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Innate pattern recognition receptors (PRRs) expressed on dendritic cells (DC) link direct recognition of pathogens to initiation of T cell responses. Here I describe evidence that Dectin-1 is a novel pattern recognition receptor involved the activation of dendritic cells by the yeast cell wall preparation, zymosan. Zymosan contains ligands for the known PRR Toll-like Receptor-2 (TLR2). Interestingly, recent work in macrophages has implicated the p-glucan receptor Dectin-1 in zymosan recognition. This thesis demonstrates that Dectin-1 can function as a PRR independently of the Toll-like Receptor system to induce DC cytokine production (IL-2, IL-10) and Notch-ligand upregulation (Jagged-1). My work has helped determine that Dectin-1 can signal via a novel HemlTAM motif to the tyrosine kinase Syk. DC stimulated with zymosan upregulate IL-10, IL-2 and Jagged-1 in a Syk-dependent manner. Indeed, IL-10 and Jagged-1 induction is independent of TLR-mediated recognition of zymosan. In addition, zymosan induced ERK activation is entirely dependent on signalling through Syk and is independent of TLR signalling. I demonstrate that this ERK activation is necessary for the induction of IL-2 and IL-10 in response to zymosan. Finally I present preliminary findings on how the unusual cytokine signature of zymosan-stimulated DCs may bias Thl and Thl7 differentiation induced in vitro.

Stimulation of macrophages with LPS alone or in combination with IFN-y is termed 'innate' and 'classical' activation, respectively. This is associated with the expression of nos2 and p40 (IL-12/IL-23). Stimulation of macrophages with IL-4 or IL-13 is termed 'alternative' activation and is associated with expression of argI, dectin-1, mrc-1 ,fizz1 and ym1. Recent studies demonstrate that arg1 is expressed in some innate activated macrophages.

Here presented is an account of the polarised inflammatory responses of macrophages in the absence of SOCS2 and SOCS3, which are fixed and dominant compared to wild type. In this setting, SOCS2-1- macrophages upregulate Ml genes and cytokines in response to pro- and anti-inflammatory stimuli, whilst socs3LysMcre macrophages up-regulate only M2 associated responses. Our investigations show that macrophages are unable to polarise towards an Ml phenotype in the absence of SOCS3, whilst the absence of SOCS2 enhances Ml polarisation at the expense of M2. We show these antipodal polarised responses correlates to altered cell recruitment of T-regulatory cells through distorted chemokine production. We note marked alterations in both STAT signalling and promoter binding, and NF1C~ signalling and translocation. Interestingly, the absence of SOCS2 or SOCS3 has a profound effect on Siglec-E expression on macrophages. Siglec-E is undetectable in the absence of SOCS2, whilst it is up regulated in the absence of SOCS3. As an inhibitory receptor, the increased presence or absence of Siglec-E correlated well to alterations in LPS-induced cytokine responses. This thesis indicates that a lack of Siglec-E is marked by clearly exaggerated and elevated pro-inflammatory responses. This is seen both, in the absence of SOCS2, and by shRNA knockdown in vitro. Also, we show that elevated levels of Siglec-E, as present in socs3LysMcre macrophages, or an increase in Siglec-E activation leads to a decreased pro- inflammatory responses. In order to exploit this latter point, Siglec ligand (sialic acid)-conjugated poly-lactic (eo-glycolic) acid (PLGA) nanoparticles were employed in a number of models, all of which resulted in attenuated inflammation. Preliminary investigation of Human macrophages shows similar trends on treatment with sialic acid-conjugated nanoparticles, leading to attenuation of LPS- induced endotoxin shock responses, suggesting that these nanoparticles could have wide and far- reaching applications in the treatment of inflammatory disease.

The initial aim of this study was to examine the nature of the interaction between adenovirus and transfected macrophage, and to characterise the molecular mechanisms underlying macrophage response to adenoviral infection. Results showed that adenoviral transfection activated macrophages, promoting production of pro-inflammatory mediators and priming an enhanced response to other inflammatory stimuli.  Activation was dependent on the nuclear factor kappa B (NF<span style='font-family:Symbol'>kB) signalling pathway, which was activated within two hours of transfection, and could be prevented using an inhibitory adenovirus which blocked NF<span style='font-family:Symbol'>kB signalling.  The ultimate phenotype of the transfected macrophage was determined both by non-specific viral activation and by the properties of the transgene expressed. The second aim of this research was to investigate the effect of the local cytokine milieu on transgene expression in vitro.  Results showed that transgene expression under the control of two different promoter constructs was subject to regulation by pro-inflammatory mediators, by mechanisms at least partly dependent on the NF<span style='font-family:Symbol'>kB signalling pathway.  the results have important implications for the use of these promoters in gene therapy applications where the gene of interest is delivered into an inflammatory environment. The final stage of the project focused on the use of transfected macrophages in vivo, in a rat model of glomerulonephritis.  Results showed that transfected macrophages expressing the anti-inflammatory cytokine IL-4 localised with enhanced efficiency to inflamed glomeruli after intra-renal artery injection of the mechanisms for this were examined.  Better understanding of the mechanisms which promote localisation may ultimately permit the design of genetically-modified macrophages which selectively target sites of injury for delivery of anti-inflammatory cytokines.

Pathology is often linked to inappropriate levels of immunogenicity or immunosuppression that occur within certain settings.  The ability to augment or restrict macrophage suppressive function therefore offers the potential for therapeutic intervention.  A detailed knowledge of the repertoire of macrophage suppressive mechanisms would be a pre-requisite for such manipulation.  This study was developed to provide an <i>in vitro</i> macrophage/T cell co-culture model that could provide insights into suppressive events <i>in vivo.  </i>Previous <i>in vitro</i> studies of macrophage anti-proliferative mechanisms have reported a complete dependence on nitric oxide production.  This finding offers little scope for correlation with the majority of in vivo situations, where there is evidence that suppressive relationships are less straightforward.  In contrast, the work described here showed that the overall picture is much more complex.  Over a range of different types of experiment the suppressive role of NO was highly variable, and could even at times be irrelevant, as demonstrated by the use of iNOS^-/-macrophages.  PGE₂ was shown to exert strain-dependent suppressive effects, but TGF-β did not appear to be involved.  Additional suppressive mediators were clearly implicated but remained unidentified. To summarise, the results of this work provide evidence of the multiplicity, pleiotropy, redundancy and strain-dependence that may relate to macrophage mechanisms of T cell suppression.  Inconsideration of investigations that are both described here and also indicated for future work, this model has previously undescribed potential to link <i>in </i>vitro findings with the highly complex interactions likely to be encountered <i>in vivo.</i>

Inflammatory diseases such as autoimmune and atopic diseases are a common problem worldwide. Although there have been substantial advances in medical therapies, current treatments are not able to cure these conditions. In order to develop more specific, individually targeted and efficient treatments, a better understanding of the cells, mediators and mechanisms that lead to pathology are necessary. Macrophages and T cells are major players in the human immune system. Despite the abundance of macrophages at inflammatory sites and their secretion of T cell polarising cytokines, their roles as antigen presenting cells has been overlooked until now. This PhD project aimed to determine, firstly, the contribution of differentially activated human macrophages in the regulation of adaptive immune responses in inflammation with the main focus on the pro‐inflammatory T cell subset Th17, secondly, how macrophage functions could be modified to alter T cell polarisation and thirdly, how novel alternative mechanisms using electric fields can alter T cell responses. I demonstrate for the first time that macrophages can efficiently induce T cell polarisation and Th17 differentiation in response to recall and primary antigens and that the specific macrophage activation state is essential to drive Th17 responses. This suggests that macrophages are an important stimulus contributing to pathogenic T cell responses in human inflammatory diseases. Importantly, both memory and naïve T cells gave rise to Th17 cells following culture with antigen‐loaded activated macrophages, where non‐specific effects of mitogenic activation were avoided. Targeting human macrophage signalling pathways through SOCS3, reduced their pro‐inflammatory potential and Th17 polarising ability, pointing to SOCS3 as an effective therapeutic target. As an alternative approach, I demonstrate here that small electric fields of physiological strength strongly influence immune responses and significantly dampen Th17 differentiation. This suggests that EFs have the potential to facilitate healing processes or support conventional therapies for inflammatory diseases. Overall, these data present a strong basis for the development novel treatment possibilities for inflammatory diseases that are distinct from the currently used conventional therapies.