Angela M. Green

IL-17 Production Is Dominated by γδ T Cells rather than CD4 T Cells during Mycobacterium tuberculosis Infection

IL-17 is a cytokine produced by T cells in response to IL-23. Recent data support a new subset of CD4 Th cells distinct from Th1 or Th2 cells that produce IL-17 and may contribute to inflammation. In this study, we demonstrate that, in naive mice, as well as during Mycobacterium tuberculosis infection, IL-17 production is primarily from γδ T cells and other non-CD4+CD8+ cells, rather than CD4 T cells. The production of IL-17 by these cells is stimulated by IL-23 alone, and strongly induced by the cytokines, including IL-23, produced by M. tuberculosis-infected dendritic cells. IL-23 is present in the lungs early in infection and the IL-17-producing cells, such as γδ T cells, may represent a central innate protective response to pulmonary infection.

Role of Toll-Like Receptors in Changes in Gene Expression and NF-κB Activation in Mouse Hepatocytes Stimulated with Lipopolysaccharide

ABSTRACT The liver is an important site of host-microbe interaction. Although hepatocytes have been reported to be responsive to lipopolysaccharide (LPS), the global gene expression changes by LPS and mechanism(s) by which LPS stimulates cultured hepatocytes remain uncertain. Cultures of primary mouse hepatocytes were incubated with LPS to assess its effects on the global gene expression, hepatic transcription factors, and mitogen-activated protein (MAP) kinase activation. DNA microarray analysis indicated that LPS modulates the selective expression of more than 80 genes and expressed sequence tags. We have shown previously that hepatocytes express CD14, which is required both for uptake and responsiveness to LPS. In other cells, responsiveness to microbial products requires expression of Toll-like receptors (TLR) and their associated accessory molecules. Hepatocytes expressed TLR1 through TLR9 as well as MyD88 and MD-2 transcripts, as shown by reverse transcriptase PCR analysis, indicating that hepatocytes express all known microbe recognition molecules. The MAP kinase extracellular signal-regulated kinase 1/2 was phosphorylated in response to LPS in mouse hepatocytes, and the levels of phosphorylation were lower in hepatocytes from TLR4-null mice. NF-κB activation was reduced in TLR4-mutant or -null hepatocytes compared to control hepatocytes, and this defect was partially restored by adenoviral transduction of mouse TLR4. Thus, hepatocytes respond to nanogram concentrations of LPS through a TLR4 response pathway.

Nitric-oxide production by murine mammary adenocarcinoma cells promotes tumor-cell invasiveness

The role of nitric oxide (NO) in tumor biology remains controversial and poorly understood. While a few reports indicate that the presence of NO in tumor cells or their micro‐environment is detrimental for tumor‐cell survival, and consequently their metastatic ability, a large body of data suggests that NO promotes tumor progression. The purpose of this study was to identify the source of NO in the spontaneously metastasizing C3‐L5 murine mammary‐adenocarcinoma model, the role of tumor‐derived NO in tumor‐cell invasiveness, and the mechanisms underlying the invasion‐stimulating effects of tumor‐derived NO. The source of NO was established by immunocytochemical localization of NO synthase (NOS) enzymes in C3‐L5 cells in vitro and transplanted tumors in vivo. An in vitro transwell Matrigel invasion assay was used to test the invasiveness of C3‐L5 cells in the presence or the absence of NO blocking agents or iNOS inducers (IFN‐γ and LPS). The mechanisms underlying the invasion‐stimulating effects of tumor‐derived NO were examined by measuring mRNA expression of matrix metalloproteinases (MMP)‐2 and ‐9, and tissue inhibitors of metalloproteinases (TIMP) 1, 2 and 3 in C3‐L5 cells in various experimental conditions. Results showed that C3‐L5 cells expressed high level of eNOS protein in vitro, and in vivo, both in primary and in metastatic tumors. C3‐L5 cells also expressed iNOS mRNA and protein when cultured in the presence of IFN‐γ and LPS. Constitutively produced NO promoted tumor‐cell invasiveness in vitro by down‐regulating TIMP 2 and TIMP 3. In addition, there was up‐regulation of MMP‐2, when extra NO was induced by IFN‐γ and LPS. In conclusion, NO produced by C3‐L5 cells promoted tumor‐cell invasiveness by altering the balance between MMP‐2 and its inhibitors TIMP‐2 and 3. Thus, our earlier observations of anti‐tumor and anti‐metastatic effects of NO inhibitors in vivo in this tumor model can be explained, at least in part, by reduced tumor‐cell invasiveness. Int. J. Cancer 81:889–896, 1999.

IFN-γ from CD4 T Cells Is Essential for Host Survival and Enhances CD8 T Cell Function during Mycobacterium tuberculosis Infection

IFN-γ is necessary in both humans and mice for control of Mycobacterium tuberculosis. CD4 T cells are a significant source of IFN-γ during acute infection in mice and are required for control of bacterial growth and host survival. However, several other types of cells can and do produce IFN-γ during the course of the infection. We sought to determine whether IFN-γ from sources other than CD4 T cells was sufficient to control M. tuberculosis infection and whether CD4 T cells had a role in addition to IFN-γ production. To investigate the role of IFN-γ from CD4 T cells, a murine adoptive transfer model was developed in which all cells were capable of producing IFN-γ, with the exception of CD4 T cells. Our data in this system support that CD4 T cells are essential for control of infection, but also that IFN-γ from CD4 T cells is necessary for host survival and optimal long-term control of bacterial burden. In addition, IFN-γ from CD4 T cells was required for a robust CD8 T cell response. IFN-γ from T cells inhibited intracellular replication of M. tuberculosis in macrophages, suggesting IFN-γ may be necessary for intracellular bactericidal activity. Thus, although CD4 T cells play additional roles in the control of M. tuberculosis infection, IFN-γ is a major function by which these cells participate in resistance to tuberculosis.

CD4+ Regulatory T Cells in a Cynomolgus Macaque Model of Mycobacterium tuberculosis Infection

BACKGROUND Mycobacterium tuberculosis infection in humans results in either latent infection or active tuberculosis. We sought to determine whether a higher frequency of regulatory T (T(reg)) cells predispose an individual toward active disease or whether T(reg) cells develop in response to active disease. METHODS In cynomolgus macaques infected with a low dose of M. tuberculosis, approximately 50% develop primary tuberculosis, and approximately 50% become latently infected. Forty-one animals were monitored for 6-8 months to assess the correlation of the frequency of Foxp3(+) cells in peripheral blood and airways with the outcome of infection. RESULTS In all animals, the frequency of T(reg) cells (CD4(+)Foxp3(+)) in peripheral blood rapidly decreased and simultaneously increased in the airways. Latently infected monkeys had a significantly higher frequency of T(reg) cells in peripheral blood before infection and during early infection, compared with monkeys that developed active disease. Monkeys with active disease experienced increased frequencies of T(reg) cells among peripheral blood mononuclear cells as they developed disease. CONCLUSIONS Our data suggest that increased frequencies of T(reg) cells in active disease occur in response to increased inflammation rather than act as a causative factor in progression to active disease.

The hepatocyte as a microbial product-responsive cell.

Much research has focused on the responses to microbial products of immune cells such as monocytes, macrophages, and neutrophils. Although the liver is a primary response organ in various infections, relatively little is known about the antimicrobial responses of its major cell type, the hepatocyte. It is now known that the recognition of bacteria occurs via cell-surface proteins that are members of the Toll-like receptor (TLR) family. In addition, lipopolysaccharide (LPS) is bound by circulating LPS-binding protein (LBP) and presented to cell-surface CD14, which in turn interacts with TLR and transduces an intracellular signal. We investigated the CD14 and TLR2 responses of whole liver and isolated hepatocytes, and demonstrated that these cells can be induced to express the molecules necessary for responses to both Gram-positive and Gram-negative bacteria. Our findings may have clinical implications for pathological states such as sepsis.

Aberrant TGF‐β signaling reduces T regulatory cells in ICAM‐1‐deficient mice, increasing the inflammatory response to Mycobacterium tuberculosis

Foxp3+ T regulatory cells are required to prevent autoimmune disease, but also prevent clearance of some chronic infections. While natural T regulatory cells are produced in the thymus, TGF‐β1 signaling combined with T‐cell receptor signaling induces the expression of Foxp3 in CD4+ T cells in the periphery. We found that ICAM‐1−/− mice have fewer T regulatory cells in the periphery than WT controls, due to a role for ICAM‐1 in induction of Foxp3 expression in response to TGF‐β1. Further investigation revealed a functional deficiency in the TGF‐β1‐induced translocation of phosphorylated Smad3 from the cytoplasmic compartment to the nucleus in ICAM‐1‐deficient mice. This impairment in the TGF‐β1 signaling pathway is most likely responsible for the decrease in T regulatory cell induction in the absence of ICAM‐1. We hypothesized that in the presence of an inflammatory response, reduced production of inducible T regulatory cells would be evident in ICAM‐1−/− mice. Indeed, following Mycobacterium tuberculosis infection, ICAM‐1−/− mice had a pronounced reduction in T regulatory cells in the lungs compared with control mice. Consequently, the effector T‐cell response and inflammation were greater in the lungs of ICAM‐1−/− mice, resulting in morbidity due to overwhelming pathology.