The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

The peripheral naïve T cell pool is comprised of a heterogeneous population of cells at various stages of development, which is a process that begins in the thymus and is completed after a post-thymic maturation phase in the periphery. One hallmark of naïve T cells in secondary lymphoid organs is their unique ability to produce TNF rapidly after activation and prior to acquiring other effector functions. To determine how maturation influences the licensing of naïve T cells to produce TNF, we compared cytokine profiles of CD4(+) and CD8(+) single positive (SP) thymocytes, recent thymic emigrants (RTEs) and mature-naïve (MN) T cells during TCR activation. SP thymocytes exhibited a poor ability to produce TNF when compared to splenic T cells despite expressing similar TCR levels and possessing comparable activation kinetics (upregulation of CD25 and CD69). Provision of optimal antigen presenting cells from the spleen did not fully enable SP thymocytes to produce TNF, suggesting an intrinsic defect in their ability to produce TNF efficiently. Using a thymocyte adoptive transfer model, we demonstrate that the ability of T cells to produce TNF increases progressively with time in the periphery as a function of their maturation state. RTEs that were identified in NG-BAC transgenic mice by the expression of GFP showed a significantly enhanced ability to express TNF relative to SP thymocytes but not to the extent of fully MN T cells. Together, these findings suggest that TNF expression by naïve T cells is regulated via a gradual licensing process that requires functional maturation in peripheral lymphoid organs.

Epidemiologic and clinical evidence suggests that virus infection plays an important role in human type 1 diabetes pathogenesis. We used the virus-inducible BioBreeding Diabetes Resistant (BBDR) rat to investigate the ability of sodium salicylate, a non-steroidal anti-inflammatory drug (NSAID), to modulate development of type 1 diabetes. BBDR rats treated with Kilham rat virus (KRV) and polyinosinic:polycytidylic acid (pIC, a TLR3 agonist) develop diabetes at nearly 100% incidence by ~2 weeks. We found distinct temporal profiles of the proinflammatory serum cytokines, IL-1β, IL-6, IFN-γ, IL-12, and haptoglobin (an acute phase protein) in KRV+pIC treated rats. Significant elevations of IL-1β and IL-12, coupled with sustained elevations of haptoglobin, were specific to KRV+pIC and not found in rats co-treated with pIC and H1, a non-diabetogenic virus. Salicylate administered concurrently with KRV+pIC inhibited the elevations in IL-1β, IL-6, IFN-γ and haptoglobin almost completely, and reduced IL-12 levels significantly. Salicylate prevented diabetes in a dose-dependent manner, and diabetes-free animals had no evidence of insulitis. Our data support an important role for innate immunity in virus-induced type 1 diabetes pathogenesis. The ability of salicylate to prevent diabetes in this robust animal model demonstrates its potential use to prevent or attenuate human autoimmune diabetes.

Despite increasing evidence that autoreactive CD8 T-cells are involved in both the initiation of type 1 diabetes (T1D) and the destruction of beta-cells, direct evidence for their destructive role in-vivo is lacking. To address a destructive role for autoreactive CD8 T-cells in human disease, we assessed the pathogenicity of a CD8 T-cell clone derived from a T1D donor and specific for an HLA-A2-restricted epitope of islet-specific glucose-6-phosphatase catalytic-subunit related protein (IGRP). HLA-A2/IGRP tetramer staining revealed a higher frequency of IGRP-specific CD8 T-cells in the peripheral blood of recent onset human individuals than of healthy donors. IGRP(265-273)-specific CD8 T-cells that were cloned from the peripheral blood of a recent onset T1D individual were shown to secrete IFNγ and Granzyme B after antigen-specific activation and lyse HLA-A2-expressing murine islets in-vitro. Lytic capacity was also demonstrated in-vivo by specific killing of peptide-pulsed target cells. Using the HLA-A2 NOD-scid IL2rγ(null) mouse model, HLA-A2-restricted IGRP-specific CD8 T-cells induced a destructive insulitis. Together, this is the first evidence that human HLA-restricted autoreactive CD8 T-cells target HLA-expressing beta-cells in-vivo, demonstrating the translational value of humanized mice to study mechanisms of disease and therapeutic intervention strategies.

The production of fully immunologically competent humanized mice engrafted with peripheral lymphocyte populations provides a model for in vivo testing of new vaccines, the durability of immunological memory and cancer therapies. This approach is limited, however, by the failure to efficiently engraft human B lymphocytes in immunodeficient mice. We hypothesized that this deficiency was due to the failure of the murine microenvironment to support human B cell survival. We report that while the human B lymphocyte survival factor, B lymphocyte stimulator (BLyS/BAFF) enhances the survival of human B cells ex vivo, murine BLyS has no such protective effect. Although human B cells bound both human and murine BLyS, nuclear accumulation of NF-kappaB p52, an indication of the induction of a protective anti-apoptotic response, following stimulation with human BLyS was more robust than that induced with murine BLyS suggesting a fundamental disparity in BLyS receptor signaling. Efficient engraftment of both human B and T lymphocytes in NOD rag1(-/-) Prf1(-/-) immunodeficient mice treated with recombinant human BLyS is observed after adoptive transfer of human PBL relative to PBS treated controls. Human BLyS treated recipients had on average 40-fold higher levels of serum Ig than controls and mounted a de novo antibody response to the thymus-independent antigens in pneumovax vaccine. The data indicate that production of fully immunologically competent humanized mice from PBL can be markedly facilitated by providing human BLyS.

In vivo implantation of sterile materials and devices results in a foreign body immune response leading to fibrosis of implanted material. Neutrophils, one of the first immune cells to be recruited to implantation sites, have been suggested to contribute to the establishment of the inflammatory microenvironment that initiates the fibrotic response. However, the precise numbers and roles of neutrophils in response to implanted devices remains unclear. Using a mouse model of peritoneal microcapsule implantation, we show 30-500 fold increased neutrophil presence in the peritoneal exudates in response to implants. We demonstrate that these neutrophils secrete increased amounts of a variety of inflammatory cytokines and chemokines. Further, we observe that they participate in the foreign body response through the formation of neutrophil extracellular traps (NETs) on implant surfaces. Our results provide new insight into neutrophil function during a foreign body response to peritoneal implants which has implications for the development of biologically compatible medical devices.

Inhibition of PD1-PDL1 signaling in NOD mice accelerates onset of type 1 diabetes implicating this pathway in suppressing the emergence of pancreatic beta cell reactive T-cells. However, the molecular mechanism by which PD1 signaling protects from type 1 diabetes is not clear. We hypothesized that differential susceptibility of Idd mouse strains to type 1 diabetes when challenged with anti PDL1 will identify genomic loci that collaborate with PD1 signaling in suppressing type 1 diabetes.

The centrosome is important for microtubule organization and cell cycle progression in animal cells. Recently, mutations in the centrosomal protein, pericentrin, have been linked to human microcephalic osteodysplastic primordial dwarfism (MOPD II), a rare genetic disease characterized by severe growth retardation and early onset of type 2 diabetes among other clinical manifestations. While the link between centrosomal and cell cycle defects may account for growth deficiencies, the mechanism linking pericentrin mutations with dysregulated glucose homeostasis and pre-pubertal onset of diabetes is unknown. In this report we observed abundant expression of pericentrin in quiescent pancreatic beta-cells of normal animals which led us to hypothesize that pericentrin may have a critical function in beta-cells distinct from its known role in regulating cell cycle progression. In addition to the typical centrosome localization, pericentrin was also enriched with secretory vesicles in the cytoplasm. Pericentrin overexpression in beta-cells resulted in aggregation of insulin-containing secretory vesicles with cytoplasmic, but not centrosomal, pericentriolar material and an increase in total levels of intracellular insulin. RNAi- mediated silencing of pericentrin in secretory beta-cells caused dysregulated secretory vesicle hypersecretion of insulin into the media. Together, these data suggest that pericentrin may regulate the intracellular distribution and secretion of insulin. Mice transplanted with pericentrin-depleted islets exhibited abnormal fasting hypoglycemia and inability to regulate blood glucose normally during a glucose challenge, which is consistent with our in vitro data. This previously unrecognized function for a centrosomal protein to mediate vesicle docking in secretory endocrine cells emphasizes the adaptability of these scaffolding proteins to regulate diverse cellular processes and identifies a novel target for modulating regulated protein secretion in disorders such as diabetes.