The innate immune system is designed to tightly regulate the magnitude of inflammatory responses to promote clearance of microorganisms without excessive tissue damage. Macrophages secrete cytokines and chemokines to incite inflammation in response to conserved microbial structures detected via the Toll-like receptors (TLRs). Although IL-1b and IL-18 expression is induced by TLR signaling, these cytokines are not secreted. A second signal detected by cytosolic sensors (including those in the Nod-likereceptor, or NLR family) activates caspase-1, a protease that cleaves IL-1 b and IL 18, leading to their secretion, and additionally triggers a form of programmed cell death called pyroptosis, a highly inflammatory form of cell death (Figure 1 below). Caspase-11 also triggers pyroptotic cell death. Constitutive activation of the inflammatory response, due to mutations in the cytosolic sensors, is associated with auto-inflammatory disease. Dysregulation of these responses results in high mortality in patients suffering from bacterial sepsis. Conversely, proper activation of the inflammatory response contributes to strong, balanced immune responses. Hence, targeting the inflammatory pathways can have beneficial effects for treating autoimmune disorders or in activating the immune response to augment vaccines.
We use various bacterial pathogens to probe cytosolic sensor pathways that defend against pathogens. We focus on inflammasome sensors, and several bacterial pathogens, including Salmonella, Burkholderia, and Listeria.
Caspase-1 discriminates virulent from avirulent bacteria. Caspase-1 is often activated in response to microbial virulence traits. This is one mechanism by which macrophages discriminate between virulent and avirulent bacteria. An example of a virulence trait detected through caspase 1 is the bacterial type III secretion system (T3SS) which functions to deliver bacterial effector proteins into the cytosol of host cells. These effectors reprogram host cell physiology to the benefit of the pathogen, thus it is important for the host to incite a more vigorous inflammatory response to bacteria expressing T3SS than avirluent bacteria. We have shown that T3SS is detected by host macrophages via cytosolic sensors that detect the accidental translocation of T3SS apparatus components or bacterial flagellin into the macrophage cytosol (Figure 2). (Miao et al., 2006; 2008; 2010b)
Caspase-11 discriminates cytosolic from vacuolar/extracellular bacteria. Recently, caspase-11 has also been found to trigger pyroptosis, and to contribute to septic shock. We found that caspase-11 detects bacteria that escape the phagosome into the cytosolic compartment, and is a critical defense system against cytosolic bacteria. Caspase-11 knockout mice are sensitive to infection by cytosolic bacteria, but resistant to LPS sepsis. Unraveling the mechanism by which cytosolic bacteria are detected will reveal unknown molecular mechanisms that underlie septic shock. (Aachoui et al., 2013)
Pyroptosis is a defense mechanism against intracellular bacteria. Pyroptosis is defined as programmed cell death that occurs after caspase-1 or -11 activation, and is the opposite of apoptosis. While apoptosis is non-lytic and non-inflammatory, pyroptosis is lytic and highly inflammatory. We showed that pyroptosis functions to lyse compromised macrophages and dendritic cells that harbor pathogens capable of replicating in the intracellular niche (either in the vacuole or cytosol). Bacteria released by pyroptosis are targeted to neutrophils and other extracellular defenses for destruction (Figure 3). (Miao et al., 2010a)
Shown in red are bacteria that have invaded host cells and escaped into the interior cytosolic compartment of the cell. Credit: Miao lab, UNC.
Figure 1. Inflammasomes detect cytosolic perturbations.
IL-1β and IL-18 secretion is regulated in a two step fashion. Their transcription is induced by Toll-like receptors, which detect extracellular microbe associated molecular patterns such as LPS. After transcription, pro-IL-1β and pro-IL-18 are held in reserve in the cytosol, unlike other cytokines and chemokines which are secreted after production. Inflammasomes regulate a proteolytic processing step that is required for IL-1β and IL-18 to be secreted. Inflammasomes, most of which are Nod-like receptors (NLRs) detect cytosolic perturbations. These may be microbial products that enter the cytosol (e.g. flagellin), or perturbations to normal cytosolic function caused by infection (e.g. toxins). Inflammasomes activate Caspase-1, a protease that cleaves the cytokines IL-1β and IL-18 to their mature and secreted forms, and also promotes a form of programmed cell death called pyroptosis. Adapted from Miao and Rajan, Front. Micro. 2011.
Figure 2. NLRC4 detects cytosolic flagellin and rod proteins.
Type III secretion systems (T3SS) are virulence factors used by numerous Gram-negative pathogens to deliver effector molecules to the cytosol of host cells. These effectors reprogram host cell physiology to the benefit of the pathogen. While T3SS is very efficient in selecting and transporting the dedicated effector proteins, it occasionally translocates other proteins. Two proteins that are mistakenly transferred are flagellin and the rod component of the T3SS apparatus. Macrophages and dendritic cells are able to detect T3SS activity by capitalizing on these mistakes made by the apparatus, detecting flagellin and rod protein via the cytosolic sensor NLRC4. Flagellin and rod are good targets for innate immune detection because they are relatively highly conserved and slow to evolve, unlike the effector proteins, which are variable between pathogens. Salmonella species express the SPI1 T3SS in the gut that is detected by NLRC4, but have evolved to evade this detection during the systemic phase of infection, when they replicate within macrophages. Salmonella express a different T3SS apparatus, SPI2, with a rod component that is not detected by NLRC4, and they repress flagellin expression. EC eukaryotic cytosol, PM plasma membrane, EX extracellular space, OM bacterial outer membrane, IM bacterial inner membrane, BC bacterial cytosol. Adapted from Miao and Rajan, Front Micro 2011.
Figure 3. Pyroptosis is a potent innate immune effector mechanism in vivo.
Caspase-1 activation triggers pyroptotic cell death in macrophages and dendritic cells. Like apoptosis and unlike necrosis/oncosis, pyroptosis is programmed, meaning that it requires the activation of specific caspases. Like necrosis/oncosis and unlike apoptosis, pyroptosis is lytic. Intracellular pathogens that replicate within macrophages, such as S. typhimurium, must effectively evade pyroptosis in order to stay within an infected cell. Otherwise, detection by the inflammasome activates Caspase-1 and triggers pyroptosis, which releases the pathogen to the extracellular space. Once in the extracellular environment, the pathogen is exposed to additional clearance mechanisms, including phagocytosis and killing by neutrophils. Adapted from Miao et al., Nat Immunol 2010.