Masato Uchijima

Intracytosolic Listeria monocytogenes induces cell death through caspase-1 activation in murine macrophages.

Listeria monocytogenes induces apoptosis in vitro and in vivo in a variety of cell types. However, the mechanism of cell death in L. monocytogenes‐infected macrophages was initially reported to be distinct from apoptosis. Here, we studied the mechanism of L. monocytogenes‐induced cell death using sensitive fluorescent techniques. We found that caspase‐1 activation preceded cell death of macrophages infected with L. monocytogenes, using fluorogenic substrates. Caspase‐1 activation was diminished after infection with wild‐type L. monocytogenes when cells were treated with NH4Cl, or if they were infected with a listeriolysin mutant that cannot escape from the phagolysosome. Mitochondrial membrane integrity was preserved during the infection. A particular mechanism of cell death, recently termed ‘pyroptosis’, is associated with infection by intracellular microorganisms, and has an inherent pro‐inflammatory character, due to involvement of caspase‐1 activation with consequent IL‐1β and IL‐18 production. Cell death through caspase‐1 activation would constitute a defence mechanism of macrophages which induces cell death to eliminate the bacterias intracytosolic niche and recruits early hosts defences through the secretion of inflammatory cytokines.

Induction of Protective Cellular Immunity against Mycobacterium tuberculosis by Recombinant Attenuated Self-Destructing Listeria monocytogenes Strains Harboring Eukaryotic Expression Plasmids for Antigen 85 Complex and MPB/MPT51

ABSTRACT We report here the induction of specific protective cellular immunity against Mycobacterium tuberculosis by the employment of vaccination with recombinant attenuated Listeria monocytogenes strains. We constructed self-destructing attenuated L. monocytogenes Δ2 strains carrying eukaryotic expression plasmids for the antigen 85 complex (Ag85A and Ag85B) and for MPB/MPT51 (mycobacterial protein secreted by M. bovis BCG/mycobacterial protein secreted by M. tuberculosis) molecules. Infection of these recombinant bacteria allowed expression of the genes in the J774A.1 murine macrophage cell line. Intraperitoneal vaccination of C57BL/6 mice with these recombinant bacteria was capable of inducing purified protein derivative-specific cellular immune responses, such as foot pad reactions, proliferative responses of splenocytes, and gamma interferon production from splenocytes, suggesting the efficacy of vaccination against mycobacterial infection by use of these recombinant L. monocytogenes strains. Furthermore, intravenous vaccination with recombinant bacteria carrying expression plasmids for Ag85A, Ag85B, or MPB/MPT51 in BALB/c mice elicited significant protective responses, comparable to those evoked by a live Mycobacterium bovis BCG vaccine. Notably, this is the first report to show that MPB/MPT51 is a major protective antigen in addition to Ag85A and Ag85B, which have been reported to be major mycobacterial protective antigens.

Evidence for the critical role of interleukin-12 but not interferon-γ in the pathogenesis of experimental colitis in mice

Background and Aims: The imbalance between helper T (Th)1/Th2 cytokines has been observed in human inflammatory bowel disease and various animal models. Because interleukin (IL)‐12 and interferon‐γ (IFN‐γ) productions are known to be a hallmark of Th1‐dominant intestinal inflammation such as 2,4,6‐trinitrobenzene sulfonic acid (TNBS)‐induced colitis, we strictly addressed the roles of IFN‐γ and IL‐12 in the development of colitis, employing knockout mice with IFN‐γ receptor (IFN‐γR) or IL‐12 p40 gene disruptions and mice administered with neutralizing monoclonal antibodies (mAbs) against IFN‐γ or IL‐12.

Protective CTL response is induced in the absence of CD4+ T cells and IFN-γ by gene gun DNA vaccination with a minigene encoding a CTL epitope of Listeria monocytogenes

Our work was undertaken to learn the mechanism of induction of protective cytotoxic T lymphocytes (CTL) by gene gun DNA vaccination with p91m encoding an H-2Kd-restricted T cell epitope of listeriolysin O (LLO). Vaccination with p91m induced vigorous antigen-specific CD8+ CTL that produce IFN-gamma and was able to confer partial protection against listerial challenge. However, the p91m-induced protective immunity was revealed to be independent of the IFN-gamma and CD4+ T cell help. The CTL induction is also suggested to require neither adjuvant activity of the plasmid used nor IFN-gamma. The data may be feasible for the design of CTL inducing vaccines in various immunodeficiencies.

IFN-γ overcomes low responsiveness of myeloid dendritic cells to CpG DNA

Dendritic cells (DC) are professional APC that have an extraordinary capacity to prime naive T cells. It has been reported that human DC subsets express distinct toll‐like receptor (TLR), which influences their function. In mice, we observed that plasmocytoid DC (pDC) express a higher level of TLR9 compared with myeloid DC (mDC) cultured with GM‐CSF. However, we demonstrated that stimulation with IFN‐γ is capable of upregulating TLR9 expression in mDC to a level comparable with expression in pDC. Consistent with this observation, IL‐12 p40 and IL‐6 mRNA expression and IL‐12 p70 secretion in response to CpG‐oligodeoxynucleotides are enhanced in mDC pretreated with IFN‐γ compared with untreated cells. Therefore, TLR‐mediated responses of DC subsets may be influenced not only by signals delivered by pathogens but also by regulatory signals from cytokines such as IFN‐γ.

Identification of an HLA-A*0201-Restricted T-Cell Epitope on the MPT51 Protein, a Major Secreted Protein Derived from Mycobacterium tuberculosis, by MPT51 Overlapping Peptide Screening

ABSTRACT CD8+ T cells play a pivotal role in protection against Mycobacterium tuberculosis infection. We identified a novel HLA-A*0201-restricted CD8+ T-cell epitope on a dominant secreted antigen of M. tuberculosis, MPT51, in HLA-A*0201 transgenic HHD mice. HHD mice were immunized with plasmid DNA encoding MPT51 with gene gun bombardment, and gamma interferon (IFN-γ) production by the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, the splenocytes were stimulated to produce IFN-γ by only one peptide, p51-70. Three-color flow cytometric analysis of intracellular IFN-γ and cell surface CD4 and CD8 staining revealed that the MPT51 p51-70 peptide contains an immunodominant CD8+ T-cell epitope. Further analysis using computer algorithms permitted identification of a bona fide T-cell epitope, p53-62. A major histocompatibility complex class I stabilization assay using T2 cells confirmed that this epitope binds to HLA-A*0201. The T cells were capable of lysing MPT51 p53-62 peptide-pulsed T2 cells. In addition, MPT51 p53-62-specific memory CD8+ T cells were found in tuberculin skin test-positive HLA-A*0201+ healthy individuals. Use of this HLA-A*0201-restricted CD8+ T-cell epitope for analysis of the role of MPT51-specific T cells in M. tuberculosis infection and for design of vaccines against tuberculosis is feasible.

A novel vaccine strategy to induce mycobacterial antigen-specific Th1 responses by utilizing the C-terminal domain of heat shock protein 70.

Heat shock protein 70 (HSP70) is a member of a highly conserved superfamily of intracellular chaperones called stress proteins that can activate innate and adaptive immune responses. We evaluated the effect of a fusion DNA vaccine that encoded mycobacterial HSP70 and MPT51, a major secreted protein of Mycobacterium tuberculosis. Spleen cells from mice immunized with fusion DNA of full-length HSP70 and MPT51 produced a higher amount of interferon-γ (IFN-γ) in response to the CD4+, but not the CD8+ T-cell epitope peptide on MPT51 than those from mice immunized with MPT51 DNA. Furthermore, because HSP70 comprises the N-terminal ATPase domain and the C-terminal peptide-binding domain, we attempted to identify the domain responsible for its enhancing effect. The fusion DNA vaccine that encoded the C-terminal domain of HSP70 and MPT51 induced a higher MPT51-specific IFN-γ production by CD4+ T cells than the vaccine that encoded MPT51 alone, whereas that with the N-terminal domain did not. Similar results were obtained by immunization with the fusion proteins. These results suggest that the DNA vaccine that encodes a chimeric antigen molecule fused with mycobacterial HSP70, especially with its C-terminal domain, can induce a stronger antigen-specific T-helper cell type 1 response than antigen DNA alone.

Butyrate suppresses hypoxia-inducible factor-1 activity in intestinal epithelial cells under hypoxic conditions

Interaction between the products of intestinal bacteria and the intestinal epithelial cells is a key event in understanding the biological, physiological, and pathological functions of the intestinal epithelium. Here, we examined the effect of butyrate, one of the major intestinal bacterial products, on hypoxia-inducible factor-1 (HIF-1) activity under hypoxic conditions in intestinal epithelial cells. HIF-1 activity was assessed by luciferase assay using cytoplasmic extracts of intestinal epithelial cells, Caco-2, and IEC-6 cells. These cells were transiently transfected with hypoxia response element (HRE)-luciferase reporter plasmids and cultured under hypoxic conditions in the presence or absence of sodium butyrate (NaB). The effect of NaB on HRE DNA binding activity in Caco-2 cells under hypoxic conditions was assessed by electrophoretic mobility shift assay. Expression of a hypoxia-responsive gene encoding intestinal trefoil factor (ITF) in Caco-2 cells after NaB treatment was assessed using reverse-transcription PCR. The barrier function of Caco-2 cells under hypoxic conditions was also evaluated by transepithelial electrical resistance measurement. NaB suppressed up-regulation of HIF-1 transcriptional activity under hypoxic conditions in Caco-2 and IEC-6 cells. In parallel, NaB reduced HRE DNA binding activity under the same conditions. Furthermore, NaB down-regulated enhanced transcription of ITF gene. Addition of NaB under hypoxic conditions delayed recovery of transepithelial electrical resistance of the monolayers after hypoxia-reoxygenation treatment. These findings indicate that NaB suppresses HIF-1 transcriptional activity on hypoxia-responsive genes by reducing the HRE DNA binding activity under hypoxic conditions in intestinal epithelial cells.

Intratracheal administration of third-generation lentivirus vector encoding MPT51 from Mycobacterium tuberculosis induces specific CD8+ T-cell responses in the lung.

The present study evaluates the potential of improved third-generation lentivirus vector with respect to their use as an in vivo-administered T-cell vaccine against tuberculosis. Intratracheal administration of the lentivirus vector encoding MPT51 of Mycobacterium tuberculosis could induce MPT51-specific CD8+ T cells in the mediastinal lymph nodes 2 weeks after the administration. The vaccination could generate MPT51-specific memory CD8+ T cells in the lung, but not in the lymph nodes. Further, a single intratracheal immunization of MPT51 lentiviral vaccine decreased significantly the number of virulent M. tuberculosis in the lung after intratracheal challenge of the bacillus. These findings suggest that intratracheal immunization of the third-generation lentiviral vaccines is a promising vaccination strategy against pulmonary tuberculosis.

Identification of HLA-DR4-restricted T-cell epitope on MPT51 protein, a major secreted protein derived from Mycobacterium tuberculosis using MPT51 overlapping peptides screening and DNA vaccination

We identified a novel HLA-DR4-restricted CD4+ T-cell epitope on a secreted antigen of Mycobacterium tuberculosis, MPT51, in 004149-MM HLA-DR4-transgenic mice which express HLA-DRB1*0401, but not murine MHC class II molecules. The mice were immunized with plasmid DNA encoding MPT51 using gene gun and interferon (IFN)-gamma production from the immune splenocytes was analyzed. In response to overlapping synthetic peptides covering the mature MPT51 sequence, only one peptide, p191-210, stimulated the splenocytes to produce IFN-gamma. Further analysis using flow cytometry and computer-assisted algorithm, ProPred, narrowed down the region of CD4+ T-cell epitope to p191-202. The CD4+ T-cell epitope would be feasible for vaccine design against tuberculosis as well as for analysis of MPT51-specific T-cells in M. tuberculosis infection.