Julie McIntosh

Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells

BackgroundNonstructural glycoprotein 4 (NSP4) encoded by rotavirus is the only viral protein currently believed to function as an enterotoxin. NSP4 is synthesized as an intracellular transmembrane glycoprotein and as such is essential for virus assembly. Infection of polarized Caco-2 cells with rotavirus also results in the secretion of glycosylated NSP4 apparently in a soluble form despite retention of its transmembrane domain. We have examined the structure, solubility and cell-binding properties of this secreted form of NSP4 to further understand the biochemical basis for its enterotoxic function. We show here that NSP4 is secreted as discrete detergent-sensitive oligomers in a complex with phospholipids and demonstrate that this secreted form of NSP4 can bind to glycosaminoglycans present on the surface of a range of different cell types.MethodsNSP4 was purified from the medium of infected cells after ultracentrifugation and ultrafiltration by successive lectin-affinity and ion exchange chromatography. Oligomerisation of NSP4 was examined by density gradient centrifugation and chemical crosslinking and the lipid content was assessed by analytical thin layer chromatography and flame ionization detection. Binding of NSP4 to various cell lines was measured using a flow cytometric-based assay.ResultsSecreted NSP4 formed oligomers that contained phospholipid but dissociated to a dimeric species in the presence of non-ionic detergent. The purified glycoprotein binds to the surface of various non-infected cells of distinct lineage. Binding of NSP4 to HT-29, a cell line of intestinal origin, is saturable and independent of divalent cations. Complementary biochemical approaches reveal that NSP4 binds to sulfated glycosaminoglycans on the plasma membrane.ConclusionOur study is the first to analyze an authentic (i.e. non-recombinant) form of NSP4 that is secreted from virus-infected cells. Despite retention of the transmembrane domain, secreted NSP4 remains soluble in an aqueous environment as an oligomeric lipoprotein that can bind to various cell types via an interaction with glycosaminoglycans. This broad cellular tropism exhibited by NSP4 may have implications for the pathophysiology of rotavirus disease.

Mapping the Distinctive Populations of Lymphatic Endothelial Cells in Different Zones of Human Lymph Nodes

The lymphatic sinuses in human lymph nodes (LNs) are crucial to LN function yet their structure remains poorly defined. Much of our current knowledge of lymphatic sinuses derives from rodent models, however human LNs differ substantially in their sinus structure, most notably due to the presence of trabeculae and trabecular lymphatic sinuses that rodent LNs lack. Lymphatic sinuses are bounded and traversed by lymphatic endothelial cells (LECs). A better understanding of LECs in human LNs is likely to improve our understanding of the regulation of cell trafficking within LNs, now an important therapeutic target, as well as disease processes that involve lymphatic sinuses. We therefore sought to map all the LECs within human LNs using multicolor immunofluorescence microscopy to visualize the distribution of a range of putative markers. PROX1 was the only marker that uniquely identified the LECs lining and traversing all the sinuses in human LNs. In contrast, LYVE1 and STAB2 were only expressed by LECs in the paracortical and medullary sinuses in the vast majority of LNs studied, whilst the subcapsular and trabecular sinuses lacked these molecules. These data highlight the existence of at least two distinctive populations of LECs within human LNs. Of the other LEC markers, we confirmed VEGFR3 was not specific for LECs, and CD144 and CD31 stained both LECs and blood vascular endothelial cells (BECs); in contrast, CD59 and CD105 stained BECs but not LECs. We also showed that antigen-presenting cells (APCs) in the sinuses could be clearly distinguished from LECs by their expression of CD169, and their lack of expression of PROX1 and STAB2, or endothelial markers such as CD144. However, both LECs and sinus APCs were stained with DCN46, an antibody commonly used to detect CD209.

Sphingosine‐1‐phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen‐presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141high podoplanin+, CD90+, ICAM1+, and VCAM1+ but lack endothelial and hematopoietic cell markers, or alpha‐smooth muscle actin. We then examined expression of the enzyme sphingosine‐1‐phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68+ APCs. CD68+ APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

An engineered non-toxic superantigen increases cross presentation of hepatitis B virus nucleocapsids by human dendritic cells.

Virus like particles (VLPs) are potent immunogens capable of priming strong protective antibody responses due to their repetitive structural arrangement and affinity for specific B cell receptors. By contrast, T cell responses to VLPs can be weak due to inefficient uptake and processing by antigen presenting cells. We report here a novel strategy for increasing the T cell reactivity of a VLP, the nucleocapsid of hepatitis B virus, through covalent coupling of M1, an engineered form of the Streptococcal superantigen SMEZ2, that binds MHC II with high affinity but lacks its T cell mitogenic capability. M1:HBcAg conjugates bound to dendritic cells and were efficiently endocytosed into late endosomes. Human dendritic cells pulsed with M1:HBcAgs stimulated HBV-specific CD8+ T cells more effectively than cells pulsed with native capsids indicating that the modified VLP was more effectively cross presented by APCs. Coupling of M1 was also able to induce significantly greater reactivity of human CD4+ T cells specific for a common T-helper epitope. These studies indicate the potential of recombinant superantigens to act as flexible molecular adjuvants that can be incorporated into various subunit vaccine platforms leading to enhanced T cell reactivity in humans.

Augmentation with an ovine forestomach matrix scaffold improves histological outcomes of rotator cuff repair in a rat model.

BackgroundRotator cuff tears can cause significant pain and functional impairment. Without surgical repair, the rotator cuff has little healing potential, and following surgical repair, they are highly prone to re-rupture. Augmenting such repairs with a biomaterial scaffold has been suggested as a potential solution. Extracellular matrix (ECM)-based scaffolds are the most commonly used rotator cuff augments, although to date, reports on their success are variable. Here, we utilize pre-clinical in vitro and in vivo assays to assess the efficacy of a novel biomaterial scaffold, ovine forestomach extracellular matrix (OFM), in augmenting rotator cuff repair.MethodsOFM was assessed in vitro for primary tenocyte growth and adherence, and for immunogenicity using an assay of primary human dendritic cell activation. In vivo, using a murine model, supraspinatus tendon repairs were carried out in 34 animals. Augmentation with OFM was compared to sham surgery and unaugmented control. At 6- and 12-week time points, the repairs were analysed biomechanically for strength of repair and histologically for quality of healing.ResultsOFM supported tenocyte growth in vitro and did not cause an immunogenic response. Augmentation with OFM improved the quality of healing of the repaired tendon, with no evidence of excessive inflammatory response. However, there was no biomechanical advantage of augmentation.ConclusionsThe ideal rotator cuff tendon augment has not yet been identified or clinically implemented. ECM scaffolds offer a promising solution to a difficult clinical problem. Here, we have shown improved histological healing with OFM augmentation. Identifying materials that offset the poorer mechanical properties of the rotator cuff post-injury/repair and enhance organised tendon healing will be paramount to incorporating augmentation into surgical treatment of the rotator cuff.

ECIS technology reveals that monocytes isolated by CD14+ve selection mediate greater loss of BBB integrity than untouched monocytes, which occurs to a greater extent with IL-1β activated endothelium in comparison to TNFα

Background We have previously shown that TNFα and IL-1β differentially regulate the inflammatory phenotype of human brain endothelial cells (hCMVECs). In this regard, IL-1β treatment was considerably more potent than TNFα at increasing expression of inflammatory chemokines and leukocyte adhesion molecules. We therefore hypothesised that interaction of the hCMVECs with human monocytes would also be dependent on the activation status of the endothelium. Therefore, the primary aim of this study was to assess whether brain endothelial cells activated by IL-1β or TNFα differed in their interaction with monocytes. Methods Monocyte interaction was measured using the real time, label-free impedance based ECIS technology, to evaluate endothelial barrier integrity during monocyte attachment and transendothelial migration. Results ECIS technology revealed that there was a greater loss of barrier integrity with IL-1β activation and this loss lasted for longer. This was expected and consistent with our hypothesis. However, more striking and concerning was the observation that the method of monocyte enrichment greatly influenced the extent of endothelial barrier compromise. Importantly, we observed that positively isolated monocytes (CD14+ve) caused greater reduction in barrier resistance, than the negatively selected monocytes (untouched). Analysis of the isolated monocyte populations revealed that the CD14+ve isolation consistently yields highly pure monocytes (>92%), whereas the untouched isolation was much more variable, yielding ~70% enrichment on average. These two enrichment methods were compared as it was thought that the presence of non-classical CD16hi monocytes in the untouched enrichment may mediate greater compromise than the classical CD14hi monocytes. This however, was not the case and these observations raise a number of important considerations pertaining to the enrichment strategy, which are essential for generating reliable and consistent data. Conclusions We conclude that IL-1β and TNFα differentially influence monocyte interaction with brain endothelial cells and moreover, the enrichment method also influences the monocyte response as revealed using ECIS technology.

Individual and cocktails of TLR ligands influence APCs in situ in human skin explants

CD4+Foxp3+ regulatory T cells (Tregs) are the main regulators of peripheral tolerance and prevent the development of fatal autoimmune disease in humans and mice. Furthermore, Tregs have also been implicated in suppressing anti-tumour immune responses and are often enriched at sites of primary and metastatic tumours. While studies have shown the effect of Treg ablation on the control of primary tumours, few studies have examined their contribution to metastasis progression. In this thesis I hypothesised that the depletion of Tregs could promote control over metastasis. To address this, a highly metastatic murine mammary carcinoma cell line 4T1 was injected into transgenic mice expressing the diphtheria toxin receptor in Foxp3+ cells. Foxp3+ cells were depleted by administration of diphtheria toxin and the impact of this on growth of primary tumours and metastases was assessed and measured in vitro clonogenic assays. Results of these experiments indicated that Tregdepletion led to control of primary tumour growth and in some mice to control of metastases. Control of metastases was linked to control of primary tumour growth. In order to measure metastasis in vivo, a PET/CT imaging technique was optimized. Primary tumours and large metastatic nodules were successfully imaged in mice using F18 FDG as a radiotracer. However, the studies described herein revealed that micrometastases in mouse lungs were too small to be reliably identified using PET data parameters. CT imaging did however enable detection of increases in tissue density within the lungs, which was suggestive of micrometastases. Data obtained in this way also indicated that Treg-depletion promotes control of metastasis in some mice. Collectively, the findings described in this thesis indicate that Tregdepletion can contribute to control of metastatic disease and should therefore represent an important component of novel immunotherapies.Changes in microbiome, mucosal immunity and intestinal integrity have been associated with the onset of Type 1 Diabetes (T1D) in children. Toll-like Receptors (TLR) have been associated all three factors. The role of TLR and their effects on microbiome in autoimmunity were studied by crossing TLR1,2,4,6,9 and MyD88 targeted deficiency mutations to the type 1 diabetes (T1D)-prone NOD mouse strain. While NOD.Tlr9-/- and NOD.Tlr6-/- mice were unaffected, T1D in NOD.Tlr4-/- and NOD.Tlr1-/- mice was exacerbated and that in NOD.Myd88-/- and NOD.Tlr2-/- mice ameliorated. Physical parameters of the intestines were compared; ileal weight was reduced in NOD.Tlr1-/-mice. Similarly, by histology, these mice had reduced villus length and width. The intestinal microbiomes of NOD wild-type (WT), NOD.Tlr1-/-, NOD.Tlr2-/- and NOD.Tlr4-/- mice were compared by high throughput sequencing of 16S ribosomal DNA (rDNA), in two cohorts, 18 months apart. Analysis of caecal 16S sequences clearly resolved the mouse lines and there were significant differences in beta diversity between the strains, with individual bacterial species contributing greatly to the differences in the microbiota of individual TLR-deficient strains. To test the relationship between microbiome and T1D, all strains were re-derived onto the parental NOD/Lt line and the incidence of T1D re-assessed within two generations. All rederived lines expressed an incidence of disease similar to that of the parental line. TLR deficiencies are associated with changes in microbiome; changes of microbiome are associated with T1D; the effects of TLR deficiencies on T1D appear to be mediated by their effects on gut flora.Intestinal TCRb+CD4-CD8b-CD8a+ (CD8aa) IELs alleviate T cell induced colitis and have been suggested to play a role in virus infection and cancer. Their thymic development has been elucidated to some extent, as IEL precursors (IELp) are known to be CD4-CD8-CD5+TCRb+, but is not yet fully understood. Within the thymus, mature IELp were identified based on their expression of CD122 and MHC class I. Two major phenotypic subsets exist within this mature thymic IELp population: a PD1+Tbet- population that preferentially expresses a4b7, and a PD1-Tbet+ population with preferential CD103 expression. These two populations were also distinct in their Valpha repertoire. The PD1+a4b7+ population contains clones that are strongly self-reactive as judged by Nur77GFP and their dramatic increase in Bim deficient mice, while the PD1-Tbet+ population did not show these characteristics. Both gave rise to CD8aa IELs upon adoptive transfer into RAG-/- recipients. However intrathymic labeling revealed that PD1+a4b7+ IELp were the major thymic emigrating population, and emigration was S1P1-dependent. In contrast, PD1-Tbet+ IELp expressed CXCR3, were retained, and accumulated in the thymus with age. Preliminary immunofluorescence data furthermore indicate differential thymic cortico-medullary localization of the IELp subtypes. These experiments more precisely define the behavior of IEL precursors.