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Our body is continuously exposed to a diverse array of infectious agents, foreign antigens, and host-derived danger signals. The innate immune system plays a vital role as the first line of defence against these insults by protecting the host from infection and maintaining tissue homeostasis. Families of signalling receptors (e.g. TLRs) recognize invading microbes and co-ordinate an appropriate inflammatory response via the production of pro-inflammatory cytokines and type I interferons (IFNs) to clear infection. However, inappropriate activation of innate immune receptors and unregulated production of inflammatory mediators is thought to be the basis for many acute and chronic inflammatory conditions. Indeed, excessive IFN production is implicated in the pathogenesis of lupus and atherosclerosis. Maintaining a balance between the beneficial and detrimental effects of IFNs is therefore crucial and their production must be tightly regulated. Yet these precise regulatory mechanisms remain poorly understood. We have discovered that macrophages deficient in the transcriptional modulator, ATF3, display significantly greater IFN β production following activation of TLRs and other innate immune receptors, as well as reduced LCMV viral infection. In contrast, overexpression of ATF3 in macrophages reduced the induction of IFN β and increased viral replication. Utilizing a range of molecular biology, biochemical and immunological techniques, integrated with powerful bioinformatics approaches we have investigated the molecular mechanisms responsible for ATF3-mediated regulation of IFN β . Our ATF3 ChIP-seq data has revealed that ATF3 regulates IFN β by binding directly to its promoter. Furthermore, we have found that ATF3 itself is regulated by IFNs and thus forms part of a negative feedback loop during the induction of IFN signalling. Together our findings indicate ATF3 is a critical regulator of the IFN innate immune response. A better understanding of how the innate immune response is regulated is imperative for the development of future therapeutic strategies against autoimmune and inflammatory diseases.

NLRP3 is an intracellular pattern-recognition receptor that senses many different activators including bacterial toxins and endogenous danger signals. Upon activation, the NLRP3 inflammasome is formed, leading to the secretion of pro-inflammatory cytokines and cell death. Because of its involvement in many diseases such as atherosclerosis and Alzheimer’s Disease, a better understanding of NLRP3 regulation may help to shape therapeutic approaches. The aim of this study was to identify post-translational modifications on NLRP3 and their impact on inflammasome activation. We applied mass spectrometry of NLRP3 to identify post-translational modifications. We identified ubiquitinated and phosphorylated residues. To analyze the function of the modifications, we mutated the residues in NLRP3 expression plasmids and used them for reconstitution of NLRP3-deficient macrophages or overexpression studies in HEK293T cells. Ubiquitinylated residues were found in the NACHT and LRR domains of NLRP3 and mutation of these residues resulted in hyperactive NLRP3. In addition, mutation of one of the phosphorylated residues to a phosphomimetic residue showed reduced IL-1beta release and caspase-1 cleavage upon NLRP3 inflammasome activation, whereas mutation to alanine had no effect. Further studies in HEK293T cells found that the recruitment of the downstream adaptor ASC as well as the self-interaction of NLRP3 were reduced with the phosphomimetic residue. Thus, NLRP3 is likely regulated by post-translational modification and needs to be deubiquitinylated and possibly dephosphorylated before activation. Knowledge about the modifications and the pathways involved may help shape novel strategies for treating diseases that involve NLRP3.

Sensing of nucleic acids (NAs) by Toll-like receptors (TLRs) is crucial in the host defence against viruses and bacteria. Yet, these TLRs can contribute to autoimmunity. UNC93B1 is a key-regulator of NA-sensing TLRs by controlling the translocation of TLRs from the ER into endolysosomes. Without functional UNC93B1, NA-sensing TLRs are retained in the ER, incapable of ligand recognition. Patients lacking UNC93B1 show defective signalling of endosomal TLRs and are at increased risk to develop herpes simplex virus encephalitis. To date, molecular insights into UNC93B1-mediated regulation of human NA-sensing TLRs are lacking. In this study, we aimed to investigate the role of trafficking motifs and post-translational modifications of UNC93B1 for human endosomal TLR activity. To this end, we utilised mass spectrometry in order to identify post-translational modification sites and prepared mutant forms of UNC93B1 by site-directed mutagenesis. EBV immortalized B cells from UNC93B1-deficient patients as well as UNC93B1 KO THP1s generated by CRISPR-Cas-based genome editing were reconstituted with wild type or mutant forms of UNC93B1 and assessed for endosomal TLR activity. The localization of UNC93B1 was analysed by confocal microscopy. The destruction of either trafficking motifs or post-translational modification sites resulted in receptor-, ligand-, and cell-type specific defects of human endosomal TLRs. Furthermore, mass spectrometry revealed that wild type versus mutant forms of UNC93B1 differed in terms of interaction partners and cellular compartment signatures. Our study reveals another level of complexity of UNC93B1-mediated TLR regulation and thereby contributes to the understanding of nucleic-acid sensing TLR biology.

Endosomal toll-like receptors, namely human TLR3, TLR7, TLR8, and TLR9 sense nucleic acids (NAs) derived from viruses and bacteria. Yet, these TLRs can also contribute to autoimmunity by recognizing host NAs. NA-sensing TLR activity is highly regulated by the correct subcellular localization. Experimental mislocalization of endosomal TLRs to the cell surface leads to insufficient microbial recognition and inflammatory disease due to the recognition of endogenous NAs. Under physiological conditions, efficient activation of murine TLR7 and TLR9 is restricted to acidic compartments due to the need for proteolytic processing. TLR trafficking within the endolysosomal system further determines the nature of signaling events, as type I IFN and proinflammatory responses are initiated from different compartments. UNC93B1 is a key-regulator of NA-sensing TLRs by controlling the translocation of TLRs from the endoplasmic reticulum (ER) into endolysosomes. Without functional UNC93B1, NA-sensing TLRs are retained in the ER, incapable of ligand recognition. Patients lacking UNC93B1 display defective signaling of endosomal TLRs, and are at increased risk of developing herpes simplex virus encephalitis. The precise mechanism of how UNC93B1 regulates TLR trafficking remains unknown. Recently, a highly conserved tyrosine-based motif of UNC93B1 was identified. This motif is essential for the correct localization and function of murine TLR9, but dispensable for murine TLR7, TLR11, TLR12, and TLR13. TLR11, TLR12, and TLR13 are not expressed in humans. In contrast, human TLR8 plays a crucial role in the initiation of immune responses to certain pathogens. To date, molecular insights into UNC93B1-mediated trafficking of human endosomal TLRs are missing. Our study reveals that the tyrosine-based motif of UNC93B1 differentially regulates human TLR7, TLR8, and TLR9 responses and thereby contributes to the understanding of human TLR trafficking.