Expression of TNF-R1 and TNF-R2 en CD4⁺, CD8⁺ and CD4⁺CD8⁺ (DP) thymocytes.

<p>DP and SP T cells were purified from thymuses of control and acutely infected mice (14 days post-infection) by cell sorting using flow cytometry. To quantitatively evaluate TNFR transcripts, total mRNA samples from highly purified fresh DP, CD4+ and CD8+ T cells were processed for quantitative RT-PCR. The relative gene expression of mRNA of TNF-R1 and TNF-R2 were enhanced in DP thymocytes from infected animals compared with controls. Results are presented as relative gene expression, where mRNA levels were calculated using the equation 2^−ΔCt (difference in Ct between β-actin and the target gene). Data depict a representative experiment. Statistically significant differences are indicated as *p<0.05.</p

TNF-α enhances export of CD4⁺CD8⁺ thymocytes.

<p>Control and 14 days-infected animals were intrathymically injected with FITC dissolved in PBS with or without TNF-α. Twenty-four hours later, recent thymic emigrants (RTEs) were recognized as FITC⁺/CD4⁺/CD8⁺ cells in secondary lymphoid organs (spleen and subcutaneous lymph nodes) by flow cytometry. Empty bars represent the absolute number of FITC⁺ cells (×10³) from PBS injected mice and black bars represent the absolute number of FITC⁺ cells from TNF-α injected animals. Results show that after infection, the numbers of RTEs are clearly enhanced, independently of whether intrathymic injection of TNF-α was applied or not. When considering CD4/CD8-defined subpopulations, we found that intrathymic inoculation of TNF-α further promoted a significant enhancement in the numbers of FITC⁺CD4⁺ and FITC⁺CD4⁺CD8⁺ cell in the spleen, but not in lymph nodes. Data depict a representative experiment of two similar ones; each experiment being done with 5–6 animals/group. Statistically significant differences are indicated as *p<0.05.</p

Correlation analyses for TNF-α thymic concentrations and atrophy parameters in <i>T. cruzi</i> acutely infected mice.

<p>RTW = relative thymic weight (thymic weight/corporal weight); DP = CD4⁺CD8⁺ double positive cells; rho = Spearman correlation coefficient, n = number of XY pairs.</p

Overall rate of emigration (ORE) after intrathymic inoculation.

<p>Control and 14 days-infected animals were injected intrathymically with FITC or FITC plus TNF-α. Twenty-four hours after, thymuses, spleens and subcutaneous lymph nodes were screened for FITC⁺ CD4/CD8-defined cell subsets and these data were adjusted in relation to the cellularity of each peripheral lymphoid organ and the percentage of thymus labelling, using the following formula: Overall rate of emigration (ORE) = (Absolute numbers of FITC⁺ cells×cellularity in the peripheral lymphoid organ)/(% of FITC⁺ cells in thymus). We verified an enlarged ORE values in infected animals, independently of whether they were inoculated or not with TNF-α. Data indicate that TNF-α promotes the output of immature DP⁺ cells to both spleen and subcutaneous lymph nodes. Absolute numbers of FITC⁺ cells, CD4⁺, CD8⁺ and DN cells was expressed on 1.10⁴ cells, while DP⁺ cells was expressed on 1.10³ cells. Cellularity of each peripheral lymphoid organ was expressed in 1.10⁶ cells. Values are mean ± SEM of 5–6 mice/group (one representative experiment of two independent series). Difference between Control <i>versus T. cruzi</i> group = ⁽*⁾p<0.05; ⁽**⁾p<0.02. Differences between TNF-α treated <i>versus</i> not treated group = ^(Δ)p<0.05; ^(ΔΔ)p<0.02.</p

Enhanced circulating and intrathymic contents of TNF-α parallel the thymic atrophy in <i>T. cruzi</i> acutely-infected mice.

<p><b>A</b>) Representative picture showing two thymuses from healthy control mice (Co) and the progressive thymic atrophy after 14, 17 and 21 days of acute infection; <b>B</b>) TNF-α protein detection in thymus homogenates by western blot. Representative photomicrographs showing TNF-α and β-actin expression in two control mice (<i>Co</i>) and in two infected mice after 21 days post-infection (<i>Inf</i>); <b>C</b>) TNF-α concentrations in thymic homogenates detected by ELISA at different days after infection and normalized to total protein contents. Bars represent the mean ± SEM of three pools/day); <b>D</b>) Systemic concentration of TNF-α along infection evaluated by ELISA. (n = 5–8 mice/day); <b>E</b>) Confocal microscopy showing an enhancement in TNF-α contents both in cortex and medulla of the thymic lobules, after 14 days of infection. Original magnification 400×. Small box is a representative control staining in which an unrelated primary antibody was applied; <b>F</b>) Graphs correspond to relative quantification analysis of TNF-α deposition in both cortex and medulla from 3–5 microscopic fields of thymuses from control (n = 5) or 14 days-infected animals (n = 5); <b>G</b>) Representative immunofluorescence staining showing thymuses of infected mice: TNF-α expression (in red, left picture); fibronectin (FN) deposition (in green, middle picture); co-localization of TNF-α and FN (right picture). Small boxes correspond to negative control where immune reaction was controlled by using unrelated primary antibodies; <b>H</b>) Bars correspond to the percentages of TNF-α plus FN co-localization in thymuses from control mice compared with infected counterparts. Significant differences are indicated as *p<0.05, **p<0.01, ***p<0.001.</p

TNF-α enhances <i>in vitro</i> fibronectin-driven migration of thymocytes from <i>T. cruzi</i> infected mice.

<p><b>A</b>) Thymocytes from control or infected animals were allowed to migrate in transwell chambers coated with fibronectin (FN) alone or FN plus TNF-α. Fibronectin-induced haptotactic response of thymocytes from infected animals was enhanced compared with controls in presence of TNF-α at the concentration of 25 pg/mL. Results derive from four experiments for TNF-25 pg/mL and three for TNF-250 pg/mL (each one obtained by pools of at least 3–4 animals by group) and correspond to specific migration after subtracting absolute cell numbers obtained in each well coated only with BSA. Statistically significant differences are indicated as p<0.05. <b>B</b>) Specific migration of thymocyte subpopulations was expressed as percentage of input (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034360#s2" target="_blank">Material and Methods</a> session). We observed an enhanced migratory response of CD4⁺, CD8⁺, DP and DN cells when TNF-α was applied at 25 pg/mL, as compared with FN alone in infected animals. Results derived from four experiments, each one obtained by pools of at least 3–4 animals by group. Data correspond to specific migration after subtracting absolute cell numbers obtained in each well coated only with BSA. Statistically significant differences are indicated as *p<0.05 and **p<0.01.</p

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

Intrathymic tissue-restricted antigen expression levels in atrophic thymus during <i>T cruzi</i> infection.

<p><b>(a)</b> Thymuses were collected from normal or <i>T. cruzi</i> infected BALB/c (day 15 post-infection) and expression levels of Aire and TRA genes were analyzed by real-time PCR. The expression of Aire as well as TRA genes was comparable in infected and uninfected thymus. Data are mean ± standard error. of triplicate measurements in one of two representative experiments using five mice per group. <b>(b)</b> Thymuses were stained with anti-Aire antibody and analyzed by immunofluorescence. The histological profiles show the presence of Aire-positive cells (arrows) in <b>(upper left)</b> normal thymus and <b>(upper right)</b> atrophic thymus at day 15 post-infection. The respective staining controls without the primary antibodies are represented in <b>(lower left)</b> and <b>(lower right)</b>. Inserts represent higher magnifications of Aire-positive cells (brown).</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

<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