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RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

ABSTRACT: The significance of the relationship between the nervous and immune systems with respect to disease course is increasingly apparent. Immune cells in the liver and spleen are responsible for the development of acute liver injury, yet the regulatory mechanisms of the interactions remain elusive. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, regulates innate immune activation via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. Here, we show that RAMP1 in Kupffer cells (KCs) plays a critical role in the etiology of immune-mediated hepatitis. RAMP1-deficient mice with concanavalin A (ConA)-mediated hepatitis, characterized by severe liver injury accompanied by infiltration of immune cells and increased secretion of pro-inflammatory cytokines by KCs and splenic T cells, showed poor survival. Removing KCs ameliorated liver damage, while depleting T cells or splenectomy led to partial amelioration. Adoptive transfer of splenic T cells from RAMP1-deficient mice led to a modest increase in liver injury. Co-culture of KCs with splenic T cells led to increased cytokine expression by both cells in a RAMP1-dependent manner. Thus, immune-mediated hepatitis develops via crosstalk between immune cells. RAMP1 in KCs is a key regulator of immune responses.

SUBMITTER: Inoue T

PROVIDER: S-EPMC6248891 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

Inoue Tomoyoshi T Ito Yoshiya Y Nishizawa Nobuyuki N Eshima Koji K Kojo Ken K Otaka Fumisato F Betto Tomohiro T Yamane Sakiko S Tsujikawa Kazutake K Koizumi Wasaburo W Majima Masataka M

PloS one 20181121 11

The significance of the relationship between the nervous and immune systems with respect to disease course is increasingly apparent. Immune cells in the liver and spleen are responsible for the development of acute liver injury, yet the regulatory mechanisms of the interactions remain elusive. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, regulates innate immune activation via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP recept ...[more]

PMID: 30462657

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Project description:Background: There has been a recent appreciation that some metabolic enzymes can profoundly influence the nature of the immune response produced in macrophages. However, the role of mitochondrial phosphoenolpyruvate carboxykinase (PCK2) in immune response remains unknown. This study aims to investigate the role of PCK2 in lipopolysaccharides (LPS)-induced activation in Kupffer cells. Methods: Inflammatory cytokines were determined by real-time quantitative reverse transcription-polymerase chain action (qRT-PCR) and flow cytometric analysis using a cytometric bead array. Western blotting and immunofluorescence staining were used to determine PCK2 expression and subcellular distribution under confocal laser microscopy. qRT-PCR, flow cytometry, and high-performance liquid chromatography (HPLC) were used to determine mitochondrial function. Pharmacological inhibition, knockdown, and overexpression of PCK2 were used to confirm its function. Co-immunoprecipitation (Co-IP) was performed to determine MAPK/NF-κB phosphorylation. Results: Inflammatory response was significantly increased in LPS-treated mice and Kupffer cells. During the inflammatory process, the protein level of PCK2 was significantly upregulated in Kupffer cells. Interestingly, the localization of PCK2 was mainly in cytosol rather than mitochondria after LPS stimulation. Gain-of-function and loss-of-function analyses found that PCK2 overexpression significantly upregulated the levels of inflammation markers, whereas PCK2 knockdown or inhibition significantly mitigated LPS-induced inflammatory response in Kupffer cells. Furthermore, PCK2 promoted protein phosphorylation of NF-κB and AKT/MAPK, the major signaling pathways, controlling inflammatory cascade activation. Conclusion: We identified a novel function of PCK2 in mediating LPS-induced inflammation and provided mechanistic insights into the regulation of inflammatory response in Kupffer cells. Therefore, PCK2 may serve as a novel therapeutic target for the regulation of Kupffer cells-mediated inflammatory responses.

Project description:Accelerated blood clearance (ABC) phenomenon is common in many PEGylated nanocarriers, whose mechanism has not been completely elucidated yet. In this study, the correlation between Kupffer cells (KCs) and ABC phenomenon has been studied by KCs-targeted liposomes inducing ABC phenomenon and KCs depletion. In other words, the 4-aminophenyl-?-D-mannopyranoside (APM) lipid derivative DSPE-PEG2000-APM (DPM), and 4-aminophenyl-?-L-fucopyranoside (APF) lipid derivative DSPE-PEG2000-APF (DPF) were conjugated and modified on alendronate sodium (AD) liposomes to specifically target and deplete KCs. The dual-ligand modified PEGylated liposomes (MFPL) showed stronger ability to damage KCs in vitro and in vivo, which also could indirectly illustrate that dual-ligand modification could better target KCs. Besides, the hepatic biodistribution and pharmacokinetics could directly prove that MFPL had a stronger targeting ability to KCs. In addition, in depletion rats, plasma concentration and splenic biodistribution of MFPL and PEGylated liposomes (PL) were significantly elevated and hepatic biodistribution was significantly reduced, which demonstrated that KCs played an important role on elimination of nanoparticles. What's more, ABC phenomenon of the secondary injection of PL was stronger in KCs depletion rats than that in normal rats, which indicated that depletion of KCs prolonged the circulation of PL in the first injection repeatedly stimulating B-cells in the marginal region of the spleen and causing it to secrete more IgM antibodies. This could also illustrate that anti-PEG IgM takes up a major station compared with KCs. Most important of all, KCs-targeted liposomes could induce a stronger ABC phenomenon than PL in normal rats, which declared that based on the same IgM concentration, the more the KCs were stimulated, the stronger ABC phenomenon was induced. However, in depletion rats, this difference of ABC phenomenon between PL and MFPL could no more exist, further demonstrating that KCs could participate and play a certain role in the ABC phenomenon.

Project description:Rationale: Kupffer cells (KCs) play a crucial role in liver immune homeostasis through interacting with other immune cells and liver sinusoidal endothelial cells (LSECs). However, how KCs exactly interact with these cells for maintaining the homeostasis still require the further investigation. CXCL10 is a chemokine that has been implicated in chemoattraction of monocytes, T cells, NK cells, and dendritic cells, and promotion of T cell adhesion to endothelial cells. Although CXCL10 is also known to participate in the pathogenesis of hepatic inflammation, the degree to which it is functionally involved in the crosstalk between immune cells and regulation of immune response is still unclear.Methods: To dynamically investigate the function of KCs, we used our recently developed rapid cell ablation model, intermedilysin (ILY)/human CD59 (hCD59)-mediated cell ablation tool, to selectively ablate KC pool under normal condition or concanavalin A (Con A)- induced hepatitis. At certain time points after KCs ablation, we performed flow cytometry to monitor the amount of hepatic infiltrating immune cells. mRNA array was used to detect the change of hepatic cytokines and chemokines levels. Cytokines and chemokines in the serum were further measured by LEGENDplexTM mouse proinflammatory chemokine panel and inflammation panel. Evans blue staining and transmission electron microscopy were used to investigate the interaction between KCs and LSECs in steady condition. CXCL10 neutralizing antibody and CXCL10 deficient mouse were used to study the role of CXCL10 in immune cell migration and pathogenesis of Con A-induced hepatitis.Results: At steady state, elimination of KCs results in a reduction of hepatic infiltrating monocytes, T, B, and NK cells and a list of cytokines and chemokines at transcriptional level. In the meantime, the depletion of KCs resulted in increased sinusoidal vascular permeability. In the pathological condition, the KCs elimination rescues Con A-induced acute hepatitis through suppressing proinflammatory immune responses by down-regulation of hepatitis-associated cytokines/chemokines in serum such as CXCL10, and recruitment of infiltrating immune cells (monocytes, T, B, and NK cells). We further documented that deficiency or blockade of CXCL10 attenuated the development of Con A-induced hepatitis associated with reduction of the infiltrating monocytes, especially inflammatory Ly6Chi monocytes.Conclusions: This study supports the notion that KCs actively interact with immune cells and LSECs for maintaining immune response and liver homeostasis. Our data indicate that the interplay between KCs and infiltrated monocytes via CXCL10 contribute to Con A-induced hepatitis.

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RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

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RAMP1 in Kupffer cells is a critical regulator in immune-mediated hepatitis.

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