Analysis of Innate Immune Signaling Networks

National Institute Of Allergy And Infectious Diseases

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Abstract

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. Our previous studies have highlighted the importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes. However, the control of opportunistic pathogens and especially intracellular bacteria by type I IFNs remains less appreciated. In FY2021, we published a study using the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses. Our previous work has demonstrated that JNK MAPK plays an important role in inflammasome licensing that occurs independently of the known requirement for MAPKs in the TLR-induced priming of inflammasome genes. In FY2021, we continued to develop and leverage an inflammasome activation protocol that could separate events associated with priming and later triggering steps, and used this setup to screen macrophages with specific, potent inhibitors of the three major MAPK signaling branches (ERK, p38 and JNK), to verify their effects on inflammasome assembly, IL-1 release and pyroptosis. Only on target JNK inhibition diminished the response kinetics of these outputs, while p38 inhibitors increased them. We further established multiplex MAPK kinase translocation reporter (KTR) macrophages to assess single cell signaling dynamics for the major MAPK signaling branches, and found that JNK and p38 exhibit biphasic activity during inflammasome activation and that distinct JNK isoforms are utilized at different times during this process. Further single cell imaging experiments established that the first phase of JNK activation corresponded closely with production of cellular ROS, whereas the second phase of JNK activation occurred shortly before a pre-pyroptotic intracellular calcium spike. Corroborating this observation, JNK inhibitors blunted both of these required inflammatory signals. To investigate putative regulators from upstream signaling cascades, we set up a kinetic cell death assay to screen across broadly acting MAP3K inhibitors as well as inhibitors of selected upstream receptor kinases for their impact on inflammasome-induced cell death. This has identified specific MAP3Ks regulating multiple phases of the inflammasome activation process. In additional studies of inflammasome regulation this fiscal year, we are considering not only microbial ligands, but also host-derived triggers associated with inflammatory diseases, such as the biologically abundant saturated fatty acid palmitate which is associated with metabolic syndromes. Palmitate has been described as an NLRP3 inflammasome trigger that depends on disruption of ER or lysosomal membranes to activate the inflammasome. However, the role of ROS, which are essential for NLRP3 activation by many other triggers, has remained unclear for palmitate. Consistent with previous studies, we found that classic NLRP3 triggers had a strong ROS requirement, though the most important ROS sources differ between triggers. In contrast, we found that the inflammasome response to palmitate was independent of the ROS sources that were required by ATP and nigericin, suggesting that palmitate triggering of the inflammasome is mechanistically distinct. Notably, the pattern of requirements for the palmitate-triggered inflammasome is very similar to that seen in noncanonical inflammasome activation by intracellular LPS. These observations suggest that palmitate may be an endogenous trigger of the noncanonical inflammasome, identifying an additional role for this pathway as a sensor of abnormally elevated fatty acids in the disease states that occur as part of metabolic syndromes.