Viral replication rate regulates clinical outcome and CD8 T cell responses during highly pathogenic H5N1 influenza virus infection in mice

Since the first recorded infection of humans with H5N1 viruses of avian origin in 1997, sporadic human infections continue to occur with a staggering mortality rate of >60%. Although sustained human-to-human transmission has not occurred yet, there is a growing concern that these H5N1 viruses might acquire this trait and raise the specter of a pandemic. Despite progress in deciphering viral determinants of pathogenicity, we still lack crucial information on virus/immune system interactions pertaining to severe disease and high mortality associated with human H5N1 influenza virus infections. Using two human isolates of H5N1 viruses that differ in their pathogenicity in mice, we have defined mechanistic links among the rate of viral replication, mortality, CD8 T cell responses, and immunopathology. The extreme pathogenicity of H5N1 viruses was directly linked to the ability of the virus to replicate rapidly, and swiftly attain high steady-state titers in the lungs within 48 hours after infection. The remarkably high replication rate of the highly pathogenic H5N1 virus did not prevent the induction of IFN-β or activation of CD8 T cells, but the CD8 T cell response was ineffective in controlling viral replication in the lungs and CD8 T cell deficiency did not affect viral titers or mortality. Additionally, BIM deficiency ameliorated lung pathology and inhibited T cell apoptosis without affecting survival of mice. Therefore, rapidly replicating, highly lethal H5N1 viruses could simply outpace and overwhelm the adaptive immune responses, and kill the host by direct cytopathic effects. However, therapeutic suppression of early viral replication and the associated enhancement of CD8 T cell responses improved the survival of mice following a lethal H5N1 infection. These findings suggest that suppression of early H5N1 virus replication is key to the programming of an effective host response, which has implications in treatment of this infection in humans.     

MAP kinases function downstream of HSP90 and upstream of mitochondria in TMV resistance gene N-mediated hypersensitive cell death

Although the involvement of heat shock protein 90 (HSP90), mitogen-activated protein kinase (MAPK) cascades and organelle dysfunction in plant hypersensitive cell death has been suggested, the mutual relationship among them has not been elucidated. Here, we show the molecular network of HSP90, the wound-induced protein kinase (WIPK)/salicylic acid-induced protein kinase (SIPK)-mediated MAPK cascade and mitochondrial dysfunction in tobacco mosaic virus (TMV) resistance gene N-dependent cell death. p50, the Avr component for N, NtMEK2(DD), a constitutively active form of a MAPK kinase of WIPK/SIPK, and a mammalian pro-apoptotic factor Bax were used for cell death induction. Suppression of HSP90 and treatment with geldanamycin, a specific inhibitor of HSP90, compromised p50- but not NtMEK2(DD)- or Bax-mediated cell death accompanying the reduction of NtMEK2, WIPK and SIPK activation. In WIPK/SIPK-double knockdown plants, p50- and NtMEK2(DD)- but not Bax-mediated cell death was suppressed. All three types of cell death induced mitochondrial dysfunction, but they were similarly suppressed by Bcl-xL, which is a mammalian anti-apoptotic factor, and prevents mitochondrial dysfunction in plants as it does in animals in the cell death signal pathway. Taken together with the expression profile of hypersensitive reaction marker genes, it was indicated that the MAPK cascade functions downstream of HSP90 and transduces the cell death signal to mitochondria for N gene-dependent cell death. Furthermore, we found that WIPK and SIPK are functionally redundant in cell death signaling using WIPK/SIPK single or double knockdown plants.     

Generation of insulin-secreting cells from pancreatic acinar cells of animal models of type 1 diabetes

We recently found that pancreatic acinar cells isolated from normal adult mouse can transdifferentiate into insulin-secreting cells in vitro. Using two different animal models of type 1 diabetes, we show here that insulin-secreting cells can also be generated from pancreatic acinar cells of rodents in the diabetic state with absolute insulin deficiency. When pancreatic acinar cells of streptozotocin-treated mice were cultured in suspension in the presence of epidermal growth factor and nicotinamide under low-serum condition, expressions of insulin genes gradually increased. In addition, expressions of other pancreatic hormones, including glucagon, somatostatin, and pancreatic polypeptide, were also induced. Analysis by the Cre/loxP-based direct cell lineage tracing system revealed that these newly made cells originated from amylase-expressing pancreatic acinar cells. Insulin secretion from the newly made cells was significantly stimulated by high glucose and other secretagogues. In addition, insulin-secreting cells were generated from pancreatic acinar cells of Komeda diabetes-prone rats, another animal model of type 1 diabetes. The present study demonstrates that insulin-secreting cells can be generated by transdifferentiation from pancreatic acinar cells of rodents in the diabetic state and further suggests that pancreatic acinar cells represent a potential source of autologous transplantable insulin-secreting cells for treatment of type 1 diabetes.     

Antinecrotic and antiapoptotic effects of hepatocyte growth factor on cholestatic hepatitis in a mouse model of bile-obstructive diseases

Cholestasis, an impairment of bile outflux, frequently occurs in liver diseases. In this process, an overaccumulation of bile acids causes hepatocyte necrosis and apoptosis, leading to advanced hepatitis. Hepatocyte growth factor (HGF) is mitogenic toward hepatocytes, but it is still unclear whether HGF has physiological and therapeutic functions during the progression of cholestasis. Using anti-HGF IgG or recombinant HGF in mice that had undergone bile duct ligation (BDL), we investigated the involvement of HGF in cholestasis-induced hepatitis. After the BDL surgery, HGF and c-Met mRNA levels transiently increased in livers during the progression of cholestatic hepatitis. When c-Met tyrosine phosphorylation was blocked in the livers of BDL-treated mice by anti-HGF IgG, hepatic dysfunction became evident, associated with the acceleration of hepatocyte necrosis and apoptosis. Inversely, administration of recombinant HGF into the mice led to the prevention of cholestasis-induced inflammation: HGF suppressed the hepatic expression of intracellular adhesion molecule-1 and neutrophil infiltration in BDL-treated mice. As a result, parenchymal necrosis was suppressed in the HGF-injected BDL mice. In addition, HGF supplement therapy reduced the number of apoptotic hepatocytes in cholestatic mice, associated with the early induction of Bcl-xL. The administration of HGF enhanced hepatic repair, via accelerating G1/S progression in hepatocytes. Our study showed that 1) upregulation of HGF production is required for protective mechanisms against cholestatic hepatitis and 2) enhancement of the intrinsic defense system by adding HGF may be a reasonable strategy to attenuate hepatic inflammation, necrosis, and apoptosis under bile-congestive conditions.     

Involvement of regulatory T cells in the experimental autoimmune encephalomyelitis-preventive effect of dendritic cells expressing myelin oligodendrocyte glycoprotein plus TRAIL

We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4(+)CD25(+) regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4(+)CD25(+) T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3(+) cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4(+)CD25(+) T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4(+)CD25(+) Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.     

Protein kinase Cdelta plays a non-redundant role in insulin secretion in pancreatic beta cells

Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.