Prion uptake in the gut: identification of the first uptake and replication sites.

After oral exposure, prions are thought to enter Peyer's patches via M cells and accumulate first upon follicular dendritic cells (FDCs) before spreading to the nervous system. How prions are actually initially acquired from the gut lumen is not known. Using high-resolution immunofluorescence and cryo-immunogold electron microscopy, we report the trafficking of the prion protein (PrP) toward Peyer's patches of wild-type and PrP-deficient mice. PrP was transiently detectable at 1 day post feeding (dpf) within large multivesicular LAMP1-positive endosomes of enterocytes in the follicle-associated epithelium (FAE) and at much lower levels within M cells. Subsequently, PrP was detected on vesicles in the late endosomal compartments of macrophages in the subepithelial dome. At 7-21 dpf, increased PrP labelling was observed on the plasma membranes of FDCs in germinal centres of Peyer's patches from wild-type mice only, identifying FDCs as the first sites of PrP conversion and replication. Detection of PrP on extracellular vesicles displaying FAE enterocyte-derived A33 protein implied transport towards FDCs in association with FAE-derived vesicles. By 21 dpf, PrP was observed on the plasma membranes of neurons within neighbouring myenteric plexi. Together, these data identify a novel potential M cell-independent mechanism for prion transport, mediated by FAE enterocytes, which acts to initiate conversion and replication upon FDCs and subsequent infection of enteric nerves.

Differential susceptibility of Sprague-Dawley and Fischer 344 rats to infection by Francisella tularensis.

The type A and B subspecies of Francisella tularensis cause severe disease, tularemia, in humans. However, only the former can be lethal especially if inhaled. It is likely that non-lethal infection is due at least in part to the ability of innate host defenses to control pathogen growth whilst acquired immunity develops. Most common small laboratory animals rapidly succumb to clinical strains of F. tularensis and are, therefore, poor models with which to study innate immunity. In an attempt to improve upon this situation in the present study, Sprague-Dawley and Fischer 344 rats were examined for their ability to combat challenge with type A and B strains of the pathogen. Sprague-Dawley rats were significantly more resistant than Fischer rats to infection with either subspecies. This correlated with the ability of Sprague-Dawley rats to arrest the growth of the pathogen at both the site of challenge and at sites of disseminated infection. The rapidity with which F. tularensis kills susceptible rats and the early onset of control of infection in resistant rats suggests that differences in innate immunity account for these disparate outcomes. Thus, the rat might be a more useful model for studying innate immunity to virulent F. tularensis than other small mammals.

In vivo depletion of CD11c+ cells impairs scrapie agent neuroinvasion from the intestine.

Following oral exposure, some transmissible spongiform encephalopathy (TSE) agents accumulate first upon follicular dendritic cells (DCs) in the GALT. Studies in mice have shown that TSE agent accumulation in the GALT, in particular the Peyer's patches, is obligatory for the efficient transmission of disease to the brain. However, the mechanism through which TSE agents are initially conveyed from the gut lumen to the GALT is not known. Studies have implicated migratory hemopoietic DCs in this process, but direct demonstration of their involvement in vivo is lacking. In this study, we have investigated the contribution of CD11c(+) DCs in scrapie agent neuroinvasion through use of CD11c-diptheria toxin receptor-transgenic mice in which CD11c(+) DCs can be specifically and transiently depleted. Using two distinct scrapie agent strains (ME7 and 139A scrapie agents), we show that when CD11c(+) DCs were transiently depleted in the GALT and spleen before oral exposure, early agent accumulation in these tissues was blocked. In addition, CD11c(+) cell depletion reduced susceptibility to oral scrapie challenge indicating that TSE agent neuroinvasion from the GALT was impaired. In conclusion, these data demonstrate that migratory CD11c(+) DCs play a key role in the translocation of the scrapie agent from the gut lumen to the GALT from which neuroinvasion subsequently occurs.

Assessing the involvement of migratory dendritic cells in the transfer of the scrapie agent from the immune to peripheral nervous systems.

Many transmissible spongiform encephalopathy (TSE) agents accumulate upon follicular dendritic cells (FDCs) in lymphoid tissues before spreading to the brain. How TSE agents spread from FDCs to the nervous system is not known as there is no physical FDC-nerve synapse. As FDCs form immobile networks we investigated whether other mobile cells might transfer TSE agents between FDCs and peripheral nerves. We show that scrapie-infected mononuclear cells, B cells and migratory dendritic cells (DCs) were unable to efficiently transmit disease to the peripheral nervous systems (PNSs) of FDC-deficient TNFR1(-/-) mice. These findings differed significantly from a similar study which suggested that scrapie-infected DCs could efficiently transmit disease directly to FDC-deficient RAG1(-/-) mice. Comparison of the innervation in spleens from TNFR1(-/-) mice and RAG1(-/-) mice indicated that the density of sympathetic nerves was much higher in RAG1(-/-) mice. These data imply that DCs could efficiently transmit disease directly to RAG1(-/-) mice because their spleens were highly innervated, but not to TNFR1(-/-) mice because their spleens were less densely innervated. As the density of the innervation in the spleens of wild-type mice also appeared to be much lower than that of RAG1(-/-) mice our data suggest that DCs are unlikely to play a key role in the transfer of TSE agents from FDCs to the PNS of wild-type mice.

Effects of antigen and recombinant porcine cytokines on pig dendritic cell cytokine expression in vitro.

To evaluate variables influencing in vitro immune response induction, pig monocyte-derived DCs (moDCs) were treated with putative type-1 and type-2 antigens (Ags, killed Mycobacterium tuberculosis (Mtb) and hen egg white lysozyme (HEWL)) and recombinant porcine cytokines (IL-6, IL-10, IL-12, IFN-gamma and TNF-alpha). Responses were measured as moDC cytokine mRNA expression. Treatment of moDCs with HEWL increased IL-13 but not IL-12, IFN-gamma or IL-10 mRNA, suggesting a DC2 phenotype. Addition of TNF-alpha, IFN-gamma or IL-12 to HEWL-treated moDCs increased IL-12p35 and reduced IL-13 mRNA; suggesting a DC1 phenotype. Mtb increased moDC IL-12p35, IFN-gamma and to a lesser extent IL-13 mRNA. This DC1 bias was enhanced by TNF-alpha, IFN-gamma or IL-12, which increased IL-12p35 and to a lesser extent IL-10 mRNA but reduced IL-13 mRNA. Addition of IL-10 to Mtb-pulsed moDCs reduced IL-12p35, IFN-gamma and IL-13, but increased IL-10 mRNA, suggesting diversion from DC1 to DC2. Thus porcine moDCs treated with Ag and/or cytokines alter moDC cytokine expression confirming their likely ability to initiate and steer acquired immune response.

Toll-like receptor, MHC II, B7 and cytokine expression by porcine monocytes and monocyte-derived dendritic cells in response to microbial pathogen-associated molecular patterns.

To evaluate effects of treatment with pathogen-associated molecular patterns (PAMPs) on toll-like receptor (TLR), MHC II, B7 and cytokine expression, pig monocytes and monocyte-derived DCs (moDCs) were treated with LPS, CpG, lipoteichoic acid (LTA), poly IC or peptidoglycan (Pep). Monocytes and moDCs treated with LPS, CpG, LTA, poly IC or Pep altered expression of at least one TLR (4, 5 and 9) and up-regulated MHC II and/or B7. The mRNA for IL-4 was not detected after any treatment. Treatment with LPS or LTA tended to up-regulate mRNA for TLR 4, Th-1 (IFN-gamma and IL-12p35) and Th-2 cytokines (IL-10 and IL-13). Poly IC or CpG tended to up-regulate TLR 9 and Th-1 cytokines. Porcine monocytes and moDCs like those of humans and mice responded to microbial PAMPs by altering TLR expression, up-regulating MHC II and B7 and altering cytokine expression toward Th-1 and/or Th-2, which may steer immune response. Hence, porcine moDCs and monocytes are likely able to discriminate between microorganisms using TLRs which determine cytokine expression and immune response bias.

Th-1/Th-2 type cytokine profiles of pig T-cells cultured with antigen-treated monocyte-derived dendritic cells.

To examine the effects of cytokine environment at the time of antigenic exposure on T-cell cytokine profiles following T-cell-antigen presenting cell (APC) interaction, pig monocyte-derived dendritic cells (mDCs) were treated with hen egg white lysozyme (HEWL) or killed Mycobacterium tuberculosis (Mtb) alone or with a recombinant pig cytokine (TNF-alpha, interleukin (IL)-12, IL-10, interferon (IFN)-gamma or IL-6) and then incubated with autologous T-cell-enriched lymphocytes. Messenger RNA was isolated from the T-cells and used to evaluate the effects of treatment on IL-12p35, IFN-gamma, IL-4, IL-10 and IL-13 expression using RT-PCR. T-cells exposed to HEWL-treated mDCs expressed high IL-13 and moderate IL-10 and IFN-gamma, suggesting T-helper 2 (Th-2) bias. Addition of any cytokine during HEWL treatment of mDCs reduced subsequent expression of IL-10 and IL-13 by T-cells. Added IL-12 increased IFN-gamma mRNA. T-cells exposed to Mtb-treated mDCs expressed increased IFN-gamma and decreased IL-10 suggesting Th-1 bias. Addition of cytokines to mDCs treated with Mtb altered T-cell cytokine mRNA expression such that TNF-alpha, IFN-gamma or IL-12 increased IFN-gamma; IL-12 and IFN-gamma suppressed IL-10, while IL-10 and IL-12 enhanced IL-13. Messenger RNA for IL-4 and IL-12p35 was not detected in the T-cells. Results suggest Th-1/Th-2 type response bias in pigs T-cells as a function of antigen type and that cytokine environment at the time of antigen-mDC interaction alters cytokine profiles of T-cells responding to antigen-pulsed mDCs. Hence, cytokines may allow designed steering of porcine immune response.