This invention relates to the use of Serum Amyloid P component (SAP) polypeptides for the treatment of Eurotiomycetes fungi infections, in particular for aspergillosis and invasive aspergillosis, alone or in combination with pentraxin-3 (PTX3) polypeptides.
Aspergillus fungi are representatives of the Trichocomaceae family of the Eurotiales order, which in turn belong to the Eurotiomycetes class.
Aspergillosis is an opportunistic fungus infection, most often the consequence of an Aspergillus fumigatus infection, associated with a wide spectrum of diseases in humans, ranging from severe infections to allergy in immune-compromised patients (Lionakis et al., 2018). In particular, aspergillosis is a major life-threatening infection patients with impaired phagocytosis, for instance, during chemotherapy or radiotherapy-induced neutropenia (Cunha et al., 2014), because their reduced immunity allows for the infection to spread from the lungs to other major organs, leading to a condition called invasive aspergillosis.
The innate immune system represents the first line of resistance against pathogens and a key determinant in the activation and orientation of adaptive immunity through the complementary activities of a cellular and humoral arm (Bottazzi et al., 2010). Cell-associated innate immune molecules sense pathogen-derived agonists leading to activation of different inflammatory pathways (Inohara et al., 2005; Takeda et al., 2003), which include phagosome formation (Sanjuan et al., 2009). Humoral è Pattern Recognition Molecules (PRMs) are an essential components of the innate immune response sharing functional outputs with antibodies (Bottazzi et al., 2010; Mantovani et al., 2013) including opsonisation, regulation of complement activation, agglutination and neutralization, discrimination of self versus non-self and modified-self (Bottazzi et al., 2010). Humoral PRMs in turn interact with and regulate cellular effectors (Bottazzi et al., 2010; Lu et al., 2008; Lu et al., 2012; Hajishengallis et al., 2010) collaborating to form stable pathogen recognition complexes for pathogen clearance (Bottazzi et al., 2010; Ng et al., 2007; Ma et al., 2009; Ma et al., 2011). These include complement cascade molecules (Ricklin et al., 2013; Genster et al., 2014), ficolins (Fujita et al., 2002), collectins (Holmskov et al., 2003) and pentraxins (Lu et al., 2012; Bottazzi et al., 2016; Pepys et al., 2003).
Pentraxins consists of an ancient group of proteins evolutionarily conserved from arachnids and insects to humans characterized by the presence of a 200 amino acid (aa) pentraxin domain in their carboxyl-terminal and a pentraxin signature (HxCxS/TWxS, x=any aa) (Pepys et al., 2003; Garlanda et al., 2005; Mantovani et al., 2008; Szalai et al., 1999; Du Clos et al., 2011). Human C Reactive protein (CRP, also called PTX1) and SAP (PTX2) constitute the short pentraxin arm of the superfamily Human CRP and SAP share gene localization and organization, protein structure and protein sequence identity (51% of aa identity). Human and murine SAP diverge in protein sequence (66% of aa identity) and regulation (Lu et al., 2008; Emsley et al., 1994). CRP and SAP are acute phase response proteins produced in the liver in response to infections and inflammatory cytokines, respectively in human and mouse (Casas et al., 2008; Pepys et al., 1979). Extra hepatic sources of short pentraxins have been described but without contributing to blood levels (Pepys et al., 2003).
PTX3 differs from the classical short pentraxins on the basis of gene localization and regulation, protein structure, and cellular sources (Bottazzi et al., 2016). PTX3 is highly conserved in human and mouse (92% of aa residue identity) and is similarly induced in immune cells (e.g. dendritic cells, macrophages) and stromal cells in response to local proinflammatory signals and pathogens (Bottazzi et al., 2016; Garlanda et al., 2005). PTX3 is stored in neutrophil granules and promptly released upon their activation (Jaillon et al., 2007). Studies in gene-targeted mice and in humans proved an essential role of PTX3 in innate immune responses against certain pathogens (Garlanda et al., 2002; Jaillon et al., 2014; Jeannin et al., 2005; Wojtowicz et al., 2015; Olesen et al., 2007; Magrini et al., 2016). In particular, mechanisms underlying the PTX3-mediated resistance to A. fumigatus were extensively investigated (Garlanda et al., 2002; Moalli et al., 2010). An association between genetic variants of PTX3 and occurrence of invasive aspergillosis after allogeneic hematopoietic stem-cell transplantation in humans is consolidated (Cunha et al., 2014; Cunha et al., 2015; Fisher et al., 2017; Lionakis et al., 2018).
CRP was the first pentraxin identified as a prototypic PRM in the 1940 and subsequently described to bind various microorganisms including fungi, yeasts, bacteria and parasites (Szalai et al., 2002). In vitro studies also indicate a specific interaction of SAP with a wide range of microorganisms, including Gram-positive (An et al., 2013; Yuste et al., 2007) and Gram-negative (Noursadeghi et al., 2000) bacteria and influenza virus (Andersen et al., 1997), through recognition of moieties such as phosphorylcholine (PC) (Schwalbe et al., 1992), teichoic acid (An et al., 2013) and terminal mannose or galactose glycan residues (Hind at al., 1985). CRP and SAP also interact with complement components to boost innate response to pathogens (Du Clos et al., 2011; Ma et al., 2017; Doni et al., 2012). However, because of considerable divergence in regulation between mouse and man (Pepys et al., 2003), studies on the physiological relevance of CRP and SAP are not conclusive. Indeed, SAP is constitutively found in human blood, but it does not increase upon inflammatory stimuli (Szalai et al., 1999), whereas it is the main acute-phase reactant in mice (Pepys et al., 1979). CRP is instead a major acute phase protein only in humans (Pepys et al., 2003). Thus, observations related to functions of the short pentraxins in mice are more difficult to be extrapolated (Pepys et al., 2006; Tennent et al., 2008).
The recombinant of endogenous human SAP (PRM-151) has been proposed as a novel anti-fibrotic immunomodulator in patients with Idiopathic Pulmonary Fibrosis (IPF) in placebo-controlled Phase 2 study trial (van den Blink et al., 2016)
A discrepancy between in vitro and in vivo results exists on the role of SAP in innate immunity. SAP prevented in vitro cell infection by influenza A virus (Andersen et al., 1997), and intracellular growth of mycobacteria (Singh et al., 2006) and malaria parasites (Balmer et al., 2000), thus suggesting a protective role in influenza, tuberculosis and malaria. However, in vivo relevance of SAP in influenza A infection is controversial (Herbert et al., 2002; Job et al., 2013), nor SAP effect on pulmonary innate immunity against tuberculosis or malaria is reported. SAP acted as opsonin for Streptococcus pneumonia and improved complement deposition on bacteria thus promoting phagocytosis (Yuste et al., 2007). SAP also enhanced in vitro phagocytosis of zymosan (Mold et al., 2001; Bharadwaj et al., 2001) and Staphylococcus aureus (An et al., 2013) by neutrophils and macrophages through FcγR-dependent but complement-independent mechanisms. On the other hand, SAP was not opsonic for Listeria monocytogenes though it enhanced macrophage listericidal activity (Singh et al., 1986). SAP interaction with certain microbes even resulted in anti-opsonic activity or in aiding virulence of these pathogens. SAP inhibited immune recognition of Mycobacterium tuberculosis by macrophages (Kaur et al., 2004). Interaction of SAP with S. pyogenes, Neisseria meningitides and some variants of Escherichia coli led to decreased phagocytosis and killing by macrophages and inhibition of complement activation (Noursadeghi et al., 2000), and SAP-deficient mice showed higher survival in experimental infections with S. pyogenes and E. coli (Noursadeghi et al., 2000). SAP was found in autopsy tissues of patients affected by invasive gastrointestinal candidiasis associated with fungus (Gilchrist et al., 2012). Classical and lectin pathways are both main initiators of complement activation against A. fumigatus (Rosbjerg et al., 2016). Heterocomplex of mannose-binding lectin (MBL) and SAP triggers cross-activation of complement on Candida albicans (Ma et al., 2011). SAP binds to lipopolysaccharide (LPS) but does not regulate inflammation in experimental endotoxemia (Noursadeghi et al., 2000; de Haas et al., 2000). Recent indirect evidence suggest an interaction of SAP with filamentous forms of invading fungi (Garcia-Sherman et al., 2015). SAP was indeed found in autopsy tissues of patients affected by invasive gastrointestinal candidiasis (Gilchrist et al., 2012) and aspergillosis, mucormycosis, and coccidioidomycosis (Klotz et al., 2016). Moreover, SAP administration inhibited the FcγR-mediated alternative macrophage activation dampening the allergic airway disease induced by A. fumigatus, in which an airway hyper reactivity and a TH2 cytokine profile contribute to alternative activation of macrophages that exhibit impaired clearance of fungi (Moreira et al., 2010). SAP was also suggested as ligand for DC-SIGN (CD209; mouse SIGN-R1) on neutrophils and macrophages in the context of fibrosis (Cox et al., 2015).
To our knowledge, no relevance of SAP in antifungal innate immune response is reported.
We have surprisingly found that SAP is involved in the innate immune response against Aspergillus fungi and that classical complement activation is required for the initiation of SAP-mediated phagocytosis of these fungi. By interacting with Aspergillus, SAP triggers complement-mediated inflammatory and innate responses essential for pathogen clearance. Use of SAP can trigger a complement-mediated fluid-phase innate immune response aimed at a microbicidal effect inducing assembly of the terminal membrane lytic complex on fungal surface via the classical complement activation pathway, and hence the basis of a novel therapeutic use of SAP, particularly in therapy-induced immunocompromised patients.
Accordingly, under a first aspect of this invention there is provided a SAP polypeptide or a functional fragment of such SAP polypeptide for use in the treatment of a Eurotiomycetes fungus infection.
In one embodiment, the Eurotiomycetes fungus is an Eurotiales fungus.
In a particular embodiment, the Eurotiales fungus is a Trichocomaceae fungus.
In a more particular embodiment, the Trichocomaceae fungus is infection is aspergillosis.
As used herein, the term “aspergillosis” excludes the merely inflammatory manifestations of aspergillosis like allergic bronchopulmonary aspergillosis and severe asthma sensitized to Aspergillus and includes all life-threatening generalised infections caused by Aspergillus in subjects with compromised immune systems: aspergilloma and chronic pulmonary aspergillosis in subjects previously affected by tuberculosis or sarcoidosis; aspergillus bronchitis in subjected affected by bronchiectasis or by cystic fibrosis; aspergillus sinusitis; and all of these diseases that evolve to invasive aspergillosis in subjects with low immune defenses such as in bone marrow transplant, chemotherapy for cancer treatment, AIDS, major burns, and in chronic granulomatous disease.
In a particular embodiment, the aspergillosis is an invasive aspergillosis.
In a further embodiment, the aspergillosis or invasive aspergillosis is due to an Aspergillus Fumigatus infection.
In a further embodiment, the aspergillosis or invasive aspergillosis is due to an Aspergillus Flavus infection.
As used herein “The percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The alignment in order to determine the percent of amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign software (DNASTAR). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
As used herein, a “functional fragment” of a SAP polypeptide is a portion of the SAP polypeptide that retains at least 70% native SAP activity in an assay suitable to test for its pharmacological activity, in particular a test useful for determining its activity in the treatment of a Eurotiomycetes fungus infection. In one embodiment, the SAP polypeptide functional fragment retains at least a percentage of native SAP activity selected from the list of 75%, 80%, 85%, 90% and 95%.
As used herein the term “SAP polypeptide” encompasses all functional forms, derivatives and variants of SEQ ID NO: 1, i.e. not limitedly:
In another embodiment, the SAP polypeptide comprises an amino acid sequence that is at least identical to SEQ ID NO:1 in a percentage selected from the list of 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%
In another embodiment, the SAP polypeptide comprises an amino acid sequence that is identical to SEQ ID NO:1.
We have also determined that SAP and PTX3 share ability to interact with similar microorganisms and to act as opsonins via FcγR and that deficiency of both SAP and PTX3 entails a further increase of susceptibility to Aspergillus infection compared to that observed in mice with single deficiency. Therefore, our results indicate an additive role of SAP and PTX3 in the antifungal response at crossroad between complement and FcγR-mediated recognition (Lu et al., 2008; Moalli et al., 2010).
Accordingly, in a second aspect of this invention, there is provided the combination of a SAP polypeptide or a functional fragment of such SAP polypeptide with a PTX3 polypeptide or a functional fragment of such PTX3 polypeptide for use in the treatment of a Eurotiomycetes fungus infection.
In one embodiment, the Eurotiomycetes fungus is an Eurotiales fungus.
In a particular embodiment, the Eurotiales fungus is a Trichocomaceae fungus.
In a more particular embodiment, the Trichocomaceae fungus is infection is aspergillosis.
In a particular embodiment, the aspergillosis is an invasive aspergillosis.
In a further embodiment, the aspergillosis or invasive aspergillosis is due to an Aspergillus Fumigatus infection.
In a further embodiment, the aspergillosis or invasive aspergillosis is due to an Aspergillus Flavus infection.
As used herein, a “functional fragment” of a PTX3 polypeptide is a portion of the PTX3 polypeptide that retains at least 70% native PTX3 activity, in an assay suitable to test for its pharmacological activity in combination with a SAP polypeptide or functional fragment of such SAP polypeptide, in particular a test useful for determining its activity in the treatment of a Eurotiomycetes fungus infection when used in combination with a SAP polypeptide or a functional fragment of such SAP polypeptide. In one embodiment, the PTX3 polypeptide functional fragment retains at least a percentage of native PTX3 activity selected from the list of 75%, 80%, 85%, 90% and 95%.
As used herein the term “PTX3 polypeptide” encompasses all functional forms, derivatives and variants of SEQ ID NO: 2, i.e. not limitedly:
In another embodiment, the SAP polypeptide comprises an amino acid sequence that is at least identical to SEQ ID NO:1 in a percentage selected from the list of 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.
In another embodiment, the SAP polypeptide comprises an amino acid sequence that is identical to SEQ ID NO:1.
In another embodiment, the PTX3 polypeptide comprises an amino acid sequence that is at least identical to SEQ ID NO:2 in a percentage selected from the list of 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%.
In another embodiment, the PTX3 polypeptide comprises an amino acid sequence that is identical to SEQ ID NO:2.
All embodiments may be combined.
Formulation
The polypeptides of the invention may be administered in the form of tablets or lozenges formulated in a conventional manner. For example, tablets and capsules for oral administration may contain conventional excipients including, but not limited to, binding agents, fillers, lubricants, disintegrants and wetting agents. Tablets may be coated according to methods well known in the art.
The polypeptides of the invention may also be administered as liquid formulations including, but not limited to, aqueous or oily suspensions, solutions, emulsions, syrups, and elixirs. The y may also be formulated as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may contain additives including, but not limited to, suspending agents, emulsifying agents, nonaqueous vehicles and preservatives.
The polypeptides of the invention may also be formulated as suppositories, which may contain suppository bases including, but not limited to, cocoa butter or glycerides.
The polypeptides of the invention may also be formulated for inhalation, which may be in a form including, but not limited to, a solution, suspension, or emulsion that may be administered as a dry powder or in the form of an aerosol using a propellant, such as dichlorodifluoromethane or trichlorofluoromethane.
The polypeptides of the invention may also be formulated as transdermal formulations comprising aqueous or nonaqueous vehicles including, but not limited to, creams, ointments, lotions, pastes, medicated plaster, patch, or membrane.
The polypeptides of the invention may also be formulated for parenteral administration including, but not limited to, by injection or continuous infusion. Formulations for injection may be in the form of suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulation agents including, but not limited to, suspending, stabilizing, and dispersing agents.
Administration
Administration of the compositions using the method described herein may be orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, or combinations thereof. Parenteral administration includes, but is not limited to, intravenous, intraarterial, intraperitoneal, subcutaneous, intramuscular, intrathecal, and intraarticular.
Dosage
The therapeutically effective amount required for use in therapy varies with the nature of the condition being treated, and the age/condition of the patient. The desired dose may be conveniently administered in a single dose, or as multiple doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. Multiple doses may be desired, or required.
The invention is now described by means of non-limiting examples.
Apcs−/− mice showed lethal infection with a median survival time (MST) of 3 days compared to MST>10 of wt, both when 1×108 (
SAP regulates innate immune cell activities (Cox et al., 2014; Cox et al., 2015), thus affecting inflammatory reactions. In an effort to investigate whether a different inflammatory response was the bases of the phenotype associated to SAP-deficiency, cytokines were measured in the bronchoalveolar fluids (BALFs) in infected mice (
The pre-opsonisation of A. fumigatus conidia with recombinant murine SAP (1.109/50 μg; 5×107 i.t. injected per mouse) rescued the susceptibility of Apcs−/− mice to infection, without affecting the resistance of wt mice (
In experiments conducted in whole blood, phagocytosis by neutrophils was reduced in Apcs−/− mice, both at 1 (47.0±4.0 vs. 41.3±2.4%; P=0.05) and 20 min (55.7±3.1 vs. 44.4±2.3%; P=0.01) after incubation with A. fumigatus conidia (
Classical and lectin pathways are both main initiators of complement activation against A. fumigatus (Rosbjerg et al., 2016). Heterocomplex of mannose-binding lectin (MBL) and SAP triggers cross-activation of complement on Candida albicans (Ma et al., 2011). Experiments of opsonophagoytosis were therefore performed in human sera deficient for different complement components to define the molecular mechanism responsible for SAP resistance to A. fumigatus. Phagocytosis of blood-derived human neutrophil was abolished in serum depleted for C3 (−72.0±4.7%, P<0.0001), C1q (−85.3±0.7%, P<0.0001) and MBL (−91.7±1.9%, P<0.0001) compared to normal, thus indicating importance of both classic and lectin pathways in resistance against this fungus (Rosbjerg et al., 2016). In two independent experiments using 5 and 10% of sera, opsonisation of human native SAP potentiated phagocytosis in normal (5%, 37.4±10.3%, P=0.009; 10%, 18.3±2.0%, P=0.01) and MBL-depleted serum (5%, 55.5±17.0%; 10%, 19.8±6.1%, P=0.04), but not in those depleted for C3 and C1q (
SAP was suggested as ligand for DC-SIGN (CD209; mouse SIGN-R1) on neutrophils and macrophages in the context of fibrosis (Cox et al., 2015). Genetic variation in DC-SIGN affects susceptibility to invasive aspergillosis (Fisher et al., 2017; Sainz et al., 2012). Therefore, we assessed the actual involvement of DC-SIGN in SAP-mediated A. fumigatus phagocytosis. SAP effect was similar in a monocytic cell line stably transfected for surface expression of DC-SIGN and in control cells (
Possible functional redundancy between pentraxins of systemic and local production was evaluated in mice with single or double deficiency for SAP and PTX3. As expected, an increased susceptibility to infection was observed both in Apcs−/− ( 8/9 non-survived mice at MST of 3 days; P=0.02) and Ptx3−/− ( 4/7, at MST of 3 days) mice compared to wt ( 2/9, on day 3) after injection with 1×108 of conidia (
PTX3 represents a specific marker of invasive aspergillosis (Kabbani et al., 2017; Cunha et al., 2014). In a cohort of 26 patients having A. fumigatus colonization or invasive aspergillosis median of circulating SAP (median, 15.36 μg/ml; IQR, 9.93-20.94) was not significant different (P=0.25, Mann-Whitney) than in 6 healthy control subjects (median, 10.97 μg/ml; IQR, 6.11-16.53) and no correlation with the circulating levels of PTX3 was observed (R=0.01) (
The most important risk factor for invasive aspergillosis is represented by neutropenia and monocytopenia that occur in immune-compromised patients (Cunha et al., 2014). A potential therapeutic effect of SAP was therefore determined in transiently myelosuppressed mice. Dosage of A. fumigatus conidia was newly optimized in mice after 2-day treatment with cyclophosphamide (150 mg/Kg). Human native SAP (4 mg/Kg) was intraperitoneally (i.p.) injected at 2 and 24 h after infection, a dose selected on the bases of SAP circulating levels upon exposure A. fumigatus (range of 19.2-31.7 μg/ml) Immune-compromised mice did not survive to infection with 5×107 ( 17/17; MST 4 days) (
Results prompted us to evaluate a cell-independent SAP effect on fungal killing. In a resazurin-based cell viability assay, pre-opsonisation of human SAP resulted in increase of microbicidal activity observed in plasma of wt (P=0.004) and Apcs−/− (P=0.02) mice (1 min). POC was used as antifungal control (
Heterocomplex of mannose-binding lectin (MBL) and SAP triggers cross-activation of complement on Candida albicans in vitro (Ma et al., 2011). Further experiments were therefore conducted to effectively evaluate in vivo relevance of SAP as an antifungal molecule also in Candida infections. As also reported (Ma et al., 2011), recombinant murine SAP (Sap; 10 μg/ml) bound with low affinity blastospores, yeasts and hyphae of Candida albicans. Sap binding was however competed by human SAP (50 μg/ml) (
Materials & Methods
Animals. Wild-type C57BL/6J mice between 8 and 10 weeks of age were purchased from Charles River Laboratories (Calco, Como, Italy). Apcs−/− mice were kindly provided by Professor Marina Botto (Imperial College, London, UK). Ptx3−/− mice were generated as described26. Apcs−/− Ptx3−/− mice were generated by crossing mice with single deficiency. All mice were used on a C57BL/6J genetic background. Mice were housed and bred in the SPF animal facility of Humanitas Clinical and Research Center in individually ventilated cages. Procedures involving animals and their care were conformed to protocols approved by the Clinical and Research Institute Humanitas (Rozzano, Milan, Italy) in compliance with national (4D.L. N. 116, G. U., suppl. 40, Feb. 18, 1992) and international law and policies (EEC Council Directive 2010/63/EU, O J L 276/33, 22-09-2010; National Institutes of Health Guide for the Care and Use of Laboratory Animals, U.S. National Research Council, 2011). The study was approved by the Italian Ministry of Health (approval n. 71/2012-B, issued on the Sep. 3, 2012 and 44/2015-PR issued 28 Jan. 2015). All efforts were made to minimize the number of animals used and their suffering.
Invasive pulmonary aspergillosis. Haematological samples from patients affected by fungal infection caused by Aspergillus spp. were provided by Prof. van de Veerdonk (Radboud University Medical Center, Nijmegen, The Netherlands). For patient studies, drawing of blood samples from patients was approved by the local ethical board at the Radboud University Nijmegen (Arnhem-Nijmegen Medical Ethical Committee).
A clinical strain of A. fumigatus was isolated from a patient with a fatal case of pulmonary aspergillosis was kindly provided by Dr. Giovanni Salvatori (Sigma-tau, Rome, Italy) (Pepys et al., 2012). Aspergillus flavus (#ATCC® 9643™) was obtained from ATCC.
The growth and culture conditions of A. fumigatus and A. Flavus conidia were as described (Garlanda et al., 2002). For intratracheal (i.t.) injection, mice were anesthetized by i.p. injection of ketamine (100 mg/Kg; i.p.) and xylazine (10 mg/Kg i.p.). After surgical exposure, a volume of 50 μl PBS2+, pH 7.4, containing 1×108 or 5×107 resting conidia (>95% viable, as determined by serial dilution and plating of the inoculum on Sabouraud dextrose agar) were delivered into trachea under direct vision using a catheter connected to the outlet of a micro-syringe (Terumo, Belgium). Survival to infection was daily monitored for 10d later. Dying mice were euthanized after evaluation of the following clinical parameters: body temperature dropping, intermittent respiration, solitude presence, sphere posture, fur erection, non-responsive alertness, and inability to ascent when induced.
In experiments of in vivo phagocytosis, mice were i.t. injected with 5×107 heat inactivated fluorescein isothiocyanate (FITC)-labelled conidia and euthanized 4 h later. In rescue experiments, conidia (1×109) were opsonized with murine recombinant SAP (50 μg/ml; R&D Systems) for 1 h at r.t. in PBS, pH 7.4, containing 0.01% (vol/vol) Tween-20® (Merck-Millipore). After washing of unbound protein, a volume of 50 μl (5×107 conidia) was i.t. injected.
In therapy experiments, immunosuppression was induced by i.p. injection of 150 mg/Kg cyclophosphamide (150μl per mouse of 20 mg/ml solution) 2d before infection. Native human SAP (Merck-Millipore) was dialysed in PBS (pH 7.4) in order to eliminate sodium azide and i.p. injected at the dose of 4 mg/Kg at 2 and 24 h after infection or in combination with Posaconazole (POS; 1.6 mg/Kg) at 16 h and 40 h after infection.
Disseminated candidiasis. Candida albicans was provided by Professor Marina Vai (Biotechnology and Biosciences Department, Università degli Studi di Milano-Bicocca) and routinely grown at 25° C. in rich medium [YEPD (yeast extract, peptone, dextrose), 1% (w/v) yeast extract, 2% (w/v) Bacto Peptone, and 2% (w/v) glucose] supplemented with uridine (50 mg/liter) as described (Santus et al., 2017). For survival experiments, a colony of C. albicans was collected by a culture plate and grown under rotation for 24 h at 37° C. in YEPD medium, and, once centrifuged (1000 rpm for 5 min), cells were injected into the retro-orbital plexus at 1.105/200 μl PBS. Survival of mice was monitored for two weeks.
Tissue injury. Skin wounding and chemical-induced liver injury was performed as previously described (Doni et al., 2015).
BALFs collection and analysis. BALFs were performed with 1.5 ml PBS, pH 7.4, containing protease inhibitors (Complete tablets, Roche Diagnostic; PMSF, Sigma-Aldrich) and 10 mM EDTA (Sigma-Aldrich) with a 22-gauge venous catheter. BALFs were centrifuged, and supernatants were collected for quantification of total protein content with Bradford's assay (Bio-RAD) and cytokines as described below. After erythrocyte lysis with ACK solution (pH 7.2; NH4Cl 0.15 M, KHCO3 10 mM, EDTA 0.1 mM), cells were resuspended in PBS, pH 7.4, containing 10 mM EDTA and 1% heat-inactivated fetal bovine serum (FCS; Sigma-Aldrich), stained with live and death dye (ThermoFisher Scientific-Molecular Probes) and analysed by BD FACS Canto™ II Flow Cytometer (BD Biosciences) with the following specific antibodies: peridinin chlorophyll protein complex (PerCP)—or brilliant violet (BV) 650-labelled anti-CD45 (#30-F11, IgG2b; 4 μg/ml); FITC- or phycoerythrin (PE)-CF594-labelled anti-Ly6G (#1A8, IgG2a; 4 μg/ml); allophycocyanin (APC)—or BV421-labelled anti-CD11b (#M 1/70, RUO, IgG2b; 1 μg/ml) (all from BD Biosciences).
Lung homogenates and analysis. Lungs were removed 16 h after infection and homogenized in 1 ml PBS, pH 7.4, containing 0.01% (vol/vol) Tween-20® (Merck-Millipore) and protease inhibitors. Samples were serially diluted 1:10 in PBS and plated on Sabouraud dextrose agar for blinded CFU counting. For lysate preparation, lungs were collected at 4 h and homogenized in 50 mM Tris-HCl, pH 7.5, containing 2 mM EGTA, 1 mM PMSF, 1% Triton X-100 (all from Sigma-Aldrich), and complete protease inhibitor cocktail. Total proteins were measured by DC Protein Assay, according to manufacturer's instructions (Bio-Rad Laboratories). Western blot analysis for C3 was performed after loading 10 μg of lung protein extracts on SDS-PAGE. The goat polyclonal anti-C3 (1:3000; Merck-Millipore) and HRP-conjugated donkey anti-goat IgG (1:5000; R&D Systems) were used. The monoclonal anti-vinculin (0.5 μg/ml; hVIN-1; Sigma-Aldrich) was used as loading control. C3d bands were quantified by Fiji-ImageJ (NIH, Bethesda USA) as a ratio of mean grey intensity values of each protein relative to vinculin bands.
Cells and in vitro phagocytic activity. Phagocytosis assay in whole blood of A. fumigatus conidia by mouse and human neutrophils was performed as described (Moalli et al., 2010). Briefly, conidia (1×109) were labelled (1 h, r.t.) with FITC (Sigma-Aldrich; 5 mg/ml in DMSO), and eventually opsonized (1 h, r.t) with murine SAP (100 μg/ml; 1.1 μM) and PTX3 (50 μg/ml; 1.1 μM). An amount of 1×107 FITC-conidia were added to 200 μl of mouse whole blood (collected with heparin) and incubated for 1, 5, 10, 20 or 30 min at 37° C. in an orbital shaker. Samples were immediately placed on ice and ACK lysis solution was added to lyse erythrocytes. Murine neutrophils were analysed by BD FACS Canto™ II Flow Cytometer (BD Biosciences) as previously described, and frequency and/or mean fluorescence intensity (MFI) of FITC neutrophils and CD11b expression were reported.
Human neutrophils were isolated from fresh whole blood of healthy volunteers through separation from erythrocytes by 3% dextran (GE Healthcare Life Sciences) density gradient sedimentation followed by Ficoll-Paque PLUS (GE Healthcare Life Sciences) and 62% Percoll® (Sigma-Aldrich) centrifugation as previously described (Moalli et al., 2010). Purity, determined by FACS analysis on forward scatter/side scatter parameters, was routinely >98%. 1×105 neutrophils were incubated for 1 and 30 min at 37° C. in 50 μl RPMI-1640 medium with 5 and 10% normal human serum (NS) or complement depleted sera and 1×105 FITC-labelled A. fumigatus conidia. Cells were transferred on ice and, after washing with PBS, pH 7.4, containing 10 mM EDTA and 0.2% bovine serum albumin (BSA; Sigma-Aldrich), FITC fluorescence in neutrophils (CD45-positive cells, neutrophils defined as FSC-Ahigh/SSC-Ahigh) was analysed by BD FACS Canto™ II Flow Cytometer (BD Biosciences). NS and sera depleted for C3, C1q, MBL, FB and FH were all obtained from CompTech (Complement Technology, Inc., USA).
U937 cell lines [control (ATCC® CRL-1593.2™) and transduced with human DC-SIGN (ATCC® CRL-3253™)] were cultured in RPMI-1640 medium containing 5% FCS, 2 mM L-glutamine, 0.1 mM non-essential amino acids (all from Lonza-BioWhittaker™) and 0.05 mM 2-mercaptoethanol (BIO-RAD). DC-SIGN expression was ascertained by FACS using a rabbit polyclonal Ab (#ab5715, 5 μg/ml; AbCam, UK) and an Alexa Fluor® 488-conjugated goat anti rabbit (2 μg/ml; ThermoFisher-Molecular Probes). Phagocytosis of FITC-labelled A. fumigatus conidia (1×107) by U937 cells (1×105) was performed as above described above.
Complement deposition on Aspergillus fumigatus. A volume of 10 μl PBS, pH 7.4, containing 1×107 conidia eventually opsonized with murine SAP (100 μg/ml per 1×109 conidia, 1 h at r.t.) was incubated (37° C.) in round bottom wells of Corning® 96-well polypropylene microplates for the indicated time points with 20 μl mouse plasma-heparin or 20 μl human NS and complement depleted sera (diluted in PBS at 10% and 30%). Complement deposition was blocked by addition of EDTA (10 mM final concentration) and by cooling in ice. After centrifugation (2000 rpm, 10 min at 4° C.), when indicated supernatant was collected for C5a measurement by ELISA. Conidia were washed and incubated (1 h, at 4° C.) with PBS, pH 7.4, 2 mM EDTA, 1% BSA containing the following primary antibodies: goat anti-C3 and activation fragments (1:5000; Merck-Millipore); rat anti-C1q (IgG1, #7H8, 1 μg/ml; HyCult® Biotech, Netherlands); rat anti-MBL (IgG2a, #8G6, 1 μg/ml; HyCult® Biotech, Netherlands) or rabbit anti-MBL (2 μg/ml; AbCam, UK); rabbit anti-C5b-C9 (MAC) (1:2000; Complement Technology, Inc.); or correspondent irrelevant IgGs. Conidia were then incubated (1 h, at 4° C.) with Alexa Fluor® 488 and 647-conjugated species-specific cross-adsorbed detection antibodies (2 μg/ml; ThermoFisher Scientific-Molecular Probes) and analysed by BD FACS Canto™ II Flow Cytometer (BD Biosciences) using forward and side scatter parameters to gate on at least 8,000 conidia. After each step, conidia were extensively washed with PBS, pH 7.4, 2 mM EDTA, 1% BSA. Results were expressed as frequency of conidia showing fluorescence compared with irrelevant controls and as geometric conidia MFI.
Fungal viability assay. Effect of SAP on A. fumigatus killing was evaluated by a resazurin-based cell viability assay as described (Mantovani et al., 2010). A volume of 10 μl PBS, pH 7.4, containing 1.5×105 conidia eventually opsonized with human SAP (100 μg/ml per 1×109 conidia, 1 h at r.t.) was placed into sterile round bottom Corning® 96-well polypropylene microplate and incubated for 1 and 30 min at 37° C. with 20 μl of 10 and 30% plasma-heparin from wt and Apcs−/− mice or human serum and different complement component-depleted sera. After incubation, plates were immediately cooled on ice and cold-centrifuged (2000 rpm, 10 min at 4° C.), and then supernatant removed. Conidia not incubated with plasma or serum were used as a negative control. Conidia treated with the fungicide drug Posaconazole (POC; 1 μM) were considered as positive control in the assay. Preparation of AlamarBlue™ Cell Viability Reagent and test was performed according with manufacturer's instructions (ThermoFisher Scientific-Invitrogen). A volume of 100 μl AlamarBlue™ solution (10 μl of AlamarBlue™ reagent and 90 μl of Sabouraud dextrose broth) was added to each well. After 17 h reaction at 37° C., fluorescence (excitation/emission at ≈530-560/590 nm) intensity was measured by microplate reader Synergy™ H4 (BioTek, France). Results represent ratio of fluorescence intensity values relative to those measured in negative controls. The actual killing of fungi was controlled as CFU count as previously described.
Proteins. A recombinant murine SAP from mouse myeloma cell line NSO was used (R&D Systems). Native SAP from human serum was purchased by Merck-Millipore. Recombinant murine PTX3 was purified from Chinese hamster ovary cells constitutively expressing the proteins, as described previously (Moalli et al., 2010). Purity of the recombinant protein was assessed by SDS-PAGE followed by silver staining. Recombinant PTX3 contained <0.125 endotoxin units/ml as checked by the Limulus amebocyte lysate assay (BioWhittaker®, Inc.). Blood was collected with heparin from the cava vein of anaesthetised mice. SAP levels were measured in mouse plasma by ELISA (DuoSet ELISA; R&D Systems). Murine TNF-α, CCL2, MPO were measured by ELISA (DuoSet ELISA; R&D Systems). Murine C5a was measured either in plasma-heparin or in BALFs previously stored at −80° C. by DuoSet ELISA (R&D Systems) maintaining EDTA (10 mM) throughout the assay in order to stop the activation of the complement cascade.
Binding of SAP. Conidia of A. fumigatus and A. flavus (1×108 CFU) were cultured 4 and 16 h under static condition in Sabouraud medium to respectively allow conidia swelling and germination. Blastospores and yeast of Candida albicans were prepared as previously described (Santus et al., 2017). Yeast (8×106/ml) were incubated at 37° C. for hyphal induction. Formation of hyphae was evaluated under a microscope at different time points until its amount was assessed at 95%. After washing with PBS2+, pH 7.4, containing 0.01% (vol/vol) Tween-20 ® (Merck-Millipore), Cells (1×107 CFU) were incubated (1 h, r. t.) with biotin-labelled murine SAP (R&D Systems) at concentrations ranging from 0.1 to 10 μg/ml in PBS2+, pH 7.4, containing 2% BSA (Sigma-Aldrich). In competition experiments, human SAP or CRP (50 μg/ml; Merck-Millipore) or murine PTX3 (50 μg/ml) were further added. After extensive washing, samples were incubated (30 min, r. t.) with Alexa Fluor® 647-conjugated streptavidin and binding was evaluated by FACS as frequency and MFI and visualized by confocal microscopy as described. In some experiments, a rat monoclonal anti-SAP (IgG2a, #273902; 5 μg/ml; R&D Systems) was also used.
Microscopy. 5-μm cryostat sections of mouse lungs were incubated in 5% of normal donkey (Sigma-Aldrich) serum, 2% BSA (Sigma-Aldrich), 0.1% Triton X-100 (Sigma-Aldrich) in PBS2+, pH 7.4, (blocking buffer) for 1 h at room temperature. Sections were incubated with the following primary antibodies diluted in blocking buffer for 2 h at r. t.: rabbit polyclonal anti-SAP (1:500; Merck-Millipore); goat polyclonal anti-PTX3 (2 μg/ml; R&D Systems); rat monoclonal anti-C3 (C3b/iC3b/C3d) (IgG2a, #11H9; 5 μg/ml; Hycult® Biotech). Sections were then incubated for 1 h with Dylight® and Alexa Fluor® (488, 568 and 647)-conjugated species-specific cross-adsorbed detection antibodies (ThermoFisher Scientific-Molecular Probes). For DNA detection, DAPI (300 nM; ThermoFisher Scientific-Molecular Probes) was used. After each step, sections were washed with PBS2+, pH 7.4, containing 0.01% (vol/vol) Tween-20® (Merck-Millipore). Correspondent IgG isotype controls were used. Sections were mounted with the antifade medium FluorPreserve® Reagent (Merck-Millipore) and analysed in a sequential scanning mode with a Leica TCS SP8 confocal microscope at Airy Unit 1 with an oil immersion lens 63× (N.A. 1.4). Images of SAP binding to resting or germinated conidia were obtained after z-stack acquisition using same instrument parameters and image deconvolution by Huygens Professional software (Scientific Volume Imaging B.V.) and presented as medium intensity projection (MIP).
Statistic. Student's t-tests were performed after data were confirmed to fulfil the criteria of normal distribution and equal variance. Otherwise Mann-Whitney test was applied. Log-rank (Mantel-Cox) test was performed for comparison of survival curves. Ordinary one-way Anova was performed for curve multiple comparisons. Statistical significance of multivariate frequency distribution between groups was also analysed by Fisher's Exact test. Analyses were performed with GraphPad Prism 6 software.
Number | Date | Country | Kind |
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18214853.6 | Dec 2018 | EP | regional |
Filing Document | Filing Date | Country | Kind |
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PCT/EP2019/085932 | 12/18/2019 | WO | 00 |