scholarly journals The TLR7 agonist imiquimod induces bronchodilation via a nonneuronal TLR7-independent mechanism: a possible role for quinoline in airway dilation

2016 ◽  
Vol 310 (11) ◽  
pp. L1121-L1129 ◽  
Author(s):  
Olivia J. Larsson ◽  
Martijn L. Manson ◽  
Magnus Starkhammar ◽  
Barbara Fuchs ◽  
Mikael Adner ◽  
...  
Keyword(s):  
Pulmonary Disease ◽  
Chemical Structure ◽  
Allergic Airway Inflammation ◽  
Toll Like Receptor ◽  
Tlr7 Agonist ◽  
Independent Mechanism ◽  
Intracellular Ca ◽  
Bilateral Vagotomy

Toll-like receptor (TLR) 7 agonists are known to reduce allergic airway inflammation. Their recently reported ability to rapidly relax airways has further increased their interest in the treatment of pulmonary disease. However, the mechanisms behind this effect are not fully understood. The present study, therefore, aimed to determine whether airway smooth muscle (ASM)-dependent mechanisms could be identified. TLR7 agonists were added to guinea pig airways following precontraction with carbachol in vitro or histamine in vivo. Pharmacological inhibitors were used to dissect conventional pathways of bronchodilation; tetrodotoxin was used or bilateral vagotomy was performed to assess neuronal involvement. Human ASM cells (HASMCs) were employed to determine the effect of TLR7 agonists on intracellular Ca2+([Ca2+]i) mobilization. The well-established TLR7 agonist imiquimod rapidly relaxed precontracted airways in vitro and in vivo. This relaxation was demonstrated to be independent of nitric oxide, carbon monoxide, and cAMP signaling, as well as neuronal activity. A limited role for prostanoids could be detected. Imiquimod induced [Ca2+]irelease from endoplasmic reticulum stores in HASMCs, inhibiting histamine-induced [Ca2+]i. The TLR7 antagonist IRS661 failed to inhibit relaxation, and the structurally dissimilar agonist CL264 did not relax airways or inhibit [Ca2+]i. This study shows that imiquimod acts directly on ASM to induce bronchorelaxation, via a TLR7-independent release of [Ca2+]i. The effect is paralleled by other bronchorelaxant compounds, like chloroquine, which, like imiquimod, but unlike CL264, contains the chemical structure quinoline. Compounds with quinoline moieties may be of interest in the development of multifunctional drugs to treat pulmonary disease.

Toll-Like Receptor-7 Agonist Directly Inhibits the Growth and Induces Apoptosis of MLL-AF9 Leukemia Cells in Vitro and in Vivo

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2989-2989
Author(s):  
Xueqing Liang ◽  
Ashish Kumar ◽  
Zhimei Wang ◽  
Brenda Weigel ◽  
Bruce R Blazar ◽  
...  
Keyword(s):  
Lethal Dose ◽  
Poly I:C ◽  
Leukemia Cells ◽  
Toll Like Receptor ◽  
Intracellular Staining ◽  
Tlr Agonists ◽  
Tlr7 Agonist ◽  
Regulated Expression

Abstract Leukemia involving rearrangements of the MLL gene is resistant to standard therapies and is often a fatal disease. MLL gene-rearrangements are commonly associated with infant-leukemia and secondary leukemias. New therapeutic strategies are needed to achieve longer survival and improve cure rates in patients with these and other refractory leukemias. Toll-like receptor (TLR) agonists are known as potent immune stimulatory agents that can elicit host anti-tumor effects in murine tumor models. We hypothesized that targeting TLRs expressed on leukemia cells with TLR agonists may have direct antitumor effects against leukemia cells. In this study, we investigated the effects of TLR agonists specific for TLR3, 4, 7, and 9, (i.e., polyinosine-polycytidylic acid (Poly(I:C)), monophosphoryl lipid A (MPL), imiquimod (IMQ), and CpG oligodeoxynucleotides (CpG ODN)), in MLL-AF9 knock-in mice that develop myeloid leukemia akin to human MLL-AF9 disease. In contrast to Poly(I:C), MPL, and CpG ODN, treatment of MLLAF9 cells with TLR7 agonist IMQ significantly increased the surface expression of CD40, CD54, CD80, and CD86 on MLL-AF9 cells in vitro. TLR7 mRNA and protein expression in MLL-AF9 cells were confirmed by real-time RT-PCR and intracellular staining/FACS analysis. Most importantly, TLR7 agonist strongly inhibited the in vitro MLL-AF9 cells in a drug dose- and treatment time-dependent manner. Whereas MLL-AF9 cells proliferated rapidly in culture with more than 40-fold increase of cell number in 5 days, the addition of IMQ at 5 mcg/ml fully inhibited the growth and induced profound apoptosis of MLL-AF9 cells with less than 2% of leukemia cells left at day 5 of culture. IMQ-mediated apoptotic death of MLL-AF9 was confirmed by viable cell counts, TMRE staining, Western blots and intracellular staining of the cleavage of caspases and PARP. Preincubation of MLLAF9 cells with caspases 8 and 10 inhibitors effectively blocked IMQ-induced apoptosis and sustained the proliferation of leukemia cells in cultures. To further determine the intracellular pathways engaged by IMQ, microarray gene expression profiles of 24-hour IMQ-treated vs. untreated MLL-AF9 cells were compared. Gene Set Enrichment Analysis (GSEA) showed that IMQ treatment resulted in up-regulated expression of a set of proapoptotic genes (e.g., p53, Bax, caspase 8, Apaf-1, etc) involved in apoptotic pathways. To determine whether IMQ pre-treatment of MLL-AF9 cells would prolong survival due to an apoptotic effect, cohorts of NOD-scid IL2Rgamma mice were i.p. injected with a lethal dose of MLL-AF9 cells with or without pre-incubation with IMQ. Mice receiving 5×106 untreated MLL-AF9 cells resulted in uniform lethality in 4 weeks. In contrast, mice receiving the same lethal dose of MLL-AF9 cells pretreated with IMQ had a significant prolonged survival, confirming in vitro findings that IMQ-treated MLL-AF9 cells undergo apoptosis. Administration of IMQ (daily i.p. injection at 1 mg/kg for 5 days) strongly inhibited leukemia cells growth and significantly prolonged the survival time of MLLAF9 mice. Flow cytometry results confirmed that residual MLL-AF9 cells recovered from IMQ treated mice were apoptotic and had up-regulated expression of cleaved caspases and PARP. In summary, our results demonstrate that TLR7 targeting of MLL-AF9 cells can directly induce apoptosis of MLL-AF9 cells in vitro and in vivo, providing new insights into the TLR-targeted therapy of refractory or relapsed leukemias.

Toll-Like Receptor Agonists Induce Immunogeneicity and Apoptosis of Acute Myeloid Leukemia Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 160-160
Author(s):  
Maki Shindo ◽  
Xueqing Liang ◽  
Zhimei Wang ◽  
Jeffery S. Miller ◽  
Martin Carroll ◽  
...  
Keyword(s):  
T Cell ◽  
Myeloid Leukemia ◽  
Leukemia Cells ◽  
Toll Like Receptor ◽  
Tlr Agonists ◽  
Tlr7 Agonist ◽  
Induced Apoptosis ◽  
Acute Myeloid

Abstract Acute myeloid leukemia (AML) is a common form of acute leukemia and remains a difficult disease with poor survival in patients who have failed standard therapy. New therapeutic strategies are needed to achieve longer survival and improve cure rates in AML patients. Toll-like receptor (TLR) agonists have been shown to elicit anti-leukemia effects in murine AML models. However, TLR expression profile of human AML cells is unknown. We analyzed TLR1-10 mRNA expression in purified AML cells from 41 patients with different AML subtypes (M0, M1, M2, M3, M4, or M5; n > 5 per group) by real-time RT-PCR. The majority of AML samples expressed high level of TLR2, 4, 7, 8, low level of TLR1, 5, 9, 10, and undetectable level of TLR3. Significant higher TLR4 and TLR7 expressions were detected on M4 and M5 subtypes of AML cells. Triggering TLR4 or TLR7 with specific TLR agonists (Monophosphoryl Lipid A or Imiquimod) significantly increased the surface expression of molecules essential for T cell activation (CD54, CD80, CD86) on AML M4/M5 cells and enhanced T-cell mediated proliferative responses against AML cells. Thus, TLR signaling enhances the immunogenicity of AML M4/M5 cells and makes them more suitable targets for T cell mediated attack. Most importantly, TLR7 agonist strongly induced apoptotic death of primary AML M4/M5 cells and inhibited the growth of TLR7-expressing AML cell lines (U937, HL-60, KG-1) in culture in a drug dose dependent manner. The addition of TLR7 agonist at 10 ug/ml fully induced apoptosis of AML cells and inhibited the growth of AML cell lines, as confirmed by viable cell counts and TMRE staining. Intracellular staining demonstrated that TLR7 agonist treatment significantly down-regulated the signal transducer and activator of transcription (STAT)3 and STAT5 protein expression in AML cells. These results suggest that TLR7 agonist-induced apoptosis of AML cells is likely via inhibition of STAT3 and/or STAT5 signaling pathway. To study the in vivo effects of TLR7 agonist against human AML cells, primary AML M4/M5 cells or a monocytic AML cell line (U937) were injected i.p. into NOD-SCID IL2Rgamma<null> mice with or without subsequent TLR7 agonist treatment. Mice receiving TLR7 agonist treatment (1 mg/kg daily i.p. infusion for 5 days) significantly reduced tumor burden with substantially lower numbers of engrafted leukemia cells detected in these xenograft mice. Flow cytometry results confirmed that residual AML cells recovered from mice treated with TLR7 agonist were apoptotic with down-regulated expression of TMRE and STAT3/STAT5, confirming previous in vitro findings that TLR7 agonist-treated AML cells are programmed to die. The antitumor efficacy of systemic administration of TLR7 agonist in NOD/SCID mice with established human AML is being investigated using these xenograft mouse models. In summary, our results provide the first report of TLR expression profile of human AML cells and demonstrate that TLR targeting of AML cells can increase the immunogeneicity of leukemia cells and directly induce AML apoptosis in vitro and in vivo, providing new insights into the biology and therapy of AML.

scholarly journals Targeting Toll-Like Receptor 2: Polarization of Porcine Macrophages by a Mycoplasma-Derived Pam2cys Lipopeptide

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 692
Author(s):  
Giulia Franzoni ◽  
Antonio Anfossi ◽  
Chiara Grazia De Ciucis ◽  
Samanta Mecocci ◽  
Tania Carta ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Macrophage Polarization ◽  
Surface Protein ◽  
Antimicrobial Activities ◽  
Toll Like Receptor ◽  
Activation Markers ◽  
Class I Mhc ◽  
Toll Like Receptor 2

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly activated innate immune cells, including porcine monocyte-derived macrophages (moMΦ). In this study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine moMΦ. We observed enhanced expression of activation markers (MHC class I, MHC class II DR, CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-Pam2Cys also upregulated the gene expression of several IFN-α subtypes, p65, NOS2, and molecules with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However, Mag-Pam2Cys downregulated the expression of IFN-α3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful consequences. We hope that our in vitro results will lay the foundation for the further evaluation of this diacylated lipopeptide as an immunopotentiator in vivo.

scholarly journals Serum Amyloid A1/Toll-Like Receptor-4 Axis, an Important Link between Inflammation and Outcome of TBI Patients

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 599
Author(s):  
Víctor Farré-Alins ◽  
Alejandra Palomino-Antolín ◽  
Paloma Narros-Fernández ◽  
Ana Belen Lopez-Rodriguez ◽  
Céline Decouty-Perez ◽  
...  
Keyword(s):  
Injury Severity ◽  
White Blood Cells ◽  
Serum Amyloid ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Important Link ◽  
Tlr4 Mrna ◽  
Serum Amyloid A1

Traumatic brain injury (TBI) is one of the leading causes of mortality and disability worldwide without any validated biomarker or set of biomarkers to help the diagnosis and evaluation of the evolution/prognosis of TBI patients. To achieve this aim, a deeper knowledge of the biochemical and pathophysiological processes triggered after the trauma is essential. Here, we identified the serum amyloid A1 protein-Toll-like receptor 4 (SAA1-TLR4) axis as an important link between inflammation and the outcome of TBI patients. Using serum and mRNA from white blood cells (WBC) of TBI patients, we found a positive correlation between serum SAA1 levels and injury severity, as well as with the 6-month outcome of TBI patients. SAA1 levels also correlate with the presence of TLR4 mRNA in WBC. In vitro, we found that SAA1 contributes to inflammation via TLR4 activation that releases inflammatory cytokines, which in turn increases SAA1 levels, establishing a positive proinflammatory loop. In vivo, post-TBI treatment with the TLR4-antagonist TAK242 reduces SAA1 levels, improves neurobehavioral outcome, and prevents blood–brain barrier disruption. Our data support further evaluation of (i) post-TBI treatment in the presence of TLR4 inhibition for limiting TBI-induced damage and (ii) SAA1-TLR4 as a biomarker of injury progression in TBI patients.

scholarly journals Toll-like receptor-9 stimulated plasmacytoid dendritic cell precursors suppress autoimmune neuroinflammation in a murine model of multiple sclerosis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Hélène Letscher ◽  
Viviane A. Agbogan ◽  
Sarantis Korniotis ◽  
Pauline Gastineau ◽  
Emmanuel Tejerina ◽  
...  
Keyword(s):  
Dendritic Cell ◽  
Plasmacytoid Dendritic Cell ◽  
Toll Like Receptor ◽  
Hematopoietic Progenitors ◽  
Autoimmune Encephalomyelitis ◽  
Hematopoietic Precursor ◽  
Therapeutic Properties ◽  
Experimental Autoimmune

AbstractEarly innate education of hematopoietic progenitors within the bone marrow (BM) stably primes them for either trained immunity or instead immunoregulatory functions. We herein demonstrate that in vivo or in vitro activation within the BM via Toll-like receptor-9 generates a population of plasmacytoid dendritic cell (pDC) precursors (CpG-pre-pDCs) that, unlike pDC precursors isolated from PBS-incubated BM (PBS-pre-pDCs), are endowed with the capacity to halt progression of ongoing experimental autoimmune encephalomyelitis. CpG activation enhances the selective migration of pDC precursors to the inflamed spinal cord, induces their immediate production of TGF-β, and after migration, of enhanced levels of IL-27. CpG-pre-pDC derived TGF-β and IL-27 ensure protection at early and late phases of the disease, respectively. Spinal cords of CpG-pre-pDC-protected recipient mice display enhanced percentages of host-derived pDCs expressing TGF-β as well as an accumulation of IL-10 producing B cells and of CD11c+ CD11b+ dendritic cells. These results reveal that pDC precursors are conferred stable therapeutic properties by early innate activation within the BM. They further extend to the pDC lineage promising perspectives for cell therapy of autoimmune diseases with innate activated hematopoietic precursor cells.

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

2007 ◽  
Vol 292 (4) ◽  
pp. L915-L923 ◽  
Author(s):  
Jaime Chávez ◽  
Patricia Segura ◽  
Mario H. Vargas ◽  
José Luis Arreola ◽  
Edgar Flores-Soto ◽  
...  
Keyword(s):  
Substance P ◽  
Guinea Pigs ◽  
Smooth Muscle Contraction ◽  
In Vivo Experiments ◽  
Intracellular Ca ◽  
Neurokinin 1 ◽  
Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

Chemistry of the phytochrome photoconversions

1983 ◽  
Vol 303 (1116) ◽  
pp. 377-386 ◽  
Keyword(s):  
Chemical Structure ◽  
Red Shift ◽  
Structural Differences

The photoconversions of phytochrome, P r ⇌ P fr , occur both in vivo and in vitro . Structural differences between P r and P fr are discussed for chromophore and apoprotein. The chemical structure of the P r chromophore has been established. The P fr chromophore was recently demonstrated to be the 15 E isomer. The red shift of absorption to 730 nm in native P fr is discussed as interaction between chromophore and apoprotein. The nature of this interaction is still unknown. Small changes in the apoprotein surface are of particular interest because they could be part of the signal chain in photoperception.

Abstract 1079: Prevention of Fatal Arrhythmia in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin-2 Mutation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Hiroko Wakimoto ◽  
Ronny Alcalai ◽  
Lei Song ◽  
Michael Arad ◽  
Christine E Seidman ◽  
...  
Keyword(s):  
Ventricular Tachycardia ◽  
Mouse Model ◽  
Peak Height ◽  
Mutant Mice ◽  
Catecholaminergic Polymorphic Ventricular Tachycardia ◽  
Polymorphic Ventricular Tachycardia ◽  
Study Drug Administration ◽  
Intracellular Ca

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmia syndrome caused by mutations in the ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2) genes and characterized by exercise or emotional stress-induced sudden death. Beta-adrenergic blockers are only partially effective and other agents have not been widely tested. Recent studies have shown that CPVT is mediated by increased Ca 2+ leak through the RyR2 channel. Our aim was to determine whether agents that inhibit intracellular Ca 2+ leak can effectively prevent CPVT. Methods: The efficacy of intraperitoneal (IP) propranolol (1mcg/g), Mg 2+ (0.002mEq/g), verapamil (8 mcg/g) and diltiazem (8 mcg/g) were tested both in vivo and in vitro using CASQ2 mutant mouse CPVT model. In vivo studies included ambulatory ECG recordings at rest and following epinephrine stress (0.4 mcg/g IP) at baseline and after study drug administration. Experiments for each drug were performed on separate days to avoid confounding effects. In vitro studies included intracellular Ca 2+ transient analysis on isolated cardiomyocytes from mutant mice with and without epinephrine (5.5 μM). Results: All 4 drugs restored sinus rhythm and reduced the frequency of VT episodes induced by epinephrine in CASQ2 mutant mice. Only verapamil completely prevented epinephrine-induced VT in 87% of the mice (p<0.01). Cardiomyocyte studies in basal conditions revealed that Mg 2+ and verapamil inhibited sarcomere contraction and normalized the prolonged Ca 2+ reuptake period in CASQ2 mutants, but did not decrease baseline Ca 2+ peak height. Epinephrine-stressed mutant myocytes had increased diastolic Ca 2+ levels, lower Ca 2+ peak height and spontaneous SR Ca 2+ release events that were partially prevented by verapamil and Mg 2+ . Verapamil was more effective than Mg 2+ in reducing the frequency of spontaneous Ca 2+ releases induced by epinephrine. Conclusions: All 4 agents can inhibit ventricular arrhythmia in CPVT mouse model; however verapamil appears most effective in preventing arrhythmia in vivo and in modifying intracellular abnormal calcium handling. Calcium antagonists might have therapeutic value in CPVT and other RyR2-mediated arrhythmias and should be considered for human clinical studies.

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