A Helminth Immunomodulator Exploits Host Signaling Events to Regulate Cytokine Production in Macrophages

Parasitic worms alter their host's immune system to diminish the inflammatory responses directed against them, using very efficient immunomodulating molecules. We have previously shown that the helminth immunomodulator cystatin (AvCystatin) profoundly reduces the progression of inflammatory diseases via modulation of macrophages. Here we elucidate the signaling events in macrophages triggered by AvCystatin. Labeled AvCystatin was predominantly taken up by macrophages and subsequently induced the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38. IL-10 expression induced by AvCystatin in macrophages was tyrosine kinase sensitive and dependent on activation of both MAP kinases, in clear contrast to expression of IL-12/23p40. In addition, phosphorylation of the transcription factors CREB and STAT3 was induced by AvCystatin and regulated by phospho-ERK. Chemical inhibition of phosphoinositide 3-kinase (PI3K) reduced AvCystatin-induced cytokine release; however, AKT, the downstream target of PI3K, was not activated following AvCystatin exposure. To characterize signaling elements involved in alteration of the macrophage phenotype we applied mathematical modeling. Experimental testing of the in silico generated hypotheses identified dual specificity phosphatase (DUSP) 1 and 2, as regulators in AvCystatin triggered macrophages in vitro and in vivo. In particular, DUSP1 was subsequently found to be responsible for regulation of ERK- and p38-phosphorylation and controlled the IL-10 expression in macrophages by AvCystatin. Thus, we show that AvCystatin exploits activation and deactivation pathways of MAP kinases to induce regulatory macrophages. This study provides insights into molecular mechanisms of macrophage manipulation by parasites and highlights the utility of mathematical modeling for the elucidation of regulatory circuits of immune cells. Author Summary: Helminths have the ability to interfere with their host's immune response, thus downregulating inflammatory responses. We previously demonstrated the role of helminth infections or isolated helminth proteins in suppressing bystander immune responses in mouse models of allergy and colitis via a macrophage and IL-10 dependent mechanism. The current study elucidates the signaling events induced by the parasite immunomodulator AvCystatin, leading to alteration of the macrophage phenotype. AvCystatin was predominantly taken up by macrophages and induced cytokine production by phosphorylation of mitogen-activated protein kinases (MAPK) ERK1/2 and p38. To identify molecules involved in the regulation of IL-10 production we developed a mathematical model. In silico generated data suggested a negative feedback mechanism via deactivating ERK1/2 and p38. Ensuing experiments validated the model and revealed AvCystatin-induced dual specificity phosphatases (DUSPs) as negative regulators of MAPK activation and IL-10 expression in vitro and in vivo. Taken together, the nematode immunomodulator AvCystatin targets activating and deactivating pathways of MAPK to induce regulatory macrophages.