Mycobacterium bovis Bacillus Calmette-Guérin-Infected Dendritic Cells Induce TNF-α-Dependent Cell Cluster Formation That Promotes Bacterial Dissemination through an In Vitro Model of the Blood-Brain Barrier.

CNS tuberculosis (CNSTB) is the most severe manifestation of extrapulmonary tuberculosis infection, but the mechanism of how mycobacteria cross the blood-brain barrier (BBB) is not well understood. In this study, we report a novel murine in vitro BBB model combining primary brain endothelial cells, Mycobacterium bovis bacillus Calmette-Guérin-infected dendritic cells (DCs), PBMCs, and bacterial Ag-specific CD4+ T cells. We show that mycobacterial infection limits DC mobility and also induces cellular cluster formation that has a similar composition to pulmonary mycobacterial granulomas. Within the clusters, infection from DCs disseminates to the recruited monocytes, promoting bacterial expansion. Mycobacterium-induced in vitro granulomas have been described previously, but this report shows that they can form on brain endothelial cell monolayers. Cellular cluster formation leads to cluster-associated damage of the endothelial cell monolayer defined by mitochondrial stress, disorganization of the tight junction proteins ZO-1 and claudin-5, upregulation of the adhesion molecules VCAM-1 and ICAM-1, and increased transmigration of bacteria-infected cells across the BBB. TNF-α inhibition reduces cluster formation on brain endothelial cells and mitigates cluster-associated damage. These data describe a model of bacterial dissemination across the BBB shedding light on a mechanism that might contribute to CNS tuberculosis infection and facilitate treatments.

A Novel In Vitro Mouse Model to Study Mycobacterium tuberculosis Dissemination Across Brain Vessels: A Combination Granuloma and Blood-Brain Barrier Mouse Model.

In vitro culture models of the blood-brain barrier (BBB) provide a useful platform to test the mechanisms of cellular infiltration and pathogen dissemination into the central nervous system (CNS). We present an in vitro mouse model of the BBB to test Mycobacterium tuberculosis (Mtb) dissemination across brain endothelial cells. One-third of the global population is infected with Mtb, and in 1%-2% of cases bacteria invade the CNS through a largely unknown process. The "Trojan horse" theory supports the role of a cellular carrier that engulfs bacteria and carries them to the brain without being recognized. We present for the first time a protocol for an in vitro BBB-granuloma model that supports the Trojan horse mechanism of Mtb dissemination into the CNS. Handling of bacterial cultures, in vivo and in vitro infections, isolation of primary astroglial and endothelial cells, and assembly of the in vitro BBB model is presented. These techniques can be used to analyze the interaction of adaptive and innate immune system cells with brain endothelial cells, cellular transmigration, BBB morphological and functional changes, and methods of bacterial dissemination. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Isolation of primary mouse brain astrocytes and endothelial cells Basic Protocol 2: Isolation of primary mouse bone marrow-derived dendritic cells Support Protocol 1: Validation of dendritic cell purity by flow cytometry Basic Protocol 3: Isolation of primary mouse peripheral blood mononuclear cells Support Protocol 2: Isolation of primary mouse spleen cells Support Protocol 3: Purification and validation of CD4+ T cells from PBMCs and spleen cells Basic Protocol 4: Isolation of liver granuloma supernatant and determination of organ load Support Protocol 4: In vivo and in vitro infection with mycobacteria Basic Protocol 5: Assembly of the BBB co-culture model Basic Protocol 6: Assembly of the combined in vitro granuloma and BBB model.