MyD88 is an adaptor protein, which plays an essential role in the intracellular signaling elicited by IL-1R and several TLRs. Central to its function is the ability of its Toll/IL-1R translation initiation region (TIR) domain to heterodimerize with the receptor and to homodimerize with another MyD88 molecule to favor the recruitment of downstream signaling molecules such as the serine/threonine kinases IL-1R-associated kinase 1 (IRAK1) and IRAK4. Herein, we have synthesized and tested the activity of a synthetic peptido-mimetic compound (ST2825) modeled after the structure of a heptapeptide in the BB-loop of the MyD88-TIR domain, which interferes with MyD88 signaling. ST2825 inhibited MyD88 dimerization in coimmunoprecipitation experiments. This effect was specific for homodimerization of the TIR domains and did not affect homodimerization of the death domains. Moreover, ST2825 interfered with recruitment of IRAK1 and IRAK4 by MyD88, causing inhibition of IL-1beta-mediated activation of NF-kappaB transcriptional activity. After oral administration, ST2825 dose-dependently inhibited IL-1beta-induced production of IL-6 in treated mice. Finally, we observed that ST2825 suppressed B cell proliferation and differentiation into plasma cells in response to CpG-induced activation of TLR9, a receptor that requires MyD88 for intracellular signaling. Our results indicate that ST2825 blocks IL-1R/TLR signaling by interfering with MyD88 homodimerization and suggest that it may have therapeutic potential in treatment of chronic inflammatory diseases.

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.

The long pentraxin 3 (PTX3) modulates different effector pathways involved in innate resistance to Aspergillus fumigatus, including complement activation or promotion of phagocytosis by interacting with FcγRs. However, whether and how TLRs modulate PTX3 mediates antifungal resistance is not known. In this study, we demonstrate that PTX3 binds myeloid differentiation protein 2 (MD-2) in vitro and exerts its protective antifungal activity in vivo through TLR4/MD-2–mediated signaling. Similar to Tlr4−/− mice, Md2−/− mice displayed high susceptibility to pulmonary aspergillosis, a phenotype associated with a proinflammatory cytokine profile and impaired antifungal activity of polymorphonuclear neutrophils. Treating Md2−/− mice with PTX3 failed to confer immune protection against the fungus, whereas adoptive transfer of MD-2–competent polymorphonuclear neutrophils restored it. Mechanistically, engagement of MD-2 by PTX3-opsonized Aspergillus conidia activated the TLR4/Toll/IL-1R domain-containing adapter inducing IFN-β–dependent signaling pathway converging on IL-10. Thus, we have identified a novel receptor mechanism, involving the TLR4/MD-2/Toll/IL-1R domain-containing adapter inducing IFN-β–mediated signaling, whereby PTX3 elicits antifungal resistance with limited immunopathology in A. fumigatus infection.

Pentraxin 3 (PTX3) is an acute-phase glycoprotein with a nonredundant function in the host resistance to Aspergillus fumigatus. PTX3 activity was evaluated against pulmonary aspergillosis in rats immunosuppressed with cortisone acetate. PTX3 enhanced the survival rate and reduced the lung fungal burden of infected rats in both therapeutic and prophylactic modalities. Thus, we extended the protective activity of PTX3 in pulmonary aspergillosis to corticosteroid-induced immunodeficiency, which is a relevant clinical condition in graftversus-host disease and in solid organ transplant.Pentraxin 3 (PTX3) is a multimeric protein composed of eight subunits covalently associated by disulfide bonds and N-glycosylated with complex-type oligosaccharides (7,8). PTX3 was identified as a nonredundant factor in the host resistance to the opportunistic fungus Aspergillus fumigatus (5). The effective pharmacological role of PTX3 against A. fumigatus infection was initially evaluated in mice with bone marrow transplants (5, 6). The combination of PTX3 with first-line antifungal treatments (amphotericin B [Ambisome] and voriconazole) indicated that PTX3 is able to enhance the therapeutic index of these drugs in mice (3, 6). PTX3 was also effective by itself against pulmonary aspergillosis in p47 phoxϪ/Ϫ mice, a model of chronic granulomatous disease (3).Corticosteroid treatment is one of the most relevant pharmacological conditions that predispose humans to a high risk of infection with Aspergillus (9, 10, 12). To further characterize PTX3, we evaluated the protective activity of this protein against A. fumigatus in corticosteroid-induced immunodeficiency.A rat model of invasive pulmonary aspergillosis (IPA) was set up. Immunodeficiency was induced in 8-to 10-week-old Sprague-Dawley rats (Harlan), fed with a low-protein diet (8% protein), by subcutaneous injections of cortisone acetate (CA) (Sigma Chemical Co., St. Louis, MO). CA was administered at a dose of 150 mg/kg of body weight 6 days before and then every other day up to the day of infection and was maintained after infection by administering 80 mg/kg every other day up to the end of the experiment (2). Conidia were obtained from 4-and 5-day cultures in Sabouraud agar medium at 28°C and scraped in a Sabouraud broth medium (0.05% Tween 80). Rats were intratracheally inoculated with a single administration of 5 ϫ 10 7 conidia of A. fumigatus in 0.2 ml of sterile saline (6). This inoculum led to mortality within 12 to 13 days and provided an extensive lung infection (Fig. 1). In order to establish whether this model of pulmonary infection was reversible by a clinically approved antifungal, voriconazole was administered orally (30 mg/kg) the same day of infection and then daily for 8 days. In all the experiments, voriconazole-treated rats showed a mean survival time (MST) of Ͼ28 days and a survival rate in the range of 50 to 90%.Human recombinant PTX3 (UniProtKB accession no. P26022), herein reported as PTX3, was produced in HEK293 cells and purified as described previously (11...

Infection caused by Aspergillus fumigatus remains a major therapeutic challenge in immunocompromised individuals. Innate immunity represents the first line of defense against pathogens. In the last 20 years, several proteins belonging to this arm of the immune system have been characterized as being endowed with antifungal activity. Among these, the prototype long pentraxin PTX3 has been identified as a non-redundant protective factor against infections caused by A. fumigatus. A number of relevant animal models of invasive aspergillosis have indicated that PTX3 exerts its protective activity in several conditions of immunosuppression. In this article, we review the current understanding of PTX3 mechanisms of action that might be of help in further exploration of the pharmacological activity of this protein against A. fumigatus.