Biol Blood Marrow Transplant 19(8): 1210-1219

PMC: PMC3720784

PMID: 23707854

Allogeneic transplants, Fas-signaling, and dysregulation of hepcidin

Introduction

There is evidence that iron overload, which is common in patients undergoing hematopoietic cell transplantation (HCT), has a negative impact on survival after allogeneic HCT [1,2], which extends beyond the acute post-HCT period [3]. Gordon et al. reported an increase in non-transferrin bound iron following transplant conditioning [4]. We showed in a NOD.CB17-Prkdc^scid/NcrCrl (NOD/SCID) mouse model (not requiring pre-transplant conditioning) that transplantation of allogeneic T cells resulted in hepatic iron deposition and down-regulation of the iron-regulator hepcidin (Hamp) in hepatocytes [5]. Activated (donor-derived) T lymphocytes express CD178, the cognate ligand for Fas (CD95) [6,7], and interact with Fas-expressing recipient tissues, including the liver. The liver is involved in the regulation of iron homeostasis [8] and is a major target of graft-versus-host disease (GVHD) [9], although a clear relationship between iron overload and GVHD has not been established. Fas-triggered signals typically initiate apoptosis, which is a histologic hallmark of GVHD.

Hepcidin is a peptide hormone that is essential for the regulation of iron homeostasis via its interaction with ferroportin1 [10]. Several signals affect the activation of Hamp, including interleukin (IL)-6 and STAT3 (Signal transducer and activator of transcription 3) [11]. Based on earlier data [5], we hypothesized that the balance of Hamp activating and inhibiting factors was altered via Fas signals. To circumvent signals induced by a transplant conditioning regimen, we used a model of parent (P) into F1 hybrid transplantation to investigate the effects of (semi-) allogeneic cells on iron homeostasis and Hamp expression in wild type recipients. To further characterize the relevance of the role of Fas-mediated signals we then used agonistic anti-Fas antibodies, which allowed us to exclude other signals that may be mediated by allogeneic cells, such as IL-1β, IL-2 or TNFα, and permitted a side by side comparison of the responses of murine and human hepatocytes in vitro. We determined Fas-induced expression of IL-6, Stat3 and Hamp, dependent upon inhibition or over-expression of FLIP (flice inhibitory protein), which interferes with Fas signaling [12]. Our results show profound effects of Fas signaling, not only on hepatocyte apoptosis, but also on hepcidin expression.

Materials and Methods

Reagents

Hamster anti-mouse Fas monoclonal antibody (MAB) (referred to as aFas, clone JO2) was obtained from BD Biosciences (San Diego, CA), mouse anti-human Fas MAB (clone CH11) from UPSTATE (Lake Placid, NY), rabbit anti-human-hepcidin-25 antibody (#ab 30760) from Abcam (San Francisco, CA), and rabbit anti-human GFP antibody (#2555) from Cell Signaling (Boston, MA). Duplex small interference RNA (siRNA) oligonucleotides (oligos) for the long splice variant of FLIP (FLIPL) were from Qiagen (Valencia, CA). A GFP-containing plasmid, FLIPL-IRES-GFP, and a scrambled sequence IRES-GFP control were generated in our laboratory. The plasmids contain a hepatic locus control region from the apolipoprotein gene, a liver-specific α1-antitrypsin promoter, a portion of human factor IX cDNA, and a bovine growth hormone polyadenylation signal [13]. Lipofectamine RNAiMAX and Lipofectamine LTX were from Invitrogen (Carlsbad, CA). Caspase-8 inhibitor Z-IETD-FMK was obtained from BD Pharmingen (San Diego, CA, USA).

Cell lines

Non-transformed murine and human hepatocyte cell lines, NMH (Balb/c-derived) and HH4, were developed by Jean Campbell, PhD [14,15].

Mice

C57Bl/6 [H-2b], Balb/c [H-2d], C3H [H-2k] and A/J [H-2a] mice were purchased from Jackson Laboratories (Bar Harbor, ME). Female C57BL/6 × C3H [H-2b/k] and Balb/c × A/J [H-2d/a] F1 hybrids were bred at the animal facility of the Fred Hutchinson Cancer Research Center (FHCRC). Mice were 6–8 weeks old at the time of the experiment. Experiments were approved by the Institutional Animal Care and Use Committee of the FHCRC.

Transplantation and analysis of outcome

Three × 10^{CD3+ T cells from C57BL/6 parental semi-allogeneic (referred to as allogeneic) donors were injected via the tail vein into F1 C57BL/6 × C3H recipients (low constitutive iron levels); similarly, 3 × 10}^{T cells from Balb/c donors were injected into F1 Balb/c × A/J recipients (high constitutive iron levels). Syngeneic F1 into F1 controls were carried out in parallel. Blood samples for the determination of iron levels were obtained on day 7 and 14, and mice were sacrificed on day 14.}Hamp expression and iron content in the liver were determined as describe previously [5]. Serum iron levels were measured using Quanti-Chrom Iron Assay Kit (BioAssay Systems, Hayward, CA).

In vitro transfection

Murine and human cell lines (NMH and HH4) or primary murine hepatocytes were plated in 12- (6-) well plates at 1×10^(5×10^{) cells/well in 1 mL of medium without antibiotics, grown to 90%–95% confluence, and transfected with siRNA oligos (FLIPL inhibition) or FLIPL-containing vectors for FLIPL-GFP (or control GFP) over-expression, using Lipofectamine RNAiMAX or Lipofectamine LTX.}

Hydrodynamic in vivo transfection

Based on dose-finding experiments, 150 μg of the plasmid (FLIPL-GFP or scrambled-GFP), diluted in 2 mL phosphate-buffered saline, were injected over 6–8 seconds into the tail veins of Balb/c mice [13].

Harvest of primary hepatocytes

Mice were anesthetized with avertin, a 27 G needle was inserted into the portal vein, and 50 mL of calcium-free Hank’s balanced salt solution (HBSS; Sigma, St. Louis, MO) supplemented with 0.02% ethylene glycol-bis (β-aminoethylether) N,N,N′,N′-tetraacetic acid (EGTA) (Sigma, St.Louis, MO) was infused at 37°C at 5 mL/min, followed by 50 mL of HBSS supplemented with 0.04% collagenase (Invitrogen, Carlsbad, CA). An incision in the inferior vena cava allowed for outflow of excess solution. The liver was chopped, hepatocyte suspensions were filtered through a 70 μm cell strainer, washed with phosphate-buffered saline (PBS), and cultured in adhesion medium [13].

Liver harvest for Immunohistochemistry and immunofluorescent staining

In separate experiments, livers from hydrodynamically transfected mice were harvested without preceding collagenase perfusion. Liver tissues were formalin-fixed for 72 hours and 4 μm sections were cut, deparaffinized and rehydrated in Tris-buffered saline containing 0.1% Tween-20 (TBS-T). Antigen retrieval was performed using used a Black and Decker steamer (Towson, MD) with a 20-minute exposure in preheated Trilogy buffer (Cell Marque, Rocklin CA) followed by 20-minute cooling. Slides were rinsed three times in wash buffer, and subsequent staining was performed at room temperature using a DAKO Autostainer (Carpinteria, CA). Slides were then blocked for 10 minutes in 15% horse serum (Vector Labs, Burlingame CA) in TBS containing 1% bovine serum albumin (BSA). Sections were stained with anti-GFP antibody (Cell Signaling, Boston, MA) and anti-hepcidin antibody (Abcam, San Francisco, CA) which were diluted 1:50 (0.42 μg/ml), incubated on the tissue for 60 minutes, and washed with wash buffer. Antibody staining was detected using biotinylated horse anti-mouse anti-serum (BA-2000, Vector Labs) at 1:200 for 30 minutes, followed by horseradish peroxidase-labeled strep-avidin (016-030-084, Jackson ImmunoResearch, West Grove PA) at a dilution of 1:2000 for 30 minutes. Staining was visualized with 3,3′-diaminobenzidine (DAB, DAKO) for 8 minutes, and slides were counterstained with a 1:4 dilution of hematoxylin (DAKO) for 2 minutes. An irrelevant, A control concentration-matched, isotype-stained slide was evaluated for background staining for each tissue sample. The expression of GFP and hepcidin were determined [5] using a Nikon E800 microscope (Experimental Histopathology Shared Resource, FHCRC).

Liver cell lysates for real-time PCR

Mice were euthanized by CO2 inhalation at 2, 4 or 6 hours after treatment with aFas, livers were minced through a 70 μm mesh, and total RNA was extracted as described [14].

FACS analysis

Apoptosis was determined by flow cytometry, using FITC-conjugated Annexin V as described [16]. Briefly, hepatocytes, modified or unmodified, were cultured in the presence/absence of aFas JO2 (375 ng/mL and 750 ng/mL) or aFas CH4 (500ng/mL and 1000 ng/mL), and apoptosis was determined after 24 hours.

Analysis of human and murine cytokines by real-time PCR

Quantitative reverse transcriptase polymerase chain reaction (qPCR) was carried out using the ABI PRISM 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA). cDNA was generated with the μMACS One-Step cDNA kit (Miltenyi Biotec) using a starting population of 5 × 104 hepatocytes. qRT-PCR was performed with SYBR Green Master Mix and three-step standard cycling conditions using sequence-specific primers. The melting curves were examined to ascertain that a single product was amplified. For quantitative analysis, the observed copy numbers of each gene were normalized to the expression of β-actin. Additionally, we verified that the PCR efficiencies of the ABI assays were >95% and that the slopes of the linear portion of the amplification curves varied by less than 5%.

Human probes

FLIPL (Hs00153439_m1), HAMP (Hs00221783_m1), STAT3 (Hs00374280_m1), IL-6 (Hs00985639_m1), and human β-actin (Hs99999903_m1) were purchase from Applied Biosystem (Carlsbad, CA,USA).

Murine probes

FlipL (Mm01255578_m1), Hamp (Mm04231240_s1), Stat3 (Mm01219775_m1), IL-6 (Mm00446191_m1), and mouse β-actin (Mm00607939_s1) were purchase from Applied Biosystem (Carlsbad, CA,USA).

Electrophoretic Mobility Shift Assay (EMSA)

One × 10^{cells were harvested and washed. Nuclear proteins were extracted using Nuclear Extraction Kit (Panomics, Fremont, CA) and incubated with a biotin end-labelled NF-κB oligonucleotide (5′-CCACAGTTGGGATTTCCCAA CCTGACCAG-3′) for 30 minutes at room temperature. A cold probe was included as a negative control. DNA protein complexes were separated from free oligonucleotides on 6% native polyacrylamide gels. Horseradish peroxidase was used to enhance chemoluminescence (Pierce Biotechnology, Inc.), and membranes were exposed to film.}

Western blot analysis

Liver cell lysates were prepared by sonicating hepatocytes for 4 minutes. Fractions were cleared by centrifugation at 20,000 × g for 10 min. Protein concentrations were quantified by bicinchoninic acid assay (Pierce Biotechnology Inc., Rockford, IL), and equal amounts of protein (30 μg) from each lysate were diluted in Laemmli sodium dodecyl sulfate sample buffer and resolved by electropheresis on 4%-12% Bis-Tris pre-cast NuPage gels (Invitrogen Corp., CA) in running buffer (50 mM 2-(N-morpholino) ethane sulfonic acid 50 mM Tris base, 0.1% SDS and 1 mM EDTA) as per the manufacturer’s instructions. The proteins were then transferred to polyvinylidene difluoride membranes for immunoblotting. The membranes were blocked in 5% nonfat dry milk (NFDM) diluted in Tris buffered saline containing 0.1% Tween-2 0 (TBS-T) for 1 hour at room temperature and then incubated overnight at 4°C in 5% NFDM/TBS-T containing either rabbit anti-STAT3 antibody (1:1000, Cell Signaling, Inc.), rabbit anti-phospho-STAT3 antibody (1:1000, Cell Signaling, Inc.) or anti-p65 antibody (1:1000, Cell Signaling, Inc.). Secondary goat anti-rabbit antibodies (1:1000; Cell Signaling, Inc.) conjugated to horseradish peroxidase were used for enhanced chemoluminescence (Pierce Biotechnology, Inc.), and membranes were exposed to film.

Statistical analysis

Apoptosis, quantitated as the mean ± SEM of the FITC-Annexin V fluorescent intensity, was determined by flow cytometry. Two independent sample values were compared using the two-sample t test; p<0.05 was considered significant.

Reagents

Cell lines

Non-transformed murine and human hepatocyte cell lines, NMH (Balb/c-derived) and HH4, were developed by Jean Campbell, PhD [14,15].

Mice

Transplantation and analysis of outcome

In vitro transfection

Hydrodynamic in vivo transfection

Harvest of primary hepatocytes

Liver harvest for Immunohistochemistry and immunofluorescent staining

Liver cell lysates for real-time PCR

Mice were euthanized by CO2 inhalation at 2, 4 or 6 hours after treatment with aFas, livers were minced through a 70 μm mesh, and total RNA was extracted as described [14].

FACS analysis

Analysis of human and murine cytokines by real-time PCR

Human probes

FLIPL (Hs00153439_m1), HAMP (Hs00221783_m1), STAT3 (Hs00374280_m1), IL-6 (Hs00985639_m1), and human β-actin (Hs99999903_m1) were purchase from Applied Biosystem (Carlsbad, CA,USA).

Murine probes

FlipL (Mm01255578_m1), Hamp (Mm04231240_s1), Stat3 (Mm01219775_m1), IL-6 (Mm00446191_m1), and mouse β-actin (Mm00607939_s1) were purchase from Applied Biosystem (Carlsbad, CA,USA).

Human probes

FLIPL (Hs00153439_m1), HAMP (Hs00221783_m1), STAT3 (Hs00374280_m1), IL-6 (Hs00985639_m1), and human β-actin (Hs99999903_m1) were purchase from Applied Biosystem (Carlsbad, CA,USA).

Murine probes

FlipL (Mm01255578_m1), Hamp (Mm04231240_s1), Stat3 (Mm01219775_m1), IL-6 (Mm00446191_m1), and mouse β-actin (Mm00607939_s1) were purchase from Applied Biosystem (Carlsbad, CA,USA).

Electrophoretic Mobility Shift Assay (EMSA)

Western blot analysis

Statistical analysis

Results

Iron levels and Hamp expression after allogeneic T cell infusion

As shown in Figure 1, syngeneic recipients showed stable plasma iron levels over 7–14 days after transplantation. Recipients of allogeneic T cells, in contrast, showed a biphasic response in plasma iron, more pronounced in the strain with low constitutive than in mice with high constitutive plasma iron levels. Expression of Hamp in allogeneic recipients was significantly lower than in syngeneic recipients (p<0.05). Hepatic iron content in C57BL/6 × C3H mice transplanted with allogeneic cells was higher than in syngeneic recipients (290±112 μg/g dry weight versus 194±65 μg/g dry weight; n=3, p= 0.045). Hepatic iron levels were not determined in Balb/c × A/J mice. Thus, while less pronounced than in NOD/SCID mice, changes in iron content in allogeneic recipients were distinctly different from those in syngeneic recipients.

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Figure 1

T lymphocyte transplantation, serum iron, and hepcidin expression in hepatocytes

F1 mice received semi-allogeneic P or syndenic F1 T lymphocytes. Serum iron was determined on days 0, 7, and 14, and hepcidin mRNA in hepatocytes was determined at autopsy (day 14) (p < 0.05)

FLIPL expression alters Fas-initiated apoptosis in hepatocytes

We had shown previously that hepatic responses similar to those observed with allogeneic cells were also observed with the use of agonistic anti-Fas antibody [17]. Specifically, we had shown that aFas (JO2, at 250–1000 ng/mL for murine cells; CH11, at 250–500 ng /mL for human cells) effectively induced apoptosis in murine (NMH) and human (HH4) hepatocyte lines, respectively [14]. Current results confirm those data (Figure 2A–C) and show that inhibition of FLIPL enhanced apoptosis not only in cell lines, but also in primary murine hepatocytes (Figure 2D–F). Conversely, over-expression of FLIPL decreased the extent of apoptosis (Figure 2G–I).

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Figure 2

FLIPL expression and apoptosis in murine (NMH) and human (HH4) hepatocyte cell lines, and in primary murine hepatocytes

A, B and C) Unmodified cells; D, E and F) Cells with FLIPL inhibition (siFLIPL); G,H and I) Cells overexpressing FLIPL (over-FLIPL). Unmodified cells (A,B and C) were treated with two concentrations of agonistic aFas antibody (JO2, 375 ng/ml and 750 ng/ml for murine cells, and CH11; 250ng/ml and 500 ng/ml for human cells). FLIPL modified cells (D – I) were treated with JO2, 750 ng/ml (murine cells) and CH11; 500 ng/ml (r human cells), respectively; the dose was based on findings in unmodified cells. The extent of apoptosis as determined at 24 hours by Annexin V staining is indicated on the vertical axis. Shown are the mean ± SEM of 3–5 experiments. Note different scales of the vertical axis.

In vitro hepcidin (Hamp) expression in response to inhibition or over-expression of FLIPL

Following siRNA-mediated inhibition of FLIPL in hepatocytes, IL-6, Stat3 and Hamp mRNA were down-regulated (Figure 3A–C); this was reflected in corresponding changes in protein levels (Figure 3D). In addition, p65, a component of NF-κB, was down-regulated (Figure 3D), suggesting that FLIPL-dependent signals also affected NF-κB.

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Figure 3

Gene expression in hepatocyte cell lines and primary hepatocytes transfected with FLIPL specific (siFLIPL) to inhibit FLIPL or scrambled (SCR) siRNA and treated with aFas

(A and C) mRNA expression of FlipL, Hamp, IL-6 and Stat3, in murine NMH cells and primary hepatocytes determined by RT-PCR; (B) Expression of FLIPL, HAMP, IL-6 and STAT3 in human HH4 cells. Changes in gene expression are shown as fold increase over expression in unmodified controls (mean ± SEM of three experiments). The expression pattern was altered significantly in cells transfected with FLIPL specific siRNA compared to SCR controls, in both vehicle treated controls (Veh) and in aFas (JO2; CH11) treated cells. (D) Western blots of cell lysates of murine NMH and human HH4 cells transfected with FLIPL specific (siFLIPL) or scrambled siRNA (SCR) and treated with aFas at two doses. Protein levels of FLIPL, STAT3, phospho STAT3 and NF-κB were reduced in siFLIPL cells in a dose-dependent fashion. The doses of aFas (JO2) were 375 and 750 ng/mL in NMH cells; for human HH4 cells the doses of aFas (CH11) were 250 and 500 ng/ml. β-actin served as loading control.

Responses in hepatocytes over-expressing FLIPL showed the opposite. IL-6, Stat3 and Hamp mRNA were up-regulated (Figure 4A–C), and expression of STAT3 and phospho-STAT3 protein was increased (Figure 4D). Also, levels of p65 were up-regulated following Fas signaling (Figure 4D).

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Figure 4

Gene expression in hepatocyte cell lines and primary hepatocytes over-expressing FLIPL (over-FLIPL) or a scrambled sequence (SCR) and treated with aFas

(A and C) Murine NMH cells and primary hepatocytes; (B) human HH4 cells. Over-expression of FLIPL basically resulted in a pattern opposite to that seen with inhibition of FLIPL (see Figure 1). Treatment with aFas (JO2, murine or CH11, human, respectively) resulted in up-regulation of Hamp, IL-6 and Stat 3. The pattern was similar but less prominent in vehicle treated controls. Shown are the mean ± SEM of 3 or 4 experiments. (D) Western blots of cell lysates of murine NMH and human HH4 cells over-expressing FLIPL (over-FLIPL) or a scrambled sequence (SCR). Shown are protein levels of FLIPL, STAT3, phospho STAT3 and p65. aFas antibody was used at the doses indicated (ng/mL). β-actin served as loading control. (Note different vertical scale in A, B, and C.)

Over-expression of FLIPL up-regulates hepcidin expression in vivo

Kinetics of Hamp expression following treatment with aFas JO2 in unmodified mice is illustrated in Figure 5A. aFas exposure induced a rapid increase in Hamp levels followed by a decline to below baseline by 4–6 hours.

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Figure 5

Over-expression of FLIPL in mouse liver by hydrodynamic transfection and effect on gene expression and survival after treatment with agonistic aFas antibody JO2

(A) JO2 was given at 0.4 μg/g body weight; 4 mice per time point. Changes in Hamp expression are expressed as fold increase in comparison to PBS treated controls. There was a steep increase in Hamp levels followed by a decline to below baseline levels by 4–6 hours. (B) Dose response and time course of transfection with the FLIPL-GFP construct. The highest efficiency was obtained at 16 hr with injection of 150 μg of plasmid DNA. Brown staining reflects the presence of GFP (magnification 20X). (C) Hepcidin expression in mouse liver following hydrodynamic transfection and treatment with aFas JO2. Upper row shows results in livers from mice transfected with the FLIPL-GFP containing plasmid, the lower row with the control-GFP plasmid. The left panels show staining for hepcidin (long white arrows), middle panels for GFP (short yellow arrows), and right panels the merged pictures. Strong auto-fluorescence is present (left panels). The merged picture (right panel, upper row) shows co-localization of FLIPL-GFP (light green) and hepcidin (orange) signals in the same hepatocyte. Only weak hepcidin signals are identifiable in panels in the bottom row, not showing distinct co-localization of hepcidin with control-GFP expressing hepatocytes (original magnification 40X). (D) mRNA expression of Hamp, IL-6 and Stat3 in murine hepatocytes transfected with murine FLIPL vs. a scrambled sequence (SCR) as determined by RT-PCR. At 16 hours after transfection mice were treated with aFas JO2 at 0.4 μg/g body weight, and sacrificed after 4 hours. The liver was harvested and whole liver lysates were prepared for analysis. Expression of Hamp, IL-6 and Stat3 was increased in FLIPL transfected cells, but decreased in controls (SCR). (E) Survival of unmodified mice (control) or mice with hepatic overexpression of FLIPL (over-FLIP) following treatment with aFas JO2 at 0.4μg/g body weight.

The transfection efficiency of FLIPL-GFP (or control plasmid) in liver was 30% at 16 hours following injection of 150 μg of plasmid (Figure 5B). At 16 hours, transfected mice were treated with aFas (JO2, 0.4 μg/g), and after 4 hours intact livers or hepatocytes (following collagenase perfusion) were harvested. Over-expression of FLIPL upregulated IL-6, Stat3 and Hamp as determined by real-time PCR and immunofluorescent staining (Figure 5C,D). All control mice injected with aFas died within 4 hours, while mice over-expressing FLIPL in the liver survived 2 to 3 times longer (Figure 5E) (p=0.001).

Both Fas and FLIPL activate NF-κB in hepatocytes and alter hepcidin expression

NF-κB mediates IL-6 expression [18], and FLIPL is implicated in the regulation of NF-κB [19,20]. We therefore determined the effect of aFas and FLIPL expression on NF-κB activity. aFas (JO2) induced activation of NF-κB as determined by EMSA (Figure 6, lanes 4 vs. lanes 3). Inhibition of FLIPL (Figure 6A) decreased NF-κB activity (lane 5 vs. 3), an effect partially overcome by aFa (lane 6 vs 5). Over-expression of FLIPL (Figure 6B) increased NF-κB activity modestly (lane 5 vs. 3); aFas enhanced that effect (lane 6 vs. 5). These findings are supported by the analysis of nuclear extracts (Figure 6C): p65/phosp65 levels were reduced with FLIPL inhibition, and increased with overexpression of FLIPL. Upon exposure to aFas, FLIPL expression increased and then rapidly declined (Figure 6D). P65 expression increased by 2–4 hours and then gradually decreased. If NF-κB activation was blocked by an IKK-IV inhibitor, only low p65 levels were detectable, and only faint bands of FLIPL were visualized, indicating that FLIPL expression was modified by NF-κB activity (Figure 6D). IL-6, a potent inducer of Hamp, is also regulated by NF-κB, and blockade of NF-κB with IKK inhibitor resulted in down-regulation of IL-6, Stat3 and Hamp mRNA. Comparable results were achieved via inhibition of FLIPL by siFLIPL (Figure 6E), suggesting a regulatory loop involving FLIPL and NF-κB (Figure 7).

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Figure 6

NF-κB activation in hepatocytes by aFas (JO2) treatment or over-expression of FLIPL

Electrophoretic mobility shift assays (EMSA) for NF-κB in NMH hepatocytes. (A) with inhibition of FLIPL [siFLIPL], (B) with overexpression of FLIPL [Over-FLIPL]). Fas signaling triggered by aFas JO2 resulted in activation of NF-κB (lane 4 vs. lane 3). Inhibition of FLIPL (A) decreased NF-κB activity (lane 5 vs. 3); this effect was partially overcome by exposure to aFas (lane 6 vs. lane 5). Over-expression of FLIPL (B), marginally increased NF-κB activity (lane 5 vs. lane 3); exposure to aFas enhanced that effect (lane 6 vs. lane 5). (C) Findings from A) and B) are supported by the analysis of nuclear extracts: p65/phosp65 levels were reduced with FLIPL inhibition, and increased with overexpression of FLIPL. Histone H2 served as loading control. (D) Left panel shows protein levels of intrinsic FLIPL and p65 in hepatocytes. JO2 was used at 1000 ng/mL, and cells were harvested at 0–16 hours. The right panel shows the effect of IKK inhibitor (300nM). Cells were harvested at 0–14 hours. β-actin served as loading control. (E) Changes in expression of Hamp, IL-6 and Stat3 in NMH cells exposed to JO2 (Fas), JO2 plus IKK inhibitor or JO2 + IKK inhibitor in the presence of FLIPL inhibition (siFLIPL). Changes in gene expression are shown as fold change compared to untreated controls (mean ± SEM of three experiments). The expression pattern was altered significantly in cells treated with IKK inhibitor and in cells with FLIPL inhibition (siFLIPL).

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Figure 7

Caspase 8, FLIPL and iron homeostasis – a working model

A) and B) Total caspase 8 expression by western blotting in murine (NMH) and human (HH4) hepatocyte cell lines with inhibition (siFLIPL) or over-expression of FLIPL (Over-FLIP), with or without caspase 8 expression by western blotting in murine (NMH) and human (HH4) hepatocyte cell lines with inhibition (siFLIPL) or over-expression of FLIPL (Over-FLIP), with or without exposure to aFas (JO2 and C11, respectively); SCR=scrambled control. β-actin served as loading control. C) Expression of Hamp, IL-6 and Stat3 in murine (NMH) and human (HH4) hepatocyte cell lines treated with JO2 or C11, respectively, in the absence or presence of caspase 8 inhibitor at two concentrations. Changes in gene expression are shown as fold increase over unmodified controls (mean ± SEM of three experiments). The expression pattern was altered significantly in the presence of a caspase 8 inhibitor (both at 5μM and 10μM) (JO2; CH11). D) Proposed working model. DISC = death inducing signaling complex.

Over-expression of FLIPL reduces caspase 8 levels in hepatocytes

FLIPL opposes caspase-8, thereby potently interfering with signaling through Fas and other death receptors [21–24]. Of note, over-expression of FLIPL was associated with reduced levels of total caspase 8 (inhibition of FLIPL had no significant effect) (Figure 7A and B), and direct inhibition of caspase 8 was followed by increased IL-6, Stat3 and Hamp mRNA (Figure 7C). These data suggest that, in addition to FLIPL-dependent signals, directly caspase 8-related effects may modify iron homeostasis (Figure 7).

Iron levels and Hamp expression after allogeneic T cell infusion

Figure 1

T lymphocyte transplantation, serum iron, and hepcidin expression in hepatocytes

F1 mice received semi-allogeneic P or syndenic F1 T lymphocytes. Serum iron was determined on days 0, 7, and 14, and hepcidin mRNA in hepatocytes was determined at autopsy (day 14) (p < 0.05)

FLIPL expression alters Fas-initiated apoptosis in hepatocytes

Figure 2

FLIPL expression and apoptosis in murine (NMH) and human (HH4) hepatocyte cell lines, and in primary murine hepatocytes

In vitro hepcidin (Hamp) expression in response to inhibition or over-expression of FLIPL

Figure 3

Gene expression in hepatocyte cell lines and primary hepatocytes transfected with FLIPL specific (siFLIPL) to inhibit FLIPL or scrambled (SCR) siRNA and treated with aFas

Figure 4

Gene expression in hepatocyte cell lines and primary hepatocytes over-expressing FLIPL (over-FLIPL) or a scrambled sequence (SCR) and treated with aFas

Over-expression of FLIPL up-regulates hepcidin expression in vivo

Figure 5

Over-expression of FLIPL in mouse liver by hydrodynamic transfection and effect on gene expression and survival after treatment with agonistic aFas antibody JO2

Both Fas and FLIPL activate NF-κB in hepatocytes and alter hepcidin expression

Figure 6

NF-κB activation in hepatocytes by aFas (JO2) treatment or over-expression of FLIPL

Figure 7

Caspase 8, FLIPL and iron homeostasis – a working model

Over-expression of FLIPL reduces caspase 8 levels in hepatocytes

Discussion

We observed previously that NOD/SCID mice transplanted with allogeneic T lymphocytes accumulated iron in hepatocytes while, contrary to expectations, down-regulating hepcidin and increasing the expression of ferroportin 1 [5]. In extensive experiments in wild type and transferrin receptor 2 mutant mice we also showed profound dysregulation of plasma iron levels [17] and induction of apoptosis in hepatocytes [14] in response to agonistic anti-Fas antibody JO2. Since alloactivated T cells express Fas ligand, we hypothesized that the T cell-induced alterations in mouse livers were mediated via Fas [25]. The interpretation of results in allogeneic transplants in wild type mice is complicated by the fact that generally recipients need to be conditioned, and conditioning by itself has potent effects on gene expression. Therefore, in the present study we confirmed our basic hypothesis of disruption of iron regulation by allogeneic cells in a P into F1 (wild type) model, where no recipient conditioning is required. We then further pursued our hypothesis that the observed effects are Fas dependent by using a Fas-specific agonistic MAB. This approach allowed us to exclude other potentially relevant signals derived from allogeneic cells such as IL-1β, TNFα and IL-2, or toll-like receptor dependent pathways, among others.

The current experiments show an increase in plasma iron and liver iron content in F1 hybrids transplanted with parental cells, accompanied by down regulation of Hamp, in comparison to F1 recipients of syngeneic cells, in agreement with the observation in immunodeficient mice [5], although the extent of hepatic iron deposition was less. Additional preliminary data with T cells form gld donors (lacking Fas ligand) showed an increase in Hamp and a decrease in ferroportin1 expression at 7–14 days (unpublished), in strong support of our Fas-based hypothesis. The role of Fas was, therefore, further characterized in the present studies. As expected, treatment with aFas induced apoptosis, and down-regulated Hamp expression in murine and human hepatocyte cell lines as well as in primary murine hepatocytes. Since increasing evidence demonstrates that IL-6 has a protective role during liver injury and is a major growth factor protecting against cell death [26–30], we focused on the role of the IL-6/ STAT3 pathway in Fas-initiated effects on iron homeostasis. As Fas-mediated signals are blocked or modulated by FLIPL, we aimed at determining whether alterations of FLIPL expression would also affect regulation of hepcidin. The combined results obtained with inhibition and over-expression of FLIPL strongly indicate that Fas-dependent signals impact iron homeostasis. Inhibition of FLIPL resulted in down-regulation of IL-6, Stat3 and Hamp mRNA. The converse results were observed following over-expression of FLIPL, with IL-6, Stat3 and Hamp being upregulated. This was true not only in cell lines in vitro, but also in mouse liver in vivo following hydrodynamic transfection. While manipulation of FLIPL by itself affected the expression of iron regulatory genes, this effect was amplified following exposure to aFas.

FLIP is thought to be the molecular switch that allows Fas to promote cell proliferation versus mediating apoptosis [31–33]. FLIPL, one of the three isoforms of FLIP, exerts strong anti-apoptotic function [19]. However, FLIPL also mediates activation of NF-κB by recruiting adaptor proteins, such as Fas-associated death domain (FADD) [23,34,35], and NF-κB potently promotes expression of IL-6 [18], which in turn is followed by STAT3 phosphorylation [36] and induction of hepcidin [37].

Conflicting data have been reported on NF-κB responses to aFas (JO2) signaling in hepatocytes [38,39]. Our experiments support the concept that Fas-mediated signals enhance DNA binding of NF-κB, regardless of the sensitivity or resistance to Fas-mediated cell death [40]. However, the regulation may be more complex. While others reported that FLIPL inhibits Fas-mediated NF-κB activation [35], the present data show that over-expression of FLIPL, in fact, enhanced NF-κB activity. Conceivably, the difference was dependent upon the cell types used and the method of over-expressing FLIPL.

Further, FLIPL interacts with caspase 8 and FADD, forming an apoptosis-inhibiting complex [41]. Interaction of caspase 8 with the N-terminus of FLIPL can induce robust NF-κB activation and IL-6 expression [18,42–45], supporting the notion that caspase 8 inhibition will affect Hamp expression, in agreement with the present data.

Thus, we propose the model shown in Figure 6D. aFas signals activate NF-κB, and this effect is modulated by FLIPL, which may favour either cell death (apoptosis) or survival. The survival pathway involves IL-6 and STAT3 and in turn is associated with up-regulation of hepcidin. How additional signals directly related to caspase 8 contribute to this outcome, remains to be determined. Taken together, the data support a role of Fas-mediated signals in Hamp dysregulation. We suggest that similar signals, mediated by allogeneic T lymphocytes, contribute to the disruption of iron homeostasis and iron accumulation in transplant recipients. Such a model does not preclude the involvement of additional pathways, including, for example, mitochondrial metabolism [46], or hemoxygenase 1 [47]. Also, at least one report [48] suggests that overexpression of FLIPL results in up-regulation of hypoxia-inducible factor-1α (HIFα), and thereby affects iron homeostasis [41]. In view of the requirement of iron for cell survival and the role of FLIPL in the control of cell death, redundancy of regulatory pathways would not be surprising.

Acknowledgments

We thank J. Randolph-Habecker and her team in Experimental Histopathology for assistance with immunohistochemistry and fluorescence analysis, Dr. David Hockenbery for critical input to the experiments, and Helen Crawford and Bonnie Larson for help with manuscript preparation.

Grant support: The authors are grateful for research funding from the National Institutes of Health, Bethesda, MD grants R01HL095999 and P01HL036444. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health nor its subsidiary Institutes and Centers. This work was also supported by a grant from the Community Foundation of Southeast Michigan to X.L., and a grant from the Faculty of Medicine, Siriraj Hospital, Mahidol University, Thailand, to E.K.

^{Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA}

^{School of Medicine, JiangNan University, Wu’Xi, China}

^{Shanghai Jiaotong University School of Medicine, Shanghai, China}

^{The 6}^{People’s Hospital affiliated to Shanghai Jiaotong University, Shanghai, China}

^{Seattle Children’s Hospital, Seattle, WA}

^{Department of Medicine, University of Washington, Seattle, WA}

^{Department of Pathology, University of Washington, Seattle, WA}

^{Benaroya Research Institute, Seattle, WA}

Corresponding Author: H. Joachim Deeg, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, D1-100, Seattle, WA. 98109-1024. gro.crchf@geedj

^{X.L. and F.X. contributed equally to these studies.}

^{Current address:} Department of Pharmaceutical Sciences, Washington State University, Pullman, WA,

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Abstract

Hepatic iron overload is common in patients undergoing hematopoietic cell transplantation. We showed previously in a murine model that transplantation of allogeneic T cells induced iron deposition and down-regulation of hepcidin (Hamp) in hepatocytes. We hypothesized that hepatic injury was related to disrupted iron homeostasis triggered by the interaction of Fas-ligand, expressed on activated T cells, with Fas on hepatocytes. In the current study, we determined the effects of modified expression of the Flice inhibitory protein (FLIP long [FLIPL]), which interferes with Fas signaling, on the impact of Fas-initiated signals on the expression of IL-6 and Stat3 and their down-stream target, Hamp. To exclude a possible contribution by other pathways, we used agonistic anti-Fas antibodies (rather than allogeneic T cells) to trigger Fas signaling. Inhibition of FLIPL by RNA interference resulted, as expected, in enhanced hepatocyte apoptosis in response to Fas signals, but also in decreased levels of IL-6, Stat3 and Hamp. In contrast, over-expression of FLIPL protected hepatocytes against agonistic anti-Fas antibody-mediated apoptosis, and increased the levels of IL-6 and Stat3, thereby maintaining the expression of Hamp in an NF-κB-dependent fashion. In vivo over-expression of FLIPL in the liver via hydrodynamic transfection, similarly, interfered with Fas-initiated apoptosis and prevented down-regulation of IL-6, Stat3 and Hamp. These data indicate that Fas-dependent signals alter the regulation of iron homeostasis and suggest that signals initiated by Fas may contribute to peri-transplant iron accumulation.

Abstract

Footnotes

Financial Disclosure Statement: The authors have no conflicts of interest.

Authors’ contributions:

Xiang Li: Designed and carried out experiments, wrote the manuscript.

Feng Xu: Carried out experiments.

Ekapun Karoopongse: Carried out experiments and provided input to study design.

A. Mario Marcondes: Carried out experiments, critiqued the manuscript.

Kayoung Lee: Assisted in in vivo experiments.

Kris Kowdley: Provided technical input and reagents.

Carol Miao: Provided vectors and technical input.

Grant D. Trobridge: Assisted with experimental design.

Jean Campbell: provided reagents and input into experimental design.

H. Joachim Deeg: Designed study and co-wrote the manuscript.

All co-authors read and critiqued the manuscript.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Footnotes

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