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The Journal of Immunology, 2004, 173: 1561-1570.
Copyright © 2004 by The American Association of Immunologists

In Vivo Pattern of Lipopolysaccharide and Anti-CD3-Induced NF-{kappa}B Activation Using a Novel Gene-Targeted Enhanced GFP Reporter Gene Mouse1

Scott T. Magness*, Humberto Jijon*, Nancy Van Houten Fisher*, Ned E. Sharpless*,{dagger}, David A. Brenner*,{ddagger} and Christian Jobin2,*

Departments of * Medicine, {dagger} Genetics, and {ddagger} Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599


   Abstract
Top
 Abstract
Introduction
 Materials and Methods:
 Results
 Discussion
 References
 
NF-{kappa}B is a family of transcription factors involved in regulating cell death/survival, differentiation, and inflammation. Although the transactivation ability of NF-{kappa}B has been extensively studied in vitro, limited information is available on the spatial and temporal transactivation pattern in vivo. To investigate the kinetics and cellular localization of NF-{kappa}B-induced transcription, we created a transgenic mouse expressing the enhanced GFP (EGFP) under the transcriptional control of NF-{kappa}B cis elements (cis-NF-{kappa}BEGFP). A gene-targeting approach was used to insert a single copy of a NF-{kappa}B-dependent EGFP reporter gene 5' of the X-linked hypoxanthine phosphoribosyltransferase locus in mouse embryonic stem cells. Embryonic fibroblasts, hepatic stellate cells, splenocytes, and dendritic cells isolated from cis-NF-{kappa}BEGFP mice demonstrated a strong induction of EGFP in response to LPS, anti-CD3, or TNF-{alpha} that was blocked by the NF-{kappa}B inhibitors BAY 11-0782 and NEMO-binding peptide. Chromatin immunoprecipitation analysis demonstrated RelA binding to the cis-NF-{kappa}BEGFP promoter. Adenoviral delivery of NF-{kappa}B-inducing kinase strongly induced EGFP expression in the liver of cis-NF-{kappa}BEGFP mice. Similarly, mice injected with anti-CD3 or LPS showed increased EGFP expression in mononuclear cells, lymph node, spleen, and liver as measured by flow cytometry and/or fluorescence microscopy. Using whole organ imaging, LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. Confocal analysis indicated EGFP expression was primarily found in lamina propria mononuclear cells. In summary, the cis-NF-{kappa}BEGFP mouse will serve as a valuable tool to address multiple questions regarding the cell-specific and real-time activation of NF-{kappa}B during normal and diseased states.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods:
 Results
 Discussion
 References
 
Nuclear factor {kappa}B is an inducible family of heterodimeric transcription factors comprised of cRel, RelA, RelB, p50, and p52 (synthesized as p105 and p100 precursors, respectively) (1), which drives the expression of a set of diverse genes involved in numerous biological process such as inflammation, innate/adaptive immunity, cancer, and development (2).

NF-{kappa}B association with the cytoplasmic inhibitor I{kappa}B prevents the transcription factor from binding to consensus cis elements and thus inhibits transcriptional activity. The most commonly recognized pathway to trigger NF-{kappa}B activation involves signal-induced proteasome-mediated I{kappa}B degradation (3). This mechanism requires inducible serine phosphorylation of I{kappa}B, which occurs at positions 32 and 36 for I{kappa}B{alpha} (4). The kinase responsible for inducible I{kappa}B phosphorylation is the I{kappa}B kinase (IKK)3 complex, composed of the catalytic IKK{alpha} and IKK{beta} subunits, and the regulatory element IKK{gamma} (5, 6). Phosphorylation is followed by the activation of a complex enzymatic system (E1, E2, E3) that adds multiple ubiquitin proteins at lysine residues 21 and 22 of phosphorylated I{kappa}B{alpha} (3). Ubiquinated I{kappa}B{alpha} is then selectively and rapidly degraded via the nonlysosomal, ATP-dependent, 26S proteolytic complex (3). Destruction of I{kappa}B{alpha} reveals the NF-{kappa}B nuclear localization signal and allows nuclear translocation, binding to {kappa}B-cis elements, and induction of gene transcription. In addition to subcellular localization, posttranslational modification such as phosphorylation and acetylation of some NF-{kappa}B subunits strongly modulate NF-{kappa}B transcriptional activity (7, 8, 9, 10, 11).

Because of its pivotal role in driving expression of multiple genes involved in adaptive and innate immunity, NF-{kappa}B activation has been extensively measured in various inflammatory disorders. For example, tissues from patients with rheumatoid arthritis, asthma, atherosclerosis, and inflammatory bowel diseases showed enhanced p65 nuclear localization and DNA-binding activity as measured by immunohistochemistry or EMSAs, respectively, indicating that NF-{kappa}B is associated with the inflammatory process (12, 13). Although informative, these techniques do not measure the dynamic nature of NF-{kappa}B activation and transactivation ability in vivo and are usually insufficient to identify specific cell types within tissues.

Recent findings suggest that NF-{kappa}B may have a protective role during the course of inflammation. We demonstrated that transgenic mice expressing an I{kappa}B{alpha} superrepressor restricted to intestinal epithelial cells (IEC) display an increased sensitivity to dextran sulfate sodium-mediated colitis (14), whereas mice with an intestine-specific knockout of IKK are more sensitive to intestinal ischemia/reperfusion injury (15). In related studies, mice lacking both p50 and one p65 allele are more susceptible to Helicobacter hepaticus-induced typhlocolitis compared with wild-type (WT) mice (16). Finally, the Crohn’s disease mutation leading to truncation of Nod2 results in decreased NF-{kappa}B activation in response to LPS (17). In addition, NF-{kappa}B-induced genes have been associated with the resolution phase of inflammation in vivo (18), implicating again that this transcription factor may have beneficial effects during selective phases of inflammation.

Although NF-{kappa}B has been implicated in acute and chronic inflammation as well as in tissue repair, the exact function of NF-{kappa}B is still unclear. The above-mentioned studies highlight the need for a more detailed characterization of NF-{kappa}B activation in vivo during acute and chronic phases of inflammation and repair, and demand a thorough investigation of the positive and negative impacts of manipulating signaling pathways that modulate NF-{kappa}B activation. However, limited approaches are available to directly measure NF-{kappa}B activity in vivo. In this regard, two transgenic animal models using NF-{kappa}B-driven lacZ (19, 20) or luciferase gene expression (21) have been generated to study NF-{kappa}B activation in vivo. These two models represent a clear improvement over the pre-existing in vitro approaches to study NF-{kappa}B activity; however, these models require exogenous substrate and/or posthumous analysis of prepared tissues or cells and therefore fail to address the instantaneous state of NF-{kappa}B activation in different organs. To this date, no animal models have allowed the precise measurement and location of NF-{kappa}B-activated cells during various physiological situations following treatment.

In this study, we report the generation and characterization of a novel transgenic mouse strain which utilizes NF-{kappa}B-dependent enhanced GFP (EGFP) reporter gene expression. We show that reporter gene expression is specifically induced by NF-{kappa}B, correlates with p65 localization to the transgene promoter, and responds in vivo to stimulation with LPS and anti-CD3. In addition, we use this transgenic mouse as a source of dendritic cells, splenocytes, and hepatic stellate cells (HSCs) to demonstrate the feasibility of conducting reporter assays in primary cells. Moreover, we demonstrate in vivo differences in regional and cellular NF-{kappa}B activation in the intestine following challenge with LPS. Thus, this strain can be used to study the spatial and temporal activation of NF-{kappa}B in vivo and ex vivo in response to physiological inducers.


   Materials and Methods:
Top
 Abstract
 Introduction
 Materials and Methods:
Results
 Discussion
 References
 
cis-NF-{kappa}BEGFP construct, embryonic stem (ES) cell culture, and gene targeting

To generate the cis-NF-{kappa}BEGFP targeting construct, a chimeric promoter containing three HIV NF-{kappa}B cis elements, 5' of the minimal c-fos promoter was isolated from {Delta}56FosdE-Lux (22 ; Fig. 1). The NF-{kappa}B promoter fragment was then ligated into the targeting vector (hypoxanthine phosphoribosyltransferase (HPRT)/EGFP) (23). The preceding cloning steps were also followed to generate a mutant (MT) NF-{kappa}B targeting vector (the MT NF-{kappa}B chimeric promoter (22) contains three mutated NF-{kappa}B cis elements: GCGGATTCCC). Fifteen micrograms of the targeting constructs, cis-NF-{kappa}BEGFP or MT cis-NF-{kappa}BEGFP, was linearized with PvuI, suspended in 0.5 ml of PBS containing 1 x 107 HPRT-deficient BK4 (24, 25) ES cells, and pulsed (350 V, 50 µF for 1 s) in a 4-mm gap electroporation cuvette. The mouse ES cell line BK4 (a subclone of E14TG2a) was maintained in an undifferentiated state on embryonic fibroblast feeder cells in DMEM high glucose, supplemented with 15% FBS (ES qualified; Life Technologies, Grand Island, NY), 0.1 mM 2-ME, 2 mM L-glutamine, 100 U/ml penicillin, and 1 mg/ml streptomycin. ES cells were grown at 37°C in a humidified atmosphere containing 5% CO2. Twenty-four hours after electroporation, the growth medium was replaced with new medium containing hypoxanthine/aminopterin/thymidine (HAT; final concentration of 0.1 mM hypoxanthine/0.4 µM aminopterin/16 µM thymidine). HAT-resistant clones were isolated and expanded 10 days after initiation of selection. Individual clonal populations were used in all experiments unless otherwise indicated.



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  		FIGURE 1. Generation of the cis-NF-{kappa}BEGFP ES cell line and gross anatomical analysis of EGFP expression. A, Schematic of the cis-NF-{kappa}BEGFP targeting construct. Top, Representation of the HPRT locus in BK4 ES cells. Middle, The cis-NF-{kappa}BEGFP targeting construct that contains three HIV NF-{kappa}B cis-elements driving expression of EGFP. Flanking HPRT homology contains the HPRT promoter, exon 1 and exon 2 (indicated by black boxes), which is required to reconstitute HPRT function (i.e., HAT resistance) upon homologous recombination. Bottom, Product of homologous recombination (which reconstitutes the HPRT gene function). B, Clonal HAT-resistant populations of targeted ES cells. "D" is a spontaneously differentiated ES colony and "U" represents an undifferentiated colony. Left, Brightfield image; right, EGFP fluorescence image. C, Gross anatomical assessment of cis-NF-{kappa}BEGFP transgene expression. Left, Brightfield images; right, EGFP fluorescence images.

 
Generation of cis-NF-{kappa}BEGFP mouse, breeding, and genotyping

Targeted ES cells were injected into day 3 C57BL6 strain blastocysts. Chimeric mice were crossed to C57BL6 mice and agouti F1 offspring were tested for the cis-NF-{kappa}BEGFP transgene by PCR amplification of EGFP contained in the construct (EGFPFOR, 5'-GAG CTG AAG GGC ATC GAC TTC AAG-3'; EGFPREV, 5'-GGA CTG GGT GCT CAG GTA GTG G-3': amplicon: 246 bp). For all experiments, F1 EGFP-positive heterozygotes were crossed to C57BL6 strain mice. WT littermates were used as controls for all experiments. F1 cis-NF-{kappa}BEGFP mice were genotyped by collecting a drop of blood (~5 µl) from a tail vein nick (using heparinized capillaries); placing the drop of blood on a glass microscope slide with a glass coverslip, and using fluorescence microscopy to identify EGFP-positive mononuclear cells. To conclude that "phenotype" (i.e., EGFP+ cells in blood) correlated with "genotype," we amplified EGPF cDNA from genomic DNA isolated from the tails. Using this genotyping method, we were able to determine that the cis-NF-{kappa}BEGFP transgene was inherited as a Mendelian sex-linked trait and, furthermore, genotype could be directly correlated with phenotype.

EGFP imaging

For whole organ analysis, animals were sacrificed and dissected followed immediately by image analysis. Live tissues and organs were imaged using either a Magnafire SP charge-coupled device camera (Optronics, Goleta, CA) in a light-tight imaging box with a filtered light source and emission filters specific for EGFP (LT-9900, excitation: 470 nm/40 nm; emission: 515 nm, Lightools Research, Encinitas, CA), or alternatively, an EGFP-specific filter (XF116-2; Omega Optical, Brattleboro, VT) was connected to a fiber optic light source and images were captured using a Nikon Cool-pix 990 digital camera (Nikon, Melville, NY) attached to a dissecting microscope (Lieder, Ludwigsburg, Germany). Identical exposure times were used to capture images within each experiment.

Microscopy

Epifluorescence microscopy was used to detect EGFP in live cells and tissue sections from cis-NF-{kappa}BEGFP mice. For tissue sections, liver and spleens were resected from mice following treatment with the NF-{kappa}B stimulus described in the text, fixed in 4% paraformaldehyde (PFA) for 10–24 h, washed twice in PBS, and transferred to vials containing 30% sucrose (made in PBS) for 24 h. Five- to 7-µm sections were cut on a cryostat and mounted in glycerol-based mounting medium before microscopic analysis. EGFP expression was imaged using an Olympus IX70 (Olympus, Melville, NY) fitted with EGFP-specific filters (XF116-2; Omega Optical). Images were captured using a digital SPOTM camera (Diagnostic Instruments, McHenry, IL). Identical exposure times were used for each data point within an individual experiment.

Dendritic cell isolation and treatment

RBC-depleted splenocytes from cis-NF-{kappa}BEGFP mice were cultured in ultralow adherence six-well plates (Costar, Corning, NY) in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 50 µM 2-ME, 50 µg/ml gentamicin, 10 ng/ml murine rGM-CSF (PeproTech, Rocky Hill, NJ), and 1 ng/ml human rTGF-{beta}1 (R&D Systems, Minneapolis, MN). After 7 days of culture, cells were collected, washed, and replated with fresh medium. Cultures were then monitored daily and split as the cells became dense, usually every 2–3 days. After 3 wk of culture, the resulting population was >93% CD11c+ and CD11b+ as determined by flow cytometry. For in vitro stimulation, cultured dendritic cells were harvested, washed, and resuspended in complete medium without additional cytokines. Cells were stimulated with cecal bacteria lysate (10 µg/ml) for 12 h to assess EGFP expression.

PBMC isolation and treatment

Flow cytometry was used to detect EGFP in individual live cells following in vitro or in vivo stimulation. Peripheral blood was isolated by thoracic puncture and placed into sodium-heparin vials. RBC from whole blood were lysed using RBC lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, and 0.1 mM Na2EDTA, pH 7.2), and PBMC were pelleted at 800 x g for 10 min, washed once with PBS, placed into 35-mm tissue culture dishes and stimulated with TNF-{alpha} (30 ng/ml).

For anti-CD3/anti-CD28 treatment, 200 µl (10 µg/ml) of anti-CD3 (BD PharMingen, San Diego, CA) was added to a 48-well plate and allowed to coat the plate for 2 h at room temperature. The plates were then washed twice with PBS/5% FCS and 0.5 ml of complete culture medium (RPMI 1640, 5% heat-inactivated FCS, 5 x 10–5 M 2-ME, 2 mM glutamine, and 1 mM pyruvate) was added for 30 min. PBMC were plated at ~50,000 cells/well and anti-CD28 (BD PharMingen) was added to a concentration of 2 µg/ml. At specified times following stimulation, PBMC were washed once in PBS, pelleted, and resuspended in FACS analysis buffer (0.1% BSA/0.01% azide in PBS) just before FACS analysis.

HSC and splenocyte isolation/stimulation

HSCs cells were isolated as described previously (26). Twenty-four hours after isolation, HSCs were pretreated for 2 h with WT or MT NEMO-binding peptide (NBP; 200 µg/ml) and then stimulated with TNF-{alpha} (30 ng/ml) for 18 h. HSCs were trypsinized, washed once with PBS, pelleted at 800 x g, and resuspended in FACS analysis buffer before quantification.

Splenocytes were isolated as described above and pretreated with the inhibitor of I{kappa}B phosphorylation, BAY 11-0782 (25 µM), for 20 min and then plated in 48-well tissue culture plates coated with anti-CD3/CD28 (4 and 2 µg/ml, respectively). Eighteen hours after anti-CD3/CD28 stimulation, splenocytes were washed once with PBS, pelleted at 800 x g, and resuspended in FACS analysis buffer before analysis.

In vivo stimulation, cell isolation, and Kupffer cell identification

Conventionally housed cis-NF-{kappa}BEGFP mice or WT littermate control mice were injected i.p. with LPS (5 mg/kg) or anti-CD3 (0.2 mg). At specified times, mice were euthanized using halothane and organs were resected. Splenocytes and thymocytes were isolated by mechanical mincing followed by separation through a 70-µm cell strainer (Falcon, Franklin Lakes, NJ). RBC were lysed and PBMC were isolated as described above and resuspended in FACS analysis buffer before analysis.

An established method to detect Kupffer cells (resident liver macrophages) is to elicit phagocytosis of fluorescent tracer spheres (27). One-micrometer red fluorescent spheres (F13083; Molecular Probes, Eugene, OR) were opsonized with normal rabbit serum for 30 min and the spheres were then washed five times with 1.5 ml of PBS. Fluorescent spheres (2 x 109) were suspended in 200 µl of PBS and injected into the tail vein. Two to 3 h postinjection, mice were perfused with PBS for 10 min to remove unphagocytosed spheres in capillaries. The livers were then resected and fixed in 4% PFA overnight at 4°C. The livers were then washed twice in PBS and placed in 30% sucrose for 24 h before cryostat sectioning. Spheres were imaged by fluorescent microscopy using standard rhodamine excitation and emission filters.

Immunohistochemistry

Small bowel was resected 16 h after LPS treatment (as described above) and fixed in 4% PFA for 8 h at room temperature. The tissue sections were washed twice in PBS and then placed in 30% sucrose at 4°C for at least 16 h before sectioning. For immunostaining, transverse cryosections (10 µm) of the small bowl were placed on glass slides and blocked for 1 h in "blocking solution" (1% normal goat serum (anti-CD64) or 1% FCS (anti-CD3{epsilon}), 1% Triton X-100 in PBS). The sections were then washed two times for 5 min at room temperature using 0.1x blocking buffer. Polyclonal Abs, anti-CD3{epsilon} M-20, and anti-CD64 H-250 (Santa Cruz Biotechnology, Santa Cruz, CA) were diluted 1/500 in 0.1x blocking buffer, applied to sections, and allowed to incubate for 16 h at 4°C in a humidified chamber. The sections were then washed three times with 0.1x blocking buffer at room temperature for 10 min. Either anti-goat-Cy3-conjugated (for anti-CD3{epsilon}) or anti-donkey-Cy3-conjugated (for anti-CD64) secondary Abs were diluted 1/200 in 0.1x blocking buffer, applied to sections, and allowed to incubate at room temperature for 1 h in a humidified chamber. The sections were then washed five times with 0.1x blocking buffer at room temperature for 5 min each. Immunostaining was visualized using confocal imaging as described earlier.

Flow cytometry analysis

Flow cytometry determination was performed as previously described (23). Briefly, 5 x 105 RBC-depleted splenocytes or PBMC were washed with PBS containing 0.1% BSA and 0.01% azide and EGFP expression was measured on a FACscan (BD Biosciences, Mountain View, CA) using the FL1 channel to detect EGFP fluorescence. Coexpression of the early activation marker CD69 was measured by surface staining with anti-CD69-PE (Caltag Laboratories, Burlingame, CA) for 20 min on ice followed by washing in PBS containing 0.1% BSA and 0.01% azide before FACScan analysis. All flow cytometric analyses were conducted on living cells within 30 min of isolation.

Chromatin immunoprecipitation (ChIP) and real-time PCR analysis

Mice were administered either 2.5 mg/kg body weight LPS or PBS i.p. vehicle for 2 h and splenocytes were isolated as described above. Splenocytes were resuspended in 1 ml of PBS and fixed by addition of 100 µl of formaldehyde solution (11% formaldehyde, 100 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA, and 50 mM Tris-Cl (pH8.0)). DNA was sheared and ChIP was performed as described previously (28).

Real-time PCR was used to semiquantify RelA binding to various gene promoters. The following promoter-specific oligonucleotides were used to amplify ChIP DNA: {kappa}BChIPFOR, 5'-CGAATTCTGCAGGTCGACGGAAAG-3'; {kappa}BChIPREV, 5'-CGGTGAACAGCTCCTCGCCCTTC–3'; RANTESFOR, 5'-GTGAAGACCAATGGCTTGACC-3'; RANTESREV, 5'-CATGTGCTGTCTCAGAGTCCTC-3'; MIP-2FOR, 5'-CAACAGTGTACTTACGCAGACG-3'; MIP-2REV, 5'-CTAGCTGCCTGCCTCATTCTAC-3'; IL-6FOR, 5'-GACATGCTCAAGTGCTGAGTCAC-3'; IL-6REV, 5'-AGATTGCACAATGTGACGTCG-3'; MCP-1FOR, 5'-CAGCATCTGGAGCTCACATTCC-3'; and MCP-1REV, 5'-GCATGAACAAGTTGAGAGATGCC-3'.

One microliter of immunoprecipitated DNA or total DNA input control was used in a standard real-time PCR using a LightCycler real-time PCR analyzer and LightCycler cyber green reagents (Roche, Basel, Switzerland). Three millimolar MgCl2 was required to produce specific amplification products with no primer-dimer formation. The PCR was cycled at 97°C, 10 s; 58°C, 5 s; and 72°C 30 s for 40 cycles. Linear amplification ranges were determined using LightCycler analysis software and reaction crossing points were applied to the formula described in the study of Pfaffl (29) to determine increases of RelA binding in control vs treated animals. Identical crossing points in "input" DNA from control and treated mice indicated that amounts of DNA put into the immunoprecipitation reaction were equivalent.

Adenoviral purification and in vivo infection

The adenoviral vector (Ad5NIK) encoding a WT NF-{kappa}B-inducing kinase, a strong inducer of NF-{kappa}B activity, and the control virus Ad5LacZ were amplified and purified as described elsewhere (30, 31). The viruses were dialyzed twice against PBS for 8 h and once overnight in 5% sucrose (made in PBS). Mice were injected i.v. with 200 µl of PBS or 1 x 107 or 1 x 109 PFU of virus. EGFP expression was assessed 7 days following Ad5NIK infection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods:
 Results
Discussion
 References
 
Generation of mouse line and tissue-specific expression of the cis-NF-{kappa}BEGFP transgene

To study NF-{kappa}B-dependent gene transcription in a spatial and temporal manner, we generated a transgenic mouse expressing the EGFP under the transcriptional control of NF-{kappa}B. Since NF-{kappa}B is a ubiquitous transcription factor involved in many homeostatic biological processes, it was necessary to minimize transgene expression due to basal NF-{kappa}B activation to be able to observe increases in transgene expression following inductive stimuli. Therefore, we used a gene-targeting approach to integrate a single copy of the NF-{kappa}B reporter construct, in a single locus, 5' of the mouse HPRT gene. Gene targeting was chosen over standard pronuclear injection to generate the transgenic mouse to assure the strain would harbor only a single copy of the transgene, integrated in a well-characterized locus that has permitted the appropriate expression of other transgene constructs (25, 32). The NF-{kappa}B-dependent reporter consisted of three HIV NF-{kappa}B sites, 5' of the minimal c-fos promoter, driving expression of the EGFP reporter gene, which was flanked by HPRT gene homology (Fig. 1A). Following homologous recombination into a male-derived, HPRT-deficient ES cell line (25), the transgene is integrated 5' of the promoter with concomitant rescue of the HPRT gene defect (Fig. 1A). A successful homologous recombination event is permissive for ES cell growth in HAT medium; conversely, random integration of the targeting vector does not restore HPRT gene expression, resulting in cell death in HAT medium.

Following gene targeting and HAT selection, ES cell colonies proliferated and were cloned for analysis of cis-NF-{kappa}BEGFP transgene activity. Spontaneously differentiated clonal lines of the cis-NF-{kappa}BEGFP ES cells displayed induced expression of EGFP, whereas the undifferentiated, pluripotent ES cells exhibited no EGFP expression demonstrating the inductive capability of the cis-NF-{kappa}BEGFP transgene at the cellular level (Fig. 1B). ES colonies containing mutant NF-{kappa}B sites exhibited no EGFP expression (data not shown).

Chimeric mice were generated from a single clone of cis-NF-{kappa}BEGFP ES cells and bred to C57BL6 mice to produce heterozygous female mice. A second breeding step of transgene-positive, F1 generation females to C57BL6 males was required to produce a hemizygous male. Mice were easily genotyped by the presence of EGFP-positive mononuclear cells in peripheral blood and demonstrated Mendelian sex-linked segregation of the transgene. Upon gross analysis of whole organs from cis-NF-{kappa}BEGFP mice, basal levels of EGFP expression were observed in the lymph nodes, Peyer’s patches, thymus, and epididymis, which are known to exhibit high basal levels of NF-{kappa}B activation (33, 34) (Fig. 1C).

Although the transgene was targeted to an X-linked locus, we did not observe any significant differences in the number of EGFP-expressing PBMC in males (n = 5) and heterozygous females (n = 5), suggesting this transgene is resistant to X chromosome inactivation (data not shown). We have initiated breeding to generate homozygous females to investigate the impact of transgene copy number on EGFP expression levels.

cis-NF-{kappa}BEGFP transgene up-regulation is specific to NF-{kappa}B activation

Recruitment of NF-{kappa}B subunits, especially RelA, to a defined promoter region is prerequisite for transcriptional activation. To selectively assess the recruitment of the potent transcriptional activator RelA to the cis-NF-{kappa}BEGFP promoter in the transgenic mice, we performed ChIP analysis on various well-defined NF-{kappa}B responsive genes (2) following in vivo challenge with LPS. LPS has been demonstrated to be a strong inducer of NF-{kappa}B activation by signaling through the TLR4 (35, 36, 37). Two hours after injection of LPS into cis-NF-{kappa}BEGFP-transgenic mice, splenocytes were isolated and the ChIP assay was performed. As predicted, we found enhanced RelA loading on the promoter of cis-NF-{kappa}BEGFP mice treated with LPS compared with PBS-treated mice (11.7-fold; Fig. 2), indicating the promoter of the cis-NF-{kappa}BEGFP transgene functionally binds the endogenous RelA subunit upon stimulation. Moreover, there was enhanced RelA recruitment to the endogenous promoters of the NF-{kappa}B responsive genes, IL-6, MIP-2, RANTES, and MCP-1 in LPS-treated mice compared with control-treated mice (Fig. 2). This demonstrates an effective recruitment of RelA to the cis-NF-{kappa}BEGFP promoter as well as to the promoters of other NF-{kappa}B-responsive genes in vivo.



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  		FIGURE 2. Increased RelA loading on the cis-NF-{kappa}BEGFP determined by ChIP analysis and real-time PCR. To measure RelA (or p65) loading onto the cis-NF-{kappa}BEGFP promoter and other NF-{kappa}B responsive genes following LPS stimulus, cis-NF-{kappa}BEGFP mice were injected with LPS and 2 h later, chromatin was isolated from splenocytes and immunoprecipitated using an anti-p65 Ab. Promoter-specific primers were then used to semiquantitatively PCR amplify DNA or the input control (figure represents amplicons from the cis-NF-{kappa}BEGFP allele). Amplicons were analyzed on a 2% agarose gel. The increase in p65 loading in LPS-treated mice was calculated based on the cyber green fluorescence of the ChIP DNA amplicons normalized to the cyber green fluorescence of the "input" DNA amplicons (specifically, the amplicons from the cis-NF-{kappa}BEGFP promoter in control and treated mice).

 
cis-NF-{kappa}BEGFP transgene is up-regulated in primary cell cultures by TNF-{alpha} or LPS

To determine the involvement of NF-{kappa}B signal transduction in stimuli-induced cis-NF-{kappa}BEGFP transgene expression, we specifically blocked NF-{kappa}B activation using NBP (38), an inhibitor of IKK activity, and BAY 11-0782, an inhibitor of I{kappa}B{alpha} phosphorylation (39) in two cell types isolated from cis-NF-{kappa}BEGFP mice. Primary HSCs were isolated from cis-NF-{kappa}BEGFP mice, cultured for 10 days, pretreated with the NBP, and then stimulated with the well-characterized NF-{kappa}B inducer, TNF-{alpha}, for 12 h. TNF-{alpha}-induced EGFP expression was completely blocked in NBP-treated cis-NF-{kappa}BEGFP HSCs compared with those treated with the mutated NBP control peptide (Fig. 3A and B). Likewise, the pharmacological inhibitor, BAY 11-0782, completely blocked anti-CD3-induced EGFP expression in primary splenocytes isolated from cis-NF-{kappa}BEGFP mice (Fig. 3C).



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  		FIGURE 3. NF-{kappa}B-dependent induction of the cis-NF-{kappa}BEGFP transgene is specifically inhibited by NF-{kappa}B inhibitors. A and B, Inhibition of NF-{kappa}B activation using NBP. Primary HSCs were isolated from cis-NF-{kappa}BEGFP mice, grown on plastic for 10 days, incubated with either WT or MT NBPs, followed by stimulation with 30 ng/ml TNF-{alpha}. A, EGFP fluorescence in TNF-{alpha}-treated HSCs incubated with WT and MT NBPs. B, EGFP fluorescence intensity plots derived from the gated populations of live cells following FACS analysis. Both EGFP intensity and percent cells expressing EGFP are indicated for each condition. C, IKK inhibition using BAY 11-0782. Splenocytes were isolated from cis-NF-{kappa}BEGFP mice and incubated with BAY 11-0782 followed by anti-CD3/CD28 treatment. Fluorescence intensities and numbers of EGFP-positive cells were determined using flow cytometry. Right, Images are representative of cis-NF-{kappa}BEGFP splenocytes treated with anti-CD3/CD28 with or without BAY 11-0782 before flow cytometry.

 
To assess the ability of other transgenic mouse cells to respond to NF-{kappa}B activators, embryonic fibroblasts, PBMC, or dendritic cells were isolated from the cis-NF-{kappa}BEGFP mice and stimulated with the classic NF-{kappa}B activators TNF-{alpha} and/or LPS. Both TNF-{alpha} and LPS induced EGFP expression in embryonic fibroblasts as determined by fluorescence microscopy (Fig. 4A). In addition, immature dendritic cells derived from the spleen of cis-NF-{kappa}BEGFP mice displayed a clear increase in EGFP expression following incubation with endotoxin-containing cecal bacterial lysates (24 h, 100 µg/ml; Fig. 4B). A time course was conducted to determine the earliest time point that EGFP could be visualized using fluorescence microscopy. We determined that cis-NF-{kappa}BEGFP transgene expression could be first observed in PBMC as early as 4 h following TNF-{alpha} stimulation (data not shown).



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  		FIGURE 4. The cis-NF-{kappa}BEGFP transgene is inducible in vitro. Primary embryonic fibroblasts (A) or splenic dendritic cells (B) were isolated from cis-NF-{kappa}BEGFP mice and treated with either TNF-{alpha}, LPS, or cecal lysates. EGFP expression was assessed using fluorescence microscopy.

 
The cis-NF-{kappa}BEGFP transgene is induced in vivo following stimulation with anti-CD3, LPS, or infection with Ad5NIK.

To assess the ability of the cis-NF-{kappa}BEGFP transgene to respond to stimuli in vivo, cis-NF-{kappa}BEGFP mice were treated with either anti-CD3, LPS or Ad5NIK and expression was analyzed in the lymph node cells, splenocytes, and PBMC using FACS analysis. The data indicate that the cis-NF-{kappa}BEGFP transgene was induced in all three cell types as shown by significant increases in the number of cells expressing EGFP (Fig. 5A). The induction of the cis-NF-{kappa}BEGFP transgene in splenocytes was restricted to cells that expressed the early activation marker CD69 (Fig. 5B), consistent with a NF-{kappa}B-activated cell type. Interestingly, only a subset of CD69-positive cells displayed enhanced EGFP expression, suggesting that in vivo NF-{kappa}B activation is heterogeneous in these cells.



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  		FIGURE 5. The cis-NF-{kappa}BEGFP transgene is inducible in vivo. A, WT (EGFP–) or cis-NF-{kappa}BEGFP mice (EGFP+) were treated with anti-CD3, and 8 h later, cells were isolated from lymph node, spleen, and peripheral blood. Cells were assessed for EGFP fluorescence using flow cytometry. The percentage of cells expressing EGFP are indicated in each plot. B, Anti-CD69-PE-conjugated Ab was used to identify anti-CD3-activated lymphocytes. EGFP/anti-CD69 plots indicate the percentage of cells expressing both markers.

 
To characterize the in vivo time course of anti-CD3 induction of the cis-NF-{kappa}BEGFP transgene, cis-NF-{kappa}BEGFP mice were injected with anti-CD3, and EGFP expression was assessed using FACS analysis and epifluorescence microscopy at 4, 8, 12, 24, 48, and 72 h following treatment. In PBMC and splenocytes, EGFP expression was first observed at 4 h, peaked at 8 h, and was absent by 72 h (Fig. 6A and B). In the livers of LPS-treated cis-NF-{kappa}BEGFP mice, this observation was essentially recapitulated in cells with morphologies consistent with hepatocytes and Kupffer cells (Fig. 6C) with peak EGFP expression at 12 h following i.p. LPS injection. NF-{kappa}B activation in Kupffer cells was further confirmed by the colocalization of EGFP with phagocytosed 1-µm red fluorescent spheres (Fig. 6D).



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  		FIGURE 6. In vivo time course of cis-NF-{kappa}BEGFP transgene induction. A and B, cis-NF-{kappa}BEGFP mice were treated with anti-CD3 and, at specified times, splenocytes (A) and PBMC (B) were isolated and assessed for EGFP expression using flow cytometry. C, cis-NF-{kappa}BEGFP mice were also treated with LPS and, at specified times, EGFP expression was assessed in the liver using fluorescence microscopy. D, To further characterize the cell type expressing EGFP in the liver, 1-µm fluorescent beads were injected into LPS-treated cis-NF-{kappa}BEGFP mice. Kupffer cell-mediated phagocytosis of the fluorescent beads was monitored using fluorescence microscopy.

 
To further document cis-NF-{kappa}BEGFP transgene expression in whole organs, in vivo, a specific EGFP imaging camera was used to visualize EGFP expression in the intestine and liver following challenge with inductive stimuli. First, cis-NF-{kappa}BEGFP mice were injected with LPS (25 µg/g body weight) for 0, 4, and 24 h, after which mice were euthanized and the intestinal tract was dissected and analyzed for EGFP expression. Although EGFP expression was unchanged in the colon and ileum, a strong induction was observed in the duodenum and proximal jejunum (Fig. 7A), suggesting regional specificity in NF-{kappa}B responsiveness in the intestine.



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  		FIGURE 7. Assessing transgene inducibility in the small bowel of LPS-treated cis-NF-{kappa}BEGFP mice. A, cis-NF-{kappa}BEGFP mice were treated with LPS and at either 4 or 24 h, EGFP fluorescence was assessed using digital fluorescence microscopy. Left, Colon to cecum; middle, cecum/ileum; right, duodenum. B and C, Cellular analysis of PBS-treated and 24-h LPS-treated duodenum. Transverse optical sections of villi (10.0 µm (B) or 3.0 µm (C)) were captured using confocal microscopy. C, Cells consistent with mononuclear (MN, yellow arrows) or IEC (white arrows) morphologies are indicated. D, Cell lineage of EGFP-expressing cells in LPS-treated duodenum was assessed using either the T cell marker anti-CD3 (red) or the general monocyte marker anti-CD64 (red). Arrows indicate representative cells that coexpress EGFP (green), CD3 (red), or CD64 (red). Overlays are represented in the right panels.

 
To gain more insight into the tissue distribution of EGFP-expressing cells following in vivo LPS injection, we performed confocal microscopy analysis on sections of the duodenum and proximal jejunum in cis-NF-{kappa}BEGFP mice. Interestingly, EGFP-positive cells were mainly located in the lamina propria of the intestine and displayed morphologies that were consistent with intraepithelial lymphocytes and dendritic cells (Fig. 7, B and C). To further characterize the EGFP-positive cells, we stained these sections with a T cell marker (CD3{epsilon}) or a general monocyte marker (CD64). Confocal microscopy shows colocalization of EGFP with both CD3{epsilon} and CD64 (Fig. 7D). Although sparse, EGFP-expressing IECs were detected in the intestine of LPS-injected cis-NF-{kappa}BEGFP mice (Fig. 7C). These data demonstrate the heterogeneity of the cellular NF-{kappa}B response to LPS and suggests that IECs are not the primary target of LPS stimulation under i.p. injection conditions.

Second, to selectively induce NF-{kappa}B activity, in vivo, we used an adenoviral vector encoding the NF-{kappa}B-inducing kinase (Ad5NIK). This viral vector strongly induces IKK kinase activity, I{kappa}B{alpha} serine phosphorylation, and NF-{kappa}B-dependent gene transcription (31). EGFP expression is markedly induced in a dose-dependent manner in the livers of Ad5NIK-infected cis-NF-{kappa}BEGFP mice compared with PBS-injected or Ad5LacZ control virus-injected cis-NF-{kappa}BEGFP mice (Fig. 8). Together, these data show that the cis-NF-{kappa}BEGFP transgene responds to various stimuli both in vivo and in vitro by inducing EGFP expression in organs and cells in a NF-{kappa}B-dependent manner.



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  		FIGURE 8. Hyperinducing IKK using Ad5NIK up-regulates the cis-NF-{kappa}BEGFP transgene. cis-NF-{kappa}BEGFP mice were infected with control Ad5LacZ (left panel) or Ad5NIK viruses (right panel). Doses indicated above liver as number of infectious particles injected i.v. Seven days after infection, EGFP expression was assessed using digital fluorescence microscopy.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods:
 Results
 Discussion
References
 
The transcription factor NF-{kappa}B has been extensively studied in various biological processes such as cellular proliferation, differentiation, and apoptosis. Using biochemical and pharmacological approaches, numerous studies demonstrate that induced NF-{kappa}B activity is associated with various inflammatory disorders and carcinogenesis. For example, the induction of NF-{kappa}B-dependent gene expression in various animal models of diseases such as arthritis, asthma, and inflammatory bowel and neurodegenerative diseases (13, 40, 41) directly correlates with the activity index of these diseases. Furthermore, NF-{kappa}B DNA-binding activity and nuclear translocation of some NF-{kappa}B subunits is increased during inflammatory bouts (13). However, previous methods to detect NF-{kappa}B activation fail to capture the dynamic aspect of NF-{kappa}B activity and provide limited physiological information on this complex signaling pathway.

In this study, we report the generation of the first NF-{kappa}B-responsive, enhanced GFP (EGFP)-reporter gene mouse using the single copy HPRT gene-targeting system. This transgenic mouse demonstrated increased EGFP expression in PBMC, splenic-derived dendritic cells, thymus, spleen, liver, and small intestine of anti-CD3- and LPS-, or Ad5NIK-treated transgenic mice. Using three different approaches, we demonstrated that stimuli-induced EGFP expression was specifically dependent on NF-{kappa}B activation. First, we performed ChIP assays on in vivo-stimulated cells to ascertain the recruitment/binding of RelA to the NF-{kappa}B-transgenic promoter. Using the ChIP technique, we found a strong induction of RelA loading not only on the cis-NF-{kappa}BEGFP-transgenic promoter, but also on the promoters of the NF-{kappa}B-responsive genes RANTES, MCP-1, MIP-2, and IL-6, suggesting that increased EGFP expression correlates with induction of physiologically relevant endogenous genes. Second, we used two different strategies to pharmacologically ablate NF-{kappa}B activation and assess the effects on the cis-NF-{kappa}BEGFP transgene expression. We used the NBP to selectively block IKK activity and the BAY 11-0782 to prevent I{kappa}B{alpha} phosphorylation. These two inhibitory approaches clearly prevented stimuli-induced EGFP expression in vivo and in vitro, demonstrating the essential role of NF-{kappa}B signaling in driving expression of the cis-NF-{kappa}BEGFP transgene. We also induced, in vivo, the NF-{kappa}B signaling cascade using adenoviral delivery of NIK, a kinase that increases IKK activity, and, thus, NF-{kappa}B-dependent gene transcription. Using this strategy, we observed a strong induction of EGFP in the livers of infected cis-NF-{kappa}BEGFP mice. These data clearly show the NF-{kappa}B-specific inducible nature of this transgenic mouse.

This study also demonstrates that dynamic expression of the cis-NF-{kappa}BEGFP transgene in whole tissues and organs is easily documented using digital fluorescence photography. Using this approach, we found that LPS selectively induced EGFP expression in the duodenum and proximal jejunum, but not in the ileum and colon. This differential responsiveness of the gut to LPS may reflect the differential exposure of the intestine to endogenous bacteria (or bacterial products) leading to a differential zone of susceptibility within the gastrointestinal tract. Bacterial distribution varies along the gastrointestinal tract with low amounts of bacteria in the small bowel (103) and, by contrast, very high levels in the distal ileum and colon (1012). Therefore, LPS challenge may initiate a stronger induction of the cis-NF-{kappa}BEGFP transgene in a section of the intestine not accustomed to high bacterial loads. Derivation of the cis-NF-{kappa}BEGFP mice in germfree conditions will help assess the differential intestinal response to endogenous bacteria.

At the cellular level, we found that in LPS-injected cis-NF-{kappa}BEGFP mice, EGFP-expressing cells were mostly located in the lamina propria of the intestine with minimal activation of IECs. This indicates that i.p. administered LPS primarily activates intestinal lamina propria mononuclear cells (LPMNC) and subsequently IECs. Whether these events are directly mediated by LPS, or are due to soluble mediators, remain to be determined. Of note, we reported that Bacterioides vulgatus colonization of germfree rats triggers NF-{kappa}B activation in IECs, but not in the LPMNC (28). This differential effect of bacteria compared with exogenously administered LPS on the NF-{kappa}B signaling pathway in the intestine likely reflects the delivery mode since bacteria engage IECs through the lumen, whereas exogenously administered LPS accesses the intestine (and thus LPMNC) through the microvascular circulation. As proof-of-concept, these studies demonstrate that similar macroscopic analysis of EGFP expression following various in vivo treatments may be useful to identify regional and cellular differences in EGFP expression.

The participation of NF-{kappa}B in the maintenance of host homeostasis in the face of various inflammatory and stress-induced challenges are well documented in the liver and intestine (42). Recently, tissue-specific deletion of IKK{beta} in enterocytes enhanced susceptibility of ischemia-reperfusion-induced intestinal injury (15). Although an important observation alone, a more thorough analysis is required to elucidate the role of NF-{kappa}B signaling in specific cells, in vivo, during disease states. Cross-breeding various tissue-specific NF-{kappa}B gene-deleted mice or various inflammatory gene-engineered murine models of inflammation with the cis-NF-{kappa}BEGFP mouse will provide critical information on the role of this pathway in various inflammatory conditions. For example, we are currently crossing the IL-10 gene-deficient mouse with the cis-NF-{kappa}BEGFP mouse to study the kinetics and cellular localization of NF-{kappa}B activation in the context of spontaneous colitis that results in IL-10–/– mice.

A possible limitation of the cis-NF-{kappa}BEGFP-transgenic mouse could be the inability to detect EGFP due to weak NF-{kappa}B activation leading to minimal EGFP accumulation that is beyond the detectable threshold. Conversely, it is predicted that the ability to detect EGFP during the chronic phase of inflammation will mainly be dependent on the nature of NF-{kappa}B transcriptional activity and on the stability of EGFP. Previous reports have demonstrated increased NF-{kappa}B activity in mucosal intestinal tissue from patients with chronic inflammatory diseases (12, 13, 43); therefore, one could anticipate that NF-{kappa}B activity will remain present in the chronic phase of inflammation in this model and that EGFP expression will be continuously induced since EGFP is under the transcriptional control of NF-{kappa}B. In addition, a bile duct ligation model of liver injury showed a clear increase of EGFP-positive cells in the liver 14 days after operation (S.T.M. and D.A.B., unpublished data). Together, these data strongly suggest that EGFP is expressed and can be measured during the course of chronic inflammation.

An attractive feature of this gene-targeting approach is the generation of cis-NF-{kappa}BEGFP-stable ES cells expressing EGFP. These cells could be used in a high-throughput random mutagenesis analysis to identify new genes involved in the regulation of NF-{kappa}B activity. Identification of cells showing loss or gain of NF-{kappa}B function could be used to generate a transgenic mouse strain to further study the functional impact of the mutation in the context of the whole organism. An additional unique feature of the cis-NF-{kappa}BEGFP mouse is the ability to FACS distinct populations of live, EGFP-positive cells that could be further studied at the molecular/biochemical level or used as donor cells in transplantation studies.

In summary, the cis-NF-{kappa}BEGFP mouse will allow for the first time the visualization of NF-{kappa}B transcriptional activation in the whole animal or cultured cells. We believe that this transgenic mouse will help address multiple questions regarding the state of NF-{kappa}B activation in embryonic development, inflammation, neurodegenerative disorders, and carcinogenesis and, additionally, will help to determine the effect of various in vivo treatments on this transcription factor. Moreover, pharmacokinetic and toxicology studies in these transgenic mice could be linked to in vivo NF-{kappa}B activation during diseased states to provide an accurate picture of the involvement of this transcription factor in this important cell fate pathway.


   Acknowledgments
 
We thank Dr. Carol Albright and Brigitte Allard for expert assistance with dendritic cell isolation, Chad Torrice for help with imaging EGFP in various organs, David Detweiler for animal husbandry, and Balfour Sartor for stimulating discussion.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health ROI Grants DK 47700 (to C.J.) and DK34987 (to D.A.B.) and by the Crohn’s and Colitis Foundation of America (to C.J.). N.E.S. is the recipient of the Sydney Kimmel Foundation for Cancer research award. Back

2 Address correspondence and reprint requests to Dr. Christian Jobin, Department of Medicine, University of North Carolina, CB 7032, 7341B, Medical Biomolecular Research Building, NC 27599. E-mail address: Job{at}med.unc.edu Back

3 Abbreviations used in this paper: IKK, I{kappa}B kinase; IEC, intestinal epithelial cell; EGFP, enhanced GFP; ES, embryonic stem; HPRT, hypoxanthine phosphoribosyltransferase; MT, mutant; HAT, hypoxanthine/aminopterin/thymidine; PFA, paraformaldehyde; HSC, hepatic stellate cell; WT, wild type; ChIP, chromatin immunoprecipitation; NBP, NEMO-binding peptide; LPMNC, lamina propria mononuclear cell. Back

Received for publication August 19, 2003. Accepted for publication May 18, 2004.


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 Discussion
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