Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures

Abstract

Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome.

Importance: Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in infection that was reduced at later time points. A similar expression pattern was observed in the parasites. Our analyses provide specific insights into the interplay between human macrophages and Leishmania parasites and constitute an important general resource for the study of how pathogens evade host defenses and modulate the functions of the cell to survive intracellularly.

FIG 1

Simultaneous interrogation of the parasite and host transcriptomes. The figure illustrates the study design, donors, and time points. Monocyte-derived human macrophages were infected with the metacyclic promastigote form of L. major (dark blue) or L. amazonensis (light blue) parasites. Following phagocytosis, metacyclic promastigotes transform into the amastigote form (3 to 10 hpi) and reside in membrane-bound compartments called phagosomes. As amastigotes divide, starting at ~24 hpi, each phagosome matures into a membrane-bound singular (L. major) or communal (L. amazonensis) vacuole inside a macrophage. Samples were collected from infected macrophages, macrophages that were allowed to phagocytose latex beads (red), and uninfected controls over 4 time points spanning from 4 to 72 hpi, as pictured. Total RNA was isolated from each sample and analyzed by RNA-seq. A total of 4 biological replicates were collected from 4 different human donors (H2 to H5; represented in different shades of green). A second collection from two of the donors was used as a technical replicate within the L. major/uninfected data set (H2′ and H3′) and for an L. amazonensis and latex bead experiment that constituted a later addition to the study design. The experimental design is further detailed in Data Set S1 in the supplemental material.

FIG 2

Dynamics and characteristics of M-CSF-induced human macrophage (MΦ) infection with Leishmania parasites. M-CSF-induced human macrophages were infected with either L. major (A) or L. amazonensis (B) for 4 h, washed, and further incubated until 24 hpi, 48 hpi, or 72 hpi. Samples collected at 4, 24, 48, and 72 hpi were subjected to transcriptional profiling by RNA-seq. The number of internalized parasites and percentage of infected cells were determined microscopically. Bars indicate the percentages of trimmed RNA-seq reads that mapped to the respective parasite reference genome. Red lines indicate the number of parasites per 100 macrophages, and cyan lines indicate the percent infected macrophages. The graphs incorporate data from all experiments, ± standard deviations. *, P < 0.05 (Student’s t test to compare each time point to 4-hpi results).

FIG 3

Global expression profiles of human macrophages and Leishmania parasites during infection. A comprehensive PCA was conducted to evaluate the relationships between samples across time points and to visualize sample-sample distances for human macrophages (uninfected controls, Leishmania-infected cells, and bead-containing cells) (A) and the intracellular forms of Leishmania (B and C). Each sphere represents an experimental sample, with increasing size indicating the progression of time points from 4 to 72 hpi. Sphere colors indicate sample type (shown in color key). In panel A, the clouds highlight the separation of the Leishmania-infected cluster (blue) from the uninfected/bead-containing cluster (orange). All analyses were performed after filtering out nonexpressed or lowly expressed genes, performing quantile normalization, and including experimental batch as a covariate in the statistical model. PCA plots showing the effects of batch adjustment in macrophage and parasite samples are shown in Fig. S1 and Fig. S2 in the supplemental material.

FIG 4

Differentially expressed genes in macrophages infected with Leishmania species and between parasite developmental stages. (A and B) The numbers of DE genes in L. major-infected macrophages (A) and L. amazonensis-infected macrophages (B) relative to uninfected controls both before and after accounting for the phagocytosis effect (top panels and bottom panels, respectively), depicted as horizontal bar plots for 4, 24, 48, and 72 hpi. (C and D) The numbers of parasite genes that were DE between stages/time points were also determined for macrophages infected with L. major (C) or L. amazonensis (D). The box width depicts the number of DE genes downregulated (left; purple) and upregulated (right; blue) at an adjusted P value of <0.05, with the total number of down- and upregulated genes shown. The color shading indicates the proportion of genes with >4-fold differential expression (dark), between 2- and 4-fold differential expression (medium), or 2-fold differential expression (light). The complete lists of DE genes are provided in Data Sets S2 and S5 in the supplemental material.

FIG 5

Responses of human and murine macrophages to L. major infection at 4 hpi after accounting for the phagocytosis effect. The differential expression profile of L. major-infected human macrophages was compared to that of L. major-infected murine macrophages collected at the same time point (40). Orthology mapping to mouse was done for the 3,273 human genes identified as differentially expressed at 4 hpi, after accounting for the phagocytosis effect, and the results were compared to the murine expression data set. A scatterplot showing the relationship between fold changes (log₂ transformed) in mice (x axis) and humans (y axis) is shown, with each human-mouse gene pair represented by a single point. Some genes duplicates were introduced by the orthology mapping process. Points in gray represent genes in the human data set with an ortholog that was not significantly DE in the murine data set. Points in shades of red represent genes that were significantly DE in both data sets, with those showing <2-fold difference (log₂ fold change, <1) in either/both host system(s) shown in pink and those with >2-fold difference (log₂ fold change, >1) in both host systems shown in red. The numbers of unique genes represented by the red and pink points are indicated for each quadrant. The complete list of genes that were DE in both systems is provided in Data Set S4 in the supplemental material.

FIG 6

Comparison of Leishmania spp. transcriptomes upon infection of human or murine macrophages. Scatterplots show the relationship between fold changes (log₂ transformed) for L. amazonensis (x axis) and L. major (y axis) orthologs for the metacyclic promastigote to 4-hpi intracellular transition (A) and the 4- to 24-hpi intracellular transition (B) within human macrophages. The lists of orthologs and their corresponding fold changes are provided in Data Set S6 in the supplemental material Scatterplots were also used to show a comparison of L. major parasite expression patterns in human macrophages with similar data sets produced using a murine system (40). The relationship between parasite gene fold changes (log₂) in the mouse system (x axis) and the human system (y axis) are shown for (C) the metacyclic promastigote to 4-hpi intracellular transition (C) and the 4- to 24-hpi intracellular transition (D). The lists of genes in the comparisons are provided in Data Set S7. Points in gray represent genes that were not significantly DE by L. major in either host system; points shown in shades of purple represent genes that were DE in the same direction in both systems; points shown in shades of green represent genes that were DE in different directions between the systems; points shown in shades of orange or blue represent genes significant in only one system. The numbers of DE genes corresponding to each color are included in parentheses below the graphs. NS, not significantly DE.