Unaltered TNF-α production by macrophages and monocytes in diet-induced obesity in the rat

BACKGROUND: Recent findings have established an association between obesity and immune dysfunction. However, most of the studies investigating the effects of obesity on immune function have been carried out in genetically obese rodent models. Since human obesity is mostly due to intake of a high fat diet and decreased energy expenditure, we asked whether immunological defects also occur in diet-induced obesity. Specifically, we focused on the function of monocytes and macrophages, as these cells are thought to be involved in the low-grade inflammation present in obesity. METHODS: Male Sprague-Dawley rats were fed a high-fat or a standard chow diet for either 2 or 10 weeks. At the end of the intervention period animals were anaesthetised, blood collected for determination of plasma mediator concentrations and lipopolysaccharide (LPS) stimulated production of TNF-α by monocytes. LPS stimulated production of TNF-α in alveolar macrophages was also determined. RESULTS: High-fat feeding for either 2 or 10 weeks resulted in significant increases in fat mass and serum leptin. Although increased serum leptin has previously been linked to modulation of innate immunity, we found no significant difference in the LPS stimulated production of TNF-α by either blood monocytes or alveolar macrophages between the dietary groups. Furthermore, we failed to find a significant increase in circulating TNF-α concentrations in obese animals, as reported for genetically obese animals. CONCLUSION: Our data suggest that defects in innate immune function observed in genetically obese animals are not mimicked by dietary obesity, and may more likely reflect the gross abnormality in leptin function of these models. Further work is required delineate the effects of dietary obesity on inflammatory state and immune function

Ganetespib inhibits LPS-induced lung neutrophil recruitment.

<p><b>(A)</b> Histogram showing the total number of neutrophils in bronchoalveolar lavage fluid (BALF) of Balb/c mice treated with (+) or without (-) LPS (10 µg/mouse, tn) and/or Ganetespib at the indicated dose (mg/kg). BALF was collected 24 hours after Ganetespib administration. Data are mean ± SEM. n = 8 mice per group (100 mg/kg, no LPS; n = 4).** p<0.01 compared to LPS/vehicle treated mice. <b>(B)</b> Representative FACS dot plots showing the F4/80 vs Ly6G gating strategy used to defined distinct cellular subsets in BALF of mice treated with or without LPS and/or Ganetespib. Cells are PI^- CD45⁺ and gated for large cells based on forward and side scatter properties. Plots show show 20,000–25,000 live (PI^-)CD45⁺ cells. Note the marked suppression of neutrophil influx by GIB.</p

Ganetespib inhibits net gelatinase and MMP-9 gelatinase activity in BALF.

<p>Balb/c mice were treated with Ganetespib (50 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse, tn). <b>(A)</b> 102–105 kDa (size markers not shown) MMP9 gelatinase activity of pooled BALF samples determined by gelatin zymography. Left panel, original lucent bands revealed on post-stained gel; right panel corresponding band densitometry in arbitrary units (image pixels). Pooled samples, no statistics. <b>(B)</b> Net gelatinase activity in unfractionated whole BALF collected 3 and 24 h post LPS was determined in individual mice by fluorogenic substrate assay. Data are mean ± SEM. n = 8 mice per group. (naïve; n = 4). *** p<0.001 compared to LPS/vehicle treated mice.</p

Effect of Ganetespib on cellular inflammation and mediator levels and transcripts.

<p><b>(A)</b> Balb/c mice were treated with Ganetespib (50 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse tn). BALF was collected at 3 and 24 h post-LPS and total cells/mL were counted and cellular composition was determined by manual counting microscopy using standard morphological criteria. Data are mean ± SEM. n = 8 mice per group for treatments, n = 4 naïve control. ** p < 0.01, *** p < 0.001 compared to LPS/vehicle treated mice. <b>(B)</b> Balb/c mice were treated with Ganetespib (30 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse, tn). ELISA data for TNFα, MIP-2, IL-6 and KC in BALF obtained at 3 and 24 h (LOD 15.6 pg/ml). Data are mean ± SEM. n = 8 mice per group and n = 4 naïve control. * p<0.05, *** p<0.001 compared to LPS/vehicle treated mice. <b>(C)</b> Relative mRNA transcript abundance for a panel of inflammatory mediators measured in pooled lung tissue (n = 2–3 mice per group) collected 3 h post LPS. Expression is normalized to naïve control levels. Pooled samples, no statistics.</p

HSP90 Inhibition Suppresses Lipopolysaccharide-Induced Lung Inflammation <i>In Vivo</i>

<div><p>Inflammation is an important component of cancer diathesis and treatment-refractory inflammation is a feature of many chronic degenerative lung diseases. HSP90 is a 90kDa protein which functions as an ATP-dependent molecular chaperone that regulates the signalling conformation and expression of multiple protein client proteins especially oncogenic mediators. HSP90 inhibitors are in clinical development as cancer therapies but the myeleosuppressive and neutropenic effect of first generation geldanamycin-class inhibitors has confounded studies on the effects on HSP90 inhibitors on inflammation. To address this we assessed the ability of Ganetespib, a non-geldanamycin HSP90 blocker, to suppress lipopolysaccharide (LPS)-induced cellular infiltrates, proteases and inflammatory mediator and transcriptional profiles. Ganetespib (10–100mg/kg, i.v.) did not directly cause myelosuppression, as assessed by video micrography and basal blood cell count, but it strongly and dose-dependently suppressed LPS-induced neutrophil mobilization into blood and neutrophil- and mononuclear cell-rich steroid-refractory lung inflammation. Ganetespib also suppressed B cell and NK cell accumulation, inflammatory cytokine and chemokine induction and MMP9 levels. These data identify non-myelosuppresssive HSP90 inhibitors as potential therapies for inflammatory diseases refractory to conventional therapy, in particular those of the lung.</p></div

Ganetespib does not suppress basal circulating blood neutrophils but inhibits LPS induced mobilization without causing neutrophil apoptosis.

<p>Balb/c mice were treated with ganetespib (100 mg/kg, i.v. or vehicle) and challenged with LPS (10 µg/mouse tn). <b>(A)</b> 24 h post-LPS peripheral blood counts and blood smears were obtained. Smears were manually differentiated into neutrophils, monocytes and lymphocytes. Total leukocytes were quantified and differentiated using standard morphological criteria of fixed and stained blood smears. Data are mean ± SEM. n = 6–8 mice per group (n = 3 for ganetespib alone). Note that ganetespib did not significantly reduce basal neutrophil numbers but suppressed mobilization post-LPS. <b>(B)</b> Kinetic profile of the effect of ganetespib on neutrophil survival as assayed by live cell imaging. Neutrophils were treated with ganetespib, (concentrations as shown) or 10 µM SN-38 (positive control), for 15mins before addition of 10ng/mL G-CSF or GM-CSF. Data points represent means +/- SEM of three independent samples. p<0.001 for SN-38 vs DMSO from 8h in G-CSF and GM-CSF-treated cells. p<0.05 for 250nM GIB vs DMSO at 24h in G-CSF-treated cells. p< 0.05 for 62.5, 250nM, 1000nM GIB vs DMSO from 19h in GM-CSF-treated cells.</p

BALF cell profiles and organ weights in Ganetespib treated mice.

<p>Groups of male BALB/c mice were treated with vehicle or GIB at the indicated doses. No LPS denotes no LPS treatment. LPS denotes LPS challenge (10µg/mouse, tn). <b>Cells.</b> Macrophage subpopulations were designated according to expression of CD11b vs CD11c as follows: CD11c^hiCD11b- = resident macrophages, CD11c^hiCD11b^hi = intermediate macrophages, CD11c^loCD11b^hi = monocytic macrophages. Dendritic cells (DCs) were defined as MHCII bright CD11c⁺ cells. FACS data are from pooled samples and are shown as mean ± SEM of triplicate replicates. NK cells were defined as CD45⁺CD19^-γδTCR^-TCRβ^-CD49b⁺ and NKT cells as CD45⁺CD19^-γδTCR^-TCRβ⁺CD49b⁺. Data are shown as mean ± SEM for n = 8/group (naïve; n = 4). <b>Organ weight.</b> Mice weighed approximately 22 g prior to treatment and there was no significant difference in basal weight between groups.</p><p>BALF cell profiles and organ weights in Ganetespib treated mice.</p