Understanding immunobiology of chikungunya virus disease using mouse models

Abstract

Chikungunya virus (CHIKV) is a mosquito borne alphavirus, whose primary disease manifestation in humans is acute and chronic, often debilitating polyarthritis/polyathralgia. Current treatments are often inadequate. This thesis aims to elucidate the roles of several immune components in CHIKV disease, using a mouse model of CHIKV infection and disease, which recapitulates many aspects of human disease. An improved understanding of the immunobiology of CHIKV is required for future development of new interventions for this recently re-emergent virus. CHIKV polyarthritis/polyathralgia is associated with a prolific monocytes/macrophage infiltration of musculoskeletal tissue in humans, monkeys and mice. The infection in all species is associated with high levels of chemokine (C-C motif) ligand 2 (CCL2), a chemokine that binds chemokine (C-C motif) receptor 2 (CCR2) and is involved in recruitment of monocytes to the sites of infection. Targeting CCL2 or CCR2 signalling has thus been proposed as a potential treatment option for CHIKV disease. Results herein show that CHIKV infection of mice deficient in CCR2 led to a significantly more pronounced and prolonged arthritis that was associated with cartilage damage. The prominent monocytes/macrophages infiltrate seen in wild-type mice was replaced by a prolific neutrophil infiltrate in CCR2-/- mice. The switch in the arthritic infiltrate was associated with changes in the expression of multiple inflammatory mediators, with the loss of anti- inflammatory macrophages and their activities (e.g. efferocytosis) also implicated in the exacerbated inflammatory response. These results illustrated that the recruited monocytes/macrophages in wild-type mice (although associated with acute disease) also have a protective role in CHIKV arthritis. These results suggest caution may be warranted when considering therapeutic targeting of CCR2 for treatment of CHIKV arthritis (Chapter 3). To study the role of different components of the immune system in CHIKV viraemia and disease, mice deficient in B cells, T cells, CD4+ T cells, natural killer cells and IFN-g were infected with CHIKV. Results herein confirmed that B cells are crucial for controlling CHIKV viraemia, but showed that T cells also play a role, albeit secondary to antibodies. B cell deficient mice experienced a life long viraemia with little overt pathology evident, supporting the view that CHIKV disease is largely an immunopathology. In T and B cell deficient mice, viraemia was likely controlled by chronic up-regulation of tumour necrosis factor and interferon gamma, but not interferon alpha/beta. Importantly, CD4+ T cells and interferon gamma were shown to play important roles in acute CHIKV arthritis, with T cells previously thought not to play a significant role (Chapter 4). CHIKV disease in humans is often associated with chronic arthritic symptoms that persist for months, occasionally over a year, after CHIKV infection. Persistence of CHIKV has been postulated to be responsible for the chronic arthritis. To further investigate this issue, chronic infection and disease parameters were analysed in the established wild-type mouse model of acute CHIKV arthritis. Viraemia is cleared day 4-5 post-infection and neutralising antibodies are detected as early as day 4 post-infection, and rose significantly thereafter. Despite the high levels of neutralising antibodies, replication competent CHIKV could be recovered from feet of infected mice up to day 14 post-infection. Importantly, significant levels of CHIKV RNA (including negative strand RNA) could be detected by quantitative real-time polymerase chain reaction in feet up to day 100 post-infection. Interferon stimulated gene 54, a factor that is up-regulated in response to viral infection and double stranded RNA, was found also to be persistently up-regulated in these feet, suggesting ongoing replication of viral RNA. Microarray analysis of these feet revealed that (i) many of the pathways up-regulated during acute arthritis were maintained during chronic infection, (ii) on-going local inflammatory responses were dominated by type I IFN responses, and (iii) many of the inflammatory pathways identified in chronically infected mouse feet were also found in chronic CHIKV patients. These observations suggest that chronic disease is due to ongoing inflammation stimulated by persistently replicating viral RNA and viral proteins encoded by that RNA (Chapter 5). In conclusion, this thesis has shown, that inflammatory CCR2+ monocyte can be critical for preventing excessive pathology and resolving inflammation, that distinct immune factors control CHIKV viraemia and arthritis, and that long-term persistence of replicating CHIKV RNA might be the cause of protracted arthritic manifestations in CHIKV patients. These studies will hopefully inform future development of intervention for CHIKV.