The research was approved by the Ethics Committee on Animal Experiments of Ribeirão Preto Medical School (CETEA-107-2009) and the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School (IACUC-2371-12). The study on human samples was approved by the Human Research Ethics Committee at Hospital das Clínicas of Faculdade de Medicina da Universidade de São Paulo (research protocol n°. 536/2008). After obtaining written informed consent from all of the patient's relatives, venous blood samples were collected.

Female C57BL/6 mice were used. The low-virulent ME-49 strain of T. gondii was harvested from the brains of infected mice (Benevides et al., 2008). Since our major aim was to address sepsis susceptibility in chronically infected mice, we only used the ME-49 strain that allows mice to progress to chronic phase of infection.

Sepsis was induced using a cecal ligation and puncture (CLP) model. Two punctures with sterile 21-G needles were used to standardize the sub-lethal sepsis (Benjamim et al., 2003; Nascimento et al., 2010).

The quantification of the bacterial load in the blood and peritoneal exudates was performed at 6, 12, and 24 h after CLP. For these analyses, the animals were anesthetized, and blood was collected via cardiac puncture, following which the animals were euthanized in a CO₂ chamber. The peritoneal exudates were collected via an injection of 1.5 mL of PBS/EDTA into the peritoneal cavity. After the sample collection, 10 μL of blood or peritoneal wash without dilutions were plated on Mueller-Hinton agar (Difco Laboratories, Detroit, MI, US) and incubated at 37°C under aerobic conditions for 24 h. Colony-forming units (CFUs) were expressed as Log2 of CFU/10 μL of blood or peritoneal wash. All procedures were performed under sterile conditions (Nascimento et al., 2010). Leukocyte migration and differential count were assessed 6, 12, and 24 h after the induction of CLP. The cells present in the peritoneal cavity were harvested via washing of the peritoneal cavity using 1.5 ml of phosphate buffered saline (PBS) containing EDTA (1 mM). The total leukocyte counts were obtained with a cell counter (Coulter AC T series analyser, Coulter, Miami, FL), and the differential count was performed using flow cytometry (BD Immunocytometry System, Franklin Lakes, NJ, USA). The results were expressed as the number of total leukocytes, neutrophils or lymphocytes in the peritoneal cavity (Alves-Filho et al., 2006).

For pathological analyses, the gut was washed to remove the intestinal contents, and the ileum fragment was individually wrapped in a “Swiss roll,” fixed in 10% formalin, embedded in paraffin and processed routinely for haematoxylin and eosin staining. Slides were imaged using light microscopy. The images were acquired with a digital camera (Leica DC300F, Switzerland) coupled to a microscope for histological analysis.

All antibodies used for flow cytometry were purchased from BD Biosciences or eBiosciences and used according to the manufacturer's instructions. For neutrophil staining, Ly6G (FITC, 1A8), CD11c (APC-Cy7, N418), CD11b (PE Cy7, M170) and F4/80 (Percp Cy5, BM8) were used. For intracellular cytokine staining, CD3 (FITC, 145-2C11), CD4 (PercP, RM4-5), CD8 (PE Cy7, 53-6.7), IFN- γ (APC, XMG1.2) and TNF-α (PE, MP6X722) were used. For transcription factors, CD3 (FITC, 145-2C11), CD4 (APC Cy7, RM4-5), CD44 (PercP, IM7) and Tbet (Alexa 647, 4B10) or CD3 (PE, 145-2C11), CD4 (APC Cy7, RM4-5), CD44 (PercP, IM7), RorγT (APC, Q31-378) and Gata3 (Alexa 488, L50-823) were used. For memory cells, CD3 (PE, 145-2C11), CD4 (APC Cy7, RM4-5), CCR7 (Alexa 647, 3D12) and CD62L (FITC, MEL-14) were used. Briefly, tissue-isolated cells were incubated with monoclonal antibodies in buffer containing blocking antibody. For intracellular staining, the cells were harvested and plated with PMA-ionomycin and Brefeldin A (Golgi plug BD bioscience), and stained for flow cytometry analysis. After incubation, cells were fixed and permeabilized with the BD Cytofix/Cytoperm kit (BD Biosciences and eBiosciences, CA, USA). Cell acquisition was performed on the BD CANTO II cell analyser (BD Biosciences) using FACSDiva software (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR).

The levels of mouse TNF-α, IFN-γ, IL-6, and KC were measured using DuoSet ELISA kits (R&D Systems).

Chronic dextran sodium sulfate (DSS) colitis was induced by administering 3% (w/v−1) DSS (molecular mass, 36–50 kDa; MP Biomedicals, OH) for 14 days, followed by 26 days of normal drinking water.

The cytokine IFN-γ was neutralized by intraperitoneal (i.p.) injection of 10 μg of purified mAb 24 h before CLP. The mouse anti-IFN-γ mAb was purified from the ascites of mice injected with an anti-IFN-γ hybridoma (XMG1.2). Controls received 500 μg of normal rat IgG diluted in PBS. The IgG was isolated from naïve rats using protein G column purification.

The broad-spectrum antibiotic treatment regimen was followed as described by Hand et al. (2012) with modifications. Briefly, each animal received a daily combination of 5 mg neomycin trisulfate (Sigma), 2.5 mg vancomycin (Sigma), 5 mg metronidazole (sigma) and 5 mg ampicillin sodium (sigma) diluted in 200 μL water via oral gavage for 2 weeks before and 2 weeks post-infection with T. gondii.

For cell isolation, CD4⁺ T cells were negatively selected using the EasySep kit (StemCell Technologies, Canada), and DNA extraction was subsequently performed. The epigenetic analysis was performed using the PCR kit EpiTect Methyl II PCR Arrays (Qiagen Sciences, USA) to determine the methylated CpG islands and indicate the percentage of methylated DNA. The DNA in each individual enzymatic reaction was quantified using real-time PCR with primers that flanked the promoter region of interest as follows: tbx21- Chr11, CpG-start 96975880, CpG-end 96976654; eomes- Chr9, CpG-start 1183859814, CpG-end 118386331; gata3- Chr2, CpG-start 9802719, CpG-end 9803061 and rora- Chr9, CpG-start 68501352, CpG-end 68502769.

Arterial blood pressure was non-invasively measured by determining the tail blood volume with a pressure recording sensor and an occlusion tail cuff (CODA System, Kent Scientific, CT) 24 h after CLP. The results were expressed in millimeters of mercury (mmHg).

The patients' blood was collected in the first 24 h after admission. The samples were processed and analyzed for cytokine production and Toxoplasma gondii serology. The levels of the cytokine IFN-γ were measured using DuoSet ELISA as indicated by the manufacturer's instructions (R&D Systems). The Toxoplasma gondii serology was performed by indirect immunofluorescence assay as indicated by the manufacturer's instructions (FLUOCON IgG/IgM WAMA, Belgium).

The animal survival was expressed as the percentage of surviving animals analyzed using the Mantel-Cox log-rank test (X₂, chi-squared). For comparison of multiple parametric data, the variance (ANOVA) tests were used followed by the post-hoc Tukey-Kramer test. Data are expressed as the means ± standard error of the mean (SEM). Statistical analysis and graphics were performed using the GraphPad Prism version 5.0 (GraphPad Software, San Diego, CA, USA).

Mice chronically infected with T. gondii for 40 days were subjected to sublethal sepsis induced by CLP surgery (coinfected mice). Herein, we observed increased mortality of coinfected mice compared to sublethal CLP-subjected mice (Figure 1A). These data indicate that chronic T. gondii infection aggravated polymicrobial sepsis, which was not due to toxoplasmosis reactivation (Figures S1A,B).

To investigate whether coinfected mice were able to control bacterial proliferation, the bacterial burden was quantified in the blood and peritoneum. We found that 24 h after CLP, coinfected mice were more efficient in controlling bacterial replication both systemically (Figure 1B) and locally (Figure 1C) compared to septic mice. Moreover, we counted the number of leukocytes recovered from the peritoneal cavity after CLP. The total leukocyte count strikingly decreased in the blood of coinfected mice (data not shown); however, the leukocytes increased in the peritoneum of coinfected mice compared to septic mice (Figure 1D). The neutrophil recruitment to the peritoneal cavity was increased in coinfected mice compared to CLP-subjected mice (Figure 1E), which paralleled the bacterial clearance. We also observed increased migration of lymphocytes in mice 12 h after CLP (Figure 1F). To investigate whether intense cellular recruitment to the peritoneal cavity reflects intestinal inflammation and tissue damage, we evaluated the histological features in the intestine 24 h after sepsis induction. We found extensive neutrophil infiltrate mainly in the ileum of coinfected mice along with decreased mRNA expression of occludin, a tight junction protein (Figures S2A,B), and massive intestinal tissue damage (Figure 1G). These data suggest that chronic T. gondii infection promotes a microenvironment in the intestine that favors the inflammatory response during CLP. Notably, we did not find evidence of vital organ failure in coinfected mice, which was evaluated by either histopathological analysis or biochemical biomarkers for renal, hepatic, cardiac, and muscular dysfunctions.

The major factor leading to host susceptibility during sepsis is the increased production of inflammatory cytokines, which can promote septic shock (Ebong et al., 1999). To evaluate whether the high mortality of coinfected mice mirrored the production of inflammatory mediators, we quantified the cytokines in the serum and peritoneal lavage 24 h after CLP. Notably, coinfected mice showed increased levels of IFN-γ (Figures 2A,E), TNF-α (Figures 2B,F), IL-6 (Figures 2C,G), and KC (Figures 2D,H) in both the serum and peritoneal lavage compared to the control groups. For the immuneregulatory cytokines, IL-4 was not detected and the levels of IL-10 in the peritoneal lavage were similar in all groups studied. Splenic CD4⁺ and CD8⁺ T cells of T. gondii-infected mice produced elevated levels of IFN-γ and TNF-α even in the absence of a secondary stimulus (Figures 3A–D). Although the size of the spleen from T. gondii-infected mice was similar to naïve mice, after the induction of sepsis, T. gondii-infected mice experienced a notable reduction in spleen size along with a striking emergence of IFN-γ- and TNF-α-producing CD4⁺ (Figures 3E–G) and CD8⁺ (Figures 3H–J) T cells into the peritoneum. These data suggest that inflammatory cells were leaving the spleen/bloodstream and reaching the peritoneal cavity. Thus, T. gondii-programmed CD4⁺ and CD8⁺ T cells may be recruited to the site of sepsis and are the primary source of inflammatory mediators during sepsis in infected mice.

To further gain insights into the mechanisms by which chronic T. gondii infection aggravates sepsis, we assessed whether chronic infection maintained a pool of long-lived lymphocytes that act as first responders in polymicrobial sepsis. As expected, chronic T. gondii infection maintained a pool of CD4⁺ and CD8⁺ T cells (CD4⁺CD44⁺ and CD8⁺CD44⁺ T cells, respectively) independent of sepsis (Figures S3A,B), thus confirming that such cells were activated by T. gondii. Indeed, chronic infection deeply induced the transcription of IFN-γ-related genes, which were intensified during sepsis development (Figure S3C). As long-lived T cells comprise a pool of central and effector memory cells (Wherry et al., 2003), we explored whether T. gondii exposure maintained a pool of memory T cells during its chronic phase. Chronic T. gondii infection induced an increased number of CD4⁺CD44^highCD62L⁺CCR7⁺ T lymphocytes, named here as central memory-like T lymphocytes (Figures 4A,B) that were converted to effector memory-like (CD4⁺CD44^highCD62L⁻CCR7⁻) T lymphocytes after CLP surgery (Figures 4C,D). Herein, sepsis induction reactivated long-lived T. gondii-experienced T cells to produce IFN-γ and TNF-α in both the mesenteric lymph nodes (Figures 4E,G) and peritoneum (Figures 4F,H). To evaluate the involvement of microbiota in promoting long-lived T cells, we induced colitis with a classical intestinal stressor dextran sodium sulfate (DSS) to promote bacterial translocation followed by CLP. Surprisingly, DSS-induced bacterial translocation was unable to induce central and effector memory-like T lymphocytes (Figures 4A–D) and IFN-γ and TNF-ɑ production after sepsis induction (Figures 4E–H). These results rule out a potential role of the microbiota in generating deleterious long-lived T cells that cross-react against released bacteria via CLP.

T. gondii-primed CD4⁺ T lymphocytes are polarized toward a Th1 profile (Gazzinelli et al., 1994), and recently, it was demonstrated that IFN-γ suppresses permissive chromatin remodeling in the regulatory region of the Il4 gene (Nishida et al., 2013). To test whether the microenvironment elicited by chronic toxoplasmosis could promote epigenetic reprogramming of lymphocytes, we purified splenic CD4⁺ T cells and investigated the repressive methylation status of their transcription factors. During the chronic phase of T. gondii infection, Gata3 was found significantly methylated compared to the naïve CD4⁺ T splenic cells (Figure 5A). Additionally, repressive methylation of Th17- (Rora) and Th1-associated transcription factors (Tbx21 and Eomes) were undetectable in chronically infected mice (Figure 5A). The epigenetic changes observed indicate that Th1-induced expression can be due to Gata3 repression. This provides further evidence for our hypothesis that Gata3 repression during T. gondii infection drives the immune response toward a Th1 pattern. To characterize whether CD4⁺ T lymphocytes are driven to long-lived Th1 lymphocytes during the chronic infection, we analyzed the transcription factors expressed by the Th1 lymphocytes (Tbet). We observed that the T. gondii-induced inflammatory milieu reprogrammed naïve CD4⁺ T lymphocytes into long-lived Th1 lymphocytes independent of CLP (Figure 5B). In contrast, T. gondii-primed T cells did not express significant amounts of the transcription factors Rorγt and Gata3 with or without a secondary infection, which excludes the role of long-lived Th17 and Th2 cells in our model.

Nitric oxide (NO) production and the subsequent cytokine storm induced during sepsis are major inductors of hypotension and septic shock (Bone et al., 1997). Indeed, we detected increased NO production in coinfected mice compared to CLP-subjected mice (Figure S4). Since coinfected mice also had increased inflammatory cytokines, mainly IFN-γ, we explored whether chronic T. gondii infection could aggravate sepsis by reducing systolic and diastolic arterial blood pressures. Only coinfected mice developed hypotension through the reduction of systolic and diastolic blood pressures (Figure 5C). Moreover, the partial blockage of IFN-γ prevented hypotension in these mice (Figure 5C).

Recent data have revealed that T. gondii infection promotes long-lived IFN-γ-producing microbiota-specific CD4⁺ T cells (Hand et al., 2012). To verify a possible role of the microbiota in controlling arterial blood pressure of coinfected mice, we treated them with broad-spectrum antibiotics to prevent bacterial translocation during the acute phase of T. gondii infection. We found that depletion of the microbiota did not prevent cytokine production and arterial hypotension (Figure 5C). These observations reveal that chronic T. gondii infection intensifies the plethora of cytokines during sepsis and predisposes the host to septic shock. Since the blockade of IFN-γ prevented host hypotension, we evaluated whether treatment with monoclonal anti-IFN-γ antibody could prevent the susceptibility of coinfected mice. The pretreatment with 10 μg/kg of anti-IFN-γ significantly improved the survival of these mice (Figure 5D).

To determine whether the IFN-γ-mediated mechanism described in coinfected mice is also observed in coinfected humans, we collected the blood of patients with sepsis to test the serology for toxoplasmosis and to measure IFN-γ production. Our data showed that patients with more severe clinical forms of sepsis had increased IFN-γ serum levels (Figures 6A,B), which strongly supports that IFN-γ production contributes to sepsis severity. In addition, patients serologically positive for toxoplasmosis had increased levels of IFN-γ during sepsis compared to serologically negative patients or healthy controls (Figure 6C). Similarly, previous exposure to T. gondii was deleterious to the host because mortality was increased in coinfected patients compared to patients who were serologically negative for toxoplasmosis (Figure 6D). Collectively, our data supports the hypothesis that patients with positive serology for toxoplasmosis are at risk for the development of severe sepsis.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.