Clarisse Vandebrouck

Thierry Ferreira
email: thierry.ferreira@univ-poitiers.fr

Glued in lipids: lipointoxication in Cystic Fibrosis

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CF transmembrane regulator (CFTR) gene. CFTR is located at the apical surface of epithelial cells and CFTR dysfunction leads to altered chloride transport. Improper chloride transport results in mucus dehydration, which becomes thick and sticky, causing many of the symptoms associated with CF. More than 2000 mutations grouped into 5 to 7 classes have been described to date depending on their impacts on the CFTR protein (1): Class I mutations directly impact protein production and mainly correspond to nonsense mutations; Class II mutations, which include the most prominent mutation F508del-CFTR, result in protein misfolding and affect the trafficking of the CFTR protein which is retained at the endoplasmic reticulum (ER) level by the ER quality control; Class III mutations are associated with altered gating of the CFTR channel; Class IV mutations result in a decrease in CFTR channel conductance of chloride and bicarbonate ions; Class V mutations lead to a strong decrease in the levels of the CFTR protein, mostly related to alternative splicing; Class VI mutations reduce CFTR stability at the apical surface; Class VII mutations, such as large gene deletions, are considered as unrescuable mutations, since they cannot be pharmacologically rescued per se. Even if apparently difficult to directly connect to CFTR dysfunction, at least at first glance, disturbances in lipid metabolism have been a persistent observation in CF, and this since more than 50 years, long before the identification of the CFTR gene [START_REF] Kuo | The fatty acid composition of the serum chylomicrons and adipose tissue of children with cystic fibrosis of the pancreas[END_REF][START_REF] Underwood | POLYUNSATURATED FATTY ACIDS AND TOCOPHEROL LEVELS IN PATIENTS WITH CYSTIC FIBROSIS*[END_REF]. More particularly, such disturbances are characterized by low levels of so-called Essential Fatty Acids (EFA), namely Linoleic Acid (LOE) and docosahexaenoic acid (DHA), in plasma and tissues from patients with CF (4). However, if these aspects have been of interest for many researchers until then, apparent discrepancies have emerged among studies, probably related to the analysis methods used, the kind of lipid species analyzed (if the EFA can be found in the organism as free species, they can also be esterified into complex lipids), and intrinsic variations between tissues. Moreover, due to these apparent disparities, clear connections between these lipid dysregulations and the course of the disease have been difficult to establish, raising the reasonable question of such alterations being a second-side effect or, rather, an important parameter to consider, as a potent contributor to the severity of the disease [START_REF] Al-Turkmani | Fatty acid alterations and n-3 fatty acid supplementation in cystic fibrosis[END_REF]. In this context, a first goal of the present review will be to pinpoint a general consensus from previous studies on the nature and the potential origins of the fatty acid dysregulations observed in CF, taking into consideration the lipid class and the tissue studied. In a second step, based on the literature generated with other models displaying similar lipid dysregulations, we will highlight the common impacts generated by such disorders on cell function, and their relevance to the CF phenotype. Finally, we will propose a unifying mechanism based on the effects of EFA deficiency on the properties of cellular membranes and how it could explain, at least in part, the progression of the CF disease and the limits which are reached with current treatments.

Fatty acid homeostasis: general principles

The main pathways of fatty acid homeostasis are summarized in Figure 1. Fatty acids vary one from the other on the basis of two criteria: the length of their acyl chains and the number of double-bonds, also referred to as unsaturations. The number of unsaturations is used to classify fatty acids as Saturated (Saturated Fatty Acids; SFA), Monounsaturated (Mono-Unsaturated Fatty Acids; MUFA) and Polyunsaturated (PolyUnsaturated Fatty Acids; PUFA). A Prime example of SFA is Palmitate, the main constituent of Palm oil, bearing 16 carbons in its acyl chain and no unsaturation (16:0). The most common MUFA is Oleate (18:1), which is exquisitely enriched in plant oils, such as Olive oil. If these species can be obtained from the diet, it has to be noted that mammalian cells display the machinery to build up their own SFA and MUFA. SFA are synthesized in the cytoplasm on a soluble fatty acid synthase (FAS) [START_REF] Riezman | The Long and Short of Fatty Acid Synthesis[END_REF]. SFA are then converted to their MUFA counterparts by stearoyl-CoA desaturases (SCDs), a family of ∆-9 fatty acid desaturases, which are embedded in the ER membrane, and are strictly oxygen-dependent. SCD1 appears to be the predominant isoform and is expressed ubiquitously among tissues [START_REF] Liu | Stearoyl CoA Desaturase 1: Role in Cellular Inflammation and Stress[END_REF]. SCD1-preferred substrates are Palmitate and Stearate (18:0), which are converted to Palmitoleate (16:1) and Oleate (18:1), the major MUFA of membrane phospholipids and storage-neutral lipids (Triglycerides (TG) and Steryl Esters; see below) [START_REF] Flowers | Role of stearoyl-coenzyme A desaturase in regulating lipid metabolism[END_REF]. By contrast, the body cannot synthesize PUFA "from scratch" and must therefore obtain basic blocks from the diet, which are referred to as essential fatty acids (EFA; Figure 1). There are two families of EFA, which are classified on the basis of the position of the first double-bond from the last carbon of the acyl chain (the omega (ω) carbon): ω -3 and ω-6. The ω-6 building block is linoleic acid (LOE; 18:2) and the ω -3's is α-linolenic acid (LON; 18:3). End-products are synthetized from these precursors by successive desaturation and elongation steps in the ER [START_REF] Al-Turkmani | Fatty acid alterations and n-3 fatty acid supplementation in cystic fibrosis[END_REF]. The main end-PUFA species found in Man are the ω-6 Arachidonic Acid (AA) and the ω-3 Docohexaenoic Acid (DHA). Noteworthily, both AA and DHA can also be directly obtained from the diet. For example, DHA is present in fish oils. As a consequence, cells from our body therefore dispose of a repertoire of fatty acids, which may however greatly vary depending on the organ considered (see below), originating from both exogenous (diet) and endogenous (synthesis) sources. The various fatty acids may thereafter have different fates (Figure 1). Virtually all fatty acids can enter the composition of membrane Phospholipids (PLs). Fatty acids can be esterified into PLs through two alternative pathways: the de novo pathway (or Kennedy pathway), which corresponds to the complete synthesis of a PL from elementary building blocks, and the remodeling pathway (or Lands cycle), which consists of the substitution of one acyl chain in a preformed PL [START_REF] Antonny | From zero to six double bonds: phospholipid unsaturation and organelle function[END_REF]. The acyltransferases involved in these pathways have been identified and their substrate specificity accounts for the acyl chain diversity of PLs. Systematic analysis of fatty acid distribution within the PLs of various tissues revealed that each organ displays a characteristic signature: in rats, organs can be classified as highly SFA-, MUFA-or PUFA-PL enriched [START_REF] Bacle | A comprehensive study of phospholipid fatty acid rearrangements in metabolic syndrome: correlations with organ dysfunction[END_REF]. Relevant to the present review, among all the organs studied, the lung appears as the organ displaying the lower proportion of PUFA within its PLs [START_REF] Bacle | A comprehensive study of phospholipid fatty acid rearrangements in metabolic syndrome: correlations with organ dysfunction[END_REF]. Therefore, sculpting the fatty acyl content of PLs is the result of the contribution i) of the repertoire of available fatty acids in a given cell from our body, which may compete one with the other to enter the PL synthesis/remodeling pathways, and ii) of the enzymatic machinery that is turned on in this specific cell type (e. g. specific acyltransferases). Excess fatty acids can also be stored in form of their neutral esters, Triglycerides (TG) and Steryl esters (SE), in a process that occurs in the ER [START_REF] Hapala | Is fat so bad? Modulation of endoplasmic reticulum stress by lipid droplet formation[END_REF]. However, whereas levels of TG are high in cells exposed to MUFA, SFA generally poorly accumulate within TG [START_REF] Listenberger | Triglyceride accumulation protects against fatty acid-induced lipotoxicity[END_REF]. Instead, SFA preferentially accumulate within other lipid species, including membrane PL, ceramides, or diacylglycerol (DAG), an intermediate in TG synthesis [START_REF] Listenberger | Triglyceride accumulation protects against fatty acid-induced lipotoxicity[END_REF][START_REF] Bacle | Strategies to Counter Saturated Fatty Acid (SFA)-Mediated Lipointoxication[END_REF]. The PUFA AA is also an important lipid since it is the precursor of inflammatory mediators known as eicosanoids, which include prostaglandins (PG), leukotrienes, and thromboxanes. ((5); Figure 1). Interestingly, if AA can be generated by de novo synthesis from LOE (see above), it can also be released from PLs by the selective action of Phospholipase A2 (PLA2), a family of enzymes that catalyze the hydrolysis of ester bonds at the sn-2 position of phospholipids, generating a free fatty acid and a lysophospholipid (5). To summarize, maintaining the correct balance between the various fatty acids is extraordinary complex and greatly varies depending on the available building blocks and the most operational metabolic/catabolic pathways in the cell type/tissue considered.

Constant dysregulation of fatty acid profile in CF

The first evidences describing altered fatty acid profiles in cystic fibrosis have been reported more than 50 years ago [START_REF] Kuo | The fatty acid composition of the serum chylomicrons and adipose tissue of children with cystic fibrosis of the pancreas[END_REF][START_REF] Rosenlund | Essential fatty acids in cystic fibrosis[END_REF][START_REF] Rosenlund | Dietary Essential Fatty Acids in Cystic Fibrosis[END_REF], and since then, many consecutive studies have reported similar findings. However, many discrepancies still exist between several studies. This is likely due, at least partly, to the fact that different methods for fatty acid characterization/quantification and tissue samples have been used. To establish clear comparisons, it is important to consider whether the fatty acid profile that is provided in a given study corresponds to free fatty acids (NEFA; i. e. non-esterified within complex lipids, such as TG and PLs), total fatty acids (total FA; free and esterified in various (if not all) complex lipids) or fatty acids within a given lipid species (TG or PL, for example). This is, of course, highly dependent on the methods used for lipid extraction, separation (if any) and analysis (mainly Gas Chromatography and Mass spectrometry). Moreover, if we focus on the specific case of PLs, it is well-established that the fatty acid signature greatly depends on the tissue/cell type considered, and that no extrapolation can easily be made between various tissues if the same specific determinations have not been driven in all the relevant organs with the same method (see above). Concerning cellular and animal models of CF, important parameters should be considered to avoid inappropriate extrapolations to CF patients and misinterpretations related to lipid-associated phenotypes. First, in vitro, one should keep in mind the culture conditions can greatly influence the fatty acid signature of the cells. For example, knowing that the main fatty acid desaturases are strictly oxygendependent (see above), oxygen scarcity, which is encountered in patient lungs suffering CF [START_REF] Urquhart | Assessment of hypoxia in children with cystic fibrosis[END_REF], results in an increase in SFA-enriched PLs in bronchial epithelial cells at the expense of MUFAcontaining ones [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF][START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF]. Interestingly, most experiments performed on CF bronchial epithelial cells are generally performed under normoxia, therefore leading to higher MUFA/SFA ratios than encountered in vivo. The choice of serum for culture media supplementation is also an important point to consider. A very interesting example is the case of horse serum, which displays a fatty acid profile that matches at best the one encountered in the human plasma, particularly in terms of LOE concentrations [START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF][START_REF] Andersson | Cell culture models demonstrate that CFTR dysfunction leads to defective fatty acid composition and metabolism[END_REF]. These observations suggest that great care should be taken before extrapolating the results obtained with cultured cells to in vivo situations, at least concerning the recapitulation of the fatty acid content. Concerning animal models, one should consider that the fatty acid signature in organs greatly varies among species. For example, and relevant to the present review, PUFA constitute 16 and 26 % of PC and PE in rat lungs, respectively [START_REF] Bacle | A comprehensive study of phospholipid fatty acid rearrangements in metabolic syndrome: correlations with organ dysfunction[END_REF], whereas, in human bronchial secretions, PUFA are found in very low levels in these PLs, corresponding to 0.8 and 9 % of total fatty acyl chains in PC and PE, respectively [START_REF] Gilljam | Increased mole fraction of arachidonic acid in bronchial phospholipids in patients with cystic fibrosis[END_REF]. Accordingly, the lower respiratory tract of the murine models appears to be poorly representative of the lower airways of CF individuals, therefore limiting their interest for the study of pulmonary disease progression in CF [START_REF] Lavelle | Animal Models of Cystic Fibrosis Pathology: Phenotypic Parallels and Divergences[END_REF]. Finally, in animal models, the diet greatly influences the fatty acid signature in several organs, including the lungs, therefore generating possible discrepancies in lipidomic data and/or phenotype severity among various studies [START_REF] Bacle | A comprehensive study of phospholipid fatty acid rearrangements in metabolic syndrome: correlations with organ dysfunction[END_REF][START_REF] Chu | Palmitic Acid-Rich High-Fat Diet Exacerbates Experimental Pulmonary Fibrosis by Modulating Endoplasmic Reticulum Stress[END_REF]. Concerning this latter point, the exact same caution should be taken with CF patients, who may suffer metabolic diseases with various severities (pancreatic insufficiency, CF-related diabetes (CFRD), Cystic Fibrosis-related Liver Disease (CFLD)…) with well-known impacts on the circulating fatty acid distribution, and are exposed to uncontrolled diets or various diet supplements. Moreover, chronic infection with Pseudomonas aeruginosa, which is a recurrent situation encountered in CF, has been shown to influence the lipidomic signature in plasmas from CF patients [START_REF] Ollero | Plasma lipidomics reveals potential prognostic signatures within a cohort of cystic fibrosis patients[END_REF]. Altogether, these parameters very likely influence the overall fatty acid balance, and render the identification of a homogenous definition of this balance very touchy among such an heterogenous patient population. These elements being considered, some consensus however appear to emerge within CF individuals (Table 1). First, decreased levels of the ω-3 series fatty acid DHA has been a consistent observation in various tissues/organs from CF patients. Decreased total DHA levels were observed in total FA in plasma [START_REF] Aldámiz-Echevarría | Persistence of Essential Fatty Acid Deficiency in Cystic Fibrosis Despite Nutritional Therapy[END_REF][START_REF] Farrell | Fatty Acid Abnormalities in Cystic Fibrosis[END_REF][START_REF] Guilbault | Cystic Fibrosis Fatty Acid Imbalance Is Linked to Ceramide Deficiency and Corrected by Fenretinide[END_REF][START_REF] Witters | Lung transplantation in cystic fibrosis normalizes essential fatty acid profiles[END_REF][START_REF] Witters | Lung transplantation in cystic fibrosis normalizes essential fatty acid profiles[END_REF], but also in nasal-and rectal-biopsy tissues (29) from CF patients. This decrease was also more specifically observed in the PL fractions in blood/plasma/serum [START_REF] Grothe | Plasma Phosphatidylcholine Alterations in Cystic Fibrosis Patients: Impaired Metabolism and Correlation with Lung Function and Inflammation[END_REF][START_REF] Guerrera | A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients[END_REF][START_REF] Strandvik | Essential fatty acid deficiency in relation to genotype in patients with cystic fibrosis[END_REF][START_REF] Van Biervliet | Relation between Fatty Acid Composition and Clinical Status or Genotype in Cystic Fibrosis Patients[END_REF] and cord serum samples [START_REF] Lloyd-Still | Essential fatty acid status and fluidity of plasma phospholipids in cystic fibrosis infants[END_REF]. Concerning the ω-6 series fatty acids, decreased LOE levels in the blood and tissues of CF patients have been a very consistent observation in the CF literature [START_REF] Al-Turkmani | Fatty acid alterations and n-3 fatty acid supplementation in cystic fibrosis[END_REF]. LOE decreased levels where observed in total FA extracted from plasma [START_REF] Aldámiz-Echevarría | Persistence of Essential Fatty Acid Deficiency in Cystic Fibrosis Despite Nutritional Therapy[END_REF][START_REF] Farrell | Fatty Acid Abnormalities in Cystic Fibrosis[END_REF][START_REF] Witters | Lung transplantation in cystic fibrosis normalizes essential fatty acid profiles[END_REF][START_REF] Witters | Lung transplantation in cystic fibrosis normalizes essential fatty acid profiles[END_REF], but also from Postmortem fat-, psoas muscle-, heart muscle-, liver-and lung-tissues and nasal-biopsies [START_REF] Farrell | Fatty Acid Abnormalities in Cystic Fibrosis[END_REF][START_REF] Freedman | Association of Cystic Fibrosis with Abnormalities in Fatty Acid Metabolism[END_REF]. As for DHA, this decrease was also more specifically observed in the PL fractions in blood/plasma/serum [START_REF] Farrell | Fatty Acid Abnormalities in Cystic Fibrosis[END_REF][START_REF] Van Biervliet | Relation between Fatty Acid Composition and Clinical Status or Genotype in Cystic Fibrosis Patients[END_REF][START_REF] Maqbool | Serum linoleic acid status as a clinical indicator of essential fatty acid status in children with cystic fibrosis[END_REF] and chord serum samples [START_REF] Lloyd-Still | Essential fatty acid status and fluidity of plasma phospholipids in cystic fibrosis infants[END_REF]. LOE being the precursor of AA (Figure 1), one may expect to observe either an overall decrease of this long chain PUFA, due to limitations in precursor levels, or an increase, in case of an augmented flux through the ω-6 pathway. Indeed, both observations have been made (Table 1): AA levels have been reported to be either decreased, increased or unchanged in total FA extracted in Plasma from CF individuals (24-28), unchanged in Postmortem tissues [START_REF] Farrell | Fatty Acid Abnormalities in Cystic Fibrosis[END_REF] and rectal biopsies [START_REF] Freedman | Association of Cystic Fibrosis with Abnormalities in Fatty Acid Metabolism[END_REF] and decreased in nasal biopsies [START_REF] Freedman | Association of Cystic Fibrosis with Abnormalities in Fatty Acid Metabolism[END_REF]. AA levels were either unchanged or decreased in PL fractions from blood/serum/plasma [START_REF] Farrell | Fatty Acid Abnormalities in Cystic Fibrosis[END_REF][START_REF] Grothe | Plasma Phosphatidylcholine Alterations in Cystic Fibrosis Patients: Impaired Metabolism and Correlation with Lung Function and Inflammation[END_REF][START_REF] Guerrera | A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients[END_REF][START_REF] Strandvik | Essential fatty acid deficiency in relation to genotype in patients with cystic fibrosis[END_REF][START_REF] Van Biervliet | Relation between Fatty Acid Composition and Clinical Status or Genotype in Cystic Fibrosis Patients[END_REF][START_REF] Maqbool | Serum linoleic acid status as a clinical indicator of essential fatty acid status in children with cystic fibrosis[END_REF], but were augmented in almost all PL species, except for PE, in bronchial secretions from CF patients [START_REF] Gilljam | Increased mole fraction of arachidonic acid in bronchial phospholipids in patients with cystic fibrosis[END_REF]. To conclude this part, it appears that CF is associated with a global decrease in PUFA, and particularly in DHA and LOE. However, the intensity of this decrease highly depends on the lipid species considered, and, even when considering a specific species, depending on the organ/tissue on interest (Table 1). Interestingly, parallel to this decrease in PUFA, increases in the levels of SFA, and more specifically Palmitate (16:0) and Stearate (18:0) have also been reported in CF individuals, particularly in circulating lipids, namely NEFA and Cholesteryl esters [START_REF] Rogiers | Fatty acid pattern of platelet phospholipids in cystic fibrosis[END_REF][START_REF] Rogiers | Abnormal Fatty Acid Turnover in the Phospholipids of the Red Blood Cell Membranes of Cystic Fibrosis Patients (in Vitro Study[END_REF] and in PL extracted from lung samples, i. e. bronchial secretions [START_REF] Gilljam | Increased mole fraction of arachidonic acid in bronchial phospholipids in patients with cystic fibrosis[END_REF] and freshly dissociated bronchial epithelial cells [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF][START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF]. Altogether, these cumulative processes result in an overall FA imbalance towards more saturated species.

The potential origins of these dysregulations

The main pathways which could participate in fatty acid dysregulations are displayed in Figure 2.

Malabsorption of precursors as an origin of these dysregulations

A common trait to most CF patients is exocrine pancreatic insufficiency, which results in altered fat absorption at the intestinal level and malnutrition. Such processes could account, at least in part and despite contradictory observations, in EFA deficiency [START_REF] Freedman | Association of Cystic Fibrosis with Abnormalities in Fatty Acid Metabolism[END_REF][START_REF] Coste | Influence of Pancreatic Status and Sex on Polyunsaturated Fatty Acid Profiles in Cystic Fibrosis[END_REF]. Alterations in chloride and water transport across duct cells result in a diminution in fluid flow by the gland, leading to duct obstruction [START_REF] Kopelman | Pancreatic Fluid Secretion and Protein Hyperconcentration in Cystic Fibrosis[END_REF]. On the long term, this leads to cellular damage and atrophy, resulting in a decrease in the secretion of digestive enzymes. However, even if supplementation with exogenous pancreatic enzyme displays beneficial effects, malabsorption cannot be fully circumvented in CF patients, as noted by persistent steatorrhea and malnutrition [START_REF] Carroccio | Use of famotidine in severe exocrine pancreatic insufficiency with persistent maldigestion on enzymatic replacement therapy[END_REF]. Another important determinant of EFA deficiency may be Cystic Fibrosis-associated Liver Disease (CFLD), which affects approximately 30% of patients and is now considered as the third cause of death in CF [START_REF] Kobelska-Dubiel | Liver disease in cystic fibrosis[END_REF]. The CFTR protein is embedded in the apical membrane of the cholangiocyte. At this level, it regulates the amounts of electrolytes and water in the bile by affecting the transport of sodium and chloride ions and the secretion and alkalinisation of the bile. Bile alkalinisation regulates digestive functions by playing a central role in the solubility of its organic components. Since the bile breaks down complex fats into free fatty acids, which can be taken into the body by the digestive tract, bile alterations are likely an important contributor to EFA deficiency.

Increased flux through the ω-6 pathway

Another important observation is that the metabolic conversion of LOE to ω-6 metabolites, including AA, is upregulated in CF cells, indicating increased flux through the ω-6 pathway [START_REF] Al-Turkmani | A mechanism accounting for the low cellular level of linoleic acid in cystic fibrosis and its reversal by DHA[END_REF]. This likely occurs because loss of CFTR function, related to mutation, results in the activation of AMP-activated Protein Kinase (AMPK), which, in turn, stimulates the expression and consequently the activity of key fatty acid desaturases (namely ∆5 and ∆6 desaturases) leading to the synthesis of AA from LOE in CF cells ((43); Fig. 1). Such a mechanism could account for the low levels of LOE in CF due to its increased metabolism to AA. Competitions between the ω-3 and ω-6 pathway could also account for the observed reduced levels of DHA in CF. Accordingly, medium supplementations with DHA or its precursor EPA (eicosapentaenoic acid) normalize the expression and activity of both ∆5 and ∆6 desaturases and result in a decreased production of AA in CF cells [START_REF] Njoroge | DHA and EPA reverse cystic fibrosis-related FA abnormalities by suppressing FA desaturase expression and activity[END_REF]. As a consequence to this mechanism, CF cells appear to display a deficit in both LOE and DHA within their PLs, which matches very nicely the observations made in humans ((42); Table 1).

Release of AA from PL by phospholipase A2

As already mentioned, non-esterified AA is a precursor of very active inflammatory mediators known as eicosanoids, which include PG, leukotrienes, and thromboxanes. Even if increased levels of AA in CF are subject to caution (Table 1), high AA levels may contribute to inflammation in some patients via this pathway. Non-esterified AA can be released by the hydrolysis of selective PL species, e. g. PC (36:4), bearing a SFA (Palmitate) at its sn-1 position and a AA at the sn-2, by the action of phospholipase A2 (PLA2; see above; [START_REF] Dennis | Phospholipase A2 in Eicosanoid Generation[END_REF]). Since the release of AA by phospholipases is rate-limiting for the eicosanoid synthesis, an abnormal activity of PLA2 resulting in the liberation of AA from PLs could be a possible mechanism to explain the inflammatory state in certain patients with CF (4). Relevantly, increased activity of PLA2 has been reported in plasma from patients with CF (4).

Hypoxia as an augmenting parameter

As already mentioned, mammalian cells display the machinery to build up their own MUFA from SFA, via the action of ∆-9 stearoyl-CoA desaturases (SCDs), and particularly SCD1. DHA and AA are synthesized from precursors, are metabolized to end products (including AA and DHA) by a series of alternating desaturation and elongation reactions. The desaturation steps are catalyzed by ∆-5 and ∆-6 desaturases (5). All fatty acid desaturases have in common that they are exquisitely oxygendependent [START_REF] Shanklin | Desaturases: emerging models for understanding functional diversification of diiron-containing enzymes[END_REF]. Hypoxia is a well-known situation encountered in CF [START_REF] Urquhart | Assessment of hypoxia in children with cystic fibrosis[END_REF][START_REF] Montgomery | Hypoxia and sterile inflammation in cystic fibrosis airways: mechanisms and potential therapies[END_REF]. In good agreement with potential impacts of hypoxia on the FA balance, exposing bronchial epithelial cells to oxygen scarcity results in an increase of SFA at the expense of UFA, and this, independently of the functional status of the CFTR channel [START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF]. Moreover, it has been demonstrated that lung transplantation in cystic fibrosis normalizes EFA profiles, suggesting direct correlations between the respiratory capacity of patients and the fatty acid signatures [START_REF] Witters | Lung transplantation in cystic fibrosis normalizes essential fatty acid profiles[END_REF]. Altogether, these data suggest that hypoxia may be an important contributor to EFA (and more generally UFA) deficiency in CF.

UFA deficiency and cellular membrane properties: the lipointoxication theory

Whatever the mechanism involved, which implication in the fatty acid signature may greatly vary depending on the patient considered, decrease in UFA within PLs appears to be a hallmark of CF. Moreover, a clear link has been established between the intensity of this UFA deficiency in circulating PLs and the severity of the disease [START_REF] Guerrera | A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients[END_REF]. PLs are the most abundant lipid classes whatever the organ considered [START_REF] Christie | Rapid separation and quantification of lipid classes by high performance liquid chromatography and mass (light-scattering) detection[END_REF]. A recent study performed on human bronchial tissue samples and dissociated bronchial epithelial cells showed that PLs constitute approximately 70 % of all lipid species, with PC being the most abundant species, followed by ethanolamine-containing PLs, and, to lesser extents, PI and PS [START_REF] Zehethofer | Lipid Analysis of Airway Epithelial Cells for Studying Respiratory Diseases[END_REF]. Of note, in mammalian cells, there is a gradual enrichment of SFA-containing PL species at the expense of unsaturated ones along the organelles of the secretory pathway (ER > Golgi > plasma membrane) ((50); Figure 3 A). The fact that such a subcellular gradient exists suggests that the correct balance between saturated and unsaturated PL species in the various organelles has decisive functions [START_REF] Antonny | From zero to six double bonds: phospholipid unsaturation and organelle function[END_REF]. In the studies displayed in Table 1, UFA deficiency in CF PL was paralleled by stable or increased levels of SFA, therefore resulting in an overall increase in the percentage of saturated-vsunsaturated-chains within these lipid species. In bronchial epithelial freshly dissociated from CF patients, a 2-fold increase in disaturated PC species and a 3-fold increase in disaturated PE, at the expense of unsaturated species, were observed as compared to non-CF individuals [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF].

The balance within PLs between SFA, MUFA and PUFA is known to regulate the biophysical properties of cellular membranes (Figure 3 B). Numerous studies have demonstrated that maintaining the balance between SFA, MUFA and PUFA within PL is essential to guarantee optimal membrane biophysical properties, compatible with the selective processes taking place within cellular compartments, such as vesicular budding or the trafficking and function of membrane proteins (see below; [START_REF] Antonny | From zero to six double bonds: phospholipid unsaturation and organelle function[END_REF]). Cumulative studies of biophysics and molecular dynamics simulations, have shown that different biophysical parameters are directly regulated by this ratio, such as the thickness of the membrane, its permeability, its elasticity, its plasticity or its surface roughness (surface voids) (Figure 3 B; for review, see (9)). For example, a membrane rich in PL containing SFA will be globally more organized due to the packing of the fatty acid chains, generating thick membranes, impermeable to water and to small hydrophilic molecules, but poorly elastic and malleable (Figure 3 B). In addition, it has been demonstrated that PUFA cannot simply considered as "super MUFA" but have the property of contorting to generate malleable and extremely deformable membranes [START_REF] Pinot | Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins[END_REF]. In contrast, MUFA, due to their simple double bond, tend to form kinks within the membrane, consequently generating elastic, but poorly deformable membranes [START_REF] Antonny | From zero to six double bonds: phospholipid unsaturation and organelle function[END_REF]. All of these results therefore demonstrate that the fatty acid content of PL, in a given membrane, has been scrupulously selected during evolution to meet specific physiological needs.

Considering the above, one may anticipate that perturbating the fatty acid balance within PLs cannot be without consequences on the trafficking and the structure/function of transmembrane proteins, including the CFTR channel ((52, 53); Fig. 3B). Among many examples in the literature, and relevant to the present review, it has been demonstrated that modifying membrane thickness alters the folding on transmembrane domains. Indeed, the length of transmembrane helices appears to have evolved during evolution to match the precise thickness of the membrane the protein is naturally embedded in [START_REF] Sharpe | A comprehensive comparison of transmembrane domains reveals organelle-specific properties[END_REF]. Perturbating this parameter drives transmembrane segments to adjust to these changes by either compressing/tilting (reduction in membrane thickness) or stretching (increase in membrane thickness) to avoid hydrophobic mismatch (which results from the exposition of their hydrophobic residues to the aqueous phase), with major impacts on their conformation, and, as a matter of fact, on their trafficking and function [START_REF] Killian | Hydrophobic mismatch between proteins and lipids in membranes[END_REF]. Another crucial parameter is the formation of surface voids, which correspond to cavities at the membrane surface, and which formation is intimately related to the fatty acyl chain composition of PLs ((56); Fig. 3B). Surface voids are strictly required for the attachment/recruitment of the COPI (Coat protein complex I) and COPII machineries, which are responsible for bidirectional membrane trafficking between the ER and the Golgi. Not surprisingly, affecting the SFA/UFA ratio within PLs in these organelles alters the recruitment of these machineries, with global impacts on the secretory pathway and on the trafficking of plasma membrane proteins, from their site of synthesis (the ER) to their final destination [START_REF] Payet | Saturated fatty acids alter the late secretory pathway by modulating membrane properties[END_REF][START_REF] Vanni | A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment[END_REF]. As a corollary to these observations, and due to the general nature and the conservation of the mechanisms involved, altered equilibria within SFA and UFA have been demonstrated to lead to dramatic dysfunctions in virtually all cell types tested so far (Figure 4; for review, see [START_REF] Bacle | Strategies to Counter Saturated Fatty Acid (SFA)-Mediated Lipointoxication[END_REF], including bronchial epithelial cells (see below). We refer to these fatty acid balance issues within PL under the term of lipointoxication.

The impacts of lipointoxication on the physiology of CF

The main impacts of lipointoxication on various cellular processes are summarized in Figure 4.

Lipointoxication and the ER function: the ER stress

The ER is the organelle where the biosynthesis and the folding of transmembrane and luminal proteins occurs. To maintain the correct balance between ER protein load and folding capacity, cells have developed a pathway known as the unfolded protein response (UPR) [START_REF] Hetz | The unfolded protein response: controlling cell fate decisions under ER stress and beyond[END_REF]. The UPR is under the control of integral protein sensors, including the inositol-requiring enzyme 1 (IRE1) and protein kinase-like ER kinase (PERK) [START_REF] Hetz | The unfolded protein response: controlling cell fate decisions under ER stress and beyond[END_REF], which are maintained under an inactive monomeric form by the interaction of their luminal domain with the chaperone BiP. When unfolded proteins accumulate within the ER, these repressive complexes are disrupted, leading to the oligomerization of IRE1 and PERK, and the activation of the UPR cascade. Induction of the UPR limits protein overload via a general decrease in translation initiation and coordinated upregulation of genes encoding proteins involved in folding (e.g. chaperones) and ER-associated degradation (ERAD) by the proteasome. UPR induction has been a recurrent observation in CF tissues (for recent review, see ( 60)). Since UPR activation is intimately linked to misfolded-protein overload, it may appear logical that specific mutations, i. e. Class II mutations, which result in CFTR misfolding, activate the UPR per se. However, UPR induction occurs when unfolded proteins are expressed at high levels and accumulate in the ER [START_REF] Welihinda | The cellular response to protein misfolding in the endoplasmic reticulum[END_REF]. In contrast, F508del CFTR, the main Class II mutation encountered in patients, is expressed at very low levels and quickly degraded by the proteasome [START_REF] Ward | Degradation of CFTR by the ubiquitinproteasome pathway[END_REF]. Furthermore, UPR induction is not CFTR-mutation specific, since such an induction can be observed in all CF patients, independently of the mutation. Altogether, these data suggest that the tissue environment resulting from CFTR dysfunction rather than CFTR mutations per se are responsible for UPR induction [START_REF] Bebok | Stressors and Stress Responses in Cystic Fibrosis[END_REF]. Many studies have demonstrated that the UPR is activated upon SFA overload, in virtually all cell types, including, among others, pancreatic β-cells [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic {beta}-cells[END_REF][START_REF] Ferru-Clement | Targeting surface voids to counter membrane disorders in lipointoxication-related diseases[END_REF][START_REF] Karaskov | Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis[END_REF], hepatocytes [START_REF] Achard | Lipid-Induced Endoplasmic Reticulum Stress in Liver Cells Results in Two Distinct Outcomes: Adaptation with Enhanced Insulin Signaling or Insulin Resistance[END_REF], muscle cells (67), adipocytes [START_REF] Yin | Palmitate induces endoplasmic reticulum stress and autophagy in mature adipocytes: Implications for apoptosis and inflammation[END_REF], yeast [START_REF] Pineau | Lipid-induced ER stress: synergistic effects of sterols and saturated fatty acids[END_REF], and, relevant to the present review, bronchial epithelial cells [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF][START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF]. Because exogenously supplied fatty acids are efficiently incorporated into PLs, and that the ER is naturally enriched in UFA-containing PL species (Figure 3 A), saturated PLs are likely the direct cause of ER-stress as a result of their impact on membrane properties. Membrane perturbations via excessive saturated PLs include modification of calcium transport, alterations in protein folding, and changes in the oligomerization status of IRE1 and PERK. For example, lipointoxication promotes depletion of ER calcium stores in various cell types [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic {beta}-cells[END_REF][START_REF] Gwiazda | Effects of palmitate on ER and cytosolic Ca2+ homeostasis in {beta}-cells[END_REF], including bronchial epithelial cells [START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF], in a process that could be related to direct effects of saturated PLs on sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump activity [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic {beta}-cells[END_REF]. Saturated PLs also induce the accumulation of misfolded proteins. Treating SFA-intoxicated cells with pharmacological chaperones that stabilize protein conformation alleviates SFA-induced UPR [START_REF] Pineau | Lipid-induced ER stress: synergistic effects of sterols and saturated fatty acids[END_REF][START_REF] Akerfeldt | Cytokine-Induced Β-Cell Death Is Independent of Endoplasmic Reticulum Stress Signaling[END_REF][START_REF] Ozcan | Chemical chaperones reduce ER stress and restore glucose homeostasis in a mouse model of type 2 diabetes[END_REF]. Various causes could account for misfolded protein accumulation under SFA treatment. First, the translocon, which catalyzes the translocation of newly translated proteins towards the lumen, is sensitive to membrane organization [START_REF] Nilsson | Inhibition of Protein Translocation across the Endoplasmic Reticulum Membrane by Sterols[END_REF]. Second, many ER chaperones are dependent on calcium [START_REF] Michalak | Ca2+ signaling and calcium binding chaperones of the endoplasmic reticulum[END_REF], the homeostasis of which is altered under SFA accumulation. Intriguingly, recent studies suggest that increased stiffness of the ER membrane due to saturated-PL accumulation can also induce UPR by favoring the direct clustering of IRE1 and PERK [START_REF] Volmer | Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains[END_REF]. Of course, lipointoxication-related induction of the UPR could have great impacts on CFTR function and trafficking. Indeed, reciprocally, it has been shown that induction of the UPR decreases wild-type CFTR expression at the transcriptional, translational, and maturational levels [START_REF] Bartoszewski | CFTR expression regulation by the unfolded protein response[END_REF]. When sustained, lipointoxication induces a global disorganization of the ER morphology. Indeed, under persistent lipointoxication, the ER tends to swell, and loses its ability to form a diffuse network through the cytoplasm to rather appear as stacks [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF][START_REF] Karaskov | Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis[END_REF][START_REF] Pineau | Lipid-induced ER stress: synergistic effects of sterols and saturated fatty acids[END_REF]. Under these circumstances, the UPR switches from anti-to pro-apoptotic leading to cell death in virtually all cell types (for review see [START_REF] Bacle | Strategies to Counter Saturated Fatty Acid (SFA)-Mediated Lipointoxication[END_REF]). Importantly, apoptosis as an important mechanism for airway epithelial cell loss in CF airways is recognized [START_REF] Soleti | Apoptotic process in cystic fibrosis cells[END_REF][START_REF] White | Apoptosis and the airway epithelium[END_REF]. Relevant to CF, reconstitution of the SFA/UFA ratio observed in bronchial epithelial cells from CF patients in cell lines resulted in the induction of the UPR, ER swelling/stack formation and, when most dramatic signatures were recapitulated, apoptosis [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF][START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF]. Relevant with the lipointoxication concept, increased stiffness of the ER membrane could be directly assessed in cellulo (Figure 3 C) and molecular dynamics simulations performed using CF and non-CF PL compositions revealed that SFA overload in CF PL impacts crucial cellular properties, including membrane thickness, area per lipid and lipid diffusion, all being bona fide indicators of membrane stiffening [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF].

The role of a membrane origin in such disorders was strengthened by the fact that restoring a correct SFA/UFA balance in PL by co-supplementation of the medium with UFA, or the use of a lipophilic molecules, namely Mannide Monoleate, which displays the property of inserting within the membrane and of acting as a membrane reshaper to restore central membrane parameters, both relieved the ER stress, restored normal properties of the ER membrane and countered SFA-induced apoptosis [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF][START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF].

Lipointoxication and inflammation

Another important characteristic of CF is excessive and sustained lung inflammation [START_REF] Roesch | Inflammation in cystic fibrosis: An update[END_REF]. If the roles of bacterial infection and induction of proinflammatory pathways via the production of AA-derived eicosanoids are undeniable [START_REF] Roesch | Inflammation in cystic fibrosis: An update[END_REF], direct augmenting impacts of lipointoxication on these processes should be considered. Indeed, SFA-related inflammation has been demonstrated in many studies in different cellular models, including adipocytes, cardiac fibroblast, hepatocytes, skeletal muscle cells, and pancreatic β-cells, in processes that can be fully reversed by the restoration of the SFA/UFA balance (for review, see [START_REF] Bacle | Strategies to Counter Saturated Fatty Acid (SFA)-Mediated Lipointoxication[END_REF]). This effect is manifested by SFA-activation of proinflammatory pathways, with the increased secretion of proinflammatory cytokines and chemokine, among which are tumor necrosis factor a (TNFα), Interleukin-6 (IL6), and IL8. High levels of IL8 can be detected in the bronchial lavage (BAL) fluid from patients with CF (80). To some extent, IL8 appears to derive from bronchial epithelial cells themselves, since airway epithelium plays a crucial role in initiating and augmenting host defense mechanisms. Relevantly, increased IL8 secretion was also demonstrated in bronchial epithelial cells under a situation which recapitulated the SFA/UFA imbalance observed in patients, a process which was relieved by UFA addition or treatment with a membrane reshaper [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF]. IL8 secretion has been shown to be under the dependence of p38 MAPK (mitogen-activated protein kinase) signaling pathway in several cell types, including bronchial epithelial cells [START_REF] Håversen | Induction of proinflammatory cytokines by long-chain saturated fatty acids in human macrophages[END_REF][START_REF] Saatian | Transcriptional regulation of lysophosphatidic acid-induced interleukin-8 expression and secretion by p38 MAPK and JNK in human bronchial epithelial cells[END_REF]. Accordingly, UFA and membrane reshapers both relieved SFA-induced induction of the MAPKpathway (17).

Lipointoxication and CFTR trafficking

The altered ER-function observed under lipointoxication has important impacts on protein folding (see above) but also on the trafficking of membrane-anchored proteins. Protein trafficking between the various compartments relies on the ability of the organelle membranes to bend in order to form transport vesicles, a property that is intimately related to the membrane properties and requires sufficient amounts of UFA-containing PLs [START_REF] Antonny | From zero to six double bonds: phospholipid unsaturation and organelle function[END_REF]. Relevantly, it has been demonstrated that ER-to-Golgi protein trafficking is precluded in SFA-treated mammalian β-cells [START_REF] Preston | Reduced endoplasmic reticulum (ER)-to-Golgi protein trafficking contributes to ER stress in lipotoxic mouse beta cells by promoting protein overload[END_REF]. These observations fit in vitro experiments showing that liposomes formed of saturated PL display a lower capacity to recruit proteins of the Coat Protein complex (COP II) machinery, which are essential for vesicular budding [START_REF] Matsuoka | COPII-Coated Vesicle Formation Reconstituted with Purified Coat Proteins and Chemically Defined Liposomes[END_REF]. This process is likely to contribute to the retention of misfolded proteins in the ER, therefore reinforcing the protein overload in this compartment. Later in the secretory pathway, saturated-PL overload in the Golgi apparatus also impacts the recruitment of proteins of the COP I machinery, therefore leading to a reduction in the number, and an altered morphology of secretory vesicles "en route" to the plasma-membrane [START_REF] Payet | Saturated fatty acids alter the late secretory pathway by modulating membrane properties[END_REF][START_REF] Vanni | A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment[END_REF]. Therefore, lipointoxication results in global alterations of the overall secretory pathway. The human genome is thought to code for approximately 5000 membrane proteins, most of them being synthesized and folded in the ER and further transported to their final destination along the secretory pathway, among which the CFTR and its functional partners. Therefore, lipointoxication is susceptible to impact the trafficking of hundreds of membrane proteins in the various cells of our body. If the specific impacts of SFA-overload on CFTR trafficking in epithelial cells have not been studied in details so far, the fact that correctors of the F508del-CFTR appear to be less efficient under lipointoxication is in good agreement with the hypothesis of potent effects of lipointoxication on this process (18).

Lipointoxication and CFTR function

Finally, the activity of membrane proteins is highly dependent on the properties of the bilayer they are embedded in, and changing such properties has highly deleterious impacts on their function and/or stability at their site of action [START_REF] Lee | Lipid-protein interactions in biological membranes: a structural perspective[END_REF][START_REF] Lee | How lipids affect the activities of integral membrane proteins[END_REF]. An exhaustively-studied example is the light receptor rhodopsin, which function is highly connected to the presence of PUFA within its surrounding PL [START_REF] Antonny | From zero to six double bonds: phospholipid unsaturation and organelle function[END_REF]. If the specific effects of membrane lipids on the activity of the CFTR channel have not been studied in great details so far, a pioneering study demonstrated that selective PL species are important for CFTR trafficking and contribute to the pathology of CF [START_REF] Eidelman | Role for Phospholipid Interactions in the Trafficking Defect of ΔF508-CFTR[END_REF]. More recently, it has been shown that selective lipid interactions with purified CFTR enhance its catalytic activity [START_REF] Chin | Lipid interactions enhance activation and potentiation of cystic fibrosis transmembrane conductance regulator (CFTR)[END_REF]. Moreover, it has been proposed that the CFTR potentiator VX-770 and its derivatives destabilize the pharmacologicallyrescued F508del-CFTR channel, by their potential non-specific effects on the lipid bilayer [START_REF] Chin | Lipophilicity of the Cystic Fibrosis drug, Ivacaftor, and its destabilizing effect on the major CF-causing mutation: F508del[END_REF]. Altogether, and even if additional studies will be undoutfully required, these data demonstrate that CFTR activity/stability is highly connected to its surrounding lipids and that modulating the membrane properties clearly impacts its function.

Outstanding Questions

If the mechanisms leading to fatty-acid remodeling within PL in various CF tissues are complex and likely result from the alteration of multiple mechanisms, which amplitude may vary from one patient to the other, bona fide modifications of the SFA/UFA balance have been repeatedly reported in CF organs, including the lung. Considering the known impacts of such an imbalance on membrane properties and the crucial effects of membrane homeostasis perturbations on the induction of stress pathways, organelle integrity, and membrane-protein trafficking and function, it is likely that lipointoxication plays a role in the etiology of the disease. An important question that still remains however is the contribution of this process to the course of the disease. Since the intensity of the lipointoxication signature nicely correlates with the severity of the disease [START_REF] Ollero | Plasma lipidomics reveals potential prognostic signatures within a cohort of cystic fibrosis patients[END_REF][START_REF] Guerrera | A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients[END_REF], and that lipotoxicty can account, by itself, for several traits associated to CF (including ER stress, stress pathway induction, inflammation and epithelial cell apoptosis), it is reasonable to consider lipointoxication as a potent contributor to the severity of the disease. Moreover, lipointoxication may also be a limiting parameter to the efficiency of existing treatments for CF. Even if additional studies will be required, the fact that F508del-CFTR correctors appear to be less efficient under lipointoxication reinforces this hypothesis [START_REF] Payet | Cystic Fibrosis Bronchial Epithelial Cells Are Lipointoxicated by Membrane Palmitate Accumulation[END_REF]. Considering these observations, reevaluating the efficiency of existing treatment in cellular models recapitulating at best the lipid signature encountered in vivo will likely help at understanding the discrepancies observed between the data obtained in cellulo, in non-lipointoxicated cellular models, and the results obtained in vivo, which appear most often quite deceptive as compared to the expected results from in vitro experiments [START_REF] Davies | VX-659-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles[END_REF][START_REF] Keating | VX-445-Tezacaftor-Ivacaftor in Patients with Cystic Fibrosis and One or Two Phe508del Alleles[END_REF].

Search strategy and selection criteria

Data for this review were obtained from PubMed searches and references from relevant articles using combinations of the following criteria: "Cystic Fibrosis", "Lipidomics", "Lipids", "Fatty Acids", "Phospholipids", "Essential Fatty Acid Deficiency", "ER stress", "Membrane Biophysics", "Structure/Function of Membrane Proteins", "Secretory Pathway" and "Membrane Protein Trafficking". Articles published in English from 1962 to 2020 were included with particular emphasis on studies from 2010 to 2020. The main pathways modulating the repertoire of fatty acids available to the cell and the major pathways leading to their distribution within complex lipids are displayed.

Contributors

Figure 2: The potential origins of lipointoxication in CF

Lipointoxication is defined as an imbalance among Saturated Fatty Acids (SFA) and Unsaturated Fatty Acids (UFA) within the main membrane constituents, namely Phospholipids (PL). Altered CFTR function results in several organ dysfunctions which could account for alterations in the repertoire of fatty acids available to the cells (and particularly bronchial epithelial cells), with an excess of SFA at the expense of UFA. These processes include Cystic Fibrosis-related Liver Disease (CFLD) and pancreatic insufficiency, leading to malabsorption of PolyUnsaturated Fatty Acids (PUFA), but also hypoxia, which precludes the desaturation steps required to form UFA from SFA (Figure 1). Moreover, mutations in the CFTR genes are also associated to an increased flux through the ω-6 pathway, which may account for decreased levels of LOE and DHA, and increased activity of Phospholipase A2 (PLA2), leading to a decrease in the amount of PUFA-containing PL species (Figure 1). See text for details.

Figure 3: The fatty acid composition of PL modulates membrane properties (A)

In mammalian cells, there is a gradual enrichment of Saturated Fatty Acid (SFA)-containing Phospholipid (PL) species at the expense of unsaturated ones (UFA) along the organelles of the secretory pathway (Endoplasmic Reticulum (ER) > Golgi > plasma membrane). (B) The ratio between SFA and UFA within PL (PhosphatidylCholine in the example displayed here) regulates several membrane properties including width, elasticity, fluidity and surface voids. Such impacts can be visualized using Molecular Dynamics Simulations. See text for details. (C) Bronchial epithelial cells bearing the F508del-CFTR mutation were cultivated either under normal conditions (Control) or under conditions recapitulating the SFA/UFA imbalance observed in freshly-dissociated bronchial epithelial cells obtained from CF patients (Lipointoxication). They were then incubated with the Laurdan probe which has the property of inserting into cellular membranes and displays different emission wavelengths depending on the overall organization of the lipid bilayer. A green color is characteristic of a fluid-membrane, and a blue color of a highly-ordered, rigid membrane. As shown, Lipointoxication results in a stiffening of the intracellular membranes, and particularly of the ER one. Adapted from Kadri et al. [START_REF] Kadri | Modulation of cellular membrane properties as a potential therapeutic strategy to counter lipointoxication in obstructive pulmonary diseases[END_REF].

Figure 4: Lipointoxication impacts crucial cellular processes

The impacts of Lipointoxication on several crucial cellular processes have been demonstrated (and are conserved) in many cell types, including bronchial epithelial cells. By perturbating the overall membrane organization (Figure 3), an SFA/UFA imbalance within PL results in the induction of stress pathways (Unfolded Protein Response (UPR) and MAPK pathway), altered protein folding, depletion of calcium stores and impaired vesicular budding in the ER. Later in the secretory pathway, lipointoxication is also associated with reduced vesicle formation at the Golgi level. Overall, these alterations result in a global disruption of the secretory and of the trafficking of plasma-membrane protein to their final destination. If sustained, lipotoxicty ultimately leads to cell death (apoptosis). Perturbed SFA/UFA balance has also been associated to an increased release of cytokines, including the pro-inflammatory chemokine IL8. See text for details.

Figure 1 :

 1 Figure 1: An overview of cellular Fatty Acid homeostasisThe main pathways modulating the repertoire of fatty acids available to the cell and the major pathways leading to their distribution within complex lipids are displayed. See text for details. SFA: Saturated Fatty Acid; MUFA: MonoUnsaturated Fatty Acid; PUFA: PolyUnsaturated Fatty Acid; PA: Palmitic Acid; OA: Oleic Acid; LON: Linolenic Acid; LOE: Linoleic Acid; EA: Eicosatrienoic Acid; AA: Arachidonic Acid; DHA; DocosaHexaenoic Acid; PLA2: Phospholipase A2; LPL: LysoPhosphoLipid; FFA: Free Fatty Acid; SPT: Serine PalmitoylTransferase; FAS: Fatty Acid Synthase.

Figure 1 :

 1 Figure 1: Fatty acid homeostasis

Figure 2 :Figure 4 :

 24 Figure 2: The potential origins of lipotoxicity in CF

  Drs Vandebrouck and Ferreira contributed equally to this work. Both authors read and approved the final version of the manuscript.

				DHA decrease	LOE decrease	AA increase
	Total Fatty Acids
	Plasma	Witters et al., 2013	Yes	Yes	No: decrease
			Farrell et al., 1985 Yes: influenced by the diet	Yes	No: decrease
			Guilbault et al., 2009	Yes	N.R.	Yes
	Aldamiz-Echevarria et al., 2009	Yes	Yes	No
	Fat, Psoas Muscle, Heart Farrell et al., 1985	N.R.	Yes	No
	Nasal-Biopsy Tissues
			Freedman et al., 2004	Yes; severity increases with P.I.	Yes.	Yes; severity may increase with P. I.
	Rectal-Biopsy Tissues Freedman et al., 2004	Yes	N.R	No
	Phopholipids
	Blood	Van Biervliet et al., 2007	Yes	Yes	No
	Plasma
			Guerrera et al., 2009	Yes, especially in severe patients (decrease in DHA-containing PC (i.e. PC 38:6))	Probably (decrease in LA-containing PC (PC 36:2))	No: rather a decrease in AA-containing species (i. e. PC 36:4) in severe patients
			Farrell et al., 1985	N.D.	Yes	No
			Grothe et al., 2015	Yes: decrease in DHA-containing PC	No	No: decrease in AA-containing PC species
	Serum
			Strandvik et al., 2001	Yes	Yes	No
			Maqbool et al., 2008	N.R.	Yes	No: decrease in AA-containing PL species
	Cord Serum Lloyd-Still et al., 1991	Yes	Yes	Yes
	Bronchial secretions
			Gilljam et al., 1986	N.D.	Depends on the lipid species considered: decreased in PA and PE, increased in PC, unchanged in PI	Depends on the lipid species considered: increased in all PL species except for PE
	Red Blood Cells Rogiers et al., 1984 (b)	N.D.	Yes (PC analyzed)	Yes (PC analyzed)

Table 1 :

 1 Fatty Acid alterations in tissues from patients with Cystic Fibrosis. The most recurrent hypotheses that have been formulated concerning Fatty Acid dysregulations in CF tissues are decreases in DocosaHexaenoic Acid (DHA) and Linoleic Acid (LOE) levels and an increase in Arachidonic Acid (AA). Here, the data from various studies were separated depending on the class of the lipid analyzed and the tissue considered. The green color indicates that the data obtained fit the corresponding hypothesis, and the red color that contradictory results have been observed. N.R. not reported; N.D. not detected; P.I.: Pancreatic Insufficiency

Acknowledgment

This work was supported by University of Poitiers (Poitiers, France) and the association "Mucoviscidose: ABCF 2" (Paris, France). The funders had no role in paper design, data collection, data analysis, interpretation, writing of the paper


Xavier Puéchal
email: xavier.puechal@aphp.fr

Granulomatose avec polyangéite (Wegener)

Résumé

La granulomatose avec polyangéite (Wegener) (GPA) est une vascularite nécrosante associant une inflammation de la paroi vasculaire des vaisseaux de petit calibre et une granulomatose, péri-et extravasculaire. Sur le plan clinique, elle se caractérise dans sa forme complète par des signes ORL, une atteinte pulmonaire et une atteinte rénale. La GPA s'accompagne de la présence d'ANCA, de type cytoplasmique dans environ 90 % des formes diffuses et dans 50 % des formes localisées, qui sont dirigés contre la protéinase 3 dans l'immense majorité des cas. Les cANCA PR3 sont très spécifiques de la GPA et ont donc une importante valeur diagnostique. Le traitement de la GPA repose sur l'association de corticoïdes et de cyclophosphamide ou de rituximab qui permet l'obtention de la rémission dans plus de 80 % des cas.

L'azathioprine était le traitement d'entretien le plus utilisé mais le rituximab en entretien, à faible dose semestrielle, diminue davantage les rechutes avec une tolérance satisfaisante. Des rechutes à distance surviennent dans plus de 50 % des cas. Un des problèmes thérapeutiques majeurs est lié à la survenue d'effets secondaires des traitements, dont la gravité et la fréquence sont souvent liées à une prolongation du traitement difficilement évitable.

Mots clés : Granulomatose avec polyangéite ; vascularite nécrosante ; vascularite associée aux ANCA ; immunomodulation thérapeutique ; médicaments biologiques ; rituximab. La granulomatose avec polyangéite (Wegener) est une vascularite nécrosante associant une inflammation de la paroi vasculaire et une granulomatose péri-et extravasculaire. Sur le plan clinique, dans sa forme complète, la granulomatose avec polyangéite (GPA) se caractérise par des signes ORL, une atteinte pulmonaire et rénale. D'autres manifestations systémiques peuvent survenir. La GPA est une maladie grave, mortelle en l'absence de traitement. Toutefois les thérapeutiques actuelles permettent de contrôler l'évolution, de la guérir dans la plupart des cas, même si les rechutes restent fréquentes.

Pathogenie de la maladie

Les neutrophiles sont les acteurs clés car ils produisent l'autoantigène, la protéinase 3, et leur régulation est altérée chez les patients [START_REF] Millet | Proteinase 3 on apoptotic cells disrupts immune silencing in autoimmune vasculitis[END_REF]. Les patients atteints de GPA possèdent des auto-anticorps dirigés contre la protéinase 3 localisée dans les neutrophiles. Ces cellules recrutées au site de l'inflammation ont un rôle crucial dans la défense contre les microbes mais peuvent aussi participer à la destruction tissulaire. L'autoantigène protéinase 3 exprimé à la membrane des neutrophiles apoptotiques perturbe leur élimination par les macrophages. L'expression de PR3 à la membrane des neutrophiles activés prévient la résolution de l'inflammation et est un élément capital dans la pathogénie de la GPA. Alors que la phagocytose d'une cellule apoptotique en condition physiologique a un effet anti-inflammatoire, la protéinase 3 est perçue comme un signal de danger par le macrophage, déclenchant une réponse d'alerte au système immunitaire. Cet effet subversif de la protéinase 3 va alors favoriser la mise en place d'une réponse auto-immune, en particulier en alertant les cellules dendritiques plasmacytoïdes qui, à leur tour, vont totalement abolir la génération de lymphocytes CD4+ régulateurs et, au contraire promouvoir l'émergence de lymphocytes activés ayant un profil Th9/Th2.

Anatomie pathologique

La GPA comprend, sur le plan anatomopathologique, une nécrose ischémique en carte de géographie qui se traduit par la formation d'abcès amicrobiens et une granulomatose polymorphe qui associe des polynucléaires, des lymphocytes et des cellules géantes multinucléées. La vascularite touche des vaisseaux de petit calibre (artérioles, capillaires, et veinules) et plus rarement de moyen calibre (Figure 1). Le granulome n'est pas spécifique, comportant des cellules géantes. Le diagnostic histologique peut être obtenu par des biopsies orientées (Encadré 1).

Formes generalisees et formes localisees

Au cours de la GPA, on distingue :

les formes généralisées/diffuses caractérisées par : les formes limitées/localisées, principalement respiratoires (ORL et/ou pulmonaire sans hémorragie alvéolaire), sans atteinte rénale, mais parfois aussi cutanées, sans altération de l'état général et qui n'engagent pas le pronostic vital (environ 30 % des cas de GPA).

Le passage d'une forme localisée/limitée à une forme généralisée/diffuse, et vice versa, est possible au cours de l'évolution de la maladie.

Epidemiologie

Il s'agit d'une maladie rare dont la prévalence est d'environ 3/100.000 habitants. Il existe un gradient nord-sud, avec une plus grande fréquence dans les pays nordiques. La GPA intéresse les deux sexes. L'âge moyen de survenue est de 45 ans. Des formes ont été décrites chez le sujet très âgé et chez l'enfant.

Signes cliniques

Les principales manifestations cliniques sont résumées dans le Tableau 1 (2-7).

Les manifestations ORL

Les signes ORL sont souvent révélateurs de la maladie.

Les manifestations articulaires et/ou musculaires

Des manifestations articulaires et/ou musculaires surviennent chez plus de la moitié des patients et peuvent être révélatrices. Il peut s'agir d'authentiques polyarthrites.

Autres manifestations

Les autres manifestations sont celles que l'on observe dans les autres vascularites (Tableau 1). Les manifestations neurologiques périphériques ne sont pas spécifiques de la maladie et comme les signes cutanés, elles traduisant l'atteinte des vaisseaux de petit calibre.

Examens biologiques

La GPA s'accompagne d'un élément essentiel au diagnostic et au suivi de la maladie : la présence d'ANCA, de fluorescence cytoplasmique diffuse, dirigés contre la protéinase 3 dans 75 % des cas et beaucoup plus rarement contre la myéloperoxydase [START_REF] Iudici | for the French Vasculitis Study Group : Granulomatosis with Polyangiitis: Data from the French Vasculitis Study Group Registry (abstract)[END_REF]. Ils sont présents dans environ 90 % des formes diffuses et dans 50 % des formes localisées de la maladie. Ils sont très spécifiques et ont donc une valeur diagnostique. Dans certaines conditions, associée à une clinique évocatrice, leur présence peut suffire au diagnostic.

Évolution et pronostic

Non traitée, la GPA avait une mortalité à 1 an d'environ 70 %. Sous traitement, une rémission est obtenue dans plus de 80 % des cas. Les rechutes sont fréquentes et surviennent dans plus de 50 % des cas. Malgré cela, le taux de survie atteint 75 % à 10 ans (8).

L'atteinte ORL est associée à un meilleur pronostic vital mais à davantage de rechutes. Un score pronostique FFS (Five Factor Score) a été proposé [START_REF] Guillevin | Prognostic factors in polyarteritis nodosa and Churg-Strauss syndrome. A prospective study in 342 patients[END_REF], dont la version actualisée s'applique aussi à la GPA [START_REF] Guillevin | Toumelin PL for the French Vasculitis Study Group (FVSG). The Five-Factor Score Revisited: Assessment of Prognoses of Systemic Necrotizing Vasculitides Based on the French Vasculitis Study Group (FVSG) Cohort[END_REF]. Elle tient compte de 5 manifestations associées à une surmortalité : l'existence d'un âge supérieur à 65 ans, d'une cardiomyopathie spécifique, de manifestations gastro-intestinales, d'une insuffisance rénale définie par une créatininémie stabilisée supérieure à 150 µmol/l et de l'absence de manifestations ORL, chaque item cotant pour un point. Néanmoins, si le FFS renseigne sur le pronostic de la GPA, il ne conditionne pas la thérapeutique, comme pour les autres vascularites nécrosantes [START_REF]Vascularites nécrosantes systémiques (périartérite noueuse et vascularites associées aux ANCA)[END_REF].

Au diagnostic, les facteurs associés à une rechute sont la présence d'ANCA anti-PR3, une atteinte cardiaque et une clairance de la créatinine > 60 ml/mn [START_REF] Mukhtyar | Outcomes from studies of antineutrophil cytoplasm antibody associated vasculitis: a systematic review by the European League Against Rheumatism systemic vasculitis task force[END_REF].

Au cours de l'évolution, la persistance d'ANCA est également associée à une augmentation du risque de rechute [START_REF] Karras | Randomised controlled trial of prolonged treatment in the remission phase of ANCA-associated vasculitis[END_REF][START_REF] Morgan | Negative anti-neutrophil cytoplasm antibody at switch to maintenance therapy is associated with a reduced risk of relapse[END_REF][START_REF] Terrier | Long-term efficacy of remission-maintenance regimens for ANCA-associated vasculitides[END_REF]. La disparition des ANCA ne permet cependant pas de prédire l'absence de rechute. Une augmentation significative du taux des ANCA PR3 fait craindre la survenue d'une rechute [START_REF] Mukhtyar | Outcomes from studies of antineutrophil cytoplasm antibody associated vasculitis: a systematic review by the European League Against Rheumatism systemic vasculitis task force[END_REF]. Mais, en cas d'augmentation du taux des ANCA, 29 à 60 % des patients ne rechutent pas à 1 an [START_REF] Boomsma | Prediction of relapses in Wegener's granulomatosis by measurement of antineutrophil cytoplasmic antibody levels: a prospective study[END_REF][START_REF] Finkielman | Antiproteinase 3 antineutrophil cytoplasmic antibodies and disease activity in Wegener granulomatosis[END_REF]. Ce qui signifie que si l'on avait traité sur cette seule augmentation, ce serait 29 à 60 % de patients en trop qui auraient été traités. On ne peut donc utiliser isolément le taux des ANCA comme élément d'adaptation thérapeutique. Une augmentation du titre des ANCA incite donc à rapprocher la surveillance mais pas à majorer le traitement.

Traitement de la granulomatose avec polyangéite

Le traitement de la GPA repose sur l'association de corticoïdes et d'immunosuppresseurs (Figure 4) [START_REF]Vascularites nécrosantes systémiques (périartérite noueuse et vascularites associées aux ANCA)[END_REF].

Dans les formes systémiques, les corticoïdes seuls ne permettent pas d'obtenir et de maintenir la rémission [START_REF] Walton | Giant-cell granuloma of respiratory tract (Wegener's granulomatosis)[END_REF]. Le cyclophosphamide, en association à la corticothérapie, permet des rémissions prolongées (2).

Néanmoins, des rechutes surviennent chez plus de la moitié des patients. [START_REF] Sneller | An analysis of forty-two Wegener's granulomatosis patients treated with methotrexate and prednisone[END_REF][START_REF] Langford | Use of methotrexate and glucocorticoids in the treatment of Wegener's granulomatosis. Long-term renal outcome in patients with glomerulonephritis[END_REF]. La seule étude contrôlée a comparé l'administration de 12 mois de méthotrexate ou de cyclophosphamide, en plus de la corticothérapie, chez des patients atteints de vascularite sans atteinte vitale [START_REF] De Groot | Randomized trial of cyclophosphamide versus methotrexate for induction of remission in early systemic antineutrophil cytoplasmic antibody-associated vasculitis[END_REF]. À 6 mois, il n'y avait pas de différence dans la fréquence de mise en rémission. Par contre, les patients ont rechuté plus souvent après méthotrexate qu'après cyclophosphamide. Cette étude confirme l'intérêt de ne pas interrompre l'immunosuppression à 12 mois et qu'un certain nombre de patients répondent au méthotrexate seul. Néanmoins, cette stratégie ne se conçoit que dans des GPA peu sévères initialement, à fonction rénale normale ou subnormale et avec une surveillance particulièrement attentive.

Le rituximab a bouleversé le traitement d'induction des formes réfractaires ou en rechute de la

GPA sévère

En association avec la corticothérapie, cet anti-CD20 a bouleversé le traitement des GPA sévères, surtout des formes réfractaires ou en rechute. Il représente une alternative efficace au cyclophosphamide. Il a obtenu une autorisation de mise sur le marché, au cours de la GPA sévère, en association à la corticothérapie, en perfusions de 375 mg/m 2 pendant 4 semaines consécutives. Les études ouvertes préliminaires concernaient surtout des patients atteints de GPA réfractaires [START_REF] Jones | A multicenter survey of rituximab therapy for refractory antineutrophil cytoplasmic antibody-associated vasculitis[END_REF][START_REF] Puéchal | Targeted immunotherapy strategies in ANCA-associated vasculitis[END_REF]. Elles ont toutes été positives sauf une [START_REF] Aries | Lack of efficacy of rituximab in Wegener's granulomatosis with refractory granulomatous manifestations[END_REF]. Une mise en rémission complète a ainsi été rapportée chez 75-83 % des patients traités [START_REF] Jones | A multicenter survey of rituximab therapy for refractory antineutrophil cytoplasmic antibody-associated vasculitis[END_REF][START_REF] Puéchal | Targeted immunotherapy strategies in ANCA-associated vasculitis[END_REF] [START_REF] Jones | for the European Vasculitis Study Group (EUVAS). Mycophenolate mofetil versus cyclophosphamide for remission induction in ANCA-associated vasculitis: a randomised, non-inferiority trial[END_REF]. Il n'y a pas eu moins de rémission à 6 mois chez les patients recevant le mycophénolate mofétil mais davantage de rechute ultérieure, surtout chez les patients avec ANCA anti-PR3. Il est donc peu utilisé chez les patients atteints de GPA.

Les anti-TNF ne sont pas utilisés

L'infliximab a parfois été prescrit en traitement d'induction des GPA réfractaires [START_REF] Bartolucci | Efficacy of the anti-TNFalpha antibody infliximab against refractory systemic vasculitides: an open pilot study on 10 patients[END_REF][START_REF] Lamprecht | Effectiveness of TNF-alpha blockade with infliximab in refractory Wegener's granulomatosis[END_REF][START_REF] Booth | Prospective study of TNFalpha blockade with infliximab in anti-neutrophil cytoplasmic antibody-associated systemic vasculitis[END_REF][START_REF] De Menthon | Infliximab or rituximab for refractory Wegener's granulomatosis: long-term follow up. A prospective randomised multicentre study on 17 patients[END_REF]. Son efficacité n'est néanmoins que suspensive. La survenue de complications infectieuses sévères a aussi été rapportée sous l'association d'infliximab et de cyclophosphamide [START_REF] Gause | Induction of remission with infliximab in active generalized Wegener´s granulomatosis is effective but complicated by severe infections (abstract)[END_REF].

Une étude contrôlée a comparé l'étanercept à un placebo, en plus du traitement conventionnel, chez des patients atteints de GPA [START_REF]The Wegener's Granulomatosis Etanercept Trial (WGET) Research Group : etanercept plus standard therapy for Wegener's granulomatosis[END_REF]

Les immunoglobulines

Par voie intraveineuse, elles ont été essentiellement employées chez les malades en rechute avec une certaine efficacité thérapeutique et une diminution du titre des ANCA. Néanmoins, nombre de malades rechutent à l'arrêt des perfusions limitant l'intérêt de cette thérapeutique, dans l'ensemble bien tolérée. Ce traitement peut être utile pour passer un cap chez certains patients avec une vascularite active et une sévère immunodépression. Les immunoglobulines sont administrées par voie intraveineuse à la posologie de 2 g/kg/mois en perfusion échelonnée sur 4 jours, d'autant plus que le malade souffre d'une insuffisance rénale.

les echanges plasmatiques

Des échanges plasmatiques étaient volontiers réalisés dans les formes sévères de vascularite associée aux ANCA avec insuffisance rénale rapidement progressive. Dans une étude randomisée contrôlée, ils s'étaient montrés significativement supérieurs aux bolus de méthylprednisolone, chez des patients avec une créatininémie initiale supérieure à 500 micromol/l, pour diminuer le nombre de patients dialysés à 3 et 12 mois [START_REF] Jayne | Randomized trial of plasma exchange or high-dosage methylprednisolone as adjuctive therapy for severe renal vasculitis[END_REF]. Néanmoins, ils n'affectaient pas la survie globale et le bénéfice rénal ne se maintenait pas sur le long terme. L'étude PEXIVAS, essai randomisé contrôlé, évaluant l'intérêt des échanges plasmatiques en cas d'atteinte rénale ou d'hémorragie alvéolaire, chez plus de 700 patients atteints de vascularite associée aux ANCA, n'a pas montré de bénéfice des échanges sur un critère composite associant décès ou évolution vers l'insuffisance rénale définitive [START_REF] Walsh | The effect of plasma exchange and reduced-dose oral glucocorticoids during induction of remission in patients with severe ANCAassociated vasculitis[END_REF]. Depuis les résultats de cette étude, on a tendance à ne proposer les échanges que dans les formes sévères réfractaires à un traitement médical bien conduit (en seconde intention) ou en cas d'hémorragie alvéolaire massive.

Une hémorragie alvéolaire est habituellement traitée par l'association de corticoïdes et de cyclophosphamide ou de rituximab, voire d'échanges plasmatiques si elle est très importante ou en l'absence d'amélioration avec un traitement médical bien conduit. Dans ces cas de vascularite en rechute ou réfractaire, il est conseillé de discuter chaque observation avec les centres de référence et de compétences.

place du trimethoprime-sulfamethoxazole

En plus de la prophylaxie des pneumocystoses, le triméthoprime-sulfaméthoxazole pourrait avoir un intérêt pour diminuer globalement la fréquence des infections sévères sous rituximab [START_REF] Kronbichler | Trimethoprimsulfamethoxazole prophylaxis prevents severe/life-threatening infections following rituximab in antineutrophil cytoplasm antibody-associated vasculitis[END_REF]. Il aurait peut-être aussi un rôle modeste dans le maintien des rémissions. Dans une courte étude contrôlée en double insu, l'administration de 2 comprimés quotidiens de 800 mg de sulfaméthoxazole pendant 2 ans s'est accompagnée d'une réduction du taux de rechute, essentiellement ORL, par rapport à celle d'un placebo, chez des patients en rémission complète [START_REF] Stegeman | Trimethoprim-sulfamethoxazole (cotrimoxazole) for the prevention of relapses of Wegener's granulomatosis for the Dutch Co-Trimoxazole Wegener Study Group[END_REF].

mesures adjuvantes

La prévention et le dépistage des complications thrombo-emboliques font partie de la prise en charge initiale.

Les complications cardio-vasculaires sont aussi augmentées au cours des vascularites associées aux ANCA, nécessitant un dépistage et une prise en charge des facteurs de risque cardio-vasculaire [START_REF] Morgan | Increased incidence of cardio-vascular events in patients with antineutrophil cytoplasmic antibody-associated vasculitides[END_REF][START_REF] Terrier | Factors associated with major cardiovascular events in patients with systemic necrotizing vasculitides: results of a long-term follow-up study[END_REF]. Sous traitement, un certain nombre d'autres effets secondaires peuvent survenir : infectieux à court terme, malin à long terme avec la survenue de cystopathies sous cyclophosphamide, de lymphomes et de tumeurs solides.

La prophylaxie des infections est justifiée. La surveillance régulière cutanée est importante car ces patients développent davantage de cancers non mélaniques que les contrôles, possiblement en lien avec l'exposition prolongée aux immunosuppresseurs [START_REF] Heijl | Incidence of malignancy in patients treated for ANCA-associated vasculitis : follow-up data from European Vasculitis Study Group clinical trials[END_REF]. Leur fréquence pourrait être moindre en cas d'utilisation du rituximab [START_REF] Van Daalen | Effect of rituximab on malignancy risk in patients with ANCA-associated vasculitis[END_REF].

Conclusion

La GPA reste une maladie sévère, chronique, volontiers récidivante. La biopsie de l'artère temporale est de bonne rentabilité chez les malades qui ont des céphalées. Sa pratique systématique n'est pas validée.

Tableau 1 -Caractéristiques de la granulomatose avec polyangéite (Wegener) selon les principales séries de la littérature.

------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- GPA : granulomatose avec polyangéite (Wegener) ; PAM : polyangéite microscopique ; GEPA : granulomatose éosinophilique avec polyangéite (Churg-Strauss).

LEGENDE DES FIGURES

Figure 1 .

 1 Figure 1. Nomenclature révisée de la Conférence de Consensus de Chapel Hill 2012 selon la taille des vaisseaux

Figure 2 .

 2 Figure 2. Nodule pulmonaire excavé chez un patient au cours d'une granulomatose avec polyangéite.

Figure 3 .

 3 Figure 3. Hémorragie alvéolaire sous forme d'opacités alvéolaires et en verre dépoli étendues de façon bilatérale.

Figure 4 .

 4 Figure 4. Traitement des vascularites associées aux ANCA selon le Protocole National de Diagnostic et de Soins

  1. une atteinte rénale ; 2. et/ou une hémorragie alvéolaire importante et évolutive ; 3. et/ou par l'atteinte d'un ou de plusieurs autres organes, en particulier s'il s'agit d'un organe vital, avec des signes généraux ;

2 Le cyclophosphamide représentait le traitement d'induction historique des GPA généralisées/diffuses

  Les bolus de cyclophosphamide sont très utilisés[START_REF] Nachman | Treatment response and relapse in antineutrophil cytoplasmic autoantibody-associated microscopic polyangiitis and glomerulonephritis[END_REF][START_REF] Adu | Controlled trial of pulse versus continuous prednisolone and cyclophosphamide in the treatment of systemic vasculitis[END_REF]. Après 3 perfusions à 15 jours d'intervalle de 0,6 g/m 2 , le cyclophosphamide est espacé toutes les trois semaines à 0,7 g/m 2 jusqu'à obtention d'une rémission complète[START_REF]Vascularites nécrosantes systémiques (périartérite noueuse et vascularites associées aux ANCA)[END_REF]. Habituellement, 6 bolus sont nécessaires en 3 mois avant le relais par le traitement d'entretien. L'administration discontinue intraveineuse permet ainsi à court terme d'obtenir un résultat superposable à celui obtenu grâce au cyclophosphamide par voie orale[START_REF] De Groot | Pulse versus daily oral cyclophosphamide for induction of remission in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized trial[END_REF]. Néanmoins, la tolérance est meilleure par voie veineuse en rapport avec une moindre dose cumulative. Le nombre de rechutes à distance reste élevé et possiblement supérieur en cas d'utilisation par voie veineuse que par voie orale.

	8.1 Traitement d'induction
	8.1.1 Les corticoïdes
	La prednisone, initialement prescrite à 1 mg/kg/j, est parfois précédée d'un ou plusieurs bolus de
	méthylprednisolone (15 mg/kg/j). Après un traitement initial de trois semaines, les corticoïdes sont diminués et
	le risque iatrogène incite à réduire au maximum les doses (19-21), pour une durée totale de 18 à 24 mois. Les
	points de repère essentiels de la corticothérapie sont : environ 20 mg/j à 3 mois, 10 mg/j à 6 mois et 5 mg/j à
	un an (14).

Le contrôle de l'affection nécessite un traitement long. Une des conséquences est la survenue d'effets secondaires fréquents et sévères. Avec les anciens protocoles prolongés de cyclophosphamide per os, le risque de survenue d'un cancer vésical était multiplié par 33, de lymphome par 11 et celui de tumeur solide par 2,4 (2).

Le traitement de la GPA fait maintenant appel à un traitement initial d'induction dont l'objectif est la mise en rémission du patient, obtenue dans les 3 à 6 mois dans l'immense majorité des cas

[START_REF]Vascularites nécrosantes systémiques (périartérite noueuse et vascularites associées aux ANCA)[END_REF]

. Dès que celle-ci est obtenue, un traitement d'entretien moins toxique est alors débuté dont l'objectif est le maintien de cette rémission.

L'étude PEXIVAS, essai randomisé contrôlé, a évalué une corticothérapie allégée en cas d'atteinte rénale ou d'hémorragie alvéolaire chez plus de 700 patients atteints de vascularite associée aux ANCA. Cette étude a montré la non-infériorité du bras allégé de corticoïdes par rapport à un schéma standard pour le contrôle de la vascularite, évalué sur un critère composite associant décès ou évolution vers l'insuffisance rénale définitive

[START_REF] Walsh | The effect of plasma exchange and reduced-dose oral glucocorticoids during induction of remission in patients with severe ANCAassociated vasculitis[END_REF]

. Par contre, le taux d'infections sévères était significativement réduit à 1 an chez les patients ayant reçu une corticothérapie allégée. Depuis cette étude, on a tendance à diminuer encore la dose cumulative des corticoïdes, même si des résultats complémentaires sur le contrôle de la vascularite avec un plus grand recul seront importants à analyser. L'ostéoporose cortisonique doit être combattue par une activité physique régulière, une alimentation riche en calcium, une supplémentation vitamino-calcique et un bisphosphonate, d'autant plus que la masse osseuse est initialement basse.

8.1.

Le cyclophosphamide est utilisé en traitement d'induction, en association avec les corticoïdes. La forme orale est prescrite à 2 mg/kg/j (2) mais est à adapter à l'âge, la fonction rénale, la réponse thérapeutique et la survenue d'effets secondaires.

8.1.3 Le méthotrexate est proposé dans les formes limitées/localisées de GPA

Son efficacité est inférieure à celle du cyclophosphamide. En traitement d'induction, le méthotrexate représente une alternative au cyclophosphamide, dans des formes de GPA ne menaçant le pronostic vital

  . Un délai de 3 mois est parfois nécessaire pour obtenir un effet thérapeutique maximal. Les patients atteints de granulomatose rétro-

	orbitaire ne répondent pas toujours ni ceux avec sténose sous glottique (29,31,32). Un traitement d'entretien
	est nécessaire après obtention d'une rémission. L'étude de la repopulation des cellules B et/ou de la
	réascension des ANCA ne suffit pour définir le meilleur moment pour envisager un retraitement par le
	rituximab (29). Deux études contrôlées ont comparé le rituximab au cyclophosphamide, en traitement
	d'induction, associé à une corticothérapie conventionnelle. Le taux de mise en rémission n'est pas moindre

avec le rituximab qu'avec le traitement classique de cyclophosphamide avec un profil de tolérance comparable à court terme

[START_REF] Stone | Rituximab versus cyclophosphamide for ANCA-associated vasculitis[END_REF][START_REF] Jones | Rituximab versus cyclophosphamide in ANCA-associated renal vasculitis[END_REF][START_REF] Specks | Efficacy of remission-induction regimens for ANCA-associated vasculitis[END_REF]

. Par contre, en cas de rechute, le rituximab est significativement associé à un meilleur taux de mise en rémission que le cyclophosphamide

[START_REF] Specks | Efficacy of remission-induction regimens for ANCA-associated vasculitis[END_REF]

. Il doit donc être privilégié par rapport au cyclophosphamide en traitement d'induction des formes actives sévères chez les patients en rechute ou les femmes en âge de procréer. Une analyse post-hoc a montré que le rituximab serait plus efficace que le cyclophosphamide pour l'obtention d'une rémission chez les patients avec ANCA anti-PR3

[START_REF] Unizony | Clinical outcomes of treatment of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis based on ANCA type[END_REF]

. Il est également très intéressant dans les GPA réfractaires, en cas d'échec d'un traitement d'induction par le cyclophosphamide, où il permet l'obtention d'une rémission dans plus de la majorité des cas

[START_REF] Miloslavsky | for the Rituximab in ANCA-Associated Vasculitis-Immune Tolerance Network Research Group. Clinical outcomes of remission induction therapy for severe antineutrophil cytoplasmic antibody-associated vasculitis[END_REF]

. Une prophylaxie contre la pneumocystose est indispensable. 8.1.5 Le mycophénolate mofétil a fait l'objet d'une étude de non-infériorité contre le cyclophosphamide en traitement d'induction des vascularites associées aux ANCA

3 Le rituximab s'est imposé comme le meilleur traitement d'entretien.

  . Aucun bénéfice n'a été obtenu sous étanercept comme traitement d'entretien ni peut-être d'induction. Par contre, les patients ayant reçu l'etanercept en association avec le cyclophosphamide ont développé davantage de néoplasies solides que ceux ayant reçu le placebo. Il a obtenu une extension d'autorisation de mise sur le marché dans cette indication fin 2018 en Europe et aux USA sur les données de l'étude MAINRITSAN[START_REF] Guillevin | Rituximab versus azathioprine for maintenance in ANCA-associated vasculitis[END_REF]. Il est débuté à la mise en rémission, dans le mois suivant la dernière perfusion de cyclophosphamide ou 4 à 6 mois après le début du traitement d'induction par le rituximab. Administré à 500 mg à J1 et J15 puis tous les 6 mois pendant 18 mois (5 perfusions en 18 mois), il représente une véritable avancée thérapeutique. Dans une étude contrôlée, après mise en rémission par le cyclophosphamide et la corticothérapie, il a permis de diviser par 6 la probabilité de rechute sévère au 28 e mois par rapport à un traitement d'entretien classique d'azathioprine poursuivi pendant 22 mois[START_REF] Guillevin | Rituximab versus azathioprine for maintenance in ANCA-associated vasculitis[END_REF]. La diminution du taux de rechute se maintient à 5 ans[START_REF] Terrier | Long-term efficacy of remission-maintenance regimens for ANCA-associated vasculitides[END_REF]. Il est aussi possible d'administrer le rituximab, uniquement en cas d'augmentation du titre des ANCA ou de repopulation des lymphocytes B, sans majorer significativement le nombre de rechutes par rapport à une perfusion systématique classique semestrielle[START_REF] Charles | for the French Vasculitis Study Group : Comparison of individually tailored versus fixed-schedule rituximab regimen to maintain ANCAassociated vasculitis remission: Results of a multicentre, randomised controlled, phase III trial (MAINRITSAN2)[END_REF]. Il est probable que

	8.2.
	8.2.2 Le méthotrexate représente aussi un traitement d'entretien possible, à 0,3 mg/kg/semaine, après
	obtention d'une rémission (43-46). Il permet de maintenir la rémission dans plus de 80 % des cas (43) mais les
	rechutes après l'arrêt s'observent encore chez plus de la moitié des patients (45). Dans le seul essai
	randomisé contrôlé, l'azathioprine et le méthotrexate ont montré une efficacité comparable en traitement
	d'entretien pendant 12 mois, après mise en rémission obtenue par corticothérapie et bolus de
	cyclophosphamide (46). Les résultats sont aussi comparables après 10 ans de suivi (8).

8.2 Traitements d'entretien 8.2.1 L'azathioprine était le traitement d'entretien classique de première intention. Il est efficace et bien toléré. Après obtention d'une rémission sous cyclophosphamide et corticoïdes, il n'y a pas plus de rechutes à 18 mois en cas de relais par l'azathioprine qu'en cas de poursuite du cyclophosphamide (21). L'azathioprine induit moins d'effets secondaires à long terme que le cyclophosphamide. La posologie initiale est de 2 mg/kg/j. Les rechutes restent fréquentes, en général après arrêt de l'azathioprine. Elles sont moins fréquentes lorsque la durée du traitement immunosuppresseur est de 4 ans après le diagnostic par rapport à un traitement d'entretien interrompu à 2 ans (13). la perfusion du J15 ne soit pas indispensable et que 4 perfusions administrées en 18 mois suffisent (49). Dans la pratique, après un traitement d'induction par le rituximab, son efficacité et sa tolérance comme traitement d'entretien de la GPA sont également satisfaisantes à moyen terme : à deux ans du diagnostic ou de la rechute, le taux de survie sans rechute est de 85 % et le taux de maintien thérapeutique du rituximab est de 78 % (32).

8.

2.4 Le leflunomide ou le mycophénolate mofétil ne sont pas prescrits en première intention. Ils

  ont été essayés chez un nombre limité de patients. Le mycophénolate mofétil s'est révélé moins efficace que l'azathioprine pour le maintien de la rémission au cours d'un essai contrôlé[START_REF] Hiemstra | Mycophenolate mofetil vs azathioprine for remission maintenance in antineutrophil cytoplasmic antibody-associated vasculitis: a randomized controlled trial[END_REF]. Leur utilisation ne peut pas être recommandée en première intention.

  Toutefois, grâce à un traitement adapté et prolongé, son pronostic s'est amélioré et le taux de survie de la maladie atteint maintenant 75 % à 10 ans. X.P. est investigateur d'essais thérapeutiques académiques évaluant le rituximab dans les vascularites associées aux ANCA, au cours desquels le rituximab est fourni par Roche. X.P. est investigateur d'essais Quels sites faut-il biopsier pour permettre le diagnostic ? La biopsie pulmonaire peut concerner un nodule ou le parenchyme d'un malade ayant présenté une hémorragie alvéolaire. La biopsie des nodules montre le granulome palissadique au sein duquel les vaisseaux présentent une vascularite artériolaire. En cas d'hémorragie alvéolaire, on trouve essentiellement une capillarite. La biopsie pulmonaire est orientée par vidéoscopie. La thoracotomie est un geste lourd, dont la mortalité et la morbidité ne sont pas négligeables. On peut donc discuter l'opportunité d'un tel geste, notamment chez un malade qui présente un tableau clinique très évocateur et des ANCA de type cytoplasmique anti-PR3 en ELISA.La biopsie naso-sinusienne est simple à réaliser mais nécessite une bonne technique, avec des prélèvements nombreux, en zone granulomateuse, plus qu'en zone de nécrose. Sa rentabilité est plus faible que la biopsie pulmonaire. La biopsie rénale. Elle doit être faite dès qu'il y a des anomalies du sédiment urinaire, une protéinurie et/ou une altération, même modérée de la fonction rénale. Elle permet de rechercher la glomérulonéphrite extracapillaire et les signes directs de la vascularite. Elle apprécie le degré de l'atteinte parenchymateuse et des lésions fibreuses déjà constituées ainsi qu'une éventuelle composante de nécrose tubulaire associée, permettant ainsi de donner des éléments pronostiques de récupération.La biopsie musculaire ou neuro-musculaire : utile et très rentable lorsque le malade présente une neuropathie périphérique.

	Encadré I.
	thérapeutiques évaluant l'avacopan (ChemoCentryx) et l'IFX 1 (InflaRx) dans les vascularites associées aux
	ANCA. X.P. a été investigateur d'essais thérapeutiques évaluant le mepolizumab dans la granulomatose
	éosinophilique avec polyangéite (GSK). X.P. a bénéficié d'honoraires pour des missions d'expertise (Sanofi),
	des présentations scientifiques (Boehringer Ingelheim) et de frais de participation/inscription à des congrès
	(Sanofi).
	Un des problèmes majeurs est lié à la survenue d'effets secondaires des traitements, dont la gravité et la
	fréquence sont souvent la conséquence d'une prolongation du traitement difficilement évitable.
	Conflits d'intérêts :

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