Intersections between innate immune response and gastric cancer development.

Worldwide, gastric cancer (GC) is the fifth most commonly diagnosed malignancy. It has a reduced prevalence but has maintained its poor prognosis being the fourth leading cause of deaths related to cancer. The highest mortality rates occur in Asian and Latin American countries, where cases are usually diagnosed at advanced stages. Overall, GC is viewed as the consequence of a multifactorial process, involving the virulence of the Helicobacter pylori (H. pylori) strains, as well as some environmental factors, dietary habits, and host intrinsic factors. The tumor microenvironment in GC appears to be chronically inflamed which promotes tumor progression and reduces the therapeutic opportunities. It has been suggested that inflammation assessment needs to be measured qualitatively and quantitatively, considering cell-infiltration types, availability of receptors to detect damage and pathogens, and presence or absence of aggressive H. pylori strains. Gastrointestinal epithelial cells express several Toll-like receptors and determine the first defensive line against pathogens, and have been also described as mediators of tumorigenesis. However, other molecules, such as cytokines related to inflammation and innate immunity, including immune checkpoint molecules, interferon-gamma pathway and NETosis have been associated with an increased risk of GC. Therefore, this review will explore innate immune activation in the context of premalignant lesions of the gastric epithelium and established gastric tumors.

How Toll-like receptors influence Parkinson's disease in the microbiome-gut-brain axis.

Recently, a large number of experimenters have found that the pathogenesis of Parkinson's disease may be related to the gut microbiome and proposed the microbiome-gut-brain axis. Studies have shown that Toll-like receptors, especially Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4), are key mediators of gut homeostasis. In addition to their established role in innate immunity throughout the body, research is increasingly showing that the Toll-like receptor 2 and Toll-like receptor 4 signaling pathways shape the development and function of the gut and enteric nervous system. Notably, Toll-like receptor 2 and Toll-like receptor 4 are dysregulated in Parkinson's disease patients and may therefore be identified as the core of early gut dysfunction in Parkinson's disease. To better understand the contribution of Toll-like receptor 2 and Toll-like receptor 4 dysfunction in the gut to early α-synuclein aggregation, we discussed the structural function of Toll-like receptor 2 and Toll-like receptor 4 and signal transduction of Toll-like receptor 2 and Toll-like receptor 4 in Parkinson's disease by reviewing clinical, animal models, and in vitro studies. We also present a conceptual model of the pathogenesis of Parkinson's disease, in which microbial dysbiosis alters the gut barrier as well as the Toll-like receptor 2 and Toll-like receptor 4 signaling pathways, ultimately leading to a positive feedback loop for chronic gut dysfunction, promoting α-synuclein aggregation in the gut and vagus nerve.

Small-Molecule Modulators Targeting Toll-like Receptors for Potential Anticancer Therapeutics.

Toll-like receptors (TLRs) are key components of the innate immune system and serve as a crucial link between innate and acquired immunity. In addition to immune function, TLRs are involved in other important pathological processes, including tumorigenesis. TLRs have dual regulatory effects on tumor immunity by activating nuclear factor κ-B signaling pathways, which induce tumor immune evasion or enhance the antitumor immune response. Therefore, TLRs have become a popular target for cancer prevention and treatment, and TLR agonists and antagonists offer considerable potential for drug development. The TLR7 agonist imiquimod (1) has been approved by the U.S. Food and Drug Administration as a treatment for malignant skin cancer. Herein, the structure, signaling pathways, and function of the TLR family are summarized, and the structure-activity relationships associated with TLR selective and multitarget modulators and their potential application in tumor therapy are systematically discussed.

Expression and function of toll-like receptor 2 in vertebrate.

Toll-like receptors (TLRs) are essential for identifying and detecting pathogen-associated molecular patterns (PAMPs) produced by a variety of pathogens, including viruses and bacteria. Since TLR2 is the only TLR capable of creating functional heterodimers with more than two other TLR types, it is very important for vertebrate immunity. TLR2 not only broadens the variety of PAMPs that it can recognize but has also the potential to diversify the subsequent signaling cascades. TLR2 is ubiquitous, which is consistent with the wide variety of tasks and functions it serves. Immune cells, endothelial cells, and epithelial cells have all been found to express TLR2. This review aims to gather currently available information about the preservation of this intriguing immunological molecule in the phylum of vertebrates.

Toll-like receptors in pathogenesis of neurodegenerative diseases and their therapeutic potential.

Toll-like receptors (TLRs) are a family of pattern-recognition receptors triggered by pathogen-derived and tissue-damage-related ligands. TLRs were previously believed to only be expressed in immune cells. However, it is now confirmed that they are ubiquitously expressed in cells within the body including neurons, astrocytes, and microglia of the central nervous system (CNS). Activation of TLRs is capable of inducing immunologic and inflammatory responses to injury or infection of CNS. This response is self-limiting that usually resolves once the infection has been eradicated or the tissue damage has been repaired. However, the persistence of inflammation-inducing insults or a failure in normal resolution mechanisms may result in overwhelming inflammation which may induce neurodegeneration. This implies that TLRs may play a role in mediating the link between inflammation and neurodegenerative diseases namely Alzheimer's disease, Parkinson's disease, Huntington's disease, stroke, and amyotrophic lateral sclerosis. So, new therapeutic approaches that specifically target TLRs may be developed by better understanding TLR expression mechanisms in the CNS and their connections to particular neurodegenerative disorders. Therefore, this review paper discussed the role of TLRs in neurodegenerative diseases.

Effects of dietary tryptophan on the antioxidant capacity and immune response associated with TOR and TLRs/MyD88/NF-κB signaling pathways in northern snakehead, Channa argus (Cantor, 1842).

Introduction: Dietary tryptophan (Trp) has been shown to influence fish feed intake, growth, immunity and inflammatory responses. The purpose of this study was to investigate the effect and mechanism of Trp on immune system of juvenile northern snakehead (Channa argus Cantor, 1842). Methods: A total of 540 fish (10.21 ± 0.11 g) were fed six experimental diets containing graded levels of Trp at 1.9, 3.0, 3.9, 4.8, 5.9 and 6.8 g/kg diet for 70 days, respectively. Results and Discussion: The results showed that supplementation of 1.9-4.8 g/kg Trp in diets had no effect on the hepatosomatic index (HSI) and renal index (RI), while dietary 3.9 and 4.8 g/kg Trp significantly increased spleen index (SI) of fish. Dietary 3.9, 4.8, 5.9 and 6.8 g/kg Trp enhanced the total hemocyte count (THC), the activities of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD). Malondinaldehyde (MDA) levels in the blood were significantly decreased by consuming 3.9 and 4.8 g/kg Trp. Fish fed with 3.0 and 3.9 g/kg Trp diets up-regulated interleukin 6 (il-6) and interleukin 8 (il-8) mRNA levels. The expression of tumor necrosis factor α (tnf-α) was highest in fish fed with 3.0 g/kg Trp diet, and the expression of interleukin 1ß (il-1ß) was highest in fish fed with 3.9 g/kg Trp diet. Dietary 4.8, 5.9 and 6.8 g/kg Trp significantly decreased il-6 and tnf-α mRNA levels in the intestine. Moreover, Trp supplementation was also beneficial to the mRNA expression of interleukin 22 (il-22). Additionally, the mRNA expression levels of target of rapamycin (tor), toll-like receptor-2 (tlr2), toll-like receptor-4 (tlr4), toll-like receptor-5 (tlr5) and myeloid differentiation primary response 88 (myd88) of intestine were significantly up-regulated in fish fed 1.9, 3.0 and 3.9 g/kg Trp diets, and down-regulated in fish fed 4.8, 5.9 and 6.8 g/kg Trp diets. Dietary 4.8 and 5.9 g/kg Trp significantly increased the expression of inhibitor of nuclear factor kappa B kinase beta subunit (ikkß) and decreased the expression of inhibitor of kappa B (iκbα), but inhibited nuclear transcription factor kappa B (nf-κb) mRNA level. Collectively, these results indicated that dietary 4.8 g/kg Trp could improve antioxidant capacity and alleviate intestinal inflammation associated with TOR and TLRs/MyD88/NF-κB signaling pathways.

Pattern-recognition receptors in endometriosis: A narrative review.

Endometriosis is closely associated with ectopic focal inflammation and immunosuppressive microenvironment. Multiple types of pattern recognition receptors (PRRs) are present in the innate immune system, which are able to detect pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) in both intracellular and external environments. However, the exact role of PRRs in endometriosis and the underlying molecular mechanism are unclear. PRRs are necessary for the innate immune system to identify and destroy invasive foreign infectious agents. Mammals mainly have two types of microbial recognition systems. The first one consists of the membrane-bound receptors, such as toll-like receptors (TLRs), which recognize extracellular microorganisms and activate intracellular signals to stimulate immune responses. The second one consists of the intracellular PRRs, including nod-like receptors (NLRs) and antiviral proteins retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA-5) with helix enzyme domain. In this review, we mainly focus on the key role of PRRs in the pathological processes associated with endometriosis. PRRs recognize PAMPs and can distinguish pathogenic microorganisms from self, triggering receptor ligand reaction followed by the stimulation of host immune response. Activated immune response promotes the transmission of microbial infection signals to the cells. As endometriosis is characterized by dysregulated inflammation and immune response, PRRs may potentially be involved in the activation of endometriosis-associated inflammation and immune disorders. Toll-like receptor 2 (TLR2), toll-like receptor 3 (TLR3), toll-like receptor 4 (TLR4), nod-like receptor family caspase activation and recruitment domain (CARD) domain containing 5 (NLRC5), nod-like receptor family pyrin domain containing 3 (NLRP3), and c-type lectin receptors (CLRs) play essential roles in endometriosis development by regulating immune and inflammatory responses. Absent in melanoma 2 (AIM2)-like receptors (ALRs) and retinoic acid-inducible gene I-like receptors (RLRs) may be involved in the activation of endometriosis-associated immune and inflammation disorders. PRRs, especially TLRs, may serve as potential therapeutic targets for alleviating pain in endometriosis patients. PRRs and their ligands interact with the innate immune system to enhance inflammation in the stromal cells during endometriosis. Thus, targeting PRRs and their new synthetic ligands may provide new therapeutic options for treating endometriosis.

Inflammatory response triggered by avian hepatitis E virus in vivo and in vitro.

Hepatitis E virus (HEV) is relevant to public health worldwide, and it affects a variety of animals. Big liver and spleen disease (BLS) and hepatitis-splenomegaly syndrome (HSS) associated with avian HEV (aHEV) were first reported in 1988 and in 1991, respectively. Here, cell culture-adapted aHEV genotype 3 strain, YT-aHEV (YT strain), a typical genotype isolated in China, was used for basic and applied research. We evaluated liver injury during the early stages of infection caused by the YT strain in vivo. Both in vivo and in vitro experimental data demonstrated that viral infection induces innate immunity, with mRNA expression levels of two key inflammatory factors, interleukin-1ß (IL-1ß) and IL-18, significantly upregulated. The YT strain infection was associated with the activation of Toll-like receptors (TLRs), nuclear factor kappa B (NF-κB), caspase-1, and NOD-like receptors (NLRs) in the liver and primary hepatocellular carcinoma epithelial cells (LMH). Moreover, inhibiting c-Jun N-terminal kinase, extracellular signal-regulated kinase (ERK1 or 2), P38, NF-κB, or caspase-1 activity has different effects on NLRs, and there is a mutual regulatory relationship between these signaling pathways. The results show that SB 203580, U0126, and VX-765 inhibited IL-1ß and IL-18 induced by the YT strain, whereas Pyrrolidinedithiocarbamate (PDTC) had no significant effect on the activity of IL-1ß and IL-18. Pretreatment of cells with SP600125 had an inhibitory effect on IL-18 but not on IL-1ß. The analysis of inhibition results suggests that there is a connection between Mitogen-activated protein kinase (MAPK), NF-κB, and the NLRs signaling pathways. This study explains the relationship between signaling pathway activation (TLRs, NF-κB, MAPK, and NLR-caspase-1) and viral-associated inflammation caused by YT strain infection, which will help to dynamic interaction between aHEV and host innate immunity.

The Role of Toll-like Receptors (TLRs) and Their Related Signaling Pathways in Viral Infection and Inflammation.

Toll-like receptors (TLRs) belong to a powerful system for the recognition and elimination of pathogen-associated molecular patterns (PAMPs) from bacteria, viruses, and other pathogens [...].

TLR9 Signaling Is Required for the Porphyromonas gingivalis-Induced Activation of IL-10-Expressing B Cells.

Immune cell pattern-recognition receptors such as Toll-like receptors (TLRs) play important roles in the regulation of host responses to periodontal pathogens. Our previous studies have demonstrated that immune regulatory B cells were activated by TLRs and alleviated periodontitis inflammation and bone loss. The purpose of this study is to determine the role of TLR9 signaling in the activation and IL-10 production of the primed-immune B cells in vitro. Wild-type (WT) and TLR9 knockout (TLR9KO) mice (C57BL/6 background, n = 5) were pre-immunized intraperitoneally with 1 × 108 formalin-fixed P. gingivalis and boosted once with 1 × 107 formalin-fixed P. gingivalis. Isolated splenocytes and purified B cells from each mouse were cultured with 1 × 108 formalin-fixed P. gingivalis for 48 h. Immunocytochemistry was performed to detect CD45+ IL-10+ cells. Levels of IL-10 expression and secretion in splenocytes and B cells were detected using qRT-PCR and ELISA, respectively. After stimulation with fixed P. gingivalis, the percentage of CD45+ IL-10+ B cells and the level of IL-10 expression were significantly increased (p < 0.01) in splenocytes and purified B cells isolated from WT mice. However, these changes were not observed in splenocytes and purified B cells from TLR9KO mice when the cells were treated with fixed P. gingivalis. The percentage of CD45+ IL-10+ B cells was significantly reduced in splenocytes and purified B cells from TLR9KO mice compared to those from WT mice when challenged with P. gingivalis. IL-10 expression in B cells from TLR9KO mice was significantly decreased compared to those from WT mice at both the mRNA and protein levels. Additionally, P. gingivalis-induced up-regulation of TNF-α mRNA expressions were consistently observed in B cells from both WT and TLR9KO mice. P. gingivalis-induced B10 activation and IL-10 production during adaptive responses by primed B cells requires TLR9 signaling and can be achieved independent of T-cell help.

Lindenane-Type Sesquiterpene Dimers Mitigate Lipopolysaccharide-Induced Inflammation by Inhibiting Toll-Like Receptor Signaling.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and trigger an inflammatory response via the myeloid differential factor 88 (MyD88)-dependent and toll-interleukin-1 receptor domain-containing adapter-inducing interferon-ß (TRIF)-dependent pathways. Lindenane type sesquiterpene dimers (LSDs) are characteristic metabolites of plants belonging to the genus Sarcandra (Chloranthaceae). The aim of this study was to evaluate the potential anti-inflammatory effects of the LSDs shizukaol D (1) and sarcandrolide E (2) on lipopolysaccharides (LPS)-stimulated RAW264.7 macrophages in vitro, and explore the underlying mechanisms. Both LSDs neutralized the LPS-induced morphological changes and production of nitric oxide (NO), as determined by CCK-8 assay and Griess assay, respectively. Furthermore, shizukaol D (1) and sarcandrolide E (2) downregulated interferon ß (IFNß), tumor necrosis factor α (TNFα) and interleukin-1ß (IL-1ß) mRNA levels as measured by reverse transcription polymerase chain reaction (RT-PCR), and inhibited the phosphorylation of nuclear factor kappa B p65 (p65), nuclear factor kappa-Bα (IκBα), Jun N-terminal kinase (JNK), extracellular regulated kinase (ERK), mitogen-activated protein kinase p38 (p38), MyD88, IL-1RI-associated protein kinase 1 (IRAK1), and transforming growth factor-ß-activated kinase 1 (TAK1) proteins in the Western blotting assay. In conclusion, LSDs can alleviate the inflammatory response by inhibiting the TLR/MyD88 signalling pathway.

A Toll pathway effector protects Drosophila specifically from distinct toxins secreted by a fungus or a bacterium.

The Drosophila systemic immune response against many Gram-positive bacteria and fungi is mediated by the Toll pathway. How Toll-regulated effectors actually fulfill this role remains poorly understood as the known Toll-regulated antimicrobial peptide (AMP) genes are active only against filamentous fungi and not against Gram-positive bacteria or yeasts. Besides AMPs, two families of peptides secreted in response to infectious stimuli that activate the Toll pathway have been identified, namely Bomanins and peptides derived from a polyprotein precursor known as Baramicin A (BaraA). Unexpectedly, the deletion of a cluster of 10 Bomanins phenocopies the Toll mutant phenotype of susceptibility to infections. Here, we demonstrate that BaraA is required specifically in the host defense against Enterococcus faecalis and against the entomopathogenic fungus Metarhizium robertsii, albeit the fungal burden is not altered in BaraA mutants. BaraA protects the fly from the action of distinct toxins secreted by these Gram-positive and fungal pathogens, respectively, Enterocin V and Destruxin A. The injection of Destruxin A leads to the rapid paralysis of flies, whether wild type (WT) or mutant. However, a larger fraction of wild-type than BaraA flies recovers from paralysis within 5 to 10 h. BaraAs' function in protecting the host from the deleterious action of Destruxin is required in glial cells, highlighting a resilience role for the Toll pathway in the nervous system against microbial virulence factors. Thus, in complement to the current paradigm, innate immunity can cope effectively with the effects of toxins secreted by pathogens through the secretion of dedicated peptides, independently of xenobiotics detoxification pathways.

Understanding the molecular response of non-mammalian toll-like receptor 22 (TLR22) in amphibious air-breathing catfish, Clarias magur (Hamilton, 1822) to bacterial infection or ligand stimulation through molecular cloning and expression profiling.

Toll-like receptor (TLR) 22 is a non-mammalian TLR, which is identified initially as a functional substitute of mammalian TLR3 in recognizing cell surface long dsRNA in teleosts. To understand the pathogen surveillance role played by TLR22 in an air-breathing catfish model the full-length cDNA of TLR22 was identified in Clarias magur and found to be consisted of 3597 nucleotides encoding for 966 amino acids. In the deduced amino acid sequence of C. magur TLR22 (CmTLR22) key signature domains such as one signal peptide, 13 LRRs, one transmembrane domain, one LRR_CT domain and an intracellular TIR domain could be identified. The CmTLR22 formed a separate cluster with other catfish TLR22 genes and situated within the TLR22 cluster in the phylogenetic analysis of teleost TLR groups. The CmTLR22 was constitutively expressed in all the 12 tested tissues of healthy C. magur juveniles with the highest transcript abundance in spleen followed by brain, intestine and head kidney. Following induction with the dsRNA viral analogue, poly (I:C), the level of expression of CmTLR22 was up-regulated in tissues such as kidney, spleen and gills. Whereas, in Aeromonas hydrophila-challenged C. magur, the expression levels of CmTLR22 was found to be up-regulated in gills, kidney and spleen, and down-regulated in liver. The findings of the current study suggest that the specific function of TLR22 is evolutionarily conserved in C. magur and might play a key role in mounting immune response by recognizing Gram-negative fish pathogen such as A. hydrophila and aquatic viruses in air-breathing amphibious catfishes.

Imbalanced Inflammatory Responses in Preterm and Term Cord Blood Monocytes and Expansion of the CD14+CD16+ Subset upon Toll-like Receptor Stimulation.

Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes-except for lower IL-1ß levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14+CD16+). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.

Dialog beyond the Grave: Necrosis in the Tumor Microenvironment and Its Contribution to Tumor Growth.

Damage-associated molecular patterns (DAMPs) are endogenous molecules released from the necrotic cells dying after exposure to various stressors. After binding to their receptors, they can stimulate various signaling pathways in target cells. DAMPs are especially abundant in the microenvironment of malignant tumors and are suspected to influence the behavior of malignant and stromal cells in multiple ways often resulting in promotion of cell proliferation, migration, invasion, and metastasis, as well as increased immune evasion. This review will start with a reminder of the main features of cell necrosis, which will be compared to other forms of cell death. Then we will summarize the various methods used to assess tumor necrosis in clinical practice including medical imaging, histopathological examination, and/or biological assays. We will also consider the importance of necrosis as a prognostic factor. Then the focus will be on the DAMPs and their role in the tumor microenvironment (TME). We will address not only their interactions with the malignant cells, frequently leading to cancer progression, but also with the immune cells and their contribution to immunosuppression. Finally, we will emphasize the role of DAMPs released by necrotic cells in the activation of Toll-like receptors (TLRs) and the possible contributions of TLRs to tumor development. This last point is very important for the future of cancer therapeutics since there are attempts to use TLR artificial ligands for cancer therapeutics.

The Role of Toll-like Receptor 2 (TLR2) in the Development and Progression of Hashimoto's Disease (HD): A Case Study on Female Patients in Poland.

Chronic lymphocytic thyroiditis, commonly known as HD, is one of the most common thyroid disorders. Due to the diverse factors affecting the etiopathogenesis of this disease (hormonal disorders and genetic and environmental factors), as well as the direct involvement of the immune system, scientists are increasingly willing to undertake research aimed at explaining the impact of the loss of immune tolerance and reactivity of autoantigens on the development of the disease. One of the directions of research in recent years is the role of the innate immune response, particularly Toll-like receptors (TLRs), in the pathogenesis of HD. The purpose of this study was to determine the importance of Toll-like receptor 2 (TLR2) expression on selected populations of immune cells, namely, monocytes (MONs) and dendritic cells (DCs), in the course of HD. Particular attention was paid to the analysis of TLR2's correlation with clinical parameters and the possibility its use as a potential biomarker molecule in the diagnostic process. Based on the obtained results, we found a statistically significant increase in the percentage of all analyzed populations of immune cells, i.e., mDC BDCA-1+CD19-, pDC BDCA-1+CD123, classical MONs CD14+CD16-, and non-classical MONs CD14+CD16+ showing on their surface TLR2 expression in patients diagnosed with HD compared to the healthy volunteers. Moreover, in the study group, we noted a more than 6-fold increase in the concentration of the soluble form of TLR2 in plasma compared to healthy patients. In addition, the correlation analysis showed significant positive correlations between the level of TLR2 expression on selected subpopulations of immune cells and biochemical indicators of thyroid function. Based on the obtained results, we can assume that TLR2 may be involved in the immunopathogenesis of HD.

Activation of TLRs Triggers GLP-1 Secretion in Mice.

The gastrointestinal tract constitutes a large interface with the inner body and is a crucial barrier against gut microbiota and other pathogens. As soon as this barrier is damaged, pathogen-associated molecular patterns (PAMPs) are recognized by immune system receptors, including toll-like receptors (TLRs). Glucagon-like peptide 1 (GLP-1) is an incretin that was originally involved in glucose metabolism and recently shown to be rapidly and strongly induced by luminal lipopolysaccharides (LPS) through TLR4 activation. In order to investigate whether the activation of TLRs other than TLR4 also increases GLP-1 secretion, we used a polymicrobial infection model through cecal ligation puncture (CLP) in wild-type and TLR4-deficient mice. TLR pathways were assessed by intraperitoneal injection of specific TLR agonists in mice. Our results show that CLP induces GLP-1 secretion both in wild-type and TLR4-deficient mice. CLP and TLR agonists increase gut and systemic inflammation. Thus, the activation of different TLRs increases GLP-1 secretion. This study highlights for the first time that, in addition to an increased inflammatory status, CLP and TLR agonists also strongly induce total GLP-1 secretion. Microbial-induced GLP-1 secretion is therefore not only a TLR4/LPS-cascade.

The architecture of transmembrane and cytoplasmic juxtamembrane regions of Toll-like receptors.

Toll-like receptors (TLRs) are the important participants of the innate immune response. Their spatial organization is well studied for the ligand-binding domains, while a lot of questions remain unanswered for the membrane and cytoplasmic regions of the proteins. Here we use solution NMR spectroscopy and computer simulations to investigate the spatial structures of transmembrane and cytoplasmic juxtamembrane regions of TLR2, TLR3, TLR5, and TLR9. According to our data, all the proteins reveal the presence of a previously unreported structural element, the cytoplasmic hydrophobic juxtamembrane α-helix. As indicated by the functional tests in living cells and bioinformatic analysis, this helix is important for receptor activation and plays a role, more complicated than a linker, connecting the transmembrane and cytoplasmic parts of the proteins.

Long-term effect of Toll-like receptor-2, -4, -5, -7 and NOD2 stimulation on Na+,K+-ATPase activity and expression in intestinal epithelial cells.

Inappropriate activation of Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain receptors (NOD) is involved in many chronic disorders, including inflammatory bowel disease (IBD). Altered function and/or expression of Na+,K+-ATPase (NKA) and epithelial ion channels are the main cause of electrolyte absorption imbalance in patients with IBD, leading to diarrhea. We aimed to evaluate the effect of TLRs and NOD2 stimulation upon NKA activity and expression in human intestinal epithelial cells (IECs) using RT-qPCR, Western blot, and electrophysiology techniques. TLR2, TLR4, and TLR7 activation inhibited NKA activity [(means ± SE) -20.0 ± 1.2%, -34.0 ± 1.5%, and -24.5 ± 2.0% in T84 cells; and -21.6 ± 7.4%, -37.7 ± 3.5%, and -11.0 ± 2.3% in Caco-2 cells]. On the other hand, activation of TLR5 increased NKA activity (16.2 ± 2.9% in T84 and 36.8 ± 5.2% in Caco-2 cells) and ß1-NKA mRNA levels (21.8 ± 7.8% in T84 cells). The TLR4 agonist synthetic monophosphoryl lipid A (MPLAs) reduced α1-NKA mRNA levels in both T84 and Caco-2 cells (-28.5 ± 3.6% and -18.7 ± 2.8%), and this was accompanied by a decrease in α1-NKA protein expression (-33.4 ± 11.8% and -39.4 ± 11.2%). NOD2 activation upregulated NKA activity (12.2 ± 5.1%) and α1-NKA mRNA levels (6.8 ± 1.6%) in Caco-2 cells. In summary, TLR2, TLR4, and TLR7 activation induce downregulation of NKA in IECs, whereas TLR5 and NOD2 activation has the opposite effect. A comprehensive understanding of the cross talk between TLRs, NOD2, and NKA is of utmost relevance for developing better IBD treatments.

CD36 neutralisation blunts TLR2-IRF7 but not IRF3 pathway in neonatal mouse brain and immature human microglia following innate immune challenge.

Innate immune response in neonatal brain is associated with a robust microglial activation and induction of Toll-like Receptors (TLRs). To date, the role of the scavenger receptor CD36 in TLRs modulation, particularly TLR2 signaling, has been well established in adult brain. However, the crosstalk between TLR4, TLR2 and CD36 and its immunogenic influence in the neonatal brain remains unclear. In this study, using a CD36 blocking antibody (anti-CD36) at post-natal day 8, we evaluated the response of neonates to systemic endotoxin (lipopolysaccharide; LPS) challenge. We visualized the TLR2 response by bioluminescence imaging using the transgenic mouse model bearing the dual reporter system luciferase/green fluorescent protein under transcriptional control of a murine TLR2 promoter. The anti-CD36 treatment modified the LPS induced inflammatory profile in neonatal brains, causing a significant decrease in inflammatory cytokine levels and the TLR2 and TLR3 mediated signalling.The interferon regulatory factor 3 (IRF3) pathway remained unaffected. Treatment of the LPS-challenged human immature microglia with anti-CD36 induced a marked decrease in TLR2/TLR3 expression levels while TLR4 and IRF3 expression was not affected, suggesting the shared CD36 regulatory mechanisms in human and mouse microglia. Collectively, our results indicate that blocking CD36 alters LPS-induced inflammatory profile of mouse and human microglia, suggesting its role in fine-tuning of neuroinflammation.