Interactions between tafenoquine and artemisinin-combination therapy partner drug in asexual and sexual stage Plasmodium falciparum

The 8-aminoquinoline tafenoquine (TFQ), a primaquine derivative, is currently in late-stage clinical development for the radical cure of P. vivax. Here drug interactions between TFQ and chloroquine and six artemisinin-combination therapy (ACT) partner drugs in P. falciparum asexual stages and gametocytes were investigated. TFQ was mostly synergistic with the ACT-partner drugs in asexual parasites regardless of genetic backgrounds. However, at fixed ratios of 1:3, 1:1 and 3:1, TFQ only interacted synergistically with naphthoquine, pyronaridine and piperaquine in gametocytes. This study indicated that TFQ and ACT-partner drugs will likely have increased potency against asexual stages of the malaria parasites, whereas some drugs may interfere with each other against the P. falciparum gametocytes. Keywords: Plasmodium falciparum, Tafenoquine, Malaria, Gametocyte, Drug interaction, 8-Aminoquinoline, Isobologram, AC

<i>In vivo</i> CD8+ T Cell Dynamics in the Liver of <i>Plasmodium yoelii</i> Immunized and Infected Mice

<div><p><i>Plasmodium falciparum</i> malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to <i>Plasmodium</i> liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination <i>in vivo</i>, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated <i>P. yoelii</i> 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in <i>situ</i>, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of <i>P. yoelii</i> infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and <i>P. yoelii</i> CS_280–288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.</p></div

Behavior of CD8+ T cells in the liver of naïve and immunized mice.

<p>Representative IVM images of CD8+ T cells observed in naïve and <i>P. yoelii</i> immunized mouse livers. Note that in contrast to naïve mice, the T cells from immunized mice exhibit the characteristic amoeboid shape of activated effector cells and patrol the sinusoids with a leading edge and a trailing pseudopod. (<b>A</b>) 3D representation of a naïve Tie2-GFP mouse liver with fluorescent endothelia (green), anti-CD8a-PE labeled CD8+ T cells (red), and Hoechst stained nuclei (blue). (<b>B</b> and <b>C</b>) 3D projections of anti-CD8a-PE labeled CD8+ T cells (green) in the livers of mice immunized with (<b>B</b>) Pyfabb/f(<b>−</b>)or (<b>C</b>) Py-RAS. Hepatocyte mitochondria were labeled with MitoTracker (red) and nuclei were stained with Hoechst (blue). (<b>D</b>) 2D snapshot of the liver of a mouse immunized with Pyuis4(<b>−</b>) showing CD8+ T cells labeled with anti-CD8a-PE (red). Hepatocyte mitochondria labeled with MitoTracker (green). CD8+ T cells of immunized mice were monitored 2 weeks after the second booster. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s003" target="_blank">Videos S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s004" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s005" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070842#pone.0070842.s006" target="_blank">S4</a></b> for the corresponding movies. Scale bars 20 μm.</p

Ly6C(high) monocytes become alternatively activated macrophages in schistosome granulomas with help from CD4+ cells.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1(GFP/+) mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C(low) monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C(high) and Ly6C(low) monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C(high) monocytes, but that these cells may transition through a Ly6C(low) state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C(high) monocytes via help from CD4⁺ T cells

Tracking adoptively transferred Py-RAS activated CD8+ T cells in the livers of infected mice.

<p>LS were identified in a well-immobilized area of the liver and labeled CD8+ T cells in the vicinity of the LS were monitored to calculated velocities (<b>A</b>) and arrest coefficients (<b>B</b>). Py-RAS CD8+ T cells in the liver of immunized mice (circles) were used as controls. CD8+ T cells were purified 2 weeks after the second booster from the spleens of Py-RAS immunized mice and 1 million was adoptively transferred into recipient mice 18 h (triangles) or 42 h (squares) after infection with PyXNL-GFP. Based on ANOVA on Ranks, the velocity and arrest coefficients of all adoptively transferred cells differed significantly from Py-RAS in the liver of immunized mice (p<0.05). The same test showed only a significant difference in the values between the two 18 h time points. At least nine infected mice were used per experimental condition. *  =  p<0.05, ns  =  not significant.</p

Tracking CD8+ cells in naïve and immunized mouse livers.

<p>CD8+ T cells were identified by anti-CD8a-PE labeling in the livers of naïve mice (circles) or mice immunized with Py-RAS (triangles), Pyuis4(<b>−</b>) (diamonds), or Pyfabb/f(<b>−</b>) (squares) and monitored via IVM. Velocities (<b>A</b>) and arrest coefficients (<b>B</b>) were calculated from the tracks of individual CD8+ T cell from at least 2 infected mice per group. One-way ANOVA on Ranks shows that the velocities of the CD8+ T cells from all immunized mice are significantly higher and the arrest coefficients significantly lower than those of the CD8+ T cells from naive mice (p<0.05). The same statistical analysis does not reveal any significant difference between the CD8+ T cell velocities between the groups of immunized mice (p>0.05). *  =  p<0.005, ns  =  not significant. CD8+ T cells of immunized mice were monitored at least 2 weeks after the second booster.</p

Behavior of adoptively transferred CD8+ T cells in the liver of infected mice.

<p>At least 2 weeks after the second booster, CD8+ T cells were purified from the spleens of Py-RAS immunized mice and loaded with CellTracker Red. One million CD8+ T cells were transferred into recipient mice 18 h (<b>A</b>) or 42 h (<b>B</b>) post infection with PyXNL-GFP, and immediately imaged by IVM. CD8+ T cells (red) remained immobile and failed to migrate to or make contact with hepatocytes infected with PyXNL-GFP LS (green). Hepatocyte autofluorescence is shown in green in (<b>A</b>) and red in (<b>B</b>). Nuclei were stained with Hoechst (blue) in (<b>A</b>). Scale bars 20 μm.</p

Tracking adoptively transferred Pyuis4(−) activated CD8+ T cells in the livers of infected mice.

<p>Mice were infected with 1–2 million PyXNL-GFP sporozoites. Activated CMTPX labeled CD8+ T cells were then purified 2 weeks after the second booster from the spleens of Py-RAS immunized mice and 1 million was adoptively transferred into the infected mice at 18 h (triangles) or 42 h (squares) after infection with PyXNL-GFP. Anti-mouse CD8a labeled T cells in Pyuis4(−) immunized mice (circles) were used as controls. ANOVA on Ranks show that the mean velocities (<b>A</b>) and arrest coefficients (<b>B</b>) of all adoptively transferred CD8+ T cells differ significantly from those of the CD8+ T cells in Pyuis4(−) immunized mice. At least three infected mice were used per experimental condition. *  =  p<0.05, ns  =  not significant.</p

Inhibition of LS development after infection of immunized mice.

<p>Naïve or Pyfabb/f(<b>−</b>) immunized BALB/c mice were infected with 3×10⁵ PyXNL-GFP sporozoites. Immunized mice were infected 2 weeks after the second booster. At 18 h or 42 h post-infection, livers were removed and LS parasites were quantified in fresh unfixed liver tissue. Using vibratome sections of known thickness and a region of interest of a defined size, the number of LS was counted and normalized to a volume of 1.3 cm³ (approximate liver volume of adult mice). Panels (<b>A</b>) and (<b>B</b>) show the reduction in LS number and volume, respectively, in Pyfabb/f(<b>−</b>) immunized mice compared to naïve mice. *  =  p<0.05.</p