Ph.D Nicolas Charles
email: nicolas.charles@inserm.fr

Ph.D Juan Rivera
email: juan_rivera@nih.gov

Charles & Rivera

Basophils and Autoreactive IgE in the Pathogenesis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a heterogeneous disease that can affect multiple organs.

A hallmark of this disease, like for other autoimmune diseases, is the presence of large amounts of autoantibodies. As such SLE is considered to be a B cell disease perpetuated by the expansion of autoreactive T and B cells. The T cells involved have long been considered to be Th1 and Th17 cells as these potent pro-inflammatory cells can be found in the tissues of SLE patients.

Recent advances point to a role for the Th2 environment in contributing to SLE through promotion of autoantibody production. Here we describe the recent work focusing on autoreactive IgE and the activation of basophils as promoting the production of autoantibodies in SLE. The findings, both in a mouse model of SLE and in human SLE subjects, support the concept that the activation of the basophil by autoreactive IgE-containing immune complexes serves to amplify the production of autoantibodies and contributes to the pathogenesis of disease.

We propose that therapeutic targeting of this amplification loop by reducing the levels of circulating autoreactive IgE may have benefit in SLE.

Introduction

Systemic Lupus Erythematosus (SLE) is a complex, multifactorial, autoimmune disease that can affect multiple organs [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. SLE is heterogeneous both in symptoms and in which target organs may be involved with damage occurring in the central nervous system (CNS), kidney, heart, skin, joints and vessels. It is well recognized that tissue damage is associated with immune complexes deposition and chronic inflammation [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. The immune complexes formed are generally comprised of auto-reactive antibodies, auto-antigens and complement components [START_REF] Rahman | Systemic lupus erythematosus[END_REF].

In SLE, most of the auto-reactive antibodies are raised against nuclear components. This selfimmunization has at its origin the loss of tolerance due to environmental and/or genetic factors that promote cell death and release of nucleosomal components that are a source of self-antigens.

The loss in tolerance is exacerbated through increased numbers of self-reactive T cells and B cells, ultimately leading to the persistent and prolific production of autoantibodies against double stranded DNA (dsDNA), nucleosomal proteins (Ro, La, Sm), neurotransmitter receptors (N methyl D aspartate (NMDA) receptors), plasma membrane components (phospholipids), cytoskeleton associated proteins (α-actinin), or complement components (C1q) [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. These autoreactive antibodies (which can be of IgA, IgM, and IgG subclasses) form circulating immune complexes (CIC) in the periphery when they encounter their self-target [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. They can deposit into organs, irrespective of the particular isotype of auto-reactive antibody. As a direct consequence, chronic inflammation (with inflammatory cells infiltrates and pro-inflammatory cytokine production) is established leading to symptoms of disease and tissue damage: i.e, cognitive impairment and hippocampal damage in the CNS, nephritis in the kidney, skin rashes, arthritis in the joints and fetal heart block in pregnant women [START_REF] Rahman | Systemic lupus erythematosus[END_REF].

As many autoimmune diseases, SLE has no specific treatment nor early diagnostic tools allowing disease prevention, disease control or definitive healing. Strong immunosuppressive therapy is still the preferred manner to temporarily silence the disease, with all of its accompanying side effects [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. SLE affects about 1 person in 2,500 in northern Europe and over 1 in 1,000 in the United States, thus lupus prevention and treatment is an important international challenge. Environmental and/or genetic factors as contributors to development or severity of disease are evident but their roles are poorly understood. For example, approximately 90% of SLE patients are child-bearing aged women and incidence of disease is 10 fold higher in African American women than in women of northern European. In some geographic areas within the US, the disease can affect 1 out of every 200 people [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. Thus, how genetics and environment contribute remains an enigma whose resolution may well advance treatment of this disease.

The immunological basis of SLE has allowed considerable exploration on the factors and types of immune cells involved in its pathogenesis. Animal models (mainly mouse models with some features of human disease) have allowed the study of the contribution of particular T cell subsets, B cells, monocytes, and dendritic cells in the development of lupus-like disease ( [START_REF] Perry | Murine models of systemic lupus erythematosus[END_REF]).

These models have been useful in defining that the pathogenesis of disease lies in the loss of tolerance in the T and B cell compartments [START_REF] Perry | Murine models of systemic lupus erythematosus[END_REF][START_REF] Sobel | The major murine systemic lupus erythematosus susceptibility locus Sle1 results in abnormal functions of both B and T cells[END_REF]. B cells themselves were shown to be essential for manifestation of the disease [START_REF] Chan | The central and multiple roles of B cells in lupus pathogenesis[END_REF]. Studies in human SLE subjects have also confirmed that dysregulation of tolerance in these cellular compartments is a hallmark of disease [START_REF] Lipsky | Systemic lupus erythematosus: an autoimmune disease of B cell hyperactivity[END_REF]. These advances in an immunological understanding of disease has led to a number of clinical trials aiming to disrupt the production of auto-antibodies by targeting B cells [START_REF] Calero | Targeting B cells for the treatment of SLE: the beginning of the end or the end of the beginning?[END_REF][START_REF] Navarra | Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomised, placebocontrolled, phase 3 trial[END_REF][START_REF] Looney | B cell-targeted therapies for systemic lupus erythematosus: an update on clinical trial data[END_REF]. Interestingly, depletion of B cells with an anti-CD20 monoclonal antibody (Rituximab, Rituxan®) did not show increased efficacy in alleviating refractory disease in a phase III clinical trial [START_REF] Looney | B cell-targeted therapies for systemic lupus erythematosus: an update on clinical trial data[END_REF]. In contrast, clinical trials aiming to disrupt B cell activation by B cell activating factor (BAFF) through the use of another monoclonal antibody (Belimumab, Benlysta®) showed more encouraging results [START_REF] Navarra | Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomised, placebocontrolled, phase 3 trial[END_REF][START_REF] Looney | B cell-targeted therapies for systemic lupus erythematosus: an update on clinical trial data[END_REF]. However, it should be noted that the criterion used for determining efficacy in these two clinical trials were markedly different. BAFF (or Blys, for B lymphocyte stimulator) is a member of the TNF ligand superfamily. This cytokine is able to activate B cells to mature towards antibody-secreting plasma cells and to proliferate. This cytokine is found in higher titers in the periphery of SLE patients [START_REF] Davidson | Targeting BAFF in autoimmunity[END_REF]. Thus, while some of these trials have shown some promise, it is clear that such strategies are not a panacea for all individuals with SLE and continued efforts towards gaining a further understanding of the contributing factors in SLE are essential.

Herein, we present novel insights on the underlying mechanisms contributing to SLE pathogenesis. Whereas the involvement of Th1 and Th17 cells and cytokines in SLE are well documented [START_REF] Peng | T-bet regulates IgG class switching and pathogenic autoantibody production[END_REF][START_REF] Mangini | Type I interferons inhibition of inflammatory T helper cell responses in systemic lupus erythematosus[END_REF], the contribution of the Th2 environment was poorly understood. Our recent work has demonstrated that basophils and auto-reactive IgE play a role in SLE pathogenesis [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF]. This finding, and the demonstrated interruption of SLE pathogenesis through inhibiting production of IgE or activation of basophils in a mouse model of spontaneous SLE, provides a novel strategy with therapeutic promise in disease intervention.

SLE: a B cell disease that includes IgE autoantibody production

As mentioned in the introduction, most of the pathologic features of SLE are linked to the levels and types of auto-reactive antibodies present in the serum of SLE patients. Disease activity is associated with the levels of autoantibodies. These auto-reactive antibodies are produced by plasma cells derived from auto-reactive B cells. Minimal data is available on how tolerance is broken leading to the accumulation of auto-reactive lymphocytes. However, there is considerable evidence that the innate immune response is somehow involved [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. The presence of an interferon alpha (IFNα) signature in a large proportion of SLE patients corroborates the activation of an innate immune response [START_REF] Ronnblom | The innate immune system in SLE: type I interferons and dendritic cells[END_REF]. Studies in both mouse models and human subjects also suggest a defect in T regulatory cells [START_REF] Monk | MRL/Mp CD4+,CD25-T cells show reduced sensitivity to suppression by CD4+,CD25+ regulatory T cells in vitro: a novel defect of T cell regulation in systemic lupus erythematosus[END_REF][START_REF] Miyara | Global natural regulatory T cell depletion in active systemic lupus erythematosus[END_REF]. Nonetheless, it is well accepted that SLE is primarily a B cell disease and that escape from negative selection of auto-reactive B cells is the key element leading to the production of auto-reactive antibodies that results in the presence of circulating immune complexes able to deposit in the targeted organ [START_REF] Rahman | Systemic lupus erythematosus[END_REF].

While autoantibody involvement in SLE pathogenesis is well documented, the relevance of antibody isotypes is not well understood. It is widely accepted that circulating immune complexes and immune complexes deposits contain IgM, IgG and IgA auto-reactive antibodies [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. Several studies [START_REF] Nagpal | Demonstration of IgE antibodies to nucleic acid antigens in patients with SLE[END_REF][START_REF] Permin | The prevalence of IgE antinuclear antibodies in rheumatoid arthritis and systemic lupus erythematosus[END_REF][START_REF] Atta | Immunoglobulin E and systemic lupus erythematosus[END_REF] have also shown the presence of auto-reactive IgE in such immune complexes. While IgG autoantibodies have been intimately linked to tissue damage and the pathology of SLE, there is also evidence for the involvement of IgM and IgA in lupus nephritis [START_REF] Rahman | Systemic lupus erythematosus[END_REF][START_REF] Oxelius | Immunoglobulin G (IgG) subclasses and human disease[END_REF][START_REF] Kingsmore | Measurement of circulating immune complexes containing IgG, IgM, IgA and IgE by flow cytometry: correlation with disease activity in patients with systemic lupus erythematosus[END_REF]. In some SLE patients high levels of IgE have been observed [START_REF] Atta | Immunoglobulin E and systemic lupus erythematosus[END_REF][START_REF] Elkayam | Serum IgE concentrations, disease activity, and atopic disorders in systemic lupus erythematosus[END_REF], whereas in others IgE levels were normal. Interestingly, whether allergy is associated with lupus is controversial and while a few reports [START_REF] Sequeira | Allergic disorders in systemic lupus erythematosus[END_REF] have linked these diseases there is also considerable evidence to the contrary [START_REF] Atta | Immunoglobulin E and systemic lupus erythematosus[END_REF][START_REF] Wozniacka | Allergic diseases, drug adverse reactions and total immunoglobulin E levels in lupus erythematosus patients[END_REF][START_REF] Sekigawa | Allergic diseases in systemic lupus erythematosus: prevalence and immunological considerations[END_REF]. Thus, little was known about the role of IgE in SLE. As we describe in more detail below, a key findings is the presence of auto-reactive IgE in a mouse model of spontaneous SLE and in a large proportion of SLE patients [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF]. Importantly, the levels of auto-reactive IgE was associated with disease severity. This unexpected finding provided a strong impetus for deciphering the role of auto-reactive IgE in lupus.

The Basophil

Basophils are rare immune cells (<1% of circulating leukocytes) well known for their involvement in allergic reactions and parasite infections [START_REF] Karasuyama | Nonredundant roles of basophils in immunity[END_REF] . Their ability to produce large amounts of Th2 promoting cytokines such as interleukin 4 (IL-4) and thymic stromal lymphopoietin (TSLP) provides them the ability to serve as immune regulators, able to influence T and B cells to promote and produce antibodies, respectively [START_REF] Karasuyama | Nonredundant roles of basophils in immunity[END_REF]. Recent studies showed that basophils are able to regulate humoral memory responses by producing IL-4 and IL-6 [START_REF] Denzel | Basophils enhance immunological memory responses[END_REF]. They were also shown to induce Th2 differentiation of naïve CD4 T cells in vivo [START_REF] Oh | Induction of Th2 type immunity in a mouse system reveals a novel immunoregulatory role of basophils[END_REF][START_REF] Hida | Negative control of basophil expansion by IRF-2 critical for the regulation of Th1/Th2 balance[END_REF][START_REF] Sokol | A mechanism for the initiation of allergeninduced T helper type 2 responses[END_REF][START_REF] Charles | Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation[END_REF], present antigen as professional antigen presenting cells (APCs) through expression of MHC class II [START_REF] Perrigoue | MHC class II-dependent basophil-CD4+ T cell interactions promote T(H)2 cytokine-dependent immunity[END_REF][START_REF] Yoshimoto | Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells[END_REF][START_REF] Sokol | Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response[END_REF], to promote plasma cells survival [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF][START_REF] Gomez | Basophils support the survival of plasma cells in mice[END_REF], and to organize the recruitment of other immune cells in an IgE-mediated chronic allergic inflammation model [START_REF] Mukai | Basophils play a critical role in the development of IgE-mediated chronic allergic inflammation independently of T cells and mast cells[END_REF]. Whether basophils exclusively play these roles is uncertain. For example, basophil-deficient mice were recently shown to mount normal Th2 responses as well as normal humoral responses [START_REF] Ohnmacht | Basophils orchestrate chronic allergic dermatitis and protective immunity against helminths[END_REF] suggesting that basophils are dispensable for these responses. However, one must be careful in overinterpreting experiments based on the use of a particular challenge or model. On the other hand, it is also likely that basophils do not replace dendritic cells as initiators of Th2 responses and, in fact, evidence has been presented for collaboration of these two cell types in initiating Th2 responses [START_REF] Tang | The T helper type 2 response to cysteine proteases requires dendritic cell-basophil cooperation via ROS-mediated signaling[END_REF]. Thus, it is likely that the basophil's role as an immunoregulatory cell may well be context dependent, where in some settings it may play a dominant role and in others it may be less important.

Lyn Kinase: a molecular rheostat in the development of lupus

Lyn is a Src family tyrosine kinase (SFK) that functions to promote or inhibit molecular signals downstream of multiple receptors including cytokine receptors (such as IL-4R or IL-3R), growth factor tyrosine kinase receptors (such as Kit or CD117) or Fc receptors (such as the high affinity IgE receptor, FcεRI). This kinase is expressed in the hematopoietic compartment [START_REF] Hibbs | The duplicitous nature of the Lyn tyrosine kinase in growth factor signaling[END_REF] but is not known to be expressed by T cells [START_REF] Yamanashi | Selective expression of a protein-tyrosine kinase, p56lyn, in hematopoietic cells and association with production of human Tcell lymphotropic virus type I[END_REF]. B cells express Lyn kinase and it serves as a key component of the B cell antigen receptor (BCR) through its ability to phosphorylate this receptor and initiate signal transduction upon BCR engagement [START_REF] Defranco | Positive and negative roles of the tyrosine kinase Lyn in B cell function[END_REF]. Interestingly, however, mice deficient in Lyn kinase show a B cell hyperreactivity to IL-4 and CD40L stimulation [START_REF] Janas | Genetic evidence for Lyn as a negative regulator of IL-4 signaling[END_REF][START_REF] Wang | Altered antigen receptor signaling and impaired Fas-mediated apoptosis of B cells in Lyn-deficient mice[END_REF], whereas BCR activation of Lyn-deficient B cells is impaired. Interestingly, mast cells [START_REF] Parravicini | Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation[END_REF][START_REF] Odom | Negative regulation of immunoglobulin Edependent allergic responses by Lyn kinase[END_REF][START_REF] Yamashita | Cutting edge: genetic variation influences Fc epsilonRI-induced mast cell activation and allergic responses[END_REF] and monocytes [START_REF] Baran | The inositol 5'-phosphatase SHIP-1 and the Src kinase Lyn negatively regulate macrophage colony-stimulating factorinduced Akt activity[END_REF](and more recently basophils [START_REF] Charles | Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation[END_REF]) from Lyn deficient mice have also been shown to be hyperreactive to various stimuli, thus demonstrating that Lyn kinase plays an important role in controlling cell homeostasis. It is now well recognized that this is mediated, to a large extent, through the requirement for Lyn kinase in the phosphorylation and activation of various phosphatases [START_REF] Hibbs | The duplicitous nature of the Lyn tyrosine kinase in growth factor signaling[END_REF] whose activity is important in controlling immune cell homeostasis.

Importantly, too much Lyn activity also has undesired immunological consequence and has been shown to promote uncontrolled monocytic proliferation and tumor development [START_REF] Harder | Gain-and loss-of-function Lyn mutant mice define a critical inhibitory role for Lyn in the myeloid lineage[END_REF] as well as mast cell activation [START_REF] Odom | Negative regulation of immunoglobulin Edependent allergic responses by Lyn kinase[END_REF]. Thus, the amount of Lyn kinase activity must be well controlled and it appears that Lyn kinase serves as a rheostat in controlling immune cell homeostasis.

As they age (about 12-14 weeks of age), Lyn kinase deficient mice begin to produce auto-reactive antibodies against nuclear components, such as double stranded DNA and other anti-nuclear antigens. These mice develop an autoimmune phenotype with some similarities to human lupus including the development of lupus nephritis. As early as 20 weeks of age, Lyn deficient mice show glomerular deposits of anti-dsDNA IgG antibodies and circulating immune complexes. They develop impaired renal function and ultimately die of renal failure [START_REF] Hibbs | Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease[END_REF][START_REF] Nishizumi | Impaired proliferation of peripheral B cells and indication of autoimmune disease in lyn-deficient mice[END_REF].

Interestingly, it has been shown that Lyn expression is decreased in peripheral B cells of some SLE patients [START_REF] Flores-Borja | Decreased Lyn expression and translocation to lipid raft signaling domains in B lymphocytes from patients with systemic lupus erythematosus[END_REF] and recently a single nucleotide polymorphism in the promoter region of the Lyn gene was linked with SLE patients of northern European heritage [START_REF] Lu | This work identifies single nucleotide polymorphisms in the LYN gene of some SLE patients, which is associated with the levels of autoantibodies[END_REF]. Of particular note, the described polymorphism was associated with autoantibody production. Thus, while yet undefined, the role of this Lyn polymorphism in the development of SLE is of considerable interest. Nonetheless, in animal models, the role of Lyn in controlling B cell responsiveness (and thus antibody production) is well appreciated; less is known about its contributory role in other cellular compartments that may also contribute in the pathogenesis of SLE.

Lyn kinase and basophils: Regulation of the Th2 environment

It has long been recognized that the high affinity IgE receptor (FcεRI) requires the activity of Lyn kinase to transmit signals that promote the effector responses of mast cells and basophils [START_REF] Eiseman | Engagement of the high-affinity IgE receptor activates src protein-related tyrosine kinases[END_REF]. This kinase phosphorylates FcεRI and initiates signal transduction, but it also has both positive and negative regulatory roles downstream of FcεRI phosphorylation [START_REF] Odom | Negative regulation of immunoglobulin Edependent allergic responses by Lyn kinase[END_REF]. Multiple studies have demonstrated that Lyn kinase has a dominant role as a negative regulator of cellular homeostasis [START_REF] Hibbs | The duplicitous nature of the Lyn tyrosine kinase in growth factor signaling[END_REF] and in mast cells its absence was shown to cause enhanced mast cell degranulation and cytokine production [START_REF] Odom | Negative regulation of immunoglobulin Edependent allergic responses by Lyn kinase[END_REF]. Strikingly, Lyn -/-mice developed an early life atopic-like allergic phenotype where high levels of IgE are produced and circulating histamine can be detected as well as an eosinophilia [START_REF] Parravicini | Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation[END_REF][START_REF] Odom | Negative regulation of immunoglobulin Edependent allergic responses by Lyn kinase[END_REF]. Additional studies demonstrated that these mice had an exacerbated response to challenge in an asthma model [START_REF] Beavitt | Lyn-deficient mice develop severe, persistent asthma: Lyn is a critical negative regulator of Th2 immunity[END_REF] and were generally hypersensitive to a Th2 challenge but also mounted a Th2 response to normally innocuous substances [START_REF] Charles | Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation[END_REF]. A significant proportion of CD4 + T cells from the spleen of Lyn -/-mice spontaneously produced IL-4. This Th2 bias was dependent on IL-4 and IgE since the deletion of these genes in the context of Lyn -/-mice completely ablated their Th2 skewing [START_REF] Charles | Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation[END_REF].

Running Title: Basophils in Lupus. Charles & Rivera

Since Lyn is not expressed in T cells, our initial bias was that mast cells might be driving this unexpected Th2 skewing in Lyn -/-mice. To test this possibility, mast cell deficient mice (Kit w-sh/w-sh ) were crossed to Lyn -/-mice. However, the resulting mice (Kit w-sh/w-sh Lyn -/-) showed no alteration of the Th2 bias [START_REF] Charles | Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation[END_REF]. This meant that the Th2 bias was initiated by another cell type able to produce substantial amounts of IL-4 to induce naïve CD4 + T cells to differentiate to Th2 cells. Analysis of other cell types demonstrated that Lyn -/-mice had a peripheral basophilia (up to 4 times the amount of circulating basophils when compared to wild type (WT) mice). In addition, Lyn -/-basophils could produce high levels of IL-4 being hyperresponsive to FcεRI stimulation. To assess the role of these basophils in causing Th2 skewing in the absence of Lyn kinase, we first activated basophils in vivo in the Kit w-sh/w-sh Lyn -/-double deficient mice by FcεRI stimulation. In the absence of mast cells or immune challenges, basophils are the principal cells that express FcεRI in the mouse. FcεRI stimulation of Kit w-sh/w-sh Lyn -/-double deficient mice revealed a marked increase in the Th2 skewing of these mice with as much as 25% of the CD4 + T cells in the spleen producing IL-4. The inverse experiment where basophils were depleted from Lyn -/-mice with a monoclonal antibody (clone MAR-1, anti-FcεRIα [START_REF] Sokol | A mechanism for the initiation of allergeninduced T helper type 2 responses[END_REF]) showed a complete rescue of the Th2 skewing, clearly demonstrating that basophil was promoting the Th2 bias seen in these mice. Of particular note, the CD4 + T cells from spleens of Lyn -/-mice constitutively produced only Th2 cytokines, no IFN-γ was detected in these cells. Moreover, Lyn -/-mice failed to effectively fight an infection with the prototypic Th1 parasite Toxoplasma Gondii, which requires IFN-γ production for survival from infection. Measurement of IFN-γ levels demonstrated that Lyn -/-mice did not mount a normal Th1 response. However, it should be noted that in vitro restimulation of CD4 + T cells from Lyn -/-mice with PMA and ionomycin caused increased IFN-γ production (as well as IL-4 and IL-13) when compared to WT cells [START_REF] Charles | Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation[END_REF].

This suggests that T cells from these mice are primed by the Lyn -/-environment in a manner that can result in potent Th1 or Th2 responses.

The mechanism by which basophils can induce the IL-4 production necessary for the potent Th2 skewing of Lyn -/-mice was explored in some detail. In mast cells, the hyperresponsiveness observed upon Lyn deficiency is, at least in part, through increased Fyn kinase activity [START_REF] Parravicini | Fyn kinase initiates complementary signals required for IgE-dependent mast cell degranulation[END_REF][START_REF] Yamashita | Cutting edge: genetic variation influences Fc epsilonRI-induced mast cell activation and allergic responses[END_REF][START_REF] Hernandez-Hansen | Dysregulated FcepsilonRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells[END_REF] and the loss of SHIP activity [START_REF] Hernandez-Hansen | Dysregulated FcepsilonRI signaling and altered Fyn and SHIP activities in Lyn-deficient mast cells[END_REF]. As these molecules regulate the activity or the amount of product, respectively, of phosphatidylinositide 3-OH kinase (PI3K), a kinase that generates phosphatidylinositide 3,4,5-trisphosphate (PIP 3 ), loss of regulatory control on PIP 3 production in these cells causes their hyperresponsiveness [START_REF] Damen | SHIP's C-terminus is essential for its hydrolysis of PIP3 and inhibition of mast cell degranulation[END_REF]. However, whether this mechanism also provided the underpinnings of the hyperresponsiveness of basophils was not known. Studies to address the role of PI3K in basophils revealed that its activity was necessary for the increased expression of GATA3 (a transcription factor known to regulate IL-4 production in T cells) in Lyn -/-basophils. Lyn -/-basophils showed a marked increase in GATA3 expression relative to their WT counterparts and this (as well as IL-4 production) was inhibited by pharmacological inhibition of PI3K activity [START_REF] Charles | Lyn kinase controls basophil GATA-3 transcription factor expression and induction of Th2 cell differentiation[END_REF]. In addition, basophils from Fyn -/-mice failed to upregulate GATA3 and were impaired in the production of IL-4. Thus, the Th2 skewing was linked to a Fyn/PI3K-dependent increased induction of GATA3 in Lyn -/-basophils.

The clear dominance of a Th2 environment in Lyn -/-mice and the late life development of a spontaneous lupus-like disease provided a good model system to explore the relationship between the Th2 environment and the development of a lupus-like phenotype.

Is there a link between the Th2 environment and development of a lupus-like phenotype?

Removing IgE or IL-4 genes in the context of the Lyn -/-background led to the reversion of the Th2 bias seen in Lyn -/-mice. These double deficient mice provided a model to ask the question of whether the Th2 bias was linked to the development of a lupus-like disease. Aging of such mice for greater than 35 weeks along with Lyn -/-mice revealed that the penetrance of disease was almost 100% in the latter mice whereas IgE/Lyn or IL-4/Lyn double deficient mice showed little signs of kidney disease. Indeed, the double deficient mice had a greater than 50% reduction in autoantibody titers and immune complex deposition in the glomeruli was markedly reduced with normal albumin/creatinine levels in the urine. Analysis of the extent of glomerular nephritis by scoring immune infiltrates, mesangial proliferation, morphology of the glomeruli, and other parameters revealed little evidence of kidney disease and damage. Thus, it was evident that inhibiting the Th2 skewing of Lyn -/-mice by depletion of IL-4 or IgE greatly reduced autoantibody production and ablated the development of overt kidney disease and damage [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF]. This unexpected result led us to question if basophils were contributing to the development of the lupus-like phenotype seen in Lyn -/-mice.

Basophils and autoreactive IgE in the development of lupus.

Evidence from the Lyn -/-mouse model

At the time of this work basophil deficient mice were not available. Thus, once again, we depleted basophils with the anti-FcεRIα (MAR-1 antibody) and found that basophils were needed to maintain high autoantibody (IgM, IgG, IgA) titers in the circulation of Lyn -/-mice.

Depletion of basophils caused a marked decrease in anti-ANA IgG and anti-dsDNA IgG antibodies within 6 days post-treatment. This was somewhat unexpected given the long half-life of antibodies in the blood, although immune complexes are cleared more rapidly than normal immunoglobulins by binding to cells. Nonetheless, we hypothesized that the role of basophils might extend to the support of plasma cell survival and function and in this manner could more rapidly influence control on antibody levels. This view was supported by the recent finding of others [START_REF] Denzel | Basophils enhance immunological memory responses[END_REF] showing that basophils contribute to humoral memory responses through their production of IL-4 and IL-6. Within 6 days of basophil depletion, the numbers of plasma cells (likely plasmablasts) in the spleen of Lyn -/-mice decreased by greater than 50%. These findings showed the direct involvement of basophils in supporting plasma cells in the spleen of Lyn -/- mice that can produce autoantibodies [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF]. Our findings that basophil support plasma cell survival and antibody production has recently been confirmed by others [START_REF] Gomez | Basophils support the survival of plasma cells in mice[END_REF].

Given that Lyn -/-mice had a peripheral basophilia and produced high levels of IL-4, IL-6 and TSLP, we asked: How do these basophils become activated? Our previous findings demonstrated that Lyn -/-basophils were hyper-reactive to stimulation via FcεRI. Moreover, given that IL-4/Lyn and IgE/Lyn double deficient mice (which are not Th2 skewed) did not produce high levels of auto-reactive antibodies or CIC's to induce kidney disease, we reasoned that in this model, IgE and the FcεRI were involved in the activation of the basophils and pathogenesis of disease. In part our reasoning was based on the prior description of the presence of autoreactive IgE in the circulation of SLE patients [START_REF] Atta | Immunoglobulin E and systemic lupus erythematosus[END_REF][START_REF] Kingsmore | Measurement of circulating immune complexes containing IgG, IgM, IgA and IgE by flow cytometry: correlation with disease activity in patients with systemic lupus erythematosus[END_REF]. Analysis of Lyn -/-mice for autoreactive IgE in the circulation revealed the presence of anti-dsDNA and anti-ANA autoantibodies of the IgE isotype. Compared to WT mice, large amounts of anti-ANA and anti-dsDNA IgE were found in the sera of Lyn -/-mice. Moreover, the presence of large amounts of CIC containing IgE was also detected by western blot. We then tested if IgE-containing immune complexes could induce IL-4 production from WT and Lyn -/-basophils and found that indeed these immune complexes could effectively activate both WT and Lyn -/-basophils, with the latter producing higher amounts of IL-4. In contrast, immune complexes that contained only IgG failed to induce IL-4 production from basophils of both genotypes. These findings provided a mechanistic explanation on how basophils might be continuously activated in the periphery of Lyn -/-mice.

The support of autoantibody production would require that activated basophils be in the appropriate environment to support T and/or B cell function. This would require their migration to the secondary lymphoid organs. To assess their ability to migrate to the secondary lymphoid organs (such as the lymph nodes and spleen), we measured the levels of CD62L (L-selectin) on the surface of basophils from WT and Lyn -/-mice. CD62L is recognized as a necessary molecule for leukocyte rolling and recruitment from blood stream to tissues. Measurement of CD62L expression on Lyn -/-basophils revealed a marked increase in its expression relative to WT basophils. Under normal conditions (in aged WT mice), the numbers of basophils in the lymph nodes are very small and difficult to detect. In contrast, aged Lyn -/-mice had very high numbers of basophils in peripheral lymph nodes as well as in the spleen. This placed the basophil in the appropriate environment where it could influence T and B cell function. However, the question as to whether the Lyn -/-basophil expressed the appropriate molecules to interact with T and B cells remained to be answered. Recently, in both mouse and human basophils, it has been described [START_REF] Perrigoue | MHC class II-dependent basophil-CD4+ T cell interactions promote T(H)2 cytokine-dependent immunity[END_REF][START_REF] Yoshimoto | Basophils contribute to T(H)2-IgE responses in vivo via IL-4 production and presentation of peptide-MHC class II complexes to CD4+ T cells[END_REF][START_REF] Sokol | Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response[END_REF] that these cells can express molecules of the major histocompatibility complex II (MHC-II). Whereas circulating basophils from Lyn -/-mice expressed low levels of MHC-II molecules on their surface, the basophils found in the lymph nodes of these mice were expressing high levels of MHC-II. The antigen being presented by these basophils remains an enigma, however, one might speculate that autoantigenic epitopes may well reside on the surface of these cells. Thus, these findings provide evidence that the basophil is capable of antigen presentation, since the appropriate expression of MHC-II requires it's assembly in the context of the antigenic peptide epitope [START_REF] Harding | Cellular and molecular aspects of antigen processing and the function of class II MHC molecules[END_REF]. In addition, however, a membrane-bound form of BAFF (B cell activating factor) was also detected on the surface of Lyn -/-basophils found in the lymph nodes, as has been described for human basophils in other chronic inflammatory diseases [START_REF] Chen | Immunoglobulin D enhances immune surveillance by activating antimicrobial, proinflammatory and B cell-stimulating programs in basophils[END_REF].

Interestingly, this was independent of increased BAFF receptor expression suggesting that the expressed BAFF was not bound to the receptor. Thus, these findings show that Lyn -/-basophils home to the secondary lymphoid organs and are fully equipped to support T and B cells at these sites. This is consistent with our finding that basophils support plasma cells in the spleen of Lyn - /-mice [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF]. Given that such plasmablasts require T cells for their expansion, our data suggests that basophils are also providing help to T cells in secondary lymphoid tissues.

Given the marked impairment of autoantibody production by the depletion of basophils, we asked whether the absence of basophils had any bearing on the kidney inflammation of Lyn -/- mice during the manifestation of lupus nephritis. Within 6 days of basophil depletion a marked reduction was observed in the pro-inflammatory cytokines (IL-4, IFN-γ, IL-1β, MCP-1, TNF-α and IL-6) in the kidneys of these mice [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF]. This showed that beyond their role as contributors to autoantibody production, the basophil also contributes to the pathogenesis of lupus nephritis by promoting inflammation.

The data obtained through the study of Lyn -/-mice demonstrates that in this model of SLE, the Th2 bias is contributing to the pathogenesis of disease. Perhaps, the best evidence is the requirement for IL-4 and IgE for disease manifestation in Lyn -/-mice. Our findings suggest that the basophil and autoreactive IgE's serve to amplify the role of autoreactive T and B cells in establishing the disease (Figure 1). This amplification seems to be key in determining the threshold of autoantibodies that are necessary to establish disease, presumably because the body can no longer effectively remove the high levels of autoantibodies and CIC's produced once this basophil-dependent amplification loop is fully established (Figure 1).

Basophils and autoreactive IgE in human SLE

While our findings in Lyn -/-mice clearly provide evidence of a Th2 influence in the pathogenesis of SLE, the relevance of the findings in this model to human disease must be explored. Evidence for the role of Th1 and Th17 cell subsets in human SLE is considerable [START_REF] Rahman | Systemic lupus erythematosus[END_REF]. In contrast, evidence for Th2 involvement is sparse and is primarily restricted to certain populations of SLE patients with more circulating IgE than healthy individuals [START_REF] Atta | Immunoglobulin E and systemic lupus erythematosus[END_REF][START_REF] Elkayam | Serum IgE concentrations, disease activity, and atopic disorders in systemic lupus erythematosus[END_REF]. Interestingly, the increased levels of circulating IgE were not associated with an increased incidence of atopic disease in this population of patients [START_REF] Atta | Immunoglobulin E and systemic lupus erythematosus[END_REF]. This suggested that a Th2 skewing might be seen in certain SLE patients. A more informative finding was the description of autoreactive IgE and IgE containing immune complexes in some SLE patients [START_REF] Kingsmore | Measurement of circulating immune complexes containing IgG, IgM, IgA and IgE by flow cytometry: correlation with disease activity in patients with systemic lupus erythematosus[END_REF]. While described, the role of these autoreactive or IgE containing immune complexes in disease was not known. Thus, we set out to explore whether the presence of autoreactive IgE's (anti-dsDNA and anti-ANA) might be associated with different measures of disease activity in SLE patients. We detected large amounts of autoreactive IgE in patient samples relative to healthy controls. Interestingly, the levels of both anti-dsDNA and anti-ANA IgE correlated with disease activity as determined by the SLEDAI score of the patients. Remarkably, patients with active lupus nephritis seemed to show the highest levels of anti-dsDNA IgE when compared to patients without kidney involvement [START_REF] Charles | Basophils and the T helper 2 environment can promote the development of lupus nephritis[END_REF]. These findings suggested that like shown in our mouse model, autoreactive IgE's might play a role in the pathogenesis of lupus in humans.

Whether basophils might be activated in human SLE was subsequently explored. Blood basophils from the same cohort of 42 patients and 40 healthy volunteers were analyzed for their activation status. Unlike mouse basophils, human basophils have known surface markers that are expressed upon their activation, such as CD63, CD203c or CCR3. Each of these markers reflect slightly different activation conditions. For instance, CD63 expression reflects the degranulation of the basophil, since CD63 is a granule membrane protein that becomes expressed on the cell surface only after the intracellular granule fuses with the plasma membrane. In contrast, CD203c is expressed when basophils are activated under conditions where they may or may not degranulate. Thus, we used this marker to assay for basophil activation. Analysis of CD203c expression showed that basophils from SLE patients were highly activated. In addition, peripheral basophils from SLE patients expressed higher levels of MHC-II molecules (HLA-DR) than the healthy controls, suggesting that their activation could result in basophil communication with T and B cells.

When analyzed for whether basophils could home to the secondary lymphoid organs in SLE patients, CD62L levels were found to be increased as compared to healthy controls, confirming the ability of these cells to home to these organs and their activated phenotype. To confirm that these cells were present in the lymph nodes and spleen from SLE patients, we analyzed several biopsy specimens of these tissues from SLE patients. In contrast to non-SLE biopsies (controls), basophils were found in the lymph nodes and spleens of SLE patients with their primary localization in germinal centers, in the middle of the B cell zone and in the surrounding T cell zone. No basophils were detected in control samples. Thus, basophils were shown to be in secondary lymphoid tissues, alike to our observation in Lyn -/-mice. Given that basophils were recruited to secondary lymphoid organs, one might expect that the numbers of basophils in the circulation would decrease. Both the percentage and absolute numbers of circulating basophils were reduced in SLE patients. These findings once again demonstrated that the activation of basophils in SLE caused increased expression of cell surface molecules (CD203c, CD62L, MHC-II…) that promote the recruitment of basophils to the secondary lymphoid organs and may allow their interaction with T and B cells. Collectively, the studies on human SLE patients are consistent with the conclusions from the analysis of Lyn -/-mice. They support that concept that autoreactive IgE and activated basophils are likely contributors to the pathogenesis of SLE (Figure 1).

Basophils and autoreactive IgE as therapeutic target in human SLE

The aforementioned findings suggest two new putative therapeutic targets in the treatment or prevention of lupus nephritis; namely autoreactive IgE and basophils. The involvement of these two factors, in an amplification loop that increases the levels of autoantibodies and CIC, suggests that disruption of this loop could have therapeutic benefit. Nonetheless, there are many poorly understood factors for the heterogeneity of disease. It is still not known what makes the difference between an SLE patient that develops active nephritis and one that does not. When looking at SLE patients with lupus nephritis, it is still not understood why some will develop class I or class II nephritis (with low severity, with mainly mesangial deposits) and others will develop class III or class IV nephritis (with high severity, mesangial deposits, extramembranous deposits and vasculitis). Our findings suggest that it is possible that the presence of level of activation of such amplification loops (whether basophil driven or not) might explain some of these features; based on the fact that class III and class IV states are correlated with the titers of autoantibodies and CIC. Thus targeting of basophils as one possible cell type for amplification of autoantibody production could lead to a beneficial outcome for some patients. This recent clinical trials targeting B cell activation [START_REF] Navarra | Efficacy and safety of belimumab in patients with active systemic lupus erythematosus: a randomised, placebocontrolled, phase 3 trial[END_REF] suggests that such approaches may have some clinical benefit in SLE. However, one must be cautious in developing such an approach for the basophil as the release of pro-inflammatory mediators (histamine, TNF-α, platelet activating factor…) contained in this cell could be an unwanted consequence of its depletion. In addition, one must consider the consequences of depleting a potential immune regulator and a potent effector against parasitic infections. Consideration of such therapeutic strategies requires further evaluation of short term and long term depletion of basophils in health and disease. Thus, a more tempting therapeutic target might be to inhibit the activation of the basophil through depletion of autoreactive IgE. Depletion of IgE-containing CIC would disrupt the persistent activation of basophils in SLE, blocking the amplification loop, decreasing the amounts of circulating autoantibodies. Depletion of IgE from the circulation is a strategy already in use in other diseases, such as allergic asthma. Omalizumab (Xolair®, Novartis) has shown efficacy in treatment of asthma [START_REF] Holgate | The use of omalizumab in the treatment of severe allergic asthma: A clinical experience update[END_REF]and allergic rhinitis [START_REF] Ben-Shoshan | Omalizumab: not only for asthma[END_REF] through blocking the binding of IgE to FcεRI. This monoclonal antibody was raised against the Fc portion of the IgE and binds the epitope on the IgE molecule that interacts with the FcεRIα. Once IgE is bound by this anti-IgE antibody, the IgE can no longer bind to the FcεRI. Thus, such an approach should stop basophil activation by IgE-containing CIC in SLE and may lead to interruption of the amplification loop described in Figure 1. Such studies are currently underway and should provide novel insights on the role of autoreactive IgE in basophil activation in SLE patients and beyond.

Concluding remarks

The recent advances in our understanding of the role of basophils in health and disease demonstrate that the longed ignored basophil granulocyte has an important immunomodulatory role in the immune system. The findings described herein show that, in some settings, the basophil, through its ability to communicate with T and B cells, links the Th2 environment as a contributor to the development of an autoimmune disease, like SLE. It will be of considerable interest to explore if this will translate to therapeutic benefit in disease.

Figure 1 :

 1 Figure 1: Basophil and autoreactive IgE amplify autoantbody production in SLE. SLE is a consequence of the loss of tolerance promoting autoreactive T and B cells that escape from negative selection. These autoreactive lymphocytes produce large amounts of circulating immune complexes (CICs) comprised of aggregated autoreactive antibodies, complement factors and autoantigens. Autoreactive IgGs, IgMs and IgAs comprise the majority of autoantibodies, but autoreactive IgE's also accumulate as CICs reaching a threshold that activates circulating blood basophils recruiting these cells to lymphoid organs via upregulation of CD62L on their cell surface. Once recruited to the secondary lymphoid organs, basophils can interact directly with autoreactive lymphocytes through upregulation of molecules like MHC-II and membrane bound BAFF. Together with the basophils ability to secrete large amounts of IL-4 (and IL-6), these cells can promote plasma cell survival and amplify autoantibody production, leading to the disease amplification. The presence of large amounts of autoantibody promotes the levels of pro-inflammatory cytokines found in the target organs, like the kidneys, resulting in uncontrolled inflammation and organ damage.

* The research of JR, reported herein, was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health.


A M Pourcher

E Jardé

M P Caprais

Anne Jaffrézic

M Marti

S Mieszkin

O Solecki

M Derrien

M Gourmelon

x-Subject category IDENTIFICATION OF LIVESTOCK FAECAL CONTAMINATION IN SURFACE WATERS USING CHEMICAL AND MICROBIOLOGICAL TOOLS

published or not. The documents may come   L'archive ouverte pluridisciplinaire

INTRODUCTION

Cattle and pig manure may contain pathogenic micro-organisms that can be transferred to soil through spreading on fields and thence to surface water. Such faecal pollution may pose risks to human health, especially in sensitive areas which support recreational shellfish harvesting, swimming and other uses where the public may come into direct contact with the water. Furthermore, the new European Directive on bathing waters (2006/7 /CE) requires the establishment of bathing water profiles, which require identification of sources of pollution. As the bacteria currently monitored to assess faecal pollution (E. coli, faecal coliforms and enterococci) do not distinguish between faecal pollution of water from animal and human sources, host-specific markers would be useful for the identification of faecal pollution. Recently, the concept of "Microbial source tracking" has been proposed [START_REF] Scott | Microbial Source Tracking: Current methodology and future directions[END_REF]. It includes biological and chemical methodologies that could be used to identify the dominant sources of faecal contamination in surface waters. Among specific markers, steroids, intensity peaks and their ratios of threedimensional fluorescence excitation-emission matrix (3D-EEM) spectroscopy, F+ RNA bacteriophages (FRNAPH) genotypes, host specific Bacteroidales and Lactobacillus appear to be interesting tools to distinguish human from animal faecal pollution [START_REF] Leeming | Using faecal sterols from humans and animals to distinguish faecal pollution in receiving waters[END_REF][START_REF] Seurinck | Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater[END_REF][START_REF] Blanch | Integrated analysis of established and novel microbial and chemical methods for microbial source tracking[END_REF][START_REF] Mieszkin | Estimation of pig fecal contamination in a river catchment by Real-Time PCR using two pig-specific Bacteroidales 16S rRNA genetic markers[END_REF], Naden et al., 2009[START_REF] Marti | Evaluation of Lactobacillus sobrius/amylovorus as a new microbial marker of pig manure[END_REF]. The aim of this study was to compare the suitability of chemical and microbiological markers to identify farm livestock sources of faecal contamination found in the environment. Four types of markers were tested: (i) the ratio of coprostanol/coprostanol+24-ethylcoprostanol (R1, expressed in percentage) and sitostanol/coprostanol (R2), (ii) tryptophan and fulvic-like fluorescence ratios (Bio/Geo and (V+VI/III)), (iii) genotypes of F-specific RNA bacteriophages (animal genotypes I and IV and human genotypes II and III) and (iv) bacterial markers belonging to Bacteroidales (human-specific HF183, ruminant-specific Rum-2-Bac and pig-specific Pig-2-Bac markers) and to the pig-specific Lactobacillus amylovorus.

MATERIALS AND METHODS

Sampling

Pig and bovine manure were collected from two farms located in Brittany (France). Six independent samples of field runoff water were collected after rainfall simulations on an experimental agricultural plot previously spread with either bovine or pig manure. Eight water samples were collected in Pays de la Loire (France). These include the effluents of three wastewater treatment plants (WWTP) and five samples, one of which was collected from a WWTP effluent (S1) and four (S0, S2, S3 and S4) from the Mayenne river (sample S0 was taken 50 m upstream of the WWTP effluent discharge, and three other samples taken downstream at distances of 10, 150 and 350 m, respectively). Three water samples were collected in the Daoulas river (Brittany, France) flowing through an agricultural area associated with intensive cattle pasture (B1 to B3); the distance between each point was approximately 500 m.

E. coli quantification

E. coli was counted using 3M TM Petrifilm E. coli (3M, France) and TBX medium (OXOID, France) for samples of 1 mL and 100 mL volume, respectively. Inoculated media were incubated for 24 h at 44°C. Blue colonies (Glucuronidase positive) were counted to determine the concentration of E. coli.

Steroids

Steroid concentrations were determined using the method reported by [START_REF] Jardé | Tracing and quantifying sources of fatty acids and steroids in amended cultivated soils[END_REF]. Briefly, freezedried samples were extracted by dichloromethane using an automated extractor (Dionex, ASE200). Hydrocarbons and polar molecules were separated into aliphatic hydrocarbons, aromatic hydrocarbons, and polar compounds by liquid chromatography on silica column using three successive elutions with organic solvents. Steroids compounds are present in the polar fraction. After derivatization using N,O-bis(trimethylsilyl)trifuoroacetamide (BSTFA)trimethylchlorosilane (TMSC) (99/1, v/v), quantitative analyses of steroid compounds were performed on a Shimadzu QP2010+ gas chromatograph mass spectrometer. The quantification of steroid compounds was performed by the addition of 2 H 6 -cholestane as an internal standard in the polar fraction.

Fluorescence

Fluorescence measurements were performed using a Perkin-Elmer LS-55B luminescence spectrometer, with the protocol described by [START_REF] Bilal | Discrimination of Farm Waste Contamination by Fluorescence Spectroscopy Coupled with Multivariate Analysis during a Biodegradation Study[END_REF]. A regional integration adapted from [START_REF] Chen | Fluorescence excitation-emission matrix regional integration to quantify spectra for dissolved organic matter[END_REF] was applied on 5 mg.L -1 C-normalized solution. With this technique, EEM is divided into biochemical (Bio) (I, II, IV) and geochemical (Geo) (III, V) fluorescent regions and three peak intensity zones of tryptophan, fulvic like and humic like fluorescence. Data were normalized to a daily-determined water Raman intensity (excitation 350 nm, emission 395 nm, Raman emission intensity averaged 10.5 + -0.5 (n=10)). Two ratios were considered: Bio/Geo and V+III/VI.

2.5

Bacteroidales and L. amylovorus

The bacterial markers were quantified using real-time PCR. Volumes of 50 to 200 mL water were filtered on 0.22 µm or centrifuged at 9000 g for 15 min. Filters and pellets (250 mg) were transferred into microtubes and stored at -20°C. Genomic DNA of Bacteroidales and L. amylovorus were extracted using the Fast DNA Spin Kit for Soil and the QIAamp DNA Stool Kit, respectively. The primers used to amplify the Representational Difference Analysis fragment of the pig specific L. amylovorus, and the 16S rRNA genes of HF183, Pig-2-Bac and Rum-2-Bac were described by [START_REF] Konstantinov | Representational difference analysis and real-time PCR for strainspecific quantification of Lactobacillus sobrius sp[END_REF], [START_REF] Seurinck | Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater[END_REF] and [START_REF] Mieszkin | Estimation of pig fecal contamination in a river catchment by Real-Time PCR using two pig-specific Bacteroidales 16S rRNA genetic markers[END_REF][START_REF] Mieszkin | Phylogenetic analysis of Bacteroidales 16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real-time PCR[END_REF], respectively.

F-specific RNA bacteriophages

FRNAPH were enumerated following the ISO method, either directly or after concentration by ultrafiltration Centricon Plus-70 (Millipore) for less contaminated water samples. Bacteriophage isolates were then genotyped by real time RT-PCR, with the One-Step RT-PCR kit, as in Ogorzaly et al. (2009).

RESULTS AND DISCUSSION

Values of the markers in manure, in runoff waters and in WWTP effluents

The values of the markers observed in manures, runoff waters contaminated by manure and in WWTP effluents are presented in table 1. Six of the markers appeared useful for differentiating livestock from human faecal pollution. The Pig-2-Bac and L. amylovorus markers were only detected in pig manure and runoff contaminated by pig manure whereas Rum-2-Bac and HF183 were specific to bovine and human contamination, respectively, confirming their host specificity reported by [START_REF] Seurinck | Detection and quantification of the human-specific HF183 Bacteroides 16S rRNA genetic marker with real-time PCR for assessment of human faecal pollution in freshwater[END_REF], [START_REF] Mieszkin | Estimation of pig fecal contamination in a river catchment by Real-Time PCR using two pig-specific Bacteroidales 16S rRNA genetic markers[END_REF][START_REF] Mieszkin | Phylogenetic analysis of Bacteroidales 16S rRNA gene sequences from human and animal effluents and assessment of ruminant faecal pollution by real-time PCR[END_REF] and [START_REF] Marti | Evaluation of Lactobacillus sobrius/amylovorus as a new microbial marker of pig manure[END_REF]. Steroids identified in pig manure were mainly represented by coprostanol, 24-ethylcoprostanol and cholestanol, whereas in bovine manure, the major steroids were 24-ethylepicoprostanol, sitostanol and sitosterol, in agreement with data of [START_REF] Leeming | Using faecal sterols from humans and animals to distinguish faecal pollution in receiving waters[END_REF]. As a consequence the coprostanol / coprostanol+ 24 ethylcoprostanol × 100 (R1 ratio) and sitostanol/coprostanol (R2 ratio) clearly differentiate bovine from pig contamination. Furthermore, the value of R1(> 65) and R2 (<0.3) in WWTP effluents also differentiates human from bovine pollution. The fluorescence ratios Bio/Geo and (V + III)/VI, which differed between pig and bovine manure, did not have the ability to distinguish the two types of pollution in water runoff samples. Animal genotypes (FRNAPH I and IV) were not detected in the pig manure and only FRNAPH I was detected in bovine manure with a low percentage of isolates (11.5%). These results agree with reports by [START_REF] Lee | Molecular Characterization of Bacteriophages for Microbial Source Tracking in Korea[END_REF] who observed that only 25% of cow faeces were positive for F specific coliphages. In the WWTP effluents, human genotypes (II+III) represented 67% of the isolates but animal genotypes were also detected (30.5% of isolates). As reported by [START_REF] Blanch | Integrated analysis of established and novel microbial and chemical methods for microbial source tracking[END_REF], FRNAPH I and FRNAPH IV appeared less efficient for detecting animal contamination, due to their sporadic occurrence. SD 2.9 10 5 8.4 10 3 3.1 10 6 8 0.25 0.034 0.06 a mean of three values; b standard deviation ; c limit of quantification

Values of markers in rivers impacted by human or bovine contamination

In the Mayenne river, impacted by a discharge of a WWTP effluent (S0 to S4 samples), none of the animal specific bacterial markers were found in samples upstream or downstream of the effluent discharge (table 2). Human-specific marker HF183 and FRNAPH were detected only in the most contaminated samples (S1 and S2). The human genotypes (II and III) represented 79% and 95% of the isolates in the water samples S1 and S2, respectively. The values of R1 and R2, which were specific to human pollution in the WWTP effluent, were less informative in river samples, probably due to the lower contamination (concentration of E. coli <10 3 CFU/100 mL). As the Mayenne river is located in a rural area, the presence of E. coli could be the result of a mixed bovine and human faecal contamination, which was too weak to be quantifiable using the real-time PCR. Conversely, the origin of contamination in the river flowing through intensive cattle pasture (B1 to B3 samples, table 2) was clearly identified by Rum-2 Bac, the absence of HF 183, Pig-2-Bac and L. amylovorus markers, low values of the ratio R1 (<44) and the high values of the ratio R2 (>1.3). Two of the markers failed to indicate this bovine contamination despite a high level of E. coli: FRNAPH (not detected) and the Bio/ Geo and (V+III)/VI ratios (uninterpretable). In conclusion, as previously reported by [START_REF] Blanch | Integrated analysis of established and novel microbial and chemical methods for microbial source tracking[END_REF], our study confirmed that the identification of the source of faecal contamination is improved by the use of several markers. Although contamination sources in the Mayenne river were more difficult to identify due to the low level of E. coli and the possibility of mixed contamination (bovine and human), highly contaminated samples (runoff waters contaminated by manure, Daoulas river) could be easily characterised with most of the animal markers: steroid ratios R1 and R2, and bacterial markers Rum-2-Bac, Pig-2-Bac and L. amylovorus. The suitability of the proposed markers has been demonstrated by their transfer via runoff to surface waters and their detection in water contaminated by bovine faeces.

TABLE 1 Mean concentrations (CFU, PFU, cells or copies / g or /100 mL) of microbial markers and chemical ratios in manures and waters contaminated by animal or human faecal pollution

 1

	Sample type	E. coli (CFU)	Phages (PFU)	L. amylovorus (cells)	Pig-2-Bac (copies)	Rum-2-Bac (copies)	HF183 (copies) R1 R2	Ratios Bio/Geo (V+III)/VI
	Pig manure	mean a 3 10 4	<lq c	2.6 10 7	1.2 10 7	<lq	<lq	59 0.23	0.47	1.33
	(/g)	SD b 7.1 10 3		1.2 10 7	1.3 10 5			3 0.02 0.003	0.01
	Bovine manure mean 5.1 10 4 1.2 10 2	<lq	<lq	2.2 10 7	<lq	45 2.70	0.39	1.66
	(/g)	SD 4.5 10 4				5.6 10 6		2 0.23 0.007	0.01
	Runoff pig	mean 1.4 10 4 <lq	3.5 10 7	6.8 10 5	<lq	<lq	57 0.62	0.25	1.35
	(/100 mL)	SD 6.7 10 3		1.4 10 7	3.4 10 5			1 0.01	0.06	0.06
	Runoff bovine mean 3.4 10 5 1.1 10 3	<lq	<lq	1.1 10 7	<lq	49 2.18	0.24	1.22
	(/100 mL)	SD 6.6 10 5				7.2 10 6		1 0.03	0.02	0.15
	WWTP	mean 3.1 10 5 7.4 10 3	<lq	<lq	<lq	3.1 10 6 70 0.26	0.22	0.88
	(/100 mL)

TABLE 2 Mean concentrations (CFU, PFU, cells or copies / 100 mL) of microbial markers and chemical ratios in two rivers, impacted either by a treated urban effluent (Mayenne river) or by bovine contamination (Daoulas river)

 2

	Origin of	sample	E. coli	Phages	L. amy a	Pig-2-Bac	Rum-2-Bac	HF183	Ratios
	pollution		(CFU)	(PFU)	(cells)	(copies)	(copies)	(copies) R1 R2	Bio/Geo (V+III)/VI
	Human	S0 (upstream)	5 10 1 <lq b	<lq	<lq	<lq	<LQ 56 0.76 0.11	1.16
		S1 (WWTP effluent) 6.3 10 5 1,70 10 4 <lq	<lq	<lq	4.4 10 4 70 0.24 0.24	0.94
		S2 (discharge)	3.2 10 3 320	<lq	<lq	<lq	8.1 10 4 nd c 1.01 0.11	1.15
		S3 (downstream) 5 10 2	<lq	<lq	<lq	<lq	<lq 51 0.96 0.11	1.15
		S4 (downstream) 3.3 10 2 <lq	<lq	<lq	<lq	<lq 54 0.86 0.11	1.17
	Bovine	B1	1.9 10 4 <lq	<lq	<lq	2.5 10 5	<lq 43 1.4 0.07	1.0
		B2	1.5 10 4 <lq	<lq	<lq	1.3 10 5	<lq 39 1.8 0.07	1.0
		B3	1.7 10 4 <lq	<lq	<lq	1.6 10 5	<lq 38 1.8 0.07	1.0

a L. amylovorus; b limit of quantification; c no data CONCLUSIONS

14th Ramiran International Conference, 13-15 septembre 2010, Lisbonne, PRTx-Subject category

14th Ramiran International Conference, 13-15 septembre 2010, Lisbonne, PRT

ACKNOWLEDGEMENTS

This work was partly supported by the Agence Française de Sécurité Sanitaire de l'Environnement et du Travail (AFSSET) (EST-2006/1/36).