Evaluation of immune responses in mice and sheep inoculated with a live attenuated Brucella melitensis REV1 vaccine produced in bioreactor.

The Brucella melitensis REV1 vaccine is the most widely employed vaccine for prophylaxis against brucellosis in sheep and goats. The objective of vaccination is disease control in herds or preventing infection in farms. In this study, we produced REV1 vaccine with a protocol, based on the use of liquid medium in a bioreactor, that resulted efficient, safe, relatively fast, and cost-effective. The live attenuated vaccine produced was tested in mice and sheep to investigate its immunogenicity and efficacy. Seventy-two female BALB/c mice were obtained and subdivided in 2 groups, one was stimulated with 1 × 106 colony-forming units (CFUs) of B. melitensis while the other with physiological solution alone and acting as control group. Furthermore, 25 sheep were subdivided into 5 groups: four were inoculated with a B. melitensis dose, ranging from 0.6 × 109 and 3.2 × 109 CFUs and the other was the control group. In addition, a serological diagnosis was performed for sheep by rapid serum agglutination and the complement-fixation test. Immunocompetent cells from both experiment were collected at different times post vaccination and immunostained to evaluate innate and adaptive-immune responses. In mice flow cytometry was used to detect macrophages, T lymphocytes, dendritic cells, memory cells, naïve cells, natural killer cells, major histocompatibility complex type II, B lymphocytes, regulatory T lymphocytes, T helper lymphocytes, cytotoxic T lymphocytes and recently activated CD4+ and CD8+ lymphocytes. In sheep, macrophages, T helper cells, cytotoxic T lymphocytes, regulatory T lymphocytes, dendritic cells, memory cells and naïve lymphocytes, by the same method, were analyzed. The results showed, both in mice and sheep, that the live, attenuated REV1 vaccine stimulated all immunocompetent cells tested, with a balanced innate and adaptive response. In the sheep experiment, the administered vaccine dose was very important because, at the lower doses, immunological tolerance tended to disappear, while, at the highest dose, the immunological tolerance remained active for a long period. In our experimental conditions, the optimal vaccine dose for sheep was 3.2 × 109 CFUs, although a good immune response was found using a dose of 1.6 × 109 CFUs. The vaccine produced in this study could be extensively employed in developing countries to control the brucellosis in sheep and goats.

ß-NGF and ß-NGF receptor upregulation in blood and synovial fluid in osteoarthritis.

Osteoarthritis (OA) of the knee is the most common form of non-traumatic joint disease. Previous studies have shown the involvement of ß-NGF and its receptors TrKA and p75NTR in OA-related pain, but their role in its pathogenesis is still unclear. The aim of our study was to investigate the amount of ß-NGF and the expression levels of its receptors on cells isolated from synovial fluid and blood from OA patients who had undergone total knee arthroplasty, in order to check any possible correlation with the disease staging. Our results show a progressive stage-related increase of ß-NGF and its receptors both in serum and synovial fluid. Furthermore, with respect to control subjects, OA patients show an increased amount of inflammatory monocytes along with an increased expression of ß-NGF, TrKA and p75NTR. In conclusion, our study suggests a stage-related modulation of ß-NGF and its receptors in the inflammatory process of OA.

A role for NGF and its receptors TrKA and p75NTR in the progression of COPD.

Nerve growth factor and its receptors, TrkA and p75NTR, are involved in inflammation and airways diseases, but their role in chronic obstructive pulmonary disease is still unclear and not well investigated. our data indicate the stage dependent variation of nerve growth factor and its receptors in chronic obstructive pulmonary disease progression. In fact, for the first time, this study evaluates the presence of nerve growth factor and its receptors in serum and in peripheral blood mononuclear cells of patients with different stages of chronic obstructive pulmonary disease compared to healthy subjects, non-smoker and current smoker. Serum monocyte chemoattractant protein-1, tumor necrosis factor-α, interleukin-10 and forced expiratory volume in 1 s were also analyzed. Compared to healthy subjects, chronic obstructive pulmonary disease patients presented a staging-dependent increase in serum nerve growth factor, negatively correlated to forced expiratory volume in 1 s and positively to monocyte chemoattractant Protein-1. The percentage of p75NTR+ peripheral blood mononuclear cells increased in early stages of chronic obstructive pulmonary disease (I-II), while TrKA+ peripheral blood mononuclear cells increased in late stages (III-IV). Our data demonstrate the involvement and modulation of nerve growth factor and its receptors in chronic obstructive pulmonary disease and in its staging.

LY294002 induces in vitro apoptosis and overexpression of p75NTR in human uterine leiomyosarcoma HTB 114 cells.

Uterine leiomyosarcoma is a severe neoplasia resistant to conventional therapies. In previous studies, we have shown that human SK-UT-1 (ATCC HTB114) uterine leiomyosarcoma cell line secretes nerve growth factor (NGF) and expresses its receptors tyrosine kinase A receptor (TrKA) and low affinity nerve growth factor receptor (p75NTR). Furthermore, we have demonstrated that direct chemical inhibition or IgG neutralization of TrKA receptor induce apoptosis through p75NTR. In the present study, HTB114 cells were exposed to the phosphoinositide 3-kinase (PI3K) inhibitor LY294002 with and without ß-NGF: apoptosis, cell cycle, activation of caspase-3 and protein kinase B (AKT) and TrKA/p75NTR phenotypic expression were evaluated. According to the type of exposure, LY294002 not only induced a relevant increase in apoptosis, but also produced a novel and unexpected phenotypic modulation of the NGF receptors with a downregulation of TrKA and an upregulation of p75NTR. This latter increase enhanced HTB114 apoptosis. Our study confirms that the interference on NGF transduction is a promising therapeutical approach in uterine leiomyosarcoma.

The tricyclic antidepressant amitriptyline is cytotoxic to HTB114 human leiomyosarcoma and induces p75(NTR)-dependent apoptosis.

Nerve growth factor (NGF) receptors, TrKA and p75(NTR), are being investigated in cancer therapy. Our previous data show that, in HTB114 uterine leiomyosarcoma cells, p75(NTR)-dependent apoptosis is inducible by cytotoxic drugs and can suppress nerve growth factor-dependent growth. Although amitriptyline can kill cancer cells and bind TrKA/B, its effects on p75-dependent apoptosis are unknown. The aim of this paper was to evaluate the antineoplastic potential of amitriptyline, and the role of p75(NTR)-dependent apoptosis in the chemoresistant uterine HTB114 leiomyosarcoma. Using proliferation assays and fluorescence-activated cell sorting analysis, we found that amitriptyline caused a marked reduction in HTB114 cell viability, associated with the parallel upregulation of p75(NTR) expression. This converted the TrKA⁺-proliferating cells into TrKA⁺/p75(NTR⁺), leading to downregulation of TrKA-prosurvival signaling (AKT) and activation of p75(NTR)-dependent apoptosis (through caspase-3). Overall, we provide novel evidence that HTB114 uterine leiomyosarcoma cells are highly sensitive to amitriptyline, supporting the role of p75(NTR)-dependent apoptosis as a novel cytotoxic mechanism of this drug and of p75(NTR) as an inducible stress receptor and a novel target in clinical oncology.

Novel localization of low affinity NGF receptor (p75) in the stroma of prostate cancer and possible implication in neoplastic invasion: an immunohistochemical and ultracytochemical study.

BACKGROUND: The localization of low affinity nerve growth factor receptor (p75) in prostate carcinogenesis is still unclear. Our aim was to reinvestigate the localization of p75 in normal and pathological prostate and to check a possible correlation to neoplastic grading. METHODS: Specimens from 33 prostate cancers and from normal prostatic tissue were analyzed for p75 expression at light and ultrastructural levels. RESULTS: In normal tissue p75-immunoreactivity was restricted to basal cells in the epithelial compartment and to nerves and blood vessel in stroma. During carcinogenesis, p75-immunoreactivity progressively decreased at the periphery of the foci according to the increase in malignancy. No p75-immunoreactivity was detected inside of the foci. On the contrary, in stroma we found a dramatic increase in p75-immunoreactivity correlated to an increase in malignancy. In this compartment, for the first time ultrastructural analysis identified p75-immunoreactivity in smooth muscle cells (SMC) that are p75-negative in normal conditions. CONCLUSION: The present study confirms at ultrastructural level a malignant-dependent p75 decrease in basal cells of neoplastic foci. Furthermore, we show a novel, malignant-dependent localization of p75 in SMC in the stroma around the neoplastic foci. Since p75 expression is present in muscle cells only during the earliest stages of differentiation and mature muscle cells lose this expression, we hypothesize that p75 re-expression in stromal SMC is a further mechanism related to the general de-differentiation of the stroma connected to the neoplastic invasion. According to this hypothesis, our results suggest that p75 analysis could be a novel prognostic marker for prostate cancer.

Defective tryptophan catabolism underlies inflammation in mouse chronic granulomatous disease.

Half a century ago, chronic granulomatous disease (CGD) was first described as a disease fatally affecting the ability of children to survive infections. Various milestone discoveries have since been made, from an insufficient ability of patients' leucocytes to kill microbes to the underlying genetic abnormalities. In this inherited disorder, phagocytes lack NADPH oxidase activity and do not generate reactive oxygen species, most notably superoxide anion, causing recurrent bacterial and fungal infections. Patients with CGD also suffer from chronic inflammatory conditions, most prominently granuloma formation in hollow viscera. The precise mechanisms of the increased microbial pathogenicity have been unclear, and more so the reasons for the exaggerated inflammatory response. Here we show that a superoxide-dependent step in tryptophan metabolism along the kynurenine pathway is blocked in CGD mice with lethal pulmonary aspergillosis, leading to unrestrained Vgamma1(+) gammadelta T-cell reactivity, dominant production of interleukin (IL)-17, defective regulatory T-cell activity and acute inflammatory lung injury. Although beneficial effects are induced by IL-17 neutralization or gammadelta T-cell contraction, complete cure and reversal of the hyperinflammatory phenotype are achieved by replacement therapy with a natural kynurenine distal to the blockade in the pathway. Effective therapy, which includes co-administration of recombinant interferon-gamma (IFN-gamma), restores production of downstream immunoactive metabolites and enables the emergence of regulatory Vgamma4(+) gammadelta and Foxp3(+) alphabeta T cells. Therefore, paradoxically, the lack of reactive oxygen species contributes to the hyperinflammatory phenotype associated with NADPH oxidase deficiencies, through a dysfunctional kynurenine pathway of tryptophan catabolism. Yet, this condition can be reverted by reactivating the pathway downstream of the superoxide-dependent step.

Provision of antifungal immunity and concomitant alloantigen tolerization by conditioned dendritic cells in experimental hematopoietic transplantation.

FoxP3(+) regulatory T (Treg) cells are important mediators of peripheral tolerance, and deficiency of this population is associated with autoimmune inflammation and onset of acute lethal graft-vs.-host disease in transplantation. Type I IFN-producing plasmacytoid dendritic cells (pDC) are implicated in the induction and maintenance of tolerance and contribute to engraftment facilitation and prevention of graft-vs.-host disease after allogeneic hematopoietic stem cells transplantation (HSCT). Because host DC function is impaired during the immediate period post-transplant, the administration of donor DC may be useful for the educational program of recovering T cells. Distinct DC subsets could be derived from bone marrow (murine) or peripheral CD14(+) cell (human) cultures in the presence of either GM-CSF/IL-4 (myeloid DC) or FLT3-ligand (mainly pDC). The ability of either DC subset to induce Th1/Treg cell priming against Aspergillus fumigatus as well as the relative contribution of murine DC subsets to antifungal priming upon adoptive transfer in hematopoietic transplanted mice with aspergillosis is not known. We found specialization and complementarity in priming and tolerization by the different DC subsets, with FL-DC fulfilling the requirement for (i) Th1/Treg antifungal priming; ii) tolerization toward alloantigens and (iii) diversion from alloantigen-specific to antigen-specific T cell responses in the presence of donor T lymphocytes. Interestingly, thymosin alpha1 (Talpha1), known to modulate human pDC functions trough TLR9, affects mobilization and tolerization of pDC by activating the indoleamine 2,3-dioxygenase-dependent pathway, and this resulted in Treg development and tolerization. Thus, transplantation tolerance and concomitant pathogen clearance could be achieved through the therapeutic induction of antigen-specific Treg cells via instructive immunotherapy with pathogen- or TLR-conditioned donor DC.

Functional yet balanced reactivity to Candida albicans requires TRIF, MyD88, and IDO-dependent inhibition of Rorc.

The ability of regulatory T (Treg) cells to inhibit aspects of innate and adaptive immunity is central to their protective function in fungal infections. In murine candidiasis, CD4(+)CD25(+) Treg cells prevent excessive inflammation but enable fungal persistence in the gastrointestinal tract, which underlies the onset of durable antifungal protection. In this study, we show that fungal growth, inflammatory immunity, and tolerance to the fungus were all controlled by the coordinate activation of naturally occurring Treg cells, which limited early inflammation at the sites of infection, and pathogen-induced Treg cells (that regulated the expression of adaptive Th immunity in secondary lymphoid organs). Naturally occurring Treg cells required the TRIF pathway for migration to inflamed sites, where the MyD88 pathway would then restrain their suppressive function. Subsequent inflammatory Th1-type immunity was modulated by induced Treg cells, which required the TRIF pathway as well, and acted through activation of IDO in dendritic cells and Th17 cell antagonism. In vitro, using naive CD4(+) cells from TRIF-deficient mice, tryptophan metabolites were capable of inducing the Foxp3-encoding gene transcriptionally and suppressing the gene encoding RORgammat, Th17 lineage specification factor. This is the first study to show that the same tryptophan catabolites can foster dendritic cell-supported generation of Foxp3(+) cells and mediate, at the same time, inhibition of RORgammat-expressing T cells.

Thymosin alpha1 activates the TLR9/MyD88/IRF7-dependent murine cytomegalovirus sensing for induction of anti-viral responses in vivo.

Reactivation of latent human cytomegalovirus following allogeneic transplantation is a major cause of morbidity and mortality and predisposes to severe complications. Thymosin alpha1 (Talpha1), a naturally occurring thymic peptide, is approved for treatment of some viral infections and as an immune adjuvant. Talpha1 successfully primed dendritic cells (DCs) for anti-microbial T helper type 1 resistance through Toll-like receptor (TLR) 9 signaling. We sought to determine here whether Talpha1 could play a role in murine cytomegalovirus infection (MCMV). To this purpose, susceptible, resistant and TLR-deficient mice were infected with MCMV, treated with Talpha1 and assessed for protection in term of microbiological and immunological parameters. Talpha1 protected susceptible and resistant mice from MCMV infection. The anti-viral effect of Talpha1 occurred through the activation of plasmacytoid DCs via the TLR9/myeloid differentiation primary response gene 88-dependent viral recognition sensing, leading to the activation of IFN regulatory factor 7 and the promotion of the IFN-alpha/IFN-gamma-dependent effector pathway.

IL-23 and the Th17 pathway promote inflammation and impair antifungal immune resistance.

Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases. We found here that the IL-23/IL-17 developmental pathway acted as a negative regulator of the Th1-mediated immune resistance to fungi and played an inflammatory role previously attributed to uncontrolled Th1 cell responses. Both inflammation and infection were exacerbated by a heightened Th17 response against Candida albicans and Aspergillus fumigatus, two major human fungal pathogens. IL-23 acted as a molecular connection between uncontrolled fungal growth and inflammation, being produced by dendritic cells in response to a high fungal burden and counter-regulating IL-12p70 production. Both IL-23 and IL-17 subverted the inflammatory program of neutrophils, which resulted in severe tissue inflammatory pathology associated with infection. Our data are the first demonstrating that the IL-23/IL-17 pathway promotes inflammation and susceptibility in an infectious disease model. As IL-23-driven inflammation promotes infection and impairs antifungal resistance, modulation of the inflammatory response represents a potential strategy to stimulate protective immune responses to fungi.

Thymosin alpha1: an endogenous regulator of inflammation, immunity, and tolerance.

Thymosin alpha1 (Talpha1), first described and characterized by Allan Goldstein in 1972, is used worldwide for the treatment of some immunodeficiencies, malignancies, and infections. Although Talpha1 has shown a variety of effects on cells and pathways of the immune system, its central role in modulating dendritic cell (DC) function has only recently been appreciated. As DCs have the ability to sense infection and tissue stress and to translate collectively this information into an appropriate immune response, an action on DCs would predict a central role for Talpha1 in inducing different forms of immunity and tolerance. Recent results have shown that Talpha1: (a) primed DCs for antifungal Th1 resistance through Toll-like receptor (TLR)/MyD88-dependent signaling and this translated in vivo in protection against aspergillosis; (b) activated plasmacytoid DCs (pDC) via the TLR9/MyD88-dependent viral recognition, thus leading to the activation of interferon regulatory factor 7 and the promotion of the IFN-alpha/IFN-gamma-dependent effector pathway, which resulted in vivo in protection against primary murine cytomegalovirus infection; (c) induced indoleamine 2,3-dioxygenase activity in DCs, thus affecting tolerization toward self as well as microbial non-self-antigens, and this resulted in vivo in transplantation tolerance and protection from inflammatory allergy. Talpha1 is produced in vivo by cleavage of prothymosin alpha in diverse mammalian tissues. Our data qualify Talpha1 as an endogenous regulator of immune homeostasis and suggest that instructive immunotherapy with Talpha1, via DCs and tryptophan catabolism, could be at work to control inflammation, immunity, and tolerance in a variety of clinical settings.

Thymosin alpha1 activates dendritic cell tryptophan catabolism and establishes a regulatory environment for balance of inflammation and tolerance.

Thymosin alpha1 (Talpha1), a naturally occurring thymic peptide, primes dendritic cells (DCs) for antifungal T-helper type 1 resistance through Toll-like receptor 9 (TLR9) signaling. As TLR9 signaling also activates the immuno-suppressive pathway of tryptophan catabolism via indoleamine 2,3-dioxygenase (IDO), we examined Talpha1 for possible induction of DC-dependent regulatory effects. Talpha1 affected T-helper cell priming and tolerance induction by human and murine DCs and induced IDO expression and function in the latter cells. IDO activation by Talpha1 required TLR9 and type I interferon receptor signaling and resulted in interleukin-10 production and generation of regulatory T cells. In transfer experiments, functionally distinct subsets of differentiated DCs were required for priming and tolerance to a fungal pathogen or alloantigens. In contrast, Talpha1-primed DCs fulfilled multiple requirements, including the induction of T-helper type 1 immunity within a regulatory environment. Thus, instructive immunotherapy with Talpha1 targeting IDO-competent DCs could allow for a balanced control of inflammation and tolerance.

Immunity and tolerance to Aspergillus involve functionally distinct regulatory T cells and tryptophan catabolism.

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defense without necessarily eliminating the fungus or causing unacceptable levels of host damage. In this study, we show that a division of labor occurs between functionally distinct regulatory T cells (Treg) that are coordinately activated by a CD28/B-7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation is controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of IL-10 and CTLA-4 on indoleamine 2,3-dioxygenase. The levels of IFN-gamma produced in this early phase set the subsequent adaptive stage by conditioning the indoleamine 2,3-dioxygenase-dependent tolerogenic program of dendritic cells and the subsequent activation and expansion of tolerogenic Treg, which produce IL-10 and TGF-beta, inhibit Th2 cells, and prevent allergy to the fungus. The coordinate activation of Treg may, however, be subverted by the fungus, as germinating conidia are capable of interfering with anti-inflammatory and tolerogenic Treg programs. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

Immunity and tolerance to Aspergillus fumigatus.

The inherent resistance to diseases caused by Aspergillus fumigatus suggests the occurrence of regulatory mechanisms that provide the host with adequate defence without necessarily eliminating the fungus or causing unacceptable levels of host damage. Efficient responses to the fungus require different mechanisms of immunity. Dendritic cells (DCs) are uniquely able to decode the fungus-associated information and translate it into qualitatively different T helper (Th) and regulatory (Treg) cell responses. A division of labour occurred between functionally distinct Treg that were coordinately activated by a CD28/B.7-dependent costimulatory pathway after exposure of mice to Aspergillus conidia. Early in infection, inflammation was controlled by the expansion, activation and local recruitment of CD4+CD25+ Treg capable of suppressing neutrophils through the combined actions of interleukin (IL10) and cytotoxic T lymphocyte antigen 4 (CTLA4) on indoleamine 2,3-dioxygenase (IDO). The levels of IFNgamma produced in this early phase set the subsequent adaptive stage by conditioning the IDO-dependent tolerogenic program of DCs and the subsequent activation and expansion of tolerogenic Treg, which produced IL10 and transforming growth factor (TGF)beta, inhibited Th2 cells, and prevented allergy to the fungus. Thus, regulation is an essential component of the host response in infection and allergy to the fungus, and its manipulation may allow the pathogen to overcome host resistance and promote disease.

Manipulating immunity against Aspergillus fumigatus.

Efficient response to Aspergillusfumigatus requires different mechanisms. Polymorphonuclear neutrophils (PMNs) are the predominant immune cells in the acute stage of most fungal infections and play a crucial role in determining the type of pathology associated with fungal infections in different clinical settings. Dendritic cells (DC) are able to decode the fungus-associated information and translate it into different T helper (Th) and regulatory (Treg) cell responses. Functionally distinct Treg cells are activated after exposure to Aspergillus conidia. Early in infection, inflammation/Th1 reactivity is controlled by Treg cells suppressing PMNs and the immunogenic program of DC. The levels of IFN-γ produced in this phase set the subsequent adaptive stage by conditioning the indoleamine 2, 3-dioxygenase (IDO)-dependent tolerogenic program of DC and the subsequent activation of tolerogenic Treg cells, which inhibit Th2 cells and prevent allergy to the fungus. Knowledge of the immunopathogenesis of Aspergillus infections may pave the way to promising strategies for immunotherapy.

A crucial role for tryptophan catabolism at the host/Candida albicans interface.

By mediating tryptophan catabolism, the enzyme indoleamine 2,3-dioxygenase (IDO) has a complex role in immunoregulation in infection, pregnancy, autoimmunity, transplantation, and neoplasia. We hypothesized that IDO might affect the outcome of the infection in mice infected with Candida albicans by virtue of its potent regulatory effects on inflammatory and T cell responses. IDO expression was examined in mice challenged with the fungus along with the consequences of its blockade by in vivo treatment with an enzyme inhibitor. We found that IDO activity was induced at sites of infection as well as in dendritic cells and effector neutrophils via IFN-gamma- and CTLA-4-dependent mechanisms. IDO inhibition greatly exacerbated infection and associated inflammatory pathology as a result of deregulated innate and adaptive/regulatory immune responses. However, a role for tryptophan catabolism was also demonstrated in a fungus-autonomous fashion; its blockade in vitro promoted yeast-to-hyphal transition. These results provide novel mechanistic insights into complex events that, occurring at the fungus/pathogen interface, relate to the dynamics of host adaptation to the fungus. The production of IFN-gamma may be squarely placed at this interface, where IDO activation probably exerts a fine control over fungal morphology as well as inflammatory and adaptive antifungal responses.

Liposomal amphotericin B activates antifungal resistance with reduced toxicity by diverting Toll-like receptor signalling from TLR-2 to TLR-4.

OBJECTIVES: Neutrophils play a crucial role in the control of the Aspergillus fumigatus infection and act in concert with antifungal drugs. This study was undertaken to obtain insights into the possible involvement of Toll-like receptors (TLRs) in the interaction of liposomal amphotericin B (L-AmB; AmBisome) with neutrophils in response to A. fumigatus. METHODS: For generation of bone marrow-transplanted mice, irradiated C57BL6 mice were infused with T cell-depleted allogeneic donor cells. For infection, mice were injected intranasally with Aspergillus fumigatus conidia and treated with L-Amb and deoxycholate amphotericin B prophylactically or therapeutically. For TLR-dependent antifungal functions, murine neutrophils were preincubated with antifungals or TLR ligands before the addition of Aspergillus conidia. RESULTS: The results show that: (a) neutrophil activation by Aspergillus occurs through TLR signalling pathways differently affecting the oxidative and non-oxidative mechanisms of the killing machinery; (b) by diverting signalling from TLR-2 to TLR-4, liposomes of AmBisome activate neutrophils to an antifungal state while attenuating the pro-inflammatory effects of deoxycholate amphotericin B; (c) this translates in vivo to the optimization of the AmBisome therapeutic efficacy in mice with aspergillosis. CONCLUSIONS: These results provide a putative molecular basis for the reduced infusion-related toxicity of AmBisome and suggest that TLR manipulation in vivo is amenable to the induction of optimal microbicidal activity in the absence of inflammatory cytotoxicity to host cells.

TLRs govern neutrophil activity in aspergillosis.

Polymorphonuclear neutrophils (PMNs) are essential in initiation and execution of the acute inflammatory response and subsequent resolution of fungal infection. PMNs, however, may act as double-edged swords, as the excessive release of oxidants and proteases may be responsible for injury to organs and fungal sepsis. To identify regulatory mechanisms that may balance PMN-dependent protection and immunopathology in fungal infections, the involvement of different TLR-activation pathways was evaluated on human PMNs exposed to the fungus Aspergillus fumigatus. Recognition of Aspergillus and activation of PMNs occurred through the involvement of distinct members of the TLR family, each likely activating specialized antifungal effector functions. By affecting the balance between fungicidal oxidative and nonoxidative mechanisms, pro- and anti-inflammatory cytokine production, and apoptosis vs necrosis, the different TLRs ultimately impacted on the quality of microbicidal activity and inflammatory pathology. Signaling through TLR2 promoted the fungicidal activity of PMNs through oxidative pathways involving extracellular release of gelatinases and proinflammatory cytokines while TLR4 favored the oxidative pathways through the participation of azurophil, myeloperoxidase-positive, granules and IL-10. This translated in vivo in the occurrence of different patterns of fungal clearance and inflammatory pathology. Both pathways were variably affected by signaling through TLR3, TLR5, TLR6, TLR7, TLR8, and TLR9. The ability of selected individual TLRs to restore antifungal functions in defective PMNs suggests that the coordinated outputs of activation of multiple TLRs may contribute to PMN function in aspergillosis.