The danger signal S100B integrates pathogen- and danger-sensing pathways to restrain inflammation.

Humans inhale hundreds of Aspergillus conidia without adverse consequences. Powerful protective mechanisms may ensure prompt control of the pathogen and inflammation. Here we reveal a previously unknown mechanism by which the danger molecule S100B integrates pathogen- and danger-sensing pathways to restrain inflammation. Upon forming complexes with TLR2 ligands, S100B inhibited TLR2 via RAGE, through a paracrine epithelial cells/neutrophil circuit that restrained pathogen-induced inflammation. However, upon binding to nucleic acids, S100B activated intracellular TLRs eventually resolve danger-induced inflammation via transcriptional inhibition of S100B. Thus, the spatiotemporal regulation of TLRs and RAGE by S100B provides evidence for an evolving braking circuit in infection whereby an endogenous danger protects against pathogen-induced inflammation and a pathogen-sensing mechanism resolves danger-induced inflammation.

Thymosin alpha1 to harness immunity to pathogens after haploidentical hematopoietic transplantation.

We designed a phase I/II clinical study to determine safety and efficacy of thymosin alpha1 (Talpha1) administration in recipients of one HLA haplotype (haploidentical) stem cell transplants for hematologic malignancies. Talpha1 administration did not cause acute or chronic graft versus host disease and was associated with significant improvement in polymorphonuclear (phagocytosis) and dendritic cell (phagocytosis, expression of costimulatory molecules, and cytokine production) functions. It was also associated with increased T-cell counts and earlier appearance of functional pathogen-specific T cell responses (by a sensitive limiting dilution assay that detects frequency of T cells specific for Aspergillus, Candida, CMV, ADV, VZV, HSV, Toxoplasma). Five of six haploidentical transplant recipients who received Talpha1 are alive and disease free at a median follow-up of 10 months after transplantation (range: 5-20). They experienced only a single nonlethal infectious episode and one patient developed fatal immune hemolytic anemia. At this very early stage of the clinical trial, we conclude Talpha1 administration is safe and may impact favorably on immune function. Larger numbers of patients and longer follow-up are, of course, needed to assess its impact on survival.

Exogenous pentraxin 3 restores antifungal resistance and restrains inflammation in murine chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening bacterial and fungal infections and hyperinflammation. The susceptibility to aspergillosis in experimental CGD (p47(phox-/-) mice) is associated with the failure to control the inherent inflammatory response to the fungus and to restrict the activation of inflammatory Th17 cells. We assessed whether pentraxin (PTX)3, a member of a family of multimeric pattern-recognition proteins with potent anti-Aspergillus activity, could limit pathogenic inflammation in p47(phox-/-) mice by curbing the IL-23/Th17 inflammatory axis in response to the fungus. We found that the production of PTX3 was delayed in CGD mice in infection but exogenous administration of PTX3 early in infection restored antifungal resistance and restrained the inflammatory response to the fungus. This occurred through down-regulation of IL-23 production by dendritic cells and epithelial cells which resulted in limited expansion of IL-23R+ gammadelta+ T cells producing IL-17A and the emergence of Th1/Treg responses with minimum pathology. Thus, PTX3 could be therapeutically used for the exploitation of NADPH-independent mechanism(s) of antifungal immune protection with limited immunopathology in CGD.

Immune sensing of Aspergillus fumigatus proteins, glycolipids, and polysaccharides and the impact on Th immunity and vaccination.

The ability of the fungus Aspergillus fumigatus to activate, suppress, or subvert host immune response during life cycle in vivo through dynamic changing of cell wall structure and secretion implicates discriminative immune sensing of distinct fungal components. In this study, we have comparatively assessed secreted- and membrane-anchored proteins, glycolipids, and polysaccharides for the ability to induce vaccine-dependent protection in transplanted mice and Th cytokine production by human-specific CD4(+) T cell clones. The results show that the different fungal components are endowed with the distinct capacity to activate Th cell responses in mice and humans, with secreted proteins inducing Th2 cell activation, membrane proteins Th1/Treg, glycolipids Th17, and polysaccharides mostly IL-10 production. Of interest, the side-by-side comparison revealed that at least three fungal components (a protease and two glycosylphosphatidylinositol-anchored proteins) retained their immunodominant Th1/Treg activating potential from mice to humans. This suggests that the broadness and specificity of human T cell repertoire against the fungus could be selectively exploited with defined immunoactive Aspergillus Ags.

Indoleamine 2,3-dioxygenase (IDO) in inflammation and allergy to Aspergillus.

Innate responses combine with adaptive immunity to generate the most effective form of resistance against Aspergillus fumigatus. A complex set of signaling networks initiate both innate and adaptive immunity in response to the different fungal morphotypes. In response, the fungus has developed or acquired sophisticated mechanisms to avoid, counteract and subvert sensors, signaling networks and a range of effector functions that constitute the host immune response. Host response and fungal countermeasures may contribute to the balance of pro-inflammatory and anti-inflammatory signaling that is eventually required to benefit both parties. Here we highlight the important contribution of the enzyme IDO (indoleamine 2,3-dioxygenase) and tryptophan catabolites to such a homeostatic condition in Aspergillus infection and allergy. By providing the host with immune defense mechanisms adequate for protection, without necessarily eliminating the fungus or causing an unacceptable level of tissue damage, IDO and tryptophan metabolites may prove to be potent regulators capable of taming innate and adaptive pathogenic inflammatory host responses.

Th17 cells in the setting of Aspergillus infection and pathology.

Innate and adaptive immune responses act to generate the most effective form of immunity for protection against Aspergillus fumigatus. The decision of how to respond is still primarily determined by interactions between fungi and cells of the innate immune system, but the actions of T cells will feed back into this dynamic equilibrium to regulate the balance between pro-inflammatory and anti-inflammatory signals. The enzyme indoleamine 2,3-dioxygenase, and tryptophan metabolites, acting as a bridge between dendritic cells and regulatory T cells, pivotally contribute to such a homeostatic condition by taming inflammatory responses. IL-23 and the newly described Th17 pathway, by means of negative regulation of tryptophan catabolism, play an inflammatory role previously attributed to uncontrolled Th1 response. Our data support a model in which IL-23/IL-17A/Th17-driven inflammation promotes infection and impairs antifungal immune resistance. Thus, modulation of the inflammatory response represents a potential strategy to stimulate protective immune responses to Aspergillus.

Lack of Toll IL-1R8 exacerbates Th17 cell responses in fungal infection.

TLRs contribute to the inflammatory response in fungal infections. Although inflammation is an essential component of the protective response to fungi, its dysregulation may significantly worsen fungal diseases. In this study, we tested the hypothesis that Toll IL-1R8 (TIR8)/single Ig IL-1-related receptor, a member of the IL-1R family acting as a negative regulator of TLR/IL-1R signaling, affects TLR responses in fungal infections. Genetically engineered Tir8(-/-) mice were assessed for inflammatory and adaptive Th cell responses to Candida albicans and Aspergillus fumigatus. Inflammatory pathology and susceptibility to infection were higher in Tir8(-/-) mice and were causally linked to the activation of the Th17 pathway. IL-1R signaling was involved in Th17 cell activation by IL-6 and TGF-beta in that limited inflammatory pathology and relative absence of Th17 cell activation were observed in IL-1RI(-/-) mice. These data demonstrate that TIR8 is required for host resistance to fungal infections and that it functions to negatively regulate IL-1-dependent activation of inflammatory Th17 responses. TIR8 may contribute toward fine-tuning the balance between protective immunity and immunopathology in infection.

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.

Pentraxin 3 protects from MCMV infection and reactivation through TLR sensing pathways leading to IRF3 activation.

Reactivation of latent human cytomegalovirus (HCMV) following allogeneic transplantation is a major cause of morbidity and mortality and predisposes to severe complications, including superinfection by Aspergillus species (spp). Antimicrobial polypeptides, including defensins and mannan-binding lectin, are known to block viral fusion by cross-linking sugars on cell surface. Pentraxin 3 (PTX3), a member of the long pentraxin family, successfully restored antifungal immunity in experimental hematopoietic transplantation. We assessed here whether PTX3 binds HCMV and murine virus (MCMV) and the impact on viral infectivity and superinfection in vivo. We found that PTX3 bound both viruses, reduced viral entry and infectivity in vitro, and protected from MCMV primary infection and reactivation as well as Aspergillus superinfection. This occurred through the activation of interferon (IFN) regulatory factor 3 (IRF3) in dendritic cells via the TLR9/MyD88-independent viral recognition sensing and the promotion of the interleukin-12 (IL-12)/IFN-gamma-dependent effector pathway.

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 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.

Antifungal immune reactivity in nasal polyposis.

As a fungal etiology has been proposed to underlie severe nasal polyposis, the present study was undertaken to assess local antifungal immune reactivity in nasal polyposis. For this purpose, microbial colonization, along with the pattern of T helper 1 (Th1)/Th2 cytokine production and Toll-like receptor (TLR) expression, was evaluated in patients with nasal symptoms and with and without polyposis and in healthy subjects. The results show that Th2 reactivity was a common finding for patients with nasal polyposis regardless of the presence of microbes. The production of interleukin-10 was elevated in patients with bacterial and, particularly, fungal colonization, while both TLR2 expression and TLR4 expression were locally impaired in microbe-colonized patients. Eosinophils and neutrophils, highly recruited in nasal polyposis, were found to exert potent antifungal effector activities toward conidia and hyphae of the fungus and to be positively regulated by TLR2 or TLR4 stimulation. Therefore, a local imbalance between activating and deactivating signals to effector cells may likely contribute to fungal pathogenicity and the expression of local immune reactivity in nasal polyposis.