Disease activity in rheumatoid arthritis is inversely related to cerebral TSPO binding assessed by [11C]PBR28 positron emission tomography

Reumatoid Arthritis (RA) is an autoimmune disorder characterized by peripheral joint inflammation. Recently, an engagement of the brain immune system has been proposed. The aim with the current investigation was to study the glial cell activation marker translocator protein (TSPO) in a well characterized cohort of RA patients and to relate it to disease activity, peripheral markers of inflammation and autonomic activity. Fifteen RA patients and fifteen healthy controls matched for age, sex and TSPO genotype (rs6971) were included in the study. TSPO was measured using Positron emission tomography (PET) and the radioligand [C-11] PBR28. The outcome measure was total distribution volume (V-T) estimated using Logan graphical analysis, with grey matter (GM) as the primary region of interest. Additional regions of interest analyses as well as voxel-wise analyses were also performed. Clinical evaluation of disease activity, symptom assessments, serum analyses of cytokines and heart rate variability (HRV) analysis of 24 h ambulatory ECG were performed in all subjects. There were no statistically significant group differences in TSPO binding, either when using the primary outcome V-T or when normalizing V-T to the lateral occipital cortex (p > 0.05). RA patients had numerically lower V-T values than healthy controls (Cohen's D for GM = -0.21). In the RA group, there was a strong negative correlation between [C-11]PBR28 V-T in GM and disease activity (DAS28)(r = -0.745, p = 0.002, corrected for rs6971 genotype). Higher serum levels of IFN gamma and TNF-alpha were found in RA patients compared to controls (p < 0.05) and several measures of autonomic activity showed significant differences between RA and controls (p < 0.05). However, no associations between markers of systemic inflammation or autonomic activity and cerebral TSPO binding were found. In conclusion, no statistically significant group differences in TSPO binding as measured with [C-11]PBR28 PET were detected. Within the RA group, lower cerebral TSPO binding was associated with higher disease activity, suggesting that cerebral TSPO expression may be related to disease modifying mechanisms in RA. In light of the earlier confirmed neuro-immune features of RA, these results warrant further investigations regarding neuro-immune joint-to-CNS signalling to open up for potentially new treatment strategies

Cholera toxin binds to LewisX and fucosylated glycoproteins play a functional role in human intestinal cell intoxication

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants

Cholera toxin binds to LewisX and fucosylated glycoproteins play a functional role in human intestinal cell intoxication

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (LeX) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the LeX glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to LeX. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants

GM1 ganglioside-independent intoxication by Cholera toxin

<div><p>Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (Le^X) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the Le^X glycan <i>in vitro</i> when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to Le^X. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants.</p></div

CT induced ion secretion can be inhibited by pretreating the tissue with AAL and PNA.

<p>Human jejunal mucosae were pre-incubated with or without AAL or PNA at the indicated concentrations, mounted in an Ussing chamber and exposed to CT. <b>(A)</b> Dot plot showing percent difference in I_ep to control tissue for jejunal mucosae over time. Each dot represents a mean of 4–7 donors (each treatment for each donor was tested in duplicates) with SEM error bars. Significance was calculated using a two-way-ANOVA with Tukey correction (compared to the CT). * represent CT to CT+AAL comparison and † represent CT to CT+PNA comparison (**** = p<0.0001 and ** = p<0.01). <b>(B)</b> Dot plot showing percent of start I_ep for jejunal mucosae at 180 min. AAL, PNA-treated or untreated tissue were treated with forskolin (or forskolin analog NKH477) bilaterally at 200 min. CT treated tissue were treated with bumetanide at 200 min. Each dot represents a mean of 2–3 donors (each treatment for each donor was tested in duplicates). Error bars show SEM.</p

CTB binds to Le^X-carrying proteins in HL-60 cells.

<p><b>(A)</b> Histogram from flow cytometry analysis of CTB-binding to HL-60 cells following pre-treatment of the cells with AAL (10 μg/ml) or pre-treatment of CTB with sugars (50 mM). <b>(B)</b> gMFI of CTB binding to HL-60 cells cultured with the indicated inhibitors (*** = p<0.001 and ** = p<0.01). <b>(C-D)</b> Western blot using anti-CTB of HL-60 cells co-cultured with <b>(C)</b> (NB-DGJ) or <b>(D)</b> (benzyl-α-GalNAc, kifunensine, or 2F-Fuc) and the precursor sugar Ac4ManNDAz to enable UV-crosslinking between CTB and glycosylated structures. Representative of two independent experiments. <b>(E)</b> Western blot using anti-Le^X of HL-60 cells after incubation with CTB, lysis and immunoprecipitation with anti-CTB. One representative out of two independent experiments is shown.</p

Le^X blocks binding of CTB to human granulocytes but not murine leukocytes.

<p><b>(A-B and D-E)</b> Histograms from flow cytometry analyses of CTB-, G33D- and OVA-binding to granulocytes in human peripheral blood. CTB was pretreated or not with titrated amounts of <b>(A)</b> Le^X-os, <b>(B)</b> GM1-os, <b>(D)</b> os-HSA (not titrated) and <b>(E)</b> Le^X-os and GM1-os. Graphs show the percent of gMFI of CTB binding to the cells where 100% represents CTB staining with no blocking oligosaccharide. <b>(C)</b> Histograms from flow cytometry analyses of CTB-, G33D- and OVA-binding to CD3+ T cells gated from murine splenocytes. CTB was pretreated or not with the indicated os or os-HSA. <b>(A-B)</b> n = 3, <b>(C)</b> One representative out of three independent experiments, <b>(D)</b> n = 8, <b>(E)</b> n = 4–9. Error bars show SD. Each dot represents one donor and significance was calculated using a one-way-ANOVA with Tukey correction compared to CTB without block if not indicated otherwise with bars (**** = p<0,0001, *** = p<0,005, ** = p<0,01).</p

CTB binds to Le^X linked to proteins but not ceramide.

<p><b>(A)</b> GM1- or Le^X-os (oligosaccharide) linked to ceramide and immobilized to wells were detected with ¹²⁵I labeled CTB. Relative binding is displayed as counts per minute (CPM). <b>(B-C)</b> ELISA with titrated amounts of os-linked to HSA, immobilized to wells and detected with <b>(B)</b> CTB-HRP and <b>(C)</b> G33D-HRP. Graph shows absorbance values from three independent experiments. <b>(D)</b> ELISA with os-linked to HSA, immobilized in wells and then blocked with indicated concentrations of anti-Le^X antibody HI98 or isotype control (IgM), and detected with CTB-HRP. Graph shows absorbance values from one representative out of two independent experiments. <b>(E)</b> ELISA with os-linked to HSA, immobilized in wells and detected with CTB-HRP in the presence of increasing blocking concentrations of tri-Le^X-os and GM1-os. Graphs show absorbance values from one representative out of two independent experiments.</p

CTB binding to primary human intestinal cells can be blocked by interference with fucosylated structures.

<p><b>(A)</b> Bar graph showing relative absorbance values from an ELISA with immobilized anti-Le^X, and detection with CTB-HRP. Samples as indicated from lysates of isolated human cells (2 μg protein/ml). Each dot represents a human donor (n = 5–8). <b>(B)</b> CD66 or <b>(C)</b> CD66 and Le^X expression by jejunal epithelial cells that were isolated using EDTA medium (villi) or enzymatic degradation after EDTA treatment (non-villi or crypt). Histograms from flow cytometry analyses of CTB-, G33D- and OVA-binding to the differentially enriched epithelial cells. <b>(B)</b> EpCAM⁺ cells and <b>(C)</b> EpCAM⁺Le^X+ cells. <b>(D-G)</b> Bar graph showing percent of gMFI of CTB binding to jejunal epithelial cells by pretreatment of the cells with <b>(D)</b> lectins, <b>(E)</b> sugars, <b>(F)</b> oligosaccharides and <b>(G)</b> HSA-linked oligosaccharides. Graphs show the percent of gMFI of CTB binding to the cells where 100% represents CTB staining with no blocking oligosaccharide. Each dot represents a donor in <b>(D)</b> n = 4–12, <b>(E)</b> n = 6–8, <b>(F)</b> n = 6–12, <b>(G)</b> n = 6–7. Significance was calculated using a one-way-ANOVA with Tukey correction compared to CTB without block if not indicated otherwise with bars (**** = p<0,0001, *** = p<0,005, ** = p<0,01 and * = p<0,05).</p