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

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

Proliferative responses to mycobacterial stimulation and levels of IFN-γ in 96-h culture supernatants of peripheral blood mononuclear cells from TB patients (n = 47), household contacts (HHC, n = 22) and healthy controls (n = 23).

<p>Patients were separated according to disease severity into mild (n = 10), moderate (n = 18) and severe (n = 19) cases. Box plots show 25–75 percentiles of data values in each group with maximum and minimum values. The line represents the median values. Panels A and C: lymphoproliferative response and IFN-γ production in TB patients, HHC and controls, respectively; Panels B and D: lymphoproliferative response and IFN-γ production in TB patients according to severity, respectively. Overall comparisons by the Kruskall-Wallis test revealed significant differences in lymphoproliferation and 4-day IFN-γ production (<i>p</i><0.05 and <i>p</i><0.02, respectively). Significant post hoc Bonferroni comparisons between groups and within TB patients are depicted by *, ** and ***, <i>p</i><0.05, p<0.025 and <i>p</i><0.01, respectively.</p

Correlation analysis of hormone and cytokine plasma levels with BMI in TB patients, healthy controls and household contacts (HHC).

<p>Correlation analysis of hormone and cytokine plasma levels with BMI in TB patients, healthy controls and household contacts (HHC).</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

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

Plot of two linear discrimination functions of the model differentiating the study groups.

<p>Function 1 = 0.692*BMI - 0.655*CRP + 0.436*Leptin - 0.305* IL-6 - 0.185*Ghrelin - 0.124*IL-1β + 0.059*Age + 0.309*DHEA - 0.192*Adiponectin - 0.212*Cortisol. Function 2 = 0.036*BMI - 0.075*CRP + 0.368*Leptin + 0.117*IL-6 - 0.066*Grhelin -0.053*IL-1β + 0.691*Age - 0.562*DHEA + 0.406*Adiponectin - 0.305*Cortisol.</p

Levels of cytokines, adipocytokines, CRP, ghrelin and HPA related hormones in TB patients, household contacts (HHC) and healthy controls.

<p>Values represent median (25–75 percentiles), data were analyzed by the Kruskall-Wallis non parametric analysis of variance.</p>a<p>Calculated with leptin values in pg/ml.</p>*<p>Differences remained significant when comparing TB patients either with HHC or controls, except for the levels of DHEA in the two former groups (p<0.05, post hoc Bonferroni comparisons).</p><p>¶ Significantly different from controls (<i>p</i><0.05, post hoc Bonferroni comparison).</p>§<p>Significantly different from mild patients (<i>p</i><0.05, post hoc Bonferroni comparisons within TB patients).</p>‡<p>Significantly different from mild patients (<i>p</i><0.05, post hoc Bonferroni comparisons within TB patients).</p