We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2‐expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N‐terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157‐S158 peptide bond for both wild‐type and H157Y human TREM2 and for the wild‐type murine orthologue. Crucially, we also show that the Alzheimer's disease‐associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat‐ and ADAM10‐siRNA‐independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer's and other neurological diseases.

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ObjectiveFatigue has been reported as the most disturbing symptom in a majority of patients with SLE. Depression is common and often severe. Together these symptoms cause significant morbidity and affect patients with otherwise relatively mild disease. Tryptophan and its metabolites in the kynurenine pathway are known to be important in several psychiatric conditions, for example, depression, which are often also associated with fatigue. We therefore investigated the kynurenine pathway in patients with SLE and controls.MethodsIn a cross-sectional design plasma samples from 132 well-characterised patients with SLE and 30 age-matched and gender-matched population-based controls were analysed by liquid chromatography tandem mass spectrometry to measure the levels of tryptophan and its metabolites kynurenine and quinolinic acid. Fatigue was measured with Fatigue Severity Scale and depression with Hospital Anxiety and Depression Scale. SLE disease activity was assessed with Systemic Lupus Erythematosus Disease Activity Index (SLEDAI).ResultsThe kynurenine/tryptophan ratio, as a measure of indoleamine 2,3-dioxygenase (IDO) activity, was increased in patients with SLE. Patients with active disease (SLEDAI ≥6) showed lower tryptophan levels compared with controls (54 µM, SD=19 vs 62 µM, SD=14, p=0.03), although patients with SLE overall did not differ compared with controls. Patients with SLE had higher levels of tryptophan metabolites kynurenine (966 nM, SD=530) and quinolinic acid (546 nM, SD=480) compared with controls (kynurenine: 712 nM, SD=230, p=0.0001; quinolinic acid: 380 nM, SD=150, p=0.001). Kynurenine, quinolinic acid and the kynurenine/tryptophan ratio correlated weakly with severe fatigue (rs=0.34, rs=0.28 and rs=0.24, respectively) but not with depression.ConclusionsMetabolites in the kynurenine pathway are altered in patients with SLE compared with controls. Interestingly, fatigue correlated weakly with measures of enhanced tryptophan metabolism, while depression did not. Drugs targeting enzymes in the kynurenine pathway, for example, IDO inhibitors or niacin (B12) supplementation, which suppresses IDO activity, merit further investigation as treatments in SLE.

The immune system has been recognized as a potential contributor to psychiatric disorders. In animals, lipopolysaccharide (LPS) is used to induce inflammation and behaviors analogous to some of the symptoms in these disorders. Recent data indicate that the kynurenine pathway contributes to LPS-induced aberrant behaviors. However, data are inconclusive regarding optimal LPS dose and treatment strategy. Here, we therefore aimed to evaluate the effects of single versus repeated administration of LPS on the kynurenine pathway. Adult C57BL6 mice were given 0.83 mg/kg LPS as a single or a repeated injection (LPS + LPS) and sacrificed after 24, 48, 72, or 120 h. Mice receiving LPS + LPS had significantly elevated brain kynurenine levels at 24 and 48 h, and elevated serum kynurenine at 24, 48 and 72 h. Brain kynurenic acid and quinolinic acid were significantly increased at 24 and 48 h in mice receiving LPS + LPS, whereas serum kynurenic acid levels were significantly decreased at 24 h. The increase of brain kynurenic acid by LPS + LPS was likely unrelated to the higher total dose as a separate group of mice receiving 1.66 mg/kg LPS as single injection 24 h prior to sacrifice did not show increased brain kynurenic acid. Serum quinolinic acid levels were not affected by LPS + LPS compared to vehicle. Animals given repeated injections of LPS showed a more robust induction of the kynurenine pathway in contrast to animals receiving a single injection. These results may be valuable in light of data showing the importance of the kynurenine pathway in psychiatric disorders.

Tryptophan degradation along the kynurenine pathway is of central importance for the immune function. Toll-like receptors (TLRs), representing the first line of immune defence against pathogens, are expressed in various cell types. The most abundant expression is found on monocytes, macrophages and dendritic cells. The aim of this study was to investigate whether stimulation with different TLR ligands induces the kynurenine pathway in human peripheral monocytes. Cell supernatants were analysed using a liquid chromatography/mass spectrometry to measure kynurenine, kynurenic acid (KYNA), quinolinic acid (QUIN) and tryptophan. Stimulation of TLR-2, TLR-3, TLR-4, TLR-7/8 and TLR-9 was found to induce the production of kynurenine, but only stimulation of TLR-3 increased levels of further downstream metabolites, such as KYNA and QUIN. Stimulation of TLR-1, TLR-5 and TLR-6 did not induce the kynurenine pathway. Taken together, this study provides novel evidence demonstrating that TLR activation induces a pattern of downstream tryptophan degradation along the kynurenine pathway in monocytes. The results of this study may implicate that TLRs can be used as new drug targets for the regulation of aberrant tryptophan metabolism along this pathway, a potential therapeutic strategy that may be of importance in several disorders.

During the past decade, we have witnessed an explosive increase in generation of large proteomics data sets, not least in cancer research. There is a growing need to extract and correctly interpret information from such data sets to generate biologically relevant hypotheses. A pathway search engine (PSE) has recently been developed as a novel tool intended to meet these requirements. Ionizing radiation (IR) is an anticancer treatment modality that triggers multiple signal transduction networks. In this work, we show that high linear energy transfer (LET) IR induces apoptosis in a non-small cell lung cancer cell line, U-1810, whereas low LET IR does not. PSE was applied to study changes in pathway status between high and low LET IR to find pathway candidates of importance for high LET-induced apoptosis. Such pathways are potential clinical targets, and they were further validated in vitro. We used an unsupervised shotgun proteomics approach where high resolution mass spectrometry coupled to nanoflow liquid chromatography determined the identity and relative abundance of expressed proteins. Based on the proteomics data, PSE suggested the JNK pathway (p ‫؍‬ 6⅐10 ؊6 ) as a key event in response to high LET IR. In addition, the Fas pathway was found to be activated (p ‫؍‬ 3⅐10 ؊5 ) and the p38 pathway was found to be deactivated (p ‫؍‬ 0.001) compared with untreated cells. Antibodybased analyses confirmed that high LET IR caused an increase in phosphorylation of JNK. Moreover pharmacological inhibition of JNK blocked high LET-induced apoptotic signaling. In contrast, neither an activation of p38 nor a role for p38 in high LET IR-induced apoptotic signaling was found. We conclude that, in contrast to conventional low LET IR, high LET IR can trigger activation of the JNK pathway, which in turn is critical for induction of apoptosis in these cells. Thus PSE predictions were largely confirmed, and PSE was proven to be a useful hypothesis-generating tool.

The ephrin and Eph signaling circuit has been reported as deregulated in a number of tumor types including nonsmall cell lung cancer (NSCLC). Here we show that suppression of the ephrin-familly member ephrin B3 decreases NSCLC cell proliferation and has profound effects on cell morphology. To reveal which signaling networks ephrin B3 utilize to regulate such effects on growth and morphology, differential regulation of phosphorylated proteins was analyzed in the NSCLC cell line U-1810. Using strong cat ion exchange (SCX) and TiO(2)-based fractionation followed by nano-LC and mass spectrometry analysis, we identified 1083 unique phosphorylated proteins. Out of these, 150 proteins were found only when ephrin B3 is expressed, whereas 66 proteins were found exclusively in U-1810 cells with silenced ephrin B3. Network analysis of changes in the phosphoproteome with regard to the presence or absence of ephrin B3 expression generated a hypothesis that the site specific phosphorylation on Ser-897 detected on the erythropoietin-producing hepatocellular receptor tyrosine kinase class A2 (EphA2) is critical for the survival of NSCLC cells. Upstream of the EphA2 phosphorylation, activation of Akt1 on Ser 129 was also revealed as part of the ephrin B3-mediated signaling pathway. Phosphorylation of these sites was further confirmed by immune-based strategies in combination with mass spectrometry. Moreover, by further stepwise pathway walking, annotating the phosphorylated sites and their corresponding kinases upstream, our data support the process in which a Heat shock protein 90 isoform (HSP90AA1) acts as a protector of EphA2, thereby saving it from degradation. In addition, protein kinase CK2 (CK2) is suggested as a dominant kinase, activating downstream substrates to generate the effects on NSCLC proliferation and morphology.

Increased activity of indoleamine 2,3-dioxygenase (IDO) and tryptophan hydroxylase (TPH) have been reported in individuals with chronic obstructive pulmonary disease (COPD). We therefore investigated the effect of gender stratification upon the observed levels of tryptophan metabolites in COPD. Tryptophan, serotonin, kynurenine, and kynurenic acid were quantified in serum of never-smokers (n = 39), smokers (n = 40), COPD smokers (n = 27), and COPD ex-smokers (n = 11) by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The individual metabolite associations with lung function, blood, and bronchoalveolar lavage (BAL) immune-cell composition, as well as chemokine and cytokine levels, were investigated. Stratification by gender and smoking status revealed that the observed alterations in kynurenine and kynurenic acid, and to a lesser extent serotonin, were prominent in males, irrespective of COPD status (kynurenine p = 0.005, kynurenic acid p = 0.009, and serotonin p = 0.02). Inferred serum IDO activity and kynurenine levels decreased in smokers relative to never-smokers (p = 0.005 and p = 0.004, respectively). In contrast, inferred tryptophan hydroxylase (TPH) activity and serotonin levels showed an increase with smoking that reached significance with COPD (p = 0.01 and p = 0.01, respectively). Serum IDO activity correlated with blood CXC chemokine ligand 9 (CXCL9, p = 0.0009, r = 0.93) and chemokine (C-C motif) ligand 4 (CCL4.(p = 0.04, r = 0.73) in female COPD smokers. Conversely, serum serotonin levels correlated with BAL CD4+ T-cells (%) (p = 0.001, r = 0.92) and CD8+ T-cells (%) (p = 0.002, r = −0.90) in female COPD smokers, but not in male COPD smokers (p = 0.1, r = 0.46 and p = 0.1, r = −0.50, respectively). IDO- and TPH-mediated tryptophan metabolites showed gender-based associations in COPD, which were primarily driven by smoking status.