Plasmacytoid dendritic cells do not migrate in intestinal or hepatic lymph

Plasmacytoid dendritic cells (pDCs) recognize pathogen-associated molecules, particularly viral, and represent an important mechanism in innate defense. They may however, also have roles in steady-state tolerogenic responses at mucosal sites. pDCs can be isolated from blood, mucosa, and lymph nodes (LNs). Although pDCs can express peripherally derived Ags in LNs and at mucosal sites, it is not clear whether pDCs actually migrate from the periphery in lymph or whether LN pDCs acquire Ags by other mechanisms. To determine whether pDCs migrate in lymph, intestine or liver-draining LNs were removed and thoracic duct leukocytes (TDLs) were collected. TDLs expressing MHC-II and CD45R, but not TCRalphabeta or CD45RA, were then analyzed. These enriched TDLs neither transcribe type I IFNs nor secrete inflammatory cytokines in response to viral stimuli in vitro or after a TLR7/8 stimulus in vivo. In addition, these TDLs do not express CD5, CD90, CD200, or Siglec-H, but do express Ig, and therefore represent B cells, despite their lack of CD45RA expression. Intestinal and hepatic lymph are hence devoid of bona fide pDCs under both steady-state conditions and after TLR7/8 stimulation. This shows that any role for pDCs in Ag-specific T cell activation or tolerance must differ from the roles of classical dendritic cells, because it cannot result from peripheral Ag capture, followed by migration of pDCs via lymph to the LN.     

SIGIRR promotes resistance against Pseudomonas aeruginosa keratitis by down-regulating type-1 immunity and IL-1R1 and TLR4 signaling

Pseudomonas aeruginosa keratitis destroys the cornea in susceptible Th1 responder C57BL/6 (B6), but not resistant Th2 responder (BALB/c) mice. To determine whether single Ig IL-1R-related molecule (SIGIRR) played a role in resistance, mRNA and protein expression levels were tested. Both were constitutively expressed in the cornea of the two mouse groups. A disparate mRNA and protein expression pattern was detected in the cornea of BALB/c vs B6 mice after infection. SIGIRR protein decreased significantly in BALB/c over B6 mice at 1 day postinfection. Thus, BALB/c mice were injected with an anti-SIGIRR Ab or IgG control. Anti-SIGIRR Ab over control-treated mice showed increased corneal opacity, stromal damage, and bacterial load. Corneal mRNA levels for IL-1beta, MIP-2, IL-1R1, TLR4, IL-18, and IFN-gamma and protein levels for IL-1beta and MIP-2 also were significantly up-regulated in anti-SIGIRR Ab over control mice, while no changes in polymorphonuclear cell number, IL-4, or IL-10 mRNA expression were detected. To further define the role of SIGIRR, RAW264.7 macrophage-like cells were transiently transfected with SIGIRR and stimulated with heat-killed P. aeruginosa or LPS. SIGIRR transfection significantly decreased mRNA levels for IL-1R1, TLR4, and type 1 immune response-associated cytokines (IL-12, IL-18, and IFN-gamma) as well as proinflammatory cytokines IL-1beta and MIP-2 protein expression. SIGIRR also negatively regulated IL-1 and LPS, but not poly(I:C)-mediated signaling and NF-kappaB activation. These data provide evidence that SIGIRR is critical in resistance to P. aeruginosa corneal infection by down-regulating type 1 immunity, and that it negatively regulates IL-1 and TLR4 signaling.     

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.     

Respiratory syncytial virus induces TLR3 protein and protein kinase R, leading to increased double-stranded RNA responsiveness in airway epithelial cells

Respiratory syncytial virus (RSV) preferentially infects airway epithelial cells, causing bronchiolitis, upper respiratory infections, asthma exacerbations, chronic obstructive pulmonary disease exacerbations, and pneumonia in immunocompromised hosts. A replication intermediate of RSV is dsRNA. This is an important ligand for both the innate immune receptor, TLR3, and protein kinase R (PKR). One known effect of RSV infection is the increased responsiveness of airway epithelial cells to subsequent bacterial ligands (i.e., LPS). In this study, we examined a possible role for RSV infection in increasing amounts and responsiveness of another TLR, TLR3. These studies demonstrate that RSV infection of A549 and human tracheobronchial epithelial cells increases the amounts of TLR3 and PKR in a time-dependent manner. This leads to increased NF-kappaB activity and production of the inflammatory cytokine IL-8 following a later exposure to dsRNA. Importantly, TLR3 was not detected on the cell surface at baseline but was detected on the cell surface after RSV infection. The data demonstrate that RSV, via an effect on TLR3 and PKR, sensitizes airway epithelial cells to subsequent dsRNA exposure. These findings are consistent with the hypothesis that RSV infection sensitizes the airway epithelium to subsequent viral and bacterial exposures by up-regulating TLRs and increasing their membrane localization.     

Lipidomics of cellular and secreted phospholipids from differentiated human fetal type II alveolar epithelial cells

Maturation of fetal alveolar type II epithelial cells in utero is characterized by specific changes to lung surfactant phospholipids. Here, we quantified the effects of hormonal differentiation in vitro on the molecular specificity of cellular and secreted phospholipids from human fetal type II epithelial cells using electrospray ionization mass spectrometry. Differentiation, assessed by morphology and changes in gene expression, was accompanied by restricted and specific modifications to cell phospholipids, principally enrichments of shorter chain species of phosphatidylcholine (PC) and phosphatidylinositol, that were not observed in fetal lung fibroblasts. Treatment of differentiated epithelial cells with secretagogues stimulated the secretion of functional surfactant-containing surfactant proteins B and C (SP-B and SP-C). Secreted material was further enriched in this same set of phospholipid species but was characterized by increased contents of short-chain monounsaturated and disaturated species other than dipalmitoyl PC (PC16:0/16:0), principally palmitoylmyristoyl PC (PC16:0/14:0) and palmitoylpalmitoleoyl PC (PC16:0/16:1). Mixtures of these PC molecular species, phosphatidylglycerol, and SP-B and SP-C were functionally active and rapidly generated low surface tension on compression in a pulsating bubble surfactometer. These results suggest that hormonally differentiated human fetal type II cells do not select the molecular composition of surfactant phospholipid on the basis of saturation but, more likely, on the basis of acyl chain length.     

Evidence that the SpoIIIE DNA translocase participates in membrane fusion during cytokinesis and engulfment

During Bacillus subtilis sporulation, SpoIIIE is required for translocation of the trapped forespore chromosome across the sporulation septum, for compartmentalization of cell-specific gene expression, and for membrane fusion after engulfment. We isolated mutations within the SpoIIIE membrane domain that block localization and function. One mutant protein initially localizes normally and completes DNA translocation, but shows reduced membrane fusion after engulfment. Fluorescence recovery after photobleaching experiments demonstrate that in this mutant the sporulation septum remains open, allowing cytoplasmic contents to diffuse between daughter cells, suggesting that it blocks membrane fusion after cytokinesis as well as after engulfment. We propose that SpoIIIE catalyses these topologically opposite fusion events by assembling or disassembling a proteinaceous fusion pore. Mutants defective in SpoIIIE assembly also demonstrate that the ability of SpoIIIE to provide a diffusion barrier is directly proportional to its ability to assemble a focus at the septal midpoint during DNA translocation. Thus, SpoIIIE mediates compartmentalization by two distinct mechanisms: the SpoIIIE focus first provides a temporary diffusion barrier during DNA translocation, and then mediates the completion of membrane fusion after division to provide a permanent diffusion barrier. SpoIIIE-like proteins might therefore serve to couple the final step in cytokinesis, septal membrane fusion, to the completion of chromosome segregation.     

Recycling or regulation? The role of amino-terminal modifying enzymes

Post-translational modifications are essential for a variety of functions, such as the translocation, activation, regulation, and, ultimately, degradation of proteins. The amino-terminal (N-terminal) region is a particularly active area for such alterations. Three types of reactions predominate: limited proteolysis to remove one or more amino acids; modification of the alpha-amino group; and side-chain-specific changes. The N-terminal peptidases expose penultimate residues, providing new substrates for peptidase or transferase action. These enzymes can act sequentially or competitively to influence a protein's longevity, location or activity. N-terminal modifying enzymes (NTMEs) might target a protein for ubiquitination and degradation or protect a protein from rapid turnover. The N-terminal peptidases might also have important roles in processing the peptides that are released from the proteasome. Plant NTMEs have roles in senescence, meiosis and defense, and proposed roles in polar auxin transport.     

Regulation of flagella

Flagellar gene networks are fascinating, owing to their complexity - they usually coordinate the expression of more than 40 genes - and particular wiring that elicits temporal expression coupled to organelle morphogenesis. Moreover, many of the lessons learned from flagellar regulation are generally applicable to type III secretion systems. Our understanding of flagellar networks is rapidly expanding to include diverse organisms, as well as deepening to enable the development of predictive wiring diagrams. Numerous regulators control the regulation of flagella, and one of the next challenges in the field is to integrate flagellar gene control into master blueprints of global gene expression.     

Evolution of a carbohydrate binding module into a protein-specific binder

A carbohydrate binding module, CBM4-2, derived from the xylanase (Xyn 10A) of Rhodothermus marinus has been used as a scaffold for molecular diversification. Its binding specificity has been evolved to recognise a quite different target, a human monoclonal IgG4. In order to understand the basis for this drastic change in specificity we have further investigated the target recognition of the IgG4-specific CBMs. Firstly, we defined that the structure target recognised by the selected CBM-variants was the protein and not the carbohydrates attached to the glycoprotein. We also identified key residues involved in the new specificity and/or responsible for the swap in specificity, from xylan to human IgG4. Specific changes present in all these CBMs included mutations not introduced in the design of the library from which the specific clones were selected. Reversion of such mutations led to a complete loss of binding to the target molecule, suggesting that they are critical for the recognition of human IgG4. Together with the mutations introduced at will, they had transformed the CBM scaffold into a protein binder. We have thus shown that the scaffold of CBM4-2 is able to harbour molecular recognition for either carbohydrate or protein structures.     

Calcium Additions and Microbial Nitrogen Cycle Processes in a Northern Hardwood Forest

Evaluating, and possibly ameliorating, the effects of base cation depletion in forest soils caused by acid deposition is an important topic in the northeastern United States. We added 850 kg Ca ha⁻¹ as wollastonite (CaSiO₃) to an 11.8-ha watershed at the Hubbard Brook Experimental Forest (HBEF), a northern hardwood forest in New Hampshire, USA, in fall 1999 to replace calcium (Ca) leached from the ecosystem by acid deposition over the past 6 decades. Soil microbial biomass carbon (C) and nitrogen (N) concentrations, gross and potential net N mineralization and nitrification rates, soil solution and stream chemistry, soil:atmosphere trace gas (CO₂, N₂O, CH₄) fluxes, and foliar N concentrations have been monitored in the treated watershed and in reference areas at the HBEF before and since the Ca addition. We expected that rates of microbial C and N cycle processes would increase in response to the treatment. By 2000, soil pH was increased by a full unit in the Oie soil horizon, and by 2002 it was increased by nearly 0.5 units in the Oa soil horizon. However, there were declines in the N content of the microbial biomass, potential net and gross N mineralization rates, and soil inorganic N pools in the Oie horizon of the treated watershed. Stream, soil solution, and foliar concentrations of N showed no response to treatment. The lack of stimulation of N cycling by Ca addition suggests that microbes may not be stimulated by increased pH and Ca levels in the naturally acidic soils at the HBEF, or that other factors (for example, phosphorus, or Ca binding of labile organic matter) may constrain the capacity of microbes to respond to increased pH in the treated watershed. Possible fates for the approximately 10 kg N ha⁻¹ decline in microbial and soil inorganic pools include components of the plant community that we did not measure (for example, seedlings, understory shrubs), increased fluxes of N₂ and/or N storage in soil organic matter. These results raise questions about the factors regulating microbial biomass and activity in northern hardwood forests that should be considered in the context of proposals to mitigate the depletion of nutrient cations in soil.