Pentraxins are a superfamily of conserved proteins that are characterized by a cyclic multimeric structure. The classical short pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are acute-phase proteins produced in the liver in response to inflammatory mediators. Short pentraxins regulate innate resistance to microbes and the scavenging of cellular debris and extracellular matrix components. In contrast, long pentraxins have an unrelated, long amino-terminal domain coupled to the carboxy-terminal pentraxin domain, and differ, with respect to short pentraxins, in their gene organization, chromosomal localization, cellular source, and in their stimuli-inducing and ligand-recognition ability. To investigate the in vivo function of the long pentraxin PTX3, we generated mice deficient in Ptx3 by homologous recombination. Ptx3-null mice were susceptible to invasive pulmonary aspergillosis. Ptx3 binds selected microbial agents, including conidia of Aspergillus fumigatus, and we found that susceptibility of Ptx3-null mice was associated with defective recognition of conidia by alveolar macrophages and dendritic cells, as well as inappropriate induction of an adaptive type 2 response. Thus, the long pentraxin Ptx3 is a secreted pattern-recognition receptor that has a non-redundant role in resistance to selected microbial agents, in particular to the opportunistic fungal pathogen Aspergillus fumigatus.

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.

The prototypic long pentraxin PTX3 is a unique fluid-phase pattern recognition receptor that plays a nonredundant role in innate immunity and female fertility. The PTX3 C-terminal domain is required for C1q recognition and complement activation and contains a single N-glycosylation site on Asn 220. In the present study, we characterized the structure of the human PTX3 glycosidic moiety and investigated its relevance in C1q interaction and activation of the complement classical pathway. By specific endo and exoglycosidases digestion and direct mass spectrometric analysis, we found that both recombinant and naturally occurring PTX3 were N-linked to fucosylated and sialylated complex-type sugars. Interestingly, glycans showed heterogeneity mainly in the relative amount of bi, tri, and tetrantennary structures depending on the cell type and inflammatory stimulus. Enzymatic removal of sialic acid or the entire glycosidic moiety equally enhanced PTX3 binding to C1q compared to that in the native protein, thus indicating that glycosylation substantially contributes to modulate PTX3/C1q interaction and that sialic acid is the main determinant of this contribution. BIAcore kinetic measurements returned decreasing K(off) values as sugars were removed, pointing to a stabilization of the PTX3/C1q complex. No major rearrangement of PTX3 quaternary structure was observed after desialylation or deglycosylation as established by size exclusion chromatography. Consistent with C1q binding, PTX3 desialylation enhanced the activation of the classical complement pathway, as assessed by C4 and C3 deposition. In conclusion, our results provided evidence of an involvement of the PTX3 sugar moiety in C1q recognition and complement activation.

PTX3 is an acute phase glycoprotein that plays key roles in resistance to certain pathogens and in female fertility. PTX3 exerts its functions by interacting with a number of structurally unrelated molecules, a capacity that is likely to rely on its complex multimeric structure stabilized by interchain disulfide bonds. In this study, PAGE analyses performed under both native and denaturing conditions indicated that human recombinant PTX3 is mainly composed of covalently linked octamers. The network of disulfide bonds supporting this octameric assembly was resolved by mass spectrometry and Cys to Ser site-directed mutagenesis. Here we report that cysteine residues at positions 47, 49, and 103 in the N-terminal domain form three symmetric interchain disulfide bonds stabilizing four protein subunits in a tetrameric arrangement. Additional interchain disulfide bonds formed by the C-terminal domain cysteines Cys 317 and Cys 318 are responsible for linking the PTX3 tetramers into octamers. We also identified three intrachain disulfide bonds within the C-terminal domain that we used as structural constraints to build a new three-dimensional model for this domain. Previously it has been shown that PTX3 is a key component of the cumulus oophorus extracellular matrix, which forms around the oocyte prior to ovulation, because cumuli from PTX3 ؊/؊ mice show defective matrix organization. Recombinant PTX3 is able to restore the normal phenotype ex vivo in cumuli from PTX3 ؊/؊ mice. Here we demonstrate that PTX3 Cys to Ser mutants, mainly assembled into tetramers, exhibited wild type rescue activity, whereas a mutant, predominantly composed of dimers, had impaired functionality. These findings indicate that protein oligomerization is essential for PTX3 activity within the cumulus matrix and implicate PTX3 tetramers as the functional molecular units required for cumulus matrix organization and stabilization.

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 IntroductionHuman cytomegalovirus (HCMV), a member of the Herpesviridae family, is a ubiquitous opportunistic pathogen that has an intimate lifelong relationship with its human host and establishes latency after clearance of primary infection. 1-3 Reactivation of latent virus following allogeneic transplantation immune responses results in progressive tissue damage manifesting as overt HCMV disease or complications of this infection, including acute and chronic graft rejection, graft-versus-host disease, and superinfection by other viruses, bacteria, and fungi, particularly Aspergillus species (spp). 3 Efforts have focused on the development of adoptive immunotherapeutic strategies to hasten host immune reconstruction, and cellular immunotherapy appears to be an attractive approach. 4,5 The immune control of murine CMV (MCMV) infection requires elements from both innate and adaptive immune systems. [6][7][8] Through the participation of members of the Toll-like receptors (TLRs) 9-11 and interferon (IFN) regulatory factor 3 (IRF) families, IRF3 in particular, 12,13 MCMV induces early dendritic cell (DC)-dependent type I IFN and interleukin-12 (IL-12) responses essential for mouse resistance to MCMV. 10,11,[14][15][16] The TLR9/ MyD88 signaling pathway mediates antiviral cytokine responses by plasmacytoid DCs (pDCs) that, through their unique capacity to secrete IFN-␣, and to a lesser extent IL-12 and other innate cytokines, are a cornerstone in the initiation of both innate and adaptive immune responses to MCMV. [15][16][17][18][19] However, conventional CD11b ϩ DCs also produce IFN-␣ independently of TLR9 and MyD88. 10,20 In addition to directly interfering with viral replication through ubiquitous cellular mechanisms, IFN-␣ controls natural killer (NK) cell cytotoxic activity 15 and regulates T-cell functions by activating classical DCs to more efficiently present antigens (Ags). 15 IL-12 and IL-18 secretion are instead required to prime a strong NK cell-dependent IFN-␥ response, 17,21,22 a process that is essential to counteract MCMV infection in the liver, in contrast to a perforin-dependent mechanism in the spleen. 23 Pentraxin 3 (PTX3) is a member of a superfamily of conserved proteins characterized by a cyclic mul...

Met, the high affinity receptor for hepatocyte growth factor, is one of the most frequently activated tyrosine kinases in human cancer and a validated target for cancer therapy. We previously developed a mouse monoclonal antibody directed against the extracellular portion of Met (

The collectin pentraxin 3 (PTX3) is an essential component of host resistance to pulmonary aspergillosis. Here we examined the protective effects of administration of PTX3 alone or together with deoxycholate amphotericin B (Fungizone) or liposomal amphotericin B (AmBisome) against invasive aspergillosis in a murine model of allogeneic bone marrow transplantation. PTX3, alone or in combination with the polyenes, was given intranasally or parenterally either before, in concomitance with, or after the intranasal infection with Aspergillus fumigatus conidia. Mice were monitored for resistance to infection and parameters of innate and adaptive T-helper immunity. The results showed the following: (i) complete resistance to infection and reinfection was observed in mice treated with PTX3 alone; (ii) the protective effect of PTX3 was similar or superior to that observed with liposomal amphotericin B or deoxycholate amphotericin B, respectively; (iii) protection was associated with accelerated recovery of lung phagocytic cells and T-helper-1 lymphocytes and concomitant decrease of inflammatory pathology; and (iv) PTX3 potentiated the therapeutic efficacy of suboptimal doses of either antimycotic drug. Together, these data suggest the potential therapeutic use of PTX3 either alone or as an adjunctive therapy in A. fumigatus infections.

MyD88 couples the activation of the Toll-like receptors and interleukin-1 receptor superfamily with intracellular signaling pathways. Upon ligand binding, activated receptors recruit MyD88 via its Toll-interleukin-1 receptor domain. MyD88 then allows the recruitment of the interleukin-1 receptor-associated kinases (IRAKs). We performed a site-directed mutagenesis of MyD88 residues, conserved in death domains of the homologous FADD and Pelle proteins, and analyzed the effect of the mutations on MyD88 signaling. Our studies revealed that mutation of residues 52 (MyD88 E52A ) and 58 (MyD88 Y58A ) impaired recruitment of both IRAK1 and IRAK4, whereas mutation of residue 95 (MyD88 K95A ) only affected IRAK4 recruitment. Since all MyD88 mutants were defective in signaling, recruitment of both IRAKs appeared necessary for activation of the pathway. Moreover, overexpression of a green fluorescent protein (GFP)-tagged mini-MyD88 protein (GFP-MyD88-(27-72)), comprising the Glu 52 and Tyr 58 residues, interfered with recruitment of both IRAK1 and IRAK4 by MyD88 and suppressed NF-B activation by the interleukin-1 receptor but not by the MyD88-independent TLR3. GFP-MyD88-(27-72) exerted its effect by titrating IRAK1 and suppressing IRAK1-dependent NF-B activation. These experiments identify novel residues of MyD88 that are crucially involved in the recruitment of IRAK1 and IRAK4 and in downstream propagation of MyD88 signaling.MyD88 was first discovered during studies addressing the differentiation of mouse myeloid cells in response to growthinhibitory stimuli (1). Subsequent investigations revealed that MyD88 possesses a modular organization (2), with an aminoterminal death domain (DD), 3 found in proteins involved in cell death (3, 4), and a carboxyl-terminal Toll-interleukin-1 receptor (TIR) domain, present in the intracytoplasmic tail of receptors belonging to the Toll-like receptor (TLR)/interleukin-1 receptor (IL-1R) superfamily (5). MyD88 also has an intermediate domain (ID) that is crucial in TLR signaling due to its interaction with IRAK4 (6). The role of MyD88 as a signal transducer was first shown in the pathways triggered by the activation of IL-1R (7, 8) and TLR4 (9). Further studies showed that all TLRs, with the sole exception of TLR3, and the IL-1R family utilize the adaptor protein MyD88 to initiate their signaling pathway (10).By virtue of its modular organization, MyD88 critically bridges activated receptor complexes to downstream adaptors/ effectors. Upon activation, MyD88 is recruited through its TIR domain by the homologous domain of the activated TLR/IL-1R (11, 12). MyD88, in turn, has been shown to interact with a family of downstream kinases, namely IRAK1 (13), IRAK2 (7), IRAK-M (15), and IRAK4 (16), through the interaction of its DD with the respective DDs present in the amino-terminal region of IRAKs (17). At this stage, this multimeric complex is competent to elicit the propagation of the signal downstream of the receptor(s). Although MyD88 recruits IRAK-1 via DD-DD interactions, its recruitment o...