Prolonged <i>Paracoccidioides brasiliensis</i> infection leads to thymic atrophy and intense fungal burden.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS or with only PBS (control group), intraperitoneally. One hundred and twenty days after inoculation, mice were killed and the thymus removed for analysis. A) Thymus from the 120dpi group was smaller in size and weighted less than the naive group. (B) Hematoxilin-Eosin staining showed histologic disorganization in 120dpi, and no evidence of typical cortical (C) and medullary (M) regions, while naive mice showed normal histologic distribution. In more detail below, giant cells and granuloma formation is present in 120dpi. Silver impregnation revealed massive fungal infiltration in the thymic medulla in 120dpi. Statistical analysis was carried out with Student’s t-test. **p<0.01. Results are expressed by Mean ± SEM. Images are representative of three independent experiments with similar results. The images were taken in an Olympus BX50. Magnification 40x (upper and lower panels); 100x (middle panel).</p

Increased inflammasome and caspase-1 activity in the thymus of infected mice.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Increased initiator caspase-8 gene expression on 120dpi group compared to the naive group. Increased inflammatory caspase-1 gene expression on 120dpi group compared to the naive group. Increased NLRP3 inflammasome gene expression on 120dpi group compared to the naive group. B) Increased pro-caspase-1 production and increased caspase-1 activity on 120dpi group compared to the naive group. C) Increased NLRP3 inflammasome complex assembly on 120dpi compared to the naive group. Statistical analysis was carried out with Student’s t-test. **p<0.01, ****p<0.0001. Representative data from three independent experiments with similar results. Expression levels of genes were represented as a relative copy numbers by using the method of delta threshold (2^-ΔΔCt). Image J software (NIH, MD, USA) was used for the estimation of the pro-caspase-1, the active form of caspase-1 and NLRP3 inflammasome assembly, through a GAPDH ratio.</p

Recirculating mature T cells home to infected thymuses, leading to increased frequency of cytokine producing Th17 and T CD8⁺ IFN-γ⁺.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Frequency of CD44^hiCD24^lo T cells increased among CD4⁺ and CD8⁺ subpopulations in 120dpi compared to the naive group. B) Cytokine producing T cells, Th17 and CD8⁺IFNγ⁺ was found in 120dpi, while practically absent in the naive group. Representative data from three independent experiments with similar results. At least 20000 events were analyzed with FlowJo vX.0.7 (Tree Star Inc., Ashland, OR, USA). Statistical analysis was carried out with Student’s t-test. ***p<0.001, ****p<0.0001. Representative data from three independent experiments with similar results.</p

Increased inflammatory cytokines gene expression and protein levels in the thymus of infected mice.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS (control group). One hundred and twenty days after inoculation, mice were killed and the thymus was collected and processed individually for analysis. A) Increased gene expression of IL-1β, IL-17, IL-18, IFN-γ and TNF-α from 120dpi group compared to the naive group. B) Protein levels of the inflammatory cytokines also showed significant increase in 120dpi group compared to the naive group. Statistical analysis was carried out with Student’s t test. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Representative data from three independent experiments with similar results. Expression levels of genes were represented as a relative copy numbers by using the method of delta threshold (2^-ΔΔCt).</p

<i>Paracoccidioides brasiliensis</i> infection leads to decreased thymocytes and TECs cellularity with increased cell death of CD4⁺CD8⁺ thymocytes and mTEC without cortisol contribution.

<p>Male BALB/c mice (n = 5 mice/group for each analyses per experiment replicate) were inoculated with 5x10⁶ Pb18 yeasts contained in PBS intraperitoneally or with only PBS as control group. One hundred and twenty days after inoculation mice were killed, the thymus and the blood were collected and processed individually for analysis. A) Total thymocytes numbers were lower in 120dpi compared to the naive group. B) Frequency of thymocytes subpopulations remained unchanged between naive and 120dpi groups. C) Frequency of thymic epithelial cells (TEC) subpopulations changed considerably in 120dpi, higher percentages of mTEC were found while cTEC frequency decreased in comparison to the naive group. D) Higher percentage of apoptotic double positive thymocytes from the 120dpi group compared to the naive group, but not in other thymocytes subpopulations. E) Higher percentage of apoptotic medullary thymic epithelial cells (mTEC) from the 120dpi group compared to the naive group, while no alterations in cTEC apoptosis was found. F) Cortisol production was not altered between the naive and 120dpi groups. At least 20000 events were analyzed with FlowJo vX.0.7 (Tree Star Inc., Ashland, OR, USA). Statistical analysis was carried out with Student’s t-test. *p<0.05; **p<0.01; ***p<0.001. Representative data from three independent experiments with similar results.</p

Adoptive transfer of regulatory T cells elicited after violacein treatment reduces EAE through the modulation of CNS inflammation and inhibition infiltration of cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (3.5mg/Kg) for three consecutive days. After the last dose of viola, mice were killed and spleen cells were prepared for single cell suspension. CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated using dynabeads, following manufacturer’s instructions (Life Technologies). 5x10⁵ cells were adoptively transferred into EAE-bearing mice, at the 10^th day after immunization, the clinical course was evaluated daily. At the 20^th day after immunization, mice were killed and lumbar spinal cords were removed and submitted to mRNA extraction protocols. A) The expression of FOXP3, IDO, IL-17, IL-10, IFN-γ and TNF-α was evaluated. Results show that adoptive transfer of viola-elicited Treg cells changed the CNS gene expression profile of EAE-bearing mice. B) The infiltration of cells in the CNS was evaluated as well. For that, spinal cords were collected and enriched in T cells, which were counted in hemocytometer. Results show that the treatment inhibited the infiltration of cells in the CNS of EAE-bearing mice. Representative data from two independent experiments. All values are represented as means ± standard error mean. *p<0.05.</p

Effect of viola treatment and viola-elicited Treg cells adoptive transfer on the severity of EAE.

<p>*: p<0,05, in comparison with the EAE group;</p><p>#: p<0,05, in comparison with the EAE+CD25^- group.</p><p>Analyses were performed with One-Way ANOVA followed by Bonferroni’s posttest.</p><p>Effect of viola treatment and viola-elicited Treg cells adoptive transfer on the severity of EAE.</p

Reduction in EAE severity after adoptive transfer of violacein-elicited Treg cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (3.5mg/Kg) for three consecutive days. After the last dose of viola, mice were killed and spleen cells were prepared for single cell suspension. CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated using dynabeads, following manufacturer’s instructions (Life Technologies). 5x10⁵ cells were adoptively transferred into EAE-bearing mice, at the 10^th day after immunization (arrow), the clinical course was evaluated daily and showed reduction in the severity of CD4⁺CD25⁺-transferred mice compared to the other groups. Representative data from two independent experiments. All values are represented as means from each group. **p<0.01.</p

Violacein-elicited regulatory T cells present an enhanced suppressive activity than “naïve” Treg cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (3.5mg/Kg) for three consecutive days. A) After the last dose of viola, mice were killed and spleen cells were prepared for single cell suspension. CD4⁺CD25⁺ and CD4⁺CD25^- cells were isolated using dynabeads, following manufacturer’s instructions (Life Technologies). As controls, cells were isolated from spleens from naïve mice. Treg cells were seeded in U-bottom 96-wells culture plate in increasing numbers. C57BL/6 mice (n = 3) were immunized with MOG peptide. After seven days, mice were killed and the spleens were collected and disrupted for the isolation of dendritic cells and total T cells with dynabeads. T cells were stained with CFSE (1,5 μM) and seeded to the plates containing Treg cells (5x10⁵ cells/well). Dendritic cells were isolated as well and seeded to the wells (5x10⁴ DCs/ well). As controls, encephalitogenic T cells were cultivated without Treg cells. The plates were incubated for 72h at 37°C and the suppressive activity of Treg cells was analyzed by flow cytometry. B) Treg cells were isolated from PBS- and viola-treated mice, seeded to 96-well plate and incubated for 48h at 37 ºC. The supernatants were collected and assayed for the detection of IL-10 by CBA. Representative data from two independent experiments. All values are represented as means ± standard error mean. *: p<0.05, **: p<0,01 and ***: p<0,005. Ns: not significant.</p

Violacein administration diminishes acute inflammation induced by Lipopolysaccharide injection.

<p>C57BL/6 mice (n = 3 mice/group) were treated with viola (3.5 mg/Kg) and lipopolysaccharide (1μg/mouse) through the intraperitoneal route. Three hours after LPS injection, mice were killed and the frequencies of dendritic cells (in A), T lymphocytes (in B), B cells (in C) and Neutrophils (in D) were assessed. E) The serum levels of selected cytokines and CXCL1 were determined as well. Representative data from two independent experiments.</p