The Drosophila innate immune system is one of the most widely characterised of all metozoan defense systems, and shares many similar characteristics to the innate immune systems of higher organisms. As such, Drosophila has become the model organism of choice for many researchers with regards to the study of the general mechanisms and regulatory elements of innate immunity. There are a number of mechanisms that Drosophila employ in order to combat infection, and these include both humoral and cellular responses. However, perhaps the most widely characterised of these mechanisms is the systemic production of anti-micorobial peptides (AMPs) via the activation of two specific immune signalling pathways – Toll and Imd (Lemaitre et al. 1995a; Belvin and Anderson 1996). In Drosophila, a number of recent studies have identified a role for the diffusible second messenger nitric oxide (NO) in the positive regulation of the Imd pathway, a pathway that is fundamental to host defence against Gram-negative bacteria (Lemaitre et al. 1995a; Nappi et al. 2000; Foley and O'Farrell 2003; McGettigan et al. 2005). To date, the exact mechanism by which NO is mediating its effects on the Imd pathway has not yet been determined. However, it can be suggested that this effect is mediated through activation of the cGMP signalling pathway, via interaction with one of its upstream components, soluble guanylate cyclase (sGC), the main intracellular target for NO (Marletta and Spiering 2003). Therefore, the aim of this study was to determine the potential role of the cGMP signalling pathway on regulation of the Drosophila Imd immune pathway. To do this, the Drosophila Malpighian (renal) tubule was used as a model system. The Malpighian tubule is a very well characterised, extensively studied epithelial tissue and for a number of years has comprised the model system of choice with regards to the study of the epithelial roles of signalling and transport genes (Dow and Davies 2001). The suitability of this tissue as a model system for this study is two-fold: Firstly, for many years, the NO/cGMP signalling pathway has been deemed as critical to tubule function (Dow et al. 1994a). Secondly, a recent study has identified the tubule as an important autonomous immune-sensing tissue where, upon immune challenge with Gram-negative bacteria, Imd pathway-associated AMPs are systemically produced in the tubule principle cells. Importantly, it has been demonstrated that activation of the Imd pathway in the principle cells is regulated via the autocrine production of NO (McGettigan et al. 2005). Data obtained from this study has demonstrated a completely novel role for cGMP signalling in the tubule. Expression analysis has revealed that cGMP acts to modulate the expression of Imd pathway-associated AMPs in a dose-dependent manner; whereby low nanomolar concentrations are shown to stimulate diptericin expression and higher micromolar concentrations of cGMP are shown to inhibit it. This effect does not appear to extend to the fat body, the canonical tissue involved in the systemic induction of AMPs, thus suggesting a completely tissue-specific mechanism. Importantly, it is shown here that the cognate cGMP-dependent protein kinases (cGKs), DG1 and DG2 (MacPherson et al. 2004a; 2004b), mediate differential effects on AMP production in the tubule. Targeted modulation of the expression of these kinases to the principle cells of the tubule using the GAL4/UAS system demonstrates that activation of DG1 mediates positive modulation of diptericin expression in the tubule. By contrast, negative modulation of diptericin expression is shown to occur following the activation of the two main isoforms of DG2, DG2P1 and DG2P2. These data therefore describe a completely novel role for each of these kinases. Significantly, the effects of these kinases on diptericin expression in the tubule are sufficient to impact on survival of the whole fly in response to septic infection with Gram-negative bacteria, as well as contribute significantly to bacterial clearance in the gut following natural infection with E.coli. This study has therefore revealed a critical novel role for both the tubule and cGKs in the regulation of defence mechanisms in response to both septic and natural infection in the adult fly. Interestingly, Q-PCR has revealed that DG1 mediates its effects downstream of Imd. Additionally, studies have revealed that both DG1 and DG2 act to regulate the Imd pathway via modulation of Relish activation, the NFκB transcription factor responsible for the induction of AMPs following activation of the Imd pathway (Hedengren et al. 1999). Translocation assays have demonstrated that targeted over-expression of dg1 to the principal cells of the tubule results in enhanced translocation of activated Relish into the nucleus, whereas targeted knock-down of this kinase by RNAi results inhibition of Relish activation. In contrast to DG1, overexpression of either dg2P1 or dg2P2 to the principal cells of the tubule results in inhibition of Relish activation, even in the presence of immune challenge. However, this study has not revealed the exact mechanism by which these kinases mediate their effects on Relish activation, and therefore it is not clear whether DG1 and/or DG2 are acting directly on Relish, or indirectly via phosphorylation of an, as of yet, unidentified substrate(s). Despite this, a completely novel function for each of these kinases is described here for the first time. Importantly, data described in this study also identifies that, with regards to Imd pathway regulation, DG1 and DG2 may be activated via different sources of cGMP within the cell. Data shows that stimulation of the Imd pathway in the tubule is facilitated by the activation of sGC via interaction with NO. Alternatively, inhibition of the Imd pathway in the tubule is shown to be facilitated by the activation of a receptor guanylate cyclase (rGC). Additionally, it is demonstrated by this study that cGMP-mediated inhibition of the Imd pathway in the tubule is regulated by the dual-specificity, tubule-enriched phosphodiesterase (PDE), PDE11 (Day et al. 2005), thus describing a functional role for this regulatory enzyme for the first time in Drosophila. In conclusion, this study further validates the role of the tubule as a critical immune-sensing tissue in Drosophila melanogaster. In addition, a completely novel role for the cGMP signalling pathway, as a differential regulator of Imd pathway activation in the tubule, is described here for the first time. In particular, an important novel functional role for each of the Drosophila cGKs, DG1 and DG2, is revealed. The data shown in this study therefore contributes to fuller understanding of not only Imd pathway regulation in Drosophila, but also provides a significant advance in the understanding of the complexities of cGMP signalling and its regulation of tubule function.

Moreover, significant differences were found in the ratio of FcγRIIb1/FcγRIIb2 expression between the cohorts of healthy controls and RA and SLE patients. The RA and SLE patients expressed relatively higher levels of the FcγRIIb2 isoform which promotes antigen-processing suggesting that these B cells may play some role in priming autoreactive responses in such individuals. Thus, as these inflammatory disorders constitute spectrum diseases, such defects in the regulation of B cell responses could be one of the contributing factors aggravating autoimmune disease development in some subgroups of patients.

The ability to clear infections and repair injury is dependent on the coordinated migration of immune cells, or leukocytes. These cells can directly destroy invading pathogens and also produce a variety of bioactive factors that promote pathogen clearance. Interactions between immune cells occur both at the site of inflammation and in specialised lymphoid organs throughout the body. The efficiency and specificity of these interactions relies on the production of a family of molecules called chemotactic cytokines, or chemokines, that drive leukocyte migration. Cells express specific profiles of chemokine receptors to ensure they are directed to the appropriate location to exert their immunological function. The field of chemokine biology, already complex, has been further complicated by the discovery of a subfamily of receptors, the atypical chemokine receptors. These molecules lack the ability to couple to signal transduction pathways used by the other chemokine receptors, and are proposed to act as chemokine scavengers or transport molecules. The atypical chemokine receptor CCX-CKR was discovered more than a decade ago but its function in vivo remains unclear. At the beginning of my project, information about this molecule was very limited. The murine receptor binds the CC chemokines CCL19, CCL21 and CCL25, which have well-characterised and critical roles in the development and homeostasis of the immune system as well as in the immune response to infection. Thus, identification of this new receptor, which unlike classical receptors does not induce cell migration in response to ligand binding, presented some exciting possibilities as to how these processes might be regulated in vivo. Reports describing the pattern of expression of CCX-CKR have thus far provided only limited and sometimes contradictory information. Additionally, while in vitro studies from our lab have provided some important clues as to the potential role of the receptor, published in vivo studies were, at the time of commencing this work, limited to one report describing an unvalidated EGFP reporter knock-in transgenic mouse and a conflicting online resource detailing data generated using a LacZ reporter mouse. To understand the true function of this molecule, it is critical to know where it is expressed in vivo and to explore its function on these cells. In this project I set out with the aim of identifying murine tissues and cells expressing CCX-CKR, as well as examining its potential as an in vivo scavenger of chemokine. Related to this, I hoped to uncover any impact of deletion of CCX-CKR on lymphoid tissue cellularity and/or function, both in resting and inflamed conditions. In chapter 3, I present data that identify lymphoid tissues and “barrier” tissues as sites of robustly detectable CCX-CKR mRNA expression. I describe how I have established a novel fluorescent chemokine tetramer-based protocol for the detection of CCL19 receptors, with emphasis on the application of this protocol to identify CCX-CKR activity on specific cell subsets. Using this method, I present evidence that some CD11b+ CD11c+ myeloid subsets in the inguinal lymph node exhibit CCX-CKR dependent internalisation of chemokine. I also describe attempts to fractionate tissues to identify cell populations responsible for the detected whole-tissue expression of CCX-CKR mRNA. The results described in chapter 4 provide support for the hypothesis that CCX-CKR regulates levels of its ligands in vivo, with alterations in chemokine levels in serum and inguinal lymph nodes in the absence of CCX-CKR. I also present evidence demonstrating that deletion of the receptor can influence mRNA levels of the related receptor CCR7. Following on from this, chapter 5 details my analysis of the impact of CCX-CKR on the cellularity of various lymphoid compartments. I present evidence that CCX-CKR influences lymphocyte populations in the peritoneal cavity, with both innate-like and conventional lymphocytes significantly overrepresented in this compartment. The cellularity of the inguinal lymph node, but not the spleen, is subtly altered by deletion of the receptor. Splenic leukocyte cellularity is not affected, either in number or in localisation. In chapter 6, I turn my attention to the possible role of CCX-CKR during the inflammatory response by examining various experimental parameters during a short-term model of induced cutaneous inflammation. This study shows that CCX-CKR deletion alters the cellularity of the myeloid compartment in the draining lymph node and again highlights myeloid subsets as displaying CCX-CKR dependent chemokine internalisation. Finally, I present preliminary data suggesting a protective effect of CCX-CKR deletion during a long-term model of inflammation-driven tumorigenesis. Taken together, my data provide tentative support for the theory that CCX-CKR acts as a chemokine scavenger in vivo. They further indicate that CCX-CKR is involved in regulating cellularity of various lymphoid compartments both at rest and during induced inflammation. In chapter 7 I discuss in detail the implications of my findings in the context of work published since my project began, and highlight growing evidence to suggest a role for CCX-CKR in regulating immune function.

Virus infection of human cells induces the transient expression of interferon-α and interferon-β. This thesis presents the results of investigations into the expression of these interferons in the human B-lymphobastoid cell line, Namalwa, following induction by Sendai virus. Quantitative and qualitative changes in the synthesis of these interferon mRNAs and proteins were investigated in order to determine how interferon gene expression is controlled. Interferon mRNA was assayed both by translation in Xenonus oocytes and by hybridization with cloned interferon cDNA. Interferon was measured both by bioassay and by immunoradiometric assay using a monoclonal antibody to interferon-α. In addition the effects of various treatment which perturb the normal control of interferon production have been assessed. Sendai virus infection of Namalvra cells resulted in the coordinate induction and regulation of both interferon-α and interferon-β synthesis. Although significant amounts of interferon-β were present, the cells did not produce any functional interferon-β protein. It was concluded that the interferon-ß mRNA in these cells was inactive. Production of interferon was shown to be increased by incubating cells at reduced temperatures following induction. It was concluded that the normal inactivation and degradation of interferon mRNA which occurs during the shut-off of interferon production was inhibited at the lower temperature. This resulted in increased interferon production over a prolonged period. Treatment of cells with butyrate or 5'-bromodeoxyuridine before induction caused a dose-dependant increase in the rate of interferon mRNA and interferon synthesis. These treatments appear to coordinately affect the control of both interferon-α and interferon-β gene expression, since no differences could be detected in the characteristics of interferon or interferon mRNA produced by treated and untreated cells. The effect of these treatments was relatively specific, since polyacrylamide gel electrophoresis of proteins from butyrate- and 5'-bromodeoxyuridine-treated cells failed to detect any changes which were comprable to or could account for the effect on interferon synthesis.

The Mannose receptor (MR) is a type I membrane molecule involved in both haemostasis and pathogen recognition. Its extracellular domains have broad ligand specificities: the cysteine-rich (CR) domain is involved in sulphated sugar binding, the C-type lectin-like domains (CTLDs) are responsible for the detection of sugars terminated in mannose, fucose or N-acetylglucosamine, and the fibronectin-type II (FNII) domain mediates collagen binding. Its recently discovered collagen binding ability raised the question of MR facilitating cellular adhesion which would then influence its function as an endocytic receptor in collagen-rich mammalian tissues. For this purpose, the level of MR-mediated endocytosis, and MR expression was analyzed by using bone-marrow-derived macrophages (BM-MΦ) plated on extracellular matrix (ECM) proteins including fibronectin (not a MR ligand), collagen type I or IV (MR-ligands). The results showed no difference in the level of MR-mediated endocytosis and MR expression at both mRNA and protein levels upon MΦ adhesion to collagen. This suggests that MR interaction with collagen may simply be crucial for tissue remodelling and wound healing, rather than adhesion. MR is also expressed in a soluble form (sMR) which is comprised of the extracellular region of intact cell-associated MR (cMR). Even though its precise role is not yet clear, enhanced sMR production was previously shown to help Pneumocystis carinii to evade M phagocytosis by forming a protective coat around the organism. In this work, the mechanism responsible for the fungi-induced MR-shedding was studied by treating MΦ with fungal particles in the presence and the absence of a wide-range of inhibitors. After treatment in serum-free conditions, the cell lysate and cell culture supernatants were analyzed by western blot, for cMR and sMR expression respectively. It was shown that fungi species other than P. carinii can also trigger sMR production, and that this effect mainly takes place through -glucan recognition. Using bio-active particulate -glucan, it was also demonstrated that MR cleavage upon -glucan recognition requires dectin-1-mediated signalling involving Syk, PI3K, and, partially, Raf-1 and that is mediated by a non-secreted metalloproteinase. Dectin-1-mediated MR-shedding may partially explain the contradictive data on the involvement of cMR in the development of immunity against fungi, as well as other pathogens recognised by dectin-1. The ability of pathogens to evade or activate the immune response may depend on the balance between sMR and cMR expression levels.

Dendritic cells (DC) are unique antigen presenting cells which play a major role in antigen presentation and initiation of the immune response by regulating B- and T- cell activation. Antigen targeting to DC receptors is an effective, safe and specific method for vaccine development. The mannose receptor (MR) is an endocytic receptor expressed by subpopulations of DC and antigen targeting through MR leads to enhanced antigen uptake and presentation to T -cells. This makes MR a favourite receptor for the development of vaccines against diseases that require T-cell immunity such as cancer and viral infections. This project sought to develop tools to target antigens through MR and investigate their ability to induce T-cells activation in vitro and in vivo. We have used three approaches to deliver antigen through MR; (i) MRspecific mAbs: 503 and 6C3, have been chemically linked to the melanoma epitope TRP-2, (ii) MR-specific chimeric antibodies carrying several model antigens have been generated by using genetic engineering and (iii) Glycopolymers and the suitable antigens such as a shorter version of model antigen ovalbumin (OVA), with and without N-glycosylation sites have been generated and characterised. Glycopolymer-OVA conjugates were prepared by chemical coupling but it requires further optimization. The binding efficiency of anti-MR antibodies has been assessed using ELISA and BIACORE and the glycopolymers have been tested for their interaction with MR. Immunisations were performed with anti-MR mAb-TRP2 conjugates which induced TRP-2 specific COS+ T-cells activation and improved humoral response. Due to limitations in this approach in terms of chemical coupling being an inefficient method and the potential involvement of Fc recetors (FcRs), chimeric Abs fused to model antigens and bearing mutated Fc were generated. These chimeric Abs, have been tested for their ability to induce T-cell activation in vitro and in vivo. But the progress has been hampered due to the labile nature of these reagents. In future, anti-MR chimeric Abs will be used to generate anti-MR single chain antibodies carrying OVA (ScFv-OVA) and the glycopolymer project will be taken up Dr. Manovani Giuseppe (School of Pharmacy, University of Nottingham). It will involve further optimization of chemical coupling of glycopolymers to a-glycosylated OVA-mini protein, and the in vitro Ag presentation assay to investigate whether glycopolymers mediated Ag targeting of APe enhance T-cells activation. These further studies would greatly benefit the understanding of the mechanisms associated with the elicitation of immune resposes as a result of Ag targeting through MR. Anti-MR reagents generated in this study along with appropriate adjuvant could be exploited to target malarial, cancerous and viral Ags for robust T-cell activation against these infectious diseases. On the other hand, the role of MR in homeostasis and allergy has been already established, and the anti-MR reagents generated in this study can be used to target allergens and self-Ags to APes in an attempt to induce tolerance.

Food-borne disease remains accountable for high levels of morbidity and mortality in the world. The World Health Organization (WHO) estimated that food-borne and water-borne diarrhoeal diseases kill about 2.2 million people annually (SGM, 2013). In this thesis, studies on microbial inactivation were carried out using Cold Atmospheric plasma (CAP) generated by a radiofrequency (RF) source at RF = 4.6 MHz. The operating gas used was a mixture of helium and oxygen. The study has investigated the effect of CAP on different strains of food-borne bacteria. These included strains/mutants of S. typhimurium, L. monocylogenes and E. coli. The result has shown that CAP has the capability to inactivate a wide range of food-borne bacteria. However, S. typhimurium LT2, E. coli 0157: H7 and E. coli HI 0407 showed more tolerance to CAP treatment than L. monocytogenes strains and E. coli K-12 MG 1655. This result led to further investigations into the role of lipopolysaccharide (LPS) structure in the tolerance to CAP treatment. To investigate the role of LPS structure, derivatives of E. coli K-12 which lack the O-antigen and isogenic mutants of them (core polysaccharide defective) were used. In addition to these strains and mutants, partially and fully restored LPS derivatives of E. coli K-12 MG1655 and S. typhimurium TA98 which is a mutant of S. typhimurium LT2 were also used. No significant differences in the sensitivity to CAP treatments between these mutant and their parental strains were found except between S. typhimurium TA98 and its parental strain. S. typhimurium TA 98 was missing many other genes, therefore it was not comparable to the parental strain. The results suggest no role for the LPS structure regions in the resistance to CAP treatment. The study has also investigated the role of oxidative stress in bacterial inactivation. Mutants of E. coli K-12 BW25113 were a single gene of the OxyR regulated genes has been deleted, were used. Three mutants were found significantly (P < 0.05) more sensitive to CAP treatments than other strains. These mutants were (delta dps), (delta grxA) and (delta sufC). The gene deleted from each mutant is hydrogen peroxide inducible and OxyR regulated (Zheng et al., 2001a). Since each of these genes has an important role in the resistance to oxidative stress, the result suggests the involvement of oxidative stress in inactivation by CAP treatments. To confirm these results and to find out whether the CAP treatment caused DNA damage or mutagenic effects, the Ames strains were used. Exposure to CAP treatment for 5 and 10 seconds caused a significant (P < 0.05) increase in the number of revertant colonies of S. typhimurium TA 100 and S. typhimurium TAI02. The results suggest that CAP caused base-pair substitution in the DNA of S. typhimurium TA 100 (Barnes et al., 1982) and transition/transversion mutations in the DNA of S. typhimurium TA 102 (Levin el al., I 982a) and potentially in the DNA of other bacteria. It can be concluded that CAP inactivates bacteria due to oxidative stress damage and induces mutation in the bacterial DNA. Modification of the Ames strain test was successfully validated. This finding highlights some concerns about application of CAP in dental and wound treatments.

Campylobaeter jejuni and C. coli are common causes of acute gastroenteritis in humans that are also associated with Guillain Barre and Miller Fisher syndrome. Poultry and other farm animals are the major sources of these pathogens. In this thesis it was demonstrated that hydrogen has the potential to act as an antioxidant to reduce oxidative stress caused during the growth of C. jejuni HPC5 when grown in a gas replacement jar. Growth in the absence of hydrogen in a modular atmosphere controlled system (MACS) was characterised by an intiallag that could be overcome by adding an antioxidant reagent FBP (10% ferrous sulphate, sodium pyruvate and sodium metabisulphite). Transcriptomic studies revealed that growth in the absence of hydrogen resulted in significant increases in the expression of superoxide dismutase, thiol peroxidase and ribosomal proteins. Transcriptomic studies were performed on the variants of C. jejuni HPC5 where bacteriophage predation had provoked intragenornic recombination to create second generation resistant types that are inefficient colonisers of chickens but revert to efficient colonisers and bacteriophage sensitivity when reintroduced into chickens to create third generation variants. The second generation variants were temperature sensitive, exhibited increased expression ofprophage Mu genes and low expression of motility associated genes. In contrast third generation variants showed an increase in the expression of the motility genes, an increase in the genes associated with the putative bacteriophage immunity factor CRISPR and reduced expression of Mu genes. Studies conducted on pigs demonstrated that a single pig can be colonised by campylobacters belonging to multiple genotypes and species. Comparative genomic hybridisation (CGH) of C. coli and C. jejuni isolated from the intestines of a single pig demonstrated these isolates shared plasmid and chromosomal encoded genes, and therefore may have undergone inter-species gene transfer due to cohabitation of a common intestinal niche. The aim of this thesis is to genotypically characterise Campylobaeter strains from chicken and pig in ideal atmospheric conditions. Our hypothesis is that Campylobacter can be grown in vitro both in gas replacement jar (ORJ) and in MACS and the molecular characterisation by transcriptomic analysis and CGH of the strains will be ideal in an atmospheric condition which is stress free.

The recent association between S. Enteritidis PT4 and poultry products has caused a great deal of concern from adverse publicity and with resulting national and international requirements to control the major food-poisoning Salmonella serotypes at the breeder and layer levels in order to ensure that poultry products are Salmonella-free. The exact mechanism whereby these serotypes are able to colonise the intestine of chickens is still exactly unknown. Indeed, there is increasing evidence that colonisation is not solely a metabolic function but that some form of physical association with cells or an organ in the gut is involved. Thus, invasion and fimbrial genes required for colonisation have been identified (Clayton et al., 2008, Morgan et al., 2004) suggesting physical contact was required. An alternative approach would be to analyse the patterns of gene expression by microarray analysis at the site of colonisation (caeca). This has been done for a number of niches and is now being applied to intra-cellular infection but has not so far been applied to the intestine. The S. Enteritidis transcriptome during the colonisation of the caeca of one day chicks was characterised by Agilent microarray. The microarray results were evaluated by real-time PCR with 96% compatibility. The pattern of gene transcription was different in the intestine compared with broth culture. Thirty four percent of the genes showed a significant change in level of expression. Major changes occured from adaptation to the caecal environment with up-regulation of genes required for energy generation and carbohydrate metabolism/transport, while amino acids and nucleotide metabolism, translation, replication and cell wall biogenesis genes were among the down-regulated genes. Fumarate respiratory and osmotic response genes were selected from the up-regulated genes and were mutated and tested in the lab for their inhibitory effect and for competitive growth under anaerobic and osmotic environments showing variable responses. Association between chicken colonisation phenotype and gene mutation indicated that genes associated with osmolarity was more important than tri carboxylic acid (TCA)-associated genes in their contribution to the colonisation phenotype. There is considerable scope for improvement in inactivated vaccines through a more rational approach. An inactivated vaccine prepared by formalising S. Enteritidis harvested directly from the chicken caeca was thought to be more protective than bacteria grown in vitro. Unfortunately this was not the case. Expected reasons for this failure are explained, and alternative approach to producing a proper effective inactivated vaccine is suggested.

Dysfunctional immunity which is the most common antecedent of immunological diseases is central to the common disorders like rheumatoid arthritis, psoriasis, thyroid disease, type-1 diabetes mellitus, multiple sclerosis and other autoimmune conditions that are leading causes of chronic morbidities and disabilities. The currently employed therapies suffer from serious side effects. For example, steroid therapy, being nonselective is seldom used. Myleotoxicity limits azathioprine while cyclosporine-A (CsA) and FK506 do show myleo-, nephro and neuro-toxicities. Antibody therapies cause immune mediated toxicity. Therefore, the need of developing less toxic and specific immune modulatory agents is a top priority. Recently, Pseudomonas aeruginosa quorum sensing signal molecules (QSSMs), N-(3-oxododecanoyl)-L-homoserine lactone (3O, C12-HSL) and 2-n-heptyl-3-hydroxy-4-(1H)-quinolone (PQS) have been found to modulate eukaryotic immune processes via different targets. The structure activity relationship (SAR) study of 3O, C12-HSL by Chhabra et.al, showed that L-homoserine lactone ring, 3-oxo or hydroxyl group and 12 to14-carbons long acyl chain, apparently conferring optimum lipophilicity and flexibility, are important for immune modulation. However, the new analogues including 3O, C12-HSL negated clinical usefulness as these also participated in bacterial quorum-sensing (QS) activity thus promoting bacterial growth. Also analogues with more than 12-carbons alkyl chain could not be explored by in vitro immune assays due to their limited solubility in the solvents employed. The present study addresses these issues by making further judicial structural changes in the 3O, C12-HSL in order to optimize its immune modulatory activity while losing its intrinsic QS activity. Accordingly, a series of new ring variants, 3-acetoxy, aza, thia, oxa and fluorine substituted analogues were developed. The later were largely confined in the 1, 3-dicarbonyl segment of the 3O, C12-HSL structure in order to modulate the 3-enolic content of the molecule. Also tetramic acid analogue, a non-enzymatic degradative product of 3O, C12-HSL was synthesized. After purity and identity characterization the new analogues were evaluated for their immune modulation (with murine cells proliferation assay), cytotoxicity (with Trypan dye exclusion assay) and auto-inducing or QS activity (with a specifically designed lux bacterial bio-reporter). The splenocyte proliferation assay demonstrated that immune suppression in 4-aza analogues increased with increasing alkyl chain (up to C15) yet without any solubility problems in the standard solvents used. Similar trend was observed when N4 was alkyl substituted (n-propyl, being most potent). At least one of the C2 hydrogens was found to be essential for activity since, difluoro substitution lost activity. Retention of immune suppression by N3-OH and increased potency by N3-NH2 (EC50=0.99 microM) demonstrated that C3=O can be replaced by an appropriate H-bond donor/acceptor groups. Complete loss of activity with 3-thia substitution (3-thia C12-HSL) and then regain of potency by 3-sulfoxide/sulfone derivatives further demonstrated the importance of 3-oxo (suitable H-bond acceptor/donor) in immune suppression. Insertion of oxygen at C4 retained activity but additional oxygen(s) in the side chain yielded analogues that were devoid of immune suppression. Analogue with thiazole ring instead of HSL ring retained activity. In case of hetero ring altered analogues of 3O, 4-aza C12-HSL, almost all retained immune suppressive activities except their oxa analogues. In conclusion, all the above strategies have delivered potent immune modulatory agents yet gratifyingly devoid of QS activity. Some of the most potent and promising non-inducing immune modulatory agents are 3O, 4-aza-C15-HSL (EC50 0.65 microM), 3-NH2-3-aza-C12-HSL (EC50 0.99 microM) and N, N'-dimethyl-2-(3-oxo-4-azadodecanoyl) aminobenzamide (EC50 0.7 microM) and are worthy of further study to be developed as therapeutic agents.