A combinatorial F box protein directed pathway controls TRAF adaptor stability to regulate inflammation

Abstract

Uncontrolled activation of tumor necrosis factor receptor-associated factor (TRAF) proteins may result in profound tissue injury by linking surface signals to cytokine release. Here we show that a ubiquitin E3 ligase component, Fbxo3, potently stimulates cytokine secretion from human inflammatory cells by destabilizing a sentinel TRAF inhibitor, Fbxl2. Fbxo3 and TRAF protein in circulation positively correlated with cytokine responses in subjects with sepsis, and we identified a polymorphism in human Fbxo3, with one variant being hypofunctional. A small-molecule inhibitor targeting Fbxo3 was sufficient to lessen severity of cytokine-driven inflammation in several mouse disease models. These studies identified a pathway of innate immunity that may be useful to detect subjects with altered immune responses during critical illness or provide a basis for therapeutic intervention targeting TRAF protein abundance.

Figure 1. Fbxl2 interacts with and targets TRAFs for polyubiquitination

a. TRAF1-6 and control protein immunoblot of MLE cells after control (CON) plasmid or ectopic Fbxl2 plasmid expression. b. MLE cells were transfected with an inducible Fbxl2 plasmid under control of exogenous doxycycline. Cells were treated with doxycycline (500 ng/ml) for 1–10 h, cell lysates were analyzed for proteins by immunoblotting. c. Fbxl2 was immunoprecipitated from cells followed by TRAF 1-6 and control protein immunoblotting. d. In vitro ubiquitination assays. SCF complex components were incubated with individual V5-TRAFs and with (+) or without (−) Fbxl2 and the full complement of ubiquitination components (right lane in each pair). e. Half-life determination using cycloheximide of each TRAF protein with (red) or without (blue) Fbxl2 plasmid overexpression in MLE cells is shown (n=2± S.D.). Cells were exposed to cycloheximide (40 μg/ml) for up to 10 h. Cells were collected and assayed by immunoblotting. Bands corresponding to each TRAF protein were quantified using imageJ software and graphed to calculate a degradation curve and protein half-lives are shown. f. U937 cells were transfected with an empty plasmid or an Fbxl2 plasmid, followed by LPS exposure (100 ng/ml) for 24 h prior to assays for cytokine secretion using a human cytokine array prior to generation of a heat map (n=3). Green denotes relatively low-level cytokine levels and red represents relatively high-level expression. Panel (a) is representative of n=3 separate experiments, panels (b, d) represent at least n=2 separate experiments; panel c is from an individual experiment.

Figure 2. Fbxl2 is phosphorylated and targeted by the SCF E3 ligase subunit Fbxo3

a. Scheme of potential phosphorylation sites within Fbxl2 (GPS2.1 prediction). b. Endogenous Fbxl2 was immunoprecipitated from MLE cell lysates and followed by phosphothreonine (Phos-T) immunoblotting. HC=heavy, LC=light immunoglobulin chains. c. Endogenous Fbxl2 was immunoprecipitated from MLE cell lysates followed by immunoblotting for several candidate kinases. d. In vitro GSK3β kinase assay using Fbxl2 as a substrate. MEK1, a dual specificity kinase is shown as a negative control. *heat inactivated GSK3β. e. Endogenous Fbxl2 was immunoprecipitated from MLE cell lysates followed by Fbxo3 immunoblotting. f. In vitro ubiquitination assays. Purified SCF^Fbxo3 complex components were incubated with V5-Fbxl2 and the full complement of ubiquitination reaction components (right lane) showing polyubiquitinated Fbxl2 (arrows). g. Fbxl2 half-life upon Fbxo3 knockdown (top blot) or Fbxo3 plasmid overexpression (lower blot). MLE cells were either transfected with empty or Fbxo3 plasmid for 24 h, or transfected with scrambled RNA or Fbxo3 shRNA for 48 h. Cells were then exposed to cycloheximide (40 μg/ml) for up to 4.5 h prior to harvesting and processing for immunoblotting. h. U937 cells were transfected with either control shRNA or two sets of Fbxo3 shRNA. 48 h later, cells were exposed LPS (100 ng/ml) for an additional 18 h before harvesting and assaying for Fbxo3, TRAF and actin proteins by immunoblotting. Panels b,d,e,f, and g are representative of n=2 separate experiments, panel h represents n=3 separate experiments, and panel c is from an individual experiment.

Figure 3. Fbxo3 contains a naturally occurring polymorphism at V221

a. Genomic DNA was extracted from human PBMC from sixty healthy Caucasian donors followed by SNP genotyping using the TaqMan^® SNP probes with real-time PCR. TaqMan® SNP genotyping was performed using probes containing VIC^™ dye linked to the 5′ end of the wild-type (WT) probe, and a FAM^™ dye linked to the 5′ end of the mutant probe. An increase in the VIC-dye fluorescence signal generated by real-time PCR indicates homozygosity for allele 1(+/+), whereas an increased FAM-dye fluorescence indicates homozygosity for allele 2 (−/−); an increase in both VIC-dye and FAM-dye fluorescence signal intensity indicates allele 1-allele 2 Fbxo3 heterozygosity (+/−). Shown are relative numbers of WT (blue, ++) or heterozygotes (red, +/−) identified. b–c. Eight WT human PBMC and eight PBMC containing the heterozygous V221I polymorphism were treated with LPS (2 μg/ml) for 24 h before assays for cytokine release (b) or TRAFs, Fbxl2, and Fbxo3 immunoblotting (c). Bands corresponding to indicated proteins on immunoblots were quantified using ImageJ software and results displayed graphically (c).

Figure 4. Fbxo3^V221I is a loss-of-function polymorphism of Fbxo3 in vitro

a. In vitro ubiquitination assays. Purified SCF^Fbxo3 (WT, left two lanes) or SCF^Fbxo3V221I variant complex (V221I, right two lanes) components were incubated with V5-Fbxl2 and the full complement of ubiquitination reaction components showing levels of polyubiquitinated Fbxl2 (arrows). b. U937 cells were transfected with V5-WT Fbxo3 or the V5-Fbxo3^V221I plasmids, followed by immunoblotting for V5, Fbxl2, and TRAF proteins. c–e. C57BL/6J mice were administered diluent (control, CON), intratracheal (i.t.) lenti-LacZ, lenti-Fbxo3, or lenti-Fbxo3^V221I (10⁷ pfu/mouse, n=4–6 mice/group) for 120 h, prior to inoculation with P. aeruginosa (PA103, 10⁴ cfu/mouse) for 24 h. Mice were then euthanized, lungs were lavaged with 3 ml saline, harvested, and then homogenized; lavage protein (c), cell counts (d), and cytokine secretion (e) were determined. f. Survival studies of mice administered i.t. PA103 (10⁵ cfu/mouse, n= 7 mice/group) was determined. Mice were carefully monitored over time; moribund, preterminal animals were immediately euthanized and recorded as deceased. Kaplan-Meier survival curves were generated using Prism software (P1: Fbxo3 V/I versus Fbxo3, P2: LacZ versus Fbxo3). g. H&E staining was performed on lung samples in (c–d). *P<0.05 versus Fbxo3. ^#P<0.05 versus LacZ or CON. Panel a is representative of n=2 separate experiments, panel b represents n=3 separate experiments.

Figure 5. Fbxo3 knockdown ameliorates pseudomonas-induced lung injury

C57BL/6J mice were administered diluent (control, CON), lentivirus encoding control shRNA (CON shRNA) or lenti-Fbxo3 shRNA (10⁷ pfu/mouse) (intratracheally [i.t.], n=5 mice/group) for 120 h, and mice were then inoculated with PA103 (10⁴ cfu/mouse) for 24 h. Mice were monitored on FlexiVent to measure lung mechanics (a–d). Mice were then euthanized and lungs lavaged with saline, harvested, and then homogenized. Lavage protein, cell counts, and cytokine secretion were measured in (e, f, h). g. H&E staining representative 1 of 4 samples from (a) was performed on lung tissue. i. Survival studies of mice administered PA103 (i.t. 10⁵ cfu/mouse, n=6 mice per group) was determined (time=hours). Mice were carefully monitored over time; moribund, preterminal animals were immediately euthanized and recorded as deceased. Kaplan-Meier survival curves were generated using Prism software. *P<0.05 versus CON shRNA and ^#P<0.05 versus CON.

Figure 6. F box and TRAF proteins in subjects with sepsis

a. Plasma from control (CON) subjects (n=7) and septic subjects (n=16) were assayed for TNF, IL-1β, and IL-6 using a cytokine ELISA kit. Inset shows levels of cytokines in heterozygotic (Het) subjects harboring a Fbxo3^V221I (n=2, m±SD) polymorphism and subjects harboring wild type Fbxo3 (n=14). b. Leukocytes from control subjects (n=6) and septic subjects (n=10) were assayed for TRAFs, Fbxl2 and Fbxo3 protein by immunoblotting and results quantified using ImageJ software and data displayed graphically.

Figure 7. Fbxo3 inhibitor BC-1215 reduces bacterial-induced inflammation

a. Structures of a base compound benzathine and BC-1215. b. PBMC (0.6 ml at 1.5 × 10⁶/ml) were treated with LPS (2 μg/ml) for 16 h with BC-1215 and cytokine release was monitored by ELISA. The drug concentrations that effectively reduced cytokine release were used to calculate the IC₅₀. In separate studies, U937 cells (0.6 ml at 1.5 × 10⁶/ml) were treated with BC-1215 for 16 h. Cells were then stained with trypan blue to identify dead cells to calculate the LC₅₀ (or LD₅₀). c. Mice were anesthetized prior to administration of BC-1215 (100 μg) through an intraperitoneal (i.p.) injection immediately prior to cecal ligation and puncture (CLP) . 6 h later, mice were euthanized and blood collected for assays of IL-6, TNF, and IL1-β levels. d. Peritoneal fluid was obtained for determination of bacterial counts after CLP in mice. e–h. BC-1215 (100 μg) was administered to mice though an i.p. injection, and mice were immediately challenged with P. aeruginosa (strain PA103, 10⁴ cfu/mouse, i.t.) or without (control, CON) for an additional 18 h. Mice were then euthanized and lungs were lavaged with saline, harvested, and then homogenized. Lavage protein (e), cell counts (f) and cytokine secretion (h) were measured. g. H&E staining was performed on lung samples. The data in (c, d) represent n=4–5 mice/group, *P<0.05 BC-1215 with CLP versus CLP and **P<0.001 versus SHAM. The data in e–h are representative of data from n=4–6 mice/group, *P<0.05 versus vehicle, ** P<0.05 versus CON.

Figure 8. Molecular regulation of pro-inflammatory cytokines mediated by F box proteins

Microbial infection or stimuli can robustly activate a variety of cell surface receptors linked to TRAF proteins that serve as critical intermediary signaling proteins to mediate cytokine synthesis and release. The F box protein Fbxl2 serves as a sentinel inhibitor of TRAFs by mediating their polyubiquitination (red circles) and proteasomal degradation in cells. Fbxl2 specifically targets at a conserved tryptophan domain within all TRAF 1-6. During microbial infection, another F box protein, Fbxo3, targets Fbxl2 for its ubiquitination and degradation at K²⁰¹; this process is facilitated by glycogen synthase kinase (GSK3β) phosphorylation (green circle) of Fbxl2 at T⁴⁰⁴. WT Fbxo3 in this pathway potently activates cytokine driven inflammation, whereas a naturally occurring Fbxo3^V221I polymorphism is hypofunctional. A small molecule Fbxo3 inhibitor, BC-1215, reduces inflammation by antagonizing actions of Fbxo3 on TRAF–cytokine signaling.