Overview of the Soluble and Membrane-bound Tumor Factors Limiting NK- mediated Immune Surveillance

Many evidences suggest that NK cells are effective in patrolling for and eliminating tumors in their onset phase, but hardly limit the progression of large established solid tumors. Beside the transition of tumor cells towards a more aggressive phenotype, the NK cell efficacy might be limited by a complex immunosuppressive milieu present in the tumor microenvironment. Indeed, different mechanisms damping NK cell function have been shown in these last years. These include a plethora of tumor-derived immunomodulatory soluble factors (TGF-β, MIF, adenosine, LKynurenin, PGE2) as well as soluble ligands (MICA, ULBP-2, PVR, B7-H6) that compete with membrane-bound tumor ligands for binding to activating NK receptors. During NK-tumor cell contact the NK cell function can also be inhibited by the engagement on NK cells of different inhibitory receptors. The specific ligands might be either constitutively expressed at the tumor cell surface (HLA-I, B7-H3, PVR) or de novo induced/up-regulated (PD-Ls) by immunostimulatory factors (IFN-γ, TNF-α). These are largely released during the active phases of the immune responses and exert an unwanted side effect called “tumor adaptive immune resistance”. This review aims to summarize the best-known molecular mechanisms that, at various times and in different ways, can limit the efficacy of the NK-mediated immune surveillance of tumors.

INTRODUCTION
Natural killer cells (NK) are crucial cytolytic effectors belonging to the family of innate lymphoid cell (ILC) [1,2]. Originally described as cells exerting a "natural" cytolytic activity due to their capability to kill the highly susceptible K562 eritroleukemia cell line, it is now well established that they require activation to exert optimal effector functions. Moreover, the susceptibility to NK-mediated killing of established tumor cell lines is superior to that of tumors ex-vivo isolated from patients, as occurs in bone marrow metastases that are much more resistant to NK-mediated aggression [3]. This supports the concept that an effective NK-mediated anti-cancer activity can't dispense with an optimal activation of endogenous or adoptively transferred NK cells. It is also crucial to understand which NK cell subset, once activated, can exert the most effective anti-cancer activity in a particular immunotherapeutic setting. Indeed, it has been recently stressed that a great heterogeneity in NK cell phenotype and functions exists that goes beyond the classical CD56 dim CD16 high and CD56 bright CD16 low/neg NK cell dichotomy [4]. Beside the identification and description of CD56 neg NK cells [5] that are particularly abundant in peripheral blood of virus-infected donors, several studies highlighted the great heterogeneity of peripheral blood CD56 dim NK cells, which include subpopulations characterized by different capabilities of being activated by cytokines, antibodies or tumor contact [6,7]. Thus, adoptive transferred NK cell-based therapeutic protocols should combine optimal activation strategies, the selection of the best NK cell subpopulation, consider the in vivo persistence of the in vitro activated NK cells, and, last but not least, their chemokine receptor repertoire. Indeed, the scarce attitude of cytotoxic CD56 dim NK cells to reach and invade the tumor parenchyma represents a major obstacle for the effectiveness of NK cells, especially in the therapy of solid tumors [8,9]. This might occur also in a chemokine-rich tumor milieu, due to a defective expression in NK cells of the chemokine receptors involved in their migration toward peripheral tissues. This hypothesis matches with the scenario described in neuroblastoma patients whose peripheral blood CD56dim NK cells are characterized by an unusual reduced expression of CX3CR1, the fractalkine (CX3CL1) receptor [10]. Interestingly, CX3CR1 has been shown to be deeply downregulated by transforming growth factor beta 1 (TGF-β1), a pleiotropic soluble factor released by most cell types including tumors that is capable of modulating pivotal effector functions of different immune cells [11,12]. TGF-β1 is one among the several soluble factors present in the tumor microenvironment that recently emerged as potent immune-modulators. Moreover, beside the classical HLA class Imediated inhibition of the NK cell activity, several additional inhibitory signals have been recently described that, during NK-totumor contacts, limit the NK-mediated immune-surveillance. Thus, the choice of the most effective, activated NK cell subsets should not disregard their profile in terms of the cognate inhibitory receptorligand interactions. This review summarizes the best-known tumorderived soluble factors and tumor-associated surface molecules exerting an immunomodulatory role in NK cells.

MIF, adenosine, L-Kynurenin, PGE2
In the past few years, many different tumor-associated regulatory/ suppressive mechanisms have been widely studied ( Figure 1). Among these, molecules shed from the tumor cell surface, such as the soluble ligands of NKG2D [13][14][15], DNAM-1 [16] and NKp30 [17], activating receptors crucial for T-and/or NK cell-mediated immune-surveillance [18]. sMICA, sULBP-2, sPVR and sB7-H6 compete for binding with the ligands expressed on the tumor cell surface, thus hindering the efficient target recognition. Moreover, different tumor-secreted soluble mediators have been shown to clearly suppress the efficacy of the immune system. Among these, the macrophage migration inhibitory factor (MIF, also known as Glycosylation-Inhibiting Factor) [19][20][21] and adenosine, an endogenous purine nucleoside highly produced by tumors expressing CD39 and CD73, ectonucleotidases converting ATP to adenosine. Both MIF and adenosine have been demonstrated to inhibit cytotoxicity and cytokine production in human NK cells, the second mainly through the engagement of the adenosine receptor 2A (AdoR2A), which is coupled to adenylyl cyclase via Gs protein [22,23].
In the mouse model the use of AdoR2A antagonist reduced the metastatic potential of CD73+ tumors [24] that was further reduced by blockade of AdoR2B. Since in vitro AdoR2B blockade had no significant effects on NK cell cytotoxicity, the benefit observed in tumor-injected mice treated with AdoR2 antagonist might depend on both NK cell-dependent and -independent mechanisms. Along this line, in mice selectively lacking AdoR2 expression in myeloid compartment it has been shown that adenosine can indirectly suppresses T and NK cell-mediated antitumor activity by shaping the functions of different myeloid cells. In particular, in this mouse model, a reduced melanoma growth was associated with a significant increase in MHC class II expression and IL-12 release in tumor-associated macrophages (TAM), features fitting with an M1-like proinflammatory macrophage polarization. Moreover, AdoR2neg TAM, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC) showed a clear reduction in IL-10 expression, a cytokine originally described as a negative regulator of IL-12 production in LPSstimulated peripheral blood mononuclear cells [25]. To date IL-10 cannot be definitively included among the tumor-derived cytokines limiting NK cell activity since both stimulatory and inhibitory effects on NK cell functions have been described. On the contrary, a huge number of data have demonstrated the immunomodulatory function of L-kynurenine, the tryptophan catabolite derived from indoleamine 2,3-dioxigenase 1 (IDO1) pathway [26], and of Prostaglandin E2 (PGE2). Both factors deeply affect the cytokine-mediated upregulation of the expression and function of different activating NK receptors such as NKp46, NKp44 and NKG2D [27][28][29] a mechanism that seems to involve the c-Jun N-terminal Kinase (JNK) pathway [29]. The extent of in vivo suppression mediated by L-kynurenine and PGE2 might be considerable since these factors are released by several cell types colonizing tumor microenvironment, including the cancer-associated fibroblasts (CAF) [30,31], MDSC and DC [32]. In particular, IDOexpressing DC exert a deep immune-suppressive effect by affecting not only proliferation and effector function of NK cells, but also by inducing the conversion of CD4+ T cells into CD4+ CD25+ Foxp3+ regulatory T cell (Treg) [

TGF-β1
TGF-β1 is the prototypic tumor-derived immunomodulatory soluble mediator, although many additional functions have been described over the years, highlighting its pleiotropic activity. Different studies reported a significant contribution of TGF-β1 in the epithelial to mesenchymal transition (EMT), a process that allows tumors of epithelial origin to acquire a less differentiated, invasive and prometastatic phenotype [40,41]. TGF-β1 has been shown to suppress the differentiation process and the effector functions of several immune cells [12]. In particular, it represses the development of human NK cells from CD34+ progenitors and inhibits differentiation of CD16 pos NK cells [42], TGF-β1 promotes the conversion of peripheral NK cells to a decidual NK-like phenotype [43] and, as shown in mouse salivary gland, it drives the differentiation of a particular ILC subpopulation sharing NK and ILC1 features [43]. Released as a large latent complex, TGF-β1 remains biologically unavailable until its activation in inflammatory sites such as the tumor microenvironment by signals including low pH, heat, proteases and members of the integrin receptor family [11]. Once activated, TGF-β1 shows a potent inhibitory effect on NK cells, both in vitro and in vivo, limiting the main NK cell effector functions including IFN-γ production and cytotoxicity [44][45][46][47][48]. In this context, in vitro conditioning of NK cells with recombinant TGF-β1 (rTGF-β1) caused severe downregulation of the surface expression of NKp30 and NKG2D, activating NK receptors cooperating in recognition and killing of several tumor histotypes [44]. The same occurred with rTGF-β2 [10], although knockout mice lacking TGF-β1 or TGF-β2 showed distinct phenotypic features suggesting that the two isoforms could also have specific, nonoverlapping functions [49]. Interestingly, while the TGF-β1-mediated downregulation of NKp30 occurred at the transcriptional levels no significant changes in NKG2D transcript was observed in TGF-β1treated NK cells [44]. Accordingly, it has been shown that the reduced NKG2D surface expression observed in the presence of TGF-β1 both in vitro and in vivo, is due to its capability of downregulating at transcriptional and translational level DAP10, the signaling subunit associated with NKG2D [50][51][52]. Very recently it has been shown that TGF-β1 also inhibits the IL-15-induced NK cell activation, particularly by selectively and quickly repressing the mTOR pathway [53], a crucial integrator of both pro-and anti-inflammatory signals. Recent data also suggest that the tumor-derived TGF-β1 might modify the migratory capability of NK cells. Indeed, it has been shown that neuroblastoma (NB) cell lines spontaneously release amounts of TGF-β1 capable of modulating the chemokine receptor repertoire of NK cells [10]. In particular NB-derived TGF-β1 increases CXCR4 and CXCR3 surface expression in all NK cells whereas it decreases that of CX3CR1 in the CD56 dim NK cell subset. Notably, unusual CX3CR1 low CD56 dim and CXCR3 high CD56 bright NK cell populations were observed in peripheral blood of patients with high risk NB (stage 4 or M) [10]. Thus, tumorderived TGF-β1 can affect the expression of chemokine receptors that play a key role in the bone marrow homing, egress, interaction with endothelium and recruitment into peripheral tissues of NK cells. Recently, in a mouse model of pulmonary allergic responses, it has been shown that TGF-β1 is crucial for the generation of allergic response acting as chemotactic factor recruiting ILC2 and eosinophils [54]. Importantly, it has been demonstrated that TGF-β1 modulates in tumor cells, the expression of specific microRNAs and up-regulates B7-H3 (CD276) [55], a molecule belonging to the family of immune checkpoint proteins, which acts as co-inhibitory factor and limits the function of NK cells. Overall data suggest that TGF-β1 antagonists, capable of overcoming or blocking its immunomodulatory effect might represent a valuable adjuvant therapy in the cure of different tumors. In this context, it has been recently shown that blocking of TGF-β1R in combination with antibodies targeting the NB-associated antigen GD2, potentiates the NK-mediated anti-NB activity leading to a reduced tumor growth and increased survival of mice injected with NB cell lines or patient-derived neuroblasts [56]. Moreover, ongoing clinical trials will evaluate the benefit of TGF-β or TGF-βR blockade in neoplastic patients (ClinicalTrials.gov NCT02452008; NCT02581787). It is of note however that, due to the wide homeostatic regulatory role of TGF-β, in order to obtain specific and restrained therapeutic effects, light should be made on the signaling pathways mediated by the different TGF-β isoforms and on the mechanisms regulating the immunomodulatory effects.

KIRs/HLA-I and NKG2A/HLA-E
The most powerful inhibitory pathway affecting the NK cell activity is represented by the interaction between HLA class I molecules (HLA-I) on target and specific inhibitory receptors on NK cells (Figure 1). These inhibitory receptors include Killer Ig-like Receptors (iKIRs), clonally distributed receptors distinguishing among allotypic determinants of the classical HLA-A, -B and -C, and the CD94/ NKG2A heterodimer specific for HLA-E [57][58][59]. Mature NK cells can also express the activating counterpart of these HLA-I specific inhibitory receptors, i.e. activating KIRs (aKIRs) and CD94/NKG2C [60,61]. During NK cell maturation the engagement of inhibitory receptors by their self HLA-I ligands confers functional competence to NK cells, through a process referred to as "licensing" or "education" that has been explained by different models [62][63][64]. Based on the "rheostat" model, during NK cell education NK cell reactivity is tuned by the strength of the inhibitory signal induced by self-HLA-I molecules [65]. The inhibitory receptor repertoire acquired by NK cells during maturation guarantees that in normal conditions inhibitory signals prevail on the activating ones safeguarding HLA-I + autologous healthy cells from NK cell-mediated killing. The NK cytolytic activity is unleashed in pathologic conditions such as virus infection or tumors where transformed cells increase the expression of ligands for activating NK receptors while downregulating that of HLA-I [66]. NK cell reactivity can be limited when tumor cells retain high levels of HLA-I expression, as occur in hematological malignancies such as acute lymphoblastic leukemia (ALL). However, it has been shown that in allogeneic settings such as in the context of haploidentical hematopoietic stem cell transplantation (haplo-HSCT), the differentiation of alloreactive NK cells from the donor, i.e cells expressing KIRs specific for HLA-I molecules absent in the recipient, strongly improve anti-leukemic surveillance [67,68].
The studies describing the beneficial graft versus leukemia (GvL) effect of alloreactive NK cells in haplo-HSCT has inspired the design of new immunotherapies aimed to enhance anti-tumor NK cell reactivity by blocking the interactions between HLA-I and iKIR or CD94/ NKG2A. A fully human anti-KIR mAb (1-7F9, Lirilumab, IPH2101) that recognizes iKIRs (KIR2DL1, KIRDL2 and KIR2DL3) has been generated [69], which favors the NK cell-mediated killing of HLAmatched tumor cells as documented in vitro and in vivo in phase I/II clinical trials involving Acute Myeloid Leukemia (AML) and multiple myeloma (MM) patients, [69,70]. Interestingly, Lirilumab has been successfully combined with the anti-CD20 rituximab to augment NKmediated cytotoxicity against lymphoma cells in vitro [71]. Based on the broad expression of HLA-E on both solid and hematological malignances, a novel therapeutic approach has been designed to block the CD94/NKG2A-HLA-E interaction, by using the humanized anti-NKG2A Monalizumab, which is currently in a phase I/II clinical trial [72]. This novel approach was developed upon the observation that NKG2A+ NK cells predominate in the early period of immune reconstitution after HSCT, thus representing optimal targets to potentiate NK cell-mediated anti-leukemic activity [73]. In addition, NKG2A+ NK cells express higher levels of activating receptors such as NCRs as compared to more differentiated KIR+ NKG2A-NK cells [6]. The high expression level could compensate the low cytotoxic potential displayed by these less differentiated NK cells. KIRs, CD57 and LIR represent phenotypic hallmarks of terminally differentiated NK cells, which show a good cytolytic potential but poor responsiveness to cytokines [74]. Interestingly, in individuals exposed to pathogens such as cytomegalovirus (CMV), the mature NK cell population is characterized by a very high percentage of cells expressing CD94/ NKG2C or aKIR [75][76][77][78], which show features similar to cells of adaptive immunity including clonal expansion capability, strong effector functions and longevity. This "memory-like" population represents a powerful candidate for adoptive NK cell transfer therapy in cancer patients [79]. Along this line it is crucial to define whether its efficacy might be limited during NK-to-tumor contacts by additional inhibitory or co-inhibitory signals, whose activity might be particularly relevant in the context of HLA-I low or negative tumors.

PD-1/PD-Ls, LAG-3/HLA-II and TIGIT/PVR
The PD-1/PD-Ls axis is a well-known immune checkpoint, i.e. inhibitory pathways that physiologically maintain self-tolerance and limit the duration and amplitude of T cell immune responses, thus minimizing tissue damage [80][81][82]. The PD-1 receptor (CD279) has been demonstrated to limit T proliferation and switch off the T cell functions mostly in peripheral tissues. More recent reports show the presence of PD-1 pos NK cells in both cancer patients and in a relevant proportion of healthy donors who were serologically positive for human CMV [83][84][85][86]. PD-1 expression is confined to terminally differentiated NKG2A neg KIR pos CD57 pos NK cells and their antitumor activity can be partially restored in vitro by antibodies disrupting the interaction between PD-1 and its cellular ligands PD-L1 and PD-L2 [86,87]. PD-L1 (B7-H1, CD274) and PD-L2 (B7-DC, CD273) belong to the B7 family that consists of several members including B7-H3 (see below) ( Figure 1). PD-L1 is expressed in several normal tissues, whereas PD-L2 is mainly restricted to antigen presenting cells (APC) [80,81]. Importantly, both inhibitory ligands can be expressed by tumor cells in response to immunostimulatory factors such as IFN-γ and TNFα that are released by activated T and NK cells [80,85,88]. This phenomenon called "adaptive immune resistance" [88] is a mechanism of escape by which cancer cells adapt their phenotype to the pressure of immune responses. Interestingly, it has recently been shown that PD-L1 neg metastatic cells purified from bone marrow aspirates of high risk NB patients have different capabilities of up-regulating PD-L1 in response to IFN-γ [85]. The lack of PD-Ls upregulation in some patient might contribute to a reduced clinical response to therapy with anti-PD-1. However, it should not be disregarded that the therapeutic efficacy of anti-PD1 antibodies has been observed also in patients who at the time of therapeutic decision carried PD-L1 neg tumors. Indeed, the clinical benefit might also depend on the strengthening of the crosstalk between PD-Ls+ APC and PD-1+ immune effectors including T or NK cells. IFN-γ is also a potent inducer of HLA class II molecules that can be recognized by LAG-3, an additional inhibitory mechanism that recently emerged in human NK cells together with TIGIT/PVR and TIM-3/galectin-9 interactions (Figure 1) [89][90][91][92]. Interestingly, the heterogeneous expression of the TIGIT inhibitory receptor observed in NK cells from healthy individuals inversely correlates with their capability of performing degranulation and IFN-γ release in response to IL-12 stimulation [91]. Moreover, low TIGIT expression has been described in CMV-induced terminally differentiated NK cells that appear more resistant to the inhibitory effect mediated by PVR pos MDSC. Since studies explored the cytokine-induced expression of these coinhibitory receptors mainly in long term-cultured NK cell lines such as NK92 [93], data on primary NK cells are required to better understand the relative contribution of these inhibitory pathways and the kinetic that regulates their emergence.

B7-H3R/B7-H3
Another interesting NK-to-tumor contact inhibitory pathway is mediated by the B7-H3 ligand, a tumor-associated surface molecule, also present in tumor-derived exosomes [94], which is endowed with both immune-regulatory and pro-tumoral functions. B7-H3 is capable of inhibiting the cytolytic activity of human NK cells against neuroblasts purified from bone marrow aspirates of high risk NB patients, which are characterized by reduced levels of HLA-I [18] and adhesion molecules (unpublished observation). The B7-H3-mediated inhibitory effect, which depends on its interaction with a still unknown inhibitory receptor, is particularly evident when using xenogenic B7-H3 high transfectants [95]. This suggests that this inhibitory pathway might require peculiar conditions to be unleashed, which could be represented by poor engagement of potent inhibitory NK receptors and/or by the presence of weak activating signals. It should be mentioned that in mouse B7-H3 has been described as a "friend" in tumor immunology [96]. In particular, intratumoral injection of an expression plasmid encoding mouse B7-H3 led to a complete NK-(and T-) mediated regression in approximately half of tumor-bearing mice [97]. Interestingly, the mouse B7-H3 gene codes for a molecule characterized by two Ig-like domains while human cell tissues predominantly express a four Ig-like domains isoform resulting from exon duplication [98]. While in human B7-H3 is still an orphan ligand, an activating receptor has been identified in mice that is represented by TREM-like transcript 2 (TREML2, TLT-2), expressed by activated T cells and myeloid cells [99]. Importantly however, Leitner and coworkers who extensively faced this issue did not find evidence for B7-H3/TREML2 interaction in human [100]. Considering that B7-H3 belongs to the B7 family that includes members interacting with both activating and inhibitory receptors, it can't be excluded the existence of a complex scenario resulting from the capability of B7-H3 to engage receptors with opposite signal. However, to date most in vitro and in vivo data lean toward a B7-H3 inhibitory role in human and the B7-H3R/B7-H3 axis has been included among the immune checkpoints [81,101,102]. An adjuvant therapeutic strategy in cancer might be represented by antibodies disrupting the interaction between B7-H3 and its receptor/s. Different phase I Clinical trials are ongoing with humanized anti-B7-H3 mAbs (NCT02628535; NCT02982941; NCT02475213) [81] and encouraging results have been obtained in the first in-human intrathecal injection of radioiodinated anti-B7-H3 Ab (following standard therapy) in 21 neuroblastoma patients with recurrent Central nervous system (CNS) metastasis [103]. It is of note that the therapeutic efficacy of anti-B7-H3 mAbs might depend not only by the strengthening of the NK-(and T-) mediated anti-tumor responses, but also by the weakening of the direct pro-tumoral activity of B7-H3. Indeed, studies in tumor of different histotype showed that high B7-H3 expression drives tumor cell progression through different molecular mechanisms. These include promotion of migration and invasiveness [104] and reduction of sensibility to chemotherapyinduced apoptosis, as demonstrated in breast [105] and pancreatic carcinoma [106]. Accordingly, high expression of B7-H3 is a negative prognostic factor in several tumors including neuroblastoma [18,[107][108][109][110][111]. In particular, in primary neuroblastoma high B7-H3 surface expression, in terms of both intensity and percentage of positive cells, has been correlated with poor event-free survival also in patients with localized disease (stage 1-3), suggesting that high B7-H3 expression might discriminate between low-and high-risk patients who need a more careful follow-up. NK cell-based immunotherapy is becoming a promising approach for the treatment of both hematological malignances and solid tumors.

Ready to kill or killers who require to be armed?
Originally described as cells exerting a "natural" cytolytic activity it is now well established that, to exert optimal effector functions, NK cells require activation via immunostimulatory cytokines and tumor contact.
NK cell population is heterogeneous and includes subsets characterized by different capabilities of being activated and different effector functions.
Once activated, NK cells need a chemokine receptor repertoire ideal to their recruitment in inflamed tissues such as tumors.

How tumors may dump NK cell function in vivo?
Tumor cells purified from patients are less susceptible to NKmediated killing than established tumor cell lines commonly used in vitro.
A complex immunosuppressive milieu is present in the tumor microenvironment.
A plethora of tumor-derived immunomodulatory factors exists, either soluble or membrane bound, that might limit the NK-mediated immune-surveillance in vivo.

Soluble immunomodulatory mediators
Tumor cells may release soluble ligands (sMICA, sULBP-2, sPVR and sB7-H6) that compete with membrane-bound tumor isoforms for binding to activating NK receptors. TGF-β1 is the prototypic tumor-derived immunomodulatory soluble mediator. In NK cells, it represses development from CD34+ progenitors and differentiation, limits the main effector functions (IFN-γ production and cytotoxicity), reduces the expression level of activating receptors (NKp30, NKG2D) and alters the chemokine receptor repertoire.

NK-to-tumor contact immunomodulatory signals
NK cell reactivity can be limited when tumor cells retain high levels of HLA-I expression, as occur in hematological malignancies.
Tumor cells may express ligands (PD-L1, PD-L2, B7-H3) belonging to the immune checkpoint family, i.e. inhibitory pathways that limit the duration and amplitude of anti-tumor responses.
B7-H3 not only weakens the strengthening of the NK-(and T-) mediated anti-tumor responses, but also exerts a direct pro-tumoral activity.
Tumor cells can constitutively express inhibitory ligands or increase their expression in response to immunostimulatory cytokines (IFN-γ, TNF-α) that are released by activated NK (and T) cells, a phenomenon called "tumor adaptive immune resistance".
IFN-γ, besides inducing PD-Ls expression, is also a potent inducer of HLA-II that can be recognized by LAG-3, an additional inhibitory mechanism that recently emerged together with TIGIT/PVR and TIM-3/galectin-9 interactions.

Future directions
When designing immunotherapies, we can't neglect the complexity of the immune-suppressive milieu characterizing the tumor microenvironment. Thus, NK cell-based therapeutic protocols should combine optimal activation strategies, the selection of the best NK cell subpopulation, consider the in vivo persistence of NK cells, and their chemokine receptor repertoire. Valuable adjuvants may include the use of humanized mAb to disrupt immune checkpoints pathways and/or to hinder the effects of soluble factors that in vivo may dampen the NK cell activity against tumors.


extfieldsofstudy: Biology Medicine

text:
Aberrant iPSC-derived human astrocytes in Alzheimer's disease

The pathological potential of human astroglia in Alzheimer's disease (AD) was analysed in vitro using induced pluripotent stem cell (iPSC) technology. Here, we report development of a human iPSC-derived astrocyte model created from healthy individuals and patients with either early-onset familial AD (FAD) or the late-onset sporadic form of AD (SAD). Our chemically defined and highly efficient model provides >95% homogeneous populations of human astrocytes within 30 days of differentiation from cortical neural progenitor cells (NPCs). All astrocytes expressed functional markers including glial fibrillary acidic protein (GFAP), excitatory amino acid transporter-1 (EAAT1), S100B and glutamine synthetase (GS) comparable to that of adult astrocytes in vivo. However, induced astrocytes derived from both SAD and FAD patients exhibit a pronounced pathological phenotype, with a significantly less complex morphological appearance, overall atrophic profiles and abnormal localisation of key functional astroglial markers. Furthermore, NPCs derived from identical patients did not show any differences, therefore, validating that remodelled astroglia are not as a result of defective neural intermediates. This work not only presents a novel model to study the mechanisms of human astrocytes in vitro, but also provides an ideal platform for further interrogation of early astroglial cell autonomous events in AD and the possibility of identification of novel therapeutic targets for the treatment of AD.

pathology. Nevertheless, most of our knowledge of AD derives from studies that utilise cell-and animal-based models of the clinically rare, early-onset, dominantly inherited familial AD (FAD). The cellular pathology of astroglia in the context of human AD remains enigmatic, mainly because of severe limitations of animal models, which, although reproducing some pathological features of the disease, do not mimic its progression in full. Neuronal loss and cognitive deficits, which are the hallmarks of AD in humans, are limited in animal models 15 and there is an increasing body of evidence showing that significant differences exist between rodent and human astrocytes. 16 Although induced pluripotent stem cell (iPSC) technology can be used to investigate human astrocyte development and function, only a limited number of studies have described homogeneous generation of healthy astrocytes. [17][18][19][20][21][22] Previous work has shown that neurones differentiated from SAD-and FAD-iPSCs resemble pathologically affected cells in vivo and express key disease hallmarks; [23][24][25] however, there is still a pressing need for tools to provide robust, homogeneous astrocyte populations to reveal astroglial contribution to neurodegenerative diseases. Here we report the generation of enriched mature astroglia from human iPSCs derived from patients with confirmed FAD and SAD as well as from a healthy control. We found that, although astrocytes derived from AD patients express the same canonical markers of mature healthy astrocytes, their morphological appearance and cellular phenotype is significantly distorted. Notably, we did not observe any differences in early neuronal commitment in early AD-derived neurones compared with healthy controls, indicating the cell autonomous pathological potential of astroglia and further confirming that cellular pathology does not stem from defective neural progenitors.

Results
Generation of neural cell lineages. Cortical NPCs were propagated in monolayer culture and maintained as described in ref. 24. Both FAD and SAD NPCs showed indistinguishable culture morphology to healthy NPCs (N = 5 per cell line; Figures 1a-c). Although a degree of variability in cell proliferation and viability was observed between cells from each individual, no significant differences in NPC growth rates were identified (N = 4 per cell line, two-way Kruskal-Wallis, Figures 1d and e). Phenotypic identification of NPCs showed positive expression of the transcription factors SOX1 and PAX6, and the filamentous marker, nestin (Figures 1f-n). However, no significant difference in NPC marker expression was observed irrespective of genotype (quantified by the proportion of immunoreactive nestin+ cells; N = 4 per cell line, analysis of variance (ANOVA), F (2,9) = 0.022, P = NS, Figure 1o). Furthermore, under terminal neuronal differentiation conditions for 35-40 days, all patient samples showed positive expression of the neural marker β-III-tubulin (Figures 1p-r). No significant difference was detected in the efficiency of early neuronal induction (as measured by the proportion of β-III-tubulin+ neurones) between any individual (N = 4 per cell line, ANOVA, F (2,9) = 0.128, P = NS, Figure 1s). Expression of the mature deep-layer cortical neuronal marker, CTIP2 was observed throughout the cultures from each patient, indicating maintenance of cortical identity following 40 days in culture (Figures 1t-v). In a separate study, we also show that cortical neurones derived from identical FAD and SAD patients exhibit elevated pathological Aβ-42 secretion and hyperphosphorylated tau species compared with healthy individuals, therefore validating an AD phenotype in these cells (data not shown). The results presented herein reveal that both SAD and FAD NPCs retain their characteristic morphology, expression of canonical markers and are able to generate mature cortical neurones with the same efficiency as healthy derived NPCs, Importantly, therefore, any changes in astroglia are not as a result of defective neuronal intermediates.
To independently evaluate astroglial commitment, we adapted a defined differentiation protocol 18 to generate purified astroglial cultures. We included additional culture supplementation of EGF and insulin, which have been shown to increase glial commitment, primary astrocyte proliferation and sub-culturing in vitro. 26,27 Astrocytes were generated within 30 days following induction from cortical NPCs in each cell line as confirmed by positive immunoreactivity to the canonical marker, GFAP and mature astrocyte markers; EAAT1, S100B and GS (Figures 2a-d). Quantification of GFAP, EAAT1, S100B and GS immunoreactive cells revealed near-pure expression in all astrocytes and no significant differences were observed between astrocytes derived from healthy control, FAD and SAD samples (N = 5 differentiations per cell line (minimum 120 cells per group), ANOVA, Supplementary Figures S1A-D). We also examined astrocyte proliferation and viability (day 14 following induction) in both AD and control cells. Despite some variability in growth rates from each individual, no significant differences were observed in overall astrocyte proliferation or viability (N = 3 per cell line, two-way Kruskal-Wallis, Supplementary Figures S1E and F). These results demonstrate that both PSEN1 mutant and ApoE4 +/+ NPCs are capable of generating astroglia in vitro and retain expression of markers consistent with mature astrocytes in vivo.
Altered cellular heterogeneity in FAD and SAD astrocytes. Our initial observations revealed considerable differences in astrocyte morphology in cells derived from AD-NPCs compared with healthy controls. In healthy astrocytes, GFAP staining intensities did not differ from cell to cell, but overall morphology of individual cells across the population varied significantly (Kruskal-Wallis; H (2) = 13.161, P = 0.001, N = 3 differentiations). The majority of healthy astrocytes (Figure 2g; 61.2% ± 6.64; 93/145 cells) displayed long (defined as 41 × cell body width) branching fine processes consistent with archetypal astrocyte phenotype (herein referred to as 'arborised'; Figure 2e). A significantly smaller population showed a process-devoid, fibroblast-like phenotype ('fibroblast-like'; 24.9% ± 8.45; 33/145 cells; Dunn-Bonferroni versus arborised cells, P = 0.002; Figure 2f, arrow). The remaining cells were thin, highly polarised, but essentially process-devoid cells ('polarised'; 13.9%   (2,9) = 0.128, P = NS). (t-v) Expression of the mature deep-layer cortical neuronal marker, CTIP2 was observed throughout cultures from each patient (green). Scale bars, 50 μm of each morphological cell type between the FAD and SAD astrocyte groups. Moreover, no significant difference in immunoreactive GFAP intensity was seen in any group (Kruskal-Wallis, H (2) = 4.496, P = NS; Figure 5f), nor was any loss of nuclear integrity or fragmentation of the GFAP cytoskeleton observed. Taken together, these data demonstrate that astrocytes derived from patients carrying a PSEN1 M146L mutation or both ApoE4 alleles display a significant Figure 3 Induced astrocytes derived from PSEN1 M146L FAD patient fibroblasts express normal astrocyte markers but show reduced morphological heterogeneity compared with healthy cells. Induced astrocytes were immunostained for GFAP (a), S100B (b), EAAT1 (c) and GS (d). Immunostaining is shown in green, DAPI counterstained nuclei are shown in blue; 495% of observed cells were positive for all markers tested. The significant majority of FAD astrocytes display a fibroblast-like, process-devoid appearance (summarised in e). **Po0.005, *Po0.05. Scale bars, 50 μm (inset, 20 μm) Figure 2 Induced astrocytes derived from healthy patient fibroblasts express mature astrocyte markers and exhibit varied morphologies. Induced astrocytes were generated in 30 days and confirmed by positive immunoreactivity to the functional markers: GFAP (a), S100B (b), EAAT1 (c) and GS (d). Immunostaining is shown in green, DAPI counterstained nuclei are shown in blue. Heterogeneity of morphology within the population of induced astrocytes was evident, with cells falling into three broad categories when stained for GFAP: arborised cells (e); polarised cells (f, arrowhead); or fibroblast-like, process-devoid cells (f, arrow); summarised in (g). **Po0.005, *Po0.05. Scale bars, 50 μm (inset, 20 μm) reduction in morphological heterogeneity when compared with those derived from healthy individuals.
To quantify astrocyte arborisation, we calculated the surface area to volume ratio, SA:Vol (Figure 5e). A larger SA:Vol ratio would indicate a cell with a relatively small cell body (low volume) displaying many processes (large surface area), whereas a small SA:Vol ratio would indicate a more rounded, fibroblast-like cell (large volume) lacking processes (low surface area). We found that the SA:Vol ratio was significantly higher in healthy astrocytes (2.12 ± 0.07 μm − 1 ) compared with either FAD or SAD cells (1.14 ± 0.05 μm − 1 and 1.19 ± 0.05 μm − 1 , respectively; Kruskal-Wallis; H (2) = 47.385, Po0.001, Dunn-Bonferroni pairwise comparisons: healthy versus FAD and SAD, both Po0.001; FAD versus SAD, P = NS), indicating a greater degree of arborisation in healthy over diseased cells. Again, these data reveal very little difference between FAD and SAD astrocytes, indicating that aberrant astrocyte morphology is a shared feature of both forms of the disease.
Aberrant expression and localisation of S100B, EAAT1 and GS in AD astrocytes. In healthy astrocytes, S100B expression was observed throughout the cytoplasm and, to a lesser extent, in the nucleus, consistent with normal astrocytes in vivo (Figure 6a, top panel; Figure 6b, left; Supplementary Movie S1). In contrast, both FAD and SAD astrocytes displayed multiple large accumulations of S100B localised to the nucleus, with very little staining in other cellular compartments (Figure 6a, middle and bottom panels, respectively). To confirm that S100B was confined within the nuclei of diseased cells, we utilised z-sectioning confocal microscopy and IsoSurface reconstruction to render 3D models of the staining. Renders of GFAP and DAPI staining were also made to permit comparisons. In both FAD and SAD astrocytes, S100B expression was restricted to the nucleus and confined to multiple, discrete foci following a similar pattern to that of nucleoli (Figure 6b, centre and right; Supplementary Movies S2 and S3, respectively). Fluorescence intensity analysis indicated that S100B levels were significantly reduced in both FAD and SAD compared with controls, but unchanged relative to each other (ANOVA, F (2,16) = 44.509, Po0.001; Hochberg's GT2, healthy versus FAD and SAD Po0.001, FAD versus SAD P = NS; Figure 5g). In both healthy and FAD astrocytes, EAAT1 revealed a uniformed pattern of fluorescence extended throughout the cell (Figures 2c and 3c, respectively). In contrast, SAD astrocytes exhibited confined nuclear accumulation of EAAT1 (Figure 4c). Quantification of fluorescence  Figure 5h). GS immunoreactivity in healthy astrocytes revealed a uniform pattern of fine puncta throughout the cytoplasm (Figure 2d, inset). In diseased cells, however, this smooth pattern was lost and replaced with a distinctly more clumped pattern of staining with much larger puncta (FAD, Figure 3d Table S1) using a proteomic array (Supplementary Figures S2A-C). Astrocytes, irrespective of their genotype, constitutively secreted only three cytokines: IL-8 (CXCL8), MCP-1 (CCL2) and TIMP-2. However, SAD astrocytes additionally secreted RANTES (CCL5) and MIP-1b. Quantification of expression levels using densitometry showed that IL-8 and MCP-1 secretion was significantly decreased in both non-stimulated ACM from FAD-and SAD-derived astrocytes compared with control ACM (Dunn-Bonferroni; Po0.001 for both, N = 3; Supplementary Figure S2D). These results show that human astrocytes secrete a distinct set of soluble mediators under non-stimulated physiological conditions Figure 5 Astrocytes derived from PSEN1 M146L FAD and ApoE4 +/+ SAD patients exhibit significant atrophy when compared with those from healthy patients as revealed by visual binning according to overall morphology (a). Exemplar 3D IsoSurface renders constructed from serial confocal z-stacks display clear differences in cell size and overall morphology (b). Scale bar, 10 μm. Quantification of cells using these renders by way of surface area (c), cell volume (d) and SA:Vol ratio (e) reveal significant differences in all aspects of cellular morphology between healthy and diseased astrocytes. Quantification of mean fluorescence intensity per immunoreactive cell reveals no significant difference in GFAP staining intensities between AD and control astrocytes (f) but S100B, EAAT1 and GS intensities are reduced in both FAD and SAD cells (g-i, respectively). ***Po0.001, **Po0.005, *Po0.05 in vitro and are partially compromised in both FAD and SAD astrocytes.

Discussion
Since astrocytes are the main homeostatic units of the CNS, their dysfunction may drive progression of neurologic disorders, including AD. Astrocytes contribute to non-cell autonomous mechanisms in various neurodegenerative disorders that were previously thought of as classically neuronal diseases. 28,29 Here we report novel pathological astroglial phenotypes in both FAD and SAD through exploitation of an iPSC-based human model. We show evidence to support critical astroglial contribution to AD namely: (i) mislocalisation and abnormal expression of mature astrocyte markers, (ii) compromised astrocyte heterogeneity and (iii) astroglial atrophy. Singularly, we did not observe differences in early maturation of AD-derived neurones compared to healthy controls, showing that astroglial changes are cell autonomous and do not derive from compromised neuronal intermediates (Figure 7). Generation of functional astrocytes from healthy iPSCs has previously been reported to be time-consuming and result in 20-30% contamination of unwanted cell types. [18][19][20] In contrast, we demonstrate highly efficient generation of enriched populations of mature cortical astrocytes (495%), irrespective of donor origin, within 30 days of Figure 6 S100B localisation within astrocytes is dramatically altered in FAD and SAD Healthy astrocytes were immunostained for S100B and visualised using deconvolution fluorescence microscopy (a) revealing S100B to be distributed throughout the cytoplasm (top panel). In contrast, in FAD and SAD cells, S100B appeared to be localised exclusively at the nucleus (middle and bottom panels, respectively). Scale bar, 20 μm. IsoSurface renders of S100B (red) and of DAPI-stained nuclei (blue; with a clipping plane set to permit visualisation of the inside of the nucleus) were constructed from serial confocal z-sections to further investigate the subcellular localisation of S100B (b; scale bars, 10 μm). GFAP staining (green) is shown for comparison for healthy cells to reveal the overall cell morphology. In healthy cells, as expected, S100B displayed a cytosolic distribution, similar to that of GFAP. In both FAD and SAD astrocytes, S100B is localised exclusively to discrete foci within the nucleus. Nuclei images have been zoomed in × 2 for clarity. Movies of these renders can be viewed in Supplementary Movies S1-S3 Aberrant human astrocytes in AD VC Jones et al induction. This is likely due to additional medium supplementation of EGF and insulin, both of which have been previously shown to increase glial commitment and primary astrocyte proliferation in vitro. 26,27 Owing to a lack of astrocyte markers that allow precise examination of subtypes that occur throughout the CNS, we were not able to confirm their cortical identity. However, it has been previously published that maturation of neurones or glia from regionally specified iPSC-derived NPCs maintain their identity. 30 Critically, the healthy iPSC-derived astrocytes described herein were comparable in their morphology and marker expression to those described in several previously published independent studies; validating our differentiation protocol and model. 18,19 Astrocytes exhibited 95-98% expression of GFAP, EAAT1, GS and S100B irrespective of healthy or disease origin. However, we observed significant redistribution and decreased expression of S100B in FAD and SAD astrocytes compared with healthy cultures. Although nuclear S100B expression has previously been reported, 18-20 its expression has not been shown to be exclusively restricted to the nucleus/ nucleoli. Why S100B would localise to a nuclear subcompartment in pathologically remodelled astrocytes remains to be established, but given that S100B is known to interact with various cytoskeletal components, together with the reduction in its expression levels, may represent a novel and early mechanism underlying SAD-and FAD-induced astrocytic atrophy. Mislocalisation of astrocytic GS was also observed in FAD and SAD astrocytes, however, altered subcellular EAAT1 distribution was only observed in cultures derived from SAD cells. Previous studies have shown altered glutamatergic signalling in neuronal AD degeneration. [31][32][33] Our results further reveal a previously unrecognised astroglial cell autonomous pathological phenotype in AD, although a significant reduction in the overall fluorescent intensities of EAAT1 and GS was only seen in FAD-derived astrocytes. This finding echoes our recent report of a significant decrease in GS expression in the frontal cortex of 3xTg-AD murine model, 34 and may hint that astroglia-associated glutamate turnover differs between the two forms of AD at an early stage.
We found that healthy induced astrocytes show significant morphological heterogeneity, in accordance with morphologically distinct cortical astrocytic subpopulations in vivo, 35 further validating the use of our cell-based model for the interrogation of astroglial function. In contrast, FAD and SAD astrocytes have reduced heterogeneity, are significantly smaller than their healthy counterparts and exhibit an almost complete absence of processes. This corroborates recent studies in mouse models of FAD showing reduced somata and process volumes in astrocytes together with a reduction in the number and complexity of astrocyte processes are an early feature of the disease. 10,11,36,37 Our results confirm that cell autonomous astrocytic atrophy is a feature of early-onset FAD, but also provide evidence that astrocytic aberrance is a likely characteristic of SAD.
We also show evidence for an altered basal astrocyte secretome profile in both FAD-and SAD-derived cells. Our study of non-stimulated healthy astrocytes in culture express a similar array of inflammatory mediators previously identified in cultures from highly purified human fetal and adult astrocytes. 38,39 Furthermore, these mediators have been previously shown to be direct targets of the transcription factor NF-κB indicating that this pathway may be constitutively active in human astrocytes under two-dimensional culture conditions. 38, 39 We also observed significantly compromised constitutive secretion of the pro-inflammatory mediators; IL-8 (CXCL8) and MCP-1 (CCL2) in FAD-and SAD-derived ACM, both of which have been previously shown to be the most abundantly expressed transcripts in adult healthy human astrocytes in vitro. 39 These early observations may indicate a potential glial paralysis as such, which has been postulated to be a fundamental factor in the evolution of AD. 14 Although, herein we describe the use of a single patient-derived cell line for each criteria, it is reasonably common-place that articles in this field are published whereby studies describe only a single Figure 7 Comparison of healthy control, FAD and SAD patient-derived βIII-tubulin immunoreactive neurones and GFAP immunoreactive astrocytes. Early neuronal appearance is indistinguishable across the groups, whereas AD astrocytes show markedly reduced heterogeneity of morphology and striking atrophy compared with healthy cells. Scale bar, 50 μm Aberrant human astrocytes in AD VC Jones et al patient-derived cell line due to the labour-intensive and costprohibitive nature of induced pluripotent stem cell-derived culture methods and differentiation protocols. 17,[40][41][42][43][44][45] However, it should be emphasised that cultured astrocytes utilised in the present study represent a simplified model relative to that of astrocytes in the CNS, whereby additional interactions with other cell types and matrix components are likely to influence the astrocytic phenotype. Yet, our highly purified human astrocyte culture model presents a unique system to delineate the autonomic responses of astrocytes to defined stimuli/matrix/co-cultures in both healthy and ADaffected cells.

Conclusions
Using a human in vitro iPSC model of familial and sporadic AD, we demonstrate a significant and previously unknown cell autonomous pathological phenotype of astroglia. Crucially, neuronal intermediates derived from identical iPSCs obtained from the same patients, did not show any pathological phenotype when compared with healthy controls, further corroborating recent ideas of the fundamental role of astroglia in the development of neurodegenerative diseases. We may further conjecture that early synaptic dysfunction, arising from the inhibition of astrocyte-synapse interplay and disruption of functional astroglial networks, is an early feature of AD progression, supporting the idea of astrocytic atrophy as a plausible mechanism for early cognitive impairment and thus providing a potential novel therapeutic target for AD intervention.

Materials and Methods
All reagents were purchased from ThermoFisher (Paisley, UK) unless stated otherwise.
Derivation and maintenance of neuronal cell lineages. Human iPSC-derived cortical neural progenitor cells (NPCs) were supplied by Axol Bioscience (Cambridge, UK) and generated from patient dermal fibroblasts exhibiting an M146L mutation in the presenellin-1 gene (PSEN1) from a 53-year-old male donor clinically affected with type III early-onset FAD and from an 87-year-old female clinically affected with late-onset SAD homozygous for the four allele of apolipoprotein E (ApoE4 +/+ ). The control samples were supplied by Axol Bioscience and reprogrammed from human female umbilical cord blood cells using the same method. NPCs were expanded following manufacturer guidelines and expression of known NPC markers were confirmed before astroglial differentiation.
For directed astroglial differentiation, NPCs were dissociated using accutase and attached with a substrate of poly-L-ornithine (20 μg/ml; Sigma, Dorset, UK) and mouse laminin (10 μg/ml) in chemically defined medium modified from Shaltouki et al. 18 comprising neurobasal medium containing B27 supplement (1 × ), nonessential amino acids (1 × ), L-glutamine (2 mM), penicillin/streptomycin (50 U/ml), 8 ng/ml FGF2 (Peprotech, London, UK), 5 ng/ml CNTF and 10 mg/ml BMP2 (Peprotech) with additional supplementation of 10 ng/ml EGF and 5 μg/ml insulin. NPCs were seeded at 3 × 10 4 cells/cm 2 and maintained at 37°C/5% CO 2 . The cells were cultured until 80% confluence (approximately 5 days) and passaged twice, as previously detailed. On the third passage, and all consecutive passages, the cells were plated onto non-coated tissue culture plastic to remove unwanted neurones and enrich for astroglia due to their superior adhesiveness. The cells were passaged at least three times during astroglial commitment. Astrocytes were generated a minimum of three times using independent differentiations from each patient sample.
For terminal neuronal differentiation, NPCs were seeded as above, but onto plates coated with 20 μg/ml poly-L-ornithine and 10 μg/ml laminin. The cells were maintained in a 1:1 mixture of N2:B27 media; where N2 medium comprised; DMEM/F-12 with Glutamax, N2 supplement (1 × ), 1 mM L-glutamine, non-essential amino acids (1 × ), 0.1 mM β-mercaptoethanol, 50 U/ml penicillin/streptomycin and 5 μg/ml insulin (Sigma); and B27 medium comprised Neurobasal, B27 supplement (1 × ), 200 mM L-glutamine, 50 U/ml penicillin/streptomycin. Terminally differentiated neurones were maintained for 30-40 days with medium changes every other day. The cell counts and viability were performed using Trypan blue exclusion assay and calculated using an automated cell counter. Population doubling times were calculated when viable cells exhibited exponential growth. Neurones were generated a minimum of three times using independent differentiations from each patient sample.
Fluorescence microscopy and image analysis. The fixed cells were visualised using a Zeiss Cell Observer z-sectioning fluorescence imaging system equipped Zeiss definite focus, HXP 120 V illumination and FITC, DsRed and DAPI filter sets. Fluorescence images were acquired using × 20 PL Apo (0.8 NA), × 40 EC Plan-Neofluar (1.3 NA) oil and × 63 PL Apo (1.4 NA) oil objectives, AxioCam MRm Rev.3 CCD camera and ZEN Pro software (Carl Zeiss, Cambridge, UK). The images were collected as serial optical z-sections taken at 0.28 μm intervals, which were subsequently deconvolved using a fast iterative algorithm with a maximum of five iterations. The z-stacks were processed and analysed using FIJI ImageJ. 46,47 All the images presented herein are maximum intensity projections of these z-stacks unless otherwise indicated. Morphological analysis was carried out by visually binning cells into one of three categories: fibroblast-like process-devoid cells, process bearing arborised cells and polarised cells. A minimum of 130 cells drawn from three separate batches of differentiated cells were analysed at × 20 magnification. Immunofluorescence intensity analysis was performed on a minimum of five random fields for each of three separate batches of differentiated cells of view at × 20 magnification using Fiji ImageJ. Z-stacks were flattened before the average background fluorescence, calculated from three random regions of each field of view, was subtracted. Raw integrated pixel densities of each image were calculated and divided by the number of immunoreactive cells to determine an average integrated pixel density per cell for each group.
To permit quantification of GFAP cytoskeleton volume and surface area, serial optical z-sections were collected at 0.5 μm intervals using a Leica TCS SP5 AOBS confocal microscope equipped with a × 63 HCX PL Apo (0.60-1.40 NA) oil objective, 405 nm and 488 nm laser lines and internal PMTs (Leica Microsystems (UK) Ltd, Milton Keynes, UK). Throughout all experiments, the pinhole was set to one Airy unit. The images were collected sequentially to minimise crosstalk using a bidirectional scan at 1000 Hz and three frame averages. Z-stacks were carefully created so as to include the entire thickness of each cell. Images were subsequently analysed using Imaris 7.7.2 3D visualisation software (Bitplane AG, Zurich, Switzerland) using the IsoSurface tool to create a smoothed 3D surface render of each cell by demarcating the edges of the GFAP-positive cytoskeleton and removing voxels before calculating GFAP surface area, volume and mean fluorescence intensity. A similar approach was used to determine the subcellular localisation of S100B by creating 3D surface renders of both S100B staining and DAPI-stained nuclei, before using a clipping plane to slice the DAPI channel only, revealing S100B staining within the nucleus. In each experimental group, a minimum of 21 randomly selected cells drawn from three separate differentiations were analysed. To quantify neural progenitor-, neurone-and astroglial-marker expression, the cell counts were expressed as a percentage of total cells in a field of view, where the total number of cells was identified using DAPIstained nuclei. The images were captured under standard epifluorescence on an Olympus IX71 microscope (Olympus, Southend-on-Sea, UK) and analysed using ImageJ. Five randomly chosen fields from three independent experiments were assessed.
Antibody arrays. ACM was collected following 30 days in culture and stored at − 80°C analysis of inflammatory mediators using a human antibody array kit (Abcam). ACM was collected following cell exposure for 48 h (normalised to cell number) and samples analysed in parallel to enable comparisons in relative expression levels between experiments. Each experiment represents pooled medium from three independent differentiations. Before analysis, all the samples were thawed on ice and centrifuged at 10 000 × g for 5 min at 4°C. Cytokine/ chemokine secretion was measured according to the manufacturer's instructions. Since, recombinant growth factors are present in ACM, a media-only control was also run in parallel to normalise the data. Positive immunoreactivity was visualised using chemiluminescence using a CCD camera (Bio-Rad, Hertfordshire, UK) and positive/negative membrane controls permitted densitometric analysis using Imagelab software (Bio-Rad).
Statistical analysis. All statistical analyses were carried out using IBM SPSS 22 (IBM Corp., Armonk, NY, USA). Data distributions were analysed by plotting histograms and Quantile-Quantile plots followed by Shapiro-Wilks tests to determine normality. For cell derivation and population analyses, each experiment was performed a minimum of three times and all assays were repeated in triplicate. Mean proportions of nestin+ NPCs and βIII-tubulin+ neurones were compared by one-way ANOVA. Analyses of cell viability and proliferation rates, cell morphologies and immunofluorescence intensity (with the exception of S100B) was carried out using Kruskal-Wallis tests followed by pairwise comparisons by the Dunn-Bonferroni method. S100B immunofluorescence intensity comparison was carried out by ANVOA followed by a Hochberg's GT2 post hoc. All the data are presented as mean ± standard error. An alpha level of 0.05 was considered significant throughout.

Conflict of Interest
The authors declare no conflict of interest.


s2fieldsofstudy: Biology Medicine Chemistry

text:
Preexposure of MCF-7 breast cancer cell line to dexamethasone alters the cytotoxic effect of paclitaxel but not 5-fluorouracil or epirubicin chemotherapy

Purpose Glucocorticoids (GCs) are often administered prior to any chemotherapeutics to prevent the secondary effects of anticancer agents. Glucocorticoid receptors (GRs) are expressed in several types of cancer cells, particularly in several histological types of breast cancer. Activation of GRs is not associated with any specific cellular response. Both proapoptotic and antiapoptotic responses have been observed, depending on the study or the type of breast cancer cells. Therefore, it is of relevance to investigate the possible modulation of apoptotic effect of chemotherapeutic agents when cancerous cells have previously been exposed to GCs. Methods In vitro cell growth was assayed by counting MCF-7 cells upon exposure to epirubicin (25 nM), 5-fluorouracil (5-FU) (15 µM), and paclitaxel (15 nM), either with or without prior exposure to the GC dexamethasone (Dex) (100 nM). Results Following preexposure to Dex, the antiapoptotic activity of paclitaxel was significantly reduced by 8.5% (p<0.05), but the activities of epirubicin and 5-FU remained unaltered. Conclusion In light of the finding that the response of MCF-7 cells pretreated with Dex was significantly reduced, we recommend that the function of GCs should be defined more precisely if they are to be used in conjunction with chemotherapy.

Introduction
Glucocorticoids (GCs) are steroid hormones that are critically involved in regulating and resolving inflammatory processes in mammals. They are also involved in many other essential processes, including cellular metabolism, differentiation, apoptosis, and immune response. 1 Clinically, GCs are used to treat allergic reactions and inflammatory or autoimmune diseases, to reduce soft tissue edema after solid organ transplantation, and to eliminate malignant lymphoid cells by triggering apoptotic cell death. 2 Various doses of GCs, most commonly dexamethasone (Dex), are frequently administered throughout the course of chemotherapeutic treatment for solid tumors in order to reduce toxicity and to protect normal tissue from the negative effects of continued exposure to genotoxic drugs. Dex is particularly effective in preventing chemotherapy-related hyperemesis. [3][4][5] First-line chemotherapy for early and advanced stage breast cancers is based mainly on anthracyclines (doxorubicin or epirubicin), cyclophosphamide, 5-fluorouracil (5-FU) (Merck, Darmstadt, Germany), and taxanes (primarily paclitaxel). 6,7 Taxanes, such as paclitaxel, are among the most common chemotherapeutic agents currently used for the treatment of breast cancer. Use of taxanes was initially limited because of hypersensitivity reactions, but once these could be adequately managed, largely by premedication with GC, taxane chemotherapy became part of the standard breast cancer treatment in most Western countries. 4 Glucocorticoid receptors (GRs) have been identified in several types of cancerous cells, including breast cancer cell lines such as MCF-7. [8][9][10][11] Exposure of these receptors -functional in MCF-7 cells -to Dex has been reported to inhibit cell proliferation. 12 Other in vitro studies have shown that GCs inhibit the growth of estrogen receptor (ER) α-positive (ERα-positive) (eg, MCF-7) cells by arresting cell cycle in G0/G1 phase. In contrast, proliferation of ERα-negative (eg, MDA-MB-231) cells is not inhibited by treatment with GCs, suggesting that GCs inhibit the proliferation of breast cancer cells via the ERα signaling pathway. 13 GCs and mineralocorticoids can also cross talk with progesterone receptors to induce a progesterone-like effect in breast cancer. 14 However, Dex treatment of MCF-7 cells has also been reported to promote cell proliferation by upregulating c-Myc, which is induced by the promotion of NFκB transcriptional activity. 12,15 Loss of GR activation has been observed in BRCA1-mutated breast tissue. 9,11 While the presence of GRs in specific breast cancer cell lines has been clearly established, their activation is associated with multiple and opposite effects. GCs exert an antiapoptotic effect. This antiapoptotic effect was studied further and could be mediated by the induction of the expression of other genes frequently associated with protection from cell apoptosis, such as Bcl-XL, BAk, SGK-1, and MKP-1. Concomitantly, Dex also reinforces its survival effect by downregulation of proapoptotic genes. [16][17][18] Dex induces the expression of genes associated with protection against cell apoptosis. GRs disrupt p53-mediated regulation of cell survival. Loss of p53 activity has been linked with a range of human cancers. P53 mediates cell apoptosis in case of DNA damage or hypoxia. 19 The majority of chemotherapy patients receive a preadministration of Dex. We deemed that this sequential treatment (ie, administration of Dex followed by a chemotherapeutic agent) warranted further assessment. The present study aimed to investigate what influence Dex treatment, prior to the administration of a chemotherapeutic agent (5-FU, epirubicin, or paclitaxel), had on the proliferation of MCF-7 cells.

Materials and methods
Cell culture MCF-7 cells obtained from (ATCC Bioresource Centre, Manassas, VA, USA), were maintained at 37°C in a humidified cell incubator with a 5% CO 2 atmosphere. Cells were cultured in Dulbecco's Modified Essential Medium (DMEM) containing phenol red and supplemented with 10% fetal bovine serum (FBS; Life Technologies, Paisley, UK), 2 mM l-glutamine, and 1% penicillin-streptomycin (all from Life Technologies). For the experimental procedures, cells were seeded in DMEM (phenol red-free) supplemented with 10% charcoal-stripped FBS and 100 nM 17β-estradiol.
Measurement of cell culture growth by cell counting MCF-7 cells were plated at a density of 15,000 cells/mL in 12-well dishes and then incubated at 37°C with a 5% CO 2 atmosphere. The following day, the cell cultures were given fresh medium, with or without Dex

statistical analysis
SigmaPlot ® 11 software (Systat Software Inc., Chicago, IL, USA) was used for the statistical analyses. Parametric analysis was performed by Student's t-test. A p-value <0.05 was considered statistically significant.

Results
Cell numbers decreased by more than half (p<0.001) when we incubated MCF-7 with 100 nM Dex for 30 min compared to untreated (CT) cells (Figure 1). No significant difference was seen between the samples exposed to epirubicin or to Dex and then epirubicin (Figure 2). This was also the case for the samples treated with 5-FU or Dex followed by 5-FU (Figure 3). By contrast, a statistically significant difference (p=0.038) was noted between samples treated with paclitaxel or with Dex followed by paclitaxel (Figure 4). Preexposure of MCF-7 cells to Dex before paclitaxel treatment reduced the cytotoxic effect of paclitaxel by 8.5%.

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Dexamethasone pretreatment before chemotherapy in MCF-7 cells

Discussion
GCs have been reported to have multiple modes of action, thus explaining their proapoptotic and antiapoptotic effects on ER-positive or ER-negative breast cancer cells, respectively. As GCs are administered systematically with epirubicin-and paclitaxel-based chemotherapies, the consequences of GC exposure in terms of the chemotherapeutic effect warranted further investigation.
We chose to study the apoptotic effects of epirubicin, 5-FU, and paclitaxel when MCF-7 cells were preexposed to Dex because they are the three main drugs currently used in routine chemotherapy. Since the anthracyclines doxorubicin and epirubicin have similar mechanisms of  action, we did not perform additional testing with doxorubicin. In this study, we demonstrated that the apoptotic activity of paclitaxel was significantly reduced (by 8.5%) when MCF-7 cells were preexposed to Dex. No significant effect of Dex was seen with exposure to the chemotherapeutic agent 5-FU or epirubicin. Under in vitro conditions, paclitaxel enhances the polymerization of tubulin so as to form stable microtubules. It also interacts directly with microtubules and protects them from depolymerization by cold or calcium, both of which can readily depolymerize microtubules in the absence of the drug. Paclitaxel arrests cells at the G2-M phase of the cell cycle by the stabilization of spindle microtubules. However, the exact mechanism by which paclitaxel induces apoptosis is not clear. 20 An explanation could be that taxanes act more than docetaxel Breast Cancer -Targets and Therapy 2017:9 submit your manuscript | www.dovepress.com

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Buxant et al by phosphorylation of bcl2, thus abrogating the normal antiapoptotic function of bcl2. 21 Wu et al 17,18 demonstrated that pretreatment of breast cancer cell lines by Dex inhibits chemotherapy-induced apoptosis in a GR-dependent manner and that it is associated with transcriptional induction of at least two genes identified in our screening -SGK-1 and MKP-1.
By contrast, there was no difference in cell proliferation following treatment with 5-FU and epirubicin. Their mechanisms of action, however, are different from that of paclitaxel. 5-FU needs to be converted at the nucleotide level in order to exert its effect. It can be incorporated into RNA, leading to interference with the maturation of nuclear RNA. Its conversion to 5-fluoro-2′deoxy-5′monophosphate, which leads to inhibition of thymidylate synthase and subsequently DNA synthesis, is however considered to be its main mechanism of action. 15,22 Epirubicin is an anthracycline antibiotic; its antitumor activity is caused by its ability to interfere with transcription, resulting in inhibition of RNA synthesis. 23 Under in vitro conditions, several, at times antagonistic, actions of GCs have been described. 2,3,[8][9][10][11][12][13][14] This underscores the relevance of a direct study of preexposure to GCs prior to chemotherapy. In our study, the results were dependent on the specific chemotherapeutic agent being administered, which may be explained by the different cytotoxic mechanisms of these chemotherapeutics. However, the result of this study is not sufficient to warrant changing the current therapeutic approach. Other factors most certainly influence the tumor response in vivo. Indeed, it has been proposed that the peritumoral stroma and cancer-associated fibroblasts play an important role in breast tumorigenesis. 24 The observed decrease in the efficacy of paclitaxel on MCF-7 cells previously exposed to Dex is, however, not sufficient to warrant changing the current therapeutic approach. Indeed, it has been proposed that the peritumoral stroma and cancer-associated fibroblasts play an important role in breast tumorigenesis. 17,24 For example, in tumors that exhibit an aggressive metastatic phenotype, such as luminal HER2-positive and triple-negative, metalloproteinase-2 (MMP-2) expression is significantly increased. 25 Dex could be acting at this level, by alleviating the effect of MMP-2. 19,26 More recently, we demonstrated frequent expression of GRs in breast cancer-associated fibroblasts. The presence of GRs within breast tumors and the adjacent peritumoral stroma must be taken into account when GCs are prescribed to cancer patients. As shown in this study, GCs could substantially decrease the cellular response(s) caused by exposure to chemotherapeutics. The use of GCs other than Dex could be a solution. Recently, Orqueda et al showed that the rigid steroid 21-hydroxy-6,19-epoxyprogesterone (21OH-6, 19OP), a selective GR ligand, behaves as a dissociated GC that retains anti-inflammatory action without affecting the apoptotic process triggered by chemotherapeutic drugs. Indeed, contrary to Dex, 21OH-6, 19OP neither reverts the paclitaxel-induced caspase-3 activity nor induces the antiapoptotic Bcl-X(L) gene expression in murine tumor mammary epithelial cells. 27

Conclusion
Since GCs affect the behavior of numerous cancer cell types, their function should be defined more precisely if they are to be used in conjunction with chemotherapy. Indeed, in this study we demonstrated a decrease in the response of MCF-7 cells to paclitaxel when they had been pretreated with Dex. Use of other antiemetogenic molecules should be considered in light of these results.


A Single Nucleotide Polymorphism in the FADS1 Gene is Associated with Plasma Fatty Acid and Lipid Profiles and Might Explain Gender Difference in Body Fat Distribution

Background Genotyping of the rs174547 polymorphism in the fatty acid desaturase 1 gene (FADS1) shows that it is associated with the FA composition of plasma phospholipids and lipid metabolic indices among several ethnic groups. However, this association requires further confirmation in the Chinese population, and little is known about the effect of polymorphisms in fatty acid-related genes on body fat distribution. Methods Anthropometric measurements of 951 Chinese adults aged 18–79 were obtained and body fat distribution was estimated using dual-energy X-ray absorptiometry. The FA composition of plasma phospholipids was measured by gas chromatography. Multiple linear regression assessed whether the rs174547 genotype was associated with FA composition, body fat distribution, and metabolic traits in additive, dominant, and recessive models. Results The rs174547 C minor allele was associated with a higher proportion of linoleic acid, lower arachidonic acid and docosahexaenoic acid, as well as lower delta-6-desaturase and delta-5-desaturase activity. Female C allele carriers had lower android fat percentages and lower levels of low-density lipoprotein-cholesterol, while male C allele carriers had lower gynoid fat percentages and higher triglyceride after adjusting for age, income, BMI, behavioral risk factors, and regional fat percentages. Conclusion An association of FADS1 rs174547 with the FA composition of plasma phospholipids was identified among this Chinese adult population. The association with body fat distribution and lipid metabolic indices differed between men and women, which might explain sexual differences in body fat distribution and lipid metabolism.

Background
Previous studies have identified associations between variations in genes encoding fatty acid desaturase 1 (FADS1) and 2 (FADS2) with changes in plasma FA profiles and altered desaturase activity [1][2][3][4]. For instance, FADS1 rs174547, a T/C SNP in intron 9 of FADS1, was reported to be associated with arachidonic acid to linoleic acid (AA:LA) FA ratios in both Caucasians and Asians [5,6]. Similarly, the association of this locus with fatty acid level was examined among Chinese, but the findings were inconsistent across studies [7,8].
FA metabolism has been linked to body fat accumulation and obesity [9][10][11][12]. However, it is not clear whether the polymorphisms in FA-related genes are associated with obesity and body fat distribution. A previous study of a Chinese population failed to associate FADS1 rs174547 with BMI [13], but as yet no studies have focused on body composition, especially body fat and its distribution (i.e., body fat percentage and central obesity). Although gender differences have been identified for body fat distribution [14], for example, men tend to accumulate fat in the abdominal region (apple-type obesity) and women in the hip region (pear-type obesity), few genetic variations explain this disparity so far. Moreover, while genome-wide association studies have revealed gene-by-sex interactions for BMI and waist circumference (WC) [15,16], few studies have explained gender differences in fat distribution measured by issue-specific technology such as dual-energy X-ray absorptiometry (DXA) or computed tomography [17].
FADS1 rs174547 was shown to be involved in the lipid metabolic pathway which catalyzes the biosynthesis of highly unsaturated FA. Thus, this SNP might impact on human lipid profiles. Previous studies associated the FADS1 rs174547 genotype with lipids levels in Chinese population. For instance, a Chinese study reported that the CC variant of rs174547 was significantly associated with increased TG and decreased HDL-C [18]. However, the findings were inconsistent in recent studies. In a recent study examining the association between the FADS gene cluster and coronary artery disease and lipids in northern Chinese Han population, no significant associations were found between rs174547 and lipids indicators [19].
This study aimed to examine the effect of FADS1 rs174547 on plasma FA composition, body composition, body fat indices, and plasma lipid profiles among Chinese adults. We also explored the impact of this SNP on gender differences in body fat distribution.

Study sample
A total of 1029 community residents aged from 18 to 82 years old were recruited voluntarily between November 2008 and May 2009 from two communities in Hangzhou City, a capital provincial city in eastern China. Two communities were chosen for convenience from Xiacheng and Xiaoshan districts of the city. The subjects were recruited voluntarily by posters, flyers, and community leaders. All subjects completed a standardized questionnaire survey, underwent physical anthropometry and body composition measurements, and provided blood samples at the Obesity and Body Composition Research Center of Zhejiang University School of Public Health. In 2014, 67 participants were sampled with stratification on BMI distribution, and the measurement of plasma FA was made using their stored plasma samples. In 2015, after excluding the participants aged <18 or >80 years, the FADS1 rs174547 genotype was determined in 951 participants (including the 67 participants sampled in 2014) using stored blood samples.
The flow of participants included in the analysis is shown in Fig. 1. Written informed consent was obtained from all participants and the study was approved by the Ethics Committee of the Second Affiliated Hospital of Zhejiang University.

Measurements
Weight and height were measured with subjects wearing light clothing and without shoes. Body weight was recorded to the nearest 0.1 kg (Detecto, Missouri, USA). Body height was measured with a hypsometer to the nearest 0.1 cm. All values were recorded as the mean of three measures. WC was measured at the approximate midpoint between the lower margin of the last palpable rib and the top of the iliac crest, at minimal respiration. Hip circumference was measured at the maximum circumference of the buttocks with the tape parallel to the floor. Blood pressure was obtained from the right arm of seated participants after a 5-min rest. It was measured twice by the same trained examiners with  [20]; the mean of the two measurements was used for analysis. BMI was calculated as weight in kilograms divided by height in meters squared. DXA (GE Lunar Prodigy, WI, USA with software version 11.40.004) was implemented to measure body fat distribution as previously described [21]. The bone mineral density, fat mass, and lean soft tissue mass of the whole body and specific regions of interest (limb, trunk, android region, and gynoid region) were measured. DXA was checked daily against a calibration phantom using the manufacturer's precision standards of <0.8%. Fat mass percentage (FMP), as an indication of fat distribution, was calculated as regional fat mass kg/body weight in kg*100.
Information on demographics, socioeconomic status, and lifestyle behavior was collected by face-to-face interview using a structured questionnaire, including questions on age, sex, income, smoking status, alcohol consumption, and physical activity. Participants were classified according to their smoking status as nonsmokers or current smokers, and according to their alcohol consumption as non-drinkers, current drinkers, and abstainers. Physical activity was grouped as low, moderate, or heavy based on its frequency and intensity according to the international physical activity questionnaire [22,23].

Blood biochemical analysis and DNA extraction
After an overnight fasting, venous blood specimens were collected for biochemical analysis. Fasting plasma glucose (FPG) concentrations were determined using a hexokinase method. Total cholesterol (TC) and TG concentrations were measured using COD-PAP and GPO-PAP methods, and HDL-C and LDL-C were measured using an enzymatic homogeneous assay. The levels of apolipoprotein A1, and apolipoprotein B were determined by immunoturbidimetric assay. All the reagents were obtained from Roche. Then remaining samples were stored at −80°C for further analysis. Genomic DNA was extracted from 1 to 5 mL whole blood using a commercially available DNA isolation kit (BloodGen Midi Kit, CWBIO, Beijing, China) according to the manufacturer's protocol between April and May 2015.

Measurement of plasma phospholipid fatty acid
For the analysis of FA composition, total lipids were extracted from 200 μl plasma using chloroform-methanol (2:1 v:v) according to the Folch method [24]. The homogenates were stilled at 4°C and subsequently centrifuged at 3000 xg for 5 min, then the lipid-containing chloroform phase was transferred to a new tube and dried under nitrogen gas. The extraction process was repeated with an equal volume of chloroform. The resulting free FAs were methylated by the addition of 14% boron trifluoride in methanol at 100°C for 1 h. The reaction was stopped by adding distilled water, and the contents were centrifuged at 3000 xg for 5 min and dried under nitrogen gas for phase separation. The lipid phase was evaporated under nitrogen gas, and reconstituted in hexane for analysis. FA methyl esters were separated by gas chromatography using an Agilent 7890A gas chromatograph (Agilent Technologies, Palo Alto, CA). Peaks were identified by comparison with FA methyl ester standards. The levels of plasma FAs were expressed as percent fatty acid composition: palmitic acid (C16:0), stearic acid, palmitoleic acid, oleic acid (OA), LA (C18:2n − 6), α-linolenic acid (C18:3n − 3), γ-linolenic acid (GLA; C18:3n − 6), Docosahexaenoic acid (DHA; C22:6n − 3), AA (C20:4n − 6), and eicosapentaenoic acid (C20:5n − 3). Desaturase activity was estimated using the ratio of the product FA to precursor FA, which has previously been well-established. The Δ6 desaturase (D6D) activity was estimated by dividing the % composition of DGLA by LA, and the Δ5 desaturase (D5D) activity was estimated by dividing the % composition of AA by DGLA [25].

Statistical analysis
The statistical significance of demographic characteristics was analyzed using the t-test for continuous variables or the chi-square test for categorical variables. Pearson's χ 2 test was used to examine whether rs174547 genotype frequencies were in Hardy-Weinberg equilibrium (HWE). The associations between FADS1 rs174547 and plasma phospholipid FAs, body composition indices, and lipid profile indicators were identified by multivariate linear regression models, with each stage of regression analysis performed assuming additive (variant/variant vs. variant/common vs. common/ common), dominant (variant/variant + variant/common vs. common/common), or recessive (variant/variant vs. variant/ common + common/common) models. The effect value was determined from the model with the lowest P value.
For the models identifying associations between FADS1 rs174547 and plasma phospholipid FAs in 67 participants, the first step was to adjust for sex and age, then to adjust for smoking, drinking, and BMI. For the models identifying associations between rs174547 and anthropometric measurements and lipid indicators in 951 participants, we adjusted the same covariates as above. We further adjusted the fat mass in other body regions to determine whether rs174547 independently influenced regional fat distribution. All analyses in the models of anthropometric measurements and lipid indicators were performed separately in men and women because of the interaction of gender. All statistical analyses were conducted using SAS software version 9.3 (SAS Institute, Cary, NC). A two-tailed value of P < 0.05 was considered statistically significant.

Results
The characteristics and rs174547 genotypes of the FA analysis sample (n = 67) are shown in Tables 1 and 2 shows anthropometric measurements and lipid analysis of the whole sample (n = 951). For whole sample analysis, men showed higher levels of physical activity, alcohol consumption, and smoking prevalence than women, while no sex difference was observed in age, BMI, or household income. The effect allele of rs174547 was the C allele, which had a frequency of 0.343 and conformed to HWE (P = 0.117), with a genotyping success rate of 99.5%. Correlations between the rs174547 genotype and plasma phospholipid FAs are shown in Table 3. The number of C alleles was positively correlated with the proportion of OA and LA in the additive model and recessive model when adjusting for age and sex. An increase in the number of C alleles was associated with a significantly higher OA level and LA level, and with a significantly lower AA level and D6D in the additive model and recessive model. When including age, sex, smoking, drinking, and BMI in the model, the positive association between the C allele and LA remained significant, while that between the C allele and OA was lost. The C allele was inversely associated with the concentration of AA, DHA, D6D, and D5D after adjusting the covariates.
The associations between the rs174547 genotype and anthropometric indicators among men and women are shown respectively in Tables 4 and 5. In men, no significant association between fat distribution and the rs174547 C allele was observed when only adjusting for age. However, after further adjusting the covariates and BMI, the C allele was negatively correlated with WC, gynoid FMP, and limb FMP. The gynoid FMP remained significantly associated with the C allele in the additive model and dominant model when further adjusting for other regional fat distribution.
In women, all indicators of body fat distribution except weight, BMI, WC, and weight-to-hip ratio (WHpR) showed a decreasing trend with increasing numbers of C alleles of the respective genotype after adjusting for age. When further adjusting for covariates and BMI, the association of the C allele with WHpR and android FMP remained significantly negatively correlated, while the association with other indicators of fat distribution diminished. When including gynoid FMP in the model, the android FMP remained significantly associated with the C allele in the dominant model.
The associations between the rs174547 genotype and lipid profiles among women and men are shown respectively in Tables 6 and 7. The level of TG was positively correlated with the C allele in the additive model among men when adjusting for covariates and BMI; the association remained significant after further adjusting for android FMP. In women, the C allele was negatively correlated with the concentration of LDL-C in the additive model and dominant model after further adjusting for android FMP.

Discussion
In the present study, we found that the FA metabolismrelated FADS1 SNP rs174547 was significantly associated with several lipid metabolic indices and anthropometric indicators. We also confirmed an association between rs174547 and plasma phospholipid FA, and showed that rs174547 was associated with body fat distribution with a significant gender difference.
Several studies have previously explored the association of SNPs in the FADS gene cluster with plasma phospholipid FA in various ethnicities, and report the correlation Adjusted for age and sex; b Adjusted for age, sex, smoke, drinking, and BMI; *P < 0.05, **P < 0.01, ***P < 0.0001 Table 4 The association between rs174547 and fat distribution in men (N = 365) Adjusted for age, income, smoking, drinking, physical activity and BMI; c Adjusted for age, income, smoking, drinking, physical activity, B MI, and other regional fat (For android fat mass percent, the gynoid fat mass percent was further adjusted, vice versa; for limb fat mass percent, the trunk fat mass percent was further adjusted, vice versa); *P < 0.05 Table 5 The association between rs174547 and fat distribution in women (N = 586) Adjusted for age, income, smoking, drinking, physical activity and BMI; c Adjusted for age, income, smoking, drinking, physical activity, BMI, and other regional fat (For android fat mass percent, the gynoid fat mass percent was further adjusted, vice versa; for limb fat mass percent, the trunk fat mass percent was further adjusted, vice versa); *P < 0.05, **P < 0.01 Table 6 Association between rs174547 and lipid profiles in men (N = 365) Indices Effect (SD)   Table 7 The association between rs174547 and lipid profiles in women (N = 586) Indices Effect (SD)  of particular alleles with higher levels of LA and lower levels of AA [3,7], which is consistent with our current results. Suhre et al. observed an association between D5D activity and rs174547 [4]; however, we demonstrated that rs174547 was more significantly associated with D6D. Similarly, the effect of rs174547 on dyslipidemia has been reported among many ethnic groups. The rs174547 C allele was previously found to be positively correlated with the level of TC and negatively correlated with the level of HDL-C in Chinese and other Asian populations [26][27][28]. However, Wu et al. found no significant associations of rs174547 with TG and TC levels among Chinese [19]. It has been acknowledged that the sexual dimorphism exists in general lipid profiles [29][30][31][32], so the association between the blood lipid indices and rs174547 might should be performed separately for female and male. Our results showed that rs174547 was significantly correlated with TC levels in men, and negatively correlated with LDL-C in women, which suggested that this locus may be a gender-specific SNP. Gender differences in body fat distribution are wellestablished [14], with women known to predominantly accumulate subcutaneous fat in their limbs and hips, while men accumulate visceral fat in their abdomens; they therefore have an increased risk of obesity-related metabolic diseases [33,34]. Body composition was precisely measured using DXA in our study, revealing that female C allele carriers had less abdominal fat reflected by the android FMP, and male C allele carriers had less subcutaneous fat reflected by the gynoid FMP. Thus, our findings indicated that FADS1 rs174547 might be a useful tool to elucidate the genetic basis of obesity-related disease, and it might be a predictive marker to identify high-risk individuals for abnormal body fat distribution and dyslipidemia, especially in male subjects. Besides, mutations of the same allele appeared to cause different body compositions among men and women, suggesting that gender differences in fat distribution reflect genetic diversity, which may involve differences in gene expression and function. It should be noted that the sexual differences in body fat distribution and lipid metabolism involve many genetic and environmental factors and their complex interactions. Our finding might partially explain this gender discrepancy.
A major strength of our study is that it is the first to demonstrate the impact of this FA-related SNP on gender differences in body fat distribution in a Chinese adult population. Although the influence of rs174547 on BMI has been studied previously [13], fat distribution measures derived from DXA are more precise and provide additional information to further understand the association with rs174547. We also thoroughly discussed the impact of gender differences on the association of rs174547 with blood lipid profiles. However, we acknowledge a number of limitations. First, we only focused on the effect of one SNP, and did not explore interaction effects between SNPs. Second, we did not consider the influence of environmental factors on genotype and phenotype, and most of the study participants were middle-aged adults. Finally, the study sample was from a capital city in eastern China and the subjects were not recruited through a random sampling procedure. In addition, the 951 sample size was relatively small. Thus our study sample might not be representative of the entire Chinese population. However, we focused on the association analysis between factors rather than descriptive analysis of the population in this study, the representativeness of the sample might not affect the association results. Further large and nationally representative studies were warranted to confirm the findings.

Conclusions
In summary, we investigated the relationship between FADS1 rs174547 with plasma phospholipid FA profiles, anthropometric measurements, and lipid profiles in this Chinese adult population. The rs174547 C allele was shown to correlate with the level of plasma FA. The association between the rs174547 genotype and body fat distribution and lipid indicators differed between men and women, which might partially explain sexual differences in body fat distribution and lipid metabolism. Future research should investigate the effects of FADS1 and FADS2 on body fat distribution, especially in women.