Volume 31, Number 3, March 2009 S isolate antigens stimulate APC derived IL-10 and IL-6 Figure 4 Bone marrow dendritic cells (BMDC) stimulated with S isolate excretory/secretory antigen (E/S) secrete higher levels of IL-10 and IL-6 compared to BMDC stimulated with E or J isolate E/S. Bone marrow was isolated and pooled from 3–5 naïve C57BL/6 mice and cultured into BMDCs. The BMDCs were incubated for 48 h, in vitro with medium alone – unstimulated (U), 50 μg/mL E isolate E/S (E), 50 μg/mL J isolate E/S (J), 50 μg/mL S isolate E/S (S) or 100 ng Lipopolysaccharide (LPS). Levels of IL-10, IL-12p70, MCP-1, IL-6, TNF-α and MIP-1α were measured by CBA; all cytokine data are pg/mL. Values represent the mean ± SD for four wells per treated group. *Signiﬁcant difference between the BMDCs stimulated with the S isolate E/S antigen and BMDCs stimulated with the E or J isolate E/S antigens *P < 0·05, **P < 0·001 and ***P < 0·001. Experiments were performed four times and data shown are a representative experiment. response. This capacity however, is not due to alterations in the cell surface markers, CD40, CD80, CD86 and CD54 (21). Furthermore, peritoneal macrophages produce low levels of IL-12, IL-6 and TNF-α following stimulation with ES-62, unlike LPS which elevates these cytokines dramatically. Interestingly, when ES-62 and LPS were cultured with the macrophages along with LPS, the levels of IL-12, IL-6 and TNF-α were suppressed to levels secreted by the addition of ES-62 alone, implying the importance of ES-62 in immune modulation. The studies presented here use a very heterogeneous mix of isolate E/S antigens (7) rather than any deﬁned antigen. Future work will involve fractionation of the S isolate E/S antigens, into high and low molecular weight components. This will begin to deﬁne the E/S products which provoke the macrophages and dendritic cells to secrete elevated levels of IL-10 and IL-6 and which may therefore contribute to parasite survival. In this context, B cell recognition of low molecular weight E/S antigens has previously been linked with a chronic infection outcome (7). Nevertheless, the S isolate total E/S is able to stimulate both BMMΦ and BMDCs to secrete signiﬁcantly more IL-10 and IL-6 than E/S from either the J or E isolates. The majority of BMDC and BMMΦ used in this study had cell surface markers representative of dendritic cell or macrophage phenotype – BMDCs (c. 60% CD11c+CD11b+) and BMMΦ (c. 70% F4/80+CD11b+). It is possible therefore that some of the responding cell types may be immature bone marrow precursors. However despite this possibility, clear differences in the cytokine proﬁles are seen after exposure to the different isolate antigens. The high levels of IL-10 and IL-6 secreted by these cells may underlie in part how the S isolate survives within the C57BL/6 host. Indeed, the induction of IL-10 and/or IL- 6 in vivo and in vitro has been suggested to aid parasite survival or control host pathology (31–33). IL-10 is a regulatory cytokine (34). It can dampen-down both Th1 and Th2 responses (35) and has been shown to be important in the control of pathology during a T. muris infection (36). These data would suggest that the S isolate E/S has evolved to stimulate a more regulatory environment. In addition, data from our laboratory demonstrates that the S isolate, which survives in C57BL/6 mice, induces more regulatory T cells in vivo compared to the E isolate which is readily expelled (D’Elia et al. unpublished data). The increase of IL-6 is slightly more puzzling, with the majority of IL-6 data focusing on the ability of IL-6, in con- junction with TGF-β, to induce Th17 cells. Further, IL-6 is known to inhibit the development of regulatory T cells (37). However the data here shows that BMMΦ secrete lower levels (similar to unstimulated) of TGF-β when stimulated with S isolate E/S compared to the other isolates. Therefore the balance of IL-6 and TGF-β may not be conducive for the generation of a Th17 response. There is contrasting data on the role of IL-6, with some research suggesting that IL- 6 leads to the development of a Th2 response (38) or the © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Parasite Immunology, 31, 123–131 129