Biomedical subjects
Publications and source records attributed to L Romani.
Recent observations on reciprocal regulation of humoral and cell-mediated immunity to Candida albicans are presented and their significance in the exploration of the host-fungus-relationship is discussed.
IL-12 is both required and prognostic for Th1 development in mice with Candida albicans infection. To delineate further the physiologic role of IL-12 in antifungal immunity, mice deficient for this cytokine were assessed for susceptibility to C. albicans infections, and for parameters of innate and adaptive immunity. IL-12-deficient mice were highly susceptible to gastrointestinal infection or to reinfection and showed elevated production of Candida-specific IgE and IL-4 and defective production of IFN-gamma. The failure to mount protective Th1 responses occurred despite the presence of an unimpaired innate antifungal immune response, which correlated with unaltered IFN-gamma production, but defective production of, and responsiveness to, inhibitory IL-10. IL-10 or IL-12 neutralization increased the innate antifungal resistance in wild-type mice. However, in IL-12-deficient mice, treatment with exogenous IL-12 or IL-10 impaired IL-4 production and increased resistance to infection, through a negative effect on the CTLA-4/B7-2 costimulatory pathway. These results confirm the obligatory role of IL-12 in the induction of anticandidal Th1 responses, and indicate the existence of a positive regulatory loop between IL-12 and IL-10 that may adversely affect the innate antifungal response, but is required for optimal costimulation of IL-12-dependent CD4+ Th1 cells.
To elucidate the role of IFN-gamma in antifungal CD4+ Th-dependent immunity, 129/Sv/Ev mice deficient for IFN-gamma receptor (IFN-gammaR(-/-)) were assessed for susceptibility to gastrointestinal or systemic Candida albicans infection and for parameters of innate and adaptive T helper immunity. IFN-gammaR(-/-) mice failed to mount protective Th1-mediated acquired immunity upon mucosal immunization or in response to a live vaccine strain of the yeast. The impaired Th1-mediated resistance correlated with defective IL-12 responsiveness, but not IL-12 production, and occurred in the presence of an increased innate antifungal resistance. The development of nonprotective Th2 responses was observed in IFN-gammaR(-/-) mice upon mucosal infection and subsequent reinfection. However, under experimental conditions of Th2 cell activation, the occurrence of Th2 cell responses was similar in IFN-gammaR(-/-) and in IFN-gammaR(+/+) mice. These results indicate the complex immunoregulatory role of IFN-gamma in the induction of mucosal and nonmucosal anticandidal Th cell responses; IFN-gamma is not essential for the occurrence of Th2 responses but is required for development of IL-12-dependent protective Th1-dependent immunity.
Interleukin (IL)-4-deficient mice were used to assess susceptibility to systemic or gastrointestinal Candida albicans infections, as well as parameters of innate and elicited T helper immunity. In the early stage of systemic infection with virulent C. albicans, an unopposed interferon (IFN)-gamma response renders IL-4-deficient mice more resistant than wild-type mice to infection. Yet, IL-4-deficient mice failed to efficiently control infection in the late stage and succumbed to it. Defective IFN-gamma and IL-12 production, but not IL-12 responsiveness, was observed in IL-4-deficient mice that failed to mount protective T helper type 1 cell (Th1)-mediated acquired immunity in response to a live vaccine strain of the yeast or upon mucosal immunization in vivo. In vitro, IL-4 primed neutrophils for cytokine release, including IL-12. However, late treatment with exogenous IL-4, while improving the outcome of infection, potentiated CD4(+) Th1 responses even in the absence of neutrophils. These findings indicate that endogenous IL-4 is required for the induction of CD4(+) Th1 protective antifungal responses, possibly through the combined activity on cells of the innate and adaptive immune systems.
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.
To define the immunological functions of tumor necrosis factor (TNF) in Candida albicans infection, TNF/lymphotoxin (LT)-alpha double-deficient mice were assessed for susceptibility to systemic or gastrointestinal infection and parameters of innate and adaptive Th immunity. When compared to wild-type mice, TNF/LT-alpha-deficient mice were more susceptible to either type of infection caused by virulent or low-virulence C. albicans cells. Susceptibility to infection correlated with impaired development of protective Th1 responses, in spite of the production of bioactive IL-12. The occurrence of predominant Th2 responses was associated with both impaired antifungal effector functions of neutrophils and a defective expression of co-stimulatory molecules on macrophages. All functions were improved upon administration of recombinant TNF-alpha, also resulting in increased resistance to infection. These findings indicate that the protective effect of TNF-alpha in candidiasis relies on the induction of antifungal Th1 responses, possibly occurring through stimulation of antifungal effector functions and co-stimulatory activities of phagocytic cells.
The 70-kDa recombinant Candida albicans heat shock protein (CaHsp70) and its 21-kDa C-terminal and 28-kDa N-terminal fragments (CaHsp70-Cter and CaHsp70-Nter, respectively) were studied for their immunogenicity, including proinflammatory cytokine induction in vitro and in vivo, and protection in a murine model of hematogenous candidiasis. The whole protein and its two fragments were strong inducers of both antibody (Ab; immunoglobulin G1 [IgG1] and IgG2b were the prevalent isotypes) and cell-mediated immunity (CMI) responses in mice. CaHsp70 preparations were also recognized as CMI targets by peripheral blood mononuclear cells of healthy human subjects. Inoculation of CaHsp70 preparations into immunized mice induced rapid production of interleukin-6 (IL-6) and tumor necrosis factor alpha, peaking at 2 to 5 h and declining within 24 h. CaHsp70 and CaHsp70-Cter also induced gamma interferon (IFN-gamma), IL-12, and IL-10 but not IL-4 production by CD4+ lymphocytes cocultured with splenic accessory cells from nonimmunized mice. In particular, the production of IFN-gamma was equal if not superior to that induced in the same cells by whole, heat-inactivated fungal cells or the mitogenic lectin concanavalin A. In immunized mice, however, IL-4 but not IL-12 was produced in addition to IFN-gamma upon in vitro stimulation of CD4+ cells with CaHsp70 and CaHsp70-Cter. These animals showed a decreased median survival time compared to nonimmunized mice, and their mortality was strictly associated with organ invasion by fungal hyphae. Their enhanced susceptibility was attributable to the immunization state, as it did not occur in congenitally athymic nude mice, which were unable to raise either Ab or CMI responses to CaHsp70 preparations. Together, our data demonstrate the elevated immunogenicity of CaHsp70, with which, however, no protection against but rather some enhancement of Candida infection seemed to occur in the mouse model used.
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Historically there has been controversy on the relative importance of antibody- and cell-mediated immune responses in the protection against fungal pathogens. The controversy was fuelled by the difficulties encountered in obtaining consistent results with polyclonal antibody experiments and I inducing long-lasting immune protection by vaccinations with induce stron cell-mediated responses. Recent studies indicate that both antibody- and cell-mediated immune responses can contribute to host protection against Candida albicans and C. neoformans. At the present time the major issue is not the relative importance of antibody- and cell-mediated immune responses but rather, the mechanisms by which the two arms of the immune system function and cooperate.
At the present time, it is clear that Th1 responses afford protection against the fungi; however, the development, maintenance and function of the protective immune responses are complex mechanisms and are influenced by multiple factors. The route of infection has been shown to affect initial cytokine production and, consequently, the induction of protective Th1 responses. The ability of different isolates of the same fungal agent to induce and sustain a protective response has also been emphasized. Protective immune responses have been shown to vary in genetically different mouse strains after infection. In addition, these protective responses, such as cellular influx and cytokine production, also vary within the same animal depending on the tissue infected. The functional dominance of certain cytokines over others in influencing development and maintenance of protective responses has been discussed. Certain cytokines may act differently in hosts lacking important components of their innate or immune repertoire. It is evident from these presentations that a more comprehensive understanding of the protective mechanisms against different fungal agents is emerging. However, there is still much to learn before cytokine modulatory therapy can be used effectively without risk in the human host.
Cell-mediated immunity (CMI) has been shown, over many decades of clinical observation and bench research, to be central to the outcome of invasive fungal infections. In recent years, understanding the role of messenger molecules (cytokines), in coordinating and augmenting cellular immunity has been ascendant. These studies have made it possible to consider using cytokines, now available in abundant quantities via recombinant DNA technologies, to treat fungal infections. In this symposium, the most important fungal pathogens that cause infections in humans, particularly in immunocompromised patients, are considered, with emphasis on how recent experimental work may lead to a better understanding of the role of cytokines and their use in therapy.
CAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that mediates adipocyte differentiation and the regulation of genes expressed in immune responses and inflammation, such as interleukin-6 (IL-6), IL-8, and granulocyte colony-stimulating factor (G-CSF). We investigated the role of C/EBPbeta (NF-IL6) in the generation of bone marrow B lymphocytes by taking advantage of C/EBPbeta-/- mice. We found that the expansion of bone marrow (BM) B lymphocytes was impaired in long-term lymphoid cultures from C/EBPbeta-/- mice. Consistent with this finding, the number of BM B cells was decreased in C/EBPbeta-/- mice. Both the levels of IL-7 gene expression and bioactive IL-7 from BM stromal cells were decreased in C/EBPbeta-/- mice. Furthermore, the proliferative responsiveness of BM B-cell precursors to IL-7 was also reduced as compared to wild-type mice, indicating that C/EBPbeta is required for the generation of BM B cells induced by IL-7. Accordingly, IL-7 stimulates the C/EBPbeta DNA-binding activity of normal BM pre-B lymphocytes as well as of 70Z/3 pre-B cells. These results point to C/EBPbeta as a critical signaling molecule in BM B lymphopoiesis.
Neutrophil defects predispose to severe fungal infections, yet the immunomodulatory role of these cells is poorly defined. The contribution of neutrophils to the early cytokine balance governing Th1 and Th2 cell development was examined in mice with candidiasis. Neutrophils secreted IL-12 and IL-10, correlating with the respective development of self-limiting (Th1-associated) and progressive (Th2-associated) disease. Exogenous IL-12 was effective in protecting neutropenic hosts susceptible to infection. These results suggest that 1) neutrophils, via their ability to release cytokines, play an active role in determining the qualitative development of the T cell response, and 2) their early role in anticandidal immunity can be replaced by exogenous IL-12.
To investigate the role and effect of IL-2 in the genesis of Th1 and Th2 responses to Candida albicans in vivo, we assessed the levels of IL-2 production and the Ag-specific proliferative response in mice with healing or nonhealing infection and the effects of IL-2 neutralization or administration on the course and outcome of infection and on the type of CD4+ Th immunity elicited. High levels of IL-2 production and Ag-specific proliferation in vitro correlated with disease progression in susceptible mice. In contrast, resolution of infection in resistant mice was accompanied by the induction of Ag-specific hyporesponsiveness and impaired IL-2 production. Progression of infection did not occur in susceptible mice treated with anti-IL-2 or anti-IL-2R mAbs; conversely, disease resolution was prevented in resistant mice treated with IL-2. CD4+ Th1 cell responses were present in BALB/c mice rendered resistant by IL-2 neutralization and CD4+ Th2 responses in mice rendered susceptible by IL-2 treatment. The presence of IL-2 restored Ag-specific responsiveness in vitro and correlated in vivo with the expansion of CD4+ MEL-149(low) cells capable of producing IL-2 and IL-4 both in vitro and in vivo as observed in adult thymectomized mice. These results indicate that production of IL-2 early in infection correlates with the induction of IL-4-producing CD4+ Th2 cells, while a transient loss of T cell responsiveness, such as IL-2 production, appears to be required for CD4+ Th1 occurrence in mice with candidiasis.
Granulocytes may serve immunoregulatory and effector roles in different limbs of the immune response to infection. Using live vaccine strain or virulent challenge in mucosal or systemic infection of mice with Candida albicans, we examined the effect of mAb-mediated depletion of neutrophils on the course of primary and secondary challenge and on development of CD4+ cell-dependent immunity. We obtained evidence of deleterious effects of neutrophil depletion occurring at the time of infection under all conditions of testing, both in naive and in previously immunized mice. In contrast, neutrophil depletion appeared to benefit the hosts late in the course of an overwhelming systemic infection. In an attempt to correlate neutrophil function with the nature of the T cell response, we tested the ability of neutrophils to produce cytokines associated with functionally distinct CD4+ Th cell responses to Candida. We found that neutrophils were endowed with the capacity to secrete IL-12 and IL-10 in vitro in response to the yeast. Neutrophil ablation early in the course of Th1-associated, self-limiting infection appeared to change the qualitative development of the T cell response, and rendered mice susceptible to infection. In addition to long recognized contributions to acute anti-candidal responses, these data suggest an important role for neutrophils both in initiation and in expression of Candida-specific immunity.
A variety of pathological conditions, including impaired immune function, is believed to underlie host susceptibility to fungal infections and to determine both the severity and the characteristic of the associated pathology. Although the redundancy and the interdependence of antifungal responses may not favor the proper dissection and appreciation of individual effector mechanisms, the T helper type 1/type 2 paradigm of acquired immunity to fungi is proving essential for a better understanding of the host response from a regulatory perspective. The recent understanding of the importance of the different T helper cell subsets in fungal infections and the increasing appreciation of the reciprocal regulation between the innate, humoral, and adaptive immune systems in the development of optimal antimicrobial immunity have offered us new clues which may lead to an understanding of T cell dependent immunity to fungi.
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-y, 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.
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.