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 complement components. To evaluate the macrophage response to combined microbial stimuli, we have profiled the transcriptional response of primary macrophages to single and pairwise combinations of toll-like receptor (TLR) ligands that induce either one or both of the MyD88 and TRIF branches of the TLR signaling pathway. We find predominantly less than additive levels of mRNA induction in response to ligands that both activate Myd88, likely due to saturation of shared signaling effectors in this pathway. On the other hand, in ligand combinations activating both Myd88 and TRIF, a select subset of key immune response mediators are induced to greater than additive levels. We predict that this synergistic response requires crosstalk between the adapter pathways, and that it signals to the host the presence of a more dangerous pathogenic challenge. We seek to determine the molecular mechanisms underlying this selective pattern of non-linear macrophage responses to combined ligands, as it would provide important insight to signaling pathway crosstalk during a microbial infection. In FY17, we completed and published a study to identify regulators of dual TLR ligand-induced IL-6 secretion, an important link between the innate and adaptive immune systems. The transcriptomic analysis described above identified approximately 200 genes with characteristics that could implicate them as regulators of the synergistic production of IL6 by dual TLR-activated macrophages. These genes were targeted in a focused siRNA screen, and approximately 30 putative hits were subjected to high throughput qPCR analysis to determine if they selectively affect different classes of TLR-induced inflammatory mediators. We identified four genes required for IL-6 secretion and not previously associated with dual-TLR induced cytokine expression; Zscan12, Helz2, Phf11d and Sertad3. All four genes were required to sustain expression of a wide range of TLR-induced immune effectors including Socs3, Lcn2, Edn1, Nos2, Cd40, Il27, Il6, Il12b, Il1a and Il1b. All four genes contain DNA binding motifs suggesting that they likely function to modify the macrophage chromatin to permit sustained expression of immune effectors. This study reveals key molecular details of how contemporaneous signaling through multiple TLRs, as would often be the case with complex pathogen challenges, produces biological outcomes distinct from those of single ligand exposures more typically used to characterize TLR pathways. To further 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 FY17, we have made considerable progress in a study which has identified an IRAK1-containing complex that directly links multi-TLR signaling to inflammasome activation. IRAK1 containing bodies, that were distinct from myddosomes and trifosomes, were formed on co-stimulation of TLR4 and TLR1/2 or on bacterial infection. The IRAK1 complexes regulated MAPK signaling and sequestered signaling components from IRAK2, resulting in limitation of TLR signaling flux. We found these complexes simultaneously recruited the inflammasome adaptor ASC, facilitating dual-TLR ligand-primed inflammasome activation that was diminished in Irak1-/- macrophages. In a Yersinia pseudotuberculosis infection model where IL-1 responses are required for effective host defense, we observe increased susceptibility and bacterial burden in Irak1-/- mice, suggesting a critical role for IRAK1-containing complexes in shaping the immune response to a multi-PAMP pathogen. Further insight to the transcriptional programs activated by the Myd88 and TRIF-dependent signaling branches within the TLR pathways has arisen this year from our study of hits identified in our genome-wide screens of the LPS response in human macrophages (project AI001106). Activation of the TLR4 signaling pathway by LPS leads to induction of both inflammatory and interferon-stimulated genes, however, the mechanisms through which these coordinately activated transcriptional programs are balanced to promote an optimal innate immune response remain poorly understood. In our genome-wide siRNA screen of the LPS-induced TNF-alpha response, we identified the interferon-stimulated protein IFIT1 as a negative regulator of the inflammatory gene program. Transcriptional profiling further identified an unexpected positive regulatory role for IFIT1 in type I interferon expression, implicating IFIT1 as a reciprocal modulator of different LPS-induced gene classes. We find that these effects of IFIT1 are mediated through modulation of a Sin3A-HDAC2 transcriptional regulatory complex at LPS-induced gene loci. Beyond the well-studied role of cytosolic IFIT1 in restricting viral replication, our data demonstrate an unappreciated function for nuclear IFIT1 in differential transcriptional regulation of separate branches of the LPS-induced gene program. The above discovery of the regulatory role for IFIT1 on interferon-stimulated genes (ISGs) in the LPS response suggest an important role for this gene program in the host response to Gram-negative bacterial infection. In FY17, we initiated a project to investigate this further. Using the Gram-negative bacteria Burkholderia cenocepacia, which replicates in the host cell cytoplasm, we have uncovered a negative relationship between type I IFN signaling and the replicative potential of invading bacteria. Firstly, cells infected with these bacteria produce significant levels of IFN beta as well as the products of IFN-stimulated genes (ISGs). Cells pre-treated with IFN beta are less permissive to bacterial replication, while cells from mice lacking the type I IFN receptor (Ifnar1) have increased levels of bacterial replication, and higher amounts of cell death, compared to wild-type cells. Interestingly, this phenotype is type I IFN-specific, as pre-treatment with IFN-gamma has no effect on bacterial replication. In contrast, these phenotypes are reversed in cells infected with Salmonella, which replicates within in modified vacuole. This suggests that different IFN classes (and induced ISGs), may confer host protection against microbes replicating in different cellular niches. Additionally, we find that siRNA knockdown of select ISGs, such as IFIT1, leads to increased bacterial replication within macrophages. Ongoing transcriptomic profiling of infected cells is now directing a targeted, pooled CRISPR screen against bacteria-induced ISGs in order to determine which of these genes are most important for protection of macrophages from intracellular bacterial replication.
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