Fat and Bone: An Odd Couple

In this review, we will first discuss the concept of bone strength and introduce how fat at different locations, including the bone marrow, directly or indirectly regulates bone turnover. We will then review the current literature supporting the mechanistic relationship between marrow fat and bone and our understanding of the relationship between body fat, body weight, and bone with emphasis on its hormonal regulation. Finally, we will briefly discuss the importance and challenges of accurately measuring the fat compartments using non-invasive methods. This review highlights the complex relationship between fat and bone and how these new concepts will impact our diagnostic and therapeutic approaches in the very near future.

The Concept of Bone Strength
At the National Institutes of Health (NIH) Consensus Conference in 2000, osteoporosis was defined as a skeletal disorder characterized by compromised bone strength that predisposes to an increased risk of fracture (1). Bone strength reflects the integration of two features: BMD and bone quality. BMD is one of the strongest risk factor for fractures and its measurement has long been used to define osteoporosis. Clinical risk factors have also been integrated with BMD measurements in an attempt to help clinicians better identify patients requiring osteoporosis therapy (2). The FRAX calculator is a user-friendly web-based tool that provides immediate quantification of risks and treatment decision making based on a very simple algorithm. However, it should always be interpreted within the clinical context as it does not take into account a number of important clinical variables.
The World Health Organization defines osteoporosis as two and a half SD below the peak bone mass [i.e., the maximum amount acquired post bone maturation around the age of 18 in women (3) and 20 in men (4), but bone growth can continue up to the age of 30]. It is expressed as grams of mineral per area or volume. On the other hand, bone quality reflects a combination of bone microarchitecture, bone turnover, and mineralization. Peak bone mass is therefore a critical parameter that will impact bone strength as the skeleton is aging. Genetic factors appear to account for over 50% of the variation in peak bone mass acquisition (5). As bone is progressively lost overtime, the higher the peak bone mass, the longer the skeleton could theoretically withstand damage. This progressive bone loss from peak bone mass occurs predominantly as a result of reduced bone formation from osteoblast (6,7) and resultant protein composition (8) and persists for decades thereafter. Additionally, accelerated bone resorption predominates in women as a result of estrogen deficiency at the menopause but also to a lesser extent in men after the fifth decade (6). However, postmenopausal women have the ability to produce estrogens from the peripheral conversion in fat tissues of testosterone to estradiol. Adipocytes indeed express the cytochrome P450 enzyme, aromatase, which can produce estradiol from testosterone. This peripheral production of estradiol has been proposed as a protective mechanism against bone loss in overweight women (9)(10)(11).
Bone strength is highly dependent on its structural and material properties. The balance between bone formation and resorption, also called bone turnover, greatly influences the material properties of bone such as tissue mineral density and collagen cross-linking. Enhanced bone turnover, as seen with a lack of estrogen in postmenopausal women, influences the structural and material properties that lead to bone microdamage. With aging, the reduction in bone strength is further compounded by progressive muscle weakness and the increased risk for falls due to lack of balance and coordination. Maintenance of bone mineralization within a relatively narrow range is also critical to the maintenance of bone strength (12). Poorly mineralized bone loses its stiffness, whereas excessive mineralization makes bone more brittle. Bisphosphonates, the most widely used drugs to treat osteoporosis by excessive bone turnover, also lead to increased mineralization and stiffness overtime. Impairment of microdamage repair is another potential side effect of bisphosphonates since normal bone turnover replaces old bone with new bone and protects against microdamage. Long-term use of bisphosphonates has been linked to atypical fractures, and one could hypothesize that the combination of abnormal mineralization and reduced turnover may play a role in its development.

The Components of the Bone Microenvironment
The bone microenvironment is comprised of several compartments, including hematopoietic cells, bone cells, and stromal cells (13). Bone cells, also referred to as the bone remodeling unit (BMU), are composed of bone-forming osteoblasts, boneresorbing osteoclasts, and osteocytes embedded within the bone matrix. The BMU is also in close contact to stromal elements of the marrow and the blood vessels supply (14). Osteoclasts are of hematopoietic origin, whereas osteoblasts originate from bone marrow mesenchymal stem cells (MSCs) (11,14,15). One of the most interesting occurrences in this environment is the accumulation of fat cells during aging and in some pathological conditions. The functional significance of this "marrow fat (MF)" accumulation correlates strongly and inversely with bone strength (16). However, its causal relationship to bone degradation as well as its potential for therapeutic targeting in osteoporosis remains to be determined. There is indeed a significant gap of knowledge in our understanding of the mechanistic relationships between fat and bone especially during the aging process.

The Components and Distribution of Body Fat
In humans, white adipose tissue (WAT) is principally located beneath the skin (subcutaneous fat) and around internal organs (visceral fat or abdominal fat). The main cellular component of WAT is the adipocyte but other cell types are also present, including fibroblasts, macrophages, and blood vessels. Its main function is energy storage. Adipose tissue also accounts for a significant proportion of the breast tissue and is found around other organs (such as pericardial and gonadal fat) providing protective padding. Adipocytes are also found in small amounts outside adipose tissues, including muscle, liver, pancreas, and heart, which are also referred as ectopic fat. Fat cells are also found in the bone marrow, "MF, " and have been the subject of enormous research interest to explore their relationship with the bone microenvironment.
Another form of adipose tissue is known as brown fat or brown adipose tissue (BAT) located mainly around the neck and large blood vessels of the thorax of neonates whose main function is to generate heat and protect neonates against cold (17). Recent studies indicate that BAT is also found in the neck and trunk of adults albeit in lesser amounts (18). Although this review focuses mainly on white fat, the relationship between BAT and bone will be briefly discussed.

Muscle Fat, Muscle Strength, and Bone Strength
Many studies have clearly demonstrated the positive impact of muscle strength on bone strength, but we will not cover this important area of research here. However, an interesting, but much less explored, area is the relationship between muscle fat and muscle strength and by extension its impact on bone strength. Fat accumulation in muscle may also have indirect effects on bone. Intermuscular adipose tissue accumulation occurs during aging or in pathological conditions such as Duchenne muscular dystrophy, which has been linked to decreased muscle strength, a known risk factor for osteoporosis and fractures (19). Increased intermuscular adipose tissue is associated with poor mobility (20) and increased risk of hip fractures (21). However, it is not yet known whether intermuscular adipose tissue accumulation is simply a marker of muscle dysfunction or has a direct causal effect on muscle function. The relationship between vitamin D and intermuscular adipose tissue is discussed later in this review.

BASiC UNDeRSTANDiNG OF THe MeCHANiSTiC ReLATiONSHiP BeTweeN MARROw FAT AND BONe STReNGTH
In this section, the origin, clinical significance, and the factors that influence MF accumulation will be discussed.

Clinical Observations
As we age, the cortex of the bones become thinner encircling concomitantly larger marrow cavities filled with fat, but whether this is a result of a passive accumulation of fat as bone is lost and marrow space increases or an age-related shift in MSC differentiation with predominant adipogenesis against osteoblastogenesis is difficult to elucidate.
Meunier et al. studied 81 iliac crest biopsies from elderly women and found that bone marrow samples from women with osteoporosis had a pronounced accumulation of adipocytes, relative to levels in healthy young subjects (22). Subsequent studies showed increased bone marrow adiposity in postmenopausal women with osteoporosis and a negative association between bone-MF and rate of bone formation (23-25). Investigations using magnetic resonance imaging (MRI) have shown that the accumulation of bone-MF in the vertebral bodies of older women with low bone mass confers an additional risk for compression fracture beyond that associated with low BMD (26). Further support for this notion are data showing an association between exogenous glucocorticoid use and endogenous over production of cortisol and marked bone marrow infiltration by adipocytes with a significant increase in fracture risk (27-29).
Marrow stromal cells isolated from postmenopausal osteoporotic patients express more adipocytic differentiation markers than those with normal bone mass and are more likely to enter an adipocyte than an osteoblast differentiation program (30, 31). Fat in bone marrow may also promote bone resorption since marrow adipocytes, much like fat cells elsewhere, secrete inflammatory cytokines capable of recruiting osteoclasts (32).

Role of MSC
Pluripotent bone marrow MSCs have the ability to become osteoblasts, chondrocytes, myocytes, or adipocytes under the influence of specific cell-derived differentiation factors (33). This process has been well demonstrated in vitro to control the fate of MSC into osteoblasts or adipocytes. This process is bidirectional and considerable plasticity has been observed both in vitro and in vivo in the ability of bone cells to become adipocytes and vice versa. Mechanical stimuli on the skeleton can also modify the differentiation of MSC into the cell lineages responsible for bone and fat formation (34-39) such that increases in bone strain add to increased osteogenic activity, whereas decreases favor the adipogenic differentiation. Lastly, the lack of estrogen in rats following oophorectomy has been reported to lead to profound fatty bone marrow infiltration, suggesting that estrogen must play an important role in regulating adipocyte recruitment (40).
Data from pathological specimens and imaging studies have consistently observed a reciprocal relationship between bone mass and increased marrow adiposity in elderly humans (22, 41-43). A recent study found that the accumulation of MF during aging is linked to increased expression of RANKL, a finding that could explain at least in part age-related bone loss (44). However, whether the relation between these two tissues in the elderly represents the clinical translation of preferential differentiation by MSC into the adipose cell lineage or is merely the unintended consequence of a passive accumulation of adipose tissue as bone is lost and marrow space increases has been a matter of considerable debate. To avoid this confounding effect, young subjects were examined and found that bone acquisition is tightly linked with decreases in marrow adiposity (16). The inverse relation between the amount of bone and MF is observed at all sites along the shaft of the bone in the young and the old regardless of age, gender, or anthropometric measures (45, 46) (Figure 1). Moreover, prospective longitudinal studies have found that bone acquisition in the appendicular skeleton of healthy young females is inversely related to changes in marrow adiposity (16). Consequently, one could make a strong argument that during the aging process, differentiation of MSCs into adipocytes is favored at the expense of osteoblasts, resulting in MF accumulation and decreased bone mass. However, this causal relationship has not yet been demonstrated.
In summary, mounting evidence supports a mechanistic relationship between MF accumulation and bone loss, pointing out the potential to target this pathway to prevent or even reverse the process of bone aging.

BASiC UNDeRSTANDiNG OF THe ReLATiONSHiP BeTweeN BODY FAT, BODY weiGHT, AND BONe STReNGTH
In this section, we will summarize the current knowledge and conflicting data linking body fat, bone mass, and fracture rate.

Clinical Observations
Postmenopausal women have the ability to produce estrogens from the peripheral conversion of testosterone to estradiol in fat tissues. Adipocytes express the cytochrome P450 enzyme, aromatase, which can produce estradiol from testosterone. This peripheral production of estradiol has been proposed as protective mechanism against bone loss in overweight women (9)(10)(11). There are also reports showing an inverse relationship between BMI and osteoclast activity in normal postmenopausal women (47) and an increase in bone resorption following weight loss (48). As discussed further in the next section, fat accumulation leads to hyperinsulinemia, which is anabolic to bone, and adipocytes produce estrogen and adiponectin, which have a positive effect on bone strength and could therefore explain this positive association observed clinically (49).
Bone mineral density measured by dual-energy X-ray absorptiometry (DXA) is positively related to body weight and BMI (49, 50), possibly because higher body weight may increase mechanical loading on the skeleton, a mechanism known to stimulate bone formation. However, DXA measurements are falsely elevated by increased body fat and therefore DXA may overestimate BMD in obese individuals (51-54). Indeed, other studies have found a strong positive association between lean mass and BMD in young women and a much weaker association between BMD and fat mass (50).
A meta-analysis indicates that a high BMI appears to protect against fractures at any site in both men and women (55). Similarly, a European study found that a higher BMI protects against vertebral fractures (56). In the study of osteoporotic fractures, body weight in the lowest quartile was found to double the risk of hip fracture (57). In contrast, other studies found that the risk of hip fractures is positively correlated with fat mass in a cohort of French women (58) and Chinese men (59). Interestingly, visceral adiposity has been linked to deterioration of bone structure and skeletal fragility (60,61), suggesting that fat compartments may have different effects on bone strength. In summary, clinical observations linking body fat and bone strength are inconsistent, and more mechanistic studies are needed to support the purported beneficial effect of obesity on osteoporosis.

iNTeGRATeD HORMONAL ReGULATiON OF FAT AND BONe
In this section, we will review the major hormonal regulators controlling fat and bone, with particular attention on the mechanisms underlying the reciprocal relationship between MF and bone.

Insulin
Hyperinsulinemia is a hallmark of the metabolic syndrome characterized by accumulation of visceral fat (62). Osteoblasts express insulin receptors (63), and insulin directly stimulates osteoblast proliferation (64) and differentiation in vitro (63). Furthermore, local application of insulin over the calvariae of adult male mice produces a significant increase of bone formation (65). Conversely, the glucose-dependent insulinotropic polypeptide (GIP)-receptor knockout mouse shows decreased bone size, mass, and formation rate (66). In clinical studies in patients with varying degrees of hyperinsulinemia, the risk of vertebral fracture was inversely related to insulin levels (67). Hyperinsulinemia following an oral glucose load is accompanied by suppression of parathyroid hormone (PTH) production and bone turnover and may therefore indirectly protect against bone loss (68,69).

Growth Hormone and Insulin-Like Growth Factor 1
In vitro GH induces MSC differentiation into osteoblast, while GH deficiency in mice results in decreased bone formation and increased bone marrow adiposity (70). Although IGF-1 does not have a direct effect on the differentiation of MSC in vitro (71), the PPAR-gamma 2 agonist rosiglitazone decreases IGF-1 expression in bone marrow MSC and lowers blood IGF-1 levels in mice and humans (72).

GLP-1 and GLP-2
Administration of glucagon-like peptide-1 (GLP-1) to diabetic mice results in an insulin-independent anabolic effect on bone (73). In humans, dietary fat and protein leads to reduction in bone turnover (74)(75)(76) possibly through GLP glucagon-like peptide-2 (GLP-2), a polypeptide produced by intestinal L cells in response to feeding. GLP-2 administration to humans is accompanied by a reduction in bone resorption and an increase in bone density (55).

Adipokines
Leptin Leptin is primarily produced by adipocytes and initially discovered as an appetite suppressant (77). The hypothalamus is regarded as the principal target of leptin. The arcuate nucleus (in the hypothalamus) contains anabolic neurons, which express both neuropeptide Y and agouti-related protein, the activity of which is inhibited by leptin, and neurons expressing proopiomelanocortin (POMC), which are activated by leptin. Insulin acts on both types of neurons in the same way as leptin, suggesting that these hormones reinforce each other's actions centrally, as well as peripherally (78).
However, subsequent studies demonstrated the potent effect of leptin on bone in animal studies (79,80). These studies demonstrated that in obese mice deficient in leptin (ob/ob mice) or in mice where the leptin receptor is defective (db/db mice), vertebral trabecular bone volume and bone formation are increased. Conversely, intracerebroventricular infusion of leptin decreased vertebral trabecular bone volume and bone formation (79). Further studies then demonstrated that these effects are mediated by the sympathetic nervous system acting on β-adrenergic receptors at the surface of osteoblasts inhibiting bone formation (80). These inhibitory effects on bone in vivo contrast with the in vitro effects reporting that leptin directly promotes the differentiation of osteoblasts (81)(82)(83)(84)(85). Leptin also reduces expression of RANK ligand of human bone marrow stromal cells and RANK expression of peripheral blood mononuclear cells (81,86), with a resultant inhibition of osteoclastogenesis (82,86). Furthermore, clinical studies have not consistently showed a relationship between administration of beta blockers, bone density improvement, and fracture prevention (87). This apparent contradiction could be explained by the ability of leptin to act positively and directly on peripheral tissues or negatively via central mechanisms involving activation of the sympathetic nervous system.

Adiponectin
Adiponectin is another adipokine produced by adipocytes whose role is to increase insulin sensitivity. Its blood levels are decreased in obese and diabetic individuals (88,89). In vitro treatment of osteoblasts with adiponectin enhances their differentiation (90). In humans, cross-sectional studies found an inverse association between circulating adiponectin levels and bone mass in both men and women, even after adjustment for fat mass (91,92).

Peroxisome Proliferator-Activated Receptor (PPAR) Gamma
PPARγ2 is the most important regulator of adipogenesis. In vitro PPARγ2 directs the commitment of MSCs into adipocytes and inhibits their differentiation into osteoblasts (9). Ablation of the PPARγ gene leads to enhanced osteoblastogenesis of embryonic stem cells in vitro and results in enhanced bone mass in vivo and reduced bone marrow adiposity (93). At the cellular level, ex vivo examination of MSCs shows commitment toward osteoblastogenesis and reduced adipogenesis (94). Administration of rosiglitazone, a specific activator of PPARγ, in mice decreases osteoblastogenesis and enhances adipogenesis in the bone marrow (95).
The canonical Wnt/beta-catenin pathway and non-canonical Wnt signaling have been implicated in this reciprocal regulation via PPAR-gamma 2. In the canonical pathway following ligand activation, Wnt binds to a transmembrane coreceptor complex consisting of Frizzled receptors and LRP5 to stimulate bone formation (96). Although Wnt10b, Wnt 3a, and Wnt 7 can stimulate the differentiation of MSC into osteoblast while inhibiting adipogenesis (97)(98)(99)(100)(101), only Wnt 7 has been shown to block PPAR-gamma 2 (99). Similarly, the non-canonical ligand Wnt5a was found to induce Runx2-mediated osteoblastogenesis while simultaneously suppressing adipogenesis in bone marrow MSC through the formation of a corepressor that blocks PPARgamma 2 gene transcription (102). In addition, PPAR-gamma 2 acts downstream of the Wnt receptor complex to enhance the proteosomic degradation of beta catenin, thereby acting as a direct regulator of osteoblastogenesis (103).

Cytokines
Duque et al. provided in vitro and in vivo evidence that interferon (IFN)-gamma is a potent inducer of MSC differentiation into mature osteoblasts and a key regulator of bone formation in mice and has therefore the potential to become an efficient drug target in osteoporosis (104,105). It was further demonstrated that IFNgamma inhibits adipogenesis in vitro and prevents MF infiltration in oophorectomized mice in vivo (106). In addition, IFN-gamma suppresses osteoclast differentiation by interfering with RANKL signaling (107), thus acting synergistically on bone cells to enhance bone strength. As discussed earlier and independently of IFN-gamma, MF accumulation during aging is linked to increased expression of RANKL, highlighting another mechanism linking bone loss to MF (44). It remains to be established whether other proinflammatory and anti-inflammatory cytokines could affect the balance between MF and osteoblastogenesis.

Glucocorticoids
Excessive production or supre-physiological administration of GC excess results in inhibition of osteoblastogenesis and accelerated adipogenesis (108) through suppression of Wnt signaling (109) and induction of PPAR-gamma 2 expression (110).
Vitamin D (from skin irradiation or in the diet) must be metabolically activated first by the liver 25 hydroxylase (CYP2R1) to 25hydroxyvitamin D (25OHD) and then by the kidney 1αhydroxylase to its active form 1,25dihydroxyvitamin D [1,25(OH)2D]. The role of vitamin D on bone and mineral homeostasis is well known, but its role in other tissue function including fat is still the subject of considerable debate.
The relationship between vitamin D and fat has been the subject of many studies in recent years. Several studies found a strong and inverse correlation between circulation levels of 25OHD and weight but also BMI (113)(114)(115)(116)(117) in both men and women across the ages. Furthermore, this inverse association was seen in all fat compartments but was stronger for visceral fat (113), perhaps indicative of higher cardiovascular morbidity. In support of this, several studies showed that decreased 25OHD levels impair insulin action (118)(119)(120) and are associated with the metabolic syndrome (120)(121)(122). At the cellular level, mechanistic studies also support this association. 1,25(OH)2D treatment of pre-adipocytes in culture decreases adipogenesis (123) through inhibition of C/EBPalpha and PPARγ;VDR and PPARγ act synergistically to inhibit adipogenesis (124). The effect of vitamin D on MF has also been examined in animal studies. Our group showed that continuous administration of 1,25(OH)2D in senescenceaccelerated mice (SAM-P/6) suppressed adipogenesis in the marrow and that isolated MSCs had a reduced expression of the adipogenic enhancer PPARγ (125) and accelerated differentiation into osteoblasts compared to placebo-treated animals. This was accompanied by an increase in both cortical and trabecular bone strength (126). Other studies also support our data that vitamin D enhances MSC differentiation to osteoblasts (127,128), suggesting that 1,25(OH)2D may exert a protective effect on bone aging.
However, a causal relationship supporting the role of vitamin D as a regulator of fat metabolism and distribution in humans has been difficult to prove. In support of this theory, Ortega et al. found that baseline 25OHD levels are predictive of the efficacy of weight loss regimen and that the vitamin D status potentiates the effect of low caloric diet (129). Several other studies showed that vitamin D supplementation induces a moderate effect on weight loss while others did not (130)(131)(132)(133)(134)(135). On the other hand, other clinical studies point to the evidence of fat as a reservoir for vitamin D (sequestration theory). First, it was shown that obesity is directly related to 25OHD levels: sequestration hypothesis (136,137) and second, that weight loss tends to increase 25OHD levels (138).
An unexplored effect of vitamin D action on bone could come from its effect on fat accumulation in muscle. Vitamin D is also a major determinant of skeletal muscle function (139)(140)(141)(142). A severe lack of vitamin D can cause myopathy (143,144), which tends to be more marked in the proximal muscles (145). In the elderly, vitamin D deficiency is linked to muscle weakness and increased susceptibility to falls and fractures, which improve with administration of vitamin D with calcium (146)(147)(148)(149)(150)(151)(152)(153)(154)(155)(156)(157). We recently found that in healthy young women, vitamin D levels are inversely related to the degree of fat infiltration in muscle (112), a phenotype associated with impaired muscle strength (19,20,158). Available data indicate higher muscle lipid content to also be associated with decreased muscle function in patients with neuromuscular disorders (19). Indeed, even in healthy subjects, higher muscle lipid content is associated with lower levels of muscle strength and physical performance, independent of muscle mass (20, 158). Among the different mechanisms that could explain the accumulation of fat in muscle, it is tempting to speculate that mesenchymal progenitors normally present in skeletal muscle -MSCs, muscle-derived stem cells, or muscle satellite cells -could potentially allow muscular growth and regeneration or differentiate into cells with an adipocyte phenotype, including the abilities to express adipocyte-specific genes and accumulate lipids (159)(160)(161).
Our studies showing that vitamin D is inversely related to fat infiltration in muscle (112) and positively related to muscle strength (139) in healthy young females support the notion that vitamin D may be a key determinant of muscle precursor cell (MPC) differentiation. However, whether vitamin D-mediated muscle adiposity and performance determine bone acquisition, and simultaneous decreases in marrow adiposity remains to be determined. A conceptual relationship between muscle, bone, and fat and how it could be influenced by vitamin D is shown in Figure 2.
Most vitamin D supplementation trials on muscle strength have been done in the elderly and found a reduction in falls, improvements in balance and body sway, and/or resolution of myalgia in statin-treated patients with treatment periods as short as 8-12 weeks (146,162,163). Likewise, several studies have examined the impact of vitamin D supplementation on muscle composition, primarily by assessing muscle fiber number and diameter, infiltration of fat and fibrosis, and all were in elderly subjects (152,164,165); treatment with vitamin D and calcium improved muscle composition after as short a time as 3 months. The ability to obtain tissue samples from healthy, young women undergoing surgery for sports-related injuries represents a unique approach in this field of research.
Two vitamin D supplementation studies have been done in girls; one found improvements in muscle function in the vitamin D-treated group but no significant differences in bone measures using DXA and peripheral quantitative computed tomography (CT), while the other found increases in DXA measures of lean mass and spine bone mineral content (139,166). These discrepant results likely reflect the limitations of the techniques employed. Changes in body composition influence DXA measures during growth, and peripheral quantitative CT measures in children have poor reproducibility due to large variations in bone growth (167,168). Confounding effects associated with growth and development are common when studying sexually and skeletally immature young women, using DXA or CT.

Parathyroid Hormone
Parathyroid hormone is a major regulator of calcium and bone homeostasis, but studies on its effect of fat have been so far limited. Two epidemiological studies suggest a possible positive association between circulating levels of PTH and fat mass. The first showed that circulating PTH concentrations are directly correlated with fat mass (169), and the other showed that body weight is increased in women with primary hyperparathyroidism as compared to controls (170).

BROwN ADiPOSe TiSSUe AND BONe
Much of this review focused on the interaction between white fat and bone, which is by far the most studied. In contrast, the literature on BAT and bone is almost non-existent except for two correlative studies showing that a positive relationship exists between BAT and bone volume in children and adolescent boys and girls (171), and BAT and bone size in both children and adults (171,172). The effect of BAT became insignificant when muscle mass was introduced in the model, a finding supported by a previous study showing a positive relationship between BAT and muscle mass (173). It has also been reported that young women with active BAT have higher BMD than women without BAT (174), further supporting a possible mechanistic relationship between BAT, bone growth, and bone strength. The underlying mechanism(s) remains to be established.

THe CHALLeNGeS OF FAT iMAGiNG
In this section, we will briefly summarize the recent progress in non-invasive measurement of MF and body using imaging technologies.
Studies assessing MF-bone interactions have been hindered by the difficulty of independently examining different tissues at the same site. The most commonly employed method to assess bone and body composition has been DXA, which cannot analyze muscle or MF. In contrast, CT and MRI provide accurate measures of bone, muscle, and fat independently (175)(176)(177)(178)(179). MRI has the added advantage of being able to quantify the amount of any tissue without exposing the subject to radiation. However, MRI measurements of bone, bone-MF, and muscle require state-of-the-art imaging, including Dixon capabilities. Over the past 25 years, Dixon's method has evolved significantly (19,177,(180)(181)(182)(183), and recent advances have led to more generalized algorithms (176,(184)(185)(186). We used three-point Dixon MR technique for fat quantification (182) and determined that reproducibility of the fat fraction quantifications in phantom models was excellent with a coefficient of variation of <1.5% (182). In vivo reproducibility of MF varies between 1.3 and 3% (176,177). Pixel signal intensities from the medullary canal are obtained, and total fat % is calculated by integration in slice selection direction over the imaging volume. To calculate bone structural properties from the MRI images, our group has developed a graphical user interface with Matlab (Mathworks, Natick, MA, USA) using custom algorithms. The program is designed to automatically extract endosteal and periosteal contours of the bone and to calculate geometric and structural parameters. First, the user selects a DICOM image and then crops a rectangular region of interest containing the bone of interest. The image is automatically thresholded according to bone and muscle peaks from the image histogram. Edges of the cortex are detected and contours generated. The correlation of this method with quantitative CT is excellent (187). Multiple investigators have previously evaluated properties of the femoral midshaft using tracing (188), deformable models (189), and semi-automatic algorithms.
Possible differences in the distribution of fat accumulation in children have been difficult to establish due to the limitations and the risks of the techniques used. While there are many techniques, including underwater weighing, anthropometry, body water dilution, impedance, and DXA, to estimate total body fat content, it has not been possible to differentiate between subcutaneous and visceral fat until the advent of CT and MRI (190). Both CT and MRI provide a three-dimensional assessment of body tissues (175). CT provides cross-sectional images from which the amounts and distributions of subcutaneous fat and visceral fat are well distinguished, but these determinations are areal measurements (cm 2 ) and multiple scans are necessary to obtain true volumetric values, exposing the child to radiation. In contrast, MRI allows for volume determinations and can reliably measure the amount and distribution of abdominal fat, without radiation exposure (cm 3 ) (175,179). In a study comparing MRI with five other methods (underwater weighing, O dilution, K counting, skinfold thickness, and body electrical impedance methods), MR gave the least variability and an estimate of body fat significantly closer to the mean of the five other methods than any other technique alone. MRI-based studies are likely to be less affected by individual variability and may therefore achieve higher statistical power for a given sample size (178).

CONCLUSiON
The interactions between fat and bone are complex and new emerging concepts regarding their relationship have the potential of transforming our therapeutic targeting of the skeleton. The inverse relationship between MF and bone is an enthralling area of research based on the very origin of bone and fat cell differentiation from MSC. The obesity epidemic has also brought new challenges in terms of prevention and treatment of common illnesses, such as type 2 diabetes. Here again, the interactions between body weight, body fat, and bone are much more complex, and the influence of clinical context, age, sex, and ethnicity should be considered when examining this relationship. Overall, bone and fat may not be such an odd couple but rather a very important one that deserves to be examined in all its facets as it represents a unique challenge for future health.


Identification of new miRNA biomarkers associated with HER2-positive breast cancers

Human epidermal growth factor receptor 2 (HER2) is overexpressed/amplified in ∼30% breast cancers which are associated with poor prognosis. microRNAs are small non-coding RNA which play an important role in many physiological conditions including cancer. Here we screened and identified many miRNAs which are dysregulated by HER2 overexpression. In line with our quantitative PCR analysis data, in silico analysis of microRNA expression profiles of 1302 breast tumors revealed that miR-146a-5p is up-regulated and miR-181d and miR-195-5p are down-regulated in HER2-positive tumors. Furthermore, the expression levels of these microRNAs can significantly predict patient survival and thus potentially serve as new prognostic markers for HER2-positive breast cancer.

INTRODUCTION
Breast cancer is a heterogeneous disease, comprising of multiple entities associated with distinctive histological and biological features. Based upon microarray gene expression profiles, breast cancers can be classified into biologically and clinically meaningful subgroups: luminal A, luminal B, basal-like, normal-like, and human epidermal growth factor receptor 2 (HER2) positive tumors [1]. Luminal A and luminal B breast tumors are estrogen receptor positive (ER+). Among all types of breast cancers, luminal A has the best prognosis for the patient along with the normal-like tumors. Basal-like tumors, that are negative for ER, progesterone receptor (PR) and HER2, are found in 10%-15% of all patients and showed the worst prognosis among all subtypes of breast cancers [2]. HER2 is over-expressed/amplified in approximately 20-30% breast cancers, and this type of breast cancer is known as HER2-positive breast cancer [3]. Major HER2-downstream signaling cascades include phosphoinositide 3-kinase (PI3K)/AKT and Ras/ MAPK kinase pathway which are known to regulate cell proliferation and survival [4,5]. Overexpression of HER2 is associated with relatively poor prognosis and predictive for response to trastuzumab, a monoclonal antibody that targets HER2. However, the average response rate for trastuzumab treatment was approximately 35% with response rate varying from 12-68% [6], necessitating the identification of more biomarkers for selection of treatments and prediction of clinical outcomes.
MicroRNAs (miRNA) are small non-coding RNA that negatively regulate protein-coding messenger RNAs (mRNA) at the post transcriptional level. Dysregulation of miRNAs have been found to be associated with many human diseases including cancer [7]. miRNAs can be oncogenic miRNA or tumor suppressor miRNA depending on their target genes . In addition, miRNAs have also been proposed to be used as biomarkers for cancers, for example, miR-146a-5p as a biomarker for colorectal tumor localization [8,9]. However, miRNAs' involvement in the HER2 pathway have not studied extensively, and their role in HER2 signaling remains elusive. We propose that the HER2 overexpression will lead to differential regulation of a number of miRNAs which are important in HER2 mediated tumorigenesis.

Identification of differentially expressed microRNAs in HER2-overexpressing cells using q-RT-PCR analysis
For screening purposes, immortalized human breast epithelial cells called MCF10A-Vector and its HER2 positive counterpart were used. To confirm whether HER2 transformed cells do produce HER2 receptor, a

Research Paper
western blot was performed for evidence ( Figure 1a). The MCF10A-HER2 indeed have a higher expression of the phosphorylated HER2 (p-HER2), which signifies the activation of the HER2 receptor in the cells. The activation of HER2 triggers the activation of the downstream signaling pathways. This downstream pathway activation is affirmed by the increased phosphorylation of STAT3 (p-STAT3 Y705 and S727), AKT (p-AKT) and ERK (p-ERK1/2), which are well-known downstream targets of the HER2 signaling pathway [10].
Real-time RT-PCR analysis were performed to screen for miRNAs that are differentially regulated by HER2. Three hundred and three cancer-related miRNA were selected for this screening. Considering that some primers may not work at this PCR condition, the melting curve analysis for each pair of primer was performed and the qPCR products were run on agarose gel to determine if the product of each miRNA primer is correct ( Figure 1c). For the qPCR products which didn't show single band and correct sizes or have low abundance (> 35 PCR cycles in both cells) were eliminated for further study. Using two fold change as the cutoff, we further narrowed down the list to fifty miRNAs that are differentially expressed in MCF10A-HER2 cells compared to MCF10A-Vector cells ( Figure 1b). Among these differentially expressed miRNAs, miR-146a-5p, miR-195-5p, and miR-181d are potentially involved in breast cancer development based on previous research and therefore they were selected for further statistical analysis.

In silico analysis of miRNAs expression in breast cancer
Recently, Devinge et al. have reported the miRNA expression profile of 1302 breast cancer samples by microarray analysis [11]. We first analyzed the expression statuses of the three selected miRNAs in the five subtypes of breast cancers. Both miRNAs miR-195-5p and 181d are down-regulated in HER2-positive breast epithelial cells compared to control cells ( Figure 1b). Consistently, the expression levels of these two microRNAs are significantly lower in the HER2-positve subtype compared to luminal subtypes of breast cancers ( Figure 2a and 2c). However, miR-146a-5p is up-regulated in HER2-positive breast cancer. Indeed, we found that the expression level of miR-146a-5p is higher in HER2-positive and basal subtypes compared to both luminal and normal-like subtypes of breast cancer ( Figure 2e). Given that the expression status of these selected miRNAs tend to be associated with the aggressive subtypes of breast cancer, we performed the Kaplan-Meier survival analysis to further evaluate their clinical relevance. As predicted, low expression of miR-195-5p and 181d shows an association Over 300 miRNAs were screened using qPCR analysis. The differentially expressed miRNAs were selected with the 2-fold change cutoff. c) Agarose gel picture depicting the expression of representative qPCR products from the miRNA screening. www.impactjournals.com/oncoscience with poor patient survival ( Figure 2b and 2d) whereas high expression of miR-146a-5p shows an association with poor patient survival ( Figure 2f).

DISCUSSION
Deregulated expression of miRNAs can promote tumorigenesis and contribute to a variety of cancer related phenotypes such as uncontrolled cell proliferation, invasion and metastasis. HER2-overexpressing breast cancers are generally associated with aggressive phenotypes and poor prognosis. Despite of extensive research on HER2 positive breast cancer, the involvement of miRNAs in HER2 signaling pathway is not well understood [12][13][14][15]. We screened and identified many miRNAs which were differentially expressed in HER2overexpressing breast cells compared to control cells. Furthermore, we evaluated their clinical relevance in breast cancer samples. Three miRNAs miR-146a-5p, miR-95-5p and miR-181d show their expected expression profile in HER2-positive breast cancers when compared with other subtypes of breast cancers.
Interestingly, the role of miR-146a in breast tumorigenesis remains controversial. Several studies indicated that miR-146a can function as oncomiR while few have demonstrated it as tumor suppressor miRNA in breast cancer. As an oncomiR, miR-146a could bind to the 3'-untranslated regions (UTRs) of BRCA1 and BRCA2 survival analysis based on the miR-181, miR-195-5p and miR-146a-5p expression levels. The microarray data was extracted from previous publication [11].The mean value of miRNA expression levels was used the cutoff to stratify patient into two groups. *p value < 0.05; **p value < 0.01. www.impactjournals.com/oncoscience messenger RNAs (mRNAs) and potentially modulate their mRNA expression [16,17]. On the contrary, it has been reported that miR146a negatively regulates NF-κB signaling [18,19]. Overexpression of miR-146a/b results in downregulation of IRAK1 and TRAF6 and subsequently inhibits NF-κB activation, leading to tumor suppression in breast cancer cells [18]. miR-146a has been shown to inhibit both migration, invasion, and metastasis by reducing EGFR expression [20]. It will be interesting to examine the function of miR-146a in the HER2-positive breast cancer.
miR-195-5p has been found to be down-regulated [21] and also been suggested as a possible diagnostic target for breast cancer [22]. Overexpression of miR-195-5p inhibited cell proliferation, reduced cell colony formation, suppressed cell migration and caused an accumulation of cells in the G1 phase of the cell cycle by directly targeting cyclin E1 (CCNE1) [22]. In support of the hypothesis that miR-195-5p acts as a tumor suppressor in breast cancer, we found that miR-195-5p expression is downregulated in aggressive subtypes of breast cancers, especially in HER2positive and basal types of breast cancers.
Unlike the previous two miRNAs, miR-181d has not been extensively studied. The amount of information and connection between miR-181d and cancer is scarce and it is even more in HER2 positive breast cancers. However, it has been observed that the miR-181 family (miR-181a, miR-181b, miR-181c, and miR-181d), more precisely miR-181c, is activated by HER2 expression [13]. Even though miR-181c is possibly co-expressed or repressed with miR-181d, there has not been many research done regarding this co-expression or repression. Given that miR-181d is down-regulated with the overexpression of HER2 in both cell lines and clinical samples, miR-181d may be classified as a tumor suppressor miRNA and may target an oncogene.
In summary, we presented a strategy to effectively identify novel and clinically relevant microRNA biomarkers by combining a high-throughput screening approach with in silico data-mining analysis. Further research is warranted to explore their functions in breast tumorigenesis and develop them as novel therapeutic targets.

Cell culture
MCF10A-Vector and MCF10A-HER2 cells were provided through the courtesy of Emily Wang at the Institute of City of Hope. Culturing MCF10A cells require DMEM/F12 medium from Corning cellgro™. In a 500ml bottle, 25ml of 5% horse serum from Sigma® Life Science is added to the medium along with 100μl of 500μg/ml of cholera toxin for a final concentration of 100ηg/ml. In addition, 1ml of 5mg/ml insulin for a final concentration of 10μg/ml, 63μl of 4mg/ml of hydrocortisone for a final concentration of 0.5μg/ml, and 50μl of 200μg/ml of EGF for a final concentration of 20ηg/ml are added. When detaching cells from cell culture plate, 0.25% of Trypsin acquired from Sigma® Life Science.

Western blot analysis
Protein extraction was achieved using standard protocol [23,24]. The protein concentration is then quantified and normalized to ensure equal loading into polyacrylamide gels. 8% polyacrylamide gels were used to separate the proteins. Antibodies used were acquired from Cell Signaling Technology®.

Real-time RT-PCR analysis of miRNA expression
RNA extraction from this point was done using TRIzol® protocol [25]. RNA was first poly-adenylated using E.coli poly(A) polymerase from New England Biolabs. Poly-adenylated RNA was then used for reverse transcription and the reverse-transcription polymerase chain reaction (RT-PCR). For the quantitative polymerase chain reaction (qPCR), 2μl of cDNA was mixed with 10μl of RT² SYBR® Green ROX qPCR Mastermix from Qiagen , 6μl of RNase-free water, 1μl of a universal reverse primer (5'GCG AGC ACA GAA TTA ATA CGA C3') and a forward primer specific to the miRNA. The reaction is run in an Applied Biosystem™ qPCR machine with the following parameters: Stage 1: 3 minutes hot start at 95°C. Stage 2: 15 seconds denaturing step at 95°C followed by 30 seconds annealing step at 60°C and finally the extension step is held for 35 seconds at 70°C. This stage is repeated for 45 cycles.

In silico analysis of miRNAs expression in breast cancer
The clinical effect of the gene expression profiles of microRNA was evaluated using a published data set of breast cancer patients [11]. The mean expression value was used as the cutoff to classify miRNA expression as high or low. Recurrence-free survival was estimated using the Kaplan-Meier method and compared with log-rank tests. The statistical analysis was conducted with R and GraphPad software packages (GraphPad, CA, USA). The p-values were calculated using the logrank test, and differences were considered statistically significant at p < 0.05.


extfieldsofstudy: Biology Medicine

s2fieldsofstudy: Biology Medicine

text:
Honokiol, a Lignan Biphenol Derived from the Magnolia Tree, Inhibits Dengue Virus Type 2 Infection

Dengue is the most widespread arbovirus infection and poses a serious health and economic issue in tropical and subtropical countries. Currently no licensed vaccine or compounds can be used to prevent or manage the severity of dengue virus (DENV) infection. Honokiol, a lignan biphenol derived from the Magnolia tree, is commonly used in Eastern medicine. Here we report that honokiol has profound antiviral activity against serotype 2 DENV (DENV-2). In addition to inhibiting the intracellular DENV-2 replicon, honokiol was shown to suppress the replication of DENV-2 in baby hamster kidney (BHK) and human hepatocarcinoma Huh7 cells. At the maximum non-toxic dose of honokiol treatment, the production of infectious DENV particles was reduced >90% in BHK and Huh7 cells. The underlying mechanisms revealed that the expression of DENV-2 nonstructural protein NS1/NS3 and its replicating intermediate, double-strand RNA, was dramatically reduced by honokiol treatment. Honokiol has no effect on the expression of DENV putative receptors, but may interfere with the endocytosis of DENV-2 by abrogating the co-localization of DENV envelope glycoprotein and the early endosomes. These results indicate that honokiol inhibits the replication, viral gene expression, and endocytotic process of DENV-2, making it a promising agent for chemotherapy of DENV infection.

Introduction
Dengue fever is an acute infectious disease caused by the dengue virus (DENV), an enveloped, positive-sense, single stranded RNA virus belonging to the Flaviviridae family. There are four definite serotypes of DENV (DENV-1, -2, -3, and -4) and an emerging new one has recently been reported [1]. Dengue is transmitted by mosquito vectors, principally Aedes aegypti and Aedes albopictus. It is characterized by biphasic fever, headache, pain, rash, lymphadenopathy, and leucopenia [2,3]. In most cases, the disease of dengue fever is self-limited. However, there is a risk of progressing to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), especially when cross infection of different serotypes of DENV occurs. Under such conditions, bleeding, low levels of blood platelets and blood pressure, and blood plasma leakage occur, which leads to a critical or even life-threatening situation of affected patients. Approximately 3.6 billion people living in tropical and subtropical areas are affected by dengue viruses and over 100 million infections are reported annually [4]. Dengue has become an enormous health and economic concern in these endemic countries.
Phytochemicals, which occur naturally in plants, have been a major subject in study for their health benefits and potential pharmaceutical applications. Many phytochemicals are found to have versatile effects, including antioxidant, anticancer, anti-inflammatory, anti-bacterial, and anti-viral effects [5][6][7][8]. Viruses, unlike bacterial pathogens, only exhibit biological activities when they infect host cells and utilize host cell mechanisms to replicate within them. The search for active phytochemicals against pathogenic viruses without having deleterious effects on the host has been the focus of studies in recent decades. The antiviral mechanism of these agents may be ascribed to their antioxidant/scavenging capacities, nucleic acid synthesis inhibition, viral entry suppression, or inhibition of the viral reproduction [7].
Honokiol is a lignan biphenol derived from the Magnolia tree ( Figure 1A). The bark or seed cones of the tree, such as in the Chinese herbal medicine Hou-Pu (derived from Magnolia officinalis), have been widely used in traditional medicine as analgesic, distension, or anxiety relief [9]. Honokiol is a pleiotropic compound that has a variety of pharmacological effects, including antitumorigenic [10], anti-inflammation [11], anti-thrombosis [12], anti-oxidation [13], and neuroprotective effects [14]. Being a relatively small molecule, it has been proved to be able to readily cross the blood-brain barrier and the blood-cerebrospinal fluid barrier [15]. Recently, it has been reported that honokiol can suppress the infection of hepatitis C virus in cell models by targeting viral entry and replication [16]. As a result, honokiol is a potential pharmaceutical agent with high bioavailability.

Introduction
Dengue fever is an acute infectious disease caused by the dengue virus (DENV), an enveloped, positive-sense, single stranded RNA virus belonging to the Flaviviridae family. There are four definite serotypes of DENV (DENV-1, -2, -3, and -4) and an emerging new one has recently been reported [1]. Dengue is transmitted by mosquito vectors, principally Aedes aegypti and Aedes albopictus. It is characterized by biphasic fever, headache, pain, rash, lymphadenopathy, and leucopenia [2,3]. In most cases, the disease of dengue fever is self-limited. However, there is a risk of progressing to severe dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), especially when cross infection of different serotypes of DENV occurs. Under such conditions, bleeding, low levels of blood platelets and blood pressure, and blood plasma leakage occur, which leads to a critical or even life-threatening situation of affected patients. Approximately 3.6 billion people living in tropical and subtropical areas are affected by dengue viruses and over 100 million infections are reported annually [4]. Dengue has become an enormous health and economic concern in these endemic countries.
Phytochemicals, which occur naturally in plants, have been a major subject in study for their health benefits and potential pharmaceutical applications. Many phytochemicals are found to have versatile effects, including antioxidant, anticancer, anti-inflammatory, anti-bacterial, and anti-viral effects [5][6][7][8]. Viruses, unlike bacterial pathogens, only exhibit biological activities when they infect host cells and utilize host cell mechanisms to replicate within them. The search for active phytochemicals against pathogenic viruses without having deleterious effects on the host has been the focus of studies in recent decades. The antiviral mechanism of these agents may be ascribed to their antioxidant/scavenging capacities, nucleic acid synthesis inhibition, viral entry suppression, or inhibition of the viral reproduction [7].
Honokiol is a lignan biphenol derived from the Magnolia tree ( Figure 1A). The bark or seed cones of the tree, such as in the Chinese herbal medicine Hou-Pu (derived from Magnolia officinalis), have been widely used in traditional medicine as analgesic, distension, or anxiety relief [9]. Honokiol is a pleiotropic compound that has a variety of pharmacological effects, including antitumorigenic [10], anti-inflammation [11], anti-thrombosis [12], anti-oxidation [13], and neuroprotective effects [14]. Being a relatively small molecule, it has been proved to be able to readily cross the blood-brain barrier and the blood-cerebrospinal fluid barrier [15]. Recently, it has been reported that honokiol can suppress the infection of hepatitis C virus in cell models by targeting viral entry and replication [16]. As a result, honokiol is a potential pharmaceutical agent with high bioavailability.  Currently there is no licensed vaccine for dengue, although a live attenuated chimeric vaccine is under Phase III trial [17]. The prevention of dengue fever to date is solely mounted by vector control. In addition, no anti-dengue viral agent is clinically available to treat or reduce the severity of dengue symptoms. Only supportive medical treatment is applied to patients with dengue diseases. Although DHF and DSS occur in less than 5% of all cases of dengue [18,19], severe cases that result in death are predominant in children under the age of 15 [20]. Considering the severity of dengue diseases, it is vital and urgent to develop strategies to combat DENV infection. Several phytochemicals have been reported previously to have antiviral activities [7]. Here we report that honokiol has a significant anti-DENV activity in vitro. Treatment of honokiol reduces the activity of DENV-2 replicon and suppresses the viral replication. After DENV infection, honokiol treatment suppresses the expression of viral protein NS1 and NS3 and the viral replicating intermediate, double-strand RNA (dsRNA). Honokiol may also interfere with the endocytotic process during DENV entry. These results support that honokiol intervenes in DENV-2 infection at multiple stages, making it a potent anti-DENV chemical for clinical dengue treatment.

Honokiol Effectively Inhibits the DENV Replicon
A stably transfected baby hamster kidney (BHK) cell line, BHK-D2-Fluc-SGR-Neo-1, which harbors a luciferase-reporting DENV-2 subgenomic replicon [21], was used to determine the inhibitory effect of honokiol on DENV-2 replication. The cytotoxic effect of honokiol on this BHK-DENV cell line was evaluated by a standard MTT assay with various concentrations of honokiol (1-20 µM) treatment. At 24 h, the treatment of honokiol did not have a marked effect on the viability of this cell from 1-5 µM ( Figure 1B). At 10 and 20 µM of honokiol treatment, a minor reduction of the viability of the BHK-DENV was observed, but the overall survival of cells was ě80% compared to the vehicle control. Under these conditions, honokiol was found to inhibit the luciferase activity of DENV replicon in a dose-dependent manner ( Figure 1C). At 5 µM, the reporter activity was decreased 23.3%˘3.3 compared to the vehicle controls (p = 0.006). The reduction of luciferase activity was even more prominent at 10 and 20 µM of honokiol treatment (51.6%˘2.5 and 65.1%˘0.6 reduction, respectively; p < 0.001) with a minor effect on cell viability. This result indicates that honokiol has a significant antiviral activity against DENV-2 replicon intracellularly.

Honokiol Inhibits the DENV Infection in Vitro
The DENV replicon test had indicated that honokiol has a substantial antiviral activity against DENV. To further confirm this observation, an in vitro DENV yield reduction assay was performed. Two cell lines, the BHK cell and the human hepatocarcinoma cell Huh7, were selected for the viral yield reduction assay. The cytotoxic effect at 48 h after honokiol treatment was first determined by MTT assay (Figure 2A). The half maximal cytotoxic concentration (CC 50 ) of honokiol was found to be 13.35˘1.13 µM for BHK cells and 31.19˘1.49 µM for Huh7 cells. While at 10 and 20 µM of honokiol treatment, no deleterious effect was observed in BHK and Huh7 cells, respectively, and therefore, that concentration was selected as the maximum non-toxic dose (MNTD) for each cell line in the following studies. The BHK and Huh7 cells were infected with DENV-2 and then followed by honokiol treatment with different concentrations for 48 h. The released infectious DENV particles in the cell culture supernatant post honokiol treatment was determined by fluorescence focus assay. Treatment of honokiol was found to suppress the viral production both in DENV-infected BHK and Huh7 cells ( Figure 2B,C). In BHK cells, treatment with 5 µM of honokiol did not reveal a marked reduction, while 10 µM of honokiol significantly reduced the DENV production (p < 0.001, Figure 2B). In Huh7 cells, the inhibition of DENV production was significant at 10 µM of honokiol treatment (37% reduction, p = 0.027), and was reduced further at 20 µM (p < 0.001, Figure 2C). The reduction of virus production was >90% at the MNTD (10 µM and 20 µM, respectively) of honokiol in both BHK and

Honokiol Inhibits DENV Protein Expression and Viral RNA Replication
To investigate if honokiol could inhibit the viral protein expression as well as the viral RNA replication, BHK and Huh7 cells were infected with DENV-2 and then treated with honokiol for 48 h. The expression levels of the viral non-structure protein NS1 and NS3, and the viral replicating intermediate, double-strand RNA (dsRNA), were assayed using immunofluorescence staining and analyzed by high content image analysis. In both DENV-infected BHK and Huh7 cells, the cells positive for NS1, NS3 or dsRNA were decreased after honokiol treatment, indicating their expressions were suppressed ( Figure 3A,B). The percentage of DENV-infected cells positive for the viral antigens (i.e., NS1, NS3 or dsRNA) in each experiment was quantified by the Attovision software (BD Biosciences) and the results were plotted and statistically evaluated ( Figure 3C,D). In the BHK cell model infected with DENV-2, the percentage of cells with the NS1, NS3 or dsRNA

Honokiol Inhibits DENV Protein Expression and Viral RNA Replication
To investigate if honokiol could inhibit the viral protein expression as well as the viral RNA replication, BHK and Huh7 cells were infected with DENV-2 and then treated with honokiol for 48 h. The expression levels of the viral non-structure protein NS1 and NS3, and the viral replicating intermediate, double-strand RNA (dsRNA), were assayed using immunofluorescence staining and analyzed by high content image analysis. In both DENV-infected BHK and Huh7 cells, the cells positive for NS1, NS3 or dsRNA were decreased after honokiol treatment, indicating their expressions were suppressed ( Figure 3A,B). The percentage of DENV-infected cells positive for the viral antigens (i.e., NS1, NS3 or dsRNA) in each experiment was quantified by the Attovision software (BD Biosciences) and the results were plotted and statistically evaluated ( Figure 3C,D). In the BHK cell model infected with DENV-2, the percentage of cells with the NS1, NS3 or dsRNA signal was reduced to near or below 50% by 5 µM of honokiol treatment. At 10 µM, the signal of these three viral products had almost completely vanished (reduction >90%, p < 0.001, Figure 3C). The inhibitory effect was less intense in Huh7 compared to BHK cells. At 10 µM of honokiol treatment, the viral NS3 expression showed a sensitive response to honokiol-mediated inhibition (p < 0.001, Figure 3D) as compared to the NS1 and dsRNA expression. However, a significant reduction of these three viral products was observed at 20 µM of honokiol treatment (p = 0.01 for dsRNA; p < 0.001 for NS1 and NS3). These results indicate that treatment of honokiol after DENV infection can not only inhibit the DENV protein translation (i.e., NS1 and NS3 expression) but also suppress its subsequent RNA replication (i.e., dsRNA expression), which may lead to the decreased viral production observed in previous viral yield assay ( Figure 2B,C).
Viruses 2015, 7, page-page 5 signal was reduced to near or below 50% by 5 µ M of honokiol treatment. At 10 µ M, the signal of these three viral products had almost completely vanished (reduction >90%, p < 0.001, Figure 3C). The inhibitory effect was less intense in Huh7 compared to BHK cells. At 10 µ M of honokiol treatment, the viral NS3 expression showed a sensitive response to honokiol-mediated inhibition (p < 0.001, Figure 3D) as compared to the NS1 and dsRNA expression. However, a significant reduction of these three viral products was observed at 20 µ M of honokiol treatment (p = 0.01 for dsRNA; p < 0.001 for NS1 and NS3). These results indicate that treatment of honokiol after DENV infection can not only inhibit the DENV protein translation (i.e., NS1 and NS3 expression) but also suppress its subsequent RNA replication (i.e., dsRNA expression), which may lead to the decreased viral production observed in previous viral yield assay ( Figure 2B,C).

Pretreatment with Honokiol Does Not Affect the Attachment of DENV on Host Cells
Attachment is the first step of virus entry. It is mediated by virion proteins binding to specific surface receptors such as membrane proteins, lipids, or the carbohydrate moieties present on glycoproteins or glycolipids [22,23]. To investigate if pre-treatment of honokiol inhibits the attachment of DENV to host cells, the BHK cells were first treated with 10 µ M of honokiol for 24 and 48 h prior to infection with DENV. The flow cytometry analysis showed a marked increase of fluorescence signal in BHK cells when incubated with DENV-2, confirming the attachment of DENV to the host cells ( Figure 4A,B). As expected, the presence of neutralizing antibodies 137-22 upon infection significantly reduced the fluorescence signal. Pre-treatment of honokiol did not alter the degree of DENV binding to the cell surface, regardless of treatment times. This result indicates that honokiol-mediated DENV inhibition is not related to affect the expression of DENV receptor(s) on host cells.

Pretreatment with Honokiol Does Not Affect the Attachment of DENV on Host Cells
Attachment is the first step of virus entry. It is mediated by virion proteins binding to specific surface receptors such as membrane proteins, lipids, or the carbohydrate moieties present on glycoproteins or glycolipids [22,23]. To investigate if pre-treatment of honokiol inhibits the attachment of DENV to host cells, the BHK cells were first treated with 10 µM of honokiol for 24 and 48 h prior to infection with DENV. The flow cytometry analysis showed a marked increase of fluorescence signal in BHK cells when incubated with DENV-2, confirming the attachment of DENV to the host cells ( Figure 4A,B). As expected, the presence of neutralizing antibodies 137-22 upon infection significantly reduced the fluorescence signal. Pre-treatment of honokiol did not alter the degree of DENV binding to the cell surface, regardless of treatment times. This result indicates that honokiol-mediated DENV inhibition is not related to affect the expression of DENV receptor(s) on host cells.

Pretreatment with Honokiol Does Not Affect the Attachment of DENV on Host Cells
Attachment is the first step of virus entry. It is mediated by virion proteins binding to specific surface receptors such as membrane proteins, lipids, or the carbohydrate moieties present on glycoproteins or glycolipids [22,23]. To investigate if pre-treatment of honokiol inhibits the attachment of DENV to host cells, the BHK cells were first treated with 10 µ M of honokiol for 24 and 48 h prior to infection with DENV. The flow cytometry analysis showed a marked increase of fluorescence signal in BHK cells when incubated with DENV-2, confirming the attachment of DENV to the host cells ( Figure 4A,B). As expected, the presence of neutralizing antibodies 137-22 upon infection significantly reduced the fluorescence signal. Pre-treatment of honokiol did not alter the degree of DENV binding to the cell surface, regardless of treatment times. This result indicates that honokiol-mediated DENV inhibition is not related to affect the expression of DENV receptor(s) on host cells.

Honokiol May Interfere with the Endocytic Pathway during DENV Entry
To further characterize the antiviral mechanisms of honokiol, we investigated the effect of the compound on the entry of the DENV particle into host cells. Following attachment, the DENV is internalized via receptor-mediated endocytosis [24]. Upon binding to receptors, the clathrin-mediated endocytosis is considered to be a major pathway for DENV entry [24]. In this study, honokiol was applied to Huh7 cells after DENV attachment to test if it can interfere with the viral entry process. Infection with DENV increased the amount of intracellular early endosomes ( Figure 5A,B), and the DENV E protein was found to co-localize with the early endosomes in DENV-infected cells, indicating its interactions, which is a distinct early event of endocytosis during DENV entry [25,26]. Intriguingly, the presence of honokiol (10 and 20 µ M) after DENV entry substantially suppressed the up-regulation of early endosomes, as compared to the DENV infected/mock-treated cells ( Figure 5C,D vs. 5B). Consequently, the co-localization of DENV E protein and the early endosome was markedly abrogated. This result indicates that honokiol may interfere with the endocytotic process of DENV during its entry into the host cells.

Honokiol May Interfere with the Endocytic Pathway during DENV Entry
To further characterize the antiviral mechanisms of honokiol, we investigated the effect of the compound on the entry of the DENV particle into host cells. Following attachment, the DENV is internalized via receptor-mediated endocytosis [24]. Upon binding to receptors, the clathrin-mediated endocytosis is considered to be a major pathway for DENV entry [24]. In this study, honokiol was applied to Huh7 cells after DENV attachment to test if it can interfere with the viral entry process. Infection with DENV increased the amount of intracellular early endosomes ( Figure 5A,B), and the DENV E protein was found to co-localize with the early endosomes in DENV-infected cells, indicating its interactions, which is a distinct early event of endocytosis during DENV entry [25,26]. Intriguingly, the presence of honokiol (10 and 20 µM) after DENV entry substantially suppressed the up-regulation of early endosomes, as compared to the DENV infected/mock-treated cells ( Figure 5C,D vs. 5B). Consequently, the co-localization of DENV E protein and the early endosome was markedly abrogated. This result indicates that honokiol may interfere with the endocytotic process of DENV during its entry into the host cells.

Honokiol May Interfere with the Endocytic Pathway during DENV Entry
To further characterize the antiviral mechanisms of honokiol, we investigated the effect of the compound on the entry of the DENV particle into host cells. Following attachment, the DENV is internalized via receptor-mediated endocytosis [24]. Upon binding to receptors, the clathrin-mediated endocytosis is considered to be a major pathway for DENV entry [24]. In this study, honokiol was applied to Huh7 cells after DENV attachment to test if it can interfere with the viral entry process. Infection with DENV increased the amount of intracellular early endosomes ( Figure 5A,B), and the DENV E protein was found to co-localize with the early endosomes in DENV-infected cells, indicating its interactions, which is a distinct early event of endocytosis during DENV entry [25,26]. Intriguingly, the presence of honokiol (10 and 20 µ M) after DENV entry substantially suppressed the up-regulation of early endosomes, as compared to the DENV infected/mock-treated cells ( Figure 5C,D vs. 5B). Consequently, the co-localization of DENV E protein and the early endosome was markedly abrogated. This result indicates that honokiol may interfere with the endocytotic process of DENV during its entry into the host cells.

Honokiol Mediated DENV Inhibition is Not Attributed to the Influence on Cell Cycle Progression
Studies have demonstrated that honokiol can induce G0-G1 cell cycle arrest in cancer cells [27]. In a previous study, hepatocarcinoma cell HepG2 was demonstrated to be more permissive for DENV infection and virus production in the G2 phase as compared to other phases examined [28]. Although the concentration of honokiol used in this study was equal or lower than the maximum non-toxic dose for cells, there is a possibility that the cell cycle modulation by honokiol may affect the propagation of DENV. In this study, the effect of honokiol on cell cycle regulation was examined. In BHK cells, treatment of 10 µM honokiol for 48 h did not result in any alteration of cell cycle progression ( Figure 6A and Figure S1A). In Huh7 cells, a slight increase of G0/G1 and decrease of G2 phase was observed after 48 h of 20 µM honokiol treatment ( Figure 6B and Figure S1B). However, compared to the profound inhibition of DENV propagation at 10 and 20 µM of honokiol treatment in BHK and Huh7 cells, respectively ( Figure 2B,C), the influence of cell cycle modulation by honokiol in DENV production is insignificant. This result suggests that the inhibition of DENV propagation by honokiol is not mediated mainly through altering the cell cycle phases in BHK and Huh7 cells.

Honokiol Mediated DENV Inhibition is Not Attributed to the Influence on Cell Cycle Progression
Studies have demonstrated that honokiol can induce G0-G1 cell cycle arrest in cancer cells [27]. In a previous study, hepatocarcinoma cell HepG2 was demonstrated to be more permissive for DENV infection and virus production in the G2 phase as compared to other phases examined [28]. Although the concentration of honokiol used in this study was equal or lower than the maximum non-toxic dose for cells, there is a possibility that the cell cycle modulation by honokiol may affect the propagation of DENV. In this study, the effect of honokiol on cell cycle regulation was examined. In BHK cells, treatment of 10 µ M honokiol for 48 h did not result in any alteration of cell cycle progression ( Figure 6A and Figure S1A). In Huh7 cells, a slight increase of G0/G1 and decrease of G2 phase was observed after 48 h of 20 µ M honokiol treatment ( Figure 6B and Figure S1B). However, compared to the profound inhibition of DENV propagation at 10 and 20 µ M of honokiol treatment in BHK and Huh7 cells, respectively ( Figure 2B,C), the influence of cell cycle modulation by honokiol in DENV production is insignificant. This result suggests that the inhibition of DENV propagation by honokiol is not mediated mainly through altering the cell cycle phases in BHK and Huh7 cells.

Neither NF-κB Activation Nor IFN-β Expression was Essential in Honokiol Mediated DENV Inhibition
Nuclear factor-kappa B (NF-κB) is an essential molecule in the induction of type I interferon beta (IFN-β) and cytokines in the toll-like receptor (TLR) signaling pathway [29,30]. The strain of DENV-2 PL046 used in this study has been shown to trigger a low degree of NF-κB p65 nuclear translocation [31]. Thus, the DENV inhibition by honokiol was checked to see if the NF-κB activation was involved with the use of an NF-κB-dependent luciferase reporter. It was found that the infection of DENV in Huh7 cells did not induce the activation of NF-κB, a phenomenon also observed in a previous study [32], nor did the treatment with honokiol for 48 h in mock or DENV infection ( Figure  7A). In fact, honokiol was reported to inhibit NF-κB activation in several cell types [33,34]. Type I interferon (IFN-/β) plays an important role in the generation of antiviral immune responses [35,36]. The ability of DENV to inhibit IFN-α/β signaling in cells was recognized and attributed to several DENV proteins [37][38][39][40][41]. To investigate if honokiol up-regulates the IFN-β expression to reduce the DENV infectivity, an IFN-β promoter-reporter assay was conducted in Huh7 cells. Similarly, it was found that neither honokiol treatment nor DENV infection in Huh7 cells induced the expression of IFN-β ( Figure 7B). Additionally, the nuclear translocation of interferon regulatory factor-3 (IRF-3), an upstream factor that regulates the expression of IFN-β, was not observed in either DENV infection or honokiol or poly(I:C) treatments ( Figure S2; poly(I:C) is an immunostimulant similar to dsRNA). The low/no responsiveness of the Huh7 cell line to poly(I:C) treatment was reported due to the intrinsic defect in TLR3 expression [42]. These results collectively indicate that honokiol does not exert its interference of DENV infection via the NF-κB or IFN-β pathways in this study.

Neither NF-κB Activation Nor IFN-β Expression was Essential in Honokiol Mediated DENV Inhibition
Nuclear factor-kappa B (NF-κB) is an essential molecule in the induction of type I interferon beta (IFN-β) and cytokines in the toll-like receptor (TLR) signaling pathway [29,30]. The strain of DENV-2 PL046 used in this study has been shown to trigger a low degree of NF-κB p65 nuclear translocation [31]. Thus, the DENV inhibition by honokiol was checked to see if the NF-κB activation was involved with the use of an NF-κB-dependent luciferase reporter. It was found that the infection of DENV in Huh7 cells did not induce the activation of NF-κB, a phenomenon also observed in a previous study [32], nor did the treatment with honokiol for 48 h in mock or DENV infection ( Figure 7A). In fact, honokiol was reported to inhibit NF-κB activation in several cell types [33,34]. Type I interferon (IFN-α/β) plays an important role in the generation of antiviral immune responses [35,36]. The ability of DENV to inhibit IFN-α/β signaling in cells was recognized and attributed to several DENV proteins [37][38][39][40][41]. To investigate if honokiol up-regulates the IFN-β expression to reduce the DENV infectivity, an IFN-β promoter-reporter assay was conducted in Huh7 cells. Similarly, it was found that neither honokiol treatment nor DENV infection in Huh7 cells induced the expression of IFN-β ( Figure 7B). Additionally, the nuclear translocation of interferon regulatory factor-3 (IRF-3), an upstream factor that regulates the expression of IFN-β, was not observed in either DENV infection or honokiol or poly(I:C) treatments ( Figure S2; poly(I:C) is an immunostimulant similar to dsRNA). The low/no responsiveness of the Huh7 cell line to poly(I:C) treatment was reported due to the intrinsic defect in TLR3 expression [42]. These results collectively indicate that honokiol does not exert its interference of DENV infection via the NF-κB or IFN-β pathways in this study.

Discussion
From a global health perspective, dengue virus (DENV) is the most widespread arbovirus infection in the last decades [43]. Infection with DENV leads to approximately 500,000 severe life-threatening cases and over 20,000 deaths every year [44]. Aside from attempts to control the spread of the mosquito vectors and efforts to develop a vaccine against DENV infection [45], the development of antiviral drugs for treatment of dengue fever and prevention of severe complications is an urgent priority. In this study, we report that honokiol interferes with DENV infection and propagation at multiple stages. Our study demonstrates that honokiol may be a potent anti-DENV target for antiviral therapy of dengue.
Honokiol is a component in the commonly used Chinese medicine Hou-pu from the bark of the Magnolia tree. Hou-pu has been widely used as a folk remedy for gastrointestinal disorders, anxiety, analgesic, and other diseases in Eastern medicine [9]. Honokiol has recently been shown to inhibit hepatitis C virus infection in vitro [16]. Our present study is the first study to demonstrate that honokiol possesses anti-DENV-2 activity. It would be interesting to investigate if honokiol also has an antiviral effect against other Flavivirus, such as yellow fever virus, West Nile virus, and Japanese encephalitis virus. The pharmacokinetics of honokiol has been evaluated in mice by intraperitoneal injection. At a dose of 250 mg/kg, the plasma concentration was reported to reach 1000 µ g/mL (approx. 3755 µ M) between 20-30 min after administration [46]. The pharmacokinetics of honokiol by oral administration of Hou-pu extracts had also been studied in rats. At a dose of 5 g/kg extracts (which contained 1.61 mg/kg honokiol), the maximum plasma concentration of honokiol was reported to be 1.966 mg/L (approx. 7.5 µ M) [47]. The relative small molecular weight of honokiol and its lipophilic characteristic likely contributes to its rapid distribution at multiple sites and beyond the blood-brain barrier [15]. In some severe DHF cases, encephalopathy is observed in patients with high morbidity and mortality [48]. Recent evidence has suggested that DENV is capable of central nervous system infection [49]. Since honokiol is able to readily cross the blood-brain barrier and the blood-cerebrospinal fluid barrier [15], it may serve as a potent agent for treatment of this critical complication.
The CC50 of honokiol in Huh7 cells is 31.19 µ M at 48 h (Figure 2A), and the IC50 of honokiol (by reduction of DENV infection in fluorescence focus assay) in Huh7 cells is 10.6 µ M ( Figure 2C). The selective index of the drug, when calculated by CC50/IC50, is 2.94. This relative low ratio could be attributed to the apoptosis-inducing effects of honokiol on many tumor or transformed cells [10,27]. PMA is an NF-κB activator and was used as a positive control; (B) Assay of IFN-β promoter activity. Huh7 cells transfected with IFN-β-luciferase reporters were mock or infected with DENV at MOI = 5 and then treated with honokiol at 10 and 20 µM for 48 h, followed by Firefly-Renilla luciferase assay to determine the relative expression of IFN-β promoter-driven luciferase. Poly(I:C) is a immunostimulant similar to dsRNA and was used as a control. Data indicate the average value of triplicates (mean˘SD). *** p < 0.001 as compared with the control.

Discussion
From a global health perspective, dengue virus (DENV) is the most widespread arbovirus infection in the last decades [43]. Infection with DENV leads to approximately 500,000 severe life-threatening cases and over 20,000 deaths every year [44]. Aside from attempts to control the spread of the mosquito vectors and efforts to develop a vaccine against DENV infection [45], the development of antiviral drugs for treatment of dengue fever and prevention of severe complications is an urgent priority. In this study, we report that honokiol interferes with DENV infection and propagation at multiple stages. Our study demonstrates that honokiol may be a potent anti-DENV target for antiviral therapy of dengue.
Honokiol is a component in the commonly used Chinese medicine Hou-pu from the bark of the Magnolia tree. Hou-pu has been widely used as a folk remedy for gastrointestinal disorders, anxiety, analgesic, and other diseases in Eastern medicine [9]. Honokiol has recently been shown to inhibit hepatitis C virus infection in vitro [16]. Our present study is the first study to demonstrate that honokiol possesses anti-DENV-2 activity. It would be interesting to investigate if honokiol also has an antiviral effect against other Flavivirus, such as yellow fever virus, West Nile virus, and Japanese encephalitis virus. The pharmacokinetics of honokiol has been evaluated in mice by intraperitoneal injection. At a dose of 250 mg/kg, the plasma concentration was reported to reach 1000 µg/mL (approx. 3755 µM) between 20-30 min after administration [46]. The pharmacokinetics of honokiol by oral administration of Hou-pu extracts had also been studied in rats. At a dose of 5 g/kg extracts (which contained 1.61 mg/kg honokiol), the maximum plasma concentration of honokiol was reported to be 1.966 mg/L (approx. 7.5 µM) [47]. The relative small molecular weight of honokiol and its lipophilic characteristic likely contributes to its rapid distribution at multiple sites and beyond the blood-brain barrier [15]. In some severe DHF cases, encephalopathy is observed in patients with high morbidity and mortality [48]. Recent evidence has suggested that DENV is capable of central nervous system infection [49]. Since honokiol is able to readily cross the blood-brain barrier and the blood-cerebrospinal fluid barrier [15], it may serve as a potent agent for treatment of this critical complication.
The CC 50 of honokiol in Huh7 cells is 31.19 µM at 48 h (Figure 2A), and the IC 50 of honokiol (by reduction of DENV infection in fluorescence focus assay) in Huh7 cells is 10.6 µM ( Figure 2C). The selective index of the drug, when calculated by CC 50 /IC 50 , is 2.94. This relative low ratio could be attributed to the apoptosis-inducing effects of honokiol on many tumor or transformed cells [10,27]. Honokiol had been demonstrated to selectively inhibit the proliferation of transformed cells while not affecting normal cells under the same condition. It has been reported that at 20 µM of honokiol treatment for 48 h, there is no cytocidal effect on human normal peripheral blood mononuclear cells while the inhibition of B leukemia cells is over 95% [50]. The CC 50 of Huh7 hepatoma cells was found to be 31 µM in our study (Figure 2A) and 35 µM in another study [16] under honokiol treatment, while the CC 50 of normal AML12 hepatocytes was reported to be >150 µM [51]. In another study, at concentrations up to 40 µg/mL (approx. 150 µM), honokiol had little cytotoxicity on human fibroblast cells and lymphocytes [46]. It is apparent that normal cells are much more resistant to the cytotoxic effect of honokiol than tumor cells. These results may indicate that the selective index of honokiol in anti-DENV activity would be more significant in normal cells than the results we obtained by the transformed cells. In this study, we showed that honokiol inhibits the activity of DENV-2 replicons and suppresses DENV-2 replication in BHK and Huh7 cells (Figures 1 and 2). Honokiol treatment reveals a profound and dose-dependent inhibition of DENV-2 replication, with more than 90% reduction at the maximum non-toxic dose (10 and 20 µM) as compared with the vehicle control in the focus-forming assay (Figure 2). At these concentrations, the effect of honokiol on cell cycle progression of BHK and Huh7 cells was found to be minimal ( Figure 6). These results indicate that the prominent antiviral activity of honokiol is not attributed to its cytotoxic effect on host cells. In addition, treatment with honokiol was revealed to suppress the synthesis of viral dsRNA, and NS1/NS3 proteins (Figure 3). In BHK cells, treatment with 5 µM of honokiol induced a~50% reduction of DENV dsRNA, NS1, and NS3, while at 10 µM these three viral products had almost completely subsided ( Figure 3A,C). At 20 µM of honokiol treatment, a significant suppression of DENV NS1/NS3, and dsRNA was also noted in Huh7 cells ( Figure 3B,D). The decrease of viral dsRNA by honokiol may result in reduced DENV replication observed in viral yield reduction and focus-forming assay ( Figure 2B,C). It is possible that honokiol inhibits DENV replication by interfering with the expression of DENV NS1/NS3, and consequently suppressing the synthesis of viral dsRNA through mechanisms that require future investigation. Interestingly, it was reported that patients with DENV-2 infections experienced more severe disease than those infected with other serotypes [52]. Higher titers of DENV present in the blood during the viremic phase had also been linked with increased severity of dengue [52]. Therefore, viral load reduction by the use of effective antiviral agent, such as honokiol, may conceivably decrease the chance of severe dengue complications.
Pre-treatment of honokiol did not reveal to have effects on DENV-2 infection (Figure 4). This may indicates that treatment of honokiol prior to DENV infection did not alter any host cell-related mechanisms concerning the attachment of DENV. However, honokiol did interfere with the endocytotic process of DENV-2 ( Figure 5). Subsequent to receptor-mediated endocytosis, many enveloped viruses use virus-endosome fusion to transport viral genome into the cytosol of the host cell. DENV is reported to fuse with late endosomes, while the activation of fusogenic DENV E protein is triggered at a pH-dependent characteristic for early endosomes [25,53,54]. The interaction of DENV E protein and the early endosome is an early event of DENV entry during virus-endosome fusion [25]. In our study, the presence of honokiol (10 and 20 µM) after DENV entry substantially suppressed the up-regulation of early endosomes. Moreover, the treatment of honokiol abrogated the co-localization of DENV E protein and the early endosome ( Figure 5). This observation suggests that honokiol can interfere with the process of DENV-endosome fusion by mechanisms yet to be determined. A fluorescence resonance energy transfer-based single-virus tracking assay [55] was performed to further confirm the interference of honokiol on DENV entry. Our preliminary results also supported that honokiol interferes with the endocytosis of DENV during its entry (unpublished observation). Altogether, these results indicate that honokiol can interfere with the endocytotic process of DENV-2, which may reduce the release of viral RNA into cytoplasm and inhibit the subsequent viral gene expression and viral genome replication in host cells.
In a previous study, HepG2 cell was demonstrated to be more permissive for DENV infection and replication in the G2 phase [28], suggesting that cell cycle may be a mediator of cell permissiveness for DENV. Honokiol has been demonstrated to induce G0-G1 cell cycle arrest and eventually lead to apoptosis in cancer cells [27]. The effect of honokiol on the cell cycle regulation was studied. It was found that treatment of honokiol at MNTD did not have a discernible influence on the cell cycle of BHK cells, and the effect on the Huh7 cells was fairly limited (~10%, Figure 6). At the MNTD of honokiol treatment, the inhibition of DENV-2 replication was very prominent (>90% reduction, Figure 2). These results indicate that honokiol is not likely to suppress the DENV replication by modulating cell cycles in BHK and Huh7 cells.
NF-κB is an essential molecule in the induction of interferon (IFN)-β and cytokines in the toll-like receptor (TLR) signaling pathway [29,30], and these factors play vital roles in innate immunity against DENV infection [56]. However, a recent study has indicated that NF-κB activation triggered by TLR ligands is preferentially blocked by DENV-2 PL046 infection [31]. In this study, the honokiol-mediated DENV inhibition through modulation of NF-κB activation in Huh7 cells was investigated. It was found that neither infection of DENV-2 nor honokiol treatment induced NF-κB activation in Huh7 cells in vitro ( Figure 7A). In a previous study, it was also observed that Huh7 cells infected with DENV were incapable of inducing the activation of NF-κB [32]. Rather than promoting, honokiol was reported to inhibit NF-κB activation in several cell types [33,34]. Thus, NF-κB activation is dispensable for the inhibitory effect of honokiol on DENV infection in this model. In virus-infected cells, the onset of IFN-α/β response occurs on viral entry and release or synthesis of viral components [57]. Several transcription factors, including IFN regulatory factor (IRF)-3, IRF-7, NF-κB, and activating transcription factor 2 (ATF2)/c-Jun are being activated and trigger the expression of IFN-α/β [58]. The released IFN-α/β elicits the antiviral response of the infected and neighboring cells. In this study, neither the expression of the IFN-β nor the activation of IRF-3 (an upstream regulator of IFN-β) was altered in Huh7 cells after DENV infection or honokiol treatments ( Figure 7B and Figure S2). The low/no responsiveness of the Huh7 cell line in terms of NF-κB activation and IFN-β expression to DENV infection or poly(I:C) treatment was reported due to the intrinsic defect in TLR3 expression [42]. Collectively, these results suggest that honokiol can still exert its anti-DENV response effectively in Huh7 cells without regulating the NF-κB or IFN-βpathways.
In summary, we demonstrated that honokiol, a lignan biphenol extract from the Magnolia tree, exhibits profound antiviral activity against DENV-2. Honokiol suppresses the replication of DENV-2 in infected cells in vitro. Treatment with honokiol inhibits the expression of DENV NS1, NS3, dsRNA, and interferes with the entry process of DENV. Honokiol stands as a potentially promising new therapeutic agent for anti-DENV chemotherapy.

Cell Cytotoxicity Assay
Cell viability was determined using the standard MTT assay. Briefly, cells were seeded in 96-well plates at a density of 3ˆ10 3 cells/100 µL per well overnight and then treated with honokiol at various concentrations (final volume 200 µL per well after addition of honokiol) for the times indicated. At the end of treatments, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 2.5 mg/mL; Sigma-Aldrich, St. Louis, MO, USA) was added to the culture medium and incubation continued at 37˝C for 3 h. After incubation, the supernatant was removed and the formation of dark formazan was dissolved in DMSO and measured using a microplate reader at an absorption wavelength of 570 nm. The 50% cytotoxic concentration (CC 50 ) was defined as the compound's concentration (µM) required for the reduction of cell viability by 50%, which was calculated by regression analysis.

DENV Replicon Assay
BHK-D2-Fluc-SGR-Neo-1, a stably transfected BHK cell line harboring a luciferase-reporting DENV subgenomic replicon [21], was seeded in 96-well plates at a density of 5ˆ10 3 cells/100 µL per well overnight and then treated with honokiol at various concentrations for 24 h. The culture medium was removed and cells were rinsed two times with PBS. Cells were lysed with 100 µL of lysis buffer (Promega, Madison, WI, USA), and the luciferase activity was evaluated following the manufacturer's protocol (Luciferase Assay System, Promega). The luciferase activity was measured by microplate reader at an absorption wavelength of 640 nm (EnSpire multimode plate reader, PerkinElmer, Waltham, MA, USA).

Viral Yield Reduction and Fluorescence Focus Formation Assay
The BHK and Huh7 cells were first infected with DENV-2 by incubation with medium containing the virus for 1 h. After infection, the medium was replaced with virus-free medium containing a different concentration of honokiol and incubated for 48 h. The supernatant were collected, diluted 10-fold serially with serum free medium, and added to a 96-well plate with confluent BHK cells. After 1 h of incubation, 100 µL of DMEM/FBS containing 0.8% methyl cellulose was added to each well and the cells were cultured at 37˝C with 5% CO 2 for 72 h. The resulting cells were washed with PBS, fixed with 3.7% formaldehyde/PBS, and then permeabilized with 0.1% Triton X-100/PBS. The anti-DENV-2 NS1 antibody 206-35 was added and incubated for 1.5 h. The cells were then washed and incubated with secondary HRP-conjugated goat anti-mouse IgG antibody (1:1000, GeneTex) for 1.5 h at room temperature. After extensive washes, tyramide-fluorescein [61] was added to each well and the fluorescent foci were analyzed by the Typhoon FLA 9000 Biomolecular Imager (GE Healthcare, Pittsburgh, PA, USA).

Immunofluorescence Staining
The following procedure was used for immunofluorescence staining in this study. After treatment, cells were washed twice in PBS and followed by fixation with 3.7% formaldehyde/PBS and permeabilization with 0.1% Triton X-100/PBS, except for dsRNA staining where the cells were fixed and permeabilized with ice-cold methanol for 15 min. The cells were incubated with primary antibody for 1.5 h at room temperature, and then washed and incubated with corresponding secondary antibody (1:5000) for 30 min at room temperature. Cell nuclei were stained with Hoechst 33258 (1 µg/mL) for 10 min. Immunostained cells were washed thoroughly with PBS and then examined by confocal microscopy or BD PathwayTM 435 high-content cell analyzers (BD Biosciences, Rockville, MD, USA). Antibodies against DENV-2 NS1 (clone 206-35), NS3 (clone 9-9), and envelope E protein (clone 137-22) [60], dsRNA (clone J2, English & Scientific Consulting, Bt, Szirák, Hungary), early endosome antigen-1 (EEA1, GeneTex, Irvine, CA, USA), IRF-3 (Abcam, Cambridge, UK) were used as the primary antibodies in these analyses. Anti-mouse IgG Alexa 488, anti-rabbit IgG Alexa 488, and anti-rabbit IgG Alexa 546 (Invitrogen, Eugene, Oregon), were used as secondary antibodies. For immunofluorescence assay of viral dsRNA, NS1 and NS3, cells were seeded in Cellcarrier-96well plates overnight and then infected with DENV-2 for 1 h. Honokiol was added at indicated concentration and incubated for 48 h. The resulting cells were immunofluorescence stained as described above. The images were acquired by BD PathwayTM 435 Bioimaging system and the percentage of viral dsRNA, NS1 or NS3 positive cells was analyzed using the Attovision software (BD Biosciences).

DENV Attachment Assay
To determine if pre-treatment of honokiol inhibits the attachment of DENV, BHK cells were seeded in 24-well plates at a density of 1ˆ10 5 cells per well overnight and then treated with vehicle or 10 µM honokiol for 24 and 48 h. After treatment, the cells were detached and incubated with DENV at MOI = 10 for 30 min at 4˝C. The neutralizing anti-DENV E antibody 137-22 was used as a control to block the specific DENV binding to host cells. After incubation, the cells were washed twice with PBS and then incubated with anti-DENV E antibody (137-22) for 30 min at 4˝C. The cells were then washed and incubated with anti-mouse IgG Alexa 488 and analyzed by flow cytometry (Cytomics FC500, Beckman Coulter, CA, USA).

Assay of DENV Endocytosis
Huh7 cells were first infected with DENV on ice for 1 h and then washed twice with PBS. Medium containing vehicle or honokiol was then added and the cells were incubated at 37˝C for 90 min. The cells were then fixed, stained with first and secondary antibody as described in the immunofluorescence staining section. DENV E (clone 137-22) and EEA1 antibody were used to detect DENV E protein and early endosome, respectively.

Cell Cycle Analysis
Cells were treated with honokiol for 48 h. After treatment, the cells were fixed in ice-cold 70% ethanol, washed with PBS, and then stained with propidium iodide/RNase A in PBS (Sigma-Aldrich). The cell cycle distribution of stained cells was analyzed by flow cytometry (Cytomics FC500, Beckman Coulter, CA, USA).

Statistical Analysis
Differences between multiple groups were analyzed by one-way ANOVA with Dunnett's method for pairwise comparisons. p < 0.05 was considered to be statistically significant.