Bordetella pertussis commits human dendritic cells to promote a Th1/Th17 response through the activity of adenylate cyclase toxin and MAPK-pathways

Abstract

The complex pathology of B. pertussis infection is due to multiple virulence factors having disparate effects on different cell types. We focused our investigation on the ability of B. pertussis to modulate host immunity, in particular on the role played by adenylate cyclase toxin (CyaA), an important virulence factor of B. pertussis. As a tool, we used human monocyte derived dendritic cells (MDDC), an ex vivo model useful for the evaluation of the regulatory potential of DC on T cell immune responses. The work compared MDDC functions after encounter with wild-type B. pertussis (BpWT) or a mutant lacking CyaA (BpCyaA-), or the BpCyaA- strain supplemented with either the fully functional CyaA or a derivative, CyaA*, lacking adenylate cyclase activity. As a first step, MDDC maturation, cytokine production, and modulation of T helper cell polarization were evaluated. As a second step, engagement of Toll-like receptors (TLR) 2 and TLR4 by B. pertussis and the signaling events connected to this were analyzed. These approaches allowed us to demonstrate that CyaA expressed by B. pertussis strongly interferes with DC functions, by reducing the expression of phenotypic markers and immunomodulatory cytokines, and blocking IL-12p70 production. B. pertussis-treated MDDC promoted a mixed Th1/Th17 polarization, and the activity of CyaA altered the Th1/Th17 balance, enhancing Th17 and limiting Th1 expansion. We also demonstrated that Th1 effectors are induced by B. pertussis-MDDC in the absence of IL-12p70 through an ERK1/2 dependent mechanism, and that p38 MAPK is essential for MDDC-driven Th17 expansion. The data suggest that CyaA mediates an escape strategy for the bacterium, since it reduces Th1 immunity and increases Th17 responses thought to be responsible, when the response is exacerbated, for enhanced lung inflammation and injury.

Figure 1. Cytokine production.

MDDC were either untreated (none) or treated with BpWT or BpCyaA−, the latter in the presence or absence of CyaA and CyaA* (50 ng/ml ) for 48 h. IL-12p70, IL-23, IL-1β, IL-6 and IL-10 were assessed by ELISA. Values are expressed as mean ± SE from nineteen (IL-12p70), eleven (IL-10), six (IL-23, IL-1β and IL-6) independent experiments performed with MDDC obtained from different donors, and expressed as pg/ml of cytokine released. * p<0.05 vs none; °p<0.05 vs BpWT; ^‡ p<0.05 vs BpCyaA−; ^§ p<0.05 vs BpCyaA−+CyaA*.

Figure 2. Th polarization.

MDDC either untreated (none) or treated with BpWT or BpCyaA− for 48 h were co-cultured with purified T cells as described in Methods section. On day 12, supernatants were collected and secreted cytokines were measured by ELISA. Results are expressed as mean ± SE of eight independent experiments performed with MDDC and T cells obtained from different donors. * p<0.05 vs none; ° p<0.05 vs BpWT; ^‡ p<0.05 vs BpCyaA−.

Figure 3. TLR4 and TLR2 activation.

Triggering of TLR4 and TLR2 in transfected HEK293 cells. HEK293/TLR4/p-Nifty2-SEAP and HEK293/TLR2/p-Nifty2-SEAP cells were either untreated (none) or treated with BpWT or BpCyaA− for 16 h. Positive control (PC) for TLR4 stimulation was E. coli LPS (0.1 µg/ml) and positive control for TLR2 stimulation was Pam2CSK4 (0.1 µg/ml). SEAP activity in supernatants of cell cultures was measured. Data are reported as fold increase of SEAP activity over untreated values. Mean expression ± SE of ten independent experiments is indicated. * p<0.05 vs none; ° p<0.05 vs PC.

Figure 4. Analysis of MyD88 -dependent pathway induction.

A MDDC were either untreated or treated with BpWT or BpCyaA−. Phosphorylation of p38, ERK1/2, SAP/JNK and IκB-α was determined at the indicated time-points by Western blot. A single gel was run and blotted to detect phosphorylated proteins and β tubulin to normalize the results. Data are from one representative out of four independent experiments performed with MDDC obtained from different donors. B MDDC were treated as in panel A, either in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h. Results of seven independent experiments performed with MDDC obtained from different donors are expressed as the percent of change of maturation markers (CD80, CD83, CD38) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of marker expression in MDDC not treated with inhibitors was for BpWT: CD80 (MFI) = 48±6; CD83 (%) = 23±6; CD38 (MFI) = 18±4; for BpCyaA−: CD80 (MFI) = 72±11; CD83 (%) = 36±7; CD38 (MFI) = 46±5. * p<0.05 vs control, calculated from the raw data. C MDDC were treated as in panels A and B. Results of ten (IL-12p70), eight (IL-23 and IL-10) and 5 (IL-1β) independent experiments performed with MDDC obtained from different donors, measured by ELISA, are expressed as the percent of change of cytokines with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of cytokine production (pg/ml) in MDDC not treated with inhibitors was for BpWT: IL-12p70 = 0±0; IL-23 = 310±70; IL-1β  = 330±85; IL-10 = 2034±605; for BpCyaA−: IL-12p70 = 374±43; IL-23 = 672±140; IL-1β = 600±87; IL-10 = 2477±593. * p<0.05 vs control, calculated from the raw data.

Figure 5. Analysis of MyD88-independent pathway induction.

MDDC were either untreated (none) or treated with BpWT or BpCyaA−. Phosphorylation of IRF3 was determined at the indicated time-points by Western blot. Data are from one representative out of three independent experiments performed with MDDC obtained from different donors.

Figure 6. Effects of kinase inhibition on Th polarization.

A MDDC, either untreated or treated with BpWT or BpCyaA− in the absence or presence of p38 inhibitor (SB203580), ERK1/2 inhibitor (PD98059) or PI3K inhibitor (LY294002) for 48 h, were co-cultured with purified T cells. On day 12, supernatants were collected and secreted cytokines were measured by ELISA. Results of four independent experiments performed with MDDC and T cells obtained from different donors are expressed as the percent of change of cytokines (IFNγ, IL-17 and IL-5) with respect to the corresponding stimulus in the absence of inhibitors. Mean ± SE of cytokine production (pg/ml) in MDDC not treated with inhibitors was for BpWT: IFNγ = 9013±1828; IL-17 = 900±68; IL-5 = 71±19; for BpCyaA−: IFNγ = 9834±1225; IL-17 = 423±94; IL-5 = 21±5. * p<0.05 vs control, calculated from the raw data.