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Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

Szpakowski P, Biet F, Locht C, Paszkiewicz M, Rudnicka W, Druszczyńska M, Allain F, Fol M, Pestel J, Kowalewicz-Kulbat M - J Immunol Res (2015)

Bottom Line: We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective.In contrast, both BCG strains decreased the DC-SIGN expression on human DCs.The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, University of Lodz, Banacha Street 12/19, 90-237 Lodz, Poland.

ABSTRACT
Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

No MeSH data available.


Related in: MedlinePlus

Construction of pENhIL-18 used to produce and secrete hIL-18 by rBCGhIL-18. White arrows KanR and AmpR represent the kanamycin and ampicillin resistance genes, respectively. ColE1 represents the origin of replication from E.coli (hatched boxes), and oriM represents the mycobacterial origin of replication (hatched arrows). The expression cassette contains the BCG hsp60 promoter, the ribosome-binding site, and the hsp60 initiating codon represented by the black box. The M. tuberculosis alpha-antigen signal peptide coding sequence is represented by the white box, whereas the IL-18 coding sequence is shown in black. The grey boxes indicate open reading frames of the mycobacterial plasmid, necessary for replication in BCG.
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fig1: Construction of pENhIL-18 used to produce and secrete hIL-18 by rBCGhIL-18. White arrows KanR and AmpR represent the kanamycin and ampicillin resistance genes, respectively. ColE1 represents the origin of replication from E.coli (hatched boxes), and oriM represents the mycobacterial origin of replication (hatched arrows). The expression cassette contains the BCG hsp60 promoter, the ribosome-binding site, and the hsp60 initiating codon represented by the black box. The M. tuberculosis alpha-antigen signal peptide coding sequence is represented by the white box, whereas the IL-18 coding sequence is shown in black. The grey boxes indicate open reading frames of the mycobacterial plasmid, necessary for replication in BCG.

Mentions: The M. bovis BCG vaccine strain 1173P2 (World Health Organization, Stockholm, Sweden) was genetically modified to produce mature human IL-18 (hIL-18) by transformation with pENhIL-18 (Figure 1), a pRR3 derivative [23] containing the mature hIL-18-encoding gene under the control of the BCG hsp60 promoter and modified by replacing the original signal peptide cleavage sequence with the mycobacterial signal peptide coding sequence from the BCG α-antigen. The 512 bp DNA fragment encoding hIL-18 was obtained by reverse transcription-PCR on total RNA from U-937 cells activated for 6 h with LPS (1 μg/mL). Total RNA was extracted using RNAzol (Applied Oncor) according to the manufacturer's recommendation. Reverse transcription-PCR was carried out as described previously [17] with the gene-specific primers with the following sequences: 5′-TATAGGATCCTACTTTGGCAAGCTTGAA-3′ and 5′-TATAGGTACCGGCATGAAATTTTAATAGC-3′ (Eurogentec, Liège, Belgium). The 126-bp DNA fragment encoding the α-antigen signal sequence was amplified by PCR using chromosomal BCG DNA extracted as described previously [24] with the primers 5′- GGCACAGGTCATGACAGACGTGAGCCGAAAGATTCGA-3′ and 5′-GCCGGGATCCCGCGCCCGCGGTTGCCGCTCCGCC-3′ (Eurogentec). The PCR fragment encoding hIL-18 restricted by BglII and Asp718 and the PCR fragment encoding the α-antigen signal sequence restricted by BspHI and BamHI were inserted into pUC::hsp60 [24] restricted by NcoI and Asp718, thereby generating pUC::hIL-18. The 1.32 kb PvuII fragment from pUC::hIL-18 spanning the BCG hsp60 promoter, the ribosomal binding site, the α-antigen signal peptide coding sequence, and the mature hIL-18 coding sequence, was inserted into pRR3, previously digested with ScaI. The resulting shuttle vector, pENhIL-18, was used to transform BCG, and the transformants were selected by their resistance to Kanamycin. Kanamycin-resistant BCG colonies were analyzed for their plasmid content by using electroduction [25]. The recombinant strain was named rBCGhIL-18.


Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

Szpakowski P, Biet F, Locht C, Paszkiewicz M, Rudnicka W, Druszczyńska M, Allain F, Fol M, Pestel J, Kowalewicz-Kulbat M - J Immunol Res (2015)

Construction of pENhIL-18 used to produce and secrete hIL-18 by rBCGhIL-18. White arrows KanR and AmpR represent the kanamycin and ampicillin resistance genes, respectively. ColE1 represents the origin of replication from E.coli (hatched boxes), and oriM represents the mycobacterial origin of replication (hatched arrows). The expression cassette contains the BCG hsp60 promoter, the ribosome-binding site, and the hsp60 initiating codon represented by the black box. The M. tuberculosis alpha-antigen signal peptide coding sequence is represented by the white box, whereas the IL-18 coding sequence is shown in black. The grey boxes indicate open reading frames of the mycobacterial plasmid, necessary for replication in BCG.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4539176&req=5

fig1: Construction of pENhIL-18 used to produce and secrete hIL-18 by rBCGhIL-18. White arrows KanR and AmpR represent the kanamycin and ampicillin resistance genes, respectively. ColE1 represents the origin of replication from E.coli (hatched boxes), and oriM represents the mycobacterial origin of replication (hatched arrows). The expression cassette contains the BCG hsp60 promoter, the ribosome-binding site, and the hsp60 initiating codon represented by the black box. The M. tuberculosis alpha-antigen signal peptide coding sequence is represented by the white box, whereas the IL-18 coding sequence is shown in black. The grey boxes indicate open reading frames of the mycobacterial plasmid, necessary for replication in BCG.
Mentions: The M. bovis BCG vaccine strain 1173P2 (World Health Organization, Stockholm, Sweden) was genetically modified to produce mature human IL-18 (hIL-18) by transformation with pENhIL-18 (Figure 1), a pRR3 derivative [23] containing the mature hIL-18-encoding gene under the control of the BCG hsp60 promoter and modified by replacing the original signal peptide cleavage sequence with the mycobacterial signal peptide coding sequence from the BCG α-antigen. The 512 bp DNA fragment encoding hIL-18 was obtained by reverse transcription-PCR on total RNA from U-937 cells activated for 6 h with LPS (1 μg/mL). Total RNA was extracted using RNAzol (Applied Oncor) according to the manufacturer's recommendation. Reverse transcription-PCR was carried out as described previously [17] with the gene-specific primers with the following sequences: 5′-TATAGGATCCTACTTTGGCAAGCTTGAA-3′ and 5′-TATAGGTACCGGCATGAAATTTTAATAGC-3′ (Eurogentec, Liège, Belgium). The 126-bp DNA fragment encoding the α-antigen signal sequence was amplified by PCR using chromosomal BCG DNA extracted as described previously [24] with the primers 5′- GGCACAGGTCATGACAGACGTGAGCCGAAAGATTCGA-3′ and 5′-GCCGGGATCCCGCGCCCGCGGTTGCCGCTCCGCC-3′ (Eurogentec). The PCR fragment encoding hIL-18 restricted by BglII and Asp718 and the PCR fragment encoding the α-antigen signal sequence restricted by BspHI and BamHI were inserted into pUC::hsp60 [24] restricted by NcoI and Asp718, thereby generating pUC::hIL-18. The 1.32 kb PvuII fragment from pUC::hIL-18 spanning the BCG hsp60 promoter, the ribosomal binding site, the α-antigen signal peptide coding sequence, and the mature hIL-18 coding sequence, was inserted into pRR3, previously digested with ScaI. The resulting shuttle vector, pENhIL-18, was used to transform BCG, and the transformants were selected by their resistance to Kanamycin. Kanamycin-resistant BCG colonies were analyzed for their plasmid content by using electroduction [25]. The recombinant strain was named rBCGhIL-18.

Bottom Line: We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective.In contrast, both BCG strains decreased the DC-SIGN expression on human DCs.The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Infectious Biology, Institute of Microbiology, Biotechnology and Immunology, University of Lodz, Banacha Street 12/19, 90-237 Lodz, Poland.

ABSTRACT
Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

No MeSH data available.


Related in: MedlinePlus