Antonella Mencacci

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.

Iron Overload Alters Innate and T Helper Cell Responses to Candida albicans in Mice

The effect of iron overload on susceptibility of mice to Candida albicans infection and on the type of T helper (Th) immunity elicited was investigated. Iron overload greatly increased susceptibility to disseminated infection with low-virulence C. albicans cells of exogenous origin. The candidacidal activity and the ability to release nitric oxide and bioactive interleukin (IL)-12 were greatly impaired in neutrophils and macrophages from infected mice. CD4 T cells from spleens of iron-overloaded mice were found to produce high levels of IL-4 and IL-10 and low levels of interferon-gamma. Treatment of iron-overloaded mice with the iron chelator, deferoxamine, resulted in the cure of mice from infection, restored the antifungal effector and immunomodulatory functions of the phagocytic cells, and allowed the occurrence of CD4 Th1 protective antifungal responses. These data indicate that iron overload may negatively affect CD4 Th1 development in mice with candidiasis, a function efficiently restored by therapy with deferoxamine.

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.

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.

Impaired Antifungal Effector Activity but Not Inflammatory Cell Recruitment in Interleukin-6-Deficient Mice with Invasive Pulmonary Aspergillosis

A murine model of infection, in which immunocompetent or immunosuppressed interleukin-6-deficient (IL-6(-/-)) mice were infected intranasally with Aspergillus fumigatus conidia and were monitored for parameters of fungal colonization and innate and adaptive immunity, was used to assess the role of IL-6 in invasive pulmonary aspergillosis (IPA). The results indicate that IL-6(-/-) mice were more susceptible than wild-type mice to IPA. Susceptibility was associated with increased inflammatory pathology, decreased antifungal effector functions of phagocytes, and impaired development of protective type 1 responses. Exposure to exogenous IL-6 restored antifungal effector activity.

Induction of Protective Th1 Responses to Candida albicans by Antifungal Therapy Alone or in Combination with an Interleukin-4 Antagonist

Resistance or susceptibility to disseminated and mucosal Candida albicans infections in mice correlates with the development of protective or nonprotective T helper (Th) cell responses. To determine whether immunomodulatory activity on Th cell functions is an effect beyond that provided by antifungal therapy, mice with disseminated or gastrointestinal infection were treated with amphotericin B or fluconazole and assessed for mortality, fungus burden in the organs, and parameters of Th cell ‐ dependent immunity. Both antimycotics produced protective CD4 / Th1 cell responses, as revealed by increased production of interleukin (IL)-12 and interferon-g, decreased production of IL-4, delayed-type hypersensitivity to fungal antigen, and the disappearance of antigen-specific IgE. Concomitant neutralization of endogenous IL-4 greatly increased the antifungal efficacy and the Th1-promoting activity of both agents. These results indicate that successful antifungal therapy alone or in combination with cytokine antagonists may rely on the induction of an appropriate Th antifungal cell response.

Diagnostic Performance of a Multiple Real-Time PCR Assay in Patients with Suspected Sepsis Hospitalized in an Internal Medicine Ward

ABSTRACT Early identification of causative pathogen in sepsis patients is pivotal to improve clinical outcome. SeptiFast (SF), a commercially available system for molecular diagnosis of sepsis based on PCR, has been mostly used in patients hospitalized in hematology and intensive care units. We evaluated the diagnostic accuracy and clinical usefulness of SF, compared to blood culture (BC), in 391 patients with suspected sepsis, hospitalized in a department of internal medicine. A causative pathogen was identified in 85 patients (22%). Sixty pathogens were detected by SF and 57 by BC. No significant differences were found between the two methods in the rates of pathogen detection (P = 0.74), even after excluding 9 pathogens which were isolated by BC and were not included in the SF master list (P = 0.096). The combination of SF and BC significantly improved the diagnostic yield in comparison to BC alone (P < 0.001). Compared to BC, SF showed a significantly lower contamination rate (0 versus 19 cases; P < 0.001) with a higher specificity for pathogen identification (1.00, 95% confidence interval [CI] of 0.99 to 1.00, versus 0.94, 95% CI of 0.90 to 0.96; P = 0.005) and a higher positive predictive value (1.00, 95% CI of 1.00 to 0.92%, versus 0.75, 95% CI of 0.63 to 0.83; P = 0.005). In the subgroup of patients (n = 191) who had been receiving antibiotic treatment for ≥24 h, SF identified more pathogens (16 versus 6; P = 0.049) compared to BC. These results suggest that, in patients with suspected sepsis, hospitalized in an internal medicine ward, SF could be a highly valuable adjunct to conventional BC, particularly in patients under antibiotic treatment.

Protection of Killer Antiidiotypic Antibodies against Early Invasive Aspergillosis in a Murine Model of Allogeneic T-Cell-Depleted Bone Marrow Transplantation

ABSTRACT Antiidiotypic monoclonal antibodies (MAbs) representing the internal image of a yeast killer toxin (KT) have therapeutic potential against several fungal infections. The efficacy of KT MAbs against Aspergillus fumigatus was investigated in a mouse model of T-cell-depleted allogeneic bone marrow transplantation (BMT) with invasive pulmonary aspergillosis. Mice were highly susceptible to infection at 3 days post-BMT, when profound neutropenia was observed both in the periphery and in the lungs. Treatment with KT MAbs protected the mice from infection, as judged by the long-term survival and decreased pathology associated with inhibition of fungal growth and hyphal development in the lungs. In vitro, similar to polymorphonuclear neutrophils, KT MAbs significantly inhibited the hyphal development and metabolic activity of germinated Aspergillus conidia. These results indicate that mimicking the action of neutrophils could be a strategy through which KT MAbs exert therapeutic efficacy in A. fumigatus infections.