Combating herpesvirus encephalitis by potentiating a TLR3-mTORC2 axis.

TLR3 is a sensor of double-stranded RNA that is indispensable for defense against infection with herpes simplex virus type 1 (HSV-1) in the brain. We found here that TLR3 was required for innate immune responses to HSV-1 in neurons and astrocytes. During infection with HSV-1, TLR3 recruited the metabolic checkpoint kinase complex mTORC2, which led to the induction of chemokines and trafficking of TLR3 to the cell periphery. Such trafficking enabled the activation of molecules (including mTORC1) required for the induction of type I interferons. Intracranial infection of mice with HSV-1 was exacerbated by impairment of TLR3 responses with an inhibitor of mTOR and was significantly 'rescued' by potentiation of TLR3 responses with an agonistic antibody to TLR3. These results suggest that the TLR3-mTORC2 axis might be a therapeutic target through which to combat herpes simplex encephalitis.

Structural basis for thioredoxin-mediated suppression of NLRP1 inflammasome.

Inflammasome sensors detect pathogen- and danger-associated molecular patterns and promote inflammation and pyroptosis1. NLRP1 was the first inflammasome sensor to be described, and its hyperactivation is linked to autoinflammatory disease and cancer2-6. However, the mechanism underlying the activation and regulation of NLRP1 has not been clearly elucidated4,7,8. Here we identify ubiquitously expressed endogenous thioredoxin (TRX) as a binder of NLRP1 and a suppressor of the NLRP1 inflammasome. The cryo-electron microscopy structure of human NLRP1 shows NLRP1 bound to Spodoptera frugiperda TRX. Mutagenesis studies of NLRP1 and human TRX show that TRX in the oxidized form binds to the nucleotide-binding domain subdomain of NLRP1. This observation highlights the crucial role of redox-active cysteines of TRX in NLRP1 binding. Cellular assays reveal that TRX suppresses NLRP1 inflammasome activation and thus negatively regulates NLRP1. Our data identify the TRX system as an intrinsic checkpoint for innate immunity and provide opportunities for future therapeutic intervention in NLRP1 inflammasome activation targeting this system.

Toll-like Receptor 9 Contains Two DNA Binding Sites that Function Cooperatively to Promote Receptor Dimerization and Activation.

Toll-like receptor 9 (TLR9) recognizes DNA containing CpG motifs derived from bacteria and viruses and activates the innate immune response to eliminate them. TLR9 is known to bind to CpG DNA, and here, we identified another DNA binding site in TLR9 that binds DNA containing cytosine at the second position from the 5' end (5'-xCx DNA). 5'-xCx DNAs bound to TLR9 in the presence of CpG DNA and cooperatively promoted dimerization and activation of TLR9. Binding at both sites was important for efficient activation of TLR9. The 5'-xCx DNA bound the site corresponding to the nucleoside binding site in TLR7 and TLR8 as revealed by the structural analysis. This study revealed that TLR9 recognizes two types of DNA through its two binding sites for efficient activation. This information may contribute to the development of drugs that control the activity of TLR9.

Aberrant monocytopoiesis drives granuloma development in sarcoidosis.

In sarcoidosis, granulomas develop in multiple organs including the liver and lungs. Although mechanistic target of rapamycin complex 1 (mTORC1) activation in macrophages drives granuloma development in sarcoidosis by enhancing macrophage proliferation, little is known about the macrophage subsets that proliferate and mature into granuloma macrophages. Here, we show that aberrantly increased monocytopoiesis gives rise to granulomas in a sarcoidosis model, in which Tsc2, a negative regulator of mTORC1, is conditionally deleted in CSF1R-expressing macrophages (Tsc2csf1rΔ mice). In Tsc2csf1rΔ mice, common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), common monocyte progenitors / monocyte progenitors (cMoPs / MPs), inducible monocyte progenitors (iMoPs), and Ly6Cint CX3CR1low CD14- immature monocytes (iMOs), but not monocyte-dendritic cell progenitors (MDPs) and common dendritic cell progenitors (CDPs), accumulated and proliferated in the spleen. Consistent with this, monocytes, neutrophils, and neutrophil-like monocytes increased in the spleens of Tsc2csf1rΔ mice, whereas dendritic cells did not. The adoptive transfer of splenic iMOs into wild-type mice gave rise to granulomas in the liver and lungs. In these target organs, iMOs matured into Ly6Chi classical monocytes/macrophages (cMOs). Giant macrophages (gMAs) also accumulated in the liver and lungs, which were similar to granuloma macrophages in expression of cell surface markers such as MerTK and SLAMF7. Furthermore, the gMA-specific genes were expressed in human macrophages from sarcoidosis skin lesions. These results suggest that mTORC1 drives granuloma development by promoting the proliferation of monocyte/neutrophil progenitors and iMOs predominantly in the spleen, and that proliferating iMOs mature into cMOs and then gMAs to give rise to granuloma after migration into the liver and lungs in sarcoidosis.

Structural Analysis Reveals that Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA.

Toll-like receptor 7 (TLR7) is a single-stranded RNA (ssRNA) sensor in innate immunity and also responds to guanosine and chemical ligands, such as imidazoquinoline compounds. However, TLR7 activation mechanism by these ligands remain largely unknown. Here, we generated crystal structures of three TLR7 complexes, and found that all formed an activated m-shaped dimer with two ligand-binding sites. The first site conserved in TLR7 and TLR8 was used for small ligand-binding essential for its activation. The second site spatially distinct from that of TLR8 was used for a ssRNA-binding that enhanced the affinity of the first-site ligands. The first site preferentially recognized guanosine and the second site specifically bound to uridine moieties in ssRNA. Our structural, biochemical, and mutagenesis studies indicated that TLR7 is a dual receptor for guanosine and uridine-containing ssRNA. Our findings have important implications for understanding of TLR7 function, as well as for therapeutic manipulation of TLR7 activation.

Endosomal Toll-Like Receptors as Therapeutic Targets for Autoimmune Diseases.

Nucleic acid (NA)-sensing Toll-like receptors (TLRs) reside in the endosomal compartment of innate immune cells, such as macrophages and dendritic cells. NAs transported to the endosomal compartment are degraded by DNases and RNases. Degradation products, including single-stranded DNA, oligoRNA, and nucleosides, are recognized by TLR7, TLR8, and TLR9 to drive the defense responses against pathogens. NA degradation influences endosomal TLR responses by generating and degrading TLR ligands. TLR ligand accumulation because of impaired NA degradation causes constitutive TLR activation, leading to autoinflammatory and autoimmune diseases. Furthermore, some genes associated with these diseases promote endosomal TLR responses. Therefore, endosomal TLRs are promising therapeutic targets for TLR-mediated inflammatory diseases, and novel drugs targeting TLRs are being developed.

Role of the Orphan Transporter SLC35E1 in the Nuclear Egress of Herpes Simplex Virus 1.

This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.

Dynamic control of nucleic-acid-sensing Toll-like receptors by the endosomal compartment.

Nucleic-acid (NA)-sensing Toll-like receptors (TLRs) are synthesized in the endoplasmic reticulum and mature with chaperones, such as Unc93B1 and the protein associated with TLR4 A (PRAT4A)-gp96 complex. The TLR-Unc93B1 complexes move to the endosomal compartment, where proteases such as cathepsins activate their responsiveness through proteolytic cleavage of the extracellular domain of TLRs. Without proteolytic cleavage, ligand-dependent dimerization of NA-sensing TLRs is prevented by the uncleaved loop in the extracellular domains. Additionally, the association of Unc93B1 inhibits ligand-dependent dimerization of TLR3 and TLR9 and, therefore, Unc93B1 is released from these TLRs before dimerization. Ligand-activated NA-sensing TLRs induce the production of pro-inflammatory cytokines and act on the endosomal compartment to initiate anterograde trafficking to the cell periphery for type I interferon production. In the endosomal compartment, DNA and RNA are degraded by DNases and RNases, respectively, generating degradation products. DNase 2A and RNase T2 generate ligands for TLR9 and TLR8, respectively. In this mechanism, DNases and RNases control innate immune responses to NAs in endosomal compartments. NA-sensing TLRs and the endosomal compartment work together to monitor environmental cues through endosomes and decide to launch innate immune responses.

Skewed endosomal RNA responses from TLR7 to TLR3 in RNase T2-deficient macrophages.

RNase T2, a ubiquitously expressed RNase, degrades RNAs in the endosomal compartments. RNA sensors, double-stranded RNA (dsRNA)-sensing Toll-like receptor 3 (TLR3) and single-stranded RNA (ssRNA)-sensing TLR7, are localized in the endosomal compartment in mouse macrophages. We here studied the role of RNase T2 in TLR3 and TLR7 responses in macrophages. Macrophages expressed RNase T2 and a member of the RNase A family RNase 4. RNase T2 was also expressed in plasmacytoid and conventional dendritic cells. Treatment with dsRNAs or type I interferon (IFN) up-regulated expression of RNase T2 but not RNase 4. RNase T2-deficiency in macrophages up-regulated TLR3 responses but impaired TLR7 responses. Mechanistically, RNase T2 degraded both dsRNAs and ssRNAs in vitro, and its mutants showed a positive correlation between RNA degradation and the rescue of altered TLR3 and TLR7 responses. H122A and C188R RNase T2 mutations, not H69A and E118V mutations, impaired both RNA degradation and the rescue of altered TLR3 and TLR7 responses. RNase T2 in bone marrow-derived macrophages was broadly distributed from early endosomes to lysosomes, and colocalized with the internalized TLR3 ligand poly(I:C). These results suggest that RNase T2-dependent RNA degradation in endosomes/lysosomes negatively and positively regulates TLR3 and TLR7 responses, respectively, in macrophages.

Biallelic Variants in CNPY3, Encoding an Endoplasmic Reticulum Chaperone, Cause Early-Onset Epileptic Encephalopathy.

Early-onset epileptic encephalopathies, including West syndrome (WS), are a group of neurological disorders characterized by developmental impairments and intractable seizures from early infancy. We have now identified biallelic CNPY3 variants in three individuals with WS; these include compound-heterozygous missense and frameshift variants in a family with two affected siblings (individuals 1 and 2) and a homozygous splicing variant in a consanguineous family (individual 3). All three individuals showed hippocampal malrotation. In individuals 1 and 2, electroencephalography (EEG) revealed characteristic fast waves and diffuse sharp- and slow-wave complexes. The fast waves were clinically associated with seizures. CNPY3 encodes a co-chaperone in the endoplasmic reticulum and regulates the subcellular distribution and responses of multiple Toll-like receptors. The amount of CNPY3 in lymphoblastoid cells derived from individuals 1 and 2 was severely lower than that in control cells. Cnpy3-knockout mice exhibited spastic or dystonic features under resting conditions and hyperactivity and anxiolytic behavior during the open field test. Also, their resting EEG showed enhanced activity in the fast beta frequency band (20-35 Hz), which could mimic the fast waves in individuals 1 and 2. These data suggest that CNPY3 and Cnpy3 perform essential roles in brain function in addition to known Toll-like receptor-dependent immune responses.

The impact of cell maturation and tissue microenvironments on the expression of endosomal Toll-like receptors in monocytes and macrophages.

Toll-like receptors (TLRs) impact myeloid cell responsiveness to environmental cues such as pathogen components and metabolites. Although TLR protein expression in monocytes and tissue macrophages is thought to be optimized for microenvironments in each tissue, a comprehensive study has not been reported. We here examined protein expression of endogenous TLRs in tissue-resident myeloid cells. Neutrophils in peripheral blood, spleen, liver and lung expressed TLR2, TLR4 and TLR5 in all tissues. Ly6C+ MHC II‒ classical monocytes mature into Ly6C‒ MHC II+ monocyte-derived dendritic cells (moDCs) or Ly6C‒ MHC II‒ patrolling monocytes. These subsets were found in all the tissues studied. TLR2 and TLR4 were displayed on all of these subsets, regardless of location. In contrast, expression of endosomal TLRs did vary with tissues and subsets. moDCs expressed TLR9, but much less TLR7. In contrast, TLR7, not TLR3 or TLR9, was highly expressed in classical and patrolling monocytes. Tissue macrophages such as red pulp macrophages in the spleen, Kupffer cells in the liver, microglia in the brain, alveolar macrophages in the lung and adipose tissue macrophages all expressed TLR2, TLR4 and TLR3. TLR7 was also expressed in these tissue macrophages except Kupffer cells in the liver. TLR9 expression in tissue macrophages was much lower or hard to detect. These results suggest that expression of endosomal TLRs in myeloid cells is influenced by their differentiation status and tissue-specific microenvironments.

Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9.

Innate immunity serves as the first line of defence against invading pathogens such as bacteria and viruses. Toll-like receptors (TLRs) are examples of innate immune receptors, which sense specific molecular patterns from pathogens and activate immune responses. TLR9 recognizes bacterial and viral DNA containing the cytosine-phosphate-guanine (CpG) dideoxynucleotide motif. The molecular basis by which CpG-containing DNA (CpG-DNA) elicits immunostimulatory activity via TLR9 remains to be elucidated. Here we show the crystal structures of three forms of TLR9: unliganded, bound to agonistic CpG-DNA, and bound to inhibitory DNA (iDNA). Agonistic-CpG-DNA-bound TLR9 formed a symmetric TLR9-CpG-DNA complex with 2:2 stoichiometry, whereas iDNA-bound TLR9 was a monomer. CpG-DNA was recognized by both protomers in the dimer, in particular by the amino-terminal fragment (LRRNT-LRR10) from one protomer and the carboxy-terminal fragment (LRR20-LRR22) from the other. The iDNA, which formed a stem-loop structure suitable for binding by intramolecular base pairing, bound to the concave surface from LRR2-LRR10. This structure serves as an important basis for improving our understanding of the functional mechanisms of TLR9.

Cytidine deaminase enables Toll-like receptor 8 activation by cytidine or its analogs.

Toll-like receptor 8 (TLR8), a sensor for pathogen-derived single-stranded RNA (ssRNA), binds to uridine (Uri) and ssRNA to induce defense responses. We here show that cytidine (Cyd) with ssRNA also activated TLR8 in peripheral blood leukocytes (PBLs) and a myeloid cell line U937, but not in an embryonic kidney cell line 293T. Cyd deaminase (CDA), an enzyme highly expressed in leukocytes, deaminates Cyd to Uri. CDA expression enabled TLR8 response to Cyd and ssRNA in 293T cells. CDA deficiency and a CDA inhibitor both reduced TLR8 responses to Cyd and ssRNA in U937. The CDA inhibitor also reduced PBL response to Cyd and ssRNA. A Cyd analogue, azacytidine, is used for the therapy of myelodysplastic syndrome and acute myeloid leukemia. Azacytidine with ssRNA induced tumor necrosis factor-α expression in U937 and PBLs in a manner dependent on CDA and TLR8. These results suggest that CDA enables TLR8 activation by Cyd or its analogues with ssRNA through deaminating activity. Nucleoside metabolism might impact TLR8 responses in a variety of situations such as the treatment with nucleoside analogues.

Unc93B1 restricts systemic lethal inflammation by orchestrating Toll-like receptor 7 and 9 trafficking.

Toll-like receptor-7 (TLR7) and 9, innate immune sensors for microbial RNA or DNA, have been implicated in autoimmunity. Upon activation, TLR7 and 9 are transported from the endoplasmic reticulum (ER) to endolysosomes for nucleic acid sensing by an ER-resident protein, Unc93B1. Little is known, however, about a role for sensor transportation in controlling autoimmunity. TLR9 competes with TLR7 for Unc93B1-dependent trafficking and predominates over TLR7. TLR9 skewing is actively maintained by Unc93B1 and reversed to TLR7 if Unc93B1 loses preferential binding via a D34A mutation. We here demonstrate that mice harboring a D34A mutation showed TLR7-dependent, systemic lethal inflammation. CD4(+) T cells showed marked differentiation toward T helper 1 (Th1) or Th17 cell subsets. B cell depletion abolished T cell differentiation and systemic inflammation. Thus, Unc93B1 controls homeostatic TLR7 activation by balancing TLR9 to TLR7 trafficking.

Nucleic acid-sensing TLRs and autoimmunity: novel insights from structural and cell biology.

Invasion of pathogenic microorganisms or tissue damage activates innate immune signaling receptors that sample subcellular locations for foreign molecular structures, altered host molecules, or signs of compartment breaches. Upon engagement of innate immune receptors an acute but transient inflammatory response is initiated, aimed at the clearance of pathogens and cellular debris. Among the molecules that are sensed are nucleic acids, which activate several members of the transmembrane Toll-like receptor (TLR) family. Inappropriate recognition of nucleic acids by TLRs can cause inflammatory pathologies and autoimmunity. Here, we review the mechanisms involved in triggering nucleic acid-sensing TLRs and indicate checkpoints that restrict their activation to endolysosomal compartments. These mechanisms are crucial to sample the content of endosomes for nucleic acids in the context of infection or tissue damage, yet prevent accidental activation by host nucleic acids under physiological conditions. Decoding the molecular mechanisms that regulate nucleic acid recognition by TLRs is central to understand pathologies linked to unrestricted nucleic acid sensing and to develop novel therapeutic strategies.

Mechanisms controlling nucleic acid-sensing Toll-like receptors.

Nucleic acid (NA)-sensing Toll-like receptors (TLRs) respond to DNA/RNA derived from pathogens and dead cells. Structural studies have revealed a variety of molecular mechanisms by which TLRs sense NAs. Double-stranded RNA and single-stranded DNA directly bind to TLR3 and TLR9, respectively, whereas TLR7 and TLR8 bind to nucleosides and oligoribonucleotides derived from RNAs. Activation of ligand-bound TLRs is influenced by the functional status of TLRs. Proteolytic cleavage of NA-sensing TLRs enables ligand-dependent TLR dimerization. Trafficking of ligand-activated TLRs in endosomal and lysosomal compartments is requisite for production of type I interferons. Activation of NA-sensing TLRs is required for the control of viruses such as herpes simplex virus and endogenous retroviruses. On the other hand, excessive activation of NA-sensing TLRs drives disease progression in a variety of inflammatory diseases including systemic lupus erythematosus, heart failure, arthritis and non-alcoholic steatohepatitis. NA-sensing TLRs are targets for therapeutic intervention in these diseases. We here focus on our recent progresses in our understanding of NA-sensing TLRs.

Autoinhibition and relief mechanism by the proteolytic processing of Toll-like receptor 8.

Toll-like receptor 8 (TLR8) senses single-stranded RNA (ssRNA) and initiates innate immune responses. TLR8 requires proteolytic cleavage at the loop region (Z-loop) between leucine-rich repeat (LRR) 14 and LRR15 for its activation. However, the molecular basis of Z-loop processing remains unknown. To elucidate the mechanism of Z-loop processing, we performed biochemical and structural studies of how the Z-loop affects the function of TLR8. TLR8 with the uncleaved Z-loop is unable to form a dimer, which is essential for activation, irrespective of the presence of agonistic ligands. Crystallographic analysis revealed that the uncleaved Z-loop located on the ascending lateral face prevents the approach of the dimerization partner by steric hindrance. This autoinhibition mechanism of dimerization by the Z-loop might be occurring in the proteins of the same subfamily, TLR7 and TLR9.

Epithelial membrane protein 3 (Emp3) downregulates induction and function of cytotoxic T lymphocytes by macrophages via TNF-α production.

Tetraspanin membrane protein, epithelial membrane protein 3 (Emp3), is expressed in lymphoid tissues. Herein, we have examined the Emp3 in antigen presenting cell (APC) function in the CD8+ cytotoxic T lymphocytes (CTLs) induction. Emp3-overexpressing RAW264.7 macrophage cell line derived from BALB/c mice reduced anti-C57BL/6 alloreactive CTL induction, while Emp3-knockdown RAW264.7 enhanced it compared with parent RAW267.4. Emp3-overexpressing RAW264.7 inhibited, but Emp3-knockdown RAW264.7 augmented, CD8+ T cell proliferation, interferon-γ secretion, IL-2 consumption, and IL-2Rα expression on CD8+ T cells. The supernatant from co-culture with Emp3-overexpressing RAW264.7 contained higher amount of TNF-α, and TNF- α neutralization significantly restored all these inhibitions and the alloreactive CTL induction. These results suggest that Emp3 in allogeneic APCs possesses the inhibitory function of alloreactive CTL induction by downregulation of IL-2Rα expression CD8+ T cells via an increase in TNF-α production. This demonstrates a novel mechanism for regulating CTL induction by Emp3 in APCs through TNF-α production.

Requirement of glycosylation machinery in TLR responses revealed by CRISPR/Cas9 screening.

The Toll family of receptors sense microbial products and activate a defense response. The molecular machinery required for the TLR response is not yet fully understood. In the present study, we used a clustered, regularly interspaced, short palindromic repeats (CRISPR)/CAS9 screening system to study TLR responses. We employed a cell line expressing TLR with an NF-κB-driven GFP reporter. The cell line was transduced with a guide RNA (gRNA) library and stimulated with TLR ligands. The cells impaired in GFP induction were sorted, and gRNAs were sequenced. Identified genes were ranked according to the count of sequence reads and the number of gRNA target sites. The screening system worked correctly, as molecules that were already known to be required for the TLR response were identified by the screening. Furthermore, this system revealed that the oligosaccharide transferase complex (OSTC) mediating co-translational glycosylation was required for TLR5, 7 and 9 responses. Protein expression of TLR5, but not an irrelevant molecule (CD44), was abolished by the lack of OSTC, suggesting the essential role of glycosylation in TLR5 protein stability. These results demonstrate that the screening system established here is able to reveal molecular mechanisms underlying the TLR response.

Core fucose is critical for CD14-dependent Toll-like receptor 4 signaling.

Core fucosylation, a posttranslational modification of N-glycans, modifies several growth factor receptors and impacts on their ligand binding affinity. Core-fucose-deficient mice generated by ablating the α1,6 fucosyltransferase enzyme, Fut8, exhibit severe pulmonary emphysema, partly due to impaired macrophage function, similar to aged Toll-like receptor 4 (Tlr4)-deficient mice. We therefore suspect that a lack of core fucose affects the TLR4-dependent signaling pathway. Indeed, upon lipopolysaccharide stimulation, Fut8-deficient mouse embryonic fibroblasts (MEFs) produced similar levels of interleukin-6 but markedly reduced levels of interferon-ß (IFN-ß) compared with wild-type MEFs. Lectin blot analysis of the TLR4 signaling complex revealed that core fucosylation was specifically found on CD14. Even though similar levels of TLR4/myeloid differentiation factor 2 (MD2) activation and dimerization were observed in Fut8-deficient cells after lipopolysaccharide stimulation, internalization of TLR4 and CD14 was significantly impaired. Given that internalized TLR4/MD2 induces IFN-ß production, impaired IFN-ß production in Fut8-deficient cells is ascribed to impaired TLR4/MD2 internalization. These data show for the first time that glycosylation critically regulates TLR4 signaling.