TXM peptides inhibit SARS-CoV-2 infection, syncytia formation, and lower inflammatory consequences.

After three years of the SARS-CoV-2 pandemic, the search and availability of relatively low-cost benchtop therapeutics for people not at high risk for a severe disease are still ongoing. Although vaccines and new SARS-CoV-2 variants reduce the death toll, the long COVID-19 along with neurologic symptoms can develop and persist even after a mild initial infection. Reinfections, which further increase the risk of sequelae in multiple organ systems as well as the risk of death, continue to require caution. The spike protein of SARS-CoV-2 is an important target for both vaccines and therapeutics. The presence of disulfide bonds in the receptor binding domain (RBD) of the spike protein is essential for its binding to the human ACE2 receptor and cell entry. Here, we demonstrate that thiol-reducing peptides based on the active site of oxidoreductase thioredoxin 1, called thioredoxin mimetic (TXM) peptides, can prevent syncytia formation, SARS-CoV-2 entry into cells, and infection in a mouse model. We also show that TXM peptides inhibit the redox-sensitive HIV pseudotyped viral cell entry. These results support disulfide targeting as a common therapeutic strategy for treating infections caused by viruses using redox-sensitive fusion. Furthermore, TXM peptides exert anti-inflammatory properties by lowering the activation of NF-κB and IRF signaling pathways, mitogen-activated protein kinases (MAPKs) and lipopolysaccharide (LPS)-induced cytokines in mice. The antioxidant and anti-inflammatory effects of the TXM peptides, which also cross the blood-brain barrier, in combination with prevention of viral infections, may provide a beneficial clinical strategy to lower viral infections and mitigate severe consequences of COVID-19.

Half is enough: Oxidized lysophospholipids as novel bioactive molecules.

Studies in the last decade have established the roles of oxidized phospholipids as modulators of various cellular processes, from inflammation and immunity to cell death. Oxidized lysophospholipids, formed through the activity of phospholipases and oxidative enzymes and lacking an acyl chain in comparison with parent phospholipids, are now emerging as novel bioactive lipid mediators. Their detection and structural characterization have been limited in the past due to low amounts and the complexity of their biosynthetic and removal pathways, but recent studies have unequivocally demonstrated their formation under inflammatory conditions. The involvement of oxidized lysophospholipids in immune regulation classifies them as damage-associated molecular patterns (DAMPs), which can promote sterile inflammation and contribute to autoimmune and chronic diseases as well as aging-related diseases. Their signaling pathways are just beginning to be revealed. As the first publications indicate that oxidized lysophospholipids use the same receptors as pathogen-associated molecular patterns (PAMPs), it is likely that the inhibition of signaling pathways activated by oxidized lysophospholipids would affect innate immunity per se. On the other hand, inhibition or modulation of their enzymatic formation, which would not interfere with the response to pathogens, might be beneficial and is potentially a promising new field of research.

Disruption of disulfides within RBD of SARS-CoV-2 spike protein prevents fusion and represents a target for viral entry inhibition by registered drugs.

The SARS-CoV-2 pandemic imposed a large burden on health and society. Therapeutics targeting different components and processes of the viral infection replication cycle are being investigated, particularly to repurpose already approved drugs. Spike protein is an important target for both vaccines and therapeutics. Insights into the mechanisms of spike-ACE2 binding and cell fusion could support the identification of compounds with inhibitory effects. Here, we demonstrate that the integrity of disulfide bonds within the receptor-binding domain (RBD) plays an important role in the membrane fusion process although their disruption does not prevent binding of spike protein to ACE2. Several reducing agents and thiol-reactive compounds are able to inhibit viral entry. N-acetyl cysteine amide, L-ascorbic acid, JTT-705, and auranofin prevented syncytia formation, viral entry into cells, and infection in a mouse model, supporting disulfides of the RBD as a therapeutically relevant target.

A Nanoscaffolded Spike-RBD Vaccine Provides Protection against SARS-CoV-2 with Minimal Anti-Scaffold Response.

The response of the adaptive immune system is augmented by multimeric presentation of a specific antigen, resembling viral particles. Several vaccines have been designed based on natural or designed protein scaffolds, which exhibited a potent adaptive immune response to antigens; however, antibodies are also generated against the scaffold, which may impair subsequent vaccination. In order to compare polypeptide scaffolds of different size and oligomerization state with respect to their efficiency, including anti-scaffold immunity, we compared several strategies of presentation of the RBD domain of the SARS-CoV-2 spike protein, an antigen aiming to generate neutralizing antibodies. A comparison of several genetic fusions of RBD to different nanoscaffolding domains (foldon, ferritin, lumazine synthase, and ß-annulus peptide) delivered as DNA plasmids demonstrated a strongly augmented immune response, with high titers of neutralizing antibodies and a robust T-cell response in mice. Antibody titers and virus neutralization were most potently enhanced by fusion to the small ß-annulus peptide scaffold, which itself triggered a minimal response in contrast to larger scaffolds. The ß-annulus fused RBD protein increased residence in lymph nodes and triggered the most potent viral neutralization in immunization by a recombinant protein. Results of the study support the use of a nanoscaffolding platform using the ß-annulus peptide for vaccine design.

Cleavage-Mediated Regulation of Myd88 Signaling by Inflammasome-Activated Caspase-1.

Coordination among multiple signaling pathways ensures an appropriate immune response, where a signaling pathway may impair or augment another signaling pathway. Here, we report a negative feedback regulation of signaling through the key innate immune mediator MyD88 by inflammasome-activated caspase-1. NLRP3 inflammasome activation impaired agonist- or infection-induced TLR signaling and cytokine production through the proteolytic cleavage of MyD88 by caspase-1. Site-specific mutagenesis was used to identify caspase-1 cleavage site within MyD88 intermediary segment. Different cleavage site location within MyD88 defined the functional consequences of MyD88 cleavage between mouse and human cells. LPS/monosodium urate-induced mouse inflammation model corroborated the physiological role of this mechanism of regulation, that could be reversed by chemical inhibition of NLRP3. While Toll/interleukin-1 receptor (TIR) domain released by MyD88 cleavage additionally contributed to the inhibition of signaling, Waldenström's macroglobulinemia associated MyD88L265P mutation is able to evade the caspase-1-mediated inhibition of MyD88 signaling through the ability of its TIRL265P domain to recruit full length MyD88 and facilitate signaling. The characterization of this mechanism reveals an additional layer of innate immunity regulation.

Calcium Ionophore-Induced Extracellular Vesicles Mediate Cytoprotection against Simulated Ischemia/Reperfusion Injury in Cardiomyocyte-Derived Cell Lines by Inducing Heme Oxygenase 1.

Cardioprotection against ischemia/reperfusion injury is still an unmet clinical need. The transient activation of Toll-like receptors (TLRs) has been implicated in cardioprotection, which may be achieved by treatment with blood-derived extracellular vesicles (EVs). However, since the isolation of EVs from blood takes considerable effort, the aim of our study was to establish a cellular model from which cardioprotective EVs can be isolated in a well-reproducible manner. EV release was induced in HEK293 cells with calcium ionophore A23187. EVs were characterized and cytoprotection was assessed in H9c2 and AC16 cell lines. Cardioprotection afforded by EVs and its mechanism were investigated after 16 h simulated ischemia and 2 h reperfusion. The induction of HEK293 cells by calcium ionophore resulted in the release of heterogenous populations of EVs. In H9c2 and AC16 cells, stressEVs induced the downstream signaling of TLR4 and heme oxygenase 1 (HO-1) expression in H9c2 cells. StressEVs decreased necrosis due to simulated ischemia/reperfusion injury in H9c2 and AC16 cells, which was independent of TLR4 induction, but not that of HO-1. Calcium ionophore-induced EVs exert cytoprotection by inducing HO-1 in a TLR4-independent manner.

Synergy between 15-lipoxygenase and secreted PLA2 promotes inflammation by formation of TLR4 agonists from extracellular vesicles.

Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived extracellular vesicles (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4), resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain. Hydroxy, hydroperoxy, and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry (MS), and they activated the same gene pattern as stressEVs. Extracellular PLA2 activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA2 promoted K/BxN serum-induced arthritis in mice, whereby ankle swelling was partially TLR4 dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA2 enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.

Adamantane Containing Peptidoglycan Fragments Enhance RANTES and IL-6 Production in Lipopolysaccharide-Induced Macrophages.

We report the enhancement of the lipopolysaccharide-induced immune response by adamantane containing peptidoglycan fragments in vitro. The immune stimulation was detected by Il-6 (interleukine 6) and RANTES (regulated on activation, normal T cell expressed and secreted) chemokine expression using cell assays on immortalized mouse bone-marrow derived macrophages. The most active compound was a α-D-mannosyl derivative of an adamantylated tripeptide with L-chirality at the adamantyl group attachment, whereby the mannose moiety assumed to target mannose receptors expressed on macrophage cell surfaces. The immune co-stimulatory effect was also influenced by the configuration of the adamantyl center, revealing the importance of specific molecular recognition event taking place with its receptor. The immunostimulating activities of these compounds were further enhanced upon their incorporation into lipid bilayers, which is likely related to the presence of the adamantyl group that helps anchor the peptidoglycan fragment into lipid nanoparticles. We concluded that the proposed adamantane containing peptidoglycan fragments act as co-stimulatory agents and are also suitable for the preparation of lipid nanoparticle-based delivery of peptidoglycan fragments.

Targeted Delivery of Adamantylated Peptidoglycan Immunomodulators in Lipid Nanocarriers: NMR Shows That Cargo Fragments Are Available on the Surface.

We present an in-depth investigation of the membrane interactions of peptidoglycan (PGN)-based immune adjuvants designed for lipid-based delivery systems using NMR spectroscopy. The derivatives contain a cargo peptidoglycan (PGN) dipeptide fragment and an adamantyl group, which serves as an anchor to the lipid bilayer. Furthermore, derivatives with a mannose group that can actively target cell surface receptors on immune cells are also studied. We showed that the targeting mannose group and the cargo PGN fragment are both available on the lipid bilayer surface, thereby enabling interactions with cognate receptors. We found that the nonmannosylated compounds are incorporated stronger into the lipid assemblies than the mannosylated ones, but the latter compounds penetrate deeper in the bilayer. This might be explained by stronger electrostatic interactions available for zwitterionic nonmannosylated derivatives as opposed to the compounds in which the charged N-terminus is capped by mannose groups. The higher incorporation efficiency of the nonmannosylated compounds correlated with a larger relative enhancement in immune stimulation activities upon lipid incorporation compared to that of the derivatives with the mannose group. The chirality of the adamantyl group also influenced the incorporation efficiency, which in turn correlated with membrane-associated conformations that affect possible intermolecular interactions with lipid molecules. These findings will help in improving the development of PGN-based immune adjuvants suitable for delivery in lipid nanoparticles.

Isolation of High-Purity Extracellular Vesicles by the Combination of Iodixanol Density Gradient Ultracentrifugation and Bind-Elute Chromatography From Blood Plasma.

Background: Extracellular vesicles (EVs) (isolated from blood plasma) are currently being extensively researched, both as biomarkers and for their therapeutic possibilities. One challenging aspect to this research is the efficient isolation of high-purity EVs from blood plasma in quantities sufficient for in vivo experiments. In accordance with this challenge, the aim of this study was to develop an isolation method in which to separate the majority of EVs from major impurities such as lipoprotein particles and the abundant plasma proteins albumin and fibrinogen. Methods: Samples of rat blood were centrifuged to remove cells, platelets, large EVs and protein aggregates without prior filtration. Density gradient ultracentrifugation was performed by loading plasma sample onto 50, 30, and 10% iodixanol layers and then centrifuged at 120,000 ×g for 24 h. Ten fractions (F1-10) were collected from top to bottom. Fractions with the highest EV content were further purified by ultracentrifugation, size exclusion, or bind-elute chromatography. Efficiency and purity were assessed by Western blots. Morphology and size distribution of particles were examined by dynamic light scattering and electron microscopy (EM). Results: The highest band intensities of EV markers Alix, Tsg101 and CD81 were detected by Western blot in F6 of small-scale DGUC (61.5 ± 10.4%; 48.1 ± 5.8%; 41.9 ± 3.8%, respectively) at a density of 1.128-1.174 g/mL, where the presence of vesicles with a mean diameter of 38 ± 2 nm was confirmed by EM and DLS. Only 1.4 ± 0.5% of LDL and chylomicron marker, 3.0 ± 1.3% of HDL marker, and 9.9 ± 0.4% of albumin remained in the EV-rich F6. However, 32.8 ± 1.5% of the total fibrinogen beta was found in this fraction. Second-step purification by UC or SEC did not improve EV separation, while after BEC on HiScreen Capto Core 700 albumin and lipoprotein contamination were below detection limit in EV-rich fractions. However, BEC decreased efficiency of EV isolation, and fibrinogen was still present in EV-rich fractions. Conclusion: This is the first demonstration that DGUC is able to markedly reduce the lipoprotein content of EV isolates while it separates EVs with high efficiency. Moreover, isolation of lipoprotein- and albumin-free EVs from blood plasma can be achieved by DGUC followed by BEC, however, on the expense of reduced EV yield.

Extracellular vesicle-mediated transfer of constitutively active MyD88L265P engages MyD88wt and activates signaling.

The link between inflammation and cancer is particularly strong in Waldenström macroglobulinemia (WM), a diffuse large B-cell lymphoma wherein the majority of patients harbor a constitutively active mutation in the innate immune-signaling adaptor myeloid differentiation primary response 88 (MyD88). MyD88Leu265Pro (MyD88L265P) constitutively triggers the myddosome assembly providing a survival signal for cancer cells. Here, we report detection and a functional role of MyD88 in the extracellular vesicles (EVs) shed from WM cells. MyD88L265P was transferred via EVs into the cytoplasm of the recipient mast cells and macrophages, recruiting the endogenous MyD88 that triggered the activation of proinflammatory signaling in the absence of receptor activation. Additionally, internalization of EVs containing MyD88L265P was observed in mice with an effect on the bone marrow microenvironment. MyD88-loaded EVs were detected in the bone marrow aspirates of WM patients thus establishing the physiological role of EVs for MyD88L265P transmission and shaping of the proinflammatory microenvironment. Results establish the mechanism of transmission of signaling complexes via EVs to propagate inflammation as a new mechanism of intercellular communication.

Activation of cell membrane-localized Toll-like receptor 3 by siRNA.

Small interfering RNA molecules (siRNA) are short dsRNAs that are used for different therapeutic applications. On the other hand, dsRNAs can bind to and activate cell RNA sensors and consequently trigger inflammatory response. Here we show that siRNA activates primary human endothelial cells and human lymphatic endothelial cells and that this response is inhibited by antibodies against TLR3. In contrast, the activation of human lymphatic endothelial cells by poly(I:C) was inhibited by bafilomycin but not by anti-TLR3 antibodies. Bafilomycin also inhibited poly(I:C) but not siRNA cell stimulation in TLR3-transfected HEK293. The response to siRNA required the expression of UNC93B1, which directs TLR3 to the surface of HEK293 cells. We propose that the engaged signaling pathway of TLR3 depends on the receptor localization and on the length of the dsRNA, where the activation of cell membrane TLR3 by short dsRNA leads to a predominantly proinflammatory response, whereas TLR3 activation in endosomal compartments by long dsRNA is characterized by the production of type I IFN. A molecular model suggests that the siRNA can bind to the binding sites of the TLR3 ectodomain and trigger receptor dimerization. These results contribute to understanding of the mechanism of side effects seen in the therapeutic application of naked, unmodified siRNA as a result of the activation of TLR3 localized at the plasma membrane.

Locked and proteolysis-based transcription activator-like effector (TALE) regulation.

Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE.

Toll-like receptor 4 senses oxidative stress mediated by the oxidation of phospholipids in extracellular vesicles.

Oxidative stress produced in response to infection or sterile injury activates the innate immune response. We found that extracellular vesicles (EVs) isolated from the plasma of patients with rheumatoid arthritis or secreted from cells subjected to oxidative stress contained oxidized phospholipids that stimulated cells expressing Toll-like receptor 4 (TLR4) in a manner dependent on its co-receptor MD-2. EVs from healthy subjects or reconstituted synthetic EVs subjected to limited oxidation gained the ability to stimulate TLR4-expressing cells, whereas prolonged oxidation abrogated this property. Furthermore, we found that 15-lipoxygenase generated hydro(pero)xylated phospholipids that stimulated TLR4-expressing cells. Molecular modeling suggested that the mechanism of activation of TLR4 by oxidized phospholipids in EVs was structurally similar to that of the TLR4 ligand lipopolysaccharide (LPS). This was supported by experiments showing that EV-mediated stimulation of cells required MD-2, that mutations that block LPS binding to TLR4 abrogated the stimulatory effect of EVs, and that EVs induced TLR4 dimerization. On the other hand, analysis of gene expression profiles showed that genes encoding factors that resolve inflammation were more abundantly expressed in responses to EVs than in response to LPS. Together, these data suggest that EVs act as an oxidative stress-induced endogenous danger signal that underlies the pervasive role of TLR4 in inflammatory diseases.

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Postulates for validating TLR4 agonists.

TLRs play a central role in the innate immune response, recognizing a variety of molecular structures characteristic of pathogens. Although TLR4, together with its co-receptor myeloid differentiation-2 (MD-2), recognize bacterial LPS and therefore Gram-negative bacterial infections, it also plays a key role in many other pathophysiological processes, including sterile inflammation and viral infection. Specifically, numerous endogenous agonists of TLR4 of notably diverse nature, ranging from proteins to metal ions, have been reported. Direct activation of a single receptor by such a range of molecular signals is very difficult to explain from a structural and mechanistic point of view. It is likely that only a subset of these directly activate the TLR4-MD-2 complex. We propose three postulates aimed at distinguishing the direct agonists of TLR4 from indirect activators. These postulates are as follows: (i) that the agonist requires the TLR4/MD-2 receptor complex; (ii) that agonist formed synthetically or in situ must activate the receptor complex in order to eliminate artifacts of contamination by other agonists; and (iii) that a specific molecular interaction between the agonist and TLR4/MD-2 must be identified. The same type of postulates can be applied to pattern recognition receptors in general.

A role for stefin B (cystatin B) in inflammation and endotoxemia.

Stefin B (cystatin B) is an endogenous cysteine cathepsin inhibitor, and the loss-of-function mutations in the stefin B gene were reported in patients with Unverricht-Lundborg disease (EPM1). In this study we demonstrated that stefin B-deficient (StB KO) mice were significantly more sensitive to the lethal LPS-induced sepsis and secreted higher amounts of pro-inflammatory cytokines IL-1ß and IL-18 in the serum. We further showed that increased caspase-11 gene expression and better pro-inflammatory caspase-1 and -11 activation determined in StB KO bone marrow-derived macrophages resulted in enhanced IL-1ß processing. Pretreatment of macrophages with the cathepsin inhibitor E-64d did not affect secretion of IL-1ß, suggesting that the increased cathepsin activity determined in StB KO bone marrow-derived macrophages is not essential for inflammasome activation. Upon LPS stimulation, stefin B was targeted into the mitochondria, and the lack of stefin B resulted in the increased destabilization of mitochondrial membrane potential and mitochondrial superoxide generation. Collectively, our study demonstrates that the LPS-induced sepsis in StB KO mice is dependent on caspase-11 and mitochondrial reactive oxygen species but is not associated with the lysosomal destabilization and increased cathepsin activity in the cytosol.

Inflammation-mediating proteases: structure, function in (patho) physiology and inhibition.

Proteases regulating inflammation are versatile enzymes, usually extracellular matrix degrading enzymes that are involved in wound healing, angiogenesis, coagulation, development, apoptosis and other physiological processes. Their dysregulation and increased expression during inflammation can have devastating consequences, promoting etiology of vascular diseases, inflammatory arthritis, cancer, and allograft rejection. In this review several proteases (ADAMTS, granzymes, plasmin, and kallikreins) with different mechanisms and substrates are described in addition to their physiological roles and contribution to inflammation and inflammatory diseases. Inhibition of proteases may therefore represent an attractive strategy for treatment and herein we describe physiological and engineered inhibitors.

Vanadate from air pollutant inhibits hrs-dependent endosome fusion and augments responsiveness to toll-like receptors.

There is a well-established association between exposure to air pollutants and pulmonary injuries. For example, metals found in ROFA (residual oil fly ash) increase susceptibility of mice as well as humans to microbial infections. In our research, we have found that vanadate substantially increased the response of several Toll-like receptors (TLRs) to stimulation with their ligands. Although vanadate caused generation of reactive oxygen species (ROS), the addition of ROS scavenger N-acetyl cysteine (NAC) had no effect on augmented lipopolysaccharide (LPS) stimulation. We further showed that vanadate inhibits endosome fusion. This effect was determined by measuring the size of endosomes, NF-κB activity and TLR4 degradation in Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) overexpressed cells. Moreover, we identified the role of Hrs phosphorylation in these processes. Based on our findings, we can conclude that vanadate potentiates TLR4 activity by increasing Hrs phosphorylation status, reducing the size of Hrs/TLR4-positive endosomes and impacting TLR4 degradation, thus contributing to the detrimental effects of air pollutants on human health.

The ectodomain of TLR3 receptor is required for its plasma membrane translocation.

Toll-like receptor 3 (TLR3) is a dsRNA sensing receptor that is localized in the cellular compartments but also at the plasma membrane. Overexpression of UNC93B1 promoted localization of TLR3, but not other nucleic acid sensing TLRs, to the plasma membrane. Here we show that UNC93B1 itself is localized at the plasma membrane. We investigated the role of different domains of TLR3 on cell signaling by preparing chimeric receptors between TLR3 and TLR9 where each of the transmembrane segments or cytosolic domains has been exchanged. While the ectodomain completely governs ligand specificity and the cytosolic TIR domain determines the engagement of the signaling adapters as well as the potentiation of receptor activation by UNC93B1, the ectodomain but not transmembrane segment or cytosolic domain determines plasma membrane localization of TLR3. Nevertheless, TLR3 receptor and ligand endocytosis as well as endosomal acidification are important for the robust signaling of TLR3.