Auke Verhaar

Glucocorticoids cause rapid dissociation of a T-cell-receptor-associated protein complex containing LCK and FYN.

Although glucocorticoid (GC)‐induced nongenomic effects have been reported, the underlying mechanisms remain unexplained. We previously described that lymphocyte‐specific protein tyrosine kinase (LCK) and FYN oncogene related to SRC, FGR, YES (FYN) mediate GC‐induced inhibition of T‐cell‐receptor (TCR) signalling. Here we characterize the underlying molecular mechanism. The present study shows that the GC receptor is part of a TCR‐linked multiprotein complex containing heat‐shock protein (HSP)90, LCK and FYN, which is essential for TCR‐dependent LCK/FYN activation. Experiments with cells transfected with GC‐receptor short interfering RNA (siRNA) showed that the GC receptor is an essential component of the TCR signalling complex. Short‐term GC treatment induces dissociation of this protein complex, resulting in impaired TCR signalling as a consequence of abrogated LCK/FYN activation. HSP90siRNA‐transfected cells are not able to assemble this TCR‐associated multiprotein complex, and accordingly HSP90siRNA treatment mimics GC effects on LCK/FYN activities. These observations support a model for nongenomic GC‐induced immunosuppression on the basis of dissolution of membrane‐bound GC‐receptor multiprotein complexes after GC‐receptor ligation.

Regulatory macrophages induced by infliximab are involved in healing in vivo and in vitro.

Background: Regulatory macrophages play an important role in wound healing and gut homeostasis and have antiinflammatory properties. Induction of this cell type (M&psgr;ind) by the anti‐tumor necrosis factor (TNF) antibodies, infliximab and adalimumab, has recently been shown in vitro. Also, the superiority of infliximab/azathioprine combination therapy over infliximab or azathioprine monotherapy has recently been established, but the mechanism behind this remains unclear. The aim of this study was to examine the induction of regulatory macrophages in patients with and without mucosal healing in response to infliximab. In addition, we studied the effect of infliximab/azathioprine combination treatment on the differentiation and function of regulatory macrophages. Methods: Inflammatory bowel disease (IBD) patients (n = 10) underwent endoscopy before and after first infliximab treatment. Immunohistochemical staining of CD68 and CD206 was performed in all patients. Mixed lymphocyte reactions (MLRs) were treated with infliximab, azathioprine, or both. Macrophage phenotype was evaluated by flow cytometry and inhibition of T‐cell proliferation was measured in a secondary MLR containing macrophages and third‐party lymphocytes. Results: A significant induction of regulatory macrophages was observed in patients with mucosal healing after treatment with infliximab; this induction was absent in patients without mucosal healing. In addition, M&psgr;ind have the ability to induce wound healing in an in vitro model, further suggesting a key role for infliximab‐induced macrophages in mucosal healing. Upon infliximab/azathioprine combination treatment, an increased number of regulatory macrophages was observed. These macrophages also displayed stronger immunosuppressive properties than macrophages induced by infliximab monotherapy. Conclusions: These data show that regulatory macrophages may be involved in mucosal healing and provide a rationale for the superiority of infliximab/azathioprine combination treatment observed in the clinic. (Inflamm Bowel Dis 2012;)

Autophagy Attenuates the Adaptive Immune Response by Destabilizing the Immunologic Synapse

BACKGROUND & AIMS Variants in the genes ATG16L1 and IRGM affect autophagy and are associated with the development of Crohns disease. It is not clear how autophagy is linked to loss of immune tolerance in the intestine. We investigated the involvement of the immunologic synapse-the site of contact between dendritic cells (DCs) and T cells, which contains molecules involved in antigen recognition and regulates immune response. METHODS DC autophagy was reduced using small interfering RNAs or pharmacologic inhibitors. DC phenotype and function were analyzed by confocal microscopy, time-lapse microscopy, and flow cytometry. We also examined DCs isolated from patients with Crohns disease who carried the ATG16L1 risk allele. RESULTS Immunologic synapse formation induced formation of autophagosomes in DCs; the autophagosomes were oriented toward the immunologic synapse and contained synaptic components. Knockdown of ATG16L1 and IRGM with small interfering RNAs in DCs resulted in hyperstable interactions between DCs and T cells, increased activation of T cells, and activation of a T-helper 17 cell response. LKB1 was recruited to the immunologic synapse, and induction of autophagy in DC required inhibition of mammalian target of rapamycine signaling by the LKB1-AMP activated protein kinase (AMPK) pathway. DCs from patients with Crohns disease who had an ATG16L1 risk allele had a similar hyperstability of the immunologic synapse. CONCLUSIONS Autophagy is induced upon formation of the immunologic synapse and negatively regulates T-cell activation. This mechanism might increase adaptive immunity in patients with Crohns disease who carry ATG16L1 risk alleles.

Polymorphisms near TBX5 and GDF7 are associated with increased risk for Barrett's esophagus.

Background & Aims Barretts esophagus (BE) increases the risk of esophageal adenocarcinoma (EAC). We found the risk to be BE has been associated with single nucleotide polymorphisms (SNPs) on chromosome 6p21 (within the HLA region) and on 16q23, where the closest protein-coding gene is FOXF1. Subsequently, the Barretts and Esophageal Adenocarcinoma Consortium (BEACON) identified risk loci for BE and esophageal adenocarcinoma near CRTC1 and BARX1, and within 100 kb of FOXP1. We aimed to identify further SNPs that increased BE risk and to validate previously reported associations. Methods We performed a genome-wide association study (GWAS) to identify variants associated with BE and further analyzed promising variants identified by BEACON by genotyping 10,158 patients with BE and 21,062 controls. Results We identified 2 SNPs not previously associated with BE: rs3072 (2p24.1; odds ratio [OR] = 1.14; 95% CI: 1.09–1.18; P = 1.8 × 10−11) and rs2701108 (12q24.21; OR = 0.90; 95% CI: 0.86–0.93; P = 7.5 × 10−9). The closest protein-coding genes were respectively GDF7 (rs3072), which encodes a ligand in the bone morphogenetic protein pathway, and TBX5 (rs2701108), which encodes a transcription factor that regulates esophageal and cardiac development. Our data also supported in BE cases 3 risk SNPs identified by BEACON (rs2687201, rs11789015, and rs10423674). Meta-analysis of all data identified another SNP associated with BE and esophageal adenocarcinoma: rs3784262, within ALDH1A2 (OR = 0.90; 95% CI: 0.87–0.93; P = 3.72 × 10−9). Conclusions We identified 2 loci associated with risk of BE and provided data to support a further locus. The genes we found to be associated with risk for BE encode transcription factors involved in thoracic, diaphragmatic, and esophageal development or proteins involved in the inflammatory response.

Origin and Functional Activity of the Membrane-Bound Glucocorticoid Receptor

OBJECTIVE Glucocorticoids (GCs) exert their antiinflammatory and immunosuppressive effects in humans primarily via the cytosolic GC receptor (cGR) but also via rapid, nongenomic mechanisms. Most likely, membrane-bound GRs (mGR) are involved in nongenomic GC signaling. The aim of this study was to investigate the origin and functional activity of mGR. METHODS We analyzed the origin of mGR using mGR-expressing HEK 293T cells, by transient and stable RNA interference-mediated GR reduction. GR messenger RNA (mRNA) and cGR and mGR protein levels were analyzed by real-time quantitative polymerase chain reaction, immunoblotting, and high-sensitivity immunofluorescence staining. Furthermore, we analyzed the functional activity of mGR, using membrane-impermeable bovine serum albumin (BSA)-bound dexamethasone (DEX-BSA) in human monocytes. Membrane-bound GR-expressing monocytes were treated with DEX, DEX-BSA, or BSA. Cell lysates were analyzed using PepChip arrays in order to identify kinases triggered by DEX-BSA, with validation using Bio-Plex assays and immunoblotting. RESULTS Our data showed that transient reduction of GR mRNA in HEK 293T cells decreased cGR protein levels but not mGR protein levels. However, stably transfected cells showed reduced cGR protein expression and significantly reduced mGR protein expression. Furthermore, 51 kinase substrates were identified for which phosphorylation was either reduced or increased. We observed p38 MAP kinase (MAPK) as one possible upstream kinase. Validation of these data by Bio-Plex phosphoprotein assay and immunoblotting showed increased phosphorylation of p38 MAPK after treatment with DEX-BSA. CONCLUSION Our data demonstrate that the human GR gene encodes for both cGR and mGR. Membrane-bound GR retains functional activity, as indicated by induced phosphorylation of p38 MAPK due to DEX-BSA treatment. Membrane-bound GR-mediated cellular signaling needs to be investigated further in order to clarify its therapeutic potential.

Specific Inhibition of c-Raf Activity by Semapimod Induces Clinical Remission in Severe Crohn’s Disease

There is a substantial need for novel treatment strategies in Crohn’s disease (CD), a chronic relapsing inflammatory disease of the gut. In an earlier study, we reported clinical efficacy of a 2-wk treatment with semapimod (CNI-1493) in 12 patients with therapy resistant CD. The aim of this study was to identify the cellular target underlying semapimod action. In vitro experiments with murine macrophages showed impaired MAPK signaling and decreased cytokine production due to semapimod treatment. In vitro kinase assays revealed c-Raf as a direct molecular target of semapimod, and semapimod did not affect b-Raf enzymatic activity. Immunohistochemistry performed on paired colon biopsies obtained from CD patients (n = 6) demonstrated increased expression of phospho-MEK, the substrate of Raf. Strikingly, phospho-MEK levels were significantly decreased in patients with a good clinical response to semapimod, but no decrease in phospho-MEK expression was observed in a clinically nonresponsive patient. In conclusion, this study identifies c-Raf as a molecular target of semapimod action and suggests that decreased c-Raf activity correlates with clinical benefit in CD. Our observations indicate that c-Raf inhibitors are prime candidates for the treatment of CD.

A Relay Pathway between Arginine and Tryptophan Metabolism Confers Immunosuppressive Properties on Dendritic Cells

SUMMARY Arginase 1 (Arg1) and indoleamine 2,3‐dioxygenase 1 (IDO1) are immunoregulatory enzymes catalyzing the degradation of l‐arginine and l‐tryptophan, respectively, resulting in local amino acid deprivation. In addition, unlike Arg1, IDO1 is also endowed with non‐enzymatic signaling activity in dendritic cells (DCs). Despite considerable knowledge of their individual biology, no integrated functions of Arg1 and IDO1 have been reported yet. We found that IDO1 phosphorylation and consequent activation of IDO1 signaling in DCs was strictly dependent on prior expression of Arg1 and Arg1‐dependent production of polyamines. Polyamines, either produced by DCs or released by bystander Arg1+ myeloid‐derived suppressor cells, conditioned DCs toward an IDO1‐dependent, immunosuppressive phenotype via activation of the Src kinase, which has IDO1‐phosphorylating activity. Thus our data indicate that Arg1 and IDO1 are linked by an entwined pathway in immunometabolism and that their joint modulation could represent an important target for effective immunotherapy in several disease settings. HighlightsDendritic cells (DCs) can co‐express Arg1 and IDO1 immunosuppressive enzymesArg1 activity is required for IDO1 induction by TGF‐&bgr; in DCsSpermidine, a downstream Arg1 product, but not arginine starvation, induces IDO1 in DCsArg1+ myeloid derived suppressor cells (MDSCs) can render DCs immunosuppressive via IDO1 &NA; Arginase 1 (Arg1) and indoleamine 2,3‐dioxygenase 1 (IDO1) are immunosuppressive enzymes known to operate in distinct immune cells. Mondanelli and colleagues demonstrate that Arg1 and IDO1 cooperate in conferring long‐term, immunosuppressive effects to dendritic cells.

Impaired autophagy leads to abnormal dendritic cell–epithelial cell interactions

BACKGROUND AND AIMS Dendritic cells (DC) are key players in intestinal immunity, as these cells can direct the immune response to either a tolerogenic or an immunogenic phenotype. In the intestine, DC sample and process luminal antigens by protruding dendrites through the epithelial cell layer. At the same time barrier integrity is maintained through the continuous formation of tight junctions. Aberrations in these interactions may lead to altered antigen sampling and improper immune responses. We have recently shown that autophagy, a process implicated in the pathogenesis of Crohns disease, regulates cellular interactions in the context of DC and T cells. In this study we aimed to determine whether autophagy also regulates DC-epithelial cell interactions and whether this influences the ensuing immune response. METHODS DC were generated from peripheral blood monocytes of healthy volunteers. For interaction studies, DC were co-cultured with intestinal epithelial cells on the baso-lateral side of a transwell insert. Modulation of autophagy was achieved using atg16l1 specific siRNA or pharmacological inhibitors. Intraepithelial protrusion of dendrites was determined by confocal microscopy. Luminal sampling and DC activation status were analyzed by flow cytometry. Protein expression was measured by immunoblotting and cytometric bead assay. RESULTS Adhesion molecules E-cadherin and occludin partly localized to autophagosomes and increased autophagy resulted in decreased levels of these proteins. Reduced autophagy in either DC, epithelial cells or both resulted in the decreased formation of transepithelial protrusions by DC as well as a reduction in antigen sampling. Moreover, when autophagy was inhibited in the co-culture model, DC expressed increased levels of HLA-DR and costimulatory molecule CD86. Furthermore, decreased levels of autophagy resulted in lower IL-10 production by DC and these cells induced significantly more T-cell proliferation in an allogeneic mixed lymphocyte reaction. CONCLUSIONS In intestinal DC-epithelial cell interactions, autophagy deficiency leads to decreased antigen sampling, increased DC maturation and a more pro-inflammatory type of DC.

Mesenchymal stromal cell function is not affected by drugs used in the treatment of inflammatory bowel disease

BACKGROUND AND AIMS Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I-III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays. METHODS The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-α on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied. RESULTS MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-α antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment. CONCLUSIONS This study demonstrates that, in vitro, MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-α therapy.

Comparison of Peptide Array Substrate Phosphorylation of c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8

Kinases are pivotal regulators of cellular physiology. The human genome contains more than 500 putative kinases, which exert their action via the phosphorylation of specific substrates. The determinants of this specificity are still only partly understood and as a consequence it is difficult to predict kinase substrate preferences from the primary structure, hampering the understanding of kinase function in physiology and prompting the development of technologies that allow easy assessment of kinase substrate consensus sequences. Hence, we decided to explore the usefulness of phosphorylation of peptide arrays comprising of 1176 different peptide substrates with recombinant kinases for determining kinase substrate preferences, based on the contribution of individual amino acids to total array phosphorylation. Employing this technology, we were able to determine the consensus peptide sequences for substrates of both c-Raf and Mitogen Activated Protein Kinase Kinase Kinase 8, two highly homologous kinases with distinct signalling roles in cellular physiology. The results show that although consensus sequences for these two kinases identified through our analysis share important chemical similarities, there is still some sequence specificity that could explain the different biological action of the two enzymes. Thus peptide arrays are a useful instrument for deducing substrate consensus sequences and highly homologous kinases can differ in their requirement for phosphorylation events.