Abstract
Toll-like receptor 10 (TLR10) is the only orphan receptor whose natural ligand and function are unknown among the 10 human TLRs. In this study, to test whether TLR10 recognizes some known TLR ligands, we established a stable TLR10 knockdown human monocytic cell line THP-1 using TLR10 short hairpin RNA lentiviral particle and puromycin selection. Among 60 TLR10 knockdown clones that were derived from each single transduced cell, six clones were randomly selected, and then one of those clones, named E7, was chosen for the functional study. E7 exhibited approximately 50% inhibition of TLR10 mRNA and protein expression. Of all the TLRs, only the expression of TLR10 changed significantly in this cell line. Additionally, phorbol 12-myristate 13-acetate-induced macrophage differentiation of TLR10 knockdown cells was not affected in the knockdown cells. When exposed to TLR ligands, such as synthetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin, significant induction of proinflammatory cytokine gene expression including Interleukin-8 (IL-8), Interleukin-1 beta (IL-1β), Tumor necrosis factor-alpha (TNF-α) and Chemokine (C–C Motif) Ligand 20 (CCL20) expression, was found in the control THP-1 cells, whereas the TLR10 knockdown cells exhibited a significant reduction in the expression of IL-8, IL-1β, and CCL20. TNF-α was the only cytokine for which the expression did not decrease in the TLR10 knockdown cells from that measured in the control cells. Analysis of putative binding sites for transcription factors using a binding-site-prediction program revealed that the TNF-α promoter does not have putative binding sites for AP-1 or c-Jun, comprising a major transcription factor along with NF-κB for TLR signaling. Our results suggest that TLR10 is involved in the recognition of FSL-1, LPS, and flagellin and TLR-ligand-induced expression of TNF-α does not depend on TLR10.
Highlights
Toll-like receptors (TLRs) are innate immune receptors responsible for detecting host invasion by pathogens
THP-1 cells are widely used as a model system of human monocytes and are known to express Toll-like receptor 10 (TLR10) [6,15,19]
The functional experiments carried out in this study revealed that when TLR10 knockdown cells were challenged with FSL-1, LPS, or flagellin, IL-8 and IL-1β expression was significantly reduced from that in the control cells
Summary
Toll-like receptors (TLRs) are innate immune receptors responsible for detecting host invasion by pathogens. Among the 10 TLRs discovered in humans, TLR10 is the only orphan receptor whose natural ligand and function are unknown. A viable alternative would be the use of human TLR10 knockdown immune cells for functional study. A study using computational modeling suggested that a TLR2/10 dimer recognizes triacylated lipopeptides, while a TLR1/10 dimer or a TLR10 homodimer senses diacylated. Studies with human TLR10 knockdown monocytic cell lines have suggested that TLR10 acts as an immune lipsoepnespotridfoers [t4h]e. FIunnacdtdiointiaoln, raescteupdtoyrusrinecgoggansitzriincgepiHtheelilciaolbaccetlelsr frpoymlori paltiipeonptsoilnyfseacctcehdabriydHe e(LlicPoSb)a[c7t]e.rTphyeloseripprreovpioosuesdstthuadtiTesLRal1l0sfuogrmgesstaehdettheraotdTimLRer10waitchtsTaLsR2ananimd macutsne assaenfusonrctfioornpaal trheocgepentosrmreuccohglnikizeinogthHerelTicLoRbascdteor.pylori lipopolysaccharide (LPS) [7]. Controversial issue of the potential function of TLR10, weInetshtaisbslitsuhdeyd, inaorsdtaebr lteo iTnLveRs1t0igaktenothcekdcoonwtrnovheursmiaalnissumeoonfotchyetipcotceenltliallinfuencatniodn oefxTamLRin1e0d, wtehe estparboliinshfleadmamsatatobrley TcLyRto1k0inkensocekxdporewsnsiohnumofanthmesoenoceclyltsicincerlel slipnoenasnedtoexTaLmRinleidgatnhdespsrouicnhflaams smyanttohreytic cydtoiakcinyelasteexdplripesospiornotoefinth(FesSeLc-1e)ll,sliipnorpeosplyosnascechtoarTidLeR(lLigPaSn)d, asnsducfhlaagselslyinn.thetic diacylated lipoprotein (FSL-1), lipopolysaccharide (LPS), and flagellin. Gray sshhaaddeedd aarreeaa,,nneeggaattiivveeccoonnttrroolloommiitttiinnggtthheepprriimmaarryyaannttiibbooddyy;;hhiissttooggrraammsswwitithhtthhiicckkddaasshheeddlliinneeaannddddoottteedd lliinnee,, ttwwoo iinnddeeppeennddeennttsseettss ooff TTLLRR1100 sshhRRNNAA ttrraannssdduucceedd cceelllss;; hhiissttooggrraammwwitithh ssoolliidd lliinnee,, ccoonnttrrooll sshhRRNNAA--ttrraannssdduucceeddcceellllss
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