Interaction of type II alveolar epithelial cells and dendritic cells in Mycobacterium tuberculosis infection: the role of HIF-1?

Author(s)

Tamara Silva Rodrigues

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Medicina de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-02-13

Examining board members

Vania Luiza Deperon Bonato; José Carlos Farias Alves Filho; André Luiz Barbosa Báfica; Denise Morais da Fonseca

• Advisor: Vania Luiza Deperon Bonato

Abstract

Tuberculosis (Tb) is a chronic infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). In the alveolar space, the bacillus contacts cells from the immune system, such as dendritic cells (DCs), as well as alveolar epithelial cells (AEC). In Tb, the transcription factor HIF-1? (Hypoxia-inducible factor 1- alpha) is accumulated in infected macrophages and adjacent alveolar epithelial cells. Therefore, the aim of this work was to investigate the role of HIF-1? in the proinflammatory response of type II AEC (AEC-II) and modulation of DCs function upon contact with AEC-II during Mtb infection. MLE-15 cells were infected with Mtb H37Rv (MOI10) to evaluate the permissiveness to infection (electron microscopy), killing ability (CFU) and cell activation through cytokine analysis (ELISA), nitrite (Griess assay), TLR2, TLR9 and HIF-1? (qPCR and / or Western blotting). Bone marrow derived DCs (BMDCs) modulation by AEC-II, were analyzed directly (contact) or indirectly (conditioned medium - CM) of uninfected (CM-NIC) or infected (CMIC) MLE-15. We determined cytokines (ELISA), nitrite (Griess Assay), HIF-1? expression, glycolytic enzymes, co-stimulatory molecules and CCR7 (flow cytometry). Chemotaxie assay was performed on Boyden camera. Proliferation of naive CD4 + T cells in co-culture with BMDCs and maintenance of Th17 were assessed by flow cytometry. Eventually, BMDCs were previously infected with Mtb (MOI2) and then stimulated with CM-NIC or CM-IC. MLE-15 cells were permissive to infection, failing to control the bacilli growth. Infection induced increased production of IL-6, NO2-, CCL5, S100A9 and IFN-?. Despite the initial accumulation of HIF-1?, gene expression dropped over time. The gene expression of TLR2 and TLR9 was also increased. Positive regulation of HIF-1? in epithelial cells induced a reduction of IL-6 and NO, but did not significantly interfere with the number of CFU. BMDCs stimulated with CM-IC showed higher production of IL-1?, IL-12, IL-6, and IL-10, greater GLUT1 and HK2 gene expression, in addition to the initial12 accumulation of HIF-1?, which was degraded within 24 hours, accompanied by low iNOS expression. The expression of MHC-II, CD80, CD86 and CCR7 was increased in BMDCs undergoing CM-IC, while the induction of HIF-1? accumulation through its stabilizer, DMOG, was able to negatively revert this response. The higher maturation of BMDCs resulted in a greater proliferation of naive CD4 + T cells, but hampered induction of CD4 + IFN? + T cells. However, cytokines produced favor the maintenance of IL-17 producing CD4 + cells. The phenotype of higher maturation was lost in Mtb-infected BMDC, accompanied by low TNF production and high IL-10 production. In conclusion, HIF-1? showed an anti-inflammatory function, reducing the production of proinflammatory molecules by AEC-II and negatively regulating the maturation and migration of DCs. In addition, although Mtb-infected AEC-II favor maturation and migration of DCs, Mtbinfection of DCs is capable of subverting this response (AU)

• FAPESP's process: 17/16538-0 - The role of HIF-1a in the interaction of type II alveolar epithelial cells and dendritic cells during the immune response against Mycobacterium tuberculosis
• Grantee: Tamara Silva Rodrigues
• Support Opportunities: Scholarships in Brazil - Master