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Biomedical subjects

Christopher B Wilson

Publications and source records attributed to Christopher B Wilson.

At least 19 recordsLinked to original sources
PubMed2005

Inhibition of MHC class I is a virulence factor in herpes simplex virus infection of mice.

Herpes simplex virus (HSV) has a number of genes devoted to immune evasion. One such gene, ICP47, binds to the transporter associated with antigen presentation (TAP) 1/2 thereby preventing transport of viral peptides into the endoplasmic reticulum, loading of peptides onto nascent major histocompatibility complex (MHC) class I molecules, and presentation of peptides to CD8 T cells. However, ICP47 binds poorly to murine TAP1/2 and so inhibits antigen presentation by MHC class I in mice much less efficiently than in humans, limiting the utility of murine models to address the importance of MHC class I inhibition in HSV immunopathogenesis. To address this limitation, we generated recombinant HSVs that efficiently inhibit antigen presentation by murine MHC class I. These recombinant viruses prevented cytotoxic T lymphocyte killing of infected cells in vitro, replicated to higher titers in the central nervous system, and induced paralysis more frequently than control HSV. This increase in virulence was due to inhibition of antigen presentation to CD8 T cells, since these differences were not evident in MHC class I-deficient mice or in mice in which CD8 T cells were depleted. Inhibition of MHC class I by the recombinant viruses did not impair the induction of the HSV-specific CD8 T-cell response, indicating that cross-presentation is the principal mechanism by which HSV-specific CD8 T cells are induced. This inhibition in turn facilitates greater viral entry, replication, and/or survival in the central nervous system, leading to an increased incidence of paralysis.

Journal Article↗
PubMed2005

An essential role for non-bone marrow-derived cells in control of Pseudomonas aeruginosa pneumonia.

MyD88 is an adapter protein required for the induction of proinflammatory cytokines by most Toll-like receptors (TLR), and Pseudomonas aeruginosa expresses ligands for multiple TLRs. MyD88(-/-) (KO) mice are highly susceptible to aerosolized P. aeruginosa, failing to elicit an early inflammatory response and permitting a 3-log increase in bacterial CFU in the lungs by 24 h after infection. We hypothesized that alveolar macrophages are the first cells to recognize and kill aerosolized P. aeruginosa in an MyD88-dependent fashion due to their location within the airways. To determine which cells in the lungs mediate MyD88-dependent defenses against P. aeruginosa, we generated radiation bone marrow (BM) chimeras between MyD88KO and wild-type (WT) mice. MyD88KO mice transplanted with MyD88KO BM (MyD88KO-->MyD88KO mice) displayed uncontrolled bacterial replication, whereas all other chimeras controlled the infection by 24 h. However, at 4 h, both MyD88KO-->MyD88KO and WT-->MyD88KO mice permitted intrapulmonary bacterial replication, whereas MyD88KO-->WT and WT-->WT mice did not, indicating that the source of BM had little impact on the early control of infection. Similarly, the genotype of the recipient rather than that of the BM donor determined early neutrophil recruitment to the lungs. Whereas intrapulmonary TNF-alpha and IL-1beta production were associated with WT BM, levels of the CXC chemokines MIP-2 and KC as well as GM-CSF were associated with recipient genotype. We conclude that lung parenchymal and BM-derived cells collaborate in the MyD88-dependent response to P. aeruginosa infection in the lungs in mice.

Adaptor Proteins, Signal Transducing↗
PubMed2005

T cell lineage choice and differentiation in the absence of the RNase III enzyme Dicer.

The ribonuclease III enzyme Dicer is essential for the processing of micro-RNAs (miRNAs) and small interfering RNAs (siRNAs) from double-stranded RNA precursors. miRNAs and siRNAs regulate chromatin structure, gene transcription, mRNA stability, and translation in a wide range of organisms. To provide a model system to explore the role of Dicer-generated RNAs in the differentiation of mammalian cells in vivo, we have generated a conditional Dicer allele. Deletion of Dicer at an early stage of T cell development compromised the survival of alphabeta lineage cells, whereas the numbers of gammadelta-expressing thymocytes were not affected. In developing thymocytes, Dicer was not required for the maintenance of transcriptional silencing at pericentromeric satellite sequences (constitutive heterochromatin), the maintenance of DNA methylation and X chromosome inactivation in female cells (facultative heterochromatin), and the stable shutdown of a developmentally regulated gene (developmentally regulated gene silencing). Most remarkably, given that one third of mammalian mRNAs are putative miRNA targets, Dicer seems to be dispensable for CD4/8 lineage commitment, a process in which epigenetic regulation of lineage choice has been well documented. Thus, although Dicer seems to be critical for the development of the early embryo, it may have limited impact on the implementation of some lineage-specific gene expression programs.

Animals↗
PubMed2005

DNA methylation and the expanding epigenetics of T cell lineage commitment.

During their development from progenitors, lymphocytes make a series of cell fate decisions. These decisions reflect and require changes in overall programs of gene expression. To maintain cellular identity, programs of gene expression must be iterated through mitosis in a heritable manner by epigenetic processes, which include DNA methylation, methyl-CpG-binding proteins, histone modifications, transcription factors and higher order chromatin structure. Current evidence is consistent with the notion that DNA methylation acts in concert with other epigenetic processes to limit the probability of aberrant gene expression and to stabilize, rather than to initiate, cell fate decisions. In particular, DNA methylation appears to be a non-redundant repressor of CD8 expression in TCR-gammadelta T cells and Th2 cytokine expression in Th1 and CD8 T cells, and is required to enforce clonally restricted Ly49 and KIR gene expression in NK cells. However, most of our knowledge is derived from in vitro studies, and the importance of DNA methylation in memory cell lineage fidelity in vivo remains to be shown convincingly.

Animals↗
PubMed2005

Role of B-cell proliferation in the establishment of gammaherpesvirus latency.

Murine gammaherpesvirus 68 (gammaHV68) provides a tractable small animal model with which to study the mechanisms involved in the establishment and maintenance of latency by gammaherpesviruses. Similar to the human gammaherpesvirus Epstein-Barr virus (EBV), gammaHV68 establishes and maintains latency in the memory B-cell compartment following intranasal infection. Here we have sought to determine whether, like EBV infection, gammaHV68 infection in vivo is associated with B-cell proliferation during the establishment of chronic infection. We show that gammaHV68 infection leads to significant splenic B-cell proliferation as late as day 42 postinfection. Notably, gammaHV68 latency was found predominantly in the proliferating B-cell population in the spleen on both days 16 and 42 postinfection. Furthermore, virus reactivation upon ex vivo culture was heavily biased toward the proliferating B-cell population. DNA methyltransferase 1 (Dnmt1) is a critical maintenance methyltransferase which, during DNA replication, maintains the DNA methylation patterns of the cellular genome, a process that is essential for the survival of proliferating cells. To assess whether the establishment of gammaHV68 latency requires B-cell proliferation, we characterized infections of conditional Dnmt1 knockout mice by utilizing a recombinant gammaHV68 that expresses Cre-recombinase (gammaHV68-Cre). In C57BL/6 mice, the gammaHV68-Cre virus exhibited normal acute virus replication in the lungs as well as normal establishment and reactivation from latency. Furthermore, the gammaHV68-Cre virus also replicated normally during the acute phase of infection in the lungs of Dnmt1 conditional mice. However, deletion of the Dnmt1 alleles from gammaHV68-infected cells in vivo led to a severe ablation of viral latency, as assessed on both days 16 and 42 postinfection. Thus, the studies provide direct evidence that the proliferation of latently infected B cells is critical for the establishment of chronic gammaHV68 infection.

Animals↗
PubMed2004

Cutting edge: immunity and IFN-gamma production during Listeria monocytogenes infection in the absence of T-bet.

The T-box transcription factor T-bet is an important regulator of IFN-gamma production in all cell types and is considered to be essential for the generation of CD4 Th1 T cells. IFN-gamma in turn plays a critical role in immunity to many infectious agents. In this study, we demonstrate that T-bet is not required for host resistance to primary Listeria monocytogenes (LM) infection. In the innate immune phase, control of LM replication, serum IFN-gamma, and numbers of IFN-gamma-producing NK cells were similar in T-bet-deficient and control mice. In the adaptive immune phase, there was no defect in bacterial clearance or in the numbers of LM-specific IFN-gamma-producing CD8 T cells in T-bet-deficient mice and only a modest, although significant, reduction in the numbers of Th1 CD4 T cells and IFN-gamma secretion by CD4 T cells. Thus, host resistance and the generation of IFN-gamma-producing cells in response to LM infection are not substantially compromised in the absence of T-bet.

Animals↗
PubMed2004

DNA methylation is a nonredundant repressor of the Th2 effector program.

The extent to which DNA methylation contributes to proper regulation of murine T cell effector function is unclear. In this study, we show that in the absence of the maintenance DNA methyltransferase Dnmt1, silencing of IL-4, IL-5, IL-13, and IL-10 in CD8 T cells was abolished, and expression of these Th2 cytokines increased as much as 1000-fold compared with that of control CD8 T cells. Th2 cytokine expression also increased in Dnmt1(-/-) CD4 T cells, but the increase ( approximately 20-40-fold for IL-4 and IL-10, </=5-fold for IL-5 and IL-13) was less than for CD8 T cells. As a result, both Dnmt1(-/-) CD4 and CD8 T cells expressed high and comparable amounts of Th2 cytokines. Loss of Dnmt1 had more subtle effects on IL-2 (</=5-fold increase) and IFN-gamma ( approximately 5-10-fold increase) expression and did not affect the normal bias for greater IL-2 expression by CD4 T cells and greater IFN-gamma expression by CD8 T cells, nor the exclusive expression of perforin and granzyme B by the CD8 T cells. These results indicate that Dnmt1 and DNA methylation are necessary to prevent cell autonomous Th2 cytokine expression in CD8 T cells but are not essential for maintaining proper T cell subset-specific expression of Th1 or CTL effectors. We also found that the expression of Th2 cytokines by Dnmt1(-/-) T cells was appropriately up-regulated in Th2 conditions and down-regulated in Th1 conditions, indicating that transcription factors and DNA methylation are complementary and nonredundant mechanisms by which the Th2 effector program is regulated.

Animals↗
PubMed2004

Evolutionarily conserved sequence elements that positively regulate IFN-gamma expression in T cells.

Our understanding of mechanisms by which the expression of IFN-gamma is regulated is limited. Herein, we identify two evolutionarily conserved noncoding sequence elements (IFNgCNS1 and IFNg CNS2) located approximately 5 kb upstream and approximately 18 kb downstream of the initiation codon of the murine Ifng gene. When linked to the murine Ifng gene (-3.4 to +5.6 kb) and transiently transfected into EL-4 cells, these elements clearly enhanced IFN-gamma expression in response to ionomycin and phorbol 12-myristate 13-acetate and weakly enhanced expression in response to T-bet. A DNase I hypersensitive site and extragenic transcripts at IFNgCNS2 correlated positively with the capacity of primary T cell subsets to produce IFN-gamma. Transcriptionally favorable histone modifications in the Ifng promoter, intronic regions, IFNgCNS2, and, although less pronounced, IFNgCNS1 increased as naïve T cells differentiated into IFN-gamma-producing effector CD8+ and T helper (TH) 1 T cells, but not into TH2 T cells. Like IFN-gamma expression, these histone modifications were T-bet-dependent in CD4+ cells, but not CD8+ T cells. These findings define two distal regulatory elements associated with T cell subset-specific IFN-gamma expression.

Animals↗
PubMed2004

Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin.

To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-kappa B activation in the respiratory epithelium using a mutant I kappa B-alpha construct that functioned as a dominant negative inhibitor of NF-kappa B translocation (dnI kappa B-alpha). We developed two lines of transgenic mice in which expression of dnI kappa B-alpha was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-kappa B was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-alpha, IL-1 beta, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-alpha within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-kappa B activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.

Administration, Inhalation↗
PubMed2004

Cutting edge: myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus.

Myeloid differentiation factor 88 (MyD88) is an adapter molecule required for signal transduction via Toll-like receptors (TLRs) and receptors of the IL-1 family. Consequently, MyD88-deficient mice are highly susceptible to bacterial infections, including systemic infection with Staphylococcus aureus. To determine the role of MyD88 in innate immunity to bacterial pneumonia, we exposed MyD88-deficient and wild-type mice to aerosolized Pseudomonas aeruginosa or S. aureus. As predicted, MyD88-deficient mice failed to mount an early cytokine or inflammatory response or to control bacterial replication after infection with P. aeruginosa, which resulted in necrotizing pneumonia and death. By contrast, MyD88-deficient mice controlled S. aureus infection despite blunted local cytokine and inflammatory responses. Thus, whereas MyD88-dependent signaling is integral to the initiation of cytokine and inflammatory responses to both pathogens following infection of the lower respiratory tract, MyD88 is essential for innate immunity to P. aeruginosa but not S. aureus.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Increased dendritic cell numbers impair protective immunity to intracellular bacteria despite augmenting antigen-specific CD8+ T lymphocyte responses.

Dendritic cells (DCs) reside in tissues, where they function as sentinels, providing an essential link between innate and adaptive immunity. Increasing the numbers of DCs in vivo augments T cell responses, and can cause dramatic CTL-dependent tumor regression. To determine whether greater DC numbers promoted T cell-mediated protection in the context of host defense against intracellular bacteria, we treated mice with Flt3 ligand (Flt3-L) to increase DCs in vivo and challenged them with Listeria monocytogenes. Unexpectedly, after primary challenge with Listeria, the overall control of Listeria infection was impaired in Flt3-L-treated mice, which had greater bacterial burden and mortality than controls. Similar results were obtained when DC numbers were increased by treatment with polyethylene glycol-conjugated GM-CSF rather than Flt3-L and in mice infected with Mycobacterium tuberculosis. Impaired protection was not due to dysfunctional T cell responses, as Flt3-L-treated mice had a greater frequency and absolute number of Ag-specific CD8+ T cells, which produced IFN-gamma, exhibited cytolytic activity, and transferred protection. The increased Listeria burden in Flt3-L-treated mice was preferentially associated with DCs, which were unable to kill Listeria and more resistant to CTL lysis compared with macrophages in vitro. Although we cannot exclude the possibility that other potential effects, in addition to increased numbers of DCs, are shared by Flt3-L and polyethylene glycol-conjugated GM-CSF and contributed to the increase in susceptibility observed in treated mice, these results support the notion that DC numbers must be properly controlled within physiological limits to optimize host defense to intracellular bacterial pathogens.

Adjuvants, Immunologic↗
PubMed2004

Deficient MHC class I cross-presentation of soluble antigen by murine neonatal dendritic cells.

Neonates respond suboptimally to many vaccines. The reasons for this defect are unclear, but suboptimal antigen presentation by dendritic cells has been suggested as one possibility. In this report we describe an in vitro system that allows the generation of large numbers of resting murine neonatal dendritic cells facilitating their study. Using this system, we show a clear reduction in the ability of neonatal dendritic cells to present soluble ovalbumin, while the capacity to present ovalbumin peptide is intact. This suggests a specific defect in cross-presentation of exogenous antigen via the major histocompatibility complex (MHC) class I pathway. Deficient cross-presentation may contribute to the suboptimal CD8 T-cell response to vaccines in neonates.

Age Factors↗
PubMed2004

Characterization of flagellin expression and its role in Listeria monocytogenes infection and immunity.

Flagellin is the structural component of flagella produced by many pathogenic bacteria and is a potent proinflammatory molecule that mediates these effects through Toll-like receptor (TLR) 5. In Listeria monocytogenes (LM), flagellin expression is regulated by temperature and has been described as being shut off at 37 degrees C. In this study, we demonstrate that TLR5-mediated cell activation and flagellin expression is maintained at 37 degrees C in some laboratory-adapted strains and in approximately 20% of LM clinical isolates. To determine the role of flagellin in LM infection, a targeted mutation in the structural gene for flagellin (flaA) was generated in a parental LM strain that expressed flagellin under all conditions examined. In vitro studies demonstrated that this deltaflaA mutant was (i). non-motile; (ii). not able to activate TLR5-transfected HeLa cells; and (iii). induced tumour necrosis factor (TNF)-alpha production in approximately 50% fewer CD11b+ cells in splenocytes from normal mice compared with the parental strain. However, there was no significant alteration in virulence of the deltaflaA mutant after either intravenous or oral murine infection. Similarly, there was no difference in the generation of LM-specific CD8 or CD4 T cells after intravenous or oral infection. These data indicate that flagellin is not essential for LM pathogenesis or for the induction of LM-specific adaptive immune responses in normal mice.

Animals↗
PubMed2003

Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells.

How T cells regulate interleukin 4 (IL-4) expression is not completely understood. We show here that single-positive thymocytes express IL-4, but attenuate GATA-3 expression, recruit DNA methyltransferases (Dnmts) to the Il4-Il13 locus and downregulate IL-4 expression as they mature into T cells. Type 2 polarization blocks Dnmt1 recruitment, enhances histone H3 Lys4 methylation (indicative of accessible chromatin) and initiates DNA demethylation of the locus. Dnmt1-/- CD4 and CD8 T cells derepress IL-4 expression considerably, demethylate DNA and increase H3 Lys4 methylation without affecting GATA-3 expression, demonstrating that Dnmt1 and DNA methylation are essential for proper Il4 regulation. These results indicate that Dnmts, DNA and histone methylation, and transcription factors 'collaborate' to determine appropriate Il4 expression patterns.

Animals↗
PubMed2003

Cutting edge: protective cell-mediated immunity to Listeria monocytogenes in the absence of myeloid differentiation factor 88.

In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response. The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM). In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM. Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM. These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Sequential roles of Brg, the ATPase subunit of BAF chromatin remodeling complexes, in thymocyte development.

T cells develop through distinct stages directed by a series of signals. We explored the roles of SWI/SNF-like BAF chromatin remodeling complexes in this process by progressive deletion of the ATPase subunit, Brg, through successive stages of early T cell development. Brg-deficient cells were blocked at each of the developmental transitions examined. Bcl-xL overexpression suppressed cell death without relieving the developmental blockades, leading to the accumulation of Brg-deleted cells that were unexpectedly cell cycle arrested. These defects resulted partly from the disruptions of pre-TCR and potentially Wnt signaling pathways controlling the expression of genes such as c-Kit and c-Myc critical for continued development. Our studies indicate that BAF complexes dynamically remodel chromatin to propel sequential developmental transitions in response to external signals.

Adenosine Triphosphatases↗