In vivo Imaging Method to Distinguish Acute and Chronic Inflammation

Summary

We describe a non-invasive imaging method for distinguishing inflammatory stages. Systemic delivery of luminol reveals areas of acute inflammation dependent upon MPO activity in neutrophils. In contrast, injection of lucigenin allows for visualization of chronic inflammation dependent upon Phox activity in macrophages.

Abstract

Inflammation is a fundamental aspect of many human diseases. In this video report, we demonstrate non-invasive bioluminescence imaging techniques that distinguish acute and chronic inflammation in mouse models. With tissue damage or pathogen invasion, neutrophils are the first line of defense, playing a major role in mediating the acute inflammatory response. As the inflammatory reaction progresses, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages, which mediate chronic inflammation and promote tissue repair by removing tissue debris and producing anti-inflammatory cytokines. Intraperitoneal injection of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) enables detection of acute inflammation largely mediated by tissue-infiltrating neutrophils. Luminol specifically reacts with the superoxide generated within the phagosomes of neutrophils since bioluminescence results from a myeloperoxidase (MPO) mediated reaction. Lucigenin (bis-N-methylacridinium nitrate) also reacts with superoxide in order to generate bioluminescence. However, lucigenin bioluminescence is independent of MPO and it solely relies on phagocyte NADPH oxidase (Phox) in macrophages during chronic inflammation. Together, luminol and lucigenin allow non-invasive visualization and longitudinal assessment of different phagocyte populations across both acute and chronic inflammatory phases. Given the important role of inflammation in a variety of human diseases, we believe this non-invasive imaging method can help investigate the differential roles of neutrophils and macrophages in a variety of pathological conditions.

Introduction

Inflammation is a highly regulated biological response involved in a variety of human diseases, including microbial infection ¹, wound healing ², diabetes ³, cancer ⁴, cardiovascular ⁵, neurodegenerative ⁶, and autoimmune diseases ⁷. Tissue inflammation requires proper coordination of various immune cells in order to achieve pathogen clearance, tissue repair, and disease resolution. Neutrophils and macrophages are key immune mediators of tissue inflammation. In the acute phase of inflammation, neutrophils are the first responders to various harmful stimuli and tissue damage ⁸. The neutrophils rapidly extravasate from circulation to the site of injury, where the cells inactivate invading microbes by releasing anti-microbial granules and phagocytosis. During phagocytosis, neutrophils engulf invading microbes into phagosomes, within which the cells produce high levels of superoxide (O₂·^-). Phagosomal superoxide is the primary source of many downstream reactive oxygen species (ROS). For example, superoxide can be dismutated to hydrogen peroxide (H₂O₂) by spontaneous dismutation or by superoxide dismutase (SOD) ^{9, 10}. In neutrophils, myeloperoxidase (MPO) further converts hydrogen peroxide to anti-microbial hypochlorous acid (HOCl) ¹¹. As the inflammatory responses continue, circulating monocytes gradually migrate into the site of injury and differentiate into mature macrophages ², whose phagocytic function help remove inactivated pathogens and cell debris. In addition, as a key regulator in the later phase of inflammation, macrophage promotes tissue repair by producing anti-inflammatory cytokines ¹² and by generating extracellular ROS at a lower level ⁹. The ROS generated at this later stage regulate tissue remodeling, new vessel formation, and reepithelialization ¹³.

Phagocyte NADPH oxidase (Phox) is the primary source of superoxide production in both neutrophils and macrophages ⁹. Phox is a multi-subunit complex whose assembly is tightly regulated ⁹. The holoenzyme contains several cytosolic regulatory subunits (p67^phox, p47^phox, p40^phox, and RAC) and a membrane-bound heterodimer cytochrome b₅₅₈ (consist of subunit CYBA and CYBB). Cytochrome b₅₅₈ is the reaction core within which the CYBB subunit (also known as p91^phox and NOX2) carries out the primary redox chain reaction ⁹. Interestingly, its assembly sites are different between neutrophils and macrophages. In resting neutrophils, cytochrome b₅₅₈ is mostly present in the membrane of intracellular storage granules ¹⁴. During phagocytosis, neutrophils assemble the holoenzymes at phagosomes ⁹, where high levels of MPO activity are also present. The neutrophil Phox rapidly consumes oxygen and exerts its microbicidal power by ROS production, a phenomenon termed the respiratory burst ¹¹. In contrast, macrophages have a lower level of MPO expression and cytochrome b₅₅₈ is mostly found in the plasma membrane ^{15, 16}. Thus neutrophils produce high levels of superoxide for anti-microbial activity, while macrophages generate less superoxide for regulatory functions ¹⁵.

Since inflammation is an intricate in vivo process, non-invasive imaging methods specific for different phases of inflammation would allow quantitative and longitudinal assessment of disease models. Using mechanistic studies, we have previously demonstrated the use of two chemiluminescent agents, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) and lucigenin (bis-N-methylacridinium nitrate), for non-invasive imaging of acute and late (chronic) stages of inflammation, respectively ¹⁷. Luminol enables visualization of neutrophil MPO activity in the acute phase of inflammation ^18-20, whereas lucigenin bioluminescence can be used to assess macrophage activity in association with the late phase or chronic inflammation ¹⁷. In this manuscript, we used two experimental inflammation models (s.c. PMA and s.c. LPS) to demonstrate these imaging techniques.

Protocol

Note: All animal studies were performed under approved institutional protocols and animal care guidelines.

1. Reagents and Solutions

1. PMA solution for s.c. inoculation: Prepare a stock solution of phorbol 12-myristate 13-acetate (PMA) at 5 mg/ml in DMSO. Store the stock solution at -20 °C. Before inoculation, thaw the stock solution and dilute to 1 mg/ml PMA in PBS.
2. LPS solution for s.c. inoculation: Dissolve lipopolysaccharide (LPS from Salmonella enterica serotype enteritidis) in sterile PBS at 1 mg/ml prior to s.c. inoculation.
3. Luminol solution for acute phase imaging: Dissolve luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt) in sterile normal saline (0.9% NaCl) at 10 mg/ml. The solution can be stored at -20 °C before use.
4. Lucigenin solution for chronic phase imaging: Dissolve lucigenin (bis-N-methylacridinium nitrate) in sterile normal saline at 2.5 mg/ml. The solution can be stored at -20 °C before use.

2. Subcutaneous PMA Inflammation Model

1. Anesthetize NCr nude mice in an induction chamber with 1-2% isoflurane. Confirm general anesthesia by loss of movement and a constant respiratory rate. Transfer an animal to a nose cone supplied with isoflurane-mixed gas and keep the animal under anesthesia.
2. Use an isopropyl alcohol wipe to clean and disinfect the injection site on the left flank.
3. Using sterile technique, inject 50 μl of the PMA inoculation solution (containing 50 μg of PMA) into the subcutaneous space on the left flank. Remove excess fluid from the injection site by using an isopropyl alcohol pad. Avoid the use of analgesia as it may affect inflammatory responses.
4. Move the animal back to the housing cage and closely monitor its recovery from anesthesia. In order to assist in the recovery, use a heating pad to keep the animal warm.

3. Subcutaneous LPS Inflammation Model

1. Anesthetize C57BL/6J mice with 1-2% isoflurane in an induction chamber. Once establishing general anesthesia, transfer an animal to a nose cone supplied with isoflurane gas and keep the animal under anesthesia.
2. Use an isopropyl alcohol wipe to clean the injection site on the left footpad.
3. Using sterile technique, inject 50 μl of the LPS inoculation solution (containing 50 μg of LPS) into the left footpad. Remove excess fluid from the injection site by using an isopropyl alcohol pad. Avoid the use of analgesia as it may affect inflammatory responses.
4. Move the animal back to the housing cage and closely monitor its recovery from anesthesia. Use a heating pad to keep the animal warm during recovery.

4. Bioluminescence Imaging of Inflammation

1. Anesthetize the animal in an induction chamber with 1-2% isoflurane-mixed gas. Confirm general anesthesia by loss of movement and a constant respiratory rate.
2. While the animal is still under anesthesia, intraperitoneally (i.p.) inject the luminol solution (10 mg/ml), or the lucigenin solution (2.5 mg/ml) for acute or chronic inflammation imaging, respectively. The final dosage is 100 mg/kg for luminol and 25 mg/kg for lucigenin. A lower dose of lucigenin (10-15 mg/kg) can be used to avoid possible toxicity for some mouse strains. Signs of toxicity include shortness of breath and respiratory distress. The C57BL/6J strain has lower lucigenin tolerability than the NCr nude strain.
3. Transfer the animal into the imaging chamber of a bioluminescence imaging system.
4. Perform sequential bioluminescence imaging at 1 min intervals. Each imaging step is comprised of 1 min acquisition time, f/stop = 1, binning = 16 and 0 sec delay.
5. Within the image acquisition panel, to enable sequential multi-step imaging, click on the sequence setting button. Provide sufficient imaging steps in the acquisition profile to determine maximal luminescence output (usually 15 one-minute steps will suffice). The 15-min imaging section allows sufficient time for substrate absorption and systemic circulation.
6. Remove the animal from the imaging chamber and move it back to the housing cage. In order to assist in the recovery, use a heating pad to keep the animal warm.
7. During post-acquisition analysis, use the imaging software package to calculate peak total bioluminescent signal through standardized regions of interest (ROI). The images are present as radiance in photons/sec/cm²/sr with minimal and maximal threshold indicated. Quantitative data are presented as total flux in photons per second per ROI.

Results

We performed longitudinal bioluminescence imaging to assess acute and chronic inflammation in experimental inflammation models. Phorbol 12-myristate 13-acetate (PMA) is a potent protein kinase C (PKC) agonist that activates Phox for superoxide anion production and triggers intense acute inflammatory responses ²¹. S.C. injection of 50 mg of PMA in NCr nude mice caused rapid skin irritation and acute inflammation at injection sites ^{18, 22, 23}. We performed daily imaging for 4 days with the first image...

Discussion

In this report, we demonstrate a bioluminescence method for non-invasive imaging of inflammation in living animals. Taking advantage of two luminescent substrates, luminol and lucigenin, the method can distinguish different phases of inflammation. Luminol bioluminescence is associated with neutrophils in the acute phase of inflammation, whereas lucigenin bioluminescence is mediated by macrophages in the chronic phase. Relatively small (M.W. = 177.16 g/mol) and electrically uncharged, luminol can readily penetrate both pl...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Phorbol 12-myristate 13-acetate (PMA)	Sigma-Aldrich Co., St. Louis, MO	P8139
Lipopolysaccharide from Salmonella enterica serotype enteritidis	Sigma-Aldrich Co., St. Louis, MO	L2012
Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione, sodium salt)	Sigma-Aldrich Co., St. Louis, MO	A4685
Lucigenin (bis-N-methylacridinium nitrate)	Sigma-Aldrich Co., St. Louis, MO	M8010
IVIS Spectrum imaging system with Living Imaging 4.2 software package	Caliper LS/Perkin Elmer, Hopkinton, MA