TLR4 Knockout Research Articles

Loss of TLR3 and its downstream signaling accelerates acinar cell damage in the acute phase of pancreatitis

BackgroundAcute pancreatitis is accompanied by acinar cell damage releasing potential toll-like receptor 3 (TLR3) ligands. So far, TLR3 is known as a pattern recognition receptor in the immune signaling cascade triggering a type I interferon response. In addition, TLR3 signaling contributes to programmed cell death through the activation of caspase 8. However, the functional role of TLR3 and its downstream toll-like receptor adaptor molecule 1 (TICAM1) in the inflamed pancreas is unknown. MethodsTo uncover the role of TLR3 signaling in acute pancreatitis, we induced a cerulein-mediated pancreatitis in Tlr3 and Ticam1 knockout (KO) mice and in wildtype animals. The exocrine damage was determined by blood serum analysis and histological examination. Immunohistochemistry, gene expression and immunoblot analysis were conducted to study TLR3 function. ResultsAfter the induction of an acute pancreatitis, wildtype mice showed a high endosomal TLR3 expression in acinar cells. In comparison to wildtype and Ticam1 KO mice, Tlr3 KO mice exhibited the highest severity of pancreatitis with an increased NF-κB activation and elevated expression of the pro-inflammatory cytokines Il6 and Tnf, although the amount of infiltrating immune cells was unaffected. Additionally, we detected a strong elevation of acinar cell necrosis and reduced levels of cleaved caspase 8 in Tlr3 and Ticam1 KO mice. ConclusionsTLR3 and its downstream adaptor TICAM1 are important mediators of acinar cell damage in acute pancreatitis. They possess a critical role in programmed cell death and our data suggest that TLR3 signaling controls the onset and severity of acute pancreatitis.

Angiotensin II Causes Biphasic STAT3 Activation Through TLR4 to Initiate Cardiac Remodeling

Evidence indicates that Ang II (angiotensin II) activates STAT3 (signal transducer and activator of transcription 3) in cardiomyocytes. However, the mechanisms underlying STAT3 activation and downstream responses are not fully known. In this study, we show that Ang II caused biphasic STAT3 activation in cardiomyocytes. A rapid and early activation was mediated by direct association between TLR4 (toll-like receptor-4) and STAT3. This early activation increased IL-6 (interleukin-6) production, which in turn, induced the second STAT3 activation through the IL-6/gp130 (glycoprotein 130)/JAK2 (Janus-family tyrosine kinases 2) pathway, resulting in unregulated expression of genes for cardiac remodeling. Moreover, STAT3 inhibition or TLR4 knockout in mice protected against Ang II-induced hypertrophy, fibrosis, and cardiac functional deficits. Thus, Ang II-induced STAT3 activation in cardiomyocytes was biphasic, providing a sequential induction of IL-6 and myocardial remodeling genes, respectively. This work supports a novel mechanism on STAT3 activation in Ang II-induced cardiac dysfunction and remodeling.

OK-432 Administration Inhibits Murine Allergic Rhinitis at the Induction Phase, through the Macrophage Activation with TLR2 Signaling Pathway.

OK-432, a preparation of a low-virulence strain (Su) of Streptococcus pyogenes (Group A) killed by a penicillin and lyophilized, is a stiff inducer of Th1 cytokines, and exerts anti-cancer effects in tumor-bearing mice. OK-432 has been reported to consist of many bacterial components, such as peptidoglycan, M-protein, etc. However, it is yet to be ascertained which bacterial component induces T helper 1 (Th1) responses. For the last decade, Toll-like receptor (TLR) family proteins are well elucidated to play a role in recognizing bacterial components and inducing interleukin (IL)-12 from macrophages. Above all, peptidoglycan seems to be the agonist of TLR2 rather than the obverse. In our present study, the role of TLR2 for the recognition of OK-432 by macrophages and the effects of OK-432 are examined on murine allergic rhinitis model. Interestingly, results show IL-12 production by macrophages derived from TLR2 knock-out (ko) mice was significantly decreased, in comparison with that of macrophages derived from wild-type mice. Moreover, in TLR2 ko mice, no regulatory effect of OK-432 was observed on an allergic rhinitis model. These data indicate that TLR2 signaling is involved in regulating OK-432-induced anti-T helper 2 (Th2) immunity, and may offer a new prophylactic and therapeutic approach using OK-432 to downregulate allergic disorders, such as allergic rhinitis.

The impact of the absence of Toll-like receptor-2 during Sporothrix brasiliensis infection

Sporothrix brasiliensis, a member of the Sporothrix schenckii complex, is a major cause of epidemic outbreaks of sporotrichosis due to its greater virulence and ability to evade the immune system. The absence of studies about this species led to this study, with the aim to evaluate the importance of Toll-like receptor-2 (TLR-2) during S. brasiliensis infection. In vitro assays were performed using bone marrow-derived macrophages from both wild-type (C57BL/6) and TLR-2 knockout (-/-) mice. In vivo assays were also performed, on which the mice (C57BL/6 and TLR-2-/-) were intraperitoneally infected with S. brasiliensis yeast American Type Culture Collection MYA-4831 and euthanized on days 7, 14 and 28 post infection. The following parameters were then evaluated: fungal burden in spleen, liver, kidney and brain; the production of cytokines TNF-α, IFN-γ, IL-4, IL-2, IL-6 and IL-10. The in vitro results showed that the absence of TLR-2 resulted in impaired phagocytosis, microbicide mechanisms utilizing the production of nitric oxide, and the cytokine production (TNF-α, IL-6 and IL-10). The in vivo results demonstrated that the absence of TLR-2 during experimental S. brasiliensis infection promoted increased dissemination after 14 and 28 days and suggests a polarized Th17 response in an attempt to control the infection. TLR-2 signalling appears to be important in the innate immune response against S. brasiliensis.

RIG-I Signaling via MAVS Is Dispensable for Survival in Lethal Influenza Infection In Vivo.

Retinoic acid-inducible gene I (RIG-I) is an important regulator of virus-induced antiviral interferons (IFNs) and proinflammatory cytokines. It requires interaction with an adaptor molecule, mitochondrial antiviral-signaling protein (MAVS), to activate downstream signaling pathways. To elucidate the mechanism(s) by which RIG-I-dependent recognition of IAV infection in vivo triggers innate immune responses, we infected mutant mice lacking RIG-I or MAVS with influenza A virus (IAV) and measured their innate immune responses. As has previously been demonstrated with isolated deletion of the virus recognition receptors TLR3, TLR7, and NOD2, RIG-I or MAVS knockout (KO) did not result in higher mortality and did not reduce IAV-induced cytokine responses in mice. Infected RIG-I KO animals displayed similar lung inflammation profiles as did WT mice, in terms of the protein concentration, total cell count, and inflammatory cell composition in the bronchoalveolar lavage fluid. RNA-Seq results demonstrated that all types of mice exhibited equivalent antiviral and inflammatory gene responses following IAV infection. Together, the results indicated that although RIG-I is important in innate cytokine responses in vitro, individual deletion of the genes encoding RIG-I or MAVS did not change survival or innate responses in vivo after IAV infection in mice.

Research on mechanism of PCS in damaging vascular endothelial cells and promoting formation of atherosclerosis via TLR4/TREM-1.

The study aimed to explore the effects of p-cresyl sulfate (PCS) of damaging vascular endothelial cells and promoting the formation of atherosclerosis in mice. The apolipoprotein E (ApoE)-/- mice were fed normally and with a high-fat diet; the ApoE-/- mice fed with high-fat diet were divided into two groups and treated with blank control and PCS, respectively. The aortic arch in each group was taken and underwent the oil red O staining, and the serum PCS content in each group was detected. The basic components of plaque were observed, including foam cells, lipid deposition, and cholesterol crystal. Moreover, human umbilical vein endothelial cells were cultured and divided into control group, PCS treatment group (PCS), PCS treatment with TLR4 overexpression group (PCS+TLR4+), and PCS treatment with TLR4 knock-out group (PCS+TLR4-). The degree of endothelial cell damage was detected using a cluster of differentiation CD42b-/CD31+ endothelial microparticles (EMPs), and expressions of Toll-like receptor 4 (TLR4), triggering receptor expressed on myeloid cells-1 (TREM-1), phosphorylated-endothelial nitric oxide synthase (p-eNOS), and tumor necrosis factor-α (TNF-α) in cells were detected via Polymerase Chain Reaction (PCR) and Western blotting. The serum PCS concentration in high-fat ApoE-/- mice was increased, and the aortic arch sections of ApoE-/- mice treated with PCS displayed the evident atherosclerotic plaques. Experimental results of human umbilical vein endothelial cells showed that the activity of human umbilical vein endothelial cells treated with PCS declined, the expression levels of TLR4, TREM-1, and TNF-α were increased, while that of p-eNOS was decreased. After the TLR4 knockout, the above effects of PCS were reversed. PCS damages vascular endothelial cells through TRL4/TREM-1, thereby accelerating the formation of atherosclerosis.

Endothelial Stanniocalcin 1 Maintains Mitochondrial Bioenergetics and Prevents Oxidant-Induced Lung Injury via Toll-Like Receptor 4.

Oxidant-induced endothelial injury plays a critical role in the pathogenesis of acute lung injury (ALI) and subsequent respiratory failure. Our previous studies revealed an endogenous antioxidant and protective pathway in lung endothelium mediated by heat shock protein 70 (Hsp70)-toll-like receptor 4 (TLR4) signaling. However, the downstream effector mechanisms remained unclear. Stanniocalcin 1 (STC1) has been reported to mediate antioxidant responses in tissues such as the lungs. However, regulators of STC1 expression as well as its physiological function in the lungs were unknown. We sought to elucidate the relationship between TLR4 and STC1 in hyperoxia-induced lung injury in vitro and in vivo and to define the functional role of STC1 expression in lung endothelium. We identified significantly decreased STC1 expression in TLR4 knockout mouse lungs and primary lung endothelium isolated from TLR4 knockout mice. Overexpression of STC1 was associated with endothelial cytoprotection, whereas decreased or insufficient expression was associated with increased oxidant-induced injury and death. An Hsp70-TLR4-nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) signal mediates STC1 induction in the lungs and endothelial cells. We also demonstrated a previously unrecognized role for mitochondrial-associated STC1, via TLR4, in maintaining normal glycolysis, mitochondrial bioenergetics, and mitochondrial calcium levels. To date, a physiological role for STC1 in oxidant-induced ALI has not been identified. In addition, our studies show that STC1 is regulated by TLR4 and exerts lung and endothelial protection in response to sterile oxidant-induced lung injury. Our studies reveal a novel TLR4-STC1-mediated mitochondrial pathway that has homeostatic as well as oxidant-induced cytoprotective functions in lung endothelium.

Outer Membrane Vesicles Derived From Escherichia coli Regulate Neutrophil Migration by Induction of Endothelial IL-8.

Outer membrane vesicles (OMVs) are spherical, proteolipid nanostructures that are constitutively released by Gram-negative bacteria including Escherichia coli. Although it has been shown that administration of E. coli OMVs stimulates a strong pulmonary inflammatory response with infiltration of neutrophils into the lungs in vivo, the mechanism of E. coli OMV-mediated neutrophil recruitment is poorly characterized. In this study, we observed significant infiltration of neutrophils into the mouse lung tissues in vivo, with increased expression of the neutrophil chemoattractant CXCL1, a murine functional homolog of human IL-8, on intraperitoneal administration of E. coli OMVs. In addition, OMVs and CD31-positive endothelial cells colocalized in the mouse lungs. Moreover, in vitro results showed that E. coli OMVs significantly increased IL-8 release from human microvascular endothelial cells and toll-like receptor (TLR)4 was found to be the main component for recognizing E. coli OMVs among human endothelial cell-associated TLRs. Furthermore, the transmigration of neutrophils was suppressed in the lung tissues obtained from TLR4 knockout mice treated with E. coli OMVs. Taken together, our data demonstrated that E. coli OMVs potently recruit neutrophils into the lung via the release of IL-8/CXCL1 from endothelial cells in TLR4- and NF-κB-dependent manners.

Aflatoxin B1 Promotes Influenza Replication and Increases Virus Related Lung Damage via Activation of TLR4 Signaling.

Aflatoxin B1 (AFB1), which alters immune responses to mammals, is one of the most common mycotoxins in feeds and food. Swine influenza virus (SIV) is a major pathogen of both animals and humans. However, there have been few studies about the relationship between AFB1 exposure and SIV replication. Here, for the first time, we investigated the involvement of AFB1 in SIV replication in vitro and in vivo and explored the underlying mechanism using multiple cell lines and mouse models. In vitro studies demonstrated that low concentrations of AFB1 (0.01–0.25 μg/ml) markedly promoted SIV replication as revealed by increased viral titers and matrix protein (M) mRNA and nucleoprotein (NP) levels in MDCK cells, A549 cells and PAMs. In vivo studies showed that 10–40 μg/kg of AFB1 exacerbated SIV infection in mice as illustrated by significantly higher lung virus titers, viral M mRNA levels, NP levels, lung indexes and more severe lung damage. Further study showed that AFB1 upregulated TLR4, but not other TLRs, in SIV-infected PAMs. Moreover, AFB1 activated TLR4 signaling as demonstrated by the increases of phosphorylated NFκB p65 and TNF-α release in PAMs and mice. In contrast, TLR4 knockdown or the use of BAY 11-7082, a specific inhibitor of NFκB, blocked the AFB1-promoted SIV replication and inflammatory responses in PAMs. Furthermore, a TLR4-specific antagonist, TAK242, and TLR4 knockout both attenuated the AFB1-promoted SIV replication, inflammation and lung damage in mice. We therefore conclude that AFB1 exposure aggravates SIV replication, inflammation and lung damage by activating TLR4-NFκB signaling.

Herpes Simplex Virus Type 1 Engages Toll Like Receptor 2 to Recruit Macrophages During Infection of Enteric Neurons.

Herpes simplex virus type 1 (HSV-1) is a widespread neurotropic pathogen responsible for a range of clinical manifestations. Inflammatory cell infiltrate is a common feature of HSV-1 infections and has been implicated in neurodegeneration. Therefore, viral recognition by innate immune receptors (i.e., TLR2) and the subsequent inflammatory response are now deemed key players in HSV-1 pathogenesis. In this study we infected with HSV-1 the enteric nervous system (ENS) of wild-type (WT) and TLR2 knock-out (TLR2ko) mice to investigate whether and how TLR2 participates in HSV-1 induced neuromuscular dysfunction. Our findings demonstrated viral specific transcripts suggestive of abortive replication in the ENS of both WT and TLR2ko mice. Moreover, HSV-1 triggered TLR2-MyD88 depend signaling in myenteric neurons and induced structural and functional alterations of the ENS. Gastrointestinal dysmotility was, however, less pronounced in TLR2ko as compared with WT mice. Interesting, HSV-1 caused up-regulation of monocyte chemoattractant protein-1 (CCL2) and recruitment of CD11b+ macrophages in the myenteric ganglia of WT but not TLR2ko mice. At the opposite, the myenteric plexuses of TLR2ko mice were surrounded by a dense infiltration of HSV-1 reactive CD3+CD8+INFγ+ lymphocytes. Indeed, depletion CD3+CD8+ cells by means of administration of anti-CD8 monoclonal antibody reduced neuromuscular dysfunction in TLR2ko mice infected with HSV-1. During HSV-1 infection, the engagement of TLR2 mediates production of CCL2 in infected neurons and coordinates macrophage recruitment. Bearing in mind these observations, blockage of TLR2 signaling could provide novel therapeutic strategies to support protective and specific T-cell responses and to improve neuromuscular dysfunction in pathogen-mediated alterations of the ENS.

Toxic adjuvants alter the function and phenotype of dendritic cells to initiate adaptive immune responses induced by oral Helicobacter pylori vaccines.

Toxic adjuvant is considered as an indispensable constituent for oral Helicobacter pylori (H. pylori) vaccines. However, the elaborate role of toxic adjuvant in the initiation of adaptive immune response is largely undescribed. We employed an acid-resistant HP55/PLGA nanoparticles (NPs) delivery system encapsulating three antigens (Hsp, Nap, and Lpp20) from H. pylori and accompanied with three adjuvants (LPS, CpG, and chimeric flagellum (CF)) to explore the underlying mechanism of the adjuvant constituent. H. pylori-specific antibody responses were detected by ELISA. Gastric inflammatory and Th1/Th17 responses were analyzed by flow cytometry. Expressions of inflammatory cytokines were measured by quantitative real-time PCR. In bone marrow-derived dendritic cells' (BMDCs) model, the addition of toxic adjuvants is responsible for the proinflammatory function, but not the mature phenotype of BMDCs. In vivo, intestinal loop injection with NPs+LPS, rather than NPs alone, altered the dendritic cell (DC) phenotypes in mesenteric lymph nodes and drove a local proinflammatory microenvironment. In a prophylactic vaccination model, mice immunized with NPs+adjuvants significantly reduced the gastric colonization of H.pylori, induced antigen-specific antibody responses and Th1/Th17 cell responses. After H. pylori challenge, these mice showed potent recall responses involving both neutrophil and inflammatory monocyte infiltration. Additionally, TLR4 knockout mice were immunized with NPs+LPS and NPs+CF, respectively; only the recipients of NPs+CF orchestrated a protective response to control bacterial infection. Our study indicated that toxic adjuvants within oral H.pylori vaccines altered the function and phenotype of dendritic cells and facilitated the establishment of proinflammatory microenvironment to initiate adaptive immune responses.

Lethal influenza A virus preferentially activates TLR3 and triggers a severe inflammatory response

The “cytokine storm” and excessive inflammation triggered by lethal avian influenza virus (IAV) are responsible for its high virulence and mortality. However, the molecular mechanism behind these effects is unclear. In this study, we used LA795 cells and a mouse model to assess the crucial role of TLR3 during infection with lethal avian influenza A virus and subsequent inflammation. The results showed that IAVs could replicate and proliferate well in LA795 cells and that the replication of H5N1 was more efficient than human H1N1 and lowly pathogenic avian H7N2 viruses. The TLR3 signaling pathways were activated preferentially in vitro and in vivo and a range of pro-inflammatory cytokines were released following H5N1 infection. RNAi and TLR3 knockout mice were used to validate the results. These results are the first to provide insight into the preferential involvement of TLR3 in lethal avian influenza A virus infection and inflammation compared with others such as human or lowly pathogenic avian influenza A viruses. The data will increase understanding of the pathogenesis of lethal avian influenza A virus infection and may help facilitate the development of novel therapeutic aids targeting TLR3 signaling pathways.

TLR2 and TLR4 play opposite role in autophagy associated with cisplatin-induced acute kidney injury.

Acute kidney injury (AKI) is considered an inflammatory disease in which toll-like receptors (TLRs) signaling pathways play an important role. The activation of TLRs results in production of several inflammatory cytokines leading to further renal damage. In contrast, TLRs are key players on autophagy induction, which is associated with a protective function on cisplatin-induced AKI. Hence, the present study aimed to evaluate the specific participation of TLR2 and TLR4 molecules on the development of cisplatin-induced AKI. Complementarily, we also investigated the link between TLRs and heme oxygenase-1 (HO-1), a promisor cytoprotective molecule. First, we observed that only the absence of TLR2 but not TLR4 in mice exacerbated the renal dysfunction, tissue injury and mortality rate, even under an immunologically privileged microenvironment. Second, we demonstrated that TLR2 knockout (KO) mice presented lower expression of autophagy-associated markers when compared with TLR4 KO animals. Similar parameter was confirmed in vitro, using tubular epithelial cells derived from both KO mice. To test the cross-talking between HO-1 and TLRs, hemin (an HO-1 internal inducer) was administrated in cisplatin-treated TLR2 and TLR4 KO mice and it was detected an improvement in the global renal tissue parameters. However, this protection was less evident at TLR2 KO mice. In summary, we documented that TLR2 plays a protective role in cisplatin-induced AKI progression, in part, by a mechanism associated with autophagy up-regulation, considering that its interplay with HO-1 can promote renal tissue recover.

The importance of Toll-like receptor 4 during experimental Sporothrix brasiliensis infection.

Here we investigated the importance of Toll-like receptor 4 (TLR-4) in innate immune response to Sporothrix brasiliensis, a virulent fungus of Sporothrix spp. In vitro assays, using C57Bl/6 (wild type [WT]) bone marrow-derived macrophages (BMDMs), and TLR-4 knockout (TLR-4-/-) showed that the absence of TLR-4 resulted in impaired phagocytosis and lower levels of tumor necrosis factor α (TNF-α), interleukin (IL)-6, and nitric oxide. In vivo assays were also performed, and the mice (WT and TLR-4-/-) were intraperitoneally infected with S. brasiliensis yeast ATCC MyA-4831 and euthanized on days 7, 14, and 28 postinfection, with the following parameters evaluated: fungal burden in liver, spleen, kidney, and brain, and the production of cytokines interferon γ (IFN-γ), TNF-α, IL-2, IL-4, IL-6, and IL-10. The results demonstrate the macrophages dependency on TLR-4 for inflammatory activation and in the absence of TLR-4 during experimental S. brasiliensis infection enhanced dissemination occurred after 14 and 28 days. These data show that TLR-4 signals are important for the recognition of S. brasiliensis by macrophages, and their absence promotes the persistence of the infection.

Kidney Proximal Tubular TLR9 Exacerbates Ischemic Acute Kidney Injury.

The role for kidney TLR9 in ischemic acute kidney injury (AKI) remains unclear. In this study, we tested the hypothesis that renal proximal tubular TLR9 activation exacerbates ischemic AKI by promoting renal tubular epithelial apoptosis and inflammation. To test this hypothesis, we generated mice lacking TLR9 in renal proximal tubules (TLR9fl/fl PEPCK Cre mice). Contrasting previous studies in global TLR9 knockout mice, mice lacking renal proximal tubular TLR9 were protected against renal ischemia/reperfusion (IR) injury, with reduced renal tubular necrosis, inflammation (decreased proinflammatory cytokine synthesis and neutrophil infiltration), and apoptosis (decreased DNA fragmentation and caspase activation) when compared with wild-type (TLR9fl/fl) mice. Consistent with this, a selective TLR9 agonist oligonucleotide 1668 exacerbated renal IR injury in TLR9fl/fl mice but not in renal proximal tubular TLR9-null mice. Furthermore, in cultured human and mouse proximal tubule cells, TLR9-selective ligands induced NF-κB activation, proinflammatory cytokine mRNA synthesis, as well as caspase activation. We further confirm in the present study that global TLR9 deficiency had no impact on murine ischemic AKI. Taken together, our studies show that renal proximal tubular TLR9 activation exacerbates ischemic AKI by promoting renal tubular inflammation, apoptosis as well as necrosis, after IR via NF-κB and caspase activation. Our studies further suggest the complex nature of TLR9 activation, as renal tubular epithelial TLR9 promotes cell injury and death whereas TLR9 signaling in other cell types may promote cytoprotective effects.

Astaxanthin mitigates subarachnoid hemorrhage injury primarily by increasing sirtuin 1 and inhibiting the Toll-like receptor 4 signaling pathway.

Inflammation plays a key role in the progression of subarachnoid hemorrhage (SAH). Here, we examined the effects of astaxanthin (ATX) on the inflammatory response and secondary damage after SAH and the underlying mechanisms of action. In vivo, a prechiasmatic cistern injection model was established in rats and mice. In addition, neuron-microglia cocultures were exposed to oxyhemoglobin to mimic SAH in vitro. Western blotting revealed that protein expression of TLR4 was markedly increased in microglia at 24 h after SAH, with consequent increases in the downstream molecules myeloid differentiation factor 88 and NF-кB. Treatment with ATX significantly inhibited the TLR4 activation, increased sirtuin 1 expression, and inhibited the subsequent inflammatory response both in vivo and in vitro. ATX also significantly decreased high-mobility group box 1 nuclear translocation and secretion in neurons, an effect that was reversed by the sirtuin 1-specific inhibitor sirtinol. ATX administered 4 h after SAH ameliorated cerebral inflammation, brain edema, and neuronal death and improved neurologic function. ATX reduced neuronal death but did not improve neurologic function in TLR4 knockout mice. These results suggest that ATX reduces the proinflammatory response and secondary brain injury after SAH, primarily by increasing sirtuin 1 levels and inhibiting the TLR4 signaling pathway.-Zhang, X., Lu, Y., Wu, Q., Dai, H., Li, W., Lv, S., Zhou, X., Zhang, X., Hang, C., Wang, J. Astaxanthin mitigates subarachnoid hemorrhage injury primarily by increasing sirtuin 1 and inhibiting the Toll-like receptor 4 signaling pathway.

TLR4 mediates high-fat diet induced physiological changes in mice via attenuating PPARγ/ABCG1 signaling pathway

High-fat diet (HFD) is known to promote atherosclerosis which accelerates the development of atherosclerotic cardiovascular diseases. Vascular dysfunction characterized by inflammation and lipid accumulation is common in atherosclerosis caused by HFD. The specific effects of HFD on blood vessels and the underlying mechanisms need to be further clarified. Toll-like receptor 4 (TLR4) is a key contributing factor in atherosclerosis and TLR4 deficiency protects vascular smooth muscle cells against inflammatory responses and lipid accumulation in vitro. However, the physiological significance of TLR4 signaling in HFD-induced changes is unknown. In this study, we observed that HFD feeding increased body weight, circulating inflammatory cytokines and lipid accumulation in the aorta of wild-type mice but apart from increasing body weight, did not affect the TLR4 knockout mice. TLR4 expression increased significantly in the arterial walls after receiving HFD treatment, while that of the co-localizing PPARγ and ABCG1 markedly decreased. TLR4 deficiency reversed the HFD-induced attenuation of PPARγ and ABCG1. In conclusion, TLR4 mediates HFD induced increase in body weight, inflammation and aortic lipid accumulation through, at least partly, the PPARγ/ABCG1 signaling pathway. Therefore, interfering with TLR4 signaling is a viable therapeutic option in diet induced atherosclerosis.

Abstract 4726: Novel intravenous injectable TLR7 agonist, DSP-0509, synergistically enhanced antitumor immune responses in combination with anti-PD-1 antibody

Abstract Toll-like receptors (TLRs) are a family of pattern-recognition receptors (PRR) that recognize pathogen-associated molecular patterns (PAMPs). TLR7 is mainly expressed in plasmacytoid dendritic cells (pDCs) and recognizes virus derived single-stranded RNA (ssRNA). TLR7 stimulation in pDCs induces type I interferon secretion, which results in innate immune activation. Clinical studies have shown that TLR7 agonist induced anti-tumor immune activation. Despite these signs of clinical activity, administration of TLR7 agonist is generally limited to topical application, due to the potential of immune related adverse effects. In this study, we present a novel small molecule TLR7 agonist DSP-0509 formulated for intravenous administration. TLR7 agonistic activity was evaluated by in vitro reporter assay systems and TLR7 knock out mice. Induction of interferon alpha (IFNα) and inflammatory cytokines in mice and human blood was measured as PD response with ELISA. Anti-tumor effect was evaluated in LM8-bearing syngeneic mouse model. Combination effect of DSP-0509 with anti-PD-1 antibody was evaluated in CT26-bearing mice, along with flow cytometric analysis of tumor infiltrating lymphocytes (TILs). DSP-0509 had agonistic activity on human TLR7 (EC50= 316 nM), but not on human TLR8 (EC50> 10 μM). DSP-0509 had high water solubility and rapid elimination from the body (T1/2:0.69h), partly explained by excretion via organic anion transporting peptide (OATP) transporters. Intravenous administration of DSP-0509 induced IFNα secretion in wild type mice, but not in TLR7 knock out mice. Minimum cytokine induction dose of DSP-0509 in human whole blood was lower than that of a well-known TLR7/8 agonist 852A. Intravenous administration of DSP-0509 suppressed the primary tumor growth and the number of lung metastatic nodules in LM8-bearing model. Combination of DSP-0509 with anti-PD-1 antibody significantly suppressed the tumor growth compared to treatments with each monotherapy (P<0.05). The ratio of CD8+ T cells and effector memory T cell populations (CD8+CD62L-CD127+) in TILs and the surface expression of MHC class I molecule on tumor cells was significantly increased in the combination group (P<0.05). All mice responded to combination therapy rejected re-challenged tumor growth. The study showed that DSP-0509 is a novel TLR7 agonist with an intravenous injectable profile and rapid elimination from the body (T1/2: 0.69h). DSP-0509 showed anti-tumor effect against primary tumor growth and metastasis. In addition, anti-PD-1 antibody furthermore enhanced the effect of DSP-0509. We also reported that the combination of DSP-0509 with anti-PD-1 antibody significantly induced effector memory T cells (p<0.05 vs each monotherapy) and showed durable response. Further evaluations of DSP-0509 are warranted. Citation Format: Yosuke Ota, Takeshi Otsubo, Junya Koroki, Yuko Hirose, Erina Koga-Yamakawa, Masashi Murata, Masashi Goto, Yasushi Matsuki. Novel intravenous injectable TLR7 agonist, DSP-0509, synergistically enhanced antitumor immune responses in combination with anti-PD-1 antibody [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4726.

Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality.

Adenoviral vectors (AdV) are considered promising candidates for vaccine applications. A prominent group of Toll-like receptors (TLRs) participate in the adenovirus-induced adaptive immune response, yet there is little information regarding the role of TLR4 in AdV-induced immune responses in recent literature. We investigated the function of TLR4 in both adaptive and innate immune responses to an AdV-based anthrax vaccine. By immunizing wild-type and TLR4 knockout (TLR4-KO) mice, we revealed the requirement of TLR4 in AdV-induced innate responses. We also showed that TLR4 functions are required for germinal centre responses in immunized mice, as expression of the apoptosis-related marker Fas was down-regulated on germinal centre B cells from TLR4-KO mice. Likewise, decreased expression of inducible costimulator on follicular T helper cells was observed in immunized TLR4-KO mice. Moreover, a potent protective antigen-specific humoral immune response was mimicked using an adjuvant system containing the TLR4 agonist monophosphoryl lipid A. Overall, our findings showed that very rapid antigen-specific antibody production is correlated with the TLR4-imprinted germinal centre response to AdV-based vaccine. These results provide additional evidence for the use of the AdV and a TLR agonist to induce humoral responses. Our findings offer new insights into rational vaccine design.

Role of toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production in chemotherapy-induced mucositis.

Despite significant advances in our ability to treat cancer, cytotoxic chemotherapy continues to be the mainstay treatment for many solid tumours. Chemotherapy is commonly associated with a raft of largely manageable adverse events; however, gastrointestinal (GI) toxicity (also termed mucositis) remains a significant challenge with little in the way of preventative and therapeutic options. The inability to manage GI complications likely reflects our incomplete understanding of its aetiology and the idiosyncrasies of each chemotherapeutic agent. This review highlights aims to provide a narrative for the involvement of Toll-like receptor (TLR4) in the development of chemotherapy-induced GI mucositis, an already emerging theme within this field. Particular focus will be placed upon the signalling interaction between TLR4 and interleukin (IL)-6. This parallels recent preclinical findings showing that TLR4 knockout mice, which are protected from developing severe GI mucositis, completely lack an IL-6 response. As such, we suggest that this signalling pathway presents as a novel mechanism with potential for therapeutic intervention.