Phospholipase D1 mediates lymphocyte adhesion and migration in experimental autoimmune encephalomyelitis

Lymphocyte adhesion and subsequent trafficking across endothelial barriers are essential steps in various immune‐mediated disorders of the CNS, including MS. The molecular mechanisms underlying these processes, however, are still unknown. Phospholipase D1 (PLD1), an enzyme that generates phosphatidic acid through hydrolysis of phosphatidylcholine and additionally yields choline as a product, has been described as regulator of the cell mobility. By using PLD1‐deficient mice, we investigated the functional significance of PLD1 for lymphocyte adhesion and migration in vitro and after myelin oligodendrocyte glycoprotein (MOG)_35–55‐induced EAE, a model of human MS. The lack of PLD1 reduced chemokine‐mediated static adhesion of lymphocytes to the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM‐1) and intercellular adhesion molecule 1 (ICAM‐1) in vitro, and was accompanied by a decreased migratory capacity in both blood brain barrier and cell migration models. Importantly, PLD1 is also relevant for the recruitment of immune cells into the CNS in vivo since disease severity after EAE was significantly attenuated in PLD1‐deficient mice. Furthermore, PLD1 expression could be detected on lymphocytes in MS patients. Our findings suggest a critical function of PLD1‐dependent intracellular signaling cascades in regulating lymphocyte trafficking during autoimmune CNS inflammation.     

Lack of microbiota reduces innate responses and enhances adaptive immunity against Listeria monocytogenes infection

The intestinal microbiota influences not only metabolic processes, but also the mucosal and systemic immune systems. Here, we compare innate and adaptive immune responses against the intracellular pathogen Listeria monocytogenes in germfree (GF) and conventional mice. We show that animals without endogenous microbiota are highly susceptible to primary infection with impaired activation and accumulation of phagocytes to the site of infection. Unexpectedly, secondary infection with otherwise lethal dose resulted in survival of all GF animals which cleared bacteria more rapidly and developed a stronger antilisterial CD8⁺ memory T‐cell response compared to conventional mice. In summary, lack of the intestinal microbiota impairs early innate immunity, but enhances activation and expansion of memory T cells.     

When neuroscience gets wet and hardcore: neurocognitive markers obtained during whole body water immersion

Neutral buoyancy facilities are used to prepare astronauts and cosmonauts for extra vehicular activities e.g. on-board of the International Space Station. While previous studies indicated a decrease in cognitive performance in an under water setting, they have only provided behavioural data. This study aimed to review whether recording of electro cortical activity by the use of electroencephalography (EEG) is possible in an under water setting and if so, to identify the influence of water immersion at a depth of 4 m on neurocognitive markers. Ten male subjects performed a cognitive choice-reaction times (RT) task that progressed through five levels of increasing difficulty on land and when submerged 4 m under water. N200 latency and amplitude in the occipital and frontal areas were measured, and baseline cortical activity was measured during rest in both conditions. Neither RT nor amplitude or latency of the N200 showed any significant changes between the land and the under water conditions. Also theta, alpha and beta frequencies showed no differences between the two conditions. The data provided in this study demonstrate the possibility of recording EEG even under the extreme conditions of full body water immersion. The lack of cognitive impairment in RT and N200 in the under water condition may be explained by the fact that only experienced divers participated in the study. As a proof of principle, this study generates many new experimental possibilities that will improve our understanding of cognitive processes under water.     

Gut Commensal Bacteria and Regional Wnt Gene Expression in the Proximal Versus Distal Colon

Regional expression of Wingless/Int (Wnt) genes plays a central role in regulating intestinal development and homeostasis. However, our knowledge of such regional Wnt proteins in the colon remains limited. To understand further the effect of Wnt signaling components in controlling intestinal epithelial homeostasis, we investigated whether the physiological heterogeneity of the proximal and distal colon can be explained by differential Wnt signaling. With the use of a Wnt signaling-specific PCR array, expression of 84 Wnt-mediated signal transduction genes was analyzed, and a differential signature of Wnt-related genes in the proximal versus distal murine colon was identified. Several Wnt agonists (Wnt5a, Wnt8b, and Wnt11), the Wnt receptor frizzled family receptor 3, and the Wnt inhibitory factor 1 were differentially expressed along the colon length. These Wnt signatures were associated with differential epithelial cell proliferation and migration in the proximal versus distal colon. Furthermore, reduced Wnt/β-catenin activity and decreased Wnt5a and Wnt11 expression were observed in mice lacking commensal bacteria, an effect that was reversed by conventionalization of germ-free mice. Interestingly, myeloid differentiation primary response gene 88 knockout mice showed decreased Wnt5a levels, indicating a role for Toll-like receptor signaling in regulating Wnt5a expression. Our results suggest that the morphological and physiological heterogeneity within the colon is in part facilitated by the differential expression of Wnt signaling components and influenced by colonization with bacteria.One of the fundamental aspects in the development of the gastrointestinal tract is the spatiotemporal expression of signaling molecules that regulate cell fate and differentiation. Previous studies have highlighted a central role of the evolutionarily conserved Wingless/Int (Wnt) signaling pathways as key regulators of embryonic development and epithelial homeostasis in the gut.^1–3 In development, local expression patterns of Wnt signaling components play an important role in organogenesis.^4,5 Wnt signals control important biological processes required for cell proliferation, differentiation, polarity, and movement, depending on the target cell and the cellular environment.Recent reports have highlighted the importance of understanding the role of Wnt signaling in the intestinal tract. The intestinal epithelium is highly dynamic and, depending on the species and location, is actively turned over in <1 week.⁶ Wnt/β-catenin signaling regulates intestinal epithelial cell (IEC) homeostasis and proliferation by increasing β-catenin stability in crypt epithelial cells, whereas IEC migration and differentiation are believed to be in part facilitated through noncanonical (Wnt) signaling pathways independent of β-catenin.^6,7 The renewal of intestinal epithelia requires a delicate balance of signaling proteins to control epithelial cell proliferation and migration that in turn is vital for maintaining mucosal homeostasis. Interestingly, regional differences in Wnt gene expression in small versus large intestine are observed in adult mice, suggesting the importance of differential local Wnt expression in regulation of intestinal mucosal homeostasis.⁷Although the entire colon exhibits considerable morphological and physiological heterogeneity along its length,^8–11 the expression pattern of Wnt signaling components in the different regions of the adult colon remains poorly understood. Embryologically, the cecum, ascending colon, and the proximal two-thirds of the transverse colon are derived from the midgut, whereas the distal colon originates from the hindgut. Such distinct origins of the colonic segment support specific biological characteristics and suggest that distinct regulatory factors are likely to control epithelial homeostasis in the proximal versus distal colon. In addition, important contributing factors that influence Wnt/β-catenin signaling and intestinal epithelial proliferation might be microbial communities that localize in the intestine in distinct regions.^6,12 Such a delicate physiological balance of Wnt signaling and intestinal epithelial homeostasis is further perturbed in mucosal inflammatory and neoplastic diseases,^3,13 which also indicate regional differences in the proximal versus distal colonic segments.^14–17In the present study, we investigate the regional heterogeneity of Wnt genes in the proximal versus distal colon. Given the importance of luminal microbiota in influencing intestinal epithelial homeostasis¹⁸ and to determine whether the Wnt signatures are influenced by microflora colonization, we examined expression of Wnt proteins in the colonic segments of mice raised under germ-free (GF) conditions.     

Direct acute tubular damage contributes to Shigatoxin‐mediated kidney failure

The pathogenesis and therapy of Shigatoxin 2 (Stx2)‐mediated kidney failure remain controversial. Our aim was to test whether, during an infection with Stx2‐producing E. coli (STEC), Stx2 exerts direct effects on renal tubular epithelium and thereby possibly contributes to acute renal failure. Mice represent a suitable model because they, like humans, express the Stx2‐receptor Gb3 in the tubular epithelium but, in contrast to humans, not in glomerular endothelia, and are thus free of glomerular thrombotic microangiopathy (TMA). In wild‐type mice, Stx2 caused acute tubular dysfunction with consequent electrolyte disturbance, which was most likely the cause of death. Tubule‐specific depletion of Gb3 protected the mice from acute renal failure. In vitro, Stx2 induced secretion of proinflammatory cytokines and apoptosis in human tubular epithelial cells, thus implicating a direct effect of Stx2 on the tubular epithelium. To correlate these results to human disease, kidney biopsies and outcome were analysed in patients with Stx2‐associated kidney failure (n = 11, aged 22–44 years). The majority of kidney biopsies showed different stages of an ongoing TMA; however, no glomerular complement activation could be demonstrated. All biopsies, including those without TMA, showed severe acute tubular damage. Due to these findings, patients were treated with supportive therapy without complement‐inhibiting antibodies (eculizumab) or immunoadsorption. Despite the severity of the initial disease [creatinine 6.34 (1.31–17.60) mg/dl, lactate dehydrogenase 1944 (753–2792) U/l, platelets 33 (19–124)/nl and haemoglobin 6.2 (5.2–7.8) g/dl; median (range)], all patients were discharged after 33 (range 19–43) days with no neurological symptoms and no dialysis requirement [creatinine 1.39 (range 0.84–2.86) mg/dl]. The creatinine decreased further to 0.90 (range 0.66–1.27) mg/dl after 24 months. Based on these data, one may surmise that acute tubular damage represents a separate pathophysiological mechanism, importantly contributing to Stx2‐mediated acute kidney failure. Specifically in young adults, an excellent outcome can be achieved by supportive therapy only. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Attention interference for emotional stimuli in cardiac interoceptive awareness

Previous research has shown that individuals with good perception of cardiac signals, referred to as cardiac interoceptive awareness, experience emotions more intensely. To investigate if emotional experience in high cardiac interoceptive awareness arises from biases in attention, we compared the performance of participants with high versus regular cardiac interoceptive awareness in an emotional Stroop task. Participants with high cardiac interoceptive awareness showed attention interference for negative words, whereas participants with regular cardiac interoceptive awareness showed attention facilitation for positive words. Our data provide further evidence for the emotion information process associated with cardiac interoceptive awareness. The results support the notion that better access to cardiac changes accompanying negative emotional conditions results in automatic vigilance for negative information and may interfere with cognitive processing.     

Bile acids increase the activity of the epithelial Na+ channel

The epithelial Na⁺ channel (ENaC) is a key regulator of Na⁺ absorption in various epithelia including the distal nephron and the distal colon. ENaC is a constitutively active channel, but its activity is modulated by a number of mechanisms. These include proteolytic activation, ubiquitination and cell surface expression, phosphorylation, intracellular Na⁺ concentration, and shear stress. ENaC is related to the bile acid-sensitive ion channel (BASIC), a channel that is expressed in the epithelial cells of bile ducts. BASIC is activated by millimolar concentrations of extracellular bile acids. Bile acids are synthesized by the liver and secreted into the duodenum to aid lipolysis. A large fraction of the secreted bile acids is absorbed by the ileum and recirculated into the liver, but a small fraction passes the colon and is excreted. Bile acids can influence the ion transport processes in the intestinal tract including the colon. In this study, we show that various bile acids present in rat bile potently and reversibly increase the activity of rat ENaC expressed in Xenopus oocytes, suggesting that bile acids are natural modulators of ENaC activity.     

Ca handling during excitation–contraction coupling in heart failure

In the heart, coupling between excitation of the surface membrane and activation of contractile apparatus is mediated by Ca released from the sarcoplasmic reticulum (SR). Several components of Ca machinery are perfectly arranged within the SR network and the T-tubular system to generate a regular Ca cycling and thereby rhythmic beating activity of the heart. Among these components, ryanodine receptor (RyR) and SR Ca ATPase (SERCA) complexes play a particularly important role and their dysfunction largely underlies abnormal Ca homeostasis in diseased hearts such as in heart failure. The abnormalities in Ca regulation occur at practically all main steps of Ca cycling in the failing heart, including activation and termination of SR Ca release, diastolic SR Ca leak, and SR Ca uptake. The contributions of these different mechanisms to depressed contractile function and enhanced arrhythmogenesis may vary in different HF models. This brief review will therefore focus on modifications in RyR and SERCA structure that occur in the failing heart and how these molecular modifications affect SR Ca regulation and excitation–contraction coupling.     

A splice variant of the two-pore domain potassium channel TREK-1 with only one pore domain reduces the surface expression of full-length TREK-1 channels

We have identified a novel splice variant of the human and rat two-pore domain potassium (K_2P) channel TREK-1. The splice variant TREK-1e results from skipping of exon 5, which causes a frame shift in exon 6. The frame shift produces a novel C-terminal amino acid sequence and a premature termination of translation, which leads to a loss of transmembrane domains M3 and M4 and of the second pore domain. RT-PCR experiments revealed a preferential expression of TREK-1e in kidney, adrenal gland, and amygdala. TREK-1e was nonfunctional when expressed in Xenopus oocytes. However, both the surface expression and the current density of full-length TREK-1 were reduced by co-expression of TREK-1e. Live cell imaging in COS-7 cells transfected with GFP-tagged TREK-1e showed that this splice variant was retained in the endoplasmic reticulum (ER). Attachment of the C-terminus of TREK-1e to two different reporter proteins (Kir2.1 and CD8) led to a strong reduction in the surface expression of these fusion proteins. Progressive truncation of the C-terminus of TREK-1e in these reporter constructs revealed a critical region (amino acids 198 to 205) responsible for the intracellular retention. Mutagenesis experiments indicated that amino acids I204 and W205 are key residues mediating the ER retention of TREK-1e. Our results suggest that the TREK-1e splice variant may interfere with the vesicular traffic of full-length TREK-1 channels from the ER to the plasma membrane. Thus, TREK-1e might modulate the copy number of functional TREK-1 channels at the cell surface, providing a novel mechanism for fine tuning of TREK-1 currents.