Prolactin: A versatile regulator of inflammation and autoimmune pathology Massimo Costanza, Nadine Binart, Lawrence Steinman, Rosetta Pedotti PII: DOI: Reference:

S1568-9972(14)00277-8 doi: 10.1016/j.autrev.2014.11.005 AUTREV 1644

To appear in:

Autoimmunity Reviews

Received date: Accepted date:

1 November 2014 8 November 2014

Please cite this article as: Costanza Massimo, Binart Nadine, Steinman Lawrence, Pedotti Rosetta, Prolactin: A versatile regulator of inflammation and autoimmune pathology, Autoimmunity Reviews (2014), doi: 10.1016/j.autrev.2014.11.005

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ACCEPTED MANUSCRIPT Milan, November 7th, 2014

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Prolactin: a versatile regulator of inflammation and autoimmune pathology

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Massimo Costanza a, Nadine Binart b, Lawrence Steinman c and Rosetta Pedotti a,*

Neuroimmunology and Neuromuscular Disorder Unit, Neurological Institute Foundation IRCCS

C. Besta, Milan 20133, Italy

INSERM U693, Université Paris-Sud, Faculté de Médecine Paris-Sud, Le Kremlin-Bicêtre 94276,

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France

Departments of Pediatrics, Neurology and Neurological Sciences, Stanford University, Stanford,

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CA 94305, USA

* Corresponding author: Rosetta Pedotti, MD, PhD Neuroimmunology and Neuromuscular Disorder Unit, Neurological Institute Foundation IRCCS C. Besta, via Amadeo 42, 20133 Milan, Italy [email protected]. Phone (+39) 02-23944654. Fax (+39) 02-23944708

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ACCEPTED MANUSCRIPT ABSTRACT

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Prolactin (PRL) has long been proposed as an immune-stimulating and detrimental factor in autoimmune disorders. However, recent findings have challenged this common view, showing that

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PRL does not play a crucial role in the development of experimental autoimmune encephalomyelitis, animal model for multiple sclerosis (MS), and even protects against adjuvantinduced model of rheumatoid arthritis (RA). In this review we provide a critical overview of data

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supporting a role for PRL in the regulation of immune responses. In addition, we focus on studies

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exploring the involvement of PRL in autoimmune diseases, such as systemic lupus erythematosus,

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MS and RA, in light of the recently-outlined regenerative properties of this hormone.

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Keywords: Prolactin

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Inflammation

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Systemic Lupus Erythematosus Rheumatoid Arthritis Multiple Sclerosis Tissue repair

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ACCEPTED MANUSCRIPT Abbreviations: Ag, antigen

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BCR, bromocriptine

EAE, experimental autoimmune encephalomyelitis

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CNS, central nervous system

GM-CSF, granulocyte-monocyte colony stimulating factor IFN-, interferon-

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Ig, immunoglobulin

iNOS, inducible nitric oxide synthase

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IL, interleukin

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IP-10, interferon gamma-induced protein-10 IRF-1, interferon regulatory factor-1

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JAK, Janus kinase

LFA-1, leukocyte functional antigen-1

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MAPK, mitogen-activated protein kinase

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MCP-1, monocyte chemoattractant protein-1 MHC, major histocompatibility complex MIP-1, macrophage inflammatory protein-1 MS, multiple sclerosis NK, natural killer PBMC, peripheral blood mononuclear cells PRL, prolactin PRLR, prolactin receptor RA, rheumatoid arthritis SLE, systemic lupus erythematosus STAT, signal transducer and activator of transcription 3

ACCEPTED MANUSCRIPT SVZ, subventricular zone Th, T helper

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TNF-, tumor necrosis factor-

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VLA-4, very late antigen-4

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ACCEPTED MANUSCRIPT 1. Introduction Prolactin (PRL) is a polypeptide hormone discovered more than eighty years ago as a pituitary

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factor stimulating mammary gland development and lactation in rabbits [1]. Since its first discovery, several extra-pituitary sources of PRL have been identified and a great number of other

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functions have been associated with this hormone in various vertebrate species [2]. One of the most controversial and enigmatic aspects of PRL biology is related to its role in regulating immune responses and autoimmune inflammation. Indeed, a plethora of studies since the late 70’s has

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documented the ability of PRL to stimulate the proliferation and the inflammatory activity of

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immune cells. This remarkable amount of work, along with several reports describing hyperprolactinemia in autoimmune disorders, has set the background for the general belief that PRL is a detrimental factor in autoimmunity, and has prompted to investigate in both preclinical models

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and clinical studies if PRL depletion by pharmacological treatments might ameliorate the clinical

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course of autoimmune diseases. With the exception of systemic lupus erythematosus (SLE), in diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA) these studies have led to

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inconclusive results and the actual contribution of PRL has long remained elusive. Also, the

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relevance of PRL in the immune system (and consequently in autoimmunity) has been revaluated in the last decade, since no evident immune deficits have been identified in PRL- and PRL receptor (PRLR)-deficient mouse models. The interpretation of the effect of PRL in autoimmune pathology has been further complicated since an increasing number of studies has uncovered that PRL is unexpectedly endowed with regenerative properties for several tissues, including the central nervous system (CNS) [3,4] and the bone and cartilage [5,6], which are target of autoimmune attacks in MS and RA, respectively. The aim of this review is first to provide a general overview on the main established functions of PRL in the immune system, as emerged by both in vitro and in vivo experimental approaches. Second, to discuss major studies exploring the contribution of PRL to autoimmune disorders, with specific emphasis on latest work performed in experimental models

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ACCEPTED MANUSCRIPT of SLE, MS and RA, which have shed light on new and unexpected effects exerted by PRL in

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autoimmunity.

2. The biology of PRL

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PRL is mainly produced by lactotroph cells of the anterior pituitary gland, but, in humans, an alternative promoter (also known as superdistal or extrapituitary promoter) drives PRL expression in several extra-pituitary sites, such as immune, decidual, mammary, epithelial and fat cells [7]. The

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superdistal promoter is located upstream of the pituitary promoter and generates an alternative

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transcript, 150 bp longer than the classical pituitary mRNA. Both PRL transcripts encode for an identical mature protein of 199 amino acids (23 kDa) [7]. PRL can undergo several posttranslational modifications, such as phosphorylation and glycosylation. Proteolytic cleavage

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originates 14-, 16- and 22-kDa PRL variants [8]. Other PRL isoforms have been identified in

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human sera and result from processes of polymerization or aggregation with immunoglobulins (Igs), such as “big PRL” (45-50 kDa), “big big” PRL or macroprolactin (>100 kDa). These higher

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molecular weight variants have less biological activity than the monomeric 23 kDa PRL [9].

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PRL secretion is under dual regulation by hypothalamic hormones such as thyrotropinreleasing factor and dopamine via the pituitary portal circulation [8,10]. The biological effects of PRL are mediated by its interaction with PRLR, a member of the cytokine receptor superfamily, which includes receptors for interleukin (IL)-2, IL-6, granulocyte-monocyte colony stimulating factor (GM-CSF) and leptin [11,12]. This receptor is present in nearly all organs and tissues and is particularly interesting because it can be activated by three sequence-diverse human hormones: PRL, growth hormone, and placental lactogen. Binding of PRL to its receptor [13] activates at least three signaling pathways including the Janus kinase/signal transducer and activator of transcription (JAK/STAT), the phosphoinositide 3-kinase and the mitogen-activated protein kinase (MAPK). Most current knowledge on PRL has been obtained from the study of PRL- and PRLR-deficient mice [14,15] providing strong proof of the main PRL signaling pathway. 6

ACCEPTED MANUSCRIPT The multiple biological functions of PRL have been subdivided into the following categories: water and electrolyte balance, growth and development, endocrinology and metabolism,

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brain and behaviour, reproduction, and finally immunoregulation [2]. However, the extremely wide spectrum of PRL activities must be regarded as a panel of functions that are modulated by, rather

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strictly dependent on, PRL.

3. PRL and immune functions

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3.1. In vitro studies

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Many studies have analysed the immune-modulating functions of PRL in vitro, suggesting that PRL has the ability to affect the development, survival and function of cells belonging to both innate and adaptive arms of the immune system (Figure 1) [16,17].

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PRL has been shown to sustain the phagocytic and inflammatory activities of macrophages.

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PRL enhances the release of reactive oxygen intermediates by human macrophages [18] and supports the cytotoxic activity of mouse tumor-associated macrophages against tumor cells [19].

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Murine peritoneal macrophages treated with low-to-high amounts of PRL (the optimal dose was

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100 ng/ml) display activation of p38 MAPK and STAT3 signaling pathways and produce higher quantities of nitric oxide and pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-, IL-1, interferon (IFN)- and IL-12 [20,21]. Interestingly, very high PRL concentration (1000 ng/ml) significantly enhances the secretion of the anti-inflammatory cytokine IL-10 in these cells [22]. PRL also stimulates macrophages to release several chemokines, e.g. macrophage inflammatory protein (MIP)-1, interferon gamma-induced protein (IP)-10 and monocyte chemoattractant protein (MCP)-1 [22]. Human granulocytes exposed to PRL exhibit the activation of STAT1 and p38 MAPK intracellular pathways and the upregulation of inflammation-related genes such as inducible nitric oxide synthase (iNOS) and interferon regulatory factor 1 (IRF-1) [23]. PRL also stimulates the release of IFN- by human natural killer (NK) cells and sustains their cytolytic activity [24]. In synergy with IL-15, PRL has been shown to increase the proliferation of a 7

ACCEPTED MANUSCRIPT human NK cell line and to induce the transcription of perforin gene [25]. Also dendritic cells (DCs) are affected by PRL treatment [26]. Low levels of PRL, in combination with GM-CSF, polarize

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monocytic precursors obtained from human peripheral blood mononuclear cells (PBMCs) toward an immature DC phenotype, enhancing the expression of MHC class II and co-stimulatory

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molecules such as CD80 and CD86 [27]. Further exposure of immature DCs to high concentrations of PRL promotes their maturation to functional antigen presenting cells, characterized by increased ability to stimulate the proliferation and IFN- production of T cells [27,28]. Similar findings have

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been obtained with rat thymic DCs [29].

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PRL has been reported to directly shape several lymphocyte functions. Human B cell hybridomas [30] and peripheral blood B cells [31] secrete higher levels of antibodies following treatment with PRL in a dose-dependent manner. Rat fetal thymic cultures exposed to PRL exhibit

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enhanced thymocyte proliferation and differentiation of double negative CD4-CD8- precursor cells

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to double positive CD4+CD8+ cells [32]. An anti-PRL antiserum was shown to inhibit the proliferation of a non-immortalized murine T helper (Th) cell clone in response to IL-2 [33,34].

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However, PRL was not found at either transcript or protein levels in this Th cell clone [33] and was

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suggested to derive from culture medium. In line with these data, we did not detect PRL at either mRNA or protein levels in mouse primary CD4+ T cells [35]. In human T cells, PRL has been proposed to support proliferation [36], survival [37], and to act as an autocrine factor. Indeed, an extrapituitary PRL transcript has been found in human T cells [38], and its levels are increased following stimulation with cyclic AMP [39], as well as in PBMCs and Jurkat T cell line after treatment with prostaglandin E2 [40,41]. PRL promotes the adhesion of PBMCs and Jurkat T cells to activated endothelial cells [42]. The adhesion of T cells supported by PRL is mediated by integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), and is dependent on the activation of JAK-2, STAT-3 and STAT-5 intracellular pathways [42].

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ACCEPTED MANUSCRIPT 3.2. In vivo studies 3.2.1. Early approaches investigating PRL functions in the immune system

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A major role for PRL in the immune system has originally been suggested in vivo by the observation of multiple immune system deficits in mice with primary genetic deficiencies of

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pituitary hormones, such as dwarf mice. This strain harbours a spontaneous mutation in the gene encoding the pituitary-specific transcription factor Pou1f1 (previously Pit-1), which results in primary deficiencies of PRL, growth hormone and thyroid-stimulating hormone. These mice

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display abnormal primary B cell development, while deficits in humoral and cell-mediated immune

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responses are controversial [43]. These findings have been supported by studies performed with hypophysectomized rats, which have numerous immune dysfunctions that can be restored by PRL injection. These deficits include bone marrow leukopenia [44], impaired contact hypersensitivity

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reactions [45], and decreased antibody response to T cell-dependent antigens [46]. Another

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approach to investigate PRL functions in the immune system was to reduce PRL serum levels by bromocriptine (BCR), a dopamine D2 agonist that inhibits PRL secretion from the pituitary gland

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[43]. Immune reactions such as contact hypersensitivity and antibody response to LPS are impaired

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in BCR-treated rats and restored by PRL administration [47]. In mice, hypoprolactinemia induced by BCR leads to reduced survival after infection with pathogens such as Listeria Monocytogenes, and impaired macrophage and T cell responses [48]. However, the specificity of BCR pharmacological approach has been questioned by studies demonstrating that this drug can directly suppress the proliferation and function of B and T lymphocytes, independently of PRL [49,50].

3.2.2. Studies with PRL- and PRLR-deficient strains More recently, the generation of mice carrying Prl and Prlr gene-targeted deletions has contributed to better understand the involvement of PRL in the immune system. Studies with PRL- and PRLRdeficient mice have outlined that PRL is dispensable for the development of the immune system. PRL-KO mice exhibit normal percentages of B cell progenitors (pro-B and pre-B cells) and mature 9

ACCEPTED MANUSCRIPT B cells in the bone marrow and normal percentages of T cell precursors and CD4+ and CD8+ T cells in the thymus. No alterations in the percentages of lymphocytes and myeloid cells were observed in

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secondary lymphoid organs, such as the spleen and lymph nodes [14]. These findings were confirmed and extended in PRLR-KO mice, which display no alterations in immune system

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development and mount effective immune responses to several types of immune challenges. Indeed, these mice develop normal levels of serum antibodies following immunization with a T celldependent antigen, and successfully respond to the injection with an allogeneic tumor cell line or to

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the infection with Listeria Monocytogenes [51]. Taken together, the results obtained with PRL- and

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PRLR-deficient strains have questioned the concept of PRL as a critical immune player, as suggested by early in vivo approaches. Nonetheless, these data do not rule out that PRL might contribute more significantly in vivo to immune responses not yet investigated and/or in particular

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conditions, such as stress [52] or under hyperprolactinemic states induced by either physiologic

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adaptations (e.g. pregnancy or lactation), pathological conditions (e.g. pituitary adenoma) or

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pharmacological treatments (e.g. haloperidol).

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4. Prolactin and autoimmune diseases The female prevalence of many autoimmune disorders has encouraged investigating the impact of hormonal factors to autoimmune pathology. If hormones such as estrogens [9] and leptin [53] have a recognized role in autoimmunity, PRL involvement has long been a matter of controversy. The large number of studies hypothesizing an immune-stimulating potential for PRL has contributed to the general perception that PRL plays a pathogenic role in autoimmune conditions. Also, the modulation of disease progression observed during pregnancy and post-partum period, associated with the key functions exerted by PRL in female reproduction, have indirectly reinforced this view. Hyperprolactinemia has often been associated with autoimmune conditions, such as SLE, RA, MS, autoimmune thyroiditis [54], myasthenia gravis [55] and diabetes mellitus [56], however in several cases discordant results have been provided [57-59]. In the following paragraphs we discuss in 10

ACCEPTED MANUSCRIPT detail the studies that explored the involvement of PRL in the pathogenesis of three autoimmune

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disorders, namely SLE, MS and RA.

4.1. Systemic Lupus Erythematosus

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SLE is a systemic autoimmune disease with a female:male ratio of 9:1, affecting several organs, such as kidney, skin, heart and brain [60]. SLE is characterized by flaws in B cell tolerance and the presence of circulating immune complexes and autoantibodies directed to several targets, such as

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nuclear antigens and lipoproteins, which promote tissue damage and dysfunction [61]. Other

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components of innate and adaptive immune system are involved in the pathogenesis of SLE, such as neutrophils [62], basophils [63] and T cells [64], which contribute to amplify and perpetuate the inflammatory injury in target organs.

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SLE is probably the autoimmune disease where the contribution of PRL has been most

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extensively explored. Hyperprolactinemia has been reported in 15-30% of SLE patients [59]. To our knowledge, lupus is the only autoimmune disease where serum PRL levels have been measured

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discriminating the different isoforms of PRL [65]. This kind of analysis has outlined that levels of

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serum monomeric PRL (23 kDa) are higher in patients with active disease than in patients with nonactive disease, while there is a negative correlation between disease activity index and the percentage of the so-called “big big PRL” (>100 kDa), which is believed to have less biologic activity [65]. A recent study also identified a positive correlation between high PRL levels and disease damage in SLE [66]. Stevens and colleagues have identified a G/T single nucleotide polymorphism in the extrapituitary promoter of PRL at position -1149. The alleles G and T result respectively in increased or decreased expression of PRL mRNA in peripheral blood leukocytes activated in vitro with phytohemagglutinin. Interestingly, an increased frequency of PRL-1149 G allele has been found in a cohort of SLE patients in comparison to healthy controls [67]. In line with this finding, PBMCs from SLE subjects secrete PRL in vitro at higher levels than healthy controls, and this PRL production has been ascribed to B rather than T cells [68]. Moreover, PBMCs from 11

ACCEPTED MANUSCRIPT SLE patients but not from healthy donors spontaneously produce IgG in vitro and increase their secretion after treatment with PRL. Physiological concentrations of PRL (20 ng/ml) are more

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effective than high levels (100 ng/ml) to stimulate IgG production in this setting. Also, there is a significant correlation between the amount of IgG produced by PRL-treated PBMCs and SLE

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activity, suggesting that PBMCs susceptibility to PRL is markedly associated to the disease state [69]. An indirect support for a role of PRL in the pathogenesis of SLE has been provided by two clinical trials showing reduction of disease flares after treatment with BCR [70,71].

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Based on the correlation between hyperprolactinemia and disease activity in humans, studies

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in experimental models have generally assessed the role of PRL in SLE pathogenesis under hyperprolactinemic conditions (Table 1). First, the effects of high levels of PRL (from 3- to 18-fold) obtained by pituitary transplants were tested in female NZB/W F1 mice [72], a strain characterized

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by B cell hyperactivity [73] and spontaneously developing a lupus-like syndrome [74].

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Hyperprolactinemia in these mice leads to accelerated mortality, enhanced renal dysfunction, increased serum levels of anti-DNA antibodies and immune complexes deposition [72]. These

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effects are gender independent, as also male NZB/W mice subjected to mild to severe

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hyperprolactinemia exhibit faster pathology and mortality [75]. Second, the effects of PRL were evaluated in R4A-2b mouse, a Balb/c strain transgenic for the heavy chain of an anti-DNA antibody, which does not spontaneously develop lupus [76]. In this model hyperprolactinemia (i.e. double PRL serum concentrations achieved by daily injections of PRL) induces a lupus-like phenotype, characterized by the development of anti-DNA antibodies, the expansion of transgeneexpressing B cells and the accumulation of Ig deposits in glomeruli [76]. This phenotype is associated to a marked change of B cell repertoire in the spleen, with the decrease of immature T1 transitional B cells and the increase of mature marginal zone and follicular B cells [76]. These pathological features promoted by PRL injection were no more detectable 3 months after discontinuation of hormone treatment, suggesting that the immune stimulating effects mediated by PRL have a relatively short duration [77]. Of note, PRL does not trigger the break of B cell 12

ACCEPTED MANUSCRIPT tolerance in transgenic R4A-2b Balb/c mice deficient for CD4+ T cells, suggesting that PRL actions are probably directed on T cell-dependent follicular B cell subset [76]. The same authors

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have demonstrated that high PRL serum levels interfere with several mechanisms of B cell tolerance, impairing B cell receptor-mediated clonal deletion and decreasing the threshold of

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activation of anergic B cells [78]. Interestingly, when the same PRL dose regimen used with Balb/c R4A-2b strain has been applied to R4A-2b mice on a C57BL/6 background, no lupus phenotype was observed, indicating that PRL effect is genetically determined [76]. However, further work has

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shown that in C57BL/6 mice carrying both R4A-2b transgene and the lupus susceptibility interval

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Sle3/5, PRL can induce a lupus-like syndrome [79]. Sle3 genetic locus derives from chromosome 7 of a lupus-prone strain and leads to excessive antigen presentation and the consequent expansion

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and hyperactivity of CD4+ T cells in C57BL/6 mice [80,81]. Some of the immune-stimulating effects mediated by PRL in this background might also involve DCs. Indeed, the adoptive transfer

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of DCs derived from prolactin-treated Sle3.C57BL/6 mice into normal C57BL/6 mice, results in the increase of IgG deposits, DNA-reactive B cells and activated B cells [82]. Another group has

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reported that injection of metoclopramide in MRL and MRL/lpr lupus-prone strains induces

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hyperprolactinemia and augmented levels of ant-DNA antibodies and proteinuria. Conversely, the same treatment in C57BL/6 mice induces high levels of serum PRL but does not trigger any lupus clinical sign [83].

Taken together, these data suggest that PRL can act as a tolerance-breaking factor under specific genetic basis.

4.2. Multiple Sclerosis MS is a chronic inflammatory disorder of the central nervous system (CNS), affecting 2.5 million people worldwide, with a female:male ratio of 2-3:1 [84]. The pathological hallmark of MS is the presence of multifocal areas of immune cell infiltration, demyelination and axonal damage mainly located in the white matter of the CNS [85]. MS and experimental autoimmune encephalomyelitis 13

ACCEPTED MANUSCRIPT (EAE), animal model for this disease, are generally believed to be induced by myelin-reactive CD4+ Th1/Th17 cells, which drive an inflammatory response against oligodendrocytes that form myelin

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sheath surrounding neuronal axon [85]. PRL has been classically considered a potentially detrimental factor both in MS and EAE,

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animal model for this disease. This notion dates back to 1983, when Nagy et al. reported that preventive treatment of rat EAE with BCR attenuates clinical symptoms and diminishes immune cell infiltration in CNS lesions [47]. Later, it was shown that serum PRL levels increase during the

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induction phase of rat EAE, and that BCR reduces both clinical symptoms of disease and

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proliferative response of splenocytes to myelin antigen [86]. Similar results have been obtained by using dihydroergocriptine, a dopaminergic agonist structurally related to BCR [87]. BCR was found to reduce rat EAE severity also when administered after the onset of clinical signs [88] (Table 1).

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Discordant results have been obtained by studies exploring hyperprolactinemia in MS [59]. A

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clinical report has recently described the case of a male patient with MS who suffered the first disease attack during the appearance of a PRL-secreting adenoma, which induced an upsurge of

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PRL serum levels to 38 ng/ml (normal values in males are 2-10 ng/ml). The same patient

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experienced the only two MS relapses in concomitance with adenoma recurrence and the associated hyperprolactinemia [89]. Another study has found a positive correlation between PRL serum levels and the number of anti-MOG antibody secreting cells [90]. When BCR treatment has been applied to human MS, an open label study reported no efficacy in reducing disease activity [91], while a single case report has described the control of paroxystic symptoms [92]. On the whole, these data did not allow drawing a definite conclusion on the role of PRL in CNS autoimmunity. The interest towards PRL in MS has been reactivated in recent years after two studies showed that exclusive breast-feeding (a hyperprolactinemic physiological condition) surprisingly reduces the risk of relapses occurring during postpartum [93,94], although other articles reported discordant results [95-97]. In line with these findings and contrarily to the hypothesis of a detrimental role for PRL in CNS autoimmunity, PRL has been recently demonstrated to mediate the proliferation of 14

ACCEPTED MANUSCRIPT oligodendrocyte precursor cells during pregnancy and to promote myelin repair in a spontaneously remyelinating model of lysolecithin-induced focal demyelination (Table 1) [4]. These data have

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suggested that PRL could also become a potentially therapeutic agent for MS. Moreover, the regenerative effects of PRL in the CNS have been demonstrated to be not only restricted to

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oligodendrocytes. Indeed, endogenous PRL has been shown to support neurogenesis in the subventricular zone (SVZ) of pregnant mice [3]. Also, PRL-induced neurogenesis in the SVZ and dentate gyrus is required for paternal recognition of adult offspring [98]. PRL-deficient mice give

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rise to a reduced number of hippocampal neurospheres in vitro [99] and PRL injection in

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chronically stressed mice sustains neurogenesis in the hippocampal dentate gyrus [100]. PRL administration increases survival in a model of severe spinal muscular atrophy, a neurodegenerative disease characterized by the loss of motor neurons [101]. Last, PRLR-deficient mice display retinal

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photoresponsive dysfunction and reactive gliosis, while hyperprolactinemia counteracts retinal

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degeneration associated with light damage [102]. To better clarify the contribution of PRL to CNS autoimmune pathology, we have recently

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characterized chronic EAE, an immune-mediated model of demyelination, in mice with Prl or Prlr

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gene-targeted deletions (Table 1). This study has suggested that PRL does not impact crucially on EAE expression [35]. Indeed, PRL and PRLR-deficient mice develop EAE with a slightly delayed onset compared to littermate control mice but with full clinical severity. The delayed onset of first clinical symptoms is associated with a delay in the establishment of autoreactive Th1/Th17 immune responses against the myelin antigen in draining lymph nodes. The lack of difference in EAE severity between PRL-, PRLR-deficient and wild-type mice observed in this study suggests that the absence of PRL or its receptor can be compensated by other factors during the development of the disease, and thus that PRL does not play a central role in chronic EAE. In contrast with a previous report [86], in this study we did not observe a raise of PRL concentrations during EAE. Moreover, Prl transcript could not be detected in either LNCs or CD4+ T cells isolated from both naïve mice or mice with EAE, and PRL protein could not be found in supernatants of in vitro stimulated T cells 15

ACCEPTED MANUSCRIPT [35]. These findings appear to rule out CD4+ T cells as a possible source of local PRL that could contribute in an autocrine manner to the development of autoreactive T cell responses in EAE.

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Given the robustness of the EAE model, whether or not PRL and its receptor might favour myelin repair and disease recovery in CNS autoimmune demyelination remains a question requiring further

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investigation.

4.3. Rheumatoid Arthritis

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RA is a chronic, autoimmune inflammatory disease affecting the synovial membrane, cartilage and bone [103]. In RA, the inflamed joint is characterized by a hyperplastic synovial membrane

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infiltrated by fibroblasts and immune cells, such as macrophages, B cells and T cells (“pannus”) [104]. The arthritic pannus has invasive and erosive properties toward the surrounding cartilage and

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bone, leading to joint inflammation and pathology [105]. CD4+ T cells are present in high numbers

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in the inflamed synovial membrane and are believed to play a central role in mediating immune cell infiltration and tissue destruction in RA [103].

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PRL has been detected in synovial fluid of knee joint [106], and T cells infiltrating the

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synovium of RA patients secrete PRL, which stimulates the proliferation and production of inflammatory cytokines and matrix metalloproteinases by synovial cells [107]. CD14+ monocytes isolated from RA patients display increased expression of PRLR in comparison to healthy controls and secrete higher amounts of TNF- upon in vitro exposure to PRL [108]. A large-scale population study performed on more than three-thousand RA patients and four thousand controls has found a possible correlation between decreased risk of developing RA and the PRL-1149 T polymorphism [109,110], previously characterized in lupus patients [67] and which has been associated with reduced PRL production by lymphocytes. In experimental models (Table 1), early studies have shown that hypophysectomised or BCR-treated rats develop milder adjuvant-induced arthritis [45,47]. These results, along with the human data more recently produced, have long supported the idea that PRL might promote RA pathology. 16

ACCEPTED MANUSCRIPT However, in last few years several papers started to point out that, in the context of joint tissue, PRL can also play protective and regenerative functions. Indeed, reduced bone formation

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rate and decreased bone mineral density have been found in PRLR-deficient mice [5]. PRL has been shown to inhibit in vitro chondrocyte apoptosis induced by serum starvation [111], to augment

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the proliferation of bone marrow-derived mesenchymal stem cells undergoing chondrogenic differentiation and to increase their synthesis of proteoglycans and type II collagen [6]. In accordance with these previous observations, and contrarily to the hypothesis of a pathogenic role

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of PRL in RA, a recent study has demonstrated that, surprisingly, high levels of circulating PRL

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protect against permanent joint damage and inflammation in experimental arthritis in rats [112]. In this study PRL was shown to counteract the apoptosis of rat chondrocytes induced by a mixture of

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cytokines (i.e. TNF-, IL-1, IFN-) both in vitro and in vivo via JAK2/STAT3-dependent pathway. Interestingly, PRLR-deficient mice injected with the same cytokine cocktail display

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higher chondrocyte apoptosis than wild-type mice, suggesting that, under inflammatory conditions, endogenous PRL at physiological concentrations contributes to chondrocyte survival [112].

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Administration of high-dose PRL (serum PRL levels were similar to those observed in pregnancy)

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either in a preventive or in a therapeutic regimen to rats with adjuvant-induced arthritis significantly ameliorated the severity of joint damage, reduced ankle swelling and pannus formation, and decreased transcript levels of inflammatory mediators such as TNF-, IL-1, IFN- and IL-6 [112].

5. Conclusions and future perspectives PRL has long been proposed as a triggering and/or promoting factor in autoimmune inflammation. Recent work performed in experimental models of SLE, MS and RA has highlighted how complex and heterogeneous are the effects of this hormone in autoimmune processes. Overall, the work performed so far suggests that, according to the specific pathologic context, PRL can promote, be dispensable or even protect against autoimmune pathology. Differences in immune mechanisms and in the organs that are the targets of the autoimmune process might account for differences in the 17

ACCEPTED MANUSCRIPT global effect exerted by PRL in specific autoimmune conditions. One possible interpretation of these data might be that the immune-stimulating and detrimental features of PRL prevail in B cell-

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dominated autoimmune diseases, such as SLE. However, in experimental models, the effects of PRL in SLE have been assessed under hyperprolactinemic conditions obtained by pituitary

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transplants or PRL injection. Thus, the contribution of endogenous PRL to SLE has not yet been established. Further studies should be performed to evaluate how genetic deficiencies of PRL or its receptor impact SLE development in mouse strains that spontaneously develop the disease.

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In T cell-driven autoimmune disorders, PRL appears to impact marginally on immune

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response as in chronic EAE, animal model of MS, or even to promote tissue repair as in adjuvantinduced arthritis, model of RA. In MS, PRL has even been recently proposed as a therapeutic agent, because of its pro-remyelinating effect in a lysolecithin-model of demyelination. Whether or not the

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administration of exogenous PRL might lead to amelioration (due to its pro-remyelinating effects)

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or worsening (due to its immune-stimulating properties) of EAE, still remains an open question. Moreover, further investigation is required to understand whether PRL affects CNS autoimmunity

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in physiological high-hyperprolactinemic states, such as pregnancy and breastfeeding. Last, it might

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be interesting to investigate whether PRL plays a more relevant role in EAE models other than MOG35-55-induced EAE, such as relapsing-remitting EAE, that mimics the more common clinical form of MS, or EAE induced by whole MOG protein, in which B lymphocytes and antibodies are known to play a more relevant role [113]. In RA, additional studies are necessary to understand whether the reduction of inflammatory cytokines observed in rats with adjuvant-induced arthritis treated with PRL is a direct effect of PRL on immune cells, or an indirect effect of enhanced prosurvival and reparative processes promoted by PRL in the joint. It might also be useful to evaluate the impact of PRL in other experimental models, such as collagen-induced arthritis, an extensively used model of RA involving B cells to a greater extent than adjuvant-arthritis [114].

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ACCEPTED MANUSCRIPT In conclusion, the involvement of PRL in autoimmunity appears much more complex than being solely restricted to immune stimulation, and might be the result of a fine interplay between

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the immune-modulating and regenerative properties of this hormone.

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ACCEPTED MANUSCRIPT Take-home messages 

Even though immune responses are not strictly dependent on PRL, this hormone has the

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potential to affect the phenotype and functions of cells belonging to both the innate and adaptive arms of the immune system.

PRL is also endowed with regenerative properties for several tissues and cells, such as

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

chondrocytes, oligodendrocyte precursor cells and neural stem/progenitor cells. 

The role of PRL in autoimmune disorders such as SLE, MS and RA is complex, and still

The work performed in experimental models of autoimmune disorders has revealed that

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

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there are several issues to be ascertained to understand how PRL affects these diseases.

PRL can promote, be dispensable or even protect against autoimmunity. While in experimental models of SLE increased levels of PRL (i.e. hyperprolactinemia)

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contribute to break B cell tolerance and promote autoimmune pathology, the study

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conducted in PRL- or PRLR-deficient mice suggests that PRL plays a redundant role in chronic EAE, animal model of MS.

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Hyperprolactinemia protects against adjuvant-induced arthritis, animal model of RA.

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

20

ACCEPTED MANUSCRIPT Acknowledgments

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This work has been supported by grants from Fondazione Italiana Sclerosi Multipla (FISM-AISM) (FISM cod. 2012/R/13 to R.P) and Italian Ministry of Health (GR-2009-1607206 to R.P.). M.C. has

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been supported by a training (research) fellowship from FISM (FISM Cod. 2008/B/2).

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The authors declare no conflicts of interest

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Statement of conflicts of interest

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ACCEPTED MANUSCRIPT Figure Legends.

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Figure 1. Summary of the immune-stimulating effects of prolactin based on in vitro approaches. Abbreviations: IFN-, interferon-; Ig, immunoglobulin; IL-1, interleukin-1; iNOS, inducible

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nitric oxide synthase; IP-10, interferon gamma-induced protein-10; IRF-1, interferon regulatory factor-1; LFA-1, leukocyte functional antigen-1; MAPK, mitogen-activated protein kinase; MHC, major histocompatibility complex; MIP-1, macrophage inflammatory protein-1; NK, natural

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killer; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription; TNF-

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PT

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MA

, tumor necrosis factor-; VLA-4, very late antigen-4.

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MA

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SC RI

PT

ACCEPTED MANUSCRIPT

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ACCEPTED MANUSCRIPT Table 1 PRL in the pathogenesis of experimental models of SLE, SM and RA. Rodent Model

Experimental approach

Clinical and pathological outcome

Proposed PRL effect

Refs

SLE

NZB/W F1 mouse

BCR treatment

Increased survival Reduced frequency of anti-DNA Ig

Detrimental

[72]

NZB/W F1 mouse

Pituitary transplant

Enhanced mortality Early albuminuria Increased titers of serum IgG

Detrimental

[72,75]

R4A-2b Balb/c Tg mouse

Injection of ovine or recombinant mouse PRL

Development of anti-DNA Ig Increase of mature B cells Glomerular IgG deposition

Detrimental

[76]

Sle3/5 R4A-2b C57BL/6 Tg mouse

Implantation of ovine PRL pellets

Development of anti-DNA Ig Glomerular IgG deposition Proteinuria

Detrimental

[79]

EAE in Lewis rats

Treatment with ergoline derivatives

Amelioration of clinical symptoms Reduction of proliferative T cell response to myelin Ag

Detrimental

[47,8688]

MOG35-55induced EAE

PRL-KO mice

Slightly-delayed onset and full disease severity Mildly-delayed Th1/Th17 responses to myelin Ag

Redundant

[35]

MOG35-55induced EAE

PRLR-KO mice

Slightly-delayed onset and full disease severity Mildly-delayed Th1/Th17 responses to myelin Ag

Redundant

[35]

Lysolecithininduced demyelinationa

Injection of mouse recombinant PRL

Increase of oligodendrocyte proliferation Reduction of demyelination

Protective

[4]

Rat adjuvantinduced arthritis

Hypophysectomy

Decrease of paw swelling

Detrimental

[45,47]

Rat adjuvantinduced arthritis

BCR treatment

Decrease of paw swelling

Detrimental

[47]

Rat adjuvantinduced arthritis

Implantation of osmotic pumps delivering PRL

Decrease of paw swelling Reduction of bone erosion Down-modulation of transcripts for inflammatory mediators

Protective

[112]

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RA

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MS

PT

Human Disease

Abbreviations: Ag, antigen; BCR, bromocriptine; EAE, experimental autoimmune encephalomyelitis; Ig, immunoglobulin; MOG35-55, myelin oligodendrocyte glycoprotein (amino acids 35-55); MS, multiple sclerosis; PRL, prolactin; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; Tg, transgenic; Th, T helper. a Lysolecithininduced model of demyelination is generally used for analysing processes of demyelination and remyelination in the absence of adaptive immune response.

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ACCEPTED MANUSCRIPT Highlights

PRL has an immune-modulating potential



PRL is endowed with tissue-regenerative properties



PRL can promote, be dispensable or even protect against autoimmune pathology

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

37