"VSports app下载" Abstract
To test the significance of lipid peroxidation in the development of alcoholic liver injury, an ethanol (EtOH) liquid diet was fed to male 129/SvJ mice (wild-type, WT) and glutathione S-transferase A4–4-null (GSTA4−/−) mice for 40 days. GSTA4−/− mice were crossed with peroxisome proliferator-activated receptor-α-null mice (PPAR-α−/−), and the effects of EtOH in the resulting double knockout (dKO) mice were compared with the other strains. EtOH increased lipid peroxidation in all except WT mice (P < 0. 05). Increased steatosis and mRNA expression of the inflammatory markers CXCL2, tumor necrosis factor-α (TNF-α), and α-smooth muscle actin (α-SMA) were observed in EtOH GSTA4−/− compared with EtOH WT mice (P < 0. 05). EtOH PPAR-α−/− mice had increased steatosis, serum alanine aminotransferase (ALT), and hepatic CD3+ T cell populations and elevated mRNA encoding CD14, CXCL2, TNF-α, IL-6, CD138, transforming growth factor-β, platelet-derived growth factor receptor-β (PDGFR-β), matrix metalloproteinase (MMP)-9, MMP-13, α-SMA, and collagen type 1 compared with EtOH WT mice. EtOH-fed dKO mice displayed elevation of periportal hepatic 4-hydroxynonenal adducts and serum antibodies against malondialdehyde adducts compared with EtOH feeding of GSTA4−/−, PPAR-α−/−, and WT mice (P < 0. 05). ALT was higher in EtOH dKO mice compared with all other groups (P < 0 V体育平台登录. 001). EtOH-fed dKO mice displayed elevated mRNAs for TNF-α and CD14, histological evidence of fibrosis, and increased PDGFR, MMP-9, and MMP-13 mRNAs compared with the EtOH GSTA4−/− or EtOH PPAR-α−/− genotype (P < 0. 05). These findings demonstrate the central role lipid peroxidation plays in mediating progression of alcohol-induced necroinflammatory liver injury, stellate cell activation, matrix remodeling, and fibrosis.
Keywords: alcohol, liver, lipid peroxidation, glutathione S-transferase A4–4, peroxisome proliferator-activated receptor-α, 4-hydroxynonenal
in the liver, chronic alcohol consumption produces oxidative stress, which in turn results in lipid peroxidation of membrane lipids to form highly reactive electrophilic α,β unsaturated aldehydes, such as 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA) (9, 18). These electrophilic aldehydes, which are capable of covalent interactions with protein and nucleic acids and have the potential to modify a variety of cellular functions, have been suggested to play a major role in alcoholic liver disease (ALD) progression (2, 4, 15, 41). Previously, we have demonstrated colocalization of 4-HNE and MDA adducts with hepatic microvesicular lipid deposits in female rats receiving a total enteral nutrition diet containing ethanol (EtOH) (34) V体育官网入口. The accumulation of adducts coincided with development of steatosis but occurred before a proinflammatory response. These findings suggest that lipid peroxidation/adduct formation is an early event in liver injury, and reactive aldehydes have the potential to initiate or amplify proinflammatory and profibrotic responses resulting from early injury (14, 26, 35, 40, 41). In support of these findings, several 4-HNE-mediated mechanisms have been reported, which include increased chemotaxis and recruitment of neutrophils and monocytes in response to liver injury (11, 32), induction of apoptosis via activation of JNK and p38, reduced ERK signaling (18, 45), reduced cellular proliferation, and increased fibrogenesis (45).
Glutathione S-transferase A4–4 (GSTA4) is a phase II detoxification enzyme that eliminates natural and environmental toxicants through glutathione (GSH) conjugation and is highly efficient in conjugating 4-HNE to its inactive form (4, 9, 17, 39). In GSTA4−/− knockout mice, early liver injury associated with carbon tetrachloride-induced lipid peroxidation was significantly elevated (12), suggesting that GSTA4 is a primary cellular defense mechanism against injury associated with reactive aldehydes VSports在线直播. Peroxisome proliferator-activated receptor-α (PPAR-α) is a nuclear hormone receptor and transcription factor that regulates hepatic inflammation and lipid metabolism (30). In PPAR-α−/− mice, exposure to high-fat diets or diets containing EtOH results in increased lipid accumulation, increased markers of oxidative stress, and an increase in the proinflammatory response (1, 22, 25). In this study, we examined the early progression of alcoholic liver injury after 40 days of EtOH exposure in GSTA4−/− and PPAR-α−/− mice and generated a GSTA4−/−/PPAR-α−/− double knockout mouse strain (dKO) to determine whether EtOH-associated accumulation of reactive aldehydes, particularly 4-HNE, would have a significant effect on hepatic steatosis and inflammatory and fibrogenic responses associated with the PPAR-α−/− genotype.
MATERIALS AND METHODS (VSports在线直播)
Animals and experimental design.
All the animal studies described below were approved by the Institutional Animal Care and Use Committee at the University of Arkansas for Medical Sciences (UAMS). All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals at an American Association for Accreditation of Laboratory Animal Care-approved animal facility at UAMS. Sv129/J mice and 129S4/SvJae-Pparatm1Gonz/J (PPAR-α−/− mice, stock no. 003580) were purchased from Jackson Laboratories (Bar Harbor, ME). GSTA4−/− mice were generated as previously described (13). PPAR-α/GSTA4 dKO mice were generated by breeding the single knockout strains to produce offspring heterozygous for both genes and subsequent breeding of the F1 generation. For PCR detection of the null alleles, DNA isolated from tail snips was assessed with two primer sets as follows: GSTA4−/−, a sense-strand primer 5′ tccaatacacaaaaatgcatga 3′ and two anti-sense strands, 5′ GATGGCCCTGGTCTGTGTCAGC 3′ and 5′ CTGTCCATCTGCACGAGACTAGTG 3′, specific for the WT and mutant mGSTA4 gene, respectively (13); and for PPAR-α−/−, sense-strand 5′ GAG AAG TTG CAG GAG GGG ATT GTG and anti-sense primers 5′ CCC ATT TCG GTA GCA GGT AGT CTT 3′, specific for WT gene, and 5′ GCA ATC CAT CTT GTT CAA TGG C 3′, specific mutant mPPAR-α gene. F2 pups homozygous for both mutant alleles were bred to generate a PPAR-α/GSTA4 dKO colony. For the EtOH exposure study, 13-wk-old male Sv129 WT, GSTA4−/−, PPAR-α−/−, and dKO mice (n = 6–8/group) were randomly assigned to be fed Lieber-DeCarli liquid diets containing EtOH or were pair fed (PF) Lieber-DeCarli high-fat control liquid diets for 6 wk as previously described (42). Initially, all mice received the control liquid diet, which consisted of 35% energy from fat, 18% from protein, and 47% from carbohydrate. In the EtOH groups, EtOH calories were substituted for carbohydrate calories, and mice were acclimated to the diet by increasing the percentage of EtOH slowly to a maximum of 28% of total calories (5% vol/vol) and maintained until death. All groups had ad libitum access to water. Mice given the control diet were isocalorically PF to their corresponding EtOH group based on the diet consumption of the previous day V体育2025版. Animal body weights were measured weekly. At death, liver was weighed, and pieces were formalin fixed and frozen for further analysis. Blood EtOH concentrations (BEC) were determined as previously described (30).
Lipid peroxidation.
Overall liver lipid peroxidation was assessed by a thiobarbituric acid reactive substrate (TBARS) assay as described by Ohkawa et al. (24). Liver 4-HNE adducts were detected immunohistochemically and quantified as previously described by Shearn et al. (36). Immunohistochemical characterization was performed using rabbit polyclonal anti-4-HNE and goat anti-rabbit polyclonal antibodies Vectastain ABC IHC kit (Vector Laboratories Burlingame, CA). Pictures were taken on a NIKON Eclipse TE300 at ×100 magnification using a DS-Fi2 camera. Quantification was done using NIS Elements V4. 13. 04, with three measurements per zone (centrilobular or periportal), four exposures per slide, and four animals per condition VSports. Exposure time was 24 ms, and the area of measurement was 100 × 100 pixels. Overall changes in staining were quantified by using the ratio of staining in the periportal region compared with the centrilobular region (zone 1: zone 3). Bovine serum albumin adducts with HNE and MDA were prepared as described by Mottaran et al. (21). Colocalization studies were performed in liver sections from EtOH-treated dKO mice to determine whether 4-HNE adducts occurred in Kupffer cells or stellate cells in addition to hepatocytes. Serial sections were stained for 4-HNE, F4/80 (ABD Serotec; Bio-Rad, Hercules, CA), (a Kupffer cell marker), or for the appearance of α-smooth muscle actin (α-SMA) (Sigma, St. Louis, MO) (a marker of activated stellate cells). Antibodies to adducted proteins in rat serum were measured in Microwell plates coated with modified or native BSA as described previously (21, 29, 30).
Liver pathology.
Liver samples were fixed in 10% neutral buffered formalin and processed, and paraffin-embedded sections were stained with hematoxylin and eosin (H and E). H and E-stained liver sections were scored for steatosis, inflammation (macrophage infiltration), and necrosis by a veterinary pathologist with no prior knowledge of the treatment groups. Steatosis was scored as the percentage of parenchymal cells containing fat (micro- or macrosteatosis) as <5% = 0, 5–33% = 1, >33–66%% = 2; >66% = 3 (30). Presence of contiguous patches of microsteatosis was given a weighted score of 1. The presence of inflammation based on infiltration by polymorphonuclear cells, leukocytes, and mononuclear cells was evaluated using a scale where no inflammation = 0; <2 foci of inflammatory cells per ×200 field = 1, 2–4 foci per ×200 field = 2, >4 foci per ×200 field = 3. For scoring of necrosis, the presence of necrotic foci was assessed using a scale where 0 = none, 1 = few, and 2 = many. Fibrosis was detected histologically by picrosirius red staining of collagen (20). Inflammatory cell populations were also assessed by immunohistochemical staining using F4/80 as a marker of macrophages and CD3 as a marker of thymus-derived intrahepatic T cells. F4/80 staining was done using a 1:100 dilution of rat anti-mouse antibody overnight with Vector Laboratories ABC system. CD3 staining used a Dako rabbit anti-human antibody dilution 1:500 for 60 min and Vector Laboratories IMMPRESS system. In both cases, the chromogen was diaminobenzidine. In the case of F4/80, the counterstain was Gills hematoxylin. In the case of CD3, the counterstain was Harris hematoxylin. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining using the In Situ Cell Death Detection Kit from Roche Diagnostics (Indianapolis, IN). Hepatic cellular proliferation was measured by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) expression as described by Greenwell et al VSports app下载. (15). Nuclei of S-phase cells were stained dark brown.
Biochemical and molecular assessment of liver injury.
Serum alanine aminotransferase (ALT) levels were assessed as a measure of liver damage by using the Infinity ALT liquid stable reagent (Thermo Electron, Waltham, MA). Triglycerides were extracted from liver homogenates with chloroform/methanol (2:1 vol/vol), and the triglyceride concentration was assayed using commercially available reagents (Cayman Chemical, Ann Arbor, MI). Hepatic markers of inflammation and macrophage infiltration, immune cell subclasses, matrix remodeling, and fibrosis were assessed by real-time RT-PCR V体育官网. Total RNA was isolated from tissue using Tri reagent (MRC, Cincinnati, OH) per manufacturer's protocol and reversed transcribed using iScript cDNA synthesis kit (Bio-Rad). Gene expression was determined by use of SYBR green and an ABI 7500 sequence detection system (Applied Biosystems, Foster City, CA). Results were quantified using the ΔCt method relative to 18S and then to WT PF controls. Gene-specific primers are presented in Table 1.
Table 1.
Real-time RT-PCR primer sequences
Gene | Forward Sequence (5′-3′) | Reverse Sequence (5′-3′) |
---|---|---|
Inflammation and infiltration | ||
TNF-α | GACGTGGAACTGGCAGAAGAG | GCCACAAGCAGGAATGAGAAG |
IL-6 | CTTCACAAGTCGGAGGCTTAAT | GCAAGTGCATCATCGTTGTTC |
IL-10 | ACAGCCGGGAAGACAATAAC | CAGCTGGTCCTTTGTTTGAAA |
TGF-β | GTGCGGCAGCTGTACATTGACTTT | TGTACTGTGTGTCCAGGCTCCAAA |
CD14 | CTAAGTATTGCCCAAGCACACTCA | CCCAACTCAGGGTTGTCAGACA |
CD68 | TTCTCCAGCTGTTCACCTTGACCT | GTTGCAAGAGAAACATGGCCCGAA |
CD45 | AACTTCTGGCCTTTGGATTTGCCC | TATGGTTGTGCTTGGAGGGTCAGT |
CXCL1 | GTGTCTAGTTGGTAGGGCATAAT | CAGTCCTTTGAACGTCTCTGT |
CXCL2 | TAAGCACCGAGGAGAGTAGAA | GTCCAAGGGTTACTCACAACA |
CD4 | TCCCACTCACCCTCAAGATA | ATAACCACCAGGTTCACTTC |
B220 | CCCTTCTTCTGCCTCAAAGT | CACCTGGATGATATGTGGTCTC |
IFN-γ | ATCGGCTGACCTAGAGAAGA | AGCCAAGATGCAGTGTGTAG |
CD138 | ATGCGTACAACAGGGTATGG | GAGGTGGCTATTCCACAGTATC |
Stellate cell activation, matrix remodeling, and fibrosis | ||
α−SMA | ACTACTGCCGAGCGTGAGAT | AAGGTAGACAGCGAAGCCAA |
Collagen type 1 | AGGCTTTGATGGACGCAATG | GCGGCTCCAGGAAGACC |
PDGFR | CCTCGGCCTGTGACTAGAAG | GGACGAGGGGAACAACATTA |
MMP-9 | TTCTCGAATCACGGAGGAAGCCAA | AAGGCTGAGTTCAACTTTGCAGGC |
MMP-13 | TGGCTTAGAGGTGACTGGCAAACT | TATTCACCCACATCAGGCACTCCA |
Statistical analysis.
Data are presented as means ± SE. The effects of the EtOH on WT, GSTA4−/−, PPAR-α−/−, and dKO genotype and the interaction thereof were determined using a four- by two-way ANOVA, followed by Student's Newman-Keuls post hoc analysis. Statistical significance was set at P < 0.05. SigmaPlot software package 11.0 (Systat Software, San Jose, CA) was used to perform all statistical tests. Further statistical analysis of genotype X EtOH interactions between the WT and GSTA4−/− mice was performed using two-way ANOVA followed by Student's Newman Keuls post hoc analysis.
RESULTS
Effects of genotype, high-fat diet, and alcohol on growth, body composition, and EtOH metabolism.
No significant effect of genotype was observed on body weight of PF mice after 6 wk of liquid diet feeding (Table 2). In contrast, final body weight was reduced following EtOH feeding in WT, GSTA4−/−, and dKO mice but not in PPAR-α−/− mice (P < 0.05) despite no significant genotype differences in EtOH diet intake or BEC attained (Table 2). Absolute liver weight and liver weight as a percentage of body weight were higher in PF PPAR-α−/− mice and PF dKO mice than in PF WT or PF GSTA4−/− mice (P < 0.05). EtOH treatment increased absolute weight of livers in PPAR-α−/− mice and dKO mice over PF groups of the same genotype (P < 0.05). EtOH treatment also increased relative liver weight over PF mice of all genotypes (P < 0.05). However, relative weight of livers in the EtOH PPAR-α−/− mice and EtOH dKO mice was higher than that of the EtOH WT or EtOH GSTA4−/− groups (P < 0.05) (Table 2). Increased liver weight in PF PPAR-α−/− mice and PF dKO mice was accompanied by increased accumulation of triglycerides (P < 0.05) (Table 2) and the appearance of fat in centrilobular hepatocytes (Fig. 1). EtOH treatment resulted in triglyceride accumulation and panlobular macrosteatosis in the EtOH GSTA4−/− mice compared with the PF GSTA4−/− group and EtOH WT group (P < 0.05) (Tables 2 and 3, Fig. 1). Triglyceride content and appearance of steatosis were higher in both EtOH PPAR-α−/− mice and EtOH dKO mice than in EtOH WT or EtOH GSTA4−/− mice (P < 0.05). Fat accumulation was not significantly higher in either EtOH PPAR-α−/− mice and EtOH dKO mice compared with their respective PF controls (Tables 2 and 3). However, the pattern of steatosis in the EtOH-treated PPAR-α−/− and dKO mice was shifted from centrilobular to panlobular compared with their PF controls (Fig. 1).
Table 2.
Effects of EtOH consumption on growth, body, and liver weight
Diet Intake*, ml/day | Mean BEC*, mg/dl | BW†, g | Liver Weight†, g | Liver Weight/BW†, % | Liver Triglycerides†, mg/g liver | |
---|---|---|---|---|---|---|
Group | ||||||
WT PF | 27.6 ± 0.93c | 0.97 ± 0.03a | 3.3 ± 0.13a | 104.3 ± 13.6a | ||
WT EtOH | 14.2 ± 0.19 | 171.7 ± 102.5 | 21.3 ± 0.66a | 0.86 ± 0.04a | 4.0 ± 0.13b | 133.0 ± 32.5a |
GSTA4−/− PF | 26.2 ± 0.73c | 0.80 ± 0.03a | 3.0 ± 0.06a | 65.4 ± 18.8a | ||
GSTA4−/− EtOH | 16.2 ± 0.43 | 109.2 ± 94.9 | 22.1 ± 0.40a | 0.90 ± 0.09a | 3.9 ± 0.14b | 255.0 ± 45.0b |
PPAR-α−/− PF | 29.6 ± 0.87c | 1.23 ± 0.11b | 4.1 ± 0.27b | 554.8 ± 37.7c | ||
PPAR-α−/− EtOH | 15.1 ± 0.5 | 98.5 ± 113.8 | 26.5 ± 1.33b | 1.90 ± 0.08d | 7.2 ± 0.38c | 603.9 ± 92.0c |
dKO PF | 28.6 ± 0.62c | 1.27 ± 0.18b | 4.4 ± 0.25b | 565.9 ± 78.7c | ||
dKO EtOH | 15.1 ± 0.48 | 119.4 ± 89.5 | 21.5 ± 0.80a | 1.48 ± 0.06c | 6.9 ± 0.31c | 686.7 ± 39.8c |
P Value | ||||||
Genotype | <0.001 | <0.001 | ||||
EtOH | <0.001 | <0.001 | ||||
Interaction | 0.0056 | <0.001 | <0.001 | 0.115 |
Data are means ± SE for n = 6 mice/group. Blood alcohol concentration (BEC) and hepatic triglyceride concentrations were determined as described in materials and methods.
BW, body weight; WT, wild-type; PF, pair-fed; EtOH, ethanol; GSTA4−/−, glutathione S-transferase A4-4-null; PPAR-α−/−, peroxisome proliferator-activated receptor-α-null; dKO, double knockout.
Significance was determined by 1-way ANOVA followed by Student's Newman-Keuls post hoc analysis.
Significance was determined by a 4- by 2-way ANOVA followed by Student's Newman-Keuls post hoc analysis. Groups with different letter subscripts are significant from each other, P < 0.05, a < b < c < d.
Fig. 1.
Representative hematoxylin and eosin-stained liver sections from wild-type (WT), glutathione S-transferase A4–4-null (GSTA4−/−), peroxisome proliferator-activated receptor-α-null mice (PPAR-α−/−), and double knockout (dKO mice) fed a Lieber-DeCarli ethanol (EtOH) or control pair-fed (PF) diet as described in materials and methods. Magnification, ×10. SV, 129/SvJ mice. CV, central vein; PT, portal triad.
Table 3.
Effects of EtOH consumption on nonfibrotic liver pathology
Pathology Score |
||||||
---|---|---|---|---|---|---|
Steatosis | Inflammation | Necrosis | Serum ALT, U/l | Apoptosis, % TUNEL-positive cells | PCNA, % S-phase | |
Group | ||||||
WT PF | 1.1 ± 0.26a | 0.66 ± 0.10a | 0.66 ± 0.49a | 29.4 ± 11.2a | 0.06 ± 0.03a | 0.96 ± 0.08a |
WT EtOH | 1.2 ± 0.40a | 0.35 ± 0.20a | 0.25 ± 0.25a | 38.6 ± 4.4a | 0.14 ± 0.02a | 1.30 ± 0.23a |
GSTA4−/− PF | 1.3 ± 0.13a | 0.40 ± 0.15a | 0.00 ± 0.00a | 35.5 ± 14.8a | 0.05 ± 0.03a | 0.92 ± 0.08a |
GSTA4−/− EtOH | 2.0 ± 0.01b | 0.26 ± 0.13a | 0.00 ± 0.00a | 74.8 ± 15.1a | 0.14 ± 0.06a | 2.21 ± 0.26a,b |
PPAR-α−/− PF | 2.6 ± 0.16c | 1.30 ± 0.14b | 1.90 ± 0.34b | 62.9 ± 13.5a | 0.08 ± 0.03a | 1.34 ± 0.17a |
PPAR-α−/− EtOH | 2.9 ± 0.03c | 1.20 ± 0.20b | 0.60 ± 0.22a | 190 0.3 ± 21.1b | 0.15 ± 0.03a | 2.81 ± 0.33b |
dKO PF | 2.5 ± 0.17c | 1.10 ± 0.01b | 1.33 ± 0.28b | 53.7 ± 5.5a | 0.18 ± 0.02a | 1.82 ± 0.15a |
dKO EtOH | 2.7 ± 0.17c | 0.90 ± 0.22b | 0.16 ± 0.11a | 297.0 ± 40.7c | 0.27 ± 0.08a | 2.67 ± 0.42b |
P value | ||||||
Genotype | <0.001 | <0.001 | <0.001 | 0.012 | <0.001 | |
EtOH | 0.022 | 0.090 | <0.001 | 0.011 | <0.001 | |
Interaction | 0.323 | 0.836 | 0.093 | <0.001 | 0.991 | 0.288 |
Data are means ± SE for n = 6 mice/group. Pathology score was assigned as defined under materials and methods; normal pathology score is steatosis = 0 and inflammation/necrosis = 0. ALT, alanine aminotransferase activity; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling, PCNA, proliferating cell nuclear antigen. Significance was determined by 4- by 2-way ANOVA followed by Student's Newman-Keuls post hoc analysis. Groups with different letter subscripts are significant from each other, P < 0.05.
Effects of genotype, high-fat diet, and alcohol on lipid peroxidation.
4-HNE protein adducts were measured in fixed liver by immunohistochemistry, as described previously (36). Representative slides are shown in Fig. 2A, and image quantification of lobular staining is shown in Fig. 2B. There was no discernable effect of genotype on lobular distribution of protein adducts in PF mice, and EtOH treatment did not significantly increase staining in WT mice. In contrast, EtOH treatment increased periportal 4-HNE adducts compared with PF controls in GSTA4−/− mice and especially in PPAR-α−/− mice and in dKO mice (P < 0.05). Staining was higher in the EtOH PPAR-α−/− mice than the EtOH GSTA4−/− mice and in the EtOH dKO mice than in either single knockout (P < 0.05). HNE adducts appeared to be specific for hepatocytes, and no colocalization of HNE staining was observed with staining using antibodies against F4/80, a macrophage marker, or with staining using antibodies against α-SMA, a marker of activated stellate cells in serial liver sections from EtOH-treated dKO mice (data not shown). Overall levels of liver lipid peroxidation as measured colorimetrically by TBARS were greater in PF dKO mice than in PF WT, GSTA4−/−, and PPAR-α−/− mice but did not reach significance (P = 0.06) (Fig. 2C). EtOH treatment did not significantly increase overall lipid peroxidation in WT or GSTA4−/− mice relative to their PF controls, but total lipid peroxidation products were increased in EtOH-treated PPAR-α−/− mice. In EtOH dKO mice, TBARS were greater than in EtOH GSTA4−/− or EtOH WT mice (P < 0.05) but were similar to values found in PPAR-α−/− mice and not significantly different from values found in the PF dKO mice. Circulating autoantibodies recognizing 4-HNE and MDA protein adducts were measured by ELISA, and the data are shown in Fig. 3. No genotype differences were observed between mean values for pooled samples from the PF groups, and PF data were collapsed into one group to allow statistical comparisons with EtOH treatment. EtOH increased circulating antibodies against 4-HNE protein adducts in all mouse genotypes relative to PF (P < 0.05). However, there were no genotype differences between EtOH-treated groups. In contrast, EtOH treatment increased antibody titer against MDA-adducted proteins equally in the WT, GSTA4−/−, and PPAR-α−/− groups compared with PF (P < 0.05), but these antibodies were present at higher levels in serum from EtOH dKO mice than in the other EtOH-treated groups (P < 0.05).
Fig. 2.
Immunohistochemical characterization of hydroxynonenal (4-HNE) adducts and overall lipid peroxidation in PF and EtOH-fed WT, GSTA4−/−, PPAR-α−/−, and dKO mice. A: representative sections of 4-HNE staining intensity between the groups. B: quantification of 4-HNE adducts expressed as a ratio of zone 1 to zone 3. C: thiobarbituric acid reactive substrate (TBARS). Data expressed as means ± SE. Groups with different letter subscripts are significant from each other, P < 0.05.
Fig. 3.
Accumulation of autoantibodies to malondialdehyde (MDA) and 4-HNE protein adducts in the serum of WT, GSTA4−/−, PPAR-α−/−, and dKO mice receiving EtOH. A: MDA. B: 4-HNE. Data expressed as means ± SE. Groups with different letter subscripts are significant from each other, P < 0.05.
Effects of genotype, high-fat diet, and alcohol on progression of liver injury beyond simple steatosis.
Liver pathology data are presented in Table 3. In general, despite chronic feeding of high-fat diet and EtOH, inflammation and necrosis pathology scores were low in both PF and EtOH-treated mice.
In both the PF and EtOH groups, inflammatory infiltrates as measured by histological analysis were higher in PPAR-α−/− mice and dKO mice than in the WT or GSTA4−/− groups (P < 0.05), but there were no significant differences between PF and EtOH groups within any genotype. However, in the dKO mice, EtOH did significantly increase the CD68/CD45 ratio, a marker of leukocyte activation and infiltration (23, 25), compared with all other genotypes receiving alcohol (Fig. 4). Analysis of additional immune cell subtypes and of the Th1 response in these animals is presented in Figs. 5 and 6. mRNA expression of the T-helper cell marker CD4 (43) was suppressed in PF GSTA4−/− mice relative to PF WT mice (P < 0.05) and suppressed by chronic EtOH treatment in WT and dKO mice relative to their high-fat PF groups (P < 0.05). In contrast, CD3+ T cell numbers were increased by EtOH treatment relative to the PF groups in both PPAR-α−/− and dKO mice (Fig. 6). Expression of mRNA for IFN-γ, a marker of the Th1 response (10, 29), was significantly elevated in PF PPAR-α−/− mice and PF dKO mice compared with PF WT or GSTA4−/− groups (P <0.05). However, this response was also suppressed relative to the PF groups after EtOH treatment in both groups of mice lacking PPAR-α (P < 0.05). Expression of mRNA encoding B220, a marker of B cells (6), was decreased in PF GSTA4−/− mice relative to PF WT mice but was increased in PPAR-α−/− mice and dKO mice from the PF groups compared with either PF WT or GSTA4−/− mice (P < 0.05). Expression of B220 mRNA was suppressed relative to PF in EtOH-treated WT and dKO mice (P < 0.05). Interestingly, expression of CD138 mRNA, a marker of B cell differentiation into immunoglobulin-secreting plasma cells (8), was increased in PF PPAR-α−/− mice and dKO mice from the PF groups compared with either PF WT or GSTA4−/− mice (P < 0.05) and increased by chronic EtOH treatment in all mouse genotypes relative to their PF groups (P < 0.05). Molecular markers of Kupffer cell activation and chemokine and cytokine production were also examined. CD14 mRNA expression, a marker of Kupffer cell activation (10), was significantly higher in the EtOH-treated PPAR-α−/− mice compared with the EtOH-treated WT and GSTA4−/− mice (Fig. 4) but lower compared with the EtOH-treated dKO mice (P < 0.05). Macrophage numbers as measured by immunohistochemical analysis of F4/80 staining were unchanged by EtOH in either PPAR-α−/− or dKO mice (Fig. 6). Data on hepatic expression of mRNAs encoding chemokines and cytokines are shown in Table 4. CXCL2 mRNA was significantly increased in PF PPAR-α−/− mice and PF dKO mice compared with PF WT or PF GSTA4−/− groups (P <0.05). In addition, EtOH treatment increased CXCL2 mRNA expression over PF groups in the EtOH dKO and EtOH PPAR-α−/− mice (P < 0.05). Likewise, CXCL1 mRNA was also significantly increased by EtOH treatment over PF groups in the PPAR-α−/− and dKO mice. Tumor necrosis factor-α (TNF-α) mRNA was expressed at higher levels in PF PPAR-α−/− mice and PF dKO mice compared with PF WT or PF GSTA4−/− groups (P <0.05). In addition, TNF-α mRNA expression was increased by EtOH over PF groups in GSTA4−/− and dKO mice (P < 0.05) but not in WT or PPAR-α−/− mice (Table 4). IL-6 mRNA was unchanged by genotype in the PF groups but was elevated by EtOH treatment in the PPAR-α−/− mice. In contrast, IL-10 mRNA expression was not significantly affected by genotype or EtOH treatment (Table 4).
Fig. 4.
Effects of EtOH on hepatic Kupffer cell activation and leukocyte infiltration and activation in WT, GSTA4−/−, PPAR-α−/−, and dKO mice. A: CD14 mRNA expression. B: CD68:CD45 ratio. Data expressed as means ± SE. Groups with different letter subscripts are significant from each other, P < 0.05.
Fig. 5.
Effects of high-fat PF and EtOH on molecular markers of T-helper cells (CD4 mRNA), hepatic Th-1 response (IFN-γ mRNA), B cell recruitment (B220 mRNA), and B cell differentiation to plasma cells (CD138 mRNA) in WT, GSTA4−/−, PPAR-α−/−, and dKO mice. Data expressed as means ± SE. Groups with different letter subscripts are significant from each other, P < 0.05.
Fig. 6.
Effects of EtOH on hepatic macrophage and CD3+ T cell populations in PPAR-α−/− and dKO mice. Top: representative immunohistochemical staining of liver sections from pair-fed (PF) and EtOH-treated mice. Left: macrophage staining with F4/80. Right: T cell staining with CD3. Bottom: quantitation of immunohistochemical staining in 4 random liver sections from each treatment group. Data expressed as means ± SE. EtOH groups with * are significant from PF, P < 0.05.
Table 4.
Relative hepatic cytokine and chemokine mRNA expression in WT, GSTA4−/−, PPAR-α−/−, and dKO mice following EtOH consumption
mRNA Expression | ||||||
---|---|---|---|---|---|---|
TNF-α | IL-6 | Il-10 | TGF-β | CXCL2 | CXCL1 | |
Group | ||||||
WT PF | 1.00 ± 0.14a | 1.00 ± 0.46a | 1.00 ± 0.21a | 1.00 ± 0.16a | 1.00 ± 0.16a | 1.00 ± 0.20a |
WT EtOH | 0.35 ± 0.14a | 1.00 ± 0.29a | 1.25 ± 0.64a | 0.70 ± 0.17a | 1.50 ± 0.77a | 0.94 ± 0.42a |
GSTA4−/− PF | 0.51 ± 0.23a | 0.96 ± 0.36a | 0.50 ± 0.13a | 0.72 ± 0.19a | 0.57 ± 0.18a | 0.78 ± 0.38a |
GSTA4−/− EtOH | 2.16 ± 0.83b | 1.20 ± 0.21a | 1.24 ± 0.63a | 1.00 ± 0.11a | 2.30 ± 0.93a | 1.10 ± 0.36a |
PPAR-α−/− PF | 2.26 ± 0.21b | 1.60 ± 0.46a | 2.30 ± 0.4a | 1.08 ± 0.14a | 6.30 ± 1.00b | 1.97 ± 0.23a |
PPAR-α−/− EtOH | 2.99 ± 0.63b | 6.01 ± 1.9b | 1.96 ± 0.43a | 1.92 ± 0.42b | 22.70 ± 5.21c | 3.26 ± 0.95b |
dKO PF | 1.96 ± 0.34b | 1.70 ± 0.30a | 1.00 ± 0.261a | 1.16 ± 0.25a | 5.50 ± 1.09b | 0.93 ± 0.21a |
dKO EtOH | 4.75 ± 0.87c | 1.80 ± 0.41a | 2.68 ± 1.33a | 1.80 ± 0.28b | 29.73 ± 3.50d | 2.01 ± 0.50b |
P value | ||||||
Genotype | 0.234 | 0.010 | 0.004 | |||
EtOH | 0.270 | 0.041 | 0.054 | |||
Interaction | 0.019 | 0.031 | 0.477 | 0.139 | <0.001 | 0.345 |
Data are means ± SE for n = 6 mice/group. Real-time RT-PCR data are expressed as gene expression normalized to 18 S and expressed relative to the WT PF group. Significance was determined by 4- by 2-way ANOVA. TGF, transforming growth factor. Groups with different letter subscripts are significant from each other, P < 0.05.
Numbers of necrotic foci, an indicator of cumulative injury associated with inflammatory infiltrates, were higher in PF PPAR-α−/− mice and PF dKO mice than in the PF WT or PF GSTA4−/− groups (P <0.05). However, necrotic foci numbers were actually reduced after EtOH treatment in the EtOH PPAR-α−/− mice and EtOH dKO mice compared with their respective PF groups (P < 0.05). In contrast to pathological assessment, serum ALT values assessing hepatocyte necrosis biochemically demonstrated significant elevation after EtOH treatment in the PPAR-α−/− mice and PF dKO mice compared with their respective PF controls with the highest values in the EtOH dKO group (P < 0.05) (Table 3). In general, the percentage of apoptotic cells as measured by TUNEL was very low and was not significantly affected by treatment or genotype (Table 3). Hepatic repair in response to high-fat PF or EtOH-induced injury was assessed by measurement of PCNA staining in fixed liver sections (15). EtOH treatment had no effect on the percentage of S-phase hepatocytes in WT mice. However, hepatocyte proliferation was significantly stimulated by EtOH in the PPAR-α−/− and dKO mice relative to their PF controls, P < 0.05, and a trend for increased proliferation was also observed in the EtOH-treated GSTA4−/− mice, P = 0.06, (Table 3).
Fibrosis was examined by staining fixed liver sections with picrosirius red to detect collagen (20). Positive staining was observed in the parenchyma in EtOH-treated PPAR-α−/− and dKO mice, especially the latter (Fig. 7). The cytokine transforming growth factor-β (TGF-β) has been linked to stellate cell activation and fibrosis development (2, 5). Elevated TGF-β mRNA expression was observed after EtOH treatment in the PPAR-α−/− mice and the dKO mice compared with their respective PF groups (P < 0.05) but not in WT or GSTA4−/− mice (Table 4). Analysis of additional molecular markers of hepatic stellate cell activation, matrix remodeling, and fibrosis is shown in Fig. 8. In WT and GSTA4−/− mice, EtOH had no effect on collagen type 1 mRNA relative to the PF group. In contrast, EtOH increased collagen type 1 mRNA relative to respective PF groups in both the PPAR-α−/− and dKO mice (Fig. 8A). Expression of mRNA for α-SMA was unaffected by EtOH treatment in WT mice but was increased relative to the EtOH WT group in the other three genotypes (P < 0.05, Fig. 8B). Expression of another marker of stellate cell activation, mRNA-encoding platelet-derived growth factor receptor-β (PDGFR), was only significantly elevated by EtOH treatment relative to PF controls in the PPAR-α−/− and dKO groups (P < 0.05). Moreover, PDGFR mRNA expression was greater in the EtOH dKO group than in the EtOH PPAR-α−/− group (P < 0.05, Fig. 8C). Expression of mRNAs encoding two matrix metalloproteinase (MMP) enzymes MMP-9 and MMP-13 were also measured. EtOH treatment increased expression of MMP-9 mRNA in the EtOH PPAR-α−/− mice compared with that observed in the EtOH GSTA4−/− or EtOH WT groups (P < 0.05) and then further increased in the EtOH dKO mice compared with the EtOH PPAR-α−/− group (P < 0.05) (Fig. 8D). Like MMP-9, MMP-13 mRNA expression was significantly higher in EtOH dKO mice than either EtOH GSTA4−/− or EtOH PPAR-α−/− groups (P < 0.05, Fig. 8E).
Fig. 7.
The appearance of fibrosis in EtOH-treated WT, GSTA4−/−, PPAR-α−/−, and dKO mice. Picrosirius red staining of collagen fibers. Magnification ×10.
Fig. 8.
Changes in hepatic markers of stellate cell activation, matrix remodeling, and fibrosis in WT, GSTA4−/−, PPAR-α−/−, and dKO mice receiving EtOH and PF diets. Gene expression was measured by real-time RT PCR. A: collagen type 1. B: α-smooth muscle actin (α-SMA). C: platelet-derived growth factor-β receptor (PDGFR). D: matrix metalloproteinase 9 (MMP-9). E: MMP-13. Data expressed as means ± SE. Groups with different letter subscripts are significant from each other, P < 0.05.
DISCUSSION
Oxidative stress has been shown to play a key role in progression of alcoholic liver injury (ALD) beyond simple steatosis (2, 3, 5). We have previously demonstrated that the dietary antioxidant and GSH precursor N-acetylcysteine was able to prevent necroinflammatory liver injury produced by chronic EtOH exposure in a rat model utilizing total enteral nutrition to deliver alcohol-containing diets intragastrically (29, 30). However, the molecular mechanisms whereby oxidative stress contributes to the development of inflammation, hepatocyte necrosis, stellate cell activation, and fibrosis remain the subject of continuing investigations. One consequence of oxidative stress in the presence of polyunsaturated membrane lipids is lipid peroxidation and breakdown to form reactive short-chain aldehydes, such as 4-HNE and MDA (7, 15, 19). We have demonstrated that liver 4-HNE and MDA protein adducts are formed as an early event in alcoholic liver injury (34). In vitro evidence suggests that 4-HNE-adducted proteins have impaired cellular functions, which might increase cellular injury (14, 18, 26, 33, 35, 36, 38, 45). In addition, antibodies against lipid aldehyde-adducted proteins appear in serum from human alcoholics with ALD, in patients with nonalcoholic steatohepatitis (NASH), and in rodent models of ALD and NASH, which correlate with the severity of fatty liver injury (2, 21, 29, 30). These data suggest that lipid peroxidation may also contribute significantly to autoimmune responses, which might be involved in progression of liver pathology. However, there have been no previous studies that have directly tested the importance of lipid peroxidation products in the progression of ALD in vivo. In the present study, we took advantage of two stains of genetically modified mice to manipulate the levels of lipid peroxidation produced in response to alcohol. GSTA4 conjugates GSH to α,β-unsaturated aldehydes to inactivate them. Lipid peroxidation and carbon tetrachloride-induced liver injury have been shown to be increased in GSTA4−/− mice (12). PPAR-α is a transcription factor that regulates fatty acid (FA) degradation (27). PPAR-α−/− mice become highly steatotic after high-fat feeding, and EtOH treatment and liver injury have been demonstrated to progress to steatohepatitis and fibrosis in these mice when treated with EtOH liquid diets chronically (22, 25). We hypothesized that GSTA4−/− mice would exhibit increased hepatic lipid peroxidation, 4-HNE and MDA protein adducts, autoantibodies against these adducts, and increased severity of progressive liver injury compared with WT mice fed EtOH chronically via Lieber-DeCarli liquid diets. Other than simple steatosis, no observable liver injury is produced in WT mice fed EtOH in this fashion. Therefore, to test the hypothesis that lipid peroxidation products play a role in the development of necroinflammatory injury and fibrosis, we bred GSTA4−/− mice with PPAR-α−/− mice to produce dKO mice to examine the effects of GSTA4 deletion in mice with a PPAR-α−/− background, which are already predisposed to progressive alcoholic liver injury.
Treatment of WT mice with EtOH-containing liquid diets for 40 days resulted in BECs of 100–200 mg/dl (100–200% of the U.S. legal limit for driving and readily attainable in alcoholics). There were no significant effects of genotype on BEC and thus no apparent effects on EtOH metabolism or clearance. However, despite similar levels of EtOH exposure, liver pathology differed dramatically between genotypes. A summary of the effects of genotype on liver pathology is provided in Table 5. In WT mice, feeding EtOH for 40 days at 28% total calories produced little or no liver observable injury relative to high-fat PF controls. A small amount of fat droplet accumulation in the form of macrosteatosis was accompanied by elevation of antibodies against 4-HNE and MDA adducts but with no evidence of progression to inflammation or fibrosis. As expected, 129/SvJ GSTA4-null mice had significantly increased lipid peroxidation and a periportal disposition of 4-HNE adducts after EtOH treatment compared with WT mice. Interestingly, there was a significant increase in steatosis in EtOH GSTA4−/− mice compared with EtOH WT mice. This suggests that lipid peroxidation is able to alter hepatic lipid homeostasis to enhance hepatic triglyceride accumulation. We have previously demonstrated increases in age-dependent development of obesity in 126/SvJ GSTA4−/− mice (38). This effect was associated with increased expression of acetyl-CoA carboxylase (ACC) mRNA in fat and liver and with inhibition of hepatic mitochondrial aconitase activity, resulting in increased levels of citrate, which is an allosteric activator of ACC ascribed to the effects of excess 4-HNE production. Increased ACC expression and allosteric activation resulted in increased hepatic concentrations of malonyl-CoA in these aging mice (38). Malonyl-CoA is both a FA precursor and an allosteric inhibitor of FA uptake into mitochondria, the net effect of which is to increase hepatic FA concentrations. EtOH has been shown to both increase FA transport into hepatocytes and to stimulate FA synthesis via activation of the transcription factors SREBP-1c and ChREBP (31, 36, 44). Increased steatosis in EtOH GSTA4−/− mice is consistent with additive effects of these previously described mechanisms. Despite little overt pathology, the livers from EtOH GSTA4−/− mice had some molecular indications of increased inflammation in response to EtOH treatment. Further analysis of the GSTA4−/− and WT genotypes by two-way ANOVA, followed by Student's Newman-Keuls post hoc analysis, showed significant increases in mRNA values for the inflammatory cytokine TNF-α in EtOH GSTA4−/− mice compared with PF controls and EtOH WT mice and increased expression of CD14 mRNA, an indicator of Kupffer cell activation (P <0.001). In addition, increases in expression of α-SMA in EtOH GSTA4−/− mice are an indication of stellate cell activation, two-way ANOVA, WT vs. GSTA4−/− (P < 0.05). Lack of liver pathology in these mice after EtOH treatment probably reflects ongoing repair in response to injury because EtOH GSTA4−/− mice also displayed increased hepatocyte proliferation relative to PF controls and EtOH WT mice.
VSports最新版本 - Table 5.
Summary of diet, EtOH, and genotype effects on progression of ALD
Genotype | Steatosis | HNE | Kupffer Cell Activation | Stellate Cell Activation | Immune Infiltrate | Necrosis | Proliferation | Fibrosis |
---|---|---|---|---|---|---|---|---|
PF High Fat | ||||||||
WT | − | − | − | − | − | − | − | − |
GSTA4−/− | − | − | − | − | − | − | − | − |
PPAR-α−/− | +++ | + | ± | ± | + | + | − | − |
dKO | +++ | ++ | ± | ± | + | + | − | − |
EtOH | ||||||||
WT | ± | ± | − | − | − | − | − | − |
GSTA4−/− | ++ | + | ± | ± | − | + | ± | − |
PPAR-α−/− | +++ | +++ | ++ | ++ | + | +++ | ++ | ++ |
dKO | +++ | +++ | +++ | +++ | + | ++++ | ++ | +++ |
HNE, hydroxynonenal.
Because WT 129/SvJ mice are resistant to alcohol-induced liver injury, we also tested the effects of GSTA4 deletion on a PPAR-α−/− background (dKO) because PPAR-α-null mice are a better model for the pathogenesis of human fatty liver disease and show progression of injury to hepatitis after both high-fat and EtOH feeding (1, 22, 25) and to fibrosis after chronic EtOH treatment (22, 25). Consistent with previous studies showing that PPAR-α controls FA degradation and is anti-inflammatory (27), high-fat PF by itself resulted in increased liver injury in PPAR-α mice compared with PF WT mice. Increased inflammation and necrosis pathology scores were accompanied by significant hepatomegaly, substantial steatosis, and accumulation of triglycerides. In addition, loss of PPAR-α resulted in the appearance of inflammatory infiltrates, increased molecular markers of a Th1 response and of B cell recruitment and differentiation, and significantly increased expression of mRNAs encoding chemokines, CD14, TNF-α, MMP-13, and collagen type 1 after high-fat feeding. Consistent with previous studies, EtOH treatment of these mice produced significant increases in molecular markers of Kupffer cell and stellate cell activation, matrix remodeling, and fibrosis relative to PF PPAR-α−/− mice, and serum ALT values indicative of hepatocellular necrosis were elevated threefold over EtOH WT mice. In addition, an increased hepatic population of CD3+ T cells and increased CD138 mRNA, an indicator of B cell differentiation into immunoglobulin-secreting plasma cells (8), in mice lacking PPAR-α and exposed to EtOH are consistent with an increased autoimmune response. These indications of increased progression of liver pathology in EtOH PPAR-α−/− mice were accompanied by significant increases in lipid peroxidation and 4-HNE adducts and thus might also be mediated via the toxic effects of lipid peroxidation products. Interestingly, the lobular distribution of 4-HNE adducts shifted dramatically toward zone 1 after EtOH treatment in both PPAR-α−/− and dKO mice. These data suggest that cytochrome P450 CYP2E1, which has been implicated in formation of reactive oxygen species during EtOH metabolism and which has a centrilobular zone 3 localization (2, 3, 29–31), is not involved in the formation of 4-HNE. In addition, lack of centrilobular 4-HNE adducts in EtOH-treated PPAR-α−/− and dKO mice suggests that the combination of PPAR-null genotype and EtOH stimulates metabolism of short-chain aldehydes in this region of the liver lobule by an as yet uncharacterized mechanism.
Surprisingly, there were fewer necrotic foci detected in EtOH-treated mice lacking PPAR-α compared with PF mice lacking PPAR-α even though markers of Kupffer cell activation, TNF-α mRNA expression, and serum ALTs were elevated. This likely reflects immune adaptation to EtOH exposure and the effects of ongoing injury repair because there was a reduced population of T-helper cells and smaller Th-1 response accompanied by an increase in hepatocyte proliferation after EtOH treatment for 40 days in the EtOH-treated mice compared with PF mice lacking PPAR-α. Alcohol is known to have variable tissue- and time-dependent effects on immune cell recruitment, activation, and cytokine production (10, 28). The present data are consistent with previous studies from our laboratory on the time course of hepatic immune responses to chronic EtOH exposure in rats fed EtOH liquid diets intragastrically (28). In those studies, EtOH abolished the hepatic Th-2 response throughout treatment. In contrast, after an initial acute activation of Th-1 responses over a 7–14-day period, this response was suppressed over the next 2 mo of chronic EtOH exposure (28).
Comparison of dKO mice with the PPAR-α−/− mice revealed significant further increases in overall lipid peroxidation with additional knockout of the GSTA4 gene in PF mice and further increased periportal 4-HNE adduct formation and circulating autoantibodies against MDA adducts after EtOH treatment. The EtOH dKO group displayed molecular evidence for increased leukocyte activation and infiltration (CD68/CD45 mRNA ratio), Kupffer cell activation (CD14 mRNA), increased expression of mRNA-encoding TNF-α, and evidence of increased matrix remodeling (increased mRNA expression of MMP-9 and MMP-13) relative to EtOH PPAR-α−/− mice (Table 5). These data are consistent with an additional role for lipid peroxidation products in regulation of alcohol-induced immune responses and fibrolysis.
The present data represent the first definitive in vivo study demonstrating the importance of lipid peroxidation products in mediating the early progression of ALD. They are consistent with previous alcohol studies in PPAR-α−/− mice, where protective effects of dietary polyenephosphatidylcholine against EtOH-induced inflammation and fibrosis coincided with reduced oxidative stress and levels of lipid peroxidation products (25) and with a number of in vitro studies demonstrating that HNE treatment results in nuclear protein adducts, activation of the MAP kinase JNK, and increased production of procollagen type 1A mRNA in stellate cells (45). However, the precise molecular mechanisms whereby these electrophilic products of lipid peroxidation stimulate inflammation and fibrosis require further investigation.
"VSports" GRANTS
This work was funded in part by R01 AA009300 (D. Petersen).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: M.J.J.R., P.Z., and D.R.P. conception and design of research; M.J.J.R., K.E.M., B.G., B.E., C.T.S., D.J.O., and E.A. performed experiments; M.J.J.R., K.E.M., B.G., B.E., C.T.S., D.J.O., E.A., and D.R.P. analyzed data; M.J.J.R., K.E.M., P.Z., C.T.S., E.A., T.M.B., and D.R.P. interpreted results of experiments; M.J.J.R. drafted manuscript; M.J.J.R., K.E.M., B.G., B.E., P.Z., C.T.S., D.J.O., E.A., T.M.B., and D.R.P. edited and revised manuscript; M.J.J.R., K.E.M., B.G., B.E., P.Z., C.T.S., D.J.O., E.A., T.M.B., and D.R.P. approved final version of manuscript; K.E.M., C.T.S., and D.J.O. prepared figures.
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