Different transcriptional profiles of human monocyte-derived dendritic cells infected with distinct strains of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin.

In order to analyze dendritic cells (DCs) activation following infection with different mycobacterial strains, we studied the expression profiles of 165 genes of human monocyte-derived DCs infected with H37Rv, a virulent Mycobacterium tuberculosis (MTB) laboratory strain, CMT97, a clinical MTB isolate, Mycobacterium bovis bacillus Calmette-Guérin (BCG), Aventis Pasteur, and BCG Japan, both employed as vaccine against tuberculosis. The analysis of the gene expression reveals that, despite a set of genes similarly modulated, DCs response resulted strain dependent. In particular, H37Rv significantly upregulated EBI3 expression compared with BCG Japan, while it was the only strain that failed to release a significant IL-10 amount. Of note, BCG Japan showed a marked increase in CCR7 and TNF-α expression regarding both MTB strains and it resulted the only strain failing in exponential intracellular growth. Our results suggest that DCs display the ability to elicit a tailored strain-specific immune response.

Recombinant BCG-Rv1767 amount determines, in vivo, antigen-specific T cells location, frequency, and protective outcome.

One possibility to improve the efficacy of BCG vaccine against TB is to create a recombinant BCG (r-BCG), increasing the expression of mycobacterial antigens, to ameliorate the response to BCG. Here we describe a new r-BCG expressing the gene Rv1767, induced by Mycobacterium tuberculosis during its survival in human macrophages. The r-BCG elicited a specific T cells response in Balb/c mice higher than wt BCG. The r-BCG amount used to immunise mice determined diverse Th1/Th2 equilibriums, which was not the same in spleen and Lymph Nodes. Differences in cytokines production were found for IL-10, IL-4, TNF-alpha, and Arginase-1, which, in some conditions, resulted higher in r-BCG as compared to wt BCG-immunised mice. The immunisation with r-BCG-Rv1767 induced a lesser protective activity than wt BCG in a mouse model of TB. This reduction might likely be explained by the specific T cells phenotype and setting existing before MTB challenge, induced by either the single or the triple dose of r-BCG. The use of this model may help to highlight the capacity of different M. tuberculosis antigens to induce a protective immune response, actually not necessarily embodied by an increased frequency of Antigen-specific effector memory T cells.

Profiling of Mycobacterium tuberculosis gene expression during human macrophage infection: upregulation of the alternative sigma factor G, a group of transcriptional regulators, and proteins with unknown function.

Mycobacterium tuberculosis is one of the most prolific pathogens worldwide, and its virulence resides in its capacity to survive in human macrophages. In the present study, we analyzed the gene expression profile of M. tuberculosis H37Rv in macrophages and synthetic medium at the whole genome level. Out of 3875 spots tested, 970 genes passed the statistical significance filter (t scores +/-2.5). A total of 22% of those assayed were found to be active genes (up- or downregulated), representing 5.5% of the whole MTB genome. Interestingly, 32.5% of the genes induced in our macrophage experiments are still classified as hypothetical proteins; 19.5% take part in the cell wall and processes (half of which are membrane proteins); 16% are involved in regulation and information pathways; and the PE family accounts for 3.6% of total induced genes. It is important to note that in the course of MTB replication in macrophages, we observed the upregulation of alternative sigma factor sigG and 13 MTB transcriptional regulators. The data for a selected group of upregulated genes were confirmed by real-time RT-PCR. The global MTB transcriptome described in this study suggests an intracellular MTB actively sensing its environment; it repairs and synthesizes its cell wall and DNA, so as to either repair oxidative and nitrosative damage and/or to augment its copy number and evade host cell killing. As far as we know, this is the first study describing MTB expression profiles using whole genome macroarrays during primary human macrophage infection.

An improved, real-time PCR assay for the detection of GC-rich and low abundance templates of Mycobacterium tuberculosis.

The detection of low abundance mRNA and/or GC-rich targets is very difficult using real-time PCR, often requiring laborious optimization procedures. This work shows that formamide is a useful PCR additive, increasing the sensitivity and specificity of SYBR Green real-time PCR to detect low abundance mycobacterial RNA from infected samples.

Transcriptional profile of the immune response in the lungs of patients with active tuberculosis.

Despite advances in diagnosis and treatment, Mycobacterium tuberculosis causes active disease in about 8 million people worldwide annually. The study of the interplay between the host and the pathogen at the site of infection in human TB may contribute to elucidate the pathogenesis of the disease. In this work, using macroarray technology and real-time PCR, we analyzed the modulation of 847 genes encoding immune-inflammatory mediators in BALF samples of patients affected by active pulmonary TB (PTB) and control patients affected by non-TB diseases. The data show that the PTB milieu contains a complex network of gene activation. Different genes with adhesive properties and involved in tissue repair and fibrosis were modulated. In TB patients, we observed the up-regulation of cytokines, including IFN-gamma and IFN-gamma pathway genes, of several apoptotic genes, and of potent transcriptional activators. These findings can contribute to elucidate the mechanisms of MTB pathogenicity in humans.

Gene expression profiling of human macrophages at late time of infection with Mycobacterium tuberculosis.

Macrophages play an essential role in the immune response to Mycobacterium tuberculosis (Mtb). Previous transcriptome surveys, by means of micro- and macroarrays, investigated the cellular gene expression profile during the early phases of infection (within 48 hr). However, Mtb remains within the host macrophages for a longer period, continuing to influence the macrophage gene expression and, consequently, the environment in which it persists. Therefore, we studied the transcription patterns of human macrophages for up to 7 days after infection with Mtb. We used a macroarray approach to study 858 human genes involved in immunoregulation, and we confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (q-rt RT-PCR) and by enzyme-linked immunosorbent assay the most relevant modulations. We constantly observed the up-regulation in infected macrophages versus uninfected, of the following genes: interleukin-1 beta and interleukin-8, macrophage inflammatory protein-1 alpha, growth-related oncogene-beta, epithelial cell-derived neutrophil-activating peptide-78, macrophage-derived chemokine, and matrix metalloproteinase-7; whereas macrophage colony-stimulating factor-receptor and CD4 were down-regulated in infected macrophages. Mtb is able to withstand this intense cytokine microenvironment and to survive inside the human macrophage. Therefore we simultaneously investigated by q-rt RT-PCR the modulation of five mycobacterial genes: the alternative sigma factors sigA, sigE and sigG, the alpha-crystallin (acr) and the superoxide dismutase C (sodC) involved in survival mechanisms. The identified host and mycobacterial genes that were expressed until 7 days after infection, could have a role in the interplay between the host immune defences and the bacterial escape mechanisms.

HIV-1 matrix protein p17 enhances the proliferative activity of natural killer cells and increases their ability to secrete proinflammatory cytokines.

We investigated the effects of human immunodeficiency type-1 virus (HIV-1) matrix protein p17 on freshly isolated and purified human natural killer (NK) cells. HIV-1 p17 increased the cytokines interleukin (IL) 2, IL-12 and IL-15, and induced natural killer cell proliferation, but not cytotoxicity. This effect was specific because it was abrogated by anti-p17 monoclonal antibody. Moreover, HIV-1 p17 enhanced the cytokine-induced production of tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma by NK cells. IL-4 downregulated IFN-gamma and TNF-alpha secretion in IL-2- and IL-15-treated NK cells. HIV-1 p17 restored the ability of NK cells to produce both cytokines when added to the cultures simultaneously with IL-4. The property of p17 to increase the production of TNF-alpha and IFN-gamma might be a mechanism used by HIV-1 to modulate the immune system to support its replication and spreading.

Human herpesvirus-6 modulates RANTES production in primary human endothelial cell cultures.

Human herpesvirus 6 (HHV6) is a beta-herpesvirus capable of infecting several cell types from different origins. HHV6 is the etiological agent of exantem subitum and has been associated with several diseases, all characterized by an inflammatory response triggered by chemokines. We show that strain U1102 of HHV6 is able to infect persistently human endothelial cells obtained from umbilical veins, adult aorta and adult heart microvessels, without apparent cytopathic effect. Analysis by in situ PCR showed that HHV6 sequences were present in 20% of HUVEC, 10% of aortic, and 1% of heart microvascular endothelial cells. Regardless of endothelial cell origin, HHV6 infection induced de novo synthesis of the RANTES CC-chemokine. It was found, however, that microvascular endothelial cells, despite their lower susceptibility to HHV6 infection, showed the highest RANTES expression. Chemokine production occurred also in the absence of viral DNA synthesis. Furthermore, RANTES synthesis required an active viral genome, as UV-inactivated HHV6 infection of endothelial cells did not lead to chemokine production. We investigated the expression of HHV6 U51 gene, which encodes a chemokine receptor that is already known to sequester and down modulate RANTES in epithelial cells. HHV6-infected endothelial cells co-expressed RANTES and U51 mRNAs starting from 12 hr up to 48 hr post-infection. Then, RANTES transcripts disappeared whereas U51 messages continued to be expressed. In conclusion, this study highlights the major role of HHV6 in endothelial cell biology and the development of inflammatory processes.

HHV-6 infects human aortic and heart microvascular endothelial cells, increasing their ability to secrete proinflammatory chemokines.

Endothelial cells are important targets for herpesvirus infection. To evaluate the biological effects of human herpesvirus-6 (HHV-6) infection, adult heart microvascular and aortic endothelial cells were examined for in vitro susceptibility to HHV-6 and for the alterations induced by viral infection on the production of monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8). Analysis by reverse transcription-polymerase chain reaction and by in situ polymerase chain reaction showed that HHV-6 replicates in endothelium in the absence of cytopathic effects, and that viral sequences were present in 20% umbilical vein and in 10% aortic and 1% microvascular endothelium. HHV-6 infection upregulated the production of MCP-1 and IL-8, with differences observed between aortic and microvascular endothelium. These findings demonstrate that endothelial cells represent a potential reservoir for HHV-6 infection, and the altered pattern of chemokine production can lead to attraction of immunocompetent cells and to the development of inflammatory processes.

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor.

Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor alpha and IFN-gamma released from cells stimulated by IL-2. IL-4 was found to down-regulate IFN-gamma and tumor necrosis factor alpha, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH(2)-terminal region of viral protein. Immunization of BALB/c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.