Hyunjung Min

Imiquimod Enhances Excitability of Dorsal Root Ganglion Neurons by Inhibiting Background (K2P) and Voltage-Gated (Kv1.1 and Kv1.2) Potassium Channels

BackgroundImiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.FindingsWhen IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K+ channels, Kv1.1 and Kv1.2 (voltage-gated K+ channels) and TREK1 and TRAAK (K2P channels). IQ effectively reduced the currents mediated by both K+ channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at Kv1.1 and Kv1.2.ConclusionsOur results demonstrate that IQ blocks the voltage-gated K+ channels to increase AP duration and K2P channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.

TLR4 enhances histamine-mediated pruritus by potentiating TRPV1 activity

BackgroundRecent studies have indicated that Toll-like receptor 4 (TLR4), a pathogen-recognition receptor that triggers inflammatory signals in innate immune cells, is also expressed on sensory neurons, implicating its putative role in sensory signal transmission. However, the possible function of sensory neuron TLR4 has not yet been formally addressed. In this regard, we investigated the role of TLR4 in itch signal transmission.ResultsTLR4 was expressed on a subpopulation of dorsal root ganglia (DRG) sensory neurons that express TRPV1. In TLR4-knockout mice, histamine-induced itch responses were compromised while TLR4 activation by LPS did not directly elicit an itch response. Histamine-induced intracellular calcium signals and inward currents were comparably reduced in TLR4-deficient sensory neurons. Reduced histamine sensitivity in the TLR4-deficient neurons was accompanied by a decrease in TRPV1 activity. Heterologous expression experiments in HEK293T cells indicated that TLR4 expression enhanced capsaicin-induced intracellular calcium signals and inward currents.ConclusionsOur data show that TLR4 on sensory neurons enhances histamine-induced itch signal transduction by potentiating TRPV1 activity. The results suggest that TLR4 could be a novel target for the treatment of enhanced itch sensation.

Alternatively activated brain-infiltrating macrophages facilitate recovery from collagenase-induced intracerebral hemorrhage.

BackgroundIntracerebral hemorrhage (ICH) is one of the major causes of stroke. After onset of ICH, massive infiltration of macrophages is detected in the peri-hematoma regions. Still, the function of these macrophages in ICH has not been completely elucidated.ResultsIn a collagenase-induced ICH model, CX3CR1+ macrophages accumulated in the peri-hematoma region. Characterization of these macrophages revealed expression of alternatively activated (M2) macrophage markers. In the macrophage-depleted mice, ICH-induced brain lesion volume was larger and neurological deficits were more severe compared to those of control mice, indicating a protective role of these macrophages in ICH. In the ICH-injured brain, mannose receptor-expressing macrophages increased at a delayed time point after ICH, indicating M2 polarization of the brain-infiltrating macrophages in the brain microenvironment. To explore this possibility, bone marrow-derived macrophages (BMDM) were co-cultured with mouse brain glial cells and then tested for activation phenotype. Upon co-culture with glia, the number of mannose receptor-positive M2 macrophages was significantly increased. Furthermore, treatment with glia-conditioned media increased the number of BMDM of M2 phenotype.ConclusionsIn this study, our data suggest that brain-infiltrating macrophages after ICH are polarized to the M2 phenotype by brain glial cells and thereby contribute to recovery from ICH injury.

TLR2-induced astrocyte MMP9 activation compromises the blood brain barrier and exacerbates intracerebral hemorrhage in animal models.

BackgroundThe innate immune response plays an important role in the pathogenesis of intracerebral hemorrhage (ICH). Recent studies have shown that Toll-like receptor 2 (TLR2) is involved in the innate immune response in various neurological diseases, yet neither its role in ICH nor the mechanisms by which it functions have yet been elucidated. We examined these in this study using a collagenase-induced mouse ICH model with TLR2 knock-out (KO) mice.ResultsTLR2 expression was upregulated in the ipsilateral hemorrhagic tissues of the collagenase-injected mice. Brain injury volume and neurological deficits following ICH were reduced in TLR2 KO mice compared to wild-type (WT) control mice. Heterologous blood-transfer experiments show that TLR2 signaling in brain-resident cells, but not leukocytes, contributes to the injury. In our study to elucidate underlying mechanisms, we found that damage to blood–brain barrier (BBB) integrity following ICH was attenuated in TLR2 KO mice compared to WT mice, which may be due to reduced matrix metalloproteinase-9 (MMP9) activation in astrocytes. The reduced BBB damage accompanies decreased neutrophil infiltration and proinflammatory gene expression in the injured brain parenchyma, which may account for the attenuated brain damage in TLR2 KO mice after ICH.ConclusionsTLR2 plays a detrimental role in ICH-induced brain damage by activating MMP9 in astrocytes, compromising BBB, and enhancing neutrophils infiltration and proinflammatory gene expression.

Imiquimod induces a Toll-like receptor 7-independent increase in intracellular calcium via IP(3) receptor activation.

Imiquimod is an itch-promoting, small, synthetic compound that is generally used to treat genital warts and basal cell carcinoma. The pruritogenic effect of imiquimod is considered to be due to TLR7 activation; however that idea has been challenged by our studies showing intact pruritogenic effects of imiquimod in TLR7 KO mice. Thus, the signaling pathways of imiquimod have not been completely elucidated. Here we investigated the novel effects of imiquimod on intracellular calcium ([Ca(2+)]i) signaling. We found that imiquimod induces [Ca(2+)]i increases in PC12 and F11 cells, and even in NIH-3T3 and HEK293T cells, which do not express TLR7. This [Ca(2+)]i increase was due to Ca(2+) release from the internal store without extracellular Ca(2+) influx. Neither FCCP, a mitochondrial Ca(2+) reuptake inhibitor, nor dantrolene, a ryanodine receptor inhibitor, affected the imiquimod-induced [Ca(2+)]i increase. However, 2APB, an IP3 receptor blocker, inhibited the imiquimod-induced [Ca(2+)]i increase. U73122, a PLCβ inhibitor, failed to block the imiquimod-induced [Ca(2+)]i increase. These data indicate that imiquimod triggers IP3 receptor-dependent Ca(2+) signaling independently of TLR7.

Polyamidoamine dendrimer-conjugated triamcinolone acetonide attenuates nerve injury-induced spinal cord microglia activation and mechanical allodynia

Background Accumulating evidence on the causal role of spinal cord microglia activation in the development of neuropathic pain after peripheral nerve injury suggests that microglial activation inhibitors might be useful analgesics for neuropathic pain. Studies also have shown that polyamidoamine dendrimer may function as a drug delivery vehicle to microglia in the central nervous system. In this regard, we developed polyamidoamine dendrimer-conjugated triamcinolone acetonide, a previously identified microglial activation inhibitor, and tested its analgesic efficacy in a mouse peripheral nerve injury model. Result Polyamidoamine dendrimer was delivered selectively to spinal cord microglia upon intrathecal administration. Dendrimer-conjugated triamcinolone acetonide inhibited lipoteichoic acid-induced proinflammatory gene expression in primary glial cells. In addition, dendrimer-conjugated triamcinolone acetonide administration (intrathecal) inhibited peripheral nerve injury-induced spinal cord microglial activation and the expression of pain-related genes in the spinal cord, including Nox2, IL-1β, TNF-α, and IL-6. Dendrimer-conjugated triamcinolone acetonide administration right after nerve injury almost completely reversed peripheral nerve injury-induced mechanical allodynia for up to three days. Meanwhile, dendrimer-conjugated triamcinolone acetonide administration 1.5 days post injury significantly attenuated mechanical allodynia. Conclusion Our data demonstrate that dendrimer-conjugated triamcinolone acetonide inhibits spinal cord microglia activation and attenuates neuropathic pain after peripheral nerve injury, which has therapeutic implications for the treatment of neuropathic pain.

Toll-Like Receptor 3 Contributes to Wallerian Degeneration after Peripheral Nerve Injury

Objective: It is well known that Schwann cells play an important role in Wallerian degeneration after peripheral nerve injury. Previously, we reported that toll-like receptor 3 (TLR3) is expressed on Schwann cells, implicating its role in Schwann cell activation during Wallerian degeneration. In this study, we tested this possibility using TLR3 knock-out mice. Methods: Sciatic nerve-crush injury was induced in wild-type and TLR3 knock-out mice. Histological sections of the sciatic nerve were analyzed for Wallerian degeneration on days 3 and 7 after injury. The level of macrophage infiltration was measured by real-time RT-PCR, flow cytometry and immunohistochemistry. The macrophage-recruiting chemokine gene expressions in the injured nerve were determined by real-time RT-PCR. Results: In TLR3 knock-out mice, the nerve injury-induced axonal degeneration and subsequent axonal debris clearance were reduced compared to in wild-type mice. In addition, nerve injury-induced macrophage infiltration into injury sites was attenuated in TLR3 knock-out mice and was accompanied by reduced expression of macrophage-recruiting chemokines such as CC-chemokine ligands (CCL)2/MCP-1, CCL4/MIP-1β and CCL5/RANTES. These macrophage-recruiting chemokines were induced in primary Schwann cells upon TLR3 stimulation. Finally, intraneural injection of polyinosinic-polycytidylic acid, a synthetic TLR3 agonist, induced macrophage infiltration into the sciatic nerve in vivo. Conclusion: These data show that TLR3 signaling contributes to Wallerian degeneration after peripheral nerve injury by affecting Schwann cell activation and macrophage recruitment to injured nerves.

[EXPRESS] Association of TRPV1 and TLR4 through the TIR domain potentiates TRPV1 activity by blocking activation-induced desensitization

Background We have previously reported that histamine-induced pruritus was attenuated in toll-like receptor 4 (TLR4) knockout mice due to decreased transient receptor potential V1 (TRPV1) sensitivity. Our results implied that TLR4 potentiated TRPV1 activation in sensory neurons; however, the molecular mechanism has yet to be elucidated. In this study, we investigated the molecular mechanisms of TLR4-mediated TRPV1 potentiation using TLR4-deficient sensory neurons and a heterologous expression system. Methods Primary sensory neurons were obtained from wild-type or TLR4 knockout mice, and HEK293T cells expressing TRPV1 and TLR4 were prepared by transient transfection. TRPV1 activity was analyzed by calcium imaging, fluorophotometry, and patch-clamp recording. Subcellular protein distribution was tested by immunocytochemistry and cell surface biotinylation assay. Protein interaction was assessed by western blot and immunoprecipitation assay. Results Direct association between TRPV1 and TLR4 was detected in HEK293T cells upon heterologous TRPV1 and TLR4 expression. In an immunoprecipitation assay using TLR4-deletion mutants and soluble toll/interleukin-1 receptor (TIR) protein, the cytoplasmic TIR domain of TLR4 was required for TLR4-TRPV1 association and TRPV1 potentiation. In TLR4-deficient sensory neurons, the activation-induced desensitization of TRPV1 increased, accompanied by enhanced TRPV1 clearance from the cell membrane upon activation compared to wild-type neurons. In addition, heterologous TLR4 expression inhibited activation-induced TRPV1 endocytosis and lysosomal degradation in HEK293T cells. Conclusion Our data show that direct association between TRPV1 and TLR4 through the TIR domain enhances TRPV1 activity by blocking activation-induced TRPV1 desensitization.

Heme molecule functions as an endogenous agonist of astrocyte TLR2 to contribute to secondary brain damage after intracerebral hemorrhage

Toll-like receptor 2 (TLR2) was recently shown to contribute to secondary brain damage after intracerebral hemorrhage (ICH), although the molecular mechanisms of this contribution are elusive. In this study, we tested the hypothesis that hemin functions as a TLR2 endogenous agonist, causing proinflammatory astrocyte activation and secondary brain damage after ICH. Hemin administration to the mouse brain striatum induced ICH injury and neurological deficits, however, the brain injury volume and neurological deficits due to hemin injection were significantly reduced in TLR2 knock-out (KO) mice. Hemin administration induced neutrophil infiltration and upregulated neutrophil-attracting chemokine and proinflammatory cytokine expression in wild-type (WT) mice; these effects were ameliorated in TLR2 KO mice. Likewise, ICH-induced blood-brain barrier (BBB) damage was also decreased in TLR2 KO mice. This effect was most likely due to reduced matrix metalloproteinase 9 (MMP9) activity in the TLR2 KO mice compared to WT mice. In primary astrocytes, hemin directly induced MMP9 activity as well as proinflammatory cytokine and chemokine expression in a TLR2-dependent manner. Finally, hemin-induced MMP9 activity and proinflammatory gene expression were almost completely blocked by TLR2-neutralizing antibodies. Taken together, our data propose that heme released to the brain parenchyma after ICH injury activates TLR2 in astrocytes and induces inflammatory gene expression and BBB damage, which contribute to secondary brain damage after ICH.