Transcriptomic Analysis and C-Terminal Epitope Tagging Reveal Differential Processing and Signaling of Endogenous TLR3 and TLR7

Toll-like receptor (TLR) signaling is critical for defense against pathogenic infection, as well as for modulating tissue development. Activation of different TLRs triggers common inflammatory responses such as cytokine induction. Here, we reveal differential impacts of TLR3 and TLR7 signaling on transcriptomic profiles in bone marrow-derived macrophages (BMDMs). Apart from self-regulation, TLR3, but not TLR7, induced expression of other TLRs, suggesting that TLR3 activation globally enhances innate immunity. Moreover, we observed diverse influences of TLR3 and TLR7 signaling on genes involved in methylation, caspase and autophagy pathways. We compared endogenous TLR3 and TLR7 by using CRISPR/Cas9 technology to knock in a dual Myc-HA tag at the 3’ ends of mouse Tlr3 and Tlr7. Using anti-HA antibodies to detect endogenous tagged TLR3 and TLR7, we found that both TLRs display differential tissue expression and posttranslational modifications. C-terminal tagging did not impair TLR3 activity. However, it disrupted the interaction between TLR7 and myeloid differentiation primary response 88 (MYD88), the Tir domain-containing adaptor of TLR7, which blocked its downstream signaling necessary to trigger cytokine and chemokine expression. Our study demonstrates different properties for TLR3 and TLR7, and also provides useful mouse models for further investigation of these two RNA-sensing TLRs.

TLR3 activation by Zika virus stimulates inflammatory cytokine production which dampens the antiviral response induced by RIG-I-like receptors

Infection with the Zika virus (ZIKV), a member of the Flaviviridae family, can cause serious neurological disorders, most notably microcephaly in newborns. Here we investigated the innate immune response to ZIKV infection in cells of the nervous system. In human neural progenitor cells (hNPCs), a target for ZIKV infection and likely involved in ZIKV-associated neuropathology, viral infection failed to elicit an antiviral interferon (IFN) response. However, pharmacological inhibition of TLR3 partially restored this deficit. Analogous results were obtained in human iPSC-derived astrocytes, which are capable of mounting a strong antiviral cytokine response. There, ZIKV is sensed by both RIG-I and MDA5 and induces an IFN response as well as expression of pro-inflammatory cytokines such as interleukin-6 (IL-6). Upon inhibition of TLR3, also in astrocytes the antiviral cytokine response was enhanced, whereas amounts of pro-inflammatory cytokines were reduced. To study the underlying mechanism, we used human epithelial cells as an easy to manipulate model system. We found that ZIKV is sensed in these cells by RIG-I to induce a robust IFN response and by TLR3 to trigger the expression of pro-inflammatory cytokines, including IL-6. ZIKV induced upregulation of IL-6 activated the STAT3 pathway, which decreased STAT1 phosphorylation in a SOCS-3 dependent manner, thus reducing the IFN response. In conclusion, we show that TLR3 activation by ZIKV suppresses IFN responses triggered by RIG-I-like receptors. Importance Zika virus (ZIKV) has a pronounced neurotropism and infections with this virus can cause serious neurological disorders, most notably microcephaly and the Guillain–Barré syndrome. Our studies reveal that during ZIKV infection, recognition of viral RNA by TLR3 enhances the production of inflammatory cytokines and suppresses the interferon response triggered by RIG-I-like receptors (RLR) in a SOCS3-dependent manner, thus facilitating virus replication. The discovery of this crosstalk between antiviral (RLR) and inflammatory (TLR) responses may have important implications for our understanding of ZIKV-induced pathogenesis.

N4BP1 negatively regulates NF-κB by binding and inhibiting NEMO oligomerization

Many immune responses depend upon activation of NF-κB, an important transcription factor in the elicitation of a cytokine response. Here we show that N4BP1 inhibits TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO–NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation-like) domains in N4BP1 mediate interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhances TRIF-independent (TLR2, TLR7, or TLR9-mediated) but not TRIF-dependent (TLR3 or TLR4-mediated) NF-κB activation, leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF causes activation of caspase-8, which cleaves N4BP1 distal to residues D424 and D490 and abolishes its inhibitory effect. N4bp1−/− mice also have diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase-8-dependent mechanism by which this is accomplished.

Influenza A virus enhances ciliary activity and mucociliary clearance via TLR3 in airway epithelium

Background Viral respiratory tract infections, such as influenza A virus (IAV), are common and life-threatening illnesses worldwide. The mechanisms by which viruses are removed from the respiratory tract are indispensable for airway host defense. Mucociliary clearance is an airway defense mechanism that removes pathogens from the respiratory tract. The coordination and modulation of the ciliary beating of airway epithelial cells play key roles in maintaining effective mucociliary clearance. However, the impact of respiratory virus infection on ciliary activity and mucociliary clearance remains unclear. Methods Tracheal samples were taken from wild-type (WT) and Toll-like receptor 3 (TLR3)-knockout (KO) mice. Transient organ culture of murine trachea was performed in the presence or absence of IAV, polyI:C, a synthetic TLR3 ligand, and/or reagents. Subsequently, cilia-driven flow and ciliary motility were analyzed. To evaluate cilia-driven flow, red fluorescent beads were loaded into culture media and movements of the beads onto the tracheal surface were observed using a fluorescence microscope. To evaluate ciliary motility, cilia tips were labeled with Indian ink diluted with culture medium. The motility of ink-labeled cilia tips was recorded by high-speed cameras. Results Short-term IAV infection significantly increased cilia-driven flow and ciliary beat frequency (CBF) compared with the control level in WT culture. Whereas IAV infection did not elicit any increases of cilia-driven flow and CBF in TLR3-KO culture, indicating that TLR3 was essential to elicit an increase of cilia-driven flow and CBF in response to IAV infection. TLR3 activation by polyI:C readily induced adenosine triphosphate (ATP) release from the trachea and increases of cilia-driven flow and CBF in WT culture, but not in TLR3-KO culture. Moreover, blockade of purinergic P2 receptors (P2Rs) signaling using P2R antagonist, suramin, suppressed polyI:C-mediated increases of cilia-driven flow and CBF, indicating that TLR3-mediated ciliary activation depended on released extracellular ATP and the autocrine ATP-P2R loop. Conclusions IAV infection readily increases ciliary activity and cilia-driven flow via TLR3 activation in the airway epithelium, thereby hastening mucociliary clearance and “sweeping” viruses from the airway as an initial host defense response. Mechanically, extracellular ATP release in response to TLR3 activation promotes ciliary activity through autocrine ATP-P2R loop.

Mechanism of negative regulation of NF-κB by N4BP1

Many immune responses depend upon activation of NF-κB, a key transcription factor in the elicitation of a cytokine response. Here we demonstrate that N4BP1 inhibited TLR-dependent activation of NF-κB by interacting with the NF-κB signaling essential modulator (NEMO, also known as IκB kinase γ) to attenuate NEMO-NEMO dimerization or oligomerization. The UBA-like (ubiquitin associated-like) and CUE-like (ubiquitin conjugation to ER degradation) domains in N4BP1 mediated the interaction with the NEMO COZI domain. Both in vitro and in mice, N4bp1 deficiency specifically enhanced TRIF-independent (TLR2, TLR7, or TLR9-mediated), but not TRIF-dependent (TLR3 or TLR4-mediated), NF-κB activation leading to increased production of proinflammatory cytokines. In response to TLR4 or TLR3 activation, TRIF caused activation of caspase 8, which cleaved N4BP1 distal to residues D424 and D490 and abolished its inhibitory effect. N4bp1-/- mice also exhibited diminished numbers of T cells in the peripheral blood. Our work identifies N4BP1 as an inhibitory checkpoint protein that must be overcome to activate NF-κB, and a TRIF-initiated caspase 8-dependent mechanism by which this is accomplished.

Toll-like receptor 3 mediates ischaemia/reperfusion injury after cardiac transplantation

OBJECTIVES Ischaemia and subsequent reperfusion during heart transplantation inevitably result in donor organ injury. Toll-like receptor (TLR)-3 is a pattern recognition receptor activated by viral and endogenous RNA released by injured cells. We hypothesized that ischaemia/reperfusion injury (IRI) leads to RNA release with subsequent TLR3 activation in transplanted hearts. METHODS Human endothelial cells were subjected to IRI and treated with TLR3 agonist polyinosinic–polycytidylic acid or a TLR3/double-stranded RNA complex inhibitor. TLR3 activation was analysed using reporter cells. Gene expression profiles were evaluated via next-generation sequencing. Neutrophil adhesion was assessed in vitro. Syngeneic heart transplantation of wild-type or Tlr3−/− mice was performed following 9 h of cold ischaemia. Hearts were analysed for inflammatory gene expression, cardiac damage, apoptosis and infiltrating leucocytes. RESULTS IRI resulted in RNA release with subsequent activation of TLR3. Treatment with a TLR3 inhibitor abrogated the inflammatory response upon IRI. In parallel, TLR3 stimulation caused activation of the inflammasome. Endothelial IRI resulted in TLR3-dependent adhesion of neutrophils. Tlr3−/− animals showed reduced intragraft and splenic messenger ribonucleic acid (mRNA) expression of proinflammatory cytokines, resulting in decreased myocardial damage, apoptosis and infiltrating cells. Tlr3 deficiency protected from cardiac damage, apoptosis and leucocyte infiltration after cardiac transplantation. CONCLUSIONS We uncover the release of RNA by injured cells with subsequent activation of TLR3 as a crucial pathomechanism of IRI. Our data indicate that TLR3 represents a novel target in the prevention of IRI in solid organ transplantation.