Table 1.
Primer sequences and expected amplification products.
Figure 1.
Changes in relative weight, TNF-R1 expression and morphology in endocrine glands from T. cruzi-infected mice.
The relative weight of the adrenal glands in C57BL/6 acutely infected with T. cruzi, at day 17 pi (panel A) was calculated as follows: [adrenal weight (mg)/body weight (g)]. Panel B shows western blots for the protein expression of TNF-R1 at day 17 pi. Results are expressed as mean ± SEM from 3–5 mice/group. A representative experiment from 2 independent series is shown. # p<0.05 vs. uninfected counterparts. Panel C. The cortex consists of three morphologically distinct zones. The region immediately beneath the capsule is called zone glomerulosa and constitutes about 1/5 or less of the entire thickness, this zone was only increased in Tc-WT mice. The mid portion of the cortex, called zone fasciculata, comprises about 50–65% of the thickness of the cortex in uninfected mice (WT and TNF-R1−/− mice), with Tc-WT and Tc-TNF-R1−/− mice showing a diffuse hyperplasia (the broad arrow indicates the thickness with an arbitrary vertical line). The most inner portion of the cortex, called zone reticularis, comprises about 20–25% of the cortex from uninfected mice, showing a hyperplasia in Tc-TNF-R1−/− mice. H&E stain, magnification 40 X. Scale bar: 20 µm. WT (wild type) and TNF-R1−/− (TNF receptor deficient) control mice; Tc-WT and Tc-TNF-R1−/− infected counterparts.
Table 2.
Cytokine levels in plasma of mice undergoing acute T. cruzi infection.
Table 3.
Morphometric analysis of adrenal sections of mice undergoing acute T. cruzi infection.
Figure 2.
Hormonal variations in the HPA axis from T. cruzi-infected mice.
Plasma levels of corticosterone (µg/dl, panel A) and ACTH (pg/ml, panel B) in C57BL/6 mice acutely infected with T. cruzi. Results are expressed as mean ± SEM from 3–5 mice/group. A representative experiment from 2 independent series is shown. WT (wild type) and TNF-R1−/− (TNF receptor deficient) control mice; Tc-WT and Tc-TNF-R1−/− infected counterparts. *p<0.05 vs. Tc-WT; #p<0.05 vs. uninfected counterparts.
Figure 3.
Activation of NF-κB and AP-1 in adrenal gland during T. cruzi infection.
Levels of NF-κB activity (OD 450/650, panel A) and activation of AP-1 (panel B) in nuclear extracts of adrenal glands from C57BL/6 mice acutely infected with T. cruzi. NF-κB activity assay was assessed by colorimetric determination of p50 subunit in all experimental groups. AP-1 activation was tested using direct measurement of c-jun by Western blot. Results are shown as mean ± SEM from 3–5 mice/group/day. A representative experiment from two similar rounds is shown. WT (wild type) and TNF-R1−/− (TNF receptor deficient) control mice; Tc-WT and Tc-TNF-R1−/− infected counterparts. #p<0.05 vs. uninfected counterparts; &p<0.05 vs. WT mice.
Figure 4.
Activation of MAPK pathways in adrenal glands from mice acutely infected with T.cruzi .
Blots correspond to P-p38, P-ERK and P-JNK. Blots were stripped and revealed with an anti-β-actin (panel A). Bars represent the densitometry considering WT and TNF-R1−/− as 100% (panel B). Expression is relative to β-actin. Results are expressed as mean ± SEM from 3–5 mice/group. Data correspond to one round of two representative and independent experimental rounds. WT (wild type) and TNF-R1−/− (TNF receptor deficient) control mice; Tc-WT and Tc-TNF-R1−/− infected counterparts. *p<0.05 vs. Tc-WT; #p<0.05 vs. uninfected counterparts.
Figure 5.
Steroidogenic enzymes gene expression in adrenal glands from T. cruzi infection in absence of TNF-R1.
Expression profiles of genes involved in GC synthesis/activation enzymes, StAR (panel A), CYP11A1 (panel B), CYP11B1 (panel C), 11β-HSD1 (panel D) and 11β-HSD2 (panel E) in adrenal glands from C57BL/6 or TNF-R1−/− mice acutely infected with T. cruzi. Total mRNA was purified and analyzed by Real-Time-PCR using nonsaturating conditions. The mRNA expression levels were then normalized to RPL13. Results are shown as mean ± SEM from 3–4 mice/group/day. A representative experiment from 3 independent series is shown. WT (wild type) and TNF-R1−/− (TNF receptor deficient) control mice; Tc-WT and Tc-TNF-R1−/− infected counterparts. *p<0.05 vs. Tc-WT; #p<0.05 vs. uninfected counterparts and &p<0.05 vs. WT.
Figure 6.
Gene expression of pro-inflammatory cytokines in adrenal glands from T. cruzi-infected mice.
Total RNA was extracted and the mRNA levels of TNF-α (panel A), IL-6 (panel B) and IL-1β (panel C) were assessed by quantitative PCR (normalized to RPL13). Results are shown as mean ± SEM from 3–4 mice/group/day. A representative experiment from 3 independent series is shown. WT (wild type) and TNF-R1−/− (TNF receptor deficient) control mice; Tc-WT and Tc- TNF-R1−/− infected counterparts. *p<0.05 vs. Tc-WT; #p<0.05 vs. uninfected counterparts.
Figure 7.
Expression of TNF receptor type 2 in adrenal gland from T. cruzi- infected mice.
Panel A shows frozen sections of the adrenal gland labeled with anti-TNF-R2 antibody. Confocal microscopy shows a decreased in TNF-R2 contents in cortex of the adrenal gland in Tc-WT and Tc-TNF-R1−/− (more pronounced in Tc-TNF-R1−/− group) after 17 days of infection. The small box is a representative control staining in which an unrelated primary antibody was applied. Panel B shows graphs corresponding to the relative quantification analysis of cortical TNF-R2 expression of 5–6 microscopic fields of adrenal gland from WT (wild type) and TNF-R1−/− (TNF receptor deficient) control mice and infected counterparts (Tc-WT and Tc-TNF-R1−/−). Panel C shows the gene expression of TNF-R2 in adrenal glands from T. cruzi-infected mice. Results are expressed as mean ± SEM. A representative experiment from 2 independent series is shown. The scale bar represents 50 µm (C: cortex; M: medulla). Original magnifications 200X. * p<0.05 vs. Tc-WT; # p<0.05 vs. uninfected counterparts.
Figure 8.
Detection of T. cruzi in the adrenal gland.
Panel A shows frozen sections of the adrenal gland labeled with anti–T. cruzi antiserum. Immunostaining analysis demonstrated alike deposition of parasite antigens in the adrenal glands from Tc-WT and Tc-TNF-R1−/− after 17 days of infection. The small boxes represent a control staining in which an unrelated primary antibody was applied. Panel B corresponds to the relative quantification analysis of cortical T.cruzi-labeling of 5–6 microscopic fields of adrenal glands from WT (wild type) and TNF-R1−/− (TNF receptor-1 deficient) control mice and their infected counterparts (Tc-WT and Tc-TNF-R1−/−). Panel C shows the quantitative expression of T. cruzi kDNA in adrenal glands from T. cruzi-infected mice. Results are expressed as mean ± SEM. A representative experiment from 2 independent series is shown. The scale bar represents 50 µm. Original magnifications 200X.