Rotavirus acceleration of type 1 diabetes in non-obese diabetic mice depends on type I interferon signalling.

Rotavirus infection is associated with childhood progression to type 1 diabetes. Infection by monkey rotavirus RRV accelerates diabetes onset in non-obese diabetic (NOD) mice, which relates to regional lymph node infection and a T helper 1-specific immune response. When stimulated ex vivo with RRV, plasmacytoid dendritic cells (pDCs) from naïve NOD mice secrete type I interferon, which induces the activation of bystander lymphocytes, including islet-autoreactive T cells. This is our proposed mechanism for diabetes acceleration by rotaviruses. Here we demonstrate bystander lymphocyte activation in RRV-infected NOD mice, which showed pDC activation and strong upregulation of interferon-dependent gene expression, particularly within lymph nodes. The requirement for type I interferon signalling was analysed using NOD mice lacking a functional type I interferon receptor (NOD.IFNAR1(-/-) mice). Compared with NOD mice, NOD.IFNAR1(-/-) mice showed 8-fold higher RRV titers in lymph nodes and 3-fold higher titers of total RRV antibody in serum. However, RRV-infected NOD.IFNAR1(-/-) mice exhibited delayed pDC and lymphocyte activation, no T helper 1 bias in RRV-specific antibodies and unaltered diabetes onset when compared with uninfected controls. Thus, the type I interferon signalling induced by RRV infection is required for bystander lymphocyte activation and accelerated type 1 diabetes onset in genetically susceptible mice.

Lessons from the mouse: potential contribution of bystander lymphocyte activation by viruses to human type 1 diabetes.

Viruses are considered to be potential key modulators of type 1 diabetes mellitus, with several possible mechanisms proposed for their modes of action. Here we discuss the evidence for virus involvement, including pancreatic infection and the induction of T cell-mediated molecular mimicry. A particular focus of this review is the further possibility that virus infection triggers bystander activation of pre-existing autoreactive lymphocytes. In this scenario, the virus triggers dendritic cell maturation and proinflammatory cytokine secretion by engaging pattern recognition receptors. These proinflammatory cytokines provoke bystander autoreactive lymphocyte activation in the presence of cognate autoantigen, which leads to enhanced beta cell destruction. Importantly, this mechanism does not necessarily involve pancreatic virus infection, and its virally non-specific nature suggests that it might represent a means commonly employed by multiple viruses. The ability of viruses specifically associated with type 1 diabetes, including group B coxsackievirus, rotavirus and influenza A virus, to induce these responses is also examined. The elucidation of a mechanism shared amongst several viruses for accelerating progression to type 1 diabetes would facilitate the identification of important targets for disease intervention.

NSP1 of human rotaviruses commonly inhibits NF-κB signalling by inducing ß-TrCP degradation.

Rotavirus is a leading cause of severe gastroenteritis in infants worldwide. Rotavirus nonstructural protein 1 (NSP1) is a virulence factor that inhibits innate host immune responses. NSP1 from some rotaviruses targets host interferon response factors (IRFs), leading to inhibition of type I interferon expression. A few rotaviruses encode an NSP1 that inhibits the NF-κB pathway by targeting ß-TrCP, a protein required for IκB degradation and NF-κB activation. Available evidence suggests that these NSP1 properties involve proteosomal degradation of target proteins. We show here that NSP1 from several human rotaviruses and porcine rotavirus CRW-8 inhibits the NF-κB pathway, but cannot degrade IRF3. Furthermore, ß-TrCP levels were much reduced in cells infected with these rotaviruses. This provides strong evidence that ß-TrCP degradation is required for NF-κB pathway inhibition by NSP1 and demonstrates the relevance of ß-TrCP degradation to rotavirus infection. C-terminal regions of NSP1, including a serine-containing motif resembling the ß-TrCP recognition motif of IκB, were required for NF-κB inhibition. CRW-8 infection of HT-29 intestinal epithelial cells induced significant levels of IFN-ß and CCL5 but not IL-8. This contrasts with monkey rotavirus SA11-4F, whose NSP1 inhibits IRF3 but not NF-κB. Substantial amounts of IL-8 but not IFN-ß or CCL5 were secreted from HT-29 cells infected with SA11-4F. Our results show that human rotaviruses commonly inhibit the NF-κB pathway by degrading ß-TrCP and thus stabilizing IκB. They suggest that NSP1 plays an important role during human rotavirus infection by inhibiting the expression of NF-κB-dependent cytokines, such as IL-8.

VP7 of Rhesus monkey rotavirus RRV contributes to diabetes acceleration in association with an elevated anti-rotavirus antibody response.

T cell-receptor transgenic NOD8.3 mice provide a model for spontaneous type 1 diabetes development. Infection of 5 week-old NOD8.3 mice with Rhesus monkey rotavirus (RRV) accelerates the onset of their diabetes. This acceleration requires virus replication and relates to the presence and level of serum anti-rotavirus antibodies, but the role of individual RRV genes is unknown. Here we assessed the importance for diabetes acceleration of the RRV genes encoding VP4 and VP7, by infecting NOD8.3 mice with parental and reassortant rotaviruses. Diabetes was accelerated by reassortant rotaviruses containing RRV VP7 on a UK rotavirus genetic background, but not by parental UK or a UK reassortant containing RRV VP4 without VP7. Diabetes acceleration by reassortant rotaviruses containing RRV VP7 depended on the development of a high serum anti-rotavirus antibody titer. This study shows that VP7, together with an elevated anti-rotavirus antibody response, contributes to the acceleration of diabetes onset by RRV.

Rotavirus activates lymphocytes from non-obese diabetic mice by triggering toll-like receptor 7 signaling and interferon production in plasmacytoid dendritic cells.

It has been proposed that rotavirus infection promotes the progression of genetically-predisposed children to type 1 diabetes, a chronic autoimmune disease marked by infiltration of activated lymphocytes into pancreatic islets. Non-obese diabetic (NOD) mice provide a model for the human disease. Infection of adult NOD mice with rhesus monkey rotavirus (RRV) accelerates diabetes onset, without evidence of pancreatic infection. Rather, RRV spreads to the pancreatic and mesenteric lymph nodes where its association with antigen-presenting cells, including dendritic cells, induces cellular maturation. RRV infection increases levels of the class I major histocompatibility complex on B cells and proinflammatory cytokine expression by T cells at these sites. In autoimmunity-resistant mice and human mononuclear cells from blood, rotavirus-exposed plasmacytoid dendritic cells contribute to bystander polyclonal B cell activation through type I interferon expression. Here we tested the hypothesis that rotavirus induces bystander activation of lymphocytes from NOD mice by provoking dendritic cell activation and proinflammatory cytokine secretion. NOD mouse splenocytes were stimulated with rotavirus and assessed for activation by flow cytometry. This stimulation activated antigen-presenting cells and B cells independently of virus strain and replicative ability. Instead, activation depended on virus dose and was prevented by blockade of virus decapsidation, inhibition of endosomal acidification and interference with signaling through Toll-like receptor 7 and the type I interferon receptor. Plasmacytoid dendritic cells were more efficiently activated than conventional dendritic cells by RRV, and contributed to the activation of B and T cells, including islet-autoreactive CD8+ T cells. Thus, a double-stranded RNA virus can induce Toll-like receptor 7 signaling, resulting in lymphocyte activation. Our findings suggest that bystander activation mediated by type I interferon contributes to the lymphocyte activation observed following RRV infection of NOD mice, and may play a role in diabetes acceleration by rotavirus.

Rotavirus acceleration of murine type 1 diabetes is associated with increased MHC class I-restricted antigen presentation by B cells and elevated proinflammatory cytokine expression by T cells.

Rotavirus infection has been proposed to enhance progression towards type 1 diabetes in at-risk children. Rhesus monkey rotavirus (RRV) accelerates diabetes onset in non-obese diabetic (NOD) and T cell receptor transgenic NOD8.3 mice. Infected NOD mice show virus spread to pancreatic lymph nodes (PLN) and mesenteric lymph nodes (MLN), induction of a serum T helper 1-biased specific antibody response and proinflammatory cytokine mRNA expression in PLN and islets. Here, we analysed the effects of RRV infection on intestinal responses and the activation of antigen presenting cells (APC), T cells and B cells in PLN, MLN, spleen and islets. Diabetes acceleration by RRV was associated with minimal immune activation in Peyer's patches. Increased proinflammatory cytokine expression by APC, including dendritic cells, was observed exclusively in the PLN, while cytokine expression by T cells was detected in islets, PLN, MLN and spleen. RRV infection of NOD8.3 mice increased IFNγ expression by CD8(+) T cells, which primarily recognise an islet autoantigen. A peptide corresponding to RRV VP7 amino acids 5-13, with sequence similarity to this islet autoantigen, did not induce activation or proliferation of NOD8.3 mouse T cells. RRV infection of NOD mice elevated B cell MHC I expression in PLN and MLN, and increased the B cell-mediated proliferation of islet antigen-specific CD8(+) T cells. These studies demonstrate that RRV infection of NOD mice activates APC, T cells and B cells at sites where autoreactive lymphocytes accumulate, in association with proinflammatory cytokine expression and an increased capacity to present antigen. Taken together with previous findings, these data support a possible role for bystander activation in type 1 diabetes acceleration by RRV.

Alteration of the thymic T cell repertoire by rotavirus infection is associated with delayed type 1 diabetes development in non-obese diabetic mice.

Rotaviruses are implicated as a viral trigger for the acceleration of type 1 diabetes in children. Infection of adult non-obese diabetic (NOD) mice with rotavirus strain RRV accelerates diabetes development, whereas RRV infection in infant NOD mice delays diabetes onset. In this study of infant mice, RRV titers and lymphocyte populations in the intestine, mesenteric lymph nodes (MLN) and thymus of NOD mice were compared with those in diabetes-resistant BALB/c and C57BL/6 mice. Enhanced intestinal RRV infection occurred in NOD mice compared with the other mouse strains. This was associated with increases in the frequency of CD8αß TCRαß intraepithelial lymphocytes, and their PD-L1 expression. Virus spread to the MLN and T cell numbers there also were greatest in NOD mice. Thymic RRV infection is shown here in all mouse strains, often in combination with alterations in T cell ontogeny. Infection lowered thymocyte numbers in infant NOD and C57BL/6 mice, whereas thymocyte production was unaltered overall in infant BALB/c mice. In the NOD mouse thymus, effector CD4(+) T cell numbers were reduced by infection, whereas regulatory T cell numbers were maintained. It is proposed that maintenance of thymic regulatory T cell numbers may contribute to the increased suppression of inflammatory T cells in response to a strong stimulus observed in pancreatic lymph nodes of adult mice infected as infants. These findings show that rotavirus replication is enhanced in diabetes-prone mice, and provide evidence that thymic T cell alterations may contribute to the delayed diabetes onset following RRV infection.