Angela Bacci

Dendritic Cells Transport Conidia and Hyphae of Aspergillus fumigatus from the Airways to the Draining Lymph Nodes and Initiate Disparate Th Responses to the Fungus

Aspergilli are respiratory pathogens and pulmonary infections are usually acquired through the inhalation of conidia, able to reach small airways and the alveolar space where the impaired host defense mechanisms allow hyphal germination and subsequent tissue invasion. The invasive pulmonary aspergillosis is the most common manifestation of Aspergillus fumigatus infection in immunocompromised patients and is characterized by hyphal invasion and destruction of pulmonary tissue. A Th1/Th2 dysregulation and a switch to a Th2 immune response may contribute to the development and unfavorable outcome of invasive pulmonary aspergillosis. Dendritic cells (DC) have a primary role in surveillance for pathogens at the mucosal surfaces and are recognized as the initiators of immune responses to them. In the present study, we assessed the functional activity of pulmonary DC in response to A. fumigatus conidia and hyphae, both in vitro and in vivo. We analyzed mechanisms and receptors for phagocytosis by DC as well as DC migration, maturation, and Th priming in vivo upon exposure to either form of the fungus. We found a remarkable functional plasticity of DC in response to the different forms of the fungus, as pulmonary DC were able to: 1) internalize conidia and hyphae of A. fumigatus through distinct phagocytic mechanisms and recognition receptors; 2) discriminate between the different forms in terms of cytokine production; 3) undergo functional maturation upon migration to the draining lymph nodes and spleens; and 4) instruct local and peripheral Th cell reactivity to the fungus.

B7/CD28-Dependent CD4+CD25+ Regulatory T Cells Are Essential Components of the Memory-Protective Immunity to Candida albicans

Protective immunity to the fungus Candida albicans is mediated by Ag-specific Th1 cells. Paradoxically, some Th2 cytokines are required for the maintenance of Th1-mediated immune resistance to the fungus. Therefore, in addition to the Th1/Th2 balance, other mechanisms seem to be involved in the regulation of Th1 immunity to the fungus. Here we show that CD4+CD25+ T cells, negatively regulating antifungal Th1 reactivity, are generated in mice with candidiasis. CD4+CD25+ T cells were not generated in B7-2- or CD28-deficient mice or in condition of IL-10 signaling deficiency. Accordingly, although capable of efficiently restricting the fungal growth, these mice experienced inflammatory pathology and were incapable of resistance to reinfection. CD4+CD25+ T cells poorly proliferated in vitro; were highly enriched for cells producing IL-4, IL-10, and TGF-β; and required IL-10-producing, Candida hypha-activated dendritic cells for generation. Adoptive transfer of CD4+CD25+ T cells or IL-10-producing dendritic cells restored resistance to reinfection and decreased inflammation in B7-2-deficient mice. These results show that oral tolerance induced by Candida hyphae is required for the occurrence of long-lasting protective immunity after yeast priming. The implication is that preventing reactivation rather than favoring sterilizing immunity to ubiquitous fungal pathogens may represent the ultimate expectation of vaccine-based strategies.

Cytokine- and T Helper-Dependent Lung Mucosal Immunity in Mice with Invasive Pulmonary Aspergillosis

The role of cytokine- and T helper (Th)-dependent lung mucosal antifungal immunity in murine invasive pulmonary aspergillosis (IPA) was investigated. Intact or leukopenic DBA/2 mice were resistant or highly susceptible, respectively, to infection caused by multiple intranasal injections of viable Aspergillus fumigatus conidia. Resistance was associated with unimpaired innate antifungal activity of pulmonary phagocytic cells, concomitant with high-level production of tumor necrosis factor (TNF)-alpha and interleukin (IL)-12 and the presence of interstitial lymphocytes producing interferon-gamma and IL-2. Conversely, production of TNF-alpha and IL-12 was down-regulated in highly susceptible mice, which also had defective innate antifungal immunity and high-level production of IL-4 and IL-10 by lung lymphocytes. Resistance was increased in susceptible mice upon local IL-4 or IL-10 neutralization or IL-12 administration. These results indicate that, similar to observations in mice with disseminated aspergillosis, innate and Th1-dependent immunity play an essential role in host defense against IPA.

Interleukin-4 Causes Susceptibility to Invasive Pulmonary Aspergillosis through Suppression of Protective Type I Responses

Aspergillus fumigatus, an opportunistic fungal pathogen, causes multiple allergic and nonallergic airway diseases. Invasive pulmonary aspergillosis (IPA) is a nonallergic, life-threatening disease of immunocompromised patients. In a murine model of IPA, interleukin (IL)-4-deficient (IL-4-/-) BALB/c mice were used to examine the role of IL-4 in lung pathology and immune responses. IL-4-/- mice were more resistant than wild-type mice to infection caused by multiple intranasal injections of viable A. fumigatus conidia. Resistance was associated with decreased lung inflammatory pathology, impaired T helper (Th)-2 responses (including lung eosinophilia), and an IL-12-dependent Th1 response. In contrast, development of host-detrimental antifungal Th2 cells occurred in IL-12-/- and interferon-gamma-/- mice and in IL-4-/- mice when subjected to IL-12 neutralization. These results demonstrate that IL-4 renders mice susceptible to infection with A. fumigatus by inhibition of protective Th1 responses. IL-4 appears to have a distinct role in the pathogenesis of allergic and nonallergic lung diseases caused by the fungus.

T cell vaccination in mice with invasive pulmonary aspergillosis.

Aspergillus fumigatus, an opportunistic fungal pathogen, is responsible for multiple airway diseases of an allergic and a nonallergic nature. In a murine model of invasive pulmonary aspergillosis, resistance is associated with a decreased lung inflammatory pathology and the occurrence of an IL-12-dependent Th1-type reactivity that are both impaired by IL-4. In the present study we assess the ability of Aspergillus crude culture filtrate Ags and the recombinant allergen Asp f 2 to induce protective antifungal responses in mice with invasive pulmonary aspergillosis. Similar to what occurred upon nasal exposure to viable A. fumigatus conidia, treatment of immunocompetent mice with Aspergillus crude culture filtrate Ags resulted in the development of local and peripheral protective Th1 memory responses, mediated by Ag-specific CD4+ T cells producing IFN-γ and IL-2 capable of conferring protection upon adoptive transfer to naive recipients. Protective Th1 responses could not be observed in mice deficient of IFN-γ or IL-12 and did not occur in response to Asp f 2, which, on the contrary, elicited high level production of inhibitory IL-4. The results show that Ags of Aspergillus exist with the ability to induce both Th1- and Th2-type reactivity during infection, a finding that suggests a possible mechanism through which potentially protective immune responses are inhibited in mice with the infection. However, the occurrence of Th1-mediated resistance upon vaccination with Aspergillus crude culture filtrate Ags, suggests the existence of fungal Ags useful as a candidate vaccine against invasive pulmonary aspergillosis.

Vaccination of mice against invasive aspergillosis with recombinant Aspergillus proteins and CpG oligodeoxynucleotides as adjuvants

In a murine model of invasive pulmonary aspergillosis, dendritic cells (DCs) pulsed with Aspergillus antigens induced the activation of CD4(+) Th1 cells capable of conferring resistance to the infection. Here we show that the combined, local delivery of unmethylated CpG oligodeoxynucleotides (ODNs) and the Asp f 16 Aspergillus allergen resulted in the functional maturation and activation of airway DCs capable of inducing Th1 priming and resistance to the fungus. Therefore, ODNs act as a potent adjuvant for the vaccine-induced protection against the fungus by promoting dominant Th1 response to Aspergillus antigens and allergens.

CD80 + Gr-1 + Myeloid Cells Inhibit Development of Antifungal Th1 Immunity in Mice with Candidiasis

To find out whether polymorphonuclear neutrophils (PMN), abundantly recruited in disseminated Candida albicans infection, could directly affect the activation of Th cells we addressed the issues as to whether murine PMN, like their human counterparts, express costimulatory molecules and the functional consequence of this expression in terms of antifungal immune resistance. To this purpose, we assessed 1) the expression of CD80 (B7-1) and CD86 (B7-2) molecules on peripheral, splenic, and inflammatory murine Gr-1+ PMN; 2) its modulation upon interaction with C. albicans in vitro, in vivo, and in human PMN; 3) the effect of Candida exposure on the ability of murine PMN to affect CD4+ Th1 cell proliferation and cytokine production; and 4) the mechanism responsible for this effect. Murine PMN constitutively expressed CD80 molecules on both the surface and intracellularly; however, in both murine and human PMN, CD80 expression was differentially modulated upon interaction with Candida yeasts or hyphae in vitro as well as in infected mice. The expression of the CD86 molecule was neither constitutive nor inducible upon exposure to the fungus. In vitro, Gr-1+ PMN were found to inhibit the activation of IFN-γ-producing CD4+ T cells and to induce apoptosis through a CD80/CD28-dependent mechanism. A population of CD80+Gr-1+ myeloid cells was found to be expanded in conventional as well as in bone marrow-transplanted mice with disseminated candidiasis, but its depletion increased the IFN-γ-mediated antifungal resistance. These data indicate that alternatively activated PMN expressing CD80 may adversely affect Th1-dependent resistance in fungal infections.

Dendritic Cells Pulsed with Fungal RNA Induce Protective Immunity to Candida albicans in Hematopoietic Transplantation

Immature myeloid dendritic cells (DC) phagocytose yeasts and hyphae of the fungus Candida albicans and induce different Th cell responses to the fungus. Ingestion of yeasts activates DC for production of IL-12 and Th1 priming, while ingestion of hyphae induces IL-4 production and Th2 priming. In vivo, generation of antifungal protective immunity is induced upon injection of DC ex vivo pulsed with Candida yeasts but not hyphae. In the present study we sought to determine the functional activity of DC transfected with yeast or hyphal RNA. It was found that DC, from either spleens or bone marrow, transfected with yeast, but not hyphal, RNA 1) express fungal mannoproteins on their surface; 2) undergo functional maturation, as revealed by the up-regulated expression of MHC class II Ags and costimulatory molecules; 3) produce IL-12 but no IL-4; 4) are capable of inducing Th1-dependent antifungal resistance when delivered s.c. in vivo in nontransplanted mice; and 5) provide protection against the fungus in allogeneic bone marrow-transplanted mice, by accelerating the functional recovery of Candida-specific IFN-γ-producing CD4+ donor lymphocytes. These results indicate the efficacy of DC pulsed with Candida yeasts or yeast RNA as fungal vaccines and point to the potential use of RNA-transfected DC as anti-infective vaccines in conditions that negate the use of attenuated microorganisms or in the case of poor availability of protective Ags.

Interleukin 18 Restores Defective Th1 Immunity to Candida albicans in Caspase 1-Deficient Mice

ABSTRACT Caspase 1, formerly designated interleukin 1β (IL-1β)-converting enzyme, processes pro-IL-1β and pro-IL-18 to yield active cytokines that play a pivotal role in inflammation and cell activation. We show here the effect of caspase 1 deficiency on the inflammatory and adaptive immune responses to the fungus Candida albicans. Caspase 1 deficiency did not affect susceptibility to primary systemic infection with the fungus, as revealed by survival and fungal growth. However, Th1-mediated resistance to reinfection was greatly impaired in caspase 1-deficient mice, and this correlated with low-level production of IL-12 and gamma interferon. Early in infection, production of these cytokines and that of tumor necrosis factor alpha, IL-6, and, interestingly, IL-1β occurred normally in caspase 1-deficient mice, while that of IL-18 was severely impaired. Exogenous administration of IL-18, more than IL-12, restored the Th1-mediated resistance to the infection. We conclude that, while caspase 1 is not indispensable for release of mature IL-1β in candidiasis, the caspase 1-dependent production of IL-18 may represent an important and novel pathway for the expression of sustained Th1 reactivity to the fungus.

A role for antibodies in the generation of memory antifungal immunity

Protective immunity to Candida albicans and Aspergillus fumigatus is mediated by antigen‐specific Th1 cells. To define the role of B cells and antibodies in the generation ofantifungal immune resistance, B cell‐deficient (μMT) mice were assessed for immune resistance to primary and secondary infections with both fungi. The results showed that, although passive administration of antibodies increased the fungal clearance, the innate and Th1‐mediated resistance to the primary and secondary infections were both heightened in μMT mice with candidiasis and aspergillosis. However, although capable of efficiently restricting the fungal growth, μMT mice did not survive the re‐infection with C. albicans, and this was concurrent with the failure to generate IL‐10‐producing dendritic cells and regulatory CD4+CD25+ T cells. Antifungal opsonizing antibodies restored IL‐10 production by dendritic cells from μMT mice, a finding suggesting that the availability of opsonizing antibodies may condition the nature of the dendritic cell interaction with fungi, possibly impacting on the development of long‐lasting antifungal immunity.