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Role of P2X7 receptor and CD39 ectonucleotidase in follicular regulatory T lymphocytes during experimental Malaria

Grant number: 20/01197-6
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): April 01, 2020
Effective date (End): November 30, 2021
Field of knowledge:Biological Sciences - Immunology - Cellular Immunology
Principal Investigator:Maria Regina D'Império Lima
Grantee:Paulo Henrique Lisboa Raeder
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated research grant:15/20432-8 - Intervention in signaling pathways associated with the recognition of cellular damage to reduce the pathology of severe forms of malaria and tuberculosis, AP.TEM

Abstract

Malaria is one of the most prevalent infectious diseases in human, accounting for more than 500,000 deaths annually worldwide. The immune response against plasmodium helps to control the infection but can also contribute to its severity. Necrotic death causes the release of ATP, a sign of damage to cells in the immune system that activate inflammatory and tissue repair responses. ATP can also be released by activated and apoptotic cells through a channel called panexin-1, which results in autocrine and paracrine cell signaling. The P2X7 receptor is present in cells of the immune system and activates with high concentrations of extracellular ATP. A recently published study of ours showed the involvement of ATP and the P2X7 receptor in protecting against Malaria caused by Plasmodium chabaudi AS (PcAS). This receptor contributes to the differentiation of CD4 + helper 1 lymphocytes (Th1) to the detriment of follicular CD4 + helper T lymphocytes (Tfh), which cooperate with B lymphocytes for the production of antibodies. In addition, the absence of the P2X7 receptor leads to a reduction in the population of activated regulatory T lymphocytes (Treg) (CD122+). Based on the above considerations, the objective of this project is to establish the role of the P2X7 receptor in the generation and activity of follicular regulatory T lymphocytes (Tfr) in experimental Malaria. To facilitate the analysis of Tfr lymphocytes, we will use C57BL/6 FoxP3-GFP P2X7-/- mice, whose splenic cells will be compared with those of C57BL/6 FoxP3-GFP mice. The following parameters will be evaluated in the acute and chronic phases of infection by PcAS parasites: (A) The expression of functional markers (CD25, CD39, CD44, CD62L, CD69, CD73, CD122, PD-1, CXCR5, A2A, ICOS, GITR and CTLA-4) and transcription factors (T-bet and Bcl6), as well as the production of regulatory cytokines (IL-10 and TGF-b); (B) The suppressive activity in relation to the production of IgG antibody by B lymphocytes stimulated with erythrocytes parasitized in the presence of Tfh lymphocytes, as well as the activation of B lymphocytes; (C) The expression of mRNAs related to CD4 + T lymphocyte differentiation and Tfr lymphocyte function; (D) The response to eATP, evaluating proliferation, cytokine production and regulatory activity, as well as Ca2 + entry and nuclear translocation of NFAT (nuclear factor of activated T-cells); (E) The same parameters described in the AD items obtained in C57BL/6 CD28-/- mice transferred with Tfh lymphocytes (CD4 + FoxP3-ICOS + CXCR5+), together with Tfr lymphocytes (CD4 + FoxP3 + ICOS + CXCR5+) from C57BL mice/6 Foxp3-GFP + or C57BL/6 Foxp3 + P2rx7-/- mice, which were previously infected. This last approach allows us to verify the importance of P2X7 receptor expression in TFR cells for the regulation of the immune response of Tfh and B lymphocytes. This study will contribute to understand how the damage signals affect the immune response against plasmodium. (AU)