Limits...
The B subunit of Escherichia coli heat-labile toxin alters the development and antigen-presenting capacity of dendritic cells.

Ji J, Griffiths KL, Milburn PJ, Hirst TR, O'Neill HC - J. Cell. Mol. Med. (2015)

Bottom Line: In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide.In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c(+) DC.In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells.

View Article: PubMed Central - PubMed

Affiliation: Research School of Biology, Australian National University, Canberra, ACT, Australia.

No MeSH data available.


Related in: MedlinePlus

Direct effect of EtxB on sorted DC subsets in culture. Sorted subsets of CD11c+ CD11b− CD8− (pDC) and CD11chi CD11b+ CD8− (CD8− cDC) cells were cultured for 12 hrs with either 10 μg/ml EtxB or EtxB HI, 10 ng/ml LPS, a combination of EtxB and LPS, or medium as a control. Collected cells were then stained with antibodies to CD11c, CD11b, CD8, MHC-II and CD69 for flow cytometric analysis. Isotype control antibodies were used to set gates and to determine % positive cells. Propidium iodide (PI) staining was used to detect % live (PI−) cells. MHC-II expression was determined as MFI. Expression of CD69 is shown in terms of % cells staining. Data are presented as mean ± SE of duplicates or triplicates shown as crosshairs. Results are representative of two separate experiments. Significantly different pairs of data at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 are represented by *, ** and *** respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4549052&req=5

fig05: Direct effect of EtxB on sorted DC subsets in culture. Sorted subsets of CD11c+ CD11b− CD8− (pDC) and CD11chi CD11b+ CD8− (CD8− cDC) cells were cultured for 12 hrs with either 10 μg/ml EtxB or EtxB HI, 10 ng/ml LPS, a combination of EtxB and LPS, or medium as a control. Collected cells were then stained with antibodies to CD11c, CD11b, CD8, MHC-II and CD69 for flow cytometric analysis. Isotype control antibodies were used to set gates and to determine % positive cells. Propidium iodide (PI) staining was used to detect % live (PI−) cells. MHC-II expression was determined as MFI. Expression of CD69 is shown in terms of % cells staining. Data are presented as mean ± SE of duplicates or triplicates shown as crosshairs. Results are representative of two separate experiments. Significantly different pairs of data at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 are represented by *, ** and *** respectively.

Mentions: In further experiments, T and B cell-depleted splenocytes were sorted for isolation of pure populations of pDC and CD8− cDC using marker expression as shown in Figure1. In general, CD8+ cDC were not readily recoverable in high enough numbers to be included in these experiments. Sorted pDC and CD8− cDC were therefore cultured in vitro for 12 hrs with LPS and EtxB as described above. Cells were stained with PI to determine cell viability and with an antibody to detect MHC-II and CD69 expression by flow cytometry. Results obtained were consistent with those shown in Figures3 and 4 involving cultures of heterogeneous splenic myeloid cells. Increased cell viability was seen for both cell types cultured with EtxB, with no change following treatment with LPS (Fig.5). This was associated with reduced expression of markers of activation including MHC-II and CD69 for pDC, and of MHC-II for CD8− cDC (Fig.5). Again, LPS had minimal effect on the expression of activation markers like MHC-II and CD69 over a 12 hr assay. This confirms the distinct effect of EtxB in supporting the viability of immature DC subsets as opposed to activated cDC and pDC.


The B subunit of Escherichia coli heat-labile toxin alters the development and antigen-presenting capacity of dendritic cells.

Ji J, Griffiths KL, Milburn PJ, Hirst TR, O'Neill HC - J. Cell. Mol. Med. (2015)

Direct effect of EtxB on sorted DC subsets in culture. Sorted subsets of CD11c+ CD11b− CD8− (pDC) and CD11chi CD11b+ CD8− (CD8− cDC) cells were cultured for 12 hrs with either 10 μg/ml EtxB or EtxB HI, 10 ng/ml LPS, a combination of EtxB and LPS, or medium as a control. Collected cells were then stained with antibodies to CD11c, CD11b, CD8, MHC-II and CD69 for flow cytometric analysis. Isotype control antibodies were used to set gates and to determine % positive cells. Propidium iodide (PI) staining was used to detect % live (PI−) cells. MHC-II expression was determined as MFI. Expression of CD69 is shown in terms of % cells staining. Data are presented as mean ± SE of duplicates or triplicates shown as crosshairs. Results are representative of two separate experiments. Significantly different pairs of data at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 are represented by *, ** and *** respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4549052&req=5

fig05: Direct effect of EtxB on sorted DC subsets in culture. Sorted subsets of CD11c+ CD11b− CD8− (pDC) and CD11chi CD11b+ CD8− (CD8− cDC) cells were cultured for 12 hrs with either 10 μg/ml EtxB or EtxB HI, 10 ng/ml LPS, a combination of EtxB and LPS, or medium as a control. Collected cells were then stained with antibodies to CD11c, CD11b, CD8, MHC-II and CD69 for flow cytometric analysis. Isotype control antibodies were used to set gates and to determine % positive cells. Propidium iodide (PI) staining was used to detect % live (PI−) cells. MHC-II expression was determined as MFI. Expression of CD69 is shown in terms of % cells staining. Data are presented as mean ± SE of duplicates or triplicates shown as crosshairs. Results are representative of two separate experiments. Significantly different pairs of data at P ≤ 0.05, P ≤ 0.01 and P ≤ 0.001 are represented by *, ** and *** respectively.
Mentions: In further experiments, T and B cell-depleted splenocytes were sorted for isolation of pure populations of pDC and CD8− cDC using marker expression as shown in Figure1. In general, CD8+ cDC were not readily recoverable in high enough numbers to be included in these experiments. Sorted pDC and CD8− cDC were therefore cultured in vitro for 12 hrs with LPS and EtxB as described above. Cells were stained with PI to determine cell viability and with an antibody to detect MHC-II and CD69 expression by flow cytometry. Results obtained were consistent with those shown in Figures3 and 4 involving cultures of heterogeneous splenic myeloid cells. Increased cell viability was seen for both cell types cultured with EtxB, with no change following treatment with LPS (Fig.5). This was associated with reduced expression of markers of activation including MHC-II and CD69 for pDC, and of MHC-II for CD8− cDC (Fig.5). Again, LPS had minimal effect on the expression of activation markers like MHC-II and CD69 over a 12 hr assay. This confirms the distinct effect of EtxB in supporting the viability of immature DC subsets as opposed to activated cDC and pDC.

Bottom Line: In this respect, the in vivo effect of EtxB differs from that of the highly inflammatory mediator lipopolysaccharide.In terms of the in vivo effect of EtxB on CD4 and CD8 T cell responses in mice, the interaction of EtxB directly with DC was demonstrated following conditional depletion of CD11c(+) DC.In summary, all results are consistent with EtxB displaying adjuvant ability by enhancing the turnover of DC in spleen, leading to newly mature myeloid and DC in spleen, thereby increasing DC capacity to perform as antigen-presenting cells on encounter with T cells.

View Article: PubMed Central - PubMed

Affiliation: Research School of Biology, Australian National University, Canberra, ACT, Australia.

No MeSH data available.


Related in: MedlinePlus