Mast Cell Subsets and Their Functional Modulation by the Acanthocheilonema viteae Product ES-62

Abstract

ES-62, an immunomodulator secreted by filarial nematodes, exhibits therapeutic potential in mouse models of allergic inflammation, at least in part by inducing the desensitisation of Fc ε RI-mediated mast cell responses. However, in addition to their pathogenic roles in allergic and autoimmune diseases, mast cells are important in fighting infection, wound healing, and resolving inflammation, reflecting that mast cells exhibit a phenotypic and functional plasticity. We have therefore characterised the differential functional responses to antigen (via Fc ε RI) and LPS and their modulation by ES-62 of the mature peritoneal-derived mast cells (PDMC; serosal) and those of the connective tissue-like mast cells (CTMC) and the mucosal-like mast cells derived from bone marrow progenitors (BMMC) as a first step to produce disease tissue-targeted therapeutics based on ES-62 action. All three mast cell populations were rendered hyporesponsive by ES-62 and whilst the mechanisms underlying such desensitisation have not been fully delineated, they reflect a downregulation of calcium and PKC α signalling. ES-62 also downregulated MyD88 and PKC δ in mucosal-type BMMC but not PDMC, the additional signals targeted in mucosal-type BMMC likely reflecting that these cells respond to antigen and LPS by degranulation and cytokine secretion whereas PDMC predominantly respond in a degranulation-based manner.

Figure 1

Phenotyping of mast cell subsets. Exemplar plots of mast cell phenotyping by flow cytometric analysis are shown in (a)–(d). FSC and SSC parameters of PDMC, mucosal-type BMMC, and CTMC cultured in vitro for 28 days (a) and gating (relative to isotype controls; not shown) of the consequent CD117⁺FcεR1⁺cell population (>98%; (b)) prior to the analysis of their TLR4⁺expression (c) are shown. In parallel experiments, ST2 expression of CD117⁺FcεR1⁺cells in the various populations was determined (d). Gray shaded plots (c-d) are isotype controls. In (e), exemplar images of toluidine blue staining of the mast cell populations (x10) are shown. The data are representative of at least 2 independent experiments.

Figure 2

Degranulation by mast cell subsets. Mucosal-type BMMC, CTMC and PDMC were sensitised with murine anti-DNP IgE (0.5 μg/mL) in the presence and absence of ES-62 (2 μg/mL) overnight. Cells were then stimulated with DNP (0.5 μg/mL) to induce FcεRI cross-linking (XL; (a)) or PMA plus ionomycin (both 1 μM; (b)) for 30 min at 37°C. Degranulation was determined as the %  β-hexosaminidase release relative to the total enzyme activity of the cells and the data presented are from single experiments representative of at least 2 independent experiments. PDMC were sensitised with murine anti-DNP IgE (0.5 μg/mL) in the presence and absence of ES-62 (2 μg/mL) overnight and analysed for expression of FcεRI (c), CD117 (d), and ST2 (e). Grey shaded plots (c–e) are relevant isotype controls.

Figure 3

Chemokine and cytokine release by mast cell subsets. Mucosal-type BMMC (a), (c) & (e) and CTMC (b), (d) & (f) were sensitised with murine anti-DNP IgE (0.5 μg/mL) in the presence and absence of ES-62 (2 μg/mL) overnight. Cells were then stimulated with DNP (0.5 μg/mL) to induce FcεRI cross-linking (XL) or LPS (0.5 μg/mL) for 24 h at 37°C and MCP-1 (a) & (b), IL-6 (c) & (d), and IL-13 (e) & (f) release measured by ELISA. The data presented are single experiments representative of at least 2 independent experiments apart from the IL-13 release from CTMC, which could only be detected in a single experiment.

Figure 4

Calcium mobilisation in mast cell subsets. Fura-2/AM-loaded resting, nonsensitised (a) & (b) or anti-DNP IgE (0.5 μg/mL)-sensitised (a)–(h) mucosal-type BMMC (e) & (f), CTMC (g) & (h) and PDMC (a)–(d) were stimulated at 50 s with DNP (0.5 μg/mL) to induce cross-linking (XL) of FcεR1 (a), (c), (e) & (g) or 0.5 μg/mL LPS (b), (d), (f) & (h), and intracellular calcium mobilisation and influx recorded in real time using excitation-emission ratios of 340/380 nm (a)–(h). For analysis of intracellular mobilisation alone, the cells were stimulated in calcium free HBSS supplemented with 100 μM EGTA to remove all extracellular calcium (EGTA). Calcium levels were calculated from R _max⁡ and R _min⁡ values and the data are presented as the mean calcium values of triplicate samples (base line calcium values subtracted) from a single experiment representative of at least 3 independent experiments.

Figure 5

ES-62 modulates signalling in mast cell subsets. PDMC were sensitised with murine anti-DNP IgE (0.5 μg/mL) in the presence or absence of ES-62 (2 μg/mL) overnight. Following loading with Fura-2/AM such PDMC were stimulated at 50 s with DNP (0.5 μg/mL) to induce cross-linking (XL) of FcεR1 (a) or 0.5 μg/mL LPS (b) and intracellular calcium mobilisation and influx recorded in real time using excitation-emission ratios of 340/380 nm (a) & (b). Calcium levels were calculated from R _max⁡ and R _min⁡ values and data are presented as the mean calcium values of triplicate samples from a single experiment representative of at least 3 independent experiments. PDMC and mucosal-type BMMC (c) & (e) were cultured with ES-62 (2 μg/mL) for the indicated times and expression of PKCα ((c), Cell Signalling Technology), MyD88 ((e), Abcam) and PKCδ ((e), Cell Signalling Technology) analysed by Western Blotting. In (d), following preincubation for 1 h with inhibitors of proteosomal degradation (10 μM Lactacystin, ENZO Life Sciences; LAC), caveolae/lipid raft trafficking (50 μg/mL Nystatin; NYS) and lysosomal degradation (E64d + pepstatin A both 10 μg/mL, ENZO Life Sciences), sensitised CTMC were cultured with ES-62 (2 μg/mL) for the indicated times and expression of PKCα analysed by Western Blotting. Actin was used as a loading control and ES-62-mediated downregulation of PKCα expression was observed in PDMC, Mucosal-type BMMC, and CTMC in at least 2 independent experiments.

Figure 6

Model of differential desensitisation of mast cell subsets by ES-62. Mucosal-type BMMC, CTMC, and PDMC exhibit differential functional responses to antigen (Ag)-mediated cross-linking of FcεRI and stimulation with LPS as summarised. The signalling mechanisms underlying the coupling to these differential responses and their desensitisation by ES-62 have not been fully delineated but our working model is that the FcεRI-mediated degranulation observed in PDMC involves PKCα and calcium mobilisation which are effectively targeted by ES-62. Although these cells express TLR4/MyD88, they are uncoupled from downstream functional LPS signalling by an as yet unknown mechanism. By contrast, mucosal-type BMMC exhibit both degranulation (via Ag/FcεRI) and cytokine (via FcεRI and LPS/TLR4) responses, requiring recruitment of additional signals such as PKCδ and MyD88 signalling which are both also targeted by ES-62.