Mechanisms of Enterovirus Induced Rat Insulinoma Cell Line Destruction: Role of Cytokines and Signalling Pathways

There is considerable evidence supporting an association between enterovirus (EV), infection, particularly Coxsackievirus B (CVB), and the development of type 1 diabetes. However, the mechanisms for viral induced destruction of pancreatic β-cells are not well understood and it remains unclear whether the effects are strain specific. The central hypothesis of this PhD is that EV infection induces the production of pro-inflammatory cytokine and chemokine in β-cells, causing activation of apoptotic signalling pathways and leading to immune-mediated destruction of β-cells. The major aims of the project were to determine the effect of EV infection in β-cells on (i) production of pro-inflammatory cytokine and chemokine and (ii) activation of apoptotic and anti-apoptotic pathway.The first investigation examined the effects of multiple EVs (CVB1-6; ECHO4, 6, 9, 11 and 18; and two EV71 strains) on the expression of cytokine IFN-γ, IL-1α, IL-1β, IL-5, IL-6, IL-10, IL-18, MIP-1α, EOTAXIN and GCSF) and chemokine CCL-2 and CCL5 in the rat β-cell line INS-1 and RINm5F. The second investigation examined the effects of CVB4 and CVB5 at MOI 1 and MOI 10 on the activation of apoptotic signalling factors such as p38 MAPK, ERK1/2, JNK, NF-κB, and c-Jun and the anti-apoptotic signalling factors, Akt. The third investigation examined cytokine regulation of signalling pathways in INS-1 cells transfected with either IFN-γ or IL-1β and infected with either CVB4 or CVB5. The same signalling factors were investigated in the previous study along with STAT-1. The fourth investigation examined the effects of IFN-γ and IL-1β on CVB4 and CVB5 replication in INS-1.In the first investigation, the levels of CCL-2 and CCL-5 increased in β-cells infected with CVB2, CVB4-5, CVB6, ECHO4, 6, 9 and 11 and not with two strains of EV71. The levels of IFN-γ, IL-1α, IL-1β, IL-5, IL-6, IL-10, IL-18, MIP-1α, EOTAXIN and GCSF, the majority of which are pro-inflammatory cytokines, increased in β-cells infected with CVB4 and CVB5. In the second investigation, CVB4 activated the MAPK kinase (p38 MAPK, ERK1/2 & JNK) and NF-κB pathways (NF-κB & c-Jun) However, CVB5 only activated part of the MAPK kinase (ERK1/2 & JNK) and NF-κB (NF-κB). CVB4 and CVB5 inhibited Akt pathways (Akt). In the third investigation, IFN-γ knockdown inhibited part of the MAPK kinase (ERK1/2) and NF-κB pathways in β-cells infected with CVB4. IL-1β knockdown inhibited part of p38 MAPK (ERK1/2) and NF-κB pathways (NF-κB and c-Jun) in β-cells infected with CVB4. IFN-γ knockdown inhibited part of MAPK kinase (ERK1/2) and part of NF-κB pathways (STAT-1) in β-cells infected with CVB5. Akt increased in β-cells infected with CVB4 & CVB5 and with absence of IFN-γ and IL-1β. In the fourth investigation, IFN-γ knockdown inhibited the later stage of CVB4 and CVB5 replication in β-cells. IL-1β knockdown however, inhibited CVB4 and CVB5 replication at later stage in β-cells.The results of the investigations undertaken in this thesis, contribute to our understanding of the effects of EVs infections in β-cells, in particular, the molecular mechanisms leading to β-cell apoptosis following EV infection. EVs displayed diverse effects on cell tropism, expression of immune mediators and signalling pathways in β-cells, indicating strain specific difference in β-cell tropism