Role of plasmacytoid dendritic cells in multiple sclerosis and in experimental autoimmune encephalomyelitis

Orientador: Leonilda Maria Barbosa dos SantosTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências MédicasResumo: No presente estudo demonstramos a ativação de mecanismos imunosupressores em células dendríticas plasmocitóides (pDCs) e linfócitos B pela ação do agonista de TLR-9, ODN-CpG no modelo de estudo da esclerose múltipla (EM) e na encefalomielite experimental autoimune (EAE). A EAE é o modelo experimental da esclerose múltipla. Nossos resultados demonstram que a administração in vivo de ODN-CpG reduz significativamente a gravidade de EAE. A redução da doença foi acompanhada pela diminuição da resposta proliferativa dos linfócitos encefalitogênicos e consequentemente a infiltração dessas células no sistema nervoso central. A diminuição da resposta proliferativa parece ser devido ao efeito imunoregulador das pDCs, uma vez que a depleção dessas células faz com que a resposta proliferativa retorne aos níveis normais. O tratamento com ODN-CpG induziu a expressão da enzima indoleamine 2,3 dioxigenase pelas pDCs. Essa enzima está relacionada à geração de células T reguladoras. De fato nossos resultados mostraram um aumento da porcentagem de células T CD4+CD25+ e da expressão das citocinas anti-inflamatórias IL-10 e TGF-b no grupo tratado. Adicionalmente, a transferência de pDCs ativadas isoladas é capaz de reduzir a gravidade da doença. Além das pDCs, os linfócitos B também expressam TLR-9 e podem ser ativados pelo tratamento com CpG, de fato, embora o número de células não difira do controle não tratado, a transferência de linfócitos B de animais tratados com CpG é capaz de diminuir a gravidade da doença. O efeito supressor dos linfócitos B pode ser atribuído à expressão de IL-10 nos animais tratados. Em paralelo, nós demonstramos um aumento na porcentagem de pDCs em líquido cefalorraquidiano de pacientes com EM durante a fase de surto da doença quando comparados com pacientes em remissão ou com outras doenças neurológicas não inflamatórias. Nossos resultados indicam que elas podem estar envolvidas tanto com a piora da doença, o que poderia ser explicado por uma infecção viral, ou pelo contrário, estando em maior número poderia com sua ação imunomoduladora preparar o organismo para a fase de remissão da doença. Entretanto, pacientes com EM apresentam deficiência na indução de células T naive a produzirem IL-10, mas não IFN-g, o que poderia ser explicado em parte pela deficiência da expressão de IDO obervada após ativação in vitro com CpG, quando comparadas com pDCs de indivíduos saudáveis. A deficiência da expressão de IDO pode comprometer o efeito imunomodulador das pDCs na esclerose múltiplaAbstract: In the present study we verified the activation of immunomodulatory mechanisms of plasmacytoid dendritic cells (pDCs) and B lymphocytes by the action of TLR9 agonist, CpG-ODN during multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). EAE is the experimental model of human MS. Our results provide evidence that in vivo administration of CpG-ODN significantly reduces the severity of EAE. The reduction in disease was followed by decreasing in the proliferative response of encephalitogenic lymphocytes and consequently infiltration of these cells in the central nervous system. The decrease in the proliferative response seem to be due to the immunomodulatory effect of pDCs, since depletion of these cells restored the proliferative response, returns to normal levels. Treatment with ODN-CpG induced expression of indoleamine 2,3 dioxygenase enzyme by pDCs. This enzyme is related to the enhancement of regulatory T. Indeed, our results have shown an increased of percentage of CD25+CD4+Foxp3+ cells and expression of anti-inflammatory cytokines IL-10 and TGF-b in the treated group. Moreover, the adoptive transfer of activated pDCs alone reduced the clinical course of EAE. In addition to the pDCs, B lymphocytes also express TLR9 and can be activated by treatment with CpG, in fact, although the number of cells does not differ from untreated controls, the transfer of B lymphocytes from animals treated with CpG was able to reduce the severity of the disease as well. The immunomodulatory effect of B lymphocytes may be due to expression of IL-10 in treated animals. In parallel, we were able to report an increase of pDCs percentage in cerebrospinal fluid of MS patients during relapse compared with patients in remission or other non-inflammatory neurologic diseases. This data indicate that it may be involved with the worsening of the disease, which could be explained by viral infection, or be involved in the initial immunomodulatory mechanisms responsible for the remission. However, MS patients presented deficiency to induce naive T cells to produce IL-10, but not IFN-g, which could partly be explained by the deficiency of IDO expression observed after CpG in vitro activation, when compared with pDCs from healthy individuals. The deficiency of IDO expression may compromise the immunomodulatory effect of pDCs in MS diseaseDoutoradoCiencias BasicasDoutor em Clínica Médic

Exosomes in the serum of acute myeloid leukemia patients induce dendritic cell tolerance : implications for immunotherapy

Exosomes may represent an interesting antigenic pulse for new forms of anti-tumor immunotherapy. We evaluated exosomes from serum of patients with acute myeloid leukemia (AML) as an antigenic source for dendritic cells (DC) and the effects upon antitumor cytotoxicity, assessed by the percentage of specific lysis of K562 leukemic cells in co-cultures. Surprisingly, incubation of exosomes with DCs decreased lysis of K562, which may correspond to a mechanism of tumor evasion in vivo. However, when immature DCs were pulsed with exosomes purified from K562 culture supernatants, the lysis of target cells was notably enhanced, associated with a substantial increase in the expression of the maturation marker CD83. Thus, the development of vaccines using patients' exosomes would probably add no benefits to the treatment of AML; alternately, exosomes from cultured cells may represent an effective way for maturing DCs into a cytotoxic phenotype, without the immunosuppression observed with patients' exosomes. (C) 2019 Elsevier Ltd. All rights reserved371113771383FAPESP – Fundação de Amparo à Pesquisa Do Estado De São Paulo2011/51959-

Protection against Paracoccidioides brasiliensisinfection in mice treated with modulated dendritic cells relies on inhibition of interleukin-10 production by Cd8(+) T cells

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOParacoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensis conidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are antigen-presenting cells with the unique ability to direct the adaptive immune response by the time of activation of naive T cells. This study was conducted to test whether extracts of P. brasiliensis would induce maturation of DCs. We found that DCs treated with extracts acquired an inflammatory phenotype and upon adoptive transfer conferred protection to infection. Interestingly, interleukin-10 production by CD8(+) T cells was ablated following DC transfer. Further analyses showed that lymphocytes from infected mice were high producers of interleukin-10, with CD8(+) T cells being the main source. Blockage of cross-presentation to CD8(+) T cells by modulated DCs abolished the protective effect of adoptive transfer. Collectively, our data show that adoptive transfer of P. brasiliensis-modulated DCs is an interesting approach for the control of infection in paracoccidioidomycosis.Paracoccidioidomycosis is a systemic infection prevalent in Latin American countries. Disease develops after inhalation of Paracoccidioides brasiliensisconidia followed by an improper immune activation by the host leucocytes. Dendritic cells (DCs) are ant1463486495FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP [2013/08194-9, 2011/17965-3]FAPESP [2012/22131-7, 2013/01401-9, 2014/02631-0]2013/08194-9; 2011/17965-3; 2012/22131-7; 2013/01401-9; 2014/02631-

SEMA3A partially reverses VEGF effects through binding to neuropilin-1

Cross-talk between hematopoietic stem cells (HSCs) and bone marrow stromal cells (BMSCs) is essential for HSCs regulation and leukemogenesis. Studying bone marrow of myelodysplasia patients, a pre-leukemic condition, we found mRNA overexpression of vascular endothelial growth factor A (VEGFA) in CD34+ HSCs and semaphorin 3A (SEMA3A) in BMSCs. To better understand the role of VEGFA and SEMA3A in leukemogenesis, we recruited 30 myelodysplastic syndrome (MDS) patients, 29 acute myeloid leukemia (6 secondary to MDS) patients and 12 controls. We found higher VEGFA expression in de novo AML patients (without prior MDS) group (p = 0.0073) and higher SEMA3A expression in all BMSCs patient's samples compared to control group. We then overexpressed VEGFA in an acute myelogenous leukemia cell line, KG1 cells, and in normal CD34+ cells. This overexpression increased KG1 (p = 0.045) and CD34+ cell (p = 0.042) viability and KG1 (p = 0.042) and CD34+ cell (p = 0.047) proliferation. Moreover, KG1 and CD34+ cells overexpressing VEGFA also had increased proliferation when co-cultured with human marrow stromal HS5 cells (p = 0.045 and p = 0.02, respectively). However, co-culture of these transformed cells with HS5 cells overexpressing SEMA3A reduced KG1 (p = 0.004) and CD34+ (p = 0.009) proliferation. Co-culture of KG1 transformed cells with HS27 cells overexpressing SEMA3A reduced KG1 proliferation as well (p = 0.01). To investigate whether the dominant SEMA3A effect over VEGFA could be due to competition for neuropilin1 receptor (NRP1), we performed immunoprecipitation with anti-NRP1 antibody of cell extracts of co-cultured KG1 and HS5 cells, induced or not by VEGFA and SEMA3A recombinant proteins. Results showed a preferential association of NRP1 with SEMA3A, suggesting that SEMA3A can partially reverse the effects caused by the VEGFA preventing its binding with the NRP1 receptor. Since both hematopoietic cells, leukemic and normal, showed similar behavior, we suppose that the attempt to reversion of VEGF effects by SEMA3A is a homeostatic phenomenon in the hematopoietic niche. Finally, we conclude that VEGFA overexpression confers AML cell advantages and SEMA3A may partially reverse this effect; thus, SEMA3A protein combined with VEGFA inhibitors could be beneficial for AML treatment

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

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

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

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

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