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. 2014 Mar 15;192(6):2830-2836.
doi: 10.4049/jimmunol.1300157. Epub 2014 Feb 14.

A key regulatory role for Vav1 in controlling lipopolysaccharide endotoxemia via macrophage-derived IL-6

Stefanie Zenker  1 , Julia Panteleev-Ivlev  1 , Stefan Wirtz  1 , Tadamitsu Kishimoto  2 , Maximilian J Waldner  1 , Olga Ksionda  3 , Victor L J Tybulewicz  3 , Markus F Neurath #  1 , Imke Atreya #  1
Affiliations

A key regulatory role for Vav1 in controlling lipopolysaccharide endotoxemia via macrophage-derived IL-6

Stefanie Zenker et al. J Immunol. .

Abstract

Macrophages are centrally involved in the pathogenesis of acute inflammatory diseases, peritonitis, endotoxemia, and septic shock. However, the molecular mechanisms controlling such macrophage activation are incompletely understood. In this article, we provide evidence that Vav1, a member of the RhoGEF family, plays a crucial role in macrophage activation and septic endotoxemia. Vav1-deficient mice demonstrated a significantly increased susceptibility for LPS endotoxemia that could be abrogated by anti-IL-6R Ab treatment. Subsequent studies showed that Vav1-deficient macrophages display augmented production of the proinflammatory cytokine IL-6. Nuclear Vav1 was identified as a key negative regulator of macrophage-derived IL-6 production. In fact, Vav1 formed a nuclear DNA-binding complex with heat shock transcription factor 1 at the HSE2 region of the IL-6 promoter to suppress IL-6 gene transcription in macrophages. These findings provide new insights into the pathogenesis of endotoxemia and suggest new avenues for therapy.

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Figures

Figure 1
Figure 1. LPS hypersensitivity of Vav1−/− mice
(A) Kaplan-Meier survival rate analysis of WT (closed triangle, n=30) and Vav1−/− (open triangle, n=30) mice after i.p. administration of LPS (20 mg/kg). Summarized data from 3 independent experiments. (B) 2 hours after LPS administration, serum concentrations of GLDH, AST and creatinine were quantified (n≥4). (C) Survival of LPS (10 mg/kg) exposed Rag2−/−Vav1+/+ (closed triangle, n=9) and Rag2−/−Vav1−/− (open triangle, n=5) mice was monitored. Summarized data from 2 independent experiments. (D) Macrophage depleted WT mice (Clod-lip-WT) were reconstituted with WT or Vav1−/− macrophages (n=7) before LPS injection (20 mg/kg) and monitored for survival. Combined results of 2 independent experiments are shown.
Figure 2
Figure 2. LPS hypersensitivity of Vav1−/− mice is driven by macrophage derived IL-6
Peritoneal macrophages from WT or Vav1−/− mice were isolated and stimulated with LPS ex vivo. (A) 30 min after LPS stimulation, mRNA expression of IL-6 and TNF-α were analyzed by real-time PCR. Mean values ± SEM of 6 independent experiments. (B) 2 hours after LPS stimulation, concentrations of IL-6 and TNF-α in supernatants were quantified by ELISA. Cytokine secretion of stimulated WT macrophages was defined to be 100%. Mean values ± SEM of 5 independent experiments. (C) Vav1−/− peritoneal macrophages were transiently transfected with pcDNA3.1 expression vector encoding EGFP-Vav1. 4 hours post-transfection macrophages were exposed to LPS for 1-2 hours and secreted IL-6 in supernatants was quantified by ELISA. Vav1−/− macrophages transfected with empty pcDNA3.1 vector served as control and accordant IL-6 production was set 100%. Mean values ± SEM of 3 experiments. (D) 2 hours after in vivo LPS administration, cytokine concentrations in (D) serum (n=6) and (E) peritoneal lavage (n=8) of WT or Vav1−/− mice were measured (mean values ± SEM of 4 independent experiments) and (F) blood and peritoneal lavage cells were characterized by flow cytometry. Gated CD11b+ cells were analyzed for F4/80 and Ly6C expression. The percentage of CD11b+ F4/80+ Ly6Clow cells is indicated. (G) CD11b+ F4/80+ Ly6C− macrophages from WT (black line) and Vav1−/− mice (green line) were analyzed for intracellular IL-6 expression. Control IgG isotype staining was included (grey). One representative experiment out of 3 independent experiments is shown. (H) Vav1−/− and WT mice were challenged with LPS (20 mg/kg) and after 2 and 3 hours treated with anti-IL-6R antibodies (WT close circle, n=3; Vav1−/− open circle, n=3). LPS challenged Vav1−/− mice (open triangle, n=3) and WT mice (close triangle, n=3) were included as control group. Survival was monitored. Summarized results of 2 independent experiments are shown (p=0.02).
Figure 3
Figure 3. Increased IL-6 expression in the absence of Vav1 can not be explained by classical Il-6 regulating factors
Macrophages were isolated from WT or Vav1−/− mice and stimulated with LPS ex vivo. (A) Western blot analysis of phosphorylated NFκB p65, JNK and p38 MAPK at indicated time points. (B) SOCS3 and SOCS1 expression was quantified by real-time PCR. Mean values ± SEM of 4 independent experiments are shown.
Figure 4
Figure 4. Vav1 and HSF1 containing protein complexes
Macrophages were isolated from WT or Vav1−/− mice and stimulated with LPS ex vivo. (A) Confocal imaging of cell nuclei (Hoechst 3342) Vav1 (Cy3) and HSF1 (DyLight™ 649) as well as merged images (lower panel) in WT and Vav1−/− peritoneal macrophages. Representative images and additional enlargements of indicated areas out of 3 independent experiments are shown. Arrows indicate close proximity of Vav1 and HSF1 expression in the nucleus. In addition to nuclear areas with coexpression of Vav1 and HSF1, areas with isolated Vav1 expression were noted. (B) Expression levels of Vav1 and HSF1 in macrophages were determined by Western blotting. One representative experiment out of 3 independent experiments is shown. (C) HSF1 immunoprecipitates from WT macrophages were analyzed for Vav1 by Western blot (upper panel). One representative experiment out of 3 independent experiments is shown. Specificity of detected Vav1 signals was confirmed by Western Blot analysis of HSF1 immunoprecipitates from LPS exposed Vav1 deficient macrophages (lower panel). (D) EGFP-Vav1 constructs carrying indicated mutations were transiently transfected into J774.1 macrophages. 1 day post-transfection cells were exposed to LPS stimulation for 1 hour. EGFP immunoprecipitates were analyzed for HSF1 and Vav1 by Western Blotting.
Figure 5
Figure 5. Nuclear Vav1 impacts IL-6 promoter activity
Macrophages were isolated from WT or Vav1−/− mice and stimulated with LPS ex vivo. (A) Vav1 or HSF1 bound DNA fragments were precipitated by ChIP assay from WT and Vav1−/− macrophages and analyzed by PCR for the presence of the HSE2 region of the IL-6 promoter. (B) Cellular extracts from unstimulated or LPS treated WT macrophages were incubated with biotinylated HSE probe or scrambled control probe. Vav1 or HSF1 containing DNA-protein complexes were identified by Western blot. One representative experiment out of 3 independent experiments is shown. (C) WT and Vav1−/− macrophages were transfected with reporter plasmids: IL-6 promoter (−1831 to +18) or ΔHSE2 IL6 promoter. Promoter activity was measured by luciferase reporter assay after 60 minutes of LPS stimulation. Mean values ± SEM from 3 independent experiments are shown. (D) Mechanistic model: Vav1 participates in the DNA-binding complex of HSF1 on the IL-6 promoter and thereby dampens the supportive function of HSF1 on TLR-4 triggered IL-6 expression.
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