Fig 1.
Stained PBMC were gated on cells with high FSC and SSC characteristics avoiding the lymphocyte region (A), doublets/multiplets were eliminated (B) and subsequently live cells were gated based on expression of CD14 (C). The viability channel (live/dead-L/D-) also contained CD3; therefore cells selected were also CD3- (C). CD14+ cells also expressed CD16 and HLA-DR. Therefore, these cells were classified as monocytes (D). All CD3-CD14- expressed HLA-DR and a percentage of these cells expressed either CD11c or CD123 but lacked CD19, CD66b, CD16 and CD56; therefore, these cells were classified as dendritic cells (DC) (E) (see also S4 Fig). The identity of these cells as DCs was confirmed using PBMC from healthy volunteers stained with monoclonal antibodies to BDCA-1, BDCA-2 and BDCA-3 (S4 Fig). Plots shown are from a representative volunteer. Gray histograms represent fluorescent minus one (FMO) samples.
Fig 2.
Monocyte levels in peripheral blood following infection with wt S. Typhi.
The percentages of circulating monocytes in TD (circles; n = 5) and NoTD (triangles; n = 3) participants were compared to the levels before wt S. Typhi challenge (A), as well as in subsequent days (B and C). In TD volunteers (B), the data is depicted relative to the time of typhoid diagnosis (TD) (dotted vertical line). Time points before (except for pre-challenge) and after (>+7 days) TD were depicted in narrow time frames whilst TD, TD-48h and TD-96h are shown as individual time points. Mean ± SE are presented in all graphs.
Fig 3.
Changes in CD38, CD40 and integrin α4β7 expression in monocytes following wt S. Typhi challenge.
Shown in panel A, D and G are time courses of the changes in CD38, CD40 and integrin α4β7 expression, respectively, in TD volunteers. Shown in panels B, E and H are the data in individual volunteers at pre-challenge (circles) and peak up-regulation (TD-48h) (black diamonds) for these markers in TD volunteers. The histogram insert contains an example of the up-regulation of each marker in a representative volunteer. Shown in panels C, F and I are the time courses of the expression of CD38, CD40 and integrin α4β7 in NoTD volunteers. Statistical significance compared to pre-challenge is indicated by: * p<0.05; **p<0.005 (Dunnett’s multiple comparison test).
Fig 4.
Ability of monocytes to bind S. Typhi and activation of signaling pathways following wild-type challenge.
Time courses of S. Typhi (killed-fluorescently labeled) binding to circulating monocytes from TD (A) and NoTD (D) volunteers. Shown in panels B and E are the binding data of individual volunteers at the peak time for TD (TD-48h) and NoTD (D1 Post-challenge) participants, respectively. Each volunteer is indicated by a defined symbol and color, which is also used to represent the data in panels C and F. The horizontal solid lines indicate the mean. Shown in panels C and F are the phosphorylation profiles of Erk1/2, pNFκB and p38MAPK following stimulation with S. Typhi-LPS QD-655micelles in TD and NoTD volunteers, respectively, at peak avidity. The data is presented as fold changes at peak S. Typhi binding compared to pre-challenge. The dotted line indicates no changes in phosphorylation and the horizontal lines represent the mean.
Fig 5.
DCs levels in peripheral blood following infection with wt S. Typhi.
The percentages of circulating DCs in TD (squares; n = 5) and NoTD (diamonds; n = 3) volunteers were compared to the levels before wt S. Typhi challenge (A) as well as in subsequent days (B and C). The graphs display mean ± SE.
Fig 6.
Changes in CD38, CD40 and CD21 expression in DCs following wt S. Typhi challenge.
Shown in panels A, D and G are the time courses of the changes in CD38, CD40 and CD21 expression, respectively, in TD volunteers. Shown in panels B, E and H are the data in individual volunteers at pre-challenge (squares) and peak up-regulation times (TD-0h or TD-48h) (inverted triangles) for these markers in TD volunteers. The histogram insert contains an example of the up-regulation for each marker, in a representative volunteer. Shown in panels C, F and I are the time courses of expression of CD38, CD40 and CD21 in NoTD volunteers. Statistical significance compared to pre-challenge is indicated by: * p<0.05; **p<0.005 (Dunnett’s multiple comparison test).
Fig 7.
Ability of DCs to bind S. Typhi following wild-type challenge.
Time courses of the binding ability of circulating DCs from TD (A) and NoTD (C) volunteers for S. Typhi (killed-fluorescently labeled). Shown in panels B and D are the data of individual volunteers at peak time of binding in TD (TD-48h and TD-96h) and NoTD (D1 Post-challenge) volunteers, respectively. Each volunteer is indicated by a defined symbol and color, as in Fig 4. The horizontal line indicates the means.