Chronic Toxoplasma gondii Infection Exacerbates Secondary Polymicrobial Sepsis

Abstract

Sepsis is a severe syndrome that arises when the host response to an insult is exacerbated, leading to organ failure and frequently to death. How a chronic infection that causes a prolonged Th1 expansion affects the course of sepsis is unknown. In this study, we showed that mice chronically infected with Toxoplasma gondii were more susceptible to sepsis induced by cecal ligation and puncture (CLP). Although T. gondii-infected mice exhibited efficient control of the bacterial burden, they showed increased mortality compared to the control groups. Mechanistically, chronic T. gondii infection induces the suppression of Th2 lymphocytes via Gata3-repressive methylation and simultaneously induces long-lived IFN-γ-producing CD4⁺ T lymphocytes, which promotes systemic inflammation that is harmful during CLP. Chronic T. gondii infection intensifies local and systemic Th1 cytokines as well as nitric oxide production, which reduces systolic and diastolic arterial blood pressures after sepsis induction, thus predisposing the host to septic shock. Blockade of IFN-γ prevented arterial hypotension and prolonged the host lifespan by reducing the cytokine storm. Interestingly, these data mirrored our observation in septic patients, in which sepsis severity was positively correlated to increased levels of IFN-γ in patients who were serologically positive for T. gondii. Collectively, these data demonstrated that chronic infection with T. gondii is a critical factor for sepsis severity that needs to be considered when designing strategies to prevent and control the outcome of this devastating disease.

Keywords: Toxoplasma gondii; chronic disease; coinfection; sepsis; septic shock.

Figure 1

Chronic T. gondii infection aggravates polymicrobial sepsis. Forty days after T. gondii infection, C57BL/6 mice were subjected to CLP for different analyses. The survival rate was evaluated until the 10th day post-CLP induction (A). These data are representative of three independent experiments (n = 10), and the statistical analysis was determined using the Mantel-Cox log-rank test. The bacterial load was analyzed in the blood (B) and peritoneal lavage (C) at 6, 12, or 24 h post-CLP induction. The results are expressed as the log of the colony-forming units (CFU) per microliter. The leukocytes from the peritoneum (D) were stained with May-Grünwald Giemsa, and the number of neutrophils (E) and lymphocytes (F) were determined using a cell counter. Data are presented as the means ± SEM for 4 animals per group in four independent experiments (ANOVA, followed by Tukey's test; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001). At 24 h post-CLP, histopathological features of the ileum were analyzed after staining with haematoxylin and eosin (H&E). The results are representative of three independent experiments using 4 mice per group (G).

Figure 2

The T. gondii-primed immune response promotes a storm of cytokines associated with CLP. C57BL/6 mice were infected with 5 cysts of T. gondii, and 40 days after infection, the mice were subjected to CLP. Twenty-four hours after CLP, the serum and peritoneal lavage were collected for cytokine analysis. The levels of pro-inflammatory cytokines IFN-γ (A,E), TNF-ɑ (B,F), IL-6 (C,G), and the chemokine KC (D,H) were determined using ELISA. Data are presented as the means ± SEM for 4 animals per group in three different experiments (ANOVA followed by Tukey's test; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001).

Figure 3

The T. gondii-experienced CD4+ and CD8+ T cells are programmed to produce inflammatory mediators during sepsis. Naïve or chronically T. gondii-infected mice were subjected to CLP to evaluate the frequencies of IFN-γ- or TNF-α-producing CD4⁺ and CD8⁺ T cells in the spleen (A–D). Peritoneal leukocytes were recovered from the peritoneal lavage 24 h after CLP, then stimulated with PMA-ionomycin, and stained for flow cytometry analysis (E–J). Data are presented as the means ± SEM for 4 animals in three different experiments. Statistical analysis was performed using ANOVA followed by Tukey's test; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 4

T. gondii-infected mice induce long-lived CD4⁺ T cells that are reactivated during sepsis. C57BL/6 mice were infected with 5 cysts of T. gondii or treated with 3% dextran sodium sulfate (DSS) to induce colitis. After 40 days of T. gondii infection or DSS treatment, the animals were subjected to CLP. The frequency of central memory-like (CD4⁺CD44⁺CD62L⁺ or CCR7⁺) (A,B) and effector memory-like (CD4⁺CD44⁺CD62L⁻ or CCR7⁻) (C,D) T cells was quantified in the mesenteric lymph nodes of control, coinfected, and colitis-induced mice. IFN-γ- or TNF-α-producing CD4⁺ T cells were recovered from the mesenteric lymph nodes (E,G) or peritoneal lavage and quantified (F,H). Data are presented as the means ± SEM for 4 animals in three different experiments. The lymphocytes were analyzed using flow cytometry, and statistical analysis was performed using ANOVA followed by Tukey's test; ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.

Figure 5

Treatment with anti-IFN-γ prevents hypotension and ameliorates host survival. For the methylation analysis, CD4⁺ T cells were separated from spleen cells by negative selection for DNA extraction. The pattern of DNA methylation was determined using the EpiTect Methyl II PCR Arrays kit. This analysis was performed in two independent trials, and the variation rate ≥ 10% was considered significant (A). The expression of the transcription factors T-bet, Gata-3 and RorγT was determined in CD44^highCD4⁺ T cells using flow cytometry (B). For blood pressure analysis, a group of coinfected mice was pretreated with broad-spectrum antibiotics (ATB) 2 weeks before and after T. gondii infection, and another group was treated with anti-IFN-γ antibodies 24 h before CLP induction. After 40 days of T. gondii infection, the mice were subjected to CLP, and at 24 h post-CLP, the blood pressure (mmHg) was evaluated in the animal tail using a sphygmomanometer (C). The bars represent the means ± SEM for 4 animals per group (^*p < 0.05). The survival assay was monitored for 10 days after treatment with a 10 μg/kg dose of anti-IFN-γ antibodies (D). The results are expressed as a percentage of survival, and the p-value was considered when comparing chronic T. gondii infected/CLP mice who received antibody treatment vs. animals that were not treated.

Figure 6

Patients exposed to T. gondii have increased INF-γ during sepsis. The serum concentrations of IFN-γ in septic patients or healthy individuals (healthy = 7, sepsis = 2, severe sepsis = 15, and septic shock = 11) were determined using ELISA (A–C). The data shown are the mean values of individual subjects from triplicate experiments (A–C). The statistical analysis was performed using ANOVA followed by Tukey's test; ^*p < 0.05. Linear regression analysis of the means of IFN-γ from septic patients in relationship to changes in the Sepsis-related Organ Failure Assessment (SOFA) scores are shown (r² = 0.2484; p < 0.0112) (B). The number of non-survivors is presented as a percentage (D).