Biomedical subjects

Alan Sher

Publications and source records attributed to Alan Sher.

At least 19 recordsLinked to original sources

PubMed2003

Cutting edge: in vivo induction of integrated HIV-1 expression by mycobacteria is critically dependent on Toll-like receptor 2.

Mycobacterial infection has been implicated as a possible factor in AIDS progression in populations where HIV-1 and Mycobacterium tuberculosis are coendemic. In support of this concept, we have previously shown that HIV-1-transgenic (Tg) mice infected with mycobacteria display enhanced viral gene and protein expression. In this study, we demonstrate that the induction of HIV-1 observed in this model is dependent on Toll-like receptor 2 (TLR2), a pattern recognition receptor known to be involved in mycobacteria-host interaction. Spleen cells from HIV-1-Tg mice deficient in TLR2 (Tg/TLR2(-/-)) were found to be completely defective in p24 production induced in response to live M. tuberculosis or Mycobacterium avium as well as certain mycobacterial products. Importantly, following in vivo mycobacterial infection, Tg/TLR2(-/-) mice failed to display the enhanced HIV-1 gag/env mRNA and p24 protein synthesis exhibited by wild-type Tg animals. Together, these results argue that TLR2 plays a crucial role in the activation of HIV-1 expression by mycobacterial coinfections.

Animals↗

PubMed2003

Heat-killed Brucella abortus induces TNF and IL-12p40 by distinct MyD88-dependent pathways: TNF, unlike IL-12p40 secretion, is Toll-like receptor 2 dependent.

Cattle and humans are susceptible to infection with the Gram-negative intracellular bacterium Brucella abortus. Heat-killed B. abortus (HKBA) is a strong Th1 adjuvant and carrier. Previously, we have demonstrated that dendritic cells produce IL-12 in response to HKBA stimulation. In the present study, we use knockout mice and in vitro reconstitution assays to examine the contribution of signaling by Toll-like receptors (TLRs) and their immediate downstream signaling initiator, myeloid differentiation protein MyD88, in the activation following stimulation by HKBA. Our results show that HKBA-mediated induction of IL-12p40 and TNF is dependent on the adapter molecule MyD88. To identify the TLR involved in HKBA recognition, we examined HKBA responses in TLR2- and TLR4-deficient animals. TNF responses to HKBA were TLR4 independent; however, the response in TLR2-deficient mice was significantly delayed and reduced, although not completely abolished. Studies using Chinese hamster ovary/CD14 reporter cell lines stably transfected with either human TLR2 or human TLR4 confirmed the results seen with knockout mice, namely TLR2, but not TLR4, can mediate cellular activation by HKBA. In addition, human embryonic kidney 293 cells, which do not respond to HKBA, were made responsive by transfecting TLR2, but not TLR4 or TLR9. Taken together, our data demonstrate that MyD88-dependent pathways are crucial for activation by HKBA and that TLR2 plays a role in TNF, but not IL-12p40 pathways activated by this microbial product.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Inhibition of HIV-1 infection by a CCR5-binding cyclophilin from Toxoplasma gondii.

The activation of murine dendritic cells by Toxoplasma gondii has recently been shown to depend on a parasite protein that signals through the chemokine receptor CCR5. Here we demonstrate that this molecule, cyclophilin-18 (C-18), is an inhibitor of HIV-1 cell fusion and infection with cell-free virus. T gondii C-18 efficiently blocked syncytium formation between human T cells and effector cells expressing R5 but not X4 envelopes. Neither human nor Plasmodium falciparum cyclophilins possess such inhibitory activity. Importantly, C-18 protected peripheral blood leukocytes from infection with multiple HIV-1 R5 primary isolates from several clades. C-18 bound directly to human CCR5, and this interaction was partially competed by the beta-chemokine macrophage inflammatory protein 1 beta (MIP-1 beta) and by HIV-1 R5 gp120. In contrast to several other antagonists of HIV coreceptor function, C-18 mediated inhibition did not induce beta-chemokines or cause CCR5 downmodulation, suggesting direct blocking of envelope binding to the receptor. These data support the further development of C-18 derivatives as HIV-1 inhibitors for preventing HIV-1 transmission and for postexposure prophylaxis.

Animals↗

PubMed2003

Relationships among murine CD11c(high) dendritic cell subsets as revealed by baseline gene expression patterns.

The functional relationships and properties of different subtypes of dendritic cells (DC) remain largely undefined. To better characterize these cells, we used global gene analysis to determine gene expression patterns among murine CD11c(high) DC subsets. CD4(+), CD8alpha(+), and CD8alpha(-) CD4(-) (double negative (DN)) DC were purified from spleens of normal C57/BL6 mice and analyzed using Affymetrix microarrays. The CD4(+) and CD8alpha(+) DC subsets showed distinct basal expression profiles differing by >200 individual genes. These included known DC subset markers as well as previously unrecognized, differentially expressed CD Ags such as CD1d, CD5, CD22, and CD72. Flow cytometric analysis confirmed differential expression in nine of nine cases, thereby validating the microarray analysis. Interestingly, the microarray expression profiles for DN cells strongly resembled those of CD4(+) DC, differing from them by <25 genes. This suggests that CD4(+) and DN DC are closely related phylogenetically, whereas CD8alpha(+) DC represent a more distant lineage, supporting the historical distinction between CD8alpha(+) and CD8alpha(-) DC. However, staining patterns revealed that in contrast to CD4(+) DC, the DN subset is heterogeneous and comprises at least two subpopulations. Gene Ontology and literature mining analyses of genes expressed differentially among DC subsets indicated strong associations with immune response parameters as well as cell differentiation and signaling. Such associations offer clues to possible unique functions of the CD11c(high) DC subsets that to date have been difficult to define as rigid distinctions.

Animals↗

PubMed2003

Cutting edge: bradykinin induces IL-12 production by dendritic cells: a danger signal that drives Th1 polarization.

Dendritic cells play a major role in the induction of both innate and acquired immune responses against pathogenic invaders. These cells are also able to sense endogenous activation signals liberated by injured tissues even in the absence of infection. In the present work, we demonstrate that kinins mobilize dendritic cells to produce IL-12 through activation of the B(2) bradykinin receptor subtype and that bradykinin-induced IL-12 responses are tightly regulated both by angiotensin-converting enzyme, a kinin-degrading peptidase, and by endogenous IL-10. Using a mouse model of allergic inflammation, we further show that addition of bradykinin to OVA during immunization results in decreased eosinophil infiltration on Ag challenge. The latter effect was demonstrated to be due to IL-12-driven skewing of Ag-specific T cell responses to a type 1 cytokine profile. Our data thus indicate that kinin peptides can serve as danger signals that trigger dendritic cells to produce IL-12 through activation of B(2) bradykinin receptors.

Adjuvants, Immunologic↗

PubMed2003

Toll-like receptor 2 (TLR2) and TLR9 signaling results in HIV-long terminal repeat trans-activation and HIV replication in HIV-1 transgenic mouse spleen cells: implications of simultaneous activation of TLRs on HIV replication.

Opportunistic infections are common in HIV-infected patients; they activate HIV replication and contribute to disease progression. In the present study we examined the role of Toll-like receptor 2 (TLR2) and TLR9 in HIV-long terminal repeat (HIV-LTR) trans-activation and assessed whether TLR4 synergized with TLR2 or TLR9 to induce HIV replication. Soluble Mycobacterium tuberculosis factor (STF) and phenol-soluble modulin from Staphylococcus epidermidis induced HIV-LTR trans-activation in human microvessel endothelial cells cotransfected with TLR2 cDNA. Stimulation of ex vivo spleen cells from HIV-1 transgenic mice with TLR4, TLR2, and TLR9 ligands (LPS, STF, and CpG DNA, respectively) induced p24 Ag production in a dose-dependent manner. Costimulation of HIV-1 transgenic mice spleen cells with LPS and STF or CpG DNA induced TNF-alpha and IFN-gamma production in a synergistic manner and p24 production in an additive fashion. In the THP-1 human monocytic cell line stably expressing the HIV-LTR-luciferase construct, LPS and STF also induced HIV-LTR trans-activation in an additive manner. This is the first time that TLR2 and TLR9 and costimulation of TLRs have been shown to induce HIV replication. Together these results underscore the importance of TLRs in bacterial Ag- and CpG DNA-induced HIV-LTR trans-activation and HIV replication. These observations may be important in understanding the role of the innate immune system and the molecular mechanisms involved in the increased HIV replication and HIV disease progression associated with multiple opportunistic infections.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Molecular mimicry of a CCR5 binding-domain in the microbial activation of dendritic cells.

Toxoplasma gondii releases factors that potently stimulate production of interleukin-12 (IL-12) from dendritic cells (DCs). Purification of this activity showed that cyclophilin-18 (C-18) was its principal component, and antibodies generated against recombinant C-18 inhibited tachyzoite extract-induced synthesis of IL-12. Recombinant C-18 showed high affinity for and triggered cell signaling through CCR5, a chemokine receptor important in parasite-induced IL-12 production by DCs. These findings suggest that the unusual potency of T. gondii in inducing IL-12 from DCs results from its synthesis of a unique chemokine mimic that signals through CCR5. The ability to generate this strong protective response may benefit parasite transmission by preventing the protozoan from overwhelming its intermediate hosts.

Amino Acid Sequence↗

PubMed2003

Unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites.

Monocyte-derived dendritic cells (DCs) and macrophages (Ms) generated in vitro from the same individual blood donors were exposed to 5 different pathogens, and gene expression profiles were assessed by microarray analysis. Responses to Mycobacterium tuberculosis and to phylogenetically distinct protozoan (Leishmania major, Leishmania donovani, Toxoplasma gondii) and helminth (Brugia malayi) parasites were examined, each of which produces chronic infections in humans yet vary considerably in the nature of the immune responses they trigger. In the absence of microbial stimulation, DCs and Ms constitutively expressed approximately 4000 genes, 96% of which were shared between the 2 cell types. In contrast, the genes altered transcriptionally in DCs and Ms following pathogen exposure were largely cell specific. Profiling of the gene expression data led to the identification of sets of tightly coregulated genes across all experimental conditions tested. A newly devised literature-based clustering algorithm enabled the identification of functionally and transcriptionally homogenous groups of genes. A comparison of the responses induced by the individual pathogens by means of this strategy revealed major differences in the functionally related gene profiles associated with each infectious agent. Although the intracellular pathogens induced responses clearly distinct from the extracellular B malayi, they each displayed a unique pattern of gene expression that would not necessarily be predicted on the basis of their phylogenetic relationship. The association of characteristic functional clusters with each infectious agent is consistent with the concept that antigen-presenting cells have prewired signaling patterns for use in the response to different pathogens.

Animals↗

PubMed2003

Shaping the immune response to parasites: role of dendritic cells.

Parasites represent a diverse group of pathogens that often trigger highly polarized immune responses that become tightly regulated during chronic infection. Recent studies have implicated the parasite-dendritic-cell interaction as a key determinant of the host response to these eukaryotic invaders. Dendritic cells appear to be pivotal in the initiation of cellular immunity against parasites as well as in directing Th1/Th2 effector choice. Moreover, there is increasing evidence that parasites regulate dendritic-cell function for the purposes of evading host immunity. This regulation also benefits parasites by protecting their host niche from the potentially lethal consequences of an uncontrolled inflammatory response.

Animals↗

PubMed2003

In vivo antiviral activity of novel human immunodeficiency virus type 1 nucleocapsid p7 zinc finger inhibitors in a transgenic murine model.

Control of human immunodeficiency virus through the use of inexpensive chemotherapeutics, with minimal side effects and decreased potential for engendering resistant virus, is a long-term therapeutic goal. In principle, this goal can be accomplished if viral replication in reservoirs of chronically and latently infected cells is addressed. As a first step, we have developed novel antiviral compounds based on a 2-mercaptobenzamide thioester chemotype, including the pyridinioalkanoyl thioesters, which specifically target the zinc fingers of the human immunodeficiency virus nucleocapsid protein (NCp7). Using these compounds in a murine transgenic model, in which infectious human immunodeficiency virus is induced from an integrated provirus, we show inhibition of transgenic spleen cell p24 expression with potencies comparable to acute infection assays using human peripheral blood lymphocytes. More importantly, transgenic mice treated in vivo with two 2-mercaptobenzamide thioesters expressed significantly lower plasma p24, and splenocytes from these animals produced fewer infectious virions. Thus, these thioesters may provide an effective means for inhibiting the expression of human immunodeficiency virus from integrated viral reservoirs.

Animals↗

PubMed2003

Induction and regulation of IL-12-dependent host resistance to Toxoplasma gondii.

Resistance to the intracellular protozoan parasite Toxoplasma gondii is initiated by the induction of interleukin-12 (IL-12), which stimulates interferon (IFN)-gamma synthesis by natural killer (NK) cells and T lymphocytes. This review summarizes the work of our laboratory on the mechanisms by which the parasite triggers IL-12 synthesis, and how this response is regulated to avoid the lethal effects of excessive tissue inflammation. In addition, we present an overview of our studies investigating the mechanisms by which the IFN-gamma produced as a consequence of IL-12 stimulation controls intracellular replication of the parasite in host cells.

Animals↗

PubMed2002

A critical role for IL-21 in regulating immunoglobulin production.

The cytokine interleukin-21 (IL-21) is closely related to IL-2 and IL-15, and their receptors all share the common cytokine receptor gamma chain, gammac, which is mutated in humans with X-linked severe combined immunodeficiency disease (XSCID). We demonstrate that, although mice deficient in the receptor for IL-21 (IL-21R) have normal lymphoid development, after immunization, these animals have higher production of the immunoglobulin IgE, but lower IgG1, than wild-type animals. Mice lacking both IL-4 and IL-21R exhibited a significantly more pronounced phenotype, with dysgammaglobulinemia, characterized primarily by a severely impaired IgG response. Thus, IL-21 has a significant influence on the regulation of B cell function in vivo and cooperates with IL-4. This suggests that these gammac-dependent cytokines may be those whose inactivation is primarily responsible for the B cell defect in humans with XSCID.

Animals↗

PubMed2002

Parasite-induced lipoxin A4 is an endogenous regulator of IL-12 production and immunopathology in Toxoplasma gondii infection.

The production of interleukin (IL)-12 is critical for the development of interferon (IFN)-gamma-dependent resistance to Toxoplasma gondii. Nevertheless, when this response is dysregulated, such as occurs in the absence of IL-10, the uncontrolled inflammation that results can have lethal consequences for the host. Recently, we demonstrated that lipoxin (LX)A(4), an eicosanoid mediator that depends on 5-lipoxygenase (LO) for its biosynthesis, exerts a regulatory role on dendritic cell IL-12 production triggered artificially by a T. gondii extract. We now formally establish the physiological relevance of this pathway in the systemic control of IL-12 production induced by live T. gondii infection and demonstrate its function to be distinct from that of IL-10. Thus, T. gondii-exposed wild-type, but not 5-LO-deficient animals, produced high levels of serum LXA(4) beginning at the onset of chronic infection. Moreover, 5-LO(-/-), in contrast to wild-type mice, succumbed during the same period displaying a marked encephalitis. The increased mortality of the 5-LO(-/-) animals was also associated with significant elevations of IL-12 and IFN-gamma and was completely prevented by the administration of a stable LXA(4) analogue. Together, these findings demonstrate a new pathway involving the induction of host LXs for the in vivo regulation of proinflammatory responses during microbial infection.

Animals↗

PubMed2002

Microbial recognition via Toll-like receptor-dependent and -independent pathways determines the cytokine response of murine dendritic cell subsets to CD40 triggering.

Dendritic cells (DC) can produce Th-polarizing cytokines and direct the class of the adaptive immune response. Microbial stimuli, cytokines, chemokines, and T cell-derived signals all have been shown to trigger cytokine synthesis by DC, but it remains unclear whether these signals are functionally equivalent and whether they determine the nature of the cytokine produced or simply initiate a preprogrammed pattern of cytokine production, which may be DC subtype specific. Here, we demonstrate that microbial and T cell-derived stimuli can synergize to induce production of high levels of IL-12 p70 or IL-10 by individual murine DC subsets but that the choice of cytokine is dictated by the microbial pattern recognition receptor engaged. We show that bacterial components such as CpG-containing DNA or extracts from Mycobacterium tuberculosis predispose CD8alpha(+) and CD8alpha(-)CD4(-) DC to make IL-12 p70. In contrast, exposure of CD8alpha(+), CD4(+) and CD8alpha(-)CD4(-) DC to heat-killed yeasts leads to production of IL-10. In both cases, secretion of high levels of cytokine requires a second signal from T cells, which can be replaced by CD40 ligand. Consistent with their differential effects on cytokine production, extracts from M. tuberculosis promote IL-12 production primarily via Toll-like receptor 2 and an MyD88-dependent pathway, whereas heat-killed yeasts activate DC via a Toll-like receptor 2-, MyD88-, and Toll/IL-1R domain containing protein-independent pathway. These results show that T cell feedback amplifies innate signals for cytokine production by DC and suggest that pattern recognition rather than ontogeny determines the production of cytokines by individual DC subsets.

3T3 Cells↗

PubMed2002

Mining microarray expression data by literature profiling.

BACKGROUND: The rapidly expanding fields of genomics and proteomics have prompted the development of computational methods for managing, analyzing and visualizing expression data derived from microarray screening. Nevertheless, the lack of efficient techniques for assessing the biological implications of gene-expression data remains an important obstacle in exploiting this information. RESULTS: To address this need, we have developed a mining technique based on the analysis of literature profiles generated by extracting the frequencies of certain terms from thousands of abstracts stored in the Medline literature database. Terms are then filtered on the basis of both repetitive occurrence and co-occurrence among multiple gene entries. Finally, clustering analysis is performed on the retained frequency values, shaping a coherent picture of the functional relationship among large and heterogeneous lists of genes. Such data treatment also provides information on the nature and pertinence of the associations that were formed. CONCLUSIONS: The analysis of patterns of term occurrence in abstracts constitutes a means of exploring the biological significance of large and heterogeneous lists of genes. This approach should contribute to optimizing the exploitation of microarray technologies by providing investigators with an interface between complex expression data and large literature resources.

Cluster Analysis↗

PubMed2002

ICSBP/IRF-8 retrovirus transduction rescues dendritic cell development in vitro.

Dendritic cells (DCs) develop from bone marrow (BM) progenitor cells and mature in response to external signals to elicit functions important for innate and adaptive immunity. Interferon consensus sequence binding protein (ICSBP; also called interferon regulatory factor 8 [IRF-8]) is a hematopoietic cell-specific transcription factor expressed in BM progenitor cells that contributes to myeloid cell development. In light of our earlier observation that ICSBP(-/-) mice lack CD8alpha(+) DCs, we investigated the role of ICSBP in DC development in vitro in the presence of Flt3 ligand. Immature ICSBP(-/-) DCs developed from BM progenitor cells showed assorted defects, did not mature in response to activation signals, and failed to express CD8alpha and interleukin 12 (IL-12) p40, a feature consistent with ICSBP(-/-) DCs in vivo. We show that retroviral introduction of ICSBP restores the development of immature DCs that can fully mature on activation signals. All the defects seen with ICSBP(-/-) DCs were corrected after ICSBP transduction, including the expression of CD8alpha and IL-12 p40 as well as major histocompatability complex class II and other costimulatory molecules. ICSBP is known to regulate gene expression by interacting with partner proteins PU.1 and IRFs, thereby binding to target elements ISRE and EICE. Analysis of a series of ICSBP mutants showed that the intact DNA-binding activity as well as the ability to interact with partner proteins are required for the restoration of DC development/maturation, pointing to the transcriptional function of ICSBP as a basis of restoration. Taken together, this study identifies ICSBP as a factor critical for both early differentiation and final maturation of DCs.

Animals↗

PubMed2002

Bacteria-triggered CD4(+) T regulatory cells suppress Helicobacter hepaticus-induced colitis.

We have previously demonstrated that interleukin (IL)-10-deficient (IL-10 knockout [KO]) but not wild-type (WT) mice develop colitis after infection with Helicobacter hepaticus. Here, we show that infected recombination activating gene (RAG) KO mice develop intestinal inflammation after reconstitution with CD4(+) T cells from IL-10 KO animals and that the cotransfer of CD4(+) T cells from H. hepaticus-infected but not uninfected WT mice prevents this colitis. The disease-protective WT CD4(+) cells are contained within the CD45RB(low) fraction and unexpectedly were found in both the CD25(+) and the CD25(-) subpopulations of these cells, their frequency being higher in the latter. The mechanism by which CD25(+) and CD25(-) CD45RB(low) CD4(+) cells block colitis involves IL-10 and not transforming growth factor (TGF)-beta, as treatment with anti-IL-10R but not anti-TGF-beta monoclonal antibody abrogated their protective effect. In vitro, CD45RB(low) CD4(+) cells from infected WT mice were shown to produce IL-10 and suppress interferon-gamma production by IL-10 KO CD4(+) cells in an H. hepaticus antigen-specific manner. Together, our data support the concept that H. hepaticus infection results in the induction in WT mice of regulatory T cells that prevent bacteria-induced colitis. The induction of such cells in response to gut flora may be a mechanism protecting normal individuals against inflammatory bowel disease.

Adoptive Transfer↗

PubMed2002

Essential role for ICSBP in the in vivo development of murine CD8alpha + dendritic cells.

Interferon (IFN) consensus sequence-binding protein (ICSBP) is an important transcription factor regulating proinflammatory cytokine production and the development of mononuclear phagocytes in vitro. Here we analyzed the role of ICSBP in the in vivo differentiation of 3 major subsets of murine dendritic cells (DCs). We found that ICSBP is predominantly expressed by the CD8alpha(+) subset, and more important, that ICSBP(-/-) mice have a profound and selective deficiency in CD8alpha(+) DEC205(+) DCs in lymphoid tissues. Studies using wild-type/ICSBP(-/-) chimeras revealed that this defect in CD8alpha(+) DC development is intrinsic to bone marrow-derived progenitors and not dependent on ICSBP expression in the nonhemopoietic compartment. Because DC precursor frequencies are unaltered in the bone marrow of ICSBP(-/-) mice, ICSBP appears to function by regulating CD8alpha(+) DC differentiation downstream from the generation of common DC progenitors. Although CD8alpha(-) DCs are present in normal numbers in ICSBP(-/-) animals, up-regulation of CD40, CD80, and major histocompatibility complex (MHC) class II expression was found to be impaired in this subset after in vivo microbial stimulation. Together these results demonstrate that ICSBP is critically required for the in vivo differentiation of CD8alpha(+) DCs and may also influence the functional maturation of the CD8alpha(-) subsets.

Animals↗