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

Violacein treatment up-regulates the frequency of Regulatory T cells.

<p>C57BL/6 mice (n = 6 mice/group) were treated with viola (1.75, 3.5 and 7 mg/Kg) for three consecutive days. A) Kaplan-Meier curve of survival show that mice receiving the 7mg/Kg dose died after the second administration. B) After the last dose of viola, mice were killed and spleen cells were stained for flow cytometry analysis of CD4⁺CD25⁺FOXP3⁺ (Treg) cells. Results show an increase in the frequency of Treg cells after viola treatment. C) Expression of CTLA-4 was evaluated inside the Treg cell population by flow cytometry. D) The intracellular cytokine detection was performed in spleen cells from PBS- and viola-treated mice. The spleen cells were stimulated with PMA (50 ng/mL) and Ionomycin (500 ng/mL) in the presence of Brefeldin A (1 μg/mL) for 4h at 37°C. Later cells were surface stained with antibody against CD4. Following permeabilization, cells were incubated with antibody cocktail for the detection of IL-17, IL-10 and IFN-γ and preparations were acquired in Flow Cytometer equipment. Representative data from three independent experiments. All values in bar graphs 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

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

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

Hypothesis model for the exacerbation of autoimmune neuro-inflammation.

<p>Based in our observations, we propose a model for EAE exacerbation. Malaria infection promotes thymic atrophy and the premature egress of DP-T cells to the peripheral immune system. After an inflammatory trigger, which can be infection, genetic susceptibility or chronic inflammation, these cells proliferate and migrate to the target organ where they stimulate CNS inflammation by secreting cytokines. However, there is still much to be explored, as for example, whether these DP-T cells are able to induce leukocyte recruitment, microglia and astrocyte activation, and, Blood-Brain Barrier (BBB) destruction.</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 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

Inflammation in the CNS of NK+CQ+EAE mice correlates with an increased production of inflammatory cytokines by DP-T cells.

<p>Groups of mice (n = 6 mice/group) subjected to infection and EAE induction. A) At the 10^th day after MOG-immunization, mice were killed and spinal cords were removed to analyze the gene expression of IL-17, IFN-γ, Foxp3 and IL-10 in the lumbar spinal cords of mice. Data was analyzed by One-Way Anova and post-tested with Bonferroni. B) The infiltrating cells of the CNS were enriched and stimulated by Phorbol Myristate Acetate and Ionomycin in the presence of Brefeldin A for 4 h. The frequency of IFN-γ- and IL-17-producing cells inside CD4⁺CD8⁺ T cell gate was analyzed. In all analyses, *: p<0,05. ns: not significant. Representative data of three independent experiments.</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