Maria Cristina Gagliardi

Cholera toxin induces maturation of human dendritic cells and licences them for Th2 priming

Cholera toxin (CT) is a potent mucosal adjuvant that amplifies B and T cell responses to mucosally co‐administered antigens, stimulating predominant Th2‐type responses. However, little is known about the mechanism of adjuvanticity of CT and on the influence this toxin may have on Th2 cell development during the priming of an immune response. We analyzed the effect of CT on dendritic cells (DC), which are responsible for the priming of immune responses at the systemic as well as at the mucosal level. We found that CT induces phenotypic and functional maturation of blood monocyte‐derived DC. Indeed, CT‐treated DC up‐regulate expression of HLA‐DR molecules, B7.1 and B7.2 co‐stimulatory molecules, and are able to prime naive CD4+CD45RA+ T cells in vitro, driving their polarization towards the Th2 phenotype. Furthermore, CT‐matured DC express functional chemokine receptors CCR7 and CXCR4 which may render them responsive to migratory stimuli towards secondary lymphoid organs. Interestingly, the maturation program induced by CT is unique since CT does not induce but rather inhibits cytokine (IL‐12p70 and TNF‐α) and chemokine (RANTES, MIP‐1α and MIP‐1β) secretion by lipopolysaccharide‐ or CD40 ligand‐activated DC. Our results help to elucidate the mechanism of action of CT as an adjuvant and highlight a new stimulus of bacterial origin that promotes maturation of DC.

Human Dendritic Cells following Aspergillus fumigatus Infection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response

ABSTRACT Aspergillus fumigatus is the most prevalent airborne fungal pathogen and causes fatal invasive aspergillosis in immunocompromised patients. Given the essential role of dendritic cells (DC) in initiating and regulating immune responses, we investigated the impact of A. fumigatus conidial infection on human DC. A. fumigatus conidia were rapidly internalized and induced the release of tumor necrosis factor alpha within the first 8 h. After A. fumigatus infection, the majority of DC underwent full maturation, although CCR7 expression was observed only in DC that had internalized the conidia. Additionally, the analysis of regulatory cytokines showed that infected DC simultaneously produced interleukin-12p70 (IL-12p70) and significant amounts of IL-10. IL-10 neutralization was not able to further increase IL-12p70 production from infected DC. Whereas the central role of IL-12 in the generation of Th1 cells has long been appreciated, recently two other members of the IL-12 family, IL-23 and IL-27, were reported to play important roles in the regulation of gamma interferon (IFN-γ) production from naïve and memory T cells. A. fumigatus-infected DC were also able to express high levels of IL-23p19 and low levels of IL-27p28 at later stages of infection. According to this expression pattern, A. fumigatus-infected DC were able to prime IFN-γ production of naïve T cells. Thus, this study on the expression of the new IL-12 family members controlling the Th1 response sheds light on a novel aspect of the contribution of DC to anti-Aspergillus immunity.

Cytometric detection of antigen-specific IFN-γ/IL-2 secreting cells in the diagnosis of tuberculosis

BackgroundThe purpose of this study was to further characterize the immune response to Mycobacterium tuberculosis (Mtb) antigens, in order to provide new insight into host-pathogen interactions in tuberculosis (TB), and to offer tools for a more accurate diagnosis of the different stages of TB.MethodsT-cell responses to Bacillus Calmette-Guérin (BCG), purified protein derivative (PPD), early secretory antigenic target-6 (ESAT-6) protein and culture filtrate protein-10 kDa (CFP-10) were measured in terms of interferon (IFN)-γ and interleukin (IL)-2 release, using a novel flow cytometric cell-secreting cytokine detection technique. The study was conducted on peripheral blood mononuclear cells (PBMC) obtained from active TB patients, latently TB infected individuals, and healthy donors. IL-10 and IL-17 were also measured to test their possible role as indicators of disease activity.ResultsWe confirmed that the enumeration of IFN-γ releasing cells upon Mtb-specific stimulation is sufficient to identify TB patients and that CD8+ T cells concur to IFN-γ secretion. IL-2 secreting cells were more frequently observed in latent TB infected individuals compared to active TB patients, suggesting that measurement of cells secreting this cytokine could be a marker of disease stage. No discriminating role was associated to IL-10 and IL-17 release in TB patients.ConclusionOur data indicate that the flow cytometric cytokine-secreting cell detection technique may be envisaged as an additional tool for TB diagnosis allowing the analysis of the immune response to M. tuberculosis-related antigens in the different stages of TB.

Cell wall-associated alpha-glucan is instrumental for Mycobacterium tuberculosis to block CD1 molecule expression and disable the function of dendritic cell derived from infected monocyte

We previously described an escape mechanism exploited by Mycobacterium tuberculosis (Mtb) to prevent the generation of fully competent dendritic cells (DC). We have now tested the effect of isolated mycobacterial components on human monocyte differentiation into DC and demonstrated that cell wall (CW)‐associated alpha‐glucan induces monocytes to differentiate into DC (Glu‐MoDC) with the same altered phenotype and functional behaviour of DC derived from Mtb‐infected monocytes (Mt‐MoDC). In fact, Glu‐MoDC lack CD1 molecule expression, fail to upregulate CD80 and produce IL‐10 but not IL‐12. We also showed that Glu‐MoDC are not able to prime effector T cells or present lipid antigens to CD1‐restricted T‐cell clones. Thus, we propose a mechanism of Mtb–monocyte interaction mediated by CW‐associated alpha‐glucan, which allows the bacterium to evade both innate and acquired immune responses.

Mycobacterium bovis Bacillus Calmette-Guérin infects DC-SIGN– dendritic cell and causes the inhibition of IL-12 and the enhancement of IL-10 production

The only available vaccine against tuberculosis is Mycobacterium bovis Bacillus Calmette Guérin (BCG), although its efficacy in preventing pulmonary tuberculosis is controversial. Early interactions between dendritic cells (DC) and BCG or Mycobacterium tuberculosis (Mtb) are thought to be critical for mounting a protective antimycobacterial immune response. Recent studies have shown that BCG and Mtb target the DC‐specific C‐type lectin intercellular adhesion molecule‐3‐grabbing nonintegrin (DC‐SIGN) to infect DC and inhibit their immunostimulatory function. This would occur through the interaction of the mycobacterial mannosylated lipoarabinomannan to DC‐SIGN, which would prevent DC maturation and induce the immunosuppressive cytokine interleukin (IL)‐10 synthesis. Here, we confirm that DC‐SIGN is expressed in DC derived from monocytes cultured in granulocyte macrophage‐colony stimulating factor (GM‐CSF) and IL‐4 and show that it is not expressed in DC derived from monocytes cultured in GM‐CSF and interferon‐α (IFN‐α). We also demonstrate that DC‐SIGN– DC cultured in GM‐CSF and IFN‐α are able to phagocytose BCG and to undergo a maturation program as well as DC‐SIGN+ DC cultured in IL‐4 and GM‐CSF. We also show that BCG causes the impairment of IL‐12 and the induction of IL‐10 secretion by DC, irrespective of DC‐SIGN expression. Finally, we demonstrate that the capacity to stimulate a mixed leukocyte reaction of naïve T lymphocytes is not altered by the treatment of both DC populations with BCG. These data suggest that DC‐SIGN cannot be considered as the unique DC receptor for BCG internalization, and it is more interesting that the mycobacteria‐induced immunosuppression cannot be attributed to the engagement of a single receptor.

Mycobacterium tuberculosis diverts alpha interferon-induced monocyte differentiation from dendritic cells into immunoprivileged macrophage-like host cells.

ABSTRACT Dendritic cells (DCs) are critical for initiating a pathogen-specific T-cell response. During chronic infections the pool of tissue DCs must be renewed by recruitment of both circulating DC progenitors and in loco differentiating monocytes. However, the interaction of monocytes with pathogens could affect their differentiation. Mycobacterium tuberculosis has been shown to variably interfere with the generation and function of antigen-presenting cells (APCs). In this study we found that when alpha interferon (IFN-α) is used as an inductor of monocyte differentiation, M. tuberculosis inhibits the generation of DCs, forcing the generation of immunoprivileged macrophage-like cells instead. Cells derived from M. tuberculosis-infected monocyte-derived macrophages (M. tuberculosis-infected MoMφ) retained CD14 without acquiring CD1 molecules and partially expressed B7.2 but did not up-regulate B7.1 and major histocompatibility complex (MHC) class I and II molecules. They synthesized tumor necrosis factor alpha and interleukin-10 (IL-10) but not IL-12. They also showed a reduced ability to induce proliferation and functional polarization of allogeneic T lymphocytes. Thus, in the presence of IFN-α, M. tuberculosis may hamper the renewal of potent APCs, such as DCs, generating a safe habitat for intracellular growth. M. tuberculosis-infected MoMφ, in fact, showed reduced expression of both signal 1 (CD1, MHC classes I and II) and signal 2 (B7.1 and B7.2), which are essential for mycobacterium-specific T-lymphocyte priming and/or activation. These data further suggest that M. tuberculosis has the ability to specifically interfere with monocyte differentiation. This ability may represent an effective M. tuberculosis strategy for eluding immune surveillance and persisting in the host.

Maturation of human dendritic cells induced by the adjuvant cholera toxin: role of cAMP on chemokine receptor expression

Cholera toxin (CT) is a very effective adjuvant for mucosal vaccination. It binds to cells through its B subunit and induces intracellular increase of cAMP through the A subunit. We previously showed that CT induces maturation of human dendritic cells (DCs) and this may account for its adjuvant property. Here, we investigated the role of the A subunit on DCs maturation by using forskolin, a cAMP inducer. The results show that although cAMP does not stimulate full maturation of DCs it induces upregulation of the chemokine receptors CXCR4 and CCR7.

Endogenous PGE2 promotes the induction of human Th17 responses by fungal β-glucan

The interaction of PAMPs with cells of the innate immune system shapes the adaptive host response. Here, we report that β‐glucan, a major fungal PAMP purified from Candida albicans, stimulates human DCs to secrete a pro‐Th17 cytokine pattern. Notably, β‐glucan induces PGE2 production, which has been shown to play a pivotal role in Th17 cell expansion. Inhibition of PGE2 synthesis or blockade of PGE2 receptors EP2 and EP4 drastically reduces IL‐23 production by β‐glucan‐activated DCs, suggesting that endogenous PGE2 amplifies IL‐23 synthesis in response to the C. albicans PAMP. Moreover β‐glucan promotes the expansion of Th17 cells, which is strongly decreased by EP2 and EP4 receptor blockade on DCs. Our results highlight a novel role for PGE2 in the regulation of innate and adaptive immune response triggered by recognition of a prominent, highly conserved fungal PAMP such as β‐glucan.

β-Glucan of Candida albicans cell wall causes the subversion of human monocyte differentiation into dendritic cells

The functional consequences of treating human monocytes with purified and chemically characterized Candida albicans β‐glucan—a major microbial pathogen associated molecular pattern—on their differentiation into dendritic cells (DC) were investigated. We show here that β‐glucan‐treated monocytes differentiated into mature DC (Glu‐MoDC) with altered phenotype and functional behavior, similarly to DC derived from C. albicans germ‐tubes‐infected monocytes (Gt‐MoDC). They failed to express CD1a and to up‐regulate CD80 and DR molecules. Moreover, they produced IL‐10 but not IL‐12 and primed naive T cells without inducing their functional polarization into effector cells. Since C. albicans β‐glucan is a mixture of both β‐(1,3) and β‐(1,6) glucan, we investigated their relative contribution by the use of non‐Candida β‐glucan structural analogs. We found that high molecular weight (MW) glucans β−(1,6) pustulan and β‐(1,3) curdlan totally mimicked the effect of C. albicans β‐glucan, while the low MW β‐(1,3) glucan laminarin did not have any effect. Because β‐glucan is a common constituent of all fungi and is abundantly released in vivo during systemic fungal infection, this novel effect of β‐glucan has potential implications for host‐parasite relationship in candidiasis and other mycoses. In particular, our data suggest that β‐glucan could bias noninfected, bystander monocytes, thus aggravating the general immunodeficiency, predisposing to systemic fungal infection.

Mycobacteria exploit p38 signaling to affect CD1 expression and lipid antigen presentation by human dendritic cells

ABSTRACT Group I CD1 proteins are specialized antigen-presenting molecules that present both microbial and self lipid antigens to CD1-restricted α/β T lymphocytes. The production of high levels of gamma interferon and lysis of infected macrophages by lipid-specific T lymphocytes are believed to play pivotal roles mainly in the defense against mycobacterial infections. We previously demonstrated that Mycobacterium tuberculosis and bacillus Calmette-Guérin (Mycobacterium bovis BCG) induce human monocytes to differentiate into CD1− dendritic cells (DC), which cannot present lipid antigens to specific T cells. Here, we show that in human monocytes mycobacteria trigger phosphorylation of p38 mitogen-activated protein kinase to inhibit CD1 expression in DC derived from infected monocytes. Pretreatment with a specific p38 inhibitor renders monocytes insensitive to mycobacterial subversion and allows them to differentiate into CD1+ DC, which are fully capable of presenting lipid antigens to specific T cells. We also report that one of the pathogen recognition receptors triggered by BCG to activate p38 is complement receptor 3 (CR3), as shown by reduced p38 phosphorylation and partial reestablishment of CD1 membrane expression obtained by CR3 blockade before infection. In conclusion, we propose that p38 signaling is a novel pathway exploited by mycobacteria to affect the expression of CD1 antigen-presenting cells and avoid immune recognition.