RNAi screening identifies mediators of NOD2 signaling: implications for spatial specificity of MDP recognition.

The intracellular nucleotide-binding oligomerization domain-2 (NOD2) receptor detects bacteria-derived muramyl dipeptide (MDP) and activates the transcription factor NF-κB. Here we describe the regulatome of NOD2 signaling using a systematic RNAi screen. Using three consecutive screens, we identified a set of 20 positive NF-κB regulators including the known pathway members RIPK2, RELA, and BIRC4 (XIAP) as well as FRMPD2 (FERM and PDZ domain-containing 2). FRMPD2 interacts with NOD2 via leucine-rich repeats and forms a complex with the membrane-associated protein ERBB2IP. We demonstrate that FRMPD2 spatially assembles the NOD2-signaling complex, hereby restricting NOD2-mediated immune responses to the basolateral compartment of polarized intestinal epithelial cells. We show that genetic truncation of the NOD2 leucine-rich repeat domain, which is associated with Crohn disease, impairs the interaction with FRMPD2, and that intestinal inflammation leads to down-regulation of FRMPD2. These results suggest a structural mechanism for how polarity of epithelial cells acts on intestinal NOD-like receptor signaling to mediate spatial specificity of bacterial recognition and control of immune responses.

mTNF reverse signalling induced by TNFα antagonists involves a GDF-1 dependent pathway: implications for Crohn's disease.

OBJECTIVE: Mechanisms of action (MoA) of anti-tumour necrosis factor α (TNFα) therapies in Crohn's disease (CD) may critically involve induction of immune cell apoptosis via membrane-bound TNFα (mTNFα) binding. Certolizumab pegol (CZP), which is effective in induction and maintenance of remission in CD lacks the ability to induce apoptosis. The aim of this study was to analyse transcriptomal responses of reverse signalling induced by the TNFα binding agents infliximab (IFX) and CZP in myelomonocytic cells. DESIGN: Induction of transcriptional patterns upon anti-TNFα stimulation was assessed using oligonucleotide microarrays. mRNA expression of GDF-1/ LASS1, which was identified as a shared target, was studied in inflammatory bowel disease by real-time PCR, while signalling pathways induced by growth and differentiation factor 1 (GDF-1) were investigated using western blots and ELISA. RESULTS: IFX and CZP induced a common signature of 20 transcripts that could be categorised into control of cell cycle, transcription activation and pre-mRNA processing. We selected GDF-1/LASS1 for functional follow-up, which was found to be upregulated in inflamed CD tissues. We show that downregulation of GDF-1/LASS1 depends on autocrine release of transforming growth factor ß after mTNFα ligation. We demonstrate that GDF-1 itself acts as a novel proinflammatory factor via induction of interleukin 6 and signal transducer and activator of transcription 3 and is downregulated after IFX treatment. CONCLUSION: Commonalities in the MoA of IFX and CZP comprise modulation of non-apoptotic pathways through downregulation of proinflammatory GDF-1. Further characterisation of the molecular role of GDF-1 in complex inflammatory processes in vivo is warranted to decide whether this proinflammatory molecule is a promising therapeutic target in patients with CD.

Genome-wide association analysis in sarcoidosis and Crohn's disease unravels a common susceptibility locus on 10p12.2.

BACKGROUND & AIMS: Crohn's disease (CD) and sarcoidosis (SA) are chronic inflammatory barrier diseases that share several clinical and immunological features, including the occurrence of granulomas. METHODS: A 100k genome-wide association study with 83,360 single-nucleotide polymorphisms (SNPs) was performed on 382 CD patients, 398 SA patients, and 394 control individuals. The 24 SNPs that were most strongly associated in the combined CD/SA phenotype were selected for verification in an independent sample of 1,317 patients (660 CD and 657 SA) and 1,091 controls. RESULTS: The most significant association (Bonferroni corrected P = .036) was obtained at SNP rs1398024 on chromosome 10p12.2, with an odds ratio (OR) for both diseases of 0.81 (95% confidence interval [CI], 0.69-0.96) for carriership of the rarer allele A. The P value in the overall combined sample was 4.24 x 10(-6). During further follow-up, a moderate association (OR, 0.83; 95% CI, 0.72-0.96; P = .015) was observed between rs1398024 and ulcerative colitis (1,080 patients vs 1,091 controls), the second main subphenotype of inflammatory bowel disease in addition to CD. Extensive fine mapping of the 10p12.2 locus points to yet unidentified variants in the C10ORF67 gene region as the most likely underlying risk factors. CONCLUSION: Our study demonstrates that the combined analysis of different, albeit clinically related, phenotypes can lead to the identification of common susceptibility loci.

Intraindividual Comparison of 18F-PSMA-1007 PET/CT, Multiparametric MRI, and Radical Prostatectomy Specimens in Patients with Primary Prostate Cancer: A Retrospective, Proof-of-Concept Study.

68Ga-prostate-specific membrane antigen (PSMA)-11 PET/CT represents an advanced method for the staging of primary prostate cancer (PCa) and diagnosis of recurrent or metastatic PCa. However, because of the narrow availability of 68Ga the development of alternative tracers is of high interest. The objective of this study was to examine the value of the new PET tracer 18F-PSMA-1007 for the staging of local disease by comparing it with multiparametric MRI (mpMRI) and radical prostatectomy (RP) histopathology. Methods: In 2016, 18F-PSMA-1007 PET/CT was performed in 10 men with biopsy-confirmed high-risk PCa. Nine patients underwent mpMRI in the process of primary diagnosis. Consecutively, RP was performed in all 10 men. Agreement analysis was performed retrospectively. PSMA staining was added for representative sections in RP specimen slices. Localization and agreement analysis of 18F-PSMA-1007 PET/CT, mpMRI, and RP specimens was performed by dividing the prostate into 38 sections as described in the prostate imaging reporting and data system (PI-RADS) (version 2). Sensitivity, specificity, positive predictive values, negative predictive values (NPVs), and accuracy were calculated for total and near-total agreement. Results:18F-PSMA-1007 PET/CT had an NPV of 68% and an accuracy of 75%, and mpMRI had an NPV of 88% and an accuracy of 73% for total agreement. Near-total agreement analysis resulted in an NPV of 91% and an accuracy of 93% for 18F-PSMA-1007 PET/CT and 91% and 87% for mpMRI, respectively. Retrospective combination of mpMRI and PET/CT had an accuracy of 81% for total and 93% for near-total agreement. Conclusion: Comparison with RP histopathology demonstrates that 18F-PSMA-1007 PET/CT is promising for accurate local staging of PCa.

Plastid Located WHIRLY1 Enhances the Responsiveness of Arabidopsis Seedlings Toward Abscisic Acid.

WHIRLY1 is a protein that can be translocated from the plastids to the nucleus, making it an ideal candidate for communicating information between these two compartments. Mutants of Arabidopsis thaliana lacking WHIRLY1 (why1) were shown to have a reduced sensitivity toward salicylic acid (SA) and abscisic acid (ABA) during germination. Germination assays in the presence of abamine, an inhibitor of ABA biosynthesis, revealed that the effect of SA on germination was in fact caused by a concomitant stimulation of ABA biosynthesis. In order to distinguish whether the plastid or the nuclear isoform of WHIRLY1 is adjusting the responsiveness toward ABA, sequences encoding either the complete WHIRLY1 protein or a truncated form lacking the plastid transit peptide were overexpressed in the why1 mutant background. In plants overexpressing the full-length sequence, WHIRLY1 accumulated in both plastids and the nucleus, whereas in plants overexpressing the truncated sequence, WHIRLY1 accumulated exclusively in the nucleus. Seedlings containing recombinant WHIRLY1 in both compartments were hypersensitive toward ABA. In contrast, seedlings possessing only the nuclear form of WHIRLY1 were as insensitive toward ABA as the why1 mutants. ABA was furthermore shown to lower the rate of germination of wildtype seeds even in the presence of abamine which is known to inhibit the formation of xanthoxin, the plastid located precursor of ABA. From this we conclude that plastid located WHIRLY1 enhances the responsiveness of seeds toward ABA even when ABA is supplied exogenously.

Microbial pattern recognition causes distinct functional micro-RNA signatures in primary human monocytes.

Micro-RNAs (miRNAs) are short, non-coding RNAs that regulate gene expression post transcriptionally. Several studies have demonstrated the relevance of miRNAs for a wide range of cellular mechanisms, however, the current knowledge on how miRNAs respond to relevant external stimuli, e.g. in disease scenarios is very limited. To generate a descriptive picture of the miRNA network associated to inflammatory responses, we quantified the levels of 330 miRNAs upon stimulation with a panel of pro-inflammatory components such as microbial pattern molecules (flagellin, diacylated lipopeptide lipopolysaccharide, muramyl dipeptide), infection with Listeria monocytogenes and TNF-α as pro-inflammatory control in primary human monocytes using real time PCR. As a result, we found distinct miRNA response clusters for each stimulus used. Additionally, we identified potential target genes of three selected miRNAs miR-129-5p, miR-146a and miR-378 which were part of PAMP-specific response clusters by transfecting THP1 monocytes with the corresponding pre- or anti-miRNAs and microfluidic PCR arrays. The miRNAs induced distinct transcriptomal signatures, e.g. overexpression of miRNA129-5p, which was selectively upregulated by the NOD2-elicitor MDP, led to an upregulation of DEFB1, IRAK1, FBXW7 and IKK γ (Nemo). Our findings on highly co-regulated clusters of miRNAs support the hypothesis that miRNAs act in functional groups. This study indicates that miRNAs play an important role in fine-tuning inflammatory mechanisms. Further investigation in the field of miRNA responses will help to understand their effects on gene expression and may close the regulatory gap between mRNA and protein expression in inflammatory diseases.