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Abstract

Molecular imaging methods allow the noninvasive detection and localization of specific molecules. Agents that report on molecular disease biomarkers can be used to diagnose and monitor disease. Many inflammatory diseases have molecular signatures within altered tissues. Although tissue biopsy is still the gold standard for detecting these signatures, several molecular imaging markers have been developed. Pharmacologic agents that block specific immune molecules have recently entered the clinic, and these drugs have already transformed the way we care for patients with immune-mediated diseases. The use of immunomodulatory drugs is usually guided by clinical assessment of the patient's response. Unfortunately, clinical assessment may miss the signs of inflammation, and many of the serologic markers of immune-mediated diseases correlate poorly with the underlying inflammatory activity within target tissues. Molecular imaging methods have the potential to improve our ability to detect and characterize tissue inflammation. We discuss some of the molecular signatures of immune activation and review molecular imaging methods that have been developed to detect active tissue inflammation.

THE OPTIMAL TREATMENT of chronic inflammatory conditions is plagued by many of the same difficulties as the treatment of cancer. The risks of the disease must be weighed against the toxicities of the therapies, patients with clinically silent disease may still benefit from treatment, the duration of treatment depends on the individual patient's response, and the risk of relapse may be lifelong. Given these difficulties, significant effort has been made to discover new disease biomarkers. New molecular imaging techniques offer the possibility of noninvasively detecting inflammatory diseases with high sensitivity, identifying tissue destruction, and monitoring an individual patient's response to therapy. The purpose of this article is to broadly review the inflammatory process, review those modalities that have been used for detection of molecular markers of inflammation, and consider how these applications can be employed to improve the treatment of patients with autoimmune and inflammatory diseases. These new methods may transform how we stratify patients to particular therapies. They may also allow clinicians to monitor the response to treatment and diagnose relapses. In addition, the chronic and heterogeneous nature of many inflammatory and autoimmune diseases makes clinical trials difficult to conduct.¹ Imaging biomarkers of inflammation may provide surrogate end points that will simplify and accelerate the evaluation of new drugs in a given disease.

Treatment of Autoimmune and Inflammatory Diseases

The immune system is composed of small molecules, soluble proteins (eg, antibodies, cytokines, chemokines, complement proteins), cell surface proteins (eg, receptors, adhesion molecules), and immune cells (Figure 1). This complex system is responsible for eliminating invasive pathogens and for the removal of injured cells and debris. Furthermore, the immune system provides critical surveillance against malignant cells.² The components of the immune system are often classified as belonging to the innate immune response or the adaptive immune response. The components of the innate immune response are similar among individuals and are generally engaged by conserved molecules expressed by pathogens. The adaptive immune response, in contrast, changes after the exposure of the host to foreign molecules. The repertoire of receptors of the adaptive immune system will, therefore, change over time and will be different among individuals.

Figure 1.

A diagram of inflammatory response within target tissue and key molecular targets. The inflammatory response often includes interactions of soluble and cell surface molecules as well as interactions of injured cells of the target tissue with the immune cells. This simplified diagram broadly summarizes the steps in the inflammatory response and the molecular targets that have been employed in molecular imaging. First, tissue injury is accompanied by the release of locally produced proinflammatory cytokines and chemokines, as well as vasodilation (increase in capillary lumen). The vascular permeability is increased (gaps between endothelial cells on the injured side) to allow for leakage of serum components into the injured area. Second, circulating leukocytes (phagocytes, and later T and B cells) infiltrate the tissue. Third, the reactivity from the phagocytic cells leads to the release of yet more proinflammatory mediators, as well as immune regulatory compounds, and to the phagocytosis and removal of the target antigen. Finally, depending on the nature of the inflammation, the phagocytic cells can present the antigen to T cells (through major histocompatibility complex I or II), and these in turn initiate the activation of adaptive immune responses.

An essential function of both the innate and adaptive arms of the immune system is to distinguish invasive pathogens from host cells. In general, this complex system of defense is remarkably successful at eradicating pathogens while causing minimal injury to the self. The immune system responds swiftly and effectively to infectious challenges, yet factors that limit the immune response are generated almost as soon as the immune system is activated.^3,4 Although some localized injury is common during immune responses, the degree of bystander injury is usually inconsequential compared to the importance of eliminating pathogens. Nevertheless, the cells and mediators of the immune system can cause tissue injury in a number of settings. Inflammation can be thought of, simply, as damage to the host by the immune system. In some forms of chronic infection, for example, the immune system is unsuccessful at eliminating the infection and the persistent inflammatory response can cause severe tissue destruction.

In the autoimmune diseases, the adaptive immune system responds to host epitopes as if they were foreign, and this immune response can cause devastating injury to target organs. Uncontrolled activation of the innate immune system can also lead to chronic inflammation. The term autoinflammatory diseases has been given to a group of diseases caused by activation of the innate immune system, apparently without the involvement of the adaptive immune system.⁵ These diseases can be caused by mutations in a variety of genes encoding reactive and regulatory proteins, allowing for increased activation of innate immune pathways.⁶ This diverse group of diseases includes gout, Crohn disease, and familial Mediterranean fever. Other autoinflammatory diseases are caused by defects such as protein misfolding, abnormal complement regulation, and uncontrolled macrophage activation.⁷ The innate immune system may also be activated by the altered surface of injured tissues. For example, natural antibody IgM can bind to epitopes exposed on ischemic or injured tissues and activate the complement system.^8,9 This phenomenon has been called “innate autoimmunity.”¹⁰

The cornerstone of treatment for autoimmune and autoinflammatory diseases is the use of immunomodulatory drugs. The most commonly used agents, such as corticosteroids, cause generalized suppression of the immune system. Depending on their mode of action, these drugs may also cause significant extraimmunologic side effects. More targeted agents can block the function of specific cell types. For example, cluster differentiation (CD)3-expressing T cells are depleted with muromonab-CD3 (OKT3), and CD20-expressing B cells are depleted with rituximab. More recently, drugs that block specific immune molecules have entered the clinic (sometimes termed “biologic agents”). Drugs that block a single immune factor, such as tumor necrosis factor (TNF)-α have been remarkably effective at treating inflammatory diseases, including rheumatoid arthritis (RA)¹¹ and Crohn disease.¹² Anakinra is an interleukin (IL)-1 receptor antagonist that may be effective in autoinflammatory diseases.⁶ The efficacy of therapeutic agents that target a single, specific molecule highlights the utility of noninvasive molecular imaging methods for monitoring specific molecular targets.

Need for New Imaging Biomarkers of Inflammation

Many autoimmune diseases are heterogeneous in nature. A disease such as systemic lupus erythematosus, for example, can affect many different organ systems. An individual patient's disease may remit or relapse, and the manifestations may change over time. Furthermore, the clinical signs may not reflect the activity of the underlying process. The classic signs of inflammation—tumor (swelling), dolor (pain), calor (heat), and rubor (redness)—may appear late, if at all. Left untreated, however, tissue inflammation causes irreversible damage. Tissue changes tend to start long before there is an overt loss of function, raising the possibility that early diagnosis can significantly alter the course of the disease. For example, gadolinium (Gd) complexes are used to identify demyelinating lesions in the brains of patients with multiple sclerosis, yet the lesions reflect a late-stage event in the pathologic process, substantially decreasing therapeutic potential.

Although the expanding array of immunomodulatory drugs has improved our ability to treat disease, a major drawback to all immunomodulatory therapies, even those targeting a single molecule,^13,14 is the increased risk of infection that they confer. This is compounded by the fact that the risk of recurrence of autoimmune diseases is lifelong, frequently necessitating either continued therapy or repeated courses of therapy. Clinicians may stop treatment during disease remission in an effort to minimize these toxicities. On the other hand, uncontrolled inflammation can eventually cause tissue fibrosis, and once significant fibrosis has developed, immunosuppressive drugs may no longer be of benefit.

The difficulty facing the clinician, therefore, is how to treat the patient sufficiently to suppress active tissue inflammation while minimizing the side effects of the medications. The optimal use of immunomodulatory agents requires evaluation of whether the disease is active and whether the target(s) of the drug is actively involved in the disease pathogenesis. This balance is, for the most part, maintained by clinical assessment of the patient and by following serologic biomarkers. Unfortunately, clinical assessment may miss the signs of inflammation and many of the serologic markers correlate poorly with the underlying inflammatory activity within target tissues.¹⁵ Therefore, in addition to developing new immunomodulatory agents to manipulate the immune system, it is equally important that we develop methods of monitoring disease activity and the effects of these agents. Molecular imaging methods have the potential to fill this need.

Molecular Markers of Tissue Inflammation

Many disease biomarkers are readily measured in the blood or urine. Analysis of cerebrospinal fluid or joint fluid is also essential for the evaluation of some diseases. The gold standard for evaluating inflammation is the tissue biopsy. Light microscopy of biopsied samples can reveal infiltration of the tissue by inflammatory cells, tissue edema, and fibrosis. Immunofluorescence microscopy is invaluable for detecting specific molecular markers of disease, including expression of adhesion molecules, complement and immune complex deposition, and identification of inflammatory cell subtypes. Electron microscopy can reveal ultrastructural changes in the tissue architecture.

The molecular markers of inflammation vary from disease to disease. Some are common to many forms of inflammation, such as upregulation of adhesion molecules on the vasculature.¹⁶ Other immune factors are quite specific to a particular disease, such as antibodies to the acetylcholine receptor in patients with myasthenia gravis,¹⁷ or to the glomerular basement membrane in patients with Goodpasture disease.¹⁸ Often the diagnosis of autoimmune disease requires detection of a constellation of findings. To use lupus nephritis as an example, the diagnosis and classification of the disease involve assessment of the histologic pattern of injury, immunofluorescence detection of immunoglobulin (often of multiple classes), complement fragments, and electron microscopic characterization of the location of the immune deposits.¹⁹ These findings are corroborated with serologies, such as antinuclear antibodies and perturbations in circulating levels of complement proteins.²⁰ The great promise of molecular imaging in autoimmune diseases, such as lupus nephritis, is that it may noninvasively provide the same information as is currently obtained only by tissue biopsy.

Modalities Used for Imaging Inflammation

Some of the studies discussed below employ nonspecific agents, that is, agents that enhance inflamed tissues owing to broad inflammatory changes, such as increased vascular permeability or adhesion of leukocytes to vessel walls. These changes are common to many disease processes. Targeted molecular imaging is usually performed using contrast agents linked to a vector that will specifically bind to a molecular target. The overall accuracy of a method is, therefore, a product of both the ability of the targeted contrast agent to bind the target and the specificity of the molecular target as a biomarker of inflammation. Some of the methods are of intermediate specificity. They employ contrast agents whose surface characteristics favor phagocytosis by leukocyte subsets or that bind to injured or inflamed structures. In the next few subsections, we summarize the different imaging modalities and related molecular targets that have been used for noninvasive molecular imaging of inflammation in clinical and research settings. A summary of the modalities discussed here, and their specifications, is also listed in Table 1, whereas the molecular targets employed in imaging of inflammation in various diseases are summarized in Table 2.

Table 1.

Characteristics of Modalities Used for Noninvasive Imaging of Inflammation*

Name	Spatial Resolution (mm)	Quantitative?	Radiation Exposure	Advantages	Drawbacks
Radiography	< 1	No	High	Projection imaging, rapid, widely available	High radiation, nonquantitative, nonspecific
SPECT	8–15	Yes	Low	Same as PET	Same as PET
PET	4–5	Yes	Low	Offers functional information, sensitive bone and brain imaging	Expensive, requires radionuclide preparation, poor anatomic localization of signal
SPECT/CT and PET/CT	< 1 for anatomy; 4–15 for radiotracer	Yes	Moderate to high	Merging of anatomic and functional information	Expensive, radiation exposure, signal attenuation by bones
US	< 1	Limited (with Doppler US)	None	Widely available, inexpensive, quick	Operator skill dependent, some functional information, sound waves hindered by gas and dense structures
MRI	0.1–1	Yes	None	Excellent resolution, safe, no radiation exposure, good anatomic detail, functional information, brain imaging	Expensive
Optical/FMT	0.5–1	Yes	None	Good resolution, inexpensive, sensitive	Limited tissue penetration depth, mostly preclinical applications

CT = computed tomography; FMT = fluorescence-mediated tomography; MRI = magnetic resonance imaging; PET = positron emission tomography; SPECT = single-photon emission computed tomography; US = ultrasonography.

See also Signore et al, Gotthardt et al, and Graves et al.^21,68,116

Table 2.

Imaging Modalities, Selected Molecular Targets of Inflammation, and Various Labeled Vectors Used in Targeted Molecular Imaging of Inflammation

Modality	Molecular Targets	Vectors	Applications in Humans and Animal Models	References
SPECT	CD4, CD3, E-selectin, TNF-α, IL-2 receptor, MMP	^99mTc-antibodies, ^99mTc-OKT3, ^99mTc-IL-2, ¹²³I-IL-2, ^99mTc-RP805	RA; Crohn, celiac, and thyroid diseases; type 1 diabetes; myocardial infarction	31–40, 41
PET	PBBS, TNF-α, α_vβ₃ integrin, macrophages, VAP-1, VCAM-1	¹¹C-PK11195, ⁶⁴Cu-etanercept, ⁶⁴Cu-RGD, ⁶⁴Cu-TNP, [⁶⁸Ga]DOTAVAP-P1, ¹⁸F-4V	Neuroinflammation, chronic infection, vascular inflammation, osteomyelitis	47–51, 55–57, 58, 61
US	CD11b, ICAM-1, MAdCAM-1	Albumin MB, antibody-coated MBs	Cardiac allograft rejection, IBD	73–77
MRI	VCAM-1, ICAM-1, MHC II, C3 fragments, platelets, selectins	VINP-28, VCAM-MPIO, Gd-DTPA-antibody conjugates, RT1-IO, anti-C3-IO, LIBS-MPIO, sialyl Lewis^x-Gd-DTPA	Atherosclerosis, acute brain inflammation, vasculitis, renal inflammation, thrombosis, cerebral malaria	79, 89, 95–97, 104, 106, 108, 105
Optical imaging	Vascular glycoproteins, cathepsin B, bacterial cell wall components	MECA-79- indotricarbocyanine, ProSense 680, squaraine rotaxane fluorescent probes	Joint and muscle inflammation, bacterial infection, pulmonary inflammation	122–124, 126, 128

C3 = complement component 3; CD = cluster differentiation; DOTA = 1,4,7,10-tetraazacyclododecane-N′,N″,N′″,N″″-tetraacetic acid; DTPA = diethylenetriaminepentaacetic acid; IBD = inflammatory bowel disease; ICAM = intercellular adhesion molecule; IL = interleukin; IO = iron oxide; LIBS = ligand-induced binding sites; MAdCAM = mucosal addressin cellular adhesion molecule; MB = microbubble; MECA-79 = antibody to peripheral node addressin; MHC = major histocompatibility class; MMP = matrix metalloproteinase; MPIO = microparticles of iron oxide; OKT3 = antibody to T cells; PBBS = peripheral benzodiazepine binding site; PK11195 = 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide; RA = rheumatoid arthritis; RGD = arginine (R)-glycine (G)-aspartic acid (D); RT1 = antibody to MHC; TNF = tumor necrosis factor; VAP = vascular adhesion protein; VAP-P1 = VAP selective peptide 1; VCAM = vascular cell adhesion molecule; VINP-28 = VCAM-1 internalizing nanoparticle 28.

Single-Photon Emission Computed Tomography

This scintigraphic method for detection of inflammation requires a source of gamma rays emitted from an infected or inflammatory region and registration of the emitted rays with gamma cameras. The gamma rays are derived from the deposited radiopharmaceuticals, usually labeled with gallium 67 (⁶⁷Ga), indium 111 (¹¹¹In), iodine 123 (¹²³I), or technetium 99m (^99mTc).²¹ Whereas some radionuclides are attached to their pharmaceutical by chemical conjugation methods, ¹²³I is incorporated into proteins by its direct binding to tyrosine residues.²¹ An accumulation of as little as picomolar radiotracer amounts in tissues can be detected by the gamma cameras.²²

Advantages and Drawbacks of SPECT and Its Current Clinical Applications and Applications in Development

The use of single-photon emission computed tomographic (SPECT) systems with modern detectors and image reconstruction applications offers several advantages: the use of tracers that expose patients to a relatively low dose of radioactivity (10–20 mCi) and reasonable spatial resolution (overall system resolution of 10.5 mm for low–energy, high-resolution settings).²³ Furthermore, the ability to radiolabel specific peptides, proteins, or antibodies can be used to track their accumulation at specific sites. Conceptually, this is a powerful tool for monitoring immune-mediated diseases given the wide variety of protein markers that are expressed or accumulate at sites of inflammation. The main drawbacks of SPECT imaging are the absence of structural anatomic information for localizing the site of inflammation and tissue attenuation and scatter of photons requiring attenuation correction. The former drawback is the major limitation of this detection system, whereas the latter concern can be mitigated with the image reconstruction software.

Radiolabeled proteins and large molecules accumulate in tissues with high vascular permeability and can be used to nonspecifically detect areas of inflammation. Examples include ⁶⁷Ga citrate, ¹¹¹In oxinate, ^99mTc-hexamethylpro-pylenevamine oxime (^99mTc-HMPAO), radiolabeled polyethylene glycol (PEG)-coated liposomes, and radiolabeled human polyclonal immunoglobulin. These nonspecific radiotracers are unable to distinguish between highly perfused tumors and inflammatory edema, however, and additional criteria for inflammation need to be present. Radiotracer-labeled white blood cells and nonspecific ⁶⁷Ga citrate, ^99mTc- depreotide, and ^99mTc-pertechnetate have successfully been used to detect inflammation in patients with a number of chronic inflammatory conditions, including inflammatory bowel disease (IBD),^24,25 osteomyelitis,²⁶ atherosclerosis,²⁷ and neuroinflammatory diseases.²⁸ Lymphocytes may be sensitive to damage by the radiation, however, limiting the application of this approach.²⁹

Numerous peptides and proteins have been radiolabeled to obtain more specific molecular information in various diseases. These have been used in several animal models of inflammatory disease and have also been used in small human series. For example, monoclonal antibodies to CD4 (expressed on a subset of T cells and involved in antigen recognition and full activation of T-helper cells in the synovium of RA patients³⁰) and CD3 (pan-T-cell marker) have been radiolabeled and injected into patients with RA.^31,32 Both agents were associated with increased signal in inflamed joints. Antibodies and Fab fragments to E-selectin (an adhesion molecule displayed on activated endothelium) accumulate in inflamed joints of patients with RA.^33,34 Anti-TNF-α monoclonal antibodies have also been labeled with ^99mTc and injected into patients with RA, and increased signal was seen in the joints of patients with active disease.^35,36 SPECT detection of radiolabeled antibody to TNF-α is a particularly compelling method for monitoring RA activity as this agent is also an effective treatment for the disease.

Directly labeling and injecting cytokines themselves runs the risk of exacerbating a disease, but administration of small quantities of labeled cytokines has been performed. High-affinity receptors for IL-2, for example, are expressed only on activated T cells. Binding of radiolabeled IL-2 to T cells at sites of inflammation therefore reports on both the presence and the activation state of the T cells. ¹²³I-IL-2 has been used to detect disease activity in autoimmune thyroid disease,³⁷ Crohn disease,³⁸ and celiac disease.³⁹ Scintigraphy after injection with this agent may also reveal early pancreatic inflammation in patients at risk of developing type 1 diabetes.⁴⁰ Experimental detection of matrix metalloproteinases (MMPs) in a mouse model of myocardial infarction was possible with the MMP-targeted radiotracer ^99mTc-RP805.⁴¹ Overall, the greatest potential for the use of SPECT in the imaging of inflammation is probably for high-sensitivity detection of molecules specific for a given disease.

Positron Emission Tomography

The positron emission tomography (PET) modality requires a positron-emitting radioisotope, but it registers photons. These are generated when each emitted positron is annihilated with an electron and, as a result, emits two photons at a 180° angle from each other. The emitted photons are registered on the PET scanner only when they are simultaneously detected by the external detectors. A commonly used positron-emitting radioisotope is fluorine 18 (¹⁸F). The radiopharmaceutical ¹⁸F-fluoro-D-deoxyglucose (¹⁸F-FDG) is a widely used radiotracer for PET imaging. As a glucose analogue, it is taken up by cells with a high energy demand, such as tumor cells, brain cells, or immune cells.⁴² ¹⁸F-FDG has a half-life of 110 minutes. Inside the cells, the ¹⁸F-FDG is phosphorylated to ¹⁸F-FDG-6-phosphate but is not metabolized further through the glycolytic pathway because, unlike glucose, it lacks the hydroxyl group at the second carbon atom. Instead, ¹⁸F-FDG/¹⁸F-FDG-6-phosphate continuously accumulates in cells with a high metabolic rate, allowing for the detection of its emitted photons by PET scanners. The decay of ¹⁸F at the second carbon atom to ¹⁸O⁻ and the subsequent conversion of ¹⁸O⁻ to a hydroxyl group allow the molecule to be consumed by glycolysis.⁴³

Advantages and Drawbacks of PET and Its Current Clinical Applications and Applications in Development

The advantages of PET over SPECT are its two- to threefold greater spatial resolution and the ability to quantify the amount of radiotracer present in the tissues.^42,44 For both modalities, however, the anatomic location of the radiotracer is poorly defined unless these modalities are coupled with computed tomography (CT; discussed below). The injection of RA patients with ¹⁸F-FDG causes high signal in inflamed joints by PET scanning,⁴⁵ whereas ¹⁸F-FDG-labeled leukocytes have been used for detection of infections.⁴⁶ However, specific distinction between inflammation or cancer requires concurrent clinical observations as both immune cells and cancer cells take up FDG avidly owing to their high metabolism. Therefore, the use of radiolabeled specific probes to tumor or inflammatory targets could help distinguish between the two types of tissue injury.

Targeted PET imaging probes have shown promise for the detection of macrophage infiltration, inflamed vascular walls, cytokine production, and neuroinflammation. For example, PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide] is a specific ligand of the peripheral benzodiazepine binding site (PBBS) expressed in cells of peripheral organs as well as in activated brain monocytes or macrophages. Targeting PBBS in brain is of particular interest as the high metabolic rate of brain cells does not allow PET imaging with the conventional ¹⁸F-FDG, and targeted, inflammation-specific tracers, such as PK11195, are needed. PK11195 containing the carbon 11 (¹¹C) radioisotope (¹¹C-PK11195) has been used to detect neuroinflammation in encephalitis,^47,48 multiple sclerosis,⁴⁹ Alzheimer disease,⁵⁰ Parkinson disease,⁴⁸ and amyotrophic lateral sclerosis.⁵¹ Novel PBBS-specific tracers are being developed that show greater affinity to PBBS than PK11195 and/or increased half-life of the radiotracer. Examples of these are [¹¹C]-N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide-1106 (¹¹C-DAA1106)⁵² and ¹⁸F-PBR111.⁵³ Another example of such a tracer being developed for SPECT imaging is [¹²⁵I]iodoDPA-713, which showed specific binding to PBBS in slices of rat brain with neuroinflammation.⁵⁴

The acute and chronic phases of inflammation in mice were detected by PET imaging after injection with ⁶⁴Cu-etanercept (a soluble receptor for TNF-α) or ⁶⁴Cu-labeled tetrameric RGD (arginine-glycine-aspartic acid) peptide (a ligand for the α_vβ₃ integrin), respectively.⁵⁵ Another formulation of RGD peptide, ¹⁸F-galacto-RGD, was used to detect vascular inflammation in mice.⁵⁶ Vascular adhesion protein 1 is upregulated on endothelial cells during inflammation and binds its selective peptide 1 (VAP-P1). VAP-P1, conjugated to 1,4,7,10-tetraazacyclododecane-N′,N″,N′″,N″″-tetraacetic acid (DOTA) and labeled with ⁶⁸Ga ([⁶⁸Ga] DOTAVAP-P1), was used to detect osteomyelitis in Staphylococcus aureus–infected rat tibia and distinguish it from uninfected bones.⁵⁷ A PET tracer specific for vascular cell adhesion molecule 1 (VCAM-1), ¹⁸F-4V, was successfully used with PET imaging to identify atherosclerotic plaques, or myocardial ischemia, in mice.⁵⁸ Moreover, the PET signal highly correlated with the VCAM-1 messenger ribonucleic acid levels in the injured vessels and the ischemic myocardium.⁵⁸

PET was used to detect macrophage infiltration in mouse aortic aneurysms after healthy and diseased mice were injected with dextran-coated iron oxide (IO) nanoparticles radiolabeled with ¹⁸F.⁵⁹ Vasculitis in mice was also detected noninvasively with a multimodal targeted approach using PET/magnetic resonance imaging (MRI) and detection of macrophages loaded with ⁶⁴Cu-labeled IO nanoparticles.⁶⁰ Detection of atherosclerotic plaques in mice was possible with a novel IO nanoparticle-based tracer, ⁶⁴Cu-trireporter nanoparticle (⁶⁴Cu-TNP), which allowed for PET, MRI, and optical imaging detections.⁶¹ Another compound, 2-(2-nitro-1 H-imidazol-1-yl)-N -(2,2,3,3,3-pentafluoropropyl) acetamide (EF5), was reported to specifically accumulate in hypoxic tissues. EF5 was radiolabeled with ¹⁸F ([¹⁸F]EF5) and used for the detection of atherosclerotic plaques of mice by ex vivo radiography.⁶²

As with SPECT, PET imaging to detect tissue inflammation is probably most useful in diseases where specific molecular markers of the disease have been identified.

Dual Modalities: SPECT/CT and PET/CT

A combination of SPECT or PET with CT has been used in clinical practice with a combined benefit of detecting and locating the site of inflammation.

Advantages and Drawbacks of SPECT/CT and PET/CT and Their Current Clinical Applications and Applications in Development

The combined imaging application offers sensitive detection of inflammation while also providing a superb anatomic resolution with CT. However, the dual modalities expose patients to increased radiation and are relatively expensive. In the future, the dual imaging will most likely be further improved and widely used, although its usefulness in molecular imaging of inflammation will depend on the nature of the molecular marker. The successful application of SPECT/CT or PET/CT for detecting inflammation in IBD,^63,64 carotid arterial plaque inflammation,⁶⁵ atherosclerosis,²⁴ and vasculitis⁶⁶ has been reported. In addition, PET/CT has been used for localizing the origin of inflammation in patients with fever of unknown origin.^67,68 The greatest potential for SPECT/CT and PET/CT is probably for localization of protein biomarkers, such as cytokines and antibodies. Although SPECT/CT and PET/CT provide more information than SPECT or PET alone, the combined modalities increase the cost of the studies and the dose of radiation. Whether the combined study is necessary depends, therefore, on the disease studied, the marker evaluated, and whether the anatomic localization of the molecule is diagnostically important.

Ultrasonography

Recent developments allow for molecular imaging with ultrasonography (US) and acoustically active microbubble (MB) contrast agents that are functionalized to bind molecular targets. The contrast effect of the MBs depends on the frequency of the applied US waves.⁶⁹ At high frequencies, the MBs burst, and this property could also be used for targeted delivery of therapeutics. The bursting of MBs produces a high contrast signal but destroys the contrast agent. The MBs are ordinarily confined to the intravascular space. Consequently, they must be targeted to intravascular molecules unless the disease process permits extravasation of the agent.

Advantages and Drawbacks of US and Its Current Clinical Applications and Applications in Development

The main advantages of US are its wide availability, relatively low cost, and lack of patient exposure to radiation. The drawbacks of this modality include the distortion of sound waves by gaseous media or attenuation of the waves by solid structures such as bones. Any inflammatory activity occurring beyond these structures is hard to detect. Conventional US is employed for the detection of inflammation in a number of diseases, particularly with the use of gas-filled MBs.⁷⁰ The short half-life of unbound MBs reduces the nonspecific background signal.⁷¹

MBs that contain phosphatidylserine are phagocytosed by leukocytes and have been used to detect tissue inflammation in an animal model of myocardial infarction.⁷² Similarly, albumin MBs are retained in the microcirculation of inflamed tissues.⁷³ These MBs were shown to bind CD11b on adherent leukocytes in a complement-dependent fashion.⁷⁴

Several targeting proteins have been conjugated to the surface of MBs to target them to molecular markers of inflammation. For example, an antibody to intercellular adhesion molecule 1 (ICAM-1; an adhesion molecule) was conjugated to the surface of US MBs.⁷⁵ These targeted MBs bound to activated endothelial cells and could be used to distinguish cardiac allografts undergoing rejection from those that were not. MBs functionalized with antibodies to mucosal addressin cellular adhesion molecule 1 (MAdCAM-1) were able to detect and localize ileal inflammation in a mouse model of IBD.⁷⁶ Investigators have also generated MBs with dual targeting. These MBs carried antibody to ICAM-1 as well as sialyl Lewis^x, a small molecule that binds to selectins.⁷⁷ The dual targeting increased the adhesion of the MBs in an in vitro system. Using multiple targeting vectors increases the complexity to the binding but offers the possibility of fine-tuning the sensitivity and specificity of targeted agents.

Magnetic Resonance Imaging

MRI scanners are now widely used clinically, and this modality has great versatility. Different pulse sequences can be employed to discriminate various tissues. Contrast agents have been used for functional measurements in various organs and allow the detection of changes in vascularity. Thus, conventional MRI is often used to detect inflammation. For example, MRI is a common method for diagnosing osteomyelitis.⁷⁸ The development of new contrast agents has also enabled the MRI-based detection of inflammatory tissue changes and molecular markers of inflammation such as tissue-bound C3d (a by-product of complement activation within a tissue).⁷⁹ As discussed below, some of these techniques have already been employed in humans.

Advantages and Drawbacks of MRI and Its Current Clinical Applications and Applications in Development

MRI offers outstanding resolution and provides abundant information about tissue architecture. It is widely available and does not expose patients to radiation. It is relatively expensive, however, and image acquisition can be quite long. Compared to radiolabeled SPECT and PET probes, the targeted agents used in MRI-based molecular imaging are detected with much lower sensitivity. It has been estimated that MRI probes need to reach tissue concentrations of 0.01 to 10 mM to be detected, whereas SPECT and PET can detect probes in the picomolar range.²²

As with the previous modalities discussed, contrast agents for the MRI-based detection of inflammation can report on nonspecific changes in inflamed tissues as well as specific molecular markers of inflammation. Magnetic nanoparticles have frequently been used as MRI contrast agents owing to their ability to disturb the relaxation of nearby protons, thus darkening T₂-weighted MRIs. Depending on their size, nanoparticles can be used to detect vascular leak. This principle was exploited to detect pancreatic inflammation in patients with recently diagnosed type 1 diabetes.⁸⁰ Nanoparticles are also phagocytosed by macrophages, so IO-containing formulations cause a darkening of the reticuloendothelial system in the liver and spleen on T₂-weighted images.⁸¹ Further, the particles are taken up by macrophages resident in inflamed tissues. Darkening of tissues by IO nanoparticles can therefore be employed as a marker of inflammation. This method has been used in a wide range of animal models, including a model of atherosclerosis,⁸² renal ischemia/reperfusion,⁸³ cerebral ischemia,⁸⁴ and type 1 diabetes.⁸⁵ This approach has also been used to detect inflammation in patients with renal transplant rejection,⁸⁶ atherosclerosis,⁸⁷ and multiple sclerosis.⁸⁸

Although some very novel agents have been developed, few of these have yet been tested in humans. Acute brain inflammation in mice was detected by MRI with microparticles of IO (MPIO) targeted to VCAM-1 before any pathology was detected using conventional MRI.⁸⁹ Noninvasive detection of endothelial inflammation with MRI and VCAM-1 or P-selectin-targeted MPIO was possible in animal models of atherosclerosis,⁹⁰ unilateral ischemia/reperfusion injury of kidneys,⁹¹ and cerebral ischemia.⁹² Targeted MPIO were generated to visualize platelet activation and aggregation. The MPIO were conjugating to a single-chain antibody directed toward ligand-induced binding sites (LIBS) of glycoprotein IIb/IIIa receptors of platelets, termed LIBS-MPIO.^93,94 This agent was used to detect thrombosis^95,96 and cerebral malaria⁹⁷ in mice. Macrophage infiltration in the atherosclerotic aorta of rabbits was detected noninvasively with T₂-weighted MRI and monocrystalline IO nanoparticles 47 (MION-47) and highly correlated with the histomorphometric measurements of the thickened aortic wall area.⁹⁸ MION-47 were also used for detecting and monitoring pancreatic inflammation in a mouse model of type 1 diabetes.⁹⁹ Neuroinflammation was detected in the excised brains of mouse models of multiple sclerosis with anti– ICAM-1 antibody–coated paramagnetic liposomes and high-resolution MRI.¹⁰⁰ Focal brain ischemia, blood-brain barrier breakdown, and brain inflammation were noninvasively detected with MRI and E- and P-selectin-targeted gadolinium diethylenetriaminepentaacetic acid (Gd-DTPA) conjugated with E- and P-selectin-binding sialyl Lewis^x carbohydrate antigen.^101–103 Similarly, iron-containing glyconanoparticles, coated with sialyl Lewis^x, were able to noninvasively detect demyelinating lesions in a rat model of multiple sclerosis.¹⁰⁴ VCAM-1 imaging in a mouse model of atherosclerosis was possible with VCAM-1-targeted monocrystalline IO nanoparticles VINP-28 and MRI.¹⁰⁵ Inflammation in tissues expressing ICAM-1 could be detected noninvasively with T₁-weighted MRI and anti-ICAM-1 antibodies conjugated to Gd-DTPA.¹⁰⁶ An elegant approach to monitoring dendritic cell infiltration of tumor tissue was possible in animals vaccinated against the tumor with complexes of tumor antigen and superparamagnetic iron oxide (SPIO) nanoparticles. Dendritic cells became SPIO laden when they phagocytosed the antigen-SPIO complexes. The infiltration of these SPIO-laden dendritic cells into tumors was then followed noninvasively with T₂-weighted MRI.¹⁰⁷ Major histocompatibility class (MHC) II expression in the renal medulla was detected with IO nanoparticles functionalized with the RT1 antibody to MHC II. Injection of the RT1-targted nanoparticles into wild-type rats showed significant T₂-weighted MRI signal reduction in kidneys 1 hour postinjection when compared to nanoparticles functionalized with a control antibody.¹⁰⁸ Deposition of complement C3 fragments on glomeruli in a mouse model of renal inflammation was noninvasively detected with T₂-weighted MRI and IO nanoparticles conjugated to a chimeric molecule specific to C3 fragments.⁷⁹ Acute cardiac and cerebral ischemia in mice could be noninvasively detected with repetitive proton/fluorine (¹H/¹⁹F) MRI and injection of nanoemulsions of perfluorocarbon.¹⁰⁹ Similarly, abscesses in mouse thighs could be noninvasively detected with repetitive ¹H/¹⁹F MRI after injection of cross-linked IO or perfluorocarbon nanoemulsions.¹¹⁰ Finally, a library of magnetofluorescent nanoparticle probes, capable of distinguishing distinct cell types, in different physiologic states, has been developed for noninvasive in vivo detection of these cell types with MRI and optical imaging.¹¹¹

The greatest potential for the detection of tissue inflammation using MRI-based molecular imaging is probably in diseases in which detailed anatomic information regarding the localization of the target molecule is critical to the diagnosis.

Optical Imaging

A plethora of nonradioactive and nontoxic tracers (fluorescent, bioluminescent, or near-infrared proteins and dyes) and a wide array of image acquisition instrumentation have been developed for optical imaging. Without tracers, optical imaging has been employed for nonspecific tissue evaluation in endoscopy, ophthalmology and dermatology applications. In the research setting, however, optical imaging often employs molecular tracers.^112,113 The method is particularly promising for molecular imaging as it can detect picomolar amounts of tracers at 0.5 to 1 mm resolution.^114–116 Optical imaging, including fluorescence-mediated tomography (FMT), has been comprehensively described in recent reviews.^115,117

Advantages and Drawbacks of Optical Imaging and Its Current Clinical Applications and Applications in Development

The main advantages of optical imaging are the well-developed and relatively inexpensive imaging instrumentation, the absence of ionizing radiation, and the relatively short time required for data acquisition. The primary drawback is the limited tissue penetration depth of the light beam. However, this consideration is not limiting for imaging of superficially positioned structures such as the joints in patients with RA or the skin in patients with vasculitis. Moreover, in the research setting, the use of small animals and surgical manipulations can allow for visualization of fluorescent tracers from within organs, such as the brain.¹¹⁸

Optical imaging has been successfully used to visualize inflammation within the joints of rats using the nonspecific fluorescent tracer indocyanine green¹¹⁹ or by tracking the infiltration of the joints by fluorescently labeled leukocytes.¹²⁰ An IgM antibody to vascular glycoproteins MECA-79,¹²¹ labeled with a near-infrared dye, has been employed in the optical imaging of mouse lymph nodes.¹²² Muscle inflammation in the mdx mouse, a model of Duchenne muscular dystrophy (DMD), has been detected with the use of optical imaging and near-infrared substrate (ProSense 680) of cathepsin B, an enzyme upregulated in areas of inflammation.^123,124 The resting properties of brain macrophages and their responses under activating or inflammatory conditions induced with adenosine triphosphate (ATP) stimulation have been studied in vivo with the use of fluorescently labeled macrophages of the mouse brain and two-photon microscopy.^118,125

Interestingly, a distinction of infection from sterile inflammation can be made with fluorescent dyes that bind the anionic phospholipids of bacterial envelopes. S. aureus, injected into mice, could be traced with squaraine rotaxane fluorescent probes and a whole-body imager up to 12 hours after application of the probes.¹²⁶ Tracking labeled macrophages in vivo and measuring their infiltration into inflammatory lipopolysaccharide (LPS)-containing biogel pellets in mice was conducted with FMT. The labeled macrophages were detectable in LPS-containing pellets as late as 144 hours after macrophage injection.¹²⁷ Finally, mice with pulmonary inflammation induced by instillation of LPS could be distinguished from untreated mice using the cathepsin substrate ProSense probe and FMT.¹²⁸

The greatest potential for optical imaging in the diagnosis of inflammatory diseases is probably in those diseases where the molecular target is located in dermal or mucosal surfaces and is, therefore, detectable by these methods.

Goal of Personalized Medicine

The treatment of autoimmune and autoinflammatory patients has been greatly improved by the development of biologic agents, which have a narrower range of effects than traditional immunosuppressive agents, such as corticosteroids and cytotoxic agents. The diagnosis of autoimmune disease is often uncertain, and there is great patient heterogeneity. The use of biologic agents will be improved, therefore, by methods that monitor the engagement of the particular system or the molecule targeted by the drug. Molecular imaging techniques may revolutionize the care given to these patients. These modalities offer the hope of noninvasively monitoring the systemic effects of an agent on biomarkers of disease while also potentially reporting on tissue architecture.

The ability to tailor a patient's treatment to molecular markers of inflammation within target tissues would reduce toxicities of immunosuppressive medications. Another exciting advance in the treatment of these diseases is the development of immunomodulatory agents targeted to molecular markers of tissue inflammation.¹²⁹ These agents, therefore, act on a narrow spectrum of molecular targets, with a limited anatomic distribution. A logical extension of the recent advancements in therapeutic and molecular imaging would be to develop dual-function agents that would offer simultaneous diagnostic and therapeutic applications. These agents could be directed to specific sites for reporting on the degree of disease activity while simultaneously delivering treatment therapeutics. Such advances will greatly improve our ability to offer patients individualized care.

Footnotes

Acknowledgment

Financial disclosure of authors: This work was supported by grants from the Lupus Research Institute and the Kidneeds Foundation. Dr. Thurman is a consultant for Alexion Pharmaceuticals, Inc.

Financial disclosure of reviewers: None reported.

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Molecular Imaging of Autoimmune Diseases and Inflammation

Abstract

Treatment of Autoimmune and Inflammatory Diseases

Need for New Imaging Biomarkers of Inflammation

Molecular Markers of Tissue Inflammation

Modalities Used for Imaging Inflammation

Single-Photon Emission Computed Tomography

Advantages and Drawbacks of SPECT and Its Current Clinical Applications and Applications in Development

Positron Emission Tomography

Advantages and Drawbacks of PET and Its Current Clinical Applications and Applications in Development

Dual Modalities: SPECT/CT and PET/CT

Advantages and Drawbacks of SPECT/CT and PET/CT and Their Current Clinical Applications and Applications in Development

Ultrasonography

Advantages and Drawbacks of US and Its Current Clinical Applications and Applications in Development

Magnetic Resonance Imaging

Advantages and Drawbacks of MRI and Its Current Clinical Applications and Applications in Development

Optical Imaging

Advantages and Drawbacks of Optical Imaging and Its Current Clinical Applications and Applications in Development

Goal of Personalized Medicine

Footnotes

Acknowledgment

References