Oral Route Driven Acute Trypanosoma cruzi Infection Unravels an IL-6 Dependent Hemostatic Derangement

Oral transmission of Trypanosoma cruzi, the etiologic agent of Chagas disease, is presently the most important route of infection in Brazilian Amazon. Other South American countries have also reported outbreaks of acute Chagas disease associated with food consumption. A conspicuous feature of this route of transmission is presenting symptoms such as facial and lower limbs edema, in some cases bleeding manifestations and risk of thromboembolism are evident. Notwithstanding, studies that address this route of infection are largely lacking regarding its pathogenesis and, more specifically, the crosstalk between immune and hemostatic systems. Here, BALB/c mice were orally infected with metacyclic trypomastigotes of T. cruzi Tulahuén strain and used to evaluate the cytokine response, primary and secondary hemostasis during acute T. cruzi infection. When compared with control uninfected animals, orally infected mice presented higher pro-inflammatory cytokine (TNF-α, IFN-γ, and IL-6) serum levels. The highest concentrations were obtained concomitantly to the increase of parasitemia, between 14 and 28 days post-infection (dpi). Blood counts in the oral infected group revealed concomitant leukocytosis and thrombocytopenia, the latter resulting in increased bleeding at 21 dpi. Hematological changes paralleled with prolonged activated partial thromboplastin time, Factor VIII consumption and increased D-dimer levels, suggest that oral T. cruzi infection relies on disseminated intravascular coagulation. Remarkably, blockade of the IL-6 receptor blunted hematological abnormalities, revealing a critical role of IL-6 in the course of oral infection. These results unravel that acute T. cruzi oral infection results in significant alterations in the hemostatic system and indicates the relevance of the crosstalk between inflammation and hemostasis in this parasitic disease

Oral Route Driven Acute Trypanosoma cruzi Infection Unravels an IL-6 Dependent Hemostatic Derangement

Submitted by Sandra Infurna ([email protected]) on 2019-07-04T15:19:44Z No. of bitstreams: 1 WilsonSavino_DinaAntunes_etal_IOC_2019.pdf: 2958086 bytes, checksum: 06fc5810e00b55cc4b92335c1120548b (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2019-07-04T15:30:53Z (GMT) No. of bitstreams: 1 WilsonSavino_DinaAntunes_etal_IOC_2019.pdf: 2958086 bytes, checksum: 06fc5810e00b55cc4b92335c1120548b (MD5)Made available in DSpace on 2019-07-04T15:30:53Z (GMT). No. of bitstreams: 1 WilsonSavino_DinaAntunes_etal_IOC_2019.pdf: 2958086 bytes, checksum: 06fc5810e00b55cc4b92335c1120548b (MD5) Previous issue date: 2019Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Instituto Nacional de CIência e Tecnologia em NeuroimunoModulação. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Instituto Nacional de CIência e Tecnologia em NeuroimunoModulação. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Instituto Nacional de CIência e Tecnologia em NeuroimunoModulação. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Instituto Nacional de CIência e Tecnologia em NeuroimunoModulação. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Doenças Parasitárias. Rio de Janeiro, RJ. Brasil.undação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Instituto Nacional de CIência e Tecnologia em NeuroimunoModulação. Rio de Janeiro, RJ, Brasil.undação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Instituto Nacional de CIência e Tecnologia em NeuroimunoModulação. Rio de Janeiro, RJ, Brasil.Universidade Federal do Rio de Janeiro. Instituto de Bioquímica Médica Leopoldo de Meis. Rio de Janeiro, RJ, Brasil.undação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil / Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Instituto Nacional de CIência e Tecnologia em NeuroimunoModulação. Rio de Janeiro, RJ, Brasil.Oral transmission of Trypanosoma cruzi, the etiologic agent of Chagas disease, is presently the most important route of infection in Brazilian Amazon. Other South American countries have also reported outbreaks of acute Chagas disease associated with food consumption. A conspicuous feature of this route of transmission is presenting symptoms such as facial and lower limbs edema, in some cases bleeding manifestations and risk of thromboembolism are evident. Notwithstanding, studies that address this route of infection are largely lacking regarding its pathogenesis and, more specifically, the crosstalk between immune and hemostatic systems. Here, BALB/c mice were orally infected with metacyclic trypomastigotes of T. cruzi Tulahuén strain and used to evaluate the cytokine response, primary and secondary hemostasis during acute T. cruzi infection. When compared with control uninfected animals, orally infected mice presented higher pro-inflammatory cytokine (TNF-α, IFN-γ, and IL-6) serum levels. The highest concentrations were obtained concomitantly to the increase of parasitemia, between 14 and 28 days post-infection (dpi). Blood counts in the oral infected group revealed concomitant leukocytosis and thrombocytopenia, the latter resulting in increased bleeding at 21 dpi. Hematological changes paralleled with prolonged activated partial thromboplastin time, Factor VIII consumption and increased D-dimer levels, suggest that oral T. cruzi infection relies on disseminated intravascular coagulation. Remarkably, blockade of the IL-6 receptor blunted hematological abnormalities, revealing a critical role of IL-6 in the course of oral infection. These results unravel that acute T. cruzi oral infection results in significant alterations in the hemostatic system and indicates the relevance of the crosstalk between inflammation and hemostasis in this parasitic disease

Unraveling Chagas disease transmission through the oral route: Gateways to Trypanosoma cruzi infection and target tissues

Submitted by Sandra Infurna ([email protected]) on 2017-07-20T11:15:17Z No. of bitstreams: 1 dea_Villaverde_etal_IOC_2017.pdf: 4095153 bytes, checksum: 9d778615a28eca5e7d9aecc418adb476 (MD5)Approved for entry into archive by Sandra Infurna ([email protected]) on 2017-07-20T11:38:43Z (GMT) No. of bitstreams: 1 dea_Villaverde_etal_IOC_2017.pdf: 4095153 bytes, checksum: 9d778615a28eca5e7d9aecc418adb476 (MD5)Made available in DSpace on 2017-07-20T11:38:43Z (GMT). No. of bitstreams: 1 dea_Villaverde_etal_IOC_2017.pdf: 4095153 bytes, checksum: 9d778615a28eca5e7d9aecc418adb476 (MD5) Previous issue date: 2017Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Centro Nacional de Biologia Estrutural e Bioimagem. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Biologia Molecular e Doenças Endêmicas. Rio de Janeiro, RJ. Brasil.Universidade Federal do Rio de Janeiro. Instituto de Microbiologia Paulo de Góes. Departamento de Imunologia. Rio de Janeiro, RJ, BrasilFundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Inovações em Terapias, Ensino e Bioprodutos. Rio de Janeiro, RJ, Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Laboratório de Pesquisa sobre o Timo. Rio de Janeiro, RJ. Brasil.Oral transmission of Trypanosoma cruzi, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of T. cruzi Dm28c luciferase (Dm28c-luc) parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. In vivo bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi), luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. Ex vivo analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP T. cruzi were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant T. cruzi detection and expansion in mouse nasal cavity. Moreover, T. cruzi DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral T. cruzi infection leading to parasite multiplication at the nasal cavity

Unraveling Chagas disease transmission through the oral route: Gateways to <i>Trypanosoma cruzi</i> infection and target tissues

<div><p>Oral transmission of <i>Trypanosoma cruzi</i>, the causative agent of Chagas disease, is the most important route of infection in Brazilian Amazon and Venezuela. Other South American countries have also reported outbreaks associated with food consumption. A recent study showed the importance of parasite contact with oral cavity to induce a highly severe acute disease in mice. However, it remains uncertain the primary site of parasite entry and multiplication due to an oral infection. Here, we evaluated the presence of <i>T</i>. <i>cruzi</i> Dm28c luciferase (Dm28c-luc) parasites in orally infected mice, by bioluminescence and quantitative real-time PCR. <i>In vivo</i> bioluminescent images indicated the nasomaxillary region as the site of parasite invasion in the host, becoming consistently infected throughout the acute phase. At later moments, 7 and 21 days post-infection (dpi), luminescent signal is denser in the thorax, abdomen and genital region, because of parasite dissemination in different tissues. <i>Ex vivo</i> analysis demonstrated that the nasomaxillary region, heart, mandibular lymph nodes, liver, spleen, brain, epididymal fat associated to male sex organs, salivary glands, cheek muscle, mesenteric fat and lymph nodes, stomach, esophagus, small and large intestine are target tissues at latter moments of infection. In the same line, amastigote nests of Dm28c GFP <i>T</i>. <i>cruzi</i> were detected in the nasal cavity of 6 dpi mice. Parasite quantification by real-time qPCR at 7 and 21 dpi showed predominant <i>T</i>. <i>cruzi</i> detection and expansion in mouse nasal cavity. Moreover, <i>T</i>. <i>cruzi</i> DNA was also observed in the mandibular lymph nodes, pituitary gland, heart, liver, small intestine and spleen at 7 dpi, and further, disseminated to other tissues, such as the brain, stomach, esophagus and large intestine at 21 dpi. Our results clearly demonstrated that oral cavity and adjacent compartments is the main target region in oral <i>T</i>. <i>cruzi</i> infection leading to parasite multiplication at the nasal cavity.</p></div

Course of parasite dissemination in <i>Trypanosoma cruzi</i> oral infection.

<p>Male BALB/c mice were infected in the oral cavity (<b>OI</b>) with 1x10⁶ trypomastigotes forms of <i>T</i>. <i>cruzi</i> expressing luciferase (Dm28c-luc). Representative <i>in vivo</i> bioluminescence images were acquired in the same mice (n = 6), at 7 and 21 dpi, after 15 min of D-luciferin <b>IP</b> administration (150 mg/kg), using IVIS Lumina image system (Xenogen) The scale bar for radiance (below) was correlated with the signal intensity, where red indicates higher signal and blue indicates a lower signal. Maximum and minimum signals are indicated at the right and left of the scale bar, respectively. White arrows indicate the presence of bioluminescence.</p

Hypothesis: Oral infection involves <i>Trypanosma cruzi</i> infection through the mouth into the nasal cavity, the main source of parasite replication.

<p>Nasal cavity parasites might disseminate through the olfactory nerve to the brain and also by the lymphatic and/or blood circulation to distant tissues. Moreover, as previously described by others, a small amount of parasites migrates to the gastric mucosa in initial moments of infection. <i>T</i>. <i>cruzi</i> infection in the stomach may also be associated to parasite dissemination trough the host.</p

<i>In vivo</i> bioluminescence imaging of mice infected with Dm28c-luc at 15 and 60 min of infection.

<p>Male BALB/c mice were infected in the oral cavity (<b>OI</b>) with 1x10⁶ trypomastigotes forms of <i>T</i>. <i>cruzi</i> expressing luciferase (Dm28c-luc). Evaluation of <b>OI</b> mice was performed at 15 (A and B) and 60 min (C and D) post-infection using bioluminescent imaging (IVIS Lumina system). (A) <i>In vivo</i> bioluminescence imaging at 15 min post-infection (n = 5). (B) <i>In vivo</i> quantification of luminescent signal at 15 min post-infection (n = 5). (C) <i>In vivo</i> bioluminescence imaging at 60 min post-infection (n = 3). (D) <i>In vivo</i> quantification of luminescent signal at 60 min post-infection (n = 3). The scale bar for radiance (below) was correlated with the signal intensity, where red indicates higher signal and blue indicates a lower signal. Maximum and minimum signals are indicated at the right and left of the scale bar, respectively. Numbers represent mean ± SEM. Data were analyzed using one tailed Mann-Whitney test. Statistical analysis was performed using GraphPad Prism 5. * p<0.05; ** p < 0.01.</p

Amastigote nests detection by fluorescence microscopy of nasal cavity from mice infected with Dm28c-GFP.

<p>Male BALB/c mice were infected with 1x10⁶ trypomastigotes forms of <i>T</i>. <i>cruzi</i> expressing GFP reporter gene (Dm28c-GFP). At 6 dpi, the nasal cavity tissues were removed, frozen and sections were prepared for fluorescence microscopy analysis. (A) Representative fluorescence image of the nasal cavity from uninfected mice. (B) Representative fluorescence image of the nasal cavity from infected mice. Several amastigote nests (green) can be observed in the nasal cavity. Data represent analysis from an experiment with n = 2.</p

<i>Ex vivo</i> bioluminescence images from infected mice confirm the systemic dissemination of the parasite.

<p>Male BALB/c mice were infected in the oral cavity (<b>OI</b>) with 1x10⁶ trypomastigotes forms of <i>T</i>. <i>cruzi</i> expressing luciferase (Dm28c-luc). After 10 min of D-luciferin administration <b>IP</b> (150 mg/kg), organs were harvested and images were captured using an IVIS Lumina II system. <i>Ex vivo</i> bioluminescence imaging at 7 and 21 dpi: (A) oral cavity and adjacent organs; (B) gastrointestinal tract organs; (C) others head and neck organs. Pituitary gland: inside white circle; (D) abdominal organs; (E) In the male sex organ image, testicle and epididymal fat are located at the sides and the preputial gland in the bottom; (F) thorax organs. (n = 4 palate, cheek muscle; pituitary gland, mandibular lymph nodes mesenteric fat and lymph nodes; n = 6, others organs). The scale bar for radiance (right) was correlated with the signal intensity, where red indicates higher signal and blue indicates a lower signal. Maximum and minimum signals are indicated at the top and lower region of scale bar, respectively. (G) Schematic drawing of an anatomic section from a mouse head. The septum was removed, exposing the lateral wall and some of the major structures in the head. Palate (yellow), eyes (red), brain (light and dark green), olfactory bulb (light green) and nasal cavity are shown and the distribution of surface epithelial types lining the nasal airways is represented in blue, dark-purple, orange and light-purple colors for the squamous, transitional, respiratory, and olfactory epithelium, respectively.</p

Percentage of <i>T</i>. <i>cruzi—</i>positive tissues analyzed by bioluminescence and qPCR methods.

<p>Percentage of <i>T</i>. <i>cruzi—</i>positive tissues analyzed by bioluminescence and qPCR methods.</p