An investigation into the regulatory mechanisms of neutrophil migration into lymphatic vessels in vivo

PhDNeutrophils are recognised to play a pivotal role at the interface between the innate and adaptive immune responses following their rapid recruitment to inflamed tissues and lymphoid organs. Whilst neutrophil trafficking through blood vessels has been extensively studied, the molecular mechanisms regulating their migration into the lymphatic system are still poorly understood. This thesis therefore aimed to investigate the mechanisms involved in neutrophil migration across the lymphatic endothelium during TNF- or Complete Freund’s Adjuvant + antigen (CFA+Ag)-induced inflammation of cremaster muscles in vivo. This work revealed that TNF- or CFA+Ag-stimulation induces a rapid but transient entry of tissue-infiltrated neutrophils into lymphatic vessels, a response associated with the regulation and redistribution of the lymphatic endothelial cell glycocalyx. Interestingly, antigen sensitisation resulted in the production of endogenous TNF within cremaster muscles. Using anti-TNF blocking antibodies and mice deficient in both TNF receptors (p55 and p75), endogenous TNF was demonstrated for the first time to be involved in priming and triggering the migration of neutrophils into tissue-associated lymphatic vessels upon antigen challenge. Additionally, the use of chimeric mice exhibiting neutrophils deficient in both TNFRs demonstrated that TNF directly acts on leukocytes to induce neutrophil migration into lymphatic vessels. Furthermore, the results show that TNF-induced migration of neutrophils into the lymphatic system occurs in a strictly CCR7-dependent manner; blocking CXCR4 or CXCL1 signalling does not affect this response. Finally, both TNF- or CFA+AG-stimulation induced ICAM-1 up-regulation on lymphatic vessels, allowing neutrophils to crawl along the lumen; a response that was demonstrated to be TNF-dependent. These results have provided new insights into the mechanisms that mediate neutrophil migration into lymphatic vessels and their subsequent crawling within these vessels during inflammation. In particular, a new role for TNF as a key regulator of these processes has been demonstrated. Taken together, this work has highlighted potential and effective targets to manipulate the role of neutrophils in adaptive immune responses in vivo.Institute of Bioengineering at Queen Mary University of London (QMUL) and Arthritis Research UK (ARUK

Mucosal Application of gp140 Encoding DNA Polyplexes to Different Tissues Results in Altered Immunological Outcomes in Mice

Increasing evidence suggests that mucosally targeted vaccines will enhance local humoral and cellular responses whilst still eliciting systemic immunity. We therefore investigated the capacity of nasal, sublingual or vaginal delivery of DNA-PEI polyplexes to prime immune responses prior to mucosal protein boost vaccination. Using a plasmid expressing the model antigen HIV CN54gp140 we show that each of these mucosal surfaces were permissive for DNA priming and production of antigen-specific antibody responses. The elicitation of systemic immune responses using nasally delivered polyplexed DNA followed by recombinant protein boost vaccination was equivalent to a systemic prime-boost regimen, but the mucosally applied modality had the advantage in that significant levels of antigen-specific IgA were detected in vaginal mucosal secretions. Moreover, mucosal vaccination elicited both local and systemic antigen-specific IgG(+) and IgA(+) antibody secreting cells. Finally, using an Influenza challenge model we found that a nasal or sublingual, but not vaginal, DNA prime/protein boost regimen protected against infectious challenge. These data demonstrate that mucosally applied plasmid DNA complexed to PEI followed by a mucosal protein boost generates sufficient antigen-specific humoral antibody production to protect from mucosal viral challenge

Pathological Angiogenesis Requires Syndecan-4 for Efficient VEGFA-Induced VE-Cadherin Internalization

Objective: VEGFA (Vascular endothelial growth factor A) and its receptor VEGFR2 (vascular endothelial growth factor receptor 2) drive angiogenesis in several pathologies, including diabetic retinopathy, wet age-related macular degeneration, and cancer. Studies suggest roles for HSPGs (heparan sulfate proteoglycans) in this process, although the nature of this involvement remains elusive. Here, we set to establish the role of the HSPG SDC4 (syndecan-4) in pathological angiogenesis. Approach and Results: We report that angiogenesis is impaired in mice null for SDC4 in models of neovascular eye disease and tumor development. Our work demonstrates that SDC4 is the only SDC whose gene expression is upregulated during pathological angiogenesis and is selectively enriched on immature vessels in retinas from diabetic retinopathy patients. Combining in vivo and tissue culture models, we identified SDC4 as a downstream mediator of functional angiogenic responses to VEGFA. We found that SDC4 resides at endothelial cell junctions, interacts with vascular endothelial cadherin, and is required for its internalization in response to VEGFA. Finally, we show that pathological angiogenic responses are inhibited in a model of wet age-related macular degeneration by targeting SDC4. Conclusions: We show that SDC4 is a downstream mediator of VEGFA-induced vascular endothelial cadherin internalization during pathological angiogenesis and a potential target for antiangiogenic therapies.acceptedVersionPeer reviewe

Nasal and sublingual vaccination results in protection against Influenza challenge infection.

<p>Serum-antigen-specific IgG antibody responses (n = 6 per group) were recorded one week after 3 DNA primes and a single HA boost (<b>a</b>, <b>b</b> and <b>c</b>). Specifically, a 20 µg/30 µl volume of plasmid DNA was administered three times, every two weeks, before a single 10 µg/30 µl HA protein boost. Serum anti-HA antibody concentrations are expressed as group means (ng/ml+SEM). One week after the final vaccination, mice were infected with 5HA units of Influenza X-31 in 100 µl (<b>d</b>). Body weights for DNA prime protein boost vaccinations (−) and single protein vaccination (−) groups are expressed as a mean percentage of total weight relative to day 0. Mean IFN-γ T cell responses were assessed on day 4 post infection using a commercial ELISPOT assay coated with HA protein (<b>e</b>). Results represent the means of (n = 6) IFN-γ SFU/million splenocytes on day 4 post-infection. Statistical significance was assessed using Mann Whitney U test with **<i>p</i><0.005 and *<i>p</i><0.05. Data are representative of two independent experiments.</p

A triple DNA prime, triple protein boost regimen results in antigen-specific IgG and IgA in serum samples.

<p>Serum antigen-specific IgG and IgA antibody responses (n = 8 mice per group) were assessed by ELISA one week after each immunisation until study week 13. Mice were immunised every two weeks, with either three 20 µg CN54-gp140 polyplex prime vaccinations in a final 15 µl (IN, I.Vag and SL) volume, followed by three 20 µg gp140 protein boost vaccinations, or with just three 20 µg gp140 protein vaccinations. Immunisations are indicated by vertical shaded lines. Lines represent either protein immunisation (<b>^…</b>) or DNA prime/boost vaccination groups (<b>−</b>). Antibody results are expressed as geometric group means (ug/ml+SEM). Statistical significance was assessed using Mann Whitney U test with ***<i>p</i><0.0005; **<i>p</i><0.005; *<i>p</i><0.05. Data are representative of two independent experiments.</p

Prime/boost vaccination route impacts vaginal mucosal antibody levels and antigen reactive B cells.

<p>Mucosal antigen-specific IgG (<b>a</b>) and IgA (<b>b</b>) titres from vaginal mucosal lavage were assessed (n = 8 per group) at the end of the study (week 13). Antibody results are expressed as group means (µg/ml+SEM). At the end of the study, antibody secreting IgG (<b>c</b>) and IgA (<b>d</b>) cells were obtained from excised spleens and plated at a density of 5×10⁶ cells/ml in a commercial IgG or IgA ELISPOT plate. Samples were stimulated with 5 µg/ml gp140 antigen for 16 h and developed as per the manufacturer’s instructions. Results represent group means (n = 8 mice per group) of IgG or IgA Spot Forming Units (SFU)/million antigen stimulated cells (+ SEM). Antigen-specific IgA secreting B cells from enzymatically digested and mechanically disrupted vaginal tissue (<b>e</b>) were determined using a direct ELISPOT assay at the end of the study (week 13). Results represent pooled samples and are expressed as fold increase in SFU/million antigen stimulated cells (+ SEM) as compared to negative controls. Statistical significance was assessed using Mann Whitney U test with ***<i>p</i><0.0005, **<i>p</i><0.005 and *<i>p</i><0.05. Data are representative of two independent experiments or are pooled from two experiments.</p

The route of prime/boost vaccination significantly alters serum antigen-specific antibody responses.

<p>Serum antigen-specific IgG (<b>a</b>) and IgA (<b>b</b>) antibody (µg/ml+SEM) responses (n = 8 mice per group) were assessed by ELISA one week after each immunisation until study week 13. The different prime-boost vaccination groups are compared to the IN prime-boost vaccine group for assessment of statistical significance (Wilcoxon non-parametric paired t test). Serum antigen specific IgG1 and IgG2a concentrations (<b>c</b>) were assessed during study week 13. The IgG1 and IgG2a ratios were derived by dividing the IgG1 concentration by the IgG2a concentration of each mouse. Antibody results are expressed as geometric group means (µg/ml+data set variance). Statistical significance was assessed using Mann Whitney U test with **<i>p</i><0.005; *<i>p</i><0.05 and N.A. = not applicable.</p

Pulmonary delivery of DNA vaccine constructs using deacylated PEI elicits immune responses and protects against viral challenge infection

Vaccination through mucosal surfaces has been shown to elicit antiviral immune responses against a number of mucosal pathogens. Here we demonstrate that both mucosal and systemic immune responses can be elicited against a model HIV-1 CN54gp140 antigen when cation-complexed plasmid DNA vaccines are applied topically to the murine pulmonary mucosa as an immune priming strategy. Furthermore, using an influenza challenge model we show that a plasmid DNA vaccine complexed to a less toxic form of PEI called dPEI (a nearly fully hydrolysed linear PEI with 11% additional free protonatable nitrogen atoms) can provide significant protection against a respiratory challenge infection in mice. Furthermore, we show that dPEI polyplexes have the potential to transfect not only mucosal epithelium, but also to enter deeper into tissues through the modulation of tight junction integrity. Taken together, these results demonstrate that less toxic forms of PEI can be effective delivery vehicles for plasmid DNAs to elicit cellular and humoral protective responses in vivo. Moreover, our observations suggest that these less toxic derivatives of PEI could be utilised for topical plasmid DNA vaccine delivery to human mucosal tissue surfaces, and that this application may permit dissemination of the immune responses through the linked mucosal network thus providing protective immunity at distal portals of pathogen entry