Overexpression Of Toll-like Receptors Research Articles

Overexpression of toll-like receptors and co-stimulatory molecules on immature dendritic cells of Crohn's disease

IntroductionCrohn's disease (CD) is a chronic, relapsing inflammatory disorder of the gastrointestinal tract resulting from a loss of homeostasis between the intestinal immune system and the gut microbiome. Also, evidence shows dendritic cells (DC) are the main antigen-presenting cells (APC) in the intestinal mucosa; they can regulate innate and adaptive immunity that is pivotal for maintaining immunological tolerance in the gut. The maturation of immature DC (iDCs) regulate by their surface molecules such as Toll-Like Receptors (TLRs) and co-stimulatory molecules (CD80 and CD86). The level of TLRs on iDCs plays a critical role in developing adaptive responses to antigens. Crohn's disease has a dysfunctional immune system and low tolerance of dendritic cells. This study aimed to investigate the expression of TLR2, TLR4, TLR5, and TLR9 and co-stimulatory molecules (CD80 and CD86). MethodologyHuman peripheral blood mononuclear cells (PBMCs) were collected from 15 CD patients and 12 healthy controls. PBMCs were isolated by density gradient centrifugation, and then iDCs were derived for 6 days. Flow cytometry was performed on iDCs to characterize and distinguish the phenotype of iDC populations. iDCs were harvested for RNA extraction, and the mRNA expression level of TLRs was examined by quantitative RT-PCR using SYBR Green chemistry. ResultThe results showed that TLR2, TLR4, TLR5, and TLR9 mRNA expression significantly increased in the iDCs isolated from patients with CD compared with healthy control P ≤ 0.0001. Although, TLR2 had the lowest expression among other TLRs on iDCs in CD patients. Also, the expression level of CD80 and CD86 were significantly higher in iDCs isolated from CD patients compared to healthy controls P < 0.001 OR = 0.75 CI (−8.76 to −3.17). ConclusionTLR2, TLR4, TLR5, and TLR9 expression on iDCs may be critical in the biological pathogenesis of CD. TLR alterations in the iDCs may contribute to determining dendritic cell maturation pathway lead to increases inflammatory response.

A cell-penetrating peptide blocks Toll-like receptor-mediated downstream signaling and ameliorates autoimmune and inflammatory diseases in mice

Toll-like receptors (TLRs) recognize pathogen/damage-associated molecular patterns and initiate inflammatory signaling cascades. Occasionally, overexpression of TLRs leads to the onset of numerous inflammatory diseases, necessitating the development of selective inhibitors to allow a protective yet balanced immune response. Here, we demonstrate that a novel peptide (TIP1) derived from Toll/interleukin-1 receptor (TIR) domain-containing adapter protein inhibited multiple TLR signaling pathways (MyD88-dependent and MyD88-independent) in murine and human cell lines. TIP1 also inhibited NLRP3-mediated IL-1β secretion, as we validated at both the protein and mRNA levels. Biophysical experiments confirmed that TIP1 specifically binds to the BB loop of the TLR4-TIR domain. Animal studies revealed that TIP1 inhibited the secretion of lipopolysaccharide (LPS)-induced proinflammatory cytokines in collagen-induced arthritis (CIA) and kaolin/carrageenan-induced arthritis (K/C) rodent models. TIP1 also rescued animals from sepsis and from LPS-induced kidney/liver damage. Importantly, TIP1 ameliorated the symptoms of rheumatoid arthritis in CIA and K/C rodent models, suggesting that TIP1 has therapeutic potential for the treatment of TLR-mediated autoimmune/inflammatory diseases.

Expression of Toll-Like receptors in metabolic syndrome: A systematic review

Introduction: Toll-Like Receptors (TLRs) of innate immune system have documented roles in the pathogenesis of metabolic disorders. This study aims to systematically review the expression of TLRs on metabolic syndrome (MetS). Materials and methods: We systematically searched PubMed/Medline, ISI web of Science, Scopus, Google Scholar, EMBASE, and OVID databases until February 2017. The terms ‘‘Metabolic Syndrome OR ‘‘Mets AND ‘‘Toll like receptor OR ‘‘Toll like OR ‘‘TLRs OR ‘‘TLR were used. “Expression” advertently was not used in our search and was considered in the selection process. Three steps for selecting the articles and then their qualification were conducted. Results: First, 1373 articles were found in the international databases. After removing duplicates, 963 papers remained and after two steps of selection, this number reached 410 and then 27, respectively. After full text screening and qualifying processes, we finally included 13 articles consisting of five animal and eight human studies. All human studies reported overexpression of TLRs (types 2, 4, 5, 9) in MetS, and most animal studies documented an increased TLRs expression. Conclusion: This systematic review provides evidence for the relation of innate immune system with MetS. Its findings regarding overexpression of special TLRs (e.g. types 2, 4, 5, 9) in MetS and their basic mechanisms and clinical implications might be investigated in further studies.

Increased Toll-Like Receptor-Myeloid Differentiation Factor 88 Expression at the Maternal-Fetal Interface Is Associated with Spontaneous Abortion

Aims: Toll-like receptors (TLRs) form a group of pattern recognition receptors that play a major role in maintaining innate host immunity. Myeloid differentiation factor 88 (MyD88) is an adaptor molecule that is essential for signaling via the TLR family. To analyze the mechanism of the imbalance in the innate immune system mediated by TLRs-MyD88 in spontaneous abortion, we detected the expression of TLR-2, TLR-4, TLR-7, and MyD88 in placentae and deciduas. Methods: The expression of TLR-2, TLR-4, TLR-7, and MyD88 in the placentae and deciduas of abortion-prone mice (n = 10) and normal pregnant mice (n = 10) were analyzed by immunochemistry, western blot, and real-time polymerase chain reaction. Results: There were higher protein expression levels of TLR-2, TLR-4, TLR-7, and MyD88 in the placentae and deciduas in the abortion-prone group than in the normal pregnant group. However, TLR-2, TLR-4, and TLR-7 showed lower gene expression, while MyD88 manifested higher gene expression in placentae and deciduas of abortion-prone mice matings. Conclusions: TLR-2, TLR-4, TLR-7, and MyD88 were expressed in the placenta and decidua of both the abortion-prone pregnant and normal pregnant mice. The overexpression of TLRs may excessively activate the innate immune system mediated by MyD88, which is considered to be related to the pathogenesis of spontaneous abortion.

Effects of Vaccination with the C-Strain Vaccine on Immune Cells and Cytokines of Pigs Against Classical Swine Fever Virus.

The attenuated C-strain vaccine against classical swine fever virus (CSFV) is one of the safest and most effective attenuated vaccines. However, little is known of the host immune response after vaccination with the C-strain vaccine. Blood samples from vaccinated pigs were collected to evaluate the number of immune cells, the level of specific CSFV antibody, and related cytokines induced by the vaccination of C-strain vaccine. The C-strain nucleic acid was gradually removed and specific antibody to vaccine kept increasing; the amount of the lymphocyte, Tc cell, and Th cell increased; some inflammatory cytokines such as interleukin (IL)-1 and tumor necrosis factor-α mainly showed downregulated trends, but IL-6 and IL-8 were upregulated greatly; IL-2, IL-4, IL-5, IL-12p40, IL-13, interferon (IFN)-I, and Toll-like receptors (TLRs) kept high expression level after 28 days postvaccination (dpv); IFN-γ was upregulated slightly at 5 and 9 dpv, respectively. These results suggest that the C-strain vaccine induces a Th2 cell response to produce the specific antibody. The vaccine virus replicates at very low level. C-strain vaccine burden has close relationship with the expression of TLRs. The overexpression of TLRs initiates the innate immune system to clear up the vaccine. Meanwhile, ILs expressed by immune system induce the differentiation of B cells and produce specific antibody.

Overexpression of Toll-Like Receptors 2, 3, 4, and 8 Is Correlated to the Vascular Atherosclerotic Process in the Hyperlipidemic Rabbit Model: The Effect of Statin Treatment

Background: Atherosclerosis is the major cause of cardiovascular disease; hypercholesterolemia is a major risk factor. We hypothesized that specific TLR members (TLR2, TLR3, TLR4, TLR8) may play a role in atherosclerosis progression and its accompanying inflammatory response. We determined the association of atherosclerotic lesions and TLR mRNA expression in different aortic sites. We also assessed the effects of fluvastatin (Flu) treatment on TLR expression and plaque characteristics. Methods: Male rabbits, fed with an atherogenic diet for a duration of 3 months, were screened for advanced atherosclerotic lesions in the aorta. Additional animals received normal diet or normal diet plus Flu for 1 additional month. TLR mRNA expression in various thoracic and abdominal aortic segments was assessed, together with atherosclerotic changes. Results: After high lipid diet, the atherosclerotic burden increased more in the abdominal than in the thoracic aorta; TLR2, 3, 4, and 8 also increased significantly. Flu decreased atherosclerotic plaque, calcium deposition, lipid cores, intraplaque hemorrhage, erythrocyte membranes, endothelial cells, and macrophage infiltration, while increasing smooth muscle cells in plaques of both aortic segments; it also lowered TLR2, 3, 4, and 8 expression in all aortic segments to a stronger degree than resumption of normal diet. There was a strong association between blood and tissue parameters during experimental period and finally a strong correlation found between these parameters with mRNA of TLR2, 3, 4, and 8 in various stages. Conclusion: For the first time TLR2, 3, 4, and 8 mRNA expression is prospectively explored after hypercholesterolemic diet in the rabbit model. TLR2, 3, 4, and 8 mRNA expression is strongly upregulated and correlates with the progression of atherosclerosis in the aorta. Flu significantly inhibited this progress and reduced inflammation via TLR downregulation which was strongly associated with regression of plaque morphology and atherosclerosis promoting factors.

Chronic hyperosmotic stress inhibits renal Toll-Like Receptors expression in striped catfish (Pangasianodon hypophthalmus, Sauvage) exposed or not to bacterial infection

Toll-like Receptors (TLRs) are the first innate receptors in recognizing pathogen-associated molecular patterns. In fish, upregulation of toll-like receptors during infection has been largely demonstrated while the effects of abiotic stressors on their expression remain poorly investigated. In this study, striped catfish were submitted during 20 days to three salinity profiles (freshwater, low saline water, saline water), followed by injection of a bacterial strain of Edwardsiella ictaluri. The expression of TLRs 1, 3, 4, 5, 7, 9, 19, 21, and 22 was measured in kidney at different time points in non infected and infected striped catfish. Infection induced overexpression of TLRs 1, 3, 4, 5, 7, 21 and 22. With elevated salinity, the expression of all TLRs, except TLR5, was severely decreased, particularly after bacterial infection. The TLRs responsiveness of striped catfish facing bacterial disease and salinity stress and possible consequences on striped catfish immune response's efficiency are discussed.

IMO-8400, an Antagonist of Toll-like Receptors 7, 8, and 9, in Development for Genetically Defined B-Cell Lymphomas: Safety and Activity in Phase 1 and Phase 2 Clinical Trials

Background. Toll-like receptors (TLRs) are pathogen-associated molecular pattern recognition receptors of the innate immune system. TLRs 7 and 9, which are expressed in human B-cells, respond to DNA- and RNA-based ligands by initiating a signaling cascade mediated through NF-κB, IRAK1/4, BTK, and JAK/STAT. DNA- and RNA-based ligands for TLRs 7 and 9 also are generated as damage-associated molecular patterns in certain autoimmune diseases and malignancies. MYD88 is a key adaptor molecule in TLR signaling. Recently, the oncogenic mutation MYD88 L265P mutation has been described and reported to occur in patients with activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL), Waldenström’s macroglobulinemia (WM), and other B-cell malignancies. MYD88 L265P drives over-expression of TLR 7 and 9 signaling, creating an anti-apoptotic microenvironment that promotes cancer cell survival and proliferation. Recent epidemiological data confirm a strong association between the presence of MYD88 L265P and poor outcomes in patients with DLBCL. Specific targeting of signaling initiated by TLRs 7 and 9 therefore is a novel approach to the treatment of B-cell lymphomas harboring the MYD88 L265P mutation. Preclinical data presented at AACR 2014, including from xenograft tumor studies, show that IMO-8400 inhibits cell signaling and reduces tumor growth in WM and DLBCL models harboring the MYD88 L265P mutation. Clinical data from 2 completed trials of IMO-8400 are presented here.Clinical Trial Designs. In a first-in-man Phase 1 trial in healthy subjects, IMO-8400 was administered by s.c. injection at single dosages of 0.1, 0.3, and 0.6 mg/kg and for four weeks at 0.3 and 0.6 mg/kg/week (each n=6). Goals of the Phase 1 trial included safety and tolerability evaluation through single- and multiple-dose escalation, and assessment of TLR engagement. Subsequently, a randomized, placebo-controlled Phase 2 trial was conducted with IMO-8400 in patients with moderate to severe plaque psoriasis at dosages of 0.075, 0.15, 0.3, and 0.6 mg/kg/week for 12 weeks (n=8 or 9 per dosage). Goals of the Phase 2 trial included safety and tolerability assessment over a 12-week treatment period, and evaluation of clinical proof of concept in a disease that involves TLR7 and 9 over-activation.Clinical Trial Results. IMO-8400 was well tolerated across all dose regimens in both studies, with no treatment-related severe or serious adverse events, no drug-related treatment discontinuations, and no pattern of systemic adverse events or laboratory changes. The only treatment-related adverse events in more than one subject were injection site reactions, comprised of generally mild erythema with occasional induration, pruritus, tenderness or pain. In neither trial were there adverse or consistent shifts in laboratory parameters. Specifically, any changes in white or red blood cell parameters, platelet numbers, and liver enzymes or other serum chemistry parameters were similar following IMO-8400 treatment or after placebo treatment. In the Phase 2 trial, clinical proof of mechanism was demonstrated by increased frequency of improvements in psoriasis disease activity in IMO-8400-treated patients across multiple dose levels relative to placebo-treated patients.Conclusion. Antagonism of TLRs 7 and 9 is a novel, scientifically-driven approach to the treatment of B-cell malignancies characterized by presence of the MYD88 L265P oncogenic mutation. IMO-8400, a clinical-stage drug candidate, was well tolerated in clinical trials conducted in healthy subjects and patients with psoriasis, having demonstrated no myelosuppression or any pattern of hematological or other toxicity in 12-week treatment regimens. The risk-benefit profile of IMO-8400 to date merits further clinical testing for genetically defined B-cell malignancies, as monotherapy and in combination with other agents. Based on the accumulated preclinical data, clinical safety, and demonstrated mechanism of action of IMO-8400, Phase 1/2 clinical trials of IMO-8400 are being conducted in both relapsed/refractory patients with WM and in patients with DLBCL and the MYD88 L265P mutation. DisclosuresBrenner:Idera Pharmaceuticals : Employment, Equity Ownership. Arbeit:Idera Pharmaceuticals: Employment. Sullivan:Idera Pharmaceuticals: Employment.

Benign prostatic hyperplasia: a new metabolic disease?

Benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) are conditions extremely prevalent in the aging male. Although androgens are involved in prostate growth during developmental age, their role in the pathogenesis of BPH/LUTS is debated. Recent data indicate that low testosterone and high estradiol favor disease progression. In addition, the role of other determinants, such as metabolic syndrome or prostate inflammation, is emerging. We reviewed the evidence regarding the pathogenesis of BPH/LUTS with particular attention to metabolic influence. A review of published evidence was performed using Medline. Available evidence shows that a three-hit hypothesis can be drawn. An overt, or even a subclinical, bacterial or viral infection could induce prostatic inflammation (first hit) that could be autosustained or exacerbated by the presence of an altered metabolism and in particular by hypercholesterolemia (second hit). Hypogonadism and/or hyperestrogenism could act as a third hit, favoring the maintenance of this inflammatory state. The combined action of all three hits, or even two of them, may result in overexpression of Toll-like receptors (TLRs), transformation of prostatic cells into antigen-presenting cells and activation of resident human prostate-associated lymphoid tissue ending in overproduction of growth factors which, in turn, will induce prostate remodeling and further prostate enlargement. The mechanical obstruction, along with the direct action of the unfavorable metabolic and hormonal milieu on the bladder neck, helps in generating LUTS. Inflammation, dyslipidemia and altered sex-steroid milieu mutually concur in determining BPH/LUTS.

Regulation of angiogenesis, mural cell recruitment and adventitial macrophage behavior by Toll-like receptors

The angiogenic response to injury can be studied by culturing rat or mouse aortic explants in collagen gels. Gene expression studies show that aortic angiogenesis is preceded by an immune reaction with overexpression of Toll-like receptors (TLRs) and TLR-inducible genes. TLR1, 3, and 6 are transiently upregulated at 24h whereas TLR2, 4, and 8 expression peaks at 24h but remains elevated during angiogenesis and vascular regression. Expression of TLR5, 7 and 9 steadily increases over time and is highest during vascular regression. Studies with isolated cells show that TLRs are expressed at higher levels in aortic macrophages compared to endothelial or mural cells with the exception of TLR2 and TLR9 which are more abundant in the aortic endothelium. LPS and other TLR ligands dose dependently stimulate angiogenesis and vascular endothelial growth factor production. TLR9 ligands also influence the behavior of nonendothelial cell types by blocking mural cell recruitment and inducing formation of multinucleated giant cells by macrophages. TLR9-induced mural cell depletion is associated with reduced expression of the mural cell recruiting factor PDGFB. The spontaneous angiogenic response of the aortic rings to injury is reduced in cultures from mice deficient in myeloid differentiation primary response 88 (MyD88), a key adapter molecule of TLRs, and following treatment with an inhibitor of the NFκB pathway. These results suggest that the TLR system participates in the angiogenic response of the vessel wall to injury and may play an important role in the regulation of inflammatory angiogenesis in reactive and pathologic processes.

Toll‐like receptor 4 mediates intrauterine growth restriction after systemic Campylobacter rectus infection in mice

Campylobacter rectus is associated with fetal exposure and low birthweight in humans. C. rectus also invades placental tissues and induces fetal intrauterine growth restriction (IUGR) in mice, along with overexpression of Toll-like receptors (TLR4), suggesting that TLR4 may mediate placental immunity and IUGR in mice. To test this hypothesis we examined the effect of in vitro TLR4 neutralization on trophoblastic proinflammatory activity and studied the IUGR phenotype in a congenic TLR4-mutant mouse strain after in vivo C. rectus infection. Human trophoblasts were pretreated with TLR4 neutralizing antibodies and infected with C. rectus; proinflammatory cytokine production was assessed by cytokine multiplex assays. Neutralizing TLR4 antibodies significantly impaired the production of proinflammatory cytokines in trophoblastic cells after infection in a dose-dependent manner. We used a subcutaneous chamber model to provide a C. rectus challenge in BALB/cAnPt (TLR4(Lps-d) ) and wild-type (WT) females. Females were mated with WT or TLR4(Lps-d) males once/week; pregnant mice were infected at (E)7.5 and sacrificed at (E)16.5 to establish IUGR phenotypes. Maternal C. rectus infection significantly decreased fetal weight/length in infected WT when compared with sham WT controls (P < 0.05, analysis of variance). However, infected TLR4(Lps-d -/-) mice did not show statistically significant differences in fetal weight and length when compared with WT controls (P > 0.05). Furthermore, heterozygous TLR4(Lps-d +/-) fetuses showed IUGR phenotype rescue. We conclude that TLR4 is an important mediator of trophoblastic proinflammatory responses and TLR4-deficient fetuses do not develop IUGR phenotypes after C. rectus infection, suggesting that placental cytokine activation is likely to be mediated by TLR4 during low birthweight/preterm birth pathogenesis.

Cartilage Destruction in Granulomatosis with Polyangiitis (Wegener's Granulomatosis) Is Mediated by Human Fibroblasts after Transplantation into Immunodeficient Mice

A key feature of granulomatosis with polyangiitis (GPA; or Wegener's granulomatosis) is the granulomatous inflammation of the upper respiratory tract, which leads to the subsequent destruction of adjacent tissues. The aim of our work was to study the histopathological and cellular components of tissue destruction of human GPA tissue transplanted into immunodeficient mice. Biopsy specimens from patients with active GPA (n = 10) or sinusitis (controls, n = 6) were s.c. co-implanted with healthy allogeneic human nasal cartilage into immunodeficient pfp/rag2(-/-) mice. Transplants were examined for their destructive capability of the allografted human cartilage. In addition, nasal fibroblasts from patients with GPA (n = 8) and control healthy nasal fibroblasts (n = 5) were cultured, and cell proliferation and apoptosis were quantified. mRNA and protein levels of matrix metalloproteinases and cytokines were evaluated at baseline and after proinflammatory stimulation. GPA implants showed massive destruction of the co-implanted human cartilage, whereas cartilage destruction was only marginal in control samples. Destruction was mediated by human fibroblasts and could be inhibited by corticoid treatment. The up-regulated production of matrix metalloproteinases 1, 3, and 13 and cytokines IL-6 and IL-8 was found in vivo and in vitro. Although proliferation of isolated fibroblasts was comparable between GPA and controls, GPA samples showed a significant delay of apoptosis. The destruction of nasal cartilage in GPA is mainly mediated by fibroblasts that can be blocked by corticosteroids, and this tissue destruction is not dependent on the influx of leukocytes.

Induction of tolerance to lipopolysaccharide and mycobacterial components in Chinese hamster ovary/CD14 cells is not affected by overexpression of Toll-like receptors 2 or 4.

Down-regulation of cell surface expression of Toll-like receptor (TLR) 4 following LPS stimulation has been suggested to underlie endotoxin tolerance. In this study, we examined whether overexpression of TLR2 or TLR4 would affect the ability of cells to become tolerant to LPS or the mycobacterial components, arabinose-capped lipoarabinomannan (LAM) and soluble tuberculosis factor (STF). To this end, Chinese hamster ovary/CD14 cells stably transfected with a NF-kappaB-dependent reporter construct, endothelial leukocyte adhesion molecule CD25 (the 3E10 clone), were engineered to overexpress either human TLR2 or TLR4. Transfected TLRs exhibited proper signaling functions, as evidenced by increased LPS responsiveness of 3E10/TLR4 cells and acquisition of sensitivity to TLR2-specific ligands upon transfection of TLR2 into TLR2-negative 3E10 cells. Pretreatment of cells with LPS, LAM, or STF did not modulate TLR2 or TLR4 cell surface expression. Following LPS exposure, 3E10, 3E10/TLR2, and 3E10/TLR4 cells exhibited comparable decreases in LPS-mediated NF-kappaB activation and mitogen-activated protein (MAP) kinase phosphorylation. Likewise, LPS pretreatment profoundly inhibited LPS-induced NF-kappaB translocation in Chinese hamster ovary cells that concomitantly overexpressed human TLR4 and myeloid differentiation protein-2 (MD-2), but failed to modulate TLR4 or MD-2 cell surface expression. Pretreatment of 3E10/TLR2 cells with LAM or STF decreased their NF-kappaB responses induced by subsequent stimulation with these substances or LPS. Conversely, prior exposure of 3E10/TLR2 cells to LPS led to hyporesponsiveness to LPS, LAM, and STF, indicating that LPS and mycobacterial products induce cross-tolerance. Thus, tolerance to LPS and mycobacterial components cannot be attributed solely to a decrease in TLR/MD-2 expression levels, suggesting inhibition of expression or function of other signaling intermediates.