Beta-arrestin 2 negatively regulates sepsis-induced inflammation
Introduction
Sepsis is the host-derived systemic inflammatory response to invasive infections that may result in septic shock, multiple organ failure and death.1 With increasing bacterial antibiotic resistance and the lack of new therapies, the incidence of sepsis-induced mortality remains high.12 The inflammatory process begins at the nidus of infection where bacteria proliferate and either invade the bloodstream or release various bacterial components, such as lipopolysaccharides (LPS), peptidoglycan, lipoteichoic acid, and exotoxins.3 The interaction of these microbial cellular components with macrophages, monocytes, or other host cells induces the release of inflammatory mediators that play a major role in the pathophysiology of septic shock.4
Toll-like receptors (TLRs) expressed on the host cells play critical roles in the inflammatory response. TLRs are an evolutionarily conserved family of pattern recognition receptors that mediate antimicrobial responses.5 TLR4 recognizes the Gram-negative bacteria cell wall component LPS by forming a multi-molecular protein complex with CD14 and the secreted protein myeloid differentiation factor 2 (MD-2).6 Binding to TLR4 leads to activation of a series of signalling proteins leading to activation of transcription factors and pro-inflammatory gene expression.7
Recent studies have implicated β-arrestins in TLR signalling and gene activation. β-arrestins 1 and 2 are adaptor proteins that regulate heterotrimeric guanine nucleotide binding regulatory (G) protein function by forming complexes with G-protein-coupled receptors (GPCRs). It has also been shown that β-arrestins 1 and 2 function as scaffold/adaptor proteins for GPCR activation of mitogen-activated protein (MAP) kinases including extracellular signal-regulated kinase 1/2 (ERK1/2),8 c-Jun N-terminal kinase (JNK),9 p38 kinases,10 and Src family kinases.11 In addition to MAP kinase regulation, it has been shown that β-arrestins also modulate nuclear factor (NF)-κB activity.1214 The implication of β-arrestin 2 in TLR signalling is based in part on our studies demonstrating that over-expression of β-arrestins 1 and 2 in HEK 293 cells stably expressing TLR4 attenuated LPS activation of NF-κB.1213 Wang et al.15 also demonstrated that β-arrestins directly interact with tumour necrosis factor receptor-associated factor 6 (TRAF6) following TLR or IL-1 receptor activation, preventing TRAF6-mediated signalling. In β-arrestin 2(−/−) mice, LPS also stimulated higher expression of pro-inflammatory cytokines in bone marrow-derived macrophages (BMDMs) and induced higher mortality in galactosamine-sensitized β-arrestin 2(−/−) mice.15 More recently our studies have demonstrated that β-arrestin 2 negatively regulates inflammatory responses in polymorphonuclear leucocytes.16 These composite observations support the notion that β-arrestins are essential negative regulators of innate immune activation by TLRs.
To date, the role of β-arrestin 2 in polymicrobial sepsis-induced inflammation has not been investigated. Our current studies employing β-arrestin 2(−/−) mice demonstrate that β-arrestin 2 negatively regulates caecal ligation and puncture (CLP)-induced mortality, plasma IL-6 production and caecal myeloperoxidase (MPO) activity but not hepatic MPO activity. As our previous studies demonstrated increased pulmonary MPO activity,16 we extended these studies by demonstrating increased lung injury as determined by histopathology. β-arrestin 2(−/−) mice also exhibit impaired bacterial clearance. LPS-induced tumour necrosis factor (TNF)-α, IL-6 and IL-10 production was significantly increased in the splenocytes from β-arrestin 2(−/−) mice compared with those from wild-type (WT) mice. These studies suggest that β-arrestin 2 is a negative regulator of sepsis-induced inflammation.
Materials and methods
Mice
β-arrestin 2(−/−) mice and littermate WT mice with the C57B/L6 background were generated by breeding heterozygous animals. We employed 5–8-week-old β-arrestin 2(−/−) and age-matched WT mice for all the experiments. The original heterozygous mice were obtained from Dr Robert J. Lefkowitz (Duke University Medical Center, Durham, NC). Polymerase chain reaction (PCR) was performed with genomic DNA from the tails of 4-week-old mice. The following primer pairs were used: forward, 5′-GATCAAAGCCCTCGATGATC-3′; reverse, 5′-ACAGGGTCCACTTTGTCCA-3′ and 5′-GCTAAAGCGCATGCTCCAGA-3′. The reactions were run for 35 cycles. Western blot analysis of BMDMs and splenocytes from WT and β-arrestin 2(−/−) mice confirmed the absence of β-arrestin 2 with no effect on β-arrestin 1 expression (Fig. 1). The investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and commenced with the approval of the institutional animal care and use committee.
β-arrestin 1 and 2 protein expression in bone marrow-derived macrophages (BMDMs) and splenocytes from wild-type (WT) and β-arrestin 2(−/−) mice. Bone marrow cells were cultured in the presence of macrophage colony-stimulating factor (M-CSF) for 9 days. BMDMs (a) and splenocytes (b) were lysed in the lysis buffer. Western blot analysis was performed using antibodies against β-arrestin 1 and 2. Data are representative of three independent experiments.
Cell culture and stimulation
Splenocytes and peritoneal macrophages were harvested from β-arrestin 2(−/−) mice and littermate WT mice and maintained in RPMI-1640 medium (Cellgro Mediatech Inc., Herndon, VA), supplemented with heat-inactivated 1% fetal calf serum (Sigma, St Louis, MO), 50 U/ml penicillin and 50 μg/ml streptomycin (Cellgro Mediatech Inc.) as described previously.1718 Briefly, spleen tissue was dispersed and passed through a 70-μm nylon mesh to obtain single-cell suspensions. Red blood cells were removed by adding red blood cells lysis buffer (eBioscience, San Diego, CA) for 2 min at room temperature. Peritoneal macrophages were isolated using peritoneal lavages with 10 ml of ice-cold RPMI-1640 medium and incubated at 37° for 2 hr. Non-adherent cells were washed off.
Splenocytes and peritoneal macrophages were stimulated with LPS (10 ng/ml, ULTRA PURE LPS from Escherichia coli O111:B4; List Laboratories, Campbell, CA) for 18 hr. The supernatants were collected for assay of mediator production.
BMDMs were isolated from β-arrestin 2(−/−) mice and littermate WT mice. Briefly, mice were killed under anaesthesia with ether, and both femurs were dissected free of adherent tissue. The proximal and distal femurs were removed and the marrow tissue was flushed by irrigation with culture medium. The marrow plugs were dispersed by passing a 25-gauge needle through them, and the cells were suspended by vigorous pipetting and washed. Cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS), 50 μg/ml penicillin/streptomycin and 25 ng/ml macrophage colony-stimulating factor (M-CSF; R&D System Inc., Minneapolis, MN, USA). Cells were incubated at 37° in a humidified 5% CO2 atmosphere and fresh medium with M-CSF was added every 3 days. After 9 days of culture a homogeneous population of adherent macrophages was obtained (> 90% F4/80 cells as determined by flow cytometry; data not shown). BMDMs were stimulated with LPS (1–10 ng/ml; ULTRA PURE LPS from E. coli O111:B4; List Laboratories) for 18 hr. The supernatants were collected for assay of mediator production.
Caecal ligation and puncture
CLP was performed in β-arrestin 2(−/−) mice and littermate WT mice as previously described.19 Specifically, mice were anaesthetized with ether and a midline incision was made below the diaphragm to expose the caecum. The caecum was ligated at the colon juncture with a 5-0 silk ligature suture without interrupting intestinal continuity. The caecum was punctured once with a 22-gauge needle for survival studies and punctured twice with a 22-gauge needle for other studies. The caecum was returned to the abdomen, and the incision was closed in layers with a 5-0 silk ligature suture and wound clips. After the procedure, the animals were fluid-resuscitated with sterile saline injected subcutaneously. Sham operation was performed in the same way as CLP except for the ligation and puncture of the caecum.
Fifteen per group of WT and β-arrestin 2(−/−) mice were subjected to CLP and eight per group of mice were subjected to sham operation. Mouse survival was monitored every 24 hr for a total of 120 hr. For the plasma IL-6 and tissue MPO studies, three to six mice per group were subjected to sham operation or CLP and killed after 18 hr. The plasma was collected and analysed for IL-6 production, and MPO activity was determined in the lung, liver and caecum. For bacterial load studies, four to eight mice per group were subjected to sham operation or CLP and killed after 18 hr. Bacterial load in the peritoneal cavity, blood and lung were determined by CFU count. For lung histopathology studies, three to six mice per group were subjected to sham operation or CLP and killed after 18 hr. The lung and liver were collected for histopathology.
Western blot
BMDMs and splenocytes from β-arrestin 2(−/−) and WT mice were washed and lysed with ice-cold Radio-Immunoprecipitation Assay (RIPA) lysis buffer [10 mm Tris, pH 7·4, 1% Triton X-100, 150 mm NaCl, 1 mm ethyleneglycoltetraacetic acid (EGTA), 1 mm ethylenediaminetetraacetic acid (EDTA), 1 mm phenylmethylsulphonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin A]. Cells were kept on ice for 30 min, sonicated for 3 seconds, and centrifuged for 10 min at 4° at 10 000 g. An aliquot was taken for protein determination using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA), and the remaining supernatant was stored at −20° until western blot analysis.
Lysates were added to Laemmli sample buffer and boiled for 4 min. Subsequently, protein from each sample was subjected to a 12% sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were washed with Tris-buffered saline-Tween 20 (TBST; 20 mm Tris, 500 mm NaCl and 0·1% Tween 20) and blocked with 5% milk in TBST for 1 hr. After washes with TBST, membranes were incubated with A1CT antibody (1 : 20 000 dilution) overnight at 4°. The blots were washed twice with TBST and incubated for 1 hr with horseradish peroxidase-conjugated donkey anti-rabbit-immunoglobulin G (IgG) antibody (1 : 10 000 dilution; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) in blocking buffer. Immunoreactive bands were visualized by incubation with ECL Plus detection reagents (Amersham Pharmacia Biotech, Inc.) for 5 min and development of the exposed ECL hyperfilm (Amersham Pharmacia Biotech, Inc.).
Assay for TNF-α, IL-6 and IL-10 production
TNF-α, IL-6 and IL-10 production was measured using an enzyme-linked immunosorbant assay (ELISA) with mouse TNF-α, IL-6 or IL-10 ELISA kits (eBioscience).
Measurement of MPO activity
MPO activity was determined in the lung and caecum as an index of neutrophil accumulation as previously described.20 Tissues were homogenized in a solution containing 0·5% hexa-decyl-trimethylammonium bromide dissolved in 10 mm potassium phosphate buffer (pH 7·0) and were centrifuged for 30 min at 20 000 × g at 4°. An aliquot of the supernatant was allowed to react with a solution of tetra-methyl-benzidine (1·6 mm) and 0·1 mm H2O2. The rate of change in absorbance was measured by spectrophotometry at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 μmol hydrogen peroxide/min at 37° and was expressed in units per 100 mg of tissue.
Histopathology
Lung and liver tissues were removed and fixed in phosphate-buffered saline containing 10% formalin and embedded in paraffin. Sections of the tissue organs were stained with haematoxylin and eosin (H&E) and read by a pathologist who was blinded to the experimental groups.
Bacterial load
To determine the bacterial load in the peritoneum, the peritoneal cavity was lavaged with 5 ml of sterile phosphate-buffered saline (PBS) and diluted with sterile PBS. To determine the bacterial load in the blood, 100 μl of blood was collected and diluted with sterile PBS. To determine the pulmonary bacterial load, the lungs were harvested and equal amounts of wet tissue were homogenized and diluted with PBS. One hundred microlitres of each dilution was then plated on chocolate agar plates (Fisher Scientific, Pittsburgh, PA) and incubated at 37° for 24 hr under aerobic conditions. CFU were counted. Results were expressed as CFU per millilitre or gram of wet tissue.
Statistical analysis
Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined by analysis of variance (ANOVA) with Fisher’s probable least-squares difference test or log-rank (Mantel–Cox) test using GraphPad Prism software. A value of P < 0·05 was considered statistically significant.
Mice
β-arrestin 2(−/−) mice and littermate WT mice with the C57B/L6 background were generated by breeding heterozygous animals. We employed 5–8-week-old β-arrestin 2(−/−) and age-matched WT mice for all the experiments. The original heterozygous mice were obtained from Dr Robert J. Lefkowitz (Duke University Medical Center, Durham, NC). Polymerase chain reaction (PCR) was performed with genomic DNA from the tails of 4-week-old mice. The following primer pairs were used: forward, 5′-GATCAAAGCCCTCGATGATC-3′; reverse, 5′-ACAGGGTCCACTTTGTCCA-3′ and 5′-GCTAAAGCGCATGCTCCAGA-3′. The reactions were run for 35 cycles. Western blot analysis of BMDMs and splenocytes from WT and β-arrestin 2(−/−) mice confirmed the absence of β-arrestin 2 with no effect on β-arrestin 1 expression (Fig. 1). The investigations conformed to the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and commenced with the approval of the institutional animal care and use committee.
β-arrestin 1 and 2 protein expression in bone marrow-derived macrophages (BMDMs) and splenocytes from wild-type (WT) and β-arrestin 2(−/−) mice. Bone marrow cells were cultured in the presence of macrophage colony-stimulating factor (M-CSF) for 9 days. BMDMs (a) and splenocytes (b) were lysed in the lysis buffer. Western blot analysis was performed using antibodies against β-arrestin 1 and 2. Data are representative of three independent experiments.
Cell culture and stimulation
Splenocytes and peritoneal macrophages were harvested from β-arrestin 2(−/−) mice and littermate WT mice and maintained in RPMI-1640 medium (Cellgro Mediatech Inc., Herndon, VA), supplemented with heat-inactivated 1% fetal calf serum (Sigma, St Louis, MO), 50 U/ml penicillin and 50 μg/ml streptomycin (Cellgro Mediatech Inc.) as described previously.1718 Briefly, spleen tissue was dispersed and passed through a 70-μm nylon mesh to obtain single-cell suspensions. Red blood cells were removed by adding red blood cells lysis buffer (eBioscience, San Diego, CA) for 2 min at room temperature. Peritoneal macrophages were isolated using peritoneal lavages with 10 ml of ice-cold RPMI-1640 medium and incubated at 37° for 2 hr. Non-adherent cells were washed off.
Splenocytes and peritoneal macrophages were stimulated with LPS (10 ng/ml, ULTRA PURE LPS from Escherichia coli O111:B4; List Laboratories, Campbell, CA) for 18 hr. The supernatants were collected for assay of mediator production.
BMDMs were isolated from β-arrestin 2(−/−) mice and littermate WT mice. Briefly, mice were killed under anaesthesia with ether, and both femurs were dissected free of adherent tissue. The proximal and distal femurs were removed and the marrow tissue was flushed by irrigation with culture medium. The marrow plugs were dispersed by passing a 25-gauge needle through them, and the cells were suspended by vigorous pipetting and washed. Cells were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) containing 10% fetal bovine serum (FBS), 50 μg/ml penicillin/streptomycin and 25 ng/ml macrophage colony-stimulating factor (M-CSF; R&D System Inc., Minneapolis, MN, USA). Cells were incubated at 37° in a humidified 5% CO2 atmosphere and fresh medium with M-CSF was added every 3 days. After 9 days of culture a homogeneous population of adherent macrophages was obtained (> 90% F4/80 cells as determined by flow cytometry; data not shown). BMDMs were stimulated with LPS (1–10 ng/ml; ULTRA PURE LPS from E. coli O111:B4; List Laboratories) for 18 hr. The supernatants were collected for assay of mediator production.
Caecal ligation and puncture
CLP was performed in β-arrestin 2(−/−) mice and littermate WT mice as previously described.19 Specifically, mice were anaesthetized with ether and a midline incision was made below the diaphragm to expose the caecum. The caecum was ligated at the colon juncture with a 5-0 silk ligature suture without interrupting intestinal continuity. The caecum was punctured once with a 22-gauge needle for survival studies and punctured twice with a 22-gauge needle for other studies. The caecum was returned to the abdomen, and the incision was closed in layers with a 5-0 silk ligature suture and wound clips. After the procedure, the animals were fluid-resuscitated with sterile saline injected subcutaneously. Sham operation was performed in the same way as CLP except for the ligation and puncture of the caecum.
Fifteen per group of WT and β-arrestin 2(−/−) mice were subjected to CLP and eight per group of mice were subjected to sham operation. Mouse survival was monitored every 24 hr for a total of 120 hr. For the plasma IL-6 and tissue MPO studies, three to six mice per group were subjected to sham operation or CLP and killed after 18 hr. The plasma was collected and analysed for IL-6 production, and MPO activity was determined in the lung, liver and caecum. For bacterial load studies, four to eight mice per group were subjected to sham operation or CLP and killed after 18 hr. Bacterial load in the peritoneal cavity, blood and lung were determined by CFU count. For lung histopathology studies, three to six mice per group were subjected to sham operation or CLP and killed after 18 hr. The lung and liver were collected for histopathology.
Western blot
BMDMs and splenocytes from β-arrestin 2(−/−) and WT mice were washed and lysed with ice-cold Radio-Immunoprecipitation Assay (RIPA) lysis buffer [10 mm Tris, pH 7·4, 1% Triton X-100, 150 mm NaCl, 1 mm ethyleneglycoltetraacetic acid (EGTA), 1 mm ethylenediaminetetraacetic acid (EDTA), 1 mm phenylmethylsulphonyl fluoride (PMSF), 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 μg/ml pepstatin A]. Cells were kept on ice for 30 min, sonicated for 3 seconds, and centrifuged for 10 min at 4° at 10 000 g. An aliquot was taken for protein determination using the Bio-Rad Protein Assay (Bio-Rad, Hercules, CA), and the remaining supernatant was stored at −20° until western blot analysis.
Lysates were added to Laemmli sample buffer and boiled for 4 min. Subsequently, protein from each sample was subjected to a 12% sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were washed with Tris-buffered saline-Tween 20 (TBST; 20 mm Tris, 500 mm NaCl and 0·1% Tween 20) and blocked with 5% milk in TBST for 1 hr. After washes with TBST, membranes were incubated with A1CT antibody (1 : 20 000 dilution) overnight at 4°. The blots were washed twice with TBST and incubated for 1 hr with horseradish peroxidase-conjugated donkey anti-rabbit-immunoglobulin G (IgG) antibody (1 : 10 000 dilution; Amersham Pharmacia Biotech, Inc., Piscataway, NJ) in blocking buffer. Immunoreactive bands were visualized by incubation with ECL Plus detection reagents (Amersham Pharmacia Biotech, Inc.) for 5 min and development of the exposed ECL hyperfilm (Amersham Pharmacia Biotech, Inc.).
Assay for TNF-α, IL-6 and IL-10 production
TNF-α, IL-6 and IL-10 production was measured using an enzyme-linked immunosorbant assay (ELISA) with mouse TNF-α, IL-6 or IL-10 ELISA kits (eBioscience).
Measurement of MPO activity
MPO activity was determined in the lung and caecum as an index of neutrophil accumulation as previously described.20 Tissues were homogenized in a solution containing 0·5% hexa-decyl-trimethylammonium bromide dissolved in 10 mm potassium phosphate buffer (pH 7·0) and were centrifuged for 30 min at 20 000 × g at 4°. An aliquot of the supernatant was allowed to react with a solution of tetra-methyl-benzidine (1·6 mm) and 0·1 mm H2O2. The rate of change in absorbance was measured by spectrophotometry at 650 nm. MPO activity was defined as the quantity of enzyme degrading 1 μmol hydrogen peroxide/min at 37° and was expressed in units per 100 mg of tissue.
Histopathology
Lung and liver tissues were removed and fixed in phosphate-buffered saline containing 10% formalin and embedded in paraffin. Sections of the tissue organs were stained with haematoxylin and eosin (H&E) and read by a pathologist who was blinded to the experimental groups.
Bacterial load
To determine the bacterial load in the peritoneum, the peritoneal cavity was lavaged with 5 ml of sterile phosphate-buffered saline (PBS) and diluted with sterile PBS. To determine the bacterial load in the blood, 100 μl of blood was collected and diluted with sterile PBS. To determine the pulmonary bacterial load, the lungs were harvested and equal amounts of wet tissue were homogenized and diluted with PBS. One hundred microlitres of each dilution was then plated on chocolate agar plates (Fisher Scientific, Pittsburgh, PA) and incubated at 37° for 24 hr under aerobic conditions. CFU were counted. Results were expressed as CFU per millilitre or gram of wet tissue.
Statistical analysis
Data are expressed as the mean ± standard error of the mean (SEM). Statistical significance was determined by analysis of variance (ANOVA) with Fisher’s probable least-squares difference test or log-rank (Mantel–Cox) test using GraphPad Prism software. A value of P < 0·05 was considered statistically significant.
Results
β-arrestin 2 negatively regulates CLP-induced mortality
To determine the contribution of β-arrestin 2 to polymicrobial sepsis-induced mortality, WT and β-arrestin 2(−/−) mice were subjected to CLP. CLP-induced mortality was monitored every 24 hr for a total of 120 hr. WT mice exhibited a 53% survival at 120 hr post CLP, while only 13% of β-arrestin 2(−/−) mice survived to 120 hr (P < 0·05) (Fig. 2). The animals with sham operations all survived to 120 hr.
Effect of β-arrestin 2 genetic deletion on survival in mice with caecal ligation and puncture (CLP). Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 15 per group) and sham operation (n = 8 per group). Mouse survival was monitored every 24 hr for a total of 120 hr. Statistics were determined with the log-rank (Mantel–Cox) test using GraphPad Prism software. *P < 0·05 compared with the WT CLP group.
β-arrestin 2 negatively regulates CLP-induced plasma IL-6 production
To further characterize the role of β-arrestin 2 on CLP-induced inflammatory response, WT and β-arrestin 2(−/−) mice were subjected to CLP. Eighteen hours after CLP, plasma IL-6 levels were examined. CLP-induced plasma IL-6 levels were significantly higher (25 ± 12 fold; P < 0·05) in β-arrestin 2(−/−) mice compared with WT mice (Fig. 3a).
Effect of β-arrestin 2 genetic deletion on caecal ligation and puncture (CLP)-induced plasma interleukin (IL)-6 production and caecum and liver myeloperoxidase activity. Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 6 per group) and sham operation (n = 3 per group). Eighteen hours after CLP, mice were killed. Plasma IL-6 production was measured by enzyme-linked immunosorbent assay (ELISA) (a). Caecum (b) and liver (c) myeloperoxidase activity was determined. Data represent the mean ± standard error (SE) for three to six animals. *P < 0·05 compared with the sham-operated group; #P < 0·05 compared with the WT CLP group.
β-arrestin 2 negatively regulates CLP-induced caecal but not hepatic MPO activity
We examined MPO activity as an index of neutrophil accumulation in the livers and caecums from β-arrestin 2(−/−) and WT mice. Mice were subjected to CLP, and livers and caecums were collected 18 hr after CLP. We have recently reported that CLP-induced pulmonary MPO activity was significantly increased in β-arrestin 2(−/−) mice.16 The caecum is the nidus of infection leading to sepsis in this model. Similar to increased pulmonary MPO activity as previously observed, we further observed that CLP-induced MPO activity was significantly increased in the caecum (2·4 ± 0·3 fold, P < 0·05) from β-arrestin 2(−/−) mice compared with that from WT mice (Fig. 3b). Liver MPO activity was also increased by CLP, but there was no difference between β-arrestin 2(−/−) mice and WT mice (Fig. 3c).
β-arrestin 2 negatively regulates CLP-induced lung damage
In parallel studies, CLP induced lung and liver tissue damage in β-arrestin 2(−/−) mice and WT mice. In sham-operated animals of both genotypes the lung architecture was normal. In the lungs of WT mice, histological examination revealed mild extravasation of red cells and accumulation of neutrophils in the air spaces after CLP. In β-arrestin 2(−/−) mice, lung injury appeared to be more severe and consisted of reduced alveolar air spaces, large haemorrhagic areas, infiltration of inflammatory cells, and mucus production in bronchial airways (Fig. 4). No significant liver damage was observed after CLP in both genotypes of mice (data not shown).
Effect of β-arrestin 2 genetic deletion on caecal ligation and puncture (CLP)-induced lung damage. Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 6 per group) and sham operation (n = 3 per group). Eighteen hours after CLP, mice were killed. Histopathology was performed on lung sections. The representative sections are shown at 10 and 20 times magnification.
β-arrestin 2(−/−) mice exhibit impaired bacterial clearance in response to CLP
The bacterial load in peritoneal cavity, blood and lung tissue was determined by CFU count. β-arrestin 2(−/−) mice exhibited higher bacterial loads in peritoneal cavity, blood and lung tissue (2·7 ± 0·6 fold, 2·7 ± 0·8 fold and 7·2 ± 1·6 fold, respectively; P < 0·05) compared with WT mice (Fig. 5).
Effect of β-arrestin 2 genetic deletion on bacterial clearance in response to caecal ligation and puncture (CLP). Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 7–8 per group) and sham operation (n = 4 per group). Eighteen hours after CLP, mice were killed. Bacterial loads in the peritoneal fluid (a), blood (b), and lungs (c) were examined. Data represent the mean ± standard error (SE) for four to eight animals. *P < 0·05 compared with the sham-operated group; #P < 0·05 compared with the WT CLP group.
β-arrestin 2 negatively regulates LPS-induced TNF-α, IL-6 and IL-10 production in splenocytes
In subsequent experiments we investigated the effect of β-arrestin 2 deficiency on LPS-induced pro- and anti-inflammatory cytokine production in splenocytes. The absence of β-arrestin 2 and the expression of normal levels of β-arrestin 1 in β-arrestin 2-deficient splenocytes were confirmed by western blot (Fig. 1). LPS-induced TNF-α, IL-6 and IL-10 production was significantly increased (2·2 ± 0·2 fold, 1·8 ± 0·1 fold and 2·2 ± 0·4 fold, respectively; P < 0·05) in splenocytes from β-arrestin 2(−/−) mice compared with WT mice (Fig. 6).
Effect of β-arrestin 2 genetic deletion on lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 production in splenocytes. Splenocytes were harvested from β-arrestin 2(−/−) and age-matched C57B/L6 wild-type (WT) mice and in vitro stimulated with LPS (10 ng/ml) or control medium (CON). LPS-induced TNF-α (a), IL-6 (b) and IL-10 (c) production was determined by enzyme-linked immunosorbent assay (ELISA). Data represent the mean ± standard error (SE) for three independent experiments. *P < 0·05 compared with naïve cells; #P < 0·05 compared with the stimulated WT group.
β-arrestin 2 has no effect on LPS-induced TNF-α and IL-6 production in macrophages
As macrophages are major cytokine-producing cells, we examined the effect of β-arrestin 2 deficiency on LPS-induced TNF-α and IL-6 production in peritoneal macrophages and BMDMs. The absence of β-arrestin 2 and the expression of normal levels of β-arrestin 1 in β-arrestin 2-deficient BMDMs were confirmed by western blot (Fig. 1). LPS-induced TNF-α and IL-6 production in BMDMs (Fig. 7) and peritoneal macrophages (data not shown) revealed no difference between β-arrestin 2(−/−) mice and WT mice.
Effect of β-arrestin 2 genetic deletion on lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α and interleukin (IL)-6 production in bone marrow-derived macrophages (BMDMs). BMDMs were isolated and cultured from β-arrestin 2(−/−) and age-matched C57B/L6 wild-type (WT) mice and in vitro stimulated with LPS (1–10 ng/ml) or control medium (CON). LPS-induced TNF-α (a) and IL-6 (b) production was determined by enzyme-linked immunosorbent assay (ELISA). Data represent the mean ± standard error (SE) for three independent experiments. *P < 0·05 compared with naïve cells.
β-arrestin 2 negatively regulates CLP-induced mortality
To determine the contribution of β-arrestin 2 to polymicrobial sepsis-induced mortality, WT and β-arrestin 2(−/−) mice were subjected to CLP. CLP-induced mortality was monitored every 24 hr for a total of 120 hr. WT mice exhibited a 53% survival at 120 hr post CLP, while only 13% of β-arrestin 2(−/−) mice survived to 120 hr (P < 0·05) (Fig. 2). The animals with sham operations all survived to 120 hr.
Effect of β-arrestin 2 genetic deletion on survival in mice with caecal ligation and puncture (CLP). Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 15 per group) and sham operation (n = 8 per group). Mouse survival was monitored every 24 hr for a total of 120 hr. Statistics were determined with the log-rank (Mantel–Cox) test using GraphPad Prism software. *P < 0·05 compared with the WT CLP group.
β-arrestin 2 negatively regulates CLP-induced plasma IL-6 production
To further characterize the role of β-arrestin 2 on CLP-induced inflammatory response, WT and β-arrestin 2(−/−) mice were subjected to CLP. Eighteen hours after CLP, plasma IL-6 levels were examined. CLP-induced plasma IL-6 levels were significantly higher (25 ± 12 fold; P < 0·05) in β-arrestin 2(−/−) mice compared with WT mice (Fig. 3a).
Effect of β-arrestin 2 genetic deletion on caecal ligation and puncture (CLP)-induced plasma interleukin (IL)-6 production and caecum and liver myeloperoxidase activity. Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 6 per group) and sham operation (n = 3 per group). Eighteen hours after CLP, mice were killed. Plasma IL-6 production was measured by enzyme-linked immunosorbent assay (ELISA) (a). Caecum (b) and liver (c) myeloperoxidase activity was determined. Data represent the mean ± standard error (SE) for three to six animals. *P < 0·05 compared with the sham-operated group; #P < 0·05 compared with the WT CLP group.
β-arrestin 2 negatively regulates CLP-induced caecal but not hepatic MPO activity
We examined MPO activity as an index of neutrophil accumulation in the livers and caecums from β-arrestin 2(−/−) and WT mice. Mice were subjected to CLP, and livers and caecums were collected 18 hr after CLP. We have recently reported that CLP-induced pulmonary MPO activity was significantly increased in β-arrestin 2(−/−) mice.16 The caecum is the nidus of infection leading to sepsis in this model. Similar to increased pulmonary MPO activity as previously observed, we further observed that CLP-induced MPO activity was significantly increased in the caecum (2·4 ± 0·3 fold, P < 0·05) from β-arrestin 2(−/−) mice compared with that from WT mice (Fig. 3b). Liver MPO activity was also increased by CLP, but there was no difference between β-arrestin 2(−/−) mice and WT mice (Fig. 3c).
β-arrestin 2 negatively regulates CLP-induced lung damage
In parallel studies, CLP induced lung and liver tissue damage in β-arrestin 2(−/−) mice and WT mice. In sham-operated animals of both genotypes the lung architecture was normal. In the lungs of WT mice, histological examination revealed mild extravasation of red cells and accumulation of neutrophils in the air spaces after CLP. In β-arrestin 2(−/−) mice, lung injury appeared to be more severe and consisted of reduced alveolar air spaces, large haemorrhagic areas, infiltration of inflammatory cells, and mucus production in bronchial airways (Fig. 4). No significant liver damage was observed after CLP in both genotypes of mice (data not shown).
Effect of β-arrestin 2 genetic deletion on caecal ligation and puncture (CLP)-induced lung damage. Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 6 per group) and sham operation (n = 3 per group). Eighteen hours after CLP, mice were killed. Histopathology was performed on lung sections. The representative sections are shown at 10 and 20 times magnification.
β-arrestin 2(−/−) mice exhibit impaired bacterial clearance in response to CLP
The bacterial load in peritoneal cavity, blood and lung tissue was determined by CFU count. β-arrestin 2(−/−) mice exhibited higher bacterial loads in peritoneal cavity, blood and lung tissue (2·7 ± 0·6 fold, 2·7 ± 0·8 fold and 7·2 ± 1·6 fold, respectively; P < 0·05) compared with WT mice (Fig. 5).
Effect of β-arrestin 2 genetic deletion on bacterial clearance in response to caecal ligation and puncture (CLP). Wild-type (WT) and β-arrestin 2(−/−) mice were subjected to CLP (n = 7–8 per group) and sham operation (n = 4 per group). Eighteen hours after CLP, mice were killed. Bacterial loads in the peritoneal fluid (a), blood (b), and lungs (c) were examined. Data represent the mean ± standard error (SE) for four to eight animals. *P < 0·05 compared with the sham-operated group; #P < 0·05 compared with the WT CLP group.
β-arrestin 2 negatively regulates LPS-induced TNF-α, IL-6 and IL-10 production in splenocytes
In subsequent experiments we investigated the effect of β-arrestin 2 deficiency on LPS-induced pro- and anti-inflammatory cytokine production in splenocytes. The absence of β-arrestin 2 and the expression of normal levels of β-arrestin 1 in β-arrestin 2-deficient splenocytes were confirmed by western blot (Fig. 1). LPS-induced TNF-α, IL-6 and IL-10 production was significantly increased (2·2 ± 0·2 fold, 1·8 ± 0·1 fold and 2·2 ± 0·4 fold, respectively; P < 0·05) in splenocytes from β-arrestin 2(−/−) mice compared with WT mice (Fig. 6).
Effect of β-arrestin 2 genetic deletion on lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 production in splenocytes. Splenocytes were harvested from β-arrestin 2(−/−) and age-matched C57B/L6 wild-type (WT) mice and in vitro stimulated with LPS (10 ng/ml) or control medium (CON). LPS-induced TNF-α (a), IL-6 (b) and IL-10 (c) production was determined by enzyme-linked immunosorbent assay (ELISA). Data represent the mean ± standard error (SE) for three independent experiments. *P < 0·05 compared with naïve cells; #P < 0·05 compared with the stimulated WT group.
β-arrestin 2 has no effect on LPS-induced TNF-α and IL-6 production in macrophages
As macrophages are major cytokine-producing cells, we examined the effect of β-arrestin 2 deficiency on LPS-induced TNF-α and IL-6 production in peritoneal macrophages and BMDMs. The absence of β-arrestin 2 and the expression of normal levels of β-arrestin 1 in β-arrestin 2-deficient BMDMs were confirmed by western blot (Fig. 1). LPS-induced TNF-α and IL-6 production in BMDMs (Fig. 7) and peritoneal macrophages (data not shown) revealed no difference between β-arrestin 2(−/−) mice and WT mice.
Effect of β-arrestin 2 genetic deletion on lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α and interleukin (IL)-6 production in bone marrow-derived macrophages (BMDMs). BMDMs were isolated and cultured from β-arrestin 2(−/−) and age-matched C57B/L6 wild-type (WT) mice and in vitro stimulated with LPS (1–10 ng/ml) or control medium (CON). LPS-induced TNF-α (a) and IL-6 (b) production was determined by enzyme-linked immunosorbent assay (ELISA). Data represent the mean ± standard error (SE) for three independent experiments. *P < 0·05 compared with naïve cells.
Discussion
Our studies are the first to examine the role of β-arrestin 2 in experimental polymicrobial sepsis. The data demonstrate that β-arrestin 2 negatively regulates CLP-induced polymicrobial sepsis, as shown by decreased mouse survival rate and increased plasma IL-6 production, caecal MPO activity and pulmonary, blood and peritoneal exudate bacterial load in β-arrestin 2(−/−) mice compared with WT mice. Our previous studies demonstrated increased pulmonary MPO activity in β-arrestin 2(−/−) mice following CLP, and we extended these studies by demonstrating increased histopathological lung injury and impaired bacterial clearance in β-arrestin 2(−/−) mice. In the splenocytes, β-arrestin 2 also negatively regulated LPS-induced pro- and anti- inflammatory cytokine production, as LPS-induced IL-6, TNF-α and IL-10 levels were increased in β-arrestin 2(−/−) mice. These data demonstrate that β-arrestin 2 is an important negative regulator of polymicrobial sepsis.
The mechanism by which β-arrestin 2 negatively regulates the inflammatory response in CLP remains to be determined. Both β-arrestin 1 and β-arrestin 2 play important roles in the regulation of immune responses. β-arrestin 2 associates with signalling proteins that negatively regulate the TLR-induced signalling pathway. Previous studies have demonstrated that β-arrestin 2 in association with IκBα, NF-κB1 p105 and TRAF6 negatively regulates TLR-induced signalling.131521 Our data showing that β-arrestin 2 negatively regulates LPS-induced pro- and anti-inflammatory cytokine production in splenocytes extend the previous findings. Interestingly, we found that β-arrestin 2 deficiency had no effect on LPS-induced TNF-α and IL-6 production in peritoneal macrophages or BMDMs. The later observation concerning BMDMs also differs from that reported by Wang et al.,15 who found increased cytokine production in BMDMs from β-arrestin 2(−/−) mice. The reason for this difference remains to be determined. One explanation may be that the sources and concentrations of LPS used were different. Regardless, our data suggest that splenocytes are phenotypically different from peritoneal macrophages and BMDMs with regard to β-arrestin 2 regulation.
β-arrestins have emerged as essential regulators of chemotaxis.22 Using β-arrestin 2-deficient mice, Fong et al.23 first demonstrated that β-arrestins are required for chemokine (C-X-C motif) receptor 4 (CXCR4)-mediated lymphocyte chemotaxis. Shortly thereafter, β-arrestins were demonstrated to play a role in CXCR1-, CXCR2-, CXCR3- and CCR5-mediated chemotaxis.2428In vivo studies revealed that CXCR2-mediated neutrophil recruitment to sites of inflammation was increased in β-arrestin 2(−/−) mice in the air pouch model and excisional wound model.27 However, in another in vivo study of allergic asthma, T-cell recruitment to the lung and subsequent cytokine release in response to ovalbumin were impaired in β-arrestin 2(−/−) mice.29 In the later study, neutrophil recruitment in response to LPS was unaffected in β-arrestin 2(−/−) mice.29 Collectively, these studies suggest that the role of β-arrestins in chemotaxis varies depending on the cell type, inflammatory model system and receptors.22 As the caecum is nidus of infection in the sepsis model, we examined caecal MPO activity and demonstrated that, in the β-arrestin 2-deficient mice, CLP-induced caecal MPO activity was increased compared with WT mice. Also, histopathological lung injury was increased, which is consistent with our previous finding that CLP induces increased pulmonary MPO in β-arrestin 2(−/−) mice.16 These results suggest that β-arrestin 2 negatively regulates neutrophil recruitment to inflammatory sites in CLP-induced sepsis.
Our data demonstrate that bacterial clearance in peritoneal cavity, blood and lung tissue is impaired in β-arrestin 2(−/−) mice compared with WT mice. These data may explain the increased tissue damage, neutrophil infiltration and mortality in β-arrestin 2(−/−) mice compared with WT mice. β-arrestin 2 may play a role in activation or phagocytosis of macrophages or neutrophils. However, these questions were not addressed in the current investigation.
Our study demonstrates for the first time that β-arrestin 2 negatively regulates CLP-induced inflammation. There are currently no drugs with agonist activity that selectively activate β-arrestin 2 in vivo. However, a lentiviral expression system may provide an approach to further test the hypothesis that β-arrestin 2 activation in sepsis is beneficial. These composite data demonstrate that β-arrestin 2 must be considered as one critical element in the growing list of signalling proteins that modulate sepsis-induced inflammation and require further investigation.
Abstract
β-arrestins 1 and 2 are ubiquitously expressed proteins that alter signalling by G-protein-coupled receptors. β-arrestin 2 plays an important role as a signalling adaptor and scaffold in regulating cellular inflammatory responses. We hypothesized that β-arrestin 2 is a critical modulator of inflammatory response in experimental sepsis. β-arrestin 2(−/−) and wild-type (WT) mice were subjected to caecal ligation and puncture (CLP). The survival rate was significantly decreased (P < 0·05) in β-arrestin 2(−/−) mice (13% survival) compared with WT mice (53% survival). A second group of mice were killed 18 hr after CLP for blood, peritoneal lavage and tissue sample collection. CLP-induced plasma interleukin (IL)-6 was significantly increased 25 ± 12 fold and caecal myeloperoxidase (MPO) activity was increased 2·4 ± 0·3 fold in β-arrestin 2(−/−) compared with WT mice. β-arrestin 2(−/−) mice exhibited more severe lung damage and higher bacterial loads compared with WT mice post CLP challenge as measured by histopathology and colony-forming unit count. In subsequent experiments, splenocytes, peritoneal macrophages and bone marrow-derived macrophages (BMDMs) were isolated and cultured from β-arrestin 2(−/−) and WT mice and stimulated in vitro with lipopolysaccharide (LPS). Tumour necrosis factor (TNF)-α, IL-6 and IL-10 production induced by LPS was significantly augmented (2·2 ± 0·2 fold, 1·8 ± 0·1 fold, and 2·2 ± 0·4 fold, respectively; P < 0·05) in splenocytes from β-arrestin 2(−/−) mice compared with WT mice. The splenocyte response was different from that of peritoneal macrophages or BMDMs, which exhibited no difference in TNF-α and IL-6 production upon LPS stimulation between WT and β-arrestin 2(−/−) mice. Our data demonstrate that β-arrestin 2 functions to negatively regulate the inflammatory response in polymicrobial sepsis.
Acknowledgments
This work was supported in part by NIH GM27673 (JAC), NIH GM67202 (BZ), NIH DK55524 (LML) and NIH {"type":"entrez-nucleotide","attrs":{"text":"AI079248","term_id":"3415499","term_text":"AI079248"}}AI079248 (HF).
Glossary
Abbreviations
| ANOVA | analysis of variance |
| BMDM | bone marrow-derived macrophage |
| CFU | colony-forming units |
| CLP | caecal ligation and puncture |
| FCS | fetal calf serum |
| GPCR | G-protein-coupled receptors |
| IL | interleukin |
| LPS | lipopolysaccharide |
| M-CSF | macrophage colony-stimulating factor |
| MPO | myeloperoxidase |
| TLR | Toll-like receptor |
| TNF | tumour necrosis factor |
| WT | wild type |
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