Abstract

Conclusions

Introduction

Results

Results (cont)

Chronic exposure to significant levels of lipopolysaccharide (LPS), which is one of the agents ubiqui-tously present as contaminant on airborne particles (Kline 1999; Hasday 1999), is reported to be associ-ated with the development and progression of many types of lung diseases, including asthma and chronic obstructive pulmonary disease (COPD) (Michel 1996; Vernooy 2001). Due to the pathophysi-ological complexity of chronic pulmonary diseases, certain efforts have recently been focused on identi-fying anti-inflammatory drugs that can attenuate the proinflammatory process at the very early stage of gene expression, with therapeutic inhibition of NF-κB to limit inflammatory injury of pulmonary diseases among one of the interested areas. Effort for establishing a luciferase based NF-κB responsive gene library has been made by SwitchGear Genomics and provided tools for further research of NF-κB sig-naling pathway regulation. In this study, we applied several NF-κB reporters to in vitro evaluation of anti-inflammatory agents. To exemplify an I-I bridge paradigm that has been advocated by Caliper Life Sciences as a solution for drug discovery, we explored the in vivo aspects of NF-κB activation and regu-lation pathway. We established bioluminescence reporter mice with pulmonary expression of NF-κB re-porters. With bioluminescence imaging, we were able to monitor NF-κB activation during pulmonary in-flammation. Several anti-inflammatory agents were evaluated for their effects on NF-κB activation during LPS induced lung injury and on associated pathological changes.

NF-κB activation is a critical signaling event of inflammation and has been indicated under a number of pathological conditions of lung diseases, including asthma, chronic bronchitis, and chronic obstruc-tive pulmonary disease (COPD). Intervention of this pathway may hold promises to clinical therapy of these diseases. To enable assessment of NF-κB activity in the lungs, we established a mouse model through delivery a luciferase based NF-κB reporter to the lung tissues. NF-κB activation can be monitored through bioluminescent imaging of the transfected mice, allowing sensitive quantification of the response kinetics to inflammatory stimuli. Using this model, we studied NF-κB activation during pulmonary inflammation conditions. Pulmonary exposure to LPS induced tissue injury that mimics certain aspects of COPD. In NF-κB reporter transfected mice, we observed induction of luciferase expression in the lungs following LPS challenge, establishing that bioluminescence imaging approach provides enough sensitivity for monitoring NF-κB activation in pulmonary tissue under inflammatory conditions. We further tested a number of anti-inflammatory agents, andrographolide, parthenolide, TDZD-8, MLN120B and bortezomib, of their effects on NF-κB activation. These compounds have previously been shown to inhibit NF-κB activity through either directly interfering NF-κB DNA bind-ing activity or affecting phosphorylation/degradation of IB. Pre-treatment with bortezomib or TDZD-8 inhibited LPS triggered NF-κB activation in the lungs. Inhibition of NF-κB activation was accompanied by reduced leukocytes counts in the bronchial alveolar lavage in TDZD-8 treated mice, but not in bortezomib treated mice. In summary, we have established an imaging based approach for sensitive detection of NF-κB activation and regulation during acute lung injury. This approach should potentiate further studies on NF-κB regulation under various inflammatory conditions and pre-clinical evaluation of anti-inflammation compounds.

Materials and MethodsReagents We purchased bacterial lipopolysaccharide (LPS, from Salmonella abortus equi), TDZD-8, Parthenolide and Andrographolide from Sigma-Aldrich Chemical Co., (St. Louis, MO), recombinant TNF from R&D Systems (Minneapolis, MN), and bortezomib (velcade, PS-341) from Chemietek Pharmaceuti-cals, Inc (Indianapolis, Indiana)., MLN120B from Dr. Fabio Stellari (Chiesi Pharmaceutical, Marma, Italy), Lipofectamine LTX Transfection Reagent from Invitrogen. JetPEI from VWR

Cell Based Assay of NF-κB -luc Reporters Raw264.7 cells of 70% confluency were trypsinized and washed with PBS. Subsequently 1-3 x 107 cells were suspended in 4 mL DMEM (without fetal calf serum or antibiotics). NF-κB -luc reporter DNA of 50 µg were mixed with 50 µL of Plus Reagent and incu-bated at room temperature for 5 minutes. Next 100 μL Lipofectamine LTX was added to DNA mix and in-cubated at room temp for 30 min. The DNA mixture was added to the cells together with 10 mL of media, and the mixture was transferred to a T75 flask, followed by 4 hour incubation at 37 °C with shaking. The cells were then plated to a 96 well plate with 150 μL cells/well. LPS and NF-κB inhibitors were prepared in 5X of the working concentration, and added to the plate as a 50 μL aliquot. The plate was incubated overnight in an incubator that maintained 5% CO2. After overnight incubation, the reporter activity was as-sayed through imaging with IVIS after adding 20 μL of 30 mg/mL luciferin to the each well.

In Vivo Gene Delivery We applied in vivo JetPEI (Polyplus Transfection) as a carrier for delivering DNA to the lung tissues. The DNA and JetPEI were formulated according to the product manual with a final N/P ratio of 7. Briefly, NF-κB -luc reporter of 50 µg and JetPEI of 7 μL were each diluted into 200 μL with 5% glucose. The two solutions were then mixed and incubate for 15 minutes at room temperature. The entire mixture (400 μL) was i.v. injected to C57 BL/6 albino mice and the expression of NF-κB re-porter was monitored through imaging with IVIS®.

In Vivo Bioluminescence Imaging In vivo imaging was performed using an IVIS imaging system (Caliper Life Sciences, Alameda, CA). Mice were anesthetized with isoflurane and imaged for 5 minutes following intraperitoneal injection of 150 mg/kg luciferin, Photons emitted from specific regions were quan-tified using Living Image® software (Caliper Life Sciences, Alameda, CA).

Acute Pulmonary Inflammation and the Effect of Anti-inflammation Compounds LPS was dissolved in sterile PBS to a concentration of 10 mg/mL. Mice were intratracheally delivered in a 50 μL volume with a 22-gauge intubator. The final dose of LPS was 1 mg/kg. To perform dosage study with TNF, the cytokine solution was diluted in PBS to concentrations of 0.5, 1, 2 µg in a 100 μL volume and the solutions were i.v. delivered to mice. Induction of NF-κB reporters was monitored through imag-ing with IVIS following i.p injection of luciferin at various time points. To study the effect of anti-inflamma-tion compounds, mice were administered with andrographolide (in DMSO, 5 mg/kg, i.p.), or TDZD (in DMSO, 10 mg/kg, i.p.), or bortezomib (in saline, 1 mg/kg, i.v.) at 16 hours and 1 hour prior to intratracheal delivery of LPS.

Bronchoalveolar Lavage Fluid and Tissue Harvesting Bronchoalveolar lavage (BAL) was performed as previously described (Koay 2002). Briefly, mouse tracheas were cannulated with a 18-gauge angiocath (Becton Dickinson). A 5 mL syringe was filled with 1 mL sterile saline and the lungs were instilled. This was done a total of 4 times and a total of 3 -4 mL lavage was collected. Serum was obtained intracardiacally. Lungs were harvested by resection, and tissues were immediately stored in for-malin. Total cell counts were measured in lung lavage fluid. After centrifugation at 500 g for 10 min, the BAL fluid was collected and frozen at -80 °C until analyzed for IL6, TNF, MIP2, and KC/N51 (KC) using commercial ELISA kits (R&D Systems, Minneapolis, MN). The cell pellet was resuspended in 1 mL of 1% BSA in sterile physiological saline. Total cell counts are determined using a grid hemocytometer.

Bioluminescent Mouse Models for Monitoring NF-κB Activation in theLungs and Effect

of Therapeutic Agents on Disease ProgressionNing Zhang, Dan Ansaldi, Ed Lim, Ali Akin, Jae-Boem Kim, Kevin Francis, Mark Roskey, Raj Singh, Caliper Life Sciences, Alameda, CA USA

We established a luciferase reporter based imaging approach for sensitive detection of NF-kB signaling pathway in the lung tissue under LPS induced inflammatory condition. In vivo delivery of several NF-kB reporters, NF-κB2-luc, IL-8-luc and TNFAIP3-luc all resulted in pulmonary expression of the luciferase reporter that can be detected with imaging. NF-kB mediated luciferase expression is inducible by LPS during acute lung injury. We tested several NF-kB inhibitors and showed that bortezomib and TDZD were capable of inhibiting NF-κB activation in the pulmonary tissues following LPS challenge.

While we observed good correlations between cell-based assay and in vivo results, discrepancy was also observed. TNFAIP3-luc mediated luciferase expression was induced under in vivo condition, but was suppressed in cell based assay. Thus, cell-based assay may not always predict in vivo outcome. Andrographolide showed inhibition of NF-kB activation in cell based assay but failed to in vivo at a dose of 5 mg/kg. Lack of inhibition in the in vivo study could be due to low dosage used. Cell based assay of these inhibitors indicated that andrographolide was less potent than TDZD-8 for NF-kB inhibition.

Although not shown, we also explored using a transposon system for delivering NF-kB reporters to lung tissues. In several trials, we failed to observe any advantage over the direct DNA delivery approach.

Establishment of imaging based approach for monitoring NF-kB regulation provide a platform for further screening of NF-kB inhibitors under various inflammatory disease conditions.

In Vitro Assay of Anti-Inflammation Compounds

In Vivo Comparison of NF-kB-luc Reporters

In Vivo Comparison of LPS Induced Activation of NF-kB-luc Reporters

Dose Dependent NF-kB Activation in the Lungs by TNF

Effect of Anti-inflammation Agents on NF-kB Activation

Figure 1. The selected constructs (as shown in the heatmap) represent the SwitchGear NF-kB Profiling Plate. The heatmap summarizes the untreated activity and induction response for each construct in the pathway profiling plate using one common induction strategy. On the right side of the figure, the colors represent log2 transformed ratios of treated/untreated activity according to the color scale shown at the base of the heatmap. Black indicates no change, and intensity of red or blue indicates levels of induction or repression, respectively. On the left side, the intensity of yellow parallels the activity of the promoter in the unstimulated or untreated condition. All untreated signals are relative to the highest and lowest values within the individual experiment. (“TNF”= HT1080 fibrosarcoma cells treated with 20 ng/mL TNF for 8 hours).

In Vitro Comparison of NF-kB Reporters

NF-kB Reporters

A. B.

AcknowledgementWe thank Dr. Nathan Trinklein (SwitchGear Genomics) for discussion, Dr. Stephen Oldfield for critical review of the contents.

References1. Kline, J. N., J. D. Cowden, et al. 1999. Am. J. Respir. Crit. Care Med. 160: 297-303 2. Hasday, J. D., R. Bascom, et al. 1999. Chest 115: 829-835.3. Michel, O., J. Kips, et al. 1996. Am. J. Respir. Crit. Care Med. 154: 1641-1646.4. Vernooy, J. H., M. A. Dentener, et al. 2001. Am. J. Respir. Cell Mol. Biol. 24: 569-576.

Figure 3. The effect of anti-inflammatory compounds on NF-κB activation was assayed. Four compounds, TDZD-8, andrographolide, parthenolide and MLN120B were tested at concentra-tions of 1 to 100 µM. Inhibi-tion of NF-ΚB activation was observed with all com-pounds.

Figure 5. In this study, NF-κB reporters were compared following in vivo gene delivery. All reporters showed specific luciferase expression in the lungs. The signals were the highest at day1, followed by a decline to a basal level at day7. In agreement with the in vitro data, TNFAIP3-luc showed the highest basal level activity.

Figure 6. Response of the NF-κB reporters during LPS induced lung injury was studied. Mice were imaged before and after LPS treatment. Induction of luciferase expression was observed with all three reporters. Induction of TNFAIP3 mediated luciferase expression was in contradiction to the in vitro result. Quantification of the luciferase signal from the thoracic region was shown. Following LPS treatment, we observed above 10-fold induction of in TNFAIP3 transfected mice and approximately 6-fold induction in NF-κB 2 or IL8 transfected mice.

Figure 7. With TNFAIP3 reporter transfected mice, we studied the effect of TNF treatment on ac-tivation of this reporter gene. Study was performed at 10 days post gene delivery. A dose de-pendent induction in the lung tis-sues was observed, with a peak induction at 3 hours. This induc-tion was acute, which dimin-ished at 24 hours. Quantification of the signal showed 300-500% increase at the 3 hour peak.

Figure 8. With NF-κB 2 reporter transfected mice, we studied the effect of anti-inflammation agents on NF-κB activation during LPS induced lung injury. These mice were 8 days after gene delivery and the basal level of luciferase expression was almost undetectable. Intratracheal delivery of LPS trigged a pulmonary induction of luciferase expression. Pre-treatment with TDZD-8 at 10 mg/kg (i.p.) or Bortezomib at 1 mg/kg (i.v.) totally abolished the NF-κB activation, while andrographolide at 5 mg/kg (i.p.) showed no effect. After imaging, mice were sacrificed and the Bronchoalveolar lavage (BAL) was collected and assayed for total leukocytes counts. In supporting the imaging data, mice with LPS treatment showed an increase of leukocytes infiltration into the lungs as compared to control mice. TDZD-8 treated mice showed significant decrease of leukocyte infiltration to the lung tissues, while andrographolide or bortezomib treatment produced no effect.

Figure 4. Quantification was performed to deter-mine the IC50 value of the compounds. TDZD-8 showed most potent in-hibitory effect, with an IC50 of 4-9 µM. Parthe-nolide, andrographolide and MLN120B each has an IC50 in the range of 20-30 µM.

Figure 2. A. The NF-κB -luciferase reporters were transiently transfected into the RAW264.7 cells and seeded to 96-well plates. The cells are treated with LPS (1 μg/mL) and inductions of the luciferase reporters were compared. After overnight incubation, luciferin substrate was added to the plates and luciferase activity was measured with IVIS. B. Quantification of the bioluminescence signal was shown. In untreated cells, both TNFAIP3 and NF-κB 2 reporters mediated higher basal expression of luciferase expression as compared with IL8 and IRF1. Following LPS treatment, NF-κB 2 and IL8 reporter transfected cells showed induction of luciferase expression, while IRF1 transfected cells showed no effect. In contrast, the TNFAIP3 reporter showed inhibition by LPS.

• NK-kB transcription factor control the expression of over 100 genes• SwitchGear Genomics developed a library of NF-kB reporters using the 1-3 kb human promoter fragment of NF-kB responsive genes

TNFAIP3

NFkB2

IL-8

IRF1

LPS-+

-+

-+

-+

0

20000

40000

60000

80000

100000

120000

140000

160000

TNFAIP3 NFkB-2 IL-8 IRF-1

Bio

lum

ine

sce

nce

(p

/s)

ControlLPS

0.00E+00

2.00E+04

4.00E+04

6.00E+04

8.00E+04

1.00E+05

1.20E+05

1.40E+05

0 1 3 10 30 100

Ave

rag

e R

ad

ian

ce

Amount of Compound (μM)

TNFAIP3 (SE, n=4) TDZD

Andrographolide

Parthenolide

MLN120B

0.00E+00

2.00E+04

4.00E+04

6.00E+04

8.00E+04

1.00E+05

1.20E+05

1.40E+05

1.60E+05

0 1 3 10 30 100

Ave

rag

e R

ad

ian

ce

Amount of Compound (μM)

NFkB-2 (SE, n=4) TDZD

Andrographolide

Parthenolide

MLN120B

-1.00E+04

-5.00E+03

0.00E+00

5.00E+03

1.00E+04

1.50E+04

2.00E+04

2.50E+04

3.00E+04

3.50E+04

4.00E+04

0 1 3 10 30 100

Ave

rag

e R

ad

ian

ce

Amount of Compound (μM)

IL-8 (SE, n=4) TDZD

Andrographolide

Parthenolide

MLN120B

-1.00E+04

-5.00E+03

0.00E+00

5.00E+03

1.00E+04

1.50E+04

2.00E+04

2.50E+04

3.00E+04

3.50E+04

4.00E+04

0 1 3 10 30 100

Ave

rag

e R

ad

ian

ce

Amount of Compound (μM)

IRF-1 (SE, n=4) TDZD

Andrographolide

Parthenolide

MLN120B