Bordetella pertussis and human monocyte-derived dendritic cells: a tool to identify an evading mechanism

Bordetella pertussis is the etiologic agent of pertussis or wooping cough , an highly, contagious and acute respiratory infection. B. pertussis is a strict human pathogen and transmission of disease occurs via respiratory droplets. B. pertussis posses different virulence factors classified in two categories: adhesins and toxins . Virulence factors facilitate the bacteria' s ability to adhere to respiratory cells and produce local and sistemic alterations in cell pathophysiology which contribute to microrganism replication and its capacity to evade host immune response. Adenylate cyclase toxin (ACT) is the B. pertussis factors which induces cell intoxication via cyclic AMP (cAMP) leading to inhibition of several immune functions allowing persistence of B. pertussis in the host and progression of infection. The mechanism underlying protection to B. pertussis infection is still a matter of debate. A central role in immunity is played by dendritic cells (DCs). DCs represent the initiator and modulator of the immune response that link innate and acquired immunity. DCs act as sentinels able to capture microbial antigens at the site of infection, this encounter drives the maturation process, a complex cascade of intracellular pathways that allows regulatory cytokine production, such as interleukin(IL-)12 p70 or IL- 10. After, DCs migrates to lymphonodes where they present antigens to naïve T cells and polarize the immune response. In this contest, DCs represent a tool to identify immune mechanisms which underly protection after B. pertussis infection. In the first part of the study we aimed to clarify the interaction of B. pertussis and human monocytederived DCs (MDDCs). we evaluated the ability of human MDDCs to phagocytose B. pertussis and the capacity of bacterial cells to survive intracellularly. A key point in the present study was the assessment of the ability of B. pertussis to influence MDDCs functions. In particular, we aimed to evaluate whether infected MDDCs undergo phenotypic and functional maturation, and which type of Th cells polarization is induced by infected MDDCs. The results shown that B. pertussis, while possessing a low susceptibility to be internalized by - and to survive in - MDDCs, triggers the onset of the maturation program and modulates cytokine production and antigen presenting cell functions. In particular by ELISA tests, we found that B. pertussis infected MDDCs are unable to induce the production of IL-12 p70 nevertheless driving Th1 immunity. DCs in response to pathogens can secrete not only IL-12 p70 but also a new described Th1 cytokine IL-23. IL-12 p70 and IL-23 are composed of two heterologous chains differently regulated, respectively p40/p35 and p40/p19. Quantitative RT-PCR has revealed that B. pertussis induces in MDDCs p40 and p19 (IL-23), but not p35 expression. MDDCs B. pertussis-infected drive a Th1 polarization and IL-23 may indirectly contribute to this polarization. Since ACT, as described in several studies, inhibits lipopolysaccaride (LPS)-driven IL-12 p70 production in human, to evaluate if ACT was involved in the unability of B. pertussis to induce the production of IL- 12 p70 we infected MDDCs with a wild-type strain (BpWT) and its isogenic ACT-deficient mutant (BpACT¯) in presence or absence of a cAMP analogus molecule d-butyrril-cAMP (d-cAMP). Indeed BpACT¯infection induced consistent production of IL-12 p70 and IL-12 p35 transcription. Addition of the d-cAMP abolished IL-12 p70 production and IL-12 p35 expression in BpACT¯-infected MDDCs. BpACT¯ infection induced the expression of the transcription factors interferon regulatory factor 1 (IRF- 1) and IRF-8 and of beta interferon (IFN-β), involved in IL-12 p35 regulation, and the expression of these genes was inhibited by d-cAMP addition and in BpWT-infected MDDCs. The concomitant expression of IL-12 p70 and IL-23 allowed BpACT¯ to trigger a more pronounced T helper 1 polarization compared to BpWT. Thus in conclusion the present study demonstrate that ACT-dependent cAMP induction leads to the inhibition of pathways ultimately leading to IL-12 p35 production, thus representing a mechanism for B. pertussis to escape the host immune response. Further studies could be important to recognize virulence factors or immune reactions pivotal in B. pertussis infection useful to enhance the protective capacity of the acellular pertussis vaccines

Bordetella pertussis-Infected Human Monocyte-Derived Dendritic Cells Undergo Maturation and Induce Th1 Polarization and Interleukin-23 Expression

Bordetella pertussis, the causative agent of whooping cough, is internalized by several cell types, including epithelial cells, monocytes, and neutrophils. Although its ability to survive intracellularly is still debated, it has been proven that cell-mediated immunity (CMI) plays a pivotal role in protection. In this study we aimed to clarify the interaction of B. pertussis with human monocyte-derived dendritic cells (MDDC), evaluating the ability of the bacterium to enter MDDC, to survive intracellularly, to interfere with the maturation process and functional activities, and to influence the host immune responses. The results obtained showed that B. pertussis had a low capability to be internalized by—and to survive in—MDDC. Upon contact with the bacteria, immature MDDC were induced to undergo phenotypic maturation and acquired antigen-presenting-cell functions. Despite the high levels of interleukin-10 (IL-10) and the barely detectable levels of IL-12 induced by B. pertussis, the bacterium induced maturation of MDDC and T helper 1 (Th1) polarized effector cells. Gene expression analysis of the IL-12 cytokine family clearly demonstrated that B. pertussis induced high levels of the p40 and p19 subunits of IL-23 yet failed to induce the expression of the p35 subunit of IL-12. Overall our findings show that B. pertussis, even if it survives only briefly in MDDC, promotes the synthesis of IL-23, a newly discovered Th1 polarizing cytokine. A Th1-oriented immune response is thus allowed, relevant in the induction of an adequate CMI response, and typical of protection induced by natural infection or vaccination with whole-cell vaccines

Bordetella pertussis Inhibition of Interleukin-12 (IL-12) p70 in Human Monocyte-Derived Dendritic Cells Blocks IL-12 p35 through Adenylate Cyclase Toxin-Dependent Cyclic AMP Induction

Bordetella pertussis, the causative agent of whooping cough, possesses an array of virulence factors, including adenylate cyclase toxin (ACT), relevant in the establishment of infection. Here we better define the impact of cyclic AMP (cAMP) intoxication due to the action of ACT on dendritic cell (DC)-driven immune response, by infecting monocyte-derived DC (MDDC) with an ACT-deficient B. pertussis mutant (ACT(−)18HS19) or its parental strain (WT18323). Both strains induced MDDC maturation and antigen-presenting cell functions; however, only ACT(−)18HS19 infected MDDC-induced production of interleukin-12 (IL-12) p70. Gene expression analysis of the IL-12 cytokine family subunits revealed that both strains induced high levels of p40 (protein chain communal to IL-12 p70 and IL-23) as well as p19, a subunit of IL-23. Conversely only ACT(−)18HS19 infection induced consistent transcription of IL-12 p35, a subunit of IL-12 p70. Addition of the cAMP analogous d-butyril-cAMP (d-cAMP) abolished IL-12 p70 production and IL-12 p35 expression in ACT(−)18HS19-infected MDDC. ACT(−)18HS19 infection induced the expression of the transcription factors interferon regulatory factor 1 (IRF-1) and IRF-8 and of beta interferon, involved in IL-12 p35 regulation, and the expression of these genes was inhibited by d-cAMP addition and in WT18323-infected MDDC. The concomitant expression of IL-12 p70 and IL-23 allowed ACT(−)18HS19 to trigger a more pronounced T helper 1 polarization compared to WT18323. The present study suggests that ACT-dependent cAMP induction leads to the inhibition of pathways ultimately leading to IL-12 p35 production, thus representing a mechanism for B. pertussis to escape the host immune response

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

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

Lipooligosaccharide from Bordetella pertussis induces mature human monocyte-derived dendritic cells and drives a Th2 biased response

Bordetella pertussis has a distinctive cell wall lipooligosaccharide (LOS) that is released from the bacterium during bacterial division and killing. LOS directly participates in host-bacterial interactions, in particular influencing the dendritic cells' (DC) immune regulatory ability. We analyze LOS mediated toll-like receptor (TLR) activation and dissect the role played by LOS on human monocyte-derived (MD)DC functions and polarization of the host T cell response. LOS activates TLR4-dependent signaling and induces mature MDDC able to secrete IL-10. LOS-matured MDDC enhance allogeneic presentation and skew T helper (Th) cell polarization towards a Th2 phenotype. LOS protects MDDC from undergoing apoptosis, prolonging their longevity and their functions. Compared to Escherichia coli lipopolysaccharide (LPS), the classical DC maturation stimulus, LOS was a less efficient inducer of TLR4 signaling, MDDC maturation, IL-10 secretion and allogeneic T cell proliferation and it was not able to induce IL-12p70 production in MDDC. However, the MDDC apoptosis protection exerted by LOS and LPS were comparable. In conclusion, LOS treated MDDC are able to perform antigen presentation in a context that promotes licensing of Th2 effectors. Considering these properties, the use of LOS in the formulation of acellular pertussis vaccines to potentiate protective and adjuvant capacity should be taken into consideration. © 2007 Elsevier Masson SAS. All rights reserved

TLR4 and TLR2 activation.

<p>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 <i>E. coli</i> 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. * <i>p</i><0.05 <i>vs</i> none; ° <i>p</i><0.05 <i>vs</i> PC.</p