Work package 3 - Dissection of the innate immune response in experimental models of infection

Objectives 
To define the innate immune response in two murine models of infection (chronic and multiple exposure) by:  

• Characterising the immune response profile of cultured immune cells (of lymphoid origin, and PBMCs) in terms of cytokine production and lymphocyte proliferation.
• Defining changes in the expression of key innate immune receptors and activation markers by analysis of antibody-labelled cells by flow cytometry and by real-time PCR.
• Testing the immunoregulatory functions of fractionated, partly characterised, parasite molecules (from WP4).
• Testing the stimulatory activity of parasite extracts with cell lines expressing human DC-SIGN and TLRs.

Description of work
Murine models of infection will be examined to dissect the innate immune response and its impact on immune regulation; a) multiple exposure regime, and b) chronic infection (10+weeks) regime. 

Cells will be obtained at relevant time points from the skin/sdLNs (multiple exposure) and the spleen/mesenteric LN (chronic infection).  PBMCs will be collected for comparability with studies in WP2.  Cells will be cultured with parasite ES preparations (e.g. 0-3hRP and fractions thereof from WP4), or soluble schistosome antigen preparations, either as individual preparations, or in combination, to determine the effect certain preparations have on the immune response elicited by others.  The ability to proliferate and secrete various cytokines (e.g. IFN-γ, IL-5, IL-10, IL-12, IL1α IL-13, TNF-αTGFβ in response to the different molecules will be determined to provide measures on:  immune hypo-responsiveness in the different models; how parasite ligands of innate and acquired immune response cells compares in different tissues; and the likely contributing mediators.

Cells from the tissues above will be also prepared for labelling and flow cytometric analysis, and for real-time PCR.   Cells will be analysed by flow cytometry for the expression of innate immune receptors (e.g. TLRs), and for markers of cell activation (e.g. MHCII, CD40, CD86) or regulation (FasL, PD-L1, CTLA-4). Where feasible, APCs will be ‘cell sorted’ and analysed by real time PCR using gene-specific primers.  Combined, this will determine the relative numbers of different cell types and their activation status.   If judged appropriate, cells from mice deficient in a particular immune component (e.g. TLR4, TLR2, MR) will be obtained to answer defined questions. This will address the importance of the missing component in i) the innate response in terms of cell influx, activation and cytokine expression, ii) the acquired immune response in terms of CD4+ cell reactivity and subset bias, and iii) the development of egg-induced pathology.

In parallel, experiments with cells expressing human TLR and DC-SIGN will be used to delineate the binding activity of the parasite-derived molecules. This assay is based on flow cytometric analysis of DC-SIGN.  Cell lines (HEK cells, either stable or transients). expressing TLRs when stimulated with ligands lead to cytokine production that can be measured by ELISA.