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Molecular Dissection of Immunomodulatory Function of Polysaccharides

Final Report Summary - MEDPOL (Molecular Dissection of Immunomodulatory Function of Polysaccharides)

Polysaccharides are one of the most abundant biomaterials available from many living organisms. Plants and microbes, for instance, produce polysaccharides as structural components of their cellular membranes and cell walls. Some of these polysaccharides are suggested to modulate immune function in mammals, leading to the commercialization of polysaccharide products such as anti-cancer drugs, carbohydrate vaccines, and health supplements. However, the precise mechanisms of their function are unclear and gaining such knowledge is vital for harnessing the power of immunomodulatory polysaccharides for improving human health. In this project, I aimed to identify novel immunomodulatory polysaccharides and elucidate the molecular mechanisms by which the polysaccharides elicit these health-promoting effects.

I hypothesised that immunomodulatory polysaccharides bind to glycan-binding proteins (GBP) expressed on the cell surface of immune cells. In the first year of this project, I established a cell-based assay to analyse GBP-polysaccharide interactions. Using this assay, I screened several food and microbial polysaccharides for their binding to GBPs. As a result, I identified two novel polysaccharides that bind to one GBP. In the second year, I investigated the immunostimulatory property of these two novel polysaccharides. I found that the polysaccharides activate dendritic cells (DCs), as shown by cytokine production upon co-culture of the cells with polysaccharides in vitro. Furthermore, using the receptor-deficient DCs, I was able to demonstrate that the immunomodulatory property of the polysaccharides was indeed mediated by the receptor. Overall, I successfully identified two immunomodulatory polysaccharides and addressed the molecular mechanism of the effect on DCs.

I have successfully disseminated my scientific results to the relevant scientific community at conferences. One manuscript has been submitted to a high-impact journal and a second manuscript is in preparation. Furthermore, one UK patent application has been submitted, which will encourage and potentially enable the industrial sector to commercialize these immunomodulatory polysaccharides. I was also able to disseminate my research to the general public at a local outreach event.
Through this two-year research programme, I have gained both scientific and non-scientific skills important for my career progression at the Institute of Food Research (IFR).

The specific aims of the project were to:
Aim 1: Assess whether the polysaccharide causes activation of macrophages (Macs) and DCs
Aim 2: Assess whether the polysaccharide binds to Macs and DCs
Aim 3: Identify GBPs recognizing the polysaccharides

In the first year, I focused on Aim 3, as this was the most important aim and it would provide a good starting point for the project. For this purpose, I established around 20 different reporter cells, each of which express different GBPs. Also I collected several food and microbial polysaccharides from other researchers. As such, I have achieved good interactions with a broad scientific community both in UK and beyond, including microbiologists, food scientists, and glycobiologists. These potentially collaborative links will be helpful for the development of my future research programme as well as career progression. The reporter assay results were promising. I screened around 20 different polysaccharides for their binding to GBPs (20 X 20 screening). I decided to focus on the most promising results, including a food polysaccharide, A (FPA), and a microbial lipopolysaccharide (LPS) from a human commensal bacterium, Hafnia alvei. Both polysaccharides were shown to bind to dendritic cell-associated lectin 2 (Dectin-2). Dectin-2 is a single transmembrane lectin expressed on various myeloid cells in mouse and man. Dectin-2 is originally documented as an anti-fungal receptor that recognises α-linked mannose structure as a carbohydrate ligand and induces cell activation. Thus polysaccharides activating Dectin-2 may be able to boost our immune function, which is beneficial for combatting infection and cancers.

Based on the results of the reporter assay, I developed two projects in parallel:
1. Analysis of poly-mannose O-antigen binding to Dectin-2
2. Discovery of FPA as a new Dectin-2 ligand

1. Analysis of poly-mannose O-antigen binding to Dectin-2
The newly identified polysaccharide ligand for Dectin-2 was microbial LPS from Hafnia alvei. LPS is a cell wall component of gram-negative bacteria, consisting of a relatively conserved region of lipid A and core-oligosaccharide, and a highly variable region of O-antigen polysaccharide. Hafnia alvei has a poly-mannose type O-antigen that is likely recognised by Dectin-2. Since the poly-mannose type O-antigen can be found in several gram-negative bacteria including Escherichia coli O9, I also tested whether LPS from these bacteria bind to Dectin-2. Indeed, E. coli O9 LPS bound to Dectin-2, suggesting Dectin-2 has broader specificity toward poly-mannose bearing gram-negative bacteria.

I analysed cytokine production from mouse DCs in response to Hafnia LPS, which was generated from mouse bone marrow in vitro. The cytokine production was monitored by an ELISA assay. Hafnia LPS augmented cytokine TNFα and IL-10 production. Importantly this enhanced cytokine production disappeared in Dectin-2 knock-out (KO) DCs, suggesting that Dectin-2 binding to Hafnia LPS enhances DC response to the LPS. Furthermore, I analysed intracellular signalling induced by Hafnia LPS by western-blot analysis. Hafnia LPS also stimulated human monocytes isolated from human peripheral blood, shown by TNFα production, consistent with the mouse study. These data demonstrate that Dectin-2 recognises poly-mannose bearing microbes and mediate host-microbe interaction in our body. I have generated a manuscript on this project, which has been submitted to Journal of Biological Chemistry.

2. Discovery of FPA as a new Dectin-2 ligand
I have tested whether FPA activate DCs in vitro. Mouse DCs produced TNFα and IL-10 production in response to FPA, suggesting FPA have immunostimulatory properties. Importantly the cytokine production was dependent on Dectin-2, as Dectin-2 KO DCs showed significant reduction in cytokine production. These results, for the first time, demonstrate that FPA activate DCs via Dectin-2.

In Aim 2, the detection of FPA binding to DCs via Dectin-2 proved to be challenging. Therefore, alternatively, I employed the Dectin-2 reporter assay, in order to further investigate the interaction between Dectin-2 and FPA. I was able to demonstrate that the binding was indeed mediated via the carbohydrate-recognition domain of Dectin-2, confirming the specific interaction between Dectin-2 and FPA. I have successfully submitted a UK patent application on the immunomodulatory effect of FPA. I am finalising a manuscript on the immunomodulatory effect of FPA.

These two projects have been disseminated at conferences, including a poster presentation at Gordon conference (Whistler, USA, June, 2015), an oral presentation at the 23rd international symposium on glycoconjugates (Split, Croatia, Sep 2015), and an oral presentation at Oxford Chemical Immunology Conference (Oxford, UK, April, 2016)

In summary, I have identified two novel immunomodulatory polysaccharides from food and microbes. The immunomodulatory effect relies on the interaction with Dectin-2, deciphering the molecular mechanism of the new immunomodulatory polysaccharides.

The outcomes of this project have provided new knowledge on immunomodulatory polysaccharides, which have the potential impacts as described below:
1. Applying the established research platform to other polysaccharides
The strategy used in the project (e.g. cell-based screening and immune cell-assay using receptor KO cells) can be readily applied to other type of polysaccharides. I have already provided these materials to other researchers in the EU, thus increasing the competitiveness of EU research excellence.

2. Potential product development with FPA
The Immunomodulatory effect of FPA has been submitted in a UK patent application. Once awarded, this will encourage and enable the industrial sector to pursue the commercialisation of FPA as immunomodulatory prebiotics.

3. Increased awareness of the benefits of a glycobiological approach to study host-microbe interactions. The results of this research suggest that polysaccharides are a “biological interface” enabling microbes to interact with the host immune cells. Along with the emergence of microbiota research, this research will enhance our understanding of host-microbe interactions in our body.