Exploring and Expanding the Fatty-Acid-Binding Protein Superfamily in Fasciola Species

The liver flukes Fasciola hepatica and F. gigantica infect livestock worldwide and threaten food security with climate change and problematic control measures spreading disease. Fascioliasis is also a food borne disease with up to 17 million humans infected. In the absence of vaccines, treatment depends on Triclabendazole (TCBZ) and over-use has led to widespread resistance, compromising future TCBZ control. Reductionist biology from many laboratories has predicted new therapeutic targets. To this end, the fatty acid binding protein (FABP) superfamily have proposed multi-functional roles, including functions intersecting vaccine and drug therapy, such as immune modulation and anthelmintic sequestration. Research is hindered by a lack of understanding of the full FABP superfamily complement. Although discovery studies predicted FABPs as promising vaccine candidates, it is unclear if uncharacterised FABPs are more relevant for vaccine formulations. We have coupled genome, transcriptome and EST data mining with proteomics and phylogenetics, to reveal a liver fluke FABP superfamily of 7 clades: previously identified clades I-III and newly identified clades IV-VII. All new clade FABPs were analysed using bioinformatics and cloned from both liver flukes. The extended FABP dataset will provide new study tools to research the role of FABPs in parasite biology and as therapy targets

Annexin A1 on the surface of early apoptotic cells suppresses CD8+ T cell immunity.

Prevention of an immune response against self-antigens derived from apoptotic cells is essential to preclude autoimmune and chronic inflammatory diseases. Here, we describe apoptosis induced externalization of endogenous cytosolic annexin 1 initiating an anti-inflammatory effector mechanism that suppresses the immune response against antigens of apoptotic cells. Cytosolic annexin 1 rapidly translocated to the apoptotic cell surface and inhibited dendritic cell (DC) activation induced by Toll like receptors (TLR). Annexin 1-inhibited DC showed strongly reduced secretion of pro-inflammatory cytokines (e.g. TNF and IL-12) and costimulatory surface molecules (e.g. CD40 and CD86), while anti-inflammatory mediators like PD-L1 remained unchanged. T cells stimulated by such DC lacked secretion of interferon-γ (IFN-γ) and TNF but retained IL-10 secretion. In mice, annexin 1 prevented the development of inflammatory DC and suppressed the cellular immune response against the model antigen ovalbumin (OVA) expressed in apoptotic cells. Furthermore, annexin 1 on apoptotic cells compromised OVA-specific tumor vaccination and impaired rejection of an OVA-expressing tumor. Thus, our results provide a molecular mechanism for the suppressive activity of apoptotic cells on the immune response towards apoptotic cell-derived self-antigens. This process may play an important role in prevention of autoimmune diseases and of the immune response against cancer

Annexin A1 inhibits TLR-induced signal transduction pathways.

<p>(<b>A</b>) Immature DC were incubated with annexin A1 (AnxA1, 20 µg/ml) or left untreated. Subsequently, DC were stimulated with R-848 (1 µg/ml) for the indicated time periods. TNF mRNA expression was determined by reverse transcription quantitative PCR normalized to GAPDH. Fold change was calculated relative to unstimulated DC (set to 1). Data are representative of at least 3 independent experiments. (<b>B</b>) Immature DC were treated as in (B). DNA binding of the NF-κB subunit p65 was analyzed in nuclear extracts of DC by ELISA. Error bars represent means +/− SD of duplicate measurements. Data are representative of 2 independent experiments.</p

Annexin A1 inhibits DC activation <i>in vivo</i>.

<p>Apoptotic, mOVA-expressing <i>Drosophila</i> Schneider cells (aS2-mOVA) transfected with annexin A1 (AnxA1) or a control plasmid (Ctrl) were injected into mice. After 2 days, DC in lymph nodes of naïve (−) and S2-injected mice were analyzed for CD40 and CD86 expression by flow cytometry. Shown are quantifications of the MFI for CD40 and CD86 of total lymph node cells gated on CD11c⁺ and MHC class II⁺ cells *P<0.05 (n = 7). Data are representative of 3 independent experiments.</p

Annexin A1 is exposed on the surface of early apoptotic cells.

<p>(<b>A</b>) Primary human neutrophils were analyzed for characteristic features of apoptosis during continued aging <i>in vitro</i>. Externalization of PS, loss of membrane integrity and externalization of annexin A1 (AnxA1) was measured by flow cytometry using FITC-labeled annexin 5, 7-amino-actinomycin D (7-AAD) and FITC-labeled DAC5 antibody, respectively. Fragmented DNA = nuclei with fragmented DNA. (<b>B</b>) Primary human neutrophils of several donors after 1 d <i>in vitro</i> culture were analyzed as in (A). Error bars represent means +/− SD of several donors (n = 5). ***P<0.001.</p

Annexin A1 suppresses DC activation.

<p>(<b>A</b>) Immature DC were incubated with the indicated concentrations of annexin A1 (AnxA1) or apoptotic Jurkat T cells (aJ), or left untreated. After stimulation with the indicated concentrations of R-848, TNF in culture supernatants was determined by ELISA. Error bars represent means +/− SD of duplicate cultures. ND = not detectable. (<b>B</b>) DC were treated as in (A). For several donors, the relative TNF secretion of treated DC was calculated relative to DC stimulated with R-848 only, which was set to 100%. Error bars represent means +/− SD (n = 7). ***P<0.001. (<b>C</b>) Secretion of IL-12 p40 and TGF-β from DC treated as in (A). Error bars represent means +/− SD of triplicate cultures. ND = not detectable. (<b>D</b>) Analysis of DC surface molecules. Untreated DC (iDC, dashed line) or DC stimulated by a cytokine cocktail after pre-incubation with annexin A1 (mDC+AnxA1, filled histograms) or without annexin A1 (mDC, bold lines) were analyzed by flow cytometry for the indicated molecules. Data are representative of at least 3 independent experiments. In experiments B to D, recombinant annexin A1 was used at a concentration of 5 or 10 µg/ml while R-848 was used at a concentration of 5 µg/ml.</p

Annexin A1 influences DC-mediated T cell stimulation.

<p>(<b>A</b>) Immature DC were incubated with annexin A1 (AnxA1, 20 µg/ml), apoptotic Jurkat T cells (aJ), or left untreated. 2 days after stimulation by R-848, autologous T cells together with SEB in the indicated concentrations were added to the culture. The concentration of IFN-γ in culture supernatants was determined by ELISA after 6 days. Error bars represent means +/− SD of quadruplicate cultures. (<b>B</b>) For several donors, the relative IFN-γ secretion of cultures treated as in (A) is shown. IFN-γ-secretion in cultures with treated DC was calculated relative to cultures with DC stimulated by R-848 only, which was set to 100%. Error bars represent means +/− SD (n = 7). ***P<0.001. (<b>C</b>) Cocultures were set up as in (A) and concentrations of IL-10 in culture supernatants were determined by ELISA after 4 days. Error bars represent means +/− SD of triplicate cultures. Data are representative of at least 3 independent experiments. In all experiments, R-848 was used at a concentration of 5 or 10 µg/ml.</p

Annexin A1 suppresses immune responses <i>in vivo</i>.

<p>(<b>A</b>) Apoptotic, mOVA-expressing <i>Drosophila</i> Schneider cells (aS2-mOVA) transfected with annexin A1 (AnxA1) or a control plasmid (Ctrl) were injected into mice. After 8 days, percentages of OVA-specific CD8⁺ T cells relative to total CD8⁺ T cells in lymph nodes were analyzed by flow cytometry. *P<0.05 (n = 5). (<b>B</b>) Mice were immunized as in (A) and challenged with OVA/CpG after 10 days. CD8⁺ T cell function was analyzed by an <i>in vivo</i> cytolytic assay using OVA-peptide labeled target cells (n = 4–5), *P<0.05. (<b>C</b>) Mice were immunized as in (A). After 12 days, mice were injected with OVA-expressing B16 melanoma cells. Tumor growth was monitored for 4 to 14 days. *P<0.05, **P<0.01 for tumors of mice immunized with annexin A1-expressing apoptotic S2-mOVA cells compared to mice immunized with apoptotic S2-mOVA cells (n = 6). Data are representative of 3 (A) or 2 (B, C) independent experiments.</p