Representative immunofluorescence staining images of cytospin slides of healthy peripheral blood M1 and M2 MDM.

<p>Immunofluorescence staining of cytospin slides of healthy peripheral blood MDM with the nuclear stain POPO-1 (to confirm presence of cells), TSPO, the macrophage marker CD68 and merging of the three stains. <b>(A)</b> staining of MDM stimulated with LPS plus IFN<i>-</i>γ for 24 hours; <b>(B)</b> staining of MDM stimulated with IL-4 for 24 hours. x400 magnification. Scale bar = 20μm.</p

Western blotting indicates TSPO protein expression decreases on treatment of MDM with M1 stimuli LPS plus IFN-γ.

<p>(i) representative western blot of TSPO protein with β-actin acting as loading control and (ii) densitometry normalized to β-actin in (<b>A)</b> THP-1 MDM, (<b>B)</b> healthy peripheral blood MDM and (<b>C)</b> synovial fluid MDM either unstimulated (<i>unstim</i>) or treated with 10ng/mL LPS plus 20ng/mL IFN-γ (<i>LPS</i>) for 2,4,6 and 24 hours. Densitometry data is expressed as the mean of five independent experiments ± SEM. (*<i>p</i><0.05, **<i>p</i> <0.01, ***<i>p</i>≤0.001).</p

Western blotting indicates TSPO protein expression is largely unchanged in MDM treated with M2 stimuli.

<p>(i) representative Western blots of TSPO protein with β-actin acting as loading control and (ii) densitometry normalized to β-actin in <b>(A)</b> THP-1 MDM, <b>(B)</b> healthy peripheral blood MDM and <b>(C)</b> synovial fluid MDM either unstimulated (<i>Unstim</i>) treated with 20ng/mL IL-4, 500nM dexamethasone or 3ng/mL TGF-β1 for 24 hours. Densitometry data is expressed as the mean of five independent experiments ± SEM. (*<i>p</i><0.05, **<i>p</i> <0.01, ***<i>p</i>≤0.001).</p

TSPO protein expression is higher in MDM than in monocytes.

<p>TSPO protein was assessed by Western blot with β-actin as loading control <b>(Ai)</b>, with accompanying densitometry normalised to β-actin <b>(Aii)</b>. Results shown are for THP-1 monocytes, healthy peripheral blood monocytes, synovial fluid monocytes, and corresponding MDM. <b>(B)</b> [³H]PBR28 tracer binding per 1x10⁶ cells in healthy human peripheral blood monocytes and MDM. Each experiment was run in triplicate, and data expressed as mean± SEM for five independent experiments. *** p<0.001 using Student’s <i>t</i>-test, comparing data from each MDM with that of their counterpart monocytes.</p

<i>TSPO</i> mRNA expression of monocytes compared to MDM.

<p><b>(A)</b> THP-1 monocytes and THP-1 MDM; <b>(B)</b> healthy peripheral blood monocytes and corresponding MDM <b>(C)</b> synovial fluid monocytes and corresponding MDM. Results are expressed as fold change mRNA expression (compared to <i>18s</i> RNA). Each experiment was run in triplicate, with data expressed as mean ± SEM of five independent experiments. Relative <i>TSPO</i> mRNA expression in MDM was compared to their respective monocyte counterpart whereby statistically significant differences are noted as **p<0.001 using Student’s <i>t</i>-test.</p

M1:M2 ratio of mean fold change of macrophage mRNA markers.

<p>M1:M2 ratio of mean fold change of macrophage mRNA markers.</p

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

<div><p>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 <i>in vivo</i>. 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.</p></div

Binding of the TSPO specific radio-ligand [³H]PBR28 in unstimulated, M1 and M2 healthy human peripheral blood MDM.

<p>Binding of [³H]PBR28 in unstimulated healthy peripheral blood MDM, in MDM stimulated with LPS plus IFN-γ, and MDM stimulated with IL-4 for 24 hours. Each experiment was performed in triplicate, with data expressed as mean ± SEM of five independent experiments. ** p<0.01 compared to unstimulated.</p

Expression of M1 & M2 markers in M1 and M2 MDM compared to untreated MDM.

<p>Expression of M1 & M2 markers relative to untreated healthy human peripheral blood MDM (U) in MDM treated with 10ng/mL LPS plus 20ng/mL IFN-γ (LPS) (<u>M1 stimuli</u>), or 20ng/mL IL-4 for 24 hours (IL-4) (<u>M2 stimulus</u>). <b>(A)</b> mRNA expression of M1 macrophage markers; <i>TNF-α</i>, <i>IL-12β and PTGS2</i>, in LPS plus IFN-γ or IL-4 treated MDM, compared to untreated MDM. <b>(B)</b> M2-specific mRNA expression of M2 macrophage markers; <i>IL-10</i>, <i>CD200R1</i>, <i>MRC1</i>, <i>TGM2</i>, in LPS plus IFN-γ- or IL-4-treated MDM, compared to untreated MDM. <b>(C)</b> TNF-α in supernatant of healthy human peripheral blood MDM stimulated with 10ng/mL LPS plus 20ng/mL IFN-γ, or 20ng/mL IL-4 for 24 hours, were measured using an ELISA Quantikine assay. Each experiment was performed in triplicate, with data expressed as mean ± SEM of five independent experiments. (*<i>p</i><0.05, **<i>p</i> <0.01, ***<i>p</i>≤0.001).</p

Relative <i>TSPO</i> mRNA expression in MDM from THP-1, healthy human peripheral blood, and RA synovial fluid.

<p>MDMs were stimulated with: <b>(A)</b> 10ng/mL LPS plus 20ng/mL IFN-γ, 20ng/mL IL-4, 500nM dexamethasone or 3ng/mL TGF-β1 for 24 hours, or <b>(B)</b> LPS plus IFN-γ for 2, 4, 6 and 24 hours. Each experiment was run in triplicate, with data expressed as mean of five independent experiments ± SEM and compared to unstimulated control cultures. (*<i>p</i><0.05, **<i>p</i> <0.01, ***<i>p</i>≤0.001).</p