Toxoplasma gondii chronic infection decreases visceral nociception through peripheral opioid receptor signaling

Abstract

By eliciting immune activation in the digestive tract, intestinal pathogens may perturb gut homeostasis. Some gastrointestinal infections can indeed increase the risk of developing post-infectious irritable bowel syndrome (PI-IBS). Intriguingly, the prevalent foodborne parasite Toxoplasma gondii has not been linked to the development of PI-IBS and the impact of this infection on colon homeostasis remains ill-defined. We show in a mouse model that latent T. gondii decreases visceral nociceptive responses in an opioid signaling-dependent manner. Despite the accumulation of Th1 and cytotoxic T cells in the colon of latently infected mice, the selective invalidation of enkephalin gene in T cells ruled out the involvement of T cell-derived enkephalins in hypoalgesia. These findings provide clues about how this widespread infection durably shapes the gut immune landscape and modifies intestinal physiological parameters. They suggest that in contrast to other gut microbes, T. gondii infection could be negatively associated with abdominal pain.

Fig 1. Toxoplasma gondii chronic infection decreases visceral nociceptive responses in the absence of macroscopic colonic inflammation.

(A) Experimental workflow: C57BL/6 mice were infected with Pru.GFP.GRA6-OVA T. gondii either per os with 10 cysts or by intraperitoneal injection with 200 tachyzoïtes. Experiments were performed between 9 and 11 weeks post-infection. Cartoon was modified from https://openclipart.org/detail/17558/simple-cartoon-mouse. (B) Weight measurements during the course of infection, relative to the initial body weight. Dots represent the median + /- IQR of n = 37 (ni), 22 (ip), or 29 (per os) mice. (C) Parasite burden per µg of tissue DNA measured by qPCR on genomic DNA extracted from brain and colon. Box and whisker plots show median + /- IQR. Data are from 3 pooled independent experiments with each dot representing one mouse. Statistical analysis was performed with Kruskal-Wallis test and subsequent Dunn’s correction for multiple comparisons. (D) Visceromotor response (VMR) to increasing colorectal distension pressure (15 to 60 mm Hg) measured in non-infected (ni) mice or mice infected either per os or ip. Data are shown as mean + /- SEM and correspond to 3 pooled experiments with n = 15 (ni), 6 (per os), and 12 (ip) mice per group. Statistical analysis was performed with Areas Under the Curve (AUC) using a Kruskal-Wallis test with Dunn’s correction for multiple comparisons. (E) Colon length was measured and colon was opened longitudinally to measure thickness. Data are pooled from 2 independent experiments. Statistical analysis was performed using one-way ANOVA. (F) Representative pictures of Hematoxylin/Eosin staining of distal colon section (5 µm thick) from 5 ni vs. 8 ip-infected mice (left pictures) or 5 ni vs. 6 per os-infected mice (right pictures). Scale bar = 200 µm.

Fig 2. Toxoplasma gondii infection induces long-term increase of colonic Th1 CD4+ T cells and cytotoxic CD8+ T cells.

(A-D) Flow cytometry analysis of the indicated cell subsets in the colon of non-infected (ni) vs. mice chronically ip-infected for 70 days (T. gondii). Box and whisker show median + /- IQR. Data are pooled from 2 independent experiments and each dot represents one mouse with a total of 12 (ni) and 11 (T. gondii) mice. Statistical analysis was performed using Mann-Whitney test or unpaired Student’s t test depending on the normality of the values. (A) Box and whisker plots show absolute number of CD45⁺ cells and percentage of immune cell subsets out of total CD45⁺ cells in the colon of mice infected (orange) or not (white) for 70 days. (B-D) Representative FACS plot for each condition with numbers (in blue) indicating the median percentage + /- IQR of positive cells among (B) conventional (Foxp3-) CD4 + T cells, (C) TCRαβ CD8 + T cells and (D) TCRγδ CD8 + T cells. Box and whisker plots show the absolute number (median + /- IQR) of each T cell subset in the colon of non-infected (white) vs infected (orange) mice.

Fig 3. Toxoplasma gondii infection generates Th1 CD4+ and cytotoxic CD8+ resident T cells.

(A-F) FACS plots or Box and whisker plots with median + /- IQR showing absolute number or percentage of cell subsets in the colon of non-infected (ni) or chronically ip-infected mice (T. gondii) for 70 days. Data are pooled from 2 independent experiments and each dot represents one mouse with n = 12 (ni) vs. 11 (T. gondii). Depending on the normality of the dataset, statistical analysis was performed using Mann-Whitney test or unpaired t test when comparing 2 conditions (A and B), or using Kruskal-Wallis test with Dunn’s correction for multiple comparisons (D and F). (A and B) Representative FACS plots for each condition with numbers in blue showing median percentage + /- IQR of CD49a-positive cells among (A) conventional (Foxp3-) CD4+ T cells and (B) TCRαβ+ CD8+ T cells. Corresponding frequencies of each T cell subset are represented with box and whisker plots showing median +/- IQR. (C and D) Representative FACS plot for each condition with numbers in blue indicating the median percentage +/- IQR of cytokine-positive cells out of TCRαβ CD8+ T cells (C) and the absolute number of each subset (D), represented as box and whisker plots showing median +/- IQR. (E and F) Representative FACS plots for each condition with numbers in blue indicating the median percentage +/- IQR of cytokine-positive cells out of conventional (Foxp3-) CD4+ T cells (E) and the absolute number of each subset (F), represented as box and whisker plots showing median +/- IQR.

Fig 4. Decreased visceral nociceptive responses induced by Toxoplasma gondii chronic infection relies on peripheral opioid signaling.

(A) Experimental workflow: C57BL/6 mice were infected with Pru.GFP.GRA6-OVA T. gondii either per os with 10 cysts or by intraperitoneal injection with 200 tachyzoïtes. At 10 weeks post-infection, colorectal distension was performed to assess visceral nociception of each mice 30 min after intraperitoneal injection of naloxone-methiodide or PBS. Cartoon was modified from https://openclipart.org/detail/17558/simple-cartoon-mouse. (B) Visceromotor response (VMR) to increasing colorectal distension pressure (15 to 60 mmHg) was measured in ip-infected mice before (in orange) or after (in red) naloxone methiodide injection. Data are shown as mean +/- SEM and are from 1 experiment with n = 10 mice per group. Statistical analysis was performed using a paired t test with total area under the curve. (C) Visceromotor response (VMR) to increasing colorectal distension pressure (15 to 60 mmHg) was measured in per os infected mice injected with naloxone methiodide or PBS 30 min before the colorectal distension. Data are shown as mean +/- SEM and are from 1 experiment with n = 6 mice per group. Statistical analysis was performed using a Mann-Whitney’s test with total area under the curve.