Distinct roles of apolipoprotein components within the trypanosome lytic factor complex revealed in a novel transgenic mouse model

Humans express a unique subset of high-density lipoproteins (HDLs) called trypanosome lytic factors (TLFs) that kill many Trypanosoma parasite species. The proteins apolipoprotein (apo) A-I, apoL-I, and haptoglobin-related protein, which are involved in TLF structure and function, were expressed through the introduction of transgenes in mice to explore their physiological roles in vivo. Transgenic expression of human apolipoprotein L-I alone conferred trypanolytic activity in vivo. Coexpression of human apolipoprotein A-I and haptoglobin-related protein (Hpr) had an effect on the integration of apolipoprotein L-I into HDL, and both proteins were required to increase the specific activity of TLF, which was measurable in vitro. Unexpectedly, truncated apolipoprotein L-I devoid of the serum resistance gene interacting domain, which was previously shown to kill human infective trypanosomes, was not trypanolytic in transgenic mice despite being coexpressed with human apolipoprotein A-I and Hpr and incorporated into HDLs. We conclude that all three human apolipoproteins act cooperatively to achieve maximal killing capacity and that truncated apolipoprotein L-I does not function in transgenic animals

Heterogeneity and chimerism of endothelial cells revealed by single-cell transcriptome in orthotopic liver tumors.

The liver is a common host organ for cancer, either through lesions that arise in liver epithelial cells [e.g., hepatocellular carcinoma (HCC)] or as a site of metastasis by tumors arising in other organs (e.g., colorectal cancer). However, the changes that occur in liver stromal cells in response to cancer have not been fully characterized, nor has it been determined whether the different sources of liver cancer induce distinct stromal changes. Here, we performed single-cell profiling of liver stromal cells from mouse models of induced spontaneous liver cancer or implanted colorectal liver metastases, with a focus on tumor endothelial cells (ECs). While ECs in liver tissue adjacent to cancerous lesions (so-called adjacent normal) corresponded to liver zonation phenotypes, their transcriptomes were also clearly altered by the presence of a tumor. In comparison, tumor EC transcriptomes show stronger similarities to venous than sinusoidal ECs. Further, tumor ECs, independent of tumor origin, formed distinct clusters displaying conserved "tip-like" or "stalk-like" characteristics, similar to ECs from subcutaneous tumors. However, they also carried liver-specific signatures found in normal liver ECs, suggesting an influence of the host organ on tumor ECs. Our results document gene expression signatures in ECs in liver cancer and show that the host organ, and not the site of tumor origin (liver versus colorectal), is a primary determinant of EC phenotype. In addition, primarily in tumors, we further defined a cluster of chimeric cells that expressed both myeloid and endothelial cell markers and might play a role in tumor angiogenesis

Trypanosome Lytic Factor, an Antimicrobial High-Density Lipoprotein, Ameliorates Leishmania Infection

Innate immunity is the first line of defense against invading microorganisms. Trypanosome Lytic Factor (TLF) is a minor sub-fraction of human high-density lipoprotein that provides innate immunity by completely protecting humans from infection by most species of African trypanosomes, which belong to the Kinetoplastida order. Herein, we demonstrate the broader protective effects of human TLF, which inhibits intracellular infection by Leishmania, a kinetoplastid that replicates in phagolysosomes of macrophages. We show that TLF accumulates within the parasitophorous vacuole of macrophages in vitro and reduces the number of Leishmania metacyclic promastigotes, but not amastigotes. We do not detect any activation of the macrophages by TLF in the presence or absence of Leishmania, and therefore propose that TLF directly damages the parasite in the acidic parasitophorous vacuole. To investigate the physiological relevance of this observation, we have reconstituted lytic activity in vivo by generating mice that express the two main protein components of TLFs: human apolipoprotein L-I and haptoglobin-related protein. Both proteins are expressed in mice at levels equivalent to those found in humans and circulate within high-density lipoproteins. We find that TLF mice can ameliorate an infection with Leishmania by significantly reducing the pathogen burden. In contrast, TLF mice were not protected against infection by the kinetoplastid Trypanosoma cruzi, which infects many cell types and transiently passes through a phagolysosome. We conclude that TLF not only determines species specificity for African trypanosomes, but can also ameliorate an infection with Leishmania, while having no effect on T. cruzi. We propose that TLFs are a component of the innate immune system that can limit infections by their ability to selectively damage pathogens in phagolysosomes within the reticuloendothelial system

<i>L. major</i> is sensitive to transgenic lytic HDL (TLF) <i>in vivo</i>.

<p>C57BL/6 mice (5 per group) were subjected to hydrodynamic gene delivery with a single plasmid that encodes for either apoL-I, Hpr, or both apoL-I and Hpr (apoL-I∶Hpr), or two plasmids one containing Hpr and one containing apoL-I (apoL-I+Hpr) or vector alone, before subcutaneous infection of the footpad with 1×10⁶ (A,B) or 0.5×10⁶ (C,D) <i>L. major</i> metacyclics. The size of the footpad was measured with a caliper (A,C,D). (A and B) represent the data from the same experiment, in which 19 days post-infection, mice were euthanized and footpad parasites were harvested for enumeration by serial dilution assay (B). The data shown represent the mean±SD of one typical experiment that has been repeated twice. * p<0.05, Mann Whitney test.</p

Lytic HDL does not generate NO nor requires the activation of macrophages to reduce the parasite burden.

<p>BALB/c bone-marrow derived macrophages were infected with <i>L. major</i> metacyclics at a multiplicity of infection of 3∶1, for 2 hours before the addition of bovine or human HDL (1.5 mg/ml) or treated with IFNγ and LPS; NO production in the supernatant was measured after 24 hours (A). Bone marrow derived macrophages from iNOS^−/− (B), gp91phox^−/− (C), or C57BL/6 (D) mice were infected with <i>L. major</i> metacyclics at a multiplicity of infection of 3∶1, for 2 hours, before the addition of human or bovine HDL (1.5 mg/ml). They were co-incubated for 24 hours. The data represent the mean±standard deviation (SD) of duplicate cultures of one typical experiment that has been repeated twice. ** p<0.001, *** p<0.0001 compared to bovine HDL, ANOVA test.</p

TLF surrounds parasites in the PV but is not endocytosed by <i>L. major</i>.

<p>Bone-marrow derived macrophages were infected with GFP-<i>L. major</i> parasites (green) for 2 hours before treatment with prelabelled-Alexa 594-TLF ((10 µg/ml) red) for 2 hours (A) and 24 hours (C). The grey panels are transmission light micrographs of the imaged macrophages. The lack of colocalisation of the two dyes (red and green) is revealed by the 2D cytoflurograms of 25 z-stacks (2 hours, B) and (24 hours, D). Samples were visualized and analyzed with a Leica TCS SP2 AOBS confocal laser scanning microscope.</p

<i>T. cruzi</i> is resistant to transgenic lytic HDL (TLF) <i>in vivo</i>.

<p>Swiss-Webster mice (3 per group) injected with a control plasmid, apoL-I or Hpr plasmid alone, or both apoL-I and Hpr were infected with 1×10⁶<i>T. cruzi</i> trypomasigotes IP three days after introduction of the genes by hydrodynamic gene delivery. The acute phase of the infection was followed by monitoring parasitemia daily. The data shown represent the mean±SD.</p

<i>L. major</i> and <i>L. amazonensis</i> are sensitive to lytic HDL within macrophages.

<p>Macrophages from Swiss-Webster intra-peritoneal cavities were infected with <i>L. major</i> metacyclics (A). BALB/c bone-marrow macrophages were infected with <i>L. amazonensis</i> promastigotes (B). The multiplicity of infection is 3∶1; parasites were incubated with macrophages for 2 hours, before the addition of human or bovine HDL at 1.5 mg/ml or at the indicated concentrations. Infected macrophages were co-incubated for 2, 24, or 72 hours. The data represent the mean±standard deviation (SD) of duplicate cultures of one typical experiment that has been repeated twice. * p<0.05 compared to bovine HDL at equivalent time points, ANOVA test.</p