Tuberculosis is an ancient disease that is caused by Mycobacterium tuberculosis (Mtb). Mtb has evolved and adapted to the human host over millenia, giving rise to a complex interplay between the bacterium and the human immune system. This interplay has proven hard to skew to the favor of the host, complicating the development of both vaccines and efficient treatment strategies. Tuberculosis still causes about 1,5 million deaths every year.

During active stages of the disease, Mtb causes extensive necrotic cell death and pathological inflammation in infected lung tissue, allowing the bacteria to spread. It was long thought that necrosis was an uncontrolled path to cell death, but recently several forms of regulated necrosis have been described. The in-depht understanding of these pathways in the context of Mtb could be a promising new opportunity for interfering with disease development.

Pyroptosis is a regulated necrosis pathway which was intriguing to us because it is highly inflammatory. Pyroptosis is caused by activation of innate immune sensors called inflammasomes, and leads to release of the fever-causing cytokine IL-1β. Common for the various types of necrotic cell death caused by Mtb is that they depend on a specialized Mtb secretion system known as ESX-1. ESX-1 activity is also closely correlated to virulence of Mycobacterial strains.

The most prominent effect of ESX-1 is to damage host cell membranes, in particular the phagosomal membrane surrounding Mtb after ingestion by host phagocytes. This led us to the main questions we wished to address in this work: To what extent does Mtb cause inflammasome activation, and how does phagosomal damage lead to inflammasome activation and pyroptosis? Due to the complex and heterogenous course of the Mtb infection, we wished to discern cause and effect of key events at the single-cell level. We were lucky to have a newly opened BSL3 lab equipped with a modern confocal microscope capable of long term live cell imaging. In addition, we had previously developed correlative light and electron imaging technologies for infected cells so we could better understand the ultrastructure of the processes we studied (Fig. 1). Together with engineered cell lines expressing fluorescent reporters of key processes, these technologies provided the tools necessary to visualize the interrelated processes of membrane damage, inflammasome activation and cell death.

Through our microscopic investigations, we found that Mtb activated a specific inflammasome called the NLRP3 inflammasome in human monocytes and macrophages, and that inflammasome activation was dependent on potassium efflux from the cell, common to what has been shown for most NLRP3 inflammasome activators. We saw damage to Mtb-containing phagosomes before inflammasome activation in most cells, but we were still puzzled by how this could lead to potassium leakage. Indirect explanations that have been proposed, such as damage to mitochondria or leakage of hydrolytic enzymes from damaged phagolysosomes, were also dead ends in our hands.

The breakthrough came when we decided to broaden our investigation of membrane damage to include the outer plasma membrane of the host cells. Plasma membrane damage would provide a direct explanation for how potassium could leak from the cell and activate the inflammasome. Indeed, Mtb did cause plasma membrane damage in an ESX-1 and contact-dependent manner, and plasma membrane damage was more closely correlated to inflammasome activation than damage to the phagosomal membrane. There is an ongoing battle between the host and Mtb as well, as repair processes both at the phagosome and plasma membrane are immediately initiated by the host cell, attempting to counteract the damage.

The final insight came when we visualized markers for phagosomal and plasma membrane damage simultaneously. We saw that in many cases, ingested Mtb bacteria first damaged the phagosome and then proceeded to damage the plasma membrane from inside the host cell. This chain of events explained how phagosomal damage was linked to potassium efflux, inflammasome activation and pyroptosis. Interestingly we also observed a similar course of events when our cells were treated with silica crystals. Silica is the cause of silicosis lung disease and is a well-known sterile NLRP3 activator thought to act through phagolysosomal damage, similar to Mtb.

Our new findings highlight what could be a general mechanism to NLRP3 inflammasome activation through internal damage to the plasma membrane after ingestion. Further work will be needed to see if these events also occur in vivo, and how they might be regulated by cells and altered by drug treatments. Still, a clearer understanding of how these complex events occur in single cells is a first step towards better treatment strategies for tuberculosis and potentially other diseases with shared mechanisms of immune activation and tissue damage.

Study perfomed with the authors previous group at CEMIR, NTNU