Nehal Narayan

Pro-inflammatory activation of primary microglia and macrophages increases 18 kDa translocator protein expression in rodents but not humans:

The 18kDa Translocator Protein (TSPO) is the most commonly used tissue-specific marker of inflammation in positron emission tomography (PET) studies. It is expressed in myeloid cells such as microglia and macrophages, and in rodent myeloid cells expression increases with cellular activation. We assessed the effect of myeloid cell activation on TSPO gene expression in both primary human and rodent microglia and macrophages in vitro, and also measured TSPO radioligand binding with 3H-PBR28 in primary human macrophages. As observed previously, we found that TSPO expression increases (∼9-fold) in rodent-derived macrophages and microglia upon pro-inflammatory stimulation. However, TSPO expression does not increase with classical pro-inflammatory activation in primary human microglia (fold change 0.85 [95% CI 0.58–1.12], p = 0.47). In contrast, pro-inflammatory activation of human monocyte-derived macrophages is associated with a reduction of both TSPO gene expression (fold change 0.60 [95% CI 0.45–0.74], p = 0.02) and TSPO binding site abundance (fold change 0.61 [95% CI 0.49–0.73], p < 0.0001). These findings have important implications for understanding the biology of TSPO in activated macrophages and microglia in humans. They are also clinically relevant for the interpretation of PET studies using TSPO targeting radioligands, as they suggest changes in TSPO expression may reflect microglial and macrophage density rather than activation phenotype.

The macrophage marker translocator protein (TSPO) is down-regulated on pro-inflammatory 'M1' human macrophages.

The translocator protein (TSPO) is a mitochondrial membrane protein, of as yet uncertain function. Its purported high expression on activated macrophages, has lent utility to TSPO targeted molecular imaging in the form of positron emission tomography (PET), as a means to detect and quantify inflammation in vivo. However, existing literature regarding TSPO expression on human activated macrophages is lacking, mostly deriving from brain tissue studies, including studies of brain malignancy, and inflammatory diseases such as multiple sclerosis. Here, we utilized three human sources of monocyte derived macrophages (MDM), from THP-1 monocytes, healthy peripheral blood monocytes and synovial fluid monocytes from patients with rheumatoid arthritis, to undertake a detailed investigation of TSPO expression in activated macrophages. In this work, we demonstrate a consistent down-regulation of TSPO mRNA and protein in macrophages activated to a pro-inflammatory, or ‘M1’ phenotype. Conversely, stimulation of macrophages to an M2 phenotype with IL-4, dexamethasone or TGF-β1 did not alter TSPO expression, regardless of MDM source. The reasons for this are uncertain, but our study findings add some supporting evidence for recent investigations concluding that TSPO may be involved in negative regulation of inflammatory responses in macrophages.

Advances in Positron Emission Tomography for the imaging of Rheumatoid Arthritis

Imaging techniques, such as US, are well recognized as important tools to aid early diagnosis and assess response to treatment in RA. PET offers a means of imaging the inflammatory processes of RA at a cellular level and thus may be a highly sensitive method of assessing synovitis. In this review we discuss the advantages of PET as an imaging modality for RA and summarize the existing clinical studies of PET in RA. We also highlight potentially important preclinical studies of PET in arthritis and discuss current limitations of PET as well as ongoing developments in PET technology likely to be of benefit for arthritis imaging.

Translocator protein as an imaging marker of macrophage and stromal activation in RA pannus

PET radioligands targeted to translocator protein (TSPO) offer a highly sensitive and specific means of imaging joint inflammation in rheumatoid arthritis (RA). Through high expression of TSPO on activated macrophages, TSPO PET has been widely reported in several studies of RA as a means of imaging synovial macrophages in vivo. However, this premise does not take into account the ubiquitous expression of TSPO. This study aimed to investigate TSPO expression in major cellular constituents of RA pannus—monocytes, macrophages, fibroblastlike synoviocytes (FLS cells), and CD4-positive (CD4+) T lymphocytes (T cells)—to more accurately interpret TSPO PET signal from RA synovium. Methods: Three RA patients and 3 healthy volunteers underwent PET of both knees using the TSPO radioligand 11C-PBR28. Through 3H-PBR28 autoradiography and immunostaining of synovial tissue in 6 RA patients and 6 healthy volunteers, cellular expression of TSPO in synovial tissue was evaluated. TSPO messenger RNA expression and 3H-PBR28 radioligand binding was assessed using in vitro monocytes, macrophages, FLS cells, and CD4+ T cells. Results: 11C-PBR28 PET signal was significantly higher in RA joints than in healthy joints (average SUV, 0.82 ± 0.12 vs. 0.03 ± 0.004; P < 0.01). Further, 3H-PBR28–specific binding in synovial tissue was approximately 10-fold higher in RA patients than in healthy controls. Immunofluorescence revealed TSPO expression on macrophages, FLS cells, and CD4+ T cells. The in vitro study demonstrated the highest TSPO messenger RNA expression and 3H-PBR28–specific binding in activated FLS cells, nonactivated M0 macrophages, and activated M2 reparative macrophages, with the least TSPO expression being in activated and nonactivated CD4+ T cells. Conclusion: To our knowledge, this study was the first evaluation of cellular TSPO expression in synovium, with the highest TSPO expression and PBR28 binding being found on activated synovial FLS cells and M2 macrophages. TSPO-targeted PET may therefore have a unique sensitivity in detecting FLS cells and macrophage-predominant inflammation in RA, with potential utility for assessing treatment response in trials using novel FLS-cell–targeted therapies.

Utility of [11C]PBR28 PET in the imaging of inflammatory arthritis

Abstract Background PET is a cellular imaging modality that uses radionuclides targeted to a molecule of interest. Since macrophages have a crucial role in synovitis, PET targeted to translocator protein (TSPO), a mitochondrial protein upregulated in macrophages, is postulated to be a sensitive and specific means of imaging synovitis. We aimed to ascertain whether the TSPO tracer [ 11 C]PBR28 can detect and quantify synovitis in a proof-of-principle study. Methods Ten participants with clinical evidence of synovitis in one or both knees (five with rheumatoid arthritis, five with psoriatic arthritis), and four age-matched healthy volunteers, were included. Patients underwent clinical examination, ultrasound scanning, and PET-CT of both knees, as well as synovial biopsy of one knee over 10 days, in addition to routine blood tests. Synovial tissue from biopsy was stained for TSPO, and for the macrophage markers CD68 and CD163, and scored semiquantitatively. All knees were scored according to semiquantitative synovitis severity scores on clinical examination and ultrasound by two independent assessors. Findings A significant difference was observed between average tracer uptake in knees of different synovitis severity scores on both clinical examination and ultrasound (p r =0·67, p=0·02). A positive correlation was observed between C-reactive protein and average tracer uptake in both knees ( r =0·81, p=0·03). Interpretation To our knowledge, this is the first study to indicate that [ 11 C]PBR28 PET can detect and quantify synovitis in patients with arthritis. The correlation between TSPO staining and tracer uptake implies that tracer uptake is the result of target expression and not due to confounding factors such as blood flow, but awaits confirmation with an in-vivo blocking study. Larger studies with [ 11 C]PBR28 before and after initiation of therapy will help ascertain its sensitivity as a tool for assessing response to treatment for inflammatory arthritis. Funding Imanova Academic Centre for Imaging Sciences (IMPETUS grant).