Le Syndrome de Détresse Respiratoire Aiguë (SDRA) est la forme la plus sévère d’agression alvéolaire aiguë. Il estcaractérisé par un dommage alvéolaire diffus, suivi d’une phase de réparation nécessaire à la guérison. Bien queles monocytes/macrophages soient des acteurs incontournables de la pathogénie et de la résolution du SDRA, leurpolarisation et leur rôle dans la réparation alvéolaire restent mal connus chez l’Homme. L’hypothèse défendue parcette thèse est que l’environnement alvéolaire module la différenciation et la polarisation desmonocytes/macrophages au cours du SDRA, et que les macrophages alvéolaires ainsi polarisés participentactivement à la réparation et à sa régulation. Les principaux résultats de nos travaux ont permis d’établir que : 1)l’environnement alvéolaire de SDRA inhibe la différenciation des monocytes en fibrocytes (précurseursmésenchymateux associés à la fibroprolifération et à pronostic péjoratif). L’inhibition est majoritairement due à laSerum Amyloid protein P (SAP), provenant en partie du relargage de ses stocks matriciels pulmonaires à la faveurde la lésion alvéolaire ; 2) l’environnement alvéolaire de SDRA induit une polarisation anti-inflammatoire desmacrophages se rapprochant de la polarisation induite in vitro par l’IL-10 ; 3) les macrophages anti-inflammatoirespolarisés par le lavage broncho-alvéolaire (LBA) de patients SDRA favorisent la réparation alvéolaire épithéliale viala production polarisation-dépendante d’Hepatocyte Growth Factor (HGF). Cette production macrophagique d’HGFest amplifiée via une boucle autocrine PTGS2/PGE2 chez l’Homme ; 4) ces résultats semblent étayés par lesdonnées obtenues sur une cohorte clinique qui montrent que les concentrations de sCD163 (marqueur depolarisation anti-inflammatoire) et d’HGF rapportées au nombre de macrophages alvéolaires sont plus élevéeschez les patients survivants que chez les patients décédés de SDRA. L’ensemble de nos travaux démontrent pour lapremière fois chez l’Homme le rôle bénéfique de l’environnement alvéolaire sur les monocytes/macrophages,d’une part en inhibant leur différenciation en fibrocytes contribuant ainsi à limiter la fibroprolifération pulmonaire,et d’autre part en induisant un phénotype macrophagique anti-inflammatoire, contribuant à limiter l’inflammationengendrée par la lésion alvéolaire initiale et favorisant la réparation épithéliale via la production d’HGF. Lesdonnées physiopathologiques obtenues pourraient permettre d’envisager la reprogrammation anti-inflammatoiredes macrophages comme une cible thérapeutique du SDRA en cas d’excès d’inflammation ou de fibro-proliférationavec réparation aberrante. / Acute Respiratory Distress Syndrome (ARDS) is the most severe form of acute lung injury. ARDS is characterized bydiffuse alveolar damage followed by a phase of alveolar repair necessary to recovery. Althoughmonocytes/macrophages are key actors of pathogenicity and resolution of ARDS, little is known about theirpolarization and role on alveolar repair during human ARDS. The hypothesis of our studies was that ARDS alveolarenvironment modulates differentiation and polarization of monocytes and macrophages, and that polarizedmacrophages are involved in alveolar repair and its regulation. The main results of our work have shown that: 1)ARDS alveolar environment inhibited monocytes differentiation into fibrocytes (mesenchymal progenitorsassociated with fibroprolifération and a poor prognosis), mainly through its Serum Amyloid P (SAP) content,originating, at least in part, from the release of SAP associated with lung connective tissue during ARDS; 2) ARDSalveolar environment drove an anti-inflammatory macrophage polarization, close to that induced by IL-10 in vitro;3) anti-inflammatory macrophages polarized by broncho-alveolar lavage (BAL) from ARDS patients favored alveolarepithelial repair through a polarization-dependent production of Hepatocyte Growth Factor (HGF). This HGFproduction is amplified by an autocrine PTGS2/PGE2 dependent loop in human macrophages; 4) these results mayhave clinical relevance, since sCD163 (a marker of anti-inflammatory polarization) and HGF concentrations,expressed relatively to BAL macrophage count, were higher in ARDS survivors than non-survivors. Taken together,our works demonstrate for the first time the beneficial role of the ARDS alveolar environment onmonocytes/macrophages, inhibiting their differentiation into fibrocytes, thus limiting excessive lungfibroproliferation, and inducing an anti-inflammatory macrophage polarization, thus limiting the inflammationgenerated by the initial alveolar damage and favoring epithelial repair through HGF production. The datapresented in this thesis may allow considering anti-inflammatory macrophage repolarization as a potential newtherapeutic target of ARDS with excessive inflammation or fibro-proliferation with aberrant repair.

It is a striking observation that tissue of patients invaded by the deep mycoses often lacks evidence of an inflammatory response. This lack of host response is often attributed to neutropenia secondary to chemotherapy. However, systematic studies do not support this simplistic explanation. However, invasive fungal lesions are characterized by abundant fungal functional amyloid, which in turn is bound by serum amyloid P component (SAP). We postulate that SAP is important in the local immune response in invasive fungal infections. The interaction between fungal functional amyloid, SAP, and the immune response in deep mycoses is discussed.

Diss. (sammanfattning) Umeå : Umeå universitet, 2005. / Härtill 4 uppsatser.

BACKGROUND: Serum amyloid P component (SAP) is a glycoprotein that is universally found associated with different types of amyloid deposits. It has been suggested that it stabilizes amyloid fibrils and therefore protects them from proteolytic degradation. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we show that SAP binds not only to mature amyloid fibrils but also to early aggregates of amyloidogenic mutants of the plasma protein transthyretin (TTR). It does not inhibit fibril formation of TTR mutants, which spontaneously form amyloid in vitro at physiological pH. We found that SAP prevents cell death induced by mutant TTR, while several other molecules that are also known to decorate amyloid fibrils do not have such effect. Using a Drosophila model for TTR-associated amyloidosis, we found a new role for SAP as a protective factor in inhibition of TTR-induced toxicity. Overexpression of mutated TTR leads to a neurological phenotype with changes in wing posture. SAP-transgenic flies were crossed with mutated TTR-expressing flies and the results clearly confirmed a protective effect of SAP on TTR-induced phenotype, with an almost complete reduction in abnormal wing posture. Furthermore, we found in vivo that binding of SAP to mutated TTR counteracts the otherwise detrimental effects of aggregation of amyloidogenic TTR on retinal structure. CONCLUSIONS/SIGNIFICANCE: Together, these two approaches firmly establish the protective effect of SAP on TTR-induced cell death and degenerative phenotypes, and suggest a novel role for SAP through which the toxicity of early amyloidogenic aggregates is attenuated. / <p>Epub 2013 Feb 4.</p>

Alzheimer’s Disease is the most common form of dementia, affecting 48 million people worldwide. Despite this fact, only 45% of the patients have received the diagnose. The reason behind this is the fact that the cause of the disease is still unclear. Several hypotheses have been suggested, with main focus in the imbalance between the production and the clearance of Αβ in the brain (formation of plaques) or hyperphosphorylation of the tau protein (formation of tangles). In order to have a better understanding of what is actually happening in the brain, more biomarkers need to be developed. Keeping this in mind, we tried to develop a method to monitor the protein levels of SAP in the brain. SAP is a glycoprotein, normally produced by the liver in acute phase immune responses. SAP has been correlated with AD in the 1980s and quite recently it has been shown that SAP is elevated in AD patients, but not in individuals with plaques and no dementia. For this reason, we developed a mass spectrometry based targeted quantification method for monitoring SAP in the brain, as well as C9, a blood contamination reference protein. Our method is robust enough to be further used in large studies, in order to investigate the role of SAP in AD.

C-reactive protein (CRP) is an evolutionarily conserved protein. From arthropods to humans, CRP has been found in every organism where the presence of CRP has been sought. Human CRP is a pentamer made up of five identical subunits which binds to phosphocholine (PCh) in a Ca2+-dependent manner. In various species, we define a protein as CRP if it has any two of the following three characteristics: First, it is a cyclic oligomer of almost identical subunits of molecular weight 20–30 kDa. Second, it binds to PCh in a Ca2+-dependent manner. Third, it exhibits immunological cross-reactivity with human CRP. In the arthropod horseshoe crab, CRP is a constitutively expressed protein, while in humans, CRP is an acute phase plasma protein and a component of the acute phase response. As the nature of CRP gene expression evolved from a constitutively expressed protein in arthropods to an acute phase protein in humans, the definition of CRP became distinctive. In humans, CRP can be distinguished from other homologous proteins such as serum amyloid P, but this is not the case for most other vertebrates and invertebrates. Literature indicates that the binding ability of CRP to PCh is less relevant than its binding to other ligands. Human CRP displays structure-based ligand-binding specificities, but it is not known if that is true for invertebrate CRP. During evolution, changes in the intrachain disulfide and interchain disulfide bonds and changes in the glycosylation status of CRP may be responsible for different structure-function relationships of CRP in various species. More studies of invertebrate CRP are needed to understand the reasons behind such evolution of CRP. Also, CRP evolved as a component of and along with the development of the immune system. It is important to understand the biology of ancient CRP molecules because the knowledge could be useful for immunodeficient individuals.

Amyloidoses comprise a group of gain-of-toxic function protein misfolding diseases, in which normally soluble proteins in their functional state undergo conformational changes into highly organized and generally intractable thread-like aggregates, termed amyloid fibrils. These structures accumulate predominantly in the extracellular space but growing evidence suggests that amyloids may start to form intracellularly. At least 26 different human proteins, intact or in fragmented form, are known to form amyloid, which is linked with many debilitating neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt-Jakob disease, and transthyretin (TTR)-related amyloidosis (ATTR). In this work, we focus on ATTR, which is one of the most frequent systemic amyloid diseases. A functional link was established between hereditary ATTR, a severe and fatal disorder and the enhanced propensity of the human plasma protein transthyretin (TTR) to form aggregates, caused by single point mutations in the TTR gene. The disease is heterogeneous and clinical symptoms vary from cardiomyopathy to progressing sensorimotor polyneuropathy depending on TTR variant involved and the amyloid deposition site. Despite the fact that TTR-derived amyloid accumulates in different organs such as heart, kidney, eyes, and predominantly in the peripheral nerves of ATTR patients, the exact mechanism of the disease development is not understood. In contrast to the case of AD, it has been difficult to generate an animal model for ATTR in transgenic mice that would be useful in understanding TTR aggregation processes and the mechanisms of the associated toxicity as these mice did not develop any neuropathic phenotype besides amyloid deposits. Therefore, we created a disease-model in Drosophila due to its huge repertoire of genetic techniques and easy genotype – phenotype translation, as well as its success in modeling human neurodegeneration. We have generated transgenic flies that over-express the clinical amyloidogenic variant TTRL55P, the engineered variant TTR-A (TTRV14N ⁄ V16E), and the wild-type protein. All TTR variants were found in the secreted form in the hemolymph where misfolding occurred and depending on the pool of toxic species, the fate of the fly was decided. Within a few weeks, both mutants (but not the wild-type TTR) demonstrated a time-dependent aggregation of misfolded molecules in vivo. This was associated with neurodegeneration, change in wing posture, attenuation of locomotor activity including compromised flying ability, and shortened life span. In contrast, expression of the wild-type TTR had no discernible effect on either longevity or fly behavior. In this work, we also addressed the correlation between TTR transgene dosage and thus, protein levels, with the severity of the phenotypes observed in TTR-A flies which developed a “dragged wing” phenotype. Remarkably, we established that degenerative changes such as damage to the retina strictly correlated with increased levels of mutated TTR but inversely with behavioral alterations and the dragged wing phenotype. We characterized formation of aggregates in the form of 20 nm spherules and amyloid filaments intracellularly in the thoracic adipose tissue and brain glia (both tissues that do not express the transgene). Moreover, we detected a fraction of neurotoxic TTR-A in the hemolymph of young but not old flies. We proposed that these animals counteract formation and persistence of toxic TTR-A species by removal from the circulation into intracellular compartments of glial and fat body cells and this is part of a mechanism that neutralizes the toxic effects of TTR. We validated the fly model for ATTR by applying a genetic screen during study of modifier genes. We found Serum amyloid P component (a product of the APCS gene) as a potent modifier of TTR amyloid-induced toxicity that was effective in preventing the apoptotic response in cell culture assay and capable of reducing the dragged wings when co-expressed in TTR-A flies. Finally, we optimized this fly model in order to screen for therapeutic compounds effective against ATTR. Feeding assays showed the effectiveness of several compounds among known native-state kinetic stabilizers of TTR against its aggregation. We described several early endpoints in this model, which can be used as a rapid and cost-effective method for optimizing concentrations and pre-screening of drug candidates. As the proof of principle, by feeding flies with increasing doses of diflunisal analogue (an FDA-approved Non-Steroidal Anti-Inflammatory Drug) a dose-dependent reduction of the dragged wings was observed.