FLRT2 and FLRT3 Cooperate in Maintaining the Tangential Migratory Streams of Cortical Interneurons during Development.

Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of FLRT2 and FLRT3 genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the FLRT2/FLRT3 mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons in vitro, an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion.SIGNIFICANCE STATEMENT Disruption of inhibitory cortical circuits is responsible for some aspects of psychiatric disorders such as schizophrenia or autism. These defects include interneuron migration during development. A crucial step during this process is the formation of three transient migratory streams within the developing cortex that determine the timing of interneuron final positioning and the formation of functional cortical circuits in the adult. We report that FLRT proteins are required for the proper distribution of interneurons within the cortical migratory streams and for the final laminar allocation in the postnatal cortex. These results expand the multifunctional role of FLRTs during nervous system development in addition to the role of FLRTs in axon guidance and the migration of excitatory cortical neurons.

Maturation of lymph node fibroblastic reticular cells from myofibroblastic precursors is critical for antiviral immunity.

The stromal scaffold of the lymph node (LN) paracortex is built by fibroblastic reticular cells (FRCs). Conditional ablation of lymphotoxin-ß receptor (LTßR) expression in LN FRCs and their mesenchymal progenitors in developing LNs revealed that LTßR-signaling in these cells was not essential for the formation of LNs. Although T cell zone reticular cells had lost podoplanin expression, they still formed a functional conduit system and showed enhanced expression of myofibroblastic markers. However, essential immune functions of FRCs, including homeostatic chemokine and interleukin-7 expression, were impaired. These changes in T cell zone reticular cell function were associated with increased susceptibility to viral infection. Thus, myofibroblasic FRC precursors are able to generate the basic T cell zone infrastructure, whereas LTßR-dependent maturation of FRCs guarantees full immunocompetence and hence optimal LN function during infection.

Activation of Toll-like receptor 7/8 encoded by the X chromosome alters sperm motility and provides a novel simple technology for sexing sperm.

In most mammals, the male to female sex ratio of offspring is about 50% because half of the sperm contain either the Y chromosome or X chromosome. In mice, the Y chromosome encodes fewer than 700 genes, whereas the X chromosome encodes over 3,000 genes. Although overall gene expression is lower in sperm than in somatic cells, transcription is activated selectively in round spermatids. By regulating the expression of specific genes, we hypothesized that the X chromosome might exert functional differences in sperm that are usually masked during fertilization. In this study, we found that Toll-like receptors 7/8 (TLR7/8) coding the X chromosome were expressed by approximately 50% of the round spermatids in testis and in approximately 50% of the epididymal sperm. Especially, TLR7 was localized to the tail, and TLR8 was localized to the midpiece. Ligand activation of TLR7/8 selectively suppressed the mobility of the X chromosome-bearing sperm (X-sperm) but not the Y-sperm without altering sperm viability or acrosome formation. The difference in sperm motility allowed for the separation of Y-sperm from X-sperm. Following in vitro fertilization using the ligand-selected high-mobility sperm, 90% of the embryos were XY male. Likewise, 83% of the pups obtained following embryo transfer were XY males. Conversely, the TLR7/8-activated, slow mobility sperm produced embryos and pups that were 81% XX females. Therefore, the functional differences between Y-sperm and X-sperm motility were revealed and related to different gene expression patterns, specifically TLR7/8 on X-sperm.

Variability in ß-catenin pulse dynamics in a stochastic cell fate decision in C. elegans.

During development, cell fate decisions are often highly stochastic, but with the frequency of the different possible fates tightly controlled. To understand how signaling networks control the cell fate frequency of such random decisions, we studied the stochastic decision of the Caenorhabditis elegans P3.p cell to either fuse to the hypodermis or assume vulva precursor cell fate. Using time-lapse microscopy to measure the single-cell dynamics of two key inhibitors of cell fusion, the Hox gene LIN-39 and Wnt signaling through the ß-catenin BAR-1, we uncovered significant variability in the dynamics of LIN-39 and BAR-1 levels. Most strikingly, we observed that BAR-1 accumulated in a single, 1-4 â€‹h pulse at the time of the P3.p cell fate decision, with strong variability both in pulse slope and time of pulse onset. We found that the time of BAR-1 pulse onset was delayed relative to the time of cell fusion in mutants with low cell fusion frequency, linking BAR-1 pulse timing to cell fate outcome. Overall, a model emerged where animal-to-animal variability in LIN-39 levels and BAR-1 pulse dynamics biases cell fate by modulating their absolute level at the time cell fusion is induced. Our results highlight that timing of cell signaling dynamics, rather than its average level or amplitude, could play an instructive role in determining cell fate.

MicroRNA-3473b regulates the expression of TREM2/ULK1 and inhibits autophagy in inflammatory pathogenesis of Parkinson disease.

Parkinson disease (PD) is a neurodegenerative disease characterized by selective loss of dopaminergic (DA) neurons in the midbrain. The regulatory role of a variety of microRNAs in PD has been confirmed, and our study is the first to demonstrate that miR-3473b is involved in the regulation of PD. In vitro, an miR-3473b inhibitor can inhibit the secretion of inflammatory factors (TNF-α and IL-1ß) in moues microglia cell line (BV2) cells induced by lipopolysaccharide (LPS) and promote autophagy in BV2 cells. In vivo, miR-3473b antagomir can inhibit the activation of substantia nigra pars compacta (SNpc) microglia of C57BL/6 mice induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and promote autophagy. Deletion of TREM2, one of the most highly expressed receptors in microglia, leads to the occurrence and development of PD. ULK1 is a component of the Atg1 complex. Deletion of ULK1 aggravates the pathological reaction of PD. TREM2 and ULK1 are predicted potential targets of miR-3473b by Targetscan. Then, the results of our experiments indicate that transfection with a miR-3473b mimic can inhibit the expression of TREM2 and ULK1. Data from a double luciferase experiment indicate that the 3'-UTR of TREM2, but not ULK1, is the direct target of miR-3473b. Then we aim to investigate the regulation of TREM2 and ULK1 in PD. We found that the expression of p-ULK1 was significantly increased via up-regulation of TREM2. The increased expression of p-ULK1 can promote autophagy and inhibit the expression of inflammatory factors. The regulation of ULK1 by miR-3473b may be accomplished indirectly through TREM2. Thus, miR-3473b may regulate the secretion of proinflammatory mediators by targeting TREM2/ULK1 expression to regulate the role of autophagy in the pathogenesis of inflammation in Parkinson's disease, suggesting that mir-3473b may be a potential therapeutic target to regulate the inflammatory response in PD.

Three-Dimensional Reconstructed Bone Marrow Matrix Culture Improves the Viability of Primary Myeloma Cells In-Vitro via a STAT3-Dependent Mechanism.

Primary myeloma (PM) cells are short-lived in conventional culture, which limited their usefulness as a study model. Here, we evaluated if three-dimensional (3D) culture can significantly prolong the longevity of PM cells in-vitro. We employed a previously established 3D model for culture of bone marrow mononuclear cells isolated from 15 patients. We assessed the proportion of PM cells, viability and proliferation using CD38 staining, trypan blue exclusion assays and carboxy fluorescein succinimidyl ester (CFSE) staining, respectively. We observed significantly more CD38+ viable cells in 3D than in conventional culture (65% vs. 25%, p = 0.006) on day 3. CFSE staining showed no significant difference in cell proliferation between the two culture systems. Moreover, we found that PM cells in 3D culture are more STAT3 active by measure of pSTAT3 staining (66% vs. 10%, p = 0.008). Treatment of IL6, a STAT3 activator significantly increased CD38+ cell viability (41% to 68%, p = 0.021). In comparison, inhibition of STAT3 with Stattic significantly decreased PM cell viability in 3D culture (38% to 17% p = 0.010). Neither IL6 nor Stattic affected the PM cell viability in conventional culture. This study suggests that 3D culture can significantly improve the longevity of PM cells in-vitro, and STAT3 activation can further improve their viability.

Molecular dynamics of targeting CD38 in multiple myeloma.

Multiple functions of CD38 need exploring to expand clinical application of anti-CD38 antibodies in multiple myeloma (MM). We investigated membrane dynamics of MM cells and subsequent events when CD38 is targeted by therapeutic antibodies. Human MM cells (BF01) were co-cultured in vitro with therapeutic antibody (or control immunoglobulin G) and analysed using gene expression profiling. Microvesicles from antibody-exposed cells were analysed for differential gene and microRNA (miRNA) expression, and for phenotypic characterisation. Exposure of BF01 cells to anti-CD38 antibody resulted in CD38 membrane redistribution, upregulation of metabolism-related genes and downregulation of genes involved in cell cycle processes. Microvesicles derived from antibody-exposed cells showed increased CD73 and CD39 expression, presence of programmed death-ligand 1 and significant up-/down-modulation of miRNAs. Microvesicles accumulated around immunoglobulin Fc receptor-positive (FcR+ ) cells. Upon internalisation, natural killer cells displayed significantly increased expression of genes related to activation and immune response, and downregulation of genes involved in the cell cycle. Cells may use microvesicles to transmit signals distally as part of a survival strategy. Microvesicles are equipped on their surface with enzymatic machinery leading to production of tolerogenic adenosine. Further, they are internalised in FcR+ cells with significant functional modifications. These observations have relevance for improving anti-CD38 therapeutic antibodies through targeting this mechanism and its sequelae.

miR-133-mediated regulation of the Hedgehog pathway orchestrates embryo myogenesis.

Skeletal myogenesis serves as a paradigm to investigate the molecular mechanisms underlying exquisitely regulated cell fate decisions in developing embryos. The evolutionarily conserved miR-133 family of microRNAs is expressed in the myogenic lineage, but how it acts remains incompletely understood. Here, we performed genome-wide differential transcriptomics of miR-133 knockdown (KD) embryonic somites, the source of vertebrate skeletal muscle. These analyses, performed in chick embryos, revealed extensive downregulation of Sonic hedgehog (Shh) pathway components: patched receptors, Hedgehog interacting protein and the transcriptional activator Gli1. By contrast, Gli3, a transcriptional repressor, was de-repressed and confirmed as a direct miR-133 target. Phenotypically, miR-133 KD impaired myotome formation and growth by disrupting proliferation, extracellular matrix deposition and epithelialization. Together, these observations suggest that miR-133-mediated Gli3 silencing is crucial for embryonic myogenesis. Consistent with this idea, we found that activation of Shh signalling by either purmorphamine, or KD of Gli3 by antisense morpholino, rescued the miR-133 KD phenotype. Thus, we identify a novel Shh/myogenic regulatory factor/miR-133/Gli3 axis that connects epithelial morphogenesis with myogenic fate specification.

TLR7 is expressed by support cells, but not sensory neurons, in ganglia.

BACKGROUND: Toll-like receptor 7 (TLR7) is an innate immune receptor that detects viral single-stranded RNA and triggers the production of proinflammatory cytokines and type 1 interferons in immune cells. TLR7 agonists also modulate sensory nerve function by increasing neuronal excitability, although studies are conflicting whether sensory neurons specifically express TLR7. This uncertainty has confounded the development of a mechanistic understanding of TLR7 function in nervous tissues. METHODS: TLR7 expression was tested using in situ hybridization with species-specific RNA probes in vagal and dorsal root sensory ganglia in wild-type and TLR7 knockout (KO) mice and in guinea pigs. Since TLR7 KO mice were generated by inserting an Escherichia coli lacZ gene in exon 3 of the mouse TLR7 gene, wild-type and TLR7 (KO) mouse vagal ganglia were also labeled for lacZ. In situ labeling was compared to immunohistochemistry using TLR7 antibody probes. The effects of influenza A infection on TLR7 expression in sensory ganglia and in the spleen were also assessed. RESULTS: In situ probes detected TLR7 in the spleen and in small support cells adjacent to sensory neurons in the dorsal root and vagal ganglia in wild-type mice and guinea pigs, but not in TLR7 KO mice. TLR7 was co-expressed with the macrophage marker Iba1 and the satellite glial cell marker GFAP, but not with the neuronal marker PGP9.5, indicating that TLR7 is not expressed by sensory nerves in either vagal or dorsal root ganglia in mice or guinea pigs. In contrast, TLR7 antibodies labeled small- and medium-sized neurons in wild-type and TLR7 KO mice in a TLR7-independent manner. Influenza A infection caused significant weight loss and upregulation of TLR7 in the spleens, but not in vagal ganglia, in mice. CONCLUSION: TLR7 is expressed by macrophages and satellite glial cells, but not neurons in sensory ganglia suggesting TLR7's neuromodulatory effects are mediated indirectly via activation of neuronally-associated support cells, not through activation of neurons directly. Our data also suggest TLR7's primary role in neuronal tissues is not related to antiviral immunity.

High-salt diet downregulates TREM2 expression and blunts efferocytosis of macrophages after acute ischemic stroke.

BACKGROUND: A high-salt diet (HSD) is one of the major risk factors for acute ischemic stroke (AIS). As a potential mechanism, surplus salt intake primes macrophages towards a proinflammatory phenotype. In this study, whether HSD could blunt the efferocytic capability of macrophages after ischemic stroke, thus exacerbating post-stroke neural inflammation, was investigated. METHODS: Wild-type male C57BL/6 mice were fed with fodder containing 8% sodium chloride for 4 weeks and subjected to transient middle cerebral occlusion (tMCAO). Disease severity, macrophage polarization as well as efferocytic capability were evaluated. Bone marrow-derived macrophages were cultured in vitro, and the impact of high salinity on their efferocytic activity, as well as their expression of phagocytic molecules, were analyzed. The relationships among sodium concentration, macrophage phenotype, and disease severity in AIS patients were explored. RESULTS: HSD-fed mice displayed increased infarct volume and aggravated neurological deficiency. Mice fed with HSD suffered exacerbated neural inflammation as shown by higher inflammatory mediator expression and immune cell infiltration levels. Infiltrated macrophages within stroke lesions in HSD-fed mice exhibited a shift towards proinflammatory phenotype and impaired efferocytic capability. As assessed with a PCR array, the expression of triggering receptor expressed on myeloid cells 2 (TREM2), a receptor relevant to phagocytosis, was downregulated in high-salt-treated bone marrow-derived macrophages. Enhancement of TREM2 signaling restored the efferocytic capacity and cellular inflammation resolution of macrophages in a high salinity environment in vitro and in vivo. A high concentration of urine sodium in AIS patients was found to be correlated with lower TREM2 expression and detrimental stroke outcomes. CONCLUSIONS: HSD inhibited the efferocytic capacity of macrophages by downregulating TREM2 expression, thus impeding inflammation resolution after ischemic stroke. Enhancing TREM2 signaling in monocytes/macrophages could be a promising therapeutic strategy to enhance efferocytosis and promote post-stroke inflammation resolution.

Upregulation of leukocyte immunoglobulin-like receptor B4 on interstitial macrophages in COPD; their possible protective role against emphysema formation.

BACKGROUND: Leukocyte immunoglobulin-like receptor B4 (LILRB4) is one of the inhibitory receptors in various types of immune cells including macrophages. Previous reports suggested that LILRB4 could be involved in a negative feedback system to prevent excessive inflammatory responses. However, its role has been unclear in chronic obstructive pulmonary disease (COPD), in which macrophages play a crucial role in the pathogenesis. In this study, we aimed to examine the changes of LILRB4 on macrophages both in the lung specimens of COPD patients and the lungs of a mouse emphysema model. We then tried to compare the differences in both inflammation and emphysematous changes of the model between wild-type and LILRB4-deficient mice in order to elucidate the role of LILRB4 in the pathogenesis of COPD. METHODS: We prepared single-cell suspensions of resected lung specimens of never-smokers (n = 21), non-COPD smokers (n = 16), and COPD patients (n = 14). The identification of LILRB4-expressing cells and the level of LILRB4 expression were evaluated by flow cytometry. We analyzed the relationships between the LILRB4 expression and clinical characteristics including respiratory function. In the experiments using an elastase-induced mouse model of emphysema, we also analyzed the LILRB4 expression on lung macrophages. We compared inflammatory cell accumulation and emphysematous changes induced by elastase instillation between wild-type and LILRB4-deficient mice. RESULTS: The levels of surface expression of LILRB4 are relatively high on monocyte linage cells including macrophages in the human lungs. The percentage of LILRB4+ cells in lung interstitial macrophages was increased in COPD patients compared to non-COPD smokers (p = 0.018) and correlated with the severity of emphysematous lesions detected by CT scan (rs = 0.559, p < 0.001), whereas the amount of smoking showed no correlation with LILRB4 expression. Increased LILRB4 on interstitial macrophages was also observed in elastase-treated mice (p = 0.008). LILRB4-deficient mice showed severer emphysematous lesions with increased MMP-12 expression in the model. CONCLUSIONS: LILRB4 on interstitial macrophages was upregulated both in human COPD lungs and in a mouse model of emphysema. This upregulated LILRB4 may have a protective effect against emphysema formation, possibly through decreasing MMP-12 expression in the lungs.

Antiseizure medications and fetal nutrients: Effects on choline transporters in a human placental cell line.

OBJECTIVE: Many nutrients essential to the fetus and for proper function of the placenta itself cannot freely diffuse across membrane barriers, and their transplacental transfer depends on transporters. Our previous studies provided evidence for altered expression of transporters for folic acid in trophoblasts exposed to antiseizure medications (ASMs). The goal of the current study was to explore the effects of older and newer ASMs on the expression and function of uptake transporters for choline, which interacts with folate at pathways for methyl group donation. METHODS: BeWo cells were incubated for 2 or 5 days with valproate (42, 83, or 166 µg/ml), carbamazepine (6 or 12 µg/ml), levetiracetam (10 or 30 µg/ml), lamotrigine (3 or 12 µg/ml), lacosamide (5, 10, or 20 µg/ml), or their vehicles (n = 6/treatment group). Quantitative polymerase chain reaction (PCR) analysis was utilized to study the effects of ASMs on the transcript levels of the choline transporters SLC44A1 (CTL1) and SLC44A2 (CTL2). Transporter protein expression in valproate-treated cells was assessed by western blot analysis. Choline and acetylcholine were quantified in cell lysates by a choline/acetylcholine assay kit. RESULTS: Compared with controls, valproate and levetiracetam at high therapeutic concentrations (83 and 30 µg/ml, respectively) lowered choline transporter transcript levels by up to 42% and 26%, and total choline levels by 20% and 21%, respectively (p < .05). At 83 µg/ml, valproate additionally reduced CTL1 and CTL2 protein expression, by 39 ± 21% and 61 ± 13% (mean ± SD), respectively (p < .01). Carbamazepine reduced SLC44A1 transcript levels, whereas lacosamide modestly decreased the expression of SLC44A2. Lamotrigine did not alter choline transporter expression. SIGNIFICANCE: Antiseizure medications, particularly at high therapeutic concentrations, can interfere with the placental uptake of choline. In line with current knowledge from pregnancy registries and clinical studies, the present in vitro findings further support careful adjustment of maternal ASM doses during pregnancy.

ERRγ ligand HPB2 upregulates BDNF-TrkB and enhances dopaminergic neuronal phenotype.

Brain derived neurotrophic factor (BDNF) promotes maturation of dopaminergic (DAergic) neurons in the midbrain and positively regulates their maintenance and outgrowth. Therefore, understanding the mechanisms regulating the BDNF signaling pathway in DAergic neurons may help discover potential therapeutic strategies for neuropsychological disorders associated with dysregulation of DAergic neurotransmission. Because estrogen-related receptor gamma (ERRγ) is highly expressed in both the fetal nervous system and adult brains during DAergic neuronal differentiation, and it is involved in regulating the DAergic neuronal phenotype, we asked in this study whether ERRγ ligand regulates BDNF signaling and subsequent DAergic neuronal phenotype. Based on the X-ray crystal structures of the ligand binding domain of ERRγ, we designed and synthesized the ERRγ agonist, (E)-4-hydroxy-N'-(4-(phenylethynyl)benzylidene)benzohydrazide (HPB2) (Kd value, 8.35 µmol/L). HPB2 increased BDNF mRNA and protein levels, and enhanced the expression of the BDNF receptor tropomyosin receptor kinase B (TrkB) in human neuroblastoma SH-SY5Y, differentiated Lund human mesencephalic (LUHMES) cells, and primary ventral mesencephalic (VM) neurons. HPB2-induced upregulation of BDNF was attenuated by GSK5182, an antagonist of ERRγ, and siRNA-mediated ERRγ silencing. HPB2-induced activation of extracellular-signal-regulated kinase (ERK) and phosphorylation of cAMP-response element binding protein (CREB) was responsible for BDNF upregulation in SH-SY5Y cells. HPB2 enhanced the DAergic neuronal phenotype, namely upregulation of tyrosine hydroxylase (TH) and DA transporter (DAT) with neurite outgrowth, both in SH-SY5Y and primary VM neurons, which was interfered by the inhibition of BDNF-TrkB signaling, ERRγ knockdown, or blockade of ERK activation. HPB2 also upregulated BDNF and TH in the striatum and induced neurite elongation in the substantia nigra of mice brain. In conclusion, ERRγ activation regulated BDNF expression and the subsequent DAergic neuronal phenotype in neuronal cells. Our results might provide new insights into the mechanism underlying the regulation of BDNF expression, leading to novel therapeutic strategies for neuropsychological disorders associated with DAergic dysregulation.

Antidepressant-like effect of guanosine involves activation of AMPA receptor and BDNF/TrkB signaling.

Guanosine is a purine nucleoside that has been shown to exhibit antidepressant effects, but the mechanisms underlying its effect are not well established. We investigated if the antidepressant-like effect induced by guanosine in the tail suspension test (TST) in mice involves the modulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, voltage-dependent calcium channel (VDCC), and brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB) pathway. We also evaluated if the antidepressant-like effect of guanosine is accompanied by an acute increase in hippocampal and prefrontocortical BDNF levels. Additionally, we investigated if the ability of guanosine to elicit a fast behavioral response in the novelty suppressed feeding (NSF) test is associated with morphological changes related to hippocampal synaptogenesis. The antidepressant-like effect of guanosine (0.05 mg/kg, p.o.) in the TST was prevented by DNQX (AMPA receptor antagonist), verapamil (VDCC blocker), K-252a (TrkBantagonist), or BDNF antibody. Increased P70S6K phosphorylation and higher synapsin I immunocontent in the hippocampus, but not in the prefrontal cortex, were observed 1 h after guanosine administration. Guanosine exerted an antidepressant-like effect 1, 6, and 24 h after its administration, an effect accompanied by increased hippocampal BDNF level. In the prefrontal cortex, BDNF level was increased only 1 h after guanosine treatment. Finally, guanosine was effective in the NSF test (after 1 h) but caused no alterations in dendritic spine density and remodeling in the ventral dentate gyrus (DG). Altogether, the results indicate that guanosine modulates targets known to be implicated in fast antidepressant behavioral responses (AMPA receptor, VDCC, and TrkB/BDNF pathway).

Placental Expression of Bile Acid Transporters in Intrahepatic Cholestasis of Pregnancy.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-related condition characterized by increased maternal circulating bile acids (BAs) having adverse fetal effects. We investigated whether the human placenta expresses specific regulation patterns to prevent fetal exposition to harmful amounts of BAs during ICP. Using real-time quantitative PCR, we screened placentae from healthy pregnancies (n = 12) and corresponding trophoblast cells (n = 3) for the expression of 21 solute carriers and ATP-binding cassette transporter proteins, all acknowledged as BA- and/or cholestasis-related genes. The placental gene expression pattern was compared between healthy women and ICP patients (n = 12 each). Placental SLCO3A1 (OATP3A1) gene expression was significantly altered in ICP compared with controls. The other 20 genes, including SLC10A2 (ASBT) and EPHX1 (EPOX, mEH) reported for the first time in trophoblasts, were comparably abundant in healthy and ICP placentae. ABCG5 was undetectable in all placentae. Placental SLC10A2 (ASBT), SLCO4A1 (OATP4A1), and ABCC2 mRNA levels were positively correlated with BA concentrations in ICP. Placental SLC10A2 (ASBT) mRNA was also correlated with maternal body mass index. We conclude that at the transcriptional level only a limited response of BA transport systems is found under ICP conditions. However, the extent of the transcriptional response may also depend on the severity of the ICP condition and the magnitude by which the maternal BA levels are increased.

Phenotypic and Functional Consequences of PLT Binding to Monocytes and Its Association with Clinical Features in SLE.

Platelets (PLTs) can modulate the immune system through the release of soluble mediators or through interaction with immune cells. Monocytes are the main immune cells that bind with PLTs, and this interaction is increased in several inflammatory and autoimmune conditions, including systemic lupus erythematosus (SLE). Our aim was to characterize the phenotypic and functional consequences of PLT binding to monocytes in healthy donors (HD) and in SLE and to relate it to the pathogenesis of SLE. We analyzed the phenotypic and functional features of monocytes with non-activated and activated bound PLTs by flow cytometry. We observed that monocytes with bound PLTs and especially those with activated PLTs have an up-regulated HLA-DR, CD86, CD54, CD16 and CD64 expression. Monocytes with bound PLTs also have an increased capacity for phagocytosis, though not for efferocytosis. In addition, monocytes with bound PLTs have increased IL-10, but not TNF-α, secretion. The altered phenotypic and functional features are comparable in SLE and HD monocytes and in bound PLTs. However, the percentages of monocytes with bound PLTs are significantly higher in SLE patients and are associated with undetectable levels of anti-dsDNA antibodies and hematuria, and with normal C3 and albumin/creatinine levels. Our results suggest that PLTs have a modulatory influence on monocytes and that this effect may be highlighted by an increased binding of PLTs to monocytes in autoimmune conditions.

Fat regulates expression of four-jointed reporters in vivo through a 20 bp element independently of the Hippo pathway.

Development of an organism requires accurate coordination between the growth of a tissue and orientation of cells within the tissue. The large cadherin Fat has been shown to play a role in both of these processes. Fat is involved in the establishment of planar cell polarity and regulates growth through the Hippo pathway, a developmental cascade that controls proliferation and apoptosis. Both Fat and the Hippo pathway are known to regulate transcription of four-jointed, although the nature of this regulation is unknown. In this study, we test whether Fat affects four-jointed transcription via or independently of Hippo pathway. Our analysis of the four-jointed regulatory region reveals a 1.2 kb element that functions as an enhancer for graded expression of Four-jointed in the eye imaginal disc. Within this enhancer element, we identify a 20 bp fragment that is critical for regulation by Fat but not by Hippo. Our findings suggest that Fat and the Hippo pathway control four-jointed expression independently of each other and none of the transcription factors known to function downstream of the Hippo pathway are required to regulate four-jointed expression through the 1.2 kb element.

Diesel exhaust impairs TREM2 to dysregulate neuroinflammation.

BACKGROUND: Air pollution has been linked to neurodegenerative diseases, including Alzheimer's disease (AD), and the underlying neuroimmune mechanisms remain poorly understood. TREM2 is a myeloid cell membrane receptor that is a key regulator of disease-associated microglia (DAM) cells, where loss-of-function TREM2 mutations are associated with an increased risk of AD. At present, the basic function of TREM2 in neuroinflammation is a point of controversy. Further, the impact of air pollution on TREM2 and the DAM phenotype is largely unknown. Using diesel exhaust (DE) as a model of urban air pollution exposure, we sought to address its impact on TREM2 expression, the DAM phenotype, the association of microglia with the neurovasculature, and the role of TREM2 in DE-induced neuroinflammation. METHODS: WYK rats were exposed for 4 weeks to DE (0, 50, 150, 500 µg/m3) by inhalation. DE particles (DEP) were administered intratracheally once (600 µg/mouse) or 8 times (100 µg/mouse) across 28 days to male mice (Trem2+/+, Trem2-/-, PHOX+/+, and PHOX-/-). RESULTS: Rats exposed to DE exhibited inverted-U patterns of Trem2 mRNA expression in the hippocampus and frontal cortex, while TREM2 protein was globally diminished, indicating impaired TREM2 expression. Analysis of DAM markers Cx3Cr1, Lyz2, and Lpl in the frontal cortex and hippocampus showed inverted-U patterns of expression as well, supporting dysregulation of the DAM phenotype. Further, microglial-vessel association decreased with DE inhalation in a dose-dependent manner. Mechanistically, intratracheal administration of DEP increased Tnf (TNFα), Ncf1 (p47PHOX), and Ncf2 (p67PHOX) mRNA expression in only Trem2+/+ mice, where Il1b (IL-1ß) expression was elevated in only Trem2-/- mice, emphasizing an important role for TREM2 in DEP-induced neuroinflammation. CONCLUSIONS: Collectively, these findings reveal a novel role for TREM2 in how air pollution regulates neuroinflammation and provides much needed insight into the potential mechanisms linking urban air pollution to AD.

TREM-2-p38 MAPK signaling regulates neuroinflammation during chronic cerebral hypoperfusion combined with diabetes mellitus.

BACKGROUND: Diabetes mellitus (DM) and chronic cerebral hypoperfusion(CCH)are both risk factors for cognitive impairment. However, whether DM and CCH can synergistically promote cognitive impairment and the related pathological mechanisms remain unknown. METHODS: To investigate the effect of DM and CCH on cognitive function, rats fed with high-fat diet (HFD) and injected with low-dose streptozotocin (STZ) followed by bilateral common carotid artery occlusion (BCCAO) were induced to mimic DM and CCH in vivo and mouse BV2 microglial cells were exposed to hypoxia and/or high glucose to mimic CCH complicated with DM pathologies in vitro. To further explore the underlying mechanism, TREM-2-specific small interfering RNA and TREM-2 overexpression lentivirus were used to knock out and overexpress TREM-2, respectively. RESULTS: Cognitive deficits, neuronal cell death, neuroinflammation with microglial activation, and TREM-2-MAPK signaling were enhanced when DM was superimposed on CCH both in vivo and in vitro. Manipulating TREM-2 expression levels markedly regulated the p38 MAPK signaling and the inflammatory response in vitro. TREM-2 knockout intensified while TREM-2 overexpression suppressed the p38 MAPK signaling and subsequent pro-inflammatory mediator production under high glucose and hypoxia condition. CONCLUSIONS: These results suggest that TREM-2 negatively regulates p38 MAPK-mediated inflammatory response when DM was synergistically superimposed on CCH and highlight the importance of TREM-2 as a potential target of immune regulation in DM and CCH.

DNAX Activating Protein of 12 kDa/Triggering Receptor Expressed on Myeloid Cells 2 Expression by Mouse and Human Liver Dendritic Cells: Functional Implications and Regulation of Liver Ischemia-Reperfusion Injury.

Liver interstitial dendritic cells (DCs) have been implicated in the control of ischemia-reperfusion injury (IRI) and host immune responses following liver transplantation. Mechanisms underlying these regulatory functions of hepatic DCs remain unclear. We have shown recently that the transmembrane immunoadaptor DNAX-activating protein of 12 kDa (DAP12) negatively regulates mouse liver DC maturation and proinflammatory and immune stimulatory functions. Here, we used PCR analysis and flow cytometry to characterize expression of DAP12 and its associated triggering receptor, triggering receptor expressed on myeloid cells 2 (TREM2), by mouse and human liver DCs and other immune cells compared with DCs in other tissues. We also examined the roles of DAP12 and TREM2 and their expression by liver DCs in the regulation of liver IRI. Injury was induced in DAP12-/- , TREM2-/- , or wild-type (WT) mice by 1 hour of 70% clamping and quantified following 6 hours of reperfusion. Both DAP12 and TREM2 were coexpressed at comparatively high levels by liver DCs. Mouse liver DCs lacking DAP12 or TREM2 displayed enhanced levels of nuclear factor κB and costimulatory molecule expression. Unlike normal WT liver DCs, DAP12-/- liver DC failed to inhibit proliferative responses of activated T cells. In vivo, DAP12-/- and TREM2-/- mice exhibited enhanced IRI accompanied by augmented liver DC activation. Elevated alanine aminotransferase levels and tissue injury were markedly reduced by infusion of WT but not DAP12-/- DC. Conclusion: Our data reveal a close association between DAP12 and TREM2 expression by liver DC and suggest that, by negatively regulating liver DC stimulatory function, DAP12 promotes their control of hepatic inflammatory responses; the DAP12/TREM2 signaling complex may represent a therapeutic target for control of acute liver injury/liver inflammatory disorders.