Background

Tuberculosis (TB) is an air-borne disease that encompasses pulmonary and extra-pulmonary infections by the human pathogen Mycobacterium tuberculosis (Mtb). Causing an estimated 1.5 million deaths in 2019 (WHO, 2019), TB remains the world’s deadliest infectious disease. The hallmark of the immunopathogenesis of TB is the formation of structurally organized, multicellular clusters called granulomas (Gengenbacher and Kaufmann, 2012). These structures mainly consist of a core of infected and non-infected macrophages surrounded by a rim of lymphocytes. The hostile environment within granulomas pushes Mtb to enter a slow- or non-replicating dormant state likely associated with a latent form of the disease. Consequently, Mtb dormancy leads to increased tolerance to antibiotics targeting metabolic pathways active during mycobacterial replication.

New strategies are urgently needed in order to lessen the TB death toll. Such strategies may arise from a better understanding of the infection within the granuloma’s environment. However, the complex pathophysiology of Mtb infection makes the development of relevant preclinical models particularly challenging. Despite the widespread use of animal models in the TB field, the establishment of latency and granulomatous lesions comparable to the ones seen in humans can only be observed in non-human primates. Yet, drawbacks inherent to this animal model, such as its high cost or ethical concerns, have favored the development of in vitro models (Guirado and Schlesinger, 2013). In that context, several independent groups have reported in vitro models based on the infection of human peripheral blood mononuclear cells (PBMCs) with virulent Mtb (Kapoor et al., 2013; Guirado et al., 2015; Agrawal et al., 2016; Arbués et al., 2020; Tezera et al., 2017). Despite lacking some relevant cell types (e.g., neutrophils) and the full tissue conditions (such as lung structure or vascularization), such models are able to mimic the organization of human nascent granulomas, orchestrated by relevant cytokines.

The protocol described here notably incorporates two major advantageous features. On the one hand, Mtb-infected PBMCs are embedded in a three-dimensional (3D) matrix of collagen and fibronectin, main components of the lung extracellular matrix (Stek et al., 2018). On the other hand, Mtb displays features associated with dormancy such as accumulation of intracellular triacylglycerides into lipid inclusions, loss of acid-fastness, transcriptional changes leading to a shift in carbon and energy metabolisms, and increase in antibiotic tolerance (Kapoor et al., 2013). Remarkably, this model was also able to reproduce the differential rate of latent TB reactivation observed in the clinic upon treatment with different TNF-α-neutralizing biologics (Arbués et al., 2020). Some particular limitations of this model to take into account are the difficulty of adding new immune cells for dynamic studies or its relatively low throughput.

In conclusion, the in vitro granuloma model described hereunder is particularly relevant for studies focusing on mycobacterial dormancy as well as further understanding the mechanisms involved in Mtb resuscitation.