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. 2014 Aug;184(8):2297-309.
doi: 10.1016/j.ajpath.2014.05.002. Epub 2014 Jun 17.

Nonhematopoietic β-Arrestin-1 inhibits inflammation in a murine model of polymicrobial sepsis

Deepika Sharma  1 , Nandakumar Packiriswamy  2 , Ankit Malik  1 , Peter C Lucas  3 , Narayanan Parameswaran  4
Affiliations

Nonhematopoietic β-Arrestin-1 inhibits inflammation in a murine model of polymicrobial sepsis

Deepika Sharma et al. Am J Pathol. 2014 Aug.

Abstract

β-Arrestin-1 (βArr1), a scaffolding protein critical in G-protein coupled receptor desensitization has more recently been found to be important in the pathogenesis of various inflammatory diseases. We sought to understand the role of βArr1 in sepsis pathogenesis using a mouse model of polymicrobial sepsis. Although in previous studies we established that βArr1 deficiency protects mice from endotoxemia, here we demonstrate that the absence of βArr1 remarkably renders mice more susceptible to mortality in polymicrobial sepsis. In accordance with the mortality pattern, early production of inflammatory mediators was markedly enhanced in βArr1 knockout mice systemically and locally in various organs. In addition, enhanced inflammation in the heart was associated with increased NFκB activation. Compared to these effects, immune cell infiltration, thymic apoptosis, and immune suppression during polymicrobial sepsis were unaffected by a deficiency of βArr1. Additionally, enhanced inflammation and consequent higher mortality were not observed in heterozygous mice, suggesting that one allele of βArr1 was sufficient for this protective negative regulatory role. We further demonstrate that, unexpectedly, βArr1 in nonhematopoietic cells is critical and sufficient for inhibiting sepsis-induced inflammation, whereas hematopoietic βArr1 is likely redundant. Taken together, our results reveal a novel and previously unrecognized negative regulatory role of the nonhematopoietic βArr1 in sepsis-induced inflammation.

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Figures

Figure 1
Figure 1
Role of β-arrestin-1 (βArr1) in sepsis-induced mortality and inflammation. A: Wild-type (WT), βarr1 knockout (KO) (β-Arr1−/−), and βArr1 heterozygous (β-Arr1+/−) mice were subjected to 16-guage needle single-puncture surgery and observed for mortality over 7 days. B and C: Mice from the different genotypes were subjected to cecal ligation and puncture as indicated in A, and plasma cytokine concentrations in septic mice determined at the indicated time points after surgery. n = 10 to 12 mice for each genotype (A); n = 8 to 14, with data pooled from at least two independent experiments (B and C). Error bars on the figure denote SEM. ∗∗P < 0.01 compared to WT by log-rank (Mantel Cox) test (A); ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared to WT using t-test (B and C).
Figure 2
Figure 2
Role of β-arrestin-1 (βArr1) in cellular infiltration and bacterial killing. Wild-type (WT; white bars), βArr1 knockout (KO; β-Arr1−/−; black bars), and βArr1 heterozygous (β-Arr1+/−; gray bars) mice underwent sham or cecal ligation and puncture surgeries and were euthanized at the defined time points. A: Neutrophil and macrophage infiltration in the peritoneal cavity of sham mice at 24 hours and septic mice at indicated time points after surgery were determined using flow cytometry. B: Bacterial load was represented as colony-forming units (CFU)/mL in the blood of septic mice at the indicated time points and grades of sepsis. C: Bacterial killing capacity of thioglycollate-elicited neutrophils from WT and KO mice was depicted as surviving bacteria (CFU) recovered from the extracellular medium at the indicated time points for total bacterial killing, and from cellular lysate at various time points after 20 minutes uptake for the intracellular killing assay. Data for septic mice were pooled from three independent experiments. n = 9 to 14 for septic mice and n = 3 for sham (B); n = 3 to 5 (C). Error bars denote SEM. ∗P < 0.05 using Student's t-test. 16G-SP, 16-guage needle single puncture; 20G-DP, 20-guage needle double puncture.
Figure 3
Figure 3
Role of β-arrestin-1 (βArr1) in sepsis-induced organ inflammation. A: Wild-type (WT; white bars), βArr1 knockout (β-Arr1−/−; black bars), and βArr1 heterozygous (β-Arr1+/−; gray bars) mice were subjected to 16G-single puncture surgery, and the indicated organs were collected at defined time points for analysis. IL-6 levels (pg/mg) in organ lysates (determined by ELISA) from septic mice 12 hours after surgery. B–D: Quantitative real-time PCR analysis of inflammatory mediators in heart (B), liver (C), and lung tissue (D) from septic mice 12 hours after surgery. E: Total number of neutrophils as determined by flow cytometry isolated from bronchoalveolar lavage of septic mice 24 hours after surgery. mRNA expression was normalized to HPRT before converting to fold WT. Protein and RNA data are represented as fold WT and are pooled from at least two independent experiments. n = 8 to 17 for each genotype. Error bars denote SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 using Student's t-test.
Figure 4
Figure 4
β-arrestin-1 (βArr1) inhibits cardiac NFκB signaling. Wild-type (WT; white bars) and βArr1 knockout (KO) (β-Arr1−/−; black bars) mice were subjected to 16-guage needle single-puncture surgery, and heart tissue was collected 12 hours after surgery. A–C: Representative blots (A) and quantitative presentation of phosphorylation status of IκBα (B) and ERK1/2 (C) in heart lysates from septic mice. B and C: Note that pIκBα was normalized to IκBα, and pERK1/2 to ERK2, for loading control. D: Real-time quantitative RT-PCR for NFKBIA (IκBα) mRNA expression in heart tissue from septic mice. Data are pooled from two independent experiments. n = 8 to 10 for each genotype. Error bars denote SEM. ∗P < 0.05, ∗∗P < 0.01 using Student's t-test.
Figure 5
Figure 5
Role of β-arrestin-1 (βArr1) in sepsis-induced lymphocyte apoptosis. Wild-type (WT; white bars), βArr1 knockout (β-Arr1−/−; black bars), and βArr1 heterozygous (β-Arr1+/−; dark gray bars) mice were subjected to 16-guage needle single-puncture surgery, and thymus and spleen were collected 24 hours after surgery for the indicated parameters/analysis. A and B: CD4+CD8+ T cells in thymus as determined by flow cytometry (A) and caspase-3 activity in thymic lysates of septic mice as compared to WT/sham (light gray bars) (B). C and D: CD4+ and CD8+ T cells in spleen as determined by flow cytometry (C) and caspase-3 activity in splenic lysates from septic mice as compared to WT/sham (D). Data are pooled from at least three independent experiments for septic mice. n = 4 to 6 for sham and n = 10 to 19 for septic mice for each genotype (A–C); n = 4 to 5 (D). Error bars denote SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 using Student's t-test.
Figure 6
Figure 6
Role of β-arrestin-1 (βArr1) in sepsis-induced immune-suppression. Wild-type (WT; white bars), βArr1 knockout (β-Arr1−/−; black bars), and βArr1 heterozygous (β-Arr1+/−; gray bars) mice were subjected to 16-guage needle single-puncture surgery, and spleen and peritoneal cells were collected 24 hours after surgery and processed. Cells were then plated and left untreated (control) or stimulated for 18 hours with 100 ng/mL lipopolysaccharide (LPS). A and B: Cytokine levels in the supernatants in splenocytes (A) and peritoneal cells (B) in control and LPS stimuli as determined by ELISA. Data are presented as fold change over WT control. C: Phagocytic potential and reactive oxygen species (ROS) generation in peritoneal cells from septic mice presented as mean fluorescence intensity (MFI) increase over controls. Data were pooled from three independent experiments. n = 8 to 10. Error bars denote SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 using Student's t-test (A and B).
Figure 7
Figure 7
Role of β-arrestin-1 in cytokine production in in vitro cell culture models. Spleen and resident peritoneal cells from the three genotypes were collected and processed. Equivalent number of cells were plated and treated with lipopolysaccharide (LPS) and polymicrobial culture at different concentrations and multiplicity of infection (MOI), respectively, for 18 hours. Supernatants were then assayed for IL-6 and TNFα concentrations using ELISA. A and B: Data from splenocytes (A) and peritoneal cells (B). White bars indicate wild-type; black bars, β-Arr1−/−. n = 4 to 5 mice for each genotype. Error bars denote SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 compared to WT as determined by 2-way analysis of variance followed by Bonferroni post test. WT, wild type.
Figure 8
Figure 8
Nonhematopoietic β-arrestin-1 in sepsis-induced inflammation. Bone marrow chimeras were generated. The four groups of mice were subjected to cecal ligation and puncture (CLP) with a 16-guage needle and 12 hours later euthanized for sample collection. A–D: Cytokine levels as determined by ELISA in plasma (A), peritoneal fluid (B), spleen (C), and lung and liver lysates (D). E and F: Peritoneal neutrophil infiltration (E) and blood bacterial load (F) in septic mice are shown as total count and CFU/mL, respectively. Data in A–D are presented relative to wild type (WT) for each group. The chimeric nomenclature used is donor>recipient such that the chimeric mouse has the donor's hematopoietic cells and recipient's nonhematopoietic cells. Data are pooled from at least two independent experiments, except for the KO>KO group. n = 8 to 21, WT>WT, WT>KO, and KO>WT; n = 5, KO>KO. Error bars denote SEM. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 using Student's t-test. CFU, colony-forming units; KO, knockout.
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