Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
, 87 (5)

Transcriptional Analysis Shows a Robust Host Response to Toxoplasma gondii During Early and Late Chronic Infection in Both Male and Female Mice

Affiliations
Comparative Study

Transcriptional Analysis Shows a Robust Host Response to Toxoplasma gondii During Early and Late Chronic Infection in Both Male and Female Mice

Andrew L Garfoot et al. Infect Immun.

Abstract

The long-term host effects caused by the protozoan parasite Toxoplasma gondii are poorly understood. High-throughput RNA sequencing analysis previously determined that the host response in the brain was greater and more complex at 28 days than at 10 days postinfection. Here, we analyzed the host transcriptional profile of age- and sex-matched mice during very early (21 days), early (28 days), mid (3 months), and late (6 months) chronic infection. We found that a majority of the host genes which increase in abundance at day 21 postinfection are still increased at 6 months postinfection for both male and female mice. While most of the differentially expressed genes were similar between sexes, females had far fewer genes that were significantly less abundant, which may have led to the slightly increased cyst burden in males. Transcripts for C-X-C motif chemokine ligand 13 and a C-C motif chemokine receptor 2 (CCR2) were significantly higher in females than in males during infection. As T. gondii chronic infection and profilin (PRF) confer resistance to Listeria monocytogenes infection in a CCR2-dependent manner, the differences in CCR2 expression led us to retest the protection of PRF in both sexes. Male mice were nearly as effective as female mice at reducing the bacterial burden either with a chronic infection or when treated with PRF. These data show that most of the host genes differentially expressed in response to T. gondii infection are similar between males and females. While differences in transcript abundance exist between the sexes, the infection phenotypes tested here did not show significant differences.

Keywords: RNA-seq; Toxoplasma; cerebral cortex; chronic infection; immune response; transcriptome.

Figures

FIG 1
FIG 1
RNA-seq of long-term chronic infection. (A) Timeline of infection representing RNA-seq time points, defining acute (<14 days) and very early (21 days), early (28 days), mid (90 days), and late (180 days) chronic T. gondii infection. Female cerebral cortexes (♀) were harvested 21, 28, 90, and 180 days postinfection, and male cerebral cortexes (♂) were harvested 28 and 180 days postinfection. Numbers represent the number of individual mice used for each group in the RNA-seq analysis. Arrows represent the time points used to compare acute-infection and early chronic-infection transcripts from the previous analysis (21). (B) PCA plot of samples used for RNA-seq analysis. Open symbols represent uninfected samples, and closed symbols represent infected samples. Triangle represent males, and circles represent females. Colors correspond to the time points of infection (red, 21 days; blue, 28 days; green, 90 days; purple, 180 days).
FIG 2
FIG 2
Host genes with greater abundance after infection are shared throughout infection. The numbers of female (A) and male (B) mouse genes with a greater than 2-fold increase from those of age-matched uninfected controls are shown. Shared sets are represented by overlapping regions between the 21-day (red), 28-day (green), 90-day (blue), and 180-day (yellow) time points.
FIG 3
FIG 3
Many enriched host genes are shared throughout infection and between sexes. The numbers of female (A) and male (B) mouse genes with a 20-fold increase from those of uninfected controls are shown. (C) Shared sets are represented by overlapping regions between the 28-day (green) and 180-day (yellow) time points. (D) Heat map of differential expression in infected mice compared to uninfected mice from the RNA-seq data for the 40 mouse genes from panel C that are at least 20-fold more abundant in males and females at 28 and 180 days. Bright blue indicates a 10.0 log2(FC) or 1,024-fold change, and yellow indicates no change, as seen for tubulin (TUBα1A) across the bottom. The six genes without an immune-related GO term are marked with an asterisk.
FIG 4
FIG 4
Males have a greater number of less-abundant genes than females in response to infection. (A) Total numbers of differentially expressed genes with both an FDR value of <0.05 and a 2-fold increase (solid bars) or decrease (slashed bars) compared to those of uninfected controls for females (red) and males (blue) at 28 or 180 days postinfection (DPI). (B) qPCR analysis of one of the most differentially expressed genes with greater abundance (the CD74 gene) and one of the less abundant genes in both males and females (the FCRLS gene), with the tubulin gene as a control. Data from females are in shades of red, and data from males are in shades of blue. Raw data were first normalized to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene before the fold change for infected versus uninfected was calculated.
FIG 5
FIG 5
Immunological gene ontology (GO) terms are highly enriched during infection. (A) GO term enrichment was performed using the 710 genes with 2-fold greater abundance shared among the female mouse time points (red) and the 609 genes with 2-fold greater abundance shared in the male mouse time points (blue). (B) GO term enrichment for the >20-fold more abundant genes using the 92 genes shared among the female mouse time points (red) and the 42 genes shared in the male mouse time points (blue). Terms were sorted based on their q value, and the percentages of enrichment for the top 10 most significant are shown.
FIG 6
FIG 6
Inflammatory cytokines are abundant throughout infection. Serum TNF-α (A), IFN-γ (B), IL-12 (C), IL-6 (D), IL-10 (E), and MCP1 (F) were quantified from the male (blue) and female (red) mice used for the RNA-seq analysis. Time points include 21, 28, 90, and 180 days postinfection. Circles represent cytokines from uninfected mice, and squares represent infected mice. Data are the average concentration for three mice, except for infected and uninfected males at 180 days postinfection and uninfected females at 180 days postinfection, which involved two mice each. Error bars represent the standard deviation between samples, and asterisks indicate statistically significant differences between results for infected and uninfected samples by Student's t test (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (G) Heat map differential expression for infected mice compared to uninfected mice, from the RNA-seq data for the cytokine genes. Bright blue indicates a 5.0 log2(FC) or 32-fold change. Yellow indicates no change, as seen for tubulin (TUBα1A) across the bottom. The genes for the two subunits of IL-12 are shown, as the cytometric bead array quantified the dimeric form of IL-12 (IL-12p70). (H) qPCR analysis of the IFN-γ and TNF-α cytokine genes, with the tubulin gene as a control. Data from females are in shades of red, and data from males are in shades of blue. Raw data were first normalized to the GAPDH housekeeping gene before the fold change for infected versus uninfected mice was calculated.
FIG 7
FIG 7
Outcomes of T. gondii infection are similar in male and female mice. (A) Acute-infection survival curve of male (blue, n = 6) and female (red, n = 5) mice infected with 1 × 105 parasites. (B) Chronic-infection survival curve for male (n = 28) and female (n = 26) mice infected with 1 × 104 parasites. (C) Cyst burden in brains of mice at 3 months of infection (n = 3 male, n = 3 female) and of surviving mice at 6 months postinfection (from panel B; n = 7 for males and n = 3 female). Statistical differences between male and female survival curves were calculated using the Mantel-Cox log rank test, and differences between male and female cyst burdens were determined using Student's t test (*, P < 0.05). N.D., not determined because of inadequate power.
FIG 8
FIG 8
Stimulation with T. gondii confers slightly more resistance to L. monocytogenes in female mice. (A) Heat map of transcript abundance from the RNA-seq data for the 11 genes that are more differentially expressed in females than in males in response to T. gondii infection. Bright blue indicates a 6.0 log2(FC) or 64-fold change. Yellow indicates no change, as seen for tubulin (TUBα1A) across the bottom. (B) qPCR analysis of CXCL13 and CCR2 with tubulin as a control. Data from females are in shades of red, and data from males are in shades of blue. Raw data were first normalized to the GAPDH housekeeping gene before the fold change of infected versus uninfected mice was calculated. (C) L. monocytogenes burden in spleens of male mice infected with T. gondii 28 days prior to L. monocytogenes infection (+, n = 4) compared to that of uninfected male mice (–, n = 4). (D) L. monocytogenes burden in spleens of female (red) and male (blue) mice (n = 4/group) treated intravenously with saline (–) or 10 ng of recombinant T. gondii PRF (+) 4 h prior to L. monocytogenes infection. Horizontal lines represent the mean burden for each group.

Similar articles

See all similar articles

Cited by 2 articles

Publication types

Feedback