Artículos de revistas: "Toll-like receptor 7 a 9 (TLR7"

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Conti, Fabrizio, Francesca Romana Spinelli, Simona Truglia, Francesca Miranda, Cristiano Alessandri, Fulvia Ceccarelli, Michele Bombardieri, Konstantinos Giannakakis y Guido Valesini. "Kidney Expression of Toll Like Receptors in Lupus Nephritis: Quantification and Clinicopathological Correlations". Mediators of Inflammation 2016 (2016): 1–7. http://dx.doi.org/10.1155/2016/7697592.

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Objective. The study aimed at locating and quantifying Toll Like Receptor (TLR) 3, 7, 8, and 9 expression in kidney of patients with lupus nephritis (LN) and correlating them with clinicopathological features.Methods. Kidney sections from 26 LN patients and 4 controls were analyzed by immunohistochemistry using anti-human TLR3, TLR7, TLR8, and TLR9 polyclonal antibodies; the number of TLR-positive nuclei/mm2was evaluated on digitalized images.Results. Compared to controls, LN showed a significantly higher amount of glomerular and tubulointerstitial TLR9 (p=0.003andp=0.007), whole and tubulointerstitial TLR3 (p=0.026andp=0.031), and a higher tubulointerstitial TLR7 (p=0.022). TLR9 positively correlated with activity index (p=0.0063) and tubular TLR7 with chronicity index (p=0.026). TLR9 positively correlated with Renal-SLEDAI (p=0.01).Conclusions. This is the first study quantifying kidney expressions of TLRs in LN patients; the results show an overexpression of TLR3, TLR7, and TLR9 and demonstrate a correlation with clinicopathological indices supporting a role of these mediators in the pathogenesis of LN.

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Agrawal, S. y E. R. Kandimalla. "Synthetic agonists of Toll-like receptors 7, 8 and 9". Biochemical Society Transactions 35, n.º 6 (23 de noviembre de 2007): 1461–67. http://dx.doi.org/10.1042/bst0351461.

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TLRs (Toll-like receptors) are a family of innate immune receptors that induce protective immune responses against infections. Single-stranded viral RNA and bacterial DNA containing unmethylated CpG motifs are the ligands for TLR7 and TLR8 and 9 respectively. We have carried out extensive structure–activity relationship studies of DNA- and RNA-based compounds to elucidate the impact of nucleotide motifs and structures on these TLR-mediated immune responses. These studies have led us to design novel DNA- and RNA-based compounds, which act as potent agonists of TLR9 and TLR7 and 8 respectively. These novel synthetic agonists produce different immune response profiles depending on the structures and nucleotide motifs present in them. The ability to modulate TLR-mediated immune responses with these novel DNA- and RNA-based agonists in a desired fashion may allow targeting a broad range of diseases, including cancers, asthma, allergies and infections, alone or in combination with other therapeutic agents, and their use as adjuvants with vaccines. IMO-2055, our first lead candidate, is a TLR9 agonist that is currently in clinical evaluation in oncology patients. A second candidate, IMO-2125, is also a TLR9 agonist that has been shown to induce high and sustained levels of IFN (interferon) in non-human primates and is being evaluated in HepC-infected human subjects.

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Baumann, Christoph L., Irene M. Aspalter, Omar Sharif, Andreas Pichlmair, Stephan Blüml, Florian Grebien, Manuela Bruckner et al. "CD14 is a coreceptor of Toll-like receptors 7 and 9". Journal of Experimental Medicine 207, n.º 12 (15 de noviembre de 2010): 2689–701. http://dx.doi.org/10.1084/jem.20101111.

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Recognition of pathogens by the innate immune system requires proteins that detect conserved molecular patterns. Nucleic acids are recognized by cytoplasmic sensors as well as by endosomal Toll-like receptors (TLRs). It has become evident that TLRs require additional proteins to be activated by their respective ligands. In this study, we show that CD14 (cluster of differentiation 14) constitutively interacts with the MyD88-dependent TLR7 and TLR9. CD14 was necessary for TLR7- and TLR9-dependent induction of proinflammatory cytokines in vitro and for TLR9-dependent innate immune responses in mice. CD14 associated with TLR9 stimulatory DNA in precipitation experiments and confocal imaging. The absence of CD14 led to reduced nucleic acid uptake in macrophages. Additionally, CD14 played a role in the stimulation of TLRs by viruses. Using various types of vesicular stomatitis virus, we showed that CD14 is dispensable for viral uptake but is required for the triggering of TLR-dependent cytokine responses. These data show that CD14 has a dual role in nucleic acid–mediated TLR activation: it promotes the selective uptake of nucleic acids, and it acts as a coreceptor for endosomal TLR activation.

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Fukui, Ryutaro, Shin-ichiroh Saitoh, Fumi Matsumoto, Hiroko Kozuka-Hata, Masaaki Oyama, Koichi Tabeta, Bruce Beutler y Kensuke Miyake. "Unc93B1 biases Toll-like receptor responses to nucleic acid in dendritic cells toward DNA- but against RNA-sensing". Journal of Experimental Medicine 206, n.º 6 (18 de mayo de 2009): 1339–50. http://dx.doi.org/10.1084/jem.20082316.

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Toll-like receptors (TLRs) 3, 7, and 9 recognize microbial nucleic acids in endolysosomes and initiate innate and adaptive immune responses. TLR7/9 in dendritic cells (DCs) also respond to self-derived RNA/DNA, respectively, and drive autoantibody production. Remarkably, TLR7 and 9 appear to have mutually opposing, pathogenic or protective, impacts on lupus nephritis in MRL/lpr mice. Little is known, however, about the contrasting relationship between TLR7 and 9. We show that TLR7 and 9 are inversely linked by Unc93B1, a multiple membrane-spanning endoplasmic reticulum (ER) protein. Complementation cloning with a TLR7-unresponsive but TLR9-responsive cell line revealed that amino acid D34 in Unc93B1 repressed TLR7-mediated responses. D34A mutation rendered Unc93B1-deficient DCs hyperresponsive to TLR7 ligand but hyporesponsive to TLR9 ligand, with TLR3 responses unaltered. Unc93B1 associates with and delivers TLR7/9 from the ER to endolysosomes for ligand recognition. The D34A mutation up-regulates Unc93B1 association with endogenous TLR7 in DCs, whereas Unc93B1 association with TLR9 was down-regulated by the D34A mutation. Consistently, the D34A mutation up-regulated ligand-induced trafficking of TLR7 but down-regulated that of TLR9. Collectively, TLR response to nucleic acids in DCs is biased toward DNA-sensing by Unc93B1.

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Kumar, Mahesh M., Sreenivas Adurthi, Surya Ramachandran, Geetashree Mukherjee, Omana Joy, H. Krishnamurthy, Sudhir Krishna, U. D. Bafna, Devi K. Uma y R. S. Jayshree. "Toll-Like Receptors 7, 8, and 9 Expression and Function in Primary Human Cervical Cancer Langerhans Cells: Evidence of Anergy". International Journal of Gynecologic Cancer 23, n.º 1 (enero de 2013): 184–92. http://dx.doi.org/10.1097/igc.0b013e31827a2003.

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ObjectiveHuman papillomavirus oncoproteins E6 and E7 down modulate Toll-like receptor (TLR) 9 expression in infected keratinocytes. We explored the status of expression and function of TLR7, TLR8, and TLR9 in primary human Langerhans cells (LCs) isolated from cervical tumors.MethodologySingle-cell suspensions were made from fresh tissues of squamous cell carcinoma (International Federation of Gynecology and Obstetrics stage IB2); myeloid dendritic cells were purified using CD1c magnetic activated cell separation kits. Langerhans cells were further flow sorted into CD1a+CD207+cells. Acute monocytic leukemia cell line THP-1–derived LCs (moLCs) formed the controls. mRNA from flow-sorted LCs was reverse transcribed to cDNA and TLR7, TLR8, and TLR9 amplified. Monocyte-derived Langerhans cells and cervical tumor LCs were stimulated with TLR7, TLR8, and TLR9 ligands. Culture supernatants were assayed for interleukin (IL) 1β, IL-6, IL-10, IL-12p70, interferon (IFN) α, interferon γ, and tumor necrosis factor (TNF) α by Luminex multiplex bead array. Human papillomavirus was genotyped.ResultsWe have for the first time demonstrated that the acute monocytic leukemia cell line THP-1 can be differentiated into LCs in vitro. Although these moLCs expressed all the 3 TLRs, tumor LCs expressed TLR7 and TLR8, but uniformly lacked TLR9. Also, moLCs secreted IL-6, IL-1β, and tumor necrosis factor α to TLR8 ligand and interferon α in response to TLR9 ligand; in contrast, tumor LCs did not express any cytokine to any of the 3 TLR ligands. Human papillomavirus type 16 was one of the common human papillomavirus types in all cases.ConclusionsCervical tumor LCs lacked TLR9 expression and were functionally anergic to all the 3: TLR7, TLR8, and TLR9 ligands, which may play a crucial role in immune tolerance. The exact location of block(s) in TLR7 and TLR8 signaling needs to be investigated, which would have important immunotherapeutic implications.

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Patra, Mahesh Chandra, Asma Achek, Gi-Young Kim, Suresh Panneerselvam, Hyeon-Jun Shin, Wook-Yong Baek, Wang Hee Lee et al. "A Novel Small-Molecule Inhibitor of Endosomal TLRs Reduces Inflammation and Alleviates Autoimmune Disease Symptoms in Murine Models". Cells 9, n.º 7 (9 de julio de 2020): 1648. http://dx.doi.org/10.3390/cells9071648.

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Toll-like receptors (TLRs) play a fundamental role in the inflammatory response against invading pathogens. However, the dysregulation of TLR-signaling pathways is implicated in several autoimmune/inflammatory diseases. Here, we show that a novel small molecule TLR-inhibitor (TAC5) and its derivatives TAC5-a, TAC5-c, TAC5-d, and TAC5-e predominantly antagonized poly(I:C) (TLR3)-, imiquimod (TLR7)-, TL8-506 (TLR8)-, and CpG-oligodeoxynucleotide (TLR9)-induced signaling pathways. TAC5 and TAC5-a significantly hindered the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), reduced the phosphorylation of mitogen-activated protein kinases, and inhibited the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6. Besides, TAC5-a prevented the progression of psoriasis and systemic lupus erythematosus (SLE) in mice. Interestingly, TAC5 and TAC5-a did not affect Pam3CSK4 (TLR1/2)-, FSL-1 (TLR2/6)-, or lipopolysaccharide (TLR4)-induced TNF-α secretion, indicating their specificity towards endosomal TLRs (TLR3/7/8/9). Collectively, our data suggest that the TAC5 series of compounds are potential candidates for treating autoimmune diseases such as psoriasis or SLE.

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Skert, Cristina, Manuela Fogli, Simone Perucca, Simona Fiorentini, Emirena Garrafa, Carla Filì, Annalisa Peli et al. "Expression of Toll-Like Receptors on Peripheral Blood Cells After Allogeneic Stem Cell Transplantation: Results of a Prospective Study",. Blood 118, n.º 21 (18 de noviembre de 2011): 4071. http://dx.doi.org/10.1182/blood.v118.21.4071.4071.

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Abstract Abstract 4071 Introduction. Emerging trends emphasize the importance of both innate and adaptive immune system in the response against infections, and in the pathogenesis of autoimmune and graft-versus-host (GVHD) diseases. Pattern recognition receptors such as Toll-like receptors (TLRs) play a key role in the cross-talk between innate and adaptive immune system. TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs), such as common protein, carbohydrate or DNA/RNA pattern motifs. TLRs are also receptors for endogenous ligands and damaged tissue, suggesting that both pathogen-derived molecules and products of damaged tissue can trigger signals which are responsible for the regulation of innate and adaptive immune responses. Extracellular ligands are recognized by surface TLRs (TLR1,TLR2,TLR4,TLR5, and TLR6). Intracellular TLRs (TLR3,TLR7,TLR8 and TLR9) bind mainly to foreign nucleic acids and sometimes detect self DNA/RNA. Aim of the study. Very little is known about expression and function of TLRs in vivo in patients who underwent allogeneic stem cell transplantation (SCT). The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to the onset of acute GVHD. Methods. The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and at the onset of GVHD. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls. Results. We analyzed 17 healthy donors and 34 patients. Median age was 46 years (range, 22–64) and 22 patients were male. Acute GVHD developed in 19 patients (12 with grade >=2). Clinical and transplant characteristics did not differ in patients with and without GVHD. Lymphocytes and monocytes of patients with acute GVHD showed higher levels of TLR5 (3,5±2,3 vs1,9±1,6 p=0,03; 25,8±25,9 vs 9,0±5,0 p=0,02) and a decreased expression of TLR1 (2,5±2,8 vs 4,3±2,8 p=0,02; 21,4±21,9 vs 54,9±37,4 p=0,005) and TLR9 (63,8±30,4 vs 111,1±62,9 p=0,03; 85,3±73,9 vs 164,2±90,6 p=0,01). IFN-gamma relative induction post-stimulation of TLR2,3,4 and 9 was significantly decreased in patients with acute GVHD (p< 0,04). Conclusions. TLRs show a different profile of expression in patients with acute GVHD in comparison with patients without it. These results suggest that the innate immune response via TLRs activation could be involved in the development of GVHD. In particular, a decreased expression of TLR-9 (receptor of hypomethylated DNA) on lymphocytes and monocytes can promote TLR-7 activation, inducing type I interferons and other pro-inflammatory cytokines. TLR-1 and −5, which are ligands for bacterial cell wall, could also be involved in the pathogenesis of GVHD. Moreover, acute GVHD negatively correlates with IFN-gamma production upon TLR2,3,4 and 9 activation. The assessment of a larger number of patients could be useful to understand the complex interplay among pathogens, self or non-self DNA and RNA, and the immune system. Disclosures: No relevant conflicts of interest to declare.

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Thomas, Amy, Carl Laxton, Joanne Rodman, Nisha Myangar, Nigel Horscroft y Tanya Parkinson. "Investigating Toll-Like Receptor Agonists for Potential To Treat Hepatitis C Virus Infection". Antimicrobial Agents and Chemotherapy 51, n.º 8 (4 de junio de 2007): 2969–78. http://dx.doi.org/10.1128/aac.00268-07.

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ABSTRACT Toll-like receptors (TLRs) are key mediators of innate immunity, and their activation by microbial components leads to the production of cytokines and interferons. Recombinant alpha interferon has been used to treat several viral diseases and is the current standard of care for hepatitis C virus (HCV) infection. Recently, agonists of TLR7 and TLR9 have been shown to have clinical efficacy in HCV patients, and this is correlated with their ability to induce endogenous type I interferon production. We have carried out a comprehensive study of agonists of TLRs 1 to 9 to determine if any additional TLRs can induce antiviral molecules from human peripheral blood mononuclear cells (PBMCs). The agonists were incubated with PBMCs, and the supernatant was then removed and added to HCV replicon cells to assess antiviral activity. Agonists of TLRs 3, 4, 7, 8, and 9 were found to be potent inducers of antiviral activity in PBMC supernatants, and the activity correlated with the induction of alpha interferon and the interferon-induced antiviral biomarker 2′,5′-oligoadenylate synthase. Antiviral activity of TLR7 and TLR8 agonists was blocked by an antibody that binds to the type I interferon receptor, confirming that the antiviral activity results from type I interferon induction. TLR4 and TLR8 agonists were found to strongly induce the proinflammatory cytokines interleukin 1β and tumor necrosis factor alpha at concentrations similar to those inducing antiviral activity. This raises concerns about adverse side effects if these were to be used as antiviral agents. We therefore conclude that TLRs 3, 7, and 9 represent the most attractive targets for the development of new HCV therapies.

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Shindo, Maki, Xueqing Liang, Zhimei Wang, Jeffery S. Miller, Martin Carroll y Wei Chen. "Toll-Like Receptor Agonists Induce Immunogeneicity and Apoptosis of Acute Myeloid Leukemia Cells." Blood 110, n.º 11 (16 de noviembre de 2007): 160. http://dx.doi.org/10.1182/blood.v110.11.160.160.

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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.

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Baccarella, Alyssa, Mary F. Fontana, Eunice C. Chen y Charles C. Kim. "Toll-Like Receptor 7 Mediates Early Innate Immune Responses to Malaria". Infection and Immunity 81, n.º 12 (16 de septiembre de 2013): 4431–42. http://dx.doi.org/10.1128/iai.00923-13.

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ABSTRACTInnate immune recognition of malaria parasites is the critical first step in the development of the host response. At present, Toll-like receptor 9 (TLR9) is thought to play a central role in sensing malaria infection. However, we and others have observed thatTlr9−/−mice, in contrast to mice deficient in the downstream adaptor, Myeloid differentiation primary response gene 88 (MYD88), exhibit few deficiencies in immune function during early infection with the malaria parasitePlasmodium chabaudi, implying that another MYD88-dependent receptor also contributes to the antimalarial response. Here we use candidate-based screening to identify TLR7 as a key sensor of earlyP. chabaudiinfection. We show that TLR7 mediates a rapid systemic response to infection through induction of cytokines such as type I interferons (IFN-I), interleukin 12, and gamma interferon. TLR7 is also required for induction of IFN-I by other species and strains ofPlasmodium, including an etiological agent of human disease,P. falciparum, suggesting that malaria parasites harbor a common pathogen-associated molecular pattern (PAMP) recognized by TLR7. In contrast to the nonredundant requirement for TLR7 in early immune activation, sensing through both TLR7 and TLR9 was required for proinflammatory cytokine production and immune cell activation during the peak of parasitemia. Our findings indicate that TLR7 plays a central role in early immune activation during malaria infection, whereas TLR7 and TLR9 contribute combinatorially to immune responses as infection progresses.

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Ghasiyari, Haniye, Mohammad Rostami-Nejad, Davar Amani, Kamran Rostami, Mohamad Amin Pourhoseingholi, Hamid Asadzadeh-Aghdaei y Mohammad Reza Zali. "Diverse Profiles of Toll-Like Receptors 2, 4, 7, and 9 mRNA in Peripheral Blood and Biopsy Specimens of Patients with Celiac Disease". Journal of Immunology Research 2018 (2 de julio de 2018): 1–8. http://dx.doi.org/10.1155/2018/7587095.

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Background and Aims. Both adaptive and innate immunity are involved in the development of celiac disease (CD). Altered Toll-like receptors (TLR) expression and activation may be partially responsible for the inflammation and subsequently crypt hyperplasia, but the main driver for inflammation is gliadin-reactive T-cells. Therefore, the aim of this study was to investigate the TLRs 2, 4, 7, and 9 gene expressions in both peripheral blood and intestinal mucosa of patients with celiac disease compared to healthy control (HC). Material and Methods. Blood samples from 120 confirmed active CD patients and 120 age- and sex-matched healthy volunteers served as control group were collected during 2015-2016. Also, 20 biopsy specimens from the study group were randomly collected. Total RNA was isolated using a standard commercial kit. The mRNA expression of TLRs was quantified by relative qPCR with β2 microglobulin (β2m) as a reference gene. Results. TLR4 (P=0.01) and TLR9 (P=0.02) mRNA were significantly elevated in blood samples from CD patients compared to the healthy controls. Moreover, TLR2 (P=0.03) and TLR4 (P=0.0003) expression level was increased in CD biopsy specimens compared to controls, whereas expression of TLR9 mRNA was significantly decreased in CD patients. There was no significant difference in the expression of TLR7 in biopsy and blood specimens. Conclusions. The alteration of TLR4 and TLR9 expression in the blood and biopsy samples of patients with CD supports the critical role of the innate immune system in the pathogenesis of this disease. Upregulation of TLR4 and TLR9 suggests the contribution of gut microbiota or dysregulation of the immune response to commensal flora in small bowel mucosa in celiac patients.

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Lenert, Petar S. "Classification, Mechanisms of Action, and Therapeutic Applications of Inhibitory Oligonucleotides for Toll-Like Receptors (TLR) 7 and 9". Mediators of Inflammation 2010 (2010): 1–10. http://dx.doi.org/10.1155/2010/986596.

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Our immune defense depends on two specialized armed forces. The innate force acts as an alarm mechanism that senses changes in the microenvironment through the recognition of common microbial patterns by Toll-like receptors (TLR) and NOD proteins. It rapidly generates an inflammatory response aimed at neutralizing the intruder at the mucosal checkpoint. The innate arm also communicates this message with more specialized adaptive forces represented by pathogen-specific B cells and T cells. Interestingly, B cells also express some innate sensors, like TLR7 and TLR9, and may respond to bacterial hypomethylated CpG motifs and single-stranded RNA viruses. Intracellular nucleic acid sensing TLRs play an important role in the pathogenesis of Systemic Lupus Erythematosus (SLE). In this review, we describe recent achievements in the development of oligonucleotide—(ODN)-based inhibitors of TLR9 and/or TLR7 signaling. We categorize these novel therapeutics into Classes G, R, and B based on their cellular and molecular targets. Several short ODNs have already shown promise as pathway-specific therapeutics for animal lupus. We envision their future use in human SLE, microbial DNA-dependent sepsis, and in other autoinflammatory diseases.

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Klonowska-Szymczyk, Agnieszka, Anna Wolska, Tadeusz Robak, Barbara Cebula-Obrzut, Piotr Smolewski y Ewa Robak. "Expression of Toll-Like Receptors 3, 7, and 9 in Peripheral Blood Mononuclear Cells from Patients with Systemic Lupus Erythematosus". Mediators of Inflammation 2014 (2014): 1–11. http://dx.doi.org/10.1155/2014/381418.

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Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown aetiology. The results of experimental studies point to the involvement of innate immunity receptors—toll-like receptors (TLR)—in the pathogenesis of the disease. The aim of the study was to assess the expression of TLR3, 7, and 9 in the population of peripheral blood mononuclear cells (PBMC) and in B lymphocytes (CD19+), T lymphocytes (CD4+and CD8+) using flow cytometry. The study group included 35 patients with SLE and 15 healthy controls. The patient group presented a significantly higher percentage of TLR3- and TLR9-positive cells among all PBMCs and their subpopulations (CD3+, CD4+, CD8+, and CD19+lymphocytes) as well as TLR7 in CD19+B-lymphocytes, compared to the control group. There was no correlation between the expression of all studied TLRs and the disease activity according to the SLAM scale, and the degree of organ damage according to the SLICC/ACR Damage Index. However, a correlation was observed between the percentage of various TLR-positive cells and some clinical (joint lesions) and laboratory (lymphopenia, hypogammaglobulinemia, anaemia, and higher ESR) features and menopause in women. The results of the study suggest that TLR3, 7, and 9 play a role in the pathogenesis of SLE and have an impact on organ involvement in SLE.

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Hayashi, Tomoko, Howard B. Cottam, Michael Chan, Guangyi Jin, Rommel I. Tawatao, Brian Crain, Lisa Ronacher, Karen Messer, Dennis A. Carson y Maripat Corr. "Mast cell-dependent anorexia and hypothermia induced by mucosal activation of Toll-like receptor 7". American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 295, n.º 1 (julio de 2008): R123—R132. http://dx.doi.org/10.1152/ajpregu.00527.2007.

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Systemic viral infections produce a highly regulated set of responses in sickness behavior, such as fever, anorexia, and adipsia. Toll-like receptor (TLR)7, activated by viral RNA during infection, potently stimulates the innate and adaptive immune responses that aid in viral clearance. However, the physiological consequences of TLR7 activation have not been thoroughly studied. In these experiments, we used a potent synthetic TLR7 ligand, 9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine ( SM360320 ; 1V136), to investigate the consequences of TLR7 activation in genetically defined strains of mice. Administration of the drug by the nasal, intragastric, or intraperitoneal routes caused transient hypophagia, hypodypsia, and hypothermia. Analyses of mutant mouse strains indicated that these effects were dependent on the expression of TLR7, its adaptor protein MyD88, and TNF-α, and independent of IL-1β, IL-6 and cyclo-oxygenase-1 (COX1). Partial roles were also implied for mast cells and COX2. Although plasma TNF-α levels were significantly higher after systemic drug delivery, the behavioral effects were maximal when the agent was administered to the mucosa. Tissue and mucosal mast cells are known to express high levels of TLR7 and to rapidly release TNF-α upon TLR7 ligation. Mice deficient in tissue mast cells, W/W(v), had significantly less anorexia after TLR7 activation, and this response was restored with mast cell reconstitution. Our results thus suggest that tissue mast cells may play a role in the anorexia induced by mucosal activation of TLR7.

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Pan, Zi-jian, Shannon Maier, Karen Schwarz, Jennifer Azbill, Shizuo Akira, Satoshi Uematsu y A. Darise Farris. "Toll-like receptor 7 (TLR7) modulates anti-nucleosomal autoantibody isotype and renal complement deposition in mice exposed to syngeneic late apoptotic cells". Annals of the Rheumatic Diseases 69, n.º 6 (11 de agosto de 2009): 1195–99. http://dx.doi.org/10.1136/ard.2009.108282.

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ObjectivesThe objectives of this study were to determine whether late apoptotic cell material directly induces autoantibodies characteristic of systemic lupus erythematosus (SLE) and to investigate the innate recognition pathways involved.MethodsB6, B6.MyD88–/–, B6.TLR7–/– and B6.TLR9–/– mice were subcutaneously injected with B6 syngeneic late apoptotic thymocytes (SLATs) without adjuvant on days 0, 10, 24 and 37. Sera were tested for IgG antibodies to histones and double-stranded DNA (dsDNA) by ELISA and Crithidia luciliae indirect immunofluorescence. IgG and C3 deposition in kidney glomeruli was assessed by immunostaining and fluorescence microscopy.ResultsSLAT injections induced anti-dsDNA and anti-histone antibodies of the IgG1 and IgG2b isotypes in B6 but not MyD88–/– mice. TLR7–/– and TLR9–/– mice injected with SLATs produced delayed or slightly more robust responses, respectively. SLAT injections induced IgG deposits in renal glomeruli of B6, TLR7–/– and TLR9–/– mice that were absent in MyD88–/– mice. Unlike B6 and TLR9–/– animals, TLR7–/– mice failed to exhibit IgG colocalised glomerular C3 deposits and demonstrated autoantibodies of primarily the IgG2a isotype.ConclusionsLate apoptotic cell-induced anti-histone and anti-dsDNA antibodies require MyD88 but not Toll-like receptor (TLR)9. Moreover, TLR7 promotes glomerular C3 deposition, possibly through a mechanism of altered antibody isotype switching.

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Migdał, Anna, Łukasz Migdał, Maria Oczkowicz, Adam Okólski y Anna Chełmońska-Soyta. "Influence of Age and Immunostimulation on the Level of Toll-Like Receptor Gene (TLR3, 4, and 7) Expression in Foals". Animals 10, n.º 11 (26 de octubre de 2020): 1966. http://dx.doi.org/10.3390/ani10111966.

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The aim of this study was to investigate the molecular mechanisms leading to the identification of pathogens by congenital immune receptors in foals up to 60 days of age. The study was conducted on 16 foal Polish Pony Horses (Polish Konik) divided into two study groups: control (n = 9) and experimental (n = 7). Foals from the experimental group received an intramuscular duplicate injection of 5 mL of Biotropina (Biowet) at 35 and 40 days of age. The RNA isolated from venous blood was used to evaluate the expression of theTLR3, TLR4, and TLR7 genes using RT-PCR. The results of the experiment demonstrated a statistically significant increase in the level of TLR3 gene expression and a decrease in the level ofTLR4 gene expression with foal aging. The level of TLR7 gene expression did not show age dependence. Immunostimulation with Biotropina had a significant impact on the level of the genes’ expression for Toll-like receptors. It increased the level of TLR4 expression and decreased TLR3 expression. Thus, it was concluded that the expression of theTLR3 and TLR4genes in peripheral blood cells is dependent on age. This experiment demonstrated a strong negative correlation between TLR3 and TLR4 gene expression.

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Fransen, Floris, Claire J. Boog, Jos P. van Putten y Peter van der Ley. "Agonists of Toll-Like Receptors 3, 4, 7, and 9 Are Candidates for Use as Adjuvants in an Outer Membrane Vaccine against Neisseria meningitidis Serogroup B". Infection and Immunity 75, n.º 12 (1 de octubre de 2007): 5939–46. http://dx.doi.org/10.1128/iai.00846-07.

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ABSTRACT The bacterium Neisseria meningitidis is the causative agent of meningitis and sepsis. A generally effective vaccine against N. meningitidis serogroup B is not yet available, but outer membrane vesicle vaccines are in development. These vaccines contain lipopolysaccharide (LPS). The inclusion of N. meningitidis wild-type LPS in a vaccine is controversial because of its high toxicity. Therefore, the adjuvant activity of a panel of different Toll-like receptor (TLR) agonists in combination with LPS-deficient meningococcal outer membrane complexes was compared after immunization of mice. The results demonstrate that TLR3, TLR4, TLR7, and TLR9 agonists enhance immune responses against LPS-deficient outer membrane complexes. Their adjuvant activity was characterized by higher levels of antigen-specific immunoglobulin G (IgG), IgG2a, and IgG2b; a higher IgG2a/IgG1 ratio; lower total IgE levels; and most importantly, higher serum bactericidal antibody titers compared to LPS-deficient outer membrane complexes alone.

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Paul, Santanu, Charles C. Chu, Brian A. McCarthy, Erin Boyle, Bettie M. Steinberg, Matthew Kaufman, Jonathan Kolitz, Steven L. Allen, Kanti R. Rai y Nicholas Chiorazzi. "Activation of Nucleic Acid-Sensing Toll-Like Receptors Induces Proliferation, Cytokine Production, Immunogenic Phenotype, and Plasma Cell Differentiation of CLL Cells and Immunoglobulin Production." Blood 110, n.º 11 (16 de noviembre de 2007): 1137. http://dx.doi.org/10.1182/blood.v110.11.1137.1137.

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Abstract Weak immune function of chronic lymphocytic leukemia (CLL) cells may contribute to disease progression and inhibit effective immunotherapy. Accordingly, agents that enhance the function of CLL cells may be useful in immunotherapeutic approaches to this disease. Since Toll-like receptors (TLRs) are major regulators of B-cell function in innate and adaptive immunity, we asked to what extent stimulating two nucleic acid-sensing TLRs affected the activation and differentiation of CLL B cells. Isolated CLL cells were treated with either the TLR7-activating ligand, gardiquimod, or the TLR9-activating ligand ODN 2006-G5. CLL cells were also treated with the contents of apoptotic leukemic B cells, produced by repeated freezing and thawing. Ability to induce proliferation, as defined by CFSE dilution studies, cytokine production, differentiation, and altered surface molecule expression was measured. Both TLR 7 and TLR 9 ligands individually induced high levels of proliferation and plasma cell differentiation of CLL B cells. A dramatic increase in percentage of dividing cells was seen with simultaneous TLR7 and TLR9 activation, while blocking the TLR 7/9 pathway by pre-treating CLL B cells with chloroquine or bafilomycin reduced the proliferative capacity by 90%. Furthermore, stimulating CLL B cells with TLR7 and 9 ligands significantly up-regulated activation (CD38, CD69 and CD83), adhesion (CD54), and other immunophenotypic (CD40, CD80 and CD86) markers. Enzyme immunoassays of culture supernatants after TLR 7 and TLR 9 stimulation indicated that the levels of soluble IgM increased considerably, although levels of cytoplasmic IgM were unchanged. Production of IL6 and IL10 was also induced. We propose that ligands for TLRs 9 and 7 stimulate CLL B cells to proliferate and differentiate into plasma cells in the absence of T-cell help. In addition these stimuli alter CLL cell surface phenotype to a more immunogenetic profile, providing a rationale for CpG oligonucleotides and gardiquimod as humoral vaccine adjuvants for both normal immune reactions and potentially for anti-leukemic therapy.

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Lau, Christina M., Courtney Broughton, Abigail S. Tabor, Shizuo Akira, Richard A. Flavell, Mark J. Mamula, Sean R. Christensen et al. "RNA-associated autoantigens activate B cells by combined B cell antigen receptor/Toll-like receptor 7 engagement". Journal of Experimental Medicine 202, n.º 9 (31 de octubre de 2005): 1171–77. http://dx.doi.org/10.1084/jem.20050630.

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Previous studies (Leadbetter, E.A., I.R. Rifkin, A.H. Hohlbaum, B. Beaudette, M.J. Shlomchik, and A. Marshak-Rothstein. 2002. Nature. 416:603–607; Viglianti, G.A., C.M. Lau, T.M. Hanley, B.A. Miko, M.J. Shlomchik, and A. Marshak-Rothstein. 2003. Immunity. 19:837–847) established the unique capacity of DNA and DNA-associated autoantigens to activate autoreactive B cells via sequential engagement of the B cell antigen receptor (BCR) and Toll-like receptor (TLR) 9. We demonstrate that this two-receptor paradigm can be extended to the BCR/TLR7 activation of autoreactive B cells by RNA and RNA-associated autoantigens. These data implicate TLR recognition of endogenous ligands in the response to both DNA- and RNA-associated autoantigens. Importantly, the response to RNA-associated autoantigens was markedly enhanced by IFN-α, a cytokine strongly linked to disease progression in patients with systemic lupus erythematosus (SLE). As further evidence that TLRs play a key role in autoantibody responses in SLE, we found that autoimmune-prone mice, lacking the TLR adaptor protein MyD88, had markedly reduced chromatin, Sm, and rheumatoid factor autoantibody titers.

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Sokolova, T. M. y V. V. Poloskov. "The effect of Kagocel® on gene expression of Toll-like receptors of innate immunity in THP-1 human monocytes with different levels of differentiation". BIOpreparations. Prevention, Diagnosis, Treatment 21, n.º 2 (1 de julio de 2021): 116–21. http://dx.doi.org/10.30895/2221-996x-2021-21-2-116-121.

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Kagocel® is used in Russia for the treatment of viral infections. In terms of its chemical structure, Kagocel® active ingredient is a copolymer of gossypol polyphenol and carboxymethylcellulose. The study investigated antiviral and cytokine-inducing activity of Kagocel®, as well as its toxic effects. The aim of the study was to investigate the effect of Kagoce ® active ingredient on the induction of expression of the innate immune system receptor genes (Toll-like receptors, TLR) in the THP-1 human acute monocytic leukemia cell line with different levels of differentiation. Materials and methods: the effect of Kagocel active ingredient was investigated at the concentrations of 0.2 and 2 mg/mL in the THP-1 human acute monocytic leukemia cell line with different levels of differentiation: non-differentiated monocytes, and monocytes differentiated into macrophage-like cells. Comparative analysis of the activity of TLR 2, 3, 4, 7, 8, 9 genes was carried out by quantitative RT-PCR. The study determined standard deviations of the levels of gene expression in the experimental cells (2deltaCq ± SD) relative to the expression in the control cells. Results: Kagocel active ingredient at the concentration of 0.2 mg/mL induced activation of TLR2 expression in THP-1 monocytes by 3.5 times, TLR3 by 2 times, TLR4 by 1.6 times, and at the concentration of 2 mg/mL also induced activation of TLR7 and TLR8 by 1.4 times, and TLR9 by 2 times. The levels of TLR2, TLR3, TLR9 induction were significantly higher in THP-1 monocytes partially differentiated into macrophage-like cells, and the highest stimulation level was observed for TLR2 (8 times). Conclusions: the results obtained characterise Kagocel® as a stimulator of TLR genes in the THP-1 cell line. The number of TLR genes induced in THP-1 monocytes was shown to increase with the increase in the product concentration. THP-1 monocyte differentiation into macrophage-like cells enhances susceptibility to Kagocel®. The positive regulation of TLR genes activity may account for antiviral and interferon-inducing properties of Kagocel®, and also suggests the possibility of expanding the use of the product for various immune-associated diseases.

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Keating, Sinead E., Geraldine M. Maloney, Ellen M. Moran y Andrew G. Bowie. "IRAK-2 Participates in Multiple Toll-like Receptor Signaling Pathways to NFκB via Activation of TRAF6 Ubiquitination". Journal of Biological Chemistry 282, n.º 46 (18 de septiembre de 2007): 33435–43. http://dx.doi.org/10.1074/jbc.m705266200.

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Toll-like receptor (TLR) signaling is known to involve interleukin-1 receptor-associated kinases (IRAKs), however the particular role of IRAK-2 has remained unclear. Further, although IRAK-1 was originally thought to be central for the TLR-NFκB signaling axis, recent data have shown that it is dispensable for NFκB activation for some TLRs and demonstrated an alternative role for it in interferon regulatory factor activation. Here we show that IRAK-2 is critical for the TLR-mediated NFκB activation pathway. The poxviral TLR antagonist A52 inhibited NFκB activation by TLR2, -3, -4, -5, -7, and -9 ligands, via its interaction with IRAK-2, while not affecting interferon regulatory factor activation. Knockdown of IRAK-2 expression by small interfering RNA suppressed TLR3, TLR4, and TLR8 signaling to NFκB in human cell lines, and importantly, TLR4-mediated chemokine production in primary human cells. IRAK-2 usage by different TLRs was distinct, because it acted downstream of the TLR adaptors MyD88 and Mal but upstream of TRIF. Expression of IRAK-2, but not IRAK-1, led to TRAF6 ubiquitination, an event critical for NFκB activation. Further, IRAK-2 loss-of-function mutants, which could not activate NFκB, were incapable of promoting TRAF6 ubiquitination. Thus we propose that IRAK-2 plays a more central role than IRAK-1 in TLR signaling to NFκB.

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22

Skert, Cristina, Manuela Fogli, Simone Perucca, Simona Fiorentini, Emirena Garrafa, Carla Filì, Chiara Colombi et al. "Betaherpesvirus Reactivation and Toll-Like Receptor Expression After Allogeneic Stem Cell Transplantation". Blood 118, n.º 21 (18 de noviembre de 2011): 4924. http://dx.doi.org/10.1182/blood.v118.21.4924.4924.

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Abstract Abstract 4924 Introduction. β-herpesviruses, such as CMV and HHV6, are important pathogen in transplanted patients. The morbidity because of CMV reactivation after allogeneic stem cell transplantation (SCT) has led to the monitoring of this virus and to introduction of preemptive therapy. However, CMV infection is still one of the most challenging complications, because CMV disease may occur as life-threatening pneumonitis, and may increase the risk of opportunistic infections. HHV6 reactivation has been demonstrated after SCT and this virus is recognized as important pathogen, either by direct infection or via interaction with CMV. Innate and adaptive immune response against these viruses involves the activation of Toll-like receptors (TLRs). TLRs belong to type I transmembrane glycoprotein receptor family and recognize pathogen-associated molecular patterns (PAMPs). Viral nucleic acids and viral structural proteins, such as glycoproteins, are considered as PAMPs. Endosomal TLRs (TLR3, 7, 8 and 9) recognize viral nucleic acids and some surface TLRs may be involved in the detection of structural proteins. Some clinical and experimental evidences indicate that CMV and HHV-6 can modulate the immune system and influence the immune reconstitution after SCT. However, the role of TLRs in this complex interplay remains unclear, especially in the setting of allogeneic SCT. The aim of this study was to evaluate the expression of TLRs on lymphocytes and monocytes in relation to CMV and HHV6 reactivation in the early period after allogeneic SCT. Methods. CMV and HHV6 reactivation was monitored weekly by quantitative real-time PCR until the second month after SCT. The expression of TLRs on lymphocytes and monocytes was analysed by flow cytometry as mean fluorescence intensity at day +30 and in any case before CMV or HHV6 reactivation. Functional data were obtained by ELISA assay after TLRs activation. The cell supernatants were collected and assayed for TNF-alpha, IFN-gamma and MCP-1. Relative induction of these cytokines was calculated in relation with unstimulated controls. Results. CMV reactivation within 2 months after transplantation was observed in 13 out of 33 patients. CMV pneumonitis was observed in 1 patient. HHV-6 reactivation was detected in 1 patient. Median age was 45 years (range, 22–64) and 21 patients were male. TLRs expression and function did not significantly differ in controls and patients without CMV. Lymphocytes of patients with CMV reactivation showed an increased expression of TLR5 (4,1±2,4 vs 2,0±1,7 p=0,008). TLR8 expression was lower on monocytes with CMV reactivation (0,8±0,9 vs 2,0±1,7 p=0,03). MCP-1 relative induction post-stimulation of TLR1 and 8 was significantly decreased in patients with CMV reactivation (p<0,04). Conclusions. Surface TLR2 and intracellular TLR3 and 9 are reported to recognize CMV by some authors. In our study, surface TLR5 and intracellular TLR8 seem to be involved in the interaction between CMV and the immune system of transplanted patients. In particular, TLR8 could play a protective role. MCP-1 production upon TLR1 and 8 activation negatively correlates with CMV reactivation. The defective immune system after SCT could explain these results, which could be confirmed by the assessment of a larger number of patients and the analysis of other possible interfering factors. Disclosures: No relevant conflicts of interest to declare.

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Ahonen, Cory L., Christie L. Doxsee, Sean M. McGurran, Tony R. Riter, William F. Wade, Richard J. Barth, John P. Vasilakos, Randolph J. Noelle y Ross M. Kedl. "Combined TLR and CD40 Triggering Induces Potent CD8+ T Cell Expansion with Variable Dependence on Type I IFN". Journal of Experimental Medicine 199, n.º 6 (8 de marzo de 2004): 775–84. http://dx.doi.org/10.1084/jem.20031591.

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Toll-like receptors are important in the activation of innate immunity, and CD40 is a molecule critical for many T and B cell responses. Whereas agonists for either pathway have been used as vaccine adjuvants, we show that a combination of Toll-like receptor (TLR)7 and CD40 agonists synergize to stimulate CD8+ T cell responses 10–20-fold greater than the use of either agonist alone. Antigen-specific CD8+ T cells elicited from combination CD40/TLR7 treatment demonstrated both lytic activities and interferon (IFN)γ production and an enhanced secondary response to antigenic challenge. Agonists for TLRs 2/6, 3, 4, and 9 also synergized with CD40 stimulation, demonstrating that synergy with the CD40 pathway is a property of TLR-derived stimuli in general. The CD8+ T cell expansion induced by CD40/TLR7 triggering was independent of CD4+ T cells, IFNγ, and IL-12 but dependent on B7-mediated costimulation and surprisingly on type I IFN. These studies provide the rational basis for the use of TLR and CD40 agonists together as essential adjuvants to optimize vaccines designed to elicit protective or therapeutic immunity.

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Campillo-Gimenez, Laure, Mireille Laforge, Michèle Fay, Audrey Brussel, Marie-Christine Cumont, Valérie Monceaux, Ousmane Diop et al. "Nonpathogenesis of Simian Immunodeficiency Virus Infection Is Associated with Reduced Inflammation and Recruitment of Plasmacytoid Dendritic Cells to Lymph Nodes, Not to Lack of an Interferon Type I Response, during the Acute Phase". Journal of Virology 84, n.º 4 (25 de noviembre de 2009): 1838–46. http://dx.doi.org/10.1128/jvi.01496-09.

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ABSTRACT Divergent Toll-like receptor 7 (TLR7) and TLR9 signaling has been proposed to distinguish pathogenic from nonpathogenic simian immunodeficiency virus infection in primate models. We demonstrate here that increased expression of type I interferon in pathogenic rhesus macaques compared to nonpathogenic African green monkeys was associated with the recruitment of plasmacytoid dendritic cells in the lymph nodes and the presence of an inflammatory environment early after infection, instead of a difference in the TLR7/9 response.

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Taylor, Tammi, Wilbert Derbigny, Young-June Kim y Hal E. Broxmeyer. "Expression of Functional Toll-Like Receptor 2 on Mouse Embryonic Stem Cells". Blood 112, n.º 11 (16 de noviembre de 2008): 4746. http://dx.doi.org/10.1182/blood.v112.11.4746.4746.

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Abstract Embryonic stem (ES) cells have the capacity to produce all cell types of the body. Understanding murine ES (mES) cell proliferation, survival, differentiation, and self renewal will enhance knowledge of developmental biology and essential use of ES cells. Recently, Toll Like Receptor (TLR) activation has been shown by others to play a role in influencing the differentiation of hematopoietic stem cells. Previous studies have also shown that TLR activation prevents mesenchymal stem cell differentiation into adipocytes, chondrocytes, and osteocytes and plays a role in bone repair. We hypothesized that certain TLR’s would be expressed on mES cells and that the ligands for these expressed TLR’s would induce functional activity in the mESC’s. Therefore, we wanted to determine if TLRs are expressed on mES cells and if so, are they functional. Three different mES cell lines (R1, CGR8, and E14) were used to determine if TLRs are expressed at the mRNA level using primers for murine TLR1-9 mRNA. We found that TLR’s 1, 2, 3, 6, and 9 were expressed at the mRNA level, but TLR’s 4, 5, 7, and 8 were not. Based on the availability of antibodies to TLR’s, and using flow cytometry, we found expression of TLR2 but not TLR 4 on the surface of all three mES cell lines. TLR ligands were used to treat mES cells in the presence of leukemia inhibitory factor (LIF) for an hour. Activation of TLR2 by its ligand Pam3Cys, a synthetic tri-acyl lipoprotein, on mES cells induced NF-κβ nuclear translocation when compared to ES cells not stimulated with TLR ligands. LPS, the ligand for TLR4 did not induce NF-κβ nuclear translocation on ES cells, consistent with lack of cell surface expression of TLR4 on mES cells. TLR expression and TLR ligand interaction were not associated with changes in the morphology of the mES cells or expression of Oct-4, SSEA-1, KLF-4, or Sox-2, markers for maintenance of the undifferentiated state of mES cells. This suggests that the cells remain in an undifferentiated state even after TLR activation by Pam3Cys in the presence of LIF. Thus our study has identified functionally active TLR2 on the surface of mES cells, information that may be of use to further defining a role for TLR’s on ES cells, and for manipulation of other ES cell functions.

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Moreno, María, Berber M. Mol, Silvia von Mensdorff-Pouilly, René HM Verheijen, Esther C. de Jong, B. Mary E. von Blomberg, Alfons JM van den Eertwegh, Rik J. Scheper y Hetty J. Bontkes. "Differential indirect activation of human invariant natural killer T cells by Toll-like receptor agonists". Immunotherapy 1, n.º 4 (julio de 2009): 557–70. http://dx.doi.org/10.2217/imt.09.30.

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Aim: Invariant natural killer (iNK) T cells are activated by bacterial glycosphingolipids presented by CD1d on dendritic cells (DCs). Here, it was investigated whether Toll-like receptor (TLR) ligands derived from various microorganisms can either directly or indirectly (through DC activation) activate iNKT cells. Materials & Methods: TLR expression by iNKT cells was examined and the ability of various TLR ligands to activate iNKT cells was evaluated. Results: Although human iNKT cells express all TLRs, apart from TLR8, they did not respond directly to TLR ligands. However, iNKT cells became strongly activated when total peripheral blood mononuclear cells were stimulated with TLR2/6, 7/8 and 9 ligands, but not or to a lesser extent with TLR3, 4 and 5 ligands. TLR-stimulated monocyte-derived DCs promoted iNKT cell phenotypic activation and, in turn, these activated iNKT cells further enhanced DC maturation. Conclusion: TLR agonists may act as strong adjuvants for immunotherapy by promoting combined and reciprocal activation of iNKT cells and DCs.

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Schlender, Jörg, Veit Hornung, Stefan Finke, Margit Günthner-Biller, Sabrina Marozin, Krzysztof Brzózka, Sharareh Moghim, Stefan Endres, Gunther Hartmann y Karl-Klaus Conzelmann. "Inhibition of Toll-Like Receptor 7- and 9-Mediated Alpha/Beta Interferon Production in Human Plasmacytoid Dendritic Cells by Respiratory Syncytial Virus and Measles Virus". Journal of Virology 79, n.º 9 (1 de mayo de 2005): 5507–15. http://dx.doi.org/10.1128/jvi.79.9.5507-5515.2005.

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ABSTRACT Human plasmacytoid dendritic cells (PDC) are key sentinels alerting both innate and adaptive immune responses through production of huge amounts of alpha/beta interferon (IFN). IFN induction in PDC is triggered by outside-in signal transduction pathways through Toll-like receptor 7 (TLR7) and TLR9 as well as by recognition of cytosolic virus-specific patterns. TLR7 and TLR9 ligands include single-stranded RNA and CpG-rich DNA, respectively, as well as synthetic derivatives thereof which are being evaluated as therapeutic immune modulators promoting Th1 immune responses. Here, we identify the first viruses able to block IFN production by PDC. Both TLR-dependent and -independent IFN responses are abolished in human PDC infected with clinical isolates of respiratory syncytial virus (RSV), RSV strain A2, and measles virus Schwarz, in contrast to RSV strain Long, which we previously identified as a potent IFN inducer in human PDC (Hornung et al., J. Immunol. 173:5935-5943, 2004). Notably, IFN synthesis of PDC activated by the TLR7 and TLR9 agonists resiquimod (R848) and CpG oligodeoxynucleotide 2216 is switched off by subsequent infection by RSV A2 and measles virus. The capacity of RSV and measles virus of human PDC to shut down IFN production should contribute to the characteristic features of these viruses, such as Th2-biased immune pathology, immune suppression, and superinfection.

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Yasuda, Hideo, Asada Leelahavanichkul, Shinichiro Tsunoda, James W. Dear, Yoshiyuki Takahashi, Shuichi Ito, Xuzhen Hu et al. "Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury". American Journal of Physiology-Renal Physiology 294, n.º 5 (mayo de 2008): F1050—F1058. http://dx.doi.org/10.1152/ajprenal.00461.2007.

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Mortality from sepsis has remained high despite recent advances in supportive and targeted therapies. Toll-like receptors (TLRs) sense bacterial products and stimulate pathogenic innate immune responses. Mice deficient in the common adapter protein MyD88, downstream from most TLRs, have reduced mortality and acute kidney injury (AKI) from polymicrobial sepsis. However, the identity of the TLR(s) responsible for the host response to polymicrobial sepsis is unknown. Here, we show that chloroquine, an inhibitor of endocytic TLRs (TLR3, 7, 8, 9), improves sepsis-induced mortality and AKI in a clinically relevant polymicrobial sepsis mouse model, even when administered 6 h after the septic insult. Chloroquine administration attenuated the decline in renal function, splenic apoptosis, serum markers of damage to other organs, and prototypical serum pro- and anti-inflammatory cytokines TNF-α and IL-10. An oligodeoxynucleotide inhibitor (H154) of TLR9 and TLR9-deficient mice mirror the actions of chloroquine in all functional parameters that we tested. In addition, chloroquine decreased TLR9 protein abundance in spleen, further suggesting that TLR9 signaling may be a major target for the protective actions of chloroquine. Our findings indicate that chloroquine improves survival by inhibiting multiple pathways leading to polymicrobial sepsis and that chloroquine and TLR9 inhibitors represent viable broad-spectrum and targeted therapeutic strategies, respectively, that are promising candidates for further clinical development.

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Asselin-Paturel, Carine, Géraldine Brizard, Karine Chemin, Andre Boonstra, Anne O'Garra, Alain Vicari y Giorgio Trinchieri. "Type I interferon dependence of plasmacytoid dendritic cell activation and migration". Journal of Experimental Medicine 201, n.º 7 (28 de marzo de 2005): 1157–67. http://dx.doi.org/10.1084/jem.20041930.

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Differential expression of Toll-like receptor (TLR) by conventional dendritic cells (cDCs) and plasmacytoid DC (pDCs) has been suggested to influence the type of immune response induced by microbial pathogens. In this study we show that, in vivo, cDCs and pDCs are equally activated by TLR4, -7, and -9 ligands. Type I interferon (IFN) was important for pDC activation in vivo in response to all three TLR ligands, whereas cDCs required type I IFN signaling only for TLR9- and partially for TLR7-mediated activation. Although TLR ligands induced in situ migration of spleen cDC into the T cell area, spleen pDCs formed clusters in the marginal zone and in the outer T cell area 6 h after injection of TLR9 and TLR7 ligands, respectively. In vivo treatment with TLR9 ligands decreased pDC ability to migrate ex vivo in response to IFN-induced CXCR3 ligands and increased their response to CCR7 ligands. Unlike cDCs, the migration pattern of pDCs required type I IFN for induction of CXCR3 ligands and responsiveness to CCR7 ligands. These data demonstrate that mouse pDCs differ from cDCs in the in vivo response to TLR ligands, in terms of pattern and type I IFN requirement for activation and migration.

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Horton, Christopher G., Zi-jian Pan y A. Darise Farris. "Targeting Toll-Like Receptors for Treatment of SLE". Mediators of Inflammation 2010 (2010): 1–9. http://dx.doi.org/10.1155/2010/498980.

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Toll-like receptors (TLRs) are important innate immune receptors for the identification and clearance of invading pathogens. Twelve TLRs that recognize various conserved components of microorganisms are currently known. Among these, the endosomal TLRs 3, 7/8, and 9 recognize dsRNA, ssRNA, and CpG DNA, respectively. Nucleic acid-sensing TLRs, TLR 7 in particular, have been implicated in systemic lupus erythematosus (SLE) and are thought to exacerbate disease pathology. Activation of these TLRs results in the production of inflammatory cytokines and type I interferon. Genome-wide association studies, single nucleotide polymorphism analyses as well as experimental mouse models have provided evidence of TLR signaling involvement in SLE and other autoimmune diseases. Since activation of these receptor pathways promotes autoimmune phenotypes, inhibitory drugs that target these pathways constitute important new therapeutic strategies for the treatment of systemic autoimmunity.

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McClanahan, Fabienne, Federica Calore, Nicola Zanesi, John G. Gribben y Carlo M. Croce. "Aberrant PD-L1 Expression in CLL As a Result of Adaptive Immune Resistance Mediated By Tumor-Secreted Circulating miRNA Binding to Toll-like Receptor 7". Blood 124, n.º 21 (6 de diciembre de 2014): 716. http://dx.doi.org/10.1182/blood.v124.21.716.716.

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Abstract Background: Chronic lymphocytic leukaemia (CLL) is a model cancer to study immune evasion via PD-L1/PD-1 signalling: aberrantly expressed PD-L1 on CLL and PD-1 on CD8 T cells are key mediators of poor anti-tumor immune responses, which are a major hallmark of CLL. Several preclinical studies suggest that aberrant PD-L1 expression is a result of adaptive immune resistance and is induced during immune responses within the tumor microenvironment. It has recently been proposed that specific circulating microRNAs (miRNAs) shed by malignant cells participate in the complex crosstalk between cancer cells and microenvironment, and that they activate immune cells via Toll-Like Receptor (TLR) 7 due to their structural similarity to its natural ligand. In addition, TLR signaling has been demonstrated to result in the upregulation of costimulatory molecules such as CD80, CD86 and PD-L1/2. In CLL, aberrant circulating miRNA and TLR expression patterns have been well characterized. Therefore, we hypothesized that aberrant PD-L1 expression in CLL is a result of continuous TLR7 signaling mediated by circulating miRNAs. Our aims were to demonstrate that (1) specific circulating miRNAs induce PD-L1 expression and have functional consequences, (2) miRNA/PD-L1 associations are mediated via TLR7, and (3) miR/TLR/PD-L1 interactions are subject to the dynamics of CLL development. Methods and Materials: Mononuclear cells from spleen cell suspensions from 3 month old TCL1 transgenic, wild-type (WT) or TLR7-/- mice (total n=13) were treated ex vivo for 18hours with liposomal formulations of synthetic scrambled miRNA or miRNAs -16 (negative control), -21, -29, -150 and -155, which are reported to have an effect on immune cells and to be released by CLL cells. Specific TLR7 and TLR9 agonists were included for comparison. Primary human CLL cells and healthy B cells were treated with specific TLR2/6, TLR7 and TLR9 agonists. In adoptive transfer experiments, young WT mice (n=15) were injected with 4x107CLL cells from TCL1 transgenic mice. Mice were sacrificed at days 3, 6, 9, 12 and 15, and spleen cells were treated ex vivo as above. Changes in surface PD-L1, CD69 and CD86 expression on DAPI-negative CD19+ B cells/CLL cells were determined by flow cytometry. Supernatant cytokines were screened by multiplex ELISA. Results: PD-L1 surface expression on spleen B cells from both WT and non-leukemic TCL1 mice was strongly induced by miRs -21 and -29, and moderately by -150 and -155, but not by miR-16 negative control. The degree of PD-L1 upregulation by miRs -21 and -29 was comparable to the effect of direct TLR7 and TLR9 binding by specific agonists. Similar patterns were seen for CD69 and CD86 expression. Across treatment conditions, PD-L1 expression was highly correlated with the expression of CD69 (r .7777, p<.0001) and CD86 (r .7516, p<.0001). This observation strongly suggests that PD-L1 expression after TLR engagement is a marker of activation/costimulation, and therefore a physiological adaptive immune response to TLR binding in healthy B cells. Functionally, miR treatments resulted in increased IL-6, IL-10 and TNFα, with miR-29 having the strongest effect. PD-L1, CD69 and CD86 could also be induced by TLR engagement in healthy B cells, but not in CLL patient cells, where PD-L1 was confirmed to be already aberrantly expressed. To elucidate when in the course of CLL development PD-L1 expression becomes aberrant and if it ceases to be inducible by miR treatment, we sacrificed adoptively transferred mice every 3 days to simulate tumor development. With increasing CLL the magnitude of the fold-change of PD-L1 expression following miR treatment decreased substantially, and the baseline expression of miR-untreated B cells increased consistently until day 15. Interestingly, although the PD-L1 response was substantially decreased with tumor load, it was not completely abrogated, even on day 15 when mice had a median CLL load of 71%. Importantly, miR treatment did not result in increased PD-L1, CD69 or CD86 expression in B cells from TLR7-/- mice, indicating that the miR/PD-L1 interactions are indeed mediated by TLR7. Conclusions: Our findings support that PD-L1 expression on B cells can be induced by specific miRNAs known to be produced by CLL cells, and that this effect is mediated via TLR7. Therefore, aberrant PD-L1 expression on CLL is likely to be a result of adaptive immune resistance mediated by tumor cell-produced circulating miRNAs. Disclosures Gribben: Celgene: Research Funding; Pharmacyclics: Honoraria; Roche: Honoraria.

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Allacher, Peter, Christina K. Baumgartner, Aniko G. Pordes, Rafi U. Ahmad, Hans Peter Schwarz y Birgit M. Reipert. "Stimulation and inhibition of FVIII-specific memory B-cell responses by CpG-B (ODN 1826), a ligand for Toll-like receptor 9". Blood 117, n.º 1 (6 de enero de 2011): 259–67. http://dx.doi.org/10.1182/blood-2010-06-289009.

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Abstract Factor VIII (FVIII)–specific memory B cells are essential components for regulating anamnestic antibody responses against FVIII in hemophilia A with FVIII inhibitors. We asked how stimulation and inhibition of FVIII-specific memory B cells by low and high concentrations of FVIII, respectively, are affected by concurrent activation of the innate immune system. Using CD138− spleen cells from hemophilic mice treated with FVIII to study restimulation and differentiation of memory B cells in vitro, we tested modulating activities of agonists for Toll-like receptors (TLRs) 2, 3, 4, 5, 7, and 9. Ligands for TLR7 and 9 were most effective. They not only amplified FVIII-specific memory responses in the presence of stimulating concentrations of FVIII, but also countered inhibition in the presence of inhibitory concentrations of FVIII. Notably, CpG oligodeoxynucleotide (CpG-ODN), a ligand for TLR9, expressed biphasic effects. It amplified memory responses at low concentrations and inhibited memory responses at high concentrations, both in vitro and in vivo. Both stimulatory and inhibitory activities of CpG-ODN resulted from specific interactions with TLR9. Despite their strong immunomodulatory effects in the presence of FVIII, ligands for TLR induced negligible restimulation in the absence of FVIII in vitro and no restimulation in the absence of FVIII in vivo.

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Cao, Wei, Laura Bover, Minkwon Cho, Xiaoxia Wen, Shino Hanabuchi, Musheng Bao, David B. Rosen et al. "Regulation of TLR7/9 responses in plasmacytoid dendritic cells by BST2 and ILT7 receptor interaction". Journal of Experimental Medicine 206, n.º 7 (29 de junio de 2009): 1603–14. http://dx.doi.org/10.1084/jem.20090547.

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Plasmacytoid dendritic cells (pDCs) produce copious type I interferon (IFN) upon sensing nucleic acids through Toll-like receptor (TLR) 7 and TLR9. Uncontrolled pDC activation and IFN production are implicated in lymphopenia and autoimmune diseases; therefore, a mechanism controlling pDC IFN production is essential. Human pDCs specifically express an orphan receptor, immunoglobulin-like transcript 7 (ILT7). Here, we discovered an ILT7 ligand expressed by human cell lines and identified it as bone marrow stromal cell antigen 2 (BST2; CD317). BST2 directly binds to purified ILT7 protein, initiates signaling via the ILT7–FcεRIγ complex, and strongly inhibits production of IFN and proinflammatory cytokines by pDCs. Readily induced by IFN and other proinflammatory cytokines, BST2 may modulate the human pDC’s IFN responses through ILT7 in a negative feedback fashion.

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Pfaller, Christian K. y Karl-Klaus Conzelmann. "Measles Virus V Protein Is a Decoy Substrate for IκB Kinase α and Prevents Toll-Like Receptor 7/9-Mediated Interferon Induction". Journal of Virology 82, n.º 24 (15 de octubre de 2008): 12365–73. http://dx.doi.org/10.1128/jvi.01321-08.

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ABSTRACT The central role of plasmacytoid dendritic cells (pDC) in activating host immune responses stems from their high capacity to express alpha interferon (IFN-α) after stimulation of Toll-like receptors 7 and 9 (TLR7 and -9). This involves the adapter MyD88 and the kinases interleukin-1 receptor-associated kinase 1 (IRAK1), IRAK4, and IκB kinase α (IKKα), which activates IFN regulatory factor 7 (IRF7) and is independent of the canonical kinases TBK1 and IKKε. We have recently shown that the immunosuppressive measles virus (MV) abolishes TLR7/9/MyD88-dependent IFN induction in human pDC (Schlender et al., J. Virol. 79:5507-5515, 2005), but the molecular mechanisms remained elusive. Here, we have reconstituted the pathway in cell lines and identified IKKα and IRF7 as specific targets of the MV V protein (MV-V). Binding of MV-V to IKKα resulted in phosphorylation of V on the expense of IRF7 phosphorylation by IKKα in vitro and in living cells. This corroborates the role of IKKα as the kinase phosphorylating IRF7. MV-V in addition bound to IRF7 and to phosphomimetic IRF7 and inhibited IRF7 transcriptional activity. Binding to both IKKα and IRF7 required the 68-amino-acid unique C-terminal domain of V. Inhibition of TLR/MyD88-dependent IFN induction by MV-V is unique among paramyxovirus V proteins and should contribute to the unique immunosuppressive phenotype of measles. The mechanisms employed by MV-V inspire strategies to interfere with immunopathological TLR/MyD88 signaling.

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Liang, Xueqing, Ashish Kumar, Zhimei Wang, Brenda Weigel, Bruce R. Blazar y Wei Chen. "Toll-Like Receptor-7 Agonist Directly Inhibits the Growth and Induces Apoptosis of MLL-AF9 Leukemia Cells in Vitro and in Vivo". Blood 112, n.º 11 (16 de noviembre de 2008): 2989. http://dx.doi.org/10.1182/blood.v112.11.2989.2989.

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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.

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Heil, Florian, Parviz Ahmad-Nejad, Hiroaki Hemmi, Hubertus Hochrein, Franziska Ampenberger, Tanja Gellert, Harald Dietrich et al. "The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily". European Journal of Immunology 33, n.º 11 (noviembre de 2003): 2987–97. http://dx.doi.org/10.1002/eji.200324238.

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Temburni, Sonal, Jacqueline C. Barrientos, Jonathan E. Kolitz, Steven L. Allen, Kanti R. Rai, Betty Y. Chang, Nicholas Chiorazzi y Rajendra N. Damle. "Ibrutinib Inhibits Concomitant TLR and BCR- Driven Proliferation of Chronic Lymphocytic Leukemia Cells and Overrides the Supportive Survival-Promoting Effects of Microenvironmental Signals". Blood 124, n.º 21 (6 de diciembre de 2014): 3310. http://dx.doi.org/10.1182/blood.v124.21.3310.3310.

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Abstract Chronic lymphocytic leukemia (CLL) cells interact with external stimuli via several receptors, including the B cell receptor (BCR) and cytokine/chemokine receptors. CLL cells also express varying levels of functional Toll-like receptors (TLR), including TLR7 and TLR9. In vivo, the interaction of CLL cells via BCR or TLR occurs within a supportive microenvironment that provides them with survival and proliferative signals. CLL clonal turnover, occurring as an outcome of these and other interactions might lead to the release of naked nuclear material into the microenvironment and permit an interaction with these remnants of dying/dead cells. This could lead to repeated low level T cell-independent activation, possibly resulting in disease progression. To this effect, concomitant engagement of BCR and TLR leads to differential Erk phosphorylation and apoptosis rescue in CLL cases segregated by IgV gene mutational status. Here, we have studied the impact of stromal cells on TLR-mediated activation of negatively selected CLL cells from 24 untreated cases. Marked increases in percentages of CD69+ CLL cells and density of HLA-DR expression were observed within 18 hrs after exposure to TLR7 or TLR9 agonists (Imiquimod or ODN2006, respectively). However, although CLL cells activated via their TLRs in the presence of irradiated stromal cells (HS-5) did not show further changes in CD69 and HLA-DR expression, they exhibited significantly elevated levels (p<0.01) of anti-apoptotic molecules, Survivin and Mcl-1, compared to CLL cells activated in the absence of HS-5 cells. B cells from 16 CLL cases stimulated with Imiquimod or ODN2006 in the absence/presence of HS-5 cells were assayed for rescue from apoptosis using Annexin/PI (on day 1) and cell proliferation using 3H- thymidine incorporation (on day 3). Stromal cells alone induced ~12.8% (range: 8-31%) rescue from spontaneous CLL apoptosis in culture, whereas TLR7- and TLR9-mediated activation induced ~12.2% (range: 9-44%) and ~20.1% (range: 7-41%) rescue from cell death, respectively. TLR7 and TLR9 signaling upregulated proliferation of CLL cells by 1.83 fold (range: 1.3-11.6) and 9.9 fold (range: 1.3-43.2), respectively. Co-culture with stromal cells further enhanced cell proliferation by an average of 3.54 fold (range: 1.4-7.8) for TLR7 or 2.45 fold (range: 1.1-5.6) for TLR9. Ibrutinib covalently binds and inhibits Bruton tyrosine kinase, BTK, a molecule critical for BCR signaling. In addition, ibrutinib inhibits cell signaling occurring when CXCR4 binds its ligand, stromal derived factor-1/CXCL12. However, the effects of ibrutinib on concomitant TLR+ BCR-mediated signaling in CLL have not yet been reported. We evaluated the impact of ibrutinib over a dose range of 0.1 - 25mg/ml on TLR-mediated CLL cell apoptosis and proliferation in presence/absence of stromal help, as mentioned above. Pre-incubation of CLL cells with ibrutinib for 2 hrs significantly lowered TLR-mediated induction of Survivin and Mcl-1 within 36 hrs of culture. Exposure to ibrutinib also inhibited 3H-thymidine incorporation in non-stimulated (average: 22%; range: 7-43%) and TLR7- (average: 21%; range: 7-49%) or TLR9- (average: 43%; range: 11-98%) activated CLL cells in 14/14 cases. Ibrutinib treatment resulted in an increase in TLR7-induced apoptosis in 3 of 4 CLL cases and in TLR9-induced apoptosis in 2 of 4 CLL cases. Even when CLL cells from 6 patients were cocultured with stromal cells, ibrutinib reduced cell proliferation to 45-65% of that observed in untreated cells induced via TLR7 or TLR9 alone or when combined with an anti-BCR signal. These findings reiterate the pivotal role of the microenvironment in nurturing CLL cells activated via several types of receptors. Ibrutinib, known to be efficacious in inhibiting BCR signaling, can also abrogate events downstream of TLR7 and TLR9 signaling in CLL. Importantly, inhibition of TLR 7 and 9 signaling by ibrutinib can override the protective effects from stroma-derived signals. These anti-proliferative effects of ibrutinib may be especially relevant and important for curbing concomitant activation via the BCR, TLR, and chemokine receptor pathways that likely synergize and thereby contribute to the aggressiveness of the disease. Disclosures Barrientos: Pharmacyclics, Inc: Membership on an entity's Board of Directors or advisory committees, Research Funding. Rai:Pharmacyclics, Inc: Membership on an entity's Board of Directors or advisory committees. Chang:Pharmacyclics, Inc: Employment, Equity Ownership. Chiorazzi:Pharmacyclics, Inc: Research Funding.

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Babu, Subash, R. Anuradha, N. Pavan Kumar, P. Jovvian George, V. Kumaraswami y Thomas B. Nutman. "Filarial Lymphatic Pathology Reflects Augmented Toll-Like Receptor-Mediated, Mitogen-Activated Protein Kinase-Mediated Proinflammatory Cytokine Production". Infection and Immunity 79, n.º 11 (29 de agosto de 2011): 4600–4608. http://dx.doi.org/10.1128/iai.05419-11.

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ABSTRACTLymphatic filariasis can be associated with the development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Toll-like receptors (TLRs) are thought to play a major role in the development of filarial pathology. To elucidate the role of TLRs in the development of lymphatic pathology, we examined cytokine responses to different Toll ligands in patients with chronic lymphatic pathology (CP), infected patients with subclinical pathology (INF), and uninfected, endemic-normal (EN) individuals. TLR2, -7, and -9 ligands induced significantly elevated production of Th1 and other proinflammatory cytokines in CP patients in comparison to both INF and EN patients. TLR adaptor expression was not significantly different among the groups; however, both TLR2 and TLR9 ligands induced significantly higher levels of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein (MAP) kinases (MAPK) as well as increased activation of NF-κB in CP individuals. Pharmacologic inhibition of both ERK1/2 and p38 MAP kinase pathways resulted in significantly diminished production of proinflammatory cytokines in CP individuals. Our data, therefore, strongly suggest an important role for TLR2- and TLR9-mediated proinflammatory cytokine induction and activation of both the MAPK and NF-κB pathways in the development of pathology in human lymphatic filariasis.

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Suprun, E. N., E. B. Nagovitsyna, N. I. Kuderova, S. V. Suprun y O. A. Lebed’ko. "Some groups of Toll-like receptor gene polymorphisms and their clinical and pathogenetic manifestations in children with bronchial asthma". Medical Immunology (Russia) 22, n.º 5 (1 de diciembre de 2020): 915–24. http://dx.doi.org/10.15789/1563-0625-sgo-2049.

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The prevalence of bronchial asthma has shown its steady increase in the world in recent years. Despite all the achievements of Allergology, control of the disease can be achieved only in two-thirds of patients even if all social risk factors and the influence of concomitant diseases are excluded. Thus, it is necessary to study endogenous factors that modify the pathogenesis of the disease. Toll-like receptors are the main molecules for recognizing pathogenic patterns in the human immune system. Since any Allergy is a recognition error, mutation of the genes of the recognizing molecules can have a direct and multidirectional effect on the nature of the inflammation and its clinical manifestations in bronchial asthma (BA). To detect this effect, 65 patients with BA were examined, and mutations of Toll-like receptor genes were detected: TLR2-Arg753Glu, TLR4- Asp299Gly, TLR4-Ghr399Ile, TLR9-T1237C, TLR9-A2848G, lymphocyte subpopulations CD3, CD19, CD4, CD8, CD16, phagocytosis indicators, levels of IgA, IgM, IgG, IgE and IL-6, IL-7, IL-9. The assessment of the severity of asthma and its level of control were conducted according to clinical recommendations of the Ministry of health of the Russian Federation in 2019 criteria. We have shown characteristic clinical manifestations of the studied mutations. A lighter course of the disease, more complete control over it and a better response to therapy were found in single-nucleotide substitutions in the Toll-like receptor 4 and 9 (TLR4-Asp299Gly, TLR4-Ghr399Ile, TLR9-T1237C, TLR9-A2848G). On the contrary, a heavier course and a worse response to therapy were detected in the TLR2 mutation with Arg753Glu replacement. In the studied groups, the features of immunity indicators characteristic of genotypes with a lighter and more controlled course of BA were determined: a higher absolute number of T-helpers, with multidirectional changes in the number of T-killers, but with invariably preserved higher ratio of CD4/CD8 in such genotypes. Higher levels of phagocytosis indicators (primarily characterizing chemotaxis) and IL-7, IL-9 were also detected. The exception is the TLR9-A2848G mutation, in which greater disease control and better response to therapy are combined with no changes in the studied laboratory characteristics. At the same time, a specific feature of the genotype of the studied patients with BA was revealed – a combination of Toll-like receptors 4 and 9 mutations. This suggests the presence of genetic patterns that characterize groups of patients with BA that differ in severity, response to therapy, and degree of control, which makes it possible to personalize approaches to diagnosis, prevention, and therapy of the disease.

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Tan, Ying, Amanda A. Watkins, Ian R. Rifkin y Adam Lerner. "PDE4 Inhibitors Block TLR7 and TLR9-Driven Signaling, Proliferation and Cytokine Secretion in CLL Cells As Well As Proliferation Driven by Exposure to Apoptotic Cells". Blood 120, n.º 21 (16 de noviembre de 2012): 1774. http://dx.doi.org/10.1182/blood.v120.21.1774.1774.

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Abstract Abstract 1774 A growing body of literature supports the concept that at least a subset of chronic lymphocytic leukemia (CLL) B cell-receptors (BCRs) are reactive with antigens exposed on apoptotic cells. Work done in the autoimmunity field suggests that through their BCRs, autoreactive B cells can internalize RNA and DNA-containing autoantigens, thereby activating endosomal Toll-like receptor (TLR) 7 and TLR9, respectively, with subsequent TLR-mediated signaling, proliferation and cytokine secretion. cAMP signaling modulates inflammatory signaling in immune system cells and our prior studies have shown that PKA, EPAC and CREB-mediated cAMP signaling can be potently induced in CLL cells with inhibitors of the cyclic nucleotide phosphodiesterase (PDE) PDE4 family in the absence of additional exogenous stimulators of adenylate cyclase. We therefore examined the ability of PDE4 inhibitor-mediated cAMP signaling to regulate TLR7 and 9-mediated signaling in CLL cells. Treatment of CLL cells with the TLR7 and 9 agonists R848 and CpG-B oligonucleotide, respectively, induced IRAK1 degradation and activation of three signaling pathways known to be downstream of TLR7 and 9: the transcription factors NFkB and IRF5 as well as the MAP kinase JNK. The prototypic PDE4 inhibitor rolipram inhibited R848-induced TLR7 and CpG-B-induced TLR9 signaling in CLL cells as judged by Western analysis and immunofluorescent studies of NFkB and IRF5 nuclear translocation as well as JNK phosphorylation. Rolipram also blocked R848 and CpG-B-induced CLL proliferation and TNF secretion. In contrast, rolipram augmented R848 and CpG-B-induced CLL IL-10 synthesis, in keeping with prior studies documenting cAMP response elements in the IL-10 promoter. To test whether RNA and/or DNA antigens exposed in apoptotic cells might induce proliferation in CLL cells through BCR-mediated internalization and subsequent activation of TLR7 and TLR9, we irradiated CLL cells and added them in a graded fashion to live autologous CLL cells. Such exposure to irradiated cells undergoing apoptosis drove CLL proliferation in a manner that was sensitive to TLR7 and TLR9 antagonists, IRAK4 kinase inhibitors and the PDE4 inhibitor rolipram. Our work supports the concept that BCR and TLR-driven responses to apoptotic RNA and DNA-containing complexes may drive proliferation in CLL cells and that PDE4 inhibitors and other agents that target TLR signaling may prove to be effective therapeutic agents in this disease. Disclosures: No relevant conflicts of interest to declare.

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Moreno-Eutimio, Mario Adan, Raul Aragon-Franco, Constantino Lopez-Macias, Alejandro J. Silva y Horacio Astudillo. "Toll-like receptor 7 and 9 (TLR 7 and TLR 9) expression in biopsies of in situ cervical cancer." Journal of Clinical Oncology 30, n.º 15_suppl (20 de mayo de 2012): e15584-e15584. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e15584.

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e15584 Background: In order to analyze the expression of TLR 7 and TLR 9 in cervix biopsies, we evaluated the expression of these TLRs in cervical carcinoma in situ tissue and peritumoral inflammatory tissue biopsies by immunohistochemistry. Methods: We reviewed 276 uterine cervix biopsies of Clinical Specialties Women's Health Directorate of the Ministry of National Defense (between 2009-2010); the biopsies have the following histopathological diagnosis: 124 CIN I, 68 CIN II, 34 CIN III, 21 invasive cancer and 29 cancer in situ. Results: It was observed that peritumoral inflammatory tissue receptor was expressed positively in 89.6% in cervical cancer tissue in situ, the receptor was expressed positively in 68.9% and the negative expression was 31.1%, proving that in the peritumoral inflammatory tissue compared with in situ cervical cancer was increased expression of TLR 7. In the case of the expression of TLR 9, peritumoral inflammatory tissue, expressed positively in 24 samples and 5 samples were negative expression in the tissue in situ cervical cancer samples identified 20 positive and 9 negative samples. By analyzing the intensity of the expression of TLR 7 in cervical cancer tissue in situ, we found 16 samples with focal positive (+), 3 samples with diffuse positive (+ +) and 1 sample with positive (+ + + ), while in peritumoral inflammatory tissue found 13 samples with focal positive (+), 10 samples with diffuse positive (+ +) and 3 samples with positive results (+++). By analyzing the intensity of the expression of TLR 9 in cervical cancer tissue in situ, we found 14 samples with focal positive (+), 5 samples with diffuse positive (+ +) and 1 sample with positive (+), in contrast to the peritumoral inflammatory tissue was found 7 samples with focal positive (+), 13 samples tested positive diffuse (+ +) and 4 samples with positive results (+++). Conclusions: These results show that no significant difference between the expression of TLR 7 () and TLR 9 () in the peritumoral inflammatory tissue and tissue in situ cervical cancer found a decrease in the expression of TLR 7 and TLR 9 in tissue in situ cervical cancer compared with the expression in the peritumoral tissue inflammation.

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Marklein, B., Z. Konthur, T. Haupl, A. Cope, M. Shlomchik, G. Steiner, G. Burmester y K. Skriner. "Toll-like receptor (TLR-7/9), MYOD88, TIR8, dependent and independent autoantigens". Annals of the Rheumatic Diseases 69, Suppl 2 (1 de marzo de 2010): A39. http://dx.doi.org/10.1136/ard.2010.129627j.

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Hayashi, Fumitaka, Terry K. Means y Andrew D. Luster. "Toll-like receptors stimulate human neutrophil function". Blood 102, n.º 7 (1 de octubre de 2003): 2660–69. http://dx.doi.org/10.1182/blood-2003-04-1078.

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Abstract The first immune cell to arrive at the site of infection is the neutrophil. Upon arrival, neutrophils quickly initiate microbicidal functions, including the production of antimicrobial products and proinflammatory cytokines that serve to contain infection. This allows the acquired immune system enough time to generate sterilizing immunity and memory. Neutrophils detect the presence of a pathogen through germ line-encoded receptors that recognize microbe-associated molecular patterns. In vertebrates, the best characterized of these receptors are Toll-like receptors (TLRs). We have determined the expression and function of TLRs in freshly isolated human neutrophils. Neutrophils expressed TLR1, 2, 4, 5, 6, 7, 8, 9, and 10—all the TLRs except TLR3. Granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment increased TLR2 and TLR9 expression levels. The agonists of all TLRs expressed in neutrophils triggered or primed cytokine release, superoxide generation, and L-selectin shedding, while inhibiting chemotaxis to interleukin-8 (IL-8) and increasing phagocytosis of opsonized latex beads. The response to the TLR9 agonist nonmethylated CpG-motif-containing DNA (CpG DNA) required GM-CSF pretreatment, which also enhanced the response to the other TLR agonists. Finally, using quantitative polymerase chain reaction (QPCR), we demonstrate a chemokine expression profile that suggests that TLR-stimulated neutrophils recruit innate, but not acquired, immune cells to sites of infection. (Blood. 2003;102:2660-2669)

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Dlugosz, Aldona, Katherina Zakikhany, Nathalie Acevedo, Mauro D’Amato y Greger Lindberg. "Increased Expression of Toll-Like Receptors 4, 5, and 9 in Small Bowel Mucosa from Patients with Irritable Bowel Syndrome". BioMed Research International 2017 (2017): 1–7. http://dx.doi.org/10.1155/2017/9624702.

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The aim of our study was to compare patients with irritable bowel syndrome (IBS) and healthy controls regarding the expression of toll-like receptors 2, 4, 5, and 9 (TLR2, TLR4, TLR5, and TLR9), the primary mucosal receptors of bacterial components, in small and large bowel mucosa.Methods.We analysed biopsies from jejunum and sigmoid colon of 22 patients (17 females) with IBS aged 18–66 (median: 39) years and 14 healthy volunteers (12 females) aged 22–61 (median: 42) years. Eight patients had constipation-predominant IBS (C-IBS), 7 had diarrhoea-predominant IBS (D-IBS), and 7 had IBS without predominance of constipation or diarrhoea. We analysed mRNA levels for TLRs using quantitative PCR and distribution of TLRs in mucosa using immunohistochemistry.Results.We found increased mRNA expression of TLR4 (mean fold change1.85±0.31versus1.0±0.20;p<0.05), TLR5 (1.96±0.36versus1.0±0.20;p<0.05) and TLR9 (2.00±0.24versus1.0±0.25;p<0.01) but not of TLR2 in the small bowel mucosa from patients with IBS compared to the controls. There was no significant difference in mRNA levels for TLRs in colon mucosa between patients and controls.Conclusion.Upregulation of TLR4, TLR5, and TLR9 suggests the involvement of bacteria or dysregulation of the immune response to commensal flora in small bowel mucosa in IBS patients.

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Liao, Guodong, Jia Lv, Alin Ji, Shuai Meng y Chao Chen. "TLR3 Serves as a Prognostic Biomarker and Associates with Immune Infiltration in the Renal Clear Cell Carcinoma Microenvironment". Journal of Oncology 2021 (6 de septiembre de 2021): 1–20. http://dx.doi.org/10.1155/2021/3336770.

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Background. Clear cell renal cancer (KIRC) is one of the most common cancers globally, with a poor prognosis. TLRs play a vital role in anticancer immunity and the regulation of the biological progress of tumour cells. However, the precise role of TLRs in KIRC is still ambiguous. Methods. Various bioinformatics analysis and clinical validation of tissues were performed to evaluate the prognostic value of TLRs and their correlation with immune infiltration in KIRC. Results. The expression of TLR2/3/7/8 was increased at both mRNA and protein levels in KIRC. TLRs in KIRC were involved in the activation of apoptosis, EMT, RAS/MAPK, and RTK pathways, as well as the inhibition of the cell cycle and the hormone AR pathway. Drug sensitivity analysis revealed that high expression of TLR3 and low expression of TLR7/9/10 were resistant to most of the small molecules or drugs from CTRP. Enrichment analyses showed that TLRs were mainly involved in innate immune response, toll-like receptor signalling pathway, NF-kappa B signalling pathway, and TNF signalling pathway. Furthermore, a high-level TLR3 expression was associated with a favourable prognosis in KIRC. Validation research further confirmed that TLR3 expression was increased in KIRC tissues, and high TLR3 levels were associated with poor overall survival. Moreover, TLR3 in KIRC showed a positive association with an abundance of immune cells, including B-cells, CD4+ T-cells, CD8+ T-cells, macrophage, neutrophils, and dendritic cells, and the expression of the immune biomarker sets. Several TLR3-associated kinase, miRNA, or transcription factor targets were also identified in KIRC. Conclusion. Our results indicate that TLR3 serves as a prognostic biomarker and associated with immune infiltration in KIRC. This work lays a foundation for further studies on the role of TLR3 in the carcinogenesis and progression of KIRC.

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Branchi, Vittorio, Laura Esser, Corinna Boden, Azin Jafari, Jonas Henn, Philipp Lingohr, Maria A. Gonzalez-Carmona et al. "A Combined TLR7/TLR9/GATA3 Score Can Predict Prognosis in Biliary Tract Cancer". Diagnostics 11, n.º 9 (1 de septiembre de 2021): 1597. http://dx.doi.org/10.3390/diagnostics11091597.

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Biliary tract cancer (BTC) refers to a heterogenous group of epithelial malignancies arising along the biliary tree. The highly aggressive nature combined with its silent presentation contribute to the dismal prognosis of this tumor. Tumor-infiltrating immune cells (TIICs) are frequently present in BTC and there is growing evidence regarding their role as therapeutic targets. In this study, we analyzed the immune cell infiltration in BTC and developed a promising immune signature score to predict prognosis in BTC. Immunohistochemistry (IHC) was carried out on tissue microarray sections from 45 patients with resectable cholangiocarcinoma for the detection of 6-sulfoLacNAc+ monocytes (slanMo), BDCA-2+ plasmacytoid dendritic cells (pDC), CD8+ or CD4+T-lymphocytes, CD103+ cells, GATA3+ cells, Toll-like receptor (TLR) 3, 7 and 9-expressing cells as well as programmed cell death protein 1 and programmed cell death ligand 1 positive cells. Data from the IHC staining were analyzed and correlated with clinicopathological and survival data. High expression of TLR7, TLR9, and GATA3 was associated with improved overall survival (OS, Log-rank p < 0.05). In addition, TLR9 was associated with better disease-free survival (Log-rank p < 0.05). In the multivariate Cox proportional-hazards model for OS, the TLR/TLR9/GATA3 score was found to be an independent prognostic factor for OS (“Score 2” vs. “Score 0”: HR 11.17 95% CI 2.27–54.95, p < 0.01).

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Mussari, Christopher P., Dharmpal S. Dodd, Ratna Kumar Sreekantha, Laxman Pasunoori, Honghe Wan, Shana L. Posy, David Critton et al. "Discovery of Potent and Orally Bioavailable Small Molecule Antagonists of Toll-like Receptors 7/8/9 (TLR7/8/9)". ACS Medicinal Chemistry Letters 11, n.º 9 (29 de julio de 2020): 1751–58. http://dx.doi.org/10.1021/acsmedchemlett.0c00264.

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Deering, Raquel P. y Jordan S. Orange. "Development of a Clinical Assay To Evaluate Toll-Like Receptor Function". Clinical and Vaccine Immunology 13, n.º 1 (enero de 2006): 68–76. http://dx.doi.org/10.1128/cvi.13.1.68-76.2006.

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ABSTRACT Toll-like receptors (TLRS) recognize pathogen-associated molecular patterns to enable innate immune responses. A number of genetic defects influencing the function of these receptors have been identified and are associated with recurrent and/or severe infection. Our goal was to develop a reproducible assay of TLR function in order to evaluate patients with recurrent infection who would be suspected of having a genetic defect affecting TLR signaling. We chose to study peripheral blood mononuclear cells (PBMCS) to avoid potential influences of soluble factors contained in whole blood, and we utilized ligands for TLRS 1/2, 2/6, 3, 4, 5, 6, 7, and 9. Tumor necrosis factor (TNF) production in PBMC supernatants was measured by an enzyme-linked immunosorbent assay after TLR ligand stimulation and was dependent on gene transcription and NF-κB activation. Some variables affecting the assay were assessed, including the effects of: blood anticoagulant, serum-containing media, incubation time, ligand storage, blood storage time, and cell cryopreservation. By using optimized assay conditions, effective concentrations of individual ligands and mean responses to those ligands were established for healthy control donors. Finally, three patients with a mutation in the IKBKG gene, encoding the NF-κB essential modulator (NEMO) protein, were evaluated as disease controls and were almost uniformly below the standard deviation of healthy donors for all ligands tested. Although a number of variables influence TLR ligand-induced TNF responses, this assay can be optimized for potential clinical use to screen patients with primary immunodeficiencies affecting TLR function.

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Taylor, Tammi, Young-June Kim, Wilbert Derbigny, Xuan Ou y Hal E. Broxmeyer. "Mouse Embryonic Stem Cells Express Functional Toll Like Receptor 2." Blood 114, n.º 22 (20 de noviembre de 2009): 1473. http://dx.doi.org/10.1182/blood.v114.22.1473.1473.

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Abstract Abstract 1473 Poster Board I-496 Mouse embryonic stem cells (mESCs) are unique in that they give rise to every cell type of the body. Little is known about stimuli that promote mESC differentiation and proliferation. We hypothesized that TLRs are expressed and functional, and when activated by its ligand will influence survival and proliferation of mESCs in the presence of Leukemia Inhibitory Factor (LIF). This study evaluated three mESC lines, R1, CGR8, and E14 to first determine if they express Toll Like Receptors (TLRs) at mRNA and protein levels. Next, we evaluated if the TLR ligands would induce or modulate mESC proliferation and survival of the mESCs in the presence of LIF. We then assessed the E14 mESC line to determine if the mESCs expressed MyD88 an adaptor protein molecule, known to be involved in the TLR pathway and if the TLR ligands would cause inhibitor of kinase kinase alpha/ beta (IKKα/β) phosphorylation and nuclear factor-kappa beta (NF-κβ) nuclear translocation, and cytokine production. In this study we found expression of TLRs 1, 2, 3, 5 and 6 at the mRNA level, but no mRNA expression of TLRs 4, 7, 8 and 9. We also confirmed some of these results by flow analysis. Toll Like Receptor 2 (TLR-2), but not Toll Like Receptor 4 (TLR-4), is expressed on the three mESC lines. Therefore we focused our studies on TLR-2, specifically. Pam3Cys, a synthetic triacyl-lipoprotein and a TLR-2 ligand induced a significant increase in mESC numbers on Day 3 compared to controls and also at Day 4 and Day 5 when compared to controls. Pam3Cys (10ug/ml) also enhanced the survival of mESC colony forming cells subjected to serum withdrawal and then delayed addition of serum. Next we found that E14 mESCs express molecules involved in the TLR Pathway. MyD88 was expressed in mESCs and IKKα/β phosphorylation was enhanced after 15 minute by TLR-2 ligand activation. We found a significant increase of NF-κβ nuclear translocation upon activation by Pam3Cys after 30 minutes which continued for up to an hour. Densitometry analysis of the nuclear extracts from three separate experiments shows a significant 2 fold increase in NF-κβ in the nucleus compared to control mESC nuclear extracts. Since TLR activation of leukocytes enhances cytokine production, and our group has published that mESCs produce cytokines, we studied the effect of Pam3Cys on release of cytokine from our mESC line. Cytokine release in mESCs by TLR-ligand activation was assessed by ELISA, but TLR-2 ligand stimulation did not affect cytokine release of IL-6, TNF-α, or INF-β. We found that there were no significant changes in expression of mESCs markers Oct4, KLF-4, Sox 2, and SSEA1 when compared to cells not activated by Pam3Cys. Thus the mESCs remained in a pluripotent state in the presence of LIF after activation with the TLR-2 ligand. These results demonstrate that mESCs can respond to microbial products, and TLR-2 activation enhances proliferation and survival of the mESCs. This finding expands the role of TLRs and has implications for a better understanding of the responsiveness of embryonic stem cells to certain microbial agents. Disclosures: No relevant conflicts of interest to declare.

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Hicks, Lisa, Y. Shi, J. Mena, C. Hammond, Y. Tomic, M. A. Tomai, R. L. Miller y D. E. Spaner. "The Effect of a Toll Receptor-7 Agonist (S28690) on the Immunogenicity of Chronic Lymphocytic Leukemia Cells." Blood 104, n.º 11 (16 de noviembre de 2004): 3481. http://dx.doi.org/10.1182/blood.v104.11.3481.3481.

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Abstract Background: Chronic lymphocytic leukemia (CLL) cells are weakly immunogenic, a property that may contribute to disease progression and inhibit the effectiveness of immunotherapies such as vaccines. Low surface expression of co-stimulatory molecules contributes to this poor immunogenicity. CLL cells express Toll-Like-Receptor-7 (TLR7), a powerful modulator of innate immunity. TLR7 agonists may be capable of enhancing the immunogenicity of CLL cells and thereby increasing T cell mediated killing of CLL. Methods: Circulating CLL cells were isolated directly from consenting patients by negative selection. TLR7 mRNA expression by CLL cells was demonstrated by RT-PCR. CLL cells were then incubated with S28690 (a TLR7 agonist), or with a negative control for 24–72h. Expression of the costimulatory molecules CD80, 83, 86, and 54 was determined by flow cytometry pre and post-incubation. Experiments were repeated in the presence of a NFkB inhibitor (dexamethasone), a p38 MAPK inhibitor, and a protein kinase C agonist (PDB). The effects of S28690 on phosphorylated-IkB and phosphorylated-STAT3 levels were measured by immunoblotting. The capacity of S28690-incubated CLL cells to stimulate T cell proliferation and killing was determined in mixed lymphocyte responses. Results: All tested CLL samples (n=20) expressed TLR7 mRNA, while Jurkat cells (T cell origin) did not. After incubation with S28690, CD80, 83, 86, and 54 surface expression increased on all CLL samples tested. The relative increase varied from 4 to 9-fold and was positively correlated with CD38 expression. NF-kB and p38 inhibitors decreased the effects of S28690 on co-stimulatory molecule expression while PDB amplified the effect. After incubation with S28690, IkB and STAT3 phosphorylation increased in CLL cells. S28690-incubated-CLL cells were able to stimulate moderate T cell proliferation, but did not increase T cell mediated killing of CLL cells. However, CLL cells incubated with both S28690 and PDB (a PKC agonist), exhibited much lower amounts of phosphorylated STAT3, triggered marked T cell proliferation, and stimulated T cell mediated killing of CLL cells. Conclusions: S28690 (a TLR7 agonist) causes increased expression of co-stimulatory molecules by CLL cells in vitro and transforms CLL cells into moderate stimulators of T cell proliferation. The effects of S28690 are synergistic with PKC agonists, potentially as a result of S28690-mediated NFkB activation and concurrent PKC-mediated inhibition of STAT3. These findings may find clinical application in immunotherapeutic approaches to CLL.

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