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Abstract / Cytokine 70 (2014) 28–79

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54 Helminth-induced innate type-2 cytokines protect against autoimmunity

56 Circulating fibrocytes contribute to the pathogenesis of rheumatoid arthritis

Conor M. Finlay, Anna M. Stefanska, Patrick T. Walsh, Kingston H.G. Mills, School of Biochemistry and Immunology, Trinity College Dublin, Dublin, Ireland

Carole L. Galligan 1,2, Edward C. Keystone 1,3, Eleanor N. Fish 1,2, 1 University Health Network, Toronto, Ontario, Canada, 2 Immunology, University of Toronto, Toronto, Ontario, Canada, 3 Rebecca MacDonald Centre for Arthritis & Autoimmune Diseases, Mount Sinai Hospital, Toronto, Ontario, Canada

Helminth parasites typically establish chronic infections in the mammalian host which can persist for decades. Helminths evade protective host immune responses by promoting innate and adaptive anti-inflammatory networks which can also suppress immune responses to unrelated antigens. Indeed, it has been shown parasitic infection of humans is associated with a lower incidence of allergy and autoimmune disease in rural areas of the developing world. Experimental models of autoimmunity have revealed that helminth-induced protection against disease can be mediated by the action of either Treg or Th2 cells [1]. Here we examined the immunoregulatory effects of the excretory-secretory (ES) products of the helminth parasite, Fasciola hepatica, in the mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Administration of ES delayed the onset and attenuated the clinical severity of EAE which was associated with a decreased production of auto antigen-specific IFN-c and IL-17. Unexpectedly, IL-10, TGF-b, or regulatory T cells did not play a role in the ES-induced attenuation of disease. Moreover, ES-elicited T cells did not suppress EAE and protection from disease was also retained in IL-4-deficient mice, suggesting that Th2 cells do not play a role in ES-induced amelioration of EAE. However adoptive transfer of macrophages or eosinophils, from ES-treated mice transferred protection. Furthermore, IL-5, a cytokine which mediates eosinophilia in vivo, was required for ES-induced protection against EAE. We are currently investigating the role of the epithelial cell-derived ‘alarmin’ cytokine, IL-33, in mediating the regulatory effects of ES. We have determined that IL-33 acts on mast cells to promote the alternative activation of macrophages with regulatory capacity while also promoting eosinophilia in vivo. These findings provide new insights into how helminths target innate immune cells in order to modulate the immune response thereby inhibiting the Th1 and Th17 responses that mediate inflammatory disease.

Rheumatoid arthritis (RA) is a systemic autoimmune disease resulting in joint inflammation and bone and cartilage destruction. We have identified a circulating cell population – fibrocytes – that is activated early in RA patients and mice with arthritis. As RA is characterized by autoreactive T cells, our objective was to characterize fibrocyte activation in the context of T cell activation. Using multiparameter phosphoflow FACS, we show increased numbers of activated circulating fibrocytes in the blood of RA patients, with increased TLR expression, in agreement with increased levels of phosphorylation of NFkB and IRF3. Pathogen/ligand activation of TLRs may be associated with fibrocyte activation. We observe a direct correlation between the number of circulating fibrocytes and CD4 + T cells in RA patients, with higher numbers of proinflammatory, activated Th17 cells in the circulation of RA patients compared with healthy individuals: increased phosphorylation of ZAP70, STAT3 and CD40L. Notably, activated fibrocytes in RA patients exhibit increased expression of CD40. Co-culture of naïve murine CD4 + T cells with activated fibrocytes enhances fibrocyte and T cell proliferation and polarizes the T cells to Th17 cells. Inclusion of anti-IL-6 antibodies reverses Th17 polarization and enhances Treg numbers. We infer that fibrocyte-T cell CD40-CD40L interactions result in IL-6 production that drives Th17 lineage commitment. Additionally, activated T cells secrete cytokines that enhance fibrocyte proliferation, in vitro. Gene expression analysis of activated fibrocytes and RA joint T cells reveals factors associated with a potential paracrine activation loop between fibrocytes and T cells. Altogether our data suggest that activated fibrocytes interact with T cells to promote RA pathogenesis. http://dx.doi.org/10.1016/j.cyto.2014.07.063

Reference [1] Finlay CM, Walsh KP, Mills KHG. Induction of regulatory cells by helminth parasites: exploitation for the treatment of inflammatory diseases. Immunol Rev 2014;259:206–30. http://dx.doi.org/10.1016/j.cyto.2014.07.061

55 IFN-ß regulation of glucose metabolism is PI3K/Akt dependent and important for antiviral activity against coxsackievirus B3 Daniel Burke 1, Leonidas C. Platanias 2, Eleanor N. Fish 3, 1 Immunology, University of Toronto, Toronto, Ontario, Canada, 2 Robert H. Lurie Comprehensive Cancer Center, Northwestern University Medical School, Chicago, IL, USA, 3 University Health Network, Toronto, ON, Canada An effective type I interferon (IFN)-mediated immune response requires the rapid expression of antiviral proteins that are necessary to inhibit viral replication and virus spread. We provide evidence that IFN-ß regulates metabolic events important for the induction of a rapid antiviral response. IFN-ß decreases the phosphorylation of AMPactivated protein kinase (AMPK), coincident with an increase in intracellular ATP. Our data suggest that IFN-ß regulates glucose metabolism mediated by signaling effectors similarly to activation by insulin. Our studies reveal a biphasic IFN-ß-inducible uptake of glucose by cells, mediated by PI3K/Akt, and IFN-ß-inducible regulation of GLUT4 translocation to the cell surface. Additionally, we provide evidence that IFN-ß-regulated glycolytic metabolism is important for the acute induction of an antiviral response during infection with cardiotropic coxsackievirus B3 (CVB3). Interference with IFN-ß-inducible glucose metabolism diminishes the antiviral response, whereas treatment with metformin, a drug that increases insulin sensitivity, enhances the antiviral potency of IFN-ß against CVB3 both in vitro and in vivo. Taken together, these findings highlight an important role for IFN-ß in modulating glucose metabolism during a virus infection and suggest that the use of metformin in combination with IFN-ß during acute virus infection may result in enhanced antiviral responses. http://dx.doi.org/10.1016/j.cyto.2014.07.062

57 CCL5–CCR5 interactions increase tumor cell metabolism to promote proliferation and invasion of breast cancer cells Darrin F. Gao 1,2, Eleanor N. Fish 1,2, 1 University Health Network, Toronto, Ontario, Canada, 2 Immunology, University of Toronto, Toronto, Ontario, Canada Tumor cells have acquired effectors of the innate immune system, including chemokines and their receptors, to facilitate invasion, migration and metastasis. While CCL5 may promote efficient anti-tumor immune responses, it is also associated with cancer progression and metastasis: CCL5 levels are high in more aggressive forms of breast cancer and is a predictor of rapid disease progression in stage II breast cancer. Increases in metabolic activity of cancer cells, specifically increased glycolytic activity and increased expression of glucose transporters, are associated with tumor progression. In earlier studies we provided evidence that for T cells, CCL5 activation of CCR5 results in increased glycolysis, enhanced ATP production and activation of the nutrient sensing AMPK pathway, all associated with enabling CCL5-mediated chemotaxis. Accordingly, we undertook studies to examine whether CCL5–CCR5 interactions enhance breast cancer cell metabolism to influence proliferation and invasion. We provide evidence that, at physiological levels, CCL5 enhances the proliferation of MCF-7.CCR5 breast cancer cells through mTOR-dependent increased translation of proliferation and survival proteins. We have extended these studies to include a broader panel of human breast cancer cell lines expressing CCR5, and tumor cells from PyMT-MMTV transgenic mice that spontaneously develop mammary tumors. CCL5 enhances the proliferation of the human breast cancer cell lines and the mouse mammary tumor cells, mediated by CCR5 activation. This enhanced proliferation is mTOR-dependent. Associated with enhanced proliferation we show CCL5 increases cell surface expression of GLUT1 and increases glucose uptake and ATP production. We provide evidence that increased glucose uptake is associated with enhanced glycolysis, as measured by extracellular acidification and increased glucose-6-phosphate and pryruvate. Moreover, CCL5 enhances the invasive capacity of these breast cancer cells. In vivo, we demonstrate that PyMT-MMTV mice lacking CCR5 exhibit a delayed onset of tumor formation and a lower tumor burden. Viewed altogether, these data suggest that targeting CCL5–CCR5 interactions that increase tumor metabolism may be an effective therapeutic strategy to limit tumor proliferation and tissue invasion. http://dx.doi.org/10.1016/j.cyto.2014.07.064