Search

Career situation of first and presenting author Post-doctoral fellow. Introduction Recurrent activation of synovial macrophages is considered as intrinsic factor that precedes rheumatoid arthritis (RA)-related autonomous cytokine cascades. microRNAs (miRs) and Toll-like receptors (TLRs) play a pivotal role in regulating the inflammatory response in innate immune cells and are at dysregulated levels in RA patients. Objectives Here we explored TLR-regulated miRs that tune the inflammatory response of M2-type macrophages. Methods Based on a miR-screen in TLR-stimulated human M2-macrophages we selected miR-221-3p to analyze its expression profile in clinical samples from RA, other inflammatory arthritis (OIA), osteoarthritis (OA) and healthy donors (HD) by qPCR. We challenged M1- and M2-macrophages with elevated miR-221-3p and/or miR-155-5p expression thereby mimicking RA-related conditions. Cytokine/chemokine secretion of IL-6, IL-10, IL-12, IL-8 and CXCL13 was measured using ELISA. Changes of inflammatory pathways including JAK/STAT were evaluated by Western Blot. Results TLR4-stimulation of M2-macrophages results in suppression of miR-221-3p, while in vivo expression in synovial tissue and fluids is significantly increased in RA versus OA or OIA. M2-macrophages with high levels of miR-221-3p lose their anti-inflammatory response and support a M1-like profile. miR-221-3p in combination with miR-155-5p leads to the secretion of M1-specific IL-12 and lowered IL-10. miR-221-3p is affecting the JAK/STAT pathway in M2-macrophages by downregulating JAK3 protein leading to decreased STAT3 activation. Treatment of M2-macrophages using JAK3 inhibitors display the same shift in cytokine/chemokine pattern as increased miR-221-expression. Conclusions TLR4-activated M2-macrophages exposed to elevated miR-221-3p expression diminished anti-inflammatory response partly by suppressing JAK3/STAT3 pathway. Thus, miR-221-3p is a homeostatic regulator of M2-M1 inflammatory function, which is dysregulated in RA condition. Acknowledgements Tissue RNA from RA and OA patients was kindly provided by Caroline Ospelt, University of Zurich, Center of Experimental Rheumatology, Switzerland. Disclosure of Interest None declared.

Cytokine profile of M1- and M2-polarized macrophages derived from blood of patients with rheumatoid arthritis (RA) following TLR ligand exposure and activation. M1 (GM-CSF-differentiated) and M2 (M-CSF-differentiated) macrophages were stimulated for 24 h with 300 ng/ml Pam3 or 100 ng/ml LPS. Cytokine and MMP3 release was measured by ELISA, and values are expressed as mean ± SD. n = 5–6, * p

Expression of characteristic anti-inflammatory M2 gene markers in M1 and M2 macrophages derived from blood of healthy donors (HD) or patients with rheumatoid arthritis (RA). Basal gene expression of M2 markers HMOX1, FOLR2, and SLC40A1 in M0 (monocytes), M1 (GM-CSF-differentiated), and M2 (M-CSF-differentiated) macrophages from HD (upper panel) or patients with RA (lower panel). Expression was measured by qRT-PCR. Values were normalized to UBC mRNA levels and expressed as 2−ΔCT ± SD. n = 4–5, * p

Characterization of surface markers on M0, M1-, and M2-polarized macrophages derived from peripheral blood of healthy donors (HD) compared with patients with rheumatoid arthritis (RA). For phenotypical analysis, M0 (ex vivo monocytes), M1 (GM-CSF-differentiated), and M2 (M-CSF-differentiated) macrophages derived from peripheral blood of HD or patients with RA were stained for FACS analysis with fluorescently labeled antibodies CD14-APC-Cy7, CD163-FITC, CD206-BV421, and CD86-PE. Comparison of surface marker expression on freshly isolated M0- or M1- and M2-polarized macrophages from HD (left panels) versus RA (right panels) presented with representative CD14-to-CD86 (upper panels) and CD206-to-CD163 (lower panels) density plots. n = 6. (DOCX 217 kb)

Effect of TLR or cytokine stimulation on surface marker expression in M1 versus M2 macrophages derived from peripheral monocytes of healthy donors (HD) and patients with rheumatoid arthritis (RA) based on mean fluorescence intensity (MFI) analysis. M1 (GM-CSF-differentiated) and M2 (M-CSF-differentiated) macrophages were stimulated for 24 h with 300 ng/ml Pam3, 100 ng/ml LPS, or a combination of IFN-γ/LPS. For phenotypical analysis, cells were stained for FACS analysis with fluorescently labeled antibodies CD14-APC-Cy7, CD163-FITC, CD206-BV421, CD86-PE, and CD80-FITC. Individual MFI was calculated as ΔMFI = MFIspecific surface marker − MFIcorresponding unstained control and normalized to the basal MFI of unstained control cells. n = 6, * p

Unbiased genome wide screens combined with imaging data on brain function may identify novel molecular pathways related to human cognition. Here we performed a dense genome wide screen to identify episodic memory related gene variants. A genomic locus encoding the brain expressed beta catenin like protein 1 (CTNNBL1) was significantly (P=7 × 10( 8)) associated with verbal memory performance in a cognitively healthy cohort from Switzerland (n=1073) and was replicated in a second cohort from Serbia (n=524; P=0.003). Gene expression studies showed CTNNBL1 genotype dependent differences in beta catenin like protein 1 mRNA levels in the human cortex. Functional magnetic resonance imaging in 322 subjects detected CTNNBL1 genotype dependent differences in memory related brain activations. Converging evidence from independent experiments and different methodological approaches suggests a role for CTNNBL1 in human memory.Molecular Psychiatry advance online publication 22 November 2011; doi:10.1038/mp.2011.148.

In the last decade there has been an exponential increase in knowledge about the genetic basis of complex human traits, including neuropsychiatric disorders. It is not clear, however, to what extent this knowledge can be used as a starting point for drug identification, one of the central hopes of the human genome project. The aim of the present study was to identify memory-modulating compounds through the use of human genetic information. We performed a multinational collaborative study, which included assessment of aversive memory--a trait central to posttraumatic stress disorder--and a gene-set analysis in healthy individuals. We identified 20 potential drug target genes in two genomewide-corrected gene sets: the neuroactive ligand-receptor interaction and the long-term depression gene set. In a subsequent double-blind, placebo-controlled study in healthy volunteers, we aimed at providing a proof of concept for the genome-guided identification of memory modulating compounds. Pharmacological intervention at the neuroactive ligand-receptor interaction gene set led to significant reduction of aversive memory. The findings demonstrate that genome information, along with appropriate data mining methodology, can be used as a starting point for the identification of memory-modulating compounds.

Strong memory of a traumatic event is thought to contribute to the development and symptoms of posttraumatic stress disorder (PTSD). Therefore, a genetic predisposition to build strong memories could lead to increased risk for PTSD after a traumatic event. Here we show that genetic variability of the gene encoding PKCα ( PRKCA ) was associated with memory capacity—including aversive memory—in nontraumatized subjects of European descent. This finding was replicated in an independent sample of nontraumatized subjects, who additionally underwent functional magnetic resonance imaging (fMRI). fMRI analysis revealed PRKCA genotype-dependent brain activation differences during successful encoding of aversive information. Further, the identified genetic variant was also related to traumatic memory and to the risk for PTSD in heavily traumatized survivors of the Rwandan genocide. Our results indicate a role for PKCα in memory and suggest a genetic link between memory and the risk for PTSD.