P044
In vitro modelling of the tuberculosis granuloma for antimicrobial and host-directed drug screening
B Fontes(1) R Ferreira(1) M Mandal(2) R Pinheiro(1) E Anes(2) PJG Bettencourt(1) S David(2,3) D Pires(1,2,3)
1:Center for Interdisciplinary Research in Health, Católica Medical School, Universidade Católica Portuguesa, Rio de Mouro 2635-631, Portugal; 2:Host-Pathogen Interactions Unit, Research Institute for Medicines, iMed.ULisboa, Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisbon, Portugal; 3:Departamento de Genética Humana, Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), 1649-016 Lisbon, Portugal
To accelerate the discovery of new antimicrobials or host-directed therapies, we have developed an in vitro 3D cell culture model of the TB granuloma. The model can be easily applied using commercial systems to encapsulate human peripheral blood mononuclear cells (PBMC) infected with Mycobacterium tuberculosis H37Rv (Mtb). The 3D granuloma structure exhibited a drastically lower Mtb burden with bacterial growth confined to a restricted number of cells, in contrast to the standard monolayer model. Manipulation of the macrophage and lymphocyte content showed clear distinctions in the ability to control Mtb infection, and introduction of lymphocytes helped control the infection even when the cells were physically separated. Different multiplicities of infection (MOI) didn’t affect the ability of Mtb to survive and replicate. Yet, at the lowest MOI, we observed a lower expression of proinflammatory markers and increased expression of anti-inflammatory markers. The model allowed infection assays to last up to one month, with cell viability mainly affected by the bacterial burden. The onset of bacterial replication correlated with increased cell death, primarily in the macrophage population. Some of the granulomas exhibited a distinct region of dead cells at the centre of the structure. The treatment with antibiotics showed greater resistance to isoniazid, followed by rifampicin, and increased susceptibility to pyrazinamide compared to the standard monolayer infection model.
In conclusion, the 3D model resembles some features expected in the TB granuloma, which we will explore in future drug discovery studies.