Rodgers, David T. (2013) Investigation of the therapeutic potential of ES- 62 in a murine model of SLE. PhD thesis. http://theses.gla.ac.uk/4771/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Investigation of the therapeutic potential of ES-62 in a murine model of SLE David T. Rodgers B.Sc. M.Res. Ph.D A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy The Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow. Copyright David T. Rodgers September 2013 Abstract Autoimmune inflammatory disorders such as systemic lupus erythematosus (SLE) remain debilitating conditions, as many patients are refractory to existing conventional and biologic therapies or suffer serious adverse effects, such as susceptibility to catastrophic infection. Therapies based on the actions of parasite- derived immunomodulators that dampen inflammation to promote the survival of the parasite without seriously immunocompromising the host, may therefore provide alternative strategies for the development of novel and safer drugs. One such molecule, ES-62, protects against disease in mouse models of rheumatoid arthritis and asthma; in both of these pathologies, suppression of disease is due to modulation of pathogenic IL-17A responses. As IL-17A has been implicated in the pathogenesis of SLE, in this thesis, the therapeutic potential of ES-62 is explored in the MRL/lpr mouse model of SLE. SLE is characterized by autoantibody responses to dsDNA, as well as other nuclear and cytoplasmic antigens, which result in the deposition of autoantibody- immune complexes that cause localized inflammation in tissues with dense capillary networks, most often the skin, joints and kidneys. The MRL/lpr mouse is genetically predisposed to develop lupus-like pathology displaying many of the characteristics of human disease, including the major cause of morbidity, glomerulonephritis. Twice weekly treatment of MRL/lpr mice with ES-62 significantly suppressed the development of proteinuria, a direct measure of renal dysfunction. Despite drastic improvement in renal function, the kidneys from ES-62 treated mice did not show substantial improvement in histopathology as indicated by the overall levels of glomeruloproliferation, cellular infiltration and complement or immunoglobulin deposition in the kidneys. However, exposure to ES-62 did reduce the expression of complement (C3aR and C5aR) and immunoglobulin receptors (FcγRI (CD64)), thus rendering renal cells hypo-responsive to these pro- inflammatory stimuli. Moreover, by modulating MyD88 signaling, ES-62 likely suppresses renal cell responsiveness to chronic DAMP and IL-1 signals as well as potentially promoting glomerular barrier stability. ! ii! Consistent with their hypo-responsive phenotype, renal fibroblasts from ES-62 treated mice produced less MCP-1 in response to TLR stimulation and this was associated with reduced infiltration into the kidney by effector T and B cells and granulocytes; along with the ability of the parasite product to modulate the production of the pro-inflammatory cytokines IL-17A and IL-22. ES-62 suppressed the production of IL-22 both prior to and following onset of disease suggesting a key role for this cytokine in lupus pathogenesis, a proposal confirmed by neutralization studies which demonstrated that IL-22 played an essential role in the development of disease in the MRL/lpr mouse. This was further supported by studies showing that recombinant IL-22 significantly accelerated and exacerbated disease. By contrast, despite suppressing early IL-17A responses, the production of IL-17A was significantly increased in ES-62 treated mice during the established phase of disease, suggesting that IL-17A may promote pathogenesis during the initiation of pathology, yet act to resolve aberrant inflammation in the kidney. This potential dual role for IL-17A in the regulation of kidney inflammation was corroborated by studies using neutralizing antibodies and recombinant IL-17A, as the early neutralization of IL-17A production slowed the onset and severity of proteinuria and the late administration of rIL-17A suppressed disease severity. Aberrant B cell responses drive pathogenesis both in murine models of SLE and also in human disease: reflecting this, B cell depleting therapies have proved successful in the clinic. Thus, the effects of ES-62 on the population dynamics of effector and regulatory B cell subsets were investigated and these studies revealed that ES-62 induced a hypo-responsive B cell phenotype that was associated with modulated development, migration and/or activation of pathogenic effector B cells. Furthermore, the proportion of IL-10 producing ‘regulatory’ B cells were significantly elevated in the ES-62 treated MRL/lpr mice during the established phase of disease. Crucially, the protection afforded against the development of proteinuria by ES-62 was mimicked by the adoptive transfer of B cells from ES-62 treated MRL/lpr mice: moreover, such protection was associated with modulation of the IL-17A/IL-22 axis, as observed in MRL/lpr mice treated with ES-62. ! iii! Together with previous reports on the therapeutic potential of ES-62 in arthritis and asthma, these studies suggest that therapies based on the parasite product have a future in the clinic. ES-62 itself is not suitable as a therapy, due to it immunogenic nature and the complexity of its biosynthesis: thus small molecular analogues (SMAs) of the parasite product have been synthesized. Two of these were tested in the MRL/lpr mouse and found to suppress the development of proteinuria, even when administered after the onset of pathology. This protection, as with that afforded by ES-62, was associated with a modulation of MyD88 signaling in the kidney and indicates that novel drugs, based on the safe modulation of the immune system by the parasite derived product, ES-62, have the potential to treat lupus nephritis in SLE patients. ! iv! Author’s declaration The work presented in this thesis represents the original work carried out by the author and has not been submitted in any form to another university. Where use has been made of materials provided by others, due acknowledgement has been made. Dr. David T. Rodgers B.sc M.Res Ph.D University of Glasgow September 2013 ! v! To the late nighters and weekend writers, keep fighting the good fight. ! vi! Acknowledgements First and foremost, I would like to thank my supervisor, Professor Maggie Harnett: you have been such an inspiration to work with that I feel honored to have learned my trade in your lab. I think the character and spirit of the Harnett lab at Glasgow has been, and continues to be, extremely warm and welcoming; this permeation over the years can only be a testament to the positive support and guidance that you have selflessly provided. Furthermore, I would like to thank Professor Billy Harnett for additional scientific support, mentoring, sharing a beer and taking my jokes whenever Liverpool beat Manchester United. It has been great getting to know you both and working with you over the past 4 years, I look forward to our future collaborations. My heartfelt thanks and gratitude go to the members of the Harnett labs, both old and new, who have made me feel welcome and appreciated for the entirety of my time in the lab. I’ve enjoyed my time with you in Glasgow and look forward seeing you all in the future. I would particularly like to thank those of you who have been closest to me over the years (Miguel, Rusty, Dim, Verica and Jen) as well as those non-Harnetts that have been there all the way (Vuk, Laura and Sharon) our trips to the hills, the pub and Newcastle have kept me sane over the years; although the latter may have made the rest of you insane! Special thanks has to go to the University of Glasgow for hosting he, and to the Wellcome Trust for providing an exceptional program that has allowed to me experience life and science in this great city, not to forget the summer working in the South of France. Furthermore, I would like to thank the trust for funding my scientific travel to the dull destinations of Whistler and Hawai; and for supporting my bench work, which has kept me occupied and off the streets for the past 4 years. On the topic of bench work, I would like to thank some of the most important members of the lab that are often overlooked in acknowledgements, I can’t list you all but I have to mention the Ranin electronic pipettes: without you, I couldn’t have done it. On a more serious note, there are people in the department that have provided support and technical assistance over the years: mainly Jim, Shauna, Robin and Dianne in the GBRC. I would also like to thank Tony, Sandra, Joanne and Denis at the CRF for looking after the lads. I would like to thank my family and friends, with special thanks to parents and sister, for providing me with support and inspiration; I would be nowhere today without their abiding love, dedication and tolerance of my disappearance into the world of science for weeks on end and for always phoning when the football or Strictly is on.