Preclinical Assessment of the Treatment of Second-Stage African Trypanosomiasis with Cordycepin and Deoxycoformycin

There is an urgent need to substitute the highly toxic arsenic compounds still in use for treatment of the encephalitic stage of African trypanosomiasis, a disease caused by infection with Trypanosoma brucei. We exploited the inability of trypanosomes to engage in de novo purine synthesis as a therapeutic target. Cordycepin was selected from a trypanocidal screen of a 2200-compound library. When administered together with the adenosine deaminase inhibitor deoxycoformycin, cordycepin cured mice inoculated with the human pathogenic subspecies T. brucei rhodesiense or T. brucei gambiense even after parasites had penetrated into the brain. Successful treatment was achieved by intraperitoneal, oral or subcutaneous administration of the compounds. Treatment with the doublet also diminished infection-induced cerebral inflammation. Cordycepin induced programmed cell death of the parasites. Although parasites grown in vitro with low doses of cordycepin gradually developed resistance, the resistant parasites lost virulence and showed no cross-resistance to trypanocidal drugs in clinical use. Our data strongly support testing cordycepin and deoxycoformycin as an alternative for treatment of second-stage and/or melarsoprol-resistant HAT

Bioluminescent Imaging of Trypanosoma brucei Shows Preferential Testis Dissemination Which May Hamper Drug Efficacy in Sleeping Sickness

Monitoring Trypanosoma spread using real-time imaging in vivo provides a fast method to evaluate parasite distribution especially in immunoprivileged locations. Here, we generated monomorphic and pleomorphic recombinant Trypanosoma brucei expressing the Renilla luciferase. In vitro luciferase activity measurements confirmed the uptake of the coelenterazine substrate by live parasites and light emission. We further validated the use of Renilla luciferase-tagged trypanosomes for real-time bioluminescent in vivo analysis. Interestingly, a preferential testis tropism was observed with both the monomorphic and pleomorphic recombinants. This is of importance when considering trypanocidal drug development, since parasites might be protected from many drugs by the blood-testis barrier. This hypothesis was supported by our final study of the efficacy of treatment with trypanocidal drugs in T. brucei-infected mice. We showed that parasites located in the testis, as compared to those located in the abdominal cavity, were not readily cleared by the drugs

Aicardi-Goutières syndrome gene Rnaseh2c is a metastasis susceptibility gene in breast cancer.

Breast cancer is the second leading cause of cancer-related deaths in the United States, with the majority of these deaths due to metastatic lesions rather than the primary tumor. Thus, a better understanding of the etiology of metastatic disease is crucial for improving survival. Using a haplotype mapping strategy in mouse and shRNA-mediated gene knockdown, we identified Rnaseh2c, a scaffolding protein of the heterotrimeric RNase H2 endoribonuclease complex, as a novel metastasis susceptibility factor. We found that the role of Rnaseh2c in metastatic disease is independent of RNase H2 enzymatic activity, and immunophenotyping and RNA-sequencing analysis revealed engagement of the T cell-mediated adaptive immune response. Furthermore, the cGAS-Sting pathway was not activated in the metastatic cancer cells used in this study, suggesting that the mechanism of immune response in breast cancer is different from the mechanism proposed for Aicardi-Goutières Syndrome, a rare interferonopathy caused by RNase H2 mutation. These results suggest an important novel, non-enzymatic role for RNASEH2C during breast cancer progression and add Rnaseh2c to a panel of genes we have identified that together could determine patients with high risk for metastasis. These results also highlight a potential new target for combination with immunotherapies and may contribute to a better understanding of the etiology of Aicardi-Goutières Syndrome autoimmunity

iNOS expression is associated with integrity of the BBB during infection with <i>T</i>.<i>b</i>. <i>brucei</i>.

<p>(A, D, F) IgG (A) and fibrin (D) immunolabeling and Evan’s blue extravasation (F) in the brains of WT and <i>inos</i>^<i>-/-</i> mice 23 dpi with <i>T</i>.<i>b</i>. <i>brucei</i>. (B, E, G) The mean relative integrated fluorescence densities (RIF) of IgG (B), fibrin (E) or EB (G) ± SEM in the brain parenchyma from at least 3 sections per brain and 4 animals per group are depicted. (C) IgG was also detected by Western Blot in brain lysates of <i>inos</i>^<i>-/-</i> mice 25 dpi with <i>T</i>.<i>b</i>. <i>brucei</i> but not in infected or uninfected WT mice. Differences between WT and <i>inos</i>^<i>-/-</i> mice are significant (*<i>p</i><0.05 unpaired Student’s <i>t</i> test).</p

Nitric Oxide Protects against Infection-Induced Neuroinflammation by Preserving the Stability of the Blood-Brain Barrier

<div><p>Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic <i>Trypanosoma brucei</i> parasites were compared in <i>inos</i>^<i>-/-</i> and wild-type mice. <i>Inos</i>^<i>-/-</i> mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In <i>inos</i>^<i>-/-</i> mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.</p></div

iNOS-derived NO reduces <i>T</i>.<i>b</i>. <i>brucei</i> and leukocyte penetration into the brain.

<p>(A-B) Body weight and parasitemia of WT and <i>inos</i>^<i>-/-</i> mice infected i.p. with 2x10³ <i>T</i>.<i>b</i>. <i>brucei</i>. Each point represents the mean log₁₀ parasites per ml ± SEM (n = 9 to 10 per group). Statistically significant differences in comparison with infected WT animal (*<i>p</i>< 0.05, **<i>p</i>< 0.01 two-way ANOVA). The body weights were standardized with respect to the mean of the same group before infection. One out of three independent experiments is depicted. (C-F) The mean number of <i>T</i>.<i>b</i>. <i>brucei</i> (C), and CD4+ (D), and CD8+ (E) T cells per mm² ± SEM from 6 animals per group is depicted. (F) Representative immunofluorescence images show <i>T</i>.<i>b</i>. <i>brucei</i> and cerebral endothelial cells in cerebral regions of WT and <i>inos</i>^<i>-/-</i> mice 25 dpi. A representative of three similar independent experiments is shown. Statistically significant differences in comparison to WT mice at the same dpi: (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 unpaired Student’s <i>t</i> test). (G) Representative fluorescent staining of CD45+ leukocytes and cerebral endothelial cells (Glut-1) of WT and <i>inos</i>^<i>-/-</i> mice 25 dpi. (H, I) Parasitemia (H) and weight (I) of <i>inos</i>^<i>-/-</i> mice infected i.p. with <i>T</i>. <i>brucei</i> and treated or not daily with 3.5 mg GSNO i.p. starting at 5 dpi. (J-L) The mean numbers of <i>T</i>.<i>b</i>. <i>brucei</i> (J), CD4+ (K) and CD8+ (L) cells per mm² in the brain of mice (n = 6 per group) sacrificed 23 dpi is shown (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.001 unpaired Student’s <i>t</i> test).</p

iNOS is expressed by perivascular macrophages in the brain during infection with <i>T</i>.<i>b</i>. <i>brucei</i>.

<p>(A) Concentration of NO₃ in plasma as measured by Griess assay after nitrate reductase reaction. The mean NO₃ concentration ± SEM in the plasma of infected mice (n = 5 per time point) is depicted. Differences with uninfected controls are significant (**p<0.01, ***p<0.001, unpaired Student’s <i>t</i> test). (B) Levels of S-nitrosylated molecules in plasma as measured using the 2,3-diaminonapthalene (DAN) assay. The mean relative fluorescence units (RFU) ± SEM in WT and <i>inos</i>^<i>-/-</i> infected and control animals are indicated. Differences with uninfected control and <i>inos</i>^<i>-/—</i>infected mice are significant (**<i>p</i><0.01, unpaired Student’s t test). (C) The accumulation of <i>inos</i> or <i>hprt</i> transcripts in brains sampled at various dpi with <i>T</i>.<i>b</i>. <i>brucei</i> was measured by real time PCR. The mean fold <i>inos</i> mRNA increase ± SEM in brains from infected mice (n ≥ 5 per group) is depicted. Differences with controls are significant (*<i>p</i><0.05; ***<i>p</i><0.001, unpaired Student’s <i>t</i> test). (D) Levels of S-nitrosylated proteins in brain lysates from <i>T</i>.<i>b</i>. <i>brucei</i> infected mice were measured by the biotin switch assay as described in the supplemental methods (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005442#ppat.1005442.s011" target="_blank">S1 Text</a>). GAPDH was used as a loading control. Similar results were obtained in 3 independent experiments. (E) iNOS labelling in the brain of WT mice at 0 or 30 dpi with <i>T</i>.<i>b</i>. <i>brucei</i>. (F) Immunolabelling with the activated microglia marker Iba-1 in a WT mouse. (G) FACS analysis of CD45^highCD11b⁺ macrophages (R1), CD45^dim CD11b⁺ microglia (R2) and CD45^high CD11b^- lymphocytes (R3) in the brain of WT mice was determined at 0 or 25 dpi. (H) The frequency of iNOS⁺ cells in gated populations (panel G) are depicted. Cells from <i>inos</i>^<i>-/-</i> infected mice were used as negative controls. (I) The mean percentage ± SEM of iNOS⁺ CD45^high CD11b⁺ or CD45^dim CD11b⁺ (n = 4 per group) is depicted. (J) Levels of <i>inos</i> transcripts in sorted CD45^highCD11b⁺, CD45^dimCD11b⁺ and CD45^high CD11b^- populations are shown. Three mice were pooled for each determination and at least 4 independent determinations were performed.</p

iNOS hampers T cell-mediated parasite penetration into the brain parenchyma.

<p>(A, B) Mean body weight and log₁₀ parasites per ml ± SEM of <i>rag1</i>^-/-/ <i>inos</i>^<i>-/-</i> and <i>rag1</i>^<i>-/-</i> mice (n = 9–10) infected with <i>T</i>.<i>b</i>. <i>brucei</i>. (C) Representative immunofluorescence images from the septal nuclei showing <i>T</i>.<i>b</i>. <i>brucei</i> in red and cerebral endothelial cells in green of <i>rag1</i>^<i>-/-</i>, <i>rag1</i>^-/-/ <i>inos</i>^<i>-/-</i> and <i>inos</i>^<i>-/-</i> mice at 22 dpi. (D) Quantification of <i>T</i>.<i>b</i>. <i>brucei</i> invasion in the cerebral regions <i>rag1</i>^<i>-/-</i> mice inoculated or not with 5 x10⁶ CD90⁺ T cells i.v. 7 days before infection with <i>T</i>.<i>b</i>. <i>brucei</i>. The mean number of parasites ± SEM (n = 6 per group) in T cell inoculated and non-transferred controls in one of two independent experiments is depicted. The accumulation of <i>tnf</i> (E), <i>inos</i> (F) and <i>ifng</i> (G) transcripts in infected and T cell-transferred <i>rag1</i>^<i>-/-</i> mice and controls at 23 dpi. The mean fold of mRNA increase ± SEM in brains from infected mice (n ≥ 5 per group) was calculated. Differences with controls are significant (*p<0.05 Student’s t test). (H) RNA was extracted from FACS sorted from macrophage-, microglia- and T cell-enriched brain populations from <i>T</i>. <i>brucei</i>-infected and control mice as described in materials and methods. The mean fold <i>ifng</i> mRNA increase ± SEM of 4 independent pools per group is depicted. (I) The mean fold increase of <i>ifng</i> mRNA ± SEM in RNA from brains from infected WT or <i>inos</i>^<i>-/-</i> and uninfected mice (n ≥ 4 per group) was measured. Differences with controls are significant (***p<0.001 Student’s t test).</p

iNOS protects against TNF-mediated penetration of <i>T</i>.<i>b</i>. <i>brucei</i> into the brain.

<p>(A, B) Mean body weight and log₁₀ parasites per ml of <i>inos</i>^<i>-/-</i>/ <i>tnfr1</i>^<i>-/-</i>, <i>tnfr1</i>^<i>-/-</i>, <i>inos</i>^<i>-/-</i> and WT mice infected with <i>T</i>.<i>b</i>. <i>brucei</i> ± SEM (n = 10 per group). The body weights are relative to the weight of each group before infection. Differences with infected WT animals are significant (*p<0.05, **<i>p</i>< 0.01 two-way ANOVA). One of two independent experiments is depicted. (C-E) The mean number of <i>T</i>.<i>b</i>. <i>brucei</i> (C), CD4⁺ (D), CD8⁺ T cells (E) cells per mm² in the cerebral regions of mice at 23 dpi ± SEM (n = 6). One out of two independent experiments is depicted. Differences with WT mice at the same dpi are significant (*p< 0.05, **p< 0.01, ***<i>p</i><0.001 unpaired Student’s <i>t</i> test). (F) IgG was detected by Western blot in brain lysates of <i>tnfr1</i>^<i>-/-</i>, <i>inos</i>^<i>-/-</i> <i>/tnfr1</i>^<i>-/-</i>and <i>inos</i>^<i>-/-</i> mice 23 dpi with <i>T</i>.<i>b</i>. <i>brucei</i>. (G) Accumulation of <i>inos</i> mRNA increase ± SEM in brains from WT and <i>tnfr1</i>^<i>-/-</i> mice at 23 dpi (n ≥ 5 per group) was calculated. Differences with controls are significant (**p<0.01 Student’s <i>t</i> test). (H) The concentration of NO₂ was measured in the 24 h supernatants of LPS-stimulated WT and <i>tnfr1</i>^-/- BMM using a Griess assay. The mean NO₂ levels ± SEM in triplicate cultures per condition are depicted. Differences with WT control are significant (***p<0.001 Student’s <i>t</i> test).</p