Hydroxyindole-O-methyltransferase mRNA expression in a subpopulation of photoreceptors in the chicken retina

Abstract: Hydroxyindole O -methyltransferase (HIOMT, EC 2.1.1.4) catalyzes the final step in the synthesis of melatonin in the pineal gland and retina. HIOMT mRNA was localized by in situ hybridization in the chicken retina to some, but clearly not all, photoreceptors, while in the pineal gland, most pinealocytes displayed a positive hybridization signal. The in situ hybridization localization was confirmed by immunocytochemistry, using an antibody directed against a synthetic chicken HIOMT peptide. Western blot analysis demonstrated an immunoreactive protein of about 40 kilodaltons in the pineal, but the HIOMT protein was below detectable levels in the retina. However, the HIOMT-peptide antibody did identify a modestly immunoreactive subpopulation of retinal photoreceptors. These observations suggest that, in the chicken, melatonin biosynthetic activity is located mainly in a subpopulation of retinal photoreceptors and in most pinealocytes.     

系统性硬化患者内皮祖细胞连接黏附分子-A表达

目的研究系统性硬化(SSc)患者外周血内皮祖细胞连接黏附分子-A(JAM-A)表达情况。方法收集13例SSc患者及13例对照组新鲜EDTA抗凝血2ml,采用流式细胞仪方法检测,用PerCP-CY5.5、PE、Alexa Fluor 647和FITC标记CD34、CD133、CD309和JAM-A。内皮祖细胞定义为CD34、CD133、CD309(VEGFR-2,KDR)均阳性的细胞。结果内皮祖细胞表达JAM-A,SSc患者内皮祖细胞中JAM-A表达较正常对照减少(分别为0.0775±0.0385和0.1567±0.1223,P<0.05),SSc患者内皮祖细胞数量亦较对照组少(分别为0.0817±0.0403和0.1746±0.1419,P<0.05)。结论 SSc患者内皮祖细胞中JAM-A的减少是由于患者内皮祖细胞数目减少所致。SSc患者存在血管生成障碍,JAM-A表达异常,并且在SSc发病机制中起一定作用。     

L-精氨酸抑制内皮细胞粘附分子-1表达与氧化型低密度脂蛋白促单核细胞粘附作用下降有关

用氧化型低密度脂蛋白处理培养的猪主动脉内皮细胞，以Ｆｉｃｏｌｌ－Ｈａｐａｑｕｅ分离液密度梯度离心分离人外周血单核细胞，观察Ｌ－精氨酸对内皮细胞－单核细胞粘附率的影响；采用反转录－多聚酶链反应技术检测氧化型低密度脂蛋白和Ｌ－精氨酸对内皮细胞血管细胞粘附分子－１表达的影响。结果显示：Ｌ－精氨酸具有剂量和时间依赖性地抑制氧化型低密度脂蛋白的促内皮细胞－单核细胞粘附作用。氧化型低密度脂蛋白处理内皮细胞能明显增加内皮血管细胞粘附分子。－１的表达，而Ｌ－精氨酸阻抑氧化型低密度脂蛋白的此种作用。结果提示Ｌ－精氨酸可能通过抑制血管细胞粘附分子－１表达阻抑氧化型低密度脂蛋白的促内皮细胞—单核细胞粘附作用。     

The role of GPCR signaling in cardiac Epithelial to Mesenchymal Transformation (EMT)

Congenital heart disease is the most common birth defect, affecting 1.35 million newborns every year. Heart failure is a primary cause of late morbidity and mortality after myocardial infarction. Heart development is involved in several rounds of epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET). Errors in these processes contribute to congenital heart disease, and exert deleterious effects on the heart and circulation after myocardial infarction. The identification of factors that are involved in heart development and disease, and the development of new approaches for the treatment of these disorders are of great interest. G protein coupled receptors (GPCRs) comprise 40% of clinically used drug targets, and their signaling are vital components of the heart during development, cardiac repair and in cardiac disease pathogenesis. This review focuses on the importance of EMT program in the heart, and outlines the newly identified GPCRs as potential therapeutic targets of reprogramming EMT to support cardiac cell fate during heart development and after myocardial infarction. More specifically we discuss prokineticin, serotonin, sphingosine-1-phosphate and apelin receptors in heart development and diseases. Further understanding of the regulation of EMT/MET by GPCRs during development and in the adult hearts can provide the following clinical exploitation of these pathways.     

L-精氨酸抑制内皮细胞粘附分子-1表达与氧化型低密度脂蛋白促单核细胞粘附作用下降有关

用经型低密度脂蛋白处理培养的猪主动脉内皮细胞，以Ｆｉｃｏｌｌ－Ｈａｐａｑｕｅ分离浓密度梯度离心分离人外周血单核细胞，观察Ｌ－精氨酸对内皮细胞－单核细胞粘附率的影响，采用反转录－多聚酶链反应技术检测氧化型低密度脂蛋白和Ｌ－精氨酸对内皮细胞血管细胞粘附分子－１表达的影响。结果显示：Ｌ－精氨酸具有剂量和时间依赖性地抑制氧化型低密度脂白促内皮细胞－单核细胞粘附作用，结果提示Ｌ－精氨酸可能通过抑制血管细胞粘     

The M3-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer''s disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M₁-muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M₃-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M₃-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M₃-muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M₃-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M₃-muscarinic receptor was coupled normally to G_q/11-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M₃-muscarinic receptor–dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M₃-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.     

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake

Vertebrate retinas are generally composed of rod (dim-light) and cone (bright-light) photoreceptors with distinct morphologies that evolved as adaptations to nocturnal/crepuscular and diurnal light environments. Over 70 years ago, the “transmutation” theory was proposed to explain some of the rare exceptions in which a photoreceptor type is missing, suggesting that photoreceptors could evolutionarily transition between cell types. Although studies have shown support for this theory in nocturnal geckos, the origins of all-cone retinas, such as those found in diurnal colubrid snakes, remain a mystery. Here we investigate the evolutionary fate of the rods in a diurnal garter snake and test two competing hypotheses: (i) that the rods, and their corresponding molecular machinery, were lost or (ii) that the rods were evolutionarily modified to resemble, and function, as cones. Using multiple approaches, we find evidence for a functional and unusually blue-shifted rhodopsin that is expressed in small single “cones.” Moreover, these cones express rod transducin and have rod ultrastructural features, providing strong support for the hypothesis that they are not true cones, as previously thought, but rather are modified rods. Several intriguing features of garter snake rhodopsin are suggestive of a more cone-like function. We propose that these cone-like rods may have evolved to regain spectral sensitivity and chromatic discrimination as a result of ancestral losses of middle-wavelength cone opsins in early snake evolution. This study illustrates how sensory evolution can be shaped not only by environmental constraints but also by historical contingency in forming new cell types with convergent functionality.How complex structures can arise has long fascinated evolutionary biologists, and the evolution of the eye, as noted by Charles Darwin (1), is perhaps the most famous example. Within the vertebrate eye, the light-sensing photoreceptors are complex, highly specialized cellular structures that can be divided into two general types based on their distinct morphologies and functions: cones, which are active during the day and contain cone opsin pigments, and rods, which mediate dim-light vision and contain rhodopsin (RH1) (2–4). The visual pigments contained in cone photoreceptors are classified into four different subtypes that mediate vision across the visible spectrum from the UV to the red (2). Although most vertebrate retinas are duplex, containing both cones and rods, squamate reptiles (lizards and snakes) are unusual, not only in having highly variable photoreceptor morphologies, but also for several instances of the absence of an entire class of photoreceptors, resulting in simplex retinas composed of only cones or rods (4).In a seminal book published in 1942, Walls (4) hypothesized that, during evolution, vertebrate photoreceptors could transform from one type to another, a process that he termed photoreceptor “transmutation.” As key examples of his theory, Walls (4) highlighted anatomical changes in the photoreceptors of snakes and geckos, two groups within which there have been significant shifts in diurnal and nocturnal activity patterns. Although several subsequent studies have investigated this hypothesis in geckos (5–9), whether the evolutionary transmutation of photoreceptors can happen in snakes remains an open question (10). Walls also noted a number of peculiar morphological adaptations in snake eyes, which he proposed were due to a subterranean phase early in snake evolution that led to degeneration of the ophidian visual system, resulting in loss of features common to other terrestrial vertebrates (4).Colubrid snakes are an ideal group to study Walls’s hypothesis of transmutation, due to their highly variable photoreceptor morphologies that range from all-cone in, at least some, diurnal species, such as Thamnophis (garter snakes), to all-rod in some nocturnal species, as well as species with the presumed ancestral condition of duplex retinas (4, 11). Previous studies in the diurnal colubrid Thamnophis have demonstrated an all-cone retina (4, 11–14), consisting of double cones and large single cones that express a long-wavelength pigment [presumably long wavelength-sensitive opsin (LWS)], and two classes of small single cone, one with a short-wavelength pigment [presumably short wavelength-sensitive 1 opsin (SWS1)] and the other with a middle-wavelength pigment, the identity of which is unclear (14). However, the ancestral condition for colubrids is likely to have been a duplex retina containing both rods and cones, similar to snakes such as pythons and boas, which have rods that express RH1, large single cones that express LWS, and small single cones that express SWS1 (Fig. 1) (4, 10, 11, 15, 16). The SWS2 and RH2 opsins, present ancestrally in vertebrates, appear to have been lost early in the evolution of snakes, perhaps as a result of their proposed fossorial origins (10, 17, 18).Open in a separate windowFig. 1.Illustration of evolutionary pathways for two alternative hypotheses for the evolution of an all-cone retina from a duplex ancestor in diurnal colubrids. In hypothesis 1 the rod photoreceptors, along with RH1, are lost, and an additional cone type is derived from duplication of an existing cone or retained from an ancestral condition that was lost in other snakes. In hypothesis 2 the rod photoreceptor is evolutionarily modified into a cone photoreceptor, maintaining expression of RH1 and other rod-specific phototransduction machinery.Based on these findings, we can formulate two main hypotheses for the evolution of the all-cone retina of diurnal colubrids from the duplex ancestral condition (Fig. 1). The first is that the rods were lost, and RH1 and other components of the visual transduction cascade unique to rod photoreceptors were either lost or targeted to cones. The second hypothesis is that the rods were evolutionarily modified to resemble the appearance, and presumably the function, of cones. If the rods were modified to resemble cones, we might expect a subset of cones to possess molecular components, such as RH1, and morphological features consistent with a rod ancestry. To test these hypotheses, we examined the photoreceptors and visual pigments of a diurnal garter snake (Thamnophis proximus) by combining multiple methodologies including sequencing and molecular evolutionary analyses of opsin genes, microspectrophotometry (MSP) of intact photoreceptor cells, in vitro expression of visual pigments, and scanning and transmission electron microscopy (SEM and TEM) and immunohistochemistry of T. proximus retinas. The combined results of these experiments provide strong evidence that RH1 and other components of the rod visual transduction machinery are expressed in a subset of cone-like photoreceptors with rod ultrastructural features, and that the RH1-expressing “cones” are not true cones, as previously thought, but rather are modified (i.e., “transmuted”), cone-like rods. Our results shed new light on the evolutionary origins of the all-cone retinas of diurnal colubrid snakes, demonstrating how ancestral losses can be compensated by evolutionary modification of existing cellular structures.     

TRPM1 is a component of the retinal ON bipolar cell transduction channel in the mGluR6 cascade

An essential step in intricate visual processing is the segregation of visual signals into ON and OFF pathways by retinal bipolar cells (BCs). Glutamate released from photoreceptors modulates the photoresponse of ON BCs via metabotropic glutamate receptor 6 (mGluR6) and G protein (Go) that regulates a cation channel. However, the cation channel has not yet been unequivocally identified. Here, we report a mouse TRPM1 long form (TRPM1-L) as the cation channel. We found that TRPM1-L localization is developmentally restricted to the dendritic tips of ON BCs in colocalization with mGluR6. TRPM1 null mutant mice completely lose the photoresponse of ON BCs but not that of OFF BCs. In the TRPM1-L-expressing cells, TRPM1-L functions as a constitutively active nonselective cation channel and its activity is negatively regulated by Go in the mGluR6 cascade. These results demonstrate that TRPM1-L is a component of the ON BC transduction channel downstream of mGluR6 in ON BCs.     

Expression and function of phosphoinositide 3-kinase delta in mesenchymal stromal cells from normal and leukaemic bone marrow

Within lymphoid tissues, chronic lymphocytic leukaemia (CLL) cells interact with mesenchymal stromal cells (MSC). Inhibitors of phosphoinositide 3-kinase delta (PI3Kδ) cause release of CLL cells from lymphoid tissues into blood. PI3Kδ inhibitors are thought to target only CLL and other immune cells because PI3Kδ expression is restricted to haematopoietic cells. We found that PI3Kδ is unexpectedly expressed in primary MSC derived from CLL patients and healthy donors. PI3Kδ inhibition in MSC using idelalisib or duvelisib significantly reduced their ability to support CLL migration and adhesion. These observations provide the first evidence that PI3Kδ is expressed and functional in CLL MSC.     

CLE-CLAVATA1 peptide-receptor signaling module regulates the expansion of plant root systems in a nitrogen-dependent manner

Morphological plasticity of root systems is critically important for plant survival because it allows plants to optimize their capacity to take up water and nutrients from the soil environment. Here we show that a signaling module composed of nitrogen (N)-responsive CLE (CLAVATA3/ESR-related) peptides and the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase is expressed in the root vasculature in Arabidopsis thaliana and plays a crucial role in regulating the expansion of the root system under N-deficient conditions. CLE1, -3, -4, and -7 were induced by N deficiency in roots, predominantly expressed in root pericycle cells, and their overexpression repressed the growth of lateral root primordia and their emergence from the primary root. In contrast, clv1 mutants showed progressive outgrowth of lateral root primordia into lateral roots under N-deficient conditions. The clv1 phenotype was reverted by introducing a CLV1 promoter-driven CLV1:GFP construct producing CLV1:GFP fusion proteins in phloem companion cells of roots. The overaccumulation of CLE2, -3, -4, and -7 in clv1 mutants suggested the amplitude of the CLE peptide signals being feedback-regulated by CLV1. When CLE3 was overexpressed under its own promoter in wild-type plants, the length of lateral roots was negatively correlated with increasing CLE3 mRNA levels; however, this inhibitory action of CLE3 was abrogated in the clv1 mutant background. Our findings identify the N-responsive CLE-CLV1 signaling module as an essential mechanism restrictively controlling the expansion of the lateral root system in N-deficient environments.Living organisms have developed dynamic strategies to explore nutrients in the environment. Morphological plasticity of plant roots and microorganisms is often compared with foraging behavior of animals. Plant roots are highly dynamic systems because they can modify their structure to reach nutrient resources in soil and optimize their nutrient uptake capacities. This strategy appears to be associated with morphological adaptation, because plants are sessile in nature and nutrient availabilities in soil are often altered by surrounding biotic and abiotic factors and climate changes. Morphological modifications of plant root systems are particularly prominent when they grow in soil environments with unbalanced nutrient availabilities (1–4). Among the essential elements required for plant growth, nitrogen (N) has a particularly strong effect on root development (1–6). Lateral roots can be developed in N-rich soil patches where adequate amounts of nitrate (NO₃⁻) or ammonium (NH₄⁺) are available, whereas this local outgrowth of lateral roots is restricted in N-deficient patches (7–9). In addition to these local N responses, lateral root growth is stimulated in response to mild N deficiency and suppressed under excess N supply by systemic plant signals carrying information on the nutritional status of distant plant organs (4, 10–13). These morphological responses are important for plant fitness and N acquisition, despite the cost for structuring the root system architecture (2, 6). However, lateral root growth is not sustained when plants are deprived of N for an extended period (4). Under such severe circumstances, the development of new lateral roots should rather be restricted to prevent the risk of extending roots into N-poor environments. Economizing the cost for root development appears to be an important morphological strategy for plant survival.To modify root traits in response to changing N availabilities, plants use various types of signaling molecules including hormones and small RNAs (10, 13–17). In particular, auxin signaling proteins and auxin transporters have been proven essential for lateral root development in response to local nitrate supplies (10, 14–17). These proteins are involved in increasing auxin sensitivity or auxin accumulation at lateral root initials or lateral root tips exposed to NO₃⁻, and the NRT1.1 nitrate transporter has been suggested to play a key role in NO₃⁻ sensing (8, 17, 18). In addition, mutations of the nitrate transporter NRT2.1 have been shown to repress or stimulate lateral root initiation depending on N conditions and sucrose supply (12, 19). Thus, N-dependent root development is apparently under control of complex mechanisms, although its signaling components have remained largely unidentified. In this study, we have identified several homologs of the CLE (CLAVATA3/ESR-related) gene family (20–24) to be up-regulated by N deficiency and involved in this yet unresolved regulatory mechanism. CLAVATA3 (CLV3) is known as a signaling peptide that binds to the CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase (LRR-RLK) and controls stem cell differentiation in the shoot apical meristem (25–32). CLE-receptor signaling modules are also known to control meristem function in the primary and lateral roots (33–35). The N-responsive CLE peptides described in the present study belong to the group of CLE peptides with the highest sequence similarity to CLAVATA3 (CLV3) (21–23) and may partly substitute for CLV3 in the shoot apical meristem (31, 36, 37). Our present findings indicate that the N-responsive CLE peptides and CLV1 are signaling components required for translating an N-deficient nutritional status into a morphological response inhibiting the outgrowth of lateral root primordia in Arabidopsis. The present study demonstrates a unique function of the CLE-CLV1 signaling module in roots and provides new insights into signaling mechanisms regulating the expansion of the plant root system in N-deficient environments.     

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca²⁺) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1–10 μM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca²⁺ influx. Yoda1 induced Ca²⁺-dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo−/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.     

Ghrelin以非经典受体依赖性方式促进人胚胎干细胞定向分化为心肌细胞的研究

目的 探讨Ghrelin对人胚胎干（hES）细胞定向分化为心肌细胞的影响. 方法 以不同浓度Ghrelin诱导hES细胞定向分化为心肌细胞,显微镜下计数搏动细胞团的比例,应用实时PCR检测心肌特异性标志物的表达.应用RT-PCR、Western blot及免疫荧光染色检测分化细胞中生长激素促分泌物受体1α（GHSR1α）的表达,添加GHS-R1α拮抗剂[D-lys3]-GHRP-6,观察其对Ghrelin促分化作用的影响. 结果 与对照组比较,10-10、10-9及10-8 mol/L Ghrelin均可增加搏动细胞团的比例[搏动比例分别为（12.9±1.4）％％,（19.5±2.2）％及（13.5±3.1）％vs（10.3±2.2）％,P＜0.05].RT-PCR分析显示,Ghrelin可上调α-肌球蛋白重链（α-MHC）和心肌肌钙蛋白I（cTnI）的表达[α-MHC：100％ vs （40.1±13.2）％,P＜0.001;cTnI：100％ vs （52.6±9.8）％,P＜0.001].RT-PCR、Western blot及免疫荧光染色均显示GHS-R1α在不同阶段的分化细胞中表达,但酰基化和非酰基化Ghrelin的促分化作用相似,且添加GHS-R1α拮抗剂不能阻断Ghrelin的促分化作用. 结论 Ghrelin可促进hES细胞定向分化为心肌细胞,该作用可能由GHS-R1α以外的信号途径所介导.     

Signal transducer and activator of transcription-3/suppressor of cytokine signaling-3 (STAT3/SOCS3) axis in myeloid cells regulates neuroinflammation

Suppressor of cytokine signaling (SOCS) proteins are feedback inhibitors of the JAK/STAT pathway. SOCS3 has a crucial role in inhibiting STAT3 activation, cytokine signaling, and inflammatory gene expression in macrophages/microglia. To determine the role of SOCS3 in myeloid cells in neuroinflammation, mice with conditional SOCS3 deletion in myeloid cells (LysMCre-SOCS3(fl/fl)) were tested for experimental autoimmune encephalomyelitis (EAE). The myeloid-specific SOCS3-deficient mice are vulnerable to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with a severe, nonresolving atypical form of disease. In vivo, enhanced infiltration of inflammatory cells and demyelination is prominent in the cerebellum of myeloid-specific SOCS3-deficient mice, as is enhanced STAT3 signaling and expression of inflammatory cytokines/chemokines and an immune response dominated by Th1 and Th17 cells. In vitro, SOCS3-deficient macrophages exhibit heightened STAT3 activation and are polarized toward the classical M1 phenotype. SOCS3-deficient M1 macrophages provide the microenvironment to polarize Th1 and Th17 cells and induce neuronal death. Furthermore, adoptive transfer of M2 macrophages into myeloid SOCS3-deficient mice leads to delayed onset and reduced severity of atypical EAE by decreasing STAT3 activation, Th1/Th17 cells, and proinflammatory mediators in the cerebellum. These findings indicate that myeloid cell SOCS3 provides protection from EAE through deactivation of neuroinflammatory responses.     

Spatial distribution of LTi-like cells in intestinal mucosa regulates type 3 innate immunity

Lymphoid tissue inducer (LTi)-like cells are tissue resident innate lymphocytes that rapidly secrete cytokines that promote gut epithelial integrity and protect against extracellular bacterial infections.Here, we report that the retention of LTi-like cells in conventional solitary intestinal lymphoid tissue (SILT) is essential for controlling LTi-like cell function and is maintained by expression of the chemokine receptor CXCR5. Deletion of Cxcr5 functionally unleashed LTi-like cells in a cell intrinsic manner, leading to uncontrolled IL-17 and IL-22 production. The elevated production of IL-22 in Cxcr5-deficient mice improved gut barrier integrity and protected mice during infection with the opportunistic pathogen Clostridium difficile. Interestingly, Cxcr5^−/− mice developed LTi-like cell aggregates that were displaced from their typical niche at the intestinal crypt, and LTi-like cell hyperresponsiveness was associated with the local formation of this unconventional SILT. Thus, LTi-like cell positioning within mucosa controls their activity via niche-specific signals that temper cytokine production during homeostasis.

Lymphoid tissue inducer (LTi)-like cells belong to a family of tissue resident innate lymphocytes that lack rearranged antigen-specific receptors and act as a first line of defense at barrier tissues. LTi-like cells, along with other group 3 innate lymphoid cells (ILC3), maintain intestinal homeostasis by producing the cytokines IL-22 and IL-17A, which promote gut epithelial cell proliferation, anti-microbial peptide production, and tight junction protein abundance (1, 2). The conditioning of epithelial cells by these cytokines contributes to balanced interactions between the host and commensal microbiota under steady-state conditions, and LTi-like cell-derived IL-22 promotes barrier integrity and protective immunity during infection with the enteric pathogenic bacteria (3).In addition to providing effector functions, LTi-like cells and their fetal LTi counterparts are required for early steps in lymphoid tissue development. Fetal LTi induce lymph node and Peyer’s patch development during gestation by activating lymphoid tissue organizer cells at primordial lymphoid organs with lymphotoxin (LT)-α1β2 (4–6). Similarly, LTi-like cells are required for the postnatal development of cryptopatches, small lymphoid aggregates in the intestine that have the potential to mature into isolated lymphoid follicles (ILF) in response to signals from microbes (7, 8). In line with their roles in lymphoid tissue organogenesis and maturation, LTi-like cells in adult mouse intestines preferentially localize in solitary intestinal lymphoid tissue (SILT). The microenvironments of these highly specialized niches are expected to support and regulate LTi-like cells; however, their impact on LTi-like cell behavior has not been fully explored.LTi-like cells express multiple G protein–coupled receptors that facilitate their migration in tissue (9–12). Among these, CXCR5 has a predominant role in the migration of LTi to developing lymphoid structures, with Cxcr5^−/− mice exhibiting defects in lymph node and Peyer’s patch development (13). Mice deficient in CXCR5 or its ligand CXCL13 also have delayed cryptopatch development and fail to convert cryptopatches to mature ILF because of impaired recruitment of B cells to these structures (14–16). Dendritic cells (DCs) have been shown to be a local source of CXCL13 in SILT (16) and thus likely retain B cells and LTi-like cells at these structures under homeostatic conditions via the CXCL13–CXCR5 signaling axis. The retention of LTi-like cells in SILT is expected to bring these cells in close proximity to activating and inhibitory signals provided by specialized myeloid cells, neurons that express the vasoactive intestinal peptide (VIP), and lymphocyte populations localized at these sites (17–20). However, the impact of CXCR5 on functions of LTi-like cells beyond those associated with lymphoid tissue maintenance and development remains unknown.In the current study, we show that CXCR5 expression regulates LTi-like cell function. Deletion of Cxcr5 led to increased numbers of LTi-like cells in the small intestine (SI) and enhanced their ability to produce IL-17A and IL-22. Cxcr5 regulated LTi-like cells via a cell-intrinsic mechanism that did not involve direct suppression by CXCL13. Heightened LTi-like cell activity in Cxcr5-deficient mice was associated with the development of abnormal LTi-like cell aggregates in the SI that were localized in villus lamina propria instead of at the intestinal crypt base. Importantly, augmented production of IL-22 in Cxcr5^−/− mice was protective during acute infection with the opportunistic pathogen Clostridium difficile. These data reveal that CXCR5-dependent migration can control innate type 3 immunity by altering the niche of LTi-like cells in intestinal lamina propria.     

Tumor suppressor function of laminin-binding α-dystroglycan requires a distinct β3-N-acetylglucosaminyltransferase

α-Dystroglycan (α-DG) represents a highly glycosylated cell surface molecule that is expressed in the epithelial cell-basement membrane (BM) interface and plays an essential role in epithelium development and tissue organization. The α-DG–mediated epithelial cell-BM interaction is often impaired in invasive carcinomas, yet roles and underlying mechanisms of such an impaired interaction in tumor progression remain unclear. We report here a suppressor function of laminin-binding glycans on α-DG in tumor progression. In aggressive prostate and breast carcinoma cell lines, laminin-binding glycans are dramatically decreased, although the amount of α-DG and β-dystroglycan is maintained. The decrease of laminin-binding glycans and consequent increased cell migration were associated with the decreased expression of β3-N-acetylglucosaminyltransferase-1 (β3GnT1). Forced expression of β3GnT1 in aggressive cancer cells restored the laminin-binding glycans and decreased tumor formation. β3GnT1 was found to be required for laminin-binding glycan synthesis through formation of a complex with LARGE, thus regulating the function of LARGE. Interaction of the laminin-binding glycans with laminin and other adhesive molecules in BM attenuates tumor cell migratory potential by antagonizing ERK/AKT phosphorylation induced by the components in the ECM. These results identify a previously undescribed role of carbohydrate-dependent cell-BM interaction in tumor suppression and its control by β3GnT1 and LARGE.