List of Publications (2011-Current)
2023
Sanjel, Babina; Shim, Won-Sik
Molecules That Channel Stimulus into Pruritus Book Chapter
In: Tsagareli, Merab G.; Follansbee, Taylor (Ed.): Histaminergic and Non-Histaminergic Itch: From Channels to Behavior, Nova Science, 2023, ISBN: 979-8-88697-757-8.
Abstract | Links | BibTeX | Tags: Ion Channel, Itch
@inbook{nokey,
title = {Molecules That Channel Stimulus into Pruritus},
author = {Babina Sanjel and Won-Sik Shim},
editor = {Merab G. Tsagareli and Taylor Follansbee},
doi = {10.52305/UCLY2326},
isbn = {979-8-88697-757-8},
year = {2023},
date = {2023-07-15},
urldate = {2023-07-15},
booktitle = {Histaminergic and Non-Histaminergic Itch: From Channels to Behavior},
issuetitle = {Chapter 4. Molecules That Channel Stimulus into Pruritus},
publisher = {Nova Science},
abstract = {Pruritus, a sensation that evokes a desire to scratch, is triggered by molecular cascades that result in the excitation of sensory neurons. In general, pruritogens or itch-inducing agents can induce alterations in the membrane potential, coordinated by the actions of distinctive molecules known as ion channels. These specialized molecules are membrane proteins that form distinct pores, through which specific ions can selectively flow when opened. To date, numerous ion channels have been discovered that are involved in the generation and/or transmission of pruritic signals in the vicinity of the skin. This chapter will summarize representative ion channels that play fundamental roles in pruritus.},
keywords = {Ion Channel, Itch},
pubstate = {published},
tppubtype = {inbook}
}
Pruritus, a sensation that evokes a desire to scratch, is triggered by molecular cascades that result in the excitation of sensory neurons. In general, pruritogens or itch-inducing agents can induce alterations in the membrane potential, coordinated by the actions of distinctive molecules known as ion channels. These specialized molecules are membrane proteins that form distinct pores, through which specific ions can selectively flow when opened. To date, numerous ion channels have been discovered that are involved in the generation and/or transmission of pruritic signals in the vicinity of the skin. This chapter will summarize representative ion channels that play fundamental roles in pruritus.
Kim, Hye In; Lee, Gi Baek; Song, Da Eun; Sanjel, Babina; Lee, Wook-Joo; Shim, Won-Sik
In: Life Sciences, vol. 325, 2023, ISSN: 0024-3205.
Abstract | Links | BibTeX | Tags: Calcium imaging, FSLLRY, Itch, MrgprC11, MRGPRX1, PAR2, Scratching behavior
@article{InKim2023,
title = {FSLLRY-NH2, a protease-activated receptor 2 (PAR2) antagonist, activates mas-related G protein-coupled receptor C11 (MrgprC11) to induce scratching behaviors in mice},
author = {Hye In Kim and Gi Baek Lee and Da Eun Song and Babina Sanjel and Wook-Joo Lee and Won-Sik Shim},
doi = {10.1016/j.lfs.2023.121786},
issn = {0024-3205},
year = {2023},
date = {2023-07-00},
urldate = {2023-07-00},
journal = {Life Sciences},
volume = {325},
publisher = {Elsevier BV},
abstract = {Aims: Protease-activated receptor 2 (PAR2), a type of G protein-coupled receptor (GPCR), plays a significant role in pathophysiological conditions such as inflammation. A synthetic peptide SLIGRL-NH2 (SLIGRL) can activate PAR2, while FSLLRY-NH2 (FSLLRY) is an antagonist. A previous study showed that SLIGRL activates both PAR2 and mas-related G protein-coupled receptor C11 (MrgprC11), a different type of GPCR expressed in sensory neurons. However, the impact of FSLLRY on MrgprC11 and its human ortholog MRGPRX1 was not verified. Hence, the present study aims to verify the effect of FSLLRY on MrgprC11 and MRGPRX1.
Methods: The calcium imaging technique was applied to determine the effect of FSLLRY in HEK293T cells expressing MrgprC11/MRGPRX1 or dorsal root ganglia (DRG) neurons. Scratching behavior was also investigated in wild-type and PAR2 knockout mice after injecting FSLLRY.
Key findings: It was surprisingly discovered that FSLLRY specifically activates MrgprC11 in a dose-dependent manner, but not other MRGPR subtypes. Furthermore, FSLLRY also moderately activated MRGPRX1. FSLLRY stimulates downstream pathways including Gαq/11, phospholipase C, IP3 receptor, and TRPC ion channels to evoke an increase in the intracellular calcium levels. The molecular docking analysis predicted that FSLLRY interacts with the orthosteric binding pocket of MrgprC11 and MRGPRX1. Finally, FSLLRY activated primary cultures of mouse sensory neurons, and induced scratching behaviors in mice.
Significance: The present study has revealed that FSLLRY is capable of triggering itch sensation through activation of MrgprC11. This finding highlights the importance of considering the unexpected activation of MRGPRs in future therapeutic approaches aimed at the inhibition of PAR2.},
keywords = {Calcium imaging, FSLLRY, Itch, MrgprC11, MRGPRX1, PAR2, Scratching behavior},
pubstate = {published},
tppubtype = {article}
}
Aims: Protease-activated receptor 2 (PAR2), a type of G protein-coupled receptor (GPCR), plays a significant role in pathophysiological conditions such as inflammation. A synthetic peptide SLIGRL-NH2 (SLIGRL) can activate PAR2, while FSLLRY-NH2 (FSLLRY) is an antagonist. A previous study showed that SLIGRL activates both PAR2 and mas-related G protein-coupled receptor C11 (MrgprC11), a different type of GPCR expressed in sensory neurons. However, the impact of FSLLRY on MrgprC11 and its human ortholog MRGPRX1 was not verified. Hence, the present study aims to verify the effect of FSLLRY on MrgprC11 and MRGPRX1.
Methods: The calcium imaging technique was applied to determine the effect of FSLLRY in HEK293T cells expressing MrgprC11/MRGPRX1 or dorsal root ganglia (DRG) neurons. Scratching behavior was also investigated in wild-type and PAR2 knockout mice after injecting FSLLRY.
Key findings: It was surprisingly discovered that FSLLRY specifically activates MrgprC11 in a dose-dependent manner, but not other MRGPR subtypes. Furthermore, FSLLRY also moderately activated MRGPRX1. FSLLRY stimulates downstream pathways including Gαq/11, phospholipase C, IP3 receptor, and TRPC ion channels to evoke an increase in the intracellular calcium levels. The molecular docking analysis predicted that FSLLRY interacts with the orthosteric binding pocket of MrgprC11 and MRGPRX1. Finally, FSLLRY activated primary cultures of mouse sensory neurons, and induced scratching behaviors in mice.
Significance: The present study has revealed that FSLLRY is capable of triggering itch sensation through activation of MrgprC11. This finding highlights the importance of considering the unexpected activation of MRGPRs in future therapeutic approaches aimed at the inhibition of PAR2.
Methods: The calcium imaging technique was applied to determine the effect of FSLLRY in HEK293T cells expressing MrgprC11/MRGPRX1 or dorsal root ganglia (DRG) neurons. Scratching behavior was also investigated in wild-type and PAR2 knockout mice after injecting FSLLRY.
Key findings: It was surprisingly discovered that FSLLRY specifically activates MrgprC11 in a dose-dependent manner, but not other MRGPR subtypes. Furthermore, FSLLRY also moderately activated MRGPRX1. FSLLRY stimulates downstream pathways including Gαq/11, phospholipase C, IP3 receptor, and TRPC ion channels to evoke an increase in the intracellular calcium levels. The molecular docking analysis predicted that FSLLRY interacts with the orthosteric binding pocket of MrgprC11 and MRGPRX1. Finally, FSLLRY activated primary cultures of mouse sensory neurons, and induced scratching behaviors in mice.
Significance: The present study has revealed that FSLLRY is capable of triggering itch sensation through activation of MrgprC11. This finding highlights the importance of considering the unexpected activation of MRGPRs in future therapeutic approaches aimed at the inhibition of PAR2.
Lee, Wook-Joo; Shim, Won-Sik
Rg3-enriched Korean red ginseng alleviates chloroquine-induced itch and dry skin pruritus in an MrgprA3-dependent manner in mice Journal Article
In: Integrative Medicine Research, vol. 12, no. 1, 2023, ISSN: 2213-4220.
Abstract | Links | BibTeX | Tags: Dry skin, Itch, Korean Red Ginseng, MrgprA3, RNA-seq, Scratching behavior, TRPA1
@article{Lee2023,
title = {Rg3-enriched Korean red ginseng alleviates chloroquine-induced itch and dry skin pruritus in an MrgprA3-dependent manner in mice},
author = {Wook-Joo Lee and Won-Sik Shim},
doi = {10.1016/j.imr.2022.100916},
issn = {2213-4220},
year = {2023},
date = {2023-03-00},
urldate = {2023-03-00},
journal = {Integrative Medicine Research},
volume = {12},
number = {1},
publisher = {Elsevier BV},
abstract = {Background: Previous studies have found that Korean red ginseng extract (KRG) has antipruritic effects, which can be attributed to the presence of Rg3, one of the most potent ginsenosides. Therefore, Rg3-enriched KRG extract (Rg3EKRG) is anticipated to have enhanced antipruritic effects. The present study was conducted to examine the effects of Rg3EKRG in acute chloroquine (CQ)-induced and chronic dry skin pruritus.
Methods: Calcium imaging technique was used in HE293T cells expressing MrgprA3 and TRPA1 ("MrgprA3/TRPA1") and in primary cultures of mouse dorsal root ganglia (DRG) neurons. Mouse scratching behavior tests were performed on dry skin models. To verify the altered expression of itch-related genes, real-time RNA sequencing analysis and PCR were performed on DRG sections obtained from dry skin models.
Results: Rg3EKRG suppressed CQ-induced intracellular calcium changes to a greater degree than KRG. Rg3EKRG dose-dependently inhibited CQ-induced responses in MrgprA3/TRPA1 cells. Rg3EKRG likely targeted MrgprA3 rather than TRPA1 to exert its inhibitory effect. Further, Rg3EKRG strongly inhibited the scratching behavior in mice induced by acute CQ injection. Importantly, DRG neurons obtained from dry skin mice models showed increased mRNA levels of MrgprA3, and treatment with Rg3EKRG alleviated chronic dry skin conditions and suppressed spontaneous scratching behaviors.
Conclusion: The results of the present study imply that Rg3EKRG has a stronger antipruritic effect than KRG, inhibiting both acute CQ-induced and chronic dry skin pruritus in an MrgprA3-dependent manner. Therefore, Rg3EKRG is a potential antipruritic agent that can suppress acute and chronic itching at the peripheral sensory neuronal level.},
keywords = {Dry skin, Itch, Korean Red Ginseng, MrgprA3, RNA-seq, Scratching behavior, TRPA1},
pubstate = {published},
tppubtype = {article}
}
Background: Previous studies have found that Korean red ginseng extract (KRG) has antipruritic effects, which can be attributed to the presence of Rg3, one of the most potent ginsenosides. Therefore, Rg3-enriched KRG extract (Rg3EKRG) is anticipated to have enhanced antipruritic effects. The present study was conducted to examine the effects of Rg3EKRG in acute chloroquine (CQ)-induced and chronic dry skin pruritus.
Methods: Calcium imaging technique was used in HE293T cells expressing MrgprA3 and TRPA1 ("MrgprA3/TRPA1") and in primary cultures of mouse dorsal root ganglia (DRG) neurons. Mouse scratching behavior tests were performed on dry skin models. To verify the altered expression of itch-related genes, real-time RNA sequencing analysis and PCR were performed on DRG sections obtained from dry skin models.
Results: Rg3EKRG suppressed CQ-induced intracellular calcium changes to a greater degree than KRG. Rg3EKRG dose-dependently inhibited CQ-induced responses in MrgprA3/TRPA1 cells. Rg3EKRG likely targeted MrgprA3 rather than TRPA1 to exert its inhibitory effect. Further, Rg3EKRG strongly inhibited the scratching behavior in mice induced by acute CQ injection. Importantly, DRG neurons obtained from dry skin mice models showed increased mRNA levels of MrgprA3, and treatment with Rg3EKRG alleviated chronic dry skin conditions and suppressed spontaneous scratching behaviors.
Conclusion: The results of the present study imply that Rg3EKRG has a stronger antipruritic effect than KRG, inhibiting both acute CQ-induced and chronic dry skin pruritus in an MrgprA3-dependent manner. Therefore, Rg3EKRG is a potential antipruritic agent that can suppress acute and chronic itching at the peripheral sensory neuronal level.
Methods: Calcium imaging technique was used in HE293T cells expressing MrgprA3 and TRPA1 ("MrgprA3/TRPA1") and in primary cultures of mouse dorsal root ganglia (DRG) neurons. Mouse scratching behavior tests were performed on dry skin models. To verify the altered expression of itch-related genes, real-time RNA sequencing analysis and PCR were performed on DRG sections obtained from dry skin models.
Results: Rg3EKRG suppressed CQ-induced intracellular calcium changes to a greater degree than KRG. Rg3EKRG dose-dependently inhibited CQ-induced responses in MrgprA3/TRPA1 cells. Rg3EKRG likely targeted MrgprA3 rather than TRPA1 to exert its inhibitory effect. Further, Rg3EKRG strongly inhibited the scratching behavior in mice induced by acute CQ injection. Importantly, DRG neurons obtained from dry skin mice models showed increased mRNA levels of MrgprA3, and treatment with Rg3EKRG alleviated chronic dry skin conditions and suppressed spontaneous scratching behaviors.
Conclusion: The results of the present study imply that Rg3EKRG has a stronger antipruritic effect than KRG, inhibiting both acute CQ-induced and chronic dry skin pruritus in an MrgprA3-dependent manner. Therefore, Rg3EKRG is a potential antipruritic agent that can suppress acute and chronic itching at the peripheral sensory neuronal level.
Kim, Jin-Cheol; Shim, Won-Sik; Kwak, In-Suk; Lee, Dong-Hun; Park, Jin-Seo; Lee, So-Yeon; Kang, Seok-Young; Chung, Bo-Young; Park, Chun-Wook; Kim, Hye-One
Pathogenesis and Treatment of Pruritus Associated with Chronic Kidney Disease and Cholestasis Journal Article
In: IJMS, vol. 24, no. 2, 2023, ISSN: 1422-0067.
Abstract | Links | BibTeX | Tags: Cholestasis, Chronic kidney disease, Itch
@article{Kim2023,
title = {Pathogenesis and Treatment of Pruritus Associated with Chronic Kidney Disease and Cholestasis},
author = {Jin-Cheol Kim and Won-Sik Shim and In-Suk Kwak and Dong-Hun Lee and Jin-Seo Park and So-Yeon Lee and Seok-Young Kang and Bo-Young Chung and Chun-Wook Park and Hye-One Kim},
doi = {10.3390/ijms24021559},
issn = {1422-0067},
year = {2023},
date = {2023-01-00},
urldate = {2023-01-00},
journal = {IJMS},
volume = {24},
number = {2},
publisher = {MDPI AG},
abstract = {Itching is an unpleasant sensation that provokes the desire to scratch. In general, itching is caused by dermatologic diseases, but it can also be caused by systemic diseases. Since itching hampers patients’ quality of life, it is important to understand the appropriate treatment and pathophysiology of pruritus caused by systemic diseases to improve the quality of life. Mechanisms are being studied through animal or human studies, and various treatments are being tested through clinical trials. We report current trends of two major systemic diseases: chronic kidney disease and cholestatic liver disease. This review summarizes the causes and pathophysiology of systemic diseases with pruritus and appropriate treatments. This article will contribute to patients’ quality of life. Further research will help understand the mechanisms and develop new strategies in the future.},
keywords = {Cholestasis, Chronic kidney disease, Itch},
pubstate = {published},
tppubtype = {article}
}
Itching is an unpleasant sensation that provokes the desire to scratch. In general, itching is caused by dermatologic diseases, but it can also be caused by systemic diseases. Since itching hampers patients’ quality of life, it is important to understand the appropriate treatment and pathophysiology of pruritus caused by systemic diseases to improve the quality of life. Mechanisms are being studied through animal or human studies, and various treatments are being tested through clinical trials. We report current trends of two major systemic diseases: chronic kidney disease and cholestatic liver disease. This review summarizes the causes and pathophysiology of systemic diseases with pruritus and appropriate treatments. This article will contribute to patients’ quality of life. Further research will help understand the mechanisms and develop new strategies in the future.
2022
Sanjel, Babina; Shim, Won-Sik
The contribution of mouse models to understanding atopic dermatitis Journal Article
In: Biochemical Pharmacology, vol. 203, 2022, ISSN: 0006-2952.
Abstract | Links | BibTeX | Tags: Animal model, Atopic dermatitis, Itch
@article{Sanjel2022,
title = {The contribution of mouse models to understanding atopic dermatitis},
author = {Babina Sanjel and Won-Sik Shim},
doi = {10.1016/j.bcp.2022.115177},
issn = {0006-2952},
year = {2022},
date = {2022-09-00},
urldate = {2022-09-00},
journal = {Biochemical Pharmacology},
volume = {203},
publisher = {Elsevier BV},
abstract = {Atopic dermatitis (AD) is a dermatological disease accompanied by dry and cracked skin with severe pruritus. Although various therapeutic strategies have been introduced to alleviate AD, it remains challenging to cure the disorder. To achieve such a goal, understanding the pathophysiological mechanisms of AD is a prerequisite, requiring mouse models that properly reflect the AD phenotypes. Currently, numerous AD mouse models have been established, but each model has its own advantages and weaknesses. In this review, we categorized and summarized mouse models of AD and described their characteristics from a researcher’s perspective.},
keywords = {Animal model, Atopic dermatitis, Itch},
pubstate = {published},
tppubtype = {article}
}
Atopic dermatitis (AD) is a dermatological disease accompanied by dry and cracked skin with severe pruritus. Although various therapeutic strategies have been introduced to alleviate AD, it remains challenging to cure the disorder. To achieve such a goal, understanding the pathophysiological mechanisms of AD is a prerequisite, requiring mouse models that properly reflect the AD phenotypes. Currently, numerous AD mouse models have been established, but each model has its own advantages and weaknesses. In this review, we categorized and summarized mouse models of AD and described their characteristics from a researcher’s perspective.
Song, Myung-Hyun; Shim, Won-Sik
Lithocholic Acid Activates Mas-Related G Protein-Coupled Receptors, Contributing to Itch in Mice Journal Article
In: Biomol Ther (Seoul), vol. 30, no. 1, pp. 38–47, 2022, ISSN: 2005-4483.
Abstract | Links | BibTeX | Tags: Bile acid, Calcium imaging, Itch, Lithocholic acid, MrgprA1, MrgprB2, MRGPRX4
@article{Song2022,
title = {Lithocholic Acid Activates Mas-Related G Protein-Coupled Receptors, Contributing to Itch in Mice},
author = {Myung-Hyun Song and Won-Sik Shim},
doi = {10.4062/biomolther.2021.059},
issn = {2005-4483},
year = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Biomol Ther (Seoul)},
volume = {30},
number = {1},
pages = {38--47},
publisher = {The Korean Society of Applied Pharmacology},
abstract = {The present study focused on lithocholic acid (LCA), a secondary bile acid that contributes to cholestatic pruritus. Although recent studies have found that LCA acts on MAS-related G protein-coupled receptor family member X4 (MRGPRX4) in humans, it is unclear which subtypes of MRGPRs are activated by LCA in mice since there is no precise ortholog of human MRGPRX4 in the mouse genome. Using calcium imaging, we found that LCA could activate mouse Mrgpra1 when transiently expressed in HEK293T cells. Moreover, LCA similarly activates mouse Mrgprb2. Importantly, LCA-induced responses showed dose-dependent effects through Mrgpra1 and Mrgprb2. Moreover, treatment with QWF (an antagonist of Mrgpra1 and Mrgprb2), YM254890 (Gαq inhibitor), and U73122 (an inhibitor of phospholipase C) significantly suppressed the LCA-induced responses, implying that the LCA-induced responses are indeed mediated by Mrgpra1 and Mrgprb2. Furthermore, LCA activated primary cultures of mouse sensory neurons and peritoneal mast cells, suggesting that Mrgpra1 and Mrgprb2 contribute to LCA-induced pruritus. However, acute injection of LCA did not induce noticeable differences in scratching behavior, implying that the pruritogenic role of LCA may be marginal in non-cholestatic conditions. In summary, the present study identified for the first time that LCA can activate Mrgpra1 and Mrgprb2. The current findings provide further insight into the similarities and differences between human and mouse MRGPR families, paving a way to understand the complex roles of these pruriceptors.},
keywords = {Bile acid, Calcium imaging, Itch, Lithocholic acid, MrgprA1, MrgprB2, MRGPRX4},
pubstate = {published},
tppubtype = {article}
}
The present study focused on lithocholic acid (LCA), a secondary bile acid that contributes to cholestatic pruritus. Although recent studies have found that LCA acts on MAS-related G protein-coupled receptor family member X4 (MRGPRX4) in humans, it is unclear which subtypes of MRGPRs are activated by LCA in mice since there is no precise ortholog of human MRGPRX4 in the mouse genome. Using calcium imaging, we found that LCA could activate mouse Mrgpra1 when transiently expressed in HEK293T cells. Moreover, LCA similarly activates mouse Mrgprb2. Importantly, LCA-induced responses showed dose-dependent effects through Mrgpra1 and Mrgprb2. Moreover, treatment with QWF (an antagonist of Mrgpra1 and Mrgprb2), YM254890 (Gαq inhibitor), and U73122 (an inhibitor of phospholipase C) significantly suppressed the LCA-induced responses, implying that the LCA-induced responses are indeed mediated by Mrgpra1 and Mrgprb2. Furthermore, LCA activated primary cultures of mouse sensory neurons and peritoneal mast cells, suggesting that Mrgpra1 and Mrgprb2 contribute to LCA-induced pruritus. However, acute injection of LCA did not induce noticeable differences in scratching behavior, implying that the pruritogenic role of LCA may be marginal in non-cholestatic conditions. In summary, the present study identified for the first time that LCA can activate Mrgpra1 and Mrgprb2. The current findings provide further insight into the similarities and differences between human and mouse MRGPR families, paving a way to understand the complex roles of these pruriceptors.
Kim, Hyesu; Kim, Hyungsup; Cho, Hawon; Lee, Byeongjun; Lu, Huan-Jun; Kim, Kyungmin; Chung, Sooyoung; Shim, Won-Sik; Shin, Young Kee; Dong, Xinzhong; Wood, John N.; Oh, Uhtaek
Anoctamin 1/TMEM16A in pruritoceptors is essential for Mas-related G protein receptor–dependent itch Journal Article
In: PAIN, vol. 163, no. 11, pp. 2172–2184, 2022, ISSN: 1872-6623.
Abstract | Links | BibTeX | Tags: Anoctamin 1, Itch, MrgprA3, MrgprC11
@article{Kim2022,
title = {Anoctamin 1/TMEM16A in pruritoceptors is essential for Mas-related G protein receptor–dependent itch},
author = {Hyesu Kim and Hyungsup Kim and Hawon Cho and Byeongjun Lee and Huan-Jun Lu and Kyungmin Kim and Sooyoung Chung and Won-Sik Shim and Young Kee Shin and Xinzhong Dong and John N. Wood and Uhtaek Oh},
doi = {10.1097/j.pain.0000000000002611},
issn = {1872-6623},
year = {2022},
date = {2022-00-00},
urldate = {2022-00-00},
journal = {PAIN},
volume = {163},
number = {11},
pages = {2172--2184},
publisher = {Ovid Technologies (Wolters Kluwer Health)},
abstract = {<jats:title>Abstract</jats:title>
<jats:p>Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that anoctamin 1 (ANO1), a Ca<jats:sup>2+</jats:sup>-activated chloride channel, is a transduction channel mediating Mrgpr-dependent itch signals. Genetic ablation of <jats:italic toggle="yes">Ano1</jats:italic> in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgpr-dependent itch models and the epidermal hyperplasia induced by dry skin. In vivo Ca<jats:sup>2+</jats:sup> imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgprs excited DRG neurons via ANO1. More importantly, the overexpression of <jats:italic toggle="yes">Ano1</jats:italic> in DRG neurons of <jats:italic toggle="yes">Ano1</jats:italic>-deficient mice rescued the impaired itching observed in <jats:italic toggle="yes">Ano1</jats:italic>-deficient mice. These results demonstrate that ANO1 mediates the Mrgpr-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.</jats:p>},
keywords = {Anoctamin 1, Itch, MrgprA3, MrgprC11},
pubstate = {published},
tppubtype = {article}
}
<jats:title>Abstract</jats:title>
<jats:p>Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that anoctamin 1 (ANO1), a Ca<jats:sup>2+</jats:sup>-activated chloride channel, is a transduction channel mediating Mrgpr-dependent itch signals. Genetic ablation of <jats:italic toggle="yes">Ano1</jats:italic> in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgpr-dependent itch models and the epidermal hyperplasia induced by dry skin. In vivo Ca<jats:sup>2+</jats:sup> imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgprs excited DRG neurons via ANO1. More importantly, the overexpression of <jats:italic toggle="yes">Ano1</jats:italic> in DRG neurons of <jats:italic toggle="yes">Ano1</jats:italic>-deficient mice rescued the impaired itching observed in <jats:italic toggle="yes">Ano1</jats:italic>-deficient mice. These results demonstrate that ANO1 mediates the Mrgpr-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.</jats:p>
<jats:p>Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that anoctamin 1 (ANO1), a Ca<jats:sup>2+</jats:sup>-activated chloride channel, is a transduction channel mediating Mrgpr-dependent itch signals. Genetic ablation of <jats:italic toggle="yes">Ano1</jats:italic> in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgpr-dependent itch models and the epidermal hyperplasia induced by dry skin. In vivo Ca<jats:sup>2+</jats:sup> imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgprs excited DRG neurons via ANO1. More importantly, the overexpression of <jats:italic toggle="yes">Ano1</jats:italic> in DRG neurons of <jats:italic toggle="yes">Ano1</jats:italic>-deficient mice rescued the impaired itching observed in <jats:italic toggle="yes">Ano1</jats:italic>-deficient mice. These results demonstrate that ANO1 mediates the Mrgpr-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.</jats:p>
2021
Lee, Wook-Joo; Shim, Won-Sik
Cutaneous Neuroimmune Interactions of TSLP and TRPV4 Play Pivotal Roles in Dry Skin-Induced Pruritus Journal Article
In: Front. Immunol., vol. 12, 2021, ISSN: 1664-3224.
Abstract | Links | BibTeX | Tags: Calcium imaging, Dorsal root ganglia, Dry skin, Itch, TSLP
@article{Lee2021,
title = {Cutaneous Neuroimmune Interactions of TSLP and TRPV4 Play Pivotal Roles in Dry Skin-Induced Pruritus},
author = {Wook-Joo Lee and Won-Sik Shim},
doi = {10.3389/fimmu.2021.772941},
issn = {1664-3224},
year = {2021},
date = {2021-12-02},
urldate = {2021-12-02},
journal = {Front. Immunol.},
volume = {12},
publisher = {Frontiers Media SA},
abstract = {<jats:p>Dry skin is a symptom of skin barrier dysfunction that evokes pruritus; however, the cutaneous neuroimmune interactions underlying dry skin-induced pruritus remain unclear. Therefore, we aimed to elucidate the mechanisms underlying dry skin-induced pruritus. To this end, an acetone/ethanol/water (AEW)-induced mouse model of dry skin was used in this study. We observed that the production of thymic stromal lymphopoietin (TSLP) significantly increased in the keratinocytes of AEW mice. Importantly, treatment with an antagonist of transient receptor potential cation channel subfamily V member 4 (TRPV4), HC067047, ameliorated dry skin conditions in AEW mice. The symptoms of dry skin were significantly reduced in <jats:italic>Trpv4</jats:italic> knockout (KO) mice following treatment with AEW. The increase in the intracellular calcium levels by TSLP in the dorsal root ganglia (DRG) of <jats:italic>Trpv4</jats:italic> KO mice was also significantly attenuated. The spontaneous scratching bouts were significantly decreased in both the HC067047-treated and <jats:italic>Trpv4</jats:italic> KO AEW mice. Importantly, the TSLP-dependent release of tryptase from the mast cells was significantly reduced in both the HC067047-treated mice and <jats:italic>Trpv4</jats:italic> KO AEW mice. Notably, inhibition of the TSLP-induced signaling pathway in DRG selectively reduced the spontaneous scratching bouts in AEW mice. Overall, the results demonstrated that the cutaneous neuroimmune interactions of TSLP and TRPV4 play pivotal roles in dry skin-induced pruritus.</jats:p>},
keywords = {Calcium imaging, Dorsal root ganglia, Dry skin, Itch, TSLP},
pubstate = {published},
tppubtype = {article}
}
<jats:p>Dry skin is a symptom of skin barrier dysfunction that evokes pruritus; however, the cutaneous neuroimmune interactions underlying dry skin-induced pruritus remain unclear. Therefore, we aimed to elucidate the mechanisms underlying dry skin-induced pruritus. To this end, an acetone/ethanol/water (AEW)-induced mouse model of dry skin was used in this study. We observed that the production of thymic stromal lymphopoietin (TSLP) significantly increased in the keratinocytes of AEW mice. Importantly, treatment with an antagonist of transient receptor potential cation channel subfamily V member 4 (TRPV4), HC067047, ameliorated dry skin conditions in AEW mice. The symptoms of dry skin were significantly reduced in <jats:italic>Trpv4</jats:italic> knockout (KO) mice following treatment with AEW. The increase in the intracellular calcium levels by TSLP in the dorsal root ganglia (DRG) of <jats:italic>Trpv4</jats:italic> KO mice was also significantly attenuated. The spontaneous scratching bouts were significantly decreased in both the HC067047-treated and <jats:italic>Trpv4</jats:italic> KO AEW mice. Importantly, the TSLP-dependent release of tryptase from the mast cells was significantly reduced in both the HC067047-treated mice and <jats:italic>Trpv4</jats:italic> KO AEW mice. Notably, inhibition of the TSLP-induced signaling pathway in DRG selectively reduced the spontaneous scratching bouts in AEW mice. Overall, the results demonstrated that the cutaneous neuroimmune interactions of TSLP and TRPV4 play pivotal roles in dry skin-induced pruritus.</jats:p>
Kim, J; Kim, H; Shim, W; Kwak, I; Chung, B; Kang, S; Park, C; Kim, H
Activation of Transient Receptor Potential Vanilloid-3 Channels in Keratinocytes Induces Pruritus in Humans Journal Article
In: Acta Derm Venereol, vol. 101, no. 8, 2021, ISSN: 1651-2057.
Abstract | Links | BibTeX | Tags: Itch, TRPV3, TSLP
@article{Kim2021,
title = {Activation of Transient Receptor Potential Vanilloid-3 Channels in Keratinocytes Induces Pruritus in Humans},
author = {J Kim and H Kim and W Shim and I Kwak and B Chung and S Kang and C Park and H Kim},
doi = {10.2340/00015555-3855},
issn = {1651-2057},
year = {2021},
date = {2021-08-00},
urldate = {2021-08-00},
journal = {Acta Derm Venereol},
volume = {101},
number = {8},
publisher = {Medical Journals Sweden AB},
abstract = {Carvacrol, a natural transient receptor potential vanilloid-3 activator, has been reported to cause pruritus in mice. This study aimed to evaluate the effects of carvacrol and various antipruritic agents in humans. A stimulation test with carvacrol, β-alanine, and histamine was performed. After application of the pruritic solutions, the skin was stimulated with pinpricks. In inhibition test A, Forsythia suspensa extract, containing forsythoside B (a transient receptor potential vanilloid-3 inhibitor), was applied by pricking prior to stimulation with pruritogens. In inhibition test B, olopatadine solution, tacrolimus ointment, and Scutellaria baicalensis root extract were applied, and carvacrol was applied to the same region. Carvacrol induces moderate pruritus in humans. The pruritus was relieved by Forsythia suspensa extract and olopatadine solution after 20 min of application and by tacrolimus ointment and Scutellaria baicalenis extract after 24 h of application. These results suggest that carvacrol is a pruritogen in humans, and that carvacrol-induced pruritus is inhibited by various antipruritic agents.},
keywords = {Itch, TRPV3, TSLP},
pubstate = {published},
tppubtype = {article}
}
Carvacrol, a natural transient receptor potential vanilloid-3 activator, has been reported to cause pruritus in mice. This study aimed to evaluate the effects of carvacrol and various antipruritic agents in humans. A stimulation test with carvacrol, β-alanine, and histamine was performed. After application of the pruritic solutions, the skin was stimulated with pinpricks. In inhibition test A, Forsythia suspensa extract, containing forsythoside B (a transient receptor potential vanilloid-3 inhibitor), was applied by pricking prior to stimulation with pruritogens. In inhibition test B, olopatadine solution, tacrolimus ointment, and Scutellaria baicalensis root extract were applied, and carvacrol was applied to the same region. Carvacrol induces moderate pruritus in humans. The pruritus was relieved by Forsythia suspensa extract and olopatadine solution after 20 min of application and by tacrolimus ointment and Scutellaria baicalenis extract after 24 h of application. These results suggest that carvacrol is a pruritogen in humans, and that carvacrol-induced pruritus is inhibited by various antipruritic agents.
2020
Sanjel, Babina; Shim, Won-Sik
Recent advances in understanding the molecular mechanisms of cholestatic pruritus: A review Journal Article
In: Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, vol. 1866, no. 12, 2020, ISSN: 0925-4439.
Abstract | Links | BibTeX | Tags: Animal model, Cholestasis, Itch
@article{Sanjel2020,
title = {Recent advances in understanding the molecular mechanisms of cholestatic pruritus: A review},
author = {Babina Sanjel and Won-Sik Shim},
doi = {10.1016/j.bbadis.2020.165958},
issn = {0925-4439},
year = {2020},
date = {2020-12-00},
urldate = {2020-12-00},
journal = {Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease},
volume = {1866},
number = {12},
publisher = {Elsevier BV},
abstract = {Cholestasis, a condition characterized by an abnormal decrease in bile flow, is accompanied by various symptoms such as pruritus. Although cholestatic pruritus is a prominent condition, its precise mechanisms have largely been elusive. Recently, advancements have been made for understanding the etiology and pathogenesis of cholestatic pruritus. The current review therefore focuses on summarizing the overall progress made in the elucidation of its molecular mechanisms. We have reviewed the available animal models on cholestasis to compare the differences between them, characterized potential pruritogens involved in cholestatic pruritus, and have summarized the receptor and ion channels implicated in the condition. Finally, we have discussed the available treatment options for alleviation of cholestatic pruritus. As our understanding of the mechanisms of cholestatic pruritus deepens, novel strategies to cure this condition are awaited.},
keywords = {Animal model, Cholestasis, Itch},
pubstate = {published},
tppubtype = {article}
}
Cholestasis, a condition characterized by an abnormal decrease in bile flow, is accompanied by various symptoms such as pruritus. Although cholestatic pruritus is a prominent condition, its precise mechanisms have largely been elusive. Recently, advancements have been made for understanding the etiology and pathogenesis of cholestatic pruritus. The current review therefore focuses on summarizing the overall progress made in the elucidation of its molecular mechanisms. We have reviewed the available animal models on cholestasis to compare the differences between them, characterized potential pruritogens involved in cholestatic pruritus, and have summarized the receptor and ion channels implicated in the condition. Finally, we have discussed the available treatment options for alleviation of cholestatic pruritus. As our understanding of the mechanisms of cholestatic pruritus deepens, novel strategies to cure this condition are awaited.
Choi, Da-Som; Ji, Yeounjung; Jang, Yongwoo; Lee, Wook-Joo; Shim, Won-Sik
In: Biomolecules & Therapeutics, vol. 28, no. 6, pp. 569–575, 2020, ISSN: 2005-4483.
Abstract | Links | BibTeX | Tags: Calcium imaging, Chloroquine, H1R, Histamine, Itch, MrgprA3, TRPA1, TRPV1
@article{Choi2020,
title = {Crotamiton, an Anti-Scabies Agent, Suppresses Histamine- and Chloroquine-Induced Itch Pathways in Sensory Neurons and Alleviates Scratching in Mice},
author = {Da-Som Choi and Yeounjung Ji and Yongwoo Jang and Wook-Joo Lee and Won-Sik Shim},
doi = {10.4062/biomolther.2020.063},
issn = {2005-4483},
year = {2020},
date = {2020-11-01},
urldate = {2020-11-01},
journal = {Biomolecules & Therapeutics},
volume = {28},
number = {6},
pages = {569--575},
publisher = {The Korean Society of Applied Pharmacology},
abstract = {Crotamiton is an anti-scabies drug, but it was recently found that crotamiton also suppresses non-scabietic itching in mice. However, the underlying mechanism is largely unclear. Therefore, aim of the study is to investigate mechanisms of the anti-pruritic effect of crotamiton for non-scabietic itching. Histamine and chloroquine are used as non-scabietic pruritogens. The effect of crotamiton was identified using fluorometric intracellular calcium assays in HEK293T cells and primary cultured dorsal root ganglion (DRG) neurons. Further in vivo effect was evaluated by scratching behavior tests. Crotamiton strongly inhibited histamine-induced calcium influx in HEK293T cells, expressing both histamine receptor 1 (H1R) and transient receptor potential vanilloid 1 (TRPV1), as a model of histamine-induced itching. Similarly, it also blocked chloroquine-induced calcium influx in HEK293T cells, expressing both Mas-related G-protein-coupled receptor A3 (MRGPRA3) and transient receptor potential A1 (TRPA1), as a model of histamine-independent itching. Furthermore, crotamiton also suppressed both histamine- and chloroquine-induced calcium influx in primary cultures of mouse DRG. Additionally, crotamiton strongly suppressed histamine- and chloroquine-induced scratching in mice. Overall, it was found that crotamiton has an anti-pruritic effect against non-scabietic itching by histamine and chloroquine. Therefore, crotamiton may be used as a general anti-pruritic agent, irrespective of the presence of scabies.},
keywords = {Calcium imaging, Chloroquine, H1R, Histamine, Itch, MrgprA3, TRPA1, TRPV1},
pubstate = {published},
tppubtype = {article}
}
Crotamiton is an anti-scabies drug, but it was recently found that crotamiton also suppresses non-scabietic itching in mice. However, the underlying mechanism is largely unclear. Therefore, aim of the study is to investigate mechanisms of the anti-pruritic effect of crotamiton for non-scabietic itching. Histamine and chloroquine are used as non-scabietic pruritogens. The effect of crotamiton was identified using fluorometric intracellular calcium assays in HEK293T cells and primary cultured dorsal root ganglion (DRG) neurons. Further in vivo effect was evaluated by scratching behavior tests. Crotamiton strongly inhibited histamine-induced calcium influx in HEK293T cells, expressing both histamine receptor 1 (H1R) and transient receptor potential vanilloid 1 (TRPV1), as a model of histamine-induced itching. Similarly, it also blocked chloroquine-induced calcium influx in HEK293T cells, expressing both Mas-related G-protein-coupled receptor A3 (MRGPRA3) and transient receptor potential A1 (TRPA1), as a model of histamine-independent itching. Furthermore, crotamiton also suppressed both histamine- and chloroquine-induced calcium influx in primary cultures of mouse DRG. Additionally, crotamiton strongly suppressed histamine- and chloroquine-induced scratching in mice. Overall, it was found that crotamiton has an anti-pruritic effect against non-scabietic itching by histamine and chloroquine. Therefore, crotamiton may be used as a general anti-pruritic agent, irrespective of the presence of scabies.
2019
Kim, Bo Hyun; Lee, Wook-Joo; Sanjel, Babina; Cho, Kyohee; Son, Youn Kyoung; Park, Hye Yoon; Kim, Sun Yeou; Shim, Won-Sik
Extracts of the leaves of Pyrus ussuriensis Maxim. Alleviate itch sensation via TSLP-dependent manner in mouse models of atopic dermatitis Journal Article
In: Physiology & Behavior, vol. 210, 2019, ISSN: 0031-9384.
Abstract | Links | BibTeX | Tags: Animal model, Atopic dermatitis, Calcium imaging, Itch, Scratching behavior, TSLP
@article{Kim2019,
title = {Extracts of the leaves of Pyrus ussuriensis Maxim. Alleviate itch sensation via TSLP-dependent manner in mouse models of atopic dermatitis},
author = {Bo Hyun Kim and Wook-Joo Lee and Babina Sanjel and Kyohee Cho and Youn Kyoung Son and Hye Yoon Park and Sun Yeou Kim and Won-Sik Shim},
doi = {10.1016/j.physbeh.2019.112624},
issn = {0031-9384},
year = {2019},
date = {2019-10-00},
urldate = {2019-10-00},
journal = {Physiology & Behavior},
volume = {210},
publisher = {Elsevier BV},
abstract = {Pyrus ussuriensis Maxim. commonly known as “Sandolbae” in Korean is a pear tree widely distributed across East Asia. Recent studies indicate that P. ussuriensis Maxim. leaves (PUL) have antipruritic effects. This study aimed to determine the effects of PUL extract and its fractions in decreasing the itch sensation and skin lesions in two distinct animal models of atopic dermatitis (AD) induced by dinitrofluorobenzene (DNFB) or house dust mite(HDM). Our results showed that the total ethanol extract of PUL decreased the scratching behavior in mice withDNFB- and HDM-induced AD. Moreover, the ethyl acetate fraction of PUL significantly improved the overallcondition of the mice with AD induced by HDM. Further, we used HEK293T cells that express receptors and ion channels for thymic stromal lymphopoietin (TSLP), a potent pruritogen for AD, to determine the mechanismsunderlying the antipruritic effects of PUL extract/fractions. Specific subfractions of the PUL strongly inhibited the increase in calcium levels induced by TSLP. In addition, the specific subfraction of PUL inhibited the TSLP-induced increase in calcium levels in cultured mouse dorsal root ganglia neurons. Thus, our results showed thatthe PUL extract could be effective for alleviating pruritus, and the antipruritic effects were exerted probably viathe inhibition of the TSLP pathway in peripheral sensory neurons governing the itch sensation in AD.},
keywords = {Animal model, Atopic dermatitis, Calcium imaging, Itch, Scratching behavior, TSLP},
pubstate = {published},
tppubtype = {article}
}
Pyrus ussuriensis Maxim. commonly known as “Sandolbae” in Korean is a pear tree widely distributed across East Asia. Recent studies indicate that P. ussuriensis Maxim. leaves (PUL) have antipruritic effects. This study aimed to determine the effects of PUL extract and its fractions in decreasing the itch sensation and skin lesions in two distinct animal models of atopic dermatitis (AD) induced by dinitrofluorobenzene (DNFB) or house dust mite(HDM). Our results showed that the total ethanol extract of PUL decreased the scratching behavior in mice withDNFB- and HDM-induced AD. Moreover, the ethyl acetate fraction of PUL significantly improved the overallcondition of the mice with AD induced by HDM. Further, we used HEK293T cells that express receptors and ion channels for thymic stromal lymphopoietin (TSLP), a potent pruritogen for AD, to determine the mechanismsunderlying the antipruritic effects of PUL extract/fractions. Specific subfractions of the PUL strongly inhibited the increase in calcium levels induced by TSLP. In addition, the specific subfraction of PUL inhibited the TSLP-induced increase in calcium levels in cultured mouse dorsal root ganglia neurons. Thus, our results showed thatthe PUL extract could be effective for alleviating pruritus, and the antipruritic effects were exerted probably viathe inhibition of the TSLP pathway in peripheral sensory neurons governing the itch sensation in AD.
2018
Ji, Yeounjung; Jang, Yongwoo; Lee, Wook Joo; Yang, Young Duk; Shim, Won-Sik
Different perception levels of histamine-induced itch sensation in young adult mice Journal Article
In: Physiology & Behavior, vol. 188, pp. 188–193, 2018, ISSN: 0031-9384.
Abstract | Links | BibTeX | Tags: Dorsal root ganglia, H1R, Histamine, Itch, Scratching behavior, TLR4, TRPV1
@article{Ji2018,
title = {Different perception levels of histamine-induced itch sensation in young adult mice},
author = {Yeounjung Ji and Yongwoo Jang and Wook Joo Lee and Young Duk Yang and Won-Sik Shim},
doi = {10.1016/j.physbeh.2018.02.015},
issn = {0031-9384},
year = {2018},
date = {2018-05-00},
urldate = {2018-05-00},
journal = {Physiology & Behavior},
volume = {188},
pages = {188--193},
publisher = {Elsevier BV},
abstract = {Itch is an unpleasant sensation that evokes behavioral responses such as scratching the skin. Interestingly, it isconceived that the perception of itch sensation is influenced by age. Indeed, accumulating evidence supports theidea that even children or younger adults show distinctive itch sensation depending on age. This evidence implies the presence of a mechanism that regulates the perception of itch sensation in an age-dependent fashion. Therefore, the purpose of the present study was to investigate a putative mechanism for the age-dependent perception of itch sensation by comparing histamine-induced scratching behaviors in 45-day old (D45) and 75-day old male “young adult” mice. The results indicated that, following histamine administration, the D75 mice spent a longer time scratching than D45 mice. However, the intensity of the calcium influx induced by histamine in primary culture of dorsal root ganglia (DRG) neurons was not different between D45 and D75 mice. Moreover, no apparent difference was observed in mRNA levels of a characteristic His-related receptor and ion channel. Incontrast, the mRNA levels of Toll-Like Receptor 4 (TLR4) were increased approximately by two-fold in D75 DRG compared with D45 DRG. Additionally, D75-derived DRG neurons exhibited enhanced intracellular calcium increase by lipopolysaccharide (LPS, a TLR4 agonist) than those of D45 mice. Furthermore, intensities of calciuminflux induced by histamine were significantly potentiated when co-treated with LPS in D75 DRG neurons, butnot in those of D45 mice. Thus, it appears that D75 mice showed enhanced histamine-induced scratching behaviors not by increased expression levels of histamine-related genes, but probably due to augmented TLR4},
keywords = {Dorsal root ganglia, H1R, Histamine, Itch, Scratching behavior, TLR4, TRPV1},
pubstate = {published},
tppubtype = {article}
}
Itch is an unpleasant sensation that evokes behavioral responses such as scratching the skin. Interestingly, it isconceived that the perception of itch sensation is influenced by age. Indeed, accumulating evidence supports theidea that even children or younger adults show distinctive itch sensation depending on age. This evidence implies the presence of a mechanism that regulates the perception of itch sensation in an age-dependent fashion. Therefore, the purpose of the present study was to investigate a putative mechanism for the age-dependent perception of itch sensation by comparing histamine-induced scratching behaviors in 45-day old (D45) and 75-day old male “young adult” mice. The results indicated that, following histamine administration, the D75 mice spent a longer time scratching than D45 mice. However, the intensity of the calcium influx induced by histamine in primary culture of dorsal root ganglia (DRG) neurons was not different between D45 and D75 mice. Moreover, no apparent difference was observed in mRNA levels of a characteristic His-related receptor and ion channel. Incontrast, the mRNA levels of Toll-Like Receptor 4 (TLR4) were increased approximately by two-fold in D75 DRG compared with D45 DRG. Additionally, D75-derived DRG neurons exhibited enhanced intracellular calcium increase by lipopolysaccharide (LPS, a TLR4 agonist) than those of D45 mice. Furthermore, intensities of calciuminflux induced by histamine were significantly potentiated when co-treated with LPS in D75 DRG neurons, butnot in those of D45 mice. Thus, it appears that D75 mice showed enhanced histamine-induced scratching behaviors not by increased expression levels of histamine-related genes, but probably due to augmented TLR4