Analysis of Innate Immune Signaling Networks

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Innate immune cells constantly evaluate host mucosal surfaces and peripheral tissues for signs of infection or injury. The host must find a balance between tolerance of beneficial microorganisms and minor non-pathological microbial encounter vs. the development of a robust immune response to more serious infections. Emerging evidence suggests that this decision is made by the cell based on the strength and combination of signals it receives from its engagement with microorganisms and endogenous stimuli. These signals are sensed primarily by various classes of pattern recognition receptors (PRR), and while there has been remarkable progress in characterizing the individual signaling pathways induced by these receptors, relatively few studies have addressed how immune cells integrate combined PRR inputs and the combination of these signals with others arising from soluble host derived substances such as cytokines, lipids, and metabolites. To address how the TLR signaling network might mediate responses specific to combined TLR stimuli, we have investigated the localization dynamics of proximal TLR pathway components in response to single vs. combined ligands. In FY2020 we have submitted a study which finds that upon dual-TLR ligand stimulation, IRAK1 generates cytosolic clusters that lack other myddosome components, form after the initial steps of TLR signaling, and permit canonical signaling and transcription to proceed normally. These IRAK1 clusters selectively recruit TRAF6, TBK1, MKK7 and its downstream MAPK JNK, as well as components of the NLRP3 inflammasome. This allows for signal flux redistribution from TLRs to inflammasomes and facilitates inflammasome licensing through an MKK7-JNK axis, which is defective in Irak1-/- mice. Furthermore, this defect in Irak1-/- mice manifests in increased susceptibility to inflammasomesensitive pathogens and diminished IL-1 production from inflammasomes after co-TLR priming. Thus, IRAK1 forms a coordinating hub for coincidence detection of microbial signals, which diversifies combined TLR input signals to recruit and activate additional PRR pathways in response to increasingly complex pathogenic stimuli. This uncovers a strategy that may be employed by innate immune cells as a threat assessment and thresholding mechanism for inflammasome activation (Vayttaden et al. Submitted. bioRxiv doi.org/10.1101/2019.1112.1126.888776. (2019)). In additional studies of the macrophage response to single and pairwise combinations of TLR ligands, we have previously identified characteristics of signaling pathway synergy and antagonism in signaling and cytokine outputs (Lin et al (2017) Cell Syst. 5: 25; Gottchalk et al (2016) Cell Syst. 2: 378). These studies emphasized the importance of feedback control in regulation of signaling flux, and in FY 2020 we published a collaborative study with Ron Germains Lymphocyte Biology Section of the LISB, wherein a rigorous analysis of macrophages exposed to a matrix of increasing concentrations of paired TLR ligands identified an acute negative feedback phenomenon that is selectively engaged during gram-negative bacterial infection. This anti-inflammatory feedback control is dependent on specific negative regulatory genes that are induced by type-I IFN, and has features that are independent of the established IL-10-dependent feedback loop (Gottschalk et al, Elife 8. https://doi.org/10.7554/eLife.46836. (2020)). 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. Recent studies, including the work above, 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 FY2020, we have used 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 (Dorrington et al, submitted). Our IRAK1 study described earlier 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 FY2020, we established 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. As two very distinct JNK activation states were associated with two separate but critical signaling events for manifestation of cellular inflammation, we sought to investigate putative regulators from upstream signaling cascades. To do so, 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. We find that few MAP3K inhibitors delay cell permeation following inflammasome activation, with the exception of MAP3K5. Our multi KTR macrophages treated with MAP3K5 inhibitors exhibited normal ROS generation, but exhibited dampened and delayed calcium flux and second phase JNK activation. Interestingly, in a parallel collaborative study with Aleksandra Nita-Lazars Functional Cellular Networks Section, we identified MAP3K5 as an important regulatory node in modules of ADP-Ribosylated proteins which undergo dynamic modulation of their protein interactions in response to LPS stimulation. This highlights a potentially central role for this kinase in coordinating multiple aspects of the TLR and inflammasome signaling pathways (Daniels et al, J Proteome Res, In press. (2020)).