Additional file 1: of Paracoccidioides brasiliensis infection promotes thymic disarrangement and premature egress of mature lymphocytes expressing prohibitive TCRs

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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

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

<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

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

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

Exacerbation of Autoimmune Neuro-Inflammation in Mice Cured from Blood-Stage <i>Plasmodium berghei</i> Infection

<div><p>The thymus plays an important role shaping the T cell repertoire in the periphery, partly, through the elimination of inflammatory auto-reactive cells. It has been shown that, during <i>Plasmodium berghei</i> infection, the thymus is rendered atrophic by the premature egress of CD4⁺CD8⁺ double-positive (DP) T cells to the periphery. To investigate whether autoimmune diseases are affected after <i>Plasmodium berghei</i> NK65 infection, we immunized C57BL/6 mice, which was previously infected with <i>P.berghei</i> NK65 and treated with chloroquine (CQ), with MOG_35–55 peptide and the clinical course of Experimental Autoimmune Encephalomyelitis (EAE) was evaluated. Our results showed that NK65+CQ+EAE mice developed a more severe disease than control EAE mice. The same pattern of disease severity was observed in MOG_35–55-immunized mice after adoptive transfer of <i>P.berghei</i>-elicited splenic DP-T cells. The higher frequency of IL-17⁺- and IFN-γ⁺-producing DP lymphocytes in the Central Nervous System of these mice suggests that immature lymphocytes contribute to disease worsening. To our knowledge, this is the first study to integrate the possible relationship between malaria and multiple sclerosis through the contribution of the thymus. Notwithstanding, further studies must be conducted to assert the relevance of malaria-induced thymic atrophy in the susceptibility and clinical course of other inflammatory autoimmune diseases.</p></div

Central Nervous System of malaria-cured EAE mice show increased cellular infiltration of DP-T cells.

<p>C57BL/6 mice (n = 6 mice/group) were intraperitoneally (i.p.) infected with 1×10⁶<i>P.berghei</i>-infected Red Blood Cells and treated with chloroquine (CQ, 5 mg/Kg) for five consecutive days starting at the 10^th day after infection. Three days after the last dose of CQ, EAE was induced. As controls, naïve mice were treated with CQ or vehicle before EAE induction. The spinal cords of EAE-inflicted mice were collected fourteen days after MOG-immunization. Frozen thin sections (12 µm) were made and fixed in formalin. Cells were stained with FITC-conjugated anti-CD4 and PE-conjugated anti-CD8 and analyzed in epifluorescence microscope. Figures are representative of three independent experiments. Magnification: 200X.</p

Analysis of the cellular infiltration of the CNS show reduced IFN-γ and IL-17 producing cells in CQ treated EAE mice.

<p>(A) CQ treated-mice presented reduced infiltration of inflammatory cells. (B) The percentage of IFN-γ- and IL-17-producing cells infiltrating the brain was reduced while the frequency of IL-10- producing cells was found augmented in brain of mice treated with CQ. (C, D and E) Gene expression of IFN-γ, IL-17 and IL-10 in the CNS followed the same pattern, respectively. (F) The expression of FOXP3 was evaluated in the CNS by RT-PCR. (G) The frequency of CD25⁺Foxp3⁺ cells was evaluated in spleens of mice. Results are representative of two independent experiments and are expressed as mean ± SEM for at least five animals. p<0,05 (*) and p<0.01 (**).</p

Aggravation of EAE in mice cured from malaria correlates with increased cellular immune response towards myelin.

<p>C57BL/6 mice (n = 6 mice/group) were intraperitoneally (i.p.) infected with 1×10⁶<i>P.berghei</i>-infected Red Blood Cells and treated with chloroquine (CQ, 5 mg/Kg) for five consecutive days starting at the 10^th day after infection. Three days after the last dose of CQ, mice were immunized with 100 µg of MOG_35–55 peptide and Pertussis toxin was administrated (via i.p.) at 0 and 48h after peptide immunization for EAE induction. A) The clinical course of EAE was then monitored. Linear regression analyses are exposed in the side panels, thinner lines indicate 95% confidence interval. B) At the 10^th day after MOG-immunization, the spleens of mice were collected and dissociated. Total leukocytes (5×10⁵/well) were CFSE-stained (2,5 µM) and cultured in the presence of MOG_35–55 (10µg/mL) peptide for 96h. At the end of culture period, the cells were surface stained with anti-CD3/CD4/CD8 antibody cocktail and events were acquired in a flow cytometer. The proliferation was analyzed inside each T cell population. C) The culture supernatants were assayed for the secreted cytokines IL-10, IL-4, IL-6, IL-17, TNF-α and IFN-γ. Data was analyzed by One-Way Anova and post-tested with Bonferroni. In all analyses, *: p<0,05; ns: not significant. Representative data of three independent experiments.</p