Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling

doi:10.1016/S0006-291X(02)00380-7

Biochemical and Biophysical Research Communications

Volume 293, Issue 5, 24 May 2002, Pages 1364-1369

https://doi.org/10.1016/S0006-291X(02)00380-7 Get rights and content

Abstract

A monoclonal antibody (mAb) against human Toll-like receptor (TLR) 3 was established and its effect on TLR3-mediated responses was tested using human fibroblast cell lines expressing TLR3 on the cell surface. Fibroblasts are known to produce IFN-β upon viral infection or treatment with double-stranded RNA (dsRNA) through distinct signaling pathways. Here, we show the mAb to TLR3 suppressed poly(I):poly(C)-mediated IFN-β production by human fibroblasts naturally expressing TLR3 on their surface. By reporter gene assay using HEK293 cells transfected with a human TLR3 expression vector, TLR3 recognized dsRNA to activate NF-κB and the IFN-β promoter. TLR3 signaling was not elicited by either single-stranded RNA (ssRNA) or dsDNA. Thus, specific recognition of dsRNA by extracellular TLR3 is essential for induction of type I IFN: the interassociation between dsRNA and TLR3, regardless of direct or indirect binding, should be disrupted by mAb being attached to TLR3. The mAb against TLR3 reported herein may serve as a regulator for virus-mediated immune response via an alternative pathway involving the dsRNA–TLR3 recognition which might occur on host cells.

Section snippets

Materials and methods

Cell culture and reagents. The normal human lung fibroblast MRC-5 was obtained from Riken Cell Bank (Tsukuba, Japan) and maintained in MEM supplemented with 10% heat-inactivated FCS (JRH Biosciences) and antibiotics. The human foreskin FS-4 fibroblasts [11] and human embryonic kidney (HEK) 293 cells were maintained in DMEM supplemented with 10% FCS and antibiotics. The IL-3 dependent murine cell line Ba/F3 was cultured in RPMI containing 10% FCS, 5 ng/ml murine IL-3, 100 μM 2-ME, and antibiotics.

Results and discussion

Two criteria were employed to identify mAbs recognizing human TLR3 in the supernatants of hybridomas. First, the mAbs were screened by flow cytometry that allowed the shift of the peak of fluorescence by reacting with TLR3-expressing BaF3 cells but not other TLR-expressing cells (Fig. 1A). Second, these mAbs were further selected by immunoprecipitation using lysate of BaF3 cells expressing Flag-tagged TLR3, the expression of which was judged by anti-Flag antibody (Fig. 1B). After a number of

Acknowledgements

We thank Drs. K. Toyoshima and H. Koyama for support of this work; Mr. N. Inoue for cloning human TLR3, Drs. N. Inoue, N.A. Begum, H. Oshiumi (Osaka Medical Center), T. Fujita (The Tokyo Metropolitan Institute of Medical Science), A. Yamamoto (Kansai Medical University) for providing reagents and helpful discussions. We also thank Dr. T. Taniguchi (University of Tokyo) for providing the p-125 luc reporter plasmid, Dr. S. Akashi (Institute of Medical Science, University of Tokyo) for anti-TLR4

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The existence of extracellular miRNAs (ex-miRNAs) was first described in plasma and soon demonstrated in virtually all biological fluids. Ex-miRNAs are remarkably stable in harsh conditions, including high nuclease concentrations and extreme pH variations. This largely depends on their association with proteins protecting them from degradation, such as those of the AGO family, and/or on their loading into extracellular vesicles (EV-miRNAs) and apoptotic bodies. Ex-miRNAs can be released upon cell damage or death, so possibly representing remnants devoid of any particular function. However, the fact that cells can also operate specific sorting of secreted miRNAs, suggested that ex-miRNAs may work as soluble messengers regulating cell-to-cell communications. Indeed, ample experimental evidence demonstrates that EV-miRNAs penetrate into target cells, where they exert posttranscriptional regulation of specific target messenger RNAs. In addition, we and others have proposed another regulatory function of EV-miRNAs, namely, the triggering of innate immune receptors. In this scenario, ex-miRNAs would regulate the responses of neighboring cells by inducing cytokine secretion which, in turn, would trigger “sterile” inflammation and immune activation.
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In addition, the chapter will also provide a brief outline of noncanonical subcellular locations and of other unconventional functions attributed to miRNAs, both suggesting that we have only begun to touch upon the complexity and versatility of the regulatory functions exerted by these short noncoding RNAs.
Genome-wide identification, characterization, and expression of the Toll-like receptors in Japanese flounder (Paralichthys olivaceus)
2021, Aquaculture
Toll-like receptor (TLR) family is a key ancient component of pattern recognition receptors, conserved from insects to mammals, which plays significant roles in immune defense against pathogens invasion. Teleost-specific TLR family members have lost some of the mammalian TLR orthologues or produced duplicates, particularly all bacterial could recognize cell surface TLRs. Edwardsiella tarda is an important pathogen of Japanese flounder (Paralichthys olivaceus) resulting in great economic losses. However, there is little integrated information of the roles about TLRs in mediating immune response to E.tarda infection in Japanese flounder. Here, we performed data-mining on the P. olivaceus genome to extract eleven TLR members: TLR1, TLR2, TLR3, TLR5M, TLR5S, TLR7, TLR8, TLR9, TLR18, TLR21 and TLR22. The TLR family motifs, such as Toll/interleukin (IL)-1 receptor (TIR) domain and leucine rich repeat (LRR) are conserved in TLR1-3, 5M, 7-9, 18, 21 and 22. Phylogenetic analysis verified their identities and supported the classification of TLRs into six subfamilies as in other vertebrates. And then, the expression of P. olivaceus TLRs (PoTLRs) were widely detected in immune-related tissues including intestine, skin, spleen, kidney, liver, and gill. Subsequently, the PoTLRs were exhibited differential regulation in immune-related tissues, such as intestine, spleen, liver and kidney following bacterial challenge by infection with E.tarda, indicating their roles in the immune response. The syntenic analysis showed neighboring genes of TLR1, 7, 8 and 18 were relatively conserved among P.olivaceus, O.latipes, and C.semilaevis. Meanwhile, protein and protein interaction network (PPI) results explained that TLR genes interacted with immune-related mediating factors including Interleukin-related genes, e.g. Il1 and Il10, MyD88-dependent pathway, Sarm1 and NF-κB signaling pathway, which could bind to TIR domain. These analyses will contribute to the functional characterization of P. olivaceus TLRs in further study.
Recent advances in immunotherapy, immunoadjuvant, and nanomaterial-based combination immunotherapy
2021, Coordination Chemistry Reviews
Cancer immunotherapy has emerged as the fourth major treatment modality for cancer in addition to chemotherapy, radiotherapy, and surgery. There are various types of cancer immunotherapies that can be generally divided into five categories, namely vaccine immunotherapy, cytokine immunotherapy, checkpoint blockade immunotherapy, adoptive cell transfer immunotherapy, and small molecule immunotherapy. In this review, we describe these therapeutic approaches along with their limitations. Since many immunotherapies rely on immunoadjuvants and nanoplatforms, we summarize different classes of immunoadjuvants used in cancer immunotherapies, including toll-like receptor agonists, plant-derived immunoadjuvants, aluminum salt adjuvants, and other immunoadjuvants. Finally, we provide an in-depth description of nanomaterial-based approaches for the combination of immunotherapy with chemotherapy, phototherapy, radiotherapy, as well as other therapies.
Innate and adaptive immunity in cancer
2021, Cancer Immunology and Immunotherapy: Volume 1 of Delivery Strategies and Engineering Technologies in Cancer Immunotherapy
Tumor heterogeneity can be attributed to the varying growth rate, ability to metastasize, drug resistance, and failure in therapy among the many characteristics of them being of an aggressive phenotype. Yet for cancer to propagate further with its heterogeneity, it has to face the task of being recognized by immune cells as foreign. The innate and adaptive immune cell types with their effector mechanisms and pathways can orchestrate an antitumor response leading to their eradication, thus having a crucial role in generating responses. This interplay between tumors and immune cells can lead to immune system stimulation as well as its suppression. The ability to understand this intricate interplay will allow us to harness the immune system for potential long-lasting tumor control.
Nicotinamide mononucleotide (NMN) alleviates the poly(I:C)-induced inflammatory response in human primary cell cultures
2023, Scientific Reports
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^☆

Abbreviations: DCs, dendritic cells; dsRNA, double-stranded RNA; IRF, interferon regulatory factor; LRRs, leucine-rich repeats; MEFs, murine embryonic fibroblasts; PKR, dsRNA-dependent protein kinase; poly(I):poly(C), polyinosinic acid:polycytidylic acid, double stranded; ssRNA, single-stranded RNA; TIR, Toll/IL-1 receptor homology domain; TLR, Toll-like receptor.

This work was partly reported in the Protein Research Institute Seminar held on September 14, 2001.

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Biochemical and Biophysical Research Communications

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgements

References (22)

Innate immunity: the virtues of non-clonal system of recognition

Cell

The IL-1 receptor/Toll-like receptor superfamily: crucial receptors for inflammation and host defense

Immunol. Today

Toll-like receptors: key mediators of microbe detection

Curr. Opin. Immunol.

Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-α/β gene induction

Immunity

Targeted disruption of IRF-1 or IRF-2 results in abnormal type I IFN gene induction and aberrant lymphocyte development

Cell

Toll-like receptors in the induction of the innate immune response

Nature

A human homologue of the Drosophila Toll protein signals activation of adaptive immunity

Nature

A family of human receptors structurally related to Drosophila Toll

Proc. Natl. Acad. Sci. USA

Toll-like receptors: critical proteins linking innate and acquired immunity

Nat. Immunol.

Differential expression and regulation of Toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells

J. Immunol.

Differential alteration in intestinal epithelial cell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease

Infect. Immun.

Cited by (388)

Unconventional functions of miRNAs

Genome-wide identification, characterization, and expression of the Toll-like receptors in Japanese flounder (Paralichthys olivaceus)

Recent advances in immunotherapy, immunoadjuvant, and nanomaterial-based combination immunotherapy

Innate and adaptive immunity in cancer

Nicotinamide mononucleotide (NMN) alleviates the poly(I:C)-induced inflammatory response in human primary cell cultures

Role of toll-like receptors in the pathogenesis of COVID-19: Current and future perspectives

Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling☆

Abstract

Section snippets

Materials and methods

Results and discussion

Acknowledgements

Cell

Immunol. Today

Curr. Opin. Immunol.

Immunity

Cell

Toll-like receptors in the induction of the innate immune response

Nature

A human homologue of the Drosophila Toll protein signals activation of adaptive immunity

Nature

A family of human receptors structurally related to Drosophila Toll

Proc. Natl. Acad. Sci. USA

Toll-like receptors: critical proteins linking innate and acquired immunity

Nat. Immunol.

Differential expression and regulation of Toll-like receptors (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells

J. Immunol.

Differential alteration in intestinal epithelial cell expression of Toll-like receptor 3 (TLR3) and TLR4 in inflammatory bowel disease

Infect. Immun.