>.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref058" target="_blank">58</a>].</p><p>*Value obtained from Tang, <i>et al</i>.[<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref059" target="_blank">59</a>].</p><p>>200, sufficient inhibition was not observed for an IC₅₀ calculation.</p><p>Tb: <i>Trypanosama brucei</i>.</p><p>The effect of PDE inhibitors on particulate fractions isolated from <i>P</i>. <i>falciparum</i> parasites.</p

Viability of 3D7 stage V GIE treated with different inhibitors.

<p>3D7 Stage V GIE were treated with the inhibitors for the indicated times, then washed and their viability evaluated with the colorimetric pLDH assay either immediately (time 0) or after 72 h incubation at 37°C, as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004815#ppat.1004815.ref056" target="_blank">56</a>]. The data are expressed as percent of untreated controls and are the mean of two experiments in quadruplicate.</p><p>Viability of 3D7 stage V GIE treated with different inhibitors.</p

Interfering with cAMP levels impairs mature GIE filterability.

<p><b>A.</b> Stage V GIE were harvested by magnetic isolation and incubated at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast, or 0.1% DMSO (Control). The total intracellular cAMP concentration was measured on aliquots of 6.10⁶ cells in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. *Significant differences in retention rates compared to control (<i>P <</i> 0.05). <b>B.</b> Retention in microsphilters of stages V GIEs (light grey), or uninfected red blood cells (uRBCs, dark grey). GIEs were pre-incubated at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast or 0,1% DMSO (control). ***Highly significant differences in retention rates compared to control (DMSO) (<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control. n: number of experiments. <b>C.</b> Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C for 30 min with 150 μM forskolin, 65 μM zaprinast or 0.1% DMSO (control).</p

GIE filterability is dependent on cAMP concentration.

<p><b>A.</b> cAMP concentration drops in mature GIE. The total intracellular cAMP concentration was measured in stage III and V GIE using a competitive immunoassay for the quantitative determination of cAMP. GIE were harvested by magnetic isolation and aliquots of 6.10⁶ cells were assayed in duplicate wells. The assay was carried out three times. Error bars denote the standard error of the mean. **Highly significant difference compared to stage III GIE (<i>P <</i> 0.01). <b>B</b>. Western-blot analysis of PKA-R expression in MACS-purified stage III and stage V GIE (5.10e6 parasites/lane). Immunoblots were probed with rabbit polyclonal antibodies directed against <i>Pf</i>PKA-R and with a mAb directed against <i>Pf</i>HSP70 to normalize expression. Quantity One (BioRad) analysis shows that <i>Pf</i>PKA-R levels were not significantly different between stage III and stage V. <b>C</b>. Retention rates in microsphilters of stage III GIE (dark grey), stage V GIE (light grey) and uninfected red blood cells (uRBC, black) pre-incubated 15 min at 37°C with different concentrations of 8Br-cAMP. Error bars denote the standard error of the mean. Outliers are shown as open circles. **Highly significant differences in retention rates compared to control without 8Br-cAMP (<i>P <</i> 0.01); ns: non-significant differences in retention rates compared to control; n: number of experiments.</p

Phosphorylation events contribute to the switch in deformability.

<p><b>A.</b> Western-blot analysis of Phospho-PKA site (RRXS*/T*) expression in uninfected red blood cells (uRBCs), MACS-purified stage III (stIII) and stage V (stV) GIE from the B10 clone treated (+Calyc) or not (-Calyc) with 50 nM calyculin during 2h at 37°C. Analysis was performed on membrane extracts recovered by centrifugation after 1% Triton X100 treatment. Immunoblot was probed with rabbit monoclonal antibody directed against phospho-PKA sites (RRX*S/*T) and with mouse mAb directed against Band 3 to normalize expression. Red arrows show bands with more intense phosphorylation signal in stage III GIE than in stage V GIE. Green arrow show band with less intense phosphorylation signal in stage III GIE than in stage V GIE. The experiment has been performed three times. Error bars denote the standard error of the mean. ***Highly significant differences in phosphorylation signal (<i>P <</i> 0.001). <b>B.</b> Quantitation of signal intensities in panel D using Quantity One software (BioRad). The strength of the phosphorylation signal in untreated uninfected red blood cells lysate was set to 1 and all other signals are relative to that. <b>C.</b> Retention in microsphilters of stages V GIEs (light grey), or uninfected red blood cells (uRBCs, dark grey). GIEs were pre-incubated at 37°C for 2 h with 50 nM calyculin, or 0.5% DMSO (control). ***Highly significant differences in retention rates compared to control (<i>P <</i> 0.001); ns: non-significant differences in retention rates compared to control; n: number of experiments. <b>D</b>. Graphical representation for the proportion of GIE showing a regular (light green), or deformed (dark green) shape in a population of paraformaldehyde-fixed GIE, as they flow through the microsphilters after pre-incubation at 37°C 2 h with 50 nM calyculin, or 0.5% DMSO (control). <b>E.</b> Differential interference contrast images of paraformaldehyde-fixed GIE, as they flow through the microsphilters. A majority of DMSO-treated GIE (left panel) are twisted and deformed, whereas inhibitor-treated GIE (right panel) keep a regular shape, unlike uninfected erythrocytes (red star).</p