Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes - PubMed (original) (raw)

Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10-bearing chemotactic extracellular vesicles from lipotoxic hepatocytes

Samar H Ibrahim et al. Hepatology. 2016 Mar.

Erratum in

Abstract

Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage-associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage-driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C-X-C motif) ligand 10 (CXCL10)-laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC-treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)-tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)-tagged EV marker, CD63, after LPC treatment of cotransfected Huh-7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone-marrow-derived macrophages with lipotoxic hepatocyte-derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10-neutralizing antisera. MLK3-deficient mice fed a NASH-inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild-type mice.

Conclusions: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10-bearing vesicles from hepatocytes, which are chemotactic for macrophages.

© 2015 by the American Association for the Study of Liver Diseases.

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Conflict of interest statement

Conflict of Interest: The authors report no conflict of interest.

Figures

Figure 1

Figure 1. LPC-induced EV generation is MLK3-dependent

(A) Representative images of nanoparticle tracking analysis (NTA) profile for particle size per concentration of extracellular vesicles (EVs) released from vehicle & lysophosphatidylcholine (LPC)-treated primary mouse hepatocytes (PMH) from wild type (WT) (left panel) & mixed lineage kinase (Mlk3)−/− mice (right panel). (B) Representative transmission electron photomicrograph of EVs derived from vehicle and LPC-treated WT PMH and LPC-treated Mlk3−/− PMH, and isolated by ultracentrifugation. Fold increase above vehicle in EVs release by WT and Mlk3−/− PMH treated with 20 μM LPC for 4 hour, and isolated by (C) ultracentrifugation or (D) by polymer based isolation using a commercially available kit, and quantified by NTA. (E) Huh7 cells were treated with vehicle, or 20 μM LPC with or without 1 μM of one of the MLK3 inhibitors URMC099 (URMC), and CLFB1134 (CLFB), or 20 μM of C-JUN N-terminal kinase (JNK) inhibitor SP600125. EVs were isolated by ultracentrifugation, quantified using NTA, and expressed as percentage of induced LPC release. (F) PMH were treated with OA and PA (both 400 μM for 16 hours), EVs were isolated by ultracentrifugation, and quantified using NTA. Bar columns represent mean ± S.E.M. ** P < .01, * P < .05 compared to vehicle treatment

Figure 2

Figure 2. CXCL10 is highly expressed in lipotoxic EVs in an MLK3-dependent manner

(A) C-X-C ligand 10 (CXCL10) protein levels in extracellular vesicles (EVs) derived from wild type (WT) & mixed lineage kinase (Mlk3)−/− primary mouse hepatocytes (PMH) treated with either vehicle, or 20 μM lysophosphatidylcholine (LPC), and from Huh7 cells treated with either vehicle, or 20 μM LPC with or without 1 μM of one of the MLK3 inhibitors URMC099 (URMC) and CLFB1134 (CLFB), were assessed by western blot. Tumor susceptibility gene 101 (TSG101), cluster of differentiation (CD) 63, and ALG-2-interacting protein (Alix), were employed as EV marker and Cytochrome P450 2E1 (CYP2E1) as a marker of EVs of hepatocytes origin. CXCL10, Phospho-p38, Phospho-signal transducer and activator of transcription (PSTAT) 1 & STAT1 protein levels were also assessed by western blot on whole cell lysate from PMH and Huh7 treated with vehicle, LPC alone, or LPC in the presence of either 1 μM of one of the MLK3 inhibitors URMC, and CLFB or 100 μM of the STAT1 inhibitor Fludarabine (flud). CXCL10, P-STAT1, and STAT1 protein levels were also assessed on whole cell lysate from PMH isolated from chow, and high fat, fructose, and cholesterol diet (FFC)-fed mice, actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as loading control. (B) Immunogold reactivity for CXCL10 on EVs derived from WT & Mlk3−/− PMH was assessed by immunogold electron microscopy. (C) Total RNA was extracted from Huh7 cells treated with either vehicle, or 20 μM LPC with or without 1 μM of one of the MLK3 inhibitors URMC099, and CLFB1134, and mRNA CXCL10 expression was evaluated by real-time qPCR. CXCL10 content was assessed (D) in the whole conditioned media of LPC-treated Huh7 cells, the supernatants obtained after ultracentrifugation and the EVs by enzyme linked immunosorbent assay (ELISA), (E) and also in the serum of FFC-fed mice, the supernatants obtained after ultracentrifugation and the EVs by ELISA. Bar columns represent mean ± S.E.M. NS (non-significant) p> .05, *** P < .001.

Figure 3

Figure 3. MLK3 mediates CXCL10 trafficking by a JNK dependent mechanism

(A) Huh7 cells were co-transfected with green fluorescent protein (GFP)-tagged C-X-C ligand chemokine 10 (CXCL10) and red fluorescent protein (RFP)-tagged cluster of differentiation (CD) 63. Co-localization of both CXCL10 and CD63 on the cell surface and in the extracellular vesicles, in response to LPC treatment with or without one of the MLK3 inhibitors URMC099, and CLFB1134, was assessed respectively by total internal reflectance microscopy (TIRF) and confocal microscopy. (B) CXCL10 protein levels in whole Huh7 cell lysate, treated with either vehicle, or 20 μM lysophosphatidylcholine (LPC) with or without 40 μM of the C-JUN N-terminal kinase (JNK), inhibitor SP600125 (SP) and in extracellular vesicles (EVs) derived from Huh 7 cells under the conditions above were assessed by western blot. Tumor susceptibility gene 101 (TSG101) was employed as an EV marker, and actin as a loading control. (*Irrelevant bands omitted).

Figure 4

Figure 4. Lipotoxic EVs induce macrophage chemotaxis in a CXCL10-dependent manner

(A) Chemokine (C-X-C motif) receptor 3 (CXCR3) the (C-X-C motif) ligand 10 (CXCL10) cognate receptor expression was assessed on the bone marrow derived macrophages (BMDM) cell surface by flow cytometry. (B) Using a modified Boyden chamber, BMDM chemotaxis was assessed by quantification of migrating cells toward either control (media alone), extracellular vesicles (EVs) derived from lysophosphatidylcholine (LPC) treated wild type (WT) primary mouse hepatocytes (PMH) at a concentration of 1011 particle/ml or CXCL10 recombinant mouse protein at a concentration of 500 ng/ml with or without the CXCL10 neutralizing antibody at 25 μg/ml.(C) BMDM chemotaxis was also assessed by quantification of migrating BMDM toward either EVs derived from LPC-treated WT or Mlk3−/− PMH at a concentration of 1011 EV/ml. (D) 1011 EV/ml contain an equivalent concentration of 100 pg/ml of recombinant mouse CXCL10 (rmCXCL10) as measured by enzyme linked immunosorbent assay (ELISA). The chemotactic potency of 0.5 × 1011 EV/ml and 1 × 1011EVs/ml were compared to an equivalent concentration of rmCXCL10 of 50 pg/ml and 100 pg/ml respectively. Bar columns represent mean ± S.E.M. *** P < .001, ** P < .01 compared to vehicle treatment.

Figure 5

Figure 5. Mlk3−/− mice are protected against high fat, fructose and cholesterol (FFC) diet-induced liver injury and inflammation and have reduced plasma level of CXCL10 enriched extracellular vesicles (EVs)

Wild type (WT) & mixed lineage kinase (Mlk3)−/− mice were fed either chow or FFC diet. (A) Plasma alanine aminotransferase (ALT) levels were measured. Total RNA was extracted from liver tissue and mRNA expression of (B) surface macrophage markers cluster of differentiation (CD) 68 and F4/80, (C) macrophage cytokines tumor necrosis factor (TNF) α, and interleukin (IL) 6, and (E) CXCL10 expression were evaluated by real-time qPCR. (D) Enzyme linked immunosorbant assay (ELISA) for (C-X-C motif) ligand 10 (CXCL10) was performed on (E) the whole plasma and EVs isolated from the plasma of FFC and chow fed WT and Mlk3−/− mice. Bar columns represent mean ± S.E.M. *** P < .001, ** P < .01, * P < .05 compared to WT chow-fed mice

Figure 6

Figure 6. Schematic diagram illustrating the link between lipotoxic hepatocytes and macrophages trafficking to the liver in NASH

Lipotoxicity during NASH promotes the release of (C-X-C motif) ligand 10 (CXCL10)-bearing extracellular vesicles (EVs) from hepatocytes by a mixed lineage kinase (MLK)3-dependant mechanism that involves CXCL10 induction through a STAT1-dependent mechanism, and CXCL10 trafficking into EVs through a C-JUN N-terminal kinase (JNK)-dependent mechanism. CXCL10-enriched EVs mediate macrophage chemotaxis to the liver. MLK3 inhibition prevents CXCL10-enriched EVs release from hepatocytes and thus attenuates macrophage chemotaxis.

Comment in

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