Co-localization of Middle East respiratory syndrome coronavirus (MERS-CoV) and dipeptidyl peptidase-4 in the respiratory tract and lymphoid tissues of pigs and llamas.

This study investigated the co-localization of the Middle East respiratory syndrome coronavirus (MERS-CoV) and its receptor dipeptidyl peptidase-4 (DPP4) by immunohistochemistry (IHC) across respiratory and lymphoid organs of experimentally MERS-CoV infected pigs and llamas. Also, scanning electron microscopy was performed to assess the ciliary integrity of respiratory epithelial cells in both species. In pigs, on day 2 post-inoculation (p.i.), DPP4-MERS-CoV co-localization was detected in medial turbinate epithelium. On day 4 p.i., the virus/receptor co-localized in frontal and medial turbinate epithelial cells in pigs, and epithelial cells distributed unevenly through the whole nasal cavity and in the cervical lymph node in llamas. MERS-CoV viral nucleocapsid was mainly detected in upper respiratory tract sites on days 2 and 4 p.i. in pigs and day 4 p.i. in llamas. No MERS-CoV was detected on day 24 p.i. in any tissue by IHC. While pigs showed severe ciliary loss in the nasal mucosa both on days 2 and 4 p.i. and moderate loss in the trachea on days 4 and 24 p.i., ciliation of respiratory organs in llamas was not significantly affected. Obtained data confirm the role of DPP4 for MERS-CoV entry in respiratory epithelial cells of llamas. Notably, several nasal epithelial cells in pigs were found to express viral antigen but not DPP4, suggesting the possible existence of other molecule/s facilitating virus entry or down regulation of DPP4 upon infection.

Camel whey protein improves oxidative stress and histopathological alterations in lymphoid organs through Bcl-XL/Bax expression in a streptozotocin-induced type 1 diabetic mouse model.

Type I diabetes (T1D) is a characterized by the inflammation of pancreatic islets and destruction of ß cells. Long and persistent uncontrolled diabetes tends to degenerate the immune system and increase the incidence of infections in diabetic individuals. Most serious diabetic complications are mediated by the free radicals, which damage multiple cellular components through direct effects of the cell cycle regulatory proteins. Camel whey protein (CWP) has antioxidant activity and decreases the effects of free radicals. However, the effects of CWP on lymphoid organs have not been studied in the context of diabetes. Therefore, the present study was designed to investigate the dietary influence of CWP supplementation on the lymphoid organs in streptozotocin (STZ)-induced type 1 diabetic mouse model. Three experimental groups were used: non diabetic control mice, diabetic mice, and diabetic mice treated with CWP. Induction of diabetes was associated with a marked reduction in glutathione (GSH) levels; decreased activities of GSH peroxidase (GSH Px), manganese superoxide dismutase (MnSOD) and catalase; increased reactive oxygen species (ROS) levels and iNOS activity in plasma and lymphoid organs. Furthermore, diabetic mice exhibited alterations in the expression of Bax and Bcl-XL, and subsequently pathological alterations in the architecture of the bone marrow, pancreas, thymus, and spleen. Interestingly, treatment of diabetic mice with CWP robustly restored glucose, insulin, GSH, and ROS levels and the activities of GSH Px, MnSOD, catalase and iNOS. Additionally, supplementation of diabetic mice with CWP improvement in the architecture of lymphoid tissues and rescued from apoptosis through direct effects on the Bax and Bcl-XL proteins. These data revealed the therapeutic potential of CWP against diabetic complications mediated damages of lymphoid organs.

ILK Induction in Lymphoid Organs by a TNFα-NF-κB-Regulated Pathway Promotes the Development of Chronic Lymphocytic Leukemia.

The proliferation of chronic lymphocytic leukemia (CLL) cells requires communication with the lymphoid organ microenvironment. Integrin-linked kinase (ILK) is a multifunctional intracellular adaptor protein that transmits extracellular signals to regulate malignant cell motility, metastasis, and cell-cycle progression, but is poorly characterized in hematologic malignancies. In this study, we investigated the role of ILK in the context of CLL and observed high ILK expression in patient samples, particularly in tumor cells harboring prognostic high-risk markers such as unmutated IGHV genes, high Zap70, or CD38 expression, or a signature of recent proliferation. We also found increased numbers of Ki67 (MKI67)-positive cells in regions of enhanced ILK expression in lymph nodes from CLL patients. Using coculture conditions mimicking the proliferative lymph node microenvironment, we detected a parallel induction of ILK and cyclin D1 (CCND1) expression in CLL cells that was dependent on the activation of NF-κB signaling by soluble TNFα. The newly synthesized ILK protein colocalized to centrosomal structures and was required for correct centrosome clustering and mitotic spindle organization. Furthermore, we established a mouse model of CLL in which B-cell-specific genetic ablation of ILK resulted in decelerated leukemia development due to reduced organ infiltration and proliferation of CLL cells. Collectively, our findings describe a TNFα-NF-κB-mediated mechanism by which ILK expression is induced in the lymph node microenvironment and propose that ILK promotes leukemogenesis by enabling CLL cells to cope with centrosomal defects acquired during malignant transformation. Cancer Res; 76(8); 2186-96. ©2016 AACR.

PI3-Kinase-γ Has a Distinct and Essential Role in Lung-Specific Dendritic Cell Development.

Development of dendritic cells (DCs) commences in the bone marrow, from where pre-DCs migrate to peripheral organs to differentiate into mature DCs in situ. However, the factors that regulate organ-specific differentiation to give rise to tissue-specific DC subsets remain unclear. Here we show that the Ras-PI3Kγ-Akt-mTOR signaling axis acted downstream of FLT3L signaling and was required for development of lung CD103(+) DCs and, to a smaller extent, for lung CD11b(+) DCs, but not related DC populations in other non-lymphoid organs. Furthermore, we show that in lymphoid organs such as the spleen, DCs depended on a similar signaling network to respond to FLT3 ligand with overlapping and partially redundant roles for kinases PI3Kγ and PI3Kδ. Thus we identified PI3Kγ as an essential organ-specific regulator of lung DC development and discovered a signaling network regulating tissue-specific DC development mediated by FLT3.

Evolution of vertebrate adaptive immunity: immune cells and tissues, and AID/APOBEC cytidine deaminases.

All surviving jawed vertebrate representatives achieve diversity in immunoglobulin-based B and T cell receptors for antigen recognition through recombinatorial rearrangement of V(D)J segments. However, the extant jawless vertebrates, lampreys and hagfish, instead generate three types of variable lymphocyte receptors (VLRs) through a template-mediated combinatorial assembly of different leucine-rich repeat (LRR) sequences. The clonally diverse VLRB receptors are expressed by B-like lymphocytes, while the VLRA and VLRC receptors are expressed by lymphocyte lineages that resemble αß and γδ T lymphocytes, respectively. These findings suggest that three basic types of lymphocytes, one B-like and two T-like, are an essential feature of vertebrate adaptive immunity. Around 500 million years ago, a common ancestor of jawed and jawless vertebrates evolved a genetic program for the development of prototypic lymphoid cells as a foundation for an adaptive immune system. This acquisition preceded the convergent evolution of alternative types of clonally diverse receptors for antigens in all vertebrates, as reviewed in this article.

12/15-Lipoxygenase-mediated enzymatic lipid oxidation regulates DC maturation and function.

DCs are able to undergo rapid maturation, which subsequently allows them to initiate and orchestrate T cell-driven immune responses. DC maturation must be tightly controlled in order to avoid random T cell activation and development of autoimmunity. Here, we determined that 12/15-lipoxygenase-meditated (12/15-LO-mediated) enzymatic lipid oxidation regulates DC activation and fine-tunes consecutive T cell responses. Specifically, 12/15-LO activity determined the DC activation threshold via generation of phospholipid oxidation products that induced an antioxidative response dependent on the transcription factor NRF2. Deletion of the 12/15-LO-encoding gene or pharmacologic inhibition of 12/15-LO in murine or human DCs accelerated maturation and shifted the cytokine profile, thereby favoring the differentiation of Th17 cells. Exposure of 12/15-LO-deficient DCs to 12/15-LO-derived oxidized phospholipids attenuated both DC activation and the development of Th17 cells. Analysis of lymphatic tissues from 12/15-LO-deficient mice confirmed enhanced maturation of DCs as well as an increased differentiation of Th17 cells. Moreover, experimental autoimmune encephalomyelitis in mice lacking 12/15-LO resulted in an exacerbated Th17-driven autoimmune disease. Together, our data reveal that 12/15-LO controls maturation of DCs and implicate enzymatic lipid oxidation in shaping the adaptive immune response.

Role of monochromatic light on development of cecal tonsil in young broilers.

Previously, the different monochromatic lights have been demonstrated to affect splenocyte proliferation and melatonin (MEL) secretion in broilers. The present study was designed to evaluate the effects of different monochromatic lights on the development and immune function of broiler cecal tonsils, and to disclose the mechanisms underlying these phenomena. A total of 185 broilers (P0) including intact, sham-operated, and pinealectomized groups were exposed to blue light (BL), green light (GL), red light (RL), and white light (WL) by a light-emitting diode system for 14 days. Compared with RL groups, the GL in the intact and sham-operated groups showed larger follicle areas (66.70%), higher percentages of proliferating cell nuclear antigen (PCNA)-positive cells (33.33%), increased numbers of IgA(+) cells (48.60%), and increased antioxidase activity (33.33%-61.37%), whereas, the density of iNOS and MDA content in GL were lower (43.63%-54.43%) than that of RL. In contrast, after pinealectomy, the area of follicles, the percentage of PCNA-positive cells, the number of IgA(+) cells, and the antioxidase activity decreased in the different light treatments, but the density of iNOS and MDA content increased substantially. There was no significant difference in these parameters between broilers exposed to GL and other lights (P = 0.085-1.000). The results suggested that the enhanced effects of GL on the development and immune function of cecal tonsils in young broilers were mediated by elevated antioxidative status via up-regulation of MEL.

Contributions of ocular surface components to matrix-metalloproteinases (MMP)-2 and MMP-9 in feline tears following corneal epithelial wounding.

PURPOSE: This study investigated ocular surface components that contribute to matrix-metalloproteinase (MMP)-2 and MMP-9 found in tears following corneal epithelial wounding. METHODS: Laboratory short-haired cats underwent corneal epithelial debridement in one randomly chosen eye (n = 18). Eye-flush tears were collected at baseline and during various healing stages. Procedural control eyes (identical experimental protocol as wounded eyes except for wounding, n = 5) served as controls for tear analysis. MMP activity was analyzed in tears using gelatin zymography. MMP staining patterns were evaluated in ocular tissues using immunohistochemistry and used to determine MMP expression sites responsible for tear-derived MMPs. RESULTS: The proMMP-2 and proMMP-9 activity in tears was highest in wounded and procedural control eyes during epithelial migration (8 to 36 hours post-wounding). Wounded eyes showed significantly higher proMMP-9 in tears only during and after epithelial restratification (day 3 to 4 and day 7 to 28 post-wounding, respectively) as compared to procedural controls (p<0.05). Tears from wounded and procedural control eyes showed no statistical differences for pro-MMP-2 and MMP-9 (p>0.05). Immunohistochemistry showed increased MMP-2 and MMP-9 expression in the cornea during epithelial migration and wound closure. The conjunctival epithelium exhibited highest levels of both MMPs during wound closure, while MMP-9 expression was reduced in conjunctival goblet cells during corneal epithelial migration followed by complete absence of the cells during wound closure. The immunostaining for both MMPs was elevated in the lacrimal gland during corneal healing, with little/no change in the meibomian glands. Conjunctival-associated lymphoid tissue (CALT) showed weak MMP-2 and intense MMP-9 staining. CONCLUSIONS: Following wounding, migrating corneal epithelium contributed little to the observed MMP levels in tears. The major sources assessed in the present study for tear-derived MMP-2 and MMP-9 following corneal wounding are the lacrimal gland and CALT. Other sources included stromal keratocytes and conjunctiva with goblet cells.

Lymphoid tissue phospholipase A2 group IID resolves contact hypersensitivity by driving antiinflammatory lipid mediators.

Resolution of inflammation is an active process that is mediated in part by antiinflammatory lipid mediators. Although phospholipase A2 (PLA2) enzymes have been implicated in the promotion of inflammation through mobilizing lipid mediators, the molecular entity of PLA2 subtypes acting upstream of antiinflammatory lipid mediators remains unknown. Herein, we show that secreted PLA2 group IID (PLA2G2D) is preferentially expressed in CD11c(+) dendritic cells (DCs) and macrophages and displays a pro-resolving function. In hapten-induced contact dermatitis, resolution, not propagation, of inflammation was compromised in skin and LNs of PLA2G2D-deficient mice (Pla2g2d(-/-)), in which the immune balance was shifted toward a proinflammatory state over an antiinflammatory state. Bone marrow-derived DCs from Pla2g2d(-/-) mice were hyperactivated and elicited skin inflammation after intravenous transfer into mice. Lipidomics analysis revealed that PLA2G2D in the LNs contributed to mobilization of a pool of polyunsaturated fatty acids that could serve as precursors for antiinflammatory/pro-resolving lipid mediators such as resolvin D1 and 15-deoxy-Δ(12,14)-prostaglandin J2, which reduced Th1 cytokine production and surface MHC class II expression in LN cells or DCs. Altogether, our results highlight PLA2G2D as a "resolving sPLA2" that ameliorates inflammation through mobilizing pro-resolving lipid mediators and points to a potential use of this enzyme for treatment of inflammatory disorders.

Vitamin D-1α-hydroxylase and vitamin D-24-hydroxylase mRNA studies in chickens.

A series of experiments were conducted to study the basal amounts of vitamin D-1α-hydroxylase and vitamin D-24-hydroxylase mRNA amounts in different organs and the effect of immune stimulation on 1α- and 24-hydroxylase mRNA amounts in chickens. At day of hatch, kidneys had an approximately 66-fold higher amount of 1α-hydroxylase and 550-fold higher amount of 24-hydroxylase mRNA, thigh and breast muscles had an approximately 20-fold higher amount of 1α-hydroxylase mRNA, and the thymus had an approximately 41-fold higher amount of 24-hydroxylase mRNA than the liver. An in vivo LPS injection did not alter the amount of 1α-hydroxylase mRNA in the breast muscle (P=0.60) or in the kidneys (P=0.39). An in vivo LPS injection decreased (P=0.01) the amount of 24-hydroxylase mRNA in the breast muscle at 3 d post-LPS injection. An in vivo LPS injection increased (P=0.01) the amount of 24-hydroxylase mRNA in the kidneys at 2, 3, and 6 d post-LPS injection. An in vitro stimulation altered amounts of 1α- (P=0.01) and 24-hydroxylase (P=0.04) mRNA in CD4+ cells. In conclusion, the distribution of 1α- and 24-hydroxylase mRNA amounts was similar to mammals, and an immune stimulation altered the amounts of 1α- and 24-hydroxylase mRNA in chickens.

Ectopic lymphoid neogenesis and lymphoid chemokines in Sjogren's syndrome: at the interplay between chronic inflammation, autoimmunity and lymphomagenesis.

It has long been demonstrated that a subset of patients with Sjogren's syndrome (SS) develop ectopic lymphoid structures (ELS) in the salivary glands (SG). These structures are characterised by periductal clusters of T and B lymphocytes, development of high endothelial venules and differentiation of follicular dendritic cells (FDC) networks. Evidence in patients with and animal models of SS demonstrated that the formation and maintenance of ELS in the SG is critically dependent on the ectopic expression of lymphotoxins (LT) and lymphoid chemokines CXCL13, CCL19, CCL21 and CXCL12. Several cell types, including resident epithelial, stromal and endothelial cells as well as different subsets of infiltrating immune cells, have been shown to be capable of producing some of these factors during chronic inflammation in SS. In this review we focus on the cellular and molecular mechanisms regulating the formation of ELS in SS SG, with particular emphasis on the role of lymphoid chemokines. In addition, we summarise accumulating data in support of the notion that ELS in SS represent functional niches whereby autoreactive B cells undergo affinity maturation, clonal selection and differentiation into autoantibody producing cells, thus contributing to autoimmunity over and above secondary lymphoid organs. Furthermore, we review the emerging role of ELS and lymphoid chemokines in driving extranodal B cell lymphomagenesis in SS and we focus on recent evidence suggesting that ELS identify subsets of SS patients at increased risk of developing systemic manifestations and lymphoma.

Molecular cloning and characterization of the NADPH oxidase from the kuruma shrimp, Marsupenaeus japonicus: early gene up-regulation after Vibrio penaeicida and poly(I:C) stimulations in vitro.

Free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) are involved in many physiological processes. In humans, there are 5 homologs of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) that generate superoxide (O(2)(-)), which can dismute to produce ROS, and play significant roles in innate immunity and cell proliferation. Though Noxes have been identified in vertebrates (humans and fishes) and some insects, there are very few reports investigating Noxes in crustaceans. In the present study, we describe the entire cDNA sequence (4216 bp) of Marsupenaeus japonicus (kuruma shrimp) Nox (MjNox) generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjNox encodes a protein of 1280 amino acids with an estimated mass of 146 kDa that has 46.8% sequence homology with the Nox gene of the fruit fly, Drosophila melanogaster. Highly conserved amino acid sequences were observed in the NADPH binding domain. Transcriptional analysis revealed that MjNox mRNA is highly expressed in the lymphoid organ, hepatopancreas and hemocytes of the healthy kuruma shrimp. In the hemocytes, MjNox expression reached its peak 4 h after stimulation with either Vibrio penaeicida or poly(I:C) and decreased to its normal level after 12 h.This study is the first to identify and clone a Nox family member (MjNox) from a crustacean species.

A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp.

BACKGROUND: From 2001-2003 monodon slow growth syndrome (MSGS) caused severe economic losses for Thai shrimp farmers who cultivated the native, giant tiger shrimp, and this led them to adopt exotic stocks of the domesticated whiteleg shrimp as the species of cultivation choice, despite the higher value of giant tiger shrimp. In 2008, newly discovered Laem-Singh virus (LSNV) was proposed as a necessary but insufficient cause of MSGS, and this stimulated the search for the additional component cause(s) of MSGS in the hope that discovery would lead to preventative measures that could revive cultivation of the higher value native shrimp species. RESULTS: Using a universal shotgun cloning protocol, a novel RNA, integrase-containing element (ICE) was found in giant tiger shrimp from MSGS ponds (GenBank accession number FJ498866). In situ hybridization probes and RT-PCR tests revealed that ICE and Laem-Singh virus (LSNV) occurred together in lymphoid organs (LO) of shrimp from MSGS ponds but not in shrimp from normal ponds. Tissue homogenates of shrimp from MSGS ponds yielded a fraction that gave positive RT-PCR reactions for both ICE and LSNV and showed viral-like particles by transmission electron microscopy (TEM). Bioassays of this fraction with juvenile giant tiger shrimp resulted in retarded growth with gross signs of MSGS, and in situ hybridization assays revealed ICE and LSNV together in LO, eyes and gills. Viral-like particles similar to those seen in tissue extracts from natural infections were also seen by TEM. CONCLUSIONS: ICE and LSNV were found together only in shrimp from MSGS ponds and only in shrimp showing gross signs of MSGS after injection with a preparation containing ICE and LSNV. ICE was never found in the absence of LSNV although LSNV was sometimes found in normal shrimp in the absence of ICE. The results suggest that ICE and LSNV may act together as component causes of MSGS, but this cannot be proven conclusively without single and combined bioassays using purified preparations of both ICE and LSNV. Despite this ambiguity, it is recommended in the interim that ICE be added to the agents such as LSNV already listed for exclusion from domesticated stocks of the black tiger shrimp.

Early occurrence of apoptosis in lymphoid tissues from chickens infected with strains of Newcastle disease virus of varying virulence.

Newcastle disease virus (NDV), the causative agent of Newcastle disease, is a prevalent problem in the poultry industry and often the cause of severe economic loss. There are many strains of the virus and these have varying virulence. The most virulent strains cause systemic lesions of lymphoid tissues, with necrosis and severe lymphoid depletion. Less virulent strains do not cause as much necrosis, but may predispose to secondary infection with other pathogens. Apoptosis or programmed cell death, has been demonstrated to play a role in the pathogenesis of other paramyxovirus infections, notably those caused by measles and canine distemper viruses. To investigate the role of apoptosis in lymphoid organs during NDV infection, immunohistochemistry for determination of expression of caspase-3, a marker of imminent apoptosis, was performed on formalin-fixed paraffin wax-embedded tissues (spleen, thymus, caecal tonsils and bursa of Fabricius) from 4-week-old chickens infected with NDV strains of varying virulence 2 days previously. The amount of apoptosis was proportional to the severity of the clinical disease elicited by the strains.

A double WAP domain-containing protein PmDWD from the black tiger shrimp Penaeus monodon is involved in the controlling of proteinase activities in lymphoid organ.

A homolog of mammalian secretory leucocyte proteinase inhibitor or SLPI known as a double WAP domain (DWD) protein has been found in penaeid shrimp and believed to play an important role in innate immune system of the shrimp. The PmDWD identified from the Penaeus monodon EST database was investigated for its expression under pathogen infection. Infections by Vibrio harveyi and white spot syndrome virus (WSSV) up-regulated the expression of the PmDWD, which was peaked at about 24 h post infection and, then, subsided to more or less normal level. The PmDWD was expressed in various tissues of normal, 24-h WSSV-injected and leg-amputated shrimp, predominantly in the hemocytes. The expression was dramatically increased in lymphoid organ upon WSSV infection and leg amputation. The recombinant PmDWD (rPmDWD) was not active against the commercial proteinases: trypsin, chymotrypsin, elastase and subtilisin while its mutant rPmDWD_F70R was active against the subtilisin. By using agar diffusion assay, the rPmDWD inhibited the crude proteinases from lymphoid organs of leg-amputated and WSSV-infected shrimp. It inhibited the crude proteinases from Bacillus subtilis as well. Unlike the mammalian SLPIs, the rPmDWD had no antimicrobial activity against various bacteria.

Inhibition of the IL-2-inducible tyrosine kinase (Itk) activity: a new concept for the therapy of inflammatory skin diseases.

T-cell-mediated processes play an essential role in the pathogenesis of several inflammatory skin diseases such as atopic dermatitis, allergic contact dermatitis and psoriasis. The aim of this study was to investigate the role of the IL-2-inducible tyrosine kinase (Itk), an enzyme acting downstream of the T-cell receptor (TCR), in T-cell-dependent skin inflammation using three approaches. Itk knockout mice display significantly reduced inflammatory symptoms in mouse models of acute and subacute contact hypersensitivity (CHS) reactions. Systemic administration of a novel small molecule Itk inhibitor, Compound 44, created by chemical optimization of an initial high-throughput screening hit, inhibited Itk's activity with an IC50 in the nanomolar range. Compound 44 substantially reduced proinflammatory immune responses in vitro and in vivo after systemic administration in two acute CHS models. In addition, our data reveal that human Itk, comparable to its murine homologue, is expressed mainly in T cells and is increased in lesional skin from patients with atopic dermatitis and allergic contact dermatitis. Finally, silencing of Itk by RNA interference in primary human T cells efficiently blocks TCR-induced lymphokine secretion. In conclusion, Itk represents an interesting new target for the therapy of T-cell-mediated inflammatory skin diseases.

Roles of GlcNAc-6-O-sulfotransferases in lymphoid and nonlymphoid tissues.

Recent studies using sulfotransferase-deficient mice have revealed various physiological functions of sulfated glycans. Studies using gene-targeted mice deficient in both N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 showed that these sulfotransferases play critical roles in lymphocyte homing. Recent studies indicated that GlcNAc6ST-2 is expressed not only in lymph node high endothelial venules but also in the colonic epithelial cells in mice, and that this sulfotransferase plays a critical role in GlcNAc-6-O-sulfation of the colonic-mucins, as revealed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry of the colonic-mucin O-glycans from wild-type (WT) and GlcNAc6ST-2-deficient mice. After induction of colitis by dextran sulfate sodium, significantly more leukocyte infiltration was observed in the colon of GlcNAc6ST-2-deficient mice than in that of WT mice. These studies demonstrate that GlcNAc-6-O-sulfotransferases play important roles not only in lymphoid tissues but also in nonlymphoid tissues. This chapter describes experimental procedures for assessing the functions of GlcNAc-6-O-sulfotransferases using gene-targeted mice.

Impaired lymphoid organ development in mice lacking the heparan sulfate modifying enzyme glucuronyl C5-epimerase.

The development of lymphoid organs depends on cross talk between hematopoietic cells and mesenchymal stromal cells and on vascularization of the lymphoid primordia. These processes are orchestrated by cytokines, chemokines, and angiogenic factors that require tight spatiotemporal regulation. Heparan sulfate (HS) proteoglycans are molecules designed to specifically bind and regulate the bioactivity of soluble protein ligands. Their binding capacity and specificity are controlled by modification of the HS side chain by HS-modifying enzymes. Although HS proteoglycans have been implicated in the morphogenesis of several organ systems, their role in controlling lymphoid organ development has thus far remained unexplored. In this study, we report that modification of HS by the HS-modifying enzyme glucuronyl C5-epimerase (Glce), which controls HS chain flexibility, is required for proper lymphoid organ development. Glce(-/-) mice show a strongly reduced size of the fetal spleen as well as a spectrum of defects in thymus and lymph node development, ranging from dislocation to complete absence of the organ anlage. Once established, however, the Glce(-/-) primordia recruited lymphocytes and developed normal architectural features. Furthermore, Glce(-/-) lymph node anlagen transplanted into wild-type recipient mice allowed undisturbed lymphocyte maturation. Our results indicate that modification of HS by Glce is required for controlling the activity of molecules that are instructive for early lymphoid tissue morphogenesis but may be dispensable at later developmental stages and for lymphocyte maturation and differentiation.

Redistribution of sphingosine 1-phosphate by sphingosine kinase 2 contributes to lymphopenia.

Sphingosine kinases (SKs) 1 and 2 produce high concentrations of sphingosine 1-phosphate (S1P) in blood and lymph. In contrast, S1P concentrations in lymphoid tissues are kept low by the S1P-degrading activity of the S1P-lyase. These differences in S1P concentrations drive lymphocyte circulation. Inhibition of the S1P-lyase prevents lymphocyte egress and causes lymphopenia because of increased S1P levels in lymphoid tissues. In this study, we investigated the source of this accumulating S1P in lymphoid tissues by using SK2-deficient (SK2(-/-)) mice. In contrast to wild-type mice, SK2(-/-) mice exhibited attenuated lymphopenia after S1P-lyase inhibition by 4-deoxypyridoxine (DOP). Consistently, S1P concentrations were only modestly increased in lymphoid tissues of SK2(-/-) mice compared with a significantly higher increase in wild-type mice after DOP treatment. Low S1P concentrations in lymphoid tissues of DOP-treated SK2(-/-) mice were accompanied by higher S1P concentrations in blood, suggesting that SK2(-/-) mice display defective S1P transport from blood into lymphoid tissues. To investigate this potential new role of SK2, RBCs loaded with traceable C17-S1P were transfused into wild-type and SK2(-/-) mice, resulting in much higher C17-S1P concentrations in blood of SK2(-/-) mice compared with wild-type mice 2 h after transfusion. Moreover, cocultures of RBCs with mouse splenocytes and endothelial cells demonstrated that SK2 regulated cellular uptake of S1P from RBCs. Collectively, our data suggest that S1P in lymphoid tissues derives from blood and point to an essential role of SK2 in S1P transport.