Neutralization of pro-inflammatory monocytes by targeting TLR2 dimerization ameliorates colitis.

Toll-Like Receptor Signaling in Liver Diseases

Epidermal Growth Factor Partially Restores Colonic Ion Transport Responses in Mouse Models of Chronic Colitis

Dysbiosis and accumulation of reactive oxygen species (ROS) are hallmarks of ulcerative colitis (UC) and have been associated with tumor progression. We have previously shown that UC patient biopsies and colorectal adenomas overexpress toll-like receptor 4 (TLR4) in intestinal epithelial cells (IECs). A potential mechanism of TLR4-mediated tumorigenesis is by providing cancer stem cells with redox signals that they efficiently use to outgrow their non-transformed counterparts. We test the hypothesis that epithelial signaling via TLR4 induces the production of ROS through the NADPH oxidases Nox1 and Duox2. We also explore the main redox-sensitive cascades that participate in TLR4-driven tumorigenesis. Colonoids from wild-type (WT), TLR4-KO, and Nox1-KO mice were stimulated with LPS and tested for H2O2 production in the presence of the NADPH oxidase inhibitor, diphenyleneiodonium. IECs isolated from villin-TLR4 mice (which have constitutive activation of epithelial TLR4) and their WT littermates were analyzed for H2O2 production (Amplex Red) and expression of the NADPH oxidases Nox1 and Duox2 (qPCR). Villin-TLR4 and their littermates underwent a model of CAC by azoxymethane injection followed by 2 cycles of 3% dextran sulfate sodium (1.5% in villin-TLR4 mice). Cancer stem cells (aldehyde dehydrogenase positive) were sorted for RNA sequencing. LPS incubation enhanced the expression of Nox1 and Duox2 in WT but not TLR4-KO colonoids, demonstrating that these oxidative enzymes are regulated by TLR4. Consistently, LPS stimulation elicited H2O2 release by WT organoids in a NADPH oxidase-dependent manner. Nox1-KO colonoids showed attenuated production of H2O2 upon LPS stimulation, confirming the participation of this NADPH oxidase in TLR4-mediated production of ROS. Constitutive activation of TLR4 induced the production of H2O2 in IECs, which was accompanied by a marked upregulation of Nox1, Duox2, and DuoxA2. RNAseq done on cancer stem cells shows that cancer stem cells of villin-TLR4 tumors have increased expression of NADPH oxidases, as well as enrichment of redox-sensitive pathways, including PI3K, myc, Rac1, and NF-κB. All of these pathways have been implicated in the promotion of proliferative and antiapoptotic signals. TLR4 controls the expression and function of oxidative enzymes in IECs, enhancing redox signals in cancer stem cells upon bacterial challenge. In the context of inflammation-associated dysbiosis, we speculate that overexpression of TLR4 provides additional activation of proliferative and antiapoptotic pathways, accelerating tumor development and growth. The results of our studies highlight how we might target critical signaling pathways in inflammatory-driven cancer stem cells.

OBJECTIVEInsulin resistance in diet-induced obesity (DIO) is associated with a chronic systemic low-grade inflammation, and Toll–like receptor 4 (TLR4) plays an important role in the link among insulin resistance, inflammation, and obesity. The current study aimed to analyze the effect of exercise on TLR4 expression and activation in obese rats and its consequences on insulin sensitivity and signaling.RESEARCH DESIGN AND METHODSThe effect of chronic and acute exercise was investigated on insulin sensitivity, insulin signaling, TLR4 activation, c-Jun NH2-terminal kinase (JNK) and IκB kinase (IKKβ) activity, and lipopolysaccharide (LPS) serum levels in tissues of DIO rats.RESULTSThe results showed that chronic exercise reduced TLR4 mRNA and protein expression in liver, muscle, and adipose tissue. However, both acute and chronic exercise blunted TLR4 signaling in these tissues, including a reduction in JNK and IKKβ phosphorylation and IRS-1 serine 307 phosphorylation, and, in parallel, improved insulin-induced IR, IRS-1 tyrosine phosphorylation, and Akt serine phosphorylation, and reduced LPS serum levels.CONCLUSIONSOur results show that physical exercise in DIO rats, both acute and chronic, induces an important suppression in the TLR4 signaling pathway in the liver, muscle, and adipose tissue, reduces LPS serum levels, and improves insulin signaling and sensitivity. These data provide considerable progress in our understanding of the molecular events that link physical exercise to an improvement in inflammation and insulin resistance.

Core fucosylation, catalysed by α-1, 6 fucosyltransferase (FUT8), regulates growth factor receptors in immune function. Although core fucose regulates many immune cell types, few reports confront the association between core fucose activity and an innate immune reaction. Here, we have investigated the function of core fucose in macrophages in vivo and in vitro using Fut8-deficient mice and cells. Following lipopolysaccharide (LPS) stimulation, inflammatory cytokine production in Fut8-deficient (Fut8-/-) macrophages was suppressed in both in vivo and in vitro experiments. Because LPS is recognized by Toll-like receptor 4 (TLR4), which induces the signalling cascade, TLR4 signalling was assumed to be impaired in Fut8-/- cells. Flow cytometry analyses revealed, however, that a lack of core fucose reduced the expression of, not TLR4, but CD14, which is necessary for TLR4 endocytosis. Because CD14 is necessary for TLR2 signalling, the immune response of TLR2 was also impaired in Fut8-/- macrophages. Moreover, in the dextran sodium sulphate (DSS)-induced murine colitis model, the mice grafted with Fut8-/- bone marrow cells exhibited higher resistance to inflammation than those grafted with Fut8+/+ bone marrow cells. These findings indicate that core fucose is essential for CD14-dependent TLR4 and TLR2 signalling in murine macrophage activity, leading to DSS-induced experimental colitis.

The Significance of TLR4 in Cisplatin-Induced Ototoxicity

SHP limits TLR signaling, an inducible transcriptional corepressor

The Lipid-Modifying Enzyme SMPDL3B Negatively Regulates Innate Immunity.

Harnessing innate immunity to eliminate SARS-CoV-2 and ameliorate COVID-19 disease.

Stress-Induced Neuroinflammation: Role of the Toll-Like Receptor-4 Pathway

18β-glycyrrhetinic acid inhibited mitochondrial energy metabolism and gastric carcinogenesis through methylation-regulated TLR2 signaling pathway.

The innate immune system detects a diverse range of pathogen- and host-derived molecules, such as cell wall components, nucleic acids and heat shock proteins via the Toll-like receptors (TLRs). TLRs are present in the central nervous system (CNS), predominantly expressed in microglia, the major immune cells of the brain. Increasing evidence suggests that TLRs play a crucial role in CNS pathologies, such as infection, stroke and classical neurodegenerative diseases. This thesis describes the consequences of TLR activation in the CNS. Highly specific ligands for TLR2, TLR4, TLR7 and TLR9 were used to address the TLR's individual impact on neuroinflammation and –degeneration. In addition, combined TLR activation was assessed, as it can be assumed that in both physiological and pathological states of the CNS, more than one TLR ligand is present and multiple TLRs are activated. In vitro, activation of TLR2, TLR4, TLR7 and TLR9 with their specific exogenous ligands resulted in the microglia- mediated release of inflammatory molecules. Combined TLR stimulation of TLR4 with TLR2 and TLR4 with TLR9 resulted in an increased release of TNF-alpha, IL-6, IL-10 and nitric oxide compared to activation of the respective single TLR. In contrast, additional activation of TLR7 suppressed the inflammatory response mediated by TLR2, TLR4, or TLR9, indicating that TLRs individually modulate the immune response. In co-cultures of neurons and microglia activation of TLRs resulted in neuronal cell injury, whereas only the combination of TLR4 with TLR2 led to significantly more neuronal cell death compared to activation of each TLR alone. When replacing the exogenous ligand of TLR4 with the endogenous ligand heat shock protein 60 (HSP60), combined activation of TLR4 with TLR7 resulted in an increased loss of neurons compared to single TLR activation, indicating diverging events subsequent of TLR4 activation. In vivo, the impact of TLR activation in the murine brain was evaluated after intrathecal injection of the exogenous ligands for TLR2, TLR4, TLR7 or TLR9. Activation of each TLR led to a distinct change in mRNA expression levels of TLR1-9. Activation of TLR2, TLR7 and TLR9 caused axonal and neuronal cell loss in the cerebral cortex, in part accompanied by microglia displaying an activated morphology, by the influx of leukocytes into the cerebrospinal fluid (CSF) and by elevated levels of TNF-alpha and IL-1beta mRNA. Intrathecal injection of HSP60 caused TLR4-dependent neuronal cell death and axonal injury, as well as demyelination and loss of oligodendrocytes in the cerebral cortex. In contrast to the exogenous ligand of TLR4, intrathecal HSP60 did not induce a measurable inflammatory response in the murine brain. Immunohistological analysis of brain sections from naïve mice revealed an expression of HSP60, predominantly in neurons. Moreover, elevated levels of HSP60 were detected in the CSF of mice subjected to a middle cerebral artery occlusion, indicating a release of HSP60 during brain injury and a mechanism by which TLR4 is exposed to HSP60 in CNS pathologies. These findings demonstrate that sole and combined activation of TLRs modify the pattern and extent of TLR expression, of inflammation and of neuronal cell injury in the CNS, mechanisms by which the intrinsic innate immunity might co-determine the diversity of CNS diseases. Further, the release of endogenous TLR ligands during CNS cell damage bi-directionally links CNS injury to neurodegeneration and demyelination in vivo.

Ocular surface microbiota (OSM) dysbiosis is associated with dry eye disease (DED), but the underlying mechanisms remain poorly understood. This study investigated the role of lipoteichoic acid (LTA), a Gram-positive bacterial product, in DED pathogenesis. A cross-sectional study was conducted involving 277 participants to analyze OSM composition via 16S rRNA sequencing and quantify tear LTA levels. In vitro experiments using human corneal epithelial cells (HCE-T) and in vivo mouse models were used to explore LTA-induced inflammation. The involvement of Toll-like receptor 2 (TLR2) and platelet-activating factor receptor (PAFR) was assessed using siRNAs and the PAFR antagonist Apafant. Patients with DED showed functional enrichment in LTA synthesis-related pathways in their OSM. Tear LTA levels were significantly elevated in patients with DED and correlated positively with disease risk and clinical severity. Mechanistically, LTA activated p38 MAPK and NF-κB signaling pathways via TLR2 and PAFR, upregulating pro-inflammatory cytokine expression. In mice, topical LTA administration induced corneal epithelial damage, reduced tear secretion, and caused histopathological changes. Treatment with Apafant significantly alleviated LTA-induced corneal inflammation, improved tear secretion, and preserved epithelial integrity. Our findings establish a mechanistic link between OSM-derived LTA and DED. LTA promotes ocular surface inflammation and dysfunction through TLR2 and PAFR signaling, suggesting that targeting microbial products or their receptors, such as PAFR, may offer a novel therapeutic strategy for DED.

Endoplasmic reticulum (ER) stress contribute to inflammatory bowel disease (IBD). However, the mechanistic link between toll-like receptor 4 (TLR4) and ER stress in IBD remains elusive. This study aimed to investigate the mechanism by which ER stress and TLR4 promote inflammation in IBD.IBD mouse model was established by the induction of TNBS, and Grp78 and TLR4 in intestine tissues were detected by immunohistochemistry. THP-1 cells were treated with lipopolysaccharides (LPS), ER stress inducer or inhibitor tauroursodeoxycholic acid (TUDCA), or p38 MAPK inhibitor. The activation of MAPK signaling was detected by Western blot, and the production and secretion of inflammatory factors were detected by PCR and ELISA. We found that the expression levels of TLR4 and GRP78 were significantly higher in the intestine of IBD model mice compared with control mice but were significantly lower in the intestine of IBD model mice treated with ER stress inhibitor TUDCA. ER stress inducer significantly increased while ER stress inhibitor TUDCA significantly decreased the expression and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated by LPS. Only p38 MAPK signaling was activated in THP-1 cells treated by ER stress inducer. Furthermore, p38 inhibitor SB203580 inhibited the production and secretion of TNF-α, IL-1β and IL-8 in THP-1 cells treated with LPS. In conclusion, TLR4 promotes ER stress induced inflammation in IBD, and the effects may be mediated by p38 MAPK signaling. TLR4 and p38 MAPK signaling are novel therapeutic targets for IBD.

The innate immune system allows for rapid recognition of pathogens. Toll-like receptor (TLR) signaling is a key aspect of the innate immune response, and interleukin-1 receptor-associated kinase 4 (IRAK4) plays a vital role in the TLR signaling cascade. Each TLR recognizes a distinct set of pathogen-associated molecular patterns (PAMPs) that encompass conserved microbial components such as lipopolysaccharides and flagellin. Upon binding of PAMPs and TLR activation, TLR intracellular domains initiate the oligomerization of the myeloid differentiation primary response 88 (MyD88), IRAK1, IRAK2, and IRAK4 signaling platform known as the Myddosome complex while also triggering the Toll/IL-1R domain-containing adaptor-inducing IFN-β (TRIF)-dependent pathway. The Myddosome complex initiates signal transduction pathways enabling the activation of NF-κB and mitogen-activated protein kinase (MAPK) transcription factors and the subsequent production of inflammatory cytokines. Human IRAK4 deficiency is an autosomal recessive inborn error of immunity that classically presents with blunted or delayed inflammatory response to infection and susceptibility to a narrow spectrum of pyogenic bacteria, particularly Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa. We describe a case of IRAK4 deficiency in an 11-mo-old boy with concurrent S. pneumoniae bacteremia and S. aureus cervical lymphadenitis with a blunted inflammatory response to invasive infection. Although initial clinical immune profiling was unremarkable, a high degree of suspicion for an innate immune defect prompted genetic sequencing. Genetic testing revealed a novel variant in the IRAK4 gene (c.1049delG, p.(Gly350Glufs*15)) predicted to be likely pathogenic. Functional testing showed a loss of IRAK4 protein expression and abolished TLR signaling, confirming the pathogenicity of this novel IRAK4 variant.