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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

Immunoblot analysis of rBCGhIL-18. Lane 1 contains 100 ng of mature IL-18. Whole-cell extracts of nonrecombinant BCG (lane 2) or rBCGhIL-18 (lane 3), each corresponding to an optical density at 600 nm of 0.2 were subjected to SDS-PAGE and analyzed by immunoblotting using rabbit anti-IL-18 antibodies. The sizes of the molecular weight markers are indicated in the left margin, and the presence of mature IL-18 and pro-IL-18 is indicated by arrows.
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fig2: Immunoblot analysis of rBCGhIL-18. Lane 1 contains 100 ng of mature IL-18. Whole-cell extracts of nonrecombinant BCG (lane 2) or rBCGhIL-18 (lane 3), each corresponding to an optical density at 600 nm of 0.2 were subjected to SDS-PAGE and analyzed by immunoblotting using rabbit anti-IL-18 antibodies. The sizes of the molecular weight markers are indicated in the left margin, and the presence of mature IL-18 and pro-IL-18 is indicated by arrows.

Mentions: To construct rBCGhIL-18, the hIL-18-encoding cDNA was prepared as described previously [19] and modified by substituting the original IL-18 signal peptide coding sequence with the mycobacterial secretion signal sequence from the BCG α-antigen [29]. The modified hIL-18 cDNA was inserted into the expression vector pRR3 [23] (Figure 1) and then introduced into BCG. The recombinant BCG construction, named rBCGhIL-18, was found to produce human IL-18, as shown by immunoblot analyses using an anti-human IL-18 rabbit antiserum. Immunoreactive proteins were detected in the lysate of rBCG producing hIL-18, but not in the lysate of the nonrecombinant control strain (Figure 2). The rBCGhIL-18 extract contained an immune-reactive protein with a size expected for noncleaved pro-IL-18, as well as bands whose Mr are similar to that of mature IL-18, indicating that the pro-hIL-18 in rBCGhIL-18 is partially processed into the mature form, similarly to our previous study describing the expression and immunological activity of murine IL-18 produced by recombinant BCG [19]. The IL-18 expression by recombinant BCG was checked during the entire study period, always with the same results. The concentration of hIL-18 was quantified by ELISA in cell culture supernatants of BCG- or rBCGhIL-18-stimulated DC. For the three healthy individuals-tested, cultures contained 5–10 pg/mL and 18–22 pg/mL hIL-18, after 24 h stimulation with BCG or rBCGhIL-18, respectively. Unstimulated cultures contained only 3–7 pg/mL hIL-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)

Immunoblot analysis of rBCGhIL-18. Lane 1 contains 100 ng of mature IL-18. Whole-cell extracts of nonrecombinant BCG (lane 2) or rBCGhIL-18 (lane 3), each corresponding to an optical density at 600 nm of 0.2 were subjected to SDS-PAGE and analyzed by immunoblotting using rabbit anti-IL-18 antibodies. The sizes of the molecular weight markers are indicated in the left margin, and the presence of mature IL-18 and pro-IL-18 is indicated by arrows.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: Immunoblot analysis of rBCGhIL-18. Lane 1 contains 100 ng of mature IL-18. Whole-cell extracts of nonrecombinant BCG (lane 2) or rBCGhIL-18 (lane 3), each corresponding to an optical density at 600 nm of 0.2 were subjected to SDS-PAGE and analyzed by immunoblotting using rabbit anti-IL-18 antibodies. The sizes of the molecular weight markers are indicated in the left margin, and the presence of mature IL-18 and pro-IL-18 is indicated by arrows.
Mentions: To construct rBCGhIL-18, the hIL-18-encoding cDNA was prepared as described previously [19] and modified by substituting the original IL-18 signal peptide coding sequence with the mycobacterial secretion signal sequence from the BCG α-antigen [29]. The modified hIL-18 cDNA was inserted into the expression vector pRR3 [23] (Figure 1) and then introduced into BCG. The recombinant BCG construction, named rBCGhIL-18, was found to produce human IL-18, as shown by immunoblot analyses using an anti-human IL-18 rabbit antiserum. Immunoreactive proteins were detected in the lysate of rBCG producing hIL-18, but not in the lysate of the nonrecombinant control strain (Figure 2). The rBCGhIL-18 extract contained an immune-reactive protein with a size expected for noncleaved pro-IL-18, as well as bands whose Mr are similar to that of mature IL-18, indicating that the pro-hIL-18 in rBCGhIL-18 is partially processed into the mature form, similarly to our previous study describing the expression and immunological activity of murine IL-18 produced by recombinant BCG [19]. The IL-18 expression by recombinant BCG was checked during the entire study period, always with the same results. The concentration of hIL-18 was quantified by ELISA in cell culture supernatants of BCG- or rBCGhIL-18-stimulated DC. For the three healthy individuals-tested, cultures contained 5–10 pg/mL and 18–22 pg/mL hIL-18, after 24 h stimulation with BCG or rBCGhIL-18, respectively. Unstimulated cultures contained only 3–7 pg/mL hIL-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