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Microbes were hypothesized to play a key role in the progression of type 1 diabetes (T1D). We used the LEW1.WR1 rat model of Kilham rat virus (KRV)-induced T1D to test the hypothesis that the intestinal microbiota is involved in the mechanism leading to islet destruction. Treating LEW1.WR1 rats with KRV and a combination of trimethoprim and sulfamethoxazole (Sulfatrim) beginning on the day of infection protected the rats from insulitis and T1D. Pyrosequencing of bacterial 16S rRNA and quantitative RT-PCR indicated that KRV infection resulted in a transient increase in the abundance of Bifidobacterium spp. and Clostridium spp. in fecal samples from day 5- but not day 12-infected versus uninfected animals. Similar alterations in the gut microbiome were observed in the jejunum of infected animals on day 5. Treatment with Sulfatrim restored the level of intestinal Bifidobacterium spp. and Clostridium spp. We also observed that virus infection induced the expression of KRV transcripts and the rapid upregulation of innate immune responses in Peyer's patches and pancreatic lymph nodes. However, antibiotic therapy reduced the virus-induced inflammation as reflected by the presence of lower amounts of proinflammatory molecules in both the Peyer's patches and pancreatic lymph nodes. Finally, Sulfatrim treatment reduced the number of B cells in Peyer's patches and downmodulated adaptive immune responses to KRV, but did not interfere with antiviral Ab responses or viral clearance from the spleen, pancreatic lymph nodes, and serum. The data suggest that gut microbiota may be involved in promoting virus-induced T1D in the LEW1.WR1 rat model.

Viral infections are associated epidemiologically with the expression of type 1 diabetes in humans, but the mechanisms underlying this putative association are unknown. To investigate the role of viruses in diabetes, we used a model of viral induction of autoimmune diabetes in genetically susceptible biobreeding diabetes-resistant (BBDR) rats. BBDR rats do not develop diabetes in viral-Ab-free environments, but approximately 25% of animals infected with the parvovirus Kilham rat virus (KRV) develop autoimmune diabetes via a mechanism that does not involve beta cell infection. Using this model, we recently documented that TLR agonists synergize with KRV infection and increase disease penetrance. We now report that KRV itself activates innate immunity through TLR ligation. We show that KRV infection strongly stimulates BBDR splenocytes to produce the proinflammatory cytokines IL-6 and IL-12p40 but not TNF-alpha. KRV infection induces high levels of IL-12p40 by splenic B cells and Flt-3-ligand-induced bone marrow-derived dendritic cells (DCs) but only low levels of IL-12p40 production by thioglycolate-elicited peritoneal macrophages or GM-CSF plus IL-4-induced bone marrow-derived DCs. KRV-induced cytokine production is blocked by pharmacological inhibitors of protein kinase R and NF-kappaB. Genomic KRV DNA also induces BBDR splenocytes and Flt-3L-induced DCs from wild-type but not TLR9-deficient mice to produce IL-12p40; KRV-induced up-regulation of B lymphocytes can be blocked by TLR9 antagonists including inhibitory CpG and chloroquine. Administration of chloroquine to virus-infected BBDR rats decreases the incidence of diabetes and decreases blood levels of IL-12p40. Our data implicate the TLR9-signaling pathway in KRV-induced innate immune activation and autoimmune diabetes in the BBDR rat.

Virus infection is hypothesized to be an important environmental "trigger" of type 1 diabetes in humans. We used the BBDR rat model to investigate the relationship between viral infection and autoimmune diabetes. BBDR rats are diabetes-free in viral Ab-free housing, but the disease develops in approximately 30% of BBDR rats infected with Kilham rat virus (KRV) through a process that does not involve infection of pancreatic beta cells. Pretreatment with polyinosinic-polycytidylic (poly(I:C)), a ligand of TLR3, acts synergistically to induce diabetes in 100% of KRV-infected rats. The mechanisms by which KRV induces diabetes and TLR3 ligation facilitates this process are not clear. In this study, we demonstrate that activation of the innate immune system plays a crucial role in diabetes induction. We report that multiple TLR agonists synergize with KRV infection to induce diabetes in BBDR rats, as do heat-killed Escherichia coli or Staphylococcus aureus (natural TLR agonists). KRV infection increases serum IL-12 p40 in a strain-specific manner, and increases IL-12 p40, IFN-gamma-inducible protein-10, and IFN-gamma mRNA transcript levels, particularly in the pancreatic lymph nodes of BBDR rats. Infection with vaccinia virus or H-1 parvovirus induced less stimulation of the innate immune system and failed to induce diabetes in BBDR rats. Our results suggest that the degree to which the innate immune system is activated by TLRs is important for expression of virus-induced diabetes in genetically susceptible hosts.

Macrophages play a critical role in the pathogenesis of Kilham rat virus (KRV)-induced autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats. This investigation was initiated to determine the role of macrophage-derived soluble mediators, particularly NO, in the pathogenesis of KRV-induced diabetes in DR-BB rats. We found that the expression of inducible NO synthase (iNOS), an enzyme responsible for NO production, was significantly increased during the early phase of KRV infection. Inhibition of iNOS by aminoguanidine (AG) treatment resulted in the prevention of diabetes in KRV-infected animals. The expression of IL-1beta, TNF-alpha, and IL-12 was significantly decreased in the spleen of AG-treated, KRV-infected DR-BB rats compared with PBS-treated, KRV-infected control rats. Subsequent experiments revealed that AG treatment exerted its preventive effect in KRV-infected rats by maintaining the finely tuned immune balance normally disrupted by KRV, evidenced by a significant decrease in the expression of IFN-gamma, but not IL-4, and a decrease in Th1-type chemokine receptors CCR5, CXCR3, and CXCR4. We also found that iNOS inhibition by AG decreased the KRV-induced expression of MHC class II molecules and IL-2R alpha-chain, resulting in the suppression of T cell activation, evidenced by the decreased cytolytic activity of CD8(+) T cells. We conclude that NO plays a critical immunoregulatory role by up-regulating macrophage-derived proinflammatory cytokines, up-regulating the Th1 immune response, and activating T cells, leading to type 1 diabetes after KRV infection, whereas suppression of NO production by AG treatment prevents KRV-induced autoimmune diabetes in DR-BB rats.

Viruses are believed to contribute to the pathogenesis of autoimmune type 1A diabetes in humans. This pathogenic process can be modeled in the BBDR rat, which develops pancreatic insulitis and type 1A-like diabetes after infection with Kilham's rat virus (RV). The mechanism is unknown, but does not involve infection of the pancreatic islets. We first documented that RV infection of BBDR rats induces diabetes, whereas infection with its close homologue H-1 does not. Both viruses induced similar humoral and cellular immune responses in the host, but only RV also caused a decrease in splenic CD4(+)CD25(+) T cells in both BBDR rats and normal WF rats. Surprisingly, RV infection increased CD4(+)CD25(+) T cells in pancreatic lymph nodes of BBDR but not WF rats. This increase appeared to be due to the accumulation of nonproliferating CD4(+)CD25(+) T cells. The results imply that the reduction in splenic CD4(+)CD25(+) cells observed in RV-infected animals is virus specific, whereas the increase in pancreatic lymph node CD4(+)CD25(+) cells is both virus and rat strain specific. The data suggest that RV but not H-1 infection alters T cell regulation in BBDR rats and permits the expression of autoimmune diabetes. More generally, the results suggest a mechanism that could link an underlying genetic predisposition to environmental perturbation and transform a "regulated predisposition" into autoimmune diabetes, namely, failure to maintain regulatory CD4(+)CD25(+) T cell function.

Kilham rat virus (KRV) causes autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats; however, the mechanism by which KRV induces autoimmune diabetes without the direct infection of beta cells is not well understood. We first asked whether molecular mimicry, such as a common epitope between a KRV-specific peptide and a beta cell autoantigen, is involved in the initiation of KRV-induced autoimmune diabetes in DR-BB rats. We found that KRV peptide-specific T cells generated in DR-BB rats infected with recombinant vaccinia virus expressing KRV-specific structural and nonstructural proteins could not induce diabetes, indicating that molecular mimicry is not the mechanism by which KRV induces autoimmune diabetes. Alternatively, we asked whether KRV infection of DR-BB rats could disrupt the finely tuned immune balance and activate autoreactive T cells that are cytotoxic to beta cells, resulting in T cell-mediated autoimmune diabetes. We found that both Th1-like CD45RC+CD4+ and cytotoxic CD8+ T cells were up-regulated, whereas Th2-like CD45RC-CD4+ T cells were down-regulated, and that isolated and activated CD45RC+CD4+ and CD8+ T cells from KRV-infected DR-BB rats induced autoimmune diabetes in young diabetes-prone BioBreeding (DP-BB) rats. We conclude that KRV-induced autoimmune diabetes in DR-BB rats is not due to molecular mimicry, but is due to a breakdown of the finely tuned immune balance of Th1-like CD45RC+CD4+ and Th2-like CD45RC-CD4+ T cells, resulting in the selective activation of beta cell-cytotoxic effector T cells.

Inoculation of the UMass strain of rat virus (RV-UMass) into adult immunocompetent rats results in a prolonged subclinical infection that is resolved in 4 to 8 wk. Co-labeling studies, using in situ hybridization (ISH) and immunohistochemistry (IHC), confirmed that RV-UMass was lymphocytotropic and capable of infecting CD4+ and CD8+ T cells as well as B cells. ISH studies also revealed that virus replication was restricted in unstimulated cells but was productive in concanavalin A-stimulated lymphocytes. A corollary of productive infection of lymphocytes was the suppression of lymphocyte functions. Although RV-UMass did not appear to induce phenotypic changes during the course of infection, cells from infected rats had diminished proliferation and cytolytic responses. Both peripheral and mesenteric lymph node cells exhibited only partial recovery of their proliferative and cytolytic capacities one month after infection. Furthermore, RV-UMass-infected tissue culture maintained alloreactive CD4+ T cells in vitro, and a nonlethal infection of this T cell line inhibited Ag- and IL-2-induced proliferation. Because parvoviruses are widespread among laboratory rodents, these findings emphasize the importance of identifying and excluding parvovirus infection in rodents and in cultures of rat T lymphocytes.