Terrence Town

Toll-like receptor 3 mediates West Nile virus entry into the brain causing lethal encephalitis.

West Nile virus (WNV), a mosquito-borne single-stranded (ss)RNA flavivirus, causes human disease of variable severity. We investigated the involvement of Toll-like receptor (Tlr) 3, which recognizes viral double-stranded (ds)RNA, on WNV infection. Tlr3-deficient (Tlr3−/−) mice were more resistant to lethal WNV infection and had impaired cytokine production and enhanced viral load in the periphery, whereas in the brain, viral load, inflammatory responses and neuropathology were reduced compared to wild-type mice. Peripheral WNV infection led to a breakdown of the blood-brain barrier and enhanced brain infection in wild-type but not in Tlr3−/− mice, although both groups were equally susceptible upon intracerebroventricular administration of the virus. Tumor necrosis factor-α receptor 1 signaling is vital for blood-brain barrier compromise upon Tlr3 stimulation by dsRNA or WNV. Collectively, WNV infection leads to a Tlr3-dependent inflammatory response, which is involved in brain penetration of the virus and neuronal injury.

Oxidized Mitochondrial DNA Activates the NLRP3 Inflammasome during Apoptosis

We report that in the presence of signal 1 (NF-κB), the NLRP3 inflammasome was activated by mitochondrial apoptotic signaling that licensed production of interleukin-1β (IL-1β). NLRP3 secondary signal activators such as ATP induced mitochondrial dysfunction and apoptosis, resulting in release of oxidized mitochondrial DNA (mtDNA) into the cytosol, where it bound to and activated the NLRP3 inflammasome. The antiapoptotic protein Bcl-2 inversely regulated mitochondrial dysfunction and NLRP3 inflammasome activation. Mitochondrial DNA directly induced NLRP3 inflammasome activation, because macrophages lacking mtDNA had severely attenuated IL-1β production, yet still underwent apoptosis. Both binding of oxidized mtDNA to the NLRP3 inflammasome and IL-1β secretion could be competitively inhibited by the oxidized nucleoside 8-OH-dG. Thus, our data reveal that oxidized mtDNA released during programmed cell death causes activation of the NLRP3 inflammasome. These results provide a missing link between apoptosis and inflammasome activation, via binding of cytosolic oxidized mtDNA to the NLRP3 inflammasome.

Interchromosomal associations between alternatively expressed loci

The T-helper-cell 1 and 2 (TH1 and TH2) pathways, defined by cytokines interferon-γ (IFN-γ) and interleukin-4 (IL-4), respectively, comprise two alternative CD4+ T-cell fates, with functional consequences for the host immune system. These cytokine genes are encoded on different chromosomes. The recently described TH2 locus control region (LCR) coordinately regulates the TH2 cytokine genes by participating in a complex between the LCR and promoters of the cytokine genes Il4, Il5 and Il13. Although they are spread over 120 kilobases, these elements are closely juxtaposed in the nucleus in a poised chromatin conformation. In addition to these intrachromosomal interactions, we now describe interchromosomal interactions between the promoter region of the IFN-γ gene on chromosome 10 and the regulatory regions of the TH2 cytokine locus on chromosome 11. DNase I hypersensitive sites that comprise the TH2 LCR developmentally regulate these interchromosomal interactions. Furthermore, there seems to be a cell-type-specific dynamic interaction between interacting chromatin partners whereby interchromosomal interactions are apparently lost in favour of intrachromosomal ones upon gene activation. Thus, we provide an example of eukaryotic genes located on separate chromosomes associating physically in the nucleus via interactions that may have a function in coordinating gene expression.

A protective function for interleukin 17A in T cell–mediated intestinal inflammation

Interleukin 23 (IL-23) and IL-17 have been linked to the pathogenesis of several chronic inflammatory disorders, including inflammatory bowel disease. Yet as an important function for IL-23 is emerging, the function of IL-17 in inflammatory bowel disease remains unclear. Here we demonstrate IL-17A-mediated protection in the CD45RBhi transfer model of colitis. An accelerated wasting disease elicited by T cells deficient in IL-17A correlated with higher expression of genes encoding T helper type 1–type cytokines in colon tissue. IL-17A also modulated T helper type 1 polarization in vitro. Furthermore, T cells deficient in the IL-17 receptor elicited an accelerated, aggressive wasting disease relative to that elicited by wild-type T cells in recipient mice. Our data demonstrate a protective function for IL-17 and identify T cells as not only the source but also a target of IL-17 in vivo.

Green Tea Epigallocatechin-3-Gallate (EGCG) Modulates Amyloid Precursor Protein Cleavage and Reduces Cerebral Amyloidosis in Alzheimer Transgenic Mice

Alzheimers disease (AD) is a progressive neurodegenerative disorder pathologically characterized by deposition of β-amyloid (Aβ) peptides as senile plaques in the brain. Recent studies suggest that green tea flavonoids may be used for the prevention and treatment of a variety of neurodegenerative diseases. Here, we report that (-)-epigallocatechin-3-gallate (EGCG), the main polyphenolic constituent of green tea, reduces Aβ generation in both murine neuron-like cells (N2a) transfected with the human “Swedish” mutant amyloid precursor protein (APP) and in primary neurons derived from Swedish mutant APP-overexpressing mice (Tg APPsw line 2576). In concert with these observations, we find that EGCG markedly promotes cleavage of the α-C-terminal fragment of APP and elevates the N-terminal APP cleavage product, soluble APP-α. These cleavage events are associated with elevated α-secretase activity and enhanced hydrolysis of tumor necrosis factor α-converting enzyme, a primary candidate α-secretase. As a validation of these findings in vivo, we treated Tg APPsw transgenic mice overproducing Aβ with EGCG and found decreased Aβ levels and plaques associated with promotion of the nonamyloidogenic α-secretase proteolytic pathway. These data raise the possibility that EGCG dietary supplementation may provide effective prophylaxis for AD.

Control of TH17 cells occurs in the small intestine

Interleukin (IL)-17-producing T helper cells (TH17) are a recently identified CD4+ T cell subset distinct from T helper type 1 (TH1) and T helper type 2 (TH2) cells. TH17 cells can drive antigen-specific autoimmune diseases and are considered the main population of pathogenic T cells driving experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis. The factors that are needed for the generation of TH17 cells have been well characterized. However, where and how the immune system controls TH17 cells in vivo remains unclear. Here, by using a model of tolerance induced by CD3-specific antibody, a model of sepsis and influenza A viral infection (H1N1), we show that pro-inflammatory TH17 cells can be redirected to and controlled in the small intestine. TH17-specific IL-17A secretion induced expression of the chemokine CCL20 in the small intestine, facilitating the migration of these cells specifically to the small intestine via the CCR6/CCL20 axis. Moreover, we found that TH17 cells are controlled by two different mechanisms in the small intestine: first, they are eliminated via the intestinal lumen; second, pro-inflammatory TH17 cells simultaneously acquire a regulatory phenotype with in vitro and in vivo immune-suppressive properties (rTH17). These results identify mechanisms limiting TH17 cell pathogenicity and implicate the gastrointestinal tract as a site for control of TH17 cells.

Blocking TGF-β–Smad2/3 innate immune signaling mitigates Alzheimer-like pathology

Alzheimers disease is the most common dementia and is pathologically characterized by deposition of amyloid-β peptide (Aβ) into β-amyloid plaques, neuronal injury and low-level, chronic activation of brain immunity. Transforming growth factor-βs (TGF-βs) are pleiotropic cytokines that have key roles in immune cell activation, inflammation and repair after injury. We genetically interrupted TGF-β and downstream Smad2/3 signaling (TGF-β–Smad2/3) in innate immune cells by inducing expression of CD11c promoter–driven dominant-negative TGF-β receptor type II in C57BL/6 mice (CD11c-DNR), crossed these mice with mice overexpressing mutant human amyloid precursor protein, the Tg2576 Alzheimers disease mouse model, and evaluated Alzheimers disease-like pathology. Aged double-transgenic mice showed complete mitigation of Tg2576-associated hyperactivity and partial mitigation of defective spatial working memory. Brain parenchymal and cerebrovascular β-amyloid deposits and Aβ abundance were markedly (up to 90%) attenuated in Tg2576–CD11c-DNR mice. This was associated with increased infiltration of Aβ-containing peripheral macrophages around cerebral vessels and β-amyloid plaques. In vitro, cultures of peripheral macrophages, but not microglia, from CD11c-DNR mice showed blockade of classical TGF-β–activated Smad2/3 but also showed hyperactivation of alternative bone morphogenic protein–activated Smad1/5/8 signaling and increased Aβ phagocytosis. Similar effects were noted after pharmacological inhibition of activin-like kinase-5, a type I TGF-β receptor. Taken together, our results suggest that blockade of TGF-β–Smad2/3 signaling in peripheral macrophages represents a new therapeutic target for Alzheimers disease.

Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling

Primary cilia are present on mammalian neurons and glia, but their function is largely unknown. We generated conditional homozygous mutant mice for a gene we termed Stumpy. Mutants lack cilia and have conspicuous abnormalities in postnatally developing brain regions, including a hypoplasic hippocampus characterized by a primary deficiency in neural stem cells known as astrocyte-like neural precursors (ALNPs). Previous studies suggested that primary cilia mediate sonic hedgehog (Shh) signaling. Here, we find that loss of ALNP cilia leads to abrogated Shh activity, increased cell cycle exit, and morphological abnormalities in ALNPs. Processing of Gli3, a mediator of Shh signaling, is also altered in the absence of cilia. Further, key mediators of the Shh pathway localize to ALNP cilia. Thus, selective targeting of Shh machinery to primary cilia confers to ALNPs the ability to differentially respond to Shh mitogenic signals compared to neighboring cells. Our data suggest these organelles are cellular “antennae” critically required to modulate ALNP behavior.

Role of CD40 ligand in amyloidosis in transgenic Alzheimer's mice

We have shown that interaction of CD40 with CD40L enables microglial activation in response to amyloid-β peptide (Aβ), which is associated with Alzheimers disease (AD)-like neuronal tau hyperphosphorylation in vivo. Here we report that transgenic mice overproducing Aβ, but deficient in CD40L, showed decreased astrocytosis and microgliosis associated with diminished Aβ levels and β-amyloid plaque load. Furthermore, in the PSAPP transgenic mouse model of AD, a depleting antibody against CD40L caused marked attenuation of Aβ/β-amyloid pathology, which was associated with decreased amyloidogenic processing of amyloid precursor protein (APP) and increased circulating levels of Aβ. Conversely, in neuroblastoma cells overexpressing wild-type human APP, the CD40–CD40L interaction resulted in amyloidogenic APP processing. These findings suggest several possible mechanisms underlying mitigation of AD pathology in response to CD40L depletion, and validate the CD40–CD40L interaction as a target for therapeutic intervention in AD.

Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9†

Apoptosis of hepatocytes results in the development of liver fibrosis, but the molecular signals mediating this are poorly understood. Degradation and modification of nuclear DNA is a central feature of apoptosis, and DNA from apoptotic mammalian cells is known to activate immune cells via Toll‐like receptor 9 (TLR9). We tested if DNA from apoptotic hepatocytes can induce hepatic stellate cell (HSC) differentiation. Our data show that apoptotic hepatocyte DNA and cytidine‐phosphate‐guanosine oligonucleotides induced up‐regulation of transforming growth factor β1 and collagen 1 messenger RNA both in the human HSC line LX‐2 and in primary mouse HSCs. These effects were opposed by TLR9 antagonists. We have recently shown that adenosine inhibits HSC chemotaxis, and we now show that apoptotic hepatocyte DNA also inhibits platelet‐derived growth factor (PDGF)‐mediated HSC chemotaxis. Inhibition of HSC chemotaxis by PDGF was blocked by TLR9 antagonists, and was absent in primary HSCs from mice deficient in TLR9 or the TLR adaptor molecule MyD88. Stimulation of TLR9 on HSCs blocked signaling by the PDGF signaling molecule inositol 1,4,5‐triphosphate and reduced PDGF‐mediated increase in cytosolic Ca2+. Conclusion: DNA from apoptotic hepatocytes acts as an important mediator of HSC differentiation by (1) providing a stop signal to mobile HSCs when they have reached an area of apoptosing hepatocytes and (2) inducing a stationary phenotype‐associated up‐regulation of collagen production. (HEPATOLOGY 2007.)