Gene Dosage-Dependent Negative Regulatory Role of β-Arrestin-2 in Polymicrobial Infection-Induced Inflammation

Abstract

␤-arrestin-2 (␤-arr2) is a scaffolding protein of the arrestin family with a wide variety of cellular functions. Recent studies have demonstrated differential roles for ␤-arr2 in inflammation following endotoxemia and cecal ligation and puncture (CLP) models of sepsis. Because CLP-induced inflammation involves response to fecal contents and necrotic cecum in addition to microbial challenge, in this study, we examined the role of ␤-arr2 in an exclusively polymicrobial infection (PMI) model. In addition, we examined the role of gene dosage of ␤-arr2 in polymicrobial sepsis. Our studies demonstrate that ␤-arr2 is a negative regulator of systemic inflammation in response to polymicrobial infection and that one allele is sufficient for this process. Our results further reveal that loss of ␤-arr2 leads to increased neutrophil sequestration and overt inflammation specifically in the lungs following polymicrobial infection. Consistent with this, specific NF-B and mitogen-activated protein kinase (MAPK) signaling pathways were differentially activated in the ␤-arr2 knockout (KO) mice lungs compared to the wild type (WT) following PMI. Associated with enhanced inflammation in the KO mice, PMI-induced mortality was also significantly higher in KO mice than in WT mice. To understand the differential role of ␤-arr2 in different sepsis models, we used cell culture systems to evaluate inflammatory cytokine production following endotoxin and polymicrobial stimulation. Our results demonstrate cell-type-as well as stimulus-specific roles for ␤-arr2 in inflammation. Taken together, our results reveal a negative regulatory role for ␤-arr2 in polymicrobial infection-induced inflammation and further demonstrate that one allele of ␤-arr2 is sufficient to mediate most of these effects.

A Genome-wide Small Interfering RNA (siRNA) Screen Reveals Nuclear Factor-κB (NF-κB)-independent Regulators of NOD2-induced Interleukin-8 (IL-8) Secretion

“…For example, RIPK2, RELA, and ELF4, which encodes a transcription factor that binds to the IL-8 promoter (42), were each confirmed as positive regulators of IL-8 secretion, whereas silencing TNFAIP3, a known negative regulator of NOD2 signaling (43), enhanced IL-8 secretion. In addition, knockdown of MKNK1 (MAPK-interacting Ser/Thr kinase 1), which acts downstream of p38 to increase protein translation (44,45), decreased IL-8 secretion, whereas ARRB2 silencing increased IL-8 secretion, consistent with previous reports implicating it as a negative regulator of other proinflammatory cytokines (46,47). In summary, the use of independent siRNA reagents confirmed several known regulators, highlighted above, and provides a list of dozens of high confidence IL-8 regulators for future in vivo characterization.…”

Paroxetine differentially modulates LPS-induced TNFα and IL-6 production in mouse macrophages

“…Supernatants from the treated cells were collected and cytokine analysis for IL-6, and TNFα performed according to manufacturer's instructions using ELISA Kits (eBioscience, San Diego, CA) and as described before [14]. Optical density measurements were taken at 450 nm in an Infinite M1000 PRO plate reader (Tecan, Mannedorf, Switzerland).…”

Regulation of Macrophage Biology by Lithium: A New Look at an Old Drug

“…Differentiated macrophages were pretreated with NaCl (30 mM), or LiCl (30 mM) for 12 hours followed by either no treatment or treatment with lipopolysaccharide (2 ng/ml) for another 9 hours (for RNA) or 18 hours (for ELISA). After treatment, RNA was extracted and mRNA levels determined using quantitative real-time RT-PCR as described before (Packiriswamy et al, 2013; Sharma et al, 2013). Supernatants were analyzed for CCL2 levels using ELISA from eBiosciences Inc. as described before (Irwin et al, 2013; Lee et al, 2013).…”